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Epidemiological Study links NDMA to Cancer

Wilmington Massachusetts is a leafy suburb just north of Boston. With classic New England homes and maple trees it provides an idyllic setting for family life. Nevertheless, in the 1990s, mothers started noticing that many of their children had cancer. When a proper study was done, it was discovered that 24 children out of a town of approximately 18,000 had cancer, which is six times higher than the national average. People living in Wilmington wanted to know what had caused so many cases of childhood cancer in their town, and they turned their attention to their drinking water. Knowing that the Olin chemical company had operated in Wilmington for many years, and that the site was used for many other chemical companies prior to Olin, town members wondered if it was possible that chemical waste had made its way from the Olin site to their drinking water wells.

5Olin Site Outline 300x300When town members went to local officials to express their concern about the safety of their drinking water, their concerns were largely dismissed, and they were told that their water was safe. However, local residents did not give up. Remarkably, residents combed through historic documents trying to figure out if dangerous chemicals could have made their way into their water. They learned that in fact, chemical waste had been dumped into unlined pools and lagoons adjacent to the factory raising the possibility that millions of gallons of waste may have seeped into the earth. With no formal training in chemistry, Debbie Duggan and Suzanne Sullivan started wondering if N-nitrosodimethylamine (NDMA) was in their water. They were worried about NDMA because it was well-established that NDMA is a potent carcinogen in animal models. They insisted that the town well water be tested for the presence of NDMA. When testing was finally done, it was discovered that indeed, NDMA was in the town well water that had been drunk by thousands of people for many years.

Although officials did not necessarily agree that NDMA had been a health problem, the wells were nevertheless closed in 2003. Soon thereafter, concerned about contaminated municipal water, the EPA designated the Olin site as a Superfund site. However, people living in Wilmington wanted more research. They wanted a public health study to be done to determine if NDMA was linked to cancer in their children. The study took quite some time.

Meanwhile, there was a serendipitous event. At a committee meeting at MIT, J. E. From Wilmington mass met John Essigmann, a well-recognized Prof. of toxicology and chemistry. She explained to Prof. Essigmann that people living in Wilmington were worried that a Superfund site was responsible for a childhood cancer cluster. When she said that the primary concern was NDMA, Prof. Essigmann was stunned. NDMA is a chemical that is closely related to other chemicals that he had been studying for more than 20 years. Furthermore, there were nearly half a dozen other faculty at MIT who also were studying chemicals related to NDMA. This gave Prof. Essigmann the idea that perhaps a team could be assembled to address the needs of the community and so he recruited fellow faculty and other leaders to put forth the concept of a new Superfund research program at MIT. At a meeting with William Suk and Michelle Heacock (Director and member of the NIEHS Superfund research program leadership team, respectively), the MIT team was encouraged to apply for a Superfund grant. In 2017, the MIT Superfund research program was created with Prof. Bevin Engelward and Prof. John Essigmann as the director, and codirector of the program. Later, Noelle Selin agreed to contribute as a codirector.

5NDMA 300x200The focus of the MIT Superfund research program is on alkylating agents. These are chemicals that create adducts on DNA, disrupting its structure and thus promoting mutagenesis and toxicity. In particular, the MIT team studies NDMA and polycyclic aromatic hydrocarbons (PAHs). In terms of NDMA, the team has made progress in creating sensors. Dr. Maggie He, member of the swagger lab, created a carbon nanotube sensor for NDMA in air. More recently, Jessica Beard from the Swagger lab has been developing a sensor for NDMA in water. In parallel, biologists applied their skills to study the mutagenic and carcinogenic effects of NDMA, with an emphasis on gene environment interactions that might modulate susceptibility. Jennifer Kay, Joshua Corrigan, and Amanda Armijo (from the Engelward and Essigmann laboratories) made the discovery that a particular DNA repair enzyme has a profound effect on the biological consequences of NDMA. (See When too low, animals suffer from DNA damage induced mutations in cancer, and when too high, animals suffer from DNA damage induced toxicity. This remarkable discovery paves the way for human studies, wherein the role of this repair enzyme can be studied in terms of its relationship to vulnerability to NDMA exposure.

