Digestive Tract

Digestive Tract

C H A P T E R 56 Digestive Tract Timothy A. Bertram1, John W. Ludlow1, Joydeep Basu1, Sureshkumar Muthupalani2 1 Tengion Inc., Winston-Salem, NC, US...

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56 Digestive Tract Timothy A. Bertram1, John W. Ludlow1, Joydeep Basu1, Sureshkumar Muthupalani2 1

Tengion Inc., Winston-Salem, NC, USA, 2Massachusetts Institute of Technology, Cambridge, MAs, USA

O U T L I N E 1. Introduction 2. Structure and Function of the Gastrointestinal Tract 2.1. Macroscopic and Microscopic Structure and Function 2.2. Enteric Lymphoid System 2.3. Enteric Nervous System 2.4. Biotransformation 2.5. Enterohepatic Circulation 2.6. Bacterial Metabolism 2.7. Gut Microflora and Microbiology 3. Evaluation of Gastrointestinal Toxicity 3.1. In Vitro Strategies 3.2. In Vivo Strategies 3.3. Molecular Pathology 3.4. Morphological Methods 3.5. Animal Models 3.6. Microfloral Impact on Pharmacologic and Toxicity Studies

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5. Mechanisms of Gastrointestinal Toxicity 5.1. Intestinal Barrier Function 5.2. Intestinal Malabsorption 5.3. Hypoxia 5.4. Mucosal Barrier Damage and Cytotoxicity 5.5. Hypersensitivity 5.6. Acetylcholinesterase Inhibitors 5.7. Microfloral Effects 5.8. Carcinogenicity

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Suggested Reading



1. INTRODUCTION The purpose of this chapter is to discuss the mechanisms by which toxic xenobiotics produce their deleterious effects and to determine the consequences of this toxicity on the integrity of gastrointestinal structure and function. The intrinsic ability of the gastrointestinal tract to resist toxic chemicals has led to a paucity of data regarding gastrointestinal toxicology, yet Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Third Edition. http://dx.doi.org/10.1016/B978-0-12-415759-0.00056-X

4. Response of the Gastrointestinal Tract to Injury 4.1. Pathophysiological Responses 4.2. Inflammatory Response 4.3. Mucosal Response 4.4. Organ-Specific Response 4.5. Regenerative Response

this organ system can be readily perturbed, leading to easily identified toxic responses such as emesis or diarrhea. Other perturbations such as insufficiency of some enzymes (e.g. lactase, lipase); the presence of localized inflammation, polyps, or neoplasms; changes in function such as excess production of mucus or delayed gastric emptying; or structural damage such as ulcers are more difficult to identify and attribute to toxicologic processes. For these reasons, it is


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necessary to identify those functions and structures of the gastrointestinal tract that are subject to direct or indirect chemical toxicity. When considering the potential toxic activity of various agents on the gastrointestinal tract, a number of signalments are possible. Acute effects may result from direct irritants (e.g., strong acids and bases), whereas chronic effects may be observed as increased muscular layer thickness from bulking agents. Importantly, delayed effects can be expressed years after exposure to ulcerogenic or carcinogenic agents. In addition to the array of tissue responses, interpretation of functional and morphological alterations can be complex. For example, increased mucosal thickness can occur when toxic compounds induce cellular proliferation and hyperplasia (e.g., enterochromaffin cell-like hyperplasia) or when non-toxic foodstuffs such as fiber induce generalized mucosal growth. The principal functions of the gastrointestinal tract that are subject to toxic effects of chemicals include storage, propulsion, digestion, absorption, secretion, barrier activity, and elimination. Due to the importance of nervous reflexes and hormones in regulation of the gastrointestinal tract, this organ system is relatively unusual in that toxic effects of chemicals at one site (e.g., stomach) may be expressed at another site (e.g., colon). The gastrointestinal tract is the entry site into the body of orally administered compounds that may be highly toxic to other internal organs yet have little or no noticeable effect on the gastrointestinal tract. A distinctive feature of the gastrointestinal tract is the high proliferative and metabolic rate of the mucosa. Because of its high rate of mitotic and metabolic activities, the gastrointestinal tract is more susceptible to toxicant-mediated injury than are most other organ systems. In addition, the gastrointestinal tract mucosa is a complex barrier that must exclude bacteria and their molecular toxins and, at the same time, absorb nutrient molecules that are vital for homeostasis. Furthermore, this organ system cannot sustain widespread toxicity without serious direct and indirect consequences to the rest of the body, if for no other reason than nutrient malabsorption with consequential malnutrition or starvation. The gastrointestinal tract is the only internal organ system that contains many endogenous

biotransforming and toxigenic bacteria, as well as inert drug-binding materials. Consequently, when a compound is present in the gastrointestinal milieu, the ultimate toxicity to this organ system will be determined by interactions of the chemical with bacterial and mammalian enzymes, and by the extent of respective detoxification or activation processes. The ability to evaluate genomic, proteomic, biochemical, or morphological changes in the gastrointestinal tract can be complicated because of the matrix of interactions and its exquisite sensitivity to autolysis and post-mortem alterations. Many subtle toxicologic events that occur at cellular and subcellular levels may only be observed by careful and proper handling of the gastrointestinal tissues immediately after death. The focus of this chapter is the examination of developmental, structural, and functional components of the gastrointestinal tract that are important in understanding mechanisms involved in the toxicologic pathology of this organ system. A major part of the chapter stresses both basic mechanisms of toxicologic damage and how the gastrointestinal tract responds to toxicologic insult. Also discussed are approaches that can be used to study selected pathologic mechanisms of toxicological significance, and how microbiology of the gut can affect toxicology. The latter part of the chapter covers the regenerative biology of the gastrointestinal tract, with an emphasis on tissue engineering of the small intestine as a means to overcome the effects of injury or toxicity.

2. STRUCTURE AND FUNCTION OF THE GASTROINTESTINAL TRACT A brief overview of the anatomy and physiology of the gastrointestinal tract, with important species differences, is provided to give a foundation for the understanding of gastrointestinal toxicological pathology.

2.1. Macroscopic and Microscopic Structure and Function Although many of the basic features of the gastrointestinal tract are similar for various species (Figures 56.1, 56.2), major interspecies variations are present in the fore- and hindgut




FIGURE 56.1 Epithelial cells of the gastrointestinal tract are derived from clonal stem cells. Toxic injury to these stem cells will disrupt the functional and structural development of the crypt–villus unit. Figure reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 1, p. 123, with permission.

(Table 56.1). This notwithstanding, within each major macroscopic variation the cell types composing the mucosal lining of the gastrointestinal tract are remarkably similar (Figure 56.3; Table 56.2). Esophagus The function of the esophagus in all species is to act as a conduit for food materials to leave the oral cavity and enter the gastrointestinal tract. Interspecies esophageal variations occur regarding the presence and extent of smooth and striated muscle, characteristics of the gastroesophageal junction, and sacculated adaptations (forestomachs). In all species, the esophagus is lined by stratified squamous epithelium with varying degrees of keratinization of the surface layers. The extent of keratinization of the esophageal and non-glandular gastric epithelium is dependent on the amount and type of dry foodstuff

ingested in the diet. Consequently, hyperkeratosis (cornification) of the mucosa can indicate anorexia or an increase in roughage content of the feed in both ruminants and rodents. This keratinized epithelium imparts a whitish color when the esophagus is viewed macroscopically. Keratinization is also a normal feature of the non-glandular portion of the rodent stomach. The tunica muscularis of the esophagus has two muscle layers composed of striated, smooth, or a mixture of both types of muscle, depending on the species. Variations in esophageal musculature account for the ability or inability of an animal to vomit or regurgitate. The absence of significant amounts of striated muscle in the esophagus of rats and the presence of a limiting ridge (margo plicatus) in the stomach explains why these animals are unable to vomit. The ability of dogs and guinea pigs to vomit and




ruminants to regurgitate is dependent upon the presence of striated muscle in the esophagus. Regurgitation, not vomiting, in non-ruminants indicates esophageal dysfunction or obstruction. If the esophagus is perforated by caustic compounds, chronic drainage of saliva and ingested foodstuffs into the submucosa or intrathoracic regions leads to severe inflammatory reactions, fibrosis, and strictures. The esophagus does not heal as rapidly as other portions of the gastrointestinal tract because of a marginal blood supply and a minimal amount of adventitial and serosal connective tissue.

Crypt of Lieberkühn Lymph nodule Villi Gland in submucosa Epithelium Lamina propria Muscularis mucosa

Auerbach’s plexus

Stomach Anatomical and functional variations are important considerations when designing animal studies, since compound absorption and enzyme exposure (e.g., ruminal bacteria or inflammatory cell proteases) often vary from species to species, and sites of storage (e.g., non-glandular stomach) may provide prolonged contact between the host mucosa and a toxic compound.

Mucosa Submucosa Meissner’s plexus Tunica muscularis Serosa

FIGURE 56.2 Schematic drawing of the intestinal tract. This basic tissue organization demonstrates the general organization of the entire gastrointestinal tract. Brunner’s glands are located in the submucosa of the duodenum only. Villi are present in the small intestine only. Figure reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 2, p. 124, with permission.


The stomach functions to store and macerate food – the necessary beginning of the early phases of food digestion. Some phylogenetic orders have a highly sacculated forestomach (e.g., some artiodactyls and primates) aiding in food digestion. In ruminants, the ruminal portion (rumen) of the forestomach, which is actually a modification of the esophagus, is highly permeable to volatile

TABLE 56.1 Variations in Dietary Consumption Related to Gastrointestinal Structure in Various Mammalsa Stomach Sacculated

Stratified squamous epithelium


Sacculated colon

























Lagomorpha Primates


Artiodactyla Marsupialia Perissodactyla


Large intestine


Table adapted from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Table I, p. 124, with permission.



fatty acids released from the microbial metabolism of complex carbohydrates, and is capable of active sodium and chloride absorption. Several orders, including rodents and perissodactyls (horse and swine), have a non-glandular stratified squamous portion of the stomach adjacent to fundic or cardiac mucosa. This squamous portion of the stomach is separated from the glandular stomach by a limiting ridge (margo plicatus) and serves as a storage organ for ingested material. Various inflammatory cells (lymphocytes, plasma cells, and eosinophils) may be present in the lamina propria of the limiting ridge of rodents, being a normal characteristic, and should not be interpreted as an inflammatory process. STRUCTURE

The topographical organization of the gastric mucosa varies widely among species. As monogastric simple-stomached mammals, humans and dogs have the cardia as the first glandular portion of the stomach following the esophagus or squamous forestomach; the cardiac mucosa is macroscopically red. This portion of the stomach has foveolae (gastric pits) and tortuous mucous glands. The fundus is the next glandular region, consisting of mucosal convolutions called rugae. The distal portion of the stomach, the pylorus, also has rugae, but they are smaller than those of the fundus and are arranged obliquely in the direction of the antrum. Unlike other portions of the stomach, the foveolae of the antrum are deeper and make up as much as 50% of the mucosal thickness. In spite of many macroscopic variations, the microscopic arrangement of the stomach is similar in all species. The mucosa rests on the submucosa, and these two layers are surrounded by a muscular coat (tunica muscularis) that is covered by the single mesothelial cell layer of the serosa. The fundic mucosa contains glands that are composed of mucous surface neck cells, parietal (oxyntic) cells, chief (zymogen) cells, and enteroendocrine (enterochromaffin) cells (Table 56.2). Chief cells are cuboidal and have a basally placed nucleus. The apical portion of the cytoplasm is filled with pepsinogen-filled zymogen granules. The primary function of the chief cell is to release enzymes into the gastric lumen to begin the process of gastric digestion. Parietal cells are larger but generally less numerous


than chief cells. These cells have a centrally located nucleus, and the smooth endoplasmic reticulum and mitochondria-laden cytoplasm stains intensely eosinophilic. Parietal cells release hydrochloric acid (HCl), to maintain the gastric pH, and rennin in young animals, to facilitate digestion of milk. Carbonic anhydrase acting on CO2 produces carbonic acid that dissociates to provide Hþ for excretion. Both Cl and Hþ are actively secreted into the lumen with water following the osmotic gradient. Food material in the stomach, vagus nerve stimulation, gastric distension, and gastrin released from G cells in the glands stimulates the parietal cells to release Hþ. Stimulation of chief cells to release pepsinogen comes from a combination of vagal nerve stimulation, Hþ concentrations, gastrin, and duodenal secretin-cell released secretin. The majority of gastric glands produce acid and enzymes (e.g., pepsin), but in the antrum the mucosal glands produce mucus. Gastric mucus represents a composite of mucin, aqueous electrolytes, sloughed off cells, enzymes, nucleic acids, lipids, plasma proteins, secretory immunoglobulins and bacteria, and associated bacterial metabolites. Between 90% and 95% of gastric mucus is water, 5–10% mucin, 1% electrolytes, and approximately 5% all other components. Mucin, the principal component of gastric mucus, is synthesized by and secreted from mucus-producing cells resident within mammalian gastric mucosa. MUC5AC and MUC6 are the main mucins secreted by surface or glandular mucous cells of the human stomach. These mucins vary in their primary sequence and associated carbohydrate moieties. Mucins are high molecular weight polymers composed of glycoprotein subunits joined by disulfide bridges. Each glycoprotein subunit is composed of a central peptide core flanked by carbohydrate side chains. Hydrogel formation by aqueous mucin polymers leads to formation of a protective layer over the gastric mucosa, which further assists in lubrication of the mucosal surface and digestion. Cells of the gastric glands also release arachidonic acid metabolites (e.g., prostaglandins of the E series) that facilitate protection of the mucosa from the acid and digestive enzymes in the lumen. Cellular composition of gastric glands in the fundic mucosa varies among animal species. Intermixed with the gastric gland epithelium






TABLE 56.2


Cells Composing the Epithelial Lining of the Gastrointestinal Tracta

Cell type




Absorptive cell


Small intestine to colon

Nutrient absorption

Mucous cell

Cuboidal to columnar

Stomach to rectum


Chief cell


Stomach (fundic)

Pepsin, rennin, lipase

Enterochromaffin cells


Stomach to rectum

Endocrine (at least 10 types)

Goblet cell


Small intestine to rectum


M cell


Dome of Peyer’s patches

Processed antigens

Paneth cell


Small intestine

Lysozyme, peptidase

Parietal cell


Stomach (fundic)

HCl, intrinsic factor

Undifferentiated crypt cell


Small intestine to rectum

Progenitor cell

Vacuolated cell


Colon to rectum

Progenitor cell


The number of these individual type cells in any given anatomical location in the gastrointestinal tract can vary with animal species and diet. Additionally, toxicologic agents can markedly influence the distribution and relative ratios of each cell type. Table adapted from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Table II, p. 128, with permission.

are enteroendocrine cells, a population of cells of neural crest origin. Enteroendocrine cells are usually located between the basement membrane and chief cells. The granules of enteroendocrine cells can be visualized by silver stain histochemical technologies. These cells synthesize, store, and secrete hormones in response to autonomic and intralumenal stimuli. There are at least 10 different enteroendocrine cell populations in the mucosa. Enteroendocrine cells secrete serotonin, histamine, enteroglucagon (A cells), and gastrin (G cells).

Replication of the mucosal cells in the stomach is somewhat different from that of the rest of the gastrointestinal tract. Unlike the replication of cryptal cells of the intestines and basal cells of the esophagus, gastric mucosal cell replication occurs in the neck of the gastric glands. The next layer of the gastric mucosa, immediately below the epithelium, is the lamina propria. This layer is separated from the gastric epithelial cells by a basement membrane. The lamina propria of the cardia and pylorus contains high numbers of lymphocytes and plasma cells. These


FIGURE 56.3 Normal histology of the rat GI tract. (A) Non-glandular (squamous) portion of the stomach (S) from a rat at the junction (limiting ridge) with the fundus (F) with its deep gastric glands. The stratified squamous epithelium is covered by a layer of keratin (K) and the underlying lamina propria (L) is infiltrated by a resident population of inflammatory and immune cells. (B) Fundic (oxyntic) mucosa of the glandular stomach. Gastric pits (arrows) are lined by columnar epithelium and are the outlet for fundic gland (ellipses) secretions. (C) In a highermagnification view of the fundus, eosinophilic pyramidal parietal cells (large arrows) as well as smaller, more basophilic chief cells (small arrows) lining the gastric glands are indicated. Identification of the enteroendocrine cells that are also present in lower numbers requires special staining techniques (e.g., Grimelius stain). (D) Duodenal portion of the small intestine. Villi (V) are very long in relation to the crypts (C). Tunica muscularis (TM) and attached pancreas (P) are indicated. (E) Jejunal portion of the small intestine. Villi (V) are lined by columnar epithelial cells and have a central lacteal. Crypts (C) are composed of proliferating epithelial cells. (F) A section of ileum illustrates the shortness of the villi (V) in relation to the crypts (C). Numerous goblet cells (arrows) are also evident. (G) The cecal mucosa in the rat (as well as other species) is relatively thin, with only crypts (C) and numerous goblet cells (arrows). The submucosa (SM) is rather “loose” and the tunica muscularis (TM) is thin. (H) Colonic mucosa in the rat is highly folded and has abundant goblet cells (arrows). Colonocytes are not as tall as enterocytes and no villi are present, only crypts (C). Figure reproduced from Fundamentals of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 8.2A–H, pp. 166–167, with permission.




cells of the immune system are abundant throughout the gastric mucosa and submucosa, and the pyloric lamina propria may contain numerous lymphoid follicles in the healthy animal. This lymphoid tissue can markedly enlarge in disease states that involve antigenic stimulation (Figure 56.4). The lamina muscularis mucosae separates the mucosa from the submucosa. The submucosa,

is composed of a loose connective tissue matrix supporting many nerves and blood and lymphatic vessels. Three smooth muscle layers constitute the tunica muscularis, which encircles the stomach and functions to mix food and move contents from a storage site into intestinal segments for continued digestion and nutrient absorption. Small Intestine FUNCTION

This segment is primarily responsible for secretion and absorption of nutrients. In addition, the small intestine functions to biotransform compounds, resulting in bioactivation or detoxification (Table 56.3); as a barrier to luminal contents (bacteria and non-absorbed compounds); and as a conduit for ingesta to pass out of the body. Numerous anatomical modifications increase the functional capacity of the small intestine, including its long length, linear TABLE 56.3 Mucosal Metabolic Conjugation of Selected Chemicalsa Chemical






Anthranilic acid










þ þ


FIGURE 56.4 Lymphoid nodules in the mucosa and submucosa of an inflamed porcine stomach. Lymphoid proliferation can occur in regions where gut-associated lymphoid tissue is not well developed but where antigenic stimulation of the mucosal immune system is localized. Lymphoid nodules are not normally present in the mucosa or submucosa of the stomach. Follicular (F) and parafollicular (P) regions are well differentiated. Bar ¼ 500 mm. Figure reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 4, p. 129, with permission.














Salicyclic acid



Thyroxine analogs







Modified from Watkins and Klaassen (1997). Table adapted from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Table III, p. 129, with permission.



plicae, circular plicae (valves of Kerkring), villi, and microvilli. These characteristics influence mucosal surface area and can modify the transit time of a compound through the gastrointestinal tract. Relative to the stomach and large intestine, passage time through the small intestine is relatively rapid (i.e., a few hours). Between the proximal and distal small intestine a functional gradient of ion and water transport occurs, which controls the movement of fluids and electrolytes. In the proximal small intestine, passive movement of sodium and water is from the blood to the gastrointestinal tract lumen. In contrast, fluid and sodium movement is from the lumen to the blood in the distal small intestine. Net secretion occurs in the ileum and jejunum of guinea pigs, the ileum of rabbits, and the proximal portion of the jejunum in neonatal swine. The jejunum absorbs sodium, chloride, and bicarbonate against an electrochemical gradient; however, this decreases as the animal ages. Bile salts are primarily absorbed in the ileum. STRUCTURE

The small intestine constitutes the majority of the gastrointestinal tract’s length. Major structural features of the small intestine vary little among different species. The general microscopic organization of the small intestine is similar to that of the stomach, with three distinct layers (mucosa, submucosa, and tunica muscularis) surrounded by the serosa. Small-intestinal morphology reflects its absorptive function and can be artificially divided into two zones: villi for absorptive and enzyme release, and crypts for secretion and replication (Figure 56.2). Crypt-depth to villus-height ratios vary from species to species, and can be used to assess the degree of intestinal damage resulting from a toxic compound. The distance from the base of the crypt to the tip of the villus is divided by a “shoulder” at the crypt–villus junction (Figure 56.3D). This juncture demarcates the transition between the crypt and villus. The cryptdepth to villus-height ratio in the proximal small intestine ranges from a small 1 : 7 ratio in the pig to a larger 1 : 2 ratio in the dog. This ratio also will vary with the extent of food material in the lumen, lumenal distension, and diet. Villus height progressively decreases from proximal to distal small intestine. Villi are covered


by mature and senescent cells that have migrated along the basement membrane and are supported by a connective tissue core, the lamina propria. The center of the villus has blind-ended lymph vessels or lacteals (Figure 56.3D) that are surrounded by an elaborate capillary bed subjacent to the epithelial basement membrane. These lymphatic vessels serve to carry fat-soluble compounds to the systemic circulation, thus bypassing hepatic metabolism. Cells lining the mucosa of the small intestine are primarily composed of simple columnar epithelium on the villi and a cuboidal epithelium in the crypts (Table 56.2). Villous epithelial cells have a thick microvillar membrane (approximately 11 nm) and have multiple biotransforming and metabolizing enzymes on the luminal surface. The thickness and composition of this enzyme-rich apical membrane is maintained by cytoskeletal elements (microtubules) and the presence of tight junctions at the lateral membrane–junctional complexes. Enterocytes absorb simple carbohydrates, amino acids, and some xenobiotics and then actively transport them, with little processing, into subjacent capillaries for, ultimately, transportation to the liver. Each villus consists of 2000–8000 epithelial cells and is surrounded by 6–14 crypts of Lieberkuhn. The crypt is the proliferative unit of the intestinal mucosa, as cell division is confined to the crypts. Each crypt generates four types of terminally differentiated cells: enterocytes, goblet cells (secrete mucus), enteroendocrine cells, and Paneth cells (produce lysozymes), all serving as a barrier to bacteria. Unlike the other cells, the Paneth cells remain anchored in the lower portion of the crypt and live for approximately 21 days. Each crypt produces 300–400 cells per day, and each epithelial cell has an average life of 3 days. Each crypt has stem cells that divide rapidly to produce daughter cells. Daughter cells may themselves divide several times in the lower and middle portions of the crypts. The cells differentiate and mature during an orderly and rapid migration from the crypt to the apex of a surrounding villus. As the cells migrate up the villus, they differentiate both structurally and functionally. The differentiated cells generated by this process are of four distinct types: absorptive, goblet, enteroendocrine, and Paneth cells. Absorptive cells, also known as columnar cells or enterocytes, are the majority




cell type; the other three classes are all secretory. Absorptive cells present a brush border on their apical surface. Goblet cells secrete mucus, and their apical cytoplasm is typically distended with mucus-filled secretory granules. Enteroendocrine cells (themselves composed of many individual subspecies) are smaller and secrete various gastric hormones, such as catecholamines. Paneth cells secrete antibacterial proteins (lysozyme, defensins) and differ from other differentiated cell types by their location at the base of the intestinal crypts. Once the cells reach the apex of a villus, they are exfoliated. This process of proliferation, upward migration, and subsequent exfoliation is completed in 2–5 days. Intestinal crypt cells secrete fluids and electrolytes, which are reabsorbed by intestinal villus cells. The transport of electrolytes across epithelial cell apical membranes occurs by multiple mechanisms. Uniport mechanisms move a single ion (e.g., sodium), symport systems move two ions simultaneously in the same direction (e.g., sodium and chloride), and antiports are ion exchangers which move two ions in opposite directions (e.g., sodium and hydrogen). The sodium-chloride symport system can be blocked by acetazolamide and is sensitive to agents stimulating adenylate cyclase. These systems frequently require ATP and are stimulated by cAMP, cGMP, or increased levels of intracellular calcium. Fluid transport is also modulated by neurotransmitters such as serotonin (increases secretion) and neuropeptide Y (increases absorption). Toxicologic damage to membrane-bound proteins can influence the viability of the mucosal epithelial cells and nutritional status of the animal. Within the apical membrane are proteins that consist of intimately membrane-associated calcium–magnesium dependent ATPases and alkaline phosphatases, and less tightly held lactases, sucrases, maltases, and leucine aminopeptidases. The less tightly held enzymes are responsible for digestive processes, while the more tightly held ATPases control cellular homeostasis and viability. In the thinner lateral membranes (approximately 7 nm thick), ouabainsensitive sodium–potassium ATPase is found in concentrations that are higher than in the apical surface. This enzyme is tightly linked to glucose absorption. Absorptive epithelium of the mucosa is replaced by dividing cells in the crypts of the small

intestine (Crypts of Lieberkuhn) (Figure 56.5). Most extensive replication occurs in the cells immediately above the bottom four to six cells. Negative feedback mechanisms coordinate the rate of cell proliferation in the crypts with the rate of mature-cell loss at the villus tip, resulting in epithelial cells of the small and large intestine being replaced every 2–4 days. Other less numerous cells, such as goblet cells, are scattered among the absorptive columnar cells of the villi. The numbers of goblet cells increase in the villus mucosa from proximal to distal small intestine. Lysozyme- and peptidase-rich Paneth cells are found near the base of the crypts, associated with the proliferating cells, but there is no known dietary or environmental factor that

FIGURE 56.5 Crypt cells form the proliferating unit in the small intestine portion of the gastrointestinal tract. Bromodeoxyuridine incorporation into the proliferating S-phase cells (S). Bar ¼ 50 mm. Figure reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 5, p. 131, with permission.



controls this distribution (Table 56.2). Paneth cells are found in monkeys, mice, rats, hamsters, guinea pigs, ruminants, and horses, but not in dogs, cats, swine, or raccoons. Paneth cell numbers increase in number from duodenum to ileum. The Paneth cell secretes mercury and other heavy metals into the intestinal lumen. These cells become necrotic in chronic methylmercury intoxication of primates. M cells are located in the surface epithelium overlying the lymphoid tissues of the intestinal tract. These cells are recognized histomorphologically by the microfolds on their lumenal surface (folds are not present in rats), and are highly phagocytic. They are responsible for sampling antigens from the lumen contents and for transfer of the antigen to T lymphocytes and dendritic macrophages. Macrophages can also present antigens to T lymphocytes in the dome region of the follicle. The M cells not only serve to shuttle antigenic material from the lumen to the mucosal immune system, but can also function as an access route for pathogenic microbes and particulate toxic agents (e.g., asbestos). Absorptive epithelial cells may also function as antigen-presenting cells, especially for soluble proteins. Enterocytes express Class II major histocompatability complex (MHC) antigens and are capable of stimulating T cells to activate and proliferate. Enterocytes may process soluble antigens, while M cells may be primarily responsible for processing particulate antigens. Lymphocytes, plasma cells, mucosal mast cells, and eosinophils are present throughout the lamina propria within the villi and around the crypts. The lamina propria is the neighboring loose connective tissue layer that nourishes the mucosal epithelium and its associated mucosal glands. The numbers of these cells increase with age. Cells of the lymphoid series are arranged in nodules and groups that serve to provide mucosal immunity. Although most of the cells in the lamina propria function in a similar manner to those in other regions of the body, the mucosal mast cell is functionally distinct from mast cells in other tissues (see Section 2.3, Enteric Nervous System). The tunica muscularis is usually a thin, double layer of smooth muscle tissue. Smooth muscle cells are oriented circularly or helically within the inner layer, and longitudinally in the outer layer. Contractile action by the muscularis mucosae mediates localized


folding of the mucosae that can facilitate absorption and digestion. The submucosa is found between the mucosa and muscularis externa, and is a loose connective tissue layer with comparatively large vascular and lymphatic elements. The muscularis externa is typically the most substantial layer of the intestinal wall, and is composed of an inner layer of circularly oriented smooth muscle cells and an outer layer of longitudinally oriented smooth muscle cells. Large Intestine FUNCTION

Major functions of the large intestine include storage of digesta, and water and electrolyte absorption and secretion. One of the main electrolyte absorbing processes is through the Naþ/Kþdependent ATPase pathway. Herbivores secrete large volumes of salivary, pancreatic, and biliary fluids, and in horses (perissodactyls) the large intestine secretes additional fluids equivalent to 40% of the extracellular fluid volume. However, 98% of the fluid and ions secreted in the upper gastrointestinal tract is reabsorbed in the cecum and colon. It is critical that this reabsorptive process is taken into account when attempting to investigate toxicologically induced diarrheas. The colon has protein-absorbing activity. However, relative to the small intestine, the large intestine absorbs a small amount of the total body protein needs. The large intestine instead serves as the major site of digesta retention; however, the duration and primary site of retention varies between species. The rate of passage is inversely related to the degree of colonic compartmentalization. Additionally, retrograde propulsions of the colon, associated with absorption of water and electrolytes, may help delay the passage of a toxic compound and prolong exposure of a toxicant to various biotransforming enzymes. The high concentration of bacteria in the colon facilitates roughage digestion and compound biotransformation. Although bacterial metabolism is critical for nutrition and influences toxicologic processes, the role of bacteria in colonic physiology has received limited study (Table 56.4). STRUCTURE

Macroscopic morphology of the large intestine varies widely among species. Anatomical modifications of basic structure include variations in




TABLE 56.4 Metabolic Reactions by Intestinal Microfloraa Reaction

Representative substrate

Hydrolysis Glucuronides

Bilirubin glucuronide









Dehydroxylation C-hydroxy groups

Bile acids

Reduction Nitro groups

P-Nitrobenzoic acids

Double bonds

Unsaturated fatty acids

Azo groups

Food dyes




Benzyl alcohol




Amino acids


Amino acids


Modified from Simon and Gorbach (1984). Table adapted from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Table IV, p. 132, with permission.

relative length, diameter, volume, and compartmental complexity of this organ. The secretory and absorptive capacity of the large intestine is related to both its anatomical complexity and the need for the animal to conserve water. Cecum Cecal structure varies considerably among different animal species. The cecal lumen contains many bacteria that are metabolically active in detoxifying or bioactivating ingested compounds and producing essential vitamins. Some aspects of antimicrobial toxicity are directly related to the modification of normal cecal microflora. The cecal mucosa is similar to that of the colon (see below), and the submucosa contains lymphoid tissue that functions like Peyer’s patches

of the small intestine. The primary function of the cecum is for microbial fermentation and storage of ingesta. Intralumenal ingesta can be passed back and forth from the cecum to the proximal large intestine before continuing its passage down the intestinal tract. Animals with large and functionally active ceca may have significantly different compound passage rates than do species with a rudimentary cecum (e.g., rats versus dogs). Such information must be incorporated into the design of animal model studies, and considered when interpreting toxicokinetic and drug metabolism data. Colon The mucosa of the colon and cecum is significantly different from the mucosa of the small intestine. Goblet cells are abundant in the colonic mucosa, and are responsible for adding mucus to the dehydrated ingesta. Inflammation of the colon can lead to epithelial Paneth cell metaplasia, which reduces mucus production and renders the mucosa prone to bleeding. The submucosa, tunica muscularis, and serosa of the large intestine are similar to those of the small intestine. The terminal end of the large intestine (rectum) is located retroperitoneally in the pelvic canal, and is not covered by a serosa.

