ATLA::Alternatives to Laboratory Animals

Volume 24, Number 2

The use of porcine proximal tubular cells for studying nephrotoxicity in vitro: the role of oxidative stress in cisplatin-induced cell death.

ATLA 24, 161-172, March/April 1996

Marieke Kruidering,¹ Bob Van De Water,¹ Emile De Heer,² Gerard J. Mulder¹ and J. Fred Nagelkerke¹

¹Division of Toxicology, Leiden/Amsterdam Center for Drug Research, P.O. Box 9503 and ²Department of Pathology, P.O. Box 9603, University of Leiden, 2300 RC Leiden, The Netherlands

SUMMARY

The effects of a widely used antitumour drug, cisplatin, on freshly isolated porcine proximal tubular cells (PPTC) in suspension were investigated. Incubation of the PPTC with 5-500 µM cisplatin resulted in a decrease in mitochondrial membrane potential (MMP) and in cell death. In addition, the formation of reactive oxygen species (ROS) was observed within 20 minutes. Prevention of ROS formation with the antioxidants diphenyl-p-phenylene-diamine (DPPD) or desferrioxamine had no effect on the cisplatin-induced effects on MMP and cell death, implying that cisplatin-induced ROS formation is not a cause of cell death. In order to investigate whether the ROS formation was related to mitochondrial damage, we determined the effects of cisplatin on the enzymatic activities of NADP:ubiquinone reductase (Complex I) and succinate:ubiquinone reductase (Complex II) of the respiratory chain. Exposure of the PPTC to cisplatin resulted in a time-dependent and dose-dependent inhibition of the activities of both Complex I and Complex 11. The inhibition of these activities and the depletion of ATP could not be prevented by the antioxidants, indicating that these effects are not a consequence of ROS formation. We propose that damage to the mitochondria could be a key event in cisplatin-induced cell death.

Keywords: nephrotoxicity, proximal tubule, oxidative stress, flow cytometry, cisplatin, pig