Glutathione-dependent toxicity

1. Recent studies show that glutathione conjugate formation is an important bioactivation mechanism for several groups of compounds with implications for organ-selective toxicity and carcinogenicity.

2. Vicinal dihaloalkanes, such as 1,2-dihaloethanes, yield S-(2-haloalkyl)glutathione conjugates that give rise to highly electrophilic episulphonium ions, which are involved in the cytotoxicity and mutagenicity of 1,2-dihaloethanes.

3. Nephrotoxic haloalkenes are metabolized to S-(haloalkenyl)- or S-(haloalkyl)-glutathione conjugates which, after metabolism to the corresponding cysteine conjugates, are bioactivated by renal cysteine conjugate β-lyase to yield cytotoxic or mutagenic metabolites.

4. Finally, hepatic glutathione conjugate formation with hydroquinones and amino-phenols yields conjugates that are directed to γ-glutamyltransferase-rich tissues, such as the kidney, where they undergo alkylation or redox cycling reactions, or both, that cause organ-selective damage.     

Chemical-induced nephrotoxicity mediated by glutathione S-conjugate formation.

Glutathione conjugation has been identified as an important detoxication reaction. However, several glutathione-dependent bioactivation reactions have been identified. Current knowledge on the mechanisms and the possible biological importance of these reactions is discussed in this article. Vicinal dihaloalkanes are transformed by glutathione S-transferase-catalyzed reactions to mutagenic and nephrotoxic S-(2-haloethyl) glutathione S-conjugates. Electrophilic episulphonium ions are the ultimate reactive intermediates formed and interact with nucleic acids. Several polychlorinated alkenes are bioactivated in a complex, glutathione-dependent pathway. The first step is hepatic glutathione S-conjugate formation followed by cleavage to the corresponding cysteine S-conjugates, and, after translocation to the kidney, metabolism by renal cystein conjugate beta-lyase. Beta-Lyase-dependent metabolism of halovinyl cysteine S-conjugates yields electrophilic thioketenes, whose covalent binding to cellular macromolecules is likely to be responsible for the observed nephrotoxicity of the parent compounds. Finally, hepatic glutathione conjugate formation with hydroquinones and aminophenols yields conjugates that are directed to gamma-glutamyltransferase-rich tissues, such as the kidney, where they cause alkylation or redox cycling reactions, or both, that cause organ-selective damage.     

Glutathione-dependent bioactivation of haloalkanes and haloalkenes

Haloalkanes and haloalkenes constitute an important group of widely used chemicals that have the potential to induce toxicity and cancer. The toxicity of haloalkanes and haloalkenes may be associated with cytochromes P450- or glutathione transferase-dependent bioactivation. This review is concerned with the glutathione- and glutathione transferase-dependent bioactivation of dihalomethanes, 1,2-dihaloalkanes, and haloalkenes. Dihalomethanes, e.g., dichloromethane, and 1,2-dihaloethanes, e.g., 1,2-dichloroethane and 1,2-dibromoethane, undergo glutathione transferase-catalyzed bioactivation to give S-(halomethyl)glutathione or glutathione episulfonium ions, respectively, as reactive intermediates. Haloalkenes, e.g., trichloroethene, hexachlorobutadiene, chlorotrifluoroethene, and tetrafluoroethene, undergo cysteine conjugate beta-lyase-dependent bioactivation to thioacylating intermediates, including thioacyl halides, thioketenes, and 2,2,3-trihalothiiranes. With all of these compounds, the formation of reactive intermediates is associated with their observed toxicity.     

Identification of the reactive glutathione conjugate S-(2-chloroethyl)glutathione in the bile of 1-bromo-2-chloroethane-treated rats by high-pressure liquid chromatography and precolumn derivatization with o-phthalaldehyde

