Mutation Research, 247 (1991) 267-281 © 1991 Elsevier Science Publishers B.V. 0027-5107/91/$03.50 ADONIS 002751079100086J
Role of genetics and drug metabolism in human cancer risk Daniel W. Nebert Department of Enoironmental Health, University of Cincinnati Medical Center, Cincinnati, OH 45267-0036 (U.S.A.)
Keywords: Genetics; Drug metabolism; Cancer, human
Summary The research field concerning responses to drugs having a hereditary basis is called 'pharmacogenetics'. At least 5 dozen pharmacogenetic polymorphisms have been described in clinical medicine; many are responsible for marked differences in genetic predisposition toward toxicity or cancer. Three are detailed here: the acetylation, the debrisoquine, and the A H locus polymorphism. All 3 are very common among the United States' population: I in 2 is a 'slow acetylator', 1 in 12 is a 'poor metabolizer' for more than 2 dozen commonly prescribed drugs in the debrisoquine panel, and the CYP1A1 and C Y P 1 A 2 (cytochromes P1450 and P3450) genes are highly inducible by cigarette smoke in 1 of 10 patients. Differences in xenobiotic metabolism between individuals in the same family can be greater than 200-fold, suggesting that occupationally hazardous chemicals, as well as prescribed drugs having a narrow therapeutic window, might cause strildngly dissimilar effects between patients of differing genotypes. Our ultimate goal is 'preventive toxicology', i.e. the development of simple, inexpensive, unequivocal and sensitive assays to predict individual risk of toxicity or cancer. These tests could help the individual in choosing a safer life style or place of work and might aid the physician in deciding which drug to prescribe.
Pharmacogenetics is the field study involving unusual (idiosyncratic) responses to drugs that have a hereditary basis (Kalow, 1962). The degrees of varying responses considered here are 10-200fold, although admittedly there are many differences in drug responses of < 10-fold. An idiosyncratic drug reaction is regarded as distinct from unanticipated responses due to accidental overdosage or allergic phenomena. Drugs in this paper are meant to include, in addition to the hundreds
Correspondence: Daniel W. Nebert, M.D., Department of Environmental Health, University of Cincinnati Medical Center, 3223 Eden Avenue, Cincinnati, OH 45267-0056
of clinically prescribed agents, the innumerable foreign chemicals and other substances to which we are exposed via our diet, life style, and place of work. The major source of human exposure to this myriad of compounds is foodstuff, although a significant increase in the uptake of chemical pollutants (via the lungs and skin) has occurred in the past several hundred years since the beginning of the industrial revolution. In some ways, pharmacogenetic disorders are similar to 'inborn errors of metabolism'. One important difference, however, lies in the fact that a pharmacogenetic disorder might never be realized in one's lifetime, unless the idiosyncratic response is precipitated by a particular drug. On the other hand, an inborn error of metabolism usually pre-
sents itself as a serious disease early in life. Another important difference between pharmacogenetic defects and inborn errors of metabolism lies in the frequency of affected individuals in the human population. Although many pharmacogenetic defects are as rare as inborn errors of metabolism (e.g. 1 in 800, or 1 in 20 000), some pharmacogenetic disorders can be quite common (Kalow, 1962; Nebert and Weber, 1990). The 3 discussed here in detail are in this latter category: the incidence of the affected individual in the acetylation polymorphism is 1 in 2; that for the debrisoquine polymorphism is 1 in 12; and that for the aryl hydrocarbon hydroxylase (AH) polymorphism, 1 in 10. When the incidence of a pharmacogenetic disorder is this frequent, it would be beneficial to make this knowledge available for the individual, the physician, the epidemiologist, and the employer. It would also be advantageous to develop simple clinical tests to determine the individual at risk. In this paper, several fundamental principles of drug metabolism are discussed first. Next, 3 common pharmacogenetic disorders are compared in detail. Finally, the double-edged-sword nature of these disorders is emphasized: an increased risk of cancer might befall one phenotype, while increased risk of toxicity might befall the other. Clearly, a better understanding of these disorders will never make them candidates for gene therapy. However, through better understanding and perhaps counseling, an improved life style and healthful living might be realized if one knows how to avoid certain risks of cancer a n d / o r idiosyncratic drug responses.
