Cisplatin- 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 was made of bifunctional DNA-adduct formation by these drugs. The kinetics of bifunctional cisplatin adduct formation were studied with DNA in vitro and in cultured Chinese hamster ovary (CHO) cells. Prior to adduct 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 monofunctional to bifunctional adducts, with t1/2 of approximately 2 h (37 degrees C), leads to maximum intrastrand adduct levels after approximately 4-6 h postincubation. This interval coincided with the period during which the cytotoxic effect of cisplatin could be reduced by a 1 h 10 mM thiourea post-incubation of the cells. The formation of interstrand crosslinks continued for approximately 7 h of post-incubation; then these amounted to approximately 2% of the total DNA adducts. When a DNA sample was dialysed against 0.1 M NH4HCO3 (16 h, 37 degrees C) immediately after cisplatin treatment, in order to block mono- to bifunctional adduct conversion, adduct levels were found similar to those after the 4-6 h post-incubation. From this it is clear that the high values reported 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 cisplatin data of CHO cells were compared with those after treatment of the cells with equitoxic doses of carboplatin. The data indicate that after 12 h post-incubation, when all bifunctional adducts are formed, the total amount of the various bifunctional adducts after cisplatin treatment (37.5 +/- 4.5 fmol/micrograms DNA) was in the same range as that after carboplatin (32.8 +/- 6.3 fmol/micrograms DNA). However, because the relative occurrences of the adducts were different, it could also be concluded that if one of the diadducts were exclusively responsible for the cytotoxic effect of these platinum antitumour drugs, Pt-AG is the only likely candidate.
Fichtinger-Schepman
A M
AM
TNO Food and Nutrition Research Institute, Rijswijk, The Netherlands.
van Dijk-Knijnenburg
H C
HC
van der Velde-Visser
S D
SD
Berends
F
F
Baan
R A
RA
D000818:Animals; D016466:CHO Cells; D016190:Carboplatin / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity*; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity*; D006224:Cricetinae; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D018736:DNA Adducts / Q000737:chemistry* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D007700:Kinetics; D013890:Thiourea / Q000494:pharmacology
D018736:DNA Adducts; C096276:carboplatin-DNA adduct; C057270:cisplatin-DNA adduct; D004247:DNA; D016190:Carboplatin; D013890:Thiourea; D002945:Cisplatin