Moving Forward in Human Cancer Risk Assessment

The goal of human risk assessment is to decide whether a given exposure level to a particular chemical or substance is acceptable to human health, and to provide risk management measures based on an evaluation and prediction of the effects of that exposure on human health. Within this framework, the current safety paradigm for assessing possible carcinogenic properties of drugs, cosmetics, industrial chemicals and environmental exposures relies mainly on in vitro genotoxicity testing followed by 2-year bioassays in mice and rats. This testing paradigm was developed 40 to 50 years ago with the initial premise that ¿mutagens are also carcinogens¿ and that the carcinogenic risk to humans can be extrapolated from the tumor incidence after lifetime exposure to maximally tolerated doses of chemicals in rodents. Genotoxicity testing is used as a surrogate for carcinogenicity testing and is required for initiation of clinical trials (Jacobs and Jacobson-Kram 2004) and for most industrial chemicals safety assessment. Although the carcinogenicity-testing paradigm has effectively protected patients and consumers from introduction of harmful carcinogens as drugs and other products, the testing paradigm is clearly not sustainable in the future. The causal link between genetic damage and carcinogenicity is well documented; however, the limitations of genotoxicity/carcinogenicity testing assays, the presence of additional non-genotoxic mechanisms, issues of species-specific effects, and the lack of mechanistic insights provide an enormous scientific challenge. The 2-year rodent carcinogenicity bioassays are associated with technical complexity, high costs, high animal burden as well as the uncertainty associated with extrapolating from rodents to humans. Additional frustrations exist because of the limited predictability of the 2-year bioassay and, in particular, with regard to the problem of the prediction of false positives. For instance, in the Carcinogenic Potency Project DataBase (CPDB) which includes results from chronic, long-term animal cancer tests with mice, rats, hamsters amounting to a total of 6540 individual experiments with 1547 chemicals, 751 of those chemicals or 51% have positive findings in rodent studies. Similarly, when one considers all chronically used human pharmaceuticals, some 50% induce tumors in rodents. Yet only 20 human pharmaceutical compounds have been identified as carcinogens in epidemiological studies, despite the fact that quite a large number of epidemiological studies have been carried out on these compounds, e.g. NSAID¿s, benzodiazepines, phenobarbital. This high incidence of tumors in bioassays has lead to questions concerning the human relevance of tumors induced in rodents (Knight et al. 2006; Ward 2008). In summary, dependency on the rodent model as a golden standard of cancer risk assessment is neglecting the high number of false positives and clearly has serious limitations. Consequently, there is a growing appeal for a paradigm change after "50 years of rats and mice". For instance, the current demands for volume of carcinogenic testing together with limitations of animal usage as initially stipulated by REACH (Combes et al. 2006) will require revolutionary change in the testing paradigm. For the purpose of developing a road map for this needed paradigm change in carcinogenicity testing, a workshop was held in August 2009 in Venice, Italy entitled ¿Genomics in Cancer Risk Assessment.¿ This workshop brought together toxicologists from academia and industry with governmental regulators and risk assessors from the US and the EU, for discussing the state-of-the-art in developing alternative testing strategies for genotoxicity and carcinogenicity, thereby focusing on the contribution from the ¿omics technologies. What follows is a highlight of the major conclusions and suggestions from this workshop as a path forward.JRC.DG.I.3-In-vitro method

Sex-specific associations between particulate matter exposure and gene expression in independent discovery and validation cohorts of middle-aged men and women