Fundamental principles of drug metabolism
(n ,--I ,< ¢3 m Z i,
mg per kg Fig. l. Hypothetical distributions of relatively narrow (top) and relatively broad (bottom) EDs0 values in the human population.
however, the distribution for a particular drug might range over more than 2 orders of magnitude (Fig. 1, bottom); this means that there would be a > 100-fold difference in EDs0 between the most sensitive and the most resistant individuals in the population. In Fig. 1, if the > 100-fold difference represents 2 alleles at the same genetic locus, it is possible that highly resistant phenotypes would exist among members of the same family. A 'clean'
The 'EDs0' of a drug is the effective dose (ED) that provides the desired therapeutic response to 50% of the human population. If a physician learns that the dosage of a particular drug in the Physician's Desk Reference is, for example, '5 mg/kg', the usual image in our minds is illustrated at the top of Fig. 1. The Gaussian distribution in this top figure is shown to be very sharp, i.e. everyone exhibits an EDs0 between about 4.7 and 5.3 m g / k g , implying that there would be < 2-fold variability among all individuals in the population. Clearly,
o lu an
mg per kg or parts per million (ppm)
Fig. 2. Hypothetical bimodal distribution of EDs0 values for a drug (mg/kg) or environmental chemical (ppm) in the human population in which the phenotype of the A a heterozygote cannot be distinguished from that of the a a homozygote.
The acetylation polymorphism 1.0 W
log mg per kg or parts per
Fig. 3. Hypothetical dose-response curves for 2 subsets of the human population that are particularly 'sensitive' and 'resistant' to a drug (mg/kg) or occupationally hazardous chemical (ppm). Arrows denote the EDs0 of each subset, examined as the 'fractional response' by probit analysis.
bimodal distribution is illustrated in Fig. 2, in which the AA genotype is shown distinctly separated from the Aa heterozygote and the aa homozygote. Experimental distributions similar to this hypothetical distribution will be discussed below for both the acetylation and the debrisoquine polymorphisms. The types of distributions shown in Figs. 1 and 2 are usual when comparing wild-type with mutant structural genes. On the contrary, differences in dose-response curves (Fig. 3) are commonly seen for variants of regulatory genes. Experimental data of this type will be discussed below for the A H locus polymorphism. Finally, it should be emphasized that genetic differences in response to a particular drug might only be detected if the drug has a sufficiently narrow 'therapeutic window'. Consider, for example, that the therapeutic ratio of a drug is 1000, i.e. toxic effects are not seen unless doses 1000 times the therapeutic dose are employed; individual differences in drug metabolism on the order of 10200-fold would not be manifest as a pharmacogenetic disorder. If, on the other hand, toxicity of a drug is seen at doses only 5 times the therapeutic dose, individual differences in drug metabolism of 10-200-fold would easily be detected.
N-Acetyltransferases occur in most mammalian tissues and exhibit an important pharmacogenetic disorder in the human population, as well as in rabbits and mice (Weber, 1987). The chemicals that undergo biological acetylation are, for the most part, either aromatic amines or hydrazines. The acetylation polymorphism was originally called the 'isoniazid acetylation polymorphism' because it was recognized in patients treated with isoniazid (Fig. 4). As discussed by Idle in the previous paper, individuals can easily be phenotyped as 'slow' or 'rapid' acetylators: slow acetylators are homozygous for the slow allele (r/r), whereas rapid acetylators are either homozygous ( R / R ) or heterozygous ( R / r ) for the rapid allele (Fig. 5). Although the distinction between rapid and slow phenotypes is fairly unambiguous, the data in Fig. 5 are not as 'clean' as the hypothetical data shown in Fig. 2. The slow acetylator allele frequency is approximately 0.72 in the United States, meaning that 1 in every 2 individuals is homozygous for the r allele. The slow acetylation phenotype frequency worldwide ranges from approx. 0.10 in Japanese populations to more than 0.90 in some Mediterranean peoples (Nebert and Weber, 1990).
II C-NH-NH 2
0 0 II II C-NH-NH-C-CH 3
acetylated isoniazid minorr o u t e ~ o
pyridine carboxylic acid Fig. 4. N-Acetylation of the antituberculosis drug isoniazid in mammalian liver is the major route of metabolism.
