Sulfur-containing amino acids decrease cisplatin cytotoxicity and uptake in renal tubule epithelial cell lines

Purpose: Nephrotoxicity is one of the major dose-limiting side-effects of cisplatin (DDP). The disproportionate accumulation of cisplatin in kidney tissue may play an important role; however, therapeutic measures to prevent this prime cause of nephrotoxicity are not available. Because certain amino acids (AAs) have been reported to modulate DDP nephrotoxicity in vivo, we explored the potential of all 20 protein AAs, N-acetylcysteine and DL-homocysteine to reduce DDP cytotoxicity and uptake in S-1, S-3 (proximal tubule), and DCT (distal convoluted tubule) cell lines. Methods: Immortalized but non-transformed renal tubule epithelial cell lines, derived from specific portions of the nephron of an SV40 transgenic mouse, were grown to confluency and exposed to various concentrations of DDP for 1 h with or without concurrent exposure to AAs in an otherwise AA-free Krebs-Ringer buffer (I(RB). After 1 h, cell layers were washed and replenished with medium for cytotoxicity assays, or processed immediately for the determination of DDP accumulation. Cytotoxicity was assessed 48 h later by an MTT assay, and DDP uptake after Ih was determined by atomic absorption spectroscopy. Results: In an initial screening where the cells were concurrently incubated with 0.25 mM DDP and 1 mM AA for I h in KRB, only cysteine (Cys), me-thionine (Met), N-acetylcysteine and DL-homocysteine reduced DDP toxicity. This effect was enhanced at 5 mM AA and most potent for Cys, which reduced DDP cytotoxicity by 79 +/- 3% in S3 cells, by 78 +/- 12.2% in DCT cells, and by 19 +/- 3.6% in S-1 cells (P < 0.05). Reduction of cytotoxicity was less for Met, DL-homocysteine, and N-acetylcysteine, in decreasing order. All four AAs also inhibited DDP uptake in renal cells, with Cys as the strongest inhibitor. Inhibition of DDP accumulation by 1 mM Cys after 1 h was 39% in S-3 cells, 38% in DCT cells, and 28% in S-1 cells. Again, reduction of uptake was less for the three other AAs. Pre-complexing of DDP with Cys for 16 h increased its uptake by 8- to 30-fold compared with native DDP, but markedly inhibited its toxicity. Thus, pre-complexing of DDP with Cys could not explain the reduced uptake of DDP, but could partly account for the reduction in cytotoxicity. Double-reciprocal Lineweaver-Burk plots of DDP concentration-versus-uptake rates at a constant concentration of Cys suggested that: Cys competitively inhibited DDP uptake in S-1 and DCT cells, and in a more complex fashion in S-3 cells. Conclusions: We conclude that Cys, Met, N-acetylcysteine, and DL-homocysteine differentially inhibit DDP toxicity and uptake in cultured S-1, S-3, and DCT cells, and that the inhibition of uptake, as well as the complexation of DDP with Cys within the cell, may prevent toxicity. The structural element R-CH(NH2)-[CH2](1-2)-S-R, which is common to all four molecules, may play a crucial role in blocking the transport of DDP, and could have future clinical applications.