Cysteine S-conjugates Research Articles

Synthesis of novel Se-substituted selenocysteine derivatives as potential kidney selective prodrugs of biologically active selenol compounds: evaluation of kinetics of beta-elimination reactions in rat renal cytosol.

Eighteen Se-substituted selenocysteine derivatives were synthesized as potential kidney selective prodrugs which can be activated by renal cysteine conjugate beta-lyase to selenium-containing chemoprotectants or antitumor agents. Selenocysteine derivatives with aliphatic and benzylic Se-substituents were synthesized by reducing selenocystine to selenocysteine followed by a reaction with the corresponding alkyl and benzyl halogenides. Selenocysteine derivatives with aromatic Se-substitutes were synthesized by reaction of beta-chloroalanine with substituted phenylselenol compounds, which were formed by reducing substituted diphenyl diselenides by NaBH4. The enzyme kinetic parameters (apparent Km and Vmax) of the beta-elimination reaction of the selenocysteine conjugates were studied in rat renal cytosol. The results suggest that Se-substituted L-selenocysteine conjugates are extremely good substrates for renal cysteine conjugate beta-lyases as indicated by low apparent Km and high Vmax values. The benzyl-substituted Se-conjugates appeared to be better substrates than the phenyl- and alkyl-substituted Se-conjugates. Corresponding L-cysteine S-conjugates were too poor substrates to obtain proper enzyme kinetics. Recently, local activation of cysteine S-conjugates by renal cysteine conjugate beta-lyases was proposed as a new strategy to target antitumor agents to the kidney. The present results show that Se-substituted selenocysteine conjugates may be more promising prodrugs because these compounds are much better substrates for beta-lyase.

Renal activation of trichloroethene and S-(1,2-dichlorovinyl)-L-cysteine and cell proliferative responses in the kidneys of F344 rats and B6C3F1 mice.

Covalent binding of reactive intermediates formed by renal beta-lyase activation of S-(1,2-dichlorovinyl)-L-cysteine (DCVC) has been suggested to be responsible for the greater renal sensitivity of rats than mice to the carcinogenic effects of chronic treatment with trichloroethene (TRI). Previous work demonstrated that the activation of DCVC results in acid-labile adducts to protein that can be distinguished from adducts formed by other pathways of TRI metabolism. By analyzing acid-labile adduct formation, the relationship between DCVC formation and activation from TRI and increases in rates of cell division in the kidneys of male F344 rats and B6C3F1 mice could be investigated. The delivered dose of DCVC from an oral dose of 1000 mg/kg TRI was approximately six times greater in rats than mice. However, renal activation of DCVC in mice was approximately 12 times greater than in rats. Therefore, the overall activation of TRI was about two times greater in mice than rats. Induction of cell replication in liver and kidney following doses of 1, 5, or 25 mg/kg DCVC or 1000 mg/kg TRI was also measured through the use of miniosmotic pumps that delivered BrdU subcutaneously for 3 d. Acid-labile adduct formation from DCVC and TRI displayed a consistent relationship with increased cell replication in mice and between mice and rats. Both cell replication and acid-labile adduct formation in rats given 25 mg/kg DCVC were approximately equal to that observed in mice given 1 mg/kg. Increased cell replication was not observed in rats receiving 1 or 5 mg/kg DCVC or 1000 mg/kg TRI, nor were there histological signs of nephrotoxicity. Thus, net activation of TRI by the cysteine S-conjugate pathway was found to be greater in mice than rats and these findings appeared related to differences in cell proliferative responses of the kidneys of the two species. Based on these data, it would appear that other factors must contribute to the greater sensitivity of the rat to the induction of renal carcinogenesis by TRI.

Fourier-transform ion cyclotron resonance mass spectrometric evidence for the formation of alpha-chloroenethiolates and thioketenes from chloroalkene-derived, cytotoxic 4-thiaalkanoates.

