Renal cysteine conjugate beta-lyase-mediated toxicity studied with primary cultures of human proximal tubular cells.
The beta-lyase pathway has been shown to mediate the nephrotoxicity of S-cysteine conjugates of a variety of haloalkenes in a number of animal models in vitro and in vivo. However, there is no information available concerning this mechanism of bioactivation in human tissues. In this investigation a well-characterized model of human proximal tubule epithelial cells, the presumed target cell, was used to investigate the toxicity of a series of glutathione and cysteine conjugates of nephrotoxic haloalkenes. Both S-(1,2-dichlorovinyl)-glutathione (DCVG) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) caused dose-dependent toxicity over a range of 25 to 500 microM. DCVC was consistently found to be more toxic than DCVG, but the inclusion of gamma-glutamyltransferase (0.5 U/ml) increased the toxicity of DCVG to that observed with an equimolar concentration of DCVC, indicating that metabolism to the cysteine conjugate is an important rate-limiting step in this in vitro model. S-(1,2,3,4,4-Pentachlorobutadienyl)-L-cysteine, S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine, and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine were also found to be toxic to human proximal tubular cells. Incubation with [35S]DCVC resulted in covalent binding of 35S-label, which increased linearly to a final level of 1.05 nmol/mg protein at 6 hr. Aminooxyacetic acid (250 microM), an inhibitor of pyridoxal phosphate-dependent enzymes such as beta-lyase, protected the cells from the toxicity of all of the cysteine conjugates and inhibited the covalent binding of 35S-label from [35S]DCVC to cellular macromolecules. The results of the present study provide the first evidence that human proximal tubular cells are sensitive to the toxicity of glutathione and/or cysteine conjugates of a variety of chloro- and fluoroalkenes which are activated via the beta-lyase pathway. The implications for human health are discussed.
Chen
J C
JC
Department of Pathology, University of Maryland School of Medicine, Baltimore 21201.
Stevens
J L
JL
Trifillis
A L
AL
Jones
T W
TW
D000293:Adolescent; D000328:Adult; D000625:Aminooxyacetic Acid / Q000378:metabolism; D002070:Butadienes / Q000633:toxicity; D013437:Carbon-Sulfur Lyases; D002478:Cells, Cultured; D002648:Child; D003545:Cysteine / Q000031:analogs & derivatives / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells; D005260:Female; D005978:Glutathione / Q000031:analogs & derivatives / Q000633:toxicity; D006801:Humans; D006845:Hydrocarbons, Fluorinated / Q000633:toxicity; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000201:enzymology*; D008190:Lyases / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D013329:Structure-Activity Relationship; D005466:Fluorocarbons
D000625:Aminooxyacetic Acid; D002070:Butadienes; D013437:Carbon-Sulfur Lyases; D003545:Cysteine; D005978:Glutathione; D006845:Hydrocarbons, Fluorinated; D008190:Lyases; C039961:S-(1,2-dichlorovinyl)cysteine; C039330:S-pentachlorobuta-1,3-dien-yl-cysteine; C064116:S-(1,1,2,2-tetrafluoroethyl)cysteine; D005466:Fluorocarbons; C047601:S-(2-chloro-1,1,2-trifluoroethyl)cysteine; C041464:S-(1,2-dichlorovinyl)glutathione; C019969:S-alkylcysteine lyase