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Ototoxicity is a typical dose-limiting side effect of cancer chemotherapy with cisplatin but much less so with carboplatin. To elucidate the underlying molecular pathological mechanisms, we have measured the formation and persistence of druginduced DNA adducts in the nuclei of inner ear cells of guinea pigs after short-term exposure to either cisplatin or carboplatin using immunofluorescence staining and quantitative image analysis. After application of carboplatin, all cells of the cochlea exhibited a similar burden of guanine-guanine intrastrand cross-links in DNA. In contrast, we observed a pronounced 3-to 5-fold accumulation of this cytotoxic adduct exclusively in the marginal cells of the stria vascularis between 8 and 48 h after treatment with cisplatin. In the kidney, the other critical target tissue of cisplatin toxicity, a similar high preferential formation of cytotoxic DNA adducts was measured in the tubular epithelial cells but not in other renal cell types. As for the ear, this excessive formation of DNA damage in a particular cell type was seen in animals treated with cisplatin but not those treated with carboplatin. Because cisplatin ototoxicity is often attributed to oxidative stress mediated by the generation of radical oxygen species (ROS), we have measured in parallel the levels of the lead DNA oxidation product 8-oxoguanine (8-oxoG) in cochlear cryosections. Compared with basal levels in untreated control cochleas, no additional formation of 8-oxoG was detectable up to 48 h after cisplatin treatment in the DNA of either inner-ear cell type. This suggests that the generation of ROS may be a secondary event in cisplatin ototoxicity.Platinum derivatives are frequently used in cancer chemotherapy for patients with malignancies of, for example, the urogenital tract, the lung, the colon, the stomach, or the head and neck region. It is widely accepted that the antineoplastic efficiency of cisplatin [cis-diamminedichloroplatinum (II)] as well as carboplatin (diammine-cyclobutane-dicarboxylatoplatinum) results from their interaction with the nuclear DNA of tumor cells (reviewed in Siddik, 2003). The drugs harboring two reactive groups initially induce monoadducts at nucleophilic sites (e.g., of guanine or adenine) and can lead subsequently to intra-and interstrand cross-links in the DNA. Once formed, these lesions can trigger apoptotic cascades (McKeage, 1995;Boulikas and Vougiouka, 2003) predominantly via the mitochondrial pathway (McDonald and Windebank, 2002;Lee et al., 2004). The vital role of DNA adducts in this process is confirmed by observations in human cells differing in their ability to repair drug-induced damage to their genome. Cells with impaired nucleotide excision repair functions are clearly more sensitive to cisplatin than their proficient counterparts (Furuta et al., 2002;Wu et al., 2003).On the other hand, the frequently dose limiting side effects of cisplatin on normal cells and tissues (e.g., in the inner ear and the kidney) were mainly ascribed until now to pathways not ...

Exposure to quartz and high concentrations of other poorly soluble particles can lead to the development of lung tumors in the rat. The mechanisms involved in particle-induced carcinogenesis seem to include inflammation-associated production of reactive oxygen species (ROS) and DNA damage. ROS induce 8-oxoguanine (8-oxoGua) and a panel of other oxidation products in DNA. In proliferating cells such DNA lesions can lead to various types of mutations, which might be critical for cancer-related genes with respect to tumor formation. Quartz is known to mediate the induction of 8-oxoGua in the nuclear DNA of lung cells when applied to the lung of rats. We have investigated the time- and dose-dependent biologic effects of quartz and, as a control, corundum, on cell proliferation and various pulmonary inflammation and toxicity markers in rat bronchoalveolar lavage fluid (BALF); on the induction of 8-oxoGua in the DNA of rat lung cells; and on the cellular levels of p53 wild-type and p53 mutant (mut) protein. Rats were exposed by intratracheal instillation to various amounts of quartz (0.3, 1.5, or 7.5 mg/rat) or corundum (0.3, 1.5, or 7.5 mg/rat) and measured at Days 7, 21, and 90 after exposure. Corundum had no adverse effects except a slight elevation of 8-oxoGua at a dose of 7.5 mg/rat. However, significant changes in the BALF were detected at all quartz doses. 8-oxoGua was significantly increased only at 1.5 and 7.5 mg quartz/rat. The amount of cells with detectable p53 wild-type protein levels was increased at 1.5 and 7.5 mg quartz/rat at 7 and 21 d. Elevated amounts of cells with enhanced p53 mut protein levels were measured at all time points after instillation of 7.5 mg quartz/rat.