270 Ra )id (R/R or R/r)
=~ 12 O"
Plasma Concentration of Isoniazid (/~g/ml)
Fig. 5. Distribution of 'rapid' and 'slow' acetylators in human population. (Modified and redrawn from D.A.P. Evans et al. (1960).) Two human N-acetyltransferase genes have recently been cloned and characterized (Grant et al., 1989; Blum et al., 1990). N A T 2 ( ' f o r m B') encodes an enzyme having a K m value for several aromatic amines at least 10 times lower than that of NA T1 (' form A'). Although the 2 genes exhibit 85% nucleotide similarity, the rapid and slow acetylator phenotypes appear to involve the NA T2 gene, but not the NA T1 gene. The slow acetylation phenotype is more prone to neurotoxicities from isoniazid, to lupus erythematosus from hydralazine and procainamide, while
sulfa drugs induce hemolytic anemia and cutaneous disorders, and phenytoin toxicity accompanies the combined use of phenytoin and isoniazid in slow acetylators. In these conditions and with other drugs listed in Table 1, the slow acetylators have higher serum concentrations of the drug than rapid acetylators at any time after drug ingestion (Weber, 1987). Many of these drug reactions are commonplace and not tri"ial. The incidence of procainamide-related lupoid hepatitis in the United States, for example, is 29% (Weber, 1987). This means that, if a physician prescribes procainamide to 10 patients, on the average 5 of these would be slow acetylators and 3 of the 5 would be expected to develop lupoid symptoms. There is a statistically significant association between the rapid acetylator phenotype and the incidence of colorectal carcinoma (Lang et al., 1986; Ilett et al., 1987). There are also highly significant correlations between the slow acetylator phenotype and the occurrence of bladder cancer (Evans et al., 1983; Cartwright, 1984; Hanssen et al., 1985) and between the slow acetylator phenotype and spontaneous idiopathic lupus erythematosus (Godeau et al., 1973; Reidenberg and Martin, 1974). In one of the earhest studies of a random hospital population of 111 patients with bladder carcinoma (Table 2), a striking excess of slow acetylators was observed in a group of chemical workers employed in the dye industry. This
TABLE 1 THE GROWING LIST OF DRUGS IN THE 'NAT PANEL' " Drug
Indication(s) for use of drug
Isoniazid Hydralazine Procainamide Phenelzine Dapsone Aminoglutethimide Sulfasalazine Sulfonamide Caffeine Nitrazepam Clonazepam Dipyrone Acebutolol
Tuberculosis Hypertension Cardiac arrhythrnias Neurotic depression Leprosy, chloroquine-resistant malaria Convulsions Inflammatory disorders Bacterial infections (?)Need of stimulant Anxiety states Convulsions Analgesia, fever, inflammation Cardiac arrhythmias
Data taken from Weber (1987).
Major undesirable side effect(s) Neurotoxicity, hepatitis Lupoid hepatitis Lupoid hepatitis Dizziness, lupoid hepatitis Peripheral neuropathy Adrenal insufficiency Hemolytic anemia Cutaneous reactions, skin rash
271 TABLE 2 ACETYLATOR PHENOTYPES AND INDUSTRIAL BLADDER CANCERa
group, which was exposed to benzidine, showed a 40% excess of slow acetylators ( P < 0.0005), compared with controls. Moreover, a higher portion of slow acetylators than fast acetylators developed generalized bladder disease both with carcinoma in situ and deeper invasion of the bladder wall. Additional support for the link between invasiveness and acetylator status was provided by studies of bladder cancer in Portugal (Cartwright, 1984).
Thiozalsulfone (promizol) is an arylamine sulfa drug that was found to produce a bimodal distribution of hemolytic anemia. This landmark observation (Dern et al., 1955), and subsequent experiments (Weber, 1987), strongly suggest that G6PD-deficient slow acetylators are most susceptible to promizol- or dapsone-induced hemolysis, compared with other pharmacogenetic phenotypes. This same combined pharmacogenetic disorder has been studied among factory workers. Hemoglobin adducts among workers exposed to aniline and acetanilide are highest in G6PD-deficient slow acetylators, as compared with other combined pharmacogenetic phenotypes (Lewalter and Korallus, 1987). Because substrates of the CYP1A2 enzyme are often arylamines, the possible combination of high C Y P I A 2 inducibility with slow acetylator phenotype or G6PD deficiency, or both, could produce even more striking differences in toxicity or cancer risk; this subject is discussed later in this paper.
Combined pharmacogenetic disorders
'Phase I' and 'phase II' drug metabolism
An idiosyncratic drug response may be. exacerbated by the unfortunate combination of 2 pharmacogenetic defects in the same individual. For example, glucose-6-phosphate dehydrogenase (G6PD) is an enzyme in the hexose monophosphate shunt, one of the principal sources of N A D P H generation in the normal red cell (Nebert and Weber, 1989). N A D P H is, in turn, the cofactor for glutathione reductase, which reduces oxidized glutathione (GS-SG) to the reduced form (GSH). A defect in G6PD can lead to severe drug-induced hemolysis in the absence of normal G S H levels in red cells. G6PD deficiency is an X-linked pharmacogenetic disorder that affects at least 200 million people worldwide (Nebert and Weber, 1990). Approx. 11% of black American males exhibit the A ( - ) type, and certain Sardinian communities exhibit the more severe 'Mediterranean type' at frequencies as high as 1 in every 3 persons. The G6PD gene has been cloned and localized to the X chromosome, and several diverse point mutations may account for the large degree of phenotypic heterogeneity seen in G6PD-deficient patients (Hirond and Beutler, 1988; Vulliamy et al., 1988).
