Project 4 - Genetic Susceptibility

 

Revealing the impact of genetic susceptibility factors for carcinogenic alkylating agents in the environment, with a focus on benzo[a]pyrene and N-nitrosodimethylamine.

 

Project Leaders: Bevin Engelward, Leona Samson

 

Residents who live near the Olin Superfund site (within the Mystic River Watershed) are worried because there was a cancer cluster in their community and their water contains N-nitrosodimethylamine (NDMA), a potent carcinogen in animal models. 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 is known about how genetic factors modulate NDMA’s effects on DNA damage, mutations, and cancer.

In this project, the researchers focus on Aag and Mgmt, two genes that together are responsible for repairing more than 80% of the NDMA-induced DNA lesions. Researchers are exploiting innovative mouse models with varied DNA repair capacity to learn how Aag and Mgmt impact susceptibility to NDMA-induced mutations and tumors. They are collaborating with Project 3 to reveal the impacts of Aag and Mgmt on high-resolution point mutation spectra, as well as analyses of homologous recombination-derived sequence rearrangement mutations. They will also work with Project 5 to reveal the impact of Aag and Mgmt in modulating the risk of short-term physiological responses, and systems level network responses. An innovative mouse model created in the Engelward laboratory enables fluorescence detection of large-scale sequence rearrangements, leading to unprecedented speed and accuracy in determining the frequency of de novo mutations.

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)

 

Damaged DNA can be repaired by using sequence information at a homologous site. Recombination at a reporter transgene in RaDR mice gives rise to a fluorescent signal, shown in the cell on the right.