Cysteine S-conjugates Research Articles

Molecular Identification of NAT8 as the Enzyme That Acetylates Cysteine S-Conjugates to Mercapturic Acids

Our goal was to identify the reaction catalyzed by NAT8 (N-acetyltransferase 8), a putative N-acetyltransferase homologous to the enzyme (NAT8L) that produces N-acetylaspartate in brain. The almost exclusive expression of NAT8 in kidney and liver and its predicted association with the endoplasmic reticulum suggested that it was cysteinyl-S-conjugate N-acetyltransferase, the microsomal enzyme that catalyzes the last step of mercapturic acid formation. In agreement, HEK293T extracts of cells overexpressing NAT8 catalyzed the N-acetylation of S-benzyl-L-cysteine and leukotriene E(4), two cysteine conjugates, but were inactive on other physiological amines or amino acids. Confocal microscopy indicated that NAT8 was associated with the endoplasmic reticulum. Neither of the two frequent single nucleotide polymorphisms found in NAT8, E104K nor F143S, changed the enzymatic activity or the expression of the protein by >or=2-fold, whereas a mutation (R149K) replacing an extremely conserved arginine suppressed the activity. Sequencing of genomic DNA and EST clones corresponding to the NAT8B gene, which resulted from duplication of the NAT8 gene in the primate lineage, disclosed the systematic presence of a premature stop codon at codon 16. Furthermore, truncated NAT8B and NAT8 proteins starting from the following methionine (Met-25) showed no cysteinyl-S-conjugate N-acetyltransferase activity when transfected in HEK293T cells. Taken together, these findings indicate that NAT8 is involved in mercapturic acid formation and confirm that NAT8B is an inactive gene in humans. NAT8 homologues are found in all vertebrate genomes, where they are often encoded by multiple, tandemly repeated genes as many other genes encoding xenobiotic metabolism enzymes.

Cysteine S-conjugate β-lyases: important roles in the metabolism of naturally occurring sulfur and selenium-containing compounds, xenobiotics and anticancer agents

Cysteine S-conjugate β-lyases are pyridoxal 5'-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess a good leaving group in the β-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate β-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze β-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate β-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate β-lyases have been reviewed in this journal previously (Cooper and Pinto in Amino Acids 30:1-15, 2006). Here, we focus on more recent findings regarding: (1) the identification of enzymes associated with high-M(r) cysteine S-conjugate β-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; (2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic β-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene); (3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; (4) the involvement of cysteine S-conjugate β-lyases in the metabolism/bioactivation of drugs and natural products; and (5) the role of cysteine S-conjugate β-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate β-lyases are biologically more important than hitherto appreciated.

Identification and Quantitation of theN-Acetyl-L-cysteineS-Conjugates of Bendamustine and Its Sulfoxides in Human Bile after Administration of Bendamustine Hydrochloride

We recently reported the detection of mercapturic acid pathway metabolites of bendamustine, namely, cysteine S-conjugates in human bile, which are supposed to subsequently undergo further metabolism. In this study, we describe the identification and quantitation of consecutive bendamustine metabolites occurring in human bile using authentic reference standards and the synthesis and structural confirmation of these compounds. Mass spectrometry data along with high-performance liquid chromatography retention data (fluorescence detection) of the synthetic reference standards were consistent with those of the metabolites found in human bile after administration of bendamustine hydrochloride to cancer patients. Analysis of the purified synthetic reference compounds showed a purity of at least 95%. Structural confirmation was achieved by one- and two-dimensional proton as well as carbon-13 NMR spectroscopy and mass spectrometry. A total of 16 bendamustine-related compounds were detected in the bile of patients, 11 of them were recovered as conjugates. Eight conjugates have been structurally confirmed as novel mercapturic acids and sulfoxides. Biliary excretion of the sulfoxides was twice that of the mercapturate precursors. Glutathione S-conjugates of bendamustine have not been detected in bile samples, indicating rapid enzymatic cleavage in humans. Both the lack of glutathione (GSH) conjugates and occurrence of diastereomeric sulfoxides emphasize species-related differences in the GSH conjugation of bendamustine between humans and rats. The total amount recovered in the bile as the sum of all conjugates over the period of 24 h after dosing averaged 5.2% of the administered dose. The question of whether the novel metabolites contribute to urinary excretion should be a target of future investigations.

