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Detection of DNA damage induced by human carcinogens in acellular assays: potential application for determining
S P Adams1, G M Laws, R D Storer
1Merck Research Laboratories, West Point, PA 19486, USA.
Abstract:
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 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 lambda 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 lambda DNA by each human carcinogen (or metabolites), except diethylstilbestrol, was sufficient to generate single-strand 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 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.
Insights
New cell-free assays detect direct DNA damage from carcinogens. These methods provide mechanistic insights into genotoxicity testing, complementing in vitro results.
Area of Science:
- Toxicology
- Molecular Biology
- Genetics
Background:
- Positive in vitro genotoxicity test results may not always stem from direct DNA damage.
- Distinguishing direct DNA damage from indirect effects is crucial for accurate risk assessment.
Purpose of the Study:
- To develop and validate cell-free assays for detecting direct DNA damage by compounds and metabolites.
- To assess the utility of these assays in identifying DNA adducts and strand breaks from known human carcinogens.
Main Methods:
- Utilized 32P-postlabeling/TLC and electrochemical detection to identify DNA adducts using calf thymus DNA.
- Employed alkaline gel electrophoresis to detect single-strand breaks in bacteriophage lambda DNA.
- Tested nine human carcinogens, some requiring metabolic activation (rat liver S9).
Main Results:
- All nine tested human carcinogens induced positive results in at least one of the cell-free assays.
- Specific carcinogens formed DNA adducts (e.g., aflatoxin B1, chlorambucil) and/or DNA strand breaks.
- Metabolic activation was necessary for adduct formation by some compounds (e.g., aflatoxin B1, diethylstilbestrol).
- Hydrogen peroxide significantly enhanced DNA damage (adducts and strand breaks) for certain compounds like potassium chromate.
Conclusions:
- The developed cell-free assays effectively detect direct DNA damage from various human carcinogens.
- These acellular methods offer valuable mechanistic insights into positive findings from cell-based genotoxicity tests.
- The assays can help elucidate the direct DNA-damaging potential of compounds and their metabolites.