Drug-metabolizing enzymes are classically divided into 2 broad categories. 'Phase I' enzymes involve almost exclusively cytochromes P450, which function by the insertion of 1 atom of atmospheric oxygen into a relatively inert substrate; 'phase II' enzymes act on the oxygenated intermediates by conjugation with various endogenous moieties (glucuronide, glutathione, sulfate) to produce extremely hydrophilic products that are easily excreted from the cell (Miners et al., 1987; Schuster, 1989; Nebert and Gonzalez, 1987). The reactive intermediates formed by P450 enzymes can be carcinogenic, mutagenic, a n d / o r toxic. The coordinate regulation of phase I and phase II genes and the architectural arrangement of phase I and II enzymes in each cell are, therefore, important factors ensuring metabolic clearance of foreign substances from the body with a minimal risk of accumulation of the oxygenated intermediates that might lead to disease.
Acetylator Chemical Neverchemical Cancer invasiveness type workers workers Low High Slow Rapid
22 (96%) 52 (59%) 1 36
Data taken from Cartwright et al. (1982).
The P450 gene superfamily The other 2 pharmacogenetic disorders to be discussed in this paper involve P450 genes. Cyto-
chromes P450 are enzymes involved in the oxidative metabolism (biosynthesis as well as degradation) of steroids, fatty acids, prostaglandins, leukotrienes, biogenic amines, pheromones, and plant metabolites. These monooxygenases also metabolize countless drugs, chemical carcinogens and mutagens, and other environmental contaminants (Miners et al., 1987; Schuster, 1989; Nebert and Gonzalez, 1987). The P450 superfamily presently comprises more than two dozen gene families, 10 of which exist in all mammals (Fig. 6). A nomenclature system based on evolution has been proposed (Nebert et al., 1991). For naming a gene the root symbol 'CYP' ('Cyp' for mouse), denoting cytochrome P450, is recommended; this is followed by an Arabic numeral designated the P450 gene family, a letter indicating the subfamily, and an Arabic numeral representing the individual gene. Current estimates (Nebert et al., 1989) of the total number of functional P450 genes in any one mammalian species range between at least 60 and perhaps more than 200. All of these genes are believed to have arisen by divergent evolution, i.e. from a common ancestral gene probably more than 2.5 billion years ago. A major driving force for the large number of gene duplications is believed to be animal-plant 'warfare' (Nebert and Gonzalez, 1987; Nelson et al., 1987; Nebert et al., 1989; Gonzalez and Nebert, 1990). A greater understanding about species variation in P450 genes is of central importance to pharmaceutical companies and regulatory agencies, since laboratory animals are frequently used to test the safety of drugs and other potentially toxic substances. Fig. 7 illustrates divergence of the human, rabbit, rat and mouse species from one another. It is easy to see that new P450 genes may appear, or old ones may become lost or altered, in a particular species following its divergence from other species. Between two species (e.g. between human and rat or between rat and mouse) there are already numerous examples of different P450 genes, and in some cases different catalytic activities, among members of the same P450 gene subfamily (Nebert and Gonzalez, 1987; Gonzalez and Nebert, 1990). It is highly likely that a particular test compound will produce an entirely different response - with regard to toxicity or cancer -
EVOLUTIONARY DISTANCE Fig. 6. Unweighted-pair-group method of analysis (UPGMA) of the P450 gene superfamily. The most recent nomenclature update has just appeared (Nebert et al., 1991). Gene families are designated by numbers, subfamilies by capital letters. A protein encoded by a gene in one family usually exhibits ~<40% amino acid sequence identity to that encoded by a gene in another family. Mammalian genes in the same subfamily encode protein > 59% similar (Nebert et al., 1989). Although 14 gene families are illustrated here, there are more than 100 total sequences presently available, which enlarges the superfamily to 17 families - - in 12 eukaryotes and 4 prokaryotes. The divergence between bacterial and eukaryotic genes (d = 2.5) has been set at 1400 million years before the present (Mybp), the date of the earliest evidence of eukaryotic microfossils. The estimations of time in the oldest part of the tree are subject to the largest error (Nebert and Gonzalez, 1987; Nelson and Strobel, 1987). Gene conversion events cause nonlinearity during P450 gene evolution and will contribute to the uncertainty of U P G M A alignments. The help of David R. Nelson with U P G M A alignment program is very much appre ciated.
273 TABLE 3 THE GROWING LIST OF DRUGS IN THE 'CYP2D PANEL' a
Antiarrhythmic agents Encainide Flecainide Propylajmaline
Antidepressants ( + )-Amiflamine Amitriptyline
fl-Adrenergic blocking agents
MILLIONS OF YEARS BEFORE THE PRESENT Fig. 7. Diagram of the approximate time scale, based on fossil records (Miyata et al., 1982), for divergence of 4 mammalian species from one another.
between a human and a laboratory animal, due to the presence or absence of a particular P450 gene in either species.
