Quantitative analysis of DNA adducts formed by some epoxides of industrial importance

Abstract: The work presented herein has focused on DNA adduct formation by three industrial epoxides, allyl glycidyl ether, propylene oxide and epichlorohydrin. Epoxides, owing to the strained oxirane ring are chemically reactive and can alkylate various sites in DNA. Such modifications of the genetic material can contribute to the risk of cancer development in humans. Analysis of DNA adducts formed in vivo can help to improve risk assessment and contribute to better understanding of the mechanisms of chemical carcinogenesis. The main parts of the thesis are characterisation of DNA adducts formed by the studied epoxides, development of quantitative methods for the analysis of epoxideinduced adducts and analysis of such adducts in animal and human tissue samples. Similar to many other simple alkylating agents, all three studied epoxides were shown to react preferentially with the N7-position of guanine. Other major adducts in DNA were detected at the N1- and the N3-positions of adenine and the N3-position of cytosine. These ring nitrogen-substituted products were expected to be more or less unstable, therefore rates of secondary transformations were estimated. The data on formation and stability of DNA adducts were used for development of highly specific and sensitive 32P-postlabelling methods for analysis of DNA adducts formed by the three epoxides. These methods were applied for quantitative analysis of biological samples of both animal and human origin and the main observations made in these studies can be summarized as follows: ' Allyl glycidyl ether-induced DNA adduct levels in exposed mice were very low following i.p. injection. After skin application adduct levels in other tissues were much lower than in the skin indicating a slow absorption and/or distribution of this chemical. ' Propylene oxide-induced adducts in exposed rats could be detected in all seven tissues analysed and the levels were considerably higher in target tissue (nose) compared to non-target tissues. Contrary to the observed in vivo persistence of the N1-adenine adduct, the N3-substituted cytosine seemed to be rapidly repaired. The loss of N7-substituted guanine in vivo occurred most probably solely by chemical depurination. ' Epichlorohydrin-induced N7-guanine adducts were detected in white blood cells from occupationally exposed humans, but not in any of the control subjects. Altogether, our results showed that N7-guanine and N1-adenine adducts could be useful as biomarkers of exposure to the studied epoxides. The major advantage of N7-substituted guanines is their high concentration relative to the concentration of other adducts formed. Although the levels of N1-substituted adenines in DNA are lower than that of N7-substituted guanines, they represent a feasible alternative due to persistence in vivo (shown for propylene oxide) and the high specificity and sensitivity 32P-postlabelling analysis. Because of the obtained high analytical sensitivity and the lack of detectable background levels, DNA adduct measurements would be useful for monitoring low-level occupational exposures to epoxides in humans.