Carcinogenic Potential Of Chemicals Research Articles

Deciphering exogenous chemical carcinogenicity through interpretable deep learning: A novel approach for evaluating atmospheric pollutant hazards

Cancer remains a significant global health concern, with millions of deaths attributed to it annually. Environmental pollutants play a pivotal role in cancer etiology and contribute to the growing prevalence of this disease. The carcinogenic assessment of these pollutants is crucial for chemical health evaluation and environmental risk assessments. Traditional experimental methods are expensive and time-consuming, prompting the development of alternative approaches such as in silico methods. In this regard, deep learning (DL) has shown potential but lacks optimal performance and interpretability. This study introduces an interpretable DL model called CarcGC for chemical carcinogenicity prediction, utilizing a graph convolutional neural network (GCN) that employs molecular structural graphs as inputs. Compared to existing models, CarcGC demonstrated enhanced performance, with the area under the receiver operating characteristic curve (AUCROC) reaching 0.808 on the test set. Due to air pollution is closely related to the incidence of lung cancers, we applied the CarcGC to predict the potential carcinogenicity of chemicals listed in the United States Environmental Protection Agency's Hazardous Air Pollutants (HAPs) inventory, offering a foundation for environmental carcinogenicity screening. This study highlights the potential of artificially intelligent methods in carcinogenicity prediction and underscores the value of CarcGC interpretability in revealing the structural basis and molecular mechanisms underlying chemical carcinogenicity.

Changes in miR-124-1, miR-212, miR-132, miR-134, and miR-155 Expression Patterns after 7,12-Dimethylbenz(a)anthracene Treatment in CBA/Ca Mice.

Specific gene and miRNA expression patterns are potential early biomarkers of harmful environmental carcinogen exposures. The aim of our research was to develop an assay panel by using several miRNAs for the rapid screening of potential carcinogens. The expression changes of miR-124-1, miR-212, miR-132, miR-134, and miR-155 were examined in the spleen, liver, and kidneys of CBA/Ca mice, following the 20 mg/bwkg intraperitoneal 7,12-dimethylbenz(a)anthracene (DMBA) treatment. After 24 h RNA was isolated, the miRNA expressions were analyzed by a real-time polymerase chain reaction and compared to a non-treated control. DMBA induced significant changes in the expression of miR-134, miR-132, and miR-124-1 in all examined organs in female mice. Thus, miR-134, miR-132, and miR-124-1 were found to be suitable biomarkers for the rapid screening of potential chemical carcinogens and presumably to monitor the protective effects of chemopreventive agents.

Deep neural network for the determination of transformed foci in Bhas 42 cell transformation assay

Bhas 42 cell transformation assay (CTA) has been used to estimate the carcinogenic potential of chemicals by exposing Bhas 42 cells to carcinogenic stimuli to form colonies, referred to as transformed foci, on the confluent monolayer. Transformed foci are classified and quantified by trained experts using morphological criteria. Although the assay has been certified by international validation studies and issued as a guidance document by OECD, this classification process is laborious, time consuming, and subjective. We propose using deep neural network to classify foci more rapidly and objectively. To obtain datasets, Bhas 42 CTA was conducted with a potent tumor promotor, 12-O-tetradecanoylphorbol-13-acetate, and focus images were classified by experts (1405 images in total). The labeled focus images were augmented with random image processing and used to train a convolutional neural network (CNN). The trained CNN exhibited an area under the curve score of 0.95 on a test dataset significantly outperforming conventional classifiers by beginners of focus judgment. The generalization performance of unknown chemicals was assessed by applying CNN to other tumor promotors exhibiting an area under the curve score of 0.87. The CNN-based approach could support the assay for carcinogenicity as a fundamental tool in focus scoring.

Dynamically monitoring cellular γ-H2AX reveals the potential of carcinogenicity evaluation for genotoxic compounds.

