Hans Ketelslegers

Comparison of HepG2 and HepaRG by Whole-Genome Gene Expression Analysis for the Purpose of Chemical Hazard Identification

Direct comparison of the hepatoma cell lines HepG2 and HepaRG has previously been performed by only evaluating a limited set of genes or proteins. In this study, we examined the whole-genome gene expression of both cell lines before and after exposure to the genotoxic (GTX) carcinogens aflatoxin B1 and benzo[a]pyrene and the nongenotoxic (NGTX) carcinogens cyclosporin A, 17beta-estradiol, and 2,3,7,8-tetrachlorodibenzo-para-dioxin for 12 and 48 h. Before exposure, this analysis revealed an extensive network of genes and pathways, which were regulated differentially for each cell line. The comparison of the basal gene expression between HepG2, HepaRG, primary human hepatocytes (PHH), and liver clearly showed that HepaRG resembles PHH and liver the most. After exposure to the GTX and NGTX carcinogens, for both cell lines, common pathways were found that are important in carcinogenesis, for example, cell cycle regulation and apoptosis. However, also clear differences between exposed HepG2 and HepaRG were observed, and these are related to common metabolic processes, immune response, and transcription processes. Furthermore, HepG2 performs better in discriminating between GTX and NGTX carcinogens. In conclusion, these results have shown that HepaRG is a more suited in vitro liver model for biological interpretations of the effects of exposure to chemicals, whereas HepG2 is a more promising in vitro liver model for classification studies using the toxicogenomics approach. Although, it should be noted that only five carcinogens were used in this study.

Contribution of new technologies to characterization and prediction of adverse effects.

Abstract Identification of the potential hazards of chemicals has traditionally relied on studies in laboratory animals where changes in clinical pathology and histopathology compared to untreated controls defined an adverse effect. In the past decades, increased consistency in the definition of adversity with chemically-induced effects in laboratory animals, as well as in the assessment of human relevance has been reached. More recently, a paradigm shift in toxicity testing has been proposed, mainly driven by concerns over animal welfare but also thanks to the development of new methods. Currently, in vitro approaches, toxicogenomic technologies and computational tools, are available to provide mechanistic insight in toxicological Mode of Action (MOA) of the adverse effects observed in laboratory animals. The vision described as Tox21c (Toxicity Testing in the 21st century) aims at predicting in vivo toxicity using a bottom-up-approach, starting with understanding of MOA based on in vitro data to ultimately predict adverse effects in humans. At present, a practical application of the Tox21c vision is still far away. While moving towards toxicity prediction based on in vitro data, a stepwise reduction of in vivo testing is foreseen by combining in vitro with in vivo tests. Furthermore, newly developed methods will also be increasingly applied, in conjunction with established methods in order to gain trust in these new methods. This confidence is based on a critical scientific prerequisite: the establishment of a causal link between data obtained with new technologies and adverse effects manifested in repeated-dose in vivo toxicity studies. It is proposed to apply the principles described in the WHO/IPCS framework of MOA to obtain this link. Finally, an international database of known MOAs obtained in laboratory animals using data-rich chemicals will facilitate regulatory acceptance and could further help in the validation of the toxicity pathway and adverse outcome pathway concepts.

Assessment of petroleum streams for thyroid toxicity

The thyroid gland, and its associated endocrine hormones, is a growing area of interest in regulatory toxicology due to its important role in metabolism, growth and development. This report presents a review of the toxicology data on chemically complex petroleum streams for thyroid hormone effects. Toxicological summaries and studies from all available published and un-published sources were considered, drawing upon the European REACH regulatory submissions for 19 petroleum streams, with in depth review of 11 individual study reports and 31 published papers on related products or environmental settings. Findings relevant to thyroid pathology or thyroid hormone homeostasis were specifically sought, summarized, and discussed. A total of 349 studies of 28-days or longer duration were considered in the review, including data on mice, rats, rabbits, dogs, humans, and fish. The thyroid was almost invariably not a target organ in these studies. Three rodent studies did find thyroid effects; one on a jet fuel product (JP-8), and two studies on a heavy fuel oil product (F-179). The JP-8 product differs from other fuels due to the presence of additives, and the finding of reduced T4 levels in mice in the study occurred at a dose that is above that expected to occur in environmental settings (e.g. 2000mg/kg). The finding for F-179 involved thyroid inflammation at 10-55mg/kg that co-occurred with liver pathology in rats, indicating a possible secondary effect with questionable relevance to humans. In the few cases where findings did occur, the polycyclic aromatic hydrocarbon (PAH) content was higher than in related substances, and, in support of one possible adverse outcome pathway, one in-vitro study reported reduced thyroid peroxidase (TPO) activity with exposure to some PAH compounds (pyrene, benzo(k)fluoranthene, and benzo(e)pyrene). However, it could not be determined from the data available for this review, whether these specific PAH compounds were substantially higher in the JP-8 or F-179 products than in studies in which thyroid effects were not observed. Thus, a few products may carry a weak potential to affect the thyroid at high doses in rodents, possibly through secondary effects on the rodent liver or possibly through a pathway involving the inhibition of TPO by specific members of the PAH family. Human epidemiology evidence found weak and inconsistent effects on the thyroid but without identification of specific chemicals involved. Two studies in petroleum workers, which found a lower rate of morbidity and mortality overall, reported a statistically significant increase in thyroid cancer, but the small number of cases could not exclude confounding variables as possible explanations for the statistical findings. Overall, the available data indicates a low potential for thyroid hormone effects from exposure to petroleum streams, especially when the aromatic content is low. Because regulatory studies for most chemicals do not include detailed thyroid function or receptor studies, it remains possible that subclinical effects on this system may exist that were not detectable using conventional pathology or hormone measurements.