Meanwhile, the epidemiological study of NDMA performed by the Massachusetts Department of Public Health was finally released some 20 years since the time when NDMA was discovered to be in the town’s drinking water. Town members were both stunned and dismayed to learn that NDMA exposure in utero was associated with an increased risk of childhood cancer, though the presence of other chemicals in the drinking water may have contributed. At the same time, town members felt vindicated having struggled for decades for both cleanup and answers. And at MIT, researchers became even more highly motivated to solve problems related to NDMA exposure. In fact, the team hopes to expand its work not only on NDMA but on related N-nitrosamines that have also been found at Superfund sites. While significant funding is being lined up for cleanup, key decisions with regard to how much cleanup should be done are pending. The MIT team aims to learn more about the biology of NDMA and related N-nitrosamines so as to contribute to informed decisions about the extent to which cleanup should be performed. In addition, MIT biological engineers aim to develop biomarkers that predict the downstream health consequences of NDMA long before disease onset. This is particularly important given that exposure to NDMA takes years to show up as cancer. The MIT team has also recruited new members who have expertise in water remediation. By bringing together a cross disciplinary team the MIT SRP has the potential to make a significant impact on public health by helping to protect people from hazardous chemicals.

mutation bars

MIT SRP Labs Collaborate on Study of NDMA Susceptibility

Congratulations to Amanda L. Armijo, Pennapa Thongararm, Bogdan I. Fedeles, Judy Yau, Jennifer E. Kay, Joshua J. Corrigan, Marisa Chancharoen, Supawadee Chawanthayatham, Leona D. Samson, Sebastian E. Carrasco, Bevin P. Engelward, James G. Fox, Robert G. Croy and John M. Essigmann on their recent publication in NAR Cancer in 2023! In this work, the authors show that there is a distinct mutation pattern when mice are exposed to N-nitrosodimethylamine, a DNA methylating agent. This pattern is strikingly similar to the pattern of mutations in tumors from patients treated with a related DNA methylating agent, temozolomide. The pattern appears to be driven largely by O6-methylguanine, a damaged base that readily mispairs with thymine to induce GC to AT mutations.

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Jessica Beard trainee

Trainee Spotlight | Jessica Beard

Congratulations to SRP trainee, Jessica Beard, and faculty member, Timothy Swager, for their publication in the Journal of Organic Chemistry! This is a pivotal paper for the MIT Superfund Research Program, both because it is tour de force in terms of the chemistry that is described, and also because it helps to inform cleanup. Importantly, it was written in response to members of the Wilmington Community who wanted to understand better whether or not N-nitrosamines are continuing to be formed at the Olin Chemical Superfund Site (located in Wilmington, MA). Being responsive to the community is key to the mission of the MIT SRP.

Jessica was also interviewed for a podcast by the Boston Museum of Science. Jessica shares her thoughts about her favorite molecule, her career aspirations, and her appreciation of the MIT Superfund Research Program. Listen to the podcast on the museum’s website website.

High Throughput Toxicity Assay

Cell survival assays are routine in many life science laboratories, yet direct measurements of cell growth are rarely performed due to the fact that the gold standard colony forming assay is slow and laborious. A novel adaptation to the traditional colony forming assay was developed by Postdoc Lizzie Ngo of the Engelward and Samson laboratories. Whereas the typical colony forming assay requires many large cell culture dishes, Dr. Ngo’s rapid assay fits into a 96-well plate format, enabling higher throughput direct testing of the ability of cells to divide. This technology was highlighted in the AAAS EurekAlertMIT News, and as an NIEHS Environmental Factor Paper of the Month. In addition, Dr. Ngo’s excellent work was highlighted in a Trainee Spotlight in the April edition of SRP e-Posted Notes.

