Loss of lysophosphatidic acid receptor 1 in hepatocytes reduces steatosis via down-regulation of CD36.

Nonalcoholic steatohepatitis is a major public health concern and is characterized by the accumulation of triglyceride in hepatocytes and inflammation in the liver. Steatosis is caused by dysregulation of the influx and efflux of lipids, lipogenesis, and mitochondrial ß-oxidation. Extracellular lysophosphatidic acid (LPA) regulates a broad range of cellular processes in development, tissue injury, and cancer. In the present study, we examined the roles of LPA in steatohepatitis induced by a methionine-choline-deficient (MCD) diet in mice. Hepatocytes express LPA receptor (Lpar) 1-3 mRNAs. Steatosis developed in mice fed the MCD diet was reduced by treatment with inhibitors for pan-LPAR or LPAR1. Hepatocyte-specific deletion of the Lpar1 gene also reduced the steatosis in the MCD model. Deletion of the Lpar1 gene in hepatocytes reduced expression of Cd36, a gene encoding a fatty acid transporter. Although LPA/LPAR1 signaling induces expression of Srebp1 mRNA in hepatocytes, LPA does not fully induce expression of SREBP1-target genes involved in lipogenesis. Human hepatocytes repopulated in chimeric mice are known to develop steatosis and treatment with an LPAR1 inhibitor reduces expression of CD36 mRNA and steatosis. Our data indicate that antagonism of LPAR1 reduces steatosis in mouse and human hepatocytes by down-regulation of Cd36.

Humanized liver mouse model with transplanted human hepatocytes from patients with ornithine transcarbamylase deficiency.

Ornithine transcarbamylase deficiency (OTCD) is a metabolic and genetic disease caused by dysfunction of the hepatocytic urea cycle. To develop new drugs or therapies for OTCD, it is ideal to use models that are more closely related to human metabolism and pathology. Primary human hepatocytes (HHs) isolated from two patients (a 6-month-old boy and a 5-year-old girl) and a healthy donor were transplanted into host mice (hemi-, hetero-OTCD mice, and control mice, respectively). HHs were isolated from these mice and used for serial transplantation into the next host mouse or for in vitro experiments. Histological, biochemical, and enzyme activity analyses were performed. Cultured HHs were treated with ammonium chloride or therapeutic drugs. Replacement rates exceeded 80% after serial transplantation in both OTCD mice. These highly humanized OTCD mice showed characteristics similar to OTCD patients that included increased blood ammonia levels and urine orotic acid levels enhanced by allopurinol. Hemi-OTCD mice showed defects in OTC expression and significantly low enzymatic activities, while hetero-OTCD mice showed residual OTC expression and activities. A reduction in ammonium metabolism was observed in cultured HHs from OTCD mice, and treatment with the therapeutic drug reduced the ammonia levels in the culture medium. In conclusion, we established in vivo OTC mouse models with hemi- and hetero-patient HHs. HHs isolated from the mice were useful as an in vitro model of OTCD. These OTC models could be a source of valuable patient-derived hepatocytes that would enable large scale and reproducible experiments using the same donor.

Culture density contributes to hepatic functions of fresh human hepatocytes isolated from chimeric mice with humanized livers: Novel, long-term, functional two-dimensional in vitro tool for developing new drugs.

Chimeric mice with humanized livers are considered a useful animal model for predicting human (h-) drug metabolism and toxicity. In this study, the characteristics of fresh h-hepatocytes (cFHHs, PXB-cells®) isolated from chimeric mice (PXB-mice®) were evaluated in vitro to confirm their utility for drug development. cFHHs cultured at high density (2.13 × 105 cells/cm2) displayed stable production of h-albumin and cytochrome P450 (CYP) 3A activities for at least 21 days. The mRNA expression levels of 10 of 13 CYP, UDP-glucuronosyltransferase (UGT), and transporters were maintained at >10% of the levels of freshly isolated cFHHs after 21 days. From 1 week, many bile canaliculi were observed between cFHHs, and the accumulation of the multidrug resistance-associated protein and bile salt export pump substrates in these bile canaliculi was clearly inhibited by cyclosporin A. Microarray analysis of cFHHs cultured at high density and at low density (0.53 × 105 cells/cm2) revealed that high density culture maintained high expressions of some transcription factors (HNF4α, PXR, and FXR) perhaps involved in the high CYP, UGT and transporter gene expressions of cFHHs. These results strongly suggest that cFHHs could be a novel in vitro tool for drug development studies.