Assessing the safety of co-exposure to food packaging migrants in food and water using the maximum cumulative ratio and an established decision tree

Food contact materials can release low levels of multiple chemicals (migrants) into foods and beverages, to which individuals can be exposed through food consumption. This paper investigates the potential for non-carcinogenic effects from exposure to multiple migrants using the Cefic Mixtures Ad hoc Team (MIAT) decision tree. The purpose of the assessment is to demonstrate how the decision tree can be applied to concurrent exposures to multiple migrants using either hazard or structural data on the specific components, i.e. based on the acceptable daily intake (ADI) or the threshold of toxicological concern. The tree was used to assess risks from co-exposure to migrants reported in a study on non-intentionally added substances (NIAS) eluting from food contact-grade plastic and two studies of water bottles: one on organic compounds and the other on ionic forms of various elements. The MIAT decision tree assigns co-exposures to different risk management groups (I, II, IIIA and IIIB) based on the hazard index, and the maximum cumulative ratio (MCR). The predicted co-exposures for all examples fell into Group II (low toxicological concern) and had MCR values of 1.3 and 2.4 (indicating that one or two components drove the majority of the mixture’s toxicity). MCR values from the study of inorganic ions (126 mixtures) ranged from 1.1 to 3.8 for glass and from 1.1 to 5.0 for plastic containers. The MCR values indicated that a single compound drove toxicity in 58% of the mixtures. MCR values also declined with increases in the hazard index for the screening assessments of exposure (suggesting fewer substances contributed as risk potential increased). Overall, it can be concluded that the data on co-exposure to migrants evaluated in these case studies are of low toxicological concern and the safety assessment approach described in this paper was shown to be a helpful screening tool.

Grouping of Petroleum Substances as Example UVCBs by Ion Mobility-Mass Spectrometry to Enable Chemical Composition-Based Read-Across

Substances of Unknown or Variable composition, Complex reaction products, and Biological materials (UVCBs), including many refined petroleum products, present a major challenge in regulatory submissions under the EU Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) and US High Production Volume regulatory regimes. The inherent complexity of these substances, as well as variability in composition obfuscates detailed chemical characterization of each individual substance and their grouping for human and environmental health evaluation through read-across. In this study, we applied ion mobility mass spectrometry in conjunction with cheminformatics-based data integration and visualization to derive substance-specific signatures based on the distribution and abundance of various heteroatom classes. We used petroleum substances from four petroleum substance manufacturing streams and evaluated their chemical composition similarity based on high-dimensional substance-specific quantitative parameters including m/z distribution, drift time, carbon number range, and associated double bond equivalents and hydrogen-to-carbon ratios. Data integration and visualization revealed group-specific similarities for petroleum substances. Observed differences within a product group were indicative of batch- or manufacturer-dependent variation. We demonstrate how high-resolution analytical chemistry approaches can be used effectively to support categorization of UVCBs based on their heteroatom composition and how such data can be used in regulatory decision-making.

The Role of Endocrine and Dioxin-Like Activity of Extracts of Petroleum Substances in Developmental Toxicity as Detected in a Panel of CALUX Reporter Gene Assays

Abstract Recent evidence suggests that the interaction of polycyclic aromatic hydrocarbons (PAHs), present in some petroleum substances (PS), with particular nuclear-hormone-receptors and/or the dioxin (aryl hydrocarbon receptor [AhR]) receptor, may play a role in the prenatal developmental toxicity (PDT) induced by these substances. To address this hypothesis, we evaluated the possible endocrine and dioxin-like activity of the dimethylsulfoxide (DMSO)-extracts of 9 PS, varying in PAH content, and 2 gas-to-liquid (GTL) products, containing no PAHs but having similar other properties as PS, using a series of Chemical Activated LUciferase gene eXpression (CALUX) assays. The results show that the extracts of PS tested in this study possess various endocrine and dioxin-like activities and these in vitro potencies are associated with the quantity and type of PAHs they contain. All tested DMSO-extracts of PS show a strong AhR agonist activity and rather weak antiprogesterone, antiandrogen, and estrogenic activities. In the assays that evaluate thyroid-related and antiestrogen activity, only minor effects of specific extracts, particularly those with a substantial amount of 4–5 ring PAHs, ie, sample No. 34, 98, and 99, were observed. None of the GTL extracts interacted with the selected receptors. Of all assays, the AhR agonist activity correlates best (R2 = 0.80) with the in vitro PDT of the substances as quantified previously in the embryonic stem cell test, suggesting an important role of the AhR in mediating this effect. Hierarchic clustering of the combined CALUX data clustered the compounds in line with their chemical characteristics, suggesting a PS class-specific effects signature in the various CALUX assays, depending on the PAH profile. To conclude, our findings indicate a high potential for endocrine and dioxin-like activity of some PS extracts which correlates with their in vitro PDT and is driven by the PAHs present in these substances.