The conjugation of glutathione with 1,2-dihaloethanes leads to the formation of S-(2-haloethyl)glutathione which, following intramolecular cyclization, produces an electrophilic thiiranium ion. The extent to which the formation of the thiiranium ion is responsible for the toxicity associated with 1,2-dihaloethanes has been difficult to determine because of the inherent instability of the compound under physiological conditions. The goal of this study was to attempt to identify a putative precursor of the thiiranium ion, S-(2-chloroethyl)glutathione (CEG), in the bile of rats treated with 1,2-dihaloethanes such as 1-bromo-2-chloroethane (BCE). In order to detect the presence of CEG, a precolumn procedure for derivatizing the amine of CEG with o-phthalaldehyde/2-mercaptoethanol (OPA/MCE) was developed. Studies with a model compound, S-ethylglutathione, indicated that the derivatization reaction between S-ethylglutathione and OPA/MCE proceeded rapidly and under mild conditions. The resulting fluorescent adduct of S-ethylglutathione was detected at low concentrations following separation by reverse-phase HPLC. Derivatization of CEG with OPA/MCE followed by preparative HPLC and mass spectral analysis revealed that the major fluorescent adduct in the reaction mixture was the expected 1-[(2-hydroxyethyl)thio]-2-substituted-isoindole derivative of CEG. Also present in the derivatization reaction mixture were small quantities of S-(2-hydroxyethyl)glutathione, the product of CEG hydrolysis, and a product involving the addition of MCE to CEG. Analysis of the bile samples obtained from bile-cannulated rats treated with BCE showed the presence of a peak corresponding to CEG. Over a 3-h interval, 2% of the BCE administered was excreted into the bile as CEG.     

Renal cysteine conjugate beta-lyase-mediated toxicity studied with primary cultures of human proximal tubular cells

The beta-lyase pathway has been shown to mediate the nephrotoxicity of S-cysteine conjugates of a variety of haloalkenes in a number of animal models in vitro and in vivo. However, there is no information available concerning this mechanism of bioactivation in human tissues. In this investigation a well-characterized model of human proximal tubule epithelial cells, the presumed target cell, was used to investigate the toxicity of a series of glutathione and cysteine conjugates of nephrotoxic haloalkenes. Both S-(1,2-dichlorovinyl)-glutathione (DCVG) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) caused dose-dependent toxicity over a range of 25 to 500 microM. DCVC was consistently found to be more toxic than DCVG, but the inclusion of gamma-glutamyltransferase (0.5 U/ml) increased the toxicity of DCVG to that observed with an equimolar concentration of DCVC, indicating that metabolism to the cysteine conjugate is an important rate-limiting step in this in vitro model. S-(1,2,3,4,4-Pentachlorobutadienyl)-L-cysteine, S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine, and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine were also found to be toxic to human proximal tubular cells. Incubation with [35S]DCVC resulted in covalent binding of 35S-label, which increased linearly to a final level of 1.05 nmol/mg protein at 6 hr. Aminooxyacetic acid (250 microM), an inhibitor of pyridoxal phosphate-dependent enzymes such as beta-lyase, protected the cells from the toxicity of all of the cysteine conjugates and inhibited the covalent binding of 35S-label from [35S]DCVC to cellular macromolecules. The results of the present study provide the first evidence that human proximal tubular cells are sensitive to the toxicity of glutathione and/or cysteine conjugates of a variety of chloro- and fluoroalkenes which are activated via the beta-lyase pathway. The implications for human health are discussed.     

Bioactivation mechanisms of haloalkene cysteine S-conjugates modeled by gas-phase, ion-molecule reactions

Glutathione conjugate formation plays important roles in the detoxification and bioactivation of xenobiotics. A range of nephrotoxic haloalkenes undergo bioactivation that involves glutathione and cysteine S-conjugate formation. The cysteine S-conjugates thus formed may undergo cysteine conjugate beta-lyase-catalyzed biotransformation to form cytotoxic thiolates or thiiranes. In the studies presented here, cysteine conjugate beta-lyase-catalyzed biotransformations were modeled by anion-induced elimination reactions of S-(2-bromo-1,1, 2-trifluoroethyl)-N-acetyl-L-cysteine methyl ester, S-(2-chloro-1,1, 2-trifluoroethyl)-N-acetyl-L-cysteine methyl ester, and S-(2-fluoro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine methyl ester in the gas phase. Examination of these processes in the gas phase allowed direct observation of the formation of cysteine S-conjugate-derived thiolates and thiiranes, whose formation is inferred from condensed-phase results. The cysteine S-conjugates of these haloethenes exhibit distinctive patterns of mutagenicity that are thought to be correlated with the nature of the products formed by their cysteine conjugate beta-lyase-catalyzed biotransformation. In particular, S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine is mutagenic, whereas the chloro and fluoro analogues are not. It has been proposed that the mutagenicity of S-(2-bromo-1,1, 2-trifluoroethyl)-L-cysteine is correlated with the greater propensity of the bromine-containing cysteine S-conjugate to form a thiirane compared with those of the chlorine- or fluorine-containing conjugates. The ease of thiirane formation is consistent with the gas-phase results presented here, which show that the bromine-containing conjugate has a greater propensity to form a thiirane on anionic base-induced elimination than the chloro- or fluoro-substituted analogues. The blocked cysteine S-conjugates were deprotonated by gas-phase ion-molecule reactions with hydroxide, methoxide, and ethoxide ions and then allowed to decompose. The mechanisms for these decompositions are discussed as well as the insights into the bioactivation of these cysteine S-conjugates provided by the further decompositions of thiolate intermediates.     

