Adnan A. Elfarra

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.

Mutagenicity of amino acid and glutathione S-conjugates in the Ames test

The mutagenicity of the glutathione S-conjugate S-(1,2-dichlorovinyl)glutathione (DCVG), the cysteine conjugates S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,2-dichlorovinyl)-DL-alpha-methylcysteine (DCVMC), and the homocysteine conjugates S-(1,2-dichlorovinyl)-L-homocysteine (DCVHC) and S-(1,2-dichlorovinyl)-DL-alpha-methylhomocysteine (DCVMHC) was investigated in Salmonella typhimurium strain TA2638 with the preincubation assay. DCVC was a strong, direct-acting mutagen; the cysteine conjugate beta-lyase inhibitor aminooxyacetic acid decreased significantly the number of revertants induced by DCVC; rat renal mitochondria (11,000 X g pellet) and cytosol (105,000 X g supernatant) with high beta-lyase activity increased DCVC mutagenicity at high DCVC concentrations. DCVG was also mutagenic without the addition of mammalian activating enzymes; the presence of low gamma-glutamyltransferase activity in bacteria, the reduction of DCVG mutagenicity by aminooxyacetic acid, and the potentiation of DCVG mutagenicity by rat kidney mitochondria and microsomes (105,000 X g pellet) with high gamma-glutamyltransferase activity indicate that gamma-glutamyltransferase and beta-lyase participate in the metabolism of DCVG to mutagenic intermediates. The homocysteine conjugate DCVHC was only weakly mutagenic in the presence of rat renal cytosol, which exhibits considerable gamma-lyase activity, this mutagenic effect was also inhibited by aminooxyacetic acid. The conjugates DCVMC and DCVMHC, which are not metabolized to reactive intermediates, were not mutagenic at concentrations up to 1 mumole/plate. The results demonstrate that gamma-glutamyltransferase and beta-lyase are the key enzymes in the biotransformation of cysteine and glutathione conjugates to reactive intermediates that interact with DNA and thereby cause mutagenicity.

Cellular effects of reactive intermediates: Nephrotoxicity of S-conjugates of amino acids

Several cysteine S-conjugates are potent nephrotoxins and require enzymatic activation to produce cytotoxicity. Strategies based on the knowledge that renal cysteine conjugate β-lyase is apparently a pyridoxal phosphate (PLP)-dependent enzyme have been exploited to test the hypothesis that a β-lyase-dependent activation is required for the expression of cysteine S-conjugate-induced toxicity. First, the toxicity of the model conjugate S-(1,2-dichlorovinyl)-L-cysteine (DCVC) is blocked both in vivo and in isolated, renal proximal tubular cells by aminooxyacetic acid, an inhibitor of PLP-dependent enzymes. Second, the nonmetabolizable α-methyl analogue S-(1,2-dichlorovinyl)-DL-α-methylcysteine is not toxic. Third, to test the hypothesis that the toxicity of DCVC is associated with the metabolic formation of a reactive thiol, S-(1,2-dichlorovinyl)-L-homocysteine (DCVHC), which may undergo a PLP-dependent γ-elimination reaction to produce an identical thiol, was studied. DCVHC is a potent nephrotoxin, and, similar to DCVC, its toxicity was blocked by aminooxyacetic acid and the α-methyl analogue S-(1,2-dichlorovinyl)-DL-α-methylhomocysteine was not toxic. Moreover, exposure of renal proximal tubular cells to propargylglycine, a suicide substrate for PLP-dependent enzymes that catalyze γ-elimination reactions, blocked the toxicity of DCVHC. Fourth, the renal mitochondrial β-lyase is localized in the outer membrane; therefore, although DCVC was toxic to mitochondria, no toxicity was produced in mitoplasts, which shows that a suborganelle site of activation is involved in the mitochondrial toxicity of DCVC. Finally, the toxicity of both DCVC and DCVHC was blocked by probenecid, indicating a role for the anion transport system. DCVC and DCVHC inhibit cellular and mitochondrial respiration, indicating that mitochondria are primary intracellular targets for nephrotoxic S-conjugates. Thus, the nephrotoxicity of cysteine and homocysteine S-conjugates is dependent on enzymatic activation to produce a reactive thiol, which is involved in the production of cytotoxicity.