Role of glutathione S-transferase Pi in cisplatin-induced nephrotoxicity

One of the dose-limiting toxicities of cisplatin is nephrotoxicity. Renal toxicity is localized to quiescent proximal tubule cells, where the formation of DNA-adducts cannot account for the dose-limiting toxicity. Our earlier results have shown that a glutathione conjugate of cisplatin is metabolized to a nephrotoxicant via gamma-glutamyl transpeptidase (GGT) and a cysteine S-conjugate beta-lyase. The present study was designed to evaluate the potential role of glutathione S-transferase Pi (GSTP) in the initial steps of the bioactivation of cisplatin. Wild-type mice and mice deficient in both murine GSTP genes ( GstP1/P2) were treated with cisplatin. Toxicity in both male and female mice was evaluated 5 days after treatment and renal damage was most severe in wild-type male mice. Wild-type males have ∼10-fold higher levels of GSTP expression in the liver than females, suggesting that hepatic GSTP in the wild-type males contributed to the formation of the nephrotoxic platinum–glutathione conjugate. In GstP1/P2 null mice the gender difference in toxicity was eliminated. Our data show that GSTP expression is a determinant in cisplatin-induced nephrotoxicity and its levels contribute to sex-dependent differences.

Metabolism of the CysteineS-Conjugate of Busulfan Involves a β-Lyase Reaction

The present work documents the first example of an enzyme-catalyzed beta-elimination of a thioether from a sulfonium cysteine S-conjugate. beta-(S-Tetrahydrothiophenium)-L-alanine (THT-A) is the cysteine S-conjugate of busulfan. THT-A slowly undergoes a nonenzymatic beta-elimination reaction at pH 7.4 and 37 degrees C to yield tetrahydrothiophene, pyruvate, and ammonia. This reaction is accelerated by 1) rat liver, kidney, and brain homogenates, 2) isolated rat liver mitochondria, and 3) pyridoxal 5'-phosphate (PLP). A PLP-dependent enzyme in rat liver cytosol that catalyzes a beta-lyase reaction with THT-A was identified as cystathionine gamma-lyase. This unusual drug metabolism pathway represents an alternate route for intermediates in the mercapturate pathway.

Bioactivation of Selenocysteine Derivatiives by β-Lyases Present in Common Gastrointestinal Bacterial Species

Studies in cell cultures and animal models have demonstrated cancer chemopreventive effects of certain selenium compounds. Here we describe the screening of cysteine S-conjugate beta-lyase activity in bacterial species that are implicated in the bio-activation of sulfur- and selenocysteine derivatives. We screened a range of bacterial species commonly found in the human intestine for beta-lyase activity on Se-p-methoxybenzylselenocysteine and the natural occurring S-methylcysteine and Se-methylselenocysteine conjugates. A high-performance liquid chromatography (HPLC)-assisted assay was established to determine specific activities of each strain. Of the 29 tested bacterial species, 22 showed specific activities towards the test compound reaching up to 10.1 U/mg protein, thereby accounting for 75% of total fecal activity (13.3. U/mg protein). Lysates of four bacterial strains (Bacteroides distasonis, bacteroides vulgatus, Enterococcus faecalis, and Enterococcus faecium), which exhibited high specific activities towards the test compound and which are known to be present at high numbers in the human intestine, were characterized further. Our results indicate that beta-lyase activity is widely distributed in human intestinal bacteria and might play a key role in the bioactivation of selenocysteine derivatives.

Quantification of CysteineS-Conjugate of 3-Sulfanylhexan-1-ol in Must and Wine of Petite Arvine Vine by Stable Isotope Dilution Analysis

Making use of a convenient synthetic approach to prepare the deuterated S-3-(hexan-1-ol)-cysteine by a Michael addition reaction, an analytical method was developed to measure the presence of the cysteine S-conjugate, precursor of 3-sulfanylhexan-1-ol (3-mercaptohexan-1-ol), in must and wine from Petite Arvine vine. The method uses a stable isotope dilution assay with a suitable one-step sample preparation and HPLC-MS detection. The method has limits of detection and quantification of 3 and 10 microg/L, respectively. A correlation between the increase of the precursor concentration and the increase of the degree of rot has been established.