The cytotoxicity of chloroalkene-derived cysteine S-conjugates is thought to be associated with the formation of alpha-chloroenethiolates and thioketenes as reactive intermediates. Recent studies indicate that the formation of 1,2-dichloroethenethiolate, which may give rise to chlorothioketene, is a key step in the bioactivation of 5,6-dichloro-4-thia-5-hexenoic acid (Fitzsimmons et al. (1995) Biochemistry 34, 4276-4286). We report here the use of Fourier-transform ion cyclotron resonance mass spectrometry to provide the first direct evidence for the formation of alpha-chloroenethiolate and thioketene species from a cytotoxic 4-thiaalkanoate. The bioactivation of 5,6-dichloro-4-thia-5-hexenoic acid involves conversion to the corresponding CoA thioester 5,6-dichloro-4-thia-5-hexenoyl-CoA and subsequent processing by the fatty acid beta-oxidation pathway. It has been proposed that the bioactivation of 5,6-dichloro-4-thia-5-hexenoyl-CoA involves loss of 1,2-dichloroethenethiolate, followed by loss of chloride to form chlorothioketene. 1,2-Dichloroethenethiolate and related alpha-chloroalkenethiolates have not been observed directly in aqueous solution. Fourier-transform ion cyclotron resonance mass spectrometric experiments show that S-propyl 5,6-dichloro-4-thia-5-hexenethioate reacts in the gas phase with base (hydroxide ion) to release 1,2-dichloroethenethiolate, which is observed directly in the mass spectrum of the products of the gas-phase reaction. Furthermore, the elimination of chloride from 1,2-dichloroethenethiolate on collision-induced decomposition is facile and provides evidence for chlorothioketene formation. Preliminary evidence for the formation of 1,2-dichloroethenethiolate and chlorothioketene from S-(1,2-dichlorovinyl)-N-acetyl-L-cysteine methyl ester was also obtained. These observations support the intermediacy of alpha-chloroenethiolates and chlorothioketenes in the bioactivation of cytotoxic, chloroalkene-derived 4-thiaalkanoates and cysteine S-conjugates and demonstrate the utility of Fourier-transform ion cyclotron mass spectrometry in studying the formation of reactive intermediates.

.alpha.-Thiolactones as Novel Intermediates in the Cysteine Conjugate .beta.-Lyase-Catalyzed Bioactivation of Bromine-Containing Cysteine S-Conjugates

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXT.alpha.-Thiolactones as Novel Intermediates in the Cysteine Conjugate .beta.-Lyase-Catalyzed Bioactivation of Bromine-Containing Cysteine S-ConjugatesMartin B. Finkelstein, Wolfgang Dekant, Andrew S. Kende, and M. W. AndersCite this: J. Am. Chem. Soc. 1995, 117, 37, 9590–9591Publication Date (Print):September 1, 1995Publication History Published online1 May 2002Published inissue 1 September 1995https://doi.org/10.1021/ja00142a038RIGHTS & PERMISSIONSArticle Views110Altmetric-Citations13LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (288 KB) Get e-AlertsSupporting Info (2)»Supporting Information Supporting Information Get e-Alerts

Mitochondrial bioactivation of cysteine S-conjugates and 4-thiaalkanoates: Implications for mitochondrial dysfunction and mitochondrial diseases

The toxicity of most drugs and chemicals is associated with their enzymatic conversion to toxic metabolites. Bioactivation reactions occur in a range of organs and organelles, including mitochondria. The toxicity of haloalkene-derived cysteine S-conjugates and related 4-thiaalkanoates is associated with their mitochondrial bioactivation. Toxic cysteine S-conjugates are formed by the glutathione S-transferase-catalyzed addition of glutathione to haloalkenes to give glutathione S-conjugates, which are hydrolyzed by γ-glutamyltransferase and dipeptidases. Mitochondrial cysteine conjugate ß-lyase-catalyzed bioactivation of cysteine S-conjugates affords unstable α-halothiolates. Haloalkene-derived 4-thiaalkanoates, which are analogs of cysteine S-conjugates that lack an α-amino group, undergo bioactivation by the enzymes of fatty acid ß-oxidation to give 3-hydroxy-4-thiaalkanoates that eliminate α-halothiolates. α-Halothiolates yield alkylating and acylating agents that interact with cellular macromolecules and thereby cause cell damage. Mitochondrial dysfunction is the hallmark of cysteine S-conjugate-induced cytotoxicity: decreased respiration, decreased ATP and total adenine nucleotide concentrations, depletion of the mitochondrial glutathione content, perturbations in cellular Ca 2+ homeostasis, and damage to the mitochondrial genome are seen with cysteine S-conjugates. Similar changes are observed with cytotoxic 4-thiaalkanoates, but inhibition of the medium-chain acyl-CoA dehydrogenase and hypoglycemia are also observed.

