Kinetics of gamma-H2AX focus formation upon treatment of cells with UV light and alkylating agents

Histone H2AX is rapidly phosphorylated in response to DNA double-strand breaks (DSBs) induced by ionizing radiation (IR). Here we show that DNA damage induced by alkylating agents [methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)] and ultraviolet light (UV-C) leads to a dose and time dependent accumulation of phosphorylated H2AX (gamma-H2AX). Time course experiments revealed that the number of gamma-H2AX foci reached peak levels 8 hr after MMS or MNNG treatment and declined to almost control values within 24 hr after exposure. Upon UV-C treatment, a biphasic response was observed with a maximum 12 hr after treatment. In 43-3B cells deficient in nucleotide excision repair (NER) the number of gamma-H2AX foci increased steadily. gamma-H2AX foci were preferentially formed in BrdU labeled cells. In proliferation compromised cells, the gamma-H2AX level was significantly reduced, indicating that most of the gamma-H2AX foci induced by UV-C and alkylating agent treatments were replication dependent. The data are in line with the view that DNA damage induced by UV-C light and simple alkylating agents, leads to the formation of DSBs during DNA replication giving rise to H2AX phosphorylation. In replicating NER defective cells, DSBs accumulate due to nonrepaired primary DNA lesions that produce a high level of DSBs during replication. The data support that gamma-H2AX foci are a useful marker of DSBs that are induced by S-phase dependent genotoxins during replication.     

Comparative DNA adduct formation and induction of colonic aberrant crypt foci in mice exposed to 2‐amino‐9H‐pyrido[2,3‐b]indole, 2‐amino‐3,4‐dimethylimidazo[4,5‐f]quinoline,and azoxymethane

Considerable evidence suggests that environmental factors, including diet and cigarette smoke, are involved in the pathogenesis of colon cancer. Carcinogenic nitroso compounds (NOC), such as N‐nitrosodimethylamine (NDMA), are present in tobacco and processed red meat, and NOC have been implicated in colon cancer. Azoxymethane (AOM), commonly used for experimental colon carcinogenesis, is an isomer of NDMA, and it produces the same DNA adducts as does NDMA. Heterocyclic aromatic amines (HAAs) formed during the combustion of tobacco and high‐temperature cooking of meats are also associated with an elevated risk of colon cancer. The most abundant carcinogenic HAA formed in tobacco smoke is 2‐amino‐9H‐pyrido[2,3‐b]indole (AαC), whereas 2‐amino‐3,4‐dimethylimidazo[4,5‐f]quinoline (MeIQ) is the most potent carcinogenic HAA formed during the cooking of meat and fish. However, the comparative tumor‐initiating potential of AαC, MeIQ, and AOM is unknown. In this report, we evaluate the formation of DNA adducts as a measure of genotoxicity, and the induction of colonic aberrant crypt foci (ACF) and dysplastic ACF, as an early measure of carcinogenic potency of these compounds in the colon of male A/J mice. Both AαC and AOM induced a greater number of DNA adducts than MeIQ in the liver and colon. AOM induced a greater number of ACF and dysplastic ACF than either AαC or MeIQ. Conversely, based on adduct levels, MeIQ‐DNA adducts were more potent than AαC‐ and AOM‐DNA adducts at inducing ACF. Long‐term feeding studies are required to relate levels of DNA adducts, induction of ACF, and colon cancer by these colon genotoxicants. Environ. Mol. Mutagen. 57:125–136, 2016. © 2016 Wiley Periodicals, Inc.