Alcohol,Vol. 14, No. 6, pp. 569-573,i997 Copyright© 1997ElsevierScienceInc. Printed in the USA.All rightsreserved 0"741-8329/97$17.00+ .00 ELSEVIER
Prenatal Exposure to Ethanol in Rats: Effects on Liver Energy Level and Antioxidant Status in Mothers, Fetuses, and Newborns GIOVANNI ADDOLORATO,* ANTONIO GASBARRINI,~" STEFANIA MARCOCCIA,* MARA SIMONCINI,§ PAOLA BACCARINI,~ GIOVANNI VAGNI,* ANTONIO GRIECO,* A L E S S A N D R O S B R I C C O L I , $ A L B E R T O G R A N A T O , : ~ G I U S E P P E F. S T E F A N I N I § AND GIOVANNI GASBARRINI*
Institutes of*Internal Medicine, t Medical Pathology, and S Anatomy, Catholic University, Rome, Italy Institutes of § Medical Pathology and ~1Pathological Anatomy, University of Bologna, Italy R e c e i v e d 18 J u n e 1996; A c c e p t e d 17 F e b r u a r y 1997 ADDOLORATO, G., A. GASBARRINI, S. MARCOCCIA, M. SIMONCINI, P. BACCARINI, G. VAGNI, A. GRIECO, A. SBRICCOLI, A. GRANATO, G. F. STEFANIN1 AND G. GASBARRINI. Prenatal exposure to ethanol in rats: Effects on liver energy level and antioxidant status in mothers, fetuses, and newborns. ALCOHOL 14(6) 569-573, 1997.--The fetal alcohol syndrome is a clinical condition that affects newborns from alcoholic mothers. It is not clear, however, whether ethanol consumption during gestation can affect liver functions of fetuses and newborns. In this study, we aimed to assess the effects of ethanol administration on body weight, liver energy level, and antioxidant status of mothers, fetuses, and newborns. Pregnant rats were exposed to ethanol during the third week of gestation. Body weight, survival, and liver concentration of gluthatione (GSH) and adenosintriphosphate (ATP) were measured. No differences were observed in body weight or in liver ATP and GSH between mothers exposed to ethanol and control animals. Conversely, fetuses from rats exposed to ethanol showed a marked decrease in GSH, ATP, and body weight when compared to those from control rats. Newborns exposed prenatally to ethanol were no different from those born to control mothers. This study suggests that an amount of ethanol that is not sufficient to determine a significant effect on mothers can, nevertheless, cause a marked decrease in growth and in liver antioxidant and energy status in fetuses. These parameters, however, return to control value one week after ethanol discontinuation. © 1997 Elsevier Science Inc. Ethanol
Fetal alcohol syndrome
F E T A L alcohol syndrome (FAS) consists of a distinctive pattern of visceral abnormalities, growth retardation, immune derangements, and brain lesions or dysfunctions that affect newborns issued from chronic alcoholic mothers (3,5,6,20,29). Although several experimental models of chronic alcoholism have been developed in pregnant animals to study intrauterine growth and the occurrence of FAS in the progeny (1,2,10,17,18,26,27,32), few studies have investigated the relationship between chronic alcohol intake during pregnancy or the quality of pre- and postnatal growth, either in fully clinically expressed or in incomplete forms of FAS (1,9,22,23,28). In particular, it is still not entirely clear whether ethanol consumption during gestation may alter pre- and postnatal maturation and metabolism of gastrointestinal organs.
Our group recently showed that newborns from rats exposed to an ethanol containing diet during the last week of pregnancy, that did not significantly affect animal survival, developed permanent changes in the thalamo-cortical circuits (16,24). The aim of this study was to assess whether the administration of a diet containing the same amount of ethanol as our previous study (16,24) was capable of affecting body weight and liver morphology, energy level, and antioxidant status of fetuses and newborns. METHOD Twenty-eight pregnant nulligravid Wistar rats were utilized for the study. Animals were mated at night and exam-
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TABLE 1 LIVER WEIGHT OF FETUSES, OFFSPRING, AND MOTHERS EXPOSED TO ETHANOL (E) OR SUCROSE (S) DURING THE THIRD WEEK OF PREGNANCY Fetuses (rag)
GD20 PN4 PN10
49.0 _+ 5.4 ---
52.3 _+ 4.8
-452 +_ 16 866 ÷ 86
-466 +_ 25 884 _+ 62
14.6 _+ 2.8 13.9 _+ 2.7 14.1 +_ 3.l
13.7 + 3.4 13.2 -+ 2.9 14.0 _ 3.8
Liver weight was evaluated at the discontinuation of the ethanol diet (GD20) and 4 (PN4) and 10 days (PNI0) after delivery. No significant differences were observed between the two groups at any of the time points studied, Values are expressed as mean +_ SEM.