Toluene-induced hearing loss in acivicin-treated rats

Toluene can be considered an ototoxic chemical compound in the rat. Outer hair cells are particularly sensitive to this aromatic organic solvent or to one of its metabolites. The objective of the present study was to evaluate the possible role played by cysteine S-conjugates in the ototoxic process in Long–Evans rats. To this end, renal and hepatic metabolism of toluene was modified by treatment with acivicin, an inhibitor of γ-glutamyl transferase (γ-GT). First, the efficacy of the acivicin treatment was established from a dose–response investigation in which urinary γ-GT was measured daily in rats exposed to 1750 ppm toluene, 6 h per day for five days. A twice weekly 5 mg/kg dose was reduced urinary γ-GT by 70–78%. In a subacute experiment, rats were exposed to 1750 ppm toluene for four consecutive weeks, in which the efficacy of the acivicin treatment was monitored by quantifying the urinary end product of the conjugate pathway: benzyl mercapturic acid (BMA). A 38.5% decrease in BMA was measured at the end of the exposure period. Hearing impairment was evaluated using auditory (inferior colliculus) evoked potentials and completed with conventional histological approaches. The toluene-exposed and the acivicin-treated rats exposed to toluene both had a 7-dB permanent auditory threshold shift at 16–20 kHz. Hair cell loss was not dependent on acivicin treatment. Therefore, the partial inhibition of γ-GT did not modify the toluene ototoxicity, suggesting that toluene-induced hearing loss is not strongly mediated by the production of cysteine S-conjugates. However, the data do not rule out the possibility that these metabolites may play a minor role.

Substrate specificity of human glutamine transaminase K as an aminotransferase and as a cysteine S-conjugate β-lyase

Rat kidney glutamine transaminase K (GTK) exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. The β-lyase reaction products are pyruvate, ammonium and a sulfhydryl-containing fragment. We show here that recombinant human GTK (rhGTK) also exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. S-(1,1,2,2-Tetrafluoroethyl)-l-cysteine is an excellent aminotransferase and β-lyase substrate of rhGTK. Moderate aminotransferase and β-lyase activities occur with the chemopreventive agent Se-methyl-l-selenocysteine. l-3-(2-Naphthyl)alanine, l-3-(1-naphthyl)alanine, 5-S-l-cysteinyldopamine and 5-S-l-cysteinyl-l-DOPA are measurable aminotransferase substrates, indicating that the active site can accommodate large aromatic amino acids. The α-keto acids generated by transamination/l-amino acid oxidase activity of the two catechol cysteine S-conjugates are unstable. A slow rhGTK-catalyzed β-elimination reaction, as measured by pyruvate formation, was demonstrated with 5-S-l-cysteinyldopamine, but not with 5-S-l-cysteinyl-l-DOPA. The importance of transamination, oxidation and β-elimination reactions involving 5-S-l-cysteinyldopamine, 5-S-l-cysteinyl-l-DOPA and Se-methyl-l-selenocysteine in human tissues and their biological relevance are discussed.

Structural Insight into the Mechanism of Substrate Specificity of Aedes Kynurenine Aminotransferase,

Aedes aegypti kynurenine aminotransferase (AeKAT) is a multifunctional aminotransferase. It catalyzes the transamination of a number of amino acids and uses many biologically relevant alpha-keto acids as amino group acceptors. AeKAT also is a cysteine S-conjugate beta-lyase. The most important function of AeKAT is the biosynthesis of kynurenic acid, a natural antagonist of NMDA and alpha7-nicotinic acetylcholine receptors. Here, we report the crystal structures of AeKAT in complex with its best amino acid substrates, glutamine and cysteine. Glutamine is found in both subunits of the biological dimer, and cysteine is found in one of the two subunits. Both substrates form external aldemines with pyridoxal 5-phosphate in the structures. This is the first instance in which one pyridoxal 5-phosphate enzyme has been crystallized with cysteine or glutamine forming external aldimine complexes, cysteinyl aldimine and glutaminyl aldimine. All the units with substrate are in the closed conformation form, and the unit without substrate is in the open form, which suggests that the binding of substrate induces the conformation change of AeKAT. By comparing the active site residues of the AeKAT-cysteine structure with those of the human KAT I-phenylalanine structure, we determined that Tyr286 in AeKAT is changed to Phe278 in human KAT I, which may explain why AeKAT transaminates hydrophilic amino acids more efficiently than human KAT I does.

