Effect of glutathione depletion on aminopyrine and formaldehyde metabolism

In previous studies, diethylmaleate (DEM)- and phorone-induced hepatic glutathione (GSH) depletion in rats was accompanied by impaired evolution of 14CO2 from the N-14C-labeled methyl groups of aminopyrine, which in turn was attributed to impaired generation of formaldehyde, its subsequent oxidation to formate, or to some combination of both. In the present study, l-buthionine sulfoximine (BSO)-induced hepatic GSH depletion was also accompanied by decreased evolution of CO2 from aminopyrine, but the extent of the fall in CO2 was less than that induced by DEM or phorone, even though the decrease in hepatic GSH was comparable with all three GSH-lowering compounds. Incubation of freshly prepared normal hepatic microsomes in vitro with the GSH-lowering agents resulted in impaired aminopyrine-N-demethylase (APDM) activity with inhibition by phorone greater than DEM greater than BSO. By contrast, hepatic microsomes prepared from rats pretreated with these compounds had normal APDM activity. 14CO2 evolution from i.p. administered [14C]formaldehyde was not impaired by any of the GSH-lowering compounds. Thus, assessment of APDM activity and formaldehyde metabolism did not unequivocally establish the mechanism(s) by which CO2 evolution from aminopyrine is depressed by DEM, phorone and BSO, although low GSH is likely to impair metabolism of formaldehyde formed in liver after demethylation of aminopyrine. Quantitative differences in the degree of depression of CO2 evolution suggest that at least DEM and phorone exert an additional inhibitory effect by a GSH-independent mechanism. This may involve inhibition of aminopyrine-N-demethylase activity.     

Effect of various non-hepatotoxic compounds on urinary and liver taurine levels in rats

Administration of compounds which alter protein synthesis or sulphur amino acid metabolism in rats results in changes in the excretion of urinary taurine. Treatment with diethylmaleate (DEM) or phorone, which will deplete glutathione (GSH), reduces taurine excretion, whereas treatment with buthionine sulphoximine (BSO), which will inhibit glutathione synthesis, increases taurine excretion. Treatment with cycloheximide, an inhibitor of protein synthesis, increases taurine excretion, whereas pretreatment with phenobarbital, which will increase protein synthesis, decreases taurine excretion. Administration of agents which damage organs other than the liver such as the kidney, heart and testes, does not increase urinary taurine.     

EVALUATION OF HEPATIC TOXICITY OF SEVEN-DAY REPEATED-DOSE GLUTATHIONE-DEPLETING REGIMENS IN RATS

Glutathione (GSH) depletion is a common method for examining the role of oxidative stress in the toxicity of novel compounds. Several treatment regimens for inducing hepatic GSH depletion in rats were examined for their suitability for use in a 7-day repeated-dose paradigm. Male Wistar rats (5/treatment) were administered either 2,6-dimethyl-2,5-heptadien-4-one (phorone, 250 mg/kg ip), diethylmaleate (DEM, 600 mg/kg ip), ethanol in drinking water (150 mL/L), or L-buthionine-SR-sulfoximine (BSO) in drinking water (4.446 g/L) with and without supplemental ip administration (890 mg/kg bid) for 7 days. Additional groups of 5 animals were given phorone or DEM and sacrificed 2 h after dose. Significant body weight gain suppression relative to control was evident in all treated groups but only animals given the ip/water BSO treatment resulted in mean weight loss (4%). Liver weights were significantly increased by 7 days of phorone treatment and decreased by ip/water BSO treatment. No clinically significant effects were noted on hepatic serum chemistry parameters. No hepatic histopathology was produced by any treatment, but phorone produced increased hepatocellular mitoses. BSO administered in the drinking water without supplemental ip administration appeared to be the most suitable model for routine assessment of hepatic GSH depletion in mechanistic models. The model was practical, did not induce hepatic pathology, and produced marked decreases in hepatic cytosolic GSH and moderate decreases in hepatic mitochondrial GSH.     