Amongst all toxicological endpoints, carcinogenicity might pose the greatest concern. Genetic damage has been considered an important underlying mechanism for the carcinogenicity of chemical substances. The demand for in vitro genotoxic tests as alternative approaches is growing rapidly with the implementation of new regulations for compounds. However, currently available in vitro genotoxicity tests are often limited by relatively high false positive rates. Moreover, few studies have explored carcinogenicity potential by in vitro genotoxicity testing due to the shortage of suitable toxicological biomarkers to link gene damage with cancer risk. γ-H2AX is a recently acknowledged attractive endpoint (biomarker) for evaluating DNA damage and can simultaneously reflect the DNA damage response and repair of cells. We previously reported an ultrasensitive and reliable method, namely stable-isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS), for detecting cellular γ-H2AX and evaluating genotoxic chemicals. More importantly, our method can dynamically monitor the specific processes of genotoxic compounds affecting DNA damage and repair reflected by the amount of γ-H2AX. To clarify the possibility of using this method to assess the potential carcinogenicity of genotoxic chemicals, we applied it to a set of 69 model compounds recommended by the European Center for the Validation of Alternative Methods (ECVAM), with already-characterized genotoxic potential. Compared to conventional in vitro genotoxicity assays, including the Ames test, the γ-H2AX assay by MS has high accuracy (94-96%) due to high sensitivity and specificity (88% and 100%, respectively). The dynamic profiles of model compounds after exposure in HepG2 cells were explored, and a mathematical approach was employed to simulate and quantitatively model the DNA repair kinetics of genotoxic carcinogens (GCs) based on γ-H2AX time-effect curves up to 8h. Two crucial parameters, i.e., k (rate of γ-H2AX decay) and t50 (time required for γ-H2AX from maximum decrease to half) estimated by the least squares method, were achieved. An open web server to help researchers calculate these two key parameters and profile simulated curves of the tested compound is available online ( http://ccb1.bmi.ac.cn:81/shiny-server/sample-apps/prediction1/ ). We detected a positive association between carcinogenic levels and k and t50 values of γ-H2AX in tested GCs, validating the potential of using this MS-based γ-H2AX in vitro assay to help preliminarily evaluate carcinogenicity and assess genotoxicity. This approach may be used alone or integrated into an existing battery of in vitro genetic toxicity tests.

Evaluation of potential carcinogenicity of organic chemicals in synthetic turf crumb rubber

Currently, there are >11,000 synthetic turf athletic fields in the United States and >13,000 in Europe. Concerns have been raised about exposure to carcinogenic chemicals resulting from contact with synthetic turf fields, particularly the infill material (“crumb rubber”), which is commonly fabricated from recycled tires. However, exposure data are scant, and the limited existing exposure studies have focused on a small subset of crumb rubber components. Our objective was to evaluate the carcinogenic potential of a broad range of chemical components of crumb rubber infill using computational toxicology and regulatory agency classifications from the United States Environmental Protection Agency (US EPA) and European Chemicals Agency (ECHA) to inform future exposure studies and risk analyses. Through a literature review, we identified 306 chemical constituents of crumb rubber infill from 20 publications. Utilizing ADMET Predictor™, a computational program to predict carcinogenicity and genotoxicity, 197 of the identified 306 chemicals met our a priori carcinogenicity criteria. Of these, 52 chemicals were also classified as known, presumed or suspected carcinogens by the US EPA and ECHA. Of the remaining 109 chemicals which were not predicted to be carcinogenic by our computational toxicology analysis, only 6 chemicals were classified as presumed or suspected human carcinogens by US EPA or ECHA. Importantly, the majority of crumb rubber constituents were not listed in the US EPA (n = 207) and ECHA (n = 262) databases, likely due to an absence of evaluation or insufficient information for a reliable carcinogenicity classification. By employing a cancer hazard scoring system to the chemicals which were predicted and classified by the computational analysis and government databases, several high priority carcinogens were identified, including benzene, benzidine, benzo(a)pyrene, trichloroethylene and vinyl chloride. Our findings demonstrate that computational toxicology assessment in conjunction with government classifications can be used to prioritize hazardous chemicals for future exposure monitoring studies for users of synthetic turf fields. This approach could be extended to other compounds or toxicity endpoints.

Making Sense of Carcinogens: A New Method for Navigating Mechanistic Data.

