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)

Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Abstract The cytotoxicity of cisplatin and cisplatin-DNA adduct formation in vitro and in vivo is clearly enhanced by hyperthermia. We investigated whether cytotoxicity and platinum-DNA adduct formation of two promising new third-generation platinum derivatives, lobaplatin [1,2-diamminomethylcyclobutane platinum(II) lactate] and oxaliplatin [oxalato-l,2-diaminocyclohexane platinum(II)], are also enhanced by hyperthermia. Cisplatin was used for comparison. SW 1573 cells were incubated with cisplatin, lobaplatin or oxaliplatin at different concentrations for 1 h at 37 ~ 41 ~ and 43~ The reproductive capacity of cells was determined by cloning experiments. Immunocytochemical detection of platinum-DNA adducts was performed with the rabbit antiserum NKI-A59. At 37~ cisplatin was the most cytotoxic, followed by oxaliplatin and lobaplatin. Hyperthermia clearly enhanced the cytotoxicity of cisplatin, lobaplatin and oxaliplatin. There was no further increase in cytotoxicity at 43~ compared to 41~ for cisplatin and oxaliplatin. A further increase in cytotoxicity at 43~ was observed for lobaplatin. At 43~ thermal enhancement was higher for lobaplatin than for oxaliplatin, with the reverse pattern at 41~ For both drugs, thermal enhancement of cytotoxicity was lower than observed for cisplatin. Immunocytochemical detection of platinum-DNA adducts was feasible for all the drugs. Adduct formation was enhanced at 43~ for cisplatin, lobaplatin and oxaliplatin with a relative increase of 410%, 170% and 180%. These results seem to confirm that an increase in platinum-DNA adduct formation is involved in the in vitro thermal enhancement of cytotoxicity. The observed thermal enhancement of cytotoxicity of lobaplatin and oxaliplatin in vitro warrants further in vivo investigations.

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We developed a sensitive 32P-postlabeling method for the detection of bifunctional intrastrand crosslinks d(Pt-GpG) and d(Pt-ApG) in DNA in vitro and in vivo. After enzymatic digestion of DNA the positively charged platinum adducts were purified from unplatinated products, using strong cation exchange chromatography. Subsequently the samples were deplatinated with cyanide, because platinated dinucleotides are very poor substrates for polynucleotide kinase. The excess of cyanide was removed using Sep-pak C18 cartridges, and the resulting dinucleoside monophosphates d(GpG) and d(ApG) were subsequently postlabelled. Analysis of the postlabelling mixture was performed by a combined TLC and HPLC-procedure. Good correlations with existing methods (AAS, immunocytochemistry and ELISA) were found in DNA samples treated in vitro and in vivo with cis- or carboplatin. The detection limit of the assay was 1 adduct/10(7) nucleotides in a 10 micrograms DNA sample.

The formation and persistence of platinum-DNA adducts were studied with immuno(cyto)chemical methods in male and female Sprague-Dawley rats treated with a single i.p. dose of carboplatin. Linear dose-effect curves were observed for kidney and liver with an immunocytochemical assay using NKI-A59 antiserum that recognizes intrastrand cross-links. With this method, no staining of the nuclei due to platinum-DNA damage could be observed in the spleen, testis, uterus, or ovary after administration of up to 80 mg/kg carboplatin. A homogeneous staining of the nuclei in the liver was observed. The nuclear staining in the kidney was somewhat more intense but less homogeneous, with small groups of intensely stained nuclei occasionally being seen in the outer cortex. An approximately 15 to 20-times lower dose of cisplatin than of carboplatin was needed to reach equal staining levels in the liver and kidney. Plateau staining levels in both tissues were reached at between approximately 8 and 48 h after administration of the carboplatin. This was followed by a significant reduction in the kidney samples, whereas the staining levels in the liver section seemed to be more persistent. No major difference was observed between male and female rats in the formation and removal of DNA damage in these tissues. The levels of the various DNA adducts were measured with a competitive ELISA in liver, kidney, spleen, testis, and combined ovary/uterus samples collected at 8 and 48 h after carboplatin administration. At both 8 and 48 h, the highest platination levels were observed in the kidney, followed--in decreasing order--by the liver, combined uterus and ovary samples, spleen, and testis. At 8 h after administration of carboplatin, the relative occurrence of the bifunctional adducts Pt-GG (34%), Pt-AG (27%), and G-Pt-G (32%), was similar in all tissues. The same held for the monoadducts that amounted to about 7% of the total DNA platination. These data indicate that in the first few hours after carboplatin treatment, no preference for the formation of Pt-GG adducts was observed, which confirms our earlier observations obtained with cultured cells. When the total DNA-platination levels (calculated from the sum of the adducts) seen at 8 and 48 h after treatment were compared, a substantial decrease in DNA platination was observed in the kidney (37%), liver (30%) and ovary/uterus (39%), whereas the repair levels in the testis (9%) and, probably, the spleen (18%) were substantially lower. In all tissues studied, only the relative occurrence of the Pt-GG adducts increased between 8 and 48 h, and as a result, at 48 h, after carboplatin administration the Pt-GG adduct was the major adduct persisting in the DNA samples.