Effect of glutathione depletion on sulfate activation and sulfate ester formation in rats

Sulfation of organic compounds requires activation of inorganic sulfate via formation of adenosine 3'-phosphate 5'-phosphosulfate (PAPS). Inorganic sulfate can be formed by sulfoxidation of cysteine, which can be derived from GSH. Thus, a decrease in hepatic GSH may impair formation of inorganic sulfate, the synthesis of PAPS, and the sulfation of chemicals. This hypothesis was tested by investigating the effect of GSH depletion on the levels of inorganic sulfate in serum and of PAPS in liver, and on the capacity to form the sulfate conjugate of harmol in rats. Phorone (2 mmol/kg, i.p.) decreased hepatic GSH (97%), serum inorganic sulfate (63%), and hepatic PAPS (48%). Diethyl maleate and vinylidene chloride (6 mmol/kg, each, i.p.) were less effective than phorone in decreasing GSH in liver and inorganic sulfate in serum, and they did not alter hepatic PAPS levels. Three hours after phorone treatment, the nadir of hepatic PAPS concentration, harmol was injected in order to assess sulfation in vivo. After administration of harmol (100 and 300 mumol/kg, i.v.), less harmol sulfate and more harmol glucuronide were found in the serum of phorone-treated rats as compared to control rats. At the higher dosage of harmol, phorone reduced the biliary excretion of harmol sulfate while increasing the biliary excretion of harmol glucuronide. These results indicate that severe GSH depletion decreases PAPS formation and sulfation of chemicals. However, an increase in glucuronidation may compensate for the impaired sulfation.     

Protection against alpha-naphthylisothiocyanate-induced liver injury by decreased hepatic non-protein sulfhydryl content.

alpha-Naphthylisothiocyanate (ANIT) injures bile duct epithelium and hepatic parenchymal cells in rats. It is commonly believed that ANIT must undergo bioactivation by hepatic, cytochrome P450-dependent mixed-function oxidases (MFO), since agents which are inducers or inhibitors of hepatic MFO activity enhance or attenuate, respectively, the liver injury associated with ANIT. Several of these agents also affect hepatic glutathione (GSH) content and/or GSH S-transferase activity in a manner to suggest a causal role for GSH in ANIT-induced hepatotoxicity. To determine whether GSH might be involved in the mechanism of injury, buthionine sulfoximine (BSO), diethyl maleate (DEM), or phorone was used to reduce hepatic non-protein sulfhydryl (NPSH) content, an indicator of GSH content. Twenty-four hours after ANIT treatment, rats exhibited cholestasis and elevations in serum of total bilirubin concentration, total bile acid concentration, aspartate aminotransferase (AST) activity, and gamma-glutamyltransferase activity. Cotreatment of rats with BSO decreased NPSH content by 70% at 24 hr and prevented the cholestasis and elevations in serum markers of liver injury caused by ANIT. Likewise, cotreatment of rats with DEM afforded protection against markers of liver injury. Phorone treatment attenuated ANIT-induced elevations in serum total bilirubin concentration and AST activity. Although BSO treatment afforded protection against ANIT-induced liver injury at 24 hr, the injury was evident at 48 hr, and it appeared to coincide with a return of hepatic NPSH content. These results suggest that GSH plays a causal or permissive role in the liver injury caused by ANIT.     

Role of glutathione in metabolic degradation of dichloromethane in rats

Dichloromethane (DCM) elimination and carboxyhemoglobin (COHb) generation were examined in adult female SD rats pretreated with a glutathione (GSH) depletor(s). Rats were treated with either buthionine sulfoximine (BSO; 2 mmol/kg, i.p.), diethylmaleate (DEM; 3 mmol/kg, i.p.), phorone (PHO; 1 mmol/kg, i.p.) or BSO plus PHO (BSO; 2 mmol/kg +PHO; 0.5 mmol/kg, i.p.). The hepatic GSH concentration was significantly reduced by each treatment. Decrease in hepatic GSH was maintained at least for 10 h after BSO treatment but recovered rapidly in rats treated with DEM or PHO. The hepatic p-nitrophenol hydroxylase activity was not affected by the GSH depletors at the dose used in this study. Rats were treated with an i.p. injection of DCM (3 mmol/kg) and the concentrations of DCM and the COHb levels in blood were monitored. In rats pretreated with a GSH depletor, the peak COHb level was significantly greater than that of rats treated with DCM only. The peak COHb level attained in each group of rats appeared to be inversely related to the magnitude of reduction in hepatic GSH levels. The half-life of DCM in blood was also increased in rats pretreated with the GSH depletor(s). The results indicate that the GSH-dependent metabolic reaction has an important role in the overall elimination of DCM as well as in the metabolic generation of carbon monoxide (CO) from this solvent.     