2.2. Enteric Lymphoid System The gastrointestinal immune response is multifactorial, and involves both cellular and humoral immune mechanisms. Immunologic response of the gastrointestinal tract is predominantly mediated by immunoglobulin isotype A (IgA) with and without secretory component (sIgA) (Figure 56.6). The gastrointestinal tract mucosa contains many IgA-producing plasma cells. Additionally, cell-mediated immune mechanisms are involved in the mucosal response to toxic compounds. Cell-mediated immunity of the mucosa is distinctly different from that of non-mucosal sites. This difference is exemplified by the tall columnar absorptive cells of the small intestine, which can function as antigen-presenting cells, IgA-antigen carriers, and activators of T lymphocytes. Consequently, immune mechanisms in the gastrointestinal tract involve multiple pathways for response to toxic compounds that may include hypersensitivity.




FIGURE 56.7 Peyer’s patch from the ileum of a dog illustrating M cells (MC) and the mixture of lymphocytes (small arrows) and plasma cells (large arrows) in the lamina propria. Figure reproduced from Fundamentals of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2007) Academic Press, Fig. 8.2I, p. 166, with permission.

FIGURE 56.6 The primary antibody response of the gastrointestinal tract is of the IgA isotype. Monoclonal antibodies to IgA and a peroxidase labeled secondary antibody allow the in situ localization of the IgA (A) present in inflamed gastric mucosal epithelial cells (E) and immunoglobulin-producing plasma cells (P). Nonimmunoglobulin-A-producing plasma cells (N) are also present. Bar ¼ 50 mm. Immunoglobulin A released by plasma cells is released in a dimeric form held together by the J piece (J). Secretory component (SC) is added to the dimer upon passage from the lamina propria to the lumen. This component allows the immunoglobulin to resist enzymatic degradation. Figure reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 6, p. 134, with permission.

Located throughout the small intestine are lymphoid aggregates called Peyer’s patches (Figure 56.7). Peyer’s patches represent the organized portion of the gastrointestinal immune system, and are part of the gastrointestinalassociated lymphoid tissue (GALT). The GALT composes over 25% of the body’s total lymphoid mass. Peyer’s patches can be composed of only a few lymphocytes or may be well-developed

lymphoid nodules with many active germinal centers (secondary follicles). The stronger the antigenic stimulus, the more extensive will be the response and development of the nodule. Nodules consist of follicular and parafollicular regions. Follicles are composed of B cell-rich germinal centers located in the lamina propria or submucosa. Germinal centers are surrounded by T cells in the parafollicular area and are capped by a dome of lymphocytes that extends into the specialized M cell-rich epithelial covering. Lymphoglandular complexes of the colon are an important part of the GALT complex, which is most extensively developed in the small intestine. Lymphoglandular complexes of the colon have germinal centers in the submucosa with deep epithelial invaginations. As in Peyer’s patches, M cells partially line the surface of these complexes and lymphocytes are closely apposed to these phagocytic cells. Epithelial cells lining Peyer’s patches of the rat colon function like M cells, but do not have the characteristic morphological appearance of microfolds on their lumenal surface. When the GALT is activated, lymphocyte traffic through Peyer’s patches increases. Primed and activated T and B lymphocytes migrate to mesenteric lymph nodes via the thoracic duct to




high-endothelial-cell lined postcapillary venules (BEV), and then into intestinal lymphoid tissue. Tissue specificity of the T and B cells is determined by interaction with the endothelial cells of the BEV. The lamina propria is rich in lymphoid and non-lymphoid cells. The ability of the immune system to respond to microbial, chemical, and dietary antigens helps prevent these agents from entering the body. Gastrointestinal tract mucosal hypersensitivity can be induced by circumventing the normal process in which a toxic compound is handled by the immune system. This can be done by coadministering a mucosa-damaging agent and the antigenic compound concomitantly. Lymph flows from the central lacteal to Peyer’s patches and then to many different lymph nodes, including mesenteric, pancreatic, gastric, hepatic, splenic, and colonic nodes. Lymph contains absorbed lipids, fat-soluble xenobiotics, and recirculating lymphocytes. Some of the circulating lymphocytes have been primed by antigen exposure and are migrating to other mucosal sites, including the respiratory or genital tracts. This allows immune cells exposed to antigens in the gastrointestinal tract to localize at other sites of the common mucosal immune system that may also be exposed to environmental toxins. The T-lymphocyte population in the lamina propria consists primarily of CD4 helper/ inducer cells. These cells play a major role in the development of the initial immunologic response to a new antigen, which is consistent with the concept that the gut represents a site of primary exposure of the host to many new antigens. Cytotoxic/suppressor (CD8) cells are less frequently seen in this location. For more specific information regarding GALT, and the immune system as a whole, to toxic substances, refer to Immune System (Chapter 49).

2.3. Enteric Nervous System The nervous tissue of the gastrointestinal tract is highly organized but diffuse in nature. Various motor and sensory neurons ramify throughout the wall of the gastrointestinal tract and form multiple plexuses. Nerve fibers emanate from these plexuses and vary in thickness, carrying information from one ganglion to another and from intrinsic to extrinsic neurons. The nervous tissue of the gastrointestinal tract differs from

other portions of the autonomic nervous system because many of its neurons do not receive direct input from the central nervous system. However, neural information does come from autonomic motor neurons that are both sympathetic and parasympathetic, and from gastrointestinal sensory neurons. This results in reflex activities that act independently of the brain or spinal cord. Yet the central nervous system can regulate the rate of turnover of gastrointestinal mucosal cells. Neurons of the parasympathetic ganglia are in both the submucosal (Meissner’s) plexus and myenteric (Auerbach’s) plexus. The myenteric plexus is responsible for the electrical rhythms of the gastrointestinal tract, but is not needed for propagation of the myoelectric complex. The myenteric and submucosal plexuses are interconnected to form a single functional unit, so integration of electrical activity occurs at multiple locations in the gastrointestinal tract. Parasympathetic stimulation of the gastrointestinal tract leads to increased blood flow, secretion, and muscular activity. Stimulation by the sympathetic nervous system has the opposite effects. The enteric nervous tissue is also composed of integrative circuits that consist of interneurons within ganglia that process information from intramural and mucosal sensory receptors. Sensory neurons detect fluidity, volume, chemical composition, and temperature of the lumenal contents. The appropriate motility of the gastrointestinal tract is effected via motor neurons. Specific motor neurons release neurotransmitters in the proximity of mucosal effectors, blood vessels, and muscle layers. In addition, receptors for neurotransmittors are present on and near epithelial cells. The spatial density of myenteric neurons decreases with age. Additionally, toxic substances such as anthraquinone injure the nerve fibers and may alter the number of neurons. Topical application to the gastrointestinal tract of cationic surfactants, such as benzalkonium chloride (a mixture of compounds) or benzyldimethyltetradecylammonium chloride (BAC), destroys intrinsic neurons in the myenteric plexus of the small intestine. Serosal application of BAC damages longitudinal muscle, circular muscle, the myenteric plexus, and extrinsic nerves. Two weeks after treatment with BAC, the number of muscle cells in both the longitudinal and circular muscle



layers returns nearly to control values, but the damage to nerves is more persistent. Thus, the loss of neuron mass may reflect an adaptational atrophy associated with the aging process or exposure to toxic substances. Capsaicin produces pharmacological effects on sensory neurons in the gastrointestinal tract. Capsaicin activates small-diameter sensory nerve fibers in the mucosa and wall of the gastrointestinal tract to induce release of neurotransmitters from both the central and peripheral terminals of these neurons. The neurotransmitters released are principally substance P and CGRP. The peptides initiate a cascade of pro-inflammatory events and transmit nociceptive information to the central nervous system. It is a characteristic of capsaicin and related compounds to induce desensitization to their effects since repeated exposure leads to diminished effects. Destruction of sensory nerves in sensitive species appears to be related to blockade of retrograde transport of nerve growth factor. Intragastric capsaicin, even in moderate doses, can produce changes in gastrointestinal function. For example, intragastric doses of 0.1 mg/kg of capsaicin in dogs produced pronounced excitatory effects on contractions of the colon. The contractile effects of intragastric capsaicin in the colon can be inhibited by muscarinic antagonists, implying that cholinergic neural pathways are involved. There are three principal motility patterns of the gastrointestinal tract: storage, mixing, and propulsion. Movement of a swallowed bolus from the mouth to the stomach and into the intestinal tract is a propulsive event caudally progressing in front of a contraction of the circular muscle. Effective gastric emptying requires coordinated propulsive contractions in the antrum that progress to the pyloric canal, as well as properly timed relaxation of the upper duodenum. Some drugs and toxins reduce the rate of gastric emptying by producing contractions of the duodenum that abolish the antral-duodenal pressure gradient required for effective emptying. Contractions of the circular muscle occur more or less randomly, but are somewhat fixed in timing and location by the electrical slow waves or electrical control activity generated initially in interstitial cells. Clusters of propulsive contractions associated with contractile rings migrate 5–30 cm caudally, thereby propelling content towards the cecum. Toxic agents that induce


excessive migrating clustered contractions would abnormally speed propulsion through the small intestine. Migrating motor complexes are bands of contractile activity that move caudally over the stomach and small intestine during fasting to sweep digested food remains out of the stomach. These motor complexes are active during periods of fasting, and continue until another meal is consumed. The central nervous system exerts some degree of control over the activity of these complexes, but the actual complexes are initiated in the enteric nervous system. Premature migrating motor complexes can be induced by opiates and erythromycin.

2.4. Biotransformation The mucosa of the gastrointestinal tract is a site of high enzymatic activity and compound conjugation. The mucosa is uniquely located; therefore, it is exposed to the highest concentration of orally administered compounds and can modify these compounds prior to their entry into the blood. The consequences of mucosal biotransformation are compound metabolism and activation or deactivation (detoxification) (Table 56.3). This process can therefore lead to an increase or decrease in a compound’s toxicity. Increased toxicity is exemplified by the activation of carcinogens. Detoxification occurs by metabolism of compound to non-toxic intermediates, absorption, or excretion in the feces. Intestinal mucosal enzymes that metabolize xenobiotics can prevent systemic absorption of many potentially toxic substances such as peptides (via peptidases), esters (via esterases), and alcohols (via alcohol dehydrogenase) present in the gastric mucosa. Xenobiotic metabolism can be carried out by luminal microorganisms; furthermore, luminal organisms can affect mucosal enzyme activity (Table 56.4). Factors affecting the metabolic activity of the intestinal microflora must be taken into account in studies of the biotransformation of orally ingested xenobiotics. Marked differences exist in microbial composition and metabolism of the gut flora of different species of animals, and environmental factors such as drugs (especially antibiotics), diet, and xenobiotics can modify microbial metabolism, and thus the toxicity, of foreign compounds. Presystemic clearance can occur




for some toxicants either within the enterocyte or within the gut lumen itself. This gut-associated first-pass effect represents the irreversible extraction and/or biotransformation of toxicants passing through enterocytes on their way into the lacteals or portal venous blood. Metabolites produced by enterocyte biotransformation can enter the intestinal lumen, the portal venous system, lacteals, or simply remain stored in the cell. Conjugated water-soluble compounds formed during transport into enterocytes tend to be excreted relatively quickly into the intestinal lumen, and therefore are cleared from the body by intestinal cells (Table 56.3). After oral administration, when the concentration of a xenobiotic within the enterocyte is very high, intestinal biotransformation reactions will generally be capacity limited.

The colon is three- to five-fold more active than the small intestine in certain enzymatic processes (e.g., demethylation). Compared with the liver, the jejunum has a higher monoamine oxidase activity. Several compounds, such as polychlorinated biphenyls and phenobarbital, increase cytochrome P450 (CYP) levels in the intestinal mucosa 2–4 days after exposure; the effect is greatest after oral administration of the compound. This augmentation of enzyme activity is similar to that which occurs in the liver. As occurs in the liver, chronic intake of ethanol will also increase the level of activity of several intestinal enzyme pathways. Several biotransforming and toxin-metabolizing gradients exist in the gastrointestinal tract (Figure 56.8). The proximal end of the small intestine requires more oxygen and has a higher

Cellular Location of Enzymes that Metabolize Compounds Mucus gel –bacterial and host enzymes Microvilli –digestive enzymes drug metaboliizing

Tight junction Intermediate junction


Terminal web


Lysosomes –glucuronidase Granular endoplasmic reticulum

Mitochondria –monamine oxidase Unattached ribosomes

small, lipophilic

weak acids

Smooth endoplasmic reticulum –cytochrome P450 –glucuronyl transferase –epoxide hydrolasse

Golgi material Intercellular space –metabolized and unmetabolized compound

Nucleus –minimal compound metabolism

Basement membrane

most compounds decreasing drug metabolizing activity compounds solubilized by bile

Lamina propria slowly absorbed compounds Capillary –distribution of drugs to systemic circulation

FIGURE 56.8 Biotransformation of compounds is a complex event involving absorption and metabolism gradients. Different sites of absorption will lead to different enzyme exposures. Compound solubility and transport mechanisms will result in contact with different enzymes. Figure reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 8, p. 138, with permission.



activity of alkaline phosphatases and disaccharidases than does the aboral end. Monooxygenase (CYPs) and UDP-glucuronyl transferase activities are higher in the upper duodenum than in the lower small or large intestine. Sulfation proceeds more rapidly in the proximal than in the distal small intestine and colon. There is also a gradient that exists from top to bottom of the villus. CYP activity in the small intestine provides the principal, initial biotransformation of ingested xenobiotics. Enzymes of the epithelial cells are fully competent to carry out oxidative, reductive, hydrolysis, and conjugation reactions. The oxidative reactions are largely catalyzed by CYP isozymes. The intestinal mucosa also contains non-specific esterases and amidases, UDP-glucuronosyltransferases, and reductases. Some enzyme activity, such as nitroreductase and dechlorinase, may be attributable to both mucosal enzymes and luminal microflora. Most CYP isozyme activity increases in enterocytes during their migration from crypt to villus. Nearly all CYP activity is attributable to villus cells, and NADPH CYP 450 reductase is expressed constitutively only in villus cells. Both glucuronidation and sulfation reactions increase solubility of xenobiotics and thus play a major role in intestinal first-pass clearance for various xenobiotics. Intestinal presystemic elimination of a dopamine prodrug N-(N-acetyl-Lmethionyl)-O,O-is(ethoxycarbonyl) dopamine indicates that catechol ester hydrolysis, amido hydrolysis, and catechol O-methylation can also occur in enterocytes. Different sites of the gastrointestinal tract contribute to the intestinal component of metabolic clearance. In guinea pigs, epithelium of the small and large intestine contributes to formation of active morphine 6glucuronide, whereas gastric, intestinal, and colonic epithelia are involved in formation of inactive morphine 3-glucuronide. A biotransformation gradient from the apical to the basal surface of the enterocyte is present; it is controlled by enzyme-rich drug-metabolizing organelles (e.g., SER) and active transport systems in the apical cell membrane. However, the gradient varies with the cellular location in the gastrointestinal tract and route of exposure. Compounds entering from the blood (basal) side can be found in the gastrointestinal tract lumen independent of enterohepatic circulation. Experiments using bile duct-ligated animals, animals with bile fistulae, and “in vitro” tissue


bath techniques have revealed that many toxicants enter the intestinal contents by direct transfer from blood or out of the enterocyte. In general, intestinal excretion is a relatively slow process that is important for chemicals having low rates of biotransformation and/or low renal or biliary clearance. Although passive diffusion is an important mechanism for intestinal excretion, active secretion of organic acids and bases has been demonstrated in the gut. The transepithelial elimination of ciprofloxacin in rabbits and rats is probably due to active transport. There is high local production and/or facilitated transport into the colonic lumen of polymeric immunoglobulin A (see Section 2.2, Enteric Lymphoid System). It has been shown that P-glycoprotein, which actively transports some antineoplastic drugs out of neoplastic cells, causing multidrug resistance, mediates efflux of etoposide out of intestinal cells, and this efflux is inhibitable with quinidine. P-glycoprotein (Pgp) is the 170-kDa product of the ABCB1 gene in humans, and is an ATP-powered efflux pump which can transport hundreds of structurally unrelated hydrophobic amphipathic compounds, including therapeutic drugs, peptides, and lipid-like compounds. An organic cation transporter, originally identified in kidney and liver, that is responsible for translocation of hydrophobic and hydrophilic organic cations of different structures has also been identified in the intestine. Whether this transport protein is highly active in translocating cationic toxicants into the intestinal lumen remains to be demonstrated unequivocally. As an adaptive response to renal failure, the intestine can begin to excrete chemicals such as oxalate. In addition, epithelial cells of the gastrointestinal tract can absorb and export compounds from the circulating blood and the intestinal lumen, indicating that many intestinal transport systems are likely “twoway streets.” Disposition of highly lipophilic chemicals in an organism often requires consideration of lipid transport. The two important mechanisms that contribute to the non-biliary, intestinal excretion of lipids are (1) exfoliation of intestinal cells, and (2) exudation of lipids across the mucosa. Many investigators have determined the concentration profile of compounds in the gastrointestinal tract with or without ligation of the intestine or in




isolated loops after intravenous administration of chemicals. Unfortunately, there have been seemingly contradictory results, probably due to limitations of the various experimental designs. Any major surgical interference with the dynamics of the gut may result in altered cell exfoliation, bile flow, residence time of intestinal contents, peristalsis, blood flow, reabsorption, and so on. Much work remains to be done in this difficult area of research. Intestinal biotransformation and intestinal excretion of water-soluble substances, such as metabolites of propachlor, are important physiological processes that occur in several species, including rats, pigs, and chickens. As a second example, the presystemic elimination of sumatriptan varies throughout the gastrointestinal tract of various species, probably due to regional differences in metabolic activity or transporters. In addition, there are “regional” differences in excretion – for example, the gastrointestinal metabolic clearance of 20 ,30 -dideoxyinosine in rats is low in the duodenum but significantly higher in the ileum. Many of these same metabolic processes described above are likely to occur in humans in the colon, where colonic mucosa expresses some CYP isozymes and UDPglucuronosyltransferases. Besides altering the biological activities of toxicants, biotransformation reactions in enterocytes may influence the post-absorptive fate of xenobiotics. Metabolites may be excreted by enterocytes into the intestinal lumen and eliminated as fecal matter, thereby permitting escape from enterohepatic circulation. Metabolites may be either excreted across the mucosal membrane, back into the lumen, or secreted across the serosal membrane into portal venous blood. Blood supply to the mucosa is a critical component of mucosal biotransformation. Provision of oxygen to the epithelium is important in oxidation and reduction reactions, and the microvascular anatomy of the mucosal villi provides a countercurrent exchange system. The countercurrent exchange can reduce entry of a toxin into the portal circulation. As the toxin is picked up in the villus and moved to the crypt, exchange with blood going to the villus occurs, resulting in slower compound absorption and increased time for biotransformation (Figure 56.9). Enhancement of carcinogenic activity by gastrointestinal mucosal biotransformation may

FIGURE 56.9 Counter-current mechanism active in the individual villi. Blood is carried into the villi from the lamina propria. A counter-current exchange mechanism is established by blood going to the villus tip and blood returning toward the crypt region of the lamina propria. The exchange operates primarily through passive diffusion and allows absorbed compounds, obtained in the lumen, to be carried back to the villus tip against a concentration gradient. This process is active for nutrients, diffusible compounds, and gases (e.g., O2 and CO2). Figure reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 9, p. 140, with permission.

reflect incomplete metabolism of a toxic initially non-toxic compound. In this case, the metabolized compound may form DNA-damaging intermediates, producing mutagenic molecules or, secondarily, superoxide anions. Dimethylhydrazine is an example of an organ-specific compound that undergoes only partial metabolism in the gastrointestinal tract, leading to induction of colon cancer. Incomplete metabolism is not the only way biotransformation can modify carcinogenic activity, since complete biotransformation can be important in the carcinogenic activity of compounds such as 2-acetylaminofluorene. Fecal excretion is a major route of elimination of many lipophilic chemicals, with most chemicals probably being transferred by passive diffusion and a number of toxicants excreted into the feces by non-biliary pathways. Direct mucosal-to-serosal transport into the feces occurs for some non-polar, lipophilic xenobiotics that undergo little or no biotransformation. However,



rapid exfoliation of intestinal cells may also contribute to fecal excretion of some toxicants. The intestinal excretion rate of some lipophilic chemicals can be substantially enhanced by increasing the lipophilicity of the gastrointestinal contents by, for example, adding mineral oil to the diet. For instance, oral dosing of squalene increases the fecal excretion of parenterally administered theophylline and reduces the serum level of the drug in rats. Squalene also accelerates the fecal elimination of parenterally administered strychnine in mice.

2.5. Enterohepatic Circulation Enterohepatic circulation allows for recycling of metabolized and non-metabolized compounds, and is of critical importance in toxicologic processes involving the gastrointestinal tract. This circulatory route is active when ingested compounds that are absorbed in the gastrointestinal tract enter the portal circulation, go to the liver, and then return to the gastrointestinal tract via biliary excretion. The enterohepatic circulatory pathway can also be utilized by dermally absorbed or inhaled materials that are excreted in the bile. A compound leaves the enterohepatic circulation if it passes in the feces before being reabsorbed, or into the urine before being cleared by the liver. The ultimate destiny of a compound is dependent on its chemical composition and the species of animal exposed to the compound. The importance of species differences is best illustrated by the non-steroidal anti-inflammatory drug (NSAID) indomethacin, which undergoes enterohepatic circulation; it is excreted in the feces of dogs but in the urine of rats. The duration of enterohepatic circulation is most extensive for this drug in dogs and rats, and least extensive in rabbits and humans. This observation impacts resulting species-specific variability in toxicologic response of the GI tract to NSAIDs, with dogs being less tolerant of NSAID administration when compared to rats, rabbits, or humans. The amount of a compound that is excreted in the feces is controlled by the lipophilicity of the chemical and the extent of metabolism that alters this lipophilic character. Processes that increase the aqueous nature of a compound include dealkylation, glucuronidation, and sulfation, and those that increase lipophilicity include glucuronide


hydrolysis. Increasing lipophilicity is associated with increased excretion of the compound in feces. The rate at which a chemical is excreted in the feces is limited by the time it takes for a compound to be excreted in the bile and reabsorbed by the intestine. Increasing metabolism of the chemical will increase the rate of excretion. Factors that modify this excretion rate include motility of the intestine, distance of the site of (re)absorption from the major duodenal papilla (site of common bile duct excretion), rate of conjugate hydrolysis by gastrointestinal bacteria, transport rate across the intestinal wall, and motility of the gall bladder in species which have this structure. With the exception of gall bladder motility, all factors influence intestinal transit time. The excretion of a compound in the urine depends on the liver’s ability to clear the compound from systemic blood circulation, the ability of a compound to pass the glomerular barrier, and the rate of intestinal or hepatic biotransformation. However, a compound may enter the systemic circulation intact from the gastrointestinal tract if it first enters the lymphatic circulation in the villus after being absorbed. When the portal burden of a compound entering from the gut exceeds the liver’s ability to extract it, the material will pass through the liver unchanged and enter the caudal vena cava. A compound’s extractability from the blood may be altered after it has been metabolized by bacteria or the enteric mucosa and is reabsorbed from the gastrointestinal tract. Consequently, upon reabsorption, such a metabolite may be less likely to be cleared by the liver. Glucuronidation or sulfation of a compound increases the possibility of urinary excretion. Combined biotransformational processes in the liver and intestine can substantially affect the toxicity of a compound. For example, the activation of diphenolic laxatives by microbial metabolism and conjugate hydrolysis has been clearly established. Bacteria can also modify dinitrotoluene by nitro-reduction and give rise to elevated hepatic levels of the carcinogenic metabolite dinitrobenzyl alcohol. Arylamines formed from the biliary metabolite of chloramphenicol may be responsible for the goitrogenic effect of this antibiotic in rats. Hydrolysis of polycyclic aromatic hydrocarbon (PAH) glucuronide




metabolites demonstrates how enterohepatic circulation can retoxify a detoxified compound. These are just a few of many instances of how enterohepatic circulation can affect toxicity. During enterohepatic circulation, compounds may interact with intestinal contents. This is demonstrated by the binding of bile salts to dietary fibers. Such binding will decrease the reabsorption of bile salts, and may be partially responsible for the healthful effects of soluble fibers. Alteration of bile acid circulation can influence the hepatobiliary level of several compounds that are bile-soluble (cholephils). In addition, taurocholate promotes motor activity in the colon, thereby reducing intestinal transit time. Bile acids also increase the transport of compounds across the intestinal mucosa, and may consequently enhance the toxic properties of a compound. Enterohepatic circulation will increase the toxicity of a compound to organs in the enterohepatic circuit if the compound remains active during circulation. The concentrating capacity of enterohepatic recycling may play an important role in the ulcerogenic effects of NSAIDs (such as indomethacin) in dogs. This same process may be important in the carcinogenic effects of colon carcinogens 3,3-dimethoxybenzidine and tris(2,3dibromopropyl)phosphate. Biliary excretion and enterohepatic circulation have a role in colon carcinogenesis of rats induced by 2,3-dimethyl-4-aminobiphenyl (DMAB). Rats treated orally with this carcinogen excrete mutagenic agents in the bile. However, rats injected subcutaneously with DMAB do not develop colonic neoplasms.

