Drug metabolizing enzyme changes after chronic buthionine sulfoximine exposure modify acetaminophen disposition in rats.

This study examined the effects of prolonged exposure to buthionine sulfoximine (BSO) on 1) the overall elimination pharmacokinetics of acetaminophen; 2) the sulfate and glucuronide conjugation processes primarily responsible for acetaminophen elimination; and 3) in vitro microsomal and cytoplasmic enzyme activities in rats. Rats imbibed drinking water containing 30 mM BSO for 6 days and then received an iv injection of acetaminophen, 150 mg/kg in a propylene glycol vehicle. Exposure to BSO, a specific inhibitor of gamma-glutamylcysteine synthetase, produced marked depletion of glutathione (GSH) and resulted in induction of hepatic UDP-glucuronosyltransferase and GSH-S-transferase enzyme activities, but not cytochrome P-450. BSO pretreatment had no effect on the total or renal clearance of acetaminophen in rats. However, BSO exposure increased the partial clearance of acetaminophen to acetaminophen glucuronide by 47% (1.29 +/- 0.08 vs. 1.90 +/- 0.23 ml/min/kg; p less than 0.01) and significantly (p less than 0.02) increased the percentage of the dose recovered as the glucuronide conjugate from 17.6 +/- 2.5 to 26.5 +/- 0.6 The partial clearance of acetaminophen to acetaminophen sulfate was decreased, although not significantly, from 4.46 +/- 0.62 to 3.39 +/- 0.82 ml/min/kg. BSO treatment increased microsomal UDP-glucuronosyltransferase activity toward three xenobiotic aglycones, p-nitrophenol, 1-naphthol, and morphine by 308, 61, and 66%, respectively (p less than 0.05), but not toward testosterone or estrone. Cytosolic GSH-S-transferase activity toward 1-chloro-2,4-dinitrobenzene was increased 52% by BSO, whereas p-nitrophenol sulfotransferase activity was not altered. Cytochrome P-450 concentration and monooxygenase activity were unchanged by BSO exposure.     

Protective effect of Aucuba japonica against carbon tetrachloride-induced liver damage in rats

Pretreatment of rats with ethanol extract from leaves of Aucuba japonica (600 mg/kg/day, po) for two days protected against CCl4-induced depression in plasma disappearance and biliary excretion of injected sulfobromophthalein (BSP) determined 24 hr after the CCl4 challenge (0.5 ml/kg, ip). Percent recovery of BSP in bile in 60 min for control, CCl4, extract + CCl4 treated rats was 66.8 +/- 1.9, 56.2 +/- 1.4, and 68.9 +/- 2.2, respectively. Pretreatment of the extract also protected CCl4-induced increased serum glutamic-pyruvic transaminase activity and liver necrosis as demonstrated by histological evaluations. However, pretreatment of the extract did not modify the intensity of CCl4-induced lipid peroxidation process or cytochrome P-450 destruction. The results suggest that ethanol extract of Aucuba japonica protects CCl4 hepatotoxicity at a site in the chain events leading to necrosis but not the activation step of CCl4 to X CCl3 and X C1 free radicals.     

Comparative study of phase II biotransformation in rabbit ocular tissues.

To assess the biotransformational capability of ocular tissues in the rabbit, representative phase II enzymes were assayed in five tissues from the eye, and in the liver, kidney, and intestine. Within the eye, the iris/ciliary body exhibited the highest glutathione S-transferase activity, whereas the cornea possessed the highest specific activities for N-acetyl-, sulfo-, and UDP-glucuronosyl-transferases. Cornea, iris/ciliary body, choroid, and retina exhibited significant activities of p-aminobenzoic acid N-acetyltransferase, 2-naphthol sulfotransferase, and 1-chloro-2,4-dinitrobenzene glutathione S-transferase. Despite its size and protein content, lens displayed little or no biotransformational activity. Only the iris/ciliary body conjugated sulfobromophthalein with glutathione. UDP-glucuronsyltransferase activity varied depending on tested substrates and tissues. When compared to liver, kidney, or intestine, N-acetyltransferase activity in the iris/ciliary body nearly matched the rate measured in kidney, glutathione S-transferase activity in cornea and iris/ciliary body was nearly 70 and 89%, respectively, of the rate in intestine, and corneal sulfotransferase activity was greater than that in kidney. These data suggest that biotransformation pathways are present in the eye, and particularly in ocular tissues having adequate blood supply or interfacing with the external environment.     