Effect of cytochrome P450 isozyme induction and glutathione depletion on the metabolism of CS2 to TTCA in rats

 Analysis of 2-thiothiazolidine-4-carboxylic acid (TTCA), a metabolite of carbon disulfide (CS₂), is used in the biological monitoring exposure to CS₂ at work. In order to clarify the metabolic reasons for individual variation in the urinary excretion of TTCA, the latter was studied in rats pretreated with model cytochrome P450 (CYP) enzyme inducers or glutathione (GSH) depletors. Ethanol, phenobarbital (PB) or 3-methylcholanthrene (MC) did not increase 24-h TTCA output following CS₂ inhalation (50 or 500 ppm, 6 h). After oral dosing (10 mg/rat), PB had an inhibiting effect on the excretion rate of TTCA. Tissue GSH depletors phorone, L-buthionine-(RS)-sulfoximine (BSO) and diethylmaleate (DEM) decreased TTCA excretion in rats given an oral dose (10 mg/rat) of CS₂. The initial inhibition by phorone and DEM was reversed after 6 h and from 12 h onward the TTCA in urine exceeded the control level, an effect not seen with BSO. The proportion of CS₂ excreted in urine as TTCA within 24 h was 1.7% in control rats and 1% after BSO treatment, 1.3% after PB, 1.7% after acetone, 1.8% after MC, 2.0% after phorone and 2.5% after DEM treatment. The amount of TTCA in urine increased with the CS₂ dose in a non-linear fashion: 1.6 μmol (50 ppm/6 h) vs. 4.9 μmol (500 ppm/6 h), and 0.2 μmol (1 mg/kg) versus 3.6 μmol (100 mg/kg). It is concluded that induction of different cytochrome P450 isoforms and transient glutathione depletion have only minor effects on the disposition of TTCA in rats following low-level CS₂ exposure persistently low glutathione level as achieved by E.G. BSO, markedly decreased the metabolism of CS₂ to TTCA; these metabolic effectors are unlikely to have a major role in the individual variation of CS₂ metabolism in exposed workers. Received: 14 June 1994/Accepted: 25 August 1994     

Elimination of Thioethers Following Administration of Naphthalene and Diethylmaleate to the Rhesus Monkey

ABSTRACT

As a measure of glutathione (GSH) conjugation, urinary, fecal and biliary excretion of thioethers and hepatic GSH content were measured in rhesus monkeys following administration of single doses of naphthalene and diethylmaleate (DEM). Naphthalene had little or no effect on hepatic GSH content and the excretion of thioethers into urine, feces or bile of rhesus monkeys which is similar to that observed in chimpanzees and humans and is in contrast to results obtained from rats. Apparently, conjugation of naphthalene and/or its metabolites with GSH does not play a major role in the metabolism of naphthalene in primates, whereas it is one of the major pathways in rodents. Rhesus monkeys, like chimpanzees, excreted about 13% of the various doses of DEM (30, 75 and 200 mg/kg) as thioethers into urine which is half of that excreted by rats. Six hrs after administration of 200 mg/kg DEM, the hepatic GSH content was decreased by 90% in the rhesus monkey. During the first day after this dose (200 mg/kg), the increase in the excretion of thioethers into bile corresponded to about 15% of the dose of DEM administered. Since fecal excretion of thioethers corresponded to only 1% of the dose and urinary excretion represented 12% of the dose, it appears that biliary thioethers of DEM are reabsorbed from the intestine and then excreted into urine. It appears that the rhesus monkey as well as the chimpanzee is, whereas the rat is not, a good animal model to study GSH-related conjugation reactions with predictive value for man.     

