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.

Nitric oxide (NO), the molecule of the year 1992, is gaining recognition as an important biological mediator. Its multitude of physiologic and pathophysiologic functions result from both a wide distribution of synthesis and diverse mechanisms of action. Besides its functions as a potent vasodilator and neurotransmitter, NO is important in inflammation and immunity. Both beneficial and detrimental consequences of induced synthesis have been discovered. Information is now accumulating on the regulation and function of induced NO. The recent cloning of human inducible NO cDNA should assist in defining the role of inducible NO in human physiology and pathophysiology.

Nitric oxide is a short-lived biologic mediator for diverse cell types. Synthesis of an inducible nitric oxide synthase (NOS) in murine macrophages is stimulated by lipopolysaccharide (LPS) and interferon y. In human hepatocytes, NOS activity is induced by treatment with a combination of tumor necrosis factor, interleukin 1, interferon y, and LPS. We now report the molecular cloning and expression of an inducible human hepatocyte NOS (hep-NOS) cDNA. hep-NOS has 80% amino acid sequence homology to macrophage NOS (mac-NOS Nitric oxide (NO) is a recently recognized messenger molecule mediating diverse functions including vasodilation, neurotransmission, and antimicrobial and antitumor activities. Different cells such as macrophages (1, 2), endothelial cells (3, 4), neurons (5, 6), smooth muscle cells (7,8), and cardiac myocytes (9) produce NO from L-arginine. Constitutive and inducible isoforms of NO synthase (NOS) differ in structure and regulation (reviewed in refs. 10 and 11). Constitutive NOS has been cloned from rat cerebellum (12) and bovine (13,14) and human (15, 16) endothelial cells, whereas inducible murine macrophage NOS (mac-NOS) has been cloned from RAW 264.7 cells (17)(18)(19).Rat hepatocytes make NO in vivo during chronic hepatic inflammation (20,21) and in vitro in response to conditioned Kupffer cell supernatant (22) or to lipopolysaccharide (LPS) and the cytokines tumor necrosis factor (TNF), interleukin 1 (IL-1), and interferon y (IFN-y) (23-25). Since rats treated with LPS manifest inducible NOS in numerous tissues with few macrophages (26), it is possible that more than one isoform of inducible NOS exists. Evidence for inducible human NOS activity has been shown in patients receiving IL-2 cancer therapy (27, 28) and during sepsis (29). Recently, human hepatocytes were stimulated to produce NO by the same combination of LPS and cytokines as rat hepatocytes, providing evidence that a specific human cell expresses inducible NOS (30). We now report the cloning and functional expression of a distinct form of inducible NOS from human hepatocytes. 11 MATERIALS AND METHODSIsolation of Human Hepatocytes. Human hepatocytes were isolated from histologically normal operative wedge resections (in accordance with institutional approval) by using a modification of an in situ collagenase procedure (type IV; Sigma) (30). Briefly, hepatocytes were separated from nonparenchymal cells by differential centrifugation four times at 50 x g. The hepatocytes were then further purified over a 30% Percoll gradient at a concentration of 106 hepatocytes per ml of Percoll to obtain a highly purified cell population (31). Hepatocyte purity by microscopy was >98%, and viability consistently exceeded 95% by trypan blue exclusion.Cell Culture. Hepatocytes (5 x 106) were plated onto 100-mm gelatin-coated Petri dishes (Coming) in 6 ml of culture medium. Medium consisted of Williams media E (GIBCO) with L-arginine (0.50 mM), insulin (1 uM), Hepes (15 mM), L-glutamine, penicillin, streptomycin, and 10%o (vol/vol) low endotox...

