Quiescent LLC-PK1 cells as a model for cis-diamminedichloroplatinum(II) nephrotoxicity and modulation by thiol rescue agents.
Confluent LLC-PK1 cells express many characteristics of renal proximal tubule epithelia. We report here the use of this cell line to investigate the possible mechanisms of cis-diamminedichloroplatinum(II) (DDP)-induced nephrotoxicity and diethyldithiocarbamate (DDTC) amelioration. Cells were exposed to platinum-based drug for 1 h and then incubated in drug-free medium until assayed. There was a 10-h delay between DDP exposure and onset of toxicity which then continued to develop. Viability at 72 h was 97 +/- 2 (SD), 68 +/- 3, 33 +/- 3, and 10 +/- 2% of control after treatment with 100, 200, 300, and 400 microM DDP, respectively. trans-Diamminedichloroplatinum(II) was 5-fold less toxic than DDP and diammine(1,1-cyclobutanedicarboxylato)platinum(II) (CBDCA) was nontoxic at concentrations up to 2.0 mM. Incubation of cells with DDTC (3 mM) for 1 h immediately following DDP exposure resulted in chelation of 43% of total intracellular platinum by DDTC and significantly increased 72 h viability; 97 +/- 2, 88 +/- 3, and 42 +/- 2% of control for 200, 300, and 400 microM DDP, respectively. DDP treatment of L1210 cells yielded equivalent total intracellular platinum levels, but Pt(DDTC)2 concentrations were one-tenth those in LLC-PK1 cells after subsequent treatment with DDTC (3 mM). Immediate reduction of protein and RNA, but not DNA, synthesis by DDP was concentration dependent over the same range as viability. DDP-induced increases in unscheduled DNA synthesis also did not correlate with cytotoxicity. The inhibition of protein synthesis was unchanged by pretreatment with the RNA synthesis inhibitor actinomycin D.DDTC (3 mM) exposure produced an immediate and persistent DDP dose modification of 1.6 in protein but not RNA synthesis. CBDCA (0.5 to 1.0 mM) had no effect on protein, RNA, or DNA synthesis and only slight stimulatory effects on unscheduled DNA synthesis. DDTC alone (3-3000 microM) caused significant reduction in DNA synthesis and unscheduled DNA synthesis. Neither sodium thiosulfate nor 2-mercaptoethane-sulfonate had any effect on DDP-induced cytotoxicity or inhibition of protein, RNA, or DNA synthesis when incubated immediately after DDP, even though these drugs achieved intracellular concentrations at which DDTC was protective. These data indicate that quiescent LLC-PK1 cells are a good in vitro model for the study of DDP-induced nephrotoxicity and its modulation by thiol rescue agents and that DDP inhibition and DDTC rescue of posttranscriptional processes correlate best with viability in LLC-PK1 cells.
Montine
T J
TJ
Department of Pharmacology, University of Rochester School of Medicine and Dentistry, New York 14642.
Borch
R F
RF
D000227:Adenine Nucleotides / Q000032:analysis; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000378:metabolism / Q000633:toxicity*; D004247:DNA / Q000096:biosynthesis; D004260:DNA Repair; D004050:Ditiocarb / Q000378:metabolism; D005978:Glutathione / Q000032:analysis; D007668:Kidney / Q000187:drug effects*; D014176:Protein Biosynthesis; D012313:RNA / Q000096:biosynthesis; D013438:Sulfhydryl Compounds / Q000378:metabolism / Q000494:pharmacology*
D000227:Adenine Nucleotides; D013438:Sulfhydryl Compounds; D012313:RNA; D004247:DNA; D004050:Ditiocarb; D005978:Glutathione; D002945:Cisplatin