Hepatic GSH Content Research Articles

Lack of Correlation betweenpara-Aminophenol Toxicityin Vivoandin Vitroin Female Sprague–Dawley Rats

The present study was designed to test the hypothesis thatpara-aminophenol (PAP) nephrotoxicity is due to autooxidation. We compared renal functional responses following PAP administration to female Sprague–Dawley rats and following incubation of renal proximal tubules with PAP. The concentrations of PAP selected forin vitroincubations produced cytotoxicity (for example, a decrease in oxygen consumption or adenine nucleotide concentration) in rat renal epithelial cells or rabbit proximal tubule suspensions. In rats, PAP (300 mg/kg ip) caused proximal tubular necrosis within 24 hr. Changes in renal function 24 hr following PAP administration included increased kidney weight and blood urea nitrogen concentration and decreased renal glutathione (GSH) content and adenine nucleotide concentrations. PAP did not cause hepatic damage. Within 2–4 hr following PAP administration, renal GSH content and adenine nucleotide concentrations were significantly decreased. In renal cortical slices prepared from PAP-treated rats, oxygen consumption and accumulation of organic ions (para-aminohippurate and tetraethylammonium) were significantly decreased compared with renal cortical slices prepared from control rats. In liver, GSH content was significantly decreased from 1 to 4 hr following PAP administration. In contrast to the effects of PAPin vivo,renal proximal tubules showed little evidence of injury when incubated with 0.1 or 0.5 mMPAP for up to 4 hr in the presence or absence of amino acids in the incubation medium. When tubules were incubated with 1 mMPAP for 4 hr in the presence of amino acids, GSH content, AMP concentration, and TEA uptake were significantly decreased. When amino acids were removed from the incubation medium, 1 mMPAP caused decreases in oxygen consumption and ATP concentration after 4 hr of incubation. Functional changes observed during incubation with PAPin vitrowere not consistent with functional changes observedin vivo.The discrepancy between PAP toxicityin vivoandin vitrosuggests that autooxidation is unlikely to be responsible for PAP nephrotoxicity and that nephrotoxicityin vivois primarily mediated by extrarenal bioactivation. Further, depletion of hepatic GSH content prior to changes in renal function suggests that PAP or a PAP metabolite may conjugate with hepatic GSH. These observations suggest that PAP nephrotoxicity may be mediated by PAP–GSH conjugates rather than autooxidation of PAP in the kidney.

Effects of taurine and Guanidinoethane Sulfonate on toxicity of the pyrrolizidine alkaloid monocrotaline

Monocrotaline (MONO), a pyrrolizidine alkaloid, causes pulmonary arterial hypertension and right ventricular hypertrophy due to hepatic metabolism to the alkylating pyrrole dehydromonocrotaline. Taurine, a sulfonic amino acid, is hepato- and cardioprotective in a variety of conditions. We have examined the effects of taurine and its amidino analog, guanidinoethane sulfonate (GES), in rats injected i.p. with MONO (65 mg/kg). Taurine and GES were given as 1% solutions in drinking water beginning 14 days before administration of MONO and continuing for 14 days thereafter, when the rats were killed. The MONO group had right ventricular hypertrophy and pulmonary hyperplasia. Compared with control, no significant changes in the right ventricle/left ventricle weight ratio, or the right ventricle/body weight ratio occurred in rats also given taurine or GES. Lung weights in these two groups were higher than in the control group, but below that of the MONO-alone group. The lethality of MONO over 14 days was decreased by taurine ( ld 50 for MONO alone 80 mg/kg; for MONO + taurine 121 mg/kg). Rats given only MONO had lower hepatic concentrations of GSH and cysteine (Cys), and higher activities of microsomal GSH transferase and γ-glutamyl transpeptidase. In rats also receiving taurine, hepatic GSH levels and GSH transferase activity were no different from control. γ-Glutamylcysteine (Glu-Cys) synthetase and γ-glutamyl transpeptidase activities were elevated. In MONO-injected rats given GES, hepatic GSH levels were higher and Cys levels were lower than in either the MONO alone or MONO + taurine groups. γ-Glu-Cys synthetase activity was depressed. Microsomal GSH transferase, GSH peroxidase and γ-glutamyl transpeptidase activities were elevated. Livers of MONO-injected animals showed higher levels of serine (reversed by both taurine and GES) and glycine (Gly; reversed by GES) and lower levels of glutamine. Compared with control rats, the following changes occurred in serum amino acids: MONO alone: increased aspartate, taurine and lysine; taurine-supplemented: increased taurine, methionine (Met) and lysine, and decreased Gly; GES-supplemented: decreased asparagine, serine, Gly, arginine, taurine, and valine. Compared with the MONO-alone group, the taurine-supplemented group had higher glutamate (Glu), Met and alanine, and the GES-supplemented group higher alanine and lower serine, Gly, arginine and valine. We conclude that taurine protects against MONO-induced lethality and right ventricular hypertrophy. GES also protects against right ventricular hypertrophy. However, these agents act by different mechanisms, taurine preventing many of the biochemical changes induced by MONO, with GES inducing additional changes.

