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.

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Ruminal parameters, rumen development, nutrient digestibilities, and N utilization were estimated in Holstein calves fed starch from different sources. Ground corn, ground barley, ground wheat, and crimped oats were used to formulate 4 isostarch (25% of starter dry matter) pelleted diets. These diets were randomly allocated to calves (16 calves per treatment, 8 female and 8 male) and fed ad libitum along with mixed grass hay throughout the experiment. Ruminal contents and blood were sampled at d 35, 50, and 70 of age to estimate ruminal parameters and plasma beta-hydroxybutyrate, respectively. At d 70, twenty-four male calves (6/treatment) were randomly selected, euthanized, and forestomach weight, papillae length (PL), papillae width (PW), rumen wall thickness (RWT), and papillae concentration were measured. At d 63, twenty-four female calves (6/treatment) were randomly selected and moved to metabolism stalls to estimate total tract apparent nutrient digestibilities and N utilization. Female calves were given 2 wk for adaptation to experimental facilities and then total collections of feces and urine were made from d 77 to 84 of age. Ruminal pH at d 35 of age was higher in calves fed corn and oat diets than in those fed barley and wheat diets. Ruminal pH at d 50 and 70 of age was the lowest in calves on barley diets followed by those on oat and wheat diets and then by those on the corn diet. Ruminal total volatile fatty acid concentrations at d 35 of age were greatest in calves fed corn or wheat diets followed by those fed barley and oat diets. Calves on corn and wheat diets maintained greater ruminal volatile fatty acids concentrations at d 50 and 70 of age. Ruminal ammonia, acetate, propionate, butyrate, and blood beta-hydroxybutyrate concentrations were also greater in calves on the corn and wheat diets. Full and empty weights of forestomach, PL, PW, RWT, and papillae concentrations were greater in calves on corn and wheat diets. Daily average intake of nutrients (dry matter, crude protein, neutral detergent fiber, starch, Ca, and P) was greater in calves fed corn and wheat diets than in those fed barley and oat diets. Starch source did not influence the total tract apparent digestibilities of nutrients in calves. Daily N retention (g/d) was greatest on the corn diet followed by the wheat diet and then the barley and oat diets. In conclusion, calves on a corn diet have greater ruminal capacity to accommodate feed bulk. More physically and metabolically functional rumens in calves on corn and wheat diets probably resulted in greater feed consumption and N retention.

MOON, HYUN-SEUK, CHUNG-SOO CHUNG, HONG-GU LEE, TAE-GYU KIM, YUN-JAIE CHOI, AND CHONG-SUCHO. Inhibitory effect of (Ϫ)-epigallocatechin-3-gallate on lipid accumulation of 3T3-L1 cells. Obesity. 2007;15:2571-2582. Objective: The objective of this study was to investigate the molecular mechanisms underlying the attenuating effect of (Ϫ)-epigallocatechin-3-gallate (EGCG) on proliferation and lipid accumulation of 3T3-L1 cells, with a focus on the duration of EGCG treatment. Research Methods and Procedures: Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay and diamidino-2-phenylindole staining. The anti-adipogenic effect of EGCG on 3T3-L1 cells was analyzed by glycerol-3-phosphate dehydrogenase activity and Oil red O staining. Western blot analysis was used to detect adenosine monophosphate-activated protein kinase (AMPK) activation and phosphorylation of its substrate, acetyl-CoA carboxylase (ACC), and expression of insulin (INS) receptor, INS receptor substrate-1 (IRS-1), and adipocyte marker proteins. Results: Exposure to EGCG during the early period of adipogenesis (7 days) was sufficient to prevent lipid accumulation. During this period, EGCG greatly decreased expression of the adipocyte marker proteins peroxisome proliferator-activated receptor ␥2 (PPAR␥2) and liver X receptor (LXR)-␣. Furthermore, EGCG significantly induced generation of reactive oxygen species (ROS), which led to AMPK activation, and these effects were eliminated by N-acetylcysteine (NAC) treatment. Also, EGCG increased the tyrosine phosphorylation of INS receptor and INS-1 with increasing incubation time. In contrast, EGCG treatment did not alter glycerol release in the presence or absence of 2Ј,5Ј-dideoxyadenosine (DDA), indicating that EGCG had no effect on lipolysis. Discussion: Our data demonstrate that EGCG decreased cell viability and inhibited differentiation of 3T3-L1 cells in a manner dependent on the duration of treatment. Also, we showed that inhibition of adipocyte differentiation by EGCG was associated with decreased glycerol-3-phosphate dehydrogenase (GPDH) activity accompanied by a strong inhibition of PPAR␥2-induced transcriptional activity. Furthermore, the inhibition of adipocyte differentiation by EGCG involved generation of ROS and activation of AMPK.