There’s no shortage of scientific studies that link specific environmental chemicals to human cancer. Yet until recently there was no broadly accepted method to systematically organize and evaluate mechanistic data on human carcinogens, data that are key to assessing the potential carcinogenicity of unstudied chemicals. In a review published this month in EHP, researchers apply a new method to create order among a sprawling body of mechanistic data, based on 10 key characteristics of human carcinogens. 1 As part of two 2012 workshops organized by the International Agency for Research on Cancer (IARC), several of the authors evaluated about 100 substances classified by IARC as Group 1 carcinogens, meaning they definitely cause human cancer. 2 Ultimately the experts identified 10 key characteristics shared among carcinogens: the ability to 1) act as an electrophile (i.e., metabolic activation), 2) induce DNA damage and/or mutation (genotoxicity), 3) alter DNA repair or cause genomic instability, 4) induce epigenetic changes, 5) induce oxidative stress, 6) induce chronic inflammation, 7) suppress the immune system, 8) modulate receptor-mediated effects, 9) cause cell immortalization, and 10) alter a cell’s proliferation, death, or nutrient supply. Any given carcinogen will exhibit at least one of these characteristics. 1 This flow chart shows how 1,841 mechanistic studies on benzene were assessed and categorized. (“Section 4” refers to the section of IARC Monographs where mechanistic data are discussed). For the sake of illustration, the authors indicated ... Team members, along with colleagues at IARC and the U.S. Environmental Protection Agency, used the 10 characteristics as a framework for evaluating mechanistic data gathered for benzene and polychlorinated biphenyls (PCBs) through a systematic literature review. They focused on whether exposure to the agent induced end points associated with one or more of the key characteristics. The results were subsequently sorted by population (human, animal, or cell) or by end point. More than 1,800 mechanistic studies exist for benzene, and almost 3,900 exist for PCBs. Applying the method yielded bodies of science supporting benzene as acting via 8 of the 10 mechanisms to cause cancer, and PCBs acting by as many as 7. It also indicated that the more volatile less-chlorinated PCBs act similarly to benzene via metabolic activation, whereas dioxin-like PCBs act primarily through receptor-mediated actions. 3 The method has been used elsewhere to yield mechanistic portraits for the pesticides malathion (associated with 5 of the key characteristics), 4 DDT (associated with 3 characteristics), 5 and diazinon and glyphosate (each associated with 2 characteristics). 4 The 10 characteristics “proved an incredibly useful tool for dividing up several thousand scientific papers into manageable amounts of information for people to review and opine on,” says first author Martyn Smith, a professor of toxicology at the University of California, Berkeley. For many chemicals the systematic review process uncovered areas where no research had yet been conducted, which could direct future studies. For example, “information about epigenetic changes is especially lacking, but that literature is starting to build,” says Smith. Other groups are working on ways to improve the systematic literature searches using the 10 characteristics. “We need new bioinformatic tools that use machine learning to search databases more efficiently,” Smith says. “This ground-breaking work shows us the way forward to identify and characterize key mechanistic processes across chemicals when using systematic reviews,” says Elaine Faustman, director of the Institute for Risk Analysis and Risk Communication at the University of Washington. “Until now, a clear scientific framework has been missing to allow for both identification and consistent evaluation.” Faustman was not involved in the work. Overall, the 10 characteristics offer a consistent, objective, and systematic way to identify and evaluate human carcinogens. These insights into understanding how cancer develops will help with carcinogen hazard identification and risk assessment, and could eventually guide cancer prevention and early detection.

Abstract P5-06-04: Synergistically elevated hallmarks of cancer induced in non-malignant human breast cells by mixtures of environmental chemicals

