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 test, we developed two supplemental, cell-free assays to examine the potential of compounds and metabolites to directly damage DNA. Calf thymus DNA was used as the target for the direct detection of adducts by ³²P-postlabeling/TLC and electrochemical detection, and alkaline gel electrophoresis was used to detect single-strand breakage of bacteriophage λ DNA. To show that these assays would detect damage from relevant compounds, we examined nine human carcinogens (aflatoxin B₁, busulfan, chlorambucil, cyclophosphamide, diethylstilbestrol, melphalan, 2-naphthylamine, phenacetin and potassium chromate). Each of the nine compounds produced a positive result for one or both endpoints. Using multifraction contact-transfer TLC, we detected ³²P-labeled DNA adducts produced by aflatoxin B₁, chlorambucil, diethylstilbestrol, melphalan, 2-naphthylamine, and potassium chromate (plus hydrogen peroxide). Aflatoxin B₁, diethylstilbestrol and 2-naphthylamine required metabolic activation (induced rat liver S9) to generate DNA adducts. Although potassium chromate alone induced a slight increase in the content of 8-hydroxydeoxyguanosine (a promutagenic adduct produced by reactive oxygen species), addition of hydrogen peroxide greatly increased 8-hydroxydeoxyguanosine levels. The damage to λ DNA by each human carcinogen (or metabolites), except diethylstilbestrol, was sufficient to generate single-strand breaks after neutral thermal hydrolysis at 70°C. Chromate was a weak inducer of DNA fragmentation, but adding hydrogen peroxide to the reaction mixtures dramatically increased the DNA strand breakage. Our data suggest that these non-routine, acellular tests for determining direct DNA damage may provide valuable mechanistic insight for positive responses in cell-based genetic toxicology tests.