The role of thymus-dependent T cells in hexachlorobenzene-induced inflammatory skin and lung lesions

The involvement of thymus-dependent T cells in the inflammatory skin and lung lesions and spleen effects induced by hexachlorobenzene (HCB) was investigated by using genetically athymic and euthymic WAG/Rij rats and Brown Norway (BN) rats with or without depletion of T cells by adult thymectomy, lethal irradiation, and bone marrow reconstitution. Rats were exposed to diets with no supplementation or diets supplemented with 150 or 450 mg HCB per kg diet for 4 (BN) or 6 (WAG/Rij) weeks. Skin lesion development and body weight gains were assessed during exposure and spleen and liver weights as well as histopathologic changes in skin, lung, and spleen were assessed after exposure. Oral HCB exposure of athymic and euthymic rats of both rat strains resulted in a dose-dependent increase of relative liver weight at doses of 150 and 450 mg/kg HCB and increased relative spleen weights at a dose of 450 mg/kg. HCB exposure of both strains further resulted in inflammatory changes in skin, lungs, and splenic red pulp independent of the T cell status except for skin lesions in the BN strain. HCB-exposed T cell-competent BN rats showed faster skin lesion development than the T cell-depleted rats, although qualitatively and quantitatively similar skin pathology was observed at the end of the 4-week exposure in both groups. In the WAG/Rij strain skin lesions could not be comparatively assessed due to preexistent inflammatory skin pathology in the nude rats. This study showed that thymus-derived T cells are not required for the induction of skin and lung pathology and splenic changes by HCB and therefore it is suggested that HCB acts differently from many allergenic and autoimmunogenic low molecular weight compounds that trigger pathology via thymus-dependent mechanisms. A role for mononuclear phagocytes and, in BN rats, eosinophilic granulocytes, in the HCB-induced pathology is suggested since these cells were prominently present in the HCB-induced lesions.     

Glutathione-dependent biliary excretion of arsenic

This study aimed to clarify whether glutathione (GSH) plays a role in the hepatobiliary transport of arsenic. For this purpose, the biliary excretion of 74As was measured in urethane-anaesthetized rats for 2 hr after the administration of labelled sodium arsenite (50 mumol/kg, i.v.) or arsenate (150 mumol/kg, i.v.) and under the influence of sulfobromophthalein (BSP), indocyanine green (ICG) or diethyl maleate (DEM) which are known to diminish hepatobiliary transport of GSH. Although the biliary excretion of arsenic was different after arsenite and arsenate administration in terms of quantity (19% vs 6% of dose in 2 hr, respectively) and time course, arsenic excretion responded similarly to BSP (50 mumol/kg, i.v.), ICG (25 mumol/kg, i.v.) or DEM (4 mumol/kg, i.p.) irrespective of the injected arsenical. Initially the biliary excretion of arsenic in rats with either arsenite or arsenate was significantly reduced, but then moderately increased by BSP and, more lastingly, depressed by ICG, whereas it was virtually abolished by DEM. The responses of arsenic excretion to BSP, ICG and DEM were related, both proportionally and temporally, to the effects exerted by these agents on the hepatobiliary transport of GSH, as assessed by the biliary excretion of non-protein thiols. These findings indicate that the biliary excretion of arsenic after the administration of either arsenite or arsenate is dependent on the hepatobiliary transport of GSH. Transport of arsenic as a GSH complex may account for the GSH dependence of biliary arsenic excretion.     

Changes in hepatic glutathione concentration modify cadmium-induced hepatotoxicity

Cd has a strong affinity for sulfhydryl groups and is hepatotoxic. Thus, to further understand the mechanism of Cd-induced liver injury, the effect of increased and decreased hepatic glutathione (GSH) concentration on Cd-induced liver injury was examined. Liver GSH was lowered by pretreating rats with phorone (250 mg/kg, ip) or diethyl maleate (0.85 mg/kg, ip) 2 hr prior to challenge with various doses of Cd. Ten hours after Cd (1) 40–80% of the rats pretreated with phorone or diethyl maleate and challenged with 1.0–2.0 mgCd/kg died whereas no mortality was observed in the control group; (2) plasma enzyme activities of alanine (ALT) and aspartate (AST) aminotransferase and sorbitol dehydrogenase (SDH) were markedly increased in phorone and diethyl maleate-pretreated rats challenged with Cd (0.7–2.0 mg/kg) versus control rats; and (3) moderate changes in liver histology were observed in corn oil pretreated and Cd challenged rats, while prior depletion of GSH potentiated histopathologic changes in liver produced by Cd alone. Another group of rats received cysteine (1.9 g/kg, po) 3 hr prior to injection of a lethal dose of Cd. Cysteine pretreatment increased liver GSH levels by 22% 3 hr after administration and attenuated Cd-induced liver injury as evidenced by marked decreases in plasma ALT, AST, and SDH activities. Pathological changes in liver were also reduced. These data indicate that liver reduced GSH concentration is important in modulating Cd-induced hepatotoxicity.     