Abstract BACKGROUND: Estrogenic overexposure is a well-accepted risk factor in the etiology of breast cancer. Synthetic chemicals that simulate natural estrogens, i.e., xenoestrogens (XEs), occur widely in the environment entering the human body through multiple routes. Consequently, mixtures of XEs are known to circulate in the blood at any given time. Current protocols to evaluate chemical safety rely primarily on testing chemicals individually, ignoring the fact that the general population is exposed daily to mixtures of XEs. We asked whether mixtures of low dose XEs would intensify the cellular effects of individual XEs at the same concentrations. METHODS: We exposed non-malignant breast epithelial cell cultures (HRBECs), obtained from high-risk human donors by random periareolar fine needle aspiration (RPFNA), to the high volume chemicals - bisphenol-A (BPA), methylparaben (MP) and perflourooctanoic acid (PFOA). Cells were exposed to individual XEs or mixtures of all three at a range of concentrations encompassing environmentally relevant levels. Breast cancer associated phenotypes were quantitated as test endpoints using fluorescence-activated cell sorting (FACS) and/or Western blotting. The endpoints included: total and phosphorylated estrogen receptor (ER)α, ERβ, S-phase fraction, and 4-hydoxytamoxifen (OHT) induced apoptotic fraction. We fit a log-linear dose-response model to the data generated from 3 doses for each chemical, individually and as a mixture. Evidence of synergism was tested by comparing the observed data for mixtures with that predicted by an additive-in-dose model for single doses. RESULTS: Our data demonstrates that the degree of perturbation induced by the chemical mixture tested here is not merely an additive effect of each chemical; instead it reflects considerable synergism in the mode of action. In 3/3 SNP-authenticated non-malignant HRBEC cell lines exposed to the chemical mixture at the lowest test dose, total and phosphorylated ERα levels were consistently elevated. Concurrently, ERβ level was reduced. A dose-dependent increase was observed in the S-phase fraction, together with a marked reduction of the OHT-induced apoptotic fraction in HRBECs exposed to BPA, MP, and PFOA individually (p<0.001 for each exposure). A significantly greater effect, or synergism, was detected for these endpoints upon exposure to a mixture of the chemicals at the same concentrations (p<0.001 for the combination vs. the predicted sum of individual doses). Primary HRBEC cultures from 7 additional human volunteers displayed a similar pattern of response. CONCLUSION: Routine tests conducted to assess the carcinogenic potential of chemicals overlook possible synergism among different chemicals. We show that the combined effects of low doses of various chemicals can be much stronger than predicted by addition of their effects as single agents. Despite being cost, time, and labor-intensive, current testing of individual chemicals fails to inform regulators about the safe dose range for human exposure. It is thus imperative to expand present methods to include combinatorial approaches pertaining to test chemicals, endpoints, and target cells. Citation Format: Dairkee SH, Luciani-Torres G, Moore DH, Goodson WH. Synergistically elevated hallmarks of cancer induced in non-malignant human breast cells by mixtures of environmental chemicals. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-06-04.

Transformation Assay in Bhas 42 Cells: A Model Using Initiated Cells to Study Mechanisms of Carcinogenesis and Predict Carcinogenic Potential of Chemicals

Transformation assays using cultured cells have been applied to the study of carcinogenesis. Although various cell systems exist, few cell types such as BALB/c 3T3 subclones and Syrian hamster embryo cells have been used to study chemically induced two-stage carcinogenesis. Bhas 42 cells were established as a clone by the transfection with the v-Ha-ras gene into mouse BALB/c 3T3 A31-1-1 cells and their subsequent selection based on their sensitivity to 12-O-tetradecanoylphorbol-13-acetate. Using Bhas 42 cells, transformed foci were induced by the treatment with nongenotoxic carcinogens, most of which act as tumor promoters. Therefore, Bhas 42 cells were considered to be a model of initiated cells. Subsequently, not only nongenotoxic carcinogens but also genotoxic carcinogens, most of which act as tumor initiators, were found to induce transformed foci by the modification of the protocol. Furthermore, transformation of Bhas 42 cells was induced by the transfection with genes of oncogenic potential. We interpret this high sensitivity of Bhas 42 cells to various types of carcinogenic stimuli to be related to the multistage model of carcinogenesis, as the transfection of v-Ha-ras gene further advances the parental BALB/c 3T3 A31-1-1 cells toward higher transforming potential. Thus, we propose that Bhas 42 cells are a novel and sensitive cell line for the analysis of carcinogenesis and can be used for the detection of not only carcinogenic substances but also gene alterations related to oncogenesis. This review will address characteristics of Bhas 42 cells, the transformation assay protocol, validation studies, and the various chemicals tested in this assay.

Gene expression analysis in the lung of the rasH2 transgenic mouse at week 4 prior to induction of malignant tumor formation by urethane and N-methylolacrylamide.