Conjugation reactions in hepatocytes isolated from streptozotocin-induced diabetic rats

The activities of three drug conjugation reactions, glutathione, glucuronic acid and sulphate conjugation and the synthesis of glutathione, have been measured in hepatocytes isolated from streptozotocin-induced male diabetic rats. The intracellular content of reduced glutathione (GSH) was decreased in diabetic rat hepatocytes compared with controls. Following depletion of the intracellular GSH stores with diethylmaleate, the resynthesis of GSH in the presence of 0.5 mM L-methionine, occurred faster in diabetic rat hepatocytes than in those from control rats indicating that the cystathione pathway may be more efficient in the diabetic animals. In contrast, there was no significant difference in the resynthesis of GSH between control and diabetic rat hepatocytes in the presence of L-cysteine. The GSH conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloronitrobenzene (DCNB) was deficient in diabetic rat hepatocytes, although only the effect on the former reaction was statistically significant (P less than 0.05). The Vmax for CDNB conjugation was significantly lower (P less than 0.05) in cytosolic fractions prepared from diabetic rat liver than in control rat liver fractions. This was accompanied by an increase in the affinity of the enzyme for CDNB. In contrast, the Vmax and Km for the conjugation of DCNB in cytosolic fractions were unaffected by the induced-diabetes. Glucuronic acid conjugation of both 1-naphthol and phenolphthalein was markedly deficient in diabetic rat hepatocytes. The intracellular concentrations of the cofactor for glucuronidation, UDP-glucuronic acid, were decreased in diabetic rat liver and this was thought to contribute to the defect in glucuronidation. The sulphation of 1-naphthol was not significantly altered by the induced diabetes. Deficiencies in glutathione and glucuronic acid conjugation in streptozotocin-induced diabetic rats may result in an increased susceptibility to xenobiotic induced cytotoxicity.     

Induction of rat UDP-glucuronosyltransferase and glutathione S-transferase activities by L-buthionine-S,R-sulfoximine without induction of cytochrome P-450

The effect of prolonged exposure to buthionine sulfoximine (BSO) on rat hepatic Phase I and Phase II drug-metabolizing enzymes has been examined. Exposure to 30 mM BSO in drinking water for 7 days induced hepatic microsomal UDP-glucuronosyltransferase activity (detergent-activated) toward p-nitrophenol (250%), 1-naphthol (210%), morphine (130%) and testosterone (140%), but not estrone. Glucuronosyltransferase activities were also induced after exposure for as short as 3 and as long as 13 days. When rats were returned to unsupplemented drinking water for 1 day prior to sacrifice following 6 days on 30 mM BSO, comparable induction to that seen after 7 consecutive days on the BSO solution was observed despite liver glutathione concentration having rebounded to 127% of control. Daily ingestion of BSO was similar (1 mmol/rat/day) for all periods of 30 mM BSO-drinking water exposure, with a body weight-adjusted dose range of 3.2-6.3 mmol/kg/day. An analogous inductive response caused by drinking 30 mM BSO for 3 days was elicited for p-nitrophenol and morphine glucuronidation by 6 mmol/kg doses of BSO given as single daily intraperitoneal or intragastric injections for 3 days. Intraperitoneal, intragastric and all BSO-drinking water exposures also significantly induced (130-195%) cytosolic glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene. Significant increases in UDP-glucuronosyltransferase and glutathione S-transferase activities were also observed following 3 days of exposure to BSO in the drinking water at a concentration as low as 5 mM. Cytosolic p-nitrophenol sulfotransferase activity, with one minor exception, was not enhanced by any BSO treatment regimen. Alterations in transferase activities were not accompanied by any major changes in either overall cytochrome P-450 concentration or oxidative reactions selective for two isozymes. Thus, in addition to its well-documented glutathione-depleting property, BSO also selectively induces several Phase II drug-metabolizing enzymes, an effect to be considered in studies employing extended BSO treatment.     