Effect of diethylmaleate and other glutathione depletors on protein synthesis

The alpha, beta-unsaturated carbonyl compound diethylmaleate (DEM) depletes glutathione (GSH) from liver and other tissues, and for this reason it is often used in toxicological research to study the GSH-mediated metabolism of xenobiotics. In addition to GSH depletion, however, DEM has been shown to have other nonspecific effects, such as alteration of monooxygenase activities or glycogen metabolism. In this study we found that DEM (1 ml/kg) inhibited protein synthesis in brain and liver, following in vivo administration to mice. Protein synthesis was measured as the incorporation of [3H]valine into trichloroacetic acid-precipitable material. Administration of DEM also decreased body temperature by 2-3 degrees. By increasing the environmental temperature from 22 degrees to 35 degrees the hypothermic effect of DEM was prevented, without affecting its ability to deplete GSH from brain and liver. Furthermore, when mice were maintained at 35 degrees, DEM still caused a significant decrease in protein synthesis, suggesting that this effect was only partially due to hypothermia. To test whether inhibition of protein synthesis was related to GSH depletion, groups of animals were dosed with the alpha, beta-unsaturated carbonyl phorone (diisopropylidenacetone) or the specific inhibitor of GSH synthesis, buthionine sulfoximine (BSO). Phorone decreased GSH in liver and brain; however, it had no effect on protein synthesis. BSO decreased GSH levels in liver and kidney, but not in brain, and did not have any effect on protein synthesis in any of these tissues, nor did it cause any hypothermia. Furthermore, when hepatic GSH content was decreased by in vivo administration of DEM or BSO, there was no inhibition of protein synthesis measured in vitro. These results indicate that, at the dose normally used to deplete GSH from various tissues. DEM also exerts an inhibitory effect on protein synthesis, which appears to be only partially due to its hypothermic effect, and is independent from GSH depletion. BSO, which, in our experimental conditions, lacks this and other nonspecific effects, might be a good alternative for studies aimed at characterizing the role of GSH in the metabolism and toxicity of chemicals.     

Role of glutathione in the biliary excretion of the arsenical drugs trimelarsan and melarsoprol

After administration of the inorganic sodium arsenite or arsenate to rats, the biliary excretion of arsenic is rapid, is accompanied by the biliary output of large amounts of GSH, and is completely arrested by the GSH depletor diethyl maleate (DEM). We studied the biliary excretion of trimelarsan (TMA) and melarsoprol (MAP) in rats in order to determine whether biliary excretion is also significant in the disposition of these trivalent organic arsenicals that are used as therapeutic agents and whether GSH is also involved in their hepatobiliary transport. After injection of either drug (100 micromol/kg, i.v.), arsenic was rapidly excreted in bile (up to 1 micromol/kg. min, approximately 55% of dose/100 min). Concurrently, TMA and MAP increased the biliary output of GSH 3- and 6 fold, and lowered the hepatic GSH content by 24% and 27%, respectively. In TMA-injected rats, pretreatment with DEM or buthionine sulfoximine decreased the initial biliary excretion of arsenic by 75% and 40%, respectively, whereas in MAP-injected rats these GSH depletors diminished arsenic output by 45% and 20%. Both arsenicals reacted with GSH in vitro, giving rise to the same product, which was also shown by HPLC analysis to be a major biliary metabolite of both TMA and MAP. This metabolite was sensitive to gamma-glutamyltranspeptidase in vitro and its biliary excretion was virtually prevented by the GSH depletors, confirming that it is a GSH conjugate (purportedly melarsen-diglutathione). Some TMA was excreted in the bile unchanged, whereas a significant amount of MAP also appeared there as two glucuronides. The biliary excretion of unchanged TMA and MAP glucuronides was increased by experimental depletion of GSH. These studies indicate that the biliary excretion of TMA and MAP (1) is very significant in their disposition, (2) is partially dependent on the hepatic availability of GSH, as these arsenicals are excreted in part as a GSH conjugate, and (3) is concomitant with the increased appearance of GSH in bile, probably originating from dissociation of the unstable GSH conjugate of these arsenicals. Thus, conjugation with GSH is important in the elimination of both TMA and MAP, although glucuronidation is also involved in the fate of MAP.     

