Mutagenic Effects Of Benzo Research Articles

Abstract 3088: Deciphering components of mutational signatures arising from carcinogen co-exposures: A genome-scale experimental approach

Abstract Pan-cancer analyses of tumor genomes reveal mutational signatures characteristic of particular etiologic factors. The signatures are extracted mathematically from mixed patterns typically observed by tumor sequencing. However, the components of signatures originating from complex carcinogen mixtures have not been studied in detail. Lung, head and neck and liver tumors of tobacco smokers exhibit COSMIC signature 4 marked by predominant G>N mutations, involving mainly G:C>T:A transversions with transcription strand bias, consistent with the mutagenic effects of benzo[a]pyrene (B[a]P). Additionally, A>N mutations (strand-biased A:T>T:A transversions and A:T>G:C transitions) are also prominently present, yet their origins are less understood. By using exposure-coupled clonal immortalization of human and mouse primary cells and deep sequencing, we were able to dissect ‘clean' mutational signatures of tobacco smoke carcinogens B[a]P and glycidamide (GA), a key reactive metabolite of acrylamide (ACR). Whole-genome sequencing of multiple clones derived from primary B[a]P-treated human mammary epithelial cells identified a robust mutational signature marked by strand-biased G>N mutations and increased GG>TT dinucleotides, while no apparent enrichment of A:T>T:A mutations was observed. Next, in ACR and GA-treated primary mouse embryonic fibroblasts, we established by the LC-MS/MS DNA adduct analysis that ACR exerts its mutagenic effects exclusively via GA. We then extracted from 15 treated clones the exome-scale mutational signature of GA, marked by predominant A:T>T:A transversions followed by A:T>G:C transitions and G:C>T:A transversions, all showing transcription strand bias. Similarity analysis involving known primary-cancer and experimental mutational signatures indicated that the GA mutational signature was novel. A more in-depth comparison with mutation patterns from lung adenocarcinomas of heavy smokers revealed that the GA signature, including its strand bias features, matched closely with and may thus account for the A>N mutation component of the tobacco smoking-derived signature 4. Thus, mutational signatures generated in controlled experimental settings may explain particular sub-features of cancer signatures arising from co-exposures to multiple carcinogens. Furthermore, the use of innovative in vitro systems, characterized by biological barrier bypass to mimic early steps of cell transformation, can provide revealing insights into the molecular links between mutagenesis and carcinogenesis. Funding: INCa-INSERM Plan Cancer 2015; NIH/NIEHS 1R03ES025023-01A1 Citation Format: Manuraj Pandey, Maria Zhivagui, Mona I. Churchwell, Alvin W. Ng, Liacine Bouaoun, Vincent Cahais, Martha R. Stampfer, Magali Olivier, Zdenko Herceg, Ewy Mathé, Steven G. Rozen, Frederick A. Beland, Michael Korenjak, Jiri Zavadil. Deciphering components of mutational signatures arising from carcinogen co-exposures: A genome-scale experimental approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3088.

Multifaceted Recognition of Vertebrate Rev1 by Translesion Polymerases ζ and κ

Translesion synthesis is a fundamental biological process that enables DNA replication across lesion sites to ensure timely duplication of genetic information at the cost of replication fidelity, and it is implicated in development of cancer drug resistance after chemotherapy. The eukaryotic Y-family polymerase Rev1 is an essential scaffolding protein in translesion synthesis. Its C-terminal domain (CTD), which interacts with translesion polymerase ζ through the Rev7 subunit and with polymerases κ, ι, and η in vertebrates through the Rev1-interacting region (RIR), is absolutely required for function. We report the first solution structures of the mouse Rev1 CTD and its complex with the Pol κ RIR, revealing an atypical four-helix bundle. Using yeast two-hybrid assays, we have identified a Rev7-binding surface centered at the α2-α3 loop and N-terminal half of α3 of the Rev1 CTD. Binding of the mouse Pol κ RIR to the Rev1 CTD induces folding of the disordered RIR peptide into a three-turn α-helix, with the helix stabilized by an N-terminal cap. RIR binding also induces folding of a disordered N-terminal loop of the Rev1 CTD into a β-hairpin that projects over the shallow α1-α2 surface and creates a deep hydrophobic cavity to interact with the essential FF residues juxtaposed on the same side of the RIR helix. Our combined structural and biochemical studies reveal two distinct surfaces of the Rev1 CTD that separately mediate the assembly of extension and insertion translesion polymerase complexes and provide a molecular framework for developing novel cancer therapeutics to inhibit translesion synthesis.

Nickel subsulfide is similar to potassium dichromate in protecting normal human fibroblasts from the mutagenic effects of benzo[a]pyrene diolepoxide.