Alprenolol Bopindolol Bufuralol Debrisoquine
Metroprolol Penbutolol Propranolol Timolol
Other commonlyprescribed drugs Codeine Dextromethorphan Guanoxan 4-Methoxyamphetamine
Methoxyphenamine Phenacetin Phenformin
a Data taken from Idle and Smith (1979) and Eichelbaum (1984).
The debrisoquine polymorphism Debrisoquine is a fl-adrenergic receptor blocking agent used to treat hypertension. The P450mediated 4-hydroxylation of debrisoquine and N-oxidation of sparteine (Fig. 8) occur at rates 10-200 times greater in 'extensive metabolizers ~ (EM phenotype) than 'poor metabolizers' (PM phenotype). In a British Caucasian population, H
Fig. 8. Hydroxylation of the fl-adrenergic blocking agent debrisoquine (left) and N-oxidation of the antiarrhythmic drug sparteine. Both reactions are catalyzed by the CYP2D6 enzyme.
about 8% were found to be PM individuals (Idle and Smith, 1979). When the PM phenotype (homozygous autosomal recessive) receives this drug, dizziness and hypotension are commonly observed. There is a growing list of other drugs that similarly are metabolized poorly in the PM individual (Table 3). It would therefore be extremely helpful to the physician, before prescribing a drug, to know whether that drug is a member of the 'CYP2D drug panel' and whether the patient is a PM individual. PM individuals exhibit little, if any detectable CYP2D6 protein in their livers, due to aberrant sphcing of the CYP2D6 gene transcript (Gonzalez et al., 1988). By means of restriction fragment length polymorphism (RFLP) studies, 2 distinct haplotypes were shown to account for approx. 70% of PM individuals (Skoda et al., 1988). One RFLP pattern appears to represent a single-base insertion in the first exon of the CYP2D6 gene, leading to a nonfunctional m R N A and protein. The second R F L P pattern appears to be caused by deletion of the entire CYP2D6 gene. The remaining 30% of PM individuals may be highly heterogeneous, representing perhaps 20 or more other mutant alleles.
274 8 Cancer Patients N = 59
6 >- 4 o 2 Z iii
Normal Subjects N = 123
~6 4 2
[!l IJil! 4
II , Jl , ~ _ ~ L 8
M E T A B O L I C RATIO
Fig. 9. Incidence of EM and PM phenotype among cancer patients and controls. (Modified and redrawn from data in Idle et al. (1981).)
Association of the PM phenotype with Parkinson's disease has been reported (Barbeau et al., 1985; Poirier et al., 1987). If corroborated by further epidemiologic studies, these data would suggest that the CYP2D6 enzyme might function in the metabolic clearance from the central nervous system of some potentially toxic environmental or dietary substance(s) and that PM individuals are more prone to neurotoxicity caused by the accumulation of this toxic substance. The EM phenotype, on the other hand, appears to be correlated with enhanced cancer risk. In a group of Nigerian patients with cancer of the liver and gastrointestinal tract, there was a disproportionately greater number of EM individuals (Fig. 9). In a study of 245 cigarette smokers with bronchogenic carcinoma, the cancer patients also represented a preponderance of EM phenotype individuals (Ayesh et al., 1984; Caporaso et al., 1989; Caporaso et al., 1990). Others have described an associated risk of the EM phenotype with bladder cancer (Kaisary et al., 1987). One laboratory has failed, however, to confirm the lung cancer findings (Roots et al., 1988). If confirmed, these data would suggest that the EM individual exhibits enhanced metabolism of some chemical(s) in cigarette smoke, as well as perhaps dietary substance(s), to active carcinogen(s). The A H locus polymorphism
This genetic system was named more than 15 years ago for the observation that a certain P450-
mediated activity [aryl hydrocarbon (benzopyrene) hydroxylase; AHH, now termed CYPIA1 enzyme activity] was highly inducible in some inbred mouse strains but not others (Nebert, 1989). Mammalian CYPIA1 (mouse P]-450, rat P-450c, rabbit form 6) catalyzes the oxygenation of polycyclic hydrocarbons such as benzo[a]pyrene to phenolic products and epoxides, some of which are toxic, mutagenic and carcinogenic (Fig. 10). A second, closely related mammalian enzyme, CYP]A2 (mouse P3-450, rat P-450d, rabbit form 4), metabolizes certain arylamines (Fig. 10). The lack of C y p l a l induction behaves as an autosomal recessive trait between C57BL/6 and D B A / 2 mice, and the first inducing chemicals characterized were aromatic hydrocarbons (e.g., 3methylcholanthrene and benzopyrene); hence, the name Ah locus in the mouse ( A H in humans). The A H locus encodes the cytosolic Ah receptor, which is involved in the regulation of both CYP1A1 and CYP1A2 genes. A receptor defect is responsible for the decreased C y p l a l and Cypla2 induction in D B A / 2 mice (Poland et al., 1976). The Ah receptor has been studied in detail through use of the radiolabeled potent inducer tetrachlorodibenzo-p-dioxin (TCDD) (Nebert, 1989; Poland et al., 1976) and is expected to be a member of the steroid and thyroid hormone and retinoid receptor superfamily (R.M. Evans, 1988). TCDD, a synthetic by-product of Agent Orange and hexachlorophene soap, has been implicated in several reported instances of environmental pollution during the past 30 years (Poland and Knutson, 1982). Many molecular details of the Ah locus (Fig. 11) have been elucidated in the inbred mouse and via somatic cell genetics (Poland et al., 1976; Nebert, 1989). Procarcinogens such as combustion products (e.g., benzo[a]pyrene and more than a dozen other polycyclic hydrocarbons), B-naphthoflavone and similar green plant flavones, and TCDD enter the cell passively and bind to the cytosolic Ah receptor with a theoretical K d of about 7 pM (Bradfield and Poland, 1988). The endogenous ligand is not known. During evolution, most likely plant flavones and, later, certain combustion products have appropriated the Ah receptor for stimulating their own metabolism. After the TCDD ° Ah receptor complex gains
N--C --CH 3
H ._ N/C-cH3
Fig. 10. Hydroxylation of the polycyclic aromatic hydrocarbon benzo[a]pyrene by the CYPIA1 enzyme and 2 representative arylamines by the CYP1A2 enzyme.