Protective role for type-1 metabotropic glutamate receptors against spike and wave discharges in the WAG/Rij rat model of absence epilepsy

Eight-month old WAG/Rij rats, which developed spontaneous occurring absence seizures, showed a reduced function of mGlu1 metabotropic glutamate receptors in the thalamus, as assessed by in vivo measurements of DHPG-stimulated polyphosphoinositide hydrolysis, in the presence of the mGlu5 antagonist MPEP as compared to age-matched non-epileptic control rats. These symptomatic 8-month old WAG/Rij rats also showed lower levels of thalamic mGlu1α receptors than age-matched controls and 2-month old (pre-symptomatic) WAG/Rij rats, as detected by immunoblotting. Immunohistochemical and in situ hybridization analysis indicated that the reduced expression of mGlu1 receptors found in symptomatic WAG/Rij rats was confined to an area of the thalamus that excluded the ventroposterolateral nucleus. No mGlu1 receptor mRNA was detected in the reticular thalamic nucleus. Pharmacological manipulation of mGlu1 receptors had a strong impact on absence seizures in WAG/Rij rats. Systemic treatment with the mGlu1 receptor enhancer SYN119, corresponding to compound RO0711401, reduced spontaneous spike and wave discharges spike-wave discharges (SWDs) in epileptic rats. Subcutaneous doses of 10 mg/kg of SYN119 only reduced the incidence of SWDs, whereas higher doses (30 mg/kg) also reduced the mean duration of SWDs. In contrast, treatment with the non-competitive mGlu1 receptor antagonist, JNJ16259685 (2.5 and 5 mg/kg, i.p.) increased the incidence of SWDs. These data suggest that absence epilepsy might be associated with a reduction of mGlu1 receptors in the thalamus, and that compounds that amplify the activity of mGlu1 receptors might be developed as novel anti-absence drugs. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.     

Enalapril hepatotoxicity in the rat. Effects of modulators of cytochrome P450 and glutathione.

The effects of modulators of cytochrome P450 and reduced glutathione (GSH) on the hepatotoxicity of enalapril maleate (EN) were investigated in Fischer 344 rats. Twenty-four hours following the administration of EN (1.5 to 1.8 g/kg), increased serum transaminases (ALT and AST) and hepatic necrosis were observed. Pretreatment of the animals with pregnenolone-16 alpha-carbonitrile, a selective inducer of the cytochrome P450IIIA gene subfamily, enhanced EN-induced hepatotoxicity, whereas pretreatment with the cytochrome P450 inhibitor, cobalt protoporphyrin, reduced the liver injury. Depletion of hepatic non-protein sulfhydryls (NPSHs), an indicator of GSH, by combined treatment with buthionine sulfoximine (BSO) and diethyl maleate (DEM) produced marked elevations in serum transaminases by 6 hr after EN treatment. Administered on its own, EN decreased hepatic NPSH content and when combined with the BSO/DEM pretreatment, the liver was nearly completely devoid of NPSHs. Protection from EN-induced hepatotoxicity was observed in animals administered L-2-oxothiazolidine-4-carboxylic acid, a cysteine precursor. Together, these observations suggest the involvement of cytochrome P450 in EN bioactivation and GSH in detoxification. The results corroborate previous in vitro observations pertaining to the mechanism of EN-induced cytotoxicity towards primary cultures of rat hepatocytes. Although the doses of EN used in this study were far in excess of therapeutic doses, under certain circumstances, this metabolism-mediated toxicologic mechanism could form the basis for idiosyncratic liver injury in patients receiving EN therapy.     