Methods for Measuring Cysteine S‐Conjugate β‐Lyase Activity

The cysteine conjugate β-lyase represents activities in cytoplasm and mitochondria catalyzed by at least eleven pyridoxal 5'-phosphate (PLP)-dependent enzymes in various tissues. These enzymes mediate bioactivation of cysteine S-conjugates of several haloalkanes and haloalkenes. The reaction occurs through either a direct β-elimination or a transamination followed by a retro-Michael rearrangement, resulting in the cleavage of a C-S bond. The resultant product is a reactive thiolate that rearranges to form thioacylating species. This unit presents several protocols for the assay of β-lyase activity and includes measurements of product formation and substrate loss as well as fluorescent activity stains. Support protocols describe the synthesis and high-performance liquid chromatography analysis of selected cysteine S-conjugates. Because of the diversity of enzymes that can catalyze a β-lyase reaction, each of the assays presented here may indicate only a portion of the potential β-lyase activity in a given biological preparation.

Glutathione‐Dependent Bioactivation

The classical view of the glutathione (GSH) conjugation pathway involves GSH S-transferase (GST)-dependent formation of thioether conjugates between GSH and an electrophilic substrate, processing to yield the corresponding cysteine S-conjugate, which is then converted to an N-acetylcysteine conjugate (or mercapturate). Mercapturates of most GST substrates are rendered more polar and thus readily excreted in urine. In contrast, there is a growing number of GST substrates that, rather than being detoxified, are bioactivated. These substrates include several halogenated solvents, many of which are nephrotoxic because of the tissue distribution of GSH conjugation pathway enzymes and membrane transporters, and prodrugs of certain chemotherapeutic agents. Although the initiating steps are the same regardless of whether the substrate is detoxified or bioactivated, the cysteine conjugate functions as a branch point. Bioactivated cysteine S-conjugates are metabolized in the kidneys by either cysteine conjugate β-lyase or flavin-containing monooxygenase to produce a reactive intermediate.

Chemical Toxicology of Reactive Intermediates Formed by the Glutathione-Dependent Bioactivation of Halogen-Containing Compounds

The concept that reactive intermediate formation during the biotransformation of drugs and chemicals is an important bioactivation mechanism was proposed in the 1970s and is now accepted as a major mechanism for xenobiotic-induced toxicity. The enzymology of reactive intermediate formation as well as the characterization of the formation and fate of reactive intermediates are now well-established. The mechanism by which reactive intermediates cause cell damage and death is, however, still poorly understood. Although most xenobiotic-metabolizing enzymes catalyze the bioactivation of chemicals, glutathione-dependent biotransformation has been largely associated with detoxication processes, particularly mercapturic acid formation. Abundant evidence now shows that glutathione-dependent biotransformation constitutes an important bioactivation mechanism for halogen-containing drugs and chemicals and has for many compounds been implicated in their organ-selective toxicity and in their mutagenic and carcinogenic potential. The glutathione-dependent biotransformation of haloalkenes is the first step in the cysteine S-conjugate beta-lyase pathway for the bioactivation of nephrotoxic haloalkenes. This pathway has been a rich source of reactive intermediates, including thioacyl halides, alpha-chloroalkenethiolates, 3-halo-alpha-thiolactones, 2,2,3-trihalothiiranes, halothioketenes, and vinylic sulfoxides. Glutathione-dependent bioactivation of gem-dihalomethanes and 1,2-, 1,3-, and 1,4-dihaloalkanes leads to the formation of alpha-chlorosulfides, thiiranium ions, sulfenate esters, and tetrahydrothiophenium ions, respectively, and these reactions lead to reactive intermediate formation.

Mouse Monocytes (RAW CELLS) and the Handling of Cysteine and Homocysteine S-Conjugates of Inorganic Mercury and Methylmercury