2.6. Bacterial Metabolism Ingested materials are metabolized not only by digestive and intestinal enzymes but also by resident bacteria (Table 56.4). These bacteria have metabolic activities that include reductases, hydrolases, demethylases, b-glucuronidases, and b-glucosidases. Since there are approximately 109–1012 bacteria per gram of feces in humans and animals, the potential enzymatic activities of this compartment of the gastrointestinal tract cannot be ignored. The floral composition in mammals depends on the nutritional and health status of the host, and the host’s dietary composition. Microorganisms are passive inhabitants but have an active metabolic function promoting

a wide variety of biochemical reactions important in normal physiology. Within the small intestine there is a gradual transition from the sparse gram-positive microflora of the stomach, followed by transition of gram-positive to gramnegative bacteria in the ileum and thence to the colon, where gram-negative bacteria are the principle populations. Bacterial concentrations in excess of 1012/mL of ingesta are common. Anaerobic bacteria outnumber aerobic species by a factor of 102–104. Frequently identified anaerobic microorganisms are Bacteroides, Bifidobacteria, and Eubacteria, anaerobic grampositive cocci, and Clostridium sp. Aerobic isolates include species of Enterobacteriaceae, enterococci and other streptococci, staphylococci, and Candida. The influence of intestinal microbes on the host’s nutritional status has been most clearly demonstrated using animals with and without gastrointestinal bacteria. Weight of the gastrointestinal tract and mucosal thickness are markedly reduced in animals without bacteria in their gut contents. Bacterial overgrowth can also modify lipid and carbohydrate absorption. Overgrowth of intestinal bacteria can lead to steatorrhea by hydrolyzing bile-acid conjugates and altering micelle-forming ability. Bacterial proteases also remove maltase from brush border membranes, which results in carbohydrate malabsorption. Consequently, compounds altering microbial populations can lead to altered nutritional status. Bacteria produce and release compounds that have local effects or, if absorbed, systemic impacts. Mammalian metabolic pathways generally require oxygen, so injurious compounds are generally detoxified by oxidation and conjugation pathways. However, gut bacteria are active in oxygen-free environments and thus utilize reduction and hydrolysis reactions, resulting in different metabolites with potentially harmful side effects. Furthermore, with the exception of ruminants, most bacteria are present in the lower small intestine, cecum, and colon. Consequently, the role of bacteria in modifying host responses is most marked in the lower segments of the gastrointestinal tract. An example of this can be seen with digoxin. The pharmacologic activity of digoxin is dependent upon bacterially mediated hydrolytic removal of a trisaccharide, which releases digoxigenin. In some individuals, however, there is a further reduction in the double



bond of the lactone ring, which results in the formation of a pharmacologically inactive substance, dihydrodigoxigenin. This reaction is mediated by Eubacterium lentum present in the colon of these individuals. Differences in diet appear to play a role in the presence of this bacterial species and the frequency of digoxin inactivation in humans. Additionally, the metabolism of digoxin is also less extensive in infants since infants lack specific metabolic activity which cannot be related to the absence of any particular component of the microflora, but rather appears to be a result of immaturity of bacterial enzyme systems. Bacterial deamination is another important metabolic activity that is mediated by the bacterial flora. The breakdown of urea into carbon dioxide and ammonia is catalyzed by bacterial urease. Approximately 40% of the urea synthesized by the liver is degraded by a variety of aerobic and anaerobic bacteria. The role of bacteria in gastrointestinal toxicity is most clearly defined for carcinogen activation. Many chemical carcinogens (indirect acting) require enzymatic activation before they can cause cellulartransformation.Bacterialb-glucuronidases can deconjugate glucuronides and lead to the release of carcinogenic aglycones. Additionally, fecal flora nitroreductases can activate procarcinogens. Bacteria also have a direct role in the detoxification process. Bacteria can deactivate carcinogens by N-dehydroxylation. Antibiotics can not only modify bacterial populations in the gastrointestinal tract, but also depress neuroeffector and neuromuscular transmission. In vitro studies have demonstrated that ampicillin, lincomycin, erythromycin, and clindamycin depress contractions of the muscularis mucosa. Clindamycin and erythromycin depress the responses of the muscularis mucosa to acetylcholine. As an example, impaired gastrointestinal motility after administration of oral antibiotics can facilitate the proliferation of Clostridium difficile in the lower gastrointestinal tract and lead to pseudomembranous colitis.

2.7. Gut Microflora and Microbiology The mammalian body can, on the whole, be considered as a diverse template in which multitudes of microbial organisms from five different biological kingdoms (bacteria, protozoa, fungi,


archaea, and viruses) form complex evolutionary and direct relationships that in turn define the mammalian microbial ecosystem. These microbial populations, their genetic elements (genomes), and their associated complex host–environmental interactions are collectively termed the microbiome. Microbes in any given location of the body are collectively referred to as the microbiota. The microbiome varies in its quantitative and qualitative profiles depending upon the anatomical location of the host in which they reside, and is most abundantly seen on the skin, in the mouth, gastrointestinal tract (GI), and upper respiratory tract, and in genital areas. In all hosts, including humans, rodents, dogs, and primates, the microbiome is composed predominantly of hundreds to thousands of bacterial phylotypes, and a majority of these reside in the GI tract. In fact, it is estimated that the mammalian intestine contains up to 1010– 1011 microorganisms, which is about 10 times higher than the host cell density. The richness and diversity of the gut microbiome is manifested by the presence of approximately 400– 1000 different species of bacteria. The net effect of the complex interactions between the gut microbiome and the host cells in a particular resident environment can be either beneficial or deleterious to the host, or a combination of both. The GI microbiota is important for normal gastrointestinal development and physiological functions, as well as for some GI and non-GI pathological manifestations that influence the general overall health of the host. A number of factors determine the density and type of bacterial flora that are resident in any given part of the gastrointestinal tract. Some of these modifiers include pH, peristalsis, tissue oxidation–reduction potential, type and form of diet, age, nutrient availability, mucin content, immunoglobulin levels and innate immune factors, bile secretion, bacterial adhesion and biofilm formation, cooperation or competitive exclusion strategies of different bacteria, drugs, etc. Any radical shift in the gut microbiota may transform the gut to a specific GI diseased state (dysbiosis) or impart a disease protective effect (probiosis). Gut Microflora and Topographical Anatomy Rodents are the most commonly used laboratory animals for research on gut microbiota, in addition to their prime importance for a wide




range of research applications including pharmacological and toxicity studies. In general, although there are many similarities in GI anatomy and physiology between humans and rodents, their inherent anatomo-physiological and behavioral differences are important in understanding the impact of gut microflora on research findings. The first major difference in gut anatomy between humans and rodents is the obvious size difference – a key factor in determining gut transit time. In rats, food transit time is approximately 12–35 hours, and this is influenced by the type of diet, its particle size and morphology (e.g., pellet or powder), and the level of water consumption. In humans, the GI fold transit time ranges from 33–60 hours, depending upon the type of diet, genetics, age, and a variety of other factors. In humans, due to inherent physicochemical variations between different regions, the normal GI microflora can be divided into three distinct populations: upper GI tract (stomach, duodenum, and jejunum), ileum, and colon. Adult humans do not have a fully developed cecum and instead have only a small vestigial cecal appendix, unlike mice, rats, and dogs, which have a well-developed prominent cecum. In humans, the colon compensates for the loss of cecal physiological functions. In mice and rats, the stomach is divided into a secretory glandular and non-secretory squamous part, the latter contributing to gastric expansion following food consumption. The colon is the principal site for microbial fermentation in humans, whereas in rodents the cecum is the main place for microbial fermentation and the colon mainly aids in water resorption and pellet formation. Additionally, TABLE 56.5

unlike humans, mice and rats are coprophagic and hence are continually repopulated with their own gut microflora. For example, rats may consume up to 35–50% of their fecel output, and even more if on a vitamin-depleted diet. The effect of coprophagy on GI microflora depends upon nutritional status, associated housing conditions, and environmental microflora. Any prevention of coprophagy in rodents might cause minor variations in gastric and small intestinal Lactobacillus levels, as well as negatively influence weight gain. In spite of inherent species differences, the intestines of humans, mice, rats, cats, dogs, pigs, and most other vertebrate species, including zebra fish, have been shown by microbiome studies (utilizing 16S rRNA sequences) to be populated predominantly (up to 99%) by three major bacterial phyla, namely Firmicutes, Bacteriodetes, and Actinobacteria, and to a lesser extent by two minor phyla, Proteobacteria and Fusobacteria. The remaining 1% is represented by members of the phyla Spirochaetes, Tenericutes, Verrucomicrobia, Cyanobacteria, Chlorflexi, and a few unclassified bacterial groups. However, the relative proportions of these five phyla and the type of bacterial genera within these microbiomes vary widely across different species as well among strains of the same species. Additionally, microbiome variations within a host species can be affected by housing (germ-free, conventional, SPF) and drug treatments. Of interest, only about 15% of mouse microbiome genera are typically represented in the human microbiome. The number and type of microbiota in the oral cavity, esophagus, and gastrointestinal tract is highly variable and is influenced by relative pH (Table 56.5).

Microbial Density in Different GI Compartments of Humans and Mice Human



pH gradient

Microbial mass

pH gradient

Microbial mass



102e3 cells/mL


107e9 cells/g



103e4 cells/mL


107e9 cells/g



104e5 cells/mL


107e9 cells/g



108 cells/mL


107e8 cells/g





107e8 cells/g



1011e12 cells/mL


109e10 cells/g




The esophagus in humans and most laboratory animal species harbors ingested food for only a short period, and it functions mainly as a passage tube. The esophagus is lined by stratified squamous epithelium, and is endowed with mucous glands that are variable in number and distribution across different species. The luminal surface of the squamous epithelium is the preferred site for bacterial adherence and colonization. Swallowed saliva from the mouth and secreted mucus aid in the rapid peristaltic activity of food within the esophagus. In general, the esophagus contains most of the microflora associated with the oral cavity and ingested food, but with some minor qualitative and quantitative differences. STOMACH (GLANDULAR AND NON-GLANDULAR)

The stomach in most mammalian species, due to its inherent acidic pH as well as rapid transit time and high peristaltic activity, usually contains relatively smaller numbers of bacteria compared to the rest of the GI tract. Aerobic or facultative anaerobic bacteria usually predominate in the stomach of most species. In humans, Helicobacter pylori (Hp), the etiological agent of peptic ulcer, type B gastritis, and gastric cancer, is closely associated with the gastric epithelium and is able to resist acidic pH by producing urease, an enzyme that breaks down urea to ammonia, which in turn absorbs hydrogen ions and makes the gastric pH alkaline. Some acid-resistant lactobacilli and streptococci can also colonize the stomach. Other bacterial species are able to colonize the stomach when the pH is increased due to decreased acid secretion (achlorhydria). Similarly, non-Hp Helicobacter spp. are the predominant mucosal adherent colonizers of the stomach in rats, mice, hamsters, primates, swine, dogs, and cats. Helicobacter infections of the stomach and other organs, primarily intestine and liver in both immunocompetent and immunodeficient laboratory animals, can significantly influence the outcome and interpretation of findings in biomedical research projects including pharmaco-toxicological studies. The effects of these non-Hp Helicobacters vary depending upon the geographical location of animal facilities, management practices, genetic manipulations, exposure to toxicants (e.g., carcinogens), pathogens, and antibiotic and other


drug treatments. Other gastric microbiota, including Lactobacillus spp., Streptococcus spp., and Clostridium spp., is present in the stomach of most animals. SMALL INTESTINE

In general, for all species, the small-intestinal microbial biomass is lower than that of the colon. This lower bacterial load is a result of multiple factors, such as the rapid transit time through the duodenum and jejunum, the presence of smallintestinal secretions (bile and pancreatic fluids), large amounts of secreted IgA, and the preponderance of GALT in the small intestine. An intact ileocecal valve acts as a barrier against colonic bacteria from moving into the ileum from the cecum or colon. The small intestine of humans is normally (autochthonous) colonized by different species of bacteria, such as E. coli, Klebsiella, Enterococcus, Bacteroides, Ruminococcus, Dorea, Clostridium, Coprococcus, Weisella, and Lactobacillus. In addition, bacteria might be derived externally via ingesta or from other niches. Examples of these include Granulicatella, Streptococcus mitis, Veillionella, and Lactobacillus (non-indigenous species). Metagenomic analyses indicate the importance of small-intestinal microflora in carbohydrate uptake and metabolism, as well as for the development and maintenance of mucosal and systemic immune homeostasis. LARGE INTESTINE (CECUM AND COLON)

The large intestine is highly conducive to bacterial survival, growth, and replication, due to its low pH, lower concentrations of bile salts, large volume and surface area, lower peristaltic activity, and longer transit time. Immunologically, the large intestine does not contain the high levels of secreted IgA and well-organized lymphoid tissue like GALT that are seen in the small intestine, although mucosal-associated lymphoid follicles/patches (MALT) are usually recognizable. However these lymphoid patches are not as stringent as GALT in the small intestine in terms of their bacterial immunosuppressive activity. The distal gut (cecum and colon) of humans and most laboratory animals is dominated by members of three bacterial phyla (Firmicutes, Bacteriodetes, and Actinobacteria) and, to a lesser extent, by two others (Verrucomicrobia and Proteobacteria). Microflora from the cecum of rodents is comparable to colonic microbiota in




humans, and both these organs are crucial for microbial fermentation in their respective species. Microbial fermentation is intricately linked with host nutrition, metabolism of non-degradable oligosaccharides, xenobiotic biotransformation, or destruction of mutagenic metabolites. Gut Microbiota and Host Relationships The gastrointestinal tract of all animals in laboratory, farm, or feral conditions is in constant interaction with the environmental microbial ecosystem, which in turn greatly influences the maturation and homeostasis of the gastrointestinal tract. Population dynamics, housing conditions, and geographical location play a key role in the nature of the gastrointestinal microbiota of any given host in a time-dependent manner. Gastrointestinal microbial populations are in general classified as either autochthonous (indigenous) or allochthonous (non-indigenous). For any host, the autochthonous microbiota is characterized by its ability to stably colonize most individuals of the species in one or more favored niches within the host. It usually consists of anaerobic organisms that are closely associated with the gastrointestinal epithelium and play a role in maintaining stable bacterial populations as well as the integrity of the gastrointestinal tract. Allochthonous microorganisms, on the other hand, are transient colonizers of the gut and are acquired from the environment through food or water, or from other niches of the host. Allochthonous organisms are usually excluded from the gut by the host’s immune mechanisms, or by competitive exclusion by autochthonous organisms. However, these organisms frequently colonize the gut under conditions of altered gut homeostasis. Probiotics are substances that usually contain live bacterial flora, such as Lactobacillus spp. and Bifidobacterium spp., which are believed to be beneficial for the maintenance of normal gut homeostasis. Prebiotics, on the other hand, are non-bacterial substances such as non-digestible carbohydrates that are believed specifically to stimulate the growth and proliferation of beneficial intestinal bacteria such as Bifidobacterium spp. Synbiotics are food substances that contain both probiotics and prebiotics and are believed to promote synergy between these two. Many microorganisms in the gut are endowed with important traits for their survival or fitness,

which also indirectly benefit members of another species – either the host or other autochthonous bacteria. If the beneficial effects flow in both directions, then these organisms are called symbionts or mutualistic organisms. For example, microbial fermentation of plant polysaccharides in the colon of humans and ceca of some animals results in end-products like butyrate, a process that is beneficial to both the commensal microbes and their hosts who are inherently incapable of digesting plant material to derive nutrients. Gastrointestinal microflora also inhibits the growth of pathogenic bacteria, and this mechanism is termed pathogen interference (PI) or, alternatively, colonization resistance (CR). CR can occur by direct inhibition of pathogenic bacteria by indigenous host gut microbiota, or by nutrient depletion by indigenous flora, and indirectly by stimulation of host defense mechanisms. Commensal bacteria can directly inhibit the growth of pathogenic bacteria by multiple mechanisms, including secretion of bacteriocins that are toxic to other pathogens, or release of metabolites like butyrate and acetate that are inhibitory to pathogenic bacteria, or via competition for oxygen (depleting the pathogens of oxygen) and also by competitive exclusion of pathogenic bacteria from epithelial binding sites. Because of its density, richness, and complexity, the gut microbiota inherently utilizes many “leftover” nutrients not absorbed by the host, which in turn severely restricts the availability of nutrients needed to support the colonization and growth of any ingested pathogenic bacteria. Conventionally reared animals are usually raised in conditions that favor the establishment of naturally occurring microbiota in the gut and other anatomical locations. For example, conventionally reared mice are frequently colonized by a complex and dynamic gut microflora that can include as many as 500 different bacterial species. The microflora in these animals usually confers colonization resistance to many gut pathogens – for example, Salmonella enterica serovar typhimurium. Specific pathogen-free (SPF) animals are considered to be free from a defined list of pathogens and raised in facilities that maintain their SPF state. However, their intestinal microfloral make-up is usually unknown and variable because their floral composition is dependent



TABLE 56.6

Composition of Altered Schaedler’s Flora (ASF) Description


Lactobacillus sp.


Lactobacillus sp.


Clostridium strain


Clostridium strain


Eubacterium spp.


Bacteroides spp.


Gram-positive bacterial strain


Flexispira spp.

on many factors, including the animal vendor source, genetics, and environment. Gnotobiotic animals are animals that are usually cesarianderived under aseptic conditions and raised in germ-free (GF) conditions except for experimental colonization by a set of known or defined bacterial species such as Altered Schaedler’s Flora (ASF). ASF, first developed in the mid1980s, consists of eight murine bacterial species that can be used as a substitute for conventional microbiota and can aid in the establishment of basic gut physiological functions. The components of ASF flora are shown in Table 56.6. It should be noted that mice and rats colonized by ASF do not differ greatly from germ-free (GF) counterparts in their general profiles for many microbiota-associated characteristics, as detailed in Table 56.7. However, ASF flora does not represent most of the dominant bacterial species that are usually associated with conventional mice. Germ-free animals are typically derived aseptically by hysterectomy (cesarian section), and followed by immediate transfer of the fetus into a sterile chamber for rearing in high hygienic conditions, thus preventing exposure of the fetus to maternal microflora, which usually happens via vaginal delivery. The gastrointestinal tracts of germ-free animals, if uncontaminated throughout their lives, will have different physiological, anatomical, and biochemical characteristics than their conventional counterparts. Most strikingly, GF mice and guinea pigs have an enlarged cecum


(a feature that is believed to be the result of altered gastrointestinal metabolism in the absence of gut microflora) with physiological alterations such as absence of mucin breakdown, reduced biodegradation of dietary particulate matter like fiber, and a reduced sensitivity to biogenic amines animals. Germ-free mice also have thinner intestinal walls, with slender and uniform villi, and smaller or reduced GALT/MALT foci. Further, germ-free animals tend to have a more favorable response to surgical manipulations, with reduced post-surgical intestinal adhesions in experimental studies. In certain contextual situations, GF animals administered only probiotics like lactobacilli can still retain their GF characteristics but can be induced to switch to a more conventional phenotype by administration of E. coli, Clostridium difficile, or other pathogens, thus underlying the importance of gut microbiota in shaping GI physiology and anatomy. Germ-free animals also have higher nitrogen and Vitamin K dietary requirements than conventional animals. It is now increasingly being recognized, via NMR and MS studies, that there are significant differences in the urinary metabolic profiles of germ-free mice and rats as compared to their conventionally reared counterparts (Table 56.8). GF animals are highly valuable in studying the physiological functions of gut microflora, as well its modulatory effects on many probiotics, pathogens, antibiotics, diets, toxicants, and carcinogens. Gut Microflora and Gastrointestinal Physiology The mammalian gut microbiota has co-evolved with its hosts to form a delicate equilibrium within the hosts to maintain gut physiological homeostasis. This relationship often triggers both local and systemic biological responses. The gut microbiota aids in food digestion and nutrient metabolism; promotes fat metabolism, intestinal development, and maturation; maintains epithelial integrity and homeostasis; stimulates immune cell development, differentiation, and cytokine balance (Th1 vs Th2, Th17 responses); and aids in enteric innervation pathways, promoting angiogenesis etc. AGE AND MATERNAL EFFECTS

All laboratory animals and humans are born with a more or less sterile gastrointestinal tract (especially if derived by cesarean section), but




TABLE 56.7 Physio-Biochemical Differences between Conventional and Germ-Free Micea Parameter

Conventional mice

Germ-free mice

Intestinal wall



Cell kinetics



Migration of motor complexes



Production of peptides



Sensitivity to peptides



Cecum size ( rodents)

Normal (less than 1% of total body weight)

Enlarged (> 5% of total body weight)




Colloidal osmotic pressure



Oxygen tension



Electropotential EH, mv






Bile acid metabolism

Deconjugation, dehydrogenation, and dehydroxylation occurs

No deconjugation, no dehydrogenation, and no dehydroxylation

Bilirubin metabolism

Much deconjugation, and urobilin present

Sparse deconjugation, and no urobilin



No coprostanol

Intestinal gases

Carbon dioxide

Some carbon dioxide


No methane


No hydrogen



No degradation

Short chain fatty acids

Large amounts

Lesser amounts

Tryptic activity

None to sparse


Table adapted from Gastrointestinal Microbiology, A. Ouwehand and E. E. Vaughan, eds. (2006) Taylor & Francis Group, Chapter 15, pp. 272–275.

subsequently the tract is continuously colonized by microbial populations until adult life, when the gut microflora establishes a more or less stable equilibrium with the host. In spite of many broad similarities in the diversity of gut microflora among many animal colonies/genotypes of a particular species, there is frequently a high

degree of variability in the microbiota between individual animals and within many niches in the same animal. Group housing and coprophagic behavior in rodents might have a negative impact on gut floral diversity, unlike noncoprophagic humans. In all hosts, both genetics and environment are important determinants of



TABLE 56.8


Gut Microbiome-Related Metabolic Profiles of Germ-Free and Conventional Animals

Metabolic profiles Urinary (GF state)

Observed differences in the metabolic signatures of GF animals (mice and rats) or antibiotic-treated conventional animals vs conventional animals Mice (C3H/HeJ): 3-hydroxylcinnamic acid, 4-hydroxypropionic acid, hippurate, phenylacetylglucine Rats (SD): 2-oxoglutarate, formate, trimethylamine N-oxide, hippurate, 4-hydroxypropionic acid, 3-hydroxypropionic acid

Urinary (Conventional rats following antibiotic treatment)

Lower concentrations of phenolics and other microbial metabolites, including hippurate, phenylacetylglycine, trimethylamine, dimethylamine, higher oligosaccharides and choline

Plasma (GF state)

Rats (SD): betaine, glucose, lactate

Liver (GF state)

Mice: glutathione, glycine, hypotaurine and trimethylamine N-oxide

Intestine (GF state)

Mice: alanine, aspartate, glutamate, lactate, taurine-conjugated bile acids, tyrosine and glycine

Kidney (GF state)

Betaine, glucose, ethanolamine, inosine and myo-inositol

gut microfloral diversity. Another important factor is the “maternal effect,” which is the shared exposure to maternal microflora of all the pups from the same litter that are born vaginally, and its resultant contribution to shared similarities in bacterial and immunological diversity. This effect can be discernible across multiple generations even when the offspring have been reared in different cages and environments. This effect can be largely reduced by cesarean delivery, cross-fostering, and uterine transplantation of embryos, and between different genotypes. Therefore, the final composition of any individual animal’s gut microbiome is the end result of a complex interaction between various factors, including host genetics, age, maternal effects, coprophagic behavior (if present), environment (food, other animals of same or different species), and exposure to pathogens and other factors that modify normal gut microbiota. NUTRITION AND METABOLISM

The intestinal microbiota plays an important role in host nutrition, metabolism, degradation, and excretion. Feces can contain up to 50% of degraded and viable bacterial biomass. The gut bacterial microflora is involved in the metabolism and breakdown of carbohydrates, fats, and

proteins of plant, animal, and microbial origin. It is important for the synthesis of many vitamins, for breakdown of plant fibers, and for metabolism of cholesterol, bile acids, bile salts, hormones, drugs, and toxins. Many key biotransformation reactions, like hydrolysis, reductive reactions, nitrosation, desulfation, aromatization, and removal of aromatic functional groups (dehydroxylation, decarboxylation, demethylation, deamination, and dechlorination), are performed by the intestinal microbiota by virtue of its many enzymes on a wide variety of substrates encompassing plant and animal material, host cells, secretions, enzymes, and even microbial components. The intestinal microbiota is essential for the degradation and fermentation of complex carbohydrates, such as cellulose, into much simpler soluble carbohydrate molecules that are its main sources of energy. These complex carbohydrates are not readily absorbed by the host, and thus this fermentation provides a salvage function that is beneficial to both microbe and host. Intestinal bacteria which are typically in high numbers in the colon and ileum – for example, Bacteriodes spp. – can degrade both plant polysaccharides like xylan and host-derived gylcated polysaccharides such as chondroitin sulfate, mucin, heparin,




hyaluronate, and glycosphingolipids. The end products of bacterial fermentation of carbohydrates as well as bacterial degradation of amino acids are the short chain fatty acids (SCFAs) (Table 56.9). The major SCFAs are acetate, butyrate, and propionate, and the minor ones include lactate, succinate, and formate. SCFAs, although not important for dietary needs, are important for mucosal stimulation, are readily absorbed in the colon, and facilitate colonic absorption of water and salt. Intestinal bacteria also take part in many steps of protein and amino acid metabolic pathways primarily in the large intestine by their action TABLE 56.9

on substrates comprising incompletely digested dietary proteins, intestinal epithelial structural proteins, nitrogenous components (ammonia, urea, nitrate), digestive secretions (mucins, enzymes, glycoproteins), and free amino acids/ peptides of both host and microbial origin. Gut microbiota can also produce ammonia and aid in the incorporation of ammonia into host cellular components. Many intestinal bacteria such as Bacteroides, enterobacteria, lactobacilli, clostridia, and bifidobacteria, are endowed with decarboxylases that typically act on lysine, ornithine, histidine, and tyrosine to form their respective putrefaction end products, namely

Summary of Microbial Associated or Microbial–Host Co-Evolved Metabolic Processes

Metabolite class

Metabolite examples


Short chain fatty acids (SCFA)

Acetate, butyrate, propionate

Microbial fermentation in large intestine of substrates e indigestible oligosaccharides, plant fibers, nondigested proteins and mucin


Putrescine, cadaverine

Anaerobic microbiota-driven process of putrefaction into polyamines e exerts adverse effects on the host, including genotoxicity

Methylamines and choline degradation products

Methylamine, dimethylamine, dimethylglycine, trimethylamine, trimethyl N-oxide

Microbial-induced metabolism of dietary cholines in the gut e modulated in some dietary models of obesity, diabetes and cardiovascular disease


Benzoic acid (plasma), hippurate (urine), 2- hydroxyhippurate

Low urinary hippurate is a consistent feature of obesity in humans and animal models; 2-hydroxyhippurate is also associated with colorectal cancer in humans

Chlorogenic acids

Dihydroferulic acid and its derivatives

Chlorogenic acids are known antioxidants with beneficial health effects

Tyrosine/ typtophan/ phenylalanine

4-Cresyl sulfate/indoleacetylacetate/ phenylacetylglycine

Variations of some key metabolite levels are observed in rat models of depression, diabetic patients, and in autistic children

Bile acids

Cholic acid, deoxycholic acid, ursodeoxycholic acid, glycocholic acid

Gut microbiota modifies bile acid profiles to produce secondary and tertiary bile acids; increased in diabetic patients, and with possible impacts on cholesterol metabolism


Acylglycerol, sphingomyelin, cholesterol, phosphatidylcholine, triglycerides

Gut microbiota regulates lipid synthesis and metabolism through enzymatic activities in the colon



cadavarine, putrescine, histamine, and tyramine. Intestinal bacteria can also act on complex phytochemicals like dietary lignins and isoflavones and facilitate in their degradation. Moreover, these bacteria are important players in cholesterol metabolism and bile reabsorption from the intestine, as well as de novo sterol synthesis in the gut. In the distal ileum and colon, the conjugated primary bile acids secreted in bile are acted upon by resident microflora, resulting in hydrolysis and deconjugation of secondary bile acids and subsequent release of free bile acids for intestinal reabsorption (Table 56.9). Many vitamins are synthesized from ingested food by gut bacteria, and these are utilized by both the microbes and the host cellular apparatus. These include Vitamin B12 (cobalamin), Vitamin K, pyridoxal phosphate (active form of Vitamin B6 that is important for amino acid metabolism), pantothenic acid (Vitamin B5), niacin (Vitamin B3), biotin, and folic acid. There are some differences between monogastric animals and ruminants in the bacterial metabolism of vitamins; for example, in ruminants Vitamin B12 is exclusively synthesized by colonic microflora, whereas in humans and rodents the role of bacteria in Vitamin B12 synthesis/metabolism is either insignificant or absent. Germ-free rats have an obligate requirement for dietary biotin as compared to their conventional counterparts. The microbiota is also important for absorption of many key minerals, including iron. Germ-free rats, which lack the normal intestinal microbiota, develop iron-deficient anemia when fed on a diet low in iron. DETOXIFICATION/PRODUCTION OF MUTAGENS

Microbial metabolism in the gut also serves to detoxify many toxic xenobiotics and protect or suppress the harmful effects of the xenobiotic metabolites on the host. Examples of such metabolized drugs/additives that are degraded by microflora include digoxin, diethylstilbesterol, estrogens, cyclamate, azulfidine, 3,4dihydroxyphenylalanine (DOPA), amygdalin, metronidazole, caffeine, propachlor, morphine, buprenophine, oxazepum, phenolpthalein, warfarin, and DDT. Intestinal microbiota, however, also have many bacterial enzymes that can catalyze the production of mutagens, carcinogens, and tumor


promoters; examples of these enzymes include b-glucuronidase, b-glucosidase, b-galactosidase, nitroreductase, azoreductases, tryptophanase, and 1-a-steroid dehydrogenase, to name a few. These enzymes can act on a variety of substrates, including non-nutritive plant material, as well as metabolize administered drugs/toxins and supplements/additives. Some examples of substrates that are acted upon by gut microfloral enzymes to become mutagenic include cycasin (a plant-derived b-glucosidase; carcinogenic), 2-nitofluorene, trypan blue, tryptophan, and dimethylamine. IMMUNE MODIFICATION

“Dysbiosis” of the gut microbiome is associated with many disorders in humans, such as inflammatory bowel disease (IBD), obesity, diabetes, and autoimmune disorders like multiple sclerosis. Similar disease states are also observed in laboratory animals, as well as companion and farm animals, in both natural and experimental settings. The key role played by gut bacteria in shaping the development of a robust immune system is now well established following numerous studies comparing germ-free and conventional animals. The gut microbiota is important for the optimal functioning of the humoral components of the mucosal immune system, for T-cell differentiation, and for Th1/ Th2 cytokine expression. The importance of the gut microflora is primarily understood from studies in germ-free animals, which have a suboptimal immune system as compared to conventional animals. GF animals are highly susceptible to infections by pathogenic bacteria like Shigella flexneri, Listeria monocytogenes, Clostridium difficile, and Salmonella enterica. Germfree mice, as mentioned earlier, have an enlarged cecum, and fewer gut-associated lymphocytes, plasma cells, and functional macrophages. They also exhibit a general reduction in other cellular and humoral components, including mucosal serum IgG and mucosal IgA, T-cell numbers, cytokine production, degree of vascularity, digestive enzyme activity, amounts of antimicrobial peptides, and tissue ATP levels. Intraepithelial lymphocyte (IEL) counts and expression levels of MHCII are also reduced in the intestine of germ-free mice. In addition, the colonic mucosa of these mice have a lower rCD4þ T-cell counts, and a decrease in FoxP3




regulatory T cells with a concomitant decrease in CD4þCD25þve T cells in the mesenteric lymph nodes. The sizes of Peyer’s patches, splenic lymphoid sheaths, and lymph node follicles also are smaller in GF mice as compared to conventional animals, due to lack of microbial immune stimulation. Many members of the intestinal microbiota are uncultivable, and hence very little is known about their precise roles in host immunity and mucosal barrier functions. Many commensal bacteria in the gut can induce either pro-inflammatory or anti-inflammatory states, or both, and can also promote or inhibit the growth of other bacteria, depending on different environmental or dietary settings. As an example, Helicobacter hepaticus, a normal inhabitant of the intestine of most mice, rarely causes disease in immunocompetent animals but frequently induces colitis and/or hepatitis in immunodeficient animals (for example, in IL-10 KO mice) or in instances when the anti-inflammatory effects induced by IL-10 promoting endogenous bacteria like Bacteriodetes fragilis are diminished by antibiotic therapy, toxins, or carcinogens. A more recent example of gut microbial induced immune modulation comes from observations with segmental filamentous bacteria (SFB), also known as Candidatus arthomitis. SFB are a group of well-recognized non-cultivable, autochthonous, non-pathogenic bacteria that occur in close association with the epithelium and Peyer’s patches of the ileum of mice and rats, pigs, horses, humans and other mammals, as well as chicken and fish. SFB are now recognized to be important for normal immune development through activation of both T- and B-cell responses and the resultant production of both anti- and pro-inflammatory cytokines. Specifically, SFB are recognized for their role in promoting murine T helper cell-directed Th17 differentiation and production of IL-17 and IL-22. Some of the best characterized commensal intestinal bacteria with immunomodulatory effects on pathogens are Bacteriodetes fragilis, Clostridium spp., Bifidobacterium spp., and Lactobacillus spp. A summary of some salient immunomodulatory effects of these bacteria appears in Table 56.10. It is also increasingly recognized, at least in mice, that commensal or pathogenic bacteria can immunomodulate the GI pathology associated with

other pathogenic bacteria from either the same genus or unrelated genera. An example of this phenomenon is the differential suppression or enhancement of H. pylori-induced gastric histopathological lesions in mice that have been cocolonized by enterohepatic helicobacters (EHS) such as H. muridarum or H. bilis (inhibitors of gastric inflammation) vs H. hepaticus (a proinflammatory enhancer).

