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The effects of ethanol and gamma aminobutyric acid alone and in combination on hepatic regenerative activity in the rat
Journal of Hepatology 1998; 29: 638–641 Printed in Denmark ¡ All rights reserved Munksgaard ¡ Copenhagen
Copyright C European Association for the Study of the Liver 1998
Journal of Hepatology ISSN 0168-8278
The effects of ethanol and gamma aminobutyric acid alone and in combination on hepatic regenerative activity in the rat Manna Zhang, Yuewen W. Gong and Gerry Y. Minuk Liver Diseases Unit, Departments of Medicine and Pharmacology, University of Manitoba Winnipeg, Manitoba, Canada
Background/Aims: Both ethanol and gamma aminobutyric acid (GABA) have been reported to inhibit hepatic regenerative activity in the rat. Because alcoholic beverages contain appreciable amounts of GABA, we documented whether the inhibitory effects of alcohol on the liver are derived from ethanol alone or the combination of ethanol plus GABA. Methods: Adult male Sprague-Dawley rats (nΩ6/ group) were treated with either ethanol (3 g/kg), GABA (500 mg/kg) or ethanol plus GABA (3 kg and 500 mg/kg, respectively), beginning 1 h prior to a 70% partial hepatectomy and continued every 4 h thereafter for a total of 24 h. Rats were then sacrificed and hepatic regenerative activity was documented by 3Hthymidine incorporation into hepatic DNA. Results: DNA synthesis was significantly inhibited by ethanol (ª37%, p∞0.005) and GABA (ª19%, p∞0.05). Maximum inhibition was achieved with the combination of ethanol plus GABA (ª52%, p∞0.001). To determine whether the additive effects
of ethanol plus GABA were mediated by ethanol-induced enhancement of hepatic GABAA receptor activity, additional rats (nΩ6/group) receiving the combination of ethanol plus GABA were pre-treated with a single injection of either ciprofloxacin (50 mg/kg), a GABAA receptor antagonist, or an equal volume of saline. In these experiments, ciprofloxacin pre-treatment prevented the inhibitory effects of the ethanol plus GABA combination. Conclusions: The results of this study indicate that the combination of ethanol plus GABA has a greater inhibitory effect on hepatic DNA synthesis following partial hepatectomy than ethanol alone. The clinical implication of this finding is that, when standardized for ethanol content, not all alcoholic beverages would be expected to have the same inhibitory effect on hepatic regeneration.
consumption inhibits hepatic regenerative activity (1). Whether this inhibitory effect is mediated exclusively by ethanol or contributed to by congeners present within alcoholic beverages remains to be determined. Gamma aminobutyric acid (GABA) is an inhibitory amino acid with growth regulatory properties. GABA is present in the grains and yeasts employed in the manufacture of alcoholic beverages (2,3). GABAA receptors have been identified in isolated rat hepatocytes by both standard ligand/receptor binding assays and in situ immunohistochemistry (4,5).
We have previously reported that enhanced GABAergic activity inhibits hepatic regeneration, and GABAA receptor antagonists reverse the inhibitory effects of ethanol on hepatic DNA synthesis (6,7). As a result of these findings, as well as studies in the brain documenting that ethanol potentiates GABAA receptor activity (8), we tested the hypothesis that a combination of ethanol plus GABA has a greater inhibitory effect on hepatic regeneration than either agent alone.
E
Received 11 February; revised 24 April; accepted 12 May 1998
Correspondence: Gerry Y. Minuk, Liver Diseases Unit, GF407, Health Sciences Centre, 820 Sherbrook Street, Winnipeg, Manitoba, R3A 1R9 Canada. Tel: (204) 787-4662. Fax: (204) 775-4255.
638
Key words: Alcohol; Ethanol; GABA; Hepatic regeneration; Hepatitis; Liver.