The metabolism of 3-chloro,- 3-bromo- and 3-iodoprpan-1,2-diol in rats and mice.

1. The metabolism of the 3-halopropan-1,2-diols (alpha-halohydrins) has been investigated in rats and mice. Apart from 3-chloropropan-1,2-diol (I), of which some 10% is excreted unchanged by both species, the compounds are completely degraded following intraperitoneal administration. 2. The alpha-halohydrins are detoxicated by conjugation with glutathione and produce two urinary metabolites, isolated and identified as S-(2,3-dihydroxypropyl)cysteine (VII) and the corresponding mercapturic acid N-acetyl-S-(2,3-dihydroxypropyl)cysteine (VIII). 3. When incubated with rat liver supernatant, the compounds do not conjugate with glutathione and their general chemical reactivity suggests that they react via a common intermediate proposed to be glycidol (2,3-epoxypropanol, IV). As the epoxide produces the same urinary metabolites as the alpha-halo-hydrins, and conjugates with glutathione either with or without liver supernatant to form the primary metabolite S-(2,3-dihydroxypropyl)glutathione (VI), glycidol is also proposed to be the reactive intermediate in vivo. 4. The role of epoxides in intermediary metabolism is discussed.     

Utilization of glutathione during 1,2-dihaloethane metabolism in rat hepatocytes.

The metabolism of 1,2-dihaloethanes (DHEs) to glutathione-containing metabolites by freshly isolated rat hepatocytes was investigated. 1,2-Dichloroethane (DCE), 1,2-dibromoethane (DBE), and 1-bromo-2-chloroethane (BCE) were metabolized to S-(2-hydroxyethyl)glutathione (HEG), S-(carboxymethyl)glutathione (CMG), and S,S'-(1,2-ethanediyl)bis(glutathione) (GEG). The formation of these glutathione-containing metabolites was concomitant with the depletion of intracellular glutathione (GSH) and accounted for 58%, 84%, and 71% of the DCE-, BCE-, and DBE-induced loss of intracellular GSH, respectively. The covalent binding of [14C]DBE to hepatocyte protein reached 18.7 nmol/mL of cell suspension (7.8 nmol/mg of protein) within 2.0 h of incubation. Half of this covalent binding occurred within 0.5 h of incubation (4.0 nmol/mg of protein) in the presence of high levels of intracellular GSH (30% of initial GSH level at 0.5 h). Hepatocyte metabolism of 2-chloroacetic acid produced only CMG. 2-Chloroethanol metabolism gave rise to CMG and HEG in a 11.5:1.0 ratio; 2-chloroacetaldehyde produced almost equal amounts of CMG and HEG. GEG formation was increased significantly for DBE and BCE when GSH was added to the medium during treatment, suggesting that the GSH conjugates S-(2-haloethyl)glutathione are exported from the hepatocytes. These results indicate that the glutathione S-transferase-catalyzed conjugation of GSH with the DHEs is responsible for the majority of the DHE-induced GSH depletion. The S-(2-haloethyl)glutathione conjugates appear responsible for the extensive covalent binding to protein observed during [14C]DBE metabolism.     

Glutathione-dependent bioactivation of xenobiotics

Glutathione conjugation has been identified as an important detoxication reaction. However, in recent years several glutathione-dependent bioactivation reactions have been identified. Current knowledge on the mechanisms and the possible biological importance of these reactions are discussed.

1. Dichloromethane is metabolized by glutathione conjugation to formaldehyde via S-(chloromethyl)glutathione. Both compounds are reactive intermediates and may be responsible for the dichloromethane-induced tumorigenesis in sensitive species.