The cellular response to multiple carcinogen treatment has not been extensively studied, even though the effect of individual carcinogens is, in many cases, well known. We have previously shown that potassium dichromate can protect normal human fibroblasts from the mutagenic effects of benzo[a]pyrene diolepoxide (BPDE), and that this effect may be via an oxidative stress mechanism [Tesfai et al. (1998) Mutat Res 416:159-168]. Here, we extend our previous work by showing that nickel subsulfide can produce the some effect. Normal human fibroblasts, preincubated with nickel subsulfide for 46 hr followed by a coincubation of nickel subsulfide and BPDE for 2 hr, showed a dramatic reduction in the mutant frequency of the hypoxanthine (guanine)phosphoribosyl-transferase (HPRT) gene when compared to cells treated only with BPDE. The preincubation period with nickel subsulfide was necessary to see the antagonistic effect, since it was not observed if the cells were simply incubated with both carcinogens for 2 hr. The extent of the antagonistic effect was nickel subsulfide dose-dependent and also appeared to be species-specific, since the effect was not observed when Chinese hamster fibroblasts were tested. Finally, the antagonistic effect of the nickel subsulfide was eliminated by vitamin E, suggesting that production of reactive oxygen species by the nickel may be required. This data, along with our previous work, suggest that the antagonistic effect we observe is not chromium-specific, and that it could be species-specific.

Cassia Senna Inhibits Mutagenic Activities of Benzo[a]‐pyrene, Aflatoxin B1, Shamma and Methyl Methanesulfonate

Ethanol extract of Senokot tablets (Cassia senna concentrate used as vegetable laxative), was found to be non-mutagenic while it inhibited the mutagenicity of benzo[a]pyrene, shamma, aflatoxin B1 and methyl methanesulfonate in the Ames histidine reversion assay using the Salmonella typhimurium tester strain TA98. While the Senokot extract completely inhibited the mutagenicity of promutagens (i.e. metabolic activation dependent) like benzo[a]pyrene and shamma, it reduced the mutagenic activity of the direct acting mutagen methyl methanesulfonate by only 58%. The mutagen aflatoxin B1 showed a 25-fold increase in the number of histidine revertants per plate at low concentrations (1.0-4.0 micrograms/plate) in the presence of metabolic activation system while at high concentrations (10.0-30.0 micrograms/plate) it proved to be weakly mutagenic (with a 5-fold increase in the number of histidine revertants/plate) without metabolic activation. The Senokot extract completely inhibited the mutagenic effect of low concentrations of aflatoxin B1 in the presence of metabolic activation but not that resulting from higher concentrations without metabolic activation. The results obtained with benzo[a]pyrene, shamma and aflatoxin B1 indicated that the antimutagenic effects of Senokot extract could be largely due to an interaction with the metabolic process involved in the activation of procarcinogens. However, the results obtained with methyl methanesulfonate suggested that factors in Senokot may also interact with direct mutagens to produce some antimutagenic effects. An ethanol extract of crude senna leaves found to be weakly mutagenic also inhibited (though less than Senokot) the mutagenic effect of benzo[a]pyrene suggesting that the antimutagenic principle is present in the complex plant material itself.

Xenobiotic-metabolizing enzymes and benzo[ a]pyrene metabolism in the benzo[ a]pyrene-sensitive mutant strain of Drosophila simulans

Basal levels of aryl hydrocarbon hydroxylase, epoxide hydrolase and glutathione S-transferase enzyme activities, cytochrome P-450 content and inducibility of enzymes with phenobarbital were found to be similar in the microsomes of D. simulans mutant strain 364 yv, which is sensitive to the toxic and mutagenic effects of benzo[ a]pyrene (BP), and of the wild resistant Turku strain. In contrast, increases in the rate of BP turnover per molecule of cytochrome P-450, intensity of the hemoprotein band with apparent molecular weight 56,000 and the yield of BP 7,8-dihydrodiol and 9,10-dihydrodiol occurred only in microsomes of BP-pretreated 364 yv flies but not of Turku ones. It is likely that BP induces an aberrant form of cytochrome P-450 in 364 yv flies with a rare mutation in one of the P-450 regulating genes.

Absence of mutagenic activity of benzo[a]pyrene in the soybean test system

The potential for benzo[a]pyrene to induce mutations in the soybean test system was studied. In spite of the reported high sensitivity of the soybean test system to other agents, no mutagenic effect of benzo[a]pyrene was detected even in the presence of S9 mixtures prepared from rat liver or plant tissue. Some abnormal seedling growth was observed with treatments of high concentration of the drug with rat liver S9 mixture.

Mutagenic effect of benzo[ a]pyrene in wild-type L5178Y mouse lymphoma cells

Mutagenic effect of benzo[ a]pyrene in wild-type L5178Y mouse lymphoma cells

Mutagenic effects of benzo[ a]pyrene after metabolic activation by hepatic 9000 g supernatants or intact hepatocytes

The activating capacities of isolated rat hepatocytes and 9000 g supernatant from these cells with respect to them mutagenic effect of benzo[ a]yperene on Salmonella typhimurium TA100 were investigated. No mutagenicity of benzo[ a]pyrene was found with the cell-mediated assay, unless the hepatocytes were disrupted after pre-incubation with benzo[ a]pyrene or the intracellular glutathione content was reduced. It is suggested that a retention of active metabolites and an effective detoxication may account for the absence of mutagenic response.

Detection of mutagenic polycyclic aromatic hydrocarbons in african smoked fish

The mutagenicity of six polycyclic aromatic hydrocarbons found in African smoked fish was tested using several Salmonella typhimurium strains. In the presence of fortified rat-liver post-mitochondrial fractions, mutagenic activity was observed with o-phenylenepyrene, coronene, benzo[ g,h,i]-perylene and triphenylene in the plate incorporation method, and with fluoranthene in the bacterial fluctuation test. No mutagenic effects of benzo[ b]fluoranthene towards any of the tested strains were detected.