Project 4: Genetic Susceptibility
Developing and applying novel cell-based assays and genetically engineered mice to reveal genotoxicity, cytotoxicity, mutagenicity, and carcinogenicity of alkylating agents in the environment, with a focus polycyclic aromatic hydrocarbons (PAHs) and N-nitrosodimethylamine (NDMA).
PROJECT LEADERS: BEVIN ENGELWARD, LEONA SAMSON
A major focus of the Project 4 team is gene-environment interactions, with a focus on DNA repair. Recent results point to a particular DNA repair enzyme as a pivot between seemingly opposite outcomes (cancer vs toxicity). Results of this study were published in Cell Reports and have been highlighted by the NIEHS as a Paper of the Month, as well as having been featured in the NIEHS Environmental Factor, and by the MIT Press.
In addition to studies in animals, the team has also been working on cell-based assays that have utility in studies of both NDMA and PAHs. NDMA and PAHs both create DNA adducts that are mutagenic and toxic. Support from the NIEHS SRP made it possible to bring to completion the creation of two novel high throughput assays. The HepaCometChip, which is the fastest live cell assay for detecting the ability of chemicals to form DNA adducts, and the MicroColony Chip, which is a novel cell-based platform that exploits colony size as a metric for cytotoxicity. News of the HepaCometChip manuscript (published in Nucleic Acids Research) was featured by the NIEHS, the NIEHS SRP, and the MIT Press. News of the MicroColony Chip manuscript (Cell Reports) was also featured by the NIEHS, the NIEHS SRP, and the MIT Press.
Policy decisions depend not only on knowing if NDMA can cause cancer, but also knowing how gene-environment interactions impact disease risk. While NDMA is known to be potently carcinogenic in animal models, little had been done to explore how genetic factors modulate NDMA’s effects on DNA damage, mutations, and cancer. Recent results show that the Alkyladenine DNA glycosylase (AAG) is a genetic susceptibility factor for NDMA-induced cancer in mice, wherein too little AAG increases the risk of cancer and higher than normal levels of AAG are protective. However, deeper analysis revealed that although AAG suppresses cancer, mice become susceptible to cell death, inflammation and toxicity. These results are consistent with prior results from the Samson laboratory. Specifically, AAG initiates a repair pathway by removing damaged bases. The result is that an abasic site is created, which is then cleaved in order to pave the way for a polymerase to replace the missing nucleotide. If there is extra high levels of AAG, the pathway can become unbalanced, leading to an accumulation of breaks that are highly toxic. We tracked responses to NDMA over time, and realized that there are clear patterns that can likely be used as predictive biomarkers for downstream cancer and toxicity. Being able to predict disease opens doors to identification of interventions that prevent or mitigate disease, without waiting 10 months for results. This work from the Engelward and Samson labs was published in Cell Reports and included contributions from several other MIT SRP leaders, including Forest White, John Essigmann, Robert Croy. The lead researchers included Jenny Kay (First Author), Joshua Corrigan (Second Author), as well as Amanda Armijo, Ilana Nazari and Ishwar Kohale.
Taken together, in collaboration with the other Center projects, this MIT SRP project plays the exciting role of unifying key cancer-related endpoints into an integrated whole. This project will yield insights into genetic risk factors (impacting risk assessment), give rise to deeper understanding of the mechanisms of disease progression (ultimately opening new opportunities for prevention and mitigation), to improve the understanding of the impact of NDMA on a potential window of vulnerability, yield mutational and proteomic biomarkers that predict disease susceptibility, and reveal the real-world impact of NDMA on health under conditions that reflect those that are present in the Mystic River Watershed.
One of the short term methods to detect DNA damage is based on the traditional comet assay, where damaged DNA migrates more readily in a matrix compared to undamaged DNA when electrophoresed (note the comet tail showing DNA damage in the nucleus, far right). The Engelward laboratory has developed a higher throughput version of the comet assay called the “CometChip.”
Artistic rendition of human cells in microwells in a microwell array in agarose (David M. Wood). By arraying cells, overlapping comets are avoided. Higher throughput imaging and analysis enable automated and rapid analysis 100-1000X faster than the traditional assay.
Mouse whole pancreas at 0.5 mm thickness. Yellow fluorescence indicates the presence of mutant cells. (B. Engelward)