The transformics method was developed to support the integrated testing strategy for carcinogenesis. Results showed the main role of the immune system in the transformation by 3-MCA, with initiating events related to non-genotoxic mechanisms, suggesting the involvement of the AhR receptor.

A large number of basic researches and observational studies suggested the cancer preventive activity of vitamin E, but large-scale human intervention trials have yielded disappointing results and actually showed a higher incidence of prostate cancer although the mechanisms underlying the increased risk remain largely unknown. Here we show through in vitro and in vivo studies that vitamin E produces a marked inductive effect on carcinogen-bioactivating enzymes and a pro-oxidant status promoting both DNA damage and cell transformation frequency. First, we found that vitamin E in the human prostate epithelial RWPE-1 cell line has the remarkable ability to upregulate the expression of various phase-I activating cytochrome P450 (CYP) enzymes, including activators of polycyclic aromatic hydrocarbons (PAHs), giving rise to supraphysiological levels of reactive oxygen species. Furthermore, our rat model confirmed that vitamin E in the prostate has a powerful booster effect on CYP enzymes associated with the generation of oxidative stress, thereby favoring lipid-derived electrophile spread that covalently modifies proteins. We show that vitamin E not only causes DNA damage but also promotes cell transformation frequency induced by the PAH-prototype benzo[a]pyrene. Our findings might explain why dietary supplementation with vitamin E increases the prostate cancer risk among healthy men. Prostate cancer is the most common human malignancy and the second leading cause of cancer death among men in western nations. The high global incidence of prostate cancer, the unsatisfactory outcomes of surgery and radiotherapy and the cost of curative therapies have led to a focus on primary prevention as a major public health goal 1,2. Supported by preclinical and epidemiological evidence, antioxidants from food and supplements are widely used to protect against cancer, but clinical trials do not sustain this concept 1,3-6 and actually showed a higher incidence of prostate cancer 7,8. Conceived to break through this issue, in 2001 the National Cancer Institute (NCI) launched SELECT (Selenium and vitamin E Cancer Prevention Trial), that showed a 17% increase in prostate cancer incidence in the vitamin E arm compared to placebo 7. A growth-promoting effect of vitamin E on organoids has recently been reported 9 , but its mechanism of action remains poorly understood. Since some cytochrome P-450 (CYP) isoforms have been found overexpressed in prostate cancer 10-15 , we suspected that vitamin E could have co-carcinogenic properties such as those involving carcinogen-bioactivating CYP-enzyme changes 16,17. Results and Discussion Our study stemmed from the observation that vitamin E treatment of the human non-tumorigenic prostate epithelial RWPE-1 cell line induces the gene expression of P450 enzymes such as CYP1A1 (activating, for example, polychlorinated biphenyls, aromatic amines, PHAs and alkylnitrosamines), CYP1A4 (activating polycyclic

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The Transformics Assay is an in vitro test which combines the BALB/c 3T3 Cell Transformation Assay (CTA) with microarray transcriptomics. It has been shown to improve upon the mechanistic understanding of the CTA, helping to identify mechanisms of action leading to chemical-induced transformation thanks to RNA extractions in specific time points along the process of in vitro transformation. In this study, the lowest transforming concentration of the carcinogenic benzo(a)pyrene (B(a)P) has been tested in order to find molecular signatures of initial events relevant for oncotransformation. Application of Enrichment Analysis (Metacore) to the analyses of the results facilitated key biological interpretations. After 72 h of exposure, as a consequence of the molecular initiating event of aryl hydrocarbon receptor (AhR) activation, there is a cascade of cellular events and microenvironment modification, and the immune and inflammatory responses are the main processes involved in cell response. Furthermore, pathways and processes related to cell cycle regulation, cytoskeletal adhesion and remodeling processes, cell differentiation and transformation were observed.

Alternative methods to animal testing are considered as promising tools to support the prediction of toxicological risks from environmental exposure. Among the alternative testing methods, the cell transformation assay (CTA) appears to be one of the most appropriate approaches to predict the carcinogenic properties of single chemicals, complex mixtures and environmental pollutants. The BALB/c 3T3 CTA shows a good degree of concordance with the in vivo rodent carcinogenesis tests. Whole-genome transcriptomic profiling is performed to identify genes that are transcriptionally regulated by different kinds of exposures. Its use in cell models representative of target organs may OPEN ACCESS Sustainability 2014, 6 5266 help in understanding the mode of action and predicting the risk for human health. Aiming at associating the environmental exposure to health-adverse outcomes, we used an integrated approach including the 3T3 CTA and transcriptomics on target cells, in order to evaluate the effects of airborne particulate matter (PM) on toxicological complex endpoints. Organic extracts obtained from PM 2.5 and PM 1 samples were evaluated in the 3T3 CTA in order to identify effects possibly associated with different aerodynamic diameters or airborne chemical components. The effects of the PM 2.5 extracts on human health were assessed by using whole-genome 44 K oligo-microarray slides. Statistical analysis by GeneSpring GX identified genes whose expression was modulated in response to the cell treatment. Then, modulated genes were associated with pathways, biological processes and diseases through an extensive biological analysis. Data derived from in vitro methods and omics techniques could be valuable for monitoring the exposure to toxicants, understanding the modes of action via exposure-associated gene expression patterns and to highlight the role of genes in key events related to adversity.