Preferential formation and decreased removal of cisplatin–DNA adducts in Chinese hamster ovary cell mitochondrial DNA as compared to nuclear DNA

Abstract

Levels of DNA adducts in Chinese hamster ovary (CHO) cells exposed to cis-diamminedichloroplatinum(II) (cisplatin) for 24 h, have been shown to be 4- to 6-fold higher in mitochondrial (mt) DNA as compared to nuclear (n) DNA (Olivero et al., Mutation Res., 346 (1995) 221). The aim of the present study was to understand if the preferential cisplatin binding in mtDNA is partially caused by lack of adduct removal in the mitochondria. Chinese hamster ovary cells were exposed for 6 h to 50 μM cisplatin, followed by incubation for 24 and 48 h in cisplatin-free medium. At the 30-h time point (6 h with cisplatin, 24 h without cisplatin), half of the cells from each plate were harvested and the remainder were cultured and harvested at 54 h (6 h with cisplatin, 48 h without cisplatin). The 30- and 54-h time points are called ` T 30' and ` T 54', respectively. Cisplatin–DNA adducts were measured in DNA from nuclear and mitochondrial fractions by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), a sensitive competitive microtiter-based immunoassay utilizing antiserum elicited against cisplatin-modified DNA. An initial higher level of cisplatin–DNA adducts was observed in mtDNA when compared to nDNA, at T 30. In addition, a lack of removal of adducts in mtDNA was demonstrated in cells at T 54. Dilution of DNA adducts by DNA replication was documented in pulse-chase experiments that employed [ 3H]thymidine incorporation. Adduct removal by repair-related mechanisms was considered to comprise the difference between total DNA adduct removal and adduct removal related to DNA replication. The final results demonstrated that both, higher initial binding and lack of removal of cisplatin–DNA adducts appear to contribute to the preferential cisplatin–mtDNA binding observed in CHO cells.