Nitric oxide (NO-) is a short-lived mediator which can be induced in a variety of cell types and produces many physiologic and metabolic changes in target cells. The inducible or high-output NO' synthase (NOS) pathway was first characterized in macrophages activated by lipopolysaccharide (LPS) and interferon v (IFN-y). Hepatocytes also express an inducible NOS following exposure to the combination of endotoxin (LPS) and tumor necrosis factor (TNF), interleukin 1 Following the discovery of the nitric oxide (NO-) pathway and its identification as endothelium-derived relaxing factor, a variety of cell types such as macrophages (1, 2), endothelial cells (3, 4), smooth muscle cells (5), and neurons (6, 7) have been shown to produce NO' from L-arginine. Constitutive and inducible isoforms of the NO' synthase (NOS) enzyme exist, and they differ in structure and regulation (8). The neuronal constitutive NOS is a 150-kDa protein whose activity is dependent upon calcium and calmodulin (7); the inducible macrophage NOS is a 130-kDa protein which is thought to function independently of calcium/calmodulin (9, 10). The constitutive NOS cDNA has been cloned from rat cerebellum and identifies an ==10-kilobase (kb) mRNA on Northern blot analysis (11), while the inducible murine macrophage NOS has been cloned from RAW264.7 cells by three groups and identifies an -4.4-kb mRNA (12-14). The physiologic importance of NON as a vasodilator, neurotransmitter, and antimicrobial/antitumor agent is rapidly becoming apparent. Previous work showed that rat hepatocyte/Kupffer cell cocultures stimulated with lipopolysaccharide (LPS) produce large amounts of nitrite (NO-) and nitrate (NO-), the stable end products of the NO-pathway (15). Further, it was demonstrated that hepatocytes also produce NO-in vivo during chronic hepatic inflammation (16,17) and in vitro in response to conditioned Kupffer cell supernatant (18) or to a mixture of LPS and the cytokines tumor necrosis factor (TNF), interleukin 1 (IL-1), and interferon y (IFN-y) (19). Human hepatocytes were also stimulated to produce NO' by the same combination of endotoxin and cytokines as rat hepatocytes (20). However, essentially nothing has been reported about the direct signals required for inducible NOS gene activation. Therefore, the present study was undertaken to characterize the molecular regulation of the inducible rat hepatocyte NOS by endotoxin and cytokines known to up-regulate hepatocyte NO' synthesis. MATERIALS AND METHODSIsolation of Hepatocytes. Hepatocytes were isolated from male rats (200-250 g, Harlan-Sprague-Dawley) by a modification of the in situ collagenase (Sigma) perfusion technique of Seglen (21). Hepatocytes were separated from nonparenchymal cells by differential centrifugation at 50 x g and then passed over a 30%o Percoll gradient to obtain a highly purified cell population. Hepatocyte purity assessed by microscopy was >98% and viability consistently exceeded 95% by trypan blue exclusion. For the in vivo studies, hepatocytes were harvested from rats 3...

Many drugs that are substrates of CYP3A4, the major human drug-metabolizing cytochrome P450 (CYP), show higher clearance in women than in men. Although this effect is believed to be related to drug metabolism, the underlying cause has not been elucidated. We investigated CYP3A4 in a large collection (n = 94) of well-characterized surgical liver samples and found 2-fold higher CYP3A4 levels in female compared with male samples (P <.0001) and a corresponding 50% increase in the CYP3A-dependent N-dealkylation of verapamil (P <.01). This expression difference was not due to preferential induction in women following higher drug exposure because it was even larger in a subgroup not previously exposed to drugs. Higher expression in women was also found for CYP3A4 messenger RNA (mRNA) transcripts, suggesting a pretranslational mechanism. Expression of the pregnane X receptor (PXR), which is crucially involved in CYP3A4 induction by xenobiotics, was strongly correlated to CYP3A4 at the mRNA level in all individuals as well as in the subgroup not exposed to drugs (r = 0.81; P <.0001), but no sex-dependent expression of PXR mRNA was found. The ABC transporter P-glycoprotein, which has been proposed to be implicated in the mechanism of sex-dependent drug clearance, was also not differentially expressed. The influence of drug treatment on expression was examined from patient drug histories, and strong induction of CYP3A4 by carbamazepine and St. John's wort was found. In conclusion, sex, in addition to PXR and drug exposure, is a major factor for CYP3A4 expression in humans, thus explaining many of the previous observations of sex-dependent drug clearance.

The expression of inducible nitric oxide synthase (NOS2) is complex and is regulated in part by gene transcription. In this investigation we studied the regulation of NOS2 in a human liver epithelial cell line (AKN-1) which expresses high levels of NOS2 mRNA and protein in response to tumor necrosis factor a, interleukin 113, and interferon y (cytokine mix, CM). Nuclear run-on analysis revealed that CM transcript"onally activated the human NOS2 gene. To delineate the cytokine-responsive regions of the human NOS2 promoter, we stimulated AKN-1 cells with CM following transfection of NOS2 luciferase constructs. Analysis of the first 3.8 kb upstream of the NOS2 gene demonstrated basal promoter activity but failed to show any cytokine-inducible activity. However, 3-to 5-fold inductions of luciferase activity were seen in constructs extending up to -5.8 and -7.0 kb, and a 10-fold increase was seen upon transfection of a -16 kb construct.' Further analysis of various NOS2 luciferase constructs ligated upstream of the thymidine kinase promoter identified three regions containing cytokine-responsive elements in the human NOS2 gene: -3.8 to -5.8, -5.8 to -7.0, and -7.0 to -16 kb. These results are in marked contrast with the murine macrophage NOS2 promoter in which only 1 kb of the proximal 5' flanking region is necessary to confer inducibility to lipopolysaccharide and interferon y. These data demonstrate that the human NOS2 gene is transcriptionally regulated by cytokines and identify multiple cytokineresponsive regions in the 5' flanking region of the human NOS2 gene.

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