BACKGROUND: Particulate matter (PM) exposure leads to premature death, mainly due to respiratory and cardiovascular diseases. OBJECTIVES: Identification of transcriptomic biomarkers of air pollution exposure and effect in a healthy adult population. METHODS: Microarray analyses were performed in 98 healthy volunteers (48 men, 50 women). The expression of eight sex-specific candidate biomarker genes (significantly associated with PM(10) in the discovery cohort and with a reported link to air pollution-related disease) was measured with qPCR in an independent validation cohort (75 men, 94 women). Pathway analysis was performed using Gene Set Enrichment Analysis. Average daily PM(2.5) and PM(10) exposures over 2-years were estimated for each participant’s residential address using spatiotemporal interpolation in combination with a dispersion model. RESULTS: Average long-term PM(10) was 25.9 (± 5.4) and 23.7 (± 2.3) μg/m(3) in the discovery and validation cohorts, respectively. In discovery analysis, associations between PM(10) and the expression of individual genes differed by sex. In the validation cohort, long-term PM(10) was associated with the expression of DNAJB5 and EAPP in men and ARHGAP4 (p = 0.053) in women. AKAP6 and LIMK1 were significantly associated with PM(10) in women, although associations differed in direction between the discovery and validation cohorts. Expression of the eight candidate genes in the discovery cohort differentiated between validation cohort participants with high versus low PM(10) exposure (area under the receiver operating curve = 0.92; 95% CI: 0.85, 1.00; p = 0.0002 in men, 0.86; 95% CI: 0.76, 0.96; p = 0.004 in women). CONCLUSIONS: Expression of the sex-specific candidate genes identified in the discovery population predicted PM(10) exposure in an independent cohort of adults from the same area. Confirmation in other populations may further support this as a new approach for exposure assessment, and may contribute to the discovery of molecular mechanisms for PM-induced health effects. CITATION: Vrijens K, Winckelmans E, Tsamou M, Baeyens W, De Boever P, Jennen D, de Kok TM, Den Hond E, Lefebvre W, Plusquin M, Reynders H, Schoeters G, Van Larebeke N, Vanpoucke C, Kleinjans J, Nawrot TS. 2017. Sex-specific associations between particulate matter exposure and gene expression in independent discovery and validation cohorts of middle-aged men and women. Environ Health Perspect 125:660–669; http://dx.doi.org/10.1289/EHP37

Persistence of Epigenomic Effects After Recovery From Repeated Treatment With Two Nephrocarcinogens

The discovery of the epigenetic regulation of transcription has provided a new source of mechanistic understanding to long lasting effects of chemicals. However, this information is still seldom exploited in a toxicological context and studies of chemical effect after washout remain rare. Here we studied the effects of two nephrocarcinogens on the human proximal tubule cell line RPTEC/TERT1 using high-content mRNA microarrays coupled with miRNA, histone acetylation (HA) and DNA methylation (DM) arrays and metabolomics during a 5-day repeat-dose exposure and 3 days after washout. The mycotoxin ochratoxin A (OTA) was chosen as a model compound for its known impact on HA and DM. The foremost effect observed was the modulation of thousands of mRNAs and histones by OTA during and after exposure. In comparison, the oxidant potassium bromate (KBrO3) had a milder impact on gene expression and epigenetics. However, there was no strong correlation between epigenetic modifications and mRNA changes with OTA while with KBrO3 the gene expression data correlated better with HA for both up- and down-regulated genes. Even when focusing on the genes with persistent epigenetic modifications after washout, only half were coupled to matching changes in gene expression induced by OTA, suggesting that while OTA causes a major effect on the two epigenetic mechanisms studied, these alone cannot explain its impact on gene expression. Mechanistic analysis confirmed the known activation of Nrf2 and p53 by KBrO3, while OTA inhibited most of the same genes, and genes involved in the unfolded protein response. A few miRNAs could be linked to these effects of OTA, albeit without clear contribution of epigenetics to the modulation of the pathways at large. Metabolomics revealed disturbances in amino acid balance, energy catabolism, nucleotide metabolism and polyamine metabolism with both chemicals. In conclusion, the large impact of OTA on transcription was confirmed at the mRNA level but also with two high-content epigenomic methodologies. Transcriptomic data confirmed the previously reported activation (by KBrO3) and inhibition (by OTA) of protective pathways. However, the integration of omic datasets suggested that HA and DM were not driving forces in the gene expression changes induced by either chemical

Phosphorylation of eIF2 alpha promotes cell survival in response to benzo[a]pyrene exposure

