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Mutagenesis and DNA adduct formation by 1-nitropyrene in Chinese hamster ovary cells without exogenous metabolic
J R Thornton-Manning1, B A Smith, F A Beland
1National Center for Toxicological Research, Jefferson, Arkansas 72079.
Abstract:
The direct-acting mutagenicity of 1-nitropyrene (1-NP), a tumorigenic environmental pollutant and model nitropolycyclic aromatic hydrocarbon, was studied in Chinese hamster ovary (CHO) cells. Previous reports indicated that 1-NP, a potent direct-acting mutagen in Salmonella typhimurium, was mutagenic in CHO cells only in the presence of an exogenous activation system. In this study, a DNA-repair-deficient CHO cell line, CHO-UV5, and the repair-proficient CHO-K1-BH4 cell line were used to measure the direct-acting mutagenicity of highly purified samples of 1-NP. Exposure of CHO-K1-BH4 cells for 5, 12, and 24 hr did not increase mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (p greater than 0.1; paired t test; n = 5-6), while treatment of CHO-UV5 cells for 24 hr produced a statistically significant induction of mutants (22 +/- 6 mutants per 10(6) clonable cells vs a solvent control value of 9 +/- 3 per 10(6) cells; p less than 0.01; n = 6). 32P-Postlabeling analysis of the DNA adducts formed in CHO-UV5 cells following 1-NP exposure revealed the presence of a single major adduct. Based on its chromatographic properties, its sensitivity to nuclease P1 digestion, and its resistance to hydrazine treatment, the adduct appeared to be N-(deoxyguanosin-8-yl)-1-aminopyrene, which was presumably produced by nitroreduction of 1-NP to N-hydroxy-1-aminopyrene. These results suggest that the use of extended treatment times and DNA-repair-deficient cells may be required to assess adequately the mutagenicity of nitropolycyclic aromatic hydrocarbons in CHO cells.
Insights
1-nitropyrene (1-NP) is a mutagenic pollutant. Using DNA-repair-deficient cells revealed 1-NP directly mutates Chinese hamster ovary (CHO) cells, forming DNA adducts. Extended treatment times are crucial for assessing nitropolycyclic aromatic hydrocarbon mutagenicity.
Area of Science:
- Environmental Toxicology
- Molecular Toxicology
- Genetics
Background:
- 1-nitropyrene (1-NP) is a carcinogenic nitropolycyclic aromatic hydrocarbon pollutant.
- Previous studies showed 1-NP required exogenous activation for mutagenicity in Chinese hamster ovary (CHO) cells.
- This study investigates the direct-acting mutagenicity of 1-NP in CHO cells.
Purpose of the Study:
- To determine if 1-nitropyrene (1-NP) directly mutates Chinese hamster ovary (CHO) cells.
- To identify DNA adducts formed by 1-NP in CHO cells.
- To establish optimal conditions for assessing nitropolycyclic aromatic hydrocarbon mutagenicity in CHO cells.
Main Methods:
- Utilized DNA-repair-deficient (CHO-UV5) and repair-proficient (CHO-K1-BH4) CHO cell lines.
- Exposed cells to 1-NP for varying durations (5, 12, 24 hours).
- Quantified mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus and analyzed DNA adducts using 32P-postlabeling.
Main Results:
- 1-NP induced significant mutations in CHO-UV5 cells after 24-hour exposure, but not in CHO-K1-BH4 cells.
- A single major DNA adduct, identified as N-(deoxyguanosin-8-yl)-1-aminopyrene, was formed in CHO-UV5 cells.
- The adduct formation suggests nitroreduction of 1-NP to N-hydroxy-1-aminopyrene.
Conclusions:
- DNA-repair-deficient CHO cells and extended exposure times are necessary to detect the direct mutagenicity of 1-NP.
- 1-NP is a direct-acting mutagen in CHO cells, forming specific DNA adducts.
- This highlights the importance of considering cellular repair capacity and treatment duration in mutagenicity assessments of nitropolycyclic aromatic hydrocarbons.