chromatin-binding properties, the expression of several genes is augmented. Included among the TCDD-inducible genes are CYPIA1 and CYP1A2, and at least 1 gene of each of the following phase II enzymes: NAD(P)H:menadione oxidoreductase, aldehyde dehydrogenase, UDP glucuronosyltransferase, and glutathione transferase. The fascinating coordinate regulation of these 6 functionally related enzymes by the Ah receptor has been called the [Ah] gene battery (Nebert and Gonzalez, 1987; Nebert et al., 1990). The Cyplal and Cypla2 genes are known to be transcriptionally activated in mouse liver (Gonzalez et al., 1984), and enhanced mRNA concentrations lead to increased levels of the Cyplal and Cypla2 proteins in the endoplasmic reticulum and corresponding rises in benzo[a]pyrene and acetanilide metabolism, respectively (Negishi et al., 1981). In most cases, high Cyplal and Cypla2 inducibility leads to enhanced risk of toxicity and cancer, although these observations must be tempered by the route of administration, dose of the
test compound given, and target organ being examined (Nebert, 1989). The data for the Ah receptor gene, a regulatory gene, are similar to the hypothetical curves shown in Fig. 3. It is likely that the AH polymorphism in the human population (and Ah polymorphism amongst inbred mouse strains) will represent one or a few amino acid differences in the Ah receptor protein, leading to subsets of highly sensitive individuals (in whom low doses of inducer increase CYP1A1 and CYP1A2 activity) and highly resistant individuals (in whom only high doses of inducer increase CYP1A1 and CYP1A2 activity). A wealth of information has been gained through the use of plasmids containing mouse, rat or human CYP1A1 upstream sequences and the chloramphenicol acetyltransferase reporter gene, sometimes with a heterologous promoter (Whitlock, 1987; Nebert and Jones, 1989). These constructs have been transfected into mouse hepatoma Hepa-1 wild-type (wt) cell cultures, as well as Cyplal metabolism-deficient (P1-), receptorless
TCDD, ~ Banzola]pyrene and other environmental @ pollutants
Formation [~~=--] of excretable ~ Critical innocuous targetin
Icn°du P~r;rececPt¢ors °I
PtYnS~'i°o~ 'c I I
Inducer-reducer compl exin nucleus ~ N N ~~
\ ] /
of P1-450and P3-450genes
heaSnow "received" i
~ | • ~ransl;tionof induction-
Unknown site in nucleus
.,1"7 Reactive f , f ¢ intermediate
Toxicity and/or Initiation of Cancer
I ~/ /
i,~ Binding of reactive intermediate to critical target ¢
Fig. 11. Sequence of events by which the human CYP1A1 and CYP1A2 genes become activated, or induced, by environmental chemicals that bind to the Ah receptor. The possibility that TCDD, as an extremely potent ligand, occupies the Ah receptor such that a critical life function cannot be carried out (Poland and Knutson, 1982) has been proposed to explain the extreme toxicity of this environmental contaminant. The possible inverse relationship between TCDD toxicity and euthyroid function (McKinney et al., 1985; Hong et al., 1987) is intriguing. (Modified and redrawn from Nebert (1979).) (r), and c h r o m a t i n binding-defective ( c b ) m u t a n t lines. A b o u t 1 kb upstream from the m R N A cap site lies an aromatic hydrocarbon-re-
sponsive d o m a i n ( A h R D ) , comprising 3 aromatic hydrocarbon-responsive elements ( A h R E s ) spaced approximately 80 bp apart (Fig. 12). Gel mobility
shift and methylation interference assays have demonstrated the interaction of a functional Ah receptor with the AhRD. Another dement about 200 bp upstream from the cap site is absolutely essential for all constitutive and inducible Cyplal gene expression; this proximal dement that binds proteins is necessary but not sufficient for inducible gene expression, but interaction of these proteins with those at the distal AhRD appears to be required for full Cyplal induction by TCDD (Neuhold et al., 1989). Another element in the upstream Cyplal regulatory sequences involves a negative autoregulatory loop; in the absence of a functional Cyplal gene product in P1- cells, there is an apparent derepression of constitutive Cyplal transcription (Nebert and Gonzalez, 1987). Excluding the TATA box binding protein and RNA polymerase II, we estimate that there is a minimum of 4, and possibly 9 or more, trans-acting regulatory proteins - one of which is the Ah receptor - affecting constitutive and inducible CYPIA1 gene expression. Keeping in mind that each of these proteins is probably encoded by its own distinct gene, we suspect that our attempts to identify and characterize the number of genes contributing to CYPIA1 inducibility might lead to TATAA