Role of glutathione in reduction of arsenate and of gamma-glutamyltranspeptidase in disposition of arsenite in rats

Arsenate (AsV), the environmentally prevalent form of arsenic, is converted sequentially in the body to arsenite (AsIII), monomethylarsonic acid (MMAsV), monomethylarsonous acid (MMAsIII), and dimethylarsinic acid (DMAsV) and some trimethylated metabolites. Although the biliary excretion of arsenic in rats is known to be glutathione (GSH)-dependent, involving transport of arsenic-GSH conjugates, the role of GSH in the reduction of AsV to the more toxic AsIII in vivo has not been defined. Therefore, we studied how the fate of AsV is influenced by buthionine sulfoximine (BSO), which depletes GSH in tissues. Control and BSO-treated rats were given AsV (50 micromol/kg, i.v.) and arsenic metabolites in bile, urine, blood and tissues were analysed by HPLC-HG-AFS. BSO increased retention of AsV in blood and tissues and decreased appearance of AsIII in blood, bile (by 96%) and urine (by 63%). The biliary excretion of MMAsIII was also nearly abolished, the appearance of MMAsIII and MMAsV in the blood was delayed and the renal concentrations of these monomethylated arsenicals were decreased by BSO. Interestingly, appearance of DMAsV in blood and urine remained unchanged and the concentrations of this metabolite in the kidneys and muscle were even increased in response to BSO. To test the role of gamma-glutamyltranspeptidase (GGT) in arsenic disposition, the effect of the of the GGT inhibitor acivicin was investigated in rats injected with AsIII (50 micromol/kg, i.v.). Acivicin lowered the hepatic and renal GGT activities and increased the biliary as well as urinary excretion of GSH, but failed to alter the disposition (i.e. blood and tissue concentrations, biliary and urinary excretion) of AsIII and its metabolites. In conclusion, shortage of GSH decreases not only the hepatobiliary transport of arsenic, but also reduction of AsV and the formation of monomethylated arsenic, while not hindering the production of dimethylated arsenic. While GSH plays an important role in the disposition and toxicity of arsenic, GGT, which hydrolyses GSH and GSH conjugates, apparently does not influence the fate of the GSH-reactive trivalent arsenicals in rats.     

Effects of buthionine sulfoximine and diethyl maleate on glutathione turnover in the channel catfish.

Despite the growing use of fish in toxicological studies, little is known regarding glutathione (GSH) metabolism and turnover in these aquatic species. Therefore, we examined GSH metabolism in the liver and gills of channel catfish (Ictalurus punctatus), a commonly employed aquatic toxicological model. Treatment of channel catfish with L-buthionine-S,R-sulfoximine (BSO, 400 or 1000 mg/kg, i.p.), an inhibitor of GSH biosynthesis, did not deplete hepatic GSH in channel catfish. In addition, hepatic GSH concentrations did not fluctuate in catfish starved for 3 days, indicating relatively slow turnover of hepatic GSH. However, hepatic GSH concentrations were reduced significantly (P less than 0.05) after 7 days of starvation. Administration of the thiol alkylating agent diethyl maleate (DEM, 0.6 mL/kg, i.p.) resulted in depletion of 85% of hepatic GSH at 6 hr post-DEM, with complete GSH recovery observed at 24 hr post-DEM. Co-administration of BSO and DEM (1000 mg/kg, 0.6 mL/kg, respectively) substantially depleted gill GSH and eliminated detectable liver GSH. Following BSO/DEM, GSH recovery in hepatic mitochondria occurred more rapidly than did liver cytosolic GSH. gamma-Glutamylcysteine synthetase (GCS) activities were comparable in the 10,000 g supernatants of catfish liver and gills (204 +/- 21 and 268 +/- 20 nmol/min/mg protein, respectively) whereas gamma-glutamyltranspeptidase (GGT) activity was not detected in the 600 g post-nuclear fraction of either liver or gills. In conclusion, i.p. administration of DEM was an effective means for achieving short-term hepatic GSH depletion in channel catfish, whereas co-administration of BSO and DEM elicited prolonged and extensive hepatic GSH depletion in this species. Like rodents, channel catfish maintained physiologically distinct hepatic mitochondrial and cytosolic GSH pools, and also regulated hepatic GSH levels by in situ hepatic GSH biosynthesis. However, unlike rodents, there was no evidence for a labile hepatic cytosolic GSH pool in channel catfish. These similarities and differences need to be considered when designing toxicological studies involving the GSH pathway in channel catfish and possibly other fish species.     