3. EVALUATION OF GASTROINTESTINAL TOXICITY The ability of the gastrointestinal tract to adapt to various diets and non-toxic compounds is well established. Both adaptational and toxicologic processes can be manifested by altered structure or function. Evaluation of these processes requires a basic understanding of the mechanism or suspected pathogenesis of the toxic injury or response. Routine approaches involve in vitro and in vivo methods. Because alterations in numerous other organ systems can occur as a result of gastrointestinal toxicity, whole animal studies are generally required in order to properly interpret gastrointestinal toxicity. As a result of this complex interrelationship between organ systems and the inherent complexity of the gastrointestinal tract, animal models have been developed to study various gastrointestinal diseases and toxicities.

3.1. In Vitro Strategies In vitro studies can be used to detect alterations in the mucosal lining or in individual cellular components of the mucosa. Mucosal lining studies conducted with Ussing chambers can be used to evaluate solute transport by large regions of isolated mucosa from various isolated segments of the gastrointestinal tract. Long-term organ and explant cultures of gastrointestinal tract can be used to conduct carcinogen metabolism studies. Advances through cell culture techniques for gastrointestinal epithelial cells, isolation of membrane vesicles, and molecular biological methods have provided information about binding of agents to epithelial cells, location of enzyme systems, mechanisms of molecular transport, and genetic damage.



TABLE 56.10


Salient Examples of Gut Microbiota-Induced Host Immunomodulation


Hostebacterial interaction

Salient immunodulatory effects

Bacteriodetes fragilis

Gram-negative anaerobe, most prominent gram-negative bacteria in colon, symbiotic relationship within intact bowel, can become pathogenic in perforating conditions, contains zwitterionic polysaccharide antigens that can elicit both T cell-dependent and T cell-independent responses

Shown to afford protection against immune mediated (adoptive T-cell transfer with H. hepaticus) colitis and chemical induced colitis (TNBS, trinitrobenzene sulfonic acid)

Segmental filamentous bacteria (SFB) (Candidatus arthromitis)

Non-cultivable, non-pathogenic, attaches to epithelium of small intestine over Peyer’s patches in ileum, can induce many inflammatory genes comparable to conventional state in GF mice upon monoassociation

Pro-inflammatory response likely in combination with other bacteria, and activation of Th17 cells (1L-17/IL22) cytokine production; also some anti-inflammatory effects and regulatory T-cell promotion, effects restricted to small intestine

Clostridium spp.

Numerous species, pathogenic and non-pathogenic, that colonize the large intestine of most species

Most commensal species have antiinflammatory effects and induce expansion of regulatory FoxP3þve regulatory T cells in the intestine, effects compartmentalized to the large intestine only

Bifidobacterium spp.

Mostly colonize the colon and adhere to the epithelium; importantly, aids in digestion and nutrient uptake; used as a probiotic, and decreased in inflammatory bowel disease

Bacterial cell surface components like exopolysaccharides, bacteriocins, lipoteichoic acid, extracellular proteins, secreted factors like serine proteases (serpin, inhibits neutrophil elastase) are immunostimulatory; important for mucosal integrity and tight junctions, barrier function and colonization resistance (CR); induces mucin production, anti-inflammatory cytokine production (Treg cells, IL-10, IL-12) and antimicrobial peptides

Lactobacillus spp.

Numerous species/strains with varying immunomodulatory/ probiotic effects

Bacterial surface proteins, polysaccharides, secreted proteins induce antimicrobial peptides; competitive binding of epithelial receptors against pathogens like Salmonella typhi, Staphylococcus aureus, pathogenic E. coli, Listeria monocytogenes, etc., contributing to colonization resistance; anti-inflammatory cytokine production (Treg cells, IL-10, IL-12) and antimicrobial peptides

Cellular and organelle markers have been established to identify cells from various portions of the gastrointestinal tract. Keratin can be used as a general marker of epithelial cells and the extent of differentiation. Identification of colonic

epithelial cells can be verified using antibodies to colon-specific antigen (CSAp), colon antigen 3, or 5E-113. Disaccharidases of small-intestinal epithelium can be identified using antibody or enzyme assays. Antichromogranin, neuron-specific




enolase, argyrophilic stains, and morphology can be used to identify neuroendocrine cells. Goblet cells can be identified morphologically using Schiff reagent for mucins. A biochemical anatomy of cells in the gastrointestinal tract can be obtained through in vitro assays of enzymatic activities in biopsied mucosa. Enzyme markers can be used to detect alterations of brush border (disaccharidases), lysosomes, peroxisomes, mitochondria (NADH), and endoplasmic reticulum. Evaluation of lumenal contents is also part of the search for gastrointestinal tract toxicity. Lumenal fluid can be used to determine if altered bacterial populations and abnormal enzyme secretions are present. Occult blood can be detected in stool samples, and represents a non-invasive means of evaluating the integrity of the mucosa. Since the first organ system to be exposed to ingested xenobiotics is the gastrointestinal tract, highly sensitive assessments of genotoxic potential can be conducted using a single cell electrophoresis assay to measure DNA breakage in isolated gastrointestinal cells. This assay, also referred to as the COMET assay, is adapted from other existing methods to assess single DNA strand breaks, and can be used on a variety of cell types when the cell population is in a suspension. The technique involves immobilization of single cells in agarose gel and then using an electrophoretic approach under alkaline conditions. The DNA content of the cells is visualized by ethidium bromide or acridine orange, and the charged DNA migrates with the current so that the extent of DNA migration from the nucleus towards the anode is quantified. Cells with genetic damage exhibit longer DNA bands, giving a comet-like appearance, and the tail length of the comet is related to the number of DNA strand breaks. By isolating cells from animals exposed to the test chemical, an ex vivo approach takes into account the ability of an organ system to metabolize the test chemical. Therefore, exposure of animals to a toxicant may allow detection of direct and indirect carcinogens as well as those that are classified as syncarcinogens, co-carcinogens, or anti-carcinogens.

3.2. In Vivo Strategies Whole-animal studies require proper structural and functional evaluation of the entire

animal in addition to careful attention to the gastrointestinal tract. Attention to other organs such as liver and pancreas provides clues to gastrointestinal tract disorders of uncertain origin. Altered lipid absorption (a primary intestinal disorder) and abnormal bile acid release (a primary liver disorder) may lead to similar clinical abnormalities, but clearly have different causes. Consequently, proper in-life and post-mortem evaluations are needed to properly establish a cause-and-effect relationship. The major function of the gastrointestinal tract is nutrient absorption. The extent and rate of absorption can be quantitatively assessed by administering a test agent and determining the concentration of this agent in blood, tissues, and feces. Passive permeability of markers (e.g., 51Cr-labeled EDTA) from the intestinal lumen into the blood may be used to detect smallintestinal diseases. Additionally, various agents that are non-absorbed (e.g., polyethylene glycol 4000) or absorbed (e.g., radiolabeled amino acids) can be used to answer different questions about nutrient absorption. One example is the use of radiolabelled ferritin to show that absorption of ferritin iron is mechanistically different from iron absorption through small iron complexes/salts. Propulsive activity of the gastrointestinal tract can also serve as an indicator of toxicity. Tracking unabsorbable markers serves as a net indicator of motility. Specific aspects of motility, such as muscle contractility or electrical activity, can also be quantitated. The extent of the evaluation and the exact approach used will be dictated by the goals of the study. Organ weights and volumes, and their ratios to body weight, are sensitive allometric measurements of gastrointestinal tract response and toxicity. The ability of the stomach and intestine to adapt to various diets and compounds can be established using such methods. Since both adaptational and toxic responses can be manifested by changes in tissue volumes or weights, more refined structural and functional methods may be required (Figure 56.10). Interpretation of organ weights of the GI tract must consider the animal’s basal diet and its composition, since, for example, rats maintained on total parenteral nutrition gain body weight but the weights of the stomach, small bowel, and colon are often










3.0 2.5 2.0 1.5 1.0 0.5








0.0 AIN 76A AIN 76AF NIH 07






FIGURE 56.10 Impact of different diets on volumetric (A) and allometric (B) measurements to consist of the large intestine of rats fed each diet for 3 weeks. Adaptation to each diet is reflected as a modification in the surface area and tissue volume normalized to body weight. Starting and ending body weights were not significantly different among rats fed any of the diets. The mucosa was considered as being the epithelial lining layer, lamina propria, and lamina muscularis mucosa. Muscle was considered to consist of the tunica muscularis mucosa and associated submucosa and serosa. All animals were fed ad libitum. Abbreviations: AIN, American Institute of Nutrition; A, absence of fiber; AF, fiber (5% methyl cellulose); NIH, National Institutes of Health; Purina, 5001 diet. Figure reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 20, p. 171, with permission.

only 30–40% of comparable tissues from control animals.

3.3. Molecular Pathology Genomics and proteomics form the basis of new molecular methods that can be used to provide mechanistic information on toxicologic processes underpinning lesions produced in the gastrointestinal tract. The ability to evaluate gene activity by detecting RNA using multigene cDNA or oligonucleotide sequence microarrays allows a qualitative assessment of gene expression in samples of tissues or lesions identified throughout the GI tract. Qualification of RNA can be performed through real polymerase chain reactions, and protein profiles (i.e., proteomics) can be determined using two-dimensional polyacrylamide gels. When these procedures are coupled with in situ hybridization and immunohistochemical procedures, localization of mediators and cells producing various cytokines allows for integration of molecular and morphologic information to establish a mechanistic basis for toxicologic pathologies. Application of molecular methods has been important in defining the role of growth factors and inflammatory cytokines in cellular

proliferation, morphogenesis, and tissue repair of gastric ulceration. Epidermal growth factor (EGF) inhibits acid secretion, protects the gastric mucosa, and stimulates cell migration and proliferation in the ulcer healing process. EGF exerts its action by binding to EGF-receptor, a transmembrane protein tyrosine kinase, which causes receptor dimerization, autophosphorylation, and localization of kinase substrates. Collectively, these events lead to Ras (a GTP-binding protein) activation of the Ras/Raf/MAP kinase pathway, phosphorylation of regulatory proteins and transcription factors, and, finally, cell proliferation. Similarly, gastric mucosal lesions stimulate not only production of hepatocyte growth factor (HGF) but also serine proteases in acute and chronic gastric ulcers. HGF, a heparinbinding growth factor, is a potent stimulator of mucosal cell proliferation and differentiation. As with EGF, HGF effects are mediated by specific receptors found in gastric mucosal epithelial cells. Xenobiotics such as NSAIDs affect various growth factors and cytokines to decrease epithelial cell proliferation, angiogenesis, and maturation of granulation tissue. Modification of protective prostaglandins can change vascular endothelial cell growth factor expression from fibroblasts,




thereby reducing the level of angiogenic growth factor production, and delay ulcer healing. Multiple cytokines, growth factors and regulatory factors are regulated during tissue injury,

response, and repair. The complexity of gene regulation when characterized by multigene microarray technologies is demonstrated in Figure 56.11 and Table 56.11b. Over 600 genes

FIGURE 56.11 Molecular characterization of gastric ulcers induced by indomethacin. (A) Glandular and nonglandular stomach of rat treated with indomethacin. Boxed area contains gastric ulcer near duodenum. (B) Microscopic section of gastric ulcer taken from boxed area shown in panel A. Boxed area shows margin between reactive mucosa and ulcerated submucosa. (C) Section of reactive mucosa taken from boxed area in panel B with epithelial cell hyperplasia (E), inflammatory cells (I), and fibroplasia (F). (D) Lamina propria with blood vessels containing marginated neutrophils (I) and fibroplasia (F). (E) Thirty downregulated genes expressed in tissue samples taken from the margin of the ulcer (boxed area in panel B). (F) Thirty upregulated genes expressed in tissue samples taken from the margin of ulcer (boxed area in panel B). Differentially expressed genes are involved in inflammation, cellular adhesion, DNA repair, apoptosis, cellular metabolism, cytokines, and cell cycle control (Table 56.11A&B). Figure reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 21, pp. 172–173, with permission.



FIGURE 56.11




2312 TABLE 56.11A


Key for Figure 56.11B: Gene Expression in Rat Gastric Ulcers Induced by Indomethacin

Upregulated genes

Downregulated genes


Serotonin receptors


Fanconi anemia group-C complementing protein


Hypoxanthine phosphoribosyltransferase (HPRT)






Nerve growth cone membrane protein (GAP-43)


DNA-mismatch repair protein (PMS2)




Flavin monooxygenases form-1


Macrophage-1 antigen


Platelet protein P47




Growth arrest and DNA-damage inducible protein (GADD45)


Prohormone convertase 2 (PC2)


Fibroblast growth factor receptor-2


Creatine kinase B


GTU-mismatch DNA glycosylase


ob-protein (obesity homolog)




OxyR protein


Proliferating cell nuclear antigen (PCNA)


Phosphotransferases (alcohol group acceptor)




Cytochrome c oxidase (10)


KI-67 antigen


Late embryogenesis abundant protein


spr1 protein




Malate dehydrogenase


Nitric oxide synthase related antigen-1


Hepatocyte growth factor receptor


Atrial natriuretic factor


Vascular cell adhesion molecule-1






Retinoic acid receptor gamma




Glutathione peroxidase


Glucocorticoid receptor


Serine protease inhibitor






TATA-box binding protein


Keratinocyte growth factor


DNA-mismatch repair protein (MSH3)


Cytochrome c oxidase (7C)




rDlx-protein (Homeoprotein)




Proto-oncogene protein c-yes


Glyceraldehyde 3-phosphate dehydrogenase (G3PDH)




Cellular apoptosis susceptibility (CAS)




Heparin binding EGF-like growth factor


Organic cation transporter (OCT 2)


Monocyte chemoattractant protein-3


Growth arrest specific protein-8


Endothelial cell nitric oxide synthase



Table adapted from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Table XIIA, p. 174, with permission.




TABLE 56.11B

Functional Categorization of Genes Expressed in Gastric Ulcers Induced by Indomethacin

Inflammatory/ immunoregulatory


DNA repair/apoptosis


Vascular cell adhesion molecule-1 (17)

HPRT (2) ¼ nucleotide salvage gene

spr1 protein (14) ¼ unknown function

Platelet protein P47 (6) Integrin-A2 (18)

PMS2 (4)

Fanconi anemia group-C complementing protein (31) ¼ unknown function

Monocyte chemoattractant protein-3 (29)

GADD45 (7)

Nidogen (32) ¼ structural

Endothelial cell nitric oxide synthase (30)

GTU-mismatch DNA glycosylase (9)

Atrial natriuretic factor (46) ¼ homeostasis

Macrophage-1 antigen (35)

BRCA2 (10)

serine protease inhibitor (50) ¼ unknown

Nitric oxide synthase related antigen-1 (45)

MSH3 (23)

rDlx-protein (54) ¼ unknown

Lactoferrin (57)

Caspase-8 (25)

OCT 2 (58)

Thymopoietins (3)

Integrin-A6 (19)

CAS (27) Cell cycle

Metabolism & cellular homeostasis


PCNA (11)

Flavin monooxygenases form-1 (5) Serotonin receptors (1)

KI-67 antigen (13)

Glucose-6-phosphatase (12)

Fibroblast growth factor receptor-2 (8)

Cyclin-G (21)

Malate dehydrogenase (15)

Hepatocyte growth factor receptor (16)

TATA-box binding protein (22)

G3PDH (26)

Glucocorticoid receptor (20)

Heparin binding EGF- Dimethylallyltranstransferase (34) like growth factor (28)

FYN (24)

GAP-43 (33)

PC2 (37)

ob-protein (39)

hox-7.1 (36)

Creatine kinase B (38)

OxyR protein (40)

Late embryogenesis Phosphotransferases (41) abundant protein (43)

Retinoic acid receptor gamma (48)

HSP27 (56)

Inhibin (51)

Cytochrome c oxidase 10 (42)

Growth arrest specific CYP2A 2A7 (44) protein-8 (59) wnt3a (60)

Keratinocyte growth factor (52)

Serine-esterase (47) Glutathione peroxidase (49) Cytochrome c oxidase 7C (53) Proto-oncogene protein c-yes (55)

( ) ¼ numerical designation of genes in Table 56.11A. Table adapted from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Table XIIB, p. 175, with permission.



were evaluated, using high-density microarrays, from tissue samples taken at the ulcer margin and from an adjacent region without ulceration. At least 60 genes were found to be either up- or downregulated by a factor of two-fold or more when ulcerated tissue as compared to normal tissue. The diversity of genes changing illustrates the complexity of gene modulation, and the need to integrate proteomic and genomic findings with physiologic and pathologic observations to ensure proper interpretation and attribution of a gene’s or protein’s role in a toxicologic process.

3.4. Morphological Methods Evaluation of the gastrointestinal tract for toxicity should be conducted using macroscopic, microscopic, and ultrastructural methods. Macroscopic evaluation includes identification of ulcers, enlarged lymphoid tissues (e.g., Peyer’s patches), neoplasms, and foreign bodies. Microscopic studies should be conducted on all lesions observed macroscopically, and at preselected tissue sites. Proper tissue fixation is essential for structural studies. In an attempt to standardize communications, specialty organizations are adopting standardized nomenclature for the description of microscopic lesions in certain portions of the gastrointestinal tract (Table 56.12). The most current terminology, developed by the International Harmonization of Nomenclature for Diagnostic Criteria in Rats and Mice (INHAND), will be available on the Society of Toxicologic Pathology website (http://www. toxpath.org) when completed. Assessment of morphological alterations of the gastrointestinal tract should consist of close evaluation of the mucosa and its specializations. The mucosa consists of surface epithelium, crypts/ glands, lamina propria, and a thin layer of muscle separating the mucosa and submucosa. Specializations of the mucosa include glands of the esophagus, foveolae of the stomach, villi of the small intestine, and glands of the large intestine. The submucosa and the tunica muscularis (outer muscle layers) should also be examined for changes in thickness and cellularity. Villi should be evaluated critically when assessing small-intestinal toxicity. Since villi bend in various directions, and have shapes that vary with species (e.g., tongue-like in rats and fingerlike in humans) and location (longer in the

duodenum than in the ileum), close comparisons with control animals is required to prevent misinterpretation. Changes in the lamina propria will be detected by assessing alterations in the normal cell population. Neutrophil, eosinophil, lymphocyte, and plasma cell populations may change. An increase in any of these populations is a potential indication of an underlying toxic or disease process. Inflammatory cell infiltrates frequently occur secondary to epithelial cell toxicity. Lymphoid follicles may develop and be associated with an extensive increase in lymphocytes and plasma cells. These follicles may occur in the lamina propria or submucosa (Figure 56.4). Additionally, lymphomas may be associated with a substantial number of abnormal lymphocytes in the lamina propria. Changes in the submucosa may involve blood vessels, nerves, and lymphatics. Alterations in this region are frequently characterized by lymphangiectasia and inflammatory or neoplastic cell infiltrates. Different fixatives are used for histologic evaluation, depending on the purpose of the study and the technique being used. A routinely used multipurpose fixative is 10% neutral buffered formalin. Because of rapid post-mortem autolysis, gastrointestinal tract tissues must be placed into the fixative within 1–2 minutes of death for optimum evaluation. In some laboratories it is routine to immerse the entire segment of GI tract of interest overnight in 10% neutral buffered formalin. Upon completion of fixation, the GI tract is opened longitudinally and flushed extensively with sterile water to remove any fecal matter. Segments of the fixed GI tract may then be dissected out and embedded in polymer resin prior to sectioning and histological analysis. Ultrastructural studies can be conducted using scanning or transmission electron microscopy. Scanning electron microscopy (SEM) provides information on surface alterations. This technique is particularly useful for examining altered villus structure in the small intestine. Morphometric and stereological analyses are powerful morphological methods that can combine biochemical and morphological data. Normal cellular proliferation, differentiation, and senescence are processes that have distinctive phenotypic and genotypic characteristics. These molecular alterations can be examined



phenotypically by using plant lectins (e.g., wheat germ agglutinin) and histochemical stains (e.g., periodic acid-Schiff or Alcian blue). Membrane glycoproteins are useful biomarkers to detect epithelial cell differentiation occurring in the gastrointestinal tract. An understanding of the genetic control of these processes can be obtained using techniques such as in situ hybridization. Although not as sensitive as in vitro blotting technology, in situ hybridization allows localization of genes active in organ and tissue alterations to specific cell and tissue locations.

3.5. Animal Models General Considerations Variations in mammalian gastrointestinal tract morphology show closer correlation with diet, body weight, and the need for water consumption than with taxonomical classification. The capacity of the gastrointestinal tract to hold digesta decreases with decreasing body weight in herbivorous animals; however, the rate of metabolism increases with decreasing body weight. Smaller animals may have various strategies to compensate for this phenomenon. These adaptive mechanisms include an increase in cecal volume or the practice of coprophagy by lagomorphs and rodents. Because of these modifications, the gastrointestinal tract in these species may render them unsuitable as animal models for humans. Nutritional issues must be considered when extrapolating results from gastrointestinal studies performed in healthy animals to humans. Generally, compounds of therapeutic importance are administered to human patients that are ill and consequently undernourished. Toxicologic effects of therapeutic or other beneficial compounds are tested in healthy animals. These animals are fed nutritionally balanced commercial diets for their lifespan, and are allowed to grow and develop under ideal conditions of lighting, temperature, and humidity. Since macronutrients in the diet markedly affect the drug-metabolizing enzyme systems associated with the gastrointestinal tract, such modifications may alter the maximally tolerated dose. Evaluation of the gastrointestinal tract for toxicity must involve consideration of dietary effects on the mucosa. All diets support normal


growth and health conditions of rats, yet the absorption site (mucosa) in the animals fed a semi-purified diet varies significantly between the diets. This indicates that structural and functional results may be observed that are independent of a compound’s toxic or biological effects. Since many mammalian systems have similar mechanisms of response to toxic compounds, animals serve as ideal test systems for the evaluation of toxic potential, pathophysiological responses, and systemic complications of an ingested or injected compound. In contrast to toxicity testing of compounds for human or animal use, animal models of human diseases are generally defined only on the basis of specific pathologic criteria. The specific lesions may be subtly or markedly different, but the general character of the disease process is usually similar to that observed in the human disease; otherwise, the animal model would be discarded. The few examples below demonstrate the significance and problems of using models for investigating the toxicity and efficacy of any compound that could produce lesions in the gastrointestinal tract. Animal models allow the testing of the structure–function activity of chemical compounds, as well as the identification of chemical therapeutics for human and animal disease. Additionally, basic information on the pathogenesis of the underlying cellular and biochemical processes important to lesion development can be identified. Finally, animal testing provides a substantial and significant bridge between in vitro testing and the ultimate application of any compound for human use. Specific Models ULCERATIVE LESIONS OF THE STOMACH AND SMALL INTESTINE

Propionitrile, cysteamine, 3,4-toluenediamine, and 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP) produce acute and chronic gastric and duodenal ulcers in rodents. The morphological appearance of duodenal ulcers produced by these compounds in rats is similar to that seen in humans (Table 56.13). COLITIS AND TYPHLITIS

Experimental models of colitis have been created in a number of laboratory animals (e.g., rats and guinea pigs) with ricinoleic acid, bile acids, poligeenan, melphalan, formaldehyde,



TABLE 56.12 Location


Oral Cavity

Proliferative lesions Hyperplasia



Inflammatory and ulcerative lesions Broad areas of thickened and differentiated epithelium especially the keratin layer; which may have prominent papillary endophytic projections

Proliferative lesions Hyperkeratosis/ parakeratosis



Initial lesions frequently begin as ulcerative processes followed by neutrophilic exudative phase; established lesions have lymphocyte and plasma cell accumulation; advanced lesions can be associated with alveolar bone loss; dietary content influences extent; severity and frequency of lesion

Degenerative lesions Thickened mucosal layer with retraction of nuclei; Absence of cellular atypia

Degenerative lesions Mucosal atrophy

Results from ulceration, inflammation, mineralization, or infarction; Glandular ectasia and inflammation or fibrosis of lamina propria

Chief cell atrophy

Age related decrease in size and number of chief cells

Intestinal metaplasia

Focal crypt and villous formation with goblet cells and Paneth cells; complete forms have a small intestinal morphology while incomplete forms have a large intestinal morphology


Esophageal enlargement; degeneration of muscle and nerve cells in the wall





Neoplastic lesions of the glandular mucosa


Area of superficial necrosis of the mucosa that does not extend beyond the muscularis mucosa


Adenomatous polyps are usually located in the antrum and are composed of basophilic columnar epithelium organized into glandular structures; mass may be pedunculated or endophytic and arise in areas of reactive hyperplasia


Area of mucosal necrosis that extends to or through the muscularis mucosa


Lesions invade into the submucosa and may metastasize or locally infiltrate; cytologically, cells range from dysplastic to anaplastic with pleomorphic nuclei and increased mitotic index

Glandular dilation

Distention of the basal portion of the gastric gland; distention may be sufficiently large to form a microcyst

Neuroendocrine cell tumor

Also called carcinoids; rarely spontaneous; characteristic lesion of potent gastric antisecretory agents causing hypergastrinemia; agyrophilic cells forming the tumor are reactive for nerve specific enolase and chromagranin-A; may be intramucosal or invasive

Non-neoplastic lesions of the nonglandular mucosa Infrequent spontaneous lesion that may be associated with lymphoma of gastric mucosa or other disease; induced by antisecretory drug treatment; reversible, nonprogressive alteration only reported in rats


May be a focal or diffuse thickening characterized by increased numbers of one or more cell types (basal, spinous, or granular); focal hyperplasia is differentiated from papillioma by the absence of a connective tissue core containing blood vessels and the presence of an intact muscularis mucosa

Hyperplasia (focal)

Most common type is associated with erosions or ulcers and found primarily in the antrum; hyperplastic glands may extend through the muscularis mucosa forming persistent adenomatous diverticula


Increased thickness of nonnucleated keratin layer



Eosinophilic chief cells



Non-neoplastic lesions of the glandular mucosa








TABLE 56.12

Neoplastic lesions of the nonglandular mucosa Also called hypertrophic gastritis or adenomatous hyperplasia; associated with administration of antisecretory compounds; result of endocrine stimulation of the oxyntic region; generalized increase involving all cellular compartments


Pedunculated with a prominent connective tissue core; evidence of localized invasion through the muscularis mucosa near base of papilloma should be considered evidence of carcinoma formation

Hyperplasia (neuroendocrine)

Specific neuroendocrine cell hyperplasia in response to endocrine alterations; in rats, ECL cells are the main cell types responsive to hypergastrinemia; in mice,. basal portions of gastric glands may normally be lined by pure populations of neuroendocrine cells


Circumscribed areas of dysplastic epithelium that distorts adjacent normal mucosa and is confirmed by a basement membrane; same tumors may be sessile or pedunculated


Locally invasive lesion that is a neoplastic proliferation of squamous epithelium

Proliferative lesions

Reactive hyperplasia

Neoplastic lesions

Epithelial cells are basophilic and depleted of mucus with enlarged nuclei containing prominent nucleoli; crypt herniation may occur especially if the muscularis mucosa has been disrupted; in the small intestine reactive hyperplasia can be accompanied by villous atrophy


Circumscribed areas of dysplastic epithelium that distorts adjacent normal mucosa and is confirmed by a basement membrane; same tumors may be sessile or pedunculated



Small and Large Intestine

Hyperplasia (fundic)



Crypts are elongated and have dilated lumens and may have a tortuous contour; architecture of the adjacent mucosa is not distorted by compression; epithelial cells lining the crypts are normal to markedly dysplastic and may forma single layer or be pseudostratified with many mitotic figures; goblet cell numbers are reduced; severe inflammatory reactions may be associated with these foci


Dysplastic epithelium with clear evidence of invasion past the basement membrane into the lamina propria or submucosa; the invasive characteristics of adenocarcinomas must be differentiated from tangential cuts of glands in the lamina propria found at the base of adenomas; there may be a schirrous and inflammatory response which can help differentiate invasive epithelial nests from cryptal herniation; adenocarcinoma of the small intestine are more invasive and metastasize more frequently than those of the large intestine

Information modified from The Society of Toxicologic Pathologists Committee on Standardized Nomenclature for Rodent Lesions.