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Exposure of rats to quartz (or various other particles) can lead to the development of lung tumors. At the moment, the mechanisms involved in particle-induced tumor formation are not clarified. However, it is suggested that inflammation, in conjunction with the production of reactive oxygen species (ROS) and an enhancement of epithelial cell proliferation, may play a key role in the development of lung tumors. ROS induces 8-oxoguanine (8-oxoGua) and other mutagenic DNA oxidation products, which can be converted to mutations in proliferating cells. Mutation formation in cancer-related genes is a critical event with respect to tumor formation. In this study we investigated the effects of quartz (DQ1 2) and of the nontumorigenic dust corundum on the induction of 8-oxoGua in the DNA of rat lung cells, as well as on cell proliferation and pulmonary inflammation. Wistar rats were exposed by intratracheal instillation to quartz (2.5 mg/rat) or corundum (2.5 mg/rat) suspended in physiological saline; control animals exposed to physiological saline or left untreated. Measurements were carried out 7, 21, and 90 days after the exposures. 8-oxoGua levels were determined in lung tissue sections at the single cell level by immunocytological assay using a rabbit anti-8-oxoGua antibody. After exposure to quartz, 8-oxoGua levels were significantly increased at all time points of investigation. Additionally, we observed inflammation and an enhanced cell proliferation. Exposure to corundum had no adverse effects on the lung; neither increased 8-oxoGua levels nor enhanced cell proliferation or inflammation were detected. These observations support the suggestion that inflammation associated with increased 8-oxoGua levels in lung cells and increased cell proliferation is an important determinant for particle-induced development of lung tumors in the rat.

We report the establishment of a standardized, monoclonal antibody (Mab)-based immunocytological assay (quantitative ICA) for the visualization and quantification of low levels of specific DNA O-alkylation products in individual cells by electronically intensified, indirect or direct immunofluorescence. In terms of specific binding to alkali-denatured nuclear DNA and low background noise, 10 Mabs from a collection of 154 Mabs specific for O6-methyl-2'-deoxyguanosine (O6-MedGuo), O6-ethyl-2'-deoxyguanosine (O6-EtdGuo), O6-n-butyl-2'-deoxyguanosine (O6-BudGuo) and O4-ethyl-2'-deoxythymidine (O4-EtdThd) with antibody affinity constants ranging between 1.0 x 10(6)-3.0 x 10(10) l/mol, were found to be best suited for ICA. At present, > or = 200 O6-EtdGuo residues (corresponding to an O6-EtdGuo/dGuo molar ratio in DNA of > or = 8.4 x 10(-8)), > or = 400 O6-BudGuo residues (O6-BudGuo/dGuo, > or = 1.7 x 10(-7)), > or = 1800 O4-EtdThd residues (O4-EtdThd/dThd, > or = 7.5 x 10(-7)) and > or = 4800 O6-MedGuo residues (O6-MedGuo/dGuo, > or = 2.0 x 10(-6)), can be quantified per diploid genome. Using a SIT video camera in combination with multiparameter image digital analysis, DNA adduct-specific rhodamine fluorescence signals are measured relative to nuclear DNA content (DAPI fluorescence). Adduct-specific fluorescence recordings in three different rat cell lines (BT3Ca, Fao and NO) were in excellent agreement with the data obtained by competitive radioimmunoassay (RIA) for hydrolysates of DNA isolated from the respective cells exposed in parallel to the same alkylating carcinogens (N-methyl-, N-ethyl- and N-[n-butyl]-N-nitrosourea). Accordingly, the kinetics of O6-EtdGuo repair, as determined by ICA and RIA, respectively, were superimposable. Cell-specific, quantitative ICA can, therefore, be used for the quantification of specific, stable DNA adducts induced by alkylating carcinogens or chemotherapeutic agents and for DNA repair measurements in individual (e.g. human) cells. Work is currently underway to extend the spectrum of carcinogen--DNA adduct-specific Mabs suited for quantitative ICA.

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