Potential role of the flavin-containing monooxygenases in the metabolism of endogenous compounds

Several xenobiotics and their corresponding cysteine S-conjugates are metabolized in vivo to cysteine S-conjugate sulfoxides and/or N-acetylcysteine S-conjugate sulfoxides. Homocysteine S-conjugates, such as methionine and ethionine, are also metabolized in vivo to sulfoxides. The enzymatic basis for these metabolic reactions is not known. Recently, the rat liver and kidney S- benzyl- l- cysteine S- oxidase activities were found to be associated with flavin-containing monooxygenases that are structurally and immunochemically related to known FMO1 isoforms. Further evidence for FMO1 being the major FMO isoform involved in S- benzyl- l- cysteine sulfoxidation was obtained from kinetic studies with cDNA-expressed rabbit FMOs. Endogenous cysteine S-conjugates, e.g. cysteinylcatecholamines, cysteinylleukotrienes, lanthionine and djenkolic acid may also be substrates for FMOs, since S- benzyl- l- cysteine can be considered a model for these compounds. Methionine, an endogenous homocysteine S-conjugate, was shown to be a substrate for cDNA-expressed rabbit FMO1, FMO2, and FMO3, however, the methionine sulfoxidation reaction was preferentially catalyzed by FMO3. These results suggest that FMOs may also play a role in the in vivo metabolism of endogenous homocysteine S-conjugates.

Mercapturic Acid Formation in Cultured Opossum Kidney Cells

We investigated the last step of mercapturic acid formation, the N-acetylation of cysteine S-conjugates, in the established opossum kidney (OK) cell line which exhibits characteristics of the proximal tubule. S-Benzyl-L-cysteine was used as a model substance for such a cysteine S-conjugate. We succeeded in showing that OK cells absorb S-benzyl-L-cysteine via an active transport system which is inhibitable by phenylalanine. This transport follows Michaelis-Menten kinetics and the two characterizing parameters were determined: the Michaelis-Menten constant Km = 1.8 mmol/l, and the maximum of the difference between the intracellular and the extracellular concentration of S-benzyl-L-cysteine delta Cmax = 19.4 mmol/l. S-Benzyl-L-cysteine is converted to N-acetyl-S-benzyl-L-cysteine at a constant rate, which is independent of the extracellular S-benzyl-L-cysteine concentration. Under the tested experimental conditions this is probably due to saturation of the microsomal N-acetyltransferase catalyzing this reaction. In conclusion, we have shown that OK cells are a suitable model for studying mercapturate formation. They take up S-benzyl-L-cysteine mainly via the same carrier as phenylalanine, which is known to be transported in the rat by the high-capacity, low-affinity neutral amino acid carrier, and convert it to N-acetyl-L-benzyl-S-cysteine.

Role of renal metabolism in risk to toxic chemicals.