Focal atypical hyperplasia


2320 TABLE 56.13


Selected Characteristics Comparing Cysteamine-Induced Duodenal Ulcers of Rats to the Natural Disease of Humansa Rat duodenal ulcer

Human duodenal ulcer

Anterior and posterior wall

Anterior and posterior wall

Tendency to perforate



Occurrence of massive bleeding



Chronic and active ulcers



Pyloric ulcers



Increased gastric acid release



Elevated gastrin levels


Antisecretory agents



H2 receptor antagonists



Location of ulcer

Responds to therapy:


Modified from Cheville (1980). Table updated from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Table XIV, p. 179, with permission.

alcohol, dextran sulfate, and acetic acid. Some of the lesions observed in these models may be mediated by mechanisms involving hypersensitivity or alterations in prostaglandin synthesis pathways, and are frequently associated with colon cancer if the compound induces an inflammatory process that is longstanding (e.g., poligeenan). Intracolonic administration of the hapten trinitrobenzene sulfanic acid will also lead to an immunologically based colitis. This model is best demonstrated if damage to the mucosal barrier occurs prior to compound exposure (Figure 56.12). COLON CANCER

Colon cancer represents a leading cause of cancer-related death in Western civilization, but is relatively rare in laboratory animals. Consequently, a number of experimental models have been generated to evaluate this process. In addition, various animal species and dosing regimens have been used to produce colon cancers in animals (Figure 56.13). Both genotoxic and non-genotoxic models of colon cancer are used, but the chemically induced genotoxic models are most consistent (Table 56.14). Of the various chemical models available, the hydrazine derivatives have several properties

that make them useful for evaluating the twostep mechanisms of colon carcinogenesis. Unlike the skin, colon carcinomas can be obtained in 1,2-dimethylhydrazine-treated rats without a preliminary benign tumor stage. The incidence rate is constant and directly proportional to total dose, with an average of one carcinoma per colon per year. This hydrazine derivative produces the promutagenic DNA lesion of O-6-methylguanine. Hydrazine models demonstrate that dysplastic or adenomatous changes are not obligatory stages before cancer; however, these lesions do indicate an increased risk of cancer development. The models may also be used to determine whether the major factors in determining malignancy are ones that cause benign lesions to become larger, or ones that initiate specific cancerous changes in the DNA. The rat is widely used as a rodent model of colon carcinogenesis. The F344 rat strain has a colon with many of the same structural and histochemical features of the human colon. This animal model may be better than the mouse since the mouse colon structure and epithelial-cell histochemical composition differs from that of the human. In F344 rats, azoxymethane produces colon cancer within 3 months, and the neoplasms readily metastasize to regional lymph nodes and



the liver. This biological behavior is similar to that of certain human colorectal carcinomas. ENTEROHEPATIC CIRCULATION


a physiological regulator of small-intestinal epithelium proliferation. b-CATENIN

Species differences in enterohepatic circulation are primarily determined by variations in biliary excretion. Since the rat is a very good biliary excretor, extrapolation of toxicity data on drugs undergoing enterohepatic circulation from rats to other species may result in overestimation of a compound’s safety or toxicity. The metabolism and toxicity of drugs may be influenced by enterohepatic circulation, especially in species, such as rats, deer, and horses, that lack a gall bladder. Because of the concentrating capacity of the gall bladder, compounds may be concentrated to levels 10 times greater than in hepatic bile. Such concentrating activity, coupled with prolonged biliary retention, may favor passive reabsorption through the gall bladder mucosa. Additionally, reabsorption would be markedly enhanced by concomitant mucosal injury to the gall bladder epithelium. These factors must be taken into consideration when considering selection of a model for compounds that may enter the enterohepatic circulation pathway. Knockout/Transgenic Models Transgenic and targeted gene recombination have been used to study the basic biology and toxicology of the gut in a wide variety of systems. These animal models have been used to study cell growth and regulation, metabolism, mutagenesis, and carcinogenesis. CELL GROWTH AND REGULATION TGF-a In the small intestine and colon there is regional variation in the production of EGF and TGF-a. Transgenic mice expressing TGFa regulated by a metallothionine-inducible promoter/inducer have been created which have a phenotype similar to human Menetrier’s disease, including hypertrophic gastric folds with foveolar hyperplasia and cystic dilation, increased neutral mucin staining, and reduced basal and histamine-stimulated rates of gastric acid secretion. Overexpression of TGF-a in the mouse duodenal epithelium results in a pronounced increase in crypt epithelial cell proliferation and increase in crypt–villus dimensions, and suggests that TGF-a may be

A critical component of the Wnt/ Wingless signal transduction pathway and an important effector of cell–cell adhesion through cadherins, b-catenin has been implicated in human colorectal carcinogenesis via its association with the APC gene product. However, attempts to genetically engineer mice homozygous for the null allele resulted in early embryogenic death. Recently, chimeric mice were generated with amino-terminal truncated b-catenin to study the effects on proliferation, cell fate specification, adhesion, and migration within the intestinal epithelium. The resulting mice showed marked increases (four-fold) in cell proliferation and apoptosis in the crypts of Lieberkuhn, augmentation of cell–cell junctions, and interference of normal cellular migration along the crypt–villus axis, occasionally resulting in abnormal architecture, although elevated neoplastic transformation was not observed. MUTAGENESIS

The small-intestine epithelium has been used as a target tissue by a number of groups to validate transgenic gene mutation models. In these studies, mutation rates (spontaneous and induced) at an endogenous host locus (Dlb-1) were compared with those induced at transgenic loci in BigBlue (lacl gene) and Mutamouse (lacZ gene) mice. Somatic mutations at the host locus were quantified in epithelial cells of the small intestine by the loss of the binding site for the lectin Dolichos biflorus on the surfaces of cells of the villus. The results of these studies show that the mutations at the host and transgenic loci accumulate with time, and that on exposure to alkylating agents, the lacl and lacZ transgenes respond similarly to the host Dlb-1 locus, although the induction factor (i.e., the ratio of treated over controls) is lower. In contrast, X-rays induce few lacl mutations but many Dlb-1 mutations, reflecting the reduced sensitivity of these transgenic assays to large deletions and/or chromosome rearrangements induced by clastogens. Other studies have used transgenic gene mutation assays to consider the genotoxic risk to the gut from dietary factors such as high fat content or as a result of the







presence of mutagenic carcinogens, such as heterocyclic amines, in food. MUTAGENESIS AND CARCINOGENESIS

FIGURE 56.13 Adenocarcinoma in colon of a rat orally dosed with bromodichloromethane. (A) Neoplastic tissue has thickened the wall of the colon by penetrating through the lamina muscularis mucosa (LM) and infiltrating into the submucosa. Neoplastic tissue exposed to the lumen is ulcerated (U) and inflamed. Compare the architecture of the neoplasm to that of the more normal adjacent colonic mucosa (N). (B) Neoplastic cells (N) in a loose fibrous-tissue stroma infiltrated by inflammatory cells (E). (Figures provided courtesy of Dr. Susan Elmore, National Toxicology Program, NIEHS)


Investigations of the mutagenicity and carcinogenicity of B(a)P (75 and 125 mg/kg per os for 5 days) in transgenic lacZ mice (Mutamouse) have demonstrated that the mutation frequency (MF) was increased 37-fold in colon, followed by ileum > forestomach > bone marrow, spleen > glandular stomach > liver, lung > kidney and heart cells 14 days after the last dose of B(a)P. The main target organs for carcinogenicity in this transgenic mouse strain were the forestomach and lymphatic organs. These studies demonstrate that the mutation data reflect carcinogenicity outcome, but not all organs with high frequencies of induced mutation in the lacZ transgene develop tumors, nor does the magnitude of the induced NT in the different organs correlate with the target organs for carcinogenicity. Although there is no clear rationale for these discrepancies, possible explanations include (1) the nature of the target gene and type of mutations detected by the transgenic assay (the lacZ gene is neutral and is mainly sensitive to point mutations, which may not reflect the mutations in the cancer genes associated with these tumors); (2) inadequate selection of target cells within the target organs; and (3) the importance of factors such as cell proliferation, turnover, and apoptosis in tumorigenesis at specific organ sites. Transgenic and knockout models for a wide range of cancer genes, such as p53, APC, and ras, have also been created and used to investigate both spontaneous and environmentally induced tumorigenesis in the intestine. Although these models allow for understanding of specific gene changes within the context of a genomic environment of the model species it is clear from the complexity of gene regulation in

FIGURE 56.12 Dextran sulfate-induced ulceration and inflammation in rat large intestine. (A) Transmucosal ulceration extends to lamina muscularis (LM). Lamina propria is thickened by inflammatory cells. Surface epithelium (E) is replaced by a covering of necrotic tissue, and proteinaceous and inflammatory-cell-rich exudate (D). Mucus is depleted in epithelial cells (MD) in crypts near ulcer. Bar ¼ 200 mm. (B) Higher magnification of (A) demonstrating many neutrophils (N) and macrophages (M) that characterize the infiltrating inflammatory cells in the lamina propria. Bar ¼ 100 mm. (C) Normal colonic mucosa has a goblet cell (G) rich epithelial mucosa. Note the lack of leukocytes in the lamina propria in all segments of the GI tract of the normal animal. Bar ¼ 50 mm. Figure reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 22, pp. 180–181, with permission.


2324 TABLE 56.14


Animal Models of Colon Carcinogenesis


Dose (mg/kg)


Aflatoxin B1



3,2-Dimethyl-4-aminobiphenyl (DMAB)

20 100

Rat Hamster


1,4-bis(4-fluorophenyl)-2-propynyl-Ncyclooctylcarbamate (FPOC)



Rat Rat, mouse, guinea pig

N-methyl-N-nitro-N-nitrosoguanidine (MNNG)



Methylazoxymethanol acetate (MAMA)



1,2-Dimethylhydrazine (DMH)




Rat, mouse, hamster Rat


Dose in ppm Dose is in mg/week. Table adapted from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Table XV, p. 183, with permission. b

different species that extrapolation from one species to another requires information which is currently beyond state-of-the-art interpretation of genomic and proteomic information. METABOLISM LIPOPROTEIN METABOLISM The B apolipoproteins (apo, B) play central roles in lipoprotein metabolism (such as chylomicron formation) in the intestine, and are key components of plasma lipid. Several groups have used transgenic and/ or gene-targeted (knockout) mice to study their expression, regulation, and function, and also their contribution to human diseases such as atherosclerosis and the human apo-B deficiency syndrome. Familial hypo-b-lipoproteinemia is an autosomal recessive syndrome in humans characterized by almost complete absence of apo B-containing lipoproteins in plasma. The disease is caused by mutations in the gene for the microsomal triglyceride transfer protein (MTP), resulting in malabsorption of intestinal fat and, often, severe neurological symptoms because of Vitamin E deficiency. Humans heterozygous for MTP have been reported to have normal plasma lipid levels, and this has led to the concept that MTP is normally present in excess, so that half the normal levels of MTP, as

expected in heterozygotes, does not affect lipid homeostasis. However, recent data with MTP inhibitor drugs show that inhibition of lipoprotein secretion appears to be proportional to the extent of MTP inhibition. Mice heterozygous for the wild-type Mttp allele produce only 50% of MTP in liver and intestine, and possess reduced plasma lipoprotein synthesis. Complete deficiency in MTP causes lethal developmental abnormalities, suggesting that these may be a species-specific effect and that heterozygosity for MTP deficiency in humans could reduce LDL cholesterol and apoB levels by ~20%, as in the mouse, and that this effect may be overlooked in humans because of the extremely broad range of “normal” cholesterol levels recorded. HEAVY METALS Metallothiones are low molecular weight inducible proteins, rich in cysteine (~33%), which bind heavy metals and are associated with the homeostasis of essential heavy metals such as zinc, and also protection from exposure to toxic heavy metals such as cadmium. Metallothione knockout and transgenic mice have been developed to study the expression, distribution, regulation, and function of these proteins, and to investigate heavy metal metabolism. Mice transgenic for metallothionein have elevated



levels in many tissues, including liver and intestine, and are resistant to dietary zinc deficiency. Knockout mice, in contrast, have delayed renal development and increased sensitivity to zinc and cadmium toxicity. CYTOCHROME P450 Transgenic and knockout mice of various CYP genes (e.g. CYP1A2, CYP2E1) and the Ah receptor have been constructed to investigate the role of these proteins in phase 1 metabolism of various tissues, including intestine, and their contribution to embryonic development. Proposals have been made to use genetic modification to “humanize” rodent models with human P450s for toxicological studies in vivo.

3.6. Microfloral Impact on Pharmacologic and Toxicity Studies As most GLP and non-GLP pharmacological and toxicity studies in industry and academia are carried out in well-controlled clean environmental settings, and are usually experimentally well designed with appropriate controls, the direct impact of these inherent intestinal microflora on the data and their interpretation is largely minimized. However, any observed variability in expected results in studies that are conducted in different species (mice, rats, nonhuman primates, dogs) after incorporation of inherent species-specific physiological variations should also take into account the possible impacts of gut microflora on data generated in each individual species, along with the associated impacts of environmental microflora/ housing conditions of the different test species. Within a particular species, if studies are repeated in different locations or animals are sourced from different vendors it is worth considering the possible confounding effect of gut microfloral variation in these different settings. In general, the effect of gut microflora in toxicity studies may be manifested only when the mucosal barrier function is compromised in instances of antibiotic therapy, oral administration of toxic doses of drugs, or drugs that alter GI motility or administration of chemical carcinogens. In many instances, the beneficial or detrimental effects of gut microbiota might be masked by effects attributed to the


administered test substances. In addition, in biomarker discovery studies with animals the possible role of gut microflora should also be taken into account, as it is an important player in the metabolism and degradation of food and xenobiotics. Microflora can induce the production of bacterial enzymes/secondary metabolites, many of which are genotoxic and carcinogenic, and hence the modulatory effect of the administered test compounds on microbial metabolism, if any, would determine the final outcome of the results with any test compound in pharmaco-toxicological studies.

4. RESPONSE OF THE GASTROINTESTINAL TRACT TO INJURY Most responses that occur as a result of gastrointestinal intoxication are ulcerative, proliferative, or inflammatory (Table 56.15). Many of the processes involved in gastrointestinal tract ulceration are discussed in the sections on cytotoxicity of mucosal epithelium, animal models, and ulcerative lesions of the stomach and intestine. Because of variations in structure and function, each segment of the gastrointestinal tract is affected by toxic compounds in a slightly different manner. In addition, each segment has a different range of pathophysiological responses to a toxic compound. Clinically, most alterations of the gastrointestinal tract are manifested as abnormal function, such as vomiting (if the animal is capable of such activity), diarrhea, constipation, or nutrient malabsorption. Additionally, occult or large amounts of blood may be present in the stool. Of these possible manifestations, diarrhea is one of the most commonly observed and can be life-threatening. Responses of the gastrointestinal tract to toxic compounds are considered on an organ, tissue, and cellular basis. Two specific processes, the inflammatory response and the immune response, are discussed in detail here because many of the disease processes resulting from chemical injury involve these two host reactions. Finally, the response of the enteric nervous tissue is discussed. Although the gastrointestinal tract has a large amount of nervous tissue, most neural responses are manifested only on a functional basis.


2326 TABLE 56.15


Gastrointestinal Reaction to Various Chemical and Elemental Toxins

Toxic agents

Gastrointestinal pathology

Corrosive agents Mineral acids Iodine Sodium fluoride Strong bases Phenol

Ulcerative gastritis and esophagitis

Volatile organic agents Ethanol Methanol Chloroform Gasoline and kerosene

Gastritis and gastric ulceration

Non-volatile organic agents Dimethylhydrazines Nitrosoguanidines Nitrosamines

Esophageal, stomach, and intestinal cancer, and/or acute gastritis

Metallic inorganic agents Arsenic Bismuth Copper salts Gold salts Iron salts Lead Manganese Mercury Nickel Thallium Vanadium Zinc salts

Ulcerative, hemorrhagic, and necrotic gastroenteritis and/ or colitis

Non-metallic inorganic agents Phosphorus Nitrites

Ulcerative, hemorrhagic, and necrotic gastroenteritis

Table adapted from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Table XI, p. 155, with permission.

4.1. Pathophysiological Responses Diarrhea Identification of mechanisms responsible for diarrhea should focus on the small and large intestine separately and collectively. Small-intestinal diarrheas are generally associated with increased mucosal permeability, hypersecretion, or malabsorption. Major mechanisms of large-intestinal

diarrhea include hypersecretion, large-bowel malabsorption, or colonic mucosal injury. Smallintestinal malabsorption allows fermentable nutrients to enter the colon, where bacteria generate osmotically active products. Mucosal damage and inflammation lead to release of PGE2, which stimulates electrolyte secretion, and activation of mast cells, with subsequent release of histamine. Inflammatory cells can also release mediators that stimulate nerve activity and may lead to localized gastrointestinal tract hypermotility. Consequently, the pathogenesis of diarrhea can be generally divided into four major categories: (1) increased mucosal permeability and exudation; (2) hypersecretion; (3) malabsorption; and (4) abnormal gastrointestinal motility. The pathogenesis of diarrhea can vary, but some of the most injurious mechanisms involve toxins that affect the gastrointestinal tract’s ability to transport fluid. Since a major function of the gastrointestinal tract is to absorb and secrete large amounts of fluid, such intoxications are lifethreatening even though little morphological damage may be present. This process, and the extent of the resulting diarrhea, is best demonstrated with cholera toxin. Cholera toxin activates adenylate cyclase, resulting in the secretion of large amounts of fluid into the small-intestinal lumen, overloading the large intestine’s ability to absorb lumenal water. This process leads to severe diarrhea and death, with little morphological evidence of mucosal damage. The consequences of diarrhea are systemic in nature, and include dehydration, acidosis, and electrolyte alterations. Direct loss of bicarbonate in the feces may be the major cause of acidosis. Intracellular hydrogen ion concentrations increase and potassium concentrations decrease. This electrolyte imbalance leads to improper maintenance of intracellular pH ranges, and reduces the activity of multiple enzyme systems. Reduced intracellular potassium is the result of a failure in cellular electrolyte transport. Consequently, there is inadequate maintenance of electrochemical gradients, which leads to increased extracellular potassium and mass excretion of this electrolyte in the urine and feces. Vomiting Vomiting is a clinical response that occurs in some animal species. Vomiting requires the presence of skeletal muscle in the wall of the



esophagus. Vomiting may be stimulated by direct mucosal irritation or by stimulation of the vomiting center in the central nervous system. Vomiting may also occur if the esophageal lumen is obstructed by a scar or neoplasm. Toxic substances can act on the mucosa to induce emesis via activations of sensory nerves that travel over vagal and sympathetic afferent pathways to brain medullary centers that control vomiting. One mucosal sensory emetic pathway involves activation of 5-HT3 receptors at the peripheral ends of these sensory nerves in the mucosa. 5-HT3 receptors are ligand-gated ion channels that mediate depolarization of nerves, and the vagus nerve is densely populated with 5-HT3 receptors. Cyclophosphamide, carmustine, dactinomycin, and cisplatin interact with mucosal enterochromaffin cells to promote release of large quantities of 5-HT, which leads to emesis. Bilateral abdominal vagotomy and bilateral splanchnic nerve section completely inhibit emesis induced by these anticancer drugs. Emesis induced by cancer chemotherapeutic agents can be reduced or prevented by administration of 5-HT3 antagonists, such as granisetron, or those that block both dopamine D2 receptors and 5-HT3 receptors, such as metoclopramide. However, 5-HT3 antagonists are not effective against some other emetogenic substances, such as copper sulfate, protoveratrine, or apomorphine. Cisplatin appears to act by effects on the central nervous system that promote release of 5-HT and subsequent activation of 5-HT3 receptors. Evidence for this mode of action comes from observations that combined vagal and splanchnic nerve transections do not completely prevent vomiting in response to peripherally administered cisplatin, whereas emesis is reduced by administration of 5-HT3 antagonists. The highest concentration of 5-HT3 receptors in the mammalian brainstem is located in the area subpostrema of the brain. Activation of vagal sensory neurons by 5-HT3 receptor-mediated events causes release of proemetic neurotransmitters from the central terminals of sensory fibers in the solitary nucleus and in the area subpostrema. 5-HT3 receptors in the brainstem appear to be associated with presynaptic sites and serve primarily to modulate release of neurotransmitters. Presumably, activation of the central 5-HT3 receptors enhances release of


proemetic substances, thereby activating the chemoreceptor trigger zone of the area postrema and the nearby emetic center. Agonists of dopamine D2 receptors such as apomorphine, L-dopa, and bromocryptine act in the chemoreceptor trigger zone of the brainstem area postrema. Phenothiazine drugs with significant dopamine D2 antagonists properties, such as chlorpromazine and promethazine, block the emetic actions of dopamine D2 agonists. Emetine, the principle ingredient of ipecac, and opiates, such as morphine, act non-specifically at the chemoreceptor trigger zone to initiate an emetic response. The area postrema is unprotected by a complete blood–brain barrier, thus allowing chemicals in blood to penetrate into this brain area. Constipation Constipation is a structural and functional disease. Compounds that bulk the stools (e.g., fiber) may lead to constipation if there is concomitant reduction in water intake. Polyps and neoplastic masses induced by carcinogenic agents may result in physical obstruction. The constipating effects of certain analgesic compounds (e.g., morphine) have a neurological component to their pathogenesis. Nervous Tissue and Motility Nervous tissue responses are generally identified by functional abnormalities that are initially detected clinically. Normal intestinal motility consists of peristaltic activity, which moves intralumenal contents down the gastrointestinal tract. This activity represents contraction of both longitudinal and circular muscle layers. Neuronal networks involved in peristalsis are complex and incompletely understood; however, cholinergic excitation mechanisms play a major role. Such coordinated peristaltic activity can be altered by bulking agents. These compounds cause intestinal distension and elicit contractions that occur in either direction along the bowel. Opiate (e.g., morphine) toxicity is manifested clinically as a non-propulsive and constipating pattern of segmentation motility. Segmentation movements accomplish mixing of intraluminal contents. This activity involves reciprocal neural inhibition and disinhibition of adjacent muscle segments; it may be preprogrammed into the internuncial circuitry of the gastrointestinal




nervous system. Endogenous opioid peptides may be involved in segmentation motility, since morphine locks the intestine into a continuous segmentation pattern of motility. In morphinedependent rats, diarrhea, which is the opposite of the acute effects of morphine, is a primary withdrawal event. Prostaglandin E2 and 5-hydroxytryptamine may also contribute to a secretory type of diarrhea. Extrinsic nervous input includes both stimulatory and inhibitory nerve fibers. Both vagal and sacral innervation to the large intestine is especially active during defecation. Sympathetic nerves are active in reducing blood flow and motility of the gastrointestinal tract. Inhibitory nerve input into the gastrointestinal tract leads to reduction or cessation of muscle motor activity (ileus). Tonically active inhibitory neurons can account for a low responsiveness of circular muscles to myogenic pacemakers. A model toxicity of inhibitory nerve input that leads to suppression of both cholinergic and serotoninergic synaptic transmission is norepinephrine overdose. Peritoneal irritation can also cause this effect and lead to ileus. Spasm is a functional disorder, but is the opposite of ileus, and consists of accelerated activity of circular muscles with no activity on inhibitory neurons. Intoxication by various cholinergic agents can cause spasms of the gastrointestinal tract. The model for this disorder is aganglionic megacolon of piebald mice. In these animals, the terminal segment of the large intestine lacks inhibitory neurons.

4.2. Inflammatory Response Because of the high number of bacteria and the physicochemical nature of the luminal contents, inflammation is frequently involved in many lesions of the gastrointestinal tract regardless of the underlying mechanism of injury. However, the inflammatory response is generally less severe in primary toxicologic lesions than in primary bacterial diseases. Inflammation of the stomach (gastritis) is essentially a process that is restricted to the mucosa. Gastritis is usually catarrhal (with large amounts of mucus), and may involve ulceration, hemorrhage, and lymphoid hyperplasia. “Gastritis glandulan’s,” a disease of primates, is characterized by mucosal hyperplasia and mucus-filled cysts

in the mucosa and submucosa. This lesion may be induced by ingestion of polychlorinated biphenyls. Inflammation of any part of the intestinal tract can be termed enteritis. However, this term is frequently used to designate only smallintestinal inflammation; in contrast, the term colitis is used to designate large-intestinal inflammation. Directly irritating compounds usually cause more severe inflammation of the proximal intestine (duodenum) and less inflammation of the distal tract (ileum and large intestine). Mercury can cause lesions of the large-intestinal mucosa as a result of transport from the blood into the colonic lumen. Chronic inflammatory reactions can be a primary or secondary effect in toxicologically mediated lesions. Immune-mediated responses are characterized by accumulations of chronic inflammatory cells (lymphocytes, plasma cells, and macrophages) and may have an active cellular component consisting of neutrophils and eosinophils. Diseases that result in chronic inflammation or injury to the lamina propria or lymphatic vessels cause malabsorption of fatty acids and weight loss. Fatty acids and monoglycerides are packaged into chylomicrons by the enterocytes before being exported to the central lacteal and into the lymphatic circulation. Consequently, damage to the lymphatic circulation that is longstanding can result in significant malabsorption-related disorders. Systemic complications such as septicemia and bacteremia may develop as a result of chronic inflammation and ulceration of the gastrointestinal tract. Secondary lesions may be present in the liver (e.g., abscesses), skin (e.g., perianal ulcers developing secondary to chronic diarrhea), or urinary tract (e.g., females can develop an ascending infection of the urethra from malabsorption-induced diarrhea).

