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)

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The reaction products of c i~-P t c l , ( N H )~)~ with several deoxyribonucleotides containing d(ApG) and/or d(GpA) have been studied. The various reaction products were separated by high-performance liquid chromatography and characterized by means of absorbance at 254 nm in combination with atomic absorption spectroscopy and 300-MHz 'H-NMR (pH dependence of the non-exchangeable base-protons, TI relaxation time determinations). For the larger fragments the results from these techniques were confirmed by enzymatic degradation studies of the platinated fragments.The (2)] were found, 4% of the reacted material consisted of a 1 mol Pt/2 mol dinucleotide product, and 3% of an unidentified 1 : 1 product. From the main product two rotamers were found to occur: at room temperature, 81% anti,unti and 19% anti,syn product is present. With d(GpA) about equal amounts of N1,N7and N7,N7 products were found; for both products the anti,unti and anti,syn conformations were found, respectively. Upon reaction of C~S -P~C I~( N H~)~ with d(pApG) and d(pGpA) only the N7,N7 products were found;at room temperature and pH > 1.5 these products were present in anti,anti conformation. However, for the d(pApG)-platinum chelate at -20°C a small amount (< 5%) of a second product could be observed in NMR. For the d(pGpA)-platinum chelate a second N7,N7-coordinated product was observed when the pH of the NMR sample was lowered to 1.1 (at this pH the free 5'-phosphate group is protonated). With the larger fragments d(ApGpA), d(pApGpA) and d(TpApGpApT) the intra-molecular competition between the formation of the d(ApG) or the d(GpA) chelates could be studied. Using these nucleotides no NZcoordinated products or rotamers were observed. In the case of d(ApGpA) and d(TpApGpApT) the d(GpA) chelate (67% and 75% respectively) was favoured over the d(ApG) chelate, while with d(pApGpA) about equal amounts of both chelates were formed. Since Rosenberg's discovery [l] of the antitumor activity of cisplatin [cis-PtC12(NH3)Z, also abbreviated as cis-Pt] much research has been aimed at elucidating its working mechanism [2,3]. At this moment there is a wide consensus about cellular DNA being the ultimate target [4]. This prompted many researchers to investigate the reaction products formed between cis-Pt and DNA in vitrn [5 -71, in living cells [S, 91, as well as in material obtained from cisplatin-treated patients [ 101. The major product formed appeared to be cis-Pt coordinated via the N7 atoms of a d(GpG) sequence (over 60% of the total Pt was coordinated in this mode), followed by the d(ApG)N7,N7 chelate (20%). Less frequently occurring lesions are d(GpNpG) chelates [5,6, 111, in which cis-Pt is coordinated to two guanines separated by a third base, a cross-link between two guanines of opposite strands [12] and Correspondence to J. Recdijk, Gorlaeus Laboratoria, Rijks-