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Benzo[a]pyrene--DNA adduct formation in target cells in a cell-mediated mutation assay
Abstract:
In order to analyze the adducts formed in V79 Chinese hamster target cells in a Syrian hamster embryo (HE) cell-mediated mutation assay, a procedure was developed in which the HE cells are differentially killed when the mixed cell suspension is treated with antiserum to Syrian HE cells and complement. The V79 cell suspension, separated from lysed HE cells by gradient centrifugation, is greater than 90% HE cell-free. When the carcinogen--DNA interaction products formed in these two cell types were analyzed after exposure to [3H]benzo[a]pyrene (B[a]P) for 24 h under the conditions of a cell-mediated mutation assay, the major B[a]P--DNA adducts in both cell types resulted from reaction of the anti and syn isomers of B[a]P-7,8-diol-9,10-epoxide with DNA. The amount of B[a]P bound/mg DNA in the target cells was only 30% less than in the activator cells, and the relative proportions of the adducts of the syn and anti isomers were similar in the two cell types. The target cells, which do not metabolize B[a]P, were also unable to metabolically activate B[a]P-7,8-diol to DNA-binding metabolites. Thus, the transfer of activated B[a]P metabolites from activator cells to the DNA of target cells is a relatively efficient process that appears to be independent of the relative reactivity of specific metabolites with cellular nucleophiles. The immunological cell separation procedure we describe can be adapted to the analysis of carcinogen--cell interaction products formed in cell-mediated assays in which other types of activator and target cells are used to measure either mutation or another biological endpoint.
Insights
A new method separates target cells from activator cells to study carcinogen-DNA adducts. This shows efficient transfer of activated benzo[a]pyrene (B[a]P) metabolites to target cell DNA.
Area of Science:
- Toxicology
- Molecular Biology
- Cell Biology
Background:
- Cell-mediated mutation assays are crucial for understanding carcinogen effects.
- Analyzing carcinogen-DNA adducts in target cells requires effective separation from activator cells.
- Benzo[a]pyrene (B[a]P) is a known environmental carcinogen requiring detailed study of its metabolic activation and DNA interaction.
Purpose of the Study:
- To develop and validate an immunological cell separation technique for analyzing carcinogen-DNA adducts in a cell-mediated mutation assay.
- To investigate the formation and transfer of B[a]P-DNA adducts between Syrian hamster embryo (HE) activator cells and V79 Chinese hamster target cells.
Main Methods:
- Developed an immunological method using antiserum and complement to selectively lyse HE activator cells.
- Utilized gradient centrifugation to separate V79 target cells, achieving >90% HE cell-free suspension.
- Analyzed [3H]benzo[a]pyrene (B[a]P) and its DNA adducts in both cell types after exposure.
Main Results:
- The major B[a]P-DNA adducts in both cell types were formed by the anti and syn isomers of B[a]P-7,8-diol-9,10-epoxide.
- Target cells showed only 30% less B[a]P bound per milligram of DNA compared to activator cells.
- The transfer of activated B[a]P metabolites from activator to target cells was efficient and independent of specific metabolite reactivity.
Conclusions:
- The developed immunological cell separation method effectively isolates target cells for adduct analysis.
- Efficient transfer of activated B[a]P metabolites occurs from HE activator cells to V79 target cells.
- This methodology is adaptable for studying carcinogen-cell interactions in various cell-mediated assays.