The kidneys are capable of carrying out extensive oxidation, reduction, hydrolysis, and conjugation reactions. Renal cortex has high activities of cytochrome P450 and glutathione (GSH) S-transferase. In contrast, renal medulla has high activity of prostaglandin synthetase, which can catalyze co-oxidation of xenobiotics. While these pathways are found in many tissues and at higher activities than in kidney, several key enzymes of the mercapturic acid pathway are found at especially high activities in cells of the renal proximal tubule. Investigations over the last two decades demonstrated that GSH conjugation is not only a mechanism for detoxification of reactive electrophiles. Rather, metabolism of GSH S-conjugates to the corresponding cysteine S-conjugates represents a branch point: cysteine S-conjugates may be metabolized by the cysteine S-conjugate N-acetyl-transferase to mercapturic acids, which are nontoxic and are excreted, or they may be substrates for the pyridoxal phosphate-dependent cysteine conjugate beta-lyase, which catalyzes either a beta-elimination or a transamination reaction to produce unstable thiols. These thiols rearrange to form potent acylating species that can covalently bind to cellular macromolecules, thereby producing cytotoxicity, mutagenicity, and carcinogenicity. In addition to the beta-lyase, two other renal enzymes, L-2-amino (2-hydroxy) acid oxidase and cysteine conjugate S-oxidase, can bioactivate chemicals to produce nephrotoxic species. Several halogenated alkanes and alkenes are bioactivated by these pathways. These findings show that mammalian kidney is highly active in bioactivation of xenobiotics. Although the properties of the corresponding enzymes in humans may differ, it is clear that renal metabolism can be a critical determinant of risk to chemical injury.

Metabolism of tetrachloroethene in rats: identification of N epsilon-(dichloroacetyl)-L-lysine and N epsilon-(trichloroacetyl)-L-lysine as protein adducts.

Tetrachloroethene causes renal tumors in male rats after inhalation exposure. Tetrachloroethene is metabolized by cytochrome P-450 and by glutathione conjugation. Cytochrome P-450-dependent oxidation results in the formation of trichloroacetyl chloride, which may acylate cellular nucleophiles; glutathione conjugation results in the formation of S-(1,2,2-trichlorovinyl)glutathione, which is metabolized to the corresponding cysteine S-conjugate. S-(1,2,2-Trichlorovinyl)-L-cysteine is activated by renal cysteine conjugate beta-lyase to give dichlorothioketene. Covalent binding of this electrophile is presumably responsible for the renal toxicity of tetrachloroethene. In this report, we demonstrate the formation of protein adducts formed from tetrachloroethene using SDS-PAGE and immunochemical detection with rabbit anti-trifluoroacetyl serum. This serum recognizes dichloroacetylated rabbit serum albumin prepared by chemical modification of rabbit serum albumin with S-ethyl dichlorothioacetate and exhibited a high specificity for N epsilon-(dichloroacetyl)-L-lysine residues in proteins as shown by competitive ELISA. In the liver of [14C]tetrachloroethene-treated rats, the antibody recognized several modified proteins in microsomes. A protein adduct in rat liver identified by GC/MS after hydrolysis was N epsilon-(trichloroacetyl)-L-lysine. Western blots of renal fractions from rats treated with [14C]tetrachloroethene (200 mg/kg) or S-(1,2,2-trichlorovinyl)-L-cysteine (40 mumol/kg, iv) suggested the presence of modified mitochondrial and cytosolic proteins; no modified proteins were detected in microsomes. Proteins of identical molecular weight were modified by tetrachloroethene and by S-(1,2,2-trichlorovinyl)-L-cysteine in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamine Transaminase K and ω‐Amidase Activities in Primary Cultures of Astrocytes and Neurons and in Embryonic Chick Forebrain: Marked Induction of Brain Glutamine Transaminase K at Time of Hatching

Glutamine transaminase K and omega-amidase activities are present in the chick brain and in the brains of adult mice, rats, and humans. However, the activity of glutamine transaminase K in adult mouse brain is relatively low. In the chick embryo, cerebral glutamine transaminase K activity is low between embryonic days 5 and 17, but by day 23 (day of hatching) activity rises dramatically (> 15-fold). Cerebral omega-amidase activity is relatively high at embryonic day 5 but lower between days 5 and 17; at embryonic day 23 the activity rises to a maximum. Both glutamine transaminase K and omega-amidase are present in cultured chick, rat, and mouse astrocytes and neurons. For each species, the activity of glutamine transaminase K is higher in the astrocytes than in the neurons. The activity of omega-amidase is about the same in the cultured chick astrocytes and neurons but significantly higher in rat astrocytes than in rat neurons. The data suggest that the rise in brain glutamine transaminase K activity in the chick embryo at hatching correlates with maturation of astrocytes. Glutamine transaminase K may be involved in glutamine cycling in astrocytes. Glutamine transaminase K appears to be a major cysteine S-conjugate beta-lyase of the brain and may play a role in the neurotoxicity associated with exposure to dichloroacetylene and perhaps to other toxins.