Activities of several phase I and phase II xenobiotic biotransformation enzymes in cultured hepatocytes from male and female rats

Hepatocytes were isolated from adult male and female rats and maintained in monolayer culture for up to 24 hr. The degree of preservation of representative phase I and phase II xenobiotic biotransformation enzymes was studied in these cells immediately after isolation, after attachment in culture, and after 24 hr in culture. Regarding phase I pathways, hepatocytes during 24 hr lost 50% of cytochrome P-450, but maintained high mixed function oxidase activities; 75% of aryl hydrocarbon hydroxylase and 65% of benzphetamine demethylase activities were preserved in hepatocytes from males, whereas in hepatocytes from females 70 and 50% of these activities, respectively, were maintained. Of phase II pathways, glutathione transferase activity after 24 hr, tested toward 1,2-dichloro-4-nitrobenzene as substrate, was diminished in male hepatocytes to 20% of the initial liver activity and in female cells, to 35%, whereas the activity tested toward 1-chloro-2,4-dinitrobenzene as substrate was stable. UDP-glucuronosyltransferase activities, tested toward p-nitrophenol and phenolphthalein as substrates, were slightly increased during 24 hr of culture of hepatocytes to levels higher than in liver before perfusion. The level of UDP-glucuronic acid, the endogenous substrate for the enzyme, was reduced after isolation to only 6% of the initial liver value, and then increased during culture to a level approximately 60% of normal. Thus, the changes in xenobiotic biotransformation enzymes and associated constituents in cultured hepatocytes were not uniform, although biotransformation capability remained reasonably intact.     

Structure-activity relationships in the induction of hepatic drug metabolism by azo compounds

Lipophilic azo compounds possessing 1-phenylazo-2-naphthol or 1-phenylazo-2-naphthylamine moieties induced cytochrome P-448 and related mono-oxygenase activities, UDP-glucuronyltransferase activity towards p-nitrophenol, glutathione-S-transferase activity towards 1-chloro-2,4-dinitrobenzene, aldehyde dehydrogenase, and menadione reductase activities. This pattern of induction by azo dyes is very similar to that by 3-methylcholanthrene. None of the hydrophilic azo compounds tested and none of the other lipophilic azo compounds tested including 4-phenylazo-1-naphthol induced these activities. It is suggested that the formation of a third six-membered ring fused to naphthalene in a phenanthrene-like arrangement by hydrogen bonding between the phenolic hydroxyl and azo nitrogen is required for induction.     

Differential regulation of male rat liver glutathione S-transferases: Effects of orchidectomy and hormone replacement

The effects of orchidectomy and hormone replacement on glutathione S-transferase activities in adult male rat liver were investigated. Due to the overlapping yet distinct substrate specificities of the hepatic glutathione S-transferases, we measured activity in cytosol toward four substrates: 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, trans-4-phenylbut-3-en-2-one and p-nitrobenzyl chloride. Orchidectomy resulted in a decrease in transferase activity toward 1-chloro-2,4-dinitrobenzene, trans-4-phenylbut-3-en-2-one and p-nitrobenzyl chloride to 76, 64 and 70% of control. In contrast, transferase activity toward 1,2-dichloro-4-nitrobenzene was increased to 137% of control. To determine the role of specific androgens in the hormonal dependence of the glutathione S-transferases, rats were subcutaneously implanted for 4 weeks with either blank or steroid-filled sustained-release capsules at the time of orchidectomy. Transferase activities toward 1-chloro-2,4-dinitrobenzene or p-nitrobenzyl chloride were increased to control levels by testosterone but not by any of its 5α-reduced metabolites. Transferase activity toward trans-4-phenylbut-3-en-2-one was increased to control level by either dihydrotestosterone or 5α-androstan-3-α, 17β-diol. Activity toward 1,2-dichloro-4-nitrobenzene was decreased to control level by all of the androgens studied, testosterone, dihydrotestosterone, 5α-androstan-3β,17β-diol or 5α-androstan-3α,17β-diol. Thus, the hepatic glutathione S-transferases are under separate control and are differentially regulated by testosterone and its 5α-reduced metabolites.     

Structure-activity relationships in the induction of hepatic drug metabolism by azo compounds

1. Lipophilic azo compounds possessing 1-phenylazo-2-naphthol or 1-phenylazo-2-naphthylamine moieties induced cytochrome P-448 and related mono-oxygenase activities, UDP-glucuronyltransferase activity towards p-nitrophenol, glutathione-S-transferase activity towards 1-chloro-2,4-dinitrobenzene, aldehyde dehydrogenase, and menadione reductase activities. This pattern of induction by azo dyes is very similar to that by 3-methylcholanthrene.

