Assessment of global and gene-specific DNA methylation in rat liver and kidney in response to non-genotoxic carcinogen exposure.

Altered expression of tumor suppressor genes and oncogenes, which is regulated in part at the level of DNA methylation, is an important event involved in non-genotoxic carcinogenesis. This may serve as a marker for early detection of non-genotoxic carcinogens. Therefore, we evaluated the effects of non-genotoxic hepatocarcinogens, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), hexachlorobenzene (HCB), methapyrilene (MPY) and male rat kidney carcinogens, d-limonene, p-dichlorobenzene (DCB), chloroform and ochratoxin A (OTA) on global and CpG island promoter methylation in their respective target tissues in rats. No significant dose-related effects on global DNA hypomethylation were observed in tissues of rats compared to vehicle controls using LC-MS/MS in response to short-term non-genotoxic carcinogen exposure. Initial experiments investigating gene-specific methylation using methylation-specific PCR and bisulfite sequencing, revealed partial methylation of p16 in the liver of rats treated with HCB and TCDD. However, no treatment related effects on the methylation status of Cx32, e-cadherin, VHL, c-myc, Igfbp2, and p15 were observed. We therefore applied genome-wide DNA methylation analysis using methylated DNA immunoprecipitation combined with microarrays to identify alterations in gene-specific methylation. Under the conditions of our study, some genes were differentially methylated in response to MPY and TCDD, whereas d-limonene, DCB and chloroform did not induce any methylation changes. 90-day OTA treatment revealed enrichment of several categories of genes important in protein kinase activity and mTOR cell signaling process which are related to OTA nephrocarcinogenicity.

Adverse Outcome Pathways can drive non-animal approaches for safety assessment.

Adverse Outcome Pathways (AOPs) provide an opportunity to develop new and more accurate safety assessment processes for drugs and other chemicals, and may ultimately play an important role in regulatory decision making. Not only can the development and application of AOPs pave the way for the development of improved evidence-based approaches for hazard and risk assessment, there is also the promise of a significant impact on animal welfare, with a reduced reliance on animal-based methods. The establishment of a useable and coherent knowledge framework under which AOPs will be developed and applied has been a first critical step towards realizing this opportunity. This article explores how the development of AOPs under this framework, and their application in practice, could benefit the science and practice of safety assessment, while in parallel stimulating a move away from traditional methods towards an increased acceptance of non-animal approaches. We discuss here the key areas where current, and future initiatives should be focused to enable the translation of AOPs into routine chemical safety assessment, and lasting 3Rs benefits.

Integrating omic technologies into aquatic ecological risk assessment and environmental monitoring: hurdles, achievements, and future outlook.

BACKGROUND: In this commentary we present the findings from an international consortium on fish toxicogenomics sponsored by the U.K. Natural Environment Research Council (Fish Toxicogenomics-Moving into Regulation and Monitoring, held 21-23 April 2008 at the Pacific Environmental Science Centre, Vancouver, BC, Canada). OBJECTIVES: The consortium from government agencies, academia, and industry addressed three topics: progress in ecotoxicogenomics, regulatory perspectives on roadblocks for practical implementation of toxicogenomics into risk assessment, and dealing with variability in data sets. DISCUSSION: Participants noted that examples of successful application of omic technologies have been identified, but critical studies are needed to relate molecular changes to ecological adverse outcome. Participants made recommendations for the management of technical and biological variation. They also stressed the need for enhanced interdisciplinary training and communication as well as considerable investment into the generation and curation of appropriate reference omic data. CONCLUSIONS: The participants concluded that, although there are hurdles to pass on the road to regulatory acceptance, omics technologies are already useful for elucidating modes of action of toxicants and can contribute to the risk assessment process as part of a weight-of-evidence approach.

Changes in gene expression and assessment of DNA methylation in primary human hepatocytes and HepG2 cells exposed to the environmental contaminants-Hexabromocyclododecane and 17-beta oestradiol.

We evaluated the effects of two putative non-genotoxic hepatic carcinogens, hexabromocyclododecane (HBCD) and 17-beta oestradiol (E(2)) on global and CpG promoter DNA methylation in both primary human hepatocytes and hepatocellular carcinoma (HepG2) cells. The mRNA gene expression levels of genes involved particularly in cell cycle were also evaluated and potential correlation with DNA methylation status examined. HBCD at 0.03 and 0.3 ng/mL did not produce statistically significant differences in global genomic methylation. However, E(2) (0.1 ng/mL) significantly lowered global DNA methylation levels in HepG2 cells by approximately 65% (P<0.01). In primary hepatocytes, the promoter regions of N-cym and ERalpha were methylated in both control and treated groups, signifying lack of promoter demethylation by both HBCD and E(2). Furthermore, CpG promoter methylation of RB1 was observed in HepG2 cells but this was unaffected by treatments. The remaining genes (p16, C-myc, H-ras, THRalpha, histone H3, TBK1 and TNFRalpha) were unmethylated in their CpG promoter regions in both test systems. Quantitative RT-PCR showed that HBCD at 0.03 ng/mL up-regulated the expression of N-cym whereas E(2) up-regulated the expression of ERalpha and THRalpha genes in primary hepatocytes. In HepG2 cells, the mRNA gene expression levels of p16, RB1 and N-cym were significantly down regulated by HBCD (0.03 ng/mL) and E(2) (0.1 ng/mL) while HBCD at 0.3 ng/mL, significantly down regulated the expression levels of N-cym, ERalpha and ERbeta genes. Thus, while both HBCD and E(2) may alter the expression of certain genes involved in proliferation, the mechanisms appear unrelated to DNA methylation.

A quantitative inverse relationship between connexin32 expression and cell proliferation in a rat hepatoma cell line.

Gap junctions comprised of connexin proteins are involved in direct intercellular communication and the regulation of cell behaviour and homeostasis. Reduced connexin expression and loss of gap junction function is a characteristic of many cancer cells and of the effect of many non-genotoxic carcinogens that induce cell proliferation. Moreover, when certain cancer cell lines are transfected with specific connexin genes, cells can regain control over proliferation. We have employed RNA interference and dexamethasone to modulate connexin32 expression in MH(1)C(1) cells to a range of concentrations. This allowed the determination of the quantitative relationship between connexin32 protein expression and cell proliferation. The magnitude of cell proliferation, measured by bromodeoxyuridine incorporation, was inversely proportional to the level of connexin32 expression. Q-PCR indicated a lack of change of expression of a range of cell cycle-related genes at 24h. The inverse relationship between Cx32 expression and proliferation was continuous, and a threshold level of reduction of connexin32 was not observable for an influence on proliferation.

Identification of functional regulatory regions of the connexin32 gene promoter.

Connexin32 (Cx32) is the predominant gap junction protein expressed in adult rat hepatocytes. This study investigated transcriptional regulation of the rat Cx32 gene in MH(1)C(1) rat hepatoma cells using transient expression assays in conjunction with promoter mutagenesis and 5' nested deletion analysis. Site-directed mutagenesis of the -736 and -187 hepatocyte nuclear factor-1 (HNF-1) sites, the -196 and -116 Sp1 sites, and the -729 and -329 Yin Yang 1 (YY1) sites all significantly reduced promoter activity. We have defined the contribution of each individual site to promoter activity in the intact cell. A novel upstream region of the Cx32 promoter (-1042 to -758) was cloned and shown to contain negative regulatory elements. The transcription factors HNF-1 and Sp1 have important functional roles in the transcriptional regulation of basal and cell-specific Cx32 expression. The multifunctional transcription factor YY1 is also implicated.