Bioactivation mechanism of cytotoxic homocysteine S-conjugates☆

S-(1,2-Dichlorovinyl)-L-homocysteine is a much more potent nephrotoxin than the corresponding cysteine S-conjugate S-(1,2-dichlorovinyl)-L-cysteine (A. A. Elfarra, L. H. Lash, and M. W. Anders (1986) Proc. Natl. Acad. Sci. USA 83, 2667-2671). The objective of the present experiments was to test the hypothesis that the increased toxicity of homocysteine S-conjugates may be associated with the formation of the reactive metabolite 2-oxo-3-butenoic acid, which may arise via a nonenzymatic retro-Michael elimination reaction from the 2-oxo acid metabolites of homocysteine S-conjugates. S-(2-Benzothiazolyl)-L-homocysteine, which was a substrate for purified bovine kidney cysteine conjugate beta-lyase (glutamine transaminase K) and whose metabolism was dependent on the presence of a 2-oxo acid, was cytotoxic in isolated rat kidney cells and was toxic to rat renal mitochondria, whereas the cysteine S-conjugate S-(2-benzothiazolyl)-L-cysteine had little effect. L-Methionine sulfoximine, L-canavanine, and the Michael acceptor methyl vinyl ketone were cytotoxic. The 2-hydroxy acid analogs of S-(1,2-dichlorovinyl)-L-homocysteine and 2-oxo-3-butenoic acid, S-(1,2-dichlorovinyl)-2-hydroxy-4-mercaptobutanoic acid and 2-hydroxy-3-butenoic acid, respectively, which are expected to be metabolized by rat renal L-2-hydroxy (L-amino) acid oxidase to yield 2-oxo-3-butenoic acid, were also cytotoxic. To obtain evidence for the formation of 2-oxo-3-butenoic acid as a product of the metabolism of L-homocysteine S-conjugates and analogs, trapping experiments were conducted. S-(2-Benzothiazolyl)-L-homocysteine, S-(1,2-dichlorovinyl)-L-homocysteine, L-methionine sulfoximine, and L-canavanine were converted by snake venom L-amino acid oxidase to 2-oxo-3-butenoic acid, which was trapped by the nucleophile methanethiol to yield 4-methylthio-2-oxobutanoic acid; the trapped product was derivatized with 2,4-dinitrophenylhydrazine and was identified by its electronic absorption spectrum and by high-performance liquid chromatography. Similar trapping experiments conducted with kidney homogenates and purified beta-lyase were not successful. The data indicate that the bioactivation of homocysteine S-conjugates and analogs involves the enzymatic formation of the corresponding 2-oxo acids followed by a nonenzymatic retro-Michael elimination reaction to yield the Michael acceptor 2-oxo-3-butenoic acid, which may contribute to the observed cytotoxicity of homocysteine S-conjugates.

Nephrotoxic amino acid and glutathione S-conjugates: formation and renal activation.

The mechanisms by which chemicals produce tissue damage include, for example, biotransformation to reactive, electrophilic metabolites, initiation of lipid peroxidation, and formation of toxic, reduced oxygen metabolites. The liver, because of its abundant capacity for biotransformation, is frequently the target organ for chemicals whose toxicity is associated with bioactivation. Extrahepatic target organs are well known, and this toxicity may also be associated with target organ bioactivation.

Metabolism of S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine to hydrogen sulfide and the role of hydrogen sulfide in S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine-induced mitochondrial toxicity

The nephrotoxic cysteine S-conjugate S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC) is metabolized by kidney homogenates and subcellular fractions to pyruvate and a reactive thiol, which is cytotoxic and partially decomposes to yield hydrogen sulfide and thiosulfate. Although hydrogen sulfide is a potent mitochondrial poison, the mitochondrial toxicity of CTFC is not attributable to hydrogen sulfide formation, as shown by different sites of inhibition of mitochondrial respiration by CTFC and hydrogen sulfide. The efficient mitochondrial oxidation of hydrogen sulfide apparently serves to protect mitochondria against the toxic effects of hydrogen sulfide generated from CTFC.