Crotonaldehyde is a genotoxic, mutagenic and carcinogenic alpha,beta-unsaturated carbonyl compound which forms 1,N2-propanodeoxyguanosine adducts. Humans are exposed to this compound at work places, and from tobacco
smoke and air pollution, but also from food and beverages. Therefore
crotonaldehyde can play a significant role in
carcinogenesis. Since in vivo measurement of
DNA adducts of
crotonaldehyde can improve
cancer risk assessment and contribute to the clarification of the role of
crotonaldehyde in carcinogenicity, we developed, adapted and optimized a 32P-postlabelling technique for the adducts of
crotonaldehyde based on nuclease P1 enrichment and on a
polyethylene imine modified
cellulose TLC to provide a detection sensitivity of three adducts per 10(9)
nucleotides and a labelling efficiency of 80-90%. We also report a readily performable synthesis of adduct standards and demonstrated that
DNA is completely digested to the 3'-monophosphate
nucleotides under the conditions of our enzymatic
DNA hydrolysis. We showed that the postlabelling method developed is appropriate for in vivo
DNA-binding studies. Female Fischer 344 rats were treated by gavage with
crotonaldehyde at doses of 200 and 300 mg/kg
body weight, and 20 h
after treatment adduct levels 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 found in liver
DNA of untreated rats with our method at the detection limit of 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 clarification of this discrepancy is of importance for the elucidation of the role of
crotonaldehyde in carcinogenicity, and both groups have decided to clarify this in cooperation in the near future.