Materials and Methods GABA analyses in alcoholic beverages (commercially available wines, spirits and beer) were performed in triplicate by ion exchange chromatography as previously described (9). Adult, male Sprague-Dawley rats (200–250 g) were housed in an animal-holding area and allowed free access to food and water, until the day prior to surgery
Ethanol, GABA and hepatic regeneration
when food but not water was withdrawn. On the day of surgery, the rats were randomized into four groups (nΩ6/group) and each rat underwent a 70% partial hepatectomy while under ether anaesthesia, as described by Higgins & Anderson (10). Group 1 received 1 ml of sterile saline by intraperitoneal (i.p.) injection, beginning 1 h prior to surgery and repeated every 4 h thereafter for a total of 24 h. Group 2 received 1 ml of ethanol (3 g/kg) in sterile saline by the same route and schedule as outlined for group 1 rats. Group 3 received 1 ml of GABA (500 mg/kg) dissolved in sterile saline, as outlined for groups 1 and 2. Group 4 received a mixture of ethanol plus GABA as outlined above. In an additional series of experiments, two groups of rats (nΩ6/group) received the combination of ethanol plus GABA with ciprofloxacin pre-treatment (50 mg/kg, i.p., 1 h prior to the first ethanol plus GABA injection) or an equal volume of saline on one occasion only. The dosage of ethanol employed in these experiments was based on the results of previously published studies documenting the effects of ethanol on hepatic regenerative activity (11). The dosage of GABA was also based on previous data documenting the effects of GABA on hepatic regenerative activity, the results of GABA determinations in alcoholic beverages and the equivalent amount of GABA required by rats to approximate that consumed by humans drinking alcohol to excess (2,12,13). Peak hepatic regenerative activity was documented by determining [3H]-thymidine incorporation into hepatic DNA, as described by Luk (14). Briefly, 1 h prior to death, animals received 10 mCi [3H]-thymidine/200 g body weight (specific activity, 42 Ci/mmol [1 CiΩ37 GBq]; Amersham, Oakville, Ontario, Canada) by intraperitoneal injection. At the time of death by exsanguination, the livers were excised and homogenized in 2.5 mmol/l ethylenediaminetetraacetic acid, pH 7.4 buffer. Aliquots of liver homogenate were precipitated with trichloroacetic acid (final concentration 5%) and centrifuged (800 g for 15 min) to pellets. The precipitates were washed, recentrifuged and the radioactivity in the solubilized pellet was determined in an LKB 1219 liquid scintillation counter (Wallac, Turku, Finland). DNA content in homogenates was measured after reaction with 3,5-diaminobenzoic acid at 37æC. [3H]-thymidine incorporation was expressed as disintegrations per minute incorporated per milligram hepatic DNA. Statistical analyses were performed using one-way ANOVA and the Student t-tests or Wilcoxon rank sum tests for parametric and nonparametric data, respectively, where appropriate. The results reported represent means∫SD. p-values less than 0.05 were con-
Fig. 1. The effects of saline, GABA (500 mg/kg), ethanol (3 g/kg) and GABA plus ethanol on hepatic regeneration 24 h post partial hepatectomy in rats (nΩ6/group).
Fig. 2. The effect of pretreatment with ciprofloxacin (50 mg/kg) on GABA (500 mg/kg) plus ethanol (3 g/kg) induced inhibition of hepatic regeneration (nΩ6/group).
sidered significant. This study was approved by the University of Manitoba Animal Ethics Review Committee.
Results The mean GABA concentration in commercially available alcoholic beverages was 0.38∫0.15 pmol/ml (range: 0.28 [wine]–0.55 [beer]). The results of [3H]-thymidine incorporation into hepatic DNA in the initial four study groups are given in Fig. 1. When compared to saline-treated controls, both ethanol and GABA individually inhibited hepatic DNA synthesis rates (ª37% and ª19%, p∞0.005 and 0.05, respectively). However, the most striking inhibitory effect was observed in rats treated with the combi-
639
M. Zhang et al.
nation of ethanol plus GABA, where a 52% inhibition in DNA synthesis was observed (p∞0.001). The differences between GABA alone and ethanol alone were also significant when compared to the combination of ethanol plus GABA (p∞0.05, respectively). As shown in Fig. 2, pre-treatment with ciprofloxacin was effective in preventing the inhibitory effects of the ethanol plus GABA combination. Indeed, DNA synthesis rates were not statistically significantly different from those obtained in saline-treated controls.