Fig. 12. Illustration of the distal and proximal DNA elements about 1 kb and 200 bp, respectively, upstream from the C Y P I A 1 mRNA cap site. These DNA elements are highly conserved in the mouse, rat and human upstream regions. 3 classes of regulatory proteins that interact with the distal element have been defined: I, presumably the Ali receptor (closed triangle); II, depicted by 3 bands on gel mobility shift assays, is competed by SV40 enhancer and heat-stable; III, depicted by another 3 bands on gel mobility shift assays, is not competed by SV40 DNA and is thermolabile. The proximal element binds 2 factors that are not the Ah receptor, one having properties similar to the class II regulatory protein(s) described above (Neuhold et al., 1989). The data for TCDD inducibility are consistent with the DNA 'loop-out' hypothesis for transcriptional activation of the murine C Y P I A 1 gene (Nebert and Jones, 1989).
A O I-< at:
iii (n ,< >..J.
)< O r,>o .
< !-o : c3 "' n-
,,, n3: •.J >-
O Q: 3: (J O I->o .1i~ <
•I wv |
OTHER PULMONARY DISEASE (N = 29)
(N = 21 )
Fig. 13. Incidence of 'high' and 'low' Ah inducibility phenotype among cigarette-smoking lung cancer patients and controis. (Modified and redrawn from data in Kouri et al. (1982).)
the elucidation of very complex regulatory systems. There appears to be a significant correlation between high CYP1A1 inducibility and enhanced risk of bronchogenic carcinoma in cigarette smokers (Fig. 13). The original report of a rdationship between cigarette-induced lung cancer and high CYP1A1 inducibility (Kellermann et al., 1973) could not be substantiated for several years in many laboratories (Paigen et al., 1977). The use Of a more recent assay that is substantially more reproducible, however, has led to the conclusion that the cigarette-smoking Ah-responsive individual is several times more prone to bronchogenic carcinoma than the Ah-nonresponsive smoker (Kouri et al., 1982). This increased risk of polycyclic hydrocarbon-caused cancer for the high Cyplal inducibility phenotype has also been demonstrated in numerous studies with inbred mice (Nebert, 1989). It is likely that enhanced cancer risk due to high CYP1A1 inducibility will represent differences in the human (as well as the mouse) Ah receptor gene, rather than differences in the CYPIA1 or CYPIA2 structural genes per se. This laboratory and others have cloned and sequenced
278 the human CYP1A1 and CYP1A2 genes and flanking regions (Jaiswal et al., 1985; Ikeya et al., 1989; Quattrochi and Tukey, 1989). In early experiments, we were unable to correlate human CYP1A1 restriction fragment length polymorphism (RFLP) patterns with the Ah phenotype in family studies and population screening studies (Jaiswal et al., 1985; Jaiswal and Nebert, 1986). However, recent results with a large 3-generation family have been promising, suggesting an association between CYP1A1 inducibility and an R F L P pattern of the CYP1A1 gene; in contrast, we have not yet found any informative RFLP patterns with the human CYP1A2 gene and flanking regions (Petersen et al., 1991). Among 12 human liver samples, > 15-fold differences in CYPIA2 mRNA levels were observed (Ikeya et al., 1989), suggesting that important differences in CYP1A2 gene expression (as has already been well established for CYP1A1 gene expression) could play an important role in individual risk of environmental toxicity or cancer. A recent study indicated that measurement of hemoglobin adducts of 4-aminobiphenyl and other aromatic amines might be useful in understanding the biochemical basis for increased risk of bladder cancer among cigarette smokers (Bryant et al., 1988). It is likely that these foreign chemicals are substrates for the human CYPIA2 enzyme and that individual differences in CYP1A2 gene expression could help explain some of the genetic 'noise' encountered in such epidemiological studies. Because the substrates of CYP1A2 are arylamines, it should be emphasized that individual differences in CYP1A2 gene expression might play a role in the combined pharmacogenetic phenotypes of slow acetylator and G6PD deficiency described above (Dern et al., 1955; Lewalter and Korallus, 1985). For these reasons, we hope to develop a simple, inexpensive, highly reproducible clinical test that will predict human CYP1A1 and CYP1A2 inducibility and, therefore, individual risk of certain environmentally caused toxicity and cancers. Conclusions
Each of the 3 pharmacogenetic disorders detailed in this paper shows a correlation with
