Project description:Purpose: To generate a reference long-read transcriptomic data set for use in developing new analysis pipelines and comparing their performance with existing methods. Synthetic “sequin” RNA standards (Hardwick et al. 2016) were sequenced using the Oxford Nanopore Technologies (ONT) GridION platform.
Project description:This analysis utilised the \"Transformics Assay\" and evaluated the effect of cell exposure to 0.02 μg/ml of Benzo(a)pyrene, the lowest transforming concentration, using the BALB / c 3T3 A31-1-1 cell line. The Transformic assay is an integrated approach combining the Cell Transformation Assay (CTA), with the high throughput technology of transcriptomics. The CTA is a widely used in vitro assay that has been designed to evaluate the effects of chemicals on the growth of specific cell types and their potential progression through a transformation process from being normal cells to fully malignant cells. It has the potential to detect both genotoxic and some non-genotoxic carcinogens and shows a good correlation with rodent bioassay data. However lack of understanding of the mechanistic basis of the assays, and particularly of the mechanisms leading to cell malignancy, has limited its acceptance for regulatory purposes. The Transformics Assay introduces the RNA extraction and microarray experiment performed at specific time points along the process of in vitro transformation, helping to identify mechanisms of action leading to chemical-induced transformation. The experiment was conducted testing the carcinogen Benzo(a)pyrene (CAS No.50-32-8), which is a polycyclic aromatic hydrocarbon (PHA) and classified as carcinogenic to humans (Group 1) by IARC. Particularly Benzo(a)pyrene is a prototypical member of this class of chemicals and its toxic effects has been extensively studied, both in laboratory animals and human populations. Benzo(a)pyrene and others PAHs-induced carcinogenesis is a multi-step process, which since the last decade, was considered to be initiated and substantially sustained by the reaction and binding to DNA (oncogenic targets). Indeed, as other PHAs, Benzo(a)pyrene is metabolically activated to a series of reactive intermediates by CYP450 and related enzymes under control of the aryl-hydrocarbon receptor (AhR). These events support the genotoxic mode of carcinogenic action. Nevertheless it is now widely recognized that Benzo(a)pyrene can act via non-genotoxic modes of action too, including epigenetic mechanisms involving the AhR signaling pathway, oxidative stress, disruption of mitogenic and other cellular signaling. This study aimed to explore the molecular and cellular events necessary to sustain the transforming activity of Benzo(a)pyrene, at the lowest in vitro concentration capable of sustaining the process of oncotransformation. In order to find the lowest transforming concentration, a preliminary CTA has been conducted according to the EURL-ECVAM (EU Reference Laboratory European Centre for the Validation of Alternative Methods) validated protocol. The exposure time recommended by EURL-ECVAM, 72 hrs, represents both the exposure time of cells and the RNA extraction time point. According to previous studies 72 hrs exposure could be a critical timing to study the cell transformation process, during which differences in adverse and adaptive cell responses began to become visible at the transcriptional level.