Discussion The results of this study support previously published reports documenting that individually, both ethanol and GABA have inhibitory effects on hepatic regenerative activity in the rat (1,6,12). The results also indicate that these inhibitory effects are additive, in that ethanol and GABA alone resulted in 37% and 19% inhibition of DNA synthesis rates, respectively, whereas a combination of ethanol plus GABA inhibited DNA synthesis rates by 52%. Whether the inhibitory effects observed with the ethanol plus GABA combination represent enhancement of GABAA receptor activity in the liver, as has been described in the brain (15), or whether the two agents act independently via separate pathways, remains to be determined. That pretreatment with ciprofloxacin, a potent GABAA receptor antagonist (16), prevented the inhibitory effect of ethanol plus GABA on hepatic regeneration suggests that a common pathway is more likely. However, prevention was not complete and the use of additional GABAA receptor antagonists and dose-response studies are required to support this interpretation. If confirmed in humans, the results of this study could have important clinical implications for the management of patients with alcohol-induced liver disease. Specifically, recalcitrant alcoholics or patients with non-alcohol-related forms of liver disease might be counselled to avoid those alcoholic beverages with the highest GABA contents per serving (beer±spirits± wine). Unfortunately, the fact that GABA is a ubiquitous component of the grains utilized in the manufacture of alcohol renders complete elimination of GABA from these beverages either unfeasible or a costly proposition (S. Frederickson, personal communication). Ideally, rat exposure to ethanol in this study would have occurred by voluntary oral consumption rather than intraperitoneal injections. However, voluntary consumption of ethanol by rats is limited, variable and requires a prolonged adaptation phase. The latter is of particular concern because prolonged ethanol exposure does not appear to have the same inhibitory
640
effects on hepatic regeneration as does acute ethanol exposure (17). To avoid these confounding variables, alternative routes of ethanol exposure were considered. The options were limited to repeated gastric gavages or instillations of ethanol and/or GABA by the intraperitoneal route. While gastric gavages may more closely mimick oral ethanol consumption, regurgitation of gastric contents following gavage and the stress associated with repeated gavages over a 24-h period were of sufficient concern to preclude that route (18). Moreover, because GABA is rapidly cleared from the circulation (19), the slower delivery associated with intraperitoneal instillations was felt to approximate more closely the pattern of hepatic exposure to GABA that would be predicted in alcoholics drinking to excess. In conclusion, the results of this study indicate that the inhibitory effect of ethanol on hepatic DNA synthesis is enhanced in the presence of GABA, a congener present in alcoholic beverages. Additional studies are warranted to determine whether these findings are applicable to humans and, if so, whether alterations in GABA content and/or the use of GABA receptor antagonists are of therapeutic benefit in the treatment of patients with alcohol-induced liver disease.
Acknowledgements The authors wish to thank Ms. D. Byron for her prompt and accurate typing of the manuscript. This study was supported by a grant from the Medical Research Council of Canada. Dr. Gong is the recipient of a Canadian Liver Foundation Fellowship Award.
References 1. Wands JR, Carter EA, Bucher NL, Isselbacher KJ. Inhibition of hepatic regeneration in rats by acute and chronic ethanol intoxication. Gastroenterology 1979; 77: 528–31. 2. Andrews RP, Condon GD. The LKB 4151 alpha plus amino acid analyser applications using a five-buffer lithium citrate system. Protein Chemistry Notes PCN31 LKB Biochrom (unpublished data). 3. Holdsworth ES, Neville E. Extracts of Brewer’s yeast contain GABA which enhances activation of glycogen synthetase by insulin in isolated rat hepatocytes. Biochem Int 1988; 17: 1107–16. 4. Minuk GY, Bear CE, Sarjeant EJ. Sodium-independent bicuculline-sensitive [3H]GABA binding to isolated rat hepatocytes. Am J Physiol 1987; 252: 642–7. 5. Hassard PV, Krantis A, Staines W. Visualization of GABAA receptors and GABA transaminase (GABA-T) in the rat liver. Can J Gastroenterol 1998; 12: 24A. 6. Minuk GY, Gauthier T. The effect of gamma aminobutyric acid (GABA) on hepatic regenerative activity following partial hepatectomy in rats. Gastroenterology 1993; 104: 217–21. 7. Minuk GY, Gauthier T, Zhang XK, Wang GQ, Pettigrew NM, Burczynski FJ. Ciprofloxacin prevents the inhibitory effects of acute ethanol exposure on hepatic regeneration in the rat. Hepatology 1995; 22: 1797–800.
