This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.

An adverse outcome pathway (AOP) describes the causal linkage between initial molecular events and an adverse outcome at individual or population levels. Whilst there has been considerable momentum in AOP development, far less attention has been paid to how AOPs might be practically applied for different regulatory purposes. This paper proposes a scientific confidence framework (SCF) for evaluating and applying a given AOP for different regulatory purposes ranging from prioritizing chemicals for further evaluation, to hazard prediction, and ultimately, risk assessment. The framework is illustrated using three different AOPs for several typical regulatory applications. The AOPs chosen are ones that have been recently developed and/or published, namely those for estrogenic effects, skin sensitisation, and rodent liver tumor promotion. The examples confirm how critical the data-richness of an AOP is for driving its practical application. In terms of performing risk assessment, human dosimetry methods are necessary to inform meaningful comparisons with human exposures; dosimetry is applied to effect levels based on non-testing approaches and in vitro data. Such a comparison is presented in the form of an exposure:activity ratio (EAR) to interpret biological activity in the context of exposure and to provide a basis for product stewardship and regulatory decision making.

There is increasing interest in the use of tiered approaches in risk assessment of mixtures or co-exposures to chemicals for prioritization. One possible screening-level risk assessment approach is the threshold of toxicological concern (TTC). To date, default assumptions of dose or response additivity have been used to characterize the toxicity of chemical mixtures. Before a screening-level approach could be used, it is essential to know whether synergistic interactions can occur at low, environmentally relevant exposure levels. Studies demonstrating synergism in mammalian test systems were identified from the literature, with emphasis on studies performed at doses close to the points of departure (PODs) for individual chemicals. This search identified 90 studies on mixtures. Few included quantitative estimates of low-dose synergy; calculations of the magnitude of interaction were included in only 11 papers. Quantitative methodology varied across studies in terms of the null hypothesis, response measured, POD used to test for synergy, and consideration of the slope of the dose-response curve. It was concluded that consistent approaches should be applied for quantification of synergy, including that synergy be defined in terms of departure from dose additivity; uniform procedures be developed for assessing synergy at low exposures; and the method for determining the POD for calculating synergy be standardized. After evaluation of the six studies that provided useful quantitative estimates of synergy, the magnitude of synergy at low doses did not exceed the levels predicted by additive models by more than a factor of 4.

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Other than possibly an increased risk of non-Hodgkin lymphoma, we find no other cause of death related to the mixture of the dioxin contaminants found in PCP.

A toxicogenomics approach was used to qualitatively and quantitatively compare the gene expression changes in human and rat primary hepatocytes exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Hepatocytes from five individual rats and five individual humans were exposed for 24 h to 11 concentrations of TCDD ranging from 0.00001 to 100nM and a vehicle control. Gene expression changes were analyzed using whole-genome microarrays containing 13,002 orthologs. Significant changes in expression of individual orthologs at any concentration (fold change [FC] ± 1.5 and false discovery rate < 0.05) were higher in the rat (1547) compared with human hepatocytes (475). Only 158 differentially expressed orthologs were common between rats and humans. Enrichment analysis was performed on the differentially expressed orthologs in each species with 49 and 34 enriched human and rat pathways, respectively. Only 12 enriched pathways were shared between the two species. The results demonstrate significant cross-species differences in expression at both the gene and pathway level. Benchmark dose analysis of gene expression changes showed an average 18-fold cross-species difference in potency among differentially expressed orthologs with the rat more sensitive than the human. Similar cross-species differences in potency were observed for signaling pathways. Using the maximum FC in gene expression as a measure of efficacy, the human hepatocytes showed on average a 20% lower efficacy among the individual orthologs showing differential expression. The results provide evidence for divergent cross-species gene expression changes in response to TCDD and are consistent with epidemiological and clinical evidence showing humans to be less sensitive to TCDD-induced hepatotoxicity.

The toxic equivalency factor (TEF) approach recommended by the World Health Organization is used to quantify dioxin-like exposure concentrations for mixtures of polychlorinated dibenzo-dioxins, -furans, and polychlorinated biphenyls (PCBs), including 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Whole-genome microarrays were used to evaluate the hepatic gene expression potency of TCDF and PCB126 relative to TCDD with complementary histopathology, tissue level analysis, and ethoxyresorufin-O-deethylase (EROD) assay results. Immature ovariectomized C57BL/6 mice were gavaged with 0.001, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, and 300 μg/kg TCDD and TEF-adjusted doses (TEF for TCDF and PCB126 is 0.1) of TCDF or PCB126 (1, 3, 10, 30, 100, 300, 1000, and 3000 μg/kg of TCDF or PCB126) or sesame oil vehicle and sacrificed 24 h post dose. In general, TCDD, TCDF, and PCB126 tissue levels, as well as histopathological effects, were comparable when comparing TEF-adjusted doses. Automated dose-response modeling (ToxResponse Modeler) of the microarray data identified 210 TCDF and 40 PCB126 genes that exhibited sigmoidal dose-response curves with comparable slopes when compared with TCDD. These similar responses were used to calculate a median TCDF gene expression relative potency (REP) of 0.06 and a median PCB126 gene expression REP of 0.02. REPs of 0.02 were also calculated for EROD induction for both compounds. Collectively, these data suggest that differences in the ability of the liganded aryl hydrocarbon receptor:AhR nuclear translocator complex to elicit differential hepatic gene expression, in addition to pharmacokinetic differences between ligands, influence their potency in immature ovariectomized C57BL/6 mice.

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