Protective effect of S-(1,2-dicarboxyethyl)glutathione, an intrinsic tripeptide in liver, heart and lens, and its esters on acetaminophen-induced hepatotoxicity in rats.

The administration of acetaminophen (APAP, 500 mg/kg, i.p.) produced liver necrosis and increased aspartate aminotransaminase (AST) activity in serum. The pretreatment of S-(1,2-diethoxycarbonyl)glutathione isopropyl ester (DCE-Et-GS iPr, 0.5 mmol/kg, p.o.) prevented hepatic necrosis and the elevation of serum AST activity by 99.9%. DCE-Et-GS iPr inhibited APAP-induced hepatotoxicity much more strongly than reduced glutathione (GSH), DCE-GS and other esters of DCE-GS. To clarify this protective effect, the hepatic GSH concentration was determined 2h after APAP administration. It was found that the DCE-Et-GS iPr administration significantly inhibited the GSH depletion caused by APAP, suggesting that the protective effect of DCE-Et-GS iPr on APAP-induced hepatotoxicity was due, at least in part, to the retention of hepatic GSH level.

Lack of Correlation between para-Aminophenol Toxicity in Vivo and in Vitro in Female Sprague—Dawley Rats

The present study was designed to test the hypothesis that para-aminophenol (PAP) nephrotoxicity is due to autooxidation. We compared renal functional responses following PAP administration to female Sprague-Dawley rats and following incubation of renal proximal tubules with PAP. The concentrations of PAP selected for in vitro incubations produced cytotoxicity (for example, a decrease in oxygen consumption or adenine nucleotide concentration) in rat renal epithelial cells or rabbit proximal tubule suspensions. In rats, PAP (300 mg/kg i.p.) caused proximal tubular necrosis within 24 hr. Changes in renal function 24 hr following PAP administration included increased kidney weight and blood urea nitrogen concentration and decreased renal glutathione (GSH) content and adenine nucleotide concentrations. PAP did not cause hepatic damage. Within 2-4 hr following PAP administration, renal GSH content and adenine nucleotide concentrations were significantly decreased. In renal cortical slices prepared from PAP-treated rats, oxygen consumption and accumulation of organic ions (para-aminohippurate and tetraethylammonium) were significantly decreased compared with renal cortical slices prepared from control rats. In liver, GSH content was significantly decreased from 1 to 4 hr following PAP administration. In contrast to the effects of PAP in vivo, renal proximal tubules showed little evidence of injury when incubated with 0.1 or 0.5 mM PAP for up to 4 hr in the presence or absence of amino acids in the incubation medium. When tubules were incubated with 1 mM PAP for 4 hr in the presence of amino acids, GSH content, AMP concentration, and TEA uptake were significantly decreased. When amino acids were removed from the incubation medium, 1 mM PAP caused decreases in oxygen consumption and ATP concentration after 4 hr of incubation. Functional changes observed during incubation with PAP in vitro were not consistent with functional changes observed in vivo. The discrepancy between PAP toxicity in vivo and in vitro suggests that autooxidation is unlikely to be responsible for PAP nephrotoxicity and that nephrotoxicity in vivo is primarily mediated by extrarenal bioactivation. Further, depletion of hepatic GSH content prior to changes in renal function suggests that PAP or a PAP metabolite may conjugate with hepatic GSH. These observations suggest that PAP nephrotoxicity may be mediated by PAP-GSH conjugates rather than autooxidation of PAP in the kidney.