Ethanol, GABA and hepatic regeneration 8. Allan AM, Harris RA. Gamma-aminobutyric acid and alcohol actions: neurochemical studies. Life Sci 1986; 39: 2005– 15. 9. Minuk GY, Sarjeant EJ. The effect of (a) neomycin and lactulose treatment on systemic and portal serum GABA levels in rats and (b) pH changes on [3H] GABA binding to isolated rat hepatocytes. Clin Invest Med 1988; 11: 373–6. 10. Higgins GM, Anderson RM. Experimental pathology of liver: restoration of liver of the white rat following partial surgical removal. Arch Pathol 1931; 12: 186–202. 11. Diehl AM, Wells M, Brown ND, Thorgeirsson SS, Steer CJ. The effect of ethanol on polyamine synthesis during liver regeneration in rats. J Clin Invest 1990; 85: 385–90. 12. Minuk GY, Gauthier T, Gahary A, Murphy L. The effect of gamma-aminobutyric acid (GABA) on serum and hepatic polyamine concentrations following partial hepatectomy in rats. Hepatology 1991; 14: 685–9. 13. Vincent T, Devita JK. In: Vincent T, Devita JK, editors. Cancer: Principles and Practice of Oncology, 3rd ed. Philadelphia, PA: Lippincott; 1987. p. 292–3. 14. Luk GD. Essential role of polyamine metabolism in hepatic regeneration: inhibition of deoxyribonucleic acid and protein
15.
16.
17.
18.
19.
synthesis and tissue regeneration by difluoromethylornithine in the rat. Gastroenterology 1986; 90: 1261–7. Suzdak PD, Schwartz RD, Paul SM. Ethanol stimulates gamma-aminobutyric acid receptor-mediated chloride transport in rat brain synaptoneurosomes. Proc Natl Acad Sci U S A 1986; 83: 4071–5. Segev S, Rehavi M, Rubinstein E. Quinolones, theophylline, and diclofenac interactions with the g-aminobutyric acid receptor. Antimicrob Agents Chemother 1988; 1624–6. Minuk GY, Rockman GE, Gauthier T, Markert L, Gibson J, Benarroch. The effect of long-term, voluntary ethanol consumption on hepatic regeneration in rats. Can J Physiol Pharmacol 1991; 69: 341–5. Rockman GE, Hall AM, Markert L, Glavin GB. Ethanolstress interaction: immediate versus delayed effects of ethanol and handling on stress responses of ethanol-consuming rats. Alcohol 1987; 4: 391–4. Ferenci P, Covell D, Schafer DF, Waggoner JG, Shrager R, Jones EA. Metabolism of the inhibitory neurotransmitter gaminobutyric acid in a rabbit model of fulminant hepatic failure. Hepatology 1983; 3: 507–12.
641
Copyright C European Association for the Study of the Liver 1998
Journal of Hepatology ISSN 0168-8278
The effects of ethanol and gamma aminobutyric acid alone and in combination on hepatic regenerative activity in the rat Manna Zhang, Yuewen W. Gong and Gerry Y. Minuk Liver Diseases Unit, Departments of Medicine and Pharmacology, University of Manitoba Winnipeg, Manitoba, Canada
Background/Aims: Both ethanol and gamma aminobutyric acid (GABA) have been reported to inhibit hepatic regenerative activity in the rat. Because alcoholic beverages contain appreciable amounts of GABA, we documented whether the inhibitory effects of alcohol on the liver are derived from ethanol alone or the combination of ethanol plus GABA. Methods: Adult male Sprague-Dawley rats (nΩ6/ group) were treated with either ethanol (3 g/kg), GABA (500 mg/kg) or ethanol plus GABA (3 kg and 500 mg/kg, respectively), beginning 1 h prior to a 70% partial hepatectomy and continued every 4 h thereafter for a total of 24 h. Rats were then sacrificed and hepatic regenerative activity was documented by 3Hthymidine incorporation into hepatic DNA. Results: DNA synthesis was significantly inhibited by ethanol (ª37%, p∞0.005) and GABA (ª19%, p∞0.05). Maximum inhibition was achieved with the combination of ethanol plus GABA (ª52%, p∞0.001). To determine whether the additive effects
of ethanol plus GABA were mediated by ethanol-induced enhancement of hepatic GABAA receptor activity, additional rats (nΩ6/group) receiving the combination of ethanol plus GABA were pre-treated with a single injection of either ciprofloxacin (50 mg/kg), a GABAA receptor antagonist, or an equal volume of saline. In these experiments, ciprofloxacin pre-treatment prevented the inhibitory effects of the ethanol plus GABA combination. Conclusions: The results of this study indicate that the combination of ethanol plus GABA has a greater inhibitory effect on hepatic DNA synthesis following partial hepatectomy than ethanol alone. The clinical implication of this finding is that, when standardized for ethanol content, not all alcoholic beverages would be expected to have the same inhibitory effect on hepatic regeneration.
consumption inhibits hepatic regenerative activity (1). Whether this inhibitory effect is mediated exclusively by ethanol or contributed to by congeners present within alcoholic beverages remains to be determined. Gamma aminobutyric acid (GABA) is an inhibitory amino acid with growth regulatory properties. GABA is present in the grains and yeasts employed in the manufacture of alcoholic beverages (2,3). GABAA receptors have been identified in isolated rat hepatocytes by both standard ligand/receptor binding assays and in situ immunohistochemistry (4,5).
