32P-postlabelling analysis of complex DNA adducts in human tissues

Abstract

Complex DNA adducts in humans originate from exogenous and endogenous sources. Polycyclic aromatic hydrocarbons (PAHs) are a large group of chemicals including many potent carcinogens. PAHs are ubiquitously present in the environment and PAH-DNA adducts have been extensively studied in humans. Endogenous processes such as lipid peroxidation are known to produce adduct-forming agents. The characteristics of many human adducts have not been identified. This thesis study focused on the analysis of these human adducts by the 32P-postlabelling technique.

In order to detect the effects of air pollution resulted from engine exhaust on the aromatic adduct levels, PAH-DNA adducts were analysed in peripheral blood lymphocytes (PBL) of 53 newspaper vendors working at high and low traffic areas. No difference in adduct levels was found between the high and low exposed group. In west European cities, the airborne benzo[a]pyrene concentrations are usually 2-3 times higher in high traffic areas than in low traffic areas. Our results suggested that the effects of low-level air pollution could approach the limit that can be detected by the 32P-postlabelling assay. DNA samples isolated from PBL of 317 smoking and nonsmoking lung cancer patients and controls were analysed for the aromatic adducts in a case-control study. A significantly higher adduct level was observed in current smokers compared to never- or ex-smokers and there appeared to be a decreasing trend in adduct level with increasing time since last smoking. A slight decrease of adduct level with increasing age was shown in the entire study population, whereas a slight increase of adduct level with age was shown in current smokers. The increase was much stronger among current smoking patients. These results showed a clear effect of smoking on the lymphocytic aromatic adduct level and implied an interaction between smoking and genetic host factors. A 32P-postlabelling/HPLC (high-performance liquid chromatography) method using both nuclease P1 treatment and butanol extraction was developed and applied to the analysis of the lipophilic adducts in human tissues. Abundant adducts were detected in lung, colon, breast, skin and endometrial tissues and lymphocytes. These adducts exhibited tissue-specific and, in some tissues, complex patterns. In lung tissues, the lipophilic adduct levels were about 20 times higher than the aromatic adduct levels determined by a 32P-postlabelling/TLC (thin-layer chromatography) assay. The adduct levels in lymphocytes were shown not to be associated with smoking and occupational exposure to PAIL The adduct levels of two HPLC fractions in lymphocytes were found to be age-dependent. An adduct in lung tissue DNA was identified as a putative 2,3-epoxy-4-hydroxynonanal-induced adduct. These results suggested that at least part of the lipophilic adducts were of endogenous origin. When analysing the whole spectrum of DNA adducts which were resistant to nuclease P1 treatment in human tissues by a 32P-postlabelling/HPLC assay not using butanol extraction, more abundant DNA adducts were detected in the early eluting fractions. In these fractions, acrolein- and crotonaldehyde-induced endogenous DNA adducts were detected in human lymphocytes, and lung and colon tissues. These abundant adducts were also analysed in tumor and tumor-adjacent tissues from breast cancer patients and in the normal breast tissues of the controls. The tumor and tumor-adjacent tissues showed higher adduct levels than the normal tissues, with significance detected in three HPLC fractions. Adduct levels of three HPLC fractions were found to correlate significantly with age.