Although there is evidence indicating that mononuclear phagocytes can take up mercury by some forms of endocytosis, very little is known about the potential for the uptake of mercuric species by carrier-mediated processes. Thus, we hypothesized that monocytes also possess mechanisms allowing these cells to take up inorganic mercury (Hg2+) and/or methylmercury (CH3Hg+) as cysteine (Cys) and/or homocysteine (Hcy) S-conjugates by certain membrane transport proteins. The specific thiol S-conjugates were chosen for study because our laboratory and those of some other investigators have demonstrated that these species of mercury are indeed transportable substrates for several membrane transport proteins in certain types of epithelial cells. We chose to use RAW 264.7 cells for our experiments. These cells represent an adherent line of mouse monocytes. Kinetic analyses for the uptake of Cys-Hg-Cys, CH3Hg-Cys, Hcy-Hg-Hcy, and CH3Hg-Hcy revealed that uptake occurred by a saturable, concentration-dependent mechanism, displaying Michaelis–Menten properties. Interestingly, in the cells exposed to the Cys or Hcy S-conjugate of Hg2+, significantly more Hg2+ was taken up in the presence of 140 mM sodium chloride (NaCl) than in the presence of 140 mM N-methyl-d-glucamine (NMDG), indicating that Na-dependent processes play more of a role in the uptake of these species of Hg2+ than sodium-independent ones. With respect to the uptake of CH3Hg+, rates of uptake of the Cys and Hcy S-conjugates of CH3Hg+ were similar under both Na-dependent and Na-independent conditions, although the levels of uptake of these mercuric species far exceeded the levels of uptake of the corresponding S-conjugate of Hg2+. Uptake of Hg2+ and CH3Hg+, as the Cys or Hcy S-conjugates, was also time-dependent. We also showed that when the temperature in the bathing medium was reduced to 4°C, uptake of the Cys S-conjugates Hg2+ or CH3Hg+ was for the most part reduced to negligible levels in the RAW cells; indicating that the preponderance of uptake at 37°C was not due primarily to simple diffusion and/or non-specific binding. Overall, the present findings strongly suggest that the uptake of the Cys and Hcy S-conjugates of Hg2+ and/or CH3Hg+ occurs in monocytes by one or more mechanisms involving carrier proteins.

Structural characterization of dimeric murine aminoacylase III

Aminoacylase III (AAIII) plays an important role in deacetylation of acetylated amino acids and N-acetylated S-cysteine conjugates of halogenated alkenes and alkanes. AAIII, recently cloned from mouse kidney and partially characterized, is a mixture of tetramers and dimers. In the present work, AAIII dimers were purified and shown to be enzymatically active. Limited trypsinolysis showed two domains of ∼9 and 25 kDa. The three-dimensional structure of the dimer was studied by electron microscopy of negative stained samples and by single-particle reconstruction. A 16 Å resolution model of the AAIII dimer was created. It has an unusual, cage-like, structure. A realistic AAIII tetramer model was built from two dimers.

Specificity of Aminoacylase III-Mediated Deacetylation of Mercapturic Acids

Trichloroethylene (TCE) and other halogenated alkenes are known environmental contaminants with cytotoxic and nephrotoxic effects, and are potential carcinogens. Their metabolism via the mercapturate metabolic pathway was shown to lead to their detoxification. The final products of this pathway, mercapturic acids or N-acetyl-l-cysteine S-conjugates, are secreted into the lumen in the renal proximal tubule. The proximal tubule may also deacetylate mercapturic acids, and the resulting cysteine S-conjugates are transformed by cysteine S-conjugate beta-lyases to nephrotoxic reactive thiols. The specificity and rate of mercapturic acid deacetylation may determine the toxicity of certain mercapturic acids; however, the exact enzymologic processes involved are not known in detail. In the present study we characterized the kinetics of the recently cloned mouse aminoacylase III (AAIII) toward a wide spectrum of halogenated mercapturic acids and N-acetylated amino acids. In general, the V(max) value of AAIII was significantly larger with chlorinated and brominated mercapturic acids, whereas fluorination significantly decreased it. The enzyme deacetylated mercapturic acids derived from the TCE metabolism including N-acetyl-S-(1,2-dichlorovinyl)-l-cysteine (NA-1,2-DCVC) and N-acetyl-S-(2,2-dichlorovinyl)-l-cysteine (NA-2,2-DCVC). Both mercapturic acids induced cytotoxicity in mouse proximal tubule mPCT cells expressing AAIII, which was decreased by an inhibitor of beta-lyase, aminooxyacetate. The toxic effect of NA-2,2-DCVC was smaller than that of NA-1,2-DCVC, indicating that factors other than the intracellular activity of AAIII mediate the cytotoxicity of these mercapturic acids. Our results indicate that in proximal tubule cells, AAIII plays an important role in deacetylating several halogenated mercapturic acids, and this process may be involved in their cyto- and nephrotoxicity.