2. Vicinal dihaloalkanes are transformed by glutathione S-transferase-catalyzed reactions to mutagenic and nephrotoxic S-(2-haloethyl)glutathione S-conjugates. Electrophilic episulphonium ions are the ultimate reactive intermediates formed.

3. Several polychlorinated alkenes are bioactivated in a complex, glutathione-dependent pathway. The first step is hepatic glutathione S-conjugate formation followed by cleavage to the corresponding cysteine S-conjugates, and, after translocation to the kidney, metabolism by renal cysteine conjugate β-lyase. β-Lyase-dependent metabolism of halovinyl cysteine S-conjugates yields electrophilic thioketenes, whose covalent binding to cellular macromolecules is responsible for the observed toxicity of the parent compounds.

4. Finally, hepatic glutathione conjugate formation with hydroquinones and aminophenols yields conjugates that are directed to γ-glutamyltransferase-rich tissues, such as the kidney, where they undergo alkylation or redox cycling reactions, or both, that cause organ-selective damage.     

Mechanism of S-(1,2-dichlorovinyl)glutathione-induced nephrotoxicity

S-(1,2-Dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-DL-cysteine are potent nephrotoxins. Agents that inhibit gamma-glutamyl transpeptidase, cysteine conjugate beta-lyase, and renal organic anion transport systems, namely L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125), aminooxyacetic acid, and probenecid, respectively, protected against S-conjugate-induced nephrotoxicity. Furthermore, S-(1,2-dichlorovinyl)-DL-alpha-methylcysteine, which cannot be cleaved by cysteine conjugate beta-lyase, was not nephrotoxic. These results strongly support a role for renal gamma-glutamyl transpeptidase, cysteine conjugate beta-lyase, and organic anion transport systems in S-(1,2-dichlorovinyl)glutathione- and S-(1,2-dichlorovinyl)cysteine-induced nephrotoxicity.     

Formation of mitochondrial phospholipid adducts by nephrotoxic cysteine conjugate metabolites.

Nephrotoxic cysteine conjugates derived from a variety of halogenated alkenes are enzymatically activated via the beta-lyase pathway to yield reactive sulfur-containing metabolites which bind covalently to cellular macromolecules. Mitochondria contain beta-lyase enzymes and are primary targets for binding and toxicity. Previously, mitochondrial protein and/or DNA have been considered as molecular targets for cysteine conjugate metabolite binding. We now report that metabolites of nephrotoxic cysteine conjugates form covalent adducts with rat kidney mitochondrial phospholipids. Rat kidney mitochondria were incubated with the 35S-labeled conjugates S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC), S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC), S-(1,2-dichlorovinyl)-L-cysteine, and S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine. Quantitation of metabolite binding to whole mitochondria and to mitochondrial protein and lipid fractions revealed that as much as 42% of the 35S-label associated with the mitochondria was found in the lipid fraction. Total lipids were also extracted from 35S-treated mitochondria and separated by thin-layer chromatography. 35S-Containing metabolites were found in the lipid fractions from mitochondria treated with each of the conjugates. Lipids from both [35S]CTFC- and [35S]-TFEC-treated mitochondria contained major 35S-labeled lipid adducts which had similar mobility by thin-layer chromatography. Fatty acid analysis, 19F and 31P NMR spectroscopy, and mass spectrometric analyses confirmed that the major TFEC and CTFC adducts are thioamides of phosphatidylethanolamine.     

The effect of haloalkene cysteine conjugates on rat renal glutathione reductase and lipoyl dehydrogenase activities