4.3. Mucosal Response General The intestinal mucosa separates the body from the gastrointestinal tract’s lumenal contents (including bacteria and non-absorbed toxic compounds). This lining is also responsible for selective absorption of ingesta to obtain the proper nutrients that will maintain homeostasis.



The mucosal lining is the first site of exposure to an ingested toxicant, and the cells lining the gastrointestinal tract have the capacity to respond to these toxigenic compounds. The exposure of the rapidly proliferating gastrointestinal mucosal epithelial cells to toxins would suggest that the gastrointestinal tract should be a frequent site of toxicologic injury. The actual frequency of toxicity in commonly used laboratory animals is lower than one might expect based on the high rate of cellular proliferation. Factors that account for the gastrointestinal tract’s ability to escape damage include its capacity for compound biotransformation and the large surface area that permits extensive contact between the toxic compound and multiple toxin-metabolizing enzymes. Additionally, mixing an injurious compound with lumenal contents dilutes the toxin and its effects. Finally, mucosal barrier components (e.g., mucus) and the short half-life of gastrointestinal epithelial cells effectively protect cells or remove those that have suffered molecular damage (e.g., DNA mutations). Since the epithelium of the gastrointestinal tract is the first layer of host cells to contact ingested compounds, these cells can respond to toxic compounds before they enter the circulation. Epithelial cells can also undergo biochemical changes that allow them to functionally reconstitute mucosal integrity after a toxic insult. Modification of several enzyme pathways – including the CYP 450, alcohol dehydrogenase, monoamine oxidase, epoxide hydrolases, esterases, amidases, glucuronidases, sulfatases, and various conjugation pathways – allows epithelial cells to maintain a barrier function. Many of these same enzymes are involved in compound metabolism and biotransformation. Interestingly, transgenic mice that carry a mutant dihydrofolate reductase gene display resistance to methotrexate toxicity to the gastrointestinal tract. Methotrexate interferes with DNA replication via inhibition of DHFR bioactivity and consequent reduction of de novo thymidine and purine biosynthesis. Mucosal epithelial cells can respond to reactive compounds via both membrane-bound and cytoplasmic enzymes. Although the CYP 450 pathway can generate cell-damaging and reactive intermediate epoxides, mucosal cells can form non-toxic dihydrodiols and glucuronide conjugates from


these intermediates. This process occurs via mucosal cell enzymes, including epoxide hydrolases and glucuronyl transferases. Unique forms of adaptive mucosal protection occur after exposure of the mucosa to mild irritants. Under these conditions, increased levels of prostaglandin E2 are elaborated and protect the mucosa from strong irritant damage by increasing blood flow and stimulating bicarbonate secretion in the small intestine. Sublethally injured gastrointestinal epithelial cells are able to reseal damaged membranes and can participate in covering discontinuities in the epithelial barrier. Repaired cells at the margins of ulcers and erosions become active participants in gastrointestinal barrier restitution by migrating over denuded basal lamina. Membrane resealing is a key process in maintaining an intact epithelial layer if the injury does not cause widespread and severe loss of epithelial cells. Gastrointestinal epithelial cells are protected from injury by an apical membrane enriched with viscosity-enhancing glycosphingolipids, and a microvillar and apical membrane cytoskeleton. Resealed or healed cells may remain viable for up to 24 hours in the stomach and 48 hours in the intestine. Rapid epithelial restitution is one of the mucosa’s primary defense mechanisms throughout the gastrointestinal tract (esophagus to anus). Each segment of the gastrointestinal tract has a basal rate of mucosal proliferation that varies with species, diet, and disease state. Under normal dietary conditions and health, the range of proliferation rates for the most actively dividing mucosal cells (stomach to colon) is 3–6 days. When the intestine encounters a noxious agent, enterocyte half-life is reduced. If the damage is transient, mucosal replacement and normal microarchitecture will recover within 3 days. Arachidonic acid metabolites from the cyclooxygenase and lipoxygenase pathways are elevated during gastrointestinal inflammation and after acute colonic injury in which an inflammatory process has not yet developed. These inflammatory mediators have many effects on the structure and function of the gastrointestinal tract (see above). Bile salts (e.g., deoxycholate) induce the release of arachidonic acid and cyclooxygenase and lipoxygenase metabolites of this fatty acid, and lead to the generation of active oxygen




radicals (Figure 56.14). Bile salts also induce increased colonic secretion and permeability, but this occurs by mechanisms independent of endogenous arachidonic acid metabolism. However, prostaglandins of the E series suppress enzyme release and superoxide anion production by neutrophils. Thus, the interaction of these chemical mediators may be important in controlling the response of the gastrointestinal mucosa and the final outcome of injury. Nitric oxide (NO) can be beneficial or toxic, depending on circumstances. It is synthesized from the amino acid L-arginine by at least two

FIGURE 56.14 Restitution of gastric epithelium after gastric epithelial cell damage by bile salts. Cells migrate from edges and become flattened (arrow). Capillary damage leads to leakage of fluid and formation of subepithelial vesicles (F). Bar ¼ 50 mm. Figure reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 13, p. 160, with permission.

different nitric oxide synthases. The two major isoforms of nitric oxide synthase are a constitutive enzyme, which is calcium- and calmodulindependent, and an inducible form, which is calcium-independent. The constitutive form has been identified in endothelium, nerves, and brain. It seems to be active continuously, generating small amounts of NO. The inducible form has been identified in macrophages and is probably present in other tissues, such as gastrointestinal and vascular smooth muscle, and vascular endothelium. Nitric oxide in low concentrations is thought to protect the gastrointestinal mucosa from injury and to enhance restitution of injured mucosa. It produces vasodilation of gastric microvessels and exerts an antiaggregation effect on platelets. These actions tend to maintain adequate mucosal blood flow. It also stimulates secretion of mucus by surface mucous cells and helps maintain protection against luminal acid. Laboratory studies indicate that functional repair of the epithelial barrier after acute injury is enhanced by NO. While NO confers many benefits to the gastrointestinal tract by protection of the mucosa, maintenance of mucosal blood flow, and regulating contractions of smooth muscle and propulsion, it can also be responsible for gastrointestinal toxicity. The dual nature of NO appears to be related to the two major isoforms of NO synthase. In general, the NO produced by the constitutive form of the enzyme produces beneficial effects, while the NO produced by the inducible form of the enzyme has often been implicated in vascular or epithelial cell injury. Excess production of NO in the gastrointestinal mucosa is linked to initiation of secretory diarrhea. Diarrhea induced in rats by castor oil or a bile salt (e.g., sodium choleate) is blocked by inhibitors of NO synthase. Castor oil and bile salts also induce mucosal damage, but the damage is exaggerated by co-administration of NO synthase inhibitors. These results suggest that NO mediates, at least in part, the diarrheal effect of these compounds, presumably by increasing secretion of fluid into the intestinal lumen, but, simultaneously, NO exerts a protective effect on the intestinal mucosa. This suggests that bile acids and castor oil directly damage intestinal mucosa and activate the inducible isoform of NO synthase to produce large amounts of NO linked




to production of diarrhea. Laxatives, such as phenolphthalein and bisacodyl, are also associated with electrolyte secretion, changes in mucosal histology, and abnormal motility, but linkage to NO production remains to be established. Stomach Mucosal defense and protection, initially termed cytoprotection, was originally described as the ability of prostaglandins to prevent macroscopic evidence of gastric mucosal injury. This protective phenomenon is partially dependent on the antisecretory activity of prostaglandins, and is dose- and route-dependent. It is currently understood that several mechanisms are responsible for preventing mucosal damage by normal digestive processes and injurious compounds. These include increased amounts or modifications of mucous gel covering the mucosal epithelial surface, increased secretion of bicarbonate, increased resistance to acid backdiffusion, and increased blood flow. Several of

these processes are mediated by prostaglandin synthesis in mucosal and submucosal tissues (Figure 56.15). Additionally, mucosal protection is mediated in part by lipids (neutral lipids and phospholipids) within the mucous gel layer. These lipids increase the hydrophobicity of the mucous gel, leading to repulsion of water-soluble compounds. Mucosal damage can occur with or without mucous-layer damage. The adherent mucous gel is not extensively disrupted by mucosaldamaging agents such as dilute HCl or indomethacin (Figure 56.16). These agents permeate the mucous barrier and directly damage the underlying epithelium. Since the mucous layer remains intact, it facilitates epithelial repair in these situations. However, mucus is lost when the stomach is exposed to mucosal-damaging agents such as pepsin, bile acids, and ethanol, and mechanical trauma. With persistent mucosal damage, mucous-gel secretion is impaired and mucousgel loss will exceed production. Additionally, mucus composition can be modified by epithelial

Acid lumen -mucus gel

Luminal acid Mucus cells

Luminal membrane of surface cell

Surface Cell Cl



Protective bicarbonate flux

lamina Basement propria membrane -blood flow -immune & CI– inflammatory ?ATP response -tissue buffers ?PG


H + HCO3


H2CO3 carbonic anhydrase

Gastric pit

CO2 + H2O


Blood Flow

Fenestrated capillary

Bicarbonate flux

lumen lamina propria

Chief cells CI– + H+


Parietal Cell

Parietal cells ?ATP

Mucus cells H2O

Blood flow




FIGURE 56.15 Schematic drawing demonstrates the interactions of acid production, blood flow, and bicarbonate release. These various interactions allow the gastric mucosa to resist the damaging effects of the low pH environment and toxic compound exposure. Figure reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 10, p. 146, with permission.




FIGURE 56.16 Taurocholate (bile acid) induced gastric damage in a rat 1 hour after oral gavage. Bar ¼ 50 mm. (A) Periodic acid-Schiff (PAS) staining reaction demonstrates that glycoprotein loss in foveolar mucous-epithelial cells (E) of the fundic mucosa is one of the earliest injuries induced by agents which disrupt the mucous lining of the stomach. (B) Normal control fundic mucosa with abundant PAS staining for neutral mucosubstances is gastric surface and pit epithelial cells (arrow). Figure reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 14, p. 161, with permission.

cell metaplasia, leading to chemical changes in the mucous gel and loss of functional integrity. These types of injuries will ultimately lead to collapse of the mucous barrier because the mucous gel alone cannot protect the mucosa and support rapid recovery after epithelial cell damage. These damaging effects are manifested by loss of surface epithelial cells, reduced mucus release, vascular occlusion, and, ultimately, ulceration and scar formation (Figure 56.17). Repair of chemically or mechanically induced gastric epithelial cell discontinuities can be complete within 30–90 minutes of injury (Figure 56.14). The rate of repair will vary with

location in the gastrointestinal tract (stomach or intestine) and extent of initial damage. The gastric mucosa experimentally damaged by exposure to high concentrations of sodium chloride, which produces structural damage, can be repaired over a period of 6 hours by a gradual process of restitution of epithelial integrity via migration of cells from the gastric glands. Cellular proliferation is a key epithelial cell mechanism in maintaining mucosal barrier function. The extent of general mucosal damage resulting from an insult can be anticipated based on the amount of damage the compound inflicts on proliferating cells of the mucosa. Minor




FIGURE 56.17 Acid pH-induced gastric injury. (A) Ulceration (U) of mucosa leads to cystic dilatation of associated gastric glands (C) and marked proliferation of neutral mucosubstance (PAS stained) containing mucous cells (M). Cells that are positive for the PAS reaction at the margins of the ulcer have been demonstrated to release epidermal growth factor. These cells have been proposed to have a significant role in controlling the healing and re-epithelialization process of the ulcer. Bar ¼ 500 mm. (B) Mucous cell (M) proliferation occurs around margins of cystic glands and the transition to a more normal mucosa (N) is abrupt. Bar ¼ 500 mm. (C) The base of the ulcer contains fibrous connective tissue at the base of the ulcer, trapped nests of mucous cells (M), and few inflammatory cells. Bar ¼ 100 mm. Figure reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 15, pp. 162–163, with permission.

damage to the proliferative compartment leads to mild gastritis or enteritis, and any increased loss of surface cells is readily compensated by the undamaged proliferative cells. However, severe mucosal damage will occur when the proliferating unit of the mucosa is destroyed. Such injury develops after irradiation, exposure to some mycotoxins, or cytotoxic drug administration. One of the mechanisms by which prostaglandin E2 enhances healing of the mucosa is by protecting the cells in the isthmus of the gastric pits and allowing these replicative cells to reconstitute the surface epithelium. Modifications of epithelial cell proliferation may be the only morphological indication of

mucosal injury. Low doses of indomethacin and aspirin increase epithelial proliferation in rat gastric mucosa, but have no effect in the antrum and duodenum and do not cause inflammation. In contrast, corticosteroids depress epithelial cell proliferation in fundic, antral, and duodenal mucosa of rats, and hydrocortisone predisposes to gastric ulcers. Proliferative rates are also modified by starvation, and pharmacologic and toxicologic doses of mineralocorticoids, glucocorticoids, and ACTH. Intestines The colonic mucosa is covered by relatively flat mucus-secreting cells and crypts. Several




substances serve as growth factors that can positively stimulate epithelial growth. These include gastrin, TGF-a, and TGF-b. The influence of these growth factors is exerted on the stem cell. The ingestion and digestion of food appear to be important in maintaining growth of the intestinal mucosa. Mucosal toxicity can be exhibited by decreased cell production or increased cell loss, which can lead to atrophy or ulceration. Increased cell production can lead to hyperplasia. In the colon, epithelial restitution is associated with migration of cells at a speed of approximately 2 mm/min. Rapid epithelial restitution is now considered one of the primary defense mechanisms of the stomach, small intestine, and colon, but occurs only under conditions in which damage is confined to the superficial mucosa. Regions of the mucosa with gross hemorrhagic lesions heal by a lengthy process of tissue replacement involving cell mitosis. It is thought that maintenance of regional blood flow in the area of damage is important for prevention or repair of lesions. Indirect damage may occur when blood flow to the area of damage is compromised. Three mechanisms influence restitution of the mucosal barrier after toxic damage: (1) increased epithelial cell production rate; (2) reduced cell cycle time (the period between two successive divisions of proliferating cells); and (3) an increased proliferative compartment via an increase in the proportion of cells in the proliferative cycle or an increase in the absolute number of cells that are replicating at any given time. In contrast, under adaptive conditions (intestinal resection or dietary change) only one mechanism is operative: increased cell production rates via increased numbers of cells that are proliferating. The intestinal mucosa can also be induced to proliferate as a healing response near areas of cellular toxicity and ulceration. Prostaglandin E2 and fermentable fibers (e.g., guar) are examples of inducers of mucosal proliferation in the intestinal tract. Mechanisms of restitution after proliferative unit ablation are not well established.

ruminants), but the lining mucosa and its response to injury are similar among species. Both neoplastic and ulcerative processes can lead to a reduction in the esophageal lumen by the physical obstruction of a space-occupying mass (neoplasm) or a stricture from scar contraction. If highly caustic agents are fed to animals, severe mucosal damage is followed by ulceration, inflammation, fibroplasia, and scar formation. Ulceration can be restricted to the esophagus, or may involve the stomach, small intestine, or large intestine. Spontaneous esophageal cancer is rare in animals and humans living in western civilizations. However, esophageal cancers are commonly found in humans living in regions of China. When rats are exposed to chemical carcinogens (N-methyl-N-nitrosoaniline), the mucosa of the esophagus progresses through a sequence of hyperplasia and hyperkeratosis to dysplasia, papillomas, and, finally, carcinoma. Esophageal cancer can also be induced in rats with dihydrosafrole, and in mice with gamma irradiation. Additionally, zinc-deficient rats treated with carcinogens may develop multiple neoplasms of the esophageal mucosa. Stomach ULCERATION AND INFLAMMATION

Gastric ulceration and associated inflammation and mucus loss are responses to stress (unrelated to compound administration) and various mucolytic agents (Figure 56.18). Active ulcerogens include non-steroidal anti-inflammatory compounds, alcohol, taurocholate (bile acids), nitriles, thiols, and amines (Table 56.15). In addition to these direct gastrointestinal irritants, which affect the stomach, antimitotic and antineoplastic agents (e.g., colchicine and 5-fluorouracil) cause ulceration in various parts of the gastrointestinal tract. Mechanisms of ulceration are discussed extensively in Section 5. PROLIFERATIVE RESPONSE

4.4. Organ-Specific Response Esophagus Various species have unique anatomical adaptations of the esophagus (e.g., forestomachs of

Epithelial cells maintain normal anatomical boundaries (i.e., do not infiltrate into subepithelial tissues) in hyperplastic conditions. However, if hyperplasia is of sufficient duration, it may in certain situations increase the risk of a neoplastic process developing. Adenomas are the result of




proliferative activity, the lesion should be considered a carcinoma. A hormone-mediated proliferative response of the stomach is demonstrated by hyperplasia of enterochromaffin-like (ECL) cells and neuroendocrine-cell tumors (“carcinoids”) that develop after prolonged gastrin release (Figure 56.19). Hyperplasia of these enteroendocrine cells has been demonstrated after exposure to ranitidine and substituted benzimidazoles like omeprazole, both of which produce hypergastrinemia. Animals with gastric hyperplasia have an abnormal mucosal maturation that is characterized by a decrease in the number of cells with cytoplasmic zymogen granules, a decrease in mature surface cells, glandular atrophy, loss of regular parietal cell distribution, and an increase in cellular proliferation. The initiation of carcinoma is demonstrated by loss of cellular differentiation, abnormal gland structure, invasion of mucosa into surrounding tissue, abnormal glycoprotein expression, and displacement of normal tissue.

FIGURE 56.18 Early injury to fundic gastric mucosa (bile salts) that disrupts the mucous blanket. Coagulative necrosis (arrow-bar) of the foveolar region occurs prior to the development of ulceration. Necrotic zones can extend deep into the fundic mucosa. Bar ¼ 100 mm. Figure reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 16, p. 165, with permission.

a benign proliferative response that is neoplastic, and can potentially progress to malignancy. Determination of the malignant potential of adenomas should include evaluation for evidence of epithelial dysplasia, proliferative activity, cysts, blood capillary organization, stromal infiltration of epithelial components, and inflammation. Intestinal metaplasia can occur in adenomas, as can tissue invasion and distortion. When the neoplastic process demonstrates tissue invasion or areas of severe epithelial atypia, metaplasia, or dysplasia, with or without extensive

Small Intestine Damage to the small intestinal mucosa frequently results in villus atrophy and cryptcell hyperplasia. If ulceration occurs, an associated inflammatory reaction ensues. Duodenal sites are more frequently found to have ulcers than other small-intestinal segments. The same ulcerogens that affect the stomach frequently damage the duodenum. The distal small intestine is a frequent site of functional abnormalities, such as diarrhea, rather than a location for morphological damage. The small intestine is an infrequent site of neoplastic processes. However, lymphosarcomas may originate in the lymphoid nodules of the lamina propria and submucosa. Adenocarcinomas induced by a carcinogenic agent or natural causes may originate from the mucosal epithelium and invade the submucosa and tunica muscularis mucosa (Figure 56.20). Large Intestine ULCERATION AND INFLAMMATION

The response of the large intestine to toxic injury can be studied using models that induce acute and chronic lesions. Acute erosive injury to the colonic mucosa can be induced using the bile salt deoxycholate (15 mM, 30 minutes). Damage to the surface cells is mediated by




FIGURE 56.19 Enterochromaffin-like (ECL) cell carcinoid and hyperplasia in the glandular stomach of a F344/N male rat treated with methyleugenol. (A) Carcinoid (C) compressing the adjacent fundic (F) mucosa (arrows). (B) Higher magnification of carcinoid showing the peritheliomatous and nesting arrangements (box) of the neoplastic ECL cells (E) within a delicate fibrovascular stroma. Neoplastic cells have large round centrally located nuclei and pale cytoplasm. (Figures provided courtesy of Dr. Susan Elmore, National Toxicology Program, NIEHS)

reactive oxygen metabolites and complete ablation of the surface epithelium occurs within 8 minutes. Mucosal permeability is regained after 40 minutes, and recovery of absorptive activities occurs when the epithelium is restored to a columnar phenotype (2 hours). The reparative process of the mucosa occurs by active cell migration from the proliferative zone to the surface. This process will be delayed or is unable to take place if damage to the mucosa is severe enough to damage stem cells, thus ulceration and inflammation will occur in such situations. PROLIFERATIVE RESPONSE

One common response seen in the colon is mucosal hyperplasia and polyp formation. Hyperplastic polyps of the colon may be either inflammatory or regenerative in nature. Benign

lymphoid polyps occur in the colon or rectum as a result of lymphoid tissue hyperplasia, and can protrude into the intestinal lumen. Inflammation of lymphoid polyps is a frequent concomitant event. Proliferative polyps can be classified as adenomatous polyps or adenomas. Adenomatous polyps are composed of tubules of neoplastic epithelium with little stroma (Figure 56.21). In contrast, villous adenomas have multiple projections of epithelial-lined lamina propria. Regardless of classification, the mucus content of neoplastic epithelial cells is reduced and mitotic figures are common in these polyps. The presence of multiple polyps should be regarded as an early cancerous event in rodents, since there is an adenoma–carcinoma sequence in the colon.




FIGURE 56.20 Mucinous adenocarcinoma in a male F344/N rat treated with 2,3-dibromo-1-propanol. (A) Neoplastic tissue (arrow–bar) extends from the mucosa through the submucosa into tunica mascularis mucosa. (B) Cystic spaces (C) are lined by neoplastic cells and contain mucus (M). (Figures provided courtesy of Dr. Susan Elmore, National Toxicology Program, NIEHS)


Cecal enlargement is a response to various compounds and food additives occurring in several rodent species. These materials include antibiotics, modified starches, polyols (sorbitol and mannitol), some fibers, and lactose. Cecal enlargement is associated with increased death losses in rats fed raw potato starch. Enlargement of rodent ceca has been interpreted as both a toxic and an adaptive phenomenon. Compounds that are poorly absorbed and are osmotically active are frequently associated with cecal enlargement. The mechanism for the distension has been proposed to be the attraction of fluid into the lumen. However, when the lumenal contents are removed tissue weights remain elevated, so other mechanisms are also operative. Other processes involved in cecal enlargement

and dilatation include mucosal hypertrophy and hyperplasia. This morphologic response is associated with functional changes that lead to soft stools, diarrhea, and increased large-bowel mucosal permeability. These functional alterations are likely to be mediated by the increased osmotic activity of the cecal contents. Morphological changes probably represent an adaptational process, since the changes are reversible when the diets are returned to normal. Large-intestinal enlargement is a common change observed with incompletely digested and poorly absorbed substances that are subjected to microbial metabolism in the cecum and colon. The increased microbial metabolism leads to an increase in osmotically active material, and results in soft stools and cecal distension. One functional change in rats fed sugar alcohols and




FIGURE 56.21 Polyp in colon of a male F344/N rat treated with 0-nitroanisole. The polyp almost completely occluded the lumen (L) of the colon. The mass is supported by a mucosal stalk and is composed of multiple cystic glands (C). (Figure provided courtesy of Dr. Susan Elmore, National Toxicology Program, NIEHS)

lactose is increased absorption of calcium. Sequelae to this process are increased calcium excretion in the urine and nephrocalcinosis.

4.5. Regenerative Response Repair vs Regeneration Any interruption of morphology independent of or in conjunction with an interruption of functionality of the cells comprising a tissue is defined as an injury. Injury may be caused by physical trauma or exposure to toxins and toxicants. The tissue response to injury may be divided into two classes: repair and regeneration. Repair refers to the physiologic alteration of an organ after injury for the purpose of reinstating stability without considering exact replacement of lost or damaged tissue. Repair often results in fibrosis – the formation of excess fibrous connective tissue in an organ or tissue. The steps involved in repair do not occur in a clearly defined succession, but instead partially overlap in time. During the inflammatory step of repair, soluble factors, including cytokines and chemokines, are released, which facilitates the migration and division of cells to the site of injury. These soluble factors also include signaling molecules, which are crucial for the proliferative phase of repair that follows. During this phase, fibroblasts enter the site before the

inflammation step has ended and deposit collagen, increasing the strength of the injured tissue site. This collagen also serves as an anchor for vascular endothelial cells which form new blood vessels to supply oxygen and nutrients during the repair process. Granulation tissue then begins to appear at the injury site, comprised of new blood vessels, fibroblasts, inflammatory cells, endothelial cells, myofibroblasts, and a modified form of extracellular matrix protein. Mainly composed of fibronectin and hyaluronan, this modified matrix creates a very hydrated microenvironment which facilitates cell migration. Later on during this process, the modified extracellular matrix is replaced by a matrix more closely resembling that of uninjured tissue. Hypoxic conditions often exist at a site of injury, which may inhibit fibroblast growth and deposit of extracellular matrix proteins; this can lead to excessive fibrotic scarring. The injured site also contracts, aided by the action of differentiated fibroblasts called myofibroblasts, which contain the same type of contractile proteins found in smooth muscle cells. The injured tissue edges are pulled closer together and reinforced by collagen deposition. It should be recognized that collagen deposition is a dynamic process comprising a balance between collagen production and degradation through the activities of collagenases. Shifting the balance to one side or the other results in more collagen being deposited, or increased collagen degradation. When production and degaration are equalized, the remodeling phase of the repair process has begun. During remodeling, there is a shift towards a more stronger type of collagen which is deposited in parallel with a rearrangement and alignment along tension lines of the collagen fibers. This results in an overall increase in the tensile strength of the injured site. In contrast to repair, regeneration signifies the replacement of lost or damaged tissue with a precise duplicate, such that both morphology and functionality are completely restored. Conditions required for regeneration are sometimes in direct contrast to those favoring repair. For example, a prolonged inflammatory response needed for repair will not allow for regenerated tissue to form due to the granulation at the injured site. Likewise, contraction at the site during repair will further inhibit replacement



with duplicate tissue. Many tissues are not overtly capable of regeneration, so the only response to injury is repair. Injuries involving the inner mucosal layer of the small intestine can result from chemical and radiation exposure during treatment for cancer, changes in gut microbiology in response to antibiotics, and inflammation or necrosis due to parasitic or autoimmune disease. In contrast, blunt force injuries to the small intestine, due to automobile accidents, stabbings, or gunshot wounds, often compromise the entire structure of the organ, involving both the inner and outer tissue layers. Since the inner mucosal layer is crucial for the nutrient absorption function of the small intestine, the following subsections will provide a broad overview of some of the key components involved in repair and regeneration of the epithelial cells lining this organ. Stem Cells It is reasonable to assume that any injury to the small intestine will require repopulation of the injured area by one or more cell types. Stem cells are perfect candidates to facilitate cell repopulation and actively participate in the regeneration process. Adult stem cells are multipotent; they can differentiate into a limited number of cell types. These are capable of maintaining, generating, and replacing terminally differentiated cells within their own specific tissue in response to physiological cell turnover or tissue injury. Adult stem cells are of greater importance than embryonic stem cells with regard to the regenerative response to tissue injury in the GI tract. The multipotent properties of adult stem cells makes them easier to coax into replacing lost or damaged adult tissue with exact copies of defective cells, thereby reconstituting original function. Pluripotent stem cells from embryonic sources require more controls during the differentiation process, to reduce the occurence of neoplastic transformation. Adult stem cells are considered to be an autologous source of reparative and regenerative cells capable of differentiating into selected cell types dependent upon the cytokine context. Stem cells may differentiate into fibroblasts and myofibroblasts in response to connective tissue growth factor (CCN2) and platelet-derived growth factor, respectively, thus contributing to tissue repair of gastrointestinal epithelial layers.