Effect of several metallothionein inducers on oxidative stress defense mechanisms in rats

One mechanism by which chemicals cause cellular injury is the formation of reactive oxygen species. In vitro studies have shown that metallothionein (MT), a small metal-binding, sulfhydryl-rich, readily inducible protein, can scavenge reactive oxygen species, especially hydroxyl radicals. Nevertheless, whether or not MT protects against oxidative stress in the intact animal is not known. Experimental induction of MT could help to clarify this question, however, it is unclear whether agents that induce MT also influence known antioxidant systems. Therefore, the present study was designed to determine whether the well-known MT inducers are specific for induction of MT or whether they might also influence other hepatic systems that protect against oxidative stress. Male rats were administered cadmium chloride (Cd; 30 pmol/kg s.c.), zinc chloride (Zn; 1000 pmol/kg s.c.), α-hederin (α-H, 30 pmol/kg s.c.) or lipopolysaccharide (LPS; 1 mg/kg s.c.) 24 h prior to measurement of antioxidant systems. Zn and α-H increased hepatic GSH concentration 20% and 55%, respectively. Cd significantly increased, whereas LPS reduced, the activities of selenium-dependent glutathione peroxidase and glutathione reductase. Glutathione S-transferases were not altered by any of the inducers. Cd also increased DT-diaphorase activity. Cd, Zn and α-H all decreased catalase activity 20–35%, while the activity of Superoxide dismutase was unaffected by the inducers. The amount of total cytochrome P450 enzymes and cytochrome b 5 were decreased by LPS, Cd and α-H, while Zn appeared to have no effect. The activities of P450 enzymes towards testosterone oxidation were also decreased by LPS, Cd and α-H. In conclusion, all four MT inducers examined affect systems known to protect cells against oxidative stress. Therefore, using these chemicals to determine the in vivo role of MT in protecting against oxidative stress poses difficulties.

The Effects of Diquat and Ciprofibrate on mRNA Expression and Catalytic Activities of Hepatic Xenobiotic Metabolizing and Antioxidant Enzymes in Rat Liver

Although the mechanisms responsible for chemically induced oxidative stress are under intense investigation, little is known about the effects of prooxidant chemicals on the expression of drug-metabolizing enzymes. We examined the effects of diquat (0.1 mmol/kg, ip) and ciprofibrate (0.025% w/w, diet), chemicals which induce oxidative stress via different biochemical mechanisms, on the steady-state messenger RNA (mRNA) levels of six cytochrome P450 enzymes, seven glutathione S-transferase (GST) isoenzymes, UDP-glucuronosyl transferase 1-06 (UGT1*06), γ-glutamylcysteine synthetase (γGCS), NADP(H):quinone oxidoreductase (quinone reductase), Cu/Zn superoxide dismutase (SOD), catalase, and 18S ribosomal RNA in the livers of male Sprague-Dawley rats. Effects of chemical treatments on mRNA levels were compared to changes in catalytic activities for selected enzymes. Ciprofibrate treatment selectively decreased CYP1A2 mRNA expression, whereas both chemicals suppressed CYP3A2 mRNA expression. CYP4A1 mRNA expression and lauric acid hydroxylase activities were induced by ciprofibrate treatment, whereas diquat treatment moderately increased CYP4A1 mRNA levels without affecting lauric acid hydroxylase activities. The steady-state mRNA levels encoding constitutively expressed GST isozymes (Ya1, Ya2, Yb1, Yb2, and Yc1) were decreased by diquat exposure, and the mRNA encoding four of the five constitutively expressed GSTs (Ya1, Ya2, Yb1, and Yc1) were also decreased by ciprofibrate treatment. Nonconstitutively expressed or low constitutively expressed genes (CYP1A1, CYP2B1, CYP2B2, GST Yc2, GST Yf, and UGT1*06) were not induced by exposure to the prooxidants. Changes in isozyme-specific catalytic activities were more consistent with the observed changes in mRNA expression for the GSTs than for the P450s. Both treatments had inhibitory effects on hepatic GSH biosynthesis by decreasing γGCS large-subunit mRNA expression, γGCS catalytic activities, and hepatic GSH concentrations. Cu/Zn SOD and quinone reductase mRNA levels were increased after ciprofibrate exposure, whereas Cu/Zn SOD mRNA expression was decreased in the diquat-treated animals. The results of this study indicate that diquat and ciprofibrate can decrease the expression profile of a number of phase I, phase II, and antioxidant enzymes and inhibit GSH biosynthesis. These effects may involve the pre-translational loss of hepatic mRNAs, possibly due to accelerated production of reactive oxygen species.