An early event in the nephrotoxicity of haloalkene cysteine conjugates is their metabolism by cysteine conjugate beta-lyase to generate a reactive "thiol moiety" which binds to protein. This reactive metabolite(s) has been reported to cause mitochondrial dysfunction. We have examined the effect of three haloalkene cysteine conjugates on the activity of rat renal cortical cytosolic glutathione reductase and mitochondrial lipoyl dehydrogenase, two enzymes which have been reported to be inhibited by S-(1,2-dichlorovinyl)-L-cysteine (DCVC) in the liver. N-Acetyl-S-(1,2,3,4,4-pentachloro-1,3-butadienyl)-L- cysteine (N-acetyl PCBC) produced a time- and concentration-dependent inhibition of glutathione reductase and kinetic studies showed that the inhibition was noncompetitive with a Ki of 215 microM. The enzyme activity from male rat kidney was more sensitive to N-acetyl PCBC than that from female rat kidney. Aminooxyacetic acid, an inhibitor of cysteine conjugate beta-lyase, and bis-p-nitrophenyl phosphate, an amidase inhibitor, blocked the effect of N-acetyl PCBC on glutathione reductase, indicating that metabolism by the cytosol is required to produce enzyme inhibition. S-(1,1,2,2-Tetrafluoroethyl)-L-cysteine (TFEC) and DCVC are also noncompetitive inhibitors of glutathione reductase but are less active than N-acetyl PCBC with Ki's of 2.6 and 6.2 mM for DCVC and TFEC, respectively, DCVC produced a time- and concentration-dependent inhibition of lipoyl dehydrogenase and kinetic studies showed that the inhibition was noncompetitive with a Ki of 762 microM. TFEC and PCBC also inhibit lipoyl dehydrogenase. Aminooxyacetic acid blocked the effect of DCVC, TFEC, and PCBC on lipoyl dehydrogenase, indicating that metabolism by the mitochondrial fraction is required to produce enzyme inhibition. Glutathione reductase activity in the renal cortex of male rats treated with 200 mg/kg hexachloro-1,3-butadiene (HCBD) was inhibited as early as 1 hour after dosing, before signs of marked morphological damage. The activity of lipoyl dehydrogenase was also reduced but was only statistically significant 8 hr after dosing when there was marked renal dysfunction. These findings indicate that the reactive thiol moiety formed by cysteine conjugate beta-lyase cleavage of PCBC can inhibit both glutathione reductase and lipoyl dehydrogenase activities in vivo following HCBD administration. We suggest that such inhibition is a general phenomenon, occurring with diverse and as yet unidentified renal proteins. The critical nature of mitochondrial function and the generation of reactive metabolites within this compartment make this organelle a prime target.     

N-(3,5-dichlorophenyl)succinimide nephrotoxicity: evidence against the formation of nephrotoxic glutathione or cysteine conjugates.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity via one or more metabolites. Previous studies suggested that glutathione is important for mediating NDPS-induced nephropathy. The purpose of this study was to examine the possibility that a glutathione or cysteine conjugate of NDPS or an NDPS metabolite might be the penultimate or ultimate nephrotoxic species. In one set of experiments, male Fischer 344 rats were administered intraperitoneally (i.p.) NDPS (0.4 or 1.0 mmol/kg) 1 h after pretreatment with the gamma glutamyltranspeptidase inhibitor AT-125 (acivicin) (10 mg/kg, i.p.) and renal function was monitored at 24 and 48 h. In general, AT-125 pretreatment had few effects on NDPS-induced nephropathy. In a second set of experiments, rats were treated i.p. or orally (p.o.) with a putative glutathione (S-(2-(N-(3,5-dichlorophenyl)succinimidyl)glutathione (NDPSG), a cysteine (S-(2-(N-(3,5-dichlorophenyl)succinimidyl)cysteine (NDPSC) (as the methyl ester) or N-acetylcysteine (S-(2-(N-(3,5-dichlorophenyl)succinimidyl)-N-acetylcysteine (NDPSN) conjugate of NDPS (0.2, 0.4 or 1.0 mmol/kg) or vehicle and renal function was monitored at 24 and 48 h. An intramolecular cyclization product of NDPSC, 5-carbomethoxy-2-(N-(3,5-dichlorophenyl)carbamoylmethyl)-1,4-th iazane-3-one (NDCTO) was also examined for nephrotoxic potential. None of the compounds produced toxicologically important changes in renal function or morphology. The in vitro ability of the conjugates to alter organic ion accumulation by cortical slices was also examined. All of the conjugates tested caused a reduction in p-aminohippurate (PAH) accumulation at a conjugate bath concentration of 10(-4) M, but none of the conjugates reduced tetraethylammonium (TEA) uptake. In a third experiment, the ability of the cysteine conjugate beta-lyase inhibitor aminooxyacetic acid (AOAA) (0.5 mmol/kg, i.p.) to alter the nephrotoxicity induced by two NDPS metabolites, N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) or N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (NDHSA) (0.2 mmol/kg, i.p.), was examined. AOAA pretreatment had no effect on NDHS- or NDHSA-induced nephrotoxicity. These results do not support a role for a glutathione or cysteine conjugate of NDPS or and NDPS metabolite as being the penultimate or ultimate nephrotoxic species.     

