We have studied the formation of adducts upon carboplatin treatment of isolated DNA and in cells. The major adduct formed in vitro, determined with atomic absorption spectroscopy and enzyme-linked immunosorbent assay, was the intrastrand cross-link cis-Pt(NH3)2d(pGpG)(Pt-GG) (58%). cis-Pt-(NH3)2d(pApG) (Pt-AG) (11%), cis-Pt(NH3)2d(GMP)2 (G-Pt-G) (9%), and monofunctionally bound platinum (cis-Pt(NH3)3dGMP (Pt-G), 22%) were formed in smaller amounts. These relative occurrences of the adducts, average values found between 1 and 16 h of incubation, are comparable with those after incubation with cisplatin. The formation of carboplatin-DNA adducts was slow, and about 230-fold more carboplatin than cisplatin (molar dose) was required to obtain equal levels of platination after 4 h of incubation. However, less than 20 times more carboplatin was needed to obtain equal levels of cytotoxicity after 1 h of exposure of CHO cells. The percentages of the carboplatin-DNA adducts after 7-12 h postincubation of the cells (determined with ELISA), Pt-GG (30%), Pt-AG (16%), G-Pt-G (40%), and Pt-G (14%), were different from those of the in vitro data. After 12 h postincubation, the number of interstrand cross-links (determined by alkaline elution) amounted to about 10% of the G-Pt-G adducts and 3-4% of the total amount of adducts. The immunocytochemical detection (with antiserum NKI-A59) of the platinum-DNA modifications showed a pattern similar to that found for the various bifunctional adducts: the initially low levels slowly increased to maximum values within 7-12 h and then slowly decreased. In conclusion, carboplatin forms the same bifunctional adducts as cisplatin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult rats received a single injection of the carcinogens N-ethyl-N-nitrosourea (ENU) or N,N-dimethylnitrosamine (DMN) and were killed after various time intervals (up to 56 days). Liver DNA was analysed by h.p.l.c. for alkylated purines [3-alkyladenine (3-alkylAde), 7-alkylAde, 3-alkylguanine (3-alkylGua), O6-alkylGua, 7-alkylGua, imidazole ring-opened (ro) 7-alkylGua] and alkylated pyrimidines [O2-alkylcytosine (O2-alkylCyt), 3-alkylCyt, O2-alkylthymine (O2-alkylThy), 3-alkylThy, O4-alkylThy]. Alkylphosphotriesters were assayed by both h.p.l.c. [alkylphosphotriester of thymidyl(3'-5')thymidine, (dTp(alkyl)dT)] and alkaline sucrose sedimentation (total phosphotriesters). No significant amounts of 3-alkylCyt and ro 7-alkylGua were observed, but the presence of all other products could be established 2 h after DMN or ENU. At that moment the relative amounts of some products (O2-alkylCyt, O2-alkylThy, O4-alkylThy, O6-alkylGua), when compared with 7-alkylGua, were smaller than those observed after in vitro alkylation of isolated DNA. This suggests either a lower accessibility of some sites in DNA in situ, or (as known already for O6-alkylGua) the presence of rapidly exhausted, fast repair modes for these adducts. The in vivo stability of ethylated products was (much) higher than that of the homologous methylated products. In the case of O-alkylated thymidines this difference was impressive: apparent half-life values of 17 days for O2-ethylthymine (O2-EtThy) and 14 days for O4-EtThy were calculated, whereas the corresponding values for O2-methylthymine (O2-MeThy) and O4-MeThy were 12 h and less than 4 h, respectively. Substantial, although smaller differences were also found for O2-alkylCyt (86 versus less than 4 h), 3-alkylGua (104 versus 17 h) and dTp(alkyl)dT (32 versus 7 days), whereas the rates of removal of 7-EtGua and 7-MeGua differed by 2.5-5 times (depending on the period compared). At 56 days after ENU the two major lesions were the ethylphosphotriester and O2-EtThy, whereas only traces of 7-EtGua were observed. Parenchymal and non-parenchymal liver cells of some DMN-treated rats were separated before DNA isolation. It was found that the extents of DNA alkylation at 6 h after DMN administration were almost identical, indicating that DMN activation is the same for both cell types. The results are discussed in relation to the carcinogenic effects of methylating and ethylating agents.

Male Wistar rats received a single i.p. injection of [3H]ethylnitrosourea (140 mg/kg) and were killed after 2 h, 1, 3, 6, 28 or 56 days. DNA of the following organs was isolated and analysed for the presence of 12 different ethylated bases and ethylphosphotriesters: brain, lung, liver, spleen, kidney, intestine, testis and bone marrow. At 2 h after injection the extent of DNA ethylation was found to be heterogeneous: highest in liver and lowest (3-4 times lower) in testis and brain. The rates at which O2- and O4-ethylthymine and the ethylphosphotriester dTp(Et)dT were lost, were very low in all organs except intestine and spleen. Most likely, loss in the latter organs is exclusively due to cell turnover. The rate of O6-ethylguanine repair strongly varied from organ to organ: high in liver, very low in testis and brain and intermediate in the other organs. In none of the DNAs were significant amounts of imidazole ring-opened 7-ethylguanine found. Our results strengthen the notion that a substantial part of carcinogen-induced DNA damage is of a highly persistent nature and might contribute to the carcinogenic process long after the original exposure has occurred.

No abstract

No abstract