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Effects of the (-)-anti-11R,12S-dihydrodiol 13S,14R-epoxide of dibenzo
B Mahadevan1, A Luch, A Seidel
1Department of Environmental and Molecular Toxicology, Agricultural and Life Sciences 1011, Oregon State University, Corvallis, OR 97331-7302, USA.
Abstract:
The tumor suppressor protein p53 plays an important role in recognition of DNA damage and induction of subsequent cell cycle arrest. One of its target genes encodes the protein p21(WAF1), which is involved in mediation of growth arrest after DNA damage has occurred. Dibenzo[a,l]pyrene (DB[a,l]P) is a polycyclic aromatic hydrocarbon which is an exceptionally potent carcinogen. A reactive secondary metabolite of DB[a,l]P, the fjord region (-)-anti-11R,12S-dihydrodiol 13R,14S-epoxide [(-)-anti-DB[a,l]PDE] was used to investigate DNA damage via adduct formation and cell cycle arrest in human diploid fibroblast cell cultures (HDF). Synchronous HDF were exposed to increasing concentrations (0.014, 0.028 and 0.07 microM) of (-)-anti-DB[a,l]PDE and at 1, 12, 24 and 42 h after treatment cell pellets were analyzed for DNA adduct formation and cell cycle arrest. Exposure of HDF to 0.07 microM (-)-anti-DB[a,l]PDE caused a total DNA binding level of 113 pmol adducts/mg DNA (42 h after treatment). G(1) arrest was induced by this treatment, with 91% of the cells remaining in G(1) phase compared with the solvent-treated control cultures (50%) as analyzed by propidium iodide staining and flow cytometry. Further investigation of the percentage of cells in S phase by 5-bromo-2'-deoxyuridine incorporation confirmed the G(1) arrest in HDF treated with 0.07 microM (-)-anti-DB[a,l]PDE, with only 1.5% of the cells moving into S phase compared with 39% in the control 42 h after treatment. Induction of p53 and p21(WAF1) was demonstrated by western blot analysis.
Insights
The potent carcinogen dibenzo[a,l]pyrene epoxide induces DNA damage and cell cycle arrest in human cells. This DNA damage activates tumor suppressor p53 and its target p21(WAF1), halting cell growth.
Area of Science:
- Toxicology
- Molecular Biology
- Cell Biology
Background:
- The tumor suppressor protein p53 is crucial for DNA damage response, regulating cell cycle arrest via target genes like p21(WAF1).
- Dibenzo[a,l]pyrene (DB[a,l]P) is a potent polycyclic aromatic hydrocarbon carcinogen, with its reactive metabolite (-)-anti-DB[a,l]PDE causing DNA damage.
Purpose of the Study:
- To investigate the DNA damage and cell cycle arrest induced by the DB[a,l]P metabolite (-)-anti-DB[a,l]PDE in human diploid fibroblast cells (HDF).
- To analyze the role of p53 and p21(WAF1) in response to DB[a,l]P-induced DNA damage.
Main Methods:
- Human diploid fibroblasts (HDF) were treated with varying concentrations of (-)-anti-DB[a,l]PDE.
- DNA adduct formation was quantified using DNA binding assays.
- Cell cycle distribution was analyzed by flow cytometry (propidium iodide staining) and DNA synthesis (5-bromo-2'-deoxyuridine incorporation).
- Protein levels of p53 and p21(WAF1) were assessed via western blot analysis.
Main Results:
- Exposure to 0.07 microM (-)-anti-DB[a,l]PDE resulted in significant DNA adduct formation (113 pmol adducts/mg DNA).
- A pronounced G(1) cell cycle arrest was observed, with 91% of cells in G(1) phase compared to 50% in controls.
- DNA synthesis was markedly reduced, with only 1.5% of treated cells entering S phase versus 39% in controls.
- Western blot analysis confirmed the induction of both p53 and p21(WAF1) proteins.
Conclusions:
- The reactive metabolite of dibenzo[a,l]pyrene, (-)-anti-DB[a,l]PDE, effectively induces DNA damage and G(1) cell cycle arrest in human diploid fibroblasts.
- The observed cell cycle arrest is mediated by the activation of the p53 and p21(WAF1) pathway in response to DNA damage.