Glutathione conjugation of methazolamide and subsequent reactions in the ciliary body in vitro

Conjugation reaction of methazolamide with glutathione and its subsequent reactions were studied in vitro. Glutathione, cysteinylglycine, and cysteine conjugates of methazolamide were chemically synthesized. All of the three compounds showed absorbance below 330 nm, with maximal absorbance at approximately 300 nm. At the wavelengths below 220 nm, absorbance was proportional to the number of the amino acids each compound had. Amino acid analysis of the glutathione conjugate showed that the conjugation reaction involved the cysteine residue of glutathione. In order to identify the chemical structure of the reaction product, cysteine conjugate was subjected to infrared, proton nuclear magnetic resonance, and mass spectral analyses. These studies indicated that the cysteine conjugate was S-(5-acetylimino-4-methyl-delta 2-1,3,4-thiadiazolinyl)cysteine. The reaction with glutathione was not catalyzed by glutathione S-transferases, but proceeded in the absence of the enzyme. The glutathione conjugate was degraded by bovine ciliary body homogenate to the cysteinylglycine conjugate and then to the cysteine conjugate.     

Stereoselectivity of naphthalene epoxidation by mouse, rat, and hamster pulmonary, hepatic, and renal microsomal enzymes

Previous studies have demonstrated the formation of three glutathione conjugates during the hepatic and pulmonary microsomal metabolism of naphthalene in the presence of reduced glutathione and cytosolic enzymes containing the glutathione transferases. These glutathione conjugates now have been identified by negative ion fast atom bombardment mass spectrometry, by proton NMR spectroscopy, and by chemical synthesis from the (1S,2R)- and (1R, 2S)-naphthalene 1,2-oxide enantiomers as isomeric hydroxyglutathionyldihydronaphthalene derivatives. All three glutathione adducts yielded prominent mass spectral ions at m/z 450 (M-H)-, 432 (dehydration product), and 306 (glutathionyl moiety) which were consistent with the monoglutathionyl derivatives of hydroxydihydronaphthalene. Signals in the proton NMR spectra at 3.60 and 4.95 ppm (adduct 1) and 3.60 and 4.95 ppm (adduct 2) indicated that these conjugates were diastereomers of 1-hydroxy-2-glutathionyl-1,2-dihydronaphthalene. Corresponding signals for H1 and H2 at 4.22 and 4.45 ppm for adduct 3 showed that this isomer was generated from attack of glutathione at the 1 position of the naphthalene 1,2-oxide. Incubation of synthetic (1S, 2R)-naphthalene 1,2-oxide with glutathione in the presence of glutathione transferases resulted in the formation of adducts 1 and 3 in approximately equal proportions; under identical conditions, glutathione conjugate 2 was formed from (1R, 2S)-naphthalene 1,2-oxide. Incubation of naphthalene, glutathione, and glutathione transferases with pulmonary, hepatic, or renal microsomal preparations from mouse, rat, and hamster resulted in the formation of all three glutathione conjugates. Substantial differences in the rates of formation of the individual conjugates were observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake of the glutathione conjugate S-(1,2-dichlorovinyl)glutathione by renal basal-lateral membrane vesicles and isolated kidney cells

