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Cysteine conjugate toxicity, metabolism, and binding to macromolecules in isolated rat kidney mitochondria
1W. Alton Jones Cell Science Center, Lake Placid, New York 12946.
Abstract:
The 14C-labeled, 35S-labeled, and unlabeled nephrotoxic cysteine conjugates S-(1,2-dichlorovinyl)-L-cysteine, S-(2-chloro-1,1,2-trifluoroethyl)- L-cysteine, S-(1,1,2,2-tetrafluoroethyl)-L-cysteine, S-(1,2,3,4,4-pentachlorobutadienyl)-L- cysteine (PCBC), and S-(1,1,2,3,3,3-hexafluoropropyl)-L-cysteine were synthesized and their toxicities were compared in isolated rat renal mitochondria. Inhibition of respiration, covalent binding to macromolecules, metabolism by mitochondria, metabolism by a purified cysteine conjugate beta-lyase (beta-lyase), and octanol/water partition coefficients were studied. All of the conjugates inhibited mitochondrial state 3 respiration. Only PCBC was found to uncouple oxidative phosphorylation. (Aminooxy)acetic acid, a beta-lyase inhibitor, blocked the effects of the conjugates on state 3 respiration except for the uncoupling effect of PCBC, which was not blocked. Binding of 35S label to macromolecules was observed after treatment with each of the 35S-labeled conjugates, and (aminooxy)acetic acid blocked the binding. The relative amounts of metabolism of the conjugates did not correlate well with their relative binding and toxicities, indicating some differential reactivity of metabolites and/or selectivity for binding targets. Some of the binding from 35S-labeled conjugates was removed by treatment with the disulfide-reducing agent dithiothreitol, suggesting that some of the binding was via mixed disulfides. The amount of dithiothreitol-sensitive binding differed among the conjugates. The metabolism of PCBC by permeabilized mitochondria, but not by a purified beta-lyase, was consistent with its relative toxicity and covalent binding, suggesting the involvement of other beta-lyase enzymes in the activation of PCBC to toxic species in mitochondria.
Insights
Nephrotoxic cysteine conjugates were synthesized and tested in rat kidney mitochondria. PCBC uniquely uncoupled oxidative phosphorylation, suggesting unique metabolic activation pathways distinct from other conjugates.
Area of Science:
- Biochemistry
- Toxicology
- Renal Physiology
Background:
- Cysteine conjugates are known nephrotoxins.
- Metabolic activation is crucial for their toxicity.
- Specific enzymes like beta-lyase play a role in this activation.
Purpose of the Study:
- To synthesize and compare the toxicities of novel nephrotoxic cysteine conjugates.
- To investigate the mechanisms of toxicity, including mitochondrial respiration inhibition and covalent binding.
- To explore the role of beta-lyase and other mitochondrial enzymes in conjugate metabolism and activation.
Main Methods:
- Synthesis of 14C- and 35S-labeled cysteine conjugates.
- Assessment of mitochondrial respiration (State 3) and oxidative phosphorylation uncoupling.
- Measurement of covalent binding to mitochondrial macromolecules.
- Enzymatic metabolism studies using isolated mitochondria and purified beta-lyase.
- Determination of octanol/water partition coefficients.
Main Results:
- All synthesized conjugates inhibited mitochondrial State 3 respiration.
- S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBC) uniquely uncoupled oxidative phosphorylation.
- (Aminooxy)acetic acid, a beta-lyase inhibitor, blocked conjugate-induced respiration inhibition but not PCBC's uncoupling.
- Covalent binding of 35S-labeled conjugates to macromolecules was observed and inhibited by (aminooxy)acetic acid.
- PCBC metabolism by mitochondria, but not purified beta-lyase, correlated with its toxicity, suggesting alternative activation pathways.
Conclusions:
- Nephrotoxic cysteine conjugates exhibit varying mechanisms of toxicity in renal mitochondria.
- PCBC's unique uncoupling of oxidative phosphorylation points to distinct metabolic activation pathways, potentially involving non-beta-lyase enzymes.
- Covalent binding and respiration inhibition are common toxic effects mediated by beta-lyase, but PCBC's effects are not solely dependent on this enzyme.