Glutathione conjugation and conversion to mercapturic acids can occur as an intrahepatic process.

By catalyzing the reaction of electrophilic compounds with the sulfhydryl group of glutathione, the glutathione S-transferases play physiologically important roles in the detoxication of potential alkylating agents. The glutathione S-conjugates thus formed are transported out of cells for further metabolism by gamma-glutamyltransferase and dipeptidases, ectoproteins that catalyze the sequential removal of the glutamyl and glycyl moieties, respectively. These ectoproteins are not found in all cells, but are localized predominantly to the apical surface of epithelial tissues. The resulting cysteine S-conjugates can be reabsorbed by specific cell types, and acetylated on the amino group of the cysteinyl residue by intracellular N-acetyl-transferases, to form the corresponding mercapturic acids (N-acetylcysteine S-conjugates). Mercapturic acids are then released into the circulation and delivered to the kidney for excretion in urine, or they may undergo further metabolism. Mercapturic acid biosynthesis is generally considered to be an interorgan process, with the liver serving as the major site of glutathione conjugation, and the kidney as the primary site for conversion of glutathione conjugates to cysteine conjugates. Cysteine conjugates formed in the kidney appear to be transported back to the liver for acetylation. This interorgan model of mercapturic acid synthesis is based largely on the interorgan distribution of the enzymes involved in their formation, and in particular of the enzyme gamma-glutamyltransferase. Rats have relatively low hepatic and high renal activities of gamma-glutamyltransferase, the only protein known to initiate the breakdown of glutathione S-conjugates. The low gamma-glutamyltransferase activity in rat liver limits the hepatic degradation of glutathione S-conjugates, particularly after large doses of xenobiotic. In contrast, hepatic gamma-glutamyltransferase is significantly higher in species such as rabbit, guinea pig, and dog, and as a consequence, nearly all of the glutathione and glutathione S-conjugates released by liver cells of these species is degraded within the liver. Recent studies demonstrate that glutathione S-conjugates synthesized within hepatocytes are secreted preferentially across the canalicular membrane into bile, and are broken down within biliary spaces to form cysteine S-conjugates. The latter are then reabsorbed by the liver, N-acetylated to form mercapturic acids, and reexcreted into bile, completing an intrahepatic pathway for mercapturic acid biosynthesis. The contribution of this intrahepatic pathway to overall mercapturate formation is dependent on dose of the electrophile, route of exposure, and the physicochemical properties of the glutathione S-conjugate formed, as well as the tissue distribution and activity of gamma-glutamyltransferase.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure-mutagenicity and structure-cytotoxicity studies on bromine-containing cysteine S-conjugates and related compounds.

Glutathione and cysteine S-conjugates of several haloalkenes are nephrotoxic and cytotoxic. Chloroalkene-derived S-(1-chloroalkenyl)-L-cysteine conjugates, but not fluoroalkene-derived S-(2,2-dihalo-1,1-difluorethyl)-L-cysteine conjugates, are mutagenic in the Ames test, although both types of S-conjugates are cytotoxic and nephrotoxic. Recent studies showed that bromine-containing S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteine conjugates are mutagenic in the Ames test, thus challenging the generalization that S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteine conjugates are not mutagenic. Hence a series of bromine-containing and bromine-lacking S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteine conjugates was prepared, and their mutagenicity was assessed in the Ames test with Salmonella typhimurium TA2638 as the test strain. In addition, several indices of cytotoxicity, including cytotoxicity in LLC-PK1 cells, induction of Ca2+ release from pig kidney mitochondria, and DNA double-strand breaks in LLC-PK1 cells, were measured. The bromine-containing S-conjugates S-(2-bromo-2-chloro-1,1-difluoroethyl)-L- cysteine (BCD-FC), S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine (BTFC), and S-(2,2-dibromo-1,1-difluoroethyl)-L-cysteine (DBDFC) were mutagenic in the Ames test, whereas S-(2-chloro-1,1,2-trifluorethyl)-L-cysteine (CTFC), S-(2,2-dichloro-1,1-difluoroethyl)-L-cysteine (DCDFC), and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFC), which lack bromine, were not. BCDFC, BTFC, CTFC, DBDFC, and TFC were cytotoxic in LLC-PK1 cells, and their cytotoxicity was blocked by the cysteine conjugate beta-lyase inhibitor (aminooxy)acetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the effects of L(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) on 4-aminophenol-induced nephrotoxicity in the Fischer 344 rat.