ined the next day for a vaginal plug. T h e presence of a plug indicated conception, and that day was considered as gestional day 1 (16). Twelve pregnant rats received boluses of a 20% w/v aqueous solution of ethanol from gestational day 14 to 19; the total daily a m o u n t of ethanol (2.4 g/kg/day) was subdivided into four doses (0800 and 1100 h, 1400 and 1700 h) via acute gastric intubation (24). Each time, a curved metal needle (1.8 m m external diameter) with r o u n d e d nontraumatic tip was inserted into the m o u t h and lowered to the stomach; the ethanol solution was delivered by pressure injection (16). A control group of 12 pregnant rats received an equal v o l u m e of isocaloric sucrose solution, during the same gestational period and with the same m o d e of administration. These animals served as a nutritional control and as a control for stress associated with the intubation procedure. A n additional control group of four unhandled, nongavaged pregnant rats was utilized to assess the contribution of the stress of r e p e a t e d handling and gastric gavage. Rats exposed to ethanol were fed with a diet of rat chow and water ad lib. To avoid a possible change in the feeding pattern of the rats caused by consumption of the ethanol-containing diet, control rats were fed the average v o l u m e of water and weight of chow consumed by ethanol-fed rats of corresponding gestational age. B o d y weight and survival was monitored throughout the experimental period, F o u r mothers from each group were sacrificed on gestational day 20 (GD20) and 4 (PN4) and 10 (PN10) days after delivery. F o u r fetuses per litter from each group were sacrificed at GD20. F o u r offspring per litter from each group were sacrificed at PN4 and PN10. All animals were sacrificed at the same time of day. A t each time point studied, animals were exposed to median laparotomy and livers were isolated and weighed. Hepatic tissue was excised immediately, rinsed with saline, and fixed in 10% formaline, sectioned, and stained with hematoxylin and eosin, or stored in liquid nitrogen until used. In the frozen tissue, reduced gluthatione ( G S H ) was measured by bioluminescent methods and expressed as nmol/mg of protein; adenosintriphosphate ( A T P ) was assayed spectrophotometrically using an A T P kit obtained from Sigma (Sigma Co, St. Louis, M O ) , and expressed as pmol/mg of protein. The liver pathologic specimens were observed by light microscopy for the detection of steatosis, necrosis, inflammation and fibrosis. Steatosis (defined as the percent of liver ceils containing fat) was scored as follows: < 25% = 1+, 25-50% = 2 + , 50-75% = 3 + , > 75% = 4+. All values r e p o r t e d as m e a n + SEM, Statistically significant differences between groups were determined using either
Student's t-test or A N O V A when appropriated, A value of p < 0.05 was considered to be significant. RESULTS Mortality was not observed in any of the groups studied. Liver weight of mothers, fetuses, and offspring in the ethanol group did not differ compared to the sucrose controls (Table 1). No statistical differences were observed in antioxidant and energy status of livers obtained from mothers exposed to ethanol or sucrose at any of the time points studied (Table 2). The stress of repeated handling and gastric gavage did not affect antioxidant and energy concentrations in the liver. In the additional control group of unhandled and nongavaged pregnant rats, in fact, liver concentrations of G S H and A T P measured at G D 2 0 were no different to those observed in the sucrose group (GSH: 1.15 _+ 0.21 vs. 1.i _-_ 0.12 nmol/mg of protein and A T P : 1.03 -+ 0.13 vs. 0.99 _+ 0.10 pmol/mg of protein, respectively). Conversely, at G D 2 0 fetuses from rats exposed to ethanol showed a m a r k e d decrease in liver G S H and A T P and in body weight when c o m p a r e d to fetuses from rats exposed to sucrose ( - 3 5 % , p < 0.05; - 1 5 % , NS; - 2 2 % , p < 0.05, respectively) (Figs. 1 and 2). A f t e r delivery, however, these differences disappeared. Offspring from mothers exposed to ethanol, in fact, did not present statistically significant differences when c o m p a r e d to those from mothers exposed to sucrose either 4 or 10 days after delivery (Figs. 1 and 2).