In contrast, maintaining stem cells in an immature state, perhaps as a result of being exposed to b-catenin, needed by the resident epithelial cells for regulating growth and adhesion between cells, can promote aggressive fibromatosis. MSC may ameliorate colitis by exerting an anti-inflammatory effect of the tissue, by reducing mRNA production for inflammatory cytokines such as TNF-a, interleukin-1B (IL-1B), and cyclooxygenase-2 (COX-2). Repair of the intestinal cell layers may be observed following an episode of inflammatory bowel disease (IBD) such as ulcerative colitis, which affects the epithelial lining of the gut, or Crohn’s disease, which affects the entire gut wall. Since the epithelium of the small intestine renews itself every 2–5 days under non-diseased conditions, the stem cell compartment of the small intestine is crucial to maintaining the physical and function continuity of this layer by tissue regeneration. Activation and proliferation of stem cell reservoirs within the crypts of the intestine is modulated by Wnt signaling. Proliferation appears to be dependent upon activation of nuclear b-catenin/T-cell factor transcriptional activity. Expression of Ephrin B receptors and ligands, critical for establishing the migratory path from the crypt to the villus, is modulated by the b-catenin/T-cell factor transcriptional complex. Without Wnt signaling, b-catenin, needed by resident epithelial cells for regulating growth and adhesion between cells, becomes targeted for destruction, thereby contributing to fibromosis. Transcription factor 3 (E2A immunoglobulin enhancer-binding factors E12/E47), abbreviated TCF3, is another transcription factor regulated by Wnt signaling. TCF3 can repress nanog gene expression, potentially downregulating stem cell pluripotency and self-renewal. Regeneration of the small intestine epithelium occurs with predictable regularity following the repeated normal sloughing of these cells in response to digestive enzymes and the movement of food materials by catastalsis. Signaling Pathways Cellular signal transduction occurs when an extracellular signaling molecule interacts with a receptor on the cell surface, thereby triggering a cascade of intracellular events. Mechanistically, this receptor interaction alters one or more intracellular molecules, thus serving as a second




messenger to propagate the signal into the cell, ultimately resulting in a physiological response. In addition to Wnt, which has been discussed above, bone morphogenic protein (BMP) signaling also plays an important role in regulating intestinal development and epithelial homeostasis in normal, non-diseased tissue. As the name would suggest, this family of cytokines was originally discovered based on the ability to stimulate bone and cartilage formation. Since then, BMPs have been considered to constitute a group of pivotal morphogenetic signals, orchestrating tissue architecture, including the intestine, throughout the body. BMPs generally function as a negative regulator of cell proliferation in the small intestinal crypts. In so doing, they effectively act as a brake to intestinal epithelial cell regeneration in normal gut. A BMP antagonist, called noggin (NOG), is expressed in the submucosal region adjacent to the crypts, providing a feedback mechanism of proliferation control. NOG binding to BMP receptors removes the negative effect of BMP, resulting in an increase in intestinal stem cell proliferation, thereby removing the brake to tissue regeneration. Concomitant with this receptor blockage is an increase in b-catenin nuclear translocation, thereby linking the BMP and Wnt pathways. Under normal circumstances, BMP inhibits intestinal fibrosis resulting from IBD by downregulating TNF-a and by inhibiting TGF-b mediated epithelial to mesenchymal transition. Without such BMP inhibition, activated fibroblasts would be generated, which are a key component in tissue repair. These activated fibroblasts contribute to intestinal fibrosis characteristic of IBD pathology. The Notch signaling pathway is important for cell–cell communication as well as controlling multiple processes involved in cell differentiation through the action of a single form of transmembrane receptors. Ligand binding to the extracellular domain of the receptor releases the intracellular domain following proteolytic cleavage. This second messenger will enter the nucleus of the cell, where it effectively modifies gene expression. Stem cells residing in the intestinal crypts are maintained in an undifferentiated proliferative state through this pathway. Notch signaling has also been shown to be a crucial component for maintaining intestinal epithelium, by cooperating and interacting with other molecular pathways such as Wnt and BMP.

Like BMP, Notch plays a role in the repair of the small intestine following an episode of IBD. Notch signaling is increased in epithelial cells of IBD patients, and animal experiments have suggested that activation of Notch signaling is important for recovery from colitis. Hormones Hormones are chemical substances released by cells that bind to receptors on other cells to elicit a response. Often this response is mediated by phosphorylation and dephosphorylation of intracellular proteins and increased concentrations of cyclic AMP. The number of hormone molecules available for receptor interaction generally dictates the degree to which the signal transduction pathways are activated. Endocrine cells, which release hormones directly into the blood stream, may be found throughout the mucosa of the gastrointestinal system. Gastrointestinal hormones refer to a family of peptides secreted by such endocrine cells that line the gut. In addition to their roles in regulating secretion, absorption, digestion, and motility, these hormones also affect the pathogenesis of several gastrointestinal diseases, such as cancer. There are upwards of 50 identified gastrointestinal hormones. Of the few that have been rigorously tested for their effect on intestinal mucosal cells, the following have been shown or implicated in regulating mucosal cell growth and regenerative responses either positively or negatively. GASTRIN-RELEASING PEPTIDE AND GASTRIN

Gastrin-releasing peptide (GRP) functions primarily as a stimulator involved in regulating gastrin release and subsequent gastric acid secretion. Gastrin also stimulates GI mucosal cell growth in the small bowel. Recent evidence suggests that gastrin can modulate the function of cells involved in immune and inflammatory responses. This pro-inflammatory function must be downregulated during repair of the intestinal epithelium resulting from colitis, perhaps by affecting the activity of another pro-inflammatory factor, TNF-a. GHRELIN

Ghrelin is produced largely by cells in the stomach, and to a lesser degree by cells in the small intestine and colon. This peptide plays an important role in regulating food intake, gastric



emptying, and gastric acid secretion. Not as much is known about its mechanism of action compared to other gastric hormones; Ghrelin was only discovered in 1999. Reports in the recent literature support a role for ghrelin in suppressing inflammatory and apoptotic pathways activated by gastrointestinal injury, enhancing intestinal motility, and promoting mucosal epithelial cell proliferation. Injury to intestinal epithelial cells is a common occurrence in cancer patients who receive chemotherapy and localized doses of radiation. Ghrelin-stimulated epithelial cell proliferation may play a role in tissue regeneration of the intestine following such treatments. Mechanistically, grhelin has been shown to inhibit cellular apoptosis by regulating the ratio of the apoptotic regulator protein Bcl2 with the proapototic protein BAX . SOMATOSTATIN

Somatostatin (SST) is arguably the master controller of all gastrointestinal hormones, capable of inhibiting gastric acid secretion, motility, and mucosal cell growth. SST accomplishes this feat either directly via interaction with specific receptors, or indirectly by antagonizing the function of other trophic hormones, such as gastrin. In this latter case of antagonism, SST inhibits histamine release by ECL cells, in direct opposition to the effect of gastrin, resulting in a downregulation of acid production by parietal cells. It has been demonstrated that radiation injury of intestinal mucosal cells can be alleviated by administration of an SST analog, most likely by preventing loss of mucosal surface following radiation-induced cell death. This may imply that SST functions in a proliferative capacity to enable maintenance of the surface epithelium. This suggests that SST may function in a repair pathway by inhibiting apoptosis. Growth Factors As the name implies, growth factors are substances capable of stimulating cell growth. This growth may be defined as proliferation, whereby there is an observable increase in cell number through affecting cell division, or an increase in size (hypertrophy), where the cell increases in mass. The word “cytokine” is sometimes used synonymously with “growth factor.” Technically, “growth factor” implies a positive effect on cell growth, whereas a “cytokine” may


or may not have a positive effect. Thus, some cytokines may be considered growth factors, if they have a positive effect. The important point to remember here is that both positive and negative influences on cell growth are in play during tissue repair and regeneration. In both processes, there must be a balance between cell proliferation and apoptosis during tissue remodeling in response to injury. Feedback loops involving multiple cytokines enable achievement of this balance. EPIDERMAL GROWTH FACTOR

Epidermal growth factor (EGF) consists of a family of peptides, of which EGF and transforming growth factors alpha (TGF-a) are two of the most studied members. EGF is produced by cells located in the duodenum, while TGFa is produced by epithelial cells lining the small intestine. Both EGF and TGF-a are mitogens, whereby they stimulate cell division in multiple cell types within the GI tract. In so doing, these growth factors can enhance mucosal healing after injury by increasing cell proliferation and stimulating angiogenesis. Both EGF and TGFa bind to specific cell surface receptors. This receptor binding stimulates protein tyrosinekinase activity, which in turn initiates a signal transduction cascade. This cascade ultimately results in enhanced expression of select genes and proteins required for cell proliferation. FIBROBLAST GROWTH FACTORS

The family of fibroblast growth factors (FGFs) also regulates growth and differentiation of intestinal epithelial cells, in addition to the proliferation of stem cells during the process of tissue regeneration. Acting as a mitogen, FGFs promote mucosal and epithelial cell proliferation in the small intestine in addition to mediating angiogenesis – the growth of new blood vessels from existing ones. Fibroblast growth factors stimulate fibroblast and endothelial cells, two key building blocks for angiogenesis and granulation tissue development. Together, angiogenesis and granulation increase blood supply to the area and fill the injured site with cell mass during the tissue repair process. INSULIN-LIKE GROWTH FACTORS

There are two members of the insulin-like growth factor family (IGF), designated IGF-I,




and IGF-II. Following interaction with their receptors, these growth factors upregulate epithelial and fibroblast cell proliferation while downregulating apoptosis. During the tissue repair process, IGF also stimulates intestinal epithelial cell migration so that cells can quickly be redistributed over a damaged area. In so doing, the barrier between the intestinal lumen and submucosa is rapidly restored while epithelial cell proliferation is being initiated to more completely repair the damaged area with new cells. TRANSFORMING GROWTH FACTOR

The transforming growth factor beta (TGF-b) family functions to inhibit the growth of GI mucosal cells. Following bowel epithelial cell injury, increased levels of this growth factor can be found in the intestinal mucosa, resulting in an inhibition of epithelial cell proliferation. There appears to be a synergistic relationship between TGF-b and GRP, in that together they negatively regulate intestinal epithelial cell proliferation and differentiation better than each does individually. Similar to IGF, TGF-b also stimulates intestinal epithelial cell migration to restore a barrier quickly during tissue repair. Wound Healing by Tissue Repair or Regeneration As mentioned earlier in this section, many tissues are not overtly capable of regeneration, so the only response to injury is repair. The small intestine clearly has a reparative response to the majority of injuries it may sustain, as discussed above. The question of whether this organ can also mount a regenerative response to injury has been addressed experimentally by inducing a controlled surgical injury. After removing a section of rat small intestine, a tubular construct comprised of a biodegradable scaffold material seeded with smooth muscle cells isolated from adipose was anastomosed to the anterior and distal portions of the native intestine, forming a contiguous connection. Sections of the implanted area were removed over a period of months, and prepared for histological examination. Control animals received implants of scaffold-only, which were not seeded with any cells. Tissue section staining with hematoxylin and eosin (H&E), along with Masson’s Trichrome, reveals a thickening in the fibrovascular

stroma, as evidenced by the blue staining (Figure 56.22A) in the scaffold-only implanted animals. This thickening is indicative of a reparative process. Histological examination of tissue resulting from implants of cell-seeded scaffold (Figure 56.22B) is virtually identical to that of native tissue (Figure 56.22C), with nearly complete re-epithelialization of luminal mucosal surface and multifocal growth of muscularis layers, indicating a regenerative outcome. The conclusion here is that the small intestine is indeed capable of a regenerative response to injury, although this response may be restricted to controlled situations.

5. MECHANISMS OF GASTROINTESTINAL TOXICITY Basic functions of the gastrointestinal tract include acting as a barrier, digesting and metabolizing ingested material, secreting enzymes, and absorbing needed nutrients (including water). Any impairment of these basic functions will result in functional or structural alterations and disease (Table 56.16). Because the gastrointestinal tract is involved in transport of nutrients, it is especially prone to injury by processes that alter absorptive functions. Additional mechanisms that can cause severe gastrointestinal pathology include reduced blood supply or hypoxia, acid build-up with damage to the mucosal barrier, hypersensitivity reactions, and genotoxicity, potentially leading to cancer development. At a cellular level, injury to the plasma membrane and mitochondria represents an irreversible loss of cellular viability from which there is little likelihood of return. At the tissue level, the difference between development of a superficial or a deep mucosal lesion depends on the extent of involvement of the subepithelial capillaries. In this section, general toxicologic mechanisms are discussed and several model toxicants are used to describe these processes.

5.1. Intestinal Barrier Function Most ingested toxins enter the body through the small intestine, either by passing through the enterocytes or by passive paracellular diffusion. Contents of the gut are mainly in an




FIGURE 56.22 Repair vs regeneration: unseeded vs seeded scaffolds. Representative microphotographs of unseeded and adipose-derived smooth muscle cell-seeded small intestinal tube scaffold implants showing stromal changes occurring at 16 weeks post-procedure. (A) The mucosal thickness in the unseeded scaffold was shorter and developed interrupted epithelium. The underlying wall is thickened and predominantly comprised of a fibrovascular rich stroma without appreciable regeneration of muscular layer, consistent with a reparative healing response. In contrast, the cell-seeded scaffold (B) elicited a regenerative healing response, characterized by complete re-epithelialization of luminal mucosa with normal thickness and nearly complete regeneration of muscularis layers (longitudinal and circular). Normal, native intestinal wall components (mucosa, muscularis and serosa) are shown in (C).

aqueous phase, with a 35 mm unstirred water layer next to the mucous layer of the surface epithelial cell. The ability of a xenobiotic to traverse the mucosal barrier depends upon its solubility in water for diffusion through the unstirred water layer, its size and charge for paracellular flow, and its lipid solubility for transcellular diffusion. Large and polar molecules pass poorly through epithelial tight junctions unless the epithelial barrier is disrupted (as with high doses of ethanol). Small electroneutral molecules pass easily around the epithelial cells and into the portal circulation, but polar molecules cannot pass through the lipid barrier of the cell. Weak acids or bases are in equilibrium, with both ionized and non-ionized states present, making them simultaneously soluble in water and lipids. The non-ionized molecules can diffuse through the membrane into the enterocyte. Once in the enterocyte, a xenobiotic may be pumped out of the cell by a multipurpose transporter (e.g. P-glycoprotein) on the luminal surface of villus cells, metabolized by various enzyme systems within the

cell to either a toxic or non-toxic metabolite, or transferred into the portal blood or lymph. Intestinal metabolism may play an important role for some medications, including lidocaine and cyclosporine, or may be the site of drug interactions. Some compounds also have direct toxic effects on the enterocyte without being systemically absorbed, such as toxins produced by the blue green algae Microcystis (see Phycotoxins, Chapter 38). Substrates that do not have specific transporters are absorbed passively around epithelial cells. Tight junctions are very permeable in the proximal intestine, becoming less permeable in the ileum. Electrolytes are absorbed either by paracellular bulk flow or by electrogenic transport and exchange processes, depending upon the permeability of the tight junctions of the specific segment of the intestine. There is a net secretion of approximately 7 L of fluid into the jejunum, originating from biliary, pancreatic, and intestinal secretions in humans. Intestinal secretion is due to the paracellular flow of water drawn into the lumen of the bowel by the high




osmotic load of ingesta. This fluid is then absorbed in the ileum and colon as nutrients and electrolytes are absorbed against a concentration gradient. A variety of pumps, exchangers, and channels are involved in the electrogenic transport of electrolytes in the distal small and large intestine. As electrolytes are transported out of the gut, water is also reabsorbed passively to maintain electrochemical gradients. When TABLE 56.16

Selected Mechanisms of Toxicity to the Gastrointestinal Tract


Toxic entity

Altered absorptive function: Reduced nutrient absorption

Alcohol Antimitotic agents Cholestyramine Karacine Neomycin Heavy metals

Altered solute transport

E. coli toxins Shigatoxin Laxatives

Increased absorption of allergens

Polyvinyl chloride Metallic iron Asbestos

Decreased blood supply, hypoxic uncouple oxidative phosphorylation

NSAIDs Arsenates

Damage mucosal barrier: Block protective prostaglandins


Alter mucus synthesis

NSAIDs Steroids

Direct cytotoxicity

Bile acids Ethanol

Damage proliferating cells

Radiation T-2 toxin Ricin


Trinitrobenzene Sulfuric acid


1,2 Dimethylhydrazine Azoxymethane

Table adapted from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Table V, p. 143, with permission.

nutrients or electrolytes are not absorbed, there is an increase in luminal liquid that results in diarrhea.

5.2. Intestinal Malabsorption A number of transport pathways exist in the gastrointestinal tract to carry materials across the mucosal epithelium. These mechanisms include active transport, facilitated diffusion or solvent drag, passive diffusion, pinocytosis, and phagocytosis. Most nutrients are absorbed by active transport mechanisms, in contrast to most toxicants, which are transported by a passive diffusion process. Consequently, greater lipid solubility of a toxicant will enhance absorption, smaller molecules will diffuse more rapidly, and the non-ionized forms of acids and bases will be absorbed more rapidly than the ionized forms. A significant exception to this generalization includes the active transport of inorganic led by calcium carrier mechanisms. Malabsorption results from alterations in epithelial transport mechanisms, reduction in surface area (e.g., villus blunting from antimitotic agents), or the binding of nutrients or compounds to unabsorbed intestinal contents (e.g., modified bile salt absorption by cholestyramine) (Table 56.17). Reduced nutrient absorption can be mediated by various toxins, including heavy metals and plant extracts. Cadmium interferes with or inhibits the absorption of calcium and alters digestion of protein and fat. Tobacco-leaf extracts reduce the activity of the loosely held intestinal TABLE 56.17 Intestinal Malabsorption Induced by Drugs and Chemical Agentsa Surface active agents that block fat and vitamin absorption: alcohol, cholestyramine Antibacterial agents that block fat, protein, electrolyte, and vitamin absorption: kanamycin, neomycin, polymycin Miscellaneous agents that block fat, vitamin, protein, and carbohydrate absorption: calcium carbonate, clorfibrate, colchicine, indomethacin, methotrexate, phenformin, phenytoin, phenolphthalein, quinacrine, sulfasalazine, and triparanol a

Modified from Banwell (1979). Table adapted from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Table VI, p. 144, with permission.



brush border enzymes lactase, sucrase, maltase, and alkaline phosphatase. When enzymes involved in the metabolism of complex carbohydrates are damaged, the gastrointestinal epithelial cells are unable to absorb carbohydrate-derived nutrients. Malabsorption results in malnutrition, vitamin deficiencies, and diarrhea. Toxic compounds can alter solute transport across or between mucosal epithelial cell membranes. By damaging junctional complexes between enterocytes, interfering with hydrostatic pressure gradients, or causing high lumenal osmotic pressure, a toxic compound can contribute to net water loss from the body into the feces and lead to diarrhea. Some bacterial toxins (E. coli toxins and shigatoxin) and laxative compounds act as secretagogues and promote active water loss into the lumen. Toxic doses of these secretagogues eventually lead to diarrhea. Gastrointestinal toxicity can also be mediated by an increased absorption of nutrients or toxic compounds. Glycogen content increases in the midgut epithelium of cockroaches fed pyrethrum. Degeneration of fish intestine is observed as a consequence of water and electrolyte transport alterations which occur from exposure to DDT. Increased toxicity of organophosphates in young mice compared with older mice is the result of an increased rate of absorption of the toxic compound. Particulate materials may be taken up by pinocytosis (nanometer-sized particles) or phagocytosis. In mice, phagocytosis is limited to particles smaller than 6 mm in diameter. Particulate uptake plays an important role in pathological responses to polyvinyl chloride, metallic iron, and asbestos. The passage of these particles through the protective mucosal epithelium of the gastrointestinal tract can lead to allergic hypersensitivity reactions or the entry of unmetabolized compounds directly into the lymphatic and blood circulation.

5.3. Hypoxia Hypoxia is a key factor in the pathogenesis of gastrointestinal mucosal injury. This is typified by the development of mucosal lesions in various types of shock. The degree of mucosal damage in shock is correlated with the extent of reduction of gastrointestinal blood flow. Decreased blood flow and oxygen exchange increases the


susceptibility of the mucosa to injury. A local reduction in blood flow can occur with vascular thrombosis; this mechanism is a major process in gastric injury induced by absolute ethanol. Stomach Hemorrhagic shock in rats leads to uniform blanching of the glandular mucosa of the stomach and a generalized reduction in blood flow. Small, white, ischemic foci develop on the gastric mucosa, which will ulcerate and bleed after the restoration of blood pressure or flow. Ischemia predisposes the stomach to HClmediated mucosal lesions because blood flow is sufficiently reduced to cause a build-up of hydrogen ions in the tissue. A decrease in local blood flow or an increase in acid back-diffusion can lead to mucosal injury and erosion. However, a combination of these events causes severe mucosal damage (Figure 56.15). Arachidonic acid metabolites are inflammatory mediators that can induce gastric damage. Thromboxane A2, formed by platelets, is a potent vasoconstrictor and causes extensive mucosal damage in the presence of topical taurocholate. Platelet aggregation is also promoted by thromboxane A2 and can lead to vascular thrombosis and mucosal infarction. Both mechanisms result in tissue hypoxia and are involved in the ulcerogenic effects of thromboxane. In contrast, some prostaglandins protect the mucosa from injury. Protective processes mediated through prostaglandins are thought to be increased mucosal blood flow, and an improved supply of oxygen. Modification of blood flow, prostaglandins, and the mucosal barrier, coupled with tissue-damaging bile acid and activated neutrophils, are the basis for gastric ulceration observed with non-steroidal anti-inflammatory compounds (Table 56.18). Intestines Ulcerative mucosal lesions can develop as a result of impaired villus microcirculation during hypotension. Hypoxia develops as a result of increased mean transit time for plasma in the villus vascular loop, which increases the efficiency of the countercurrent exchange mechanism in the villi of the gastrointestinal mucosa (Figure 56.9). The time available for oxygen diffusion back into the blood is increased, resulting in reduced availability of oxygen at the villus


2346 TABLE 56.18


Chemical Agents and Drugs That Can Induce Gastrointestinal Ulcers

Anti-inflammatory agents (steroids and NSAIDs): corticosteroids, phenylbutazone, indomethacin, flunixin, oxyphenbutazone, sulindac, flurbiprofen, tolmetin, ketoprofen, fenoprofen, naproxen, and ibuprofen Inflammatory mediators: histamine, serotonin Antihypertensive agents: reserpine Hormone analogs: gastrin-like compounds Catecholamines: epinephrine Antimicrobial agents: polymyxin B Antimetabolic agents Sympatholytic agents: priscoline Antihistamines: dimaprit (H-2 blocker) Amines: ethylamine, cysteamine, and cystamine Nitriles: propionitrile and butyronitrile Short-chain alkanes and alkenes Miscellaneous agents: caffeine, KCl, gold thioglucose, and haloperidol Table adapted from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Table VII, p. 146, with permission.

tip. Rheological factors such as intravascular aggregation of erythrocytes and platelets can contribute to compromising oxygen transfer, especially when blood flow is already significantly reduced. Hypoxic injury is compounded by epithelial and intraluminal enzymes, such as trypsin, that contribute to mucosal lesion development under these conditions.

5.4. Mucosal Barrier Damage and Cytotoxicity NSAIDs NSAID-induced mucosal damage follows a temporal course of events, starting early with neutrophil-independent toxicity and progressing later with neutrophil-dependent toxicity. The time course of the cascade is approximately 6 hours. Early changes include alterations in mitochondrial oxidative functions and inhibition of cyclooxygenase (COX). Once absorbed into the

mucosa, NSAIDs interact with epithelial cell mitochondria to cause uncoupling of oxidative phosphorylation, leading to energy depletion and decreased mitochondrial enzyme activity. Energy depletion leads to disruption of ATPdependent epithelial cell junctions, thus increasing intestinal epithelial permeability. The increase in permeability from altered epithelial barrier functions reduces protection from hostile gastric and/or duodenal luminal factors such as bile acids, hydrogen ions, and bacteria. The presence of bacteria will attract and activate neutrophils, which increase the ulcerogenicity of NSAIDs. Activated neutrophils attracted to the mucosal microvessels and lamina propria release active oxygen metabolites, lysosomal proteases, and leukotriene B4. Myeloperoxidase is a hemoprotein peroxidase released by activated neutrophils into the extracellular medium, where it interacts with H2O2 to form an enzyme–substrate complex with great oxidizing potential. Activated neutrophils produce large quantities of HOCl, a powerful oxidizing agent, leading to OCl generation by means of myeloperoxidase-catalyzed oxidation of Cl. Active oxygen species and lysosomal enzymes cause direct damage to epithelial cells. The leukotriene B4, released by neutrophils, is a powerful chemoattractant for additional neutrophils. In addition, leukotriene B4 causes vasoconstriction of arterioles. As a result of this cascade, gastrointestinal epithelial cells become targets of attack by bile acids, hydrogen ions, active oxygen, and lysosomal enzymes. Non-steroidal anti-inflammatory drugs (NSAIDs) are also directly cytotoxic to the mucosal epithelium, as well as blocking cyclooxygenase activity and the synthesis of mucosal protective prostaglandins, through reduction of cytosolic ATP. NSAIDs stimulate membranebound sodium pumps and alter acid production by the gastric mucosa. Lesions occur after oral or parenteral administration of NSAIDs. These lesions caused by these compounds are erythema, hemorrhage, erosions, and ulceration of the gastrointestinal mucosa. Lesions can be found in the stomach and throughout the small intestine. When NSAIDs are administered at toxic levels, the same types of lesions are present regardless of route of administration or anatomical location. The two major COX isoforms, COX-1 and COX-2, differ in their sensitivity to inhibition



by individual NSAIDs. COX-1 is the constitutive form of the enzyme found in healthy tissues, while COX-2 is an inducible form that can be stimulated by several cytokines and mediators of inflammation. Most NSAIDs inhibit activity of both isoforms of COX. Inhibition of COX-2 may be associated with most of the beneficial effects of NSAIDs, while inhibition of COX-1 may be associated with many of their adverse effects. Inhibition of COX by NSAIDs results in two significant toxicological effects: reduction in formation of prostaglandins, and increased formation of leukotrienes. The loss of endogenous prostaglandin protection may then render the stomach prone to damage by other agents that are normally only mild ulcerogens, and leaves lipoxygenase metabolites like hydroperoxyeicosatetraenoic acid and leukotrienes without the counterbalancing effects of endogenous prostaglandins. Prostaglandin E2 and prostacyclin are vasodilators, so blockade of prostaglandin E2 and prostacyclin synthesis may favor some degree of vasoconstriction and oppose prostaglandinmediated tonic vasodilation. Increased metabolism of arachidonic acid by the 5-lipoxygenase pathway with concomitant inhibition of COX may contribute to NSAID-induced gastrointestinal toxicity, since leukotrienes C4 and D4 are vasoconstrictors and leukotriene B4 is a powerful chemoattractant of neutrophils. Leukotriene B4stimulated attraction and activation of neutrophils leads to release of lysosomal enzymes and microvascular occlusion. Cellular damage to epithelial cells is further exacerbated by a reduction in mucosal blood flow, brought about by a combination of vasoconstriction caused by leukotrienes, and occlusion (white thrombi) of microvessels by activated neutrophils. As potent vasoconstrictors, lipoxygenase metabolites indirectly deprive the mucosa of oxygen. This relative hypoxia may then predispose the mucosa to other damaging agents. Support for this pathogenesis is demonstrated by studies in which lipoxygenase and cyclooxygenase co-inhibition and antioxidant agents decrease the incidence of gastric ulcers compared with cyclooxygenase-inhibitor-only exposed controls. Another important process that contributes to damage when these agents are given by an oral route is direct mucosal irritation by the chemical


itself. NSAIDs also directly decrease mucus and bicarbonate release, independently of prostaglandin inhibition. The mucous layer and presence of bicarbonate contribute to mucosal protection from endogenously produced acids. Damage to the mucosal barrier also causes intramucosal histamine release by mucosal mast cells, with resultant vascular congestion, edema, and plasma exudation. Aspirin inhibits cyclooxygenase activity and is rapidly deacetylated to salicylate. Although both aspirin and salicylate are toxic towards mucosal epithelial cells with differing potencies, salicylate is differentially toxic to mucosal epithelial cells at different gastric sites and affects mucosal barrier function, reduces cellular ATP, stimulates sodium transport pumps, and increases proton loss. Thus, NSAIDs reduce production of protecting substances and increase production of damaging substances. Prostaglandins also decrease secretion of gastric acid by direct effects on oxyntic cell prostaglandin receptors, and lower concentrations of acid at the mucosal surface to enhance mucosal restitution. However, reduction of acid secretion alone is not sufficient to counteract the damaging effects of NSAIDs. Species differences exist with regard to NSAIDinduced gastrointestinal toxicity. Dogs are more sensitive than rats, which are more sensitive than monkeys. However, although monkeys do not develop lesions after oral exposure to ibuprofen (300 mg/kg/day), gastric ulcers occur when the same dose is given intravenously. The species differences may be related to the plasma half-life of the active compound, since the propionic acid NSAID flurbiprofen has a half-life of approximately 40 hours in dogs, 6 hours in rats, and 3 hours in monkeys, which correlates with the relationship with ulcergenic sensitivity. Alcohol (Ethanol) Ethanol, like NSAIDs, causes hemorrhagic erosions in the gastric mucosa. The rate-limiting step in this lesion development is the extent of microvascular damage. Vascular injury is the result of cell membrane injury, mast cell degranulation, leukotriene release, and increased mucosal permeability. As occurs with other mucosal damaging agents, injured epithelial cells are rapidly replaced if blood flow is maintained and the basement membrane remains intact.