Transport of the glutathione S-conjugate, S-(1,2-dichlorovinyl)glutathione (DCVG), was studied in renal basal-lateral membrane vesicles and isolated rat kidney cells. The time course of S-(1,2-dichlorovinyl)glutathione uptake in membrane vesicles exhibited an overshoot in the presence of sodium, indicating transport against a concentration gradient. The initial rate of uptake with membrane potential clamped at 0 mV was stimulated 2.5-fold by an inwardly directed gradient of 100 mM sodium chloride. Hyperpolarization of the membrane potential to -60 mV in the presence of sodium stimulated uptake another 2.7-fold, indicating that cotransport of sodium and S-(1,2-dichlorovinyl)glutathione is electrogenic. Sodium-dependent DCVG uptake was inhibited by glutathione, glutathione disulfide, and gamma-glutamylglutamate, but not by the corresponding cysteine S-conjugate, S-(1,2-dichlorovinyl)cysteine, indicating that the transport system is specific for the gamma-glutamyl moiety. Probenecid was also a potent inhibitor of sodium-dependent uptake. S-(1,2-dichlorovinyl)glutathione inhibited sodium-dependent uptake of glutathione in a concentration-dependent manner. Thus, these results show that uptake of DCVG and glutathione is mediated by the same sodium-coupled system. Uptake of S-(1,2-dichlorovinyl)glutathione was also demonstrated in isolated kidney cells; in the presence of sodium, cells accumulated approximately 4-fold more DCVG than in the absence of sodium. This basal-lateral membrane transport system can enable efficient delivery of circulating S-(1,2-dichlorovinyl)glutathione to kidney cells and may, therefore, contribute to its potent and selective nephrotoxicity. In addition, it suggests that renal clearance of glutathione conjugates may include transport from the blood through epithelial cells into the lumen as well as direct filtration through the glomerulus.     

Studies on the fate of the glutathione and cysteine conjugates of acetaminophen in mice

Studies were conducted in mice to examine the origin and fate of the amino acid-containing conjugates of acetaminophen (APAP). Collection of bile containing [14C]APAP metabolites (mainly the glutathione conjugate) in common duct-cannulated mice given a 250 mg/kg oral dose of the drug reduced by greater than 70% the urinary excretion of the cysteine and mercapturic acid conjugates of APAP. This confirmed previous reports which indicated that these urinary metabolites originated from the glutathione conjugate excreted in bile. The urinary excretion of cysteine and mercapturic acid conjugates was not altered, however, by ligation of the common bile duct in mice given APAP. Thus, biliary excretion of the glutathione conjugate is not obligatory for the appearance of cysteine and mercapturic acid conjugates in urine. Intravenous administration of purified glutathione conjugate to mice having a bile-duct cannula indicated that this conjugate did not appear in bile but appeared in urine primarily in the form of the cysteine conjugate. An identical pattern of excretion was observed after an iv dose of the purified cysteine conjugate of APAP to bile duct-cannulated mice. These results indicated that, if the glutathione conjugate leaves the liver via the blood, it is rapidly converted to the cysteine conjugate which is eliminated in urine. This conversion takes place at multiple sites in the body and evidence is presented to implicate both intestine and kidney in the process. The appearance of a small amount of glutathione conjugate in urine (16%) after an iv dose of the cysteine conjugate indicates that formation of the glutathione of APAP can occur by a route that does not involve direct conjugation of reactive metabolites of the drug with glutathione.     

Biosynthesis and biotransformation of glutathione S-conjugates to toxic metabolites

The material presented in this review deals with the hypothesis that the nephrotoxicity of certain halogenated alkanes and alkenes is associated with hepatic biosynthesis of glutathione S-conjugates, which are further metabolized to the corresponding cysteine S-conjugates. Some glutathione or cysteine S-conjugates may be direct-acting nephrotoxins, but most cysteine S-conjugates require bioactivation by renal, pyridoxal phosphate-dependent enzymes, such as cysteine conjugate beta-lyase (beta-lyase). The biosynthesis of glutathione S-conjugates is catalyzed by both the cytosolic and the microsomal glutathione S-transferases, although the latter enzyme is a better catalyst for the reaction of haloalkenes with glutathione. When glutathione S-conjugate formation yields sulfur mustards, as occurs with vicinal-dihaloethanes, the S-conjugates are direct-acting toxins. In contrast, the S-conjugates formed from fluoro- and chloroalkenes yield S-alkyl- or S-vinyl glutathione conjugates, respectively, which are metabolized to the corresponding cysteine S-conjugates by gamma-glutamyltransferase and dipeptidases; inhibition of these enzymes blocks the toxicity of the glutathione S-conjugates. The cysteine S-conjugates must be metabolized by beta-lyase for the expression of toxicity; the beta-lyase inhibitor aminooxyacetic acid blocks the toxicity of cysteine S-conjugates, and the corresponding alpha-methyl cysteine S-conjugates, which cannot be metabolized by beta-lyase, are not toxic. Moreover, probenecid, an inhibitor of renal anion transport system, blocks the toxicity of cysteine S-conjugates, which cannot be metabolized by beta-lyase, are not toxic. Moreover, probenecid, an inhibitor of renal anion transport system, blocks the toxicity of cysteine S-conjugates. Homocysteine S-conjugates are also potent cyto- and nephrotoxins. The high renal content of gamma-glutamyltransferase and the renal anion transport system are probably determinants of kidney tissue as a target site. Biochemical studies indicate that renal mitochondrial dysfunction is produced by the cysteine S-conjugates. Finally, some of the glutathione and cysteine conjugates are mutagenic in the Ames test, and reactive intermediates formed by the action of beta-lyase may contribute to the nephrocarcinogenicity of certain chloroalkenes.     