The rasH2 transgenic (Tg) mice are susceptible to genotoxic and some non-genotoxic carcinogens. In carcinogenicity studies carried out using rasH2 Tg mice, the carcinogenic potential of chemicals are evaluated over a 26-week experimental period. In the present study, we examined the comprehensive gene expressions in the lungs of Tg and non-Tg mice prior to the induction of malignant tumors. Urethane (UR), a mutagenic carcinogen, was administered for 4 weeks, and thereafter withdrawn for 22 weeks. N-methylolacrylamide (NMA), a non-mutagenic carcinogen, was administered for 26 weeks. At week 4, gene expression analysis of non-neoplastic part of the lungs demonstrated changes in the expressions of the cell-cycle and inflammation related genes following UR and NMA treatment, respectively, in both the Tg and non-Tg mice. The gene expressions of epireguline, aurora kinase B, and cyclin B1 increased in the UR-treated Tg mice. We also found an increase in the plasma carcinoembryonic antigen level in the UR-treated Tg mice. Although UR treatment induced the formation of adenomas or adenocarcinomas in the lungs in all mice, earlier induction was apparent in the Tg mice. NMA treatment was found to induce the formation of adenomas and adenocarcinomas at week 26 in the Tg mice, but not in the non-Tg mice, and no expressions of specific genes were apparent in either genotype of mice. Our results indicate that analysis of cancer-related gene expressions in the lungs and plasma biomarkers at week 4 in rasH2 Tg mice could be a screening tool for carcinogenicity, especially of mutagenic carcinogens.

Assessment of Anticarcinogenic Potential of Vitex trifolia and Triticum aestivum Linn by In Vitro Rat Liver Microsomal Degranulation.

Objective:The main objective of this preliminary study is to confirm the synergistic anticarcinogenic potential of Vitex trifolia and Triticum aestivum ethanolic extracts.Materials and Methods:Rat hepatic microsomal degranulation is a short - term technique that has been used for the detection of potential chemical carcinogens, in vitro. The present study has been carried out to study the inhibition of ribosome- membrane disruption against 3, 8-Diamino-5-ethyl-6-pheylphenanthridinium bromide (EB), as the degranulating agent, by measuring the RNA/protein ratios of microsomal membranes in the presence or absence of V.trifolia and T. aestivum extracts. These two extracts were further evaluated for cytotoxic effect in HCT 116 and A549 cell lines.Results:V. trifolia and T. aestivum protects hepatic microsomes against the degranulatory attack by the carcinogen EB showed a significant reduction in the proliferation of the HCT 116 and A549 cancer cell lines.Conclusion:The ethanolic extracts of the plants, V. trifolia and T. aestivum individually possessed anti-degranulatory potential. Importantly they act synergistically, possess appreciable anticarcinogenic properties, based on their ability to inhibit EB induced liver microsomal degranulation. Further these extracts inhibit cell proliferation of cancer cell lines.

Performance of Comet and Micronucleus Assays in Metabolic Competent HepaRG Cells to Predict In Vivo Genotoxicity

Genetic toxicity information is critical for the safety assessment of all xenobiotics. In the absence of carcinogenicity data, genetic toxicity studies may be used to draw conclusions about the carcinogenicity potential of chemicals. However, current in vitro assays have many limitations as they produce a high rate of irrelevant positive data and possible false negative data due to the weakness of the in vitro models used. Based on the knowledge that the majority of human genotoxic carcinogens require metabolic activation to become genotoxic, it is necessary to develop in vitro cell models that mimic human liver metabolism to replace the use of liver S9 fraction, which, though helpful for predicting the potential carcinogenicity of chemicals in rodents, is questionable in humans. We therefore investigate whether the recently described human hepatoma HepaRG cells, which express the major characteristics of liver functions similarly to primary human hepatocytes, could be a suitable model for human genotoxicity assessment. We determine the performance of comet and micronucleus assays in HepaRG cells to predict in vivo genotoxins based on the list of compounds published by European Centre for the Validation of Alternative Methods (ECVAM). Twenty compounds were tested in HepaRG cells with comet and micronucleus assays over a 24-h period. The specificity, the sensitivity, and the accuracy of the two tests were determined. We found that the comet assay had higher specificity (100%) than the micronucleus (MN) test (80%), whereas the latter was far more sensitive (73%) than the former (44%), resulting nonetheless in an accuracy of 72% for the comet assay and 75% for the MN test. Taken together, our data suggest that the HepaRG cell line can be of use in genetic toxicology and that efforts to develop competent human liver cell models should be increased.

