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Peroxidase-dependent bioactivation and oxidation of DNA and protein in benzo[a]pyrene-initiated micronucleus
Abstract:
Micronucleus formation initiated by benzo[a]pyrene (B[a]P) and related xenobiotics is widely believed to reflect potential carcinogenic initiation, yet neither a dependence upon bioactivation nor the critical enzymes have been demonstrated. Using rat skin fibroblasts, protein oxidation (carbonyl formation) and content of prostaglandin H synthase (PHS) and cytochrome P4501A1 (CYP1A1) protein were determined by Western blot/immunodetection with enhanced chemiluminescence. DNA oxidation as 8-hydroxy-2'-deoxyguanosine formation was quantified using high-performance liquid chromatography with electrochemical detection. Fibroblast CYP1A1 activity assessed as ethoxyresorufin-O-deethylase was not detectable, and even CYP1A1 protein was measurable only after induction with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, TCDD additionally induced prostaglandin H synthase (PHS), which also was detectable constitutively. B[a]P 10 microM initiated the oxidation of DNA and protein, and the formation of micronuclei, all of which were enhanced over 2-fold by the dual CYP1A1/PHS inducer TCDD 10 nM, as well as by other PHS inducers, 12-O-tetradecanoylphorbol-13-acetate 1 microM and interleukin-1alpha 0.625 or 1.25 ng/ml, that do not induce CYP1A1 (p < .05). Conversely, B[a]P target oxidation and micronucleus formation were abolished by 1-aminobenzotriazole 1 mM (p < .05), which was a potent inhibitor of both peroxidases and P450. These results provide the first direct evidence that B[a]P-initiated micronucleus formation, like carcinogenic initiation, requires enzymatic bioactivation, and that peroxidase-dependent, reactive oxygen species-mediated oxidation of DNA, and possibly protein, constitutes a molecular mechanism of initiation in uninduced cells. Induction of either CYP1A1 or peroxidases such as PHS substantially enhances this genotoxic initiation, which may reflect cancer risk.
Insights
Benzo[a]pyrene (B[a]P) causes DNA and protein oxidation, leading to micronucleus formation. This process requires enzymatic bioactivation, with peroxidases playing a key role in genotoxic initiation and potential cancer risk.
Area of Science:
- Biochemistry
- Toxicology
- Molecular Biology
Background:
- Micronucleus formation from benzo[a]pyrene (B[a]P) is linked to cancer initiation.
- The enzymes and bioactivation pathways involved in B[a]P-induced micronucleus formation remain unclear.
Purpose of the Study:
- To investigate the enzymatic bioactivation requirements for B[a]P-initiated micronucleus formation.
- To identify the critical enzymes and molecular mechanisms underlying B[a]P genotoxicity.
Main Methods:
- Quantified DNA and protein oxidation (8-hydroxy-2'-deoxyguanosine, carbonyl formation) in rat skin fibroblasts.
- Measured prostaglandin H synthase (PHS) and cytochrome P4501A1 (CYP1A1) protein levels via Western blot.
- Assessed CYP1A1 activity and inhibited enzymatic pathways using specific inducers and inhibitors (TCDD, 12-O-tetradecanoylphorbol-13-acetate, interleukin-1alpha, 1-aminobenzotriazole).
Main Results:
- B[a]P induced DNA oxidation, protein oxidation, and micronucleus formation in fibroblasts.
- These effects were significantly enhanced by inducers of PHS and CYP1A1 (TCDD) and by PHS-specific inducers.
- Micronucleus formation and oxidation were abolished by 1-aminobenzotriazole, a broad inhibitor of P450 and peroxidases.
- CYP1A1 activity was not detectable in uninduced cells, highlighting the role of PHS.
Conclusions:
- B[a]P-initiated micronucleus formation requires enzymatic bioactivation, primarily via peroxidase-dependent oxidation.
- Reactive oxygen species-mediated oxidation of DNA and protein is a key mechanism of genotoxic initiation.
- Induction of PHS or CYP1A1 enhances genotoxic initiation, potentially increasing cancer risk.