Biotransformation and Hepatotoxicity of HCFC-123 in the Guinea Pig: Potentiation of Hepatic Injury by Prior Glutathione Depletion

The chlorofluorocarbon substitute 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123) is a structural analog of halothane. Both are oxidatively metabolized by CYP2EI, producing a reactive trifluoroacyl acid chloride intermediate and have been shown to cause acute liver necrosis in the guinea pig. With halothane, liver injury has been associated with the degree of reactive intermediate binding to hepatic protein. This injury can be potentiated by prior glutathione (GSH) depletion. Thus, the combination of GSH depletion and HCFC-123 exposure was evaluated for its hepatotoxic potential in this species. Male outbred Hartley guinea pigs were injected with either 0.8 g/kg l-buthionine-(S,R)-sulfoximine (BSO) to deplete hepatic glutathione or vehicle control solution 24 hr before a 4-hr inhalation exposure to 1.0% (v/v) HCFC-123 with 40% O2. HCFC-123 caused minimal liver injury with only 1 of 8 exposed animals displaying confluent zone 3 necrosis. GSH depletion potentiated injury producing submassive to massive liver necrosis in some animals. This potentiation was associated with a 36% increase in covalent binding of reactive HCFC-123 intermediates to hepatic protein. These results were not due to alterations in the biotransformation of HCFC-123 as indicated by plasma concentrations of the metabolites trifluoroacetic acid and fluoride ion which were not affected by BSO pretreatment. HCFC-123 was also found to cause a decrease in liver GSH concentrations following exposure. These findings demonstrate a role for hepatic GSH in helping to prevent covalent binding by the trifluoroacyl acid chloride intermediate. Inhalation of HCFC-123 can cause acute hepatic injury in the guinea pig that is worsened by low hepatic GSH concentrations.

Glutathione conjugation of bromosulfophthalein in relation to hepatic glutathione content in the rat in vivo and in the perfused rat liver

The relation between the rate of glutathione (GSH) conjugation and hepatic GSH content was studied in the rat in vivo and the in situ single-pass-perfused rat liver preparation with bromosulfophthalein (BSP) as the model substrate. The biliary excretion of the BSP-GSH conjugate and the hepatic GSH content were monitored simultaneously during intravenous infusions with BSP in the rat in vivo, and during liver perfusions with BSPcontaining perfusion medium. Rats were pretreated with single or multiple doses of buthionine sulfoximine, an inhibitor of the de novo synthesis of GSH. Surprisingly, the excretion of the BSP-GSH conjugate was sustained at a high rate, despite a virtually complete depletion of hepatic GSH, both in the rat in vivo as well as in the perfused rat liver. The results indicate that GSH was still available for conjugation with BSP after apparent depletion of the hepatic GSH pool, presumably because of a residual de novo synthesis of GSH in the liver. Despite the multiple pretreatment with buthionine sulfoximine, the de novo GSH synthesis was sufficient to sustain a high rate of GSH conjugation of BSP. The cosubstrateK m for GSH conjugation of BSP in the liver was estimated to be very small (approximately 0.3 μmol/g): the excretion rate of the BSP-GSH conjugate was only impaired at minimal hepatic GSH levels.

Glutathione conjugation of bromosulfophthalein in relation to hepatic glutathione content in the rat in vivo and in the perfused rat liver

The relation between the rate of glutathione (GSH) conjugation and hepatic GSH content was studied in the rat in vivo and the in situ single-pass-perfused rat liver preparation with bromosulfophthalein (BSP) as the model substrate. The biliary excretion of the BSP-GSH conjugate and the hepatic GSH content were monitored simultaneously during intravenous infusions with BSP in the rat in vivo, and during liver perfusions with BSP-containing perfusion medium. Rats were pretreated with single or multiple doses of buthionine sulfoximine, an inhibitor of the de novo synthesis of GSH. Surprisingly, the excretion of the BSP-GSH conjugate was sustained at a high rate, despite a virtually complete depletion of hepatic GSH, both in the rat in vivo as well as in the perfused rat liver. The results indicate that GSH was still available for conjugation with BSP after apparent depletion of the hepatic GSH pool, presumably because of a residual de novo synthesis of GSH in the liver. Despite the multiple pretreatment with buthionine sulfoximine, the de novo GSH synthesis was sufficient to sustain a high rate of GSH conjugation of BSP. The cosubstrate-Km for GSH conjugation of BSP in the liver was estimated to be very small (approximately 0.3μmol/g): the excretion rate of the BSP-GSH conjugate was only impaired at minimal hepatic GSH levels.