Isolation and characterization of N7-guanyl adducts derived from 1,2-dibromo-3-chloropropane

1,2-Dibromo-3-chloropropane is a potent renal and testicular toxicant and has been shown to induce tumor formation in laboratory animals. The toxic effects of the compound are thought to be a result of a bioactivation step in which a glutathione conjugate is formed and subsequently reacts with cellular DNA. The L-glutathione conjugate of 1,2-dibromo-3-chloropropane was chemically synthesized and used to alkylate DNA: following incubations of the conjugate with calf thymus DNA and neutral thermal hydrolysis (to release N7-guanyl adducts) four major fluorescent products were observed. Three of these were isolated and characterized, the structures being determined as S-[bis(N7-guanylmethyl)methyl]glutathione and the two diastereomers of S-[1-(hydroxymethyl)-2-(N7-guanyl)ethyl]glutathione. The fourth fluorescent product was unstable and formed in low yield and thus could not be characterized. The formation of these N7-guanyl adducts can be explained by a mechanism that includes the formation of two consecutive episulfonium ion intermediates followed by nucleophilic attack at the unsubstituted methylene carbon. These adducts bear structural and mechanistic similarities to the major adduct derived from 1,2-dibromoethane, S-[2-(N7-guanyl)ethyl]glutathione. The same adducts were also formed when DBCP was incubated with rat liver cytosol, GSH, and DNA. In vivo experiments with DBCP yielded very low levels of the N7-guanyl adducts formed in rat liver compared to the levels seen after treatments with 1,2-dibromoethane. The bis-guanyl adduct represents a cross-linked structure that may be important in the toxicity of this compound. The conjugate was not found to be mutagenic to Salmonella typhimurium TA100 but rather showed a toxic effect toward the bacteria.     

Conjugative metabolism of 1,2-dibromoethane in mitochondria: disruption of oxidative phosphorylation and alkylation of mitochondrial DNA

1,2-Dibromoethane (DBE) is an environmental contaminant that is metabolized by glutathione S-transferases to a haloethane-glutathione conjugate. Since haloethane-glutathione conjugates are known to alkylate nuclear DNA and cytoplasmic proteins, these effects were investigated in isolated rat liver mitochondria exposed to DBE by measuring guanine adducts and several aspects of oxidative phosphorylation including respiratory control ratios, respiratory enzyme activity, and ATP levels. Mitochondrial large-amplitude swelling and glutathione status were assessed to evaluate mitochondrial membrane integrity and function. When exposed to DBE, mitochondria became uncoupled rapidly, yet no large-amplitude swelling or extramitochondrial glutathione was observed. Mitochondrial GSH was depleted to 2-53% of controls after a 60-min exposure to micromolar quantities of DBE; however, no extramitochondrial GSH or GSSG was detected. The depletion of mitochondrial glutathione corresponded to an increase of an intramitochondrial GSH-conjugate which, based on HPLC elution profiles and retention times, appeared to be S,S'-(1,2-ethanediyl)bis(glutathione). Activities of the NADH oxidase and succinate oxidase respiratory enzyme systems were inhibited 10-74% at micromolar levels of DBE, with succinate oxidase inactivation occurring at lower doses. ATP concentrations in DBE-exposed mitochondria in the presence of succinate were 5-90% lower than in the controls. The DNA adduct S-[2-(N(7)-guanyl)ethyl]glutathione was detected by HPLC in mtDNA isolated from DBE-exposed mitochondria. The results suggest that respiratory enzyme inhibition, glutathione depletion, decreased ATP levels, and DNA alkylation in DBE-exposed mitochondria occur via the formation of an S-(2-bromoethyl)glutathione conjugate, the precursor of the episulfonium ion alkylating species of DBE.