IARC Classes 1 and 2 carcinogens are successfully identified by an alternative strategy that detects DNA-reactivity and cell transformation ability of chemicals

For decades, traditional toxicology has been the ultimate source of information on the carcinogenic potential of chemicals; however with increasing demand on regulation of chemicals and decreasing resources for testing, opportunities to accept “alternative” approaches have dramatically expanded. The need for tools able to identify carcinogens in shorter times and at a lower cost in terms of animal lives and money is still an open issue, and the present strategies and regulations for carcinogenicity pre-screening do not adequately protect human health. In previous papers, we have proposed an integrated in vitro/in silico strategy that detects DNA-reactivity and tissue disorganization/disruption by chemicals, and we have shown that the combination of Salmonella and Structural Alerts for the DNA-reactive carcinogens, and in vitro cell transformation assays for nongenotoxic carcinogens permits the identification of a very large proportion (up to 95%) of rodent carcinogens, while having a considerable specificity with the rodent noncarcinogens. In the present paper we expand the previous investigation and show that this alternative strategy identifies correctly IARC Classes 1 and 2 carcinogens. If implemented, this alternative strategy can contribute to improve the protection of human health while decreasing the use of animals.

Dysregulation of pathways involved in the processing of cancer and microenvironment information in MCA + TPA transformed C3H/10T1/2 cells

The two-stage cell transformation assay is an in vitro model cell culture system to identify the ability of chemicals to act as initiators or promoters of cell transformation and also to study the cellular and molecular mechanisms of chemically induced morphological and neoplastic cell transformation. The global gene expression profiles of 3-methylcholanthrene (MCA) + 12-O-tetradecanoylphorbol-13-acetate (TPA)-transformed C3H/10T1/2 cells are not known. Therefore, we have investigated the global transcriptional profile of MCA + TPA-transformed C3H10T1/2 cells using an 8 × 60 k probe microarray. The study revealed a differential regulation of pathways and gene expressions. Multifold dysregulation was seen in pathways of cancer, phagosomal activity, and tumor cell microenvironment information processing systems, notably the neuroactive ligand-receptor interaction, actin cytoskeleton regulation, tight junction, axon guidance, and cell adhesion molecules. The genes FGF1, EIF4E1B, MAGI1, and GRIA3 showed upregulation; these encoded the pluripotent fibroblast growth factor, the translation initiation factor, the tight junction scaffolding protein, and the antiapoptotic as well as the enhancer of proliferation and migration, respectively. The genes CXCL7/CXCL5/CXCL12, H2DMB1, and HSPA1A showed downregulation; these encoded the chemotactic agent protein, the protein involved in MHC class II antigen processing/presentation or participating in cell adhesion/phagosomal activity/autoimmune disorder, and the chaperone protein stabilizing the existing as well as newly translated cytosolic/organelle proteins against aggregation, respectively. By loss or gain of function, these dysregulated genes apparently seem to reprogram cells for apoptosis or proliferation and support their transformation into the tumor cell phenotype. The observed molecular changes can be seen as molecular signatures of transformed cells and can be of use as objective evidences to C3H/10T1/2 cell transformation assay in investigations on the carcinogenic potential of chemicals and their mechanism of actions using in vitro carcinogenesis method.

Abstract 23: Functional DNA repair tests and their perspectives in molecular epidemiology (experience with subjects exposed to carcinogens and in cancer patients).

Abstract DNA repair is crucial in maintaining universal genomic stability and preserving cellular functions. Defects in the DNA repair increase vulnerability to endogenous and exogenous DNA-damaging agents, consequent accumulation of mutations in the genome leading to development of several disorders. Individuals with defective DNA repair are at severely increased risk of developing cancer and other diseases. A number of factors are hypothesized to influence individual DNA repair capacity. Measurement of DNA repair capacity, through functional assays, therefore, represents an integrated marker that incorporates the effects of epigenetic factors, genetic polymorphisms, gene expression, stability of gene product, effect of inhibitors/stimulators, environmental factors and lifestyle factors. Functional DNA repair assays reflect the capacity of an organism to cope with a chronic exposure to numerous environmental and dietary genotoxicants and may be used as predictive markers in cancer therapy. We, recently, developed functional assays to measure nucleotide excision (NER) and base excision (BER) repair capacities in human tissues. Our initial data showed that in healthy subjects, occupationally exposed to potential chemical carcinogens, BER capacity inversely correlated with the levels of DNA damage and showed an increase with increased exposure. In contrast, both BER and NER capacities were significantly lower and DNA damage higher in incident colorectal cancer patients than in matched healthy control subjects. In colorectal cancer patients, interestingly, the DNA repair capacity increased to the levels observed in healthy control subjects following the completion of chemotherapy. In our other study, BER and NER capacities were measured in blood cells, healthy mucosa and tumor tissues from 70 patients with sporadic CRC. Lymphocytes in contrast to tumor tissues exhibited highest levels of BER and NER capacities. The definition of genetic and phenotypic landscape of the disease is, thus, further extended by DNA repair functional characteristics, both on the surrogate (systemic) and target tumor (topical level) tissues. Additional studies on DNA repair efficiency to determine influence on onset of the disease and in the context of gene-environment interactions are warranted. Acknowledgement: CZ GA CR: GA P304/10/1286 and GA P304/12/1585 Citation Format: Pavel Vodicka, Jana Slyskova, Rudolf Stetina, Ludmila Vodickova, Alessio Naccarati, Rajiv Kumar, Kari Hemminki. Functional DNA repair tests and their perspectives in molecular epidemiology (experience with subjects exposed to carcinogens and in cancer patients). [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 23.