2. None of the hydrophilic azo compounds tested and none of the other lipophilic azo compounds tested including 4-phenylazo-1-naphthol induced these activities.

3. It is suggested that the formation of a third six-membered ring fused to naphthalene in a phenanthrene-Iike arrangement by hydrogen bonding between the phenolic hydroxyl and azo nitrogen is required for induction.     

Protection from chlordecone-amplified carbon tetrachloride toxicity by cyanidanol: regeneration studies.

Previous work has shown that chlordecone (CD)-amplified CCl4 hepatotoxicity and lethality can be mitigated by pretreatment with cyanidanol. These studies also revealed that stimulated hepatocellular regeneration might play an important role in the cyanidanol protection of CD-amplified CCl4 toxicity. The present studies conducted over a time course of 0 to 120 hr after CCl4 challenge describe sequential changes in hepatic [3H]thymidine incorporation into hepatocellular nuclear DNA, polyamines and related enzymes, and histomorphometry of liver sections from variously treated rats. Male Sprague-Dawley rats (125-150 g) were maintained on a control diet or on a diet contaminated with CD (10 ppm) for 15 days and/or pretreated with cyanidanol (250 mg/kg, ip) at 48, 24, and 2 hr before a single ip injection of either a standard protocol dose (100 microliters/kg) or a low dose (50 microliters/kg, L) of CCl4 on Day 16 of the dietary protocol. Cyanidanol pretreatment significantly stimulated the hepatic [3H]thymidine incorporation into hepatocellular nuclear DNA of control rats irrespective of CD pretreatment. Similarly, polyamine metabolism was altered favorably for cell division, although mitotic index (metaphase) was not increased. Cyanidanol-stimulated [3H]thymidine incorporation was highly suppressed in rats receiving the CD + CCl4 standard dose combination treatment up to 36 hr, but after this time point a marked increase was observed. Hepatocellular regeneration, quantified histomorphometrically as volume density of cells in metaphase, was progressively increased in rats protected from CD + CCl4 interaction by cyanidanol, starting at 36 hr and lasting until 72 hr. Favorably altered polyamine metabolism was evident from the stimulated ornithine decarboxylase, as well as from the stimulated interconversion of the higher polyamines to maintain increased concentration of putrescine. Challenge by the same dose of CCl4 (100 microliters/kg) to CD-pretreated rats not protected by cyanidanol failed to cause any increase in [3H]thymidine incorporation up to 36 hr and resulted in animal death starting at 36 hr. In the surviving rats, [3H]thymidine incorporation at 48 hr was increased, but was less than 50% of the increase observed in the cyanidanol group. In these rats, attenuation in the stimulation of cell division and insufficiently increased putrescine levels were observed, which are consistent with the inadequate level of hepatocellular regeneration. With rats receiving CD + CCl4(L) combination, the [3H]thymidine incorporation at 48 hr was less than 50% of the increase of cyanidanol-protected rats. Cyanidanol pretreatment to the CD + CCl4 group of rats prevented the decrease in the hepatic DNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Impairment of sulfobromophthalein biliary excretion and inhibition of glutathione S-transferase activity induced by perhexiline maleate in rats

The effect of the antianginal agent perhexiline maleate (160 mg/kg i.g., daily for 4 days) on the biliary excretion of sulfobromophthalein (BSP) and BSP-glutathione and the hepatic activity of glutathione S-transferases was investigated in Wistar rats. Perhexiline maleate caused a significant reduction in the maximal biliary excretion of BSP (-28%). The decrease corresponded to a lowered excretion of the conjugated dye whereas the excretion of the parent compound did not change significantly. Administration of the drug caused no effect on the maximal biliary excretion of infused BSP-glutathione. Liver glutathione concentrations were similar in control and treated rats. Perhexiline maleate significantly reduced liver glutathione S-transferase activities toward BSP (-25%), 3,4-dichloronitrobenzene (DCNB) (-21%) and 1-chloro-3,4-dinitrobenzene (DNCB) (-27%). Kinetic studies of the enzyme in liver cytosol showed that perhexiline maleate induced an uncompetitive inhibition for the BSP substrate with a reduced Vmax and Km. The results indicate that the reduction in glutathione S-transferase activity plays an important role as a factor determining the impairment in the hepatobiliary transport of BSP caused by perhexiline maleate.     