We have previously reported that enhanced GABAergic activity inhibits hepatic regeneration, and GABAA receptor antagonists reverse the inhibitory effects of ethanol on hepatic DNA synthesis (6,7). As a result of these findings, as well as studies in the brain documenting that ethanol potentiates GABAA receptor activity (8), we tested the hypothesis that a combination of ethanol plus GABA has a greater inhibitory effect on hepatic regeneration than either agent alone.
E
Received 11 February; revised 24 April; accepted 12 May 1998
Correspondence: Gerry Y. Minuk, Liver Diseases Unit, GF407, Health Sciences Centre, 820 Sherbrook Street, Winnipeg, Manitoba, R3A 1R9 Canada. Tel: (204) 787-4662. Fax: (204) 775-4255.
638
Key words: Alcohol; Ethanol; GABA; Hepatic regeneration; Hepatitis; Liver.
Materials and Methods GABA analyses in alcoholic beverages (commercially available wines, spirits and beer) were performed in triplicate by ion exchange chromatography as previously described (9). Adult, male Sprague-Dawley rats (200–250 g) were housed in an animal-holding area and allowed free access to food and water, until the day prior to surgery
Ethanol, GABA and hepatic regeneration
when food but not water was withdrawn. On the day of surgery, the rats were randomized into four groups (nΩ6/group) and each rat underwent a 70% partial hepatectomy while under ether anaesthesia, as described by Higgins & Anderson (10). Group 1 received 1 ml of sterile saline by intraperitoneal (i.p.) injection, beginning 1 h prior to surgery and repeated every 4 h thereafter for a total of 24 h. Group 2 received 1 ml of ethanol (3 g/kg) in sterile saline by the same route and schedule as outlined for group 1 rats. Group 3 received 1 ml of GABA (500 mg/kg) dissolved in sterile saline, as outlined for groups 1 and 2. Group 4 received a mixture of ethanol plus GABA as outlined above. In an additional series of experiments, two groups of rats (nΩ6/group) received the combination of ethanol plus GABA with ciprofloxacin pre-treatment (50 mg/kg, i.p., 1 h prior to the first ethanol plus GABA injection) or an equal volume of saline on one occasion only. The dosage of ethanol employed in these experiments was based on the results of previously published studies documenting the effects of ethanol on hepatic regenerative activity (11). The dosage of GABA was also based on previous data documenting the effects of GABA on hepatic regenerative activity, the results of GABA determinations in alcoholic beverages and the equivalent amount of GABA required by rats to approximate that consumed by humans drinking alcohol to excess (2,12,13). Peak hepatic regenerative activity was documented by determining [3H]-thymidine incorporation into hepatic DNA, as described by Luk (14). Briefly, 1 h prior to death, animals received 10 mCi [3H]-thymidine/200 g body weight (specific activity, 42 Ci/mmol [1 CiΩ37 GBq]; Amersham, Oakville, Ontario, Canada) by intraperitoneal injection. At the time of death by exsanguination, the livers were excised and homogenized in 2.5 mmol/l ethylenediaminetetraacetic acid, pH 7.4 buffer. Aliquots of liver homogenate were precipitated with trichloroacetic acid (final concentration 5%) and centrifuged (800 g for 15 min) to pellets. The precipitates were washed, recentrifuged and the radioactivity in the solubilized pellet was determined in an LKB 1219 liquid scintillation counter (Wallac, Turku, Finland). DNA content in homogenates was measured after reaction with 3,5-diaminobenzoic acid at 37æC. [3H]-thymidine incorporation was expressed as disintegrations per minute incorporated per milligram hepatic DNA. Statistical analyses were performed using one-way ANOVA and the Student t-tests or Wilcoxon rank sum tests for parametric and nonparametric data, respectively, where appropriate. The results reported represent means∫SD. p-values less than 0.05 were con-
Fig. 1. The effects of saline, GABA (500 mg/kg), ethanol (3 g/kg) and GABA plus ethanol on hepatic regeneration 24 h post partial hepatectomy in rats (nΩ6/group).