4-Aminophenol (para-aminophenol; PAP) causes selective necrosis to the S3 segment of the proximal tubule in experimental animals. The mechanism of PAP nephrotoxicity has not been fully elucidated, although it has been suggested to involve glutathione (GSH)-dependent S-conjugation followed by processing by the enzyme gamma-glutamyl transpeptidase (gamma GT) to the corresponding cysteine S-conjugate. This proposed toxicity mechanism was probed further by administering L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125), a potent gamma GT inhibitor, to Fischer 344 (F344) rats before treatment with PAP (100 mg/kg). AT-125 pretreatment did not appear to protect against PAP-induced nephrotoxicity as assessed by renal histopathology, clinical chemistry and proton nuclear magnetic resonance (1H NMR) spectroscopy of urine. These data suggest that renal gamma GT activity is not a prerequisite for PAP nephrotoxicity and that the generation of a cysteine S-conjugate is not a unique requirement for the induction of PAP nephrotoxicity.

Limited role of the intestinal microflora in the nephrotoxicity of hexachloro-1,3-butadiene in rats.

The nephrotoxicity of hexachlorobutadiene has been investigated in germ-free rats and compared to conventional rats that have a normal intestinal microflora. Presynthesized mercapturate intermediates of hexachlorobutadiene, the glutathione and the cysteine S-conjugate, were also administered. Germ-free rats appeared to be slightly more susceptible to hexachlorobutadiene by judging the extent of morphological changes compared to conventional ones. A similar response was also observed after treatment with the glutathione and cysteine S-conjugates. However, no significant difference between germ-free and conventional animals was monitored in the extent of elevation of blood urea nitrogen and plasma creatinine after treatment with hexachlorobutadiene or the glutathione and the cysteine S-conjugates. The urinary excretion of marker enzymes was monitored and showed that gamma-glutamyl transferase was significantly more increased in the germ-free rats when treated with the cysteine S-conjugate. In addition, no difference in the rate of glutathione conjugation of hexachlorobutadiene was measured between the two groups. Although a tendency toward a protective effect by the presence of an intestinal microflora was observed, the role of the intestinal microflora in detoxifying hexachlorobutadiene seems to be of limited importance in rats.

Amino acid transport and glutathione homeostasis: What is the mechanism for cysteine uptake from bile?