Enhanced hepatotoxicity and toxic outcome of thioacetamide in streptozotocin-induced diabetic rats

Diabetes is known to potentiate thioacetamide (TA)-induced liver injury via enhanced bioactivation. Little attention has been given to the role of compensatory tissue repair on ultimate outcome of hepatic injury in diabetes. The objective of this study was to investigate the effect of diabetes on TA-induced liver injury and lethality and to investigate the underlying mechanisms. We hypothesized that hepatotoxicity of TA in diabetic rats would increase due to enhanced bioactivation-mediated liver injury and also due to compromised compensatory tissue repair, consequently making a nonlethal dose of TA lethal. On day 0, male Sprague-Dawley rats (250-300 g) were injected with streptozotocin (STZ, 60 mg/kg ip) to induce diabetes. On day 10 the STZ-induced diabetic rats and the nondiabetic rats received a single dose of TA (300 mg/kg ip). This normally nonlethal dose of TA caused 90% mortality in the STZ-induced diabetic rats. At various times (0-60 h) after TA administration, liver injury was assessed by plasma alanine aminotransferase (ALT), sorbitol dehydrogenase (SDH), and liver histopathology. Liver function was evaluated by plasma bilirubin. Cell proliferation and tissue repair were evaluated by [(3)H]thymidine ((3)H-T) incorporation and proliferating cell nuclear antigen (PCNA) assays. In the nondiabetic rat, liver necrosis peaked at 24 h and declined thereafter toward normal by 60 h. In the STZ-induced diabetic rat, however, liver necrosis was significantly increased from 12 h onward and progressed, culminating in liver failure and death. Liver tissue repair studies showed that, in the liver of nondiabetic rats, S-phase DNA synthesis was increased at 36 h and peaked at 48 h following TA administration. However, DNA synthesis was approximately 50% inhibited in the liver of diabetic rats. PCNA study showed a corresponding decrease of cell-cycle progression, indicating that the compensatory tissue repair was sluggish in the diabetic rats. Further investigation of tissue repair by employing equitoxic doses (300 mg TA/kg in the non-diabetic rats; 30 mg TA/kg in the diabetic rats) revealed that, despite equal injury up to 24 h following injection, the tissue repair response in the diabetic rats was much delayed. The compromised tissue repair prolonged liver injury in the diabetic rats. These studies suggest that the increased TA hepatotoxicity in the diabetic rat is due to combined effects of increased bioactivation-mediated liver injury of TA and compromised compensatory tissue repair.     

Troglitazone enhances the hepatotoxicity of acetaminophen by inducing CYP3A in rats

Troglitazone (TRZ) is the first of a new group of oral antidiabetic drugs, the thiazolidinediones, and is proven to lower plasma glucose levels in patients with type 2 diabetes mellitus. However, the concern has been raised because of several reports, in which severe hepatic dysfunction leading to hepatic failure was demonstrated in a few patients receiving the drug. We studied the effects of TRZ on the hepatotoxicity of carbon tetrachloride (CCl(4)) and acetaminophen (APAP) in rats, both of which exert their toxic effects through bioactivation associated with cytochrome P450 3A (CYP3A) and 2E1 (CYP2E1).Male standard (Wistar/ST) and type 2 diabetic model (GK/Jal) rats were kept on a powdered chow diet containing 0, 100, 500 mg/kg/rat of TRZ. Three weeks later, the rats were either sacrificed for an in vitro metabolism study or challenged with 0.50 g/kg CCl(4) p.o. or 0.75 g/kg APAP i.p.TRZ at 100 and 500 mg/kg/rat increased the CYP3A level as well as the testosterone 6beta-hydroxylation activities in liver microsomes, but did not affect CYP2E1. TRZ also enhanced APAP hepatotoxicity, as evidenced by significantly increased levels of alanine aminotransferase, aspartate aminotransferase and alpha-glutathione S-transferase in the plasma of rats, and by significantly low hepatic glutathione concentration.Our study demonstrated that high doses of TRZ can enhance hepatotoxicity of APAP in Wistar/ST and GK/Jal by inducing hepatic CYP3A.     

