Abstract
A cell line derived from pig kidney, LLC-PK1, was grown in a culture system in which the cells express morphological and biochemical characteristics of the proximal tubule. This model was used to investigate the mechanism of S-cysteine conjugate toxicity and the role of glutathione conjugate metabolism. LLC-PK1 cells have the degradative enzymes of the mercapturate pathway, and S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)-L-glutathione are toxic. S-(1,2-Dichlorovinyl)-L-glutathione is not toxic when the cells are pretreated with AT-125, an inhibitor of gamma-glutamyl transpeptidase. The cells respond to a variety of toxic cysteine conjugates. Cysteine conjugate beta-lyase activity is not detectable by standard assays, but can be measured using radiolabeled S-(1,2-dichlorovinyl)-L-cysteine. Pyruvate stimulates the beta-elimination reaction with S-(1,2-dichlorovinyl)-L-cysteine as substrate 2-3-fold. The data suggest that a side transamination reaction regulates the flux of substrate through the beta-elimination pathway; therefore, cysteine conjugate beta-lyase in LLC-PK1 cells may be regulated by transamination, and measurement of lyase activity in some systems may require the presence of alpha-ketoacids. Aminoxyacetic acid blocks both the metabolism of S-(1,2-dichlorovinyl)-L-cysteine to a reactive species which covalently binds to cellular macromolecules and toxicity. Glutathione inhibits the binding of the sulfur containing cleavage fragment to acid insoluble material in vitro. The data provide direct evidence that S-(1,2-dichlorovinyl)-L-cysteine is metabolized to a reactive species which covalently binds to cellular macromolecules, and the binding is proportional to toxicity.
Highlights
A cell line derived frompig kidney, LLC-PK1, was one and S-(2-haloethyl)-~-cystein(3e, 4)
When LLC-PK1 cells are treated with aminoxyacetic acid, the metabolism of DCVC is inhibited as is the covalent binding of 35Slabel to acid-precipitable materials (Table VII)
A product with the retention time of authentic DCVC (10.5 min) was detected inthe incubations, but not before the appearance of another peak which chromatographed with a retention time intermediate (14min) between DCVC and DCVG (16min, data not shown), suggesting thatthe S - (1,2-dichlorovinyl) cysteinylglycine conjugate was formed as an intermediate
Summary
Val. 261, No 7, Issue of March 5, ppP. r3i3n2t5e-d33in32U, .1S9.8A6. The Role of Glutathione ConjugateMetabolism and Cysteine Conjugate @-Lyasein theMechanism of S-Cysteine Conjugate Toxicity in LLC-PKl Cells*. The data provide direct evidence that S (1,2-dichlorovinyl)-~-cysteinies metabolized to a reactive species which covalently binds to cellularmacromolecules, and the bindingis proportional to toxicity This mechanism is based on the observations of Schultze and co-workers [6,10,11]who studied the nephrotoxic S-cysteine conjugate S-(1,2-dichlorovinyl)-~-cystein(DeCVC?). No differences of gas the flask contained, and a half-molar equivalent of cysteine in morphology or results were noted between cells from the two was added through the serum stopper as an aqueous solution Other Assays-cysteine conjugate @-lyaseassays were performed with S-(2-benzothiazolyl)-~-cysteinaend DCVC as previously described [26].y-Glutamyl transpeptidase,leucine aminopeptidase, and glutathione-S-transferase were measured by publishedprocedures [27,28,29].Protein was determined by themethod of Bradford [30] using the Bio-Rad kit (Bio-Rad). S-(1,2,3,4-Pentachloro-1,3-butadienyl)-thioethers of L-cysteine and L-glutathione were toxic, as was S-(2-chloro1,1,2-trifluoroethyl)-~-cystein(eTable 11).thecells respond toseveral toxic S-cysteine and S-glutathionceonju-
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