The metabolism of S-(1,2 Dichlorovinyl)-l-cysteine by rat liver mitochondria
Abstract
Isolated rat liver mitochondria degrade DCVC to pyruvic acid and ammonia. Another metabolite of unknown structure qualitatively possesses the characteristics of an alkylating agent. Pyridoxal 5'-phosphate catalyses the non-enzymic breakdown of DCVC to pyruvic acid and ammonia. Semicarbazide, a pyridoxal 5'-phosphate trapping reagent blocks the metabolism of DCVC by rat liver mitochondria. This metabolism of DCVC by rat liver mitochondria is judged to be relevant to the inhibition of respiration of rat liver mitochondria which is delayed in onset and which was reported in the preceding paper.
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Mitochondrial bioactivation of cysteine S-conjugates and 4-thiaalkanoates: Implications for mitochondrial dysfunction and mitochondrial diseases
1995, BBA - Molecular Basis of DiseaseThe 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 Ca2+ 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.
Enzymology of Cysteine S-Conjugote β-Lyases
1994, Advances in PharmacologyThis chapter addresses recent findings concerning the identity of the cysteine S-conjugate β-lyases in mammals, the reactions catalyzed by these enzymes, and the pharmacological aspects of the cysteine S-conjugate β-lyases are addressed. It also discusses β-elimination reactions with cysteine S-conjugates in detail. Relatively little attention has been given to the role of cysteine S-conjugates in human health. Prolonged exposure to low levels of pollutants that can be activated through a glutathione conjugate may predispose certain individuals to risk of kidney disease and others perhaps to inexorable loss of brain function. From this epidemiological point of view, the cysteine S-conjugate β-lyases deserve more attention. However, these enzymes are also interesting in their own right. Cysteine S-conjugate β-lyases thus far shown to occur in mammals belong to three classes: (1) those present in enteric bacteria, (2) a liver form (kynureninase), and (3) a kidney form (glutamine transaminase K). In addition, because of its unique substrate specificity, the enzyme may be the target of prodrugs designed to deliver anticancer drugs to the kidney. Indeed, some progress has already been made in this area.
Nephrotoxicity of halogenated alkenyl cysteine-S-conjugates
1991, Life SciencesIn 1916 a relationship was postulated between the occurrence of aplastic anaemia in cattle and the soy bean meal that they had been fed, which had been extracted with trichloroethylene (1). The toxic compound was later identified as S-(1,2-dichlorovinyl)-L-cysteine (DCV-Cys) (2). In addition to effects on the hemopoietic system it also produced nephrotoxicity in calves. In rats only renal tubular necrosis was found (3). Further research demonstrated that other halogenated hydrocarbons produced similar nephrotoxicity (4). The haloalkenyl cysteine-S-conjugates (Cys-S-conjugates) have extensively been studied; this has provided new insight into the biochemical processes that lead to nephrotoxicity. It has been shown that a combination of transport processes and specific metabolic pathways, resulting in reactive intermediates that bind to cellular macromolecules, makes the kidney vulnerable to the noxious effects of the haloalkenyl Cys-S-conjugates. The first part of this review gives a brief overview of the bioactivation of the haloalkenes; in the second part the present knowledge of the underlying mechanisms of cytotoxicity will be outlined.
The effect of haloalkene cysteine conjugates on rat renal glutathione reductase and lipoyl dehydrogenase activities
1990, Toxicology and Applied PharmacologyAn early event in the nephrotoxicity of haloalkene cysteine conjugates is their metabolism by cysteine conjugate β-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 μm. 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 β-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 K'is 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 μm. 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 β-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.
Assessment of S-(1,2-dichlorovinyl)-l-cysteine induced toxic events in rabbit renal cortical slices. Biochemical and histological evaluation of uptake, covalent binding, and toxicity
1990, Chemico-Biological InteractionsA renal cortical slice system was utilized to investigate the events leading to site-specific nephrotoxicity induced by (DCVC). DCVC uptake into renal cortical slices was shown to be rapid (5–15 min) as well as time- and concentration-dependent. Of the total amount taken up at 1 h, 40% was subsequently covalently bound. These observations were confirmed by autoradiography, illustrating uptake and binding in the proximal tubule cells. Following these events, toxicity was evidenced by alterations in ATP content and O2 consumption between 4 and 8 h as well as leakage of the brush border enzymes (gamma glutamyl transpeptidase and alkaline phosphatase) as early as 4 h. Light microscopy provided a sequence of histopathological changes from an initial S3 lesion between 4 and 8 h to a lesion encompassing all proximal tubule segments (by 12 h). Electron microscopy demonstrated not only the specificity of DCVC toxicity (at 6 h) but also illustrated mitochondrial damage and loss of brush borders. A comparison of continuous versus short-term exposure to DCVC indicated that an irreversible sequence of events was initiated as early as 30 min. By utilizing an in vitro model which allows correlation of biochemical and histological changes, a sequence of events leading to DCVC induced toxicity was established.
Cysteine conjugate β-lyase of rat kidney cytosol: Characterization, immunocytochemical localization, and correlation with hexachlorobutadiene nephrotoxicity
1989, Toxicology and Applied PharmacologyCysteine conjugate β-lyase (β-lyase) was purified to electrophoretic homogeneity from the kidney cytosol of male Wistar rats. The highly purified enzyme exhibited a monomeric molecular weight of 50,000 Da and was active in the α-β elimination of cysteine conjugates including S-(1,2-dichlorovinyl)-l-cysteine (DCVC), S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (TFEC), and S-(2-benzothiazolyl)-l-cysteine, particularly toward DCVC and TFEC. The purified enzyme also exhibited glutamine transaminase K activity with phenylalanine and α-keto-γ-methiolbutyrate as substrates. An antibody was raised to the purified rat protein in sheep and the crude immune serum affinity purified, yielding a specific antibody that recognized only the β-lyase protein in whole kidney homogenates. Immunocytochemical studies on rat kidney sections stained with the purified antibody revealed that the cytosolic β-lyase enzyme was mainly localized in the pars recta of the proximal tubule in untreated rats. This localization is coincident with the site-specific kidney necrosis produced by hexachloro-1,3-butadiene (HCBD). These results indicate that the tissue localization of β-lyase in the proximal tubule plays an important role in determining the specific nephrotoxicity produced by halogenated alkenes such as HCBD.