Effect of microsomal enzyme inducers on the soluble enzymes of hepatic phase II biotransformation

Numerous xenobiotics induce microsomal enzymes such as cytochrome P-450-dependent monooxygenases, epoxide hydrolase, and UDP-glucuronyltransferase by causing an increase in enzyme synthesis. Since induction of soluble enzymes involved in phase II biotransformation has not been thoroughly studied, effects of the following microsomal enzyme inducers on three important soluble enzymes were examined: phenobarbital (PB), 3-methylcholanthrene (3-MC), butylated hydroxyanisole (BHA), isosafrole (ISF), pregnenolone-16α-carbonitrile (PCN), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and trans-stilbene oxide (TSO). Representative microsomal enzymes of phase I pathways were examined simultaneously to indicate effective induction. The inducers selected produced the expected increases in hepatic cytochrome P-450 (75–170%), ethylmorphine (200–260%), and benzphetamine (100–260%) N-demethylation, benzo[a]pyrene hydroxylation (300%), ethoxyresorufin O-deethylation (2700%), and styrene oxide hydration (100–270%). The soluble conjugative enzymes studied were glutathione S-transferase, N-acetyltransferase, and sulfotransferase. Glutathione conjugation of 1,2-dichloro-4-nitrobenzene, 1-chloro-2,4-dinitrobenzene, and sulfobromophthalein was increased by TSO (100–160%), BHA (60–80%), ISF (60–80%), and PB (60–80%). β-Naphthylamine N-acetyltransferase activity was increased by PCN and 3-MC (60 and 40%, respectively). Only PCN was able to enhance sulfotransferase. Sulfation of 2-naphthol, taurolithocholate, and dehydroepiandrosterone was increased by 28, 111, and 140%, respectively. In conclusion, while microsomal enzymes could be readily induced, activity of soluble phase II enzymes was increased to a much lesser extent. Of the inducers studied, PCN was the most effective at increasing activity of soluble enzymes.     

Type 2 diabetic rats are sensitive to thioacetamide hepatotoxicity

Previously, we reported high hepatotoxic sensitivity of type 2 diabetic (DB) rats to three dissimilar hepatotoxicants. Additional work revealed that a normally nonlethal dose of CCl4 was lethal in DB rats due to inhibited compensatory tissue repair. The present study was conducted to investigate the importance of compensatory tissue repair in determining the final outcome of hepatotoxicity in diabetes, using another structurally and mechanistically dissimilar hepatotoxicant, thioacetamide (TA), to initiate liver injury. A normally nonlethal dose of TA (300 mg/kg, ip), caused 100% mortality in DB rats. Time course studies (0 to 96 h) showed that in the non-DB rats, liver injury initiated by TA as assessed by plasma alanine or aspartate aminotransferase and hepatic necrosis progressed up to 48 h and regressed to normal at 96 h resulting in 100% survival. In the DB rats, liver injury rapidly progressed resulting in progressively deteriorating liver due to rapidly expanding injury, hepatic failure, and 100% mortality between 24 and 48 h post-TA treatment. Covalent binding of 14C-TA-derived radiolabel to liver tissue did not differ from that observed in the non-DB rats, indicating similar bioactivation-based initiation of hepatotoxicity. S-phase DNA synthesis measured by [3H]-thymidine incorporation, and advancement of cells through the cell division cycle measured by PCNA immunohistochemistry, were substantially inhibited in the DB rats compared to the non-DB rats challenged with TA. Thus, inhibited cell division and compromised tissue repair in the DB rats resulted in progressive expansion of liver injury culminating in mortality. In conclusion, it appears that similar to type 1 diabetes, type 2 diabetes also increases sensitivity to dissimilar hepatotoxicants due to inhibited compensatory tissue repair, suggesting that sensitivity to hepatotoxicity in diabetes occurs in the absence as well as presence of insulin.     