System B0,+and the Transport of Thiol-S-Conjugates of Methylmercury

Methylmercury (CH(3)Hg(+)) is a clinically relevant toxicant that is the most abundant form of mercury found in the environment. After exposure, it accumulates in the kidneys, liver, and central nervous system. The mechanisms by which this toxicant is taken up by target cells are only now beginning to be understood. Some experimental data support a hypothesis involving molecular mimicry, whereby thiol conjugates of methylmercury (especially a cysteine S-conjugate) mimic one or more amino acids and are transported into target cells by amino acid transporters. In the present study, we tested the hypothesis that Cys and homocysteine (Hcy) S-conjugates of methylmercury (CH(3)Hg-S-Cys and CH(3)Hg-S-Hcy, respectively) mimic one or more amino acids at the site of the Na(+)-dependent amino acid transporter, system B(0,+). In the kidneys, system B(0,+) is situated on the luminal plasma membrane of proximal tubular epithelial cells. To test our hypothesis, we measured uptake of CH(3)Hg-S-Cys and CH(3)Hg-S-Hcy in Xenopus laevis oocytes injected with water or capped RNA encoding mouse ATB(0,+). Analyses of time course, substrate specificity, and saturation kinetics showed that the uptake of CH(3)Hg-S-Cys and CH(3)Hg-S-Hcy was 5- to 10-fold greater in oocytes expressing ATB(0,+) than in corresponding water-injected controls. Moreover, the transport of CH(3)Hg-S-Cys and CH(3)Hg-S-Hcy was inhibited by substrates transported by system B(0,+). Finally, our data indicate that CH(3)Hg-S-Cys and CH(3)Hg-S-Hcy may mimic of one or more amino acids (e.g., methionine) that are normally transported by system B(0,+). To our knowledge, this is the first report implicating system B(0,+) in the transport of any mercuric species.

Cisplatin-Induced Toxicity Is Associated with Platinum Deposition in Mouse Kidney Mitochondria in Vivo and with Selective Inactivation of the α-Ketoglutarate Dehydrogenase Complex in LLC-PK1 Cells

The anticancer drug cisplatin is nephrotoxic and neurotoxic. Previous data support the hypothesis that cisplatin is bioactivated to a nephrotoxicant. The final step in the proposed bioactivation is the formation of a platinum-cysteine S-conjugate followed by a pyridoxal 5'-phosphate (PLP)-dependent cysteine S-conjugate beta-lyase reaction. This reaction would generate pyruvate, ammonium, and a highly reactive platinum (Pt)-thiol compound in vivo that would bind to proteins. In this work, the cellular location and identity of the PLP-dependent cysteine S-conjugate beta-lyase were investigated. Pt was shown to bind to proteins in kidneys of cisplatin-treated mice. The concentration of Pt-bound proteins was higher in the mitochondrial fraction than in the cytosolic fraction. Treatment of the mice with aminooxyacetic acid (AOAA, a PLP enzyme inhibitor), which had previously been shown to block the nephrotoxicity of cisplatin, decreased the binding of Pt to mitochondrial proteins but had no effect on the amount of Pt bound to proteins in the cytosolic fraction. These data indicate that a mitochondrial enzyme catalyzes the PLP-dependent cysteine S-conjugate beta-lyase reaction. PLP-dependent mitochondrial aspartate aminotransferase (mitAspAT) is a mitochondrial enzyme that catalyzes beta-elimination reactions with cysteine S-conjugates of halogenated alkenes. We reasoned that the enzyme might also catalyze a beta-lyase reaction with the cisplatin-cysteine S-conjugate. In this study, mitAspAT was stably overexpressed in LLC-PK(1) cells. Cisplatin was significantly more toxic in confluent monolayers of LLC-PK(1) cells that overexpressed mitAspAT than in control cells containing vector alone. AOAA completely blocked the cisplatin toxicity in confluent mitAspAT-transfected cells. The Pt-thiol compound could rapidly bind proteins and inactivate enzymes in close proximity of the PLP-dependent cysteine S-conjugate beta-lyase. Treatment with 50 or 100 microM cisplatin for 3 h, followed by removal of cisplatin from the medium for 24 h, resulted in a pronounced loss of alpha-ketoglutarate dehydrogenase complex (KGDHC) activity in both mitAspAT-transfected cells and control cells. Exposure to 100 microM cisplatin resulted in a significantly greater loss of KGDHC activity in the cells overexpressing mitAspAT than in control cells. Aconitase activity was diminished in both cell types, but only at the higher level of exposure to cisplatin. AspAT activity was also significantly decreased by cisplatin treatment. By contrast, several other enzymes (both cytosolic and mitochondrial) involved in energy/amino acid metabolism were not significantly affected by cisplatin treatment in the LLC-PK(1) cells, whether or not mitAspAT was overexpressed. The susceptibility of KGDHC and aconitase to inactivation in kidney cells exposed to cisplatin metabolites may be due to the proximity of mitAspAT to KGDHC and aconitase in mitochondria. These findings support the hypothesis that a mitochondrial cysteine S-conjugate beta-lyase converts the cisplatin-cysteine S-conjugate to a toxicant, and the data are consistent with the hypothesis that mitAspAT plays a role in the bioactivation of cisplatin.