TABLE 2 GSH AND ATP CONCENTRATION OF LIVERS OF MOTHERS EXPOSED TO ETHANOL OR SUCROSE DURING THE THIRD WEEK OF PREGNANCY GSH
GD20 PN4 PN10
0.99 _+ 0.11 1.30 _+ 0.10 1.20 +_ 0.04
1.10 + 0.12 1.20 +_ 0.11 1.14 -+ 0.09
0.95 -+ 0.09 0.85 -+ 0.04 0.90 +_ 0.11
0.99 + 0.10 0.91 :± 0.03 1.01 + 0.07
GSH and ATP were expressed as nmol/mg of protein and pmol/mg of protein, respectively. GSH and ATP were evaluated at the discontinuation of the ethanol diet (GD20) and 4 (PN4) and 10 days (PN10) after delivery. No significant differences were observed between the two groups at any of the time points studied. Values are expressed as mean +_ SEM.
P R E N A T A L A L C O H O L E X P O S U R E IN RATS
O L_ Q. O
1.0 0.9 =m
O E n
0.6 o. I--
TIME FIG. 2. Body weight of fetuses and newborns obtained from mothers exposed to ethanol (dark column) or sucrose (dashed column) during the third week of pregnancy. Body weight was evaluated on discontinuation of ethanol (GD20) and 4 (PN4) and 10 days (PNI0) after delivery. Values were expressed as mean + SEM. When compared to the sucrose control group, a significant reduction in body weight (p < 0.05) was observed in fetuses from ethanoltreated mothers (GD20). No statistically significant differences were observed in the two groups after delivery.
1.5 1.3 ~)
Time FIG. 1. GSH and ATP concentration in livers of fetuses and newborns obtained from mothers exposed to ethanol (white dots) or sucrose (black dots) during the third week of pregnancy. GSH and ATP were expressed as nmol/mg of protein and pmol/rng of protein, respectively. GSH and ATP were evaluated at the discontinuation of the ethanol diet (GD20) and 4 (PN4) and 10 days (PN10) after delivery. Values were expressed as mean + SEM. When compared to the sucrose control group, a significant reduction in GSH concentration (p < 0.05) and lower ATP levels (NS) were observed in fetuses from ethanol-treated mothers. No statistically significant differences were observed in the two groups after delivery.
Regarding liver ultrastructure, the only relevant finding observed was a mild to moderate (score 1 to 2) intracellular steatosis in fetuses from ethanol-treated mothers, which was significantly greater than that observed in fetuses obtained from mother exposed to sucrose (30 -~ 15% vs. 5 _+ 3% of cells containing fat, respectively; p < 0.01). Intracellular steatosis completely disappeared after delivery. No liver specimens obtained from control fetuses, ethanol and control offspring, or ethanol and control mothers showed steatosis. The incidence of fibrosis, necrosis, and inflammation was not significant in any of the liver specimens studied.
In a recent study, Buts et al. showed that newborns issued from ethanol-treated mothers presented a transient decrease in body weight and intestinal DNA concentration in the first hours after partum; these parameters, however, returned to control levels in 10-15 days (4). Our group has also recently demonstrated that prenatal exposure to ethanol at doses that do not reduce the animal's survival causes permanent changes in the thalamo-cortical circuits (16,24). Utilising the same experimental model, we evaluated the effects on the liver of mother, fetuses, and offspring of an amount of ethanol able to cause permanent injury to the central nervous system (CNS). The results obtained show that the dose of ethanol utilized is probably insufficient or administered too briefly to cause significant damage to the mothers. However, this amount of ethanol is sufficient to cause a significant decrease in body weight and liver antioxidant status in the fetuses; energy status was decreased too, but the reduction was not significant, probably because of the limited number of animals studied. Antioxidant defense systems are specific defense mechanisms able to neutralize the oxidative effects of oxygen and its reactive metabolites. Antioxidant mechanisms include enzymes and the safe sequestration of transition metal ions. In addition, many actual and putative physiological antioxidants act by scavenging radicals. Glutathione, in particular, is important as substrate for glutathione peroxidases, transferases, and several other enzymes and as a general "radical quencer" in cells. An ethanol-mediated reduction in glutathione levels (8) and in liver energy status has already been shown in other experimental settings, such as during acute, short-term, liver alcohol exposure (15,19). In particular, our group and others showed that cell damage increased progressively both in hepatocytes and in Sertoli cells with increasing ethanol doses; the reduction in ATP levels observed was both time and ethanol concentration dependent and paralleled the occurrence of
cell damage and the unbalance of intracellular ion homeostasis (11-14). The decrease in reduced glutathione, a potent free radical scavenger that plays a pivotal role in cell protection against electrophilic drug injury and free radical attack, may be determined by the binding of acetaldehyde to glutathione or its precursor cysteine (30), by the decrease in G S H transferase activity, or by the inhibition of G S H synthesis and production by the liver (21,31). Moreover, it should be kept in mind that the antioxidant status of the cells is strictly dependent on energy status, and the detected reduction in A T P levels could therefore be a primary determinant of the G S H depletion observed. Finally, it is interesting to observe how body weight and liver antioxidant and energy status had already fully returned to control values 1 week after ethanol discontinuation; the newborns from ethanol-treated mothers, in fact, did not present any significant differences in the parameters measured when compared to the control group. This p h e n o m e -
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non, probably determined by the relatively low dose or the short period of prenatal exposure to ethanol by the mothers, shows how a dose of the toxin capable of causing p e r m a n e n t postnatal damages to the CNS causes only a transient alteration in the energy and antioxidant status of a less sensitive organ like the liver. In conclusion, our data show that ethanol administration during pregnancy determines a marked sufferance in the liver of fetuses that is transient and completely reversible after delivery. In future studies, however, it will be important to assess the effects on the liver of the same a m o u n t of ethanol also in malnourished mothers. Previous data of our and other groups, in fact, showed how ethanol-related liver injury was significantly greater in fasted animals (7,13). ACKNOWLEDGEMENTS Supported by a grant from Ricerca in Medicina, Bologna, Italy.