Outreach Publication in Scientia

Program Director Prof. Bevin Engelward and RTC leader Dr. Jenny Kay collaborated with editors of the science outreach journal Scientia to produce an article about the MIT SRP and its biological research projects. The article describes the NIEHS Superfund Research Program and MIT SRP’s chemicals of interest, N-nitrosamines, probable human carcinogens that contaminate the groundwater of Wilmington, MA. Drs. Engelward and Kay provided an overview of NDMA-induced DNA damage and repair and the team’s approach to evaluating how varied DNA repair activity may impact susceptibility to adverse health outcomes following exposure. They also described several technologies developed in the Engelward laboratory for detecting DNA damage, toxicity, and mutations. This publication enabled dissemination of advancements at the MIT SRP to a broad audience.

Study Links Childhood Cancer to NDMA Exposure

The Olin Chemical Superfund Site in Wilmington, MA, contains high levels of NDMA, a probable human carcinogen that traveled nearly a mile underground, contaminating town wells that had been used by thousands of people. After the discovery of a childhood cancer cluster, the Massachusetts Department of Public Health did an epidemiological study, and the results show an association between exposure to NDMA in utero and cancer in children. The MIT Superfund Research Program is actively engaged in being responsive to the affected community by performing relevant research and developing technologies to help address the public health impacts of NDMA. Recent research performed by the MIT team points to the possibility that the AAG DNA repair enzyme is a susceptibility factor for NDMA-induced cancer.

Leventhal Prize Winners

Leventhal City Prize for Equitable Resilience

A team led by CEC Director Dr. Kathy Vandiver won the Norman B. Leventhal City Prize, a $100,000 award offered by MIT’s Leventhal Center. The objective of the ‘Malden River Works for Waterfront Equity and Resilience’ project is to create a public open space to improve opportunities for community recreation and health. It is envisioned the river will become a place where people can gather or walk, and also enjoy being out in nature where the surroundings are healthy for both the mind and body. The project work involves redesigning a city-owned parcel which is the home of the Dept. of Public Works (DPW). The most ambitious goal, however, is a social capacity-building one, with the aim of broadening civic participation for communities of color. For this purpose, a Steering Committee for the project has been created with representative leaders from Malden’s diverse immigrant groups, including people of color who are currently underrepresented in the city government. This project was featured in an MIT News article and video and in the NIEHS PEPH Newsletter.


HepaCometChip Enables SRP Research and Chemical Safety Testing

A blind spot for high throughput genotoxicity assays is the inability to detect bulky lesions on DNA that have the potential to be carcinogenic. To overcome this limitation, Drs. Lizzie Ngo and Norah Owiti from the Engelward laboratory developed new methodologies for the CometChip, a high throughput comet assay developed at MIT. By incorporating hepatocytes, the platform can detect bulky lesions that are formed as a consequence of metabolic activation. Another challenge is that bulky lesions are not easily detected using the traditional comet assay, which reveals the presence of strand breaks but not bulky lesions. By incorporating inhibitors of DNA synthesis, the new assay traps repair intermediates, effectively enabling the cell to convert undetectable bulky lesions into detectable strand breaks. The assay is currently being used for a collaboration between an environmental science and engineering project and a biomedical project, where the goal is to identify novel PAH breakdown products and to test their biological impact. The new platform can also be used to screen for chemical safety and is described in a manuscript published in Nucleic Acids Research (DOI: 10.1093/nar/gkz1077). This work was featured in Technology NetworksScience Daily, the MIT News, and as an NIEHS Paper of the Month.