Cellular adaptation is important to cope with various stresses induced by altered environmental conditions. By controlling mRNA translation rates cells may adapt to stress to promote survival. Phosphorylation of eIF2 alpha at serine 51 is one of the pathways controlling mRNA translation. Here we investigated the role of phosphorylated eIF2 alpha during exposure to the environmental carcinogen benzo(a)pyrene (BaP). For our study we used mouse embryonic fibroblasts with a wild type eIF2 alpha (MEF WT) and mouse embryonic fibroblasts with an eIF2 alpha S51A knock-in mutation that cannot be phosphorylated. Here, we show that eIF2 alpha phosphorylation occurs in MEF WT cells but not in MEF S51A cells. Survival of MEF S51A cells is profoundly reduced compared to MEF WT controls after BaP exposure. No differences in DNA damage or ROS production were observed between MEF WT and S51A cells. Disruption of eIF2 alpha phosphorylation caused increased levels of apoptosis in response to BaP. This work demonstrates that eIF2 alpha phosphorylation is important for reducing apoptosis and promoting cell survival in order to adapt to BaP exposure

Dose and Time Dependencies in Stress Pathway Responses during Chemical Exposure: Novel Insights from Gene Regulatory Networks

Perturbation of biological networks is often observed during exposure to xenobiotics, and the identification of disturbed processes, their dynamic traits, and dose–response relationships are some of the current challenges for elucidating the mechanisms determining adverse outcomes. In this scenario, reverse engineering of gene regulatory networks (GRNs) from expression data may provide a system-level snapshot embedded within accurate molecular events. Here, we investigate the composition of GRNs inferred from groups of chemicals with two distinct outcomes, namely carcinogenicity [azathioprine (AZA) and cyclophosphamide (CYC)] and drug-induced liver injury (DILI; diclofenac, nitrofurantoin, and propylthiouracil), and a non-carcinogenic/non-DILI group (aspirin, diazepam, and omeprazole). For this, we analyzed publicly available exposed in vitro human data, taking into account dose and time dependencies. Dose–Time Network Identification (DTNI) was applied to gene sets from exposed primary human hepatocytes using four stress pathways, namely endoplasmic reticulum (ER), NF-κB, NRF2, and TP53. Inferred GRNs suggested case specificity, varying in interactions, starting nodes, and target genes across groups. DILI and carcinogenic compounds were shown to directly affect all pathway-based GRNs, while non-DILI/non-carcinogenic chemicals only affected NF-κB. NF-κB-based GRNs clearly illustrated group-specific disturbances, with the cancer-related casein kinase CSNK2A1 being a target gene only in the carcinogenic group, and opposite regulation of NF-κB subunits being observed in DILI and non-DILI/non-carcinogenic groups. Target genes in NRF2-based GRNs shared by DILI and carcinogenic compounds suggested markers of hepatotoxicity. Finally, we indicate several of these group-specific interactions as potentially novel. In summary, our reversed-engineered GRNs are capable of revealing dose dependent, chemical-specific mechanisms of action in stress-related biological networks

Workshop report: Identifying opportunities for global integration of toxicogenomics databases, 26-27 June 2013, Research Triangle Park, NC, USA

A joint US-EU workshop on enhancing data sharing and exchange in toxicogenomics was held at the National Institute for Environmental Health Sciences. Currently, efficient reuse of data is hampered by problems related to public data availability, data quality, database interoperability (the ability to exchange information), standardization and sustainability. At the workshop, experts from universities and research institutes presented databases, studies, organizations and tools that attempt to deal with these problems. Furthermore, a case study showing that combining toxicogenomics data from multiple resources leads to more accurate predictions in risk assessment was presented. All participants agreed that there is a need for a web portal describing the diverse, heterogeneous data resources relevant for toxicogenomics research. Furthermore, there was agreement that linking more data resources would improve toxicogenomics data analysis. To outline a roadmap to enhance interoperability between data resources, the participants recommend collecting user stories from the toxicogenomics research community on barriers in data sharing and exchange currently hampering answering to certain research questions. These user stories may guide the prioritization of steps to be taken for enhancing integration of toxicogenomics databases