Fig. 2. The effect of pretreatment with ciprofloxacin (50 mg/kg) on GABA (500 mg/kg) plus ethanol (3 g/kg) induced inhibition of hepatic regeneration (nΩ6/group).
sidered significant. This study was approved by the University of Manitoba Animal Ethics Review Committee.
Results The mean GABA concentration in commercially available alcoholic beverages was 0.38∫0.15 pmol/ml (range: 0.28 [wine]–0.55 [beer]). The results of [3H]-thymidine incorporation into hepatic DNA in the initial four study groups are given in Fig. 1. When compared to saline-treated controls, both ethanol and GABA individually inhibited hepatic DNA synthesis rates (ª37% and ª19%, p∞0.005 and 0.05, respectively). However, the most striking inhibitory effect was observed in rats treated with the combi-
639
M. Zhang et al.
nation of ethanol plus GABA, where a 52% inhibition in DNA synthesis was observed (p∞0.001). The differences between GABA alone and ethanol alone were also significant when compared to the combination of ethanol plus GABA (p∞0.05, respectively). As shown in Fig. 2, pre-treatment with ciprofloxacin was effective in preventing the inhibitory effects of the ethanol plus GABA combination. Indeed, DNA synthesis rates were not statistically significantly different from those obtained in saline-treated controls.
Discussion The results of this study support previously published reports documenting that individually, both ethanol and GABA have inhibitory effects on hepatic regenerative activity in the rat (1,6,12). The results also indicate that these inhibitory effects are additive, in that ethanol and GABA alone resulted in 37% and 19% inhibition of DNA synthesis rates, respectively, whereas a combination of ethanol plus GABA inhibited DNA synthesis rates by 52%. Whether the inhibitory effects observed with the ethanol plus GABA combination represent enhancement of GABAA receptor activity in the liver, as has been described in the brain (15), or whether the two agents act independently via separate pathways, remains to be determined. That pretreatment with ciprofloxacin, a potent GABAA receptor antagonist (16), prevented the inhibitory effect of ethanol plus GABA on hepatic regeneration suggests that a common pathway is more likely. However, prevention was not complete and the use of additional GABAA receptor antagonists and dose-response studies are required to support this interpretation. If confirmed in humans, the results of this study could have important clinical implications for the management of patients with alcohol-induced liver disease. Specifically, recalcitrant alcoholics or patients with non-alcohol-related forms of liver disease might be counselled to avoid those alcoholic beverages with the highest GABA contents per serving (beer±spirits± wine). Unfortunately, the fact that GABA is a ubiquitous component of the grains utilized in the manufacture of alcohol renders complete elimination of GABA from these beverages either unfeasible or a costly proposition (S. Frederickson, personal communication). Ideally, rat exposure to ethanol in this study would have occurred by voluntary oral consumption rather than intraperitoneal injections. However, voluntary consumption of ethanol by rats is limited, variable and requires a prolonged adaptation phase. The latter is of particular concern because prolonged ethanol exposure does not appear to have the same inhibitory
640
effects on hepatic regeneration as does acute ethanol exposure (17). To avoid these confounding variables, alternative routes of ethanol exposure were considered. The options were limited to repeated gastric gavages or instillations of ethanol and/or GABA by the intraperitoneal route. While gastric gavages may more closely mimick oral ethanol consumption, regurgitation of gastric contents following gavage and the stress associated with repeated gavages over a 24-h period were of sufficient concern to preclude that route (18). Moreover, because GABA is rapidly cleared from the circulation (19), the slower delivery associated with intraperitoneal instillations was felt to approximate more closely the pattern of hepatic exposure to GABA that would be predicted in alcoholics drinking to excess. In conclusion, the results of this study indicate that the inhibitory effect of ethanol on hepatic DNA synthesis is enhanced in the presence of GABA, a congener present in alcoholic beverages. Additional studies are warranted to determine whether these findings are applicable to humans and, if so, whether alterations in GABA content and/or the use of GABA receptor antagonists are of therapeutic benefit in the treatment of patients with alcohol-induced liver disease.
Acknowledgements The authors wish to thank Ms. D. Byron for her prompt and accurate typing of the manuscript. This study was supported by a grant from the Medical Research Council of Canada. Dr. Gong is the recipient of a Canadian Liver Foundation Fellowship Award.