Effect of glutathione modulation using buthionine sulfoximine on DNA methylation by dimethylnitrosamine in the rat

An in vivo study was carried out in order to determine whether glutathione (GSH) might serve as a scavenger for the supposed electrophilic methylating fragment derived from dimethylnitrosamine (DMN) and thus function to decrease the degree of cellular macromolecule interaction, estimated by measuring the DNA methylation yield. After a 4-hr pretreatment with DL-buthionine-SR-sulfoximine (BSO), a specific inhibitor of GSH synthesis, male Sprague-Dawley rats were dosed with radiolabeled DMN (250 micrograms/kg). Four hours later the animals were killed and the livers and kidneys were excised. The DNA isolated from these organs was hydrolyzed in mild acid, and the liberated purines were quantified utilizing HPLC and liquid scintillation counting. The 70-75% GSH depletion in the liver and kidney resulting from BSO pretreatment did not have any significant effect on the degree of DNA methylation as assessed by the 7-methylguanine/guanine yield. In control experiments we found that DMN doses greater than 1 mg/kg had a marked effect on liver and kidney GSH levels after 4 hr.     

Absence epilepsy and regional blood-brain barrier permeability: the effects of pentylenetetrazole-induced convulsions.

This study was designed to evaluate the blood-brain barrier permeability characteristics of the WAG/Rij strain of rats that are an ideal model for human absence epilepsy, in controls and pentylenetetrazole-induced seizures conditioned to Evans blue-albumin. For this, WAG/Rij and Wistar rats were treated with either saline or 55 mg kg-1 pentylenetetrazole i.v. after the rats were injected with 3 ml kg-1 of 2% Evans blue. Total duration of seizure activity and regional blood-brain barrier permeability changes were determined and compared with control Wistar rats. The duration of convulsive activity which was induced by pentylenetetrazole was significantly longer in WAG/Rij rats than in Wistar rats. The blood-brain barrier opening to Evans blue was not the case in saline- injected WAG/Rij or Wistar rats, but this was clearly seen in both strains after pentylenetetrazole-induced convulsions. EB leakage was mainly seen in the cortical areas, cerebellum, pons, thalamus, hypothalamus and corpus striatum of WAG/Rij rat brain, whereas this was recorded in the preoptic area, bulbus olfactorius, midbrain, hypothalamus, corpus striatum and inferior colliculus of the Wistar rats brain. As a result, the WAG/Rij rats were more susceptible than Wistar rats to PTZ-induced generalised tonic-clonic convulsions, and a different pattern in PTZ-induced changes in BBB permeability was observed between WAG/Rij rats and Wistar rats.     

Oleanolic acid protects against myocardial ischemia-reperfusion injury by enhancing mitochondrial antioxidant mechanism mediated by glutathione and alpha-tocopherol in rats

The effect of oleanolic acid (OA) pretreatment on myocardial ischemia-reperfusion (I-R) injury was investigated using an ex vivo rat heart model. Pretreatment with OA at daily doses (0.6 and 1.2 mmol/kg) for 3 days significantly protected against I-R injury in isolated rat hearts, as evidenced by the decrease in the extent of lactate dehydrogenase (LDH) leakage and improvement in contractile force recovery. The cardioprotection was associated with a slight increase in mitochondrial reduced glutathione (GSH) level and a significant increase in mitochondrial alpha-tocopherol (alpha-TOC) level, when compared with the unpretreated I-R group. To further investigate the mechanism of myocardial protection, pretreatment with a single dose of OA (1.2 mmol/kg) produced a time-dependent protection against myocardial I-R injury as assessed by LDH leakage, with the maximum extent of protection occurring at 48 hour post-dosing. The maximum cardioprotection was associated with parallel increases in mitochondrial GSH and alpha-TOC levels in ischemic-reperfused hearts, with the stimulation of the alpha-TOC level being optimal. Furthermore, buthionine sulfoximine/phorone (BSO/PHO) treatment, while abolishing the enhancing effect of OA on mitochondrial GSH, did not completely abrogate the cardioprotection against I-R injury. The remnant cardioprotection was associated with an increase in mitochondrial alpha-TOC level, when compared with the unpretreated I-R group with BSO/PHO. The results suggest that the cardioprotection afforded by OA pretreatment against I-R injury may at least in part be attributed to the enhancement of mitochondrial antioxidant mechanism mediated by GSH and alpha-TOC, particularly under I-R conditions. Abbreviations. BSO:buthionine sulfoximine GSH:reduced glutathione I-R:ischemia-reperfusion alpha-LA:alpha-lipoic acid LDH:lactate dehydrogenase OA:oleanolic acid PHO:phorone alpha-TOC:alpha-tocopherol.