Abstract 744: Testing the carcinogenic potential of water disinfectant byproducts in a human colon mucosal culture system

Abstract Epidemiological studies have linked the consumption of disinfected surface waters to an increased risk of colorectal cancer. Approximately 600 disinfection byproducts (DBPs) have been identified for a number of disinfectants currently in use. An in-depth mechanism-based structure activity analysis undertaken identified 50 unregulated DBPs with the highest risk carcinogenic potential. Because it would be financially prohibitive to test all priority DBPs in chronic animal bioassays, we are developing an in vitro model system to identify DBPs of carcinogenic potential in normal human colonocytes. Specifically, a mixed culture of human colon mucosal cells including stem cells with the capability for differentiation (NCM460) were treated with brominated DBPs bromochloroacetic acid (BCAA), dibromonitromethane (DBNM), and tribromonitromethane (TBNM), as well as a positive colon carcinogenic control, azoxymethane. To measure cancer-related pathways being activated, global gene expression analysis of 10-day 10−6M treated NCM460 monolayers was performed on Affymetrix Human Genome U133 Plus 2.0 arrays and statistically significant genes were identified using one-way ANOVA with a false discovery rate (Benjamini-Hochberg test) of ≤ 0.05 followed by a post-hoc test (Scheffe). Pathway analysis was performed using DAVID bioinformatics resource (National Institutes of Health) which identified multiple pertinent pathways activated with all or a majority of the treatments including cell adherins, ubiquitin-mediated proteolysis and xenobiotic metabolism. Of significance, AOM, DBNM and TBNM treatments induced genes involved in the WNT/β-catenin, a well studied signaling pathway that modulates cell proliferation and differentiation and is a hallmark pathway for cancer development. As validation, immunocytochemical staining for β-catenin indicated increased nuclear translocation after treatment with all DBPs and AOM when compared to the control. In addition, real-time RT-PCR analysis measured expression of select genes known to be directly regulated by β-catenin after 7 and 10-day 10−6M AOM and TBNM treatment. MYC, AXIN2 and PPARD expression significantly increased (p<0.05, t-test) with 10, but not 7, day treatment with both chemicals, suggesting WNT/β-catenin signaling pathway activation at the later time point. Importantly, preliminary studies indicate colony transformation of these cells after DBP treatment as measured in soft agar assay. Our overall results suggest the tested water DBPs may activate cellular mechanisms that mediate colon cancer development and etiology. Additionally, we demonstrate this in vitro methodology can detect carcinogenic potential of chemicals and may serve as a useful tool to test other chemicals in a more cost-effective and expedited manner than traditional animal bioassays. [Abstract does not necessarily reflect the policies of the U.S. EPA.] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 744. doi:1538-7445.AM2012-744

Assessment and Validation of US EPA's OncoLogic® Expert System and Analysis of Its Modulating Factors for Structural Alerts

OncoLogic® is a software program able to screen chemical compounds for toxicological effects. The software predicts the potential carcinogenicity of chemicals by applying rules of structure activity relationship (SAR) analysis. To validate the predictivity of OncoLogic® (Version 7.0), 123 compounds tested with the long-term carcinogenicity bioassay on rodents were extracted from the ISSCAN database and were analyzed. The concordance between the OncoLogic® SAR analysis and the bioassay results was high. To better understand the strength of the SAR science in OncoLogic®, we investigated the influence of a select group of modulating factors on the predictions by the structural alerts.