Transport of L-cysteine and a cysteine S-conjugate, S-(1,2-dichlorovinyl)-L-cysteine (DCVC) was investigated in rat liver canalicular plasma membrane (cLPM) vesicles. Cysteine uptake into an osmotically active intravesicular space was temperature sensitive and further enhanced by an inwardly directed Na+ gradient. Na+ -dependent and -independent L-cysteine uptake exhibited saturation kinetics with apparent K m of 53 ± 0.7 and 1300 ± 300 μM and Vmax of 95 ± 21 and 1600 ± 200 pmol ± mg protein−1; 10 sec−1 for the Na+ dependent components, and an apparent K m of 207 ± 48 μM and a Vmax of 355 ± 71 pmol ± mg protein−1 ± 10 sec−1 for the Na+ -independent component. Na+-dependent uptake was inhibited by L-alanine, glycine, L-phenylalanine and L-leucine, whereas Na+-independent uptake was inhibited by L-phenylalanine, L-leucine and 2-amino-2-norbornanecarboxylic acid. Both Na+ -dependent and -independent L-cysteine transport processes were inhibited by several cysteine S-conjugates, with DCVC having the strongest effect. Inhibition of [35S]-cysteine uptake by DCVC was noncompetitive with a K, of 1.2 ± 0.1 mM. On the other hand, uptake of [35 S IDCVC by the rat cLPM vesicles was not stimulated by a Na+-gradient, but was inhibited by several other amino acids, including L-cysteine. Further investigation of [35SlDCVC uptake in rat cLPM vesicles indicated a saturable Na+ -independent process with an apparent K, of 155 f 42 μM, and a V max of 393 f 53 pmol mg protein −1 5 sec −1. Time-dependent nonspecific binding of DCVC to rat cLPM vesicles was diminished by addition of aminooxyacetic acid, an inhibitor of cysteine conjugate β-lyase activity. These findings demonstrate that cysteine and DCVC uptake into cLPM vesicles occurs by both saturable and nonsaturable transport mechanisms. These transport pathways may function in vivo for uptake and recycling of biliary L-cysteine for glutathione turnover and some cysteine S-conjugates for intrahepatic mercapturic acid biosynthesis.

Amino acid transport and glutathione homeostasis: What is the mechanism for cysteine uptake from bile?

Transport of L-cysteine and a cysteine S-conjugate, S-(1,2-dichlorovinyl)-L-cysteine (DCVC) was investigated in rat liver canalicular plasma membrane (cLPM) vesicles. Cysteine uptake into an osmotically active intravesicular space was temperature sensitive and further enhanced by an inwardly directed Na+ gradient. Na+ -dependent and -independent L-cysteine uptake exhibited saturation kinetics with apparent Km of 53 ± 0.7 and 1300 ± 300 μM and Vmax of 95 ± 21 and 1600 ± 200 pmol ± mg protein−1; 10 sec−1 for the Na+ dependent components, and an apparent Km of 207 ± 48 μM and a Vmax of 355 ± 71 pmol ± mg protein−1 ± 10 sec−1 for the Na+ -independent component. Na+-dependent uptake was inhibited by L-alanine, glycine, L-phenylalanine and L-leucine, whereas Na+-independent uptake was inhibited by L-phenylalanine, L-leucine and 2-amino-2-norbornanecarboxylic acid. Both Na+ -dependent and -independent L-cysteine transport processes were inhibited by several cysteine S-conjugates, with DCVC having the strongest effect. Inhibition of [35S]-cysteine uptake by DCVC was noncompetitive with a K, of 1.2 ± 0.1 mM. On the other hand, uptake of [35 S IDCVC by the rat cLPM vesicles was not stimulated by a Na+-gradient, but was inhibited by several other amino acids, including L-cysteine. Further investigation of [35SlDCVC uptake in rat cLPM vesicles indicated a saturable Na+ -independent process with an apparent K, of 155 f 42 μM, and a Vmax of 393 f 53 pmol mg protein −1 5 sec −1. Time-dependent nonspecific binding of DCVC to rat cLPM vesicles was diminished by addition of aminooxyacetic acid, an inhibitor of cysteine conjugate β-lyase activity. These findings demonstrate that cysteine and DCVC uptake into cLPM vesicles occurs by both saturable and nonsaturable transport mechanisms. These transport pathways may function in vivo for uptake and recycling of biliary L-cysteine for glutathione turnover and some cysteine S-conjugates for intrahepatic mercapturic acid biosynthesis.

Fatty acid beta-oxidation-dependent bioactivation of halogenated thiaalkanoic acids in isolated rat hepatocytes.