Gastrotoxic effects of alcohols like ethanol are related to their ability to increase cell membrane fluidity. Osmolality and lipid solubility are also involved, but to a lesser extent. Depletion of intracellular glutathione (GSH) has been implicated in alcohol injury to mucosal cells. The levels of GSH decline in proportion to the degree of alcohol injury, and treatment with prostaglandin E2 can essentially abolish alcohol injury. N-ethylmaleimide prevents prostaglandininduced protection against alcohol injury. Chronic administration of alcohol is also associated with enhanced expression of a number of growth factors, including epidermal growth factor (EGF) and transforming growth factoralpha (TGF-a). The growth factors are thought to protect the gastric mucosa against acute injury, and may explain the observation that adaptation of the gastric mucosa to chronic alcohol administration is associated with increased cell proliferation and increased expression of mucosal EGF and TGF-a. The ability of chronic alcohol exposure to lead to hyper-regeneration of the gastric mucosa could be responsible for the suspected carcinogenic effect of alcohol. Generation of acetaldehyde by endogenous CYP isozymes, in addition to elaboration of growth factors, has also been implicated, thus suggesting a potential role of gastric mucosal alcohol dehydrogenase (ADH) in deleterious effects of alcohol on gastrointestinal mucosa. Chronic ingestion of alcohol can also lead to proliferation of mucosal epithelium, an effect of alcohol which is also probably mediated by peptide growth factors. Alcohol increases the permeability of the mucosa and causes back-diffusion of Hþ and a rise in luminal Naþ concentrations. At low alcohol concentrations (10%), mucus synthesis and bicarbonate secretion are inhibited. At higher concentrations (12–15%), alcohol releases surface mucus, depletes intracellular mucus, and promotes leakage of bicarbonate and electrolytes toward the gastric lumen. At concentrations above 20%, the severity of gastric erosions increases with increasing concentrations of alcohol. At concentrations above 40%, there is dose-dependent damage to the mucosal blood vasculature. Steroidal Compounds Steroids, like NSAIDs, also induce gastric and large-intestinal mucosal alterations and damage

by altering cytoprotective mechanisms and the mucosal barrier. Long-term or high-dose steroids induce gastric ulceration. Dogs given toxic levels of dexamethasone, a phospholipase inhibitor, develop gastric bleeding, erosions, and melena. These findings indicate that the mechanism for steroid-induced gastric lesions is partially mediated through inhibition of prostaglandin synthesis. Since the prostaglandin synthetase (cyclooxygenase) substrate arachidonic acid is reduced by inhibiting phospholipase activity, the mucosal protection provided by prostaglandins (PGE2) is lost and gastric acid activity proceeds uninhibited. This mechanism of gastric damage is in distinct contrast to that demonstrated by cysteamine. Bile Acids Bile acids are synthesized from cholesterol, and can damage the gastrointestinal tract mucosa. Bile acids are usually ionized and occur in two forms: monomeric and micellar. Of the excreted bile acids, over 97% are reabsorbed in the ileum and returned to the liver via the enterohepatic circulation. The remaining 3% undergo bacterial degradation in the colon, and are excreted in the feces or reabsorbed in the colon. Gastrointestinal bacteria deconjugate and desulfate bile salts, leading to the production of toxic/carcinogenic metabolites. Bile salt malabsorption during certain ileal diseases is implicated in colonic mucosal damage and diarrhea. Bile salts in the stomach break down gastric mucosal permeability and solubilize the outer lipid bilayer of surface epithelium; deoxycholate inhibits active sodium transport from mucosa to submucosa. The basic mechanism of mucosal barrier damage is similar for ethanol and deoxycholic acid. Bile salts stimulate colonic epithelial cell proliferation and are capable of acting as tumor promoters in the colon. Radiomimetic Agents Radiomimetic compounds result in substantial cytotoxicity of mucosal epithelial cells, and the mitotic mucosal cells of the crypt are at high risk (see Radiation and Other Physical Agents, Chapter 44). Ingestion of a trichothecene mycotoxin, T-2 toxin, results in widespread crypt epithelial necrosis and mucosal injury that resembles the effects of radiation exposure (Figure 56.23). Contributing to the mucosal




Digoxin Gastrin, EGF, TGF-a and other endogenous growth factors can stimulate gastrointestinal crypt cells to proliferate. Mucosal hyperplasia can be associated with ingested chemicals, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and probably other related polychlorinated dioxins. TCDD binding converts the receptor (the aryl hydrocarbon [Ah] receptor) to its activated functional form, which binds to DNA of the CYP 1A1 gene. Binding of the TCDDactivated Ah receptor to the DNA increases the rate of transcription of the CYP 1A1 gene. The intestinal mucosa contains significant levels of Ah receptors and CYP 1A1. TCDD can induce mucosal hyperplasia in some animal species, possibly by exerting a disinhibitory effect on gastrin release, which can then stimulate crypt cell proliferation and result in mucosal hyperplasia. TCDD intoxication also induces a “wasting syndrome” that is characterized by hypophagia and severe loss of weight. Most species given lethal doses of TCDD die within 2 weeks.

FIGURE 56.23 (A) T-2 mycotoxin-induced loss of intestinal villous epithelium (encircled) secondary to crypt necrosis in a pig. Also present is engorgement of underlying capillaries and larger blood vessels in the underlying lamina propria. (B) Higher magnification image illustrating ongoing crypt cell apoptosis (arrows) and necrosis (circle). Figure reproduced from Fundamentals of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2007) Academic Press, Fig. 8.7, p. 180, with permission.

injury is necrosis of proliferating crypt cells. This will eventually lead to loss of the remaining mucosal epithelium as a result of continued cell senescence in the absence of replacement. Consequently, there is collapse of the mucosa, ulceration, hemorrhage, and secondary inflammation. Chemotherapeutic agents such as the fluropyrimidines floxuridine and 5-fluorouracil and plant toxins (e.g., ricin) can cause similar lesions.

Cytotoxic Agents and Heavy Metals Compounds that react by 1,4 addition at the b-olefinic carbon of some cellular nucleophiles are cytotoxic. Cytotoxicity occurs because the cell is more susceptible to the oxidation products that develop during normal metabolism. Ethyl acrylate leads to forestomach damage through depletion of sulfhydryl-bearing (cysteine) groups found in thiol-containing peptides and glutathione. Glutathione is the dominant non-protein sulfhydryl-containing constituent of epithelial cells that protects these cells from oxidative damage. An excessive oxidative burden leads to glutathione depletion followed by genetic damage and cellular lipid peroxidation, and ultimately by cell death. Cellular damage can also occur by reducing oxidative metabolism. Arsenates uncouple oxidative phosphorylation in the mitochondria, possibly by substituting for inorganic phosphate and forming unstable esters. Arsenic is stored in several body sites, one of which is the wall of the gastrointestinal tract. Arsenic toxicity is related to the rate of clearance from the body and the degree of tissue accumulation. The inorganic arsenicals cause hyperemia of the




gastrointestinal blood vessels, and this, coupled with endothelial cell damage, leads to submucosal hemorrhage. Suppression of epithelial cell proliferation accentuates the damage, and hemorrhagic enteritis develops. Cadmium causes irritation of gastrointestinal epithelial cells leading to vomiting, salivation, and diarrhea. Ingestion of ionic inorganic mercury leads to precipitation of mucosal proteins and a corrosive effect on the gastrointestinal mucosa. Acute intoxication causes vomiting.

5.5. Hypersensitivity Immune-mediated hypersensitivity mechanisms of toxicity require some form of damage to the mucosal barrier. Mucosal damage leads to inadequate clearance of an antigenic or haptenic compound by the mucosal immune system. Although multiple examples of immune-mediated toxicity exist, specific antigen models provide the clearest evidence of the interaction between the mucosal barrier and gastrointestinal tract-associated immune responses. Antigens can enter enterocytes by pinocytosis or by interactions with nutrient transport systems, or can cross the mucosal barrier by paracellular pathways to interact with immune cells in the lamina propria. Antigen-presenting cells release interleukin 1 (IL1), which activates T cells to express IL-2 and release a number of cytokines, tumor necrosis factor (TNF-a), and interferons, including IFN-g. Activated macrophages also release IL-6, which activates lymphocytes; IL-8, which attracts neutrophils; colony stimulating factors that activate immune cells; and prostaglandins that maintain blood flow. B cells are stimulated by antigens and interleukins to proliferate and differentiate into plasma cells that synthesize and secrete immunoglobulins. Immunoglobulin-E is one of a number of regulators (cytokines, complement 3a) that can activate gastrointestinal mast cells. Mast cells release neurotransmitters (substance P, CGRP), histamine, interleukins, and platelet activating factor (PAF). These pro-inflammatory and immune system regulatory factors can induce changes in mucosal transport and gastrointestinal motility. Increased fluid secretion stimulated by immune mediators may be involved in regulating stimulatory effects on enteric nerves, with subsequent neurally-mediated activation of mucosal

secretory mechanisms. Secretory products of mast cells may act both directly and by means of enteric neurons to increase contractile activity of gastrointestinal smooth muscle. Cytokines released from immune and epithelial cells during the immune response may affect mucosal blood flow, induce a chronic inflammatory response, or promote generation of reactive oxygen metabolites. Experimental hypersensitivity in the gastrointestinal tract is best exemplified by using ethanol to break down the mucosal barrier and increase permeability towards luminal antigens. By administering trinitrobenzenesulfonic acid (which acts as a hapten) after ethanol pre-administration, a severe transmural granulomatous inflammation develops in the distal colon. The inflammatory response is characterized by mucosal and submucosal infiltrations of neutrophils, macrophages, Langhans-type giant cells, lymphocytes, and mast cells, and represents an example of Type IV hypersensitivity. Such immune-mediated inflammatory responses lead to severe colonic ulceration. Once an animal is sensitized to an antigenic compound, an immune reaction can be generated without first causing damage to the mucosal barrier. Such “intact barrier” reactions are likely the result of hapten transport through the barrier by mucosal epithelial cells or leukoytes. How such low molecular weight luminal antigens gain access to the intestinal lumen across the epithelial barrier remains unclear. One mechanism of action may involve paracellular diffusion across pores in tight junctions connecting epithelial cells.

5.6. Acetylcholinesterase Inhibitors Acetylcholinesterase is an enzyme normally responsible for inactivation of the neurotransmitter, acetylcholine, at synaptic and neuroeffector endings of cholinergic motor and secretomotor neurons in the enteric nervous system. Inhibition of enzyme activity allows accumulation of acetylcholine leading to increased motor activity in the gastrointestinal tract caused by stimulation of smooth muscle M3 muscarinic receptors. The accumulated acetylcholine also acts at M1 and M3 muscarinic receptors to increase salivary, gastric, pancreatic, and intestinal secretions. Extensive inhibition of acetylcholinesterase leads to the secretion of large volumes of fluid and



electrolytes into the lumen of the intestine, which results in profuse, watery diarrhea. Drugs such as neostigmine, edrophonium, and pyridostigmine, organophosphate insecticides that include parathion, malathion, and paraoxon, and toxic nerve gases including tabun, sarin, and soman all are capable of causing severe diarrhea and death as a result of reversible or irreversible blockage of actylcholinesterase.

5.7. Microfloral Effects Perturbation of Enteric Microflora Administration of antibiotics has a profound effect on the colonic and fecal flora, depending upon the specific antimicrobial activity of the agent involved, the route of administration, and the local luminal concentration of the drug. A marked reduction in the concentration of intestinal bacteria can be achieved with oral antibiotics, although this effect is usually short-lived. The effect of antibiotics in reducing the bacterial concentrations leaves a void in the bacterial ecosystem that can be filled by pathogenic bacteria. A toxin-producing anaerobic bacteria, Clostridium difficile, colonizes the large intestine usually after prolonged treatment with antibiotics producing pseudomembranous colitis. Clindamycin and ampicillin are most frequently implicated, but virtually any antibiotic can cause this syndrome. The organism elaborates protein toxins that cause ulceration and necrosis of the intestinal mucosa. Microfloral Impacts on Disease in Intestine and Other Organ Systems The plethora of gut microbiota is mostly beneficial to the host by virtue of the various microbial symbiotic physiological associations. This association can also be detrimental to the host under conditions in which gut microbial homeostasis is disturbed, such as in immunodeficient states, after exposure to drugs, toxins/carcinogens, or co-pathogens, or after mechanical damage to the GI tract. Subtle but important differences in individual microfloral composition may determine the outcome and severity of many pathological conditions and its subsequent response to therapy. Increasingly, the GI microflora is thought to be an important determinant in the pathogenesis of many human and similar


animal-related conditions such as inflammatory bowel disease (IBD), celiac disease, type 1 (insulin-dependent) diabetes, obesity, cardiovascular disease, atherosclerosis, autoimmune disease (rheumatic disorders), allergy, cancer, and some neurological and psychiatric diseases and viral diseases. In inflammatory bowel disease (e.g., Crohn’s disease and ulcerative colitis) of humans and similar experimentally induced conditions in laboratory animals, the disruption of regulatory T-cell functions and associated abnormal mucosal immune (T-cell) responses to normal intestinal commensal bacterial flora are considered as key elements in sustaining chronic immune mediated intestinal inflammation and injury. Genetically manipulated mice such as IL-10/ KO or IL2/ KO mice are common models of chronic intestinal inflammation, and under GF conditions these mice do not develop chronic colitis, highlighting the vital role of GI microflora in aggravating immune-mediated injury. Gluten-associated celiac disease of humans is a chronic autoimmune-mediated enteric disorder, characterized by increased intraepithelial lymphocytes in the jejunum and mucosal atrophy in response to the active principle, gliadin (glycoprotein), in wheat. In this disease, the commensal microflora is thought either to aggravate (E. coli) or to inhibit (Bifidobacterium spp.) the effects of gliadin-mediated epithelial barrier dysfunction and innate immune activation (see Food and Toxicologic Pathology: An Overview, Chapter 35). Type 1 (insulin-dependent) diabetes mellitus (T1D) is an organ-specific autoimmune disease characterized by selective autoimmune destruction of pancreatic insulin-producing beta (b) cells in the pancreatic islets, and the exact basis for such autoimmune destruction is poorly understood. Increasingly, environmental factors such as exposure to microfloral components are believed to be important in the promotion of the autoimmune destruction of pancreatic islets. In well-established animal models of Type 1 diabetes, such as non-obese diabetic (NOD) mice or biobreeding rats, various studies have shown that the incidence and severity of T1D in these animals is correlated with their gut microfloral state and environmental sterility. More specifically, in experimental animals under germ-free conditions, or




in SPF mice colonized with ASF flora and treated with antibiotics, there is an associated higher incidence of T1D as compared to those animals reared under conventional conditions or exposed to parasites or bacterial antigen vaccines, thus underscoring the protective role of normal gut microflora against T1D. The gut microflora is increasingly evaluated for its role in the pathogenesis of immunemediated rheumatic diseases on the basis of the observed higher incidence of arthritis in IBD patients as well as the converse findings of increased gastrointestinal complications in juvenile idiopathic arthritis patients. A similar association has been noticed in the HLA-B27 rat, a transgenic model of spontaneous ankylosing spondylitis. These rats develop inflammatory colitis and spondylitis when reared in conventional conditions with normal gut microbiota, but lose their inflammatory states when transferred into germ-free housing conditions. In the K/B  N T cell receptor (TCR) transgenic mouse model of inflammatory arthritis, the intestinal commensals segmental filamentous bacteria (SFB) were implicated in the promotion of autoimmune arthritis through induction of TH17 responses. In the area of obesity research, data from both humans and animal models (leptin deficient obese ob/ob mice, germ-free mice) imply a role for gut microflora in the manifestation of the obesity phenotype. Specifically, the proportion of Bacteriodetes in the intestinal microbial population was significantly reduced and, conversely, Firmicutes were increased in obese humans/ mice as compared to non-obese humans/mice. Many commensal intestinal microbiota, periodontitis-related bacteria (by virtue of their biofilm production), and some pathogenic agents like Chlamydia spp. and Helicobacter pylori have been indirectly implicated or suspected to play a role in the development of cardiovascular disease and chronic heart failure in humans. In the apolipoprotein-deficient (Apo E/) mouse model of atherosclerosis, experimental data have shown, under low cholesterol standard diet conditions in different microfloral settings (conventional vs GF), that plaque development in these mice was inhibited by the protective effects of gut microflora in conventional mice as compared to the increased plague formation in germ-free mice.

The intestinal microbiota is vital for gastrointestinal physiology, including the development and functionality of the gut–brain axis, a bidirectional communication system composed of neural, immunological and endocrine mechanisms that aids the brain in monitoring and modulation of GI function. As a result, the gut microbiota is now increasingly being explored for its roles in some autoimmune neurological and demyelinating diseases like multiple sclerosis as well as Parkinson’s disease and autism. Another area of interest is the potential role of gut microflora in modulating the pathogenesis of viral infections. Experimental studies using germ-free and conventional mice have shown that intestinal microbiota can suppress (for Influenza A virus, coxsackie B virus, Friend virus) or enhance (Mouse mammary tumor virus, Theiler’s murine encephalitis, HIV virusinduced AIDS) viral replication and disease severity.

5.8. Carcinogenicity Stomach Naturally occurring tumors of the forestomach are rare in rats and mice (1%); however, hamsters can have an incidence as high as 12%. Many agents are capable of inducing or modulating forestomach neoplasia in laboratory animals. For induction of carcinogenic activity, nongenotoxic carcinogens must be in contact with the epithelium of the forestomach for extended periods of time. Morphologically, both genotoxic and nongenotoxic agents lead to dysplastic areas of the forestomach (Table 56.19). However, early lesions induced by the prototypical forestomach non-genotoxic carcinogen, BHA, are reversible, whereas those induced by genotoxic agents are irreversible. Epithelial dysplasia and metaplasia with glandular distortion is a consistent feature of chemically induced precancerous lesions in rodents. The metaplastic process is also associated with changes in epithelial cell enzymes (alkaline phosphatase, b-glucuronidase) and glycoprotein (neutral and acid mucopolysaccharides) content. Glandular atrophy occurs near neoplastic sites as a result of compression and expansion of the adjacent neoplastic process. In humans, “intestinalization” should be



TABLE 56.19

Compounds that Induce Forestomach Neoplasia in Rodentsa

TABLE 56.20


Experimental Compounds for Induction of Colon Cancera



Species affected




Butylated hydroxyanisole (BHA)

Aliphatic/aromatic hydrocarbon

Rat, hamster

Aromatic amines 4-Aminodiphenyl 3,20 -Dimethyl-4-aminodiphenyl N,N00 -2,7-Fluorenylenebisacetamide




Hydrazine derivatives


Polycyclic/aromatic hydrocarbon

Hamster, mouse

Allyl chloride

Halogenated hydrocarbon


Sodium saccharin




No genotoxic properties have been demonstrated for BHA, allyl chloride, or sodium saccharin. Table adapted from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Table VIII, p. 152, with permission.

considered a precancerous condition if it is part of a longstanding chronic process; it is not established if the same criteria exist in laboratory animals. The intestinalization process is characterized by gastric-gland neck-region elongation. These regions are replaced by a metaplastic mucosa composed of goblet cells and tall columnar absorptive-type cells of the intestine. Intestines Spontaneous intestinal tumors in laboratory rodents are rare; however, the high incidence of colon cancer in humans has led to the development of animal models that utilize chemical carcinogens to initiate colon tumors (Table 56.20). Chemically induced tumors of the colon are polypoid or sessile. Sessile tumors are usually mucinous, and can progress to malignancy that is characterized by local invasion, metastasis to mesenteric lymph nodes, lung, or liver, and intussusception. Several aromatic amines induce intestinal cancers in laboratory animals through genotoxic processes (Table 56.21). However, extensive metabolism is generally required before many of these chemicals become carcinogenic. Target specificity is associated with chemical structure

Chemical class



1,2,5,6-Dibenzanthracene 20-Methylcholanthrene

1,2-Dimethylhydrazine Methylazoxymethanol Azoxymethane 1-Methyl-2-butylhydrazine Methyl-azoxybutane

Alkylnitrosamides N-Methyl-N00 -nitrosoguanidine N-Methyl-N-nitrosourea N-Nitroso-bis(2-oxypropyl)amine Miscellaneous agents

Poligeenan Dextran sulfate Bracken fern extracts Aflatoxin B1


Modified from Maskens (1982). Table adapted from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Table IX, p. 152, with permission.

and animal species. Many nitrosamines induce tumors of small and large intestine in rats, hamsters, or guinea pigs. Additionally, the rat esophagus is sensitive to the carcinogenic effects of some nitrosamines. The organ specificity for many nitrosamines may relate to the affinity of these compounds for enterocyte or non-enterocyte receptors or sitespecific cellular biotransformation (Figure 56.24). Duration of mucosal exposure to the chemical is also critical. Azoxymethane is an alkylating agent that only methylates DNA in colonic epithelium of rats and hamsters. This alkylation may partially account for the site-specific carcinogenic activity of these compounds. In the large intestine of the rat, tumors induced by certain genotoxic carcinogens (e.g., dimethylhydrazine) are associated with lymphoid aggregates. Sessiletype adenocarcinomas, but not polypoid tumors, develop in the colonic mucosa near lymphoid structures. Consequently, at least two distinct neoplastic processes may occur in the colonic mucosa. If the target epithelial cell is not associated with a lymphoid aggregate, polypoid




TABLE 56.21

Compounds That Induce Intestinal Neoplasia in Rats
































methoxyethylurea a
























butylurea amylurea

hexylureaa 3-hydroxypropylurea


diethylureaa a


hydroxyethyl-ethylurea a

The proportion of animals that will develop tumors of the large and small intestine after exposure to the nitrosourea compounds ranges from 10 to 50%; females have the lowest and males the highest frequency of lesions. The median time to death ranges from 25 to 70 weeks. Table adapted from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Table X, p. 153, with permission.

adenocarcinomas and adenomas develop; if the target cell is near lymphoid tissue, sessile tumors develop. Organ-specificity of cancer development also relates to the sites of specific genetic mutations. An example is the dominant mutation that occurs in the germline of mice (APC min/þ) which predisposes the animal to multiple intestinal tumors. The propensity for tumor development is dependent on a single allele, and tumors develop in the duodenum, ileum, and colon. Since all cells of these mice carry the mutation and tumors occur only in the intestinal tract, somatic events are needed for neoplasia to develop. Mutation of this gene may involve loss of function at a genetic site important for normal intestinal development, gain of function in a gene that has

an unknown activity, or a complex interaction of both gene suppression and activation. Genotoxic carcinogen-induced changes in rat colonic epithelium are similar to those observed in spontaneously developing colorectal cancer in humans. In rats, sulfomucins are the primary glycoprotein of the normal colonic epithelium. Shortly after treatment with azoxymethane and N-methyl-N-nitro-N-nitrosoguanidine, cryptal epithelium mucus changes to express primarily sialomucins. Normal intestinal biopsies are characterized by the predominance of sulphomucins. These features support the de novo histogenesis of colon carcinoma. Foci of aberrant crypts referred to as aberrant crypt foci (ACF) are present in the carcinogentreated rodent colon. The two most common



FIGURE 56.24 Esophageal papilloma in a male B6C3F1 mouse treated with AZT/IFN. Connective tissue core (C) for the supportive stalk of the proliferating epithelial cells (E) which are invading subjacent tissue. Surface of papilloma is covered by a thick hyperkeratinized layer (K). (Figure provided courtesy of Dr. Susan Elmore, National Toxicology Program, NIEHS).

colon-specific carcinogens, azoxymethane and 1,2dimethylhydrazine, have been used in rats and mice to induce ACF. Aberrant crypts are observed topographically on whole mounts of colonic mucosal surfaces stained with methylene blue. These exhibit morphological atypia, and are easily distinguishable from the surrounding normal crypts by taking up increased amounts of blue stain. These crypts exhibit dilated irregular luminal opening and a thicker epithelial lining and pericryptal zone, and exist as single atypical crypts or as clusters of crypts forming atypical foci. ACF are purported to be preneoplastic lesions. This is supported by a number of studies into the biology of ACF. A carcinogenic dose of azoxymethane or 1,2 dimethythydrazine induces a large number of ACF. A systematic and sequential analysis of the number and growth features of ACF has demonstrated that ACF appear in the colon of rats and mice within 2 weeks after carcinogen injection, and that their number increases with time. At early time points, ACF contain one or two crypts (crypt multiplicity of 1 or 2); however, as time progresses many of the foci expand clonally and contain several crypts in the foci. ACF display proliferative atypia and dysplasia, a preneoplastic phenotype, and also show biological heterogeneity both among individual ACF and within a focus. Some ACF expand clonally


without exhibiting dysplasia, whereas others start exhibiting dysplasia with or without clonal expansion. Not all crypts in foci exhibit dysplasia, however. Crypt “budding” (the fission and multiplication of intestinal crypts) is evident in both types of foci. These findings support the fact that dysplasia arises in ACF as a result of clonal selection. A number of genotypic atypias occur in ACF. ACF in general, as well as those with advanced growth features, resist apoptotic cell death induced by azoxymethane, and some ACF exhibit elevated levels of glutathione-S-transferase isoforms. Compounds that are carcinogenic to the gastrointestinal tract of laboratory animals may act by direct or indirect actions. Direct-acting (genotoxic) carcinogens lead to initiated cells without prior metabolic activation, with subsequent persistence of neoplastic cells growing to morphologically verified tumors. Indirect-acting (non-genotoxic) compounds, requiring biotransformation or additional promotional interactions to be carcinogenic, may result in a prolonged stimulus of proliferation leading to a substantial increase in the number of dividing cells; intestinal stem cells may be identified by expression of the stem cell markers Lgr5 and EphB2. Single Lgr5þ stem cells isolated from intestine are capable of forming intestinal organoids recapitulating crypt/villus three-dimensional organization in vitro. Expression of Lgr5 and EphB2 has been shown to define a cancer stem cell niche within colorectal tumors, and is predictive of disease relapse in colorectal cancer patients; consequently, a tissue is more vulnerable to background initiating stimuli. The exact relationships between the genetic alterations and the phenotypic expression of cancer for non-genotoxic carcinogens are incompletely understood. Regardless of mechanism, carcinogenic compounds can act on all tissues of the gastrointestinal tract. The pathogenesis of some human colon cancers involves the loss of suppressor gene activity and oncogene activation. Tumor suppressor genes are lost from chromosomes 5, 17, and 18, and the ras oncogene is activated. Although many genetic alterations are found in neoplastic cells of human colon cancers, in certain colorectal cancers there appears to be a stepwise set of specific genetic lesions that compose the path from a normal colon cell to a metastatic cancer cell. One of the first steps is the loss of suppressor




FIGURE 56.25 Microbiota composition in stomach, cecum, and colon of H. pylori-infected male INS-GAS mice (n ¼ 3, 15 weeks post-infection) versus uninfected controls in specific pathogen-free (SPF) conditions (n ¼ 2). In Hpinfected mice, a significant increase in the relative abundance of Firmicutes and a concurrent decrease of Bacteroidetes (P < 0.05) were observed in the stomach whereas no significant changes were observed in the other parts of the gastrointestinal tract.

gene function on chromosome 5, which results in an increase in cell proliferation. Demethylation of DNA, ras oncogene mutation, and loss of suppressor genes from chromosome 18 leads to adenoma formation. Mutation on chromosome 17 leads to the development of neoplastic cells. Other chromosome abnormalities ultimately lead to metastasis. This pathogenesis demonstrates that the genome of a cancer cell has multiple mutations, and that both suppressor gene loss and oncogene activation are involved in the development of mammalian cancer. Gut Microflora and Cancer The role of gut microflora in the promotion of inflammation association cancers like Helicobacter pylori (Hp)-associated gastric cancer and IBD associated colo-rectal cancers is now an active area of research. In Hp-induced gastric ulcers, gastritis, and its subsequent promotion to gastric cancer, the role of other gastric microflora, at least in experimental models, is now believed to be to play an important role in the severity of gastric lesions. Of note, in an INS-GAS hypergastrinemic mouse model of Hp infection, the lack of commensal flora in germ-free mice resulted in a decreased severity of Hp-induced gastritis and delayed the progression to gastrointestinal intraepithelial neoplasia (GIN) as compared to their infected

conventional SPF counterparts. Interestingly, Hp-infected SPF mice showed a significant increase in the amount of Frimicutes and a decrease in the levels of Bacteroidetes in their stomach as compared to non-Hp-infected SPF mice, as shown in Figure 56.25, thus implying the potential role of gut microflora in the copromotion of Hp-associated gastritis and gastric cancer. Intestinal bacteria, as detailed earlier, are capable of degrading dietary components into toxic by-products with genotoxic, carcinogenic and tumor-promoting activity, and hence are also considered to play a role in the development of colon cancer. Germ-free rats and mice have lowered ability to activate dietary procarcinogens or chemical tumor initiators like 1,2 dimethylhydrazine (DMH) and induce DNA adduct formation as compared to their conventional counterparts, indicative of the role of the complex intestinal microbiota in carcinogenesis. The microbiota in general is important in bile metabolism and formation of secondary bile acids (possible tumor promoter) and other toxic dietary metabolites like n-nitroso compounds which are associated with an increased risk for colon cancer. Intestinal bacteria, depending upon the species, can cause intestinal tumor promotion or suppression on the basis on presence of enzymes such as b-glucuronidase, bglucosidase, and nitrate- and nitro-reductases.



Intestinal Bacteriodes, Eubacteria, and Clostridia are associated with enhanced carcinogen formation and metabolism, whereas some Lactobacillus spp. and Bifidobacterium spp. have beneficial tumor protective effects.

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