References 1. Wands JR, Carter EA, Bucher NL, Isselbacher KJ. Inhibition of hepatic regeneration in rats by acute and chronic ethanol intoxication. Gastroenterology 1979; 77: 528–31. 2. Andrews RP, Condon GD. The LKB 4151 alpha plus amino acid analyser applications using a five-buffer lithium citrate system. Protein Chemistry Notes PCN31 LKB Biochrom (unpublished data). 3. Holdsworth ES, Neville E. Extracts of Brewer’s yeast contain GABA which enhances activation of glycogen synthetase by insulin in isolated rat hepatocytes. Biochem Int 1988; 17: 1107–16. 4. Minuk GY, Bear CE, Sarjeant EJ. Sodium-independent bicuculline-sensitive [3H]GABA binding to isolated rat hepatocytes. Am J Physiol 1987; 252: 642–7. 5. Hassard PV, Krantis A, Staines W. Visualization of GABAA receptors and GABA transaminase (GABA-T) in the rat liver. Can J Gastroenterol 1998; 12: 24A. 6. Minuk GY, Gauthier T. The effect of gamma aminobutyric acid (GABA) on hepatic regenerative activity following partial hepatectomy in rats. Gastroenterology 1993; 104: 217–21. 7. Minuk GY, Gauthier T, Zhang XK, Wang GQ, Pettigrew NM, Burczynski FJ. Ciprofloxacin prevents the inhibitory effects of acute ethanol exposure on hepatic regeneration in the rat. Hepatology 1995; 22: 1797–800.
Ethanol, GABA and hepatic regeneration 8. Allan AM, Harris RA. Gamma-aminobutyric acid and alcohol actions: neurochemical studies. Life Sci 1986; 39: 2005– 15. 9. Minuk GY, Sarjeant EJ. The effect of (a) neomycin and lactulose treatment on systemic and portal serum GABA levels in rats and (b) pH changes on [3H] GABA binding to isolated rat hepatocytes. Clin Invest Med 1988; 11: 373–6. 10. Higgins GM, Anderson RM. Experimental pathology of liver: restoration of liver of the white rat following partial surgical removal. Arch Pathol 1931; 12: 186–202. 11. Diehl AM, Wells M, Brown ND, Thorgeirsson SS, Steer CJ. The effect of ethanol on polyamine synthesis during liver regeneration in rats. J Clin Invest 1990; 85: 385–90. 12. Minuk GY, Gauthier T, Gahary A, Murphy L. The effect of gamma-aminobutyric acid (GABA) on serum and hepatic polyamine concentrations following partial hepatectomy in rats. Hepatology 1991; 14: 685–9. 13. Vincent T, Devita JK. In: Vincent T, Devita JK, editors. Cancer: Principles and Practice of Oncology, 3rd ed. Philadelphia, PA: Lippincott; 1987. p. 292–3. 14. Luk GD. Essential role of polyamine metabolism in hepatic regeneration: inhibition of deoxyribonucleic acid and protein
15.
16.
17.
18.
19.
synthesis and tissue regeneration by difluoromethylornithine in the rat. Gastroenterology 1986; 90: 1261–7. Suzdak PD, Schwartz RD, Paul SM. Ethanol stimulates gamma-aminobutyric acid receptor-mediated chloride transport in rat brain synaptoneurosomes. Proc Natl Acad Sci U S A 1986; 83: 4071–5. Segev S, Rehavi M, Rubinstein E. Quinolones, theophylline, and diclofenac interactions with the g-aminobutyric acid receptor. Antimicrob Agents Chemother 1988; 1624–6. Minuk GY, Rockman GE, Gauthier T, Markert L, Gibson J, Benarroch. The effect of long-term, voluntary ethanol consumption on hepatic regeneration in rats. Can J Physiol Pharmacol 1991; 69: 341–5. Rockman GE, Hall AM, Markert L, Glavin GB. Ethanolstress interaction: immediate versus delayed effects of ethanol and handling on stress responses of ethanol-consuming rats. Alcohol 1987; 4: 391–4. Ferenci P, Covell D, Schafer DF, Waggoner JG, Shrager R, Jones EA. Metabolism of the inhibitory neurotransmitter gaminobutyric acid in a rabbit model of fulminant hepatic failure. Hepatology 1983; 3: 507–12.
641