Liver and Blood Glutathione in Male Broiler Chickens, Turkeys, and Quail

The objective of the present study was to establish age relationships for hepatic and whole blood glutathione (GSH) in male broiler chickens, quail, and turkeys utilizing lines within each poultry species with different rates of growth. Liver and blood samples were obtained from three quail lines (light, medium, and heavy) at 3 and 6 wk of age, from two turkey lines (light and heavy) at 10 and 20 wk of age, and two broiler lines (medium and heavy) at 3, 5, and 7 wk of age. With the exception of the heavy turkey line, older birds generally exhibited higher hepatic GSH concentrations than younger birds. There was no apparent age-related difference in whole blood GSH, nor was whole blood GSH correlated with hepatic GSH in any poultry species.

Influence of Diethyl Maleate and Cysteine on Tissue Glutathione and Growth in Broiler Chickens

The objectives of this study were to determine the effects of diethyl maleate (DEM) and 1-cysteine (L-Cys) on tissue glutathione (GSH) and growth in male broiler chickens. In Experiment 1, broilers were treated with DEM (0, 1.5, 3, 6, or 12.0 mmol/kg BW, i.p.). After 1 h, maximum GSH depletions were to 9, 24, 20, 19, and 35% of control (0 mmol DEM/kg) for liver, lung, kidney, heart, and brain, respectively. In Experiment 2, time-course changes following 1.5 mmol DEM/kg (i.p.) were determined; time-controls received an equal amount of corn oil (CO, .25 mL/kg BW). Levels of GSH in all tissues were low at 1 and 2 h after DEM in comparison to time-control values. Tissue GSH concentrations returned to values that were not different from controls by 5 h in liver and kidney, by 12 h in heart, and by 24 h in brain and lung. In Experiment 3, the effects of feeding a control diet (0% L-Cys) or one supplemented with 1% L-Cys from 3 to 7 wk of age with weekly i.p. injections (at 3, 4, 5, and 6 wk of age) of DEM (1.5 mmol/kg BW) or CO (.25 mL/kg BW) on growth rate and tissue GSH were determined. There were no differences in BW among treatment groups between 3 and 6 wk of age. Although there were no differences in 7-wk BW between controls (0% L-Cys/CO) and birds treated with DEM fed either diet, the 1% L-Cys/CO group was heavier (P < .05) than either the 0% or 1% L-Cys/DEM groups, and heavier (P = .066) than controls at 7 wk of age. At 5 wk of age, 1% L-Cys raised GSH concentrations in liver, kidney, lung, and duodenum, but had no effect on heart GSH in birds treated with either CO or DEM. Control hepatic GSH concentrations were higher at 7 than at 5 wk of age. With the exception of duodenal GSH in CO birds, 1% L-Cys had no effect on tissue GSH concentrations in 7-wk-old birds. The results of this study provide an initial characterization of GSH metabolism in commercial male broilers and indicate that DEM produced dose- and time-dependent changes in GSH similar to reported changes in mammals. Results of this study also indicate that increased tissue GSH may be beneficial for growth.

Hepatic glutathione after ethanol administration in rat: Effects of cimetidine and omeprazole

As a fraction of ingested ethanol (EtOH) is metabolized by gastric mucosa, different amounts of alcohol reach the liver, when the same dose is administered by oral or intravenous route. In previous experiments, we demonstrated that the decrease of hepatic reduced glutathione (GSH) is less pronounced and is followed by a quicker recovery after oral than after intraperitoneal administration of the same amount of EtOH. Therefore, the time-course of hepatic GSH concentration seems to be an indirect assay of EtOH metabolism by the liver. On the basis of these findings, any condition causing a reduced function of gastric alcohol dehydrogenase (ADH) should show up as a more severe depletion of hepatic GSH. In the same rat experimental model we determined the effects of cimetidine and omeprazole administration on gastric ADH activity and on the time-course of hepatic GSH after EtOH load. Cimetidine was shown to inhibit gastric ADH with a Ki of 0.167±0.009 mmol 1 −1, accordingly, the pretreatment with this drug (20 mg kg −1 b.w. per day for 1 week) determined, after oral EtOH load, a marked reduction of hepatic GSH, likewise after intraperitoneal administration. Omeprazole exerted only a marginal inhibition on gastric ADH and this drug (0.3 mg kg −1 b.w. per day for 1 week) did not modify the time-course of hepatic GSH concentrations after EtOH load. This study indicates that the inhibition of gastric ADH, when associated with EtOH intake, induces depletion of the hepatic GSH concentration and, therefore, possible liver damage.