Rodent cell transformation assays—A brief historical perspective

In vitro cell transformation is a process characterized by a series of progressive distinctive events that often emulate manifestations occurring in vivo and which are associated with neoplasia. Attendant cellular and sub-cellular alterations include, among others: cellular immortality, phenotypic changes, aneuploidy, genetic variability, cellular disarray, anchorage-independent growth, and tumorigenicity in vivo. Early chemically induced neoplastic transformation studies involved the use of normal diploid (Syrian) hamster embryo (SHE) cells and monitored the formation of morphologically altered colonies. Later investigations employed primarily two established mouse cell lines, i.e. the BALB/c 3T3 A31 cell line and the C3H 10T 1/2 cell line, and monitored the induction of morphologically aberrant foci. In either case, such transformed cellular clusters (colonies and foci) could induce tumors upon inoculation in vivo. Some subsequent noteworthy advancements using these systems included pH adjustments, metabolic supplementation, amplification of expression of formerly latent transformed foci, concurrent detection of mutagenesis and transformation, and use of a Bhas 42 cell line (v-Ha-ras transfected BALB/c 3T3 cells) to detect both tumor initiators and promoters. Over time, such transformation assay systems have been found useful in academic, industry and regulatory laboratories, generally for research purposes, but also occasionally as screening tools for potential chemical carcinogens. Nevertheless, to date, use of these assays for decision-making purposes in the regulatory arena remains elusive and will require comprehensive validation to gain universal acceptance.

Cell transformation assays for prediction of carcinogenic potential: state of the science and future research needs

Cell transformation assays (CTAs) have long been proposed as in vitro methods for the identification of potential chemical carcinogens. Despite showing good correlation with rodent bioassay data, concerns over the subjective nature of using morphological criteria for identifying transformed cells and a lack of understanding of the mechanistic basis of the assays has limited their acceptance for regulatory purposes. However, recent drivers to find alternative carcinogenicity assessment methodologies, such as the Seventh Amendment to the EU Cosmetics Directive, have fuelled renewed interest in CTAs. Research is currently ongoing to improve the objectivity of the assays, reveal the underlying molecular changes leading to transformation and explore the use of novel cell types. The UK NC3Rs held an international workshop in November 2010 to review the current state of the art in this field and provide directions for future research. This paper outlines the key points highlighted at this meeting.

The Use of In Vitro Systems to Assess Cancer Mechanisms and the Carcinogenic Potential of Chemicals

Carcinogenesis is a highly complex, multi-stage process that can occur over a relatively long period before its clinical manifestation. While the sequence in which a cancer cell acquires the necessary traits for tumour formation can vary, there are a number of mechanisms that are common to most, if not all, cancers across the spectrum of possible causes. Many aspects of carcinogenesis can be modelled in vitro. This has led to the development of a number of mechanistically driven, cell-based assays to assess the pro-carcinogenic and anti-carcinogenic potential of chemicals. A review is presented of the current in vitro models that can be used to study carcinogenesis, with examples of cigarette smoke testing in some of these models, in order to illustrate their potential applications. We present an overview of the assays used in regulatory genotoxicity testing, as well as those designed to model other aspects that are considered to be hallmarks of cancer. The latter assays are described with a view to demonstrating the recent advances in these areas, to a point where they should now be considered for inclusion in an overall testing strategy for chemical carcinogens.

Structural alerts for estimating the carcinogenicity of pesticides and biocides

More than 20 years ago, Ashby and Tennant showed the interest of structural alerts for the prediction of the carcinogenicity of chemicals. These structural alerts are functional groups or structural features of various sizes that are linked to the level of carcinogenicity of chemicals. Since this pioneering work it has been possible to refine the alerts over time, as more experimental results have become available and additional mechanistic insights have been gained. To date, one of the most advanced lists of structural alerts for evaluating the carcinogenic potential of chemicals is the list proposed by Benigni and Bossa and that is implemented as a rule-based system in Toxtree and in the OECD QSAR Application Toolbox. In order to gain insight into the applicability of this system to the detection of potential carcinogens we screened about 200 pesticides and biocides showing a high structural diversity. Prediction results were compared with experimental data retrieved from an extensive bibliographical review. The prediction correctness was only equal to 60.14%. Attempts were made to analyse the sources of mispredictions.