Development of a P-32-postlabeling method for the detection of 1,N-2-propanodeoxyguanosine adducts of crotonaldehyde in vivo

Crotonaldehyde is a genotoxic, mutagenic and carcinogenic alpha,beta-unsatu rated carbonyl compound which forms 1,N-2-propanodeoxyguanosine adducts. Hu mans are exposed to this compound at work places. and from tobacco smoke an d air pollution, but also from food and beverages. Therefore crotonaldehyde can play a significant role in carcinogenesis. Since in vivo measurement o f DNA adducts of crotonaldehyde can improve cancer risk assessment and cont ribute to the clarification of the role of crotonaldehyde in carcinogenicit y, we developed, adapted and optimized a P-32-postlabelling technique for t he adducts of crotonaldehyde based on nuclease P1 enrichment and on a polye thylene imine modified cellulose TLC to provide a detection sensitivity of three adducts per 10(9) nucleotides and a labelling efficiency of 80-90%. W e also report a readily performable synthesis of adduct standards and demon strated that DNA is completely digested to the 3'-monophosphate nucleotides under the conditions of our enzymatic DNA hydrolysis. We showed that the p ostlabelling method developed is appropriate for in vivo DNA-binding studie s. Female Fischer 344 rats were treated by gavage with crotonaldehyde at do ses of 200 and 300 mg/kg body weight, and 20 h after treatment adduct level s of 2.9 and 3.4 adducts per 10(8) nucleotides, respectively, were found in the liver DNA. Only 1.6 nucleotides per 10(8) nucleotides were found 12 h after treatment at 200 mg/kg body weight. Absolutely no adducts could be fo und in liver DNA of untreated rats with our method at the detection limit o f three adducts per 10(9) nucleotides. In contrast to our group, the group of Chung have reported crotonaldehyde adduct levels in the range of 2.2-22 adducts per 10(8) nucleotides in DNA of untreated Fischer 344 rats. The cla rification of this discrepancy is of importance for the elucidation of the role of crotonaldehyde ill carcinogenicity, and both groups have decided to clarify this in cooperation ill the near future.