Biliary copper excretion in the neonatal rat: Role of glutathione and metallothionein

Metallothionein (MT) and glutathione (GSH) have been implicated as two major copper-binding agents involved in the hepatobiliary copper metabolism in the adult rat. This study was designed to explore their potential role in facilitating copper export from the copper-laden hepatocyte of the newborn rat. Biliary and hepatic copper, GSH, and immunoreactive MT-1 concentrations were measured in Sprague-Dawley rats at 1, 2, 3, 4, and 8 weeks of age. Bile was collected by duct cannulation for 90 minutes before the rats were killed. Liver was removed, weighed, and freeze-dried. The bile flow rate (BFR) doubled and the liver weight increased 14-fold during maturation. Hepatic and biliary copper and MT-I concentrations were significantly higher in the suckling than in the weanling. The total biliary output of copper per 24 hours was low at 1 week and increased significantly by 8 weeks of age. MT-1—bound copper represented a maximum of only 3.4% of biliary copper at 1 week and 0.5% at 8 weeks. GSH was not detected in bile until 2 weeks and then increased 15-fold by 8 weeks, while hepatic GSH levels remained unchanged. Therefore, GSH levels did not correlate with the high biliary copper concentration at week 1, although there was a close correlation between the total daily biliary excretion of copper and GSH at 2 weeks and beyond. We conclude that the net biliary copper excretion per day is relatively low in the first week of life and is independent of MT and GSH secretion. It increases significantly once GSH is available in bile. We speculate that biliary copper excretion in the neonatal rat may involve copper binding agents as yet unidentified.

Biliary copper excretion in the neonatal rat: Role of glutathione and metallothionein

Metallothionein (MT) and glutathione (GSH) have been implicated as two major copper-binding agents involved in the hepatobiliary copper metabolism in the adult rat. This study was designed to explore their potential role in facilitating copper export from the copper-laden hepatocyte of the newborn rat. Biliary and hepatic copper, GSH, and immunoreactive MT-I concentrations were measured in Sprague-Dawley rats at 1, 2, 3, 4, and 8 weeks of age. Bile was collected by duct cannulation for 90 minutes before the rats were killed. Liver was removed, weighed, and freeze-dried. The bile flow rate (BFR) doubled and the liver weight increased 14-fold during maturation. Hepatic and biliary copper and MT-I concentrations were significantly higher in the suckling than in the weanling. The total biliary output of copper per 24 hours was low at 1 week and increased significantly by 8 weeks of age. MT-I-bound copper represented a maximum of only 3.4% of biliary copper at 1 week and 0.5% at 8 weeks. GSH was not detected in bile until 2 weeks and then increased 15-fold by 8 weeks, while hepatic GSH levels remained unchanged. Therefore, GSH levels did not correlate with the high biliary copper concentration at week 1, although there was a close correlation between the total daily biliary excretion of copper and GSH at 2 weeks and beyond. We conclude that the net biliary copper excretion per day is relatively low in the first week of life and is independent of MT and GSH secretion. It increases significantly once GSH is available in bile.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of 3 dipyridyl isomers on hepatic microsomal cytochrome P450 and heme oxygenase in rats

We compared the effects of 3 dipyridyl isomers, 2,2′-dipyridyl, 2,4′-dipyridyl and 4,4′-dipyridyl, on hepatic microsomal heme oxygenase and drug-metabolizing enzyme activities in male rats. 2,2′-Dipyridyl increased cytochrome P450 (P450) content at lower doses, but decreased with increasing dose levels. Immunoblot analysis revealed that 2,2′-dipyridyl did not induce both P450 1A1/2 and P450 2B1/2, in contrast to 2,4′- and 4,4′-dipyridyls, both of which were inducers of either P450 1A1/2 and/or P450 2B1/2. Some drug-metabolizing enzyme activities gradually declined with the increasing dose level of 2,2′-dipyridyl. 2,2′-Dipyridyl was able to induce hepatic microsomal heme oxygenase in a dose-dependent manner, but 2,4′- and 4,4′-dipyridyls did not, even at the highest dose (0.80 mmol/kg) examined. Treatment of rats with 2,2′-dipyridyl resulted in the increase of glutathione (GSH) content in a dose-dependent manner, but not 4-substituted isomers. A time course study with 2,2′-dipyridyl revealed that it produced a significant decrease in hepatic GSH content at early time periods (2–6 h) after its administration with an inverse increase in heme oxygenase activity. The present investigation has revealed that in contrast to the induction of P450 by 4-substituted dipyridyl compounds, 2,2′-dipyridyl is a novel inducer of hepatic microsomal heme oxygenase, together with the change in hepatic GSH content. This study would provide information on the differential effects of simple dipyridyl isomers on hepatic enzymes involved in heme and drug metabolism.

