(E)-2-HEXENAL-INDUCED DNA-DAMAGE AND FORMATION OF CYCLIC 1,N-2(1,3-PROPANO)-2'-DEOXYGUANOSINE ADDUCTS IN MAMMALIAN-CELLS

(E)-2-Hexenal (hexenal), a natural flavor compound, acts as directly g enotoxic agent and forms cyclic 1,N-2-propano adducts with deoxyguanos ine. Formation of this adduct in isolated DNA and in cells was studied with a modified P-32-postlabeling procedure including HPLC separation , nuclease P1 enrichment, two-dimensional TLC of adducted nucleotide b isphosphates on PEI-cellulose, and quantification of adduct spots by l iquid scintillation counting. Adduct formation with the more reactive crotonaldehyde was included for comparison. Synthesized adducted dG-3' -phosphates served as external standards for identification and quanti fication. In calf thymus DNA, hexenal (0.2 mM) shows a time dependent formation of adducts, yielding 1.55 pmol/mu mol of DNA at 5 h incubati on. With crotonaldehyde (0.2 mM) the adduct rate was about 10-fold hig her. Hexenal also generated 1,N-2-propano-dG adducts in the human lymp hoblastoid Namalva cell line (0.2 mM, 1 h, 86 fmol/mu mol of DNA) and in primary rat colon mucosa cells (0.4 mM, 30 min, 50 fmol/mu mol of D NA). In primary colon mucosa cells from rats and humans, hexenal and c rotonaldehyde (0.4 mM, 30 min) induced DNA damage, detected by single cell microgel electrophoresis (comet assay), In primary rat gastric mu cosa cells, hexenal was only weakly active, inducing detectable DNA da mage in 20% of cells at 0.8 mM concentration, In contrast, primary muc osa cells from rat esophagus were as sensitive as colon cells. After s ingle oral application of hexenal to rats (up to 320 mg/kg body wt) DN A damage was not detectable in gastrointestinal mucosa. Analysis of he xenal ill selected flavored foods revealed concentrations up to 14 ppm (0.14 mM) that are comparable to its natural occurrence in some fruit s and vegetables (up to 30 ppm). Thus, the concentration range selecte d for the toxicological studies described here clearly is relevant: He xenal, at concentrations found in food, exerts genotoxic effects in ce lls from rat and human gastrointestinal tract.