Role of System ATB 0,+ in the Transport of Cysteine S ‐Conjugates of Methylmercury

Methylmercury (CH3Hg) is a clinically relevant toxicant that is the most abundant form of mercury found in the environment. Once ingested, it accumulates in the liver, kidneys and central nervous system. The mechanisms by which this toxicant is taken up by target cells are only now beginning to be understood. Some experimental data support a hypothesis involving molecular mimicry, whereby a thiol-conjugate of methylmercury, especially a cysteine S-conjugate, mimics an amino acid, such as methionine, and is transported into the target cells by one or more amino acid transporter(s). In the present study, we tested the hypothesis that the cysteine (Cys) S-conjugate of methylmercury (CH3Hg-S-Cys) mimics an amino acid at the site of system ATB0,+, which in the kidneys, is situated on the luminal plasma membrane of proximal tubular epithelial cells. To test this hypothesis, we measured the uptake of 14C-CH3Hg-S-Cys into Xenopus laevis oocytes injected with cRNA encoding mouse ATB0,+. Time-course, substrate-specificity and saturation kinetic analyses showed that the uptake of CH3Hg-S-Cys was 5–10-fold greater in oocytes injected with ATB0,+ than in water-injected controls. The transport of CH3Hg-S-Cys was inhibited by substrates of system ATB0,+. These data indicate that CH3Hg-S-Cys may act as a mimic of an amino acid that is normally transported by system ATB0,+. To our knowledge, this is the first report implicating system ATB0,+ in the Na+-dependent transport of CH3Hg-S-Cys.

Redox-Sensitive Proteins Are Potential Targets of Garlic-Derived Mercaptocysteine Derivatives1–3

Molecular investigations support existing clinical and epidemiological data that garlic-derived allylsulfides reduce cancer risk. Various allylsulfides can diminish progression of cancer cells at either the G1/S or G2/M phase. Allylsulfide derivatives modify redox-sensitive signal pathways and cause growth inhibition, mitotic arrest, and apoptosis induction. Whether allylsulfides modify intracellular redox potentials by affecting the ratio of glutathione:glutathione disulfide and/or by interacting directly with sulfhydryl domains on regulatory or catalytic-signal proteins requires further investigation. To understand the possible biochemical mechanisms contributing to the protective effects of allylsulfides, we investigated the ability of these compounds to undergo enzyme-catalyzed transformations. In addition to catalyzing gamma-elimination reactions, gamma-cystathionase can perform beta-elimination reactions with cysteinyl S-conjugates derived from garlic extracts when the S-alkyl group (R) is larger than ethyl. The reaction products are pyruvate, ammonium, and a sulfur-containing fragment (RSH). beta-Lyase substrates of gamma-cystathionase thus far identified from garlic include: S-allyl-L-cysteine (R=CH2=CHCH2-), S-allylmercapto-L-cysteine (R=CH2=CHCH2S-), and S-propylmercapto-L-cysteine (R=CH3CH2CH2S-). Mercapto derivatives yield persulfide products (RSSH) that are potential sources of sulfane sulfur, which may modify protein function by reacting at important cysteinyl domains. Thus, beta-elimination reactions with cysteine S-conjugates in garlic may modify cancer-cell growth by targeting redox-sensitive signal proteins at sulfhydryl sites, thereby regulating cell proliferation and/or apoptotic responses. These interactions may be useful in identifying efficacy of garlic-derived compounds and/or developing other novel organosulfur compounds that may modify intracellular redox potentials or interact with thiols associated within cysteine domains in regulatory, catalytic, signal, or structural proteins.