Effects of a complex mixture of chemical contaminants on hepatic glutathione, L‐cysteine and γ‐glutamylcysteine synthetase in english sole (Pleuronectes vetulus)

AbstractThe effects of chemical contaminants on concentrations of hepatic glutathione (GSH), cysteine (L‐Cys), and the activity of γ‐glutamylcysteine synthetase (γ‐GCS) were assessed in English sole (Pleuronectes vetulus) in the laboratory and in fish from the Duwamish Waterway, a contaminated urban site in Puget Sound, Washington. In the laboratory studies, fish from a nonurban (reference) site were exposed to an organic‐solvent extract of sediment from the Duwamish Waterway. Hepatic GSH concentrations significantly increased at 3 d after exposure to the extract at 1,000 g sediment extracted/kg fish. At this time point, hepatic GSH showed a dose‐dependent increase at dosages ranging from 300 to 1,600 g sediment extracted/kg fish, whereas fish exposed to a reference sediment extract showed no increase. Moreover, the increases in GSH in fish exposed to the contaminated sediment extract were not accompanied by changes in either L‐Cys concentrations or γ‐GCS activity. Similarly, fish sampled directly from the Duwamish Waterway showed increased GSH concentrations but showed no significant differences in L‐Cys levels or γ‐GCS activity compared to fish from a reference site. These results substantiate that hepatic GSH in fish is responsive to chemical contaminant exposure and further support its use as a biomarker in environmental monitoring studies. The findings also indicate that L‐Cys availability and induction of γ‐GCS were not major factors in the increase of hepatic GSH in contaminant‐exposed English sole.

Role of renal gamma-glutamyltransferase activity in hepatic utilization of exogenous glutathione.

The importance of renal gamma-glutamyltransferase activity in the hepatic utilization of exogenous glutathione (GSH) was evaluated by injecting GSH (1.67 mmol/kg body wt) i.v. into bilaterally nephrectomized and sham-operated Sprague-Dawley rats in which endogenous hepatic GSH had been decreased (0.20 +/- 0.01 mumol/g liver vs 5.87 +/- 0.26 mumol/g liver in normal controls, mean +/- SD) by diethylmaleate (0.5 ml/kg body wt, i.p.). Hepatic GSH concentration 60 min after GSH administration was lower in the nephrectomized than in the sham-operated rats (0.87 +/- 0.25 mumol/g liver vs 3.08 +/- 0.81 mumol/g liver, P < 0.001), while plasma GSH concentration was higher in the former (4.61 +/- 1.07 mM vs 0.11 +/- 0.06 mM, P < 0.001). In rats with intact kidneys which had been given a gamma-glutamyltransferase inhibitor (acivicin, 25 mumol/kg body wt i.v.) prior to GSH administration, the hepatic GSH concentrations (1.11 +/- 0.49 mumol/g liver) were comparable to those obtained in the nephrectomized rats. When N-acetylcysteine (1.67 mmol/kg body wt, i.v.) was administered instead of GSH, the hepatic GSH concentrations were similar in nephrectomized and sham-operated rats (1.54 +/- 0.23 mumol/g liver vs 2.22 +/- 0.58 mumol/g liver, NS). The gamma-glutamyltransferase activity was much higher in the kidney than in the liver (4460 +/- 830 IU/kg body wt vs 14 +/- 7 IU/kg body wt). These results indicate that the kidney plays an essential role in the hepatic utilization of exogenous GSH through its high gamma-glutamyltransferase activity.

Relationship between glutathione concentration and metabolism of the pyrrolizidine alkaloid, monocrotaline, in the isolated, perfused liver.