1. Abel, E. L.: Consumption of alcohol during pregnancy: A review of effects on growth and development of offspring. Hum. Biol. 54:421-453; 1982. 2. Baskin, G.; Henderson, G. I.; Schenker, S.: Ethanol and hepatic regeneration. Hepatology 8(2):408-411; 1988. 3. Berman, R. F.; Hannigan, J. H.; Sperry, M. A.; Zajac, C. S.: Prenatal alcohol exposure and the effects of envormental enrichment on hippocampal dendritic spine density. Alcohol 2:209-216; 1996. 4. Buts, J. P.; Sokal, E. M.; Van Hoff, F.: Prenatal exposure to ethanol in rats: Effects on postnatal maturation of the small intestine and liver. Pediatr. Res. 32:574-579; 1992. 5. Chiappelli, F.; Wong, C. M. K.; Yrmiya, R.; Norman, D. C.; Chang, M. P.; Taylor, A. N.: Fetal alcohol exposure (FAE) and neuroimmune surveillance. In: Yrmiya, R.; Taylor, A. N., eds. Alcohol, immunity and cancer. Boca Raton, FL: CRC Press; 1992:143-155. 6. Clarren, S. K.; Smith, D. W.: The fetal alcohol syndrome. N. Engl. J. Med. 298:1063-1067; 1978. 7. Cunnigham, C. C.; Malloy, C. R.; Radda, G. K.: Effect of fasting and acute ethanol administration on the energy states of in vivo liver as measured by 21P-NMR spectroscopy. Biochem. Biophys. Acta 885:12-22; 1986. 8. Devi, B. G.; Henderson, G. I.; Frosto, T. A.; Schenker, S.: Effect of ethanol on rat fetal hepatocytes: Studies on cell replication, lipid peroxidation and glutathione. Hepatology 18(3):648--659;1993. 9. Devi, B. G.; Henderson, G. 14 Frosto, T. A.; Schenker, S.: Effect of acute ethanol exposure on cultured fetal rat hepatocytes: relation to mitochondrial function. Alcohol. Clin. Exp. Res. 18(6): 1436-1442; 1994. 10. Devi, B. G.; Perez, A.; Schenker, S.: In utero ethanol exposure elicitis oxidative stress in the rat fetus. Alcohol. Clin. Exp. Res. 19(3):714-720; 1995. 11. Farghali, H.; Caraceni, P.; Rilo, H.; Borle, A. B.; Gasbarrini, A.; Donald, W. S.; Ho, C.; Van Thiel, D. H.: Effect of ethanol on energy status and intracellular calcium of Sertoli cells: A study using immobilzed perfused cells. Endocrinology 133(6):27492755; 1993. 12. Gasbarrini, A.; Borle, A. B.; Farghali, H.; Caraceni, P.; Van Thiel D. H.: Fasting enhances the effects of anoxia on ATP, Cai2+ and cell injury in isolated rat hepatocytes. Biochem. Biophys. Acta 1178:9-19; 1993. 13. Gasbarrini, A.; Borle, A. B.; Farghali, H.; Bender, C.; Francavilla, A.; Van Thiel, D. H.: Effect of anoxia on intracellular ATP, Nai+, Cai2+, Mgi2+ and eitotoxicity in rat hepatocytes. J. Biol. Chem. 267:6654-6663; 1992 14. Gasbarrini, A.; Borle, A. B.; Caraceni, P.; Colantoni, A.; Farghali, H.; Bernardi, M.; Van Thiel, D. H.: Effect of ethanol on
15. 16. 17. 18.
