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Exposure to DNA damaging agents can lead to mutations that cause cancer. The liver is particularly vulnerable because it contains high levels of Cytochrome P450 enzymes that can convert xenobiotics into DNA reactive metabolites that form potentially carcinogenic bulky DNA adducts. As such, current requirements for preclinical testing include in vivo testing for DNA damage in the liver, which often requires many animals. Given that efforts are underway in many countries to reduce or eliminate the use of animals in research, there is a critical need for fast and robust in vitro tests to discern whether xenobiotics or potential pharmaceutical agents can damage the hepatocyte genome. One possible approach is to leverage the alkaline comet assay, which is used to assess genotoxicity based on the ability of damaged DNA to become free to migrate toward the anode during electrophoresis. The comet assay, however, has several limitations. The assay is (i) slow and (ii) vulnerable to experimental noise, (iii) it is difficult to detect bulky DNA adducts since they do not directly affect DNA migration, and (iv) cell types typically used do not have robust metabolic capacity. To address some of these concerns, we have developed the "HepaCometChip" (a.k.a. the HepaRG CometChip), wherein metabolically competent cells are incorporated into a higher throughput CometChip platform. Repair trapping is used to increase sensitivity for bulky lesions: undetectable bulky lesions are converted into repair intermediates (specifically, single-strand breaks) that can be detected with the assay. Here, we describe a protocol for performing the HepaCometChip assay that includes handling and dosing of HepaRG cells and performing the CometChip assay. With its higher throughput, ability to capture metabolic activation, and sensitivity to bulky lesions, the HepaCometChip offers a potential alternative to the use of animals for genotoxicity testing. © 2022 The Authors. Current Protocols published by Wi

© 2020 Wiley Periodicals LLC. Inflammation is a major risk factor for many types of cancer, including colorectal. There are two fundamentally different mechanisms by which inflammation can contribute to carcinogenesis. First, reactive oxygen and nitrogen species (RONS) can damage DNA to cause mutations that initiate cancer. Second, inflammatory cytokines and chemokines promote proliferation, migration, and invasion. Although it is known that inflammation-associated RONS can be mutagenic, the extent to which they induce mutations in intestinal stem cells has been little explored. Furthermore, it is now widely accepted that cancer is caused by successive rounds of clonal expansion with associated de novo mutations that further promote tumor development. As such, we aimed to understand the extent to which inflammation promotes clonal expansion in normal and tumor tissue. Using an engineered mouse model that is prone to cancer and within which mutant cells fluoresce, here we have explored the impact of inflammation on de novo mutagenesis and clonal expansion in normal and tumor tissue. While inflammation is strongly associated with susceptibility to cancer and a concomitant increase in the overall proportion of mutant cells in the tissue, we did not observe an increase in mutations in normal adjacent tissue. These results are consistent with opportunities for de novo mutations and clonal expansion during tumor growth, and they suggest protective mechanisms that suppress the risk of inflammation-induced accumulation of mutant cells in normal tissue.

Exposure to N-nitrosodimethylamine (NDMA) has recently been linked to a childhood cancer cluster in Wilmington, MA, which is home to the Olin Chemical Superfund Site. When it was discovered in the 1990's that 22 children in a town of under 22,000 people got cancer, the community took action and pressed for an investigation into the possibility that chemicals from the Olin Chemical site had contaminated their water. This led to the eventual discovery that NDMA was present in the town water supply. NDMA has long been known for its potent carcinogenicity in animal models, and so the community pointed to NDMA as a possible cause. This led to an investigation by the Massachusetts Department of Public Health, which, in 2021, released its findings showing an association between NDMA exposure in utero and childhood cancer. The mission of the NIEHS Superfund Research Program is to protect human health from hazardous substances. In 2017, in response to community concerns, a team at MIT created the MIT Superfund Research Program Center with a focus on research related to NDMA. Just 1 week prior to the release of the Department of Public Health study, the MIT Superfund Research Program Center published a manuscript in Cell Reports that identifies the Alkyladenine DNA glycosylase (AAG) as a possible genetic susceptibility factor. This commentary provides an author's perspective on the context and implications of this and related research.

Exposure to N-nitrosodimethylamine (NDMA) has recently been linked to a childhood cancer cluster in Wilmington, MA, which is home to the Olin Chemical Superfund Site. When it was discovered in the 1990's that 22 children in a town of under 22,000 people got cancer, the community took action and pressed for an investigation into the possibility that chemicals from the Olin Chemical site had contaminated their water. This led to the eventual discovery that NDMA was present in the town water supply. NDMA has long been known for its potent carcinogenicity in animal models, and so the community pointed to NDMA as a possible cause. This led to an investigation by the Massachusetts Department of Public Health, which, in 2021, released its findings showing an association between NDMA exposure in utero and childhood cancer. The mission of the NIEHS Superfund Research Program is to protect human health from hazardous substances. In 2017, in response to community concerns, a team at MIT created the MIT Superfund Research Program Center with a focus on research related to NDMA. Just 1 week prior to the release of the Department of Public Health study, the MIT Superfund Research Program Center published a manuscript in Cell Reports that identifies the Alkyladenine DNA glycosylase (AAG) as a possible genetic susceptibility factor. This commentary provides an author's perspective on the context and implications of this and related research.