5,6-Dichloro-4-thia-5-hexenoic acid (DCTH), the desamino analog of the nephrotoxic cysteine S-conjugate S-(1,2-dichlorovinyl)-L-cysteine, is toxic to liver and kidney mitochondria. The mechanism by which DCTH produces mitochondrial dysfunction has not been defined. The objective of the present experiments was to test the hypothesis that DCTH is bioactivated by the mitochondrial fatty acid beta-oxidation system to cytotoxic intermediates. Incubation of isolated rat hepatocytes with DCTH produced a time- and concentration-dependent decrease in cell viability. The even-chain, elongated analog 7,8-dichloro-6-thia-7-octenoic acid was also cytotoxic, whereas the odd-chain-length analogs 6,7-dichloro-5-thia-6-heptenoic acid and 8,9-dichloro-7-thia-8-nonenoic acid were not. Sodium benzoate reduced the cytotoxicity of DCTH, indicating a role for coenzyme A in the bioactivation of DCTH. DCTH decreased cellular ATP concentrations, the cellular energy charge, and cellular glutathione concentrations; these changes preceded the decrease in cell viability, indicating that mitochondrial dysfunction may be an early event in DCTH-induced cytotoxicity. 6-Chloro-5,5,6-trifluoro-4-thiahexanoic acid and 5,6,7,8,8-pentachloro-4-thia-5,7-octadienoic acid were also cytotoxic in isolated hepatocytes, whereas 4-(2-benzothiazolyl)-4-thiabutanoic acid was not. These data are consistent with the hypothesis that the mitochondrial fatty acid beta-oxidation system is involved in the bioactivation of DCTH and that mitochondria may be important cellular targets in DCTH-induced cytotoxicity.

Nephrotoxic and Genotoxic N-Acetyl-S-Dichlorovinyl-L-Cysteine Is a Urinary Metabolite after Occupational 1,1,2-Trichloroethene Exposure in Humans: Implications for the Risk of Trichloroethene Exposure

Excretion of mercapturic acids in the urine is indicative of the formation of electrophiles in the metabolism of xenobiotics. The determination of these mercapturic acids thus may be a useful method to estimate the exposure. We identified the nephrotoxic and mutagenic mercapturic acids N-acetyl-S-(1,2-dichlorovinyl)-L- cysteine and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine in the urine of workers exposed to 1,1,2-trichloroethene. A method to quantify these mercapturic acids by gas chromatography-mass spectrometry-selected ion monitoring was developed and appreciable amounts (2.8-3.8 mumole/L were found in human urine samples. Because deacetylation determines notably the amount of the excreted mercapturic acids, the formation of the resulting cysteine S-conjugates was comparably measured in subcellular fractions of rodent and human kidneys; significant species differences in acylase activity were found. The formation of mutagenic and nephrotoxic metabolites during 1,1,2-trichloroethene metabolism mandates a revision of the risk assessment of trichloroethene exposure.

Nephrotoxic and genotoxic N-acetyl-S-dichlorovinyl-L-cysteine is a urinary metabolite after occupational 1,1,2-trichloroethene exposure in humans: implications for the risk of trichloroethene exposure.

Excretion of mercapturic acids in the urine is indicative of the formation of electrophiles in the metabolism of xenobiotics. The determination of these mercapturic acids thus may be a useful method to estimate the exposure. We identified the nephrotoxic and mutagenic mercapturic acids N-acetyl-S-(1,2-dichlorovinyl)-L- cysteine and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine in the urine of workers exposed to 1,1,2-trichloroethene. A method to quantify these mercapturic acids by gas chromatography-mass spectrometry-selected ion monitoring was developed and appreciable amounts (2.8-3.8 mumole/L were found in human urine samples. Because deacetylation determines notably the amount of the excreted mercapturic acids, the formation of the resulting cysteine S-conjugates was comparably measured in subcellular fractions of rodent and human kidneys; significant species differences in acylase activity were found. The formation of mutagenic and nephrotoxic metabolites during 1,1,2-trichloroethene metabolism mandates a revision of the risk assessment of trichloroethene exposure.

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 beta-lyase. Beta-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 gamma-glutamyltransferase-rich tissues, such as the kidney, where they undergo alkylation or redox cycling reactions, or both, that cause organ-selective damage.