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Published on: December 19, 2019
Mdm2 as a sensitive and mechanistically informative marker for genotoxicity induced by benzo[a]pyrene and
Maria Malmlöf1, Gerd Pääjärvi, Johan Högberg
1Institute of Environmental Medicine, Karolinska Institutet, S-17177 Stockholm, Sweden.
Abstract:
Mdm2 is an oncoprotein interacting with p53 and maintaining low p53 levels in unstressed cells. Here we investigated the effect of genotoxic compounds on Mdm2 phosphorylation levels. Employing the Mdm2 2A10 antibody and phosphatase treatment we found that Mdm2 accumulated in HepG2 cells when exposed to low concentrations of genotoxic compounds such as mitomycin C, etoposide, 5-fluorouracil, and benzo[a]pyrene (BP). The low-dose responses were not accompanied by p53 accumulation and the effect of low concentrations of BP on Mdm2 was not affected by small interfering RNA for p53. In human lymphoblasts 10nM BP induced an Mdm2 response. Low concentrations of BP also induced binding of Mdm2 to chromatin in HepG2 cells, but no p53 binding or H2AX phosphorylation. The more mutagenic dibenzo[a,l]pyrene as well as higher BP concentrations instead induced gammaH2AX and p53 Ser15 association with chromatin. Acrolein potentiated the effect of BP on p53 stabilization and chromatin binding. Taken together, these data suggest that (1) Mdm2 is a sensitive biomarker for certain types of genotoxicity, and (2) that polycyclic aromatic hydrocarbons-induced Mdm2 binding to chromatin reflects repairable damage, whereas chromatin binding of p53 Ser15 and gammaH2AX indicates more persistent DNA damage. The analysis of Mdm2 and related endpoints might be useful for evaluating mutagenic potentials of DNA damages. It is suggested that patterns documented here can be used for separating BP doses that induce readily repaired DNA adducts from doses that overwhelm this capacity.
Insights
Mdm2 protein levels indicate genotoxic stress from compounds like benzo[a]pyrene. Low doses suggest repairable DNA damage, while higher doses signal persistent damage, useful for mutagenicity assessment.
Area of Science:
- Molecular Biology
- Toxicology
- Cancer Research
Background:
- Mdm2 is an oncoprotein that regulates p53 levels in unstressed cells.
- Genotoxic compounds can impact cellular pathways, including Mdm2 and p53 interactions.
- Understanding cellular responses to genotoxicity is crucial for risk assessment.
Purpose of the Study:
- To investigate the effect of genotoxic compounds on Mdm2 phosphorylation and accumulation.
- To evaluate Mdm2 as a potential biomarker for genotoxicity.
- To differentiate between repairable and persistent DNA damage based on cellular markers.
Main Methods:
- Exposure of HepG2 cells and human lymphoblasts to various genotoxic compounds (mitomycin C, etoposide, 5-fluorouracil, benzo[a]pyrene, dibenzo[a,l]pyrene, acrolein).
- Analysis of Mdm2 accumulation and phosphorylation levels using Mdm2 2A10 antibody and phosphatase treatment.
- Assessment of p53 accumulation, p53 Ser15 phosphorylation, and H2AX phosphorylation (gammaH2AX) via chromatin binding assays.
- Utilized small interfering RNA (siRNA) for p53 to investigate its role in Mdm2 response.
Main Results:
- Low concentrations of genotoxic compounds, including benzo[a]pyrene (BP), induced Mdm2 accumulation in HepG2 cells without p53 accumulation.
- BP-induced Mdm2 chromatin binding occurred at low doses, correlating with repairable DNA damage.
- Higher BP concentrations or more potent mutagens induced gammaH2AX and p53 Ser15 chromatin binding, indicating persistent DNA damage.
- Acrolein potentiated BP's effects on p53 stabilization and chromatin binding.
Conclusions:
- Mdm2 serves as a sensitive biomarker for specific types of genotoxicity.
- Mdm2 chromatin binding indicates repairable DNA damage, while p53 Ser15 and gammaH2AX binding signify persistent DNA damage.
- Analyzing Mdm2 and related markers can help evaluate mutagenic potential and distinguish between DNA adducts that are readily repaired versus those that overwhelm repair capacity.
