The bile salt export pump (BSEP) is an efflux transporter, driving the elimination of endobiotic and xenobiotic substrates from hepatocytes into the bile. More specifically, it is responsible for the elimination of monovalent, conjugated bile salts, with little or no assistance from other apical transporters. Disruption of BSEP activity through genetic disorders is known to manifest in clinical liver injury such as progressive familial intrahepatic cholestasis type 2. Drug-induced disruption of BSEP is hypothesized to play a role in the development of liver injury for several marketed or withdrawn therapeutics. Unfortunately, preclinical animal models have been poor predictors of the liver injury associated with BSEP interference observed for humans, possibly because of interspecies differences in bile acid composition, differences in hepatobiliary transporter modulation or constitutive expression, as well as other mechanisms. Thus, a BSEP-mediated liver liability may go undetected until the later stages of drug development, such as during clinical trials or even postlicensing. In the absence of a relevant preclinical test system for BSEP-mediated liver injury, the toxicological relevance of available in vitro models to human health rely on the use of benchmark compounds with known clinical outcomes, such as marketed or withdrawn drugs. In this study, membrane vesicles harvested from BSEP-transfected insect cells were used to assess the activity of more than 200 benchmark compounds to thoroughly investigate the relationship between interference with BSEP function and liver injury. The data suggest a relatively strong association between the pharmacological interference with BSEP function and human hepatotoxicity. Although the most accurate translation of risk would incorporate pharmacological potency, pharmacokinetics, clearance mechanisms, tissue distribution, physicochemical properties, indication, and other drug attributes, the additional understanding of a compound's potency for BSEP interference should help to limit or avoid BSEP-related liver liabilities in humans that are not often detected by standard preclinical animal models.

Cell adhesion molecules are glycoproteins expressed on the cell surface and play an important role in inflammatory as well as neoplastic diseases. There are four main groups: the integrin family, the immunoglobulin superfamily, selectins, and cadherins. The integrin family has eight subfamilies, designated as beta 1 through beta 8. The most widely studied subfamilies are beta 1 (CD29, very late activation [VLA] members), beta 2 (leukocyte integrins such as CD11a/CD18, CD11b/CD18, CD11c/CD18, and alpha d beta 2), beta 3 (CD61, cytoadhesions), and beta 7 (alpha 4 beta 7 and alpha E beta 7). The immunoglobulin superfamily includes leukocyte function antigen-2 (LFA-2 or CD2), leukocyte function antigen-3 (LFA-3 or CD58), intercellular adhesion molecules (ICAMs), vascular adhesion molecule-1 (VCAM-1), platelet-endothelial cell adhesion molecule-1 (PE-CAM-1), and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). The selectin family includes E-selectin (CD62E), P-selectin (CD62P), and L-selectin (CD62L). Cadherins are major cell-cell adhesion molecules and include epithelial (E), placental (P), and neural (N) subclasses. The binding sites (ligands/receptors) are different for each of these cell adhesion molecules (e.g., ICAM binds to CD11/CD18; VCAM-1 binds to VLA-4). The specific cell adhesion molecules and their ligands that may be involved in pathologic conditions and potential therapeutic strategies by modulating the expression of these molecules will be discussed.

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There is little primate risk factor data in the literature evaluating the relationship between proposed mechanisms of PPAR agonist-induced hepatocarcinogenesis at clinically relevant therapeutic exposures. These studies were conducted to characterize the hepatic effects of fenofibrate and ciprofibrate in the cynomolgus monkey. Male cynomolgus monkeys were given fenofibrate (250, 1250 or 2500 mg/kg/day) or ciprofibrate (3, 30, 150 or 400 mg/kg/day) for up to 15 days. The highest doses used were approximately 4 times (fenofibrate) and 9.4 times (ciprofibrate) the human therapeutic exposure for these agents based on AUC (area under the curve). For both compounds, there was a treatment-related increase in liver weight and periportal hepatocellular hypertrophy, which was related to increases in peroxisomes (up to 2.8 times controls) and mitochondria (up to 2.5 times controls). An increase in smooth endoplasmic reticulum probably contributed to the hypertrophy. There was no indication of cell proliferation as determined by the number of mitotic figures and this was confirmed by evaluating cell proliferation by immunohistochemical staining for the Ki-67 antigen. Consistent with the findings by light microscopy, there was no treatment-related effect on the level of mRNA for proteins known to be involved in the control of hepatocyte cell division or apoptosis (e.g. P21, Cyclin D1, PCNA, CDKN1A). Furthermore, there was minimal indication of oxidative stress. Thus, there was no evidence of lipofuscin accumulation, and there was no remarkable increase in the mRNA levels for most proteins known to respond to oxidative stress (e.g. catalase, glutathione peroxidase). A mild induction in the mRNA levels of cellular beta-oxidation and detoxification enzymes (e.g. acyl CoA oxidase, thioredoxin reductase) was observed. Collectively, the data from these studies suggest that the primate responds to PPARalpha agonists in a manner that is different from the rodent suggesting that the primate may be refractory to PPAR-induced hepatocarcinogenesis.

