Metabolism of cisplatin to a nephrotoxin in proximal tubule cells

Abstract

Cisplatin, a commonly used chemotherapeutic agent, is nephrotoxic. The mechanism by which cisplatin selectively kills the proximal tubule cells was heretofore unknown. Recent studies in mice and rats have shown that the nephrotoxicity of cisplatin can be blocked by acivicin or (aminooxy) acetic acid, the same enzyme inhibitors that block the metabolic activation of a series of nephrotoxic halogenated alkenes. In this study, it was hypothesized that cisplatin is activated in the kidney to a toxic metabolite through the same pathway that has been shown to activate the halogenated alkenes. This activation begins with the formation of a glutathione-conjugate that is metabolized to a cysteinyl-glycine-conjugate, to a cysteine-conjugate, and finally to a reactive thiol. In this study, a protocol was developed in which confluent monolayers of LLC-PK1 cells were exposed to clinically relevant concentrations of cisplatin or cisplatin-conjugate for 3 h. Cell viability was assayed at 72 h. The role of gamma-glutamyl transpeptidase (GGT) and cysteine-S-conjugate beta-lyase in the metabolism of each of the cisplatin-conjugates was investigated. Pre-incubation of cisplatin with glutathione, cysteinyl-glycine, or N-acetyl-cysteine to allow for the spontaneous formation of cisplatin-conjugates increased the toxicity of cisplatin toward LLC-PK1 cells. Inhibition of GGT activity showed that GGT was necessary only for the toxicity of the cisplatin-glutathione-conjugate. Inhibition of cysteine-S-conjugate beta-lyase reduced the toxicity of each of the cisplatin-conjugates. These data demonstrate that metabolism of cisplatin in proximal tubule cells is required for its nephrotoxicity. The elucidation of this pathway provides new targets for the inhibition of cisplatin nephrotoxicity.