Characterising novel recombination pathways at DNA adducts of the environmental mutagen BPDE

Everyone is exposed to low levels of cancer-causing agents in the environment. Benzopyrene is a general pollutant that is found in smoke from stoves such as woodburners, exhaust fumes and barbequed meat and fish. Benzopyrene is a genotoxic mutagen, meaning that it directly damages the DNA to cause cancer-promoting mutations. It is likely that benzopyrene contributes to the development of common cancers such as lung and skin cancer.

Benzopyrene causes cancer through its main active ingredient, which is called BPDE. BPDE forms DNA damages called "adducts". BPDE adducts are very mutagenic, and cause both small and large changes, or mutations, in the DNA sequence. There are models explaining how BPDE causes these mutations, but some of the pathways are still not understood. A process called translesion synthesis has been made responsible for BPDE-induced mutations, but there is a lot of evidence that another pathway called homologous recombination is also involved. Homologous recombination may sometimes be more important, especially if BPDE levels in the DNA are low (which will often be the case in real life). What exactly homologous recombination does at BPDE adducts in the DNA is not very well understood.

We have new insights showing that homologous recombination proceeds by an unusual new mechanism at BPDE adducts. Understanding this new mechanism could help explain some of the effects of benzopyrene exposure, which could help to better predict and detect negative effects of pollution. It could also help predict which individuals will be more sensitive to carcinogen exposure.

The aim of this project is to treat human cell lines with BPDE and use molecular biology methods, such as microscopy, to characterise the new homologous recombination pathway in detail. This will reveal new insights into the effects of benzopyrene exposure in the cells, which is important for understanding environmental causes of cancer and cancer development in general.

We will investigate mechanistic toxicology of BPDE in human relevant models. The proposed experiments will primarily be performed in human U2OS osteosarcoma cells. We have U2OS cells for inducible PrimPol depletion and CRISPR-deleted for PARP1 and PARP2, and will generate further U2OS lines for combined depletion/expression of PrimPol for mutagenesis studies. BPDE adducts will be induced using nanomolar concentrations of commercially available (+)-anti-BPDE and adduct levels will be quantified by HPLC.

PrimPol exerts primase-, polymerase-, and TLS activity and plasmids expressing separation-of-function variants of PrimPol will be used to distinguish the contributions of these activities to BPDE-induced recombination. PrimPol-dependent ssDNA formation and resection will be assessed by immunostaining for native BrdU, RPA, and phospho-Serine S4/8 RPA, as well as S1 endonuclease-modified fibre assay and SMART fibre assay. Impact on replication fork progression will be measured using standard DNA fibre analyses.

We will further decipher the mechanisms of ssDNA gap resection and RAD51 loading at BPDE adducts using chemical inhibitors of resection factors and recombination mediators such as MRE11 and PARP1/2. Other resection proteins and recombination mediators such as BRCA1, BRCA2, RAD52, DNA2, and Exo1 will be siRNA-targeted. Initiation of recombination will be measured by RAD51 foci formation and chromatin recruitment, as well as proximity ligation assay. iPOND may be used as backup if needed. We will investigate potential competition between PrimPol-dependent recombination and translesion synthesis by DNA polymerase kappa. This will involve siRNA depleting Pol kappa and quantify eGFP-Pol kappa foci formation.

Impacts of PrimPol-dependent recombination on mutagenesis will be measured by classic mutagenesis assays e.g. HPRT, recombination assays such as sister chromatid exchange and whole genome sequencing followed by bioinformatics analyses.

This is a basic science project that will generate new understanding of the molecular mechanisms by which human cells respond to low level BPDE exposure. BPDE is the mutagenic metabolite of the widespread and highly carcinogenic pollutant B[a]P, to which virtually everyone is exposed. This project will generate new insights into how BPDE, and therefore B[a]P, exposures contribute to disease and cancer development and how they interact with genetic factors. This can ultimately help create better environmental policies and legislation, better approaches for the assessment of individual cancer risk, and potentially improved early detection, diagnosis and treatment of cancer.

1. The general public exposed to B[a]P and BPDE.
Members of the general public are highly concerned about environmental causes of cancer. Reports about rising air pollution are frequent in the UK media, and exposure from domestic stoves is a great concern in developing countries. While most sources of outdoor air pollution are beyond the control of individuals, other exposures can be controlled, e.g. B[a]P on leafy vegetables can be removed by washing. This research will improve our understanding of the consequences of BPDE exposure, which may in the short to medium term help improve public awareness of important causes of cancer. It may also help identify individuals who are at risk of developing cancer in response to exposure to certain carcinogens.

2. Government and environmental regulatory bodies.
These will benefit from better understanding of the consequences of B[a]P exposure. Air quality is regulated at international, national, and local levels. Human-made air pollution contains B[a]P, which is found in fine particulate matter (PM2.5). The R-CoI has already been involved in regulatory work that directly led to the identification of B[a]P as a substance of very high concern (SVHC) on the European level. This is also a great concern in developing countries, where there can be high levels of B[a]P exposure from domestic stoves. Concentration-dependent investigations such as those suggested here are of utmost importance for regulatory bodies as they provide the basis for any risk assessment. The assessment and potential regulation of specific exposure scenarios in domestic and occupational settings could benefit from this research in the short to medium term.

3. Health services, e.g. NHS.
These are also major stakeholders in healthy living and cancer prevention. In the long term, understanding the biological mechanisms surrounding environmental exposure in cancer development will help with the assessment of individual risk, development of early disease biomarkers and early interventions. A better understanding of the roles of e.g. homologous recombination in cancer development may in the long term help developing targeted treatments, and use existing targeted treatments such as PARP inhibitors more effectively. Both will help improve health and reduce the cost of cancer treatment, which is a major issue for the public and the health services.

4. Occupational health.
Occupational health, e.g. in the commercial private sector, also needs to perform health surveillance and prevention. In the long term, this research could help with individual risk assessment, which could be useful for health monitoring of employees who are exposed in the workplace.

5. Charities
Charities such as the British Lung Foundation, who campaign for stricter legislation to reduce indoor and outdoor air pollution, could benefit from better information on B[a]P effects in the short to medium term.

6. All employment sectors
These will benefit in the short to medium term from the training of a research scientist, who will acquire skills in interdisciplinary working, bioinformatics, project planning and management, teaching and supervision, presentation, and public engagement.