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.

One of the unique features of ␤-cells is their relatively low expression of many antioxidant enzymes. This could render ␤-cells susceptible to oxidative damage but may also provide a system that is sensitive to reactive oxygen species as signals. In isolated mouse islets and INS-1(832/13) cells, glucose increases intracellular accumulation of H 2 O 2 . In both models, insulin secretion could be stimulated by provision of either exogenous H 2 O 2 or diethyl maleate, which raises intracellular H 2 O 2 levels. Provision of exogenous H 2 O 2 scavengers, including cell permeable catalase and N-acetyl-Lcysteine, inhibited glucose-stimulated H 2 O 2 accumulation and insulin secretion (GSIS). In contrast, cell permeable superoxide dismutase, which metabolizes superoxide into H 2 O 2 , had no effect on GSIS. Because oxidative stress is an important risk factor for ␤-cell dysfunction in diabetes, the relationship between glucose-induced H 2 O 2 generation and GSIS was investigated under various oxidative stress conditions. Acute exposure of isolated mouse islets or INS-1(832/ 13) cells to oxidative stressors, including arsenite, 4-hydroxynonenal, and methylglyoxal, led to decreased GSIS. This impaired GSIS was associated with increases in a battery of endogenous antioxidant enzymes. Taken together, these findings suggest that H 2 O 2 derived from glucose metabolism is one of the metabolic signals for insulin secretion, whereas oxidative stress may disturb its signaling function. Diabetes

Cellular oxidative and electrophilic stress triggers a protective response in mammals regulated by NRF2 (nuclear factor (erythroid-derived) 2-like; NFE2L2) binding to deoxyribonucleic acid-regulatory sequences near stress-responsive genes. Studies using Nrf2-deficient mice suggest that hundreds of genes may be regulated by NRF2. To identify human NRF2-regulated genes, we conducted chromatin immunoprecipitation (ChIP)-sequencing experiments in lymphoid cells treated with the dietary isothiocyanate, sulforaphane (SFN) and carried out follow-up biological experiments on candidates. We found 242 high confidence, NRF2-bound genomic regions and 96% of these regions contained NRF2-regulatory sequence motifs. The majority of binding sites were near potential novel members of the NRF2 pathway. Validation of selected candidate genes using parallel ChIP techniques and in NRF2-silenced cell lines indicated that the expression of about two-thirds of the candidates are likely to be directly NRF2-dependent including retinoid X receptor alpha (RXRA). NRF2 regulation of RXRA has implications for response to retinoid treatments and adipogenesis. In mouse, 3T3-L1 cells’ SFN treatment affected Rxra expression early in adipogenesis, and knockdown of Nrf2-delayed Rxra expression, both leading to impaired adipogenesis.

p62/Sqstm1 is a multifunctional protein involved in cell survival, growth and death, that is degraded by autophagy. Amplification of the p62/Sqstm1 gene, and aberrant accumulation and phosphorylation of p62/Sqstm1, have been implicated in tumour development. Herein, we reveal the molecular mechanism of p62/Sqstm1-dependent malignant progression, and suggest that molecular targeting of p62/Sqstm1 represents a potential chemotherapeutic approach against hepatocellular carcinoma (HCC). Phosphorylation of p62/Sqstm1 at Ser349 directs glucose to the glucuronate pathway, and glutamine towards glutathione synthesis through activation of the transcription factor Nrf2. These changes provide HCC cells with tolerance to anti-cancer drugs and proliferation potency. Phosphorylated p62/Sqstm1 accumulates in tumour regions positive for hepatitis C virus (HCV). An inhibitor of phosphorylated p62-dependent Nrf2 activation suppresses the proliferation and anticancer agent tolerance of HCC. Our data indicate that this Nrf2 inhibitor could be used to make cancer cells less resistant to anticancer drugs, especially in HCV-positive HCC patients.