Vol. 301, Issue 3, 884-892, June 2002

-Lyase-Dependent Attenuation of Cisplatin-Mediated Toxicity by Selenocysteine Se-Conjugates in Renal Tubular Cell Lines

Martijn Rooseboom¹ , Gerben Schaaf¹ , Jan N. M. Commandeur, Nico P. E. Vermeulen and Johanna Fink-Gremmels

Leiden/Amsterdam Center for Drug Research, Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands (M.R., J.N.M.C., N.P.E.V.); and Department of Veterinary Pharmacology, Pharmacy, and Toxicology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands (G.S., J.F.-G.)

Cisplatin [cis-diamminedichloroplatinum(II)] is a widely used antitumor drug with dose-limiting nephrotoxic side effects due to selective toxicity to the proximal tubule. In the present study, the chemoprotective potential of three selenocysteine Se-conjugates, Se-methyl-L-selenocysteine, Se-(2-methoxyphenyl)-L-selenocysteine, and Se-(2-chlorobenzyl)-L-selenocysteine, belonging to three structural classes, against the nephrotoxic effects of cisplatin was investigated. Selenocysteine Se-conjugates have previously been proposed as kidney-selective prodrugs of pharmacologically active selenols because of their active uptake and bioactivation by cysteine conjugate -lyases in the kidney. To elucidate whether chemoprotection is -lyase-dependent wild-type LLC-PK₁ cells, possessing a very low -lyase activity, and LLC-PK₁ cells stably transfected with full-length cDNA coding for rat kidney cysteine conjugate -lyase/glutamine transaminase K (R1J) were used. The results indicate that all three selenocysteine Se-conjugates were able to attenuate the cisplatin-induced loss of viability in R1J cells but not in the parental LLC-PK₁ cells, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and neutral red uptake. In addition, cisplatin-induced reactive oxygen species (ROS) production was determined using 2',7'-dichlorodihydrofluorescein diacetate. The selenocysteine Se-conjugates were able to decrease ROS levels after cisplatin exposure in both cell types. However, this ROS-protective effect was more profound in R1J cells. Se-Methyl-L-selenocysteine provided the strongest protection. The protective activity against cisplatin-induced cytotoxicity and ROS generation was blocked by aminooxyacetic acid, a selective inhibitor of pyridoxal 5'-phosphate-dependent cysteine conjugate -lyases, further supporting the role of -lyase in the observed chemoprotection. The precise molecular mechanism by which selenols, generated by -lyase, provide protection against cisplatin-induced cytotoxicity, however, remains to be established.