Dr. Amanda Armijo

Trainee Spotlight | Dr. Amanda Armijo

Amanda Armijo, a postdoctoral fellow at MIT Professor John Essigmann’s group in the Department of Biological Engineering and Dr. James Fox’s group in the Division of Comparative Medicine, is studying the genotoxic signatures caused by environmental contaminants and how these mutations result in development of liver cancer. Specifically, her research focuses on the mutational patterns induced by the probable human carcinogen, N-nitrosodimethylamine (NDMA). NDMA is key contaminant of the Superfund site in Wilmington, MA, which for many years has contaminated the drinking water from several municipal wells.  As part of the MIT Superfund Research Program, Amanda is utilizing a high-fidelity duplex consensus sequencing (DS) method to reveal early onset genetic signatures of environmental toxicant-driven human diseases that occur later in life. Identifying these mutational processes can inform strategies for Superfund site remediation as well as clinical genetic disease early-detection, intervention and prevention.

These experiments are being performed in transgenic C57Bl/6 mice that contain a reporter gene to enable a mutational assessment and high-fidelity sequencing of the changes in the DNA triplets in the reporter gene region. To accomplish this, the transgenic mice are treated with a carcinogenic regimen of NDMA and then liver DNA is analyzed 10 weeks post-exposure (prior to development of cancerous lesions) with DS to produce high resolution mutational spectra (HRMS). DS is a highly accurate method to identify rare, unique mutations present in a heterogenous genetic milieu.  Mutational spectra patterns will also be identified in lesions that have fully developed into pathological cancer induced by NDMA. Gene-environment interactions that define inter-individual variations in sensitivity to NDMA will be identified using mutational patterns.

Amanda received her Bachelor’s degree in Microbiology, Immunology, and Molecular Genetics from UCLA. After working as a laboratory technician, she entered graduate school at UCLA in the department of Molecular and Medical Pharmacology where her work focused on the link between nucleotide metabolism and DNA replication stress responses. Her graduate work subsequently led to the preclinical development of a panel of small molecule inhibitors of deoxycytidine kinase as an anti-cancer therapeutic. Amanda successfully defended her dissertation and next attended Cornell University’s College of Veterinary Medicine, receiving a Doctor of Veterinary Medicine degree. Following completion of her DVM, Amanda completed an internship in Laboratory Animal Surgery and Medicine at Tufts University. She is happy to have found a program at MIT that combines her interests in both caring for laboratory animals and in performing important research that can positively impact human health. In her spare time, she enjoys sports, volunteering for spay/neuter clinics, cooking, and spending time with her family.

In Vitro Mutagenicity Testing

Adaptation of an Animal Mutation Model to Cell Culture Enables Rapid In Vitro Mutagenicity Testing

There is much interest in understanding the mechanisms underlying the complex patterns displayed in mutational spectra, because these spectra will help to illuminate the molecular etiology of genetic diseases, such as cancer. The lambda gpt delta C57BL/6J mouse is an extraordinarily useful model for the probing underlying mechanisms of human cancer, and the mutational spectra of dozens of environmental carcinogens have been characterized using this transgenic animal model. Responding to the need for a high-throughput cell culture model derived from this mouse, Dr. Pennapa Thongararm from the Essigmann lab used lentiviral transformation to produce mouse embryo fibroblast cell lines. She then tested the utility of these cells for studies of the kind of DNA damage (specifically, methylation damage) that is produced by N-nitrosamines, environmental carcinogens that are the focus of MIT SRP research.

Studies by Dr. Thongaram et al., recently published in Chemical Research in Toxicology, show a clear induction of methylation damage-induced mutations, with the predominant mutation being G to A. Based on the chemistry of the DNA damage that is formed by methylating agents, the team predicted that these mutations might be coming from O6-methylguanine, which can mispair efficiently with thymine. To test this possibility, the team used a chemical inhibitor of the protein that repairs O6MeG and queried the resulting mutation pattern. They saw a clear enhancement of the original signature, providing direct evidence that O6MeG is the main driver of mutagenicity from methylating agents, and calling attention to the importance of DNA repair as a key player in suppressing methylation-induced mutations. This work opens doors to studies of methylation damage-induced mutations in vivo where ongoing collaborative work is uncovering the importance of gene-environment interactions as modulators of the risk of cancer from exposure to environmental contaminants. Publication DOI: 10.1021/acs.chemrestox.9b00444