19. 20. 21.
22. 23. 24. 25. 26. 27. 28.
adenosine triphosphate, cytosolic free calcium and cell injury in rat hepatocytes: time course and effect of nutritional status. Dig. Dis. Sci. 41:2204-2212; 1996. Gillam, E.; Ward, L. C.: Cellular energy charges in the hearts and liver of the rats. The effects of ethanol and acetaldehyde. Int. J. Biochem. 18:1031-1038; 1986. Granato, A.; SantareUi, M.; Sbriccoli, A.; Minciacchi, D.: Multifaceted alteration of the thalamo-cortico-thalamic loop in adult rats prenatally exposed to ethanol. Anat. Embryol. 191:11-23; 1995. Hanson, J. W.; Streissguth, A. P.; Smith, D. W.: The effects of moderate alcohol consumption during pregnancy on fetal growth and morphogenesis. J. Pediatr. 92:457-460; 1978. Henderson, G. I.; Baskin, G. S.; Horbach, J.; Porter, P.; Schenker, S.: Arrest of epidermal growth factor-dependent growth in fetal hepatocytes after ethanol exposure. J. Clin. Invest. 84(4):12871294; 1989. Henderson, G. I.; Devi, B. G.; Perez, A.; Schenker, S.: In utero ethanol exposure elicitis oxidative stress in the rat fetus. Alcohol. Clin. Exp. Res. 19(3):714-720; 1995. Jones, K. L.; Smith, D. W.: Patterns of malformation in offspring of chronic alcoholic mothers. Lancet 1:1268-1271; 1983. Kocak-Toker, N.; Uysal, M.; Aykac, G.; Sivas, A.; Yalein, S.; Oz, H.: Influence of acute ethanol administration on hepatic gluthatione peroxidase and gluthatione transferase activities in the rat. Pharmacol. Res. Commun. 17:233-239; 1985. Manzke, H.; Grosse, F. R.: Incomplete and complete alcohol syndrome. Three children of female drinker. Med. Welt. 26:709712; 1982. Merlin, L.: Early fetal growth and development in mice chronically exposed to ethanol during gestation. Growth 51:146-153; 1987. Minciacchi, D.; Granato, A.; Santarelli, M. L.; Sbriccoli, A.: Modifications of thalamo-cortical circuitry in rats prenatally exposed to ethanol. Neuroreport 4:415-418; 1993. Sanehis, R. M.; Sancho-Tello, M.; Guerri, C.: The effects of chronic alcohol consumption on pregnant rats and their offspring. Alcohol Alcohol. 21:295-305; 1986. Sanehis, R. M.; Sancho-Tello, M.; Chirivella, M.; Guerri, C.: The role of maternal alcohol damage on ethanol teratogenicity in the rat. Teratology 36:199-208; 1987. Schenker, S.; Beeker, H. C.; Randall, C. L.; Phillips, D. K.; Baskin, G. S.; Henderson, G. I.: Fetal alcohol syndrome: Current status of pathogenesis. Alcohol. Clin. Exp. Res. 14(5):635-647; 1990. Schenker, S.; Hu, Z. Q.; Johnson, R. F.; Yang, Y.; Frosto, T.; Elliot, B. D.; Henderson, G. I.; Mock, D. M.: Human placental biotin transport: Normal characteristics and effect of ethanol. Alcohol. Clin. Exp. Res. 17(3):566-575; 1993. Seri, S.; D'Alessandro, A.; Aquilio, E.; Corbacelli, A4 Seri, M.;
P R E N A T A L A L C O H O L E X P O S U R E IN R A T S Goracci, G., Facial growth deformities and gengival anomalies in the newborns of alcoholic rats. Alcologia 7:205-209; 1995. 30. Shaw, S.; Jayatilleke, E.; Lieber, C. S.: Hepatic lipid peroxidation: Potentiation by chronic alcohol feeding and attenuation by methionine. J. Lab. Clin. Med. 98:417-425; 1981. 31. Speisky, H.; MacDonald, A.; Giles, G.; Orrego, H.; Gunasekara,
573 A.; Israel, Y.: Increased loss and decreased synthesis of hepatic glutathione after acute ethanol administration. Biochem. J. 225:565572; 1985. 32. Streissguth, A. P.; Landesman-Dwyer, S.; Martin, J. C.; Smith, D. W.: Teratogenic effects of alcohol in humans and laboratory animals. Science 209:353-361; 1980.