DETECTION OF DNA-DAMAGE INDUCED BY HUMAN CARCINOGENS IN ACELLULAR ASSAYS - POTENTIAL APPLICATION FOR DETERMINING GENOTOXIC MECHANISMS

Positive outcomes of in vitro genotoxicity tests may not always occur as a consequence of direct reaction of a compound or a metabolite with DNA. To follow-up positive responses in in vitro tests, we developed two supplemental, cell-free assays to examine the potential of compoun ds and metabolites to directly damage DNA. Calf thymus DNA was used as the target for the direct detection of adducts by P-32-postlabeling/T LC and electrochemical detection, and alkaline gel electrophoresis was used to detect single-strand breakage of bacteriophage lambda DNA. To show that these assays would detect damage from relevant compounds, w e examined nine human carcinogens (aflatoxin B-i, busulfan, chlorambuc il, cyclophosphamide, diethylstilbestrol, melphalan, 2-naphthylamine, phenacetin and potassium chromate). Each of the nine compounds produce d a positive result for one or both endpoints. Using multifraction con tact-transfer TLC, we detected P-32-labeled DNA adducts produced by af latoxin B-i, chlorambucil, diethylstilbestrol, melphalan, 2-naphthylam ine, and potassium chromate (plus hydrogen peroxide). Aflatoxin B-i, d iethylstilbestrol and 2-naphthylamine required metabolic activation (i nduced rat liver S9) to generate DNA adducts. Although potassium chrom ate alone induced a slight increase in the content of 8-hydroxydeoxygu anosine (a promutagenic adduct produced by reactive oxygen species), a ddition of hydrogen peroxide greatly increased 8-hydroxydeoxyguanosine levels. The damage to lambda DNA by each human carcinogen (or metabol ites), except diethylstilbestrol, was sufficient to generate single-st rand breaks after neutral thermal hydrolysis at 70 degrees C, Chromate was a weak inducer of DNA fragmentation, but adding hydrogen peroxide to the reaction mixtures dramatically increased the DNA strand breaka ge. Our data suggest that these non-routine, acellular tests for deter mining direct DNA damage may provide valuable mechanistic insight for positive responses in cell-based genetic toxicology tests.