The influence of GSH concentration on metabolism of monocrotaline was examined in the isolated, perfused rat liver. Chloroethanol (0.37 mmol/kg), diethyl maleate (5.6 mmol/kg), and buthionine sulfoximine (72.9 mmol/kg) given in vivo reduced hepatic GSH from 3.7 mumol/g wet weight to 1.5, 0.6 and 0.9 mumol/g, respectively. Livers were then perfused in vitro for 1 hr with monocrotaline (0.5 mM). GSH depletion had no effect on the total release of pyrrolic metabolites of monocrotaline. Depletion, however, markedly affected the pattern of pyrrole release. Biliary release of 7-glutathionyl-6,7-dihydro-1-hydroxy-methyl-5H-pyrrolizine (GSDHP) was reduced by up to 72%. Pretreatment with diethyl maleate or buthionine sulfoximine increased the level of protein-bound pyrroles in the liver by 107 and 84%, respectively. Such pyrroles are probably responsible for liver toxicity. GSH depletion also led to a doubling of dehydromonocrotaline release into the perfusate. This metabolite is probably responsible for the extrahepatic toxicity of monocrotaline. Release into perfusate of the relatively nontoxic metabolite, 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) was correspondingly decreased. Hepatic GSH content was increased to 4.4 mumol/g by pretreatment with oxo-4-thiazolidine carboxylate (4.76 mmol/kg). This agent increased total pyrrolic metabolites by 54%. Biliary release of GSDHP and perfusate release of dehydromonocrotaline and DHP were all increased. Thus, hepatic GSH levels regulate the metabolism of monocrotaline and dehydromonocrotaline and, consequently, the hepatic and extrahepatic toxicity of monocrotaline. GSH depletion leads to a switch from the biliary release of the midly toxic GSDHP to the perfusate release of the highly toxic dehydromonocrotaline. GSH depletion also permits more dehydromonocrotaline in the liver to become available for macromolecular alkylation. These findings suggest that nutritional intake of sulfur-containing amino acids can influence the severity of pyrrolizidine poisoning.

Hepatic glutathione determination after ethanol administration in rat: Evidence of the first-pass metabolism of ethanol

As a fraction of ingested ethanol is metabolized by gastric mucosa, different amounts of alcohol should reach the liver when the same dose is administered by oral or intravenous route. Therefore, we investigated the time-course of hepatic reduced glutathione (GSH) concentrations after intra-peritoneal or intra-gastric load of the same amount of ethanol in the rat. The test was also performed in fasted and Cimetidine-treated rats. The oral ethanol administration was followed by a less pronounced decrease and by a quicker recovery of hepatic content of GSH than after intraperitoneal route. In the fasted rat, basal hepatic GSH significantly decreased; after alcohol administra-tion the decrease of hepatic GSH was more severe and prolonged than in the fed rat. Cimetidine was shown to be a potent inhibitor of gastric ADH. Pre-treatment with Cimetidine did not change the basal levels of hepatic GSH, but after oral ethanol load, the decrease of the hepatic GSH content was significantly (p<0.005) more pronounced than in controls. This study demonstrates the beneficial effects of gastric ethanol metabolism on the liver. The reduced gastric ethanol metabolism, induced by fasting or by Cimetidine resulted in a decreased content and delayed recovery of liver GSH content.

Effects of prolonged administration of perfluorooctanoic acid on hepatic activities of enzymes which detoxify peroxide and xenobiotic in the rat

Male and female rats were fed a diet containing 0.01% (w/w) perfluorooctanoic acid (PFOA) for 2 or 26 weeks, and the effects on enzymes that participate in the metabolism of peroxides and xenobiotics in liver were studied. Elevated activity of peroxisomal β-oxidation persisted throughout the treatment of male rats with PFOA for 26 weeks. The activity of glutathione (GSH) peroxidase towards hydrogen peroxide was depressed significantly by the prolonged administration. The long-term treatment of male rats with PFOA decreased the activity of GSH peroxidase towards cumene hydroperoxide and increased the activity of microsomal NADPH-dependent lipid peroxidation. The activities of GSH reductase and hepatic content of GSH remained unchanged. There was no difference in the content of conjugated dienes in microsomal lipid between male rats exposed to PFOA for 26 weeks and age-matched control. The activities of GSH S-transferase towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene were depressed by the short-term administration of PFOA to male rats, and this inhibition became pronounced during the prolonged treatment. Microsomal cytochrome P450 was induced by the short-term treatment of male rats with PFOA, and elevated levels persisted throughout the treatment for 26 weeks. Upon the administration of PFOA to male rats for 2 weeks, the activity of 7-ethoxycoumarin O-deethylase was increased markedly, whereas the activities of either aniline p-hydroxylase or aminopyrine N-demethylase were unchanged. Although an age-dependent decrease was observed in the activity of 7-ethoxycoumarin O-deethylase, the activity in male rats treated with PFOA for 26 weeks was higher than that of age-matched control, to the same extent as was observed with the short-term treatment. The prolonged administration of PFOA to male rats caused a significant increase in the activity of both aniline p-hydroxylase and aminopyrine N-demethylase. Little changes were found in the same parameters tested in female rats even after the prolonged administration of PFOA.