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Drug induced liver injury (DILI), a major cause of pre- and post-approval failure, is challenging to predict pre-clinically. Predicting DILI is challenging due to varied underlying direct and indirect mechanisms. Nevirapine, a non-nucleoside reverse transcriptase inhibitor (NNRTI) and Ritonavir, a protease inhibitor, are antiviral drugs that cause clinical DILI with different phenotypes via different mechanisms. Assessing DILI in vitro in hepatocyte cultures typically requires drug exposures significantly higher than clinical plasma Cmax concentrations, making clinical interpretations of mechanistic pathway changes challenging. We previously described a system that uses liver-derived hemodynamic blood flow and transport parameters to restore primary human hepatocyte biology, and drug responses at concentrations relevant to in vivo or clinical exposure levels. Using this system, primary hepatocytes from 5 human donors were exposed to approximating clinical therapeutic and supra-therapeutic concentrations levels of Nevirapine (11.3 and 175.0 μM) and Ritonavir (3.5 and 62.4 μM) for 48 hours. Whole genome transcriptomics was performed by RNAseq along with functional assays for metabolic activity and function. We observed effects at both doses, but a greater number of genes were differentially expressed with higher probability at the toxic concentrations. At the toxic doses, both drugs showed direct cholestatic potential with Nevirapine increasing bile synthesis and Ritonavir inhibiting bile acid transport. Clear differences in antigen presentation were noted, with marked activation of MHC Class I by Nevirapine and suppression by Ritonavir. This suggests CD8+ T cell involvement for Nevirapine and possibly NK Killer cells for Ritonavir. Both compounds induced several drug metabolizing genes (including CYP2B6, CYP3A4 and UGT1A1), mediated by CAR activation in Nevirapine and PXR in Ritonavir. Unlike Ritonavir, Nevirapine did not increase fatty acid synthesis or activate the respiratory electron chain with simultaneous mitochondrial uncoupling supporting clinical reports of a lower propensity for steatosis. This in vitro study offers insights into the disparate direct and immune mediated toxicity mechanisms underlying Nevirapine and Ritonavir toxicity in the clinic.

Large-scale analysis of gene expression using cDNA microarrays promises the rapid detection of the mode of toxicity for drugs and other chemicals. cDNA microarrays were used to examine chemically induced alterations of gene expression in HepG2 cells exposed to a diverse group of toxicants at an equitoxic exposure concentration. The treatments were ouabain (43 microM), lauryl sulfate (260 microM), dimethylsulfoxide (1.28 M), cycloheximide (62.5 microM), tolbutamide (12.8 mM), sodium fluoride (3 mM), diethyl maleate (1.25 mM), buthionine sulfoximine (30 mM), potassium bromate (2.5 mM), sodium selenite (30 microM), alloxan (130 mM), adriamycin (40 microM), hydrogen peroxide (4 mM), and heat stress (45 degrees C x 30 minutes). Patterns of gene expression were correlated with morphologic and biochemical indicators of toxicity. Gene expression responses were characteristically different for each treatment. Patterns of expression were consistent with cell cycle arrest, DNA damage, diminished protein synthesis, and oxidative stress. Based upon these results, we concluded that gene expression changes provide a useful indicator of oxidative stress, as assessed by the GSH:GSSG ratio. Under the conditions of this cell culture test system, oxidative stress upregulated 5 genes, HMOX1, p21(waf1/cip1), GCLM, GR, TXNR1 while downregulating CYP1A1 and TOPO2A. Primers and probes for these genes were incorporated into the design of a 7-gene plate for RT-PCR. The plate design permitted statistical analysis and allowed clear discrimination between chemicals inducing oxidative vs nonoxidative stress. A simple oxidative stress score (0-1), based on the responses by the 7 genes (including p-value) on the RT-PCR plate, was correlated with the GSH:GSSG ratio using linear regression and ranking (Pearson product) procedures. These analyses yielded correlation coefficients of 0.74 and 0.87, respectively, for the treatments tested (when 1 outlier was excluded), indicating a good correlation between the biochemical and transcriptional measures of oxidative stress. We conclude that it is essential to measure the mechanism of interest directly in the test system being used when assessing gene expression as a tool for toxicology. Tables 1-15, referenced in this paper, are not printed in this issue of Toxicologic Pathology. They are available as downloadable text files at http://taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233. To access them, click on the issue link for 30(4), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.

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