Age-associated alterations in hepatic glutathione-S-transferase activities

Age-associated alterations of hepatic cytosolic glutathione-S-transferase (GST) activities towards sulfobromophthalein sodium tetrahydrate (BSP), styrene oxide (STOX), trans-4-phenyl-3-butene-2-one (PBO), 1,2-dichloro-4-nitrobenzene (DCNB), and 1-chloro-2,4-dinitrobenzene (CDNB) were investigated in Fischer-344 rats of both sexes with ages ranging from 1.5 to 28 months. The GST activities towards PBO and DCNB in male rats increased with age till 6-12 months when maximum values were attained, and then gradually decreased till 28 months when the values became the lowest. The GST activities towards STOX and BSP did not show any significant increase after 1.5 months and stayed at this level till 12 months, followed by a gradual decrease till 28 months when the values were the lowest. In contrast, the GST activity towards CDNB in male rats did not show much of an age-associated alteration. Age-associated alterations in GST activities in females were much smaller than those observed in males. Sex differences in GST activities (significantly higher male values than female values) were observed with all the substrates examined at least at some time of the animal life. The kinetic studies of GST activities indicated that alterations in the relative abundance as well as the total quantity of GST isozymes caused the substrate selective alterations of GST activities with age.     

Deficient induction of sulfobromophthalein conjugating activity by phenobarbital in hamster liver

Administration of phenobarbital, a known inducer of glutathione S-transferase activity in rat liver, failed to stimulate sulfobromophthalein (BSP) conjugation by liver cytosol in hamsters. The latter displayed poor ability to conjugate this substrate, despite very high glutathione-conjugating activity with the broad-spectrum substrate 1-chloro-2,4-dinitrobenzene (CDNB). Of the six substrates tested, in this species, 1,2-epoxy-3-(4-nitrophenoxy)propane (ENPP) was the only one whose conjugation was greatly enhanced by phenobarbital (+172%). Nevertheless, hamsters proved as responsive to phenobarbital induction as rats, since it increased their relative liver weight and microsomal enzyme activity. The deficient induction of liver BSP-conjugating activity observed with phenobarbital is consistent with the finding that it did not affect the hepatic transport of this substrate in hamsters.     

Chronic physical activity: hepatic hypertrophy and increased total biotransformation enzyme activity.

Does chronic voluntary physical activity alter hepatic or intestinal capacities for xenobiotic biotransformation? This question was investigated by comparing biotransformation enzyme activities in liver and small intestine of active and sedentary rats. Male rats allowed unlimited access to a running wheel and fed ad lib. for 6 weeks were weight-matched to sedentary controls; the active rats ate 22% more food than the sedentary rats (P less than 0.05). Active rats ran 2.8 +/- 0.6 miles/day. Liver weights were higher in the active rats (11.2 +/- 0.2 vs 9.8 +/- 0.2 g; P less than 0.05), as were total liver protein, and liver microsomal and cytosolic protein (P less than 0.05). As a result of liver hypertrophy, the active rats showed higher total liver activity of several biotransformation enzymes, including 2-naphthol sulfotransferase, styrene oxide hydrolase, benzphetamine N-demethylase, ethacrynic acid glutathione S-transferase and morphine UDP-glucuronosyltransferase (P less than 0.05). In contrast, there was no detectable difference in total liver N-acetyltransferase activity toward p-aminobenzoic acid, 2-naphthylamine, and 2-amino-fluorene as well as, relative hepatic enzyme activity (expressed per g liver or per mg protein) and total and relative intestinal enzyme activity. We conclude that chronic voluntary physical activity, accompanied by an increased food intake, results in liver hypertrophy and potentially increases total hepatic capacity to biotransform certain xenobiotic chemicals.     

CHANGES IN HEPATIC CYTOSOLIC GLUTATHIONE S-TRANSFERASE ENZYMES INDUCED BY CLOTRIMAZOLE TREATMENT IN RATS

1. The influence of the antifungal agent clotrimazole on cytosolic glutathione S-transferase activities was studied in male Wistar rats. 2. Animals received clotrimazole by gastric lavage for 3 days (75 mg/kg per day). Hepatic glutathione S-transferase activity was determined with five different substrates: 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), p-nitro-benzyl chloride (PNBC), ethacrynic acid (EA) and trans-4-phenyl-3-buten-2-one (TPBO). 3. The largest increases in glutathione S-transferase activity were found with CDNB, DCNB and PNBC (+61%, +50% and +50%, respectively, when expressed per mg of cytosolic protein). Enzyme activity toward EA was induced to a lower extent (+33%). Changes in the formation of the conjugate of TPBO were relatively small (+22%). 4. These data indicate a differential induction of glutathione S-transferase isoenzymes and suggest that clotrimazole is a phenobarbital-type inducer of enzyme activity.