The TGx-28.65 biomarker online application for analysis of transcriptomics data to identify DNA damage-inducing chemicals in human cell cultures.

The TGx-28.65 biomarker is a 65-gene expression profile generated from testing 28 model chemicals (13 that cause DNA damage and 15 that do not) in human TK6 cells. It is used to predict whether a chemical induces DNA damage or not. We expanded availability to the biomarker by developing the online TGx-28.65 biomarker application for predicting the DNA damage inducing (DDI) potential of suspect toxicants tested in p53-proficient human cells and assessing putative mode(s) of action (MOA). Applications like this that analyse gene expression data to predict the hazard potential of test chemicals hold great promise for risk assessment paradigms. The TGx-28.65 biomarker interfaces with an analytical tool to predict the probability that a test chemical can directly or indirectly induce DNA damage. User submitted in vitro microarray data are compared to the 28-chemical x 65-gene signature profile and the probability that the data fit the profile for a DDI or a non-DDI (NDDI) chemical is calculated. The results are displayed in the Results Table, which includes the classification probability and hyperlinks to view heatmaps, hierarchical clustering, and principal component analyses of user-input data in the context of the reference profile. The heatmaps and cluster plots, along with the corresponding text data files of fold changes in gene expression and Euclidean distances can be downloaded. Review of the test chemical data in relationship to the biomarker allows rapid identification of key gene alterations associated with DNA damage as well as chemicals in the reference set that produced a similar response. Environ. Mol. Mutagen. 58:529-535, 2017. © 2017 Wiley Periodicals, Inc.

Genetic pathways to colorectal cancer.

The colorectal cancer paradigm explains how genetic and histological changes lead normal epithelial cell to transform into pre-malignant adenomas then progress to malignant carcinomas. Using the Genetic Alterations in Cancer Knowledge System intragenic allele loss and gene mutation data from approximately 9000 colorectal tumors were compared to the model of colorectal tumor development. The distribution of mutations along the TP53 codons as a function of tumorigenesis also was analyzed. Alterations of APC, KRAS and TP53 were observed in a higher percentage of adenocarcinomas compared to adenomas (P<0.05) indicating that the alterations accumulated with malignancy. Alterations in BRAF, CTNNB, HRAS and NRAS were infrequent regardless of morphology. Differences were observed in the distribution of TP53 mutations with tumorigenesis. Mutations (single base substitutions) occurred most frequently at codons 175 and 273 in both tumor types; however, in adenocarcinomas the mutation incidence at codon 248 was approximately three times that reported in adenomas. It is proposed that the higher incidence of mutation at codon 248 is a later event in colorectal tumorigenesis that occurs as the tumors become malignant.

Genetic alterations in cancer knowledge system: analysis of gene mutations in mouse and human liver and lung tumors.

Mutational incidence and spectra for genes examined in both human and mouse lung and liver tumors were analyzed using the National Institute of Environmental Health Sciences (NIEHS) Genetic Alterations in Cancer (GAC) knowledge system. GAC is a publicly available, web-based system for evaluating data obtained from peer-reviewed studies of genetic changes in tumors associated with exposure to chemical, physical, or biological agents, as well as spontaneous tumors. In mice, mutations in Kras2 and Hras-1 were the most common events reported for lung and liver tumors, respectively, whether chemically induced or spontaneous. There was a significant difference in Kras2 mutation incidence for spontaneous versus induced mouse lung tumors and in Hras-1 mutation incidence and spectrum for spontaneous versus induced mouse liver tumors. The major gene changes reported for human lung and liver tumors were in KRAS2 (lung only) and TP53. The KRAS2 mutation incidence was similar for spontaneous and asbestos-induced human lung tumors, while the TP53 mutation incidence differed significantly. Aflatoxin B1, hepatitis B virus, hepatitis C virus, and vinyl chloride all caused TP53 mutations in human liver tumors, but the mutation spectrum for each agent differed. The incidence of KRAS2 mutations in human compared to mouse lung tumors differed significantly, as did the incidence of Hras and p53 gene mutations in human compared to mouse liver tumors. Differences observed in the mutation spectra for agent-induced compared to spontaneous tumors and similarities in spectra for structurally similar agents support the concept that mutation spectra can serve as a "fingerprint" of exposure based on chemical structure.

A survey of EPA/OPP and open literature on selected pesticide chemicals. III. Mutagenicity and carcinogenicity of benomyl and carbendazim.

The known aneuploidogens, benomyl and its metabolite, carbendazim (methyl 2-benzimidazole carbamate (MBC)), were selected for the third in a series of ongoing projects with selected pesticides. Mutagenicity and carcinogenicity data submitted to the US Environmental Protection Agency's (US EPA's) Office of Pesticide Programs (OPP) as part of the registration process are examined along with data from the open literature. Mutagenicity and carcinogenicity profiles are developed to provide a complete overview and to determine whether an association can be made between benomyl- and MBC-induced mouse liver tumors and aneuploidy. Since aneuploidogens are considered to indirectly affect DNA, the framework adopted by the Agency for evaluating any mode of action (MOA) for carcinogenesis is applied to the benomyl/MBC data. Both agents displayed consistent, positive results for aneuploidy induction but mostly negative results for gene mutations. Non-linear dose responses were seen both in vitro and in vivo for aneuploidy endpoints. No evidence was found suggesting that an alternative MOA other than aneuploidy may be operative. The data show that by 14 days of benomyl treatment, events associated with liver toxicity appear to set in motion the sequence of actions that leads to neoplasms. Genetic changes (as indicated by spindle impairment leading to missegregation of chromosomes, micronucleus induction and subsequent aneuploidy in bone marrow cells) can commence within 1-24h after dosing, well within the time frame for early key events. Critical steps associated with frank tumor formation in the mouse liver include hepatotoxicity, increased liver weights, cell proliferation, hypertrophy, and other steps involving hepatocellular alteration and eventual progression to neoplasms. The analysis, however, reveals weaknesses in the data base for both agents (i.e. no studies on mouse tubulin binding, no in vivo assays of aneuploidy on the target tissue (liver), and no clear data on cell proliferation relative to dose response and time dependency). The deficiencies in defining the MOA for benomyl/MBC introduce uncertainties into the analysis; consequently, benomyl/MBC induction of aneuploidy cannot be definitively linked to mouse liver carcinogenicity at this time.