malignancy, as well as with idiosyncratic drug -responses. The slow acetylator phenotype among chemical dye workers exhibits an increased risk of bladder cancer, and the slow phenotype is also more prone to develop drug-induced neurotoxicity, lupus erythematosus, and liver disease (Weber, 1987). Recent studies suggested that the rapid acetylator phenotype is more likely to develop type I diabetes and colorectal cancer (Weber, 1987). The extensive debrisoquine metabolizer (EM) phenotype has been reported to have a disproportionately higher risk of lung cancer among cigarette smokers and cancer of the liver and gastrointestinal tract, yet the PM phenotype experiences the acute idiosyncratic response of hypotension to adrenergic blocking agents and perhaps the chronic response of Parkinson's disease. Finally, individuals of high CYP1A1 inducibility exhibit an elevated risk of cigarette smoking-induced bronchogenic carcinoma; although we have cloned and sequenced the human C Y P I A 1 and C Y P I A 2 genes, R F L P patterns have not been easy to correlate with the Ah phenotype, suggesting that the true differences in C Y P I A 1 and C Y P I A 2 gene expression may reside in a regulatory gene such as the Ah receptor gene. These observations illustrate the double-edgedsword nature of pharmacogenetic disorders. (i) The phenotype most likely to develop an acute drug response need not be the same as that most likely to develop the chronic drug response. (ii) Chronic drug responses associated with certain pharmacogenetic differences can include cancer. (iii) The same response, such as enhanced risk of lung cancer, can exist in 2 quite unrelated pharmacogenetic polymorphisms (e.g. the debrisoquine polymorphism involves a gene in the C Y P 2 D subfamily, and the Ah locus is associated with regulation of the CYP1A gene subfamily). The pharmacogenetic differences described herein range between 10- and 200-fold. If these data could be extrapolated directly to risk of human disease, we may conclude that - at any given dose of drug or environmental pollutant - one individual will be 10-200 times more sensitive to toxicity or cancer due to differences in expression of the particular gene. It is important to emphasize that all of these risks are relative. A particular drug or environmental chemical that causes toxic-
ity or cancer in one individual might never cause toxicity or cancer, even at high doses, in another individual. A different foreign chemical might be lethal, without ever causing malignancy, in one phenotype while exhibiting no apparent toxicity in another phenotype. Several such dramatic examples have been described in a number of instances with Ah-responsive and Ah-nonresponsive mice (Nebert, 1989). It is expected that simple, inexpensive clinical tests will soon be available to diagnose pharmacogenetic disorders and predict whether the individual patient, as well as employee in the work place, has an increased risk of toxicity or cancer (Dern et al., 1955; Lewalter and Korallus, 1985; Roots, 1982; Stomel et al., 1989). It goes without saying that individuals must not be required to submit to such genetic testing against their will and that such information should be informative, while never being used to harm or discriminate against the individual. Results from such assays must remain confidential and should never be used to encourage genetic uniformity or superiority. The physician will benefit from the availability of such pharmacogenetic assays in order to adjust appropriately the drug dosage and to choose the best drug in treating individual patients. For example, if the patient requires a fl-adrenergic blocking agent and is determined to have the debrisoquine PM phenotype, the physician might prefer to prescribe a fl-adrenergic blocking agent that is known not to be among the growing list of drugs that are members of the CYP2D panel (Table 3). Finally, current popular trends in our society include increased efforts to maintain and improve one's health (e.g. participating in fitness centers, aerobic dancing, jogging, and eating nutritious foods) and a desire by the individual to learn more about his/her body and the latest advances in medical research. This greater awareness of the healthy body is reflected in increased sales of health food and exercise magazines, as well as science magazines for the layman. This new knowledge will allow the individual to effect changes in his/her life style to compensate for any genetically predisposed strengths or weaknesses he/she might discover. For example, upon learn-
ing of his/her high CYPIA1 inducibility phenotype, a person might decide to stop smoking cigarettes or to discontinue a job in which there is a high daily exposure to petroleum-based combustion products.
Acknowledgment The secretarial assistance of Phuong Lam is greatly appreciated.
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