CISPLATIN-DNA-ADDUCTS AND CARBOPLATIN-DNA-ADDUCTS - IS PT-AG THE CYTOTOXIC LESION

In order to determine the nature of the cytotoxic lesion(s) formed by the antitumour drugs cisplatin and carboplatin, a comparative study wa s made of bifunctional DNA-adduct formation by these drugs. The kineti cs of bifunctional cisplatin adduct formation were studied with DNA in vitro and in cultured Chinese hamster ovary (CHO) cells. Prior to add uct measurements with AAS in in vitro platinated DNA and with ELISA in cellular DNA, the monoadducts were inactivated with thiourea (10 mM; 1 h at 37 degrees C). The data indicated that the conversion of monofu nctional to bifunctional adducts, with 1(1/2) of similar to 2 h (37 de grees C), leads to maximum intrastrand adduct levels after similar to 4-6 h postincubation, This interval coincided with the period during w hich the cytotoxic effect of cisplatin could be reduced by a 1 h 10 mM thiourea post-incubation of the cells. The formation of interstrand c rosslinks continued for similar to 7 h of post-incubation; then these amounted to similar to 2% of the total DNA adducts, When a DNA sample was dialysed against 0.1 M NH4HCO3 (16 h, 37 similar to C) immediately after cisplatin treatment, in order to block mono- to bifunctional ad duct conversion, adduct levels were found similar to those after the 4 -6 h post-incubation. From this it is clear that the high values repor ted earlier for bifunctional cisplatin adducts in such DNA samples are not correct. These values apparently represent the amounts of adducts that eventually would have been formed during post-incubation in DNA in vitro but also in cells in the absence of cellular repair. The cisp latin data of CHO cells were compared with those after treatment of th e cells with equitoxic doses of carboplatin. The data indicate that af ter 12 h postincubation, when all bifunctional adducts are formed, the total amount of the various bifunctional adducts after cisplatin trea tment (37.5 +/- 4.5 fmol/mu g DNA) was in the same range as that after carboplatin (32.8 +/- 6.3 fmol/mu g DNA). However, because the relati ve occurrences of the adducts were different, it could also be conclud ed that if one of the diadducts were exclusively responsible for the c ytotoxic effect of these platinum antitumour drugs, Pt-AG is the only likely candidate.