Detection of acute toxicity of aflatoxin B1 to human hepatocytes in vitro and in vivo using chimeric mice with humanized livers.

Aflatoxin B1 (AFB1), a mycotoxin, is acutely hepatotoxic to many animals including humans. However, there are marked interspecies differences in sensitivity to AFB1-induced toxicity depending on bioactivation by cytochrome P450s (CYPs). In the present study, we examined the applicability of chimeric mice with humanized livers and derived fresh human hepatocytes for in vivo and vitro studies on AFB1 cytotoxicity to human hepatocytes. Chimeric mice with highly humanized livers and SCID mice received daily injections of vehicle (corn oil), AFB1 (3 mg/kg), and carbon tetrachloride (50 mg/kg) for 2 days. Histological analysis revealed that AFB1 promoted hepatocyte vacuolation and inflammatory cell infiltration in the area containing human hepatocytes. A novel human alanine aminotransferase 1 specific enzyme-linked immunosorbent assay demonstrated the acute toxicity of AFB1 to human hepatocytes in the chimeric mouse livers. The sensitivity of cultured fresh human hepatocytes isolated from the humanized liver mice for AFB1 cytotoxicity was comparable to that of primary human hepatocytes. Long-term exposure to AFB1 (6 or 14 days) produced a more severe cytotoxicity. The half-maximal lethal concentration was 10 times lower in the 2-week treatment than after 2 days of exposure. Lastly, the significant reduction of AFB1 cytotoxicity by a pan-CYP inhibitor or transfection with CYP3A4 specific siRNA clearly suggested that bioactivation of AFB1 catalyzed by CYPs was essential for AFB1 cytotoxicity to the human hepatocytes in our mouse model. Collectively, our results implicate the humanized liver mice and derived fresh human hepatocytes are useful models for studies of AFB1 cytotoxicity to human hepatocytes.

Changes in Bile Acid Concentrations after Administration of Ketoconazole or Rifampicin to Chimeric Mice with Humanized Liver.

Drug-induced liver injury (DILI) is a common side effect of several medications and is considered a major factor responsible for the discontinuation of drugs during their development. Cholestasis is a DILI that results from impairment of bile acid transporters, such as the bile salt export pump (BSEP), leading to accumulation of bile acids. Both in vitro and in vivo studies are required to predict the risk of drug-induced cholestasis. In the present study, we used chimeric mice with humanized liver as a model to study drug-induced cholestasis. Administration of a single dose of ketoconazole or rifampicin, known to potentially cause cholestasis by inhibiting BSEP, did not result in elevated levels of alkaline phosphatase (ALP), which are known hepatic biomarkers. The concentration of taurodeoxycholic acid increased in the liver after ketoconazole administration, whereas rifampicin resulted in increased tauromuricholic acid and taurocholic acid (TCA) levels in the liver and plasma. Furthermore, rifampicin resulted in an increase in the uniform distribution of a compound with m/z 514.3, presumed as TCA through imaging mass spectrometry. The mRNA levels of bile acid-related genes were also altered after treatment with ketoconazole or rifampicin. We believe these observations to be a part of a feedback mechanism to decrease bile acid concentrations. The changes in bile acid concentrations results may reflect the initial responses of the human body to cholestasis. Furthermore, these findings may contribute to the screening of drug candidates, thereby avoiding drug-induced cholestasis during clinical trials and drug development.

Inhibitory effects of drugs on the metabolic activity of mouse and human aldehyde oxidases and influence on drug-drug interactions.

As aldehyde oxidase (AOX) plays an emerging role in drug metabolism, understanding its significance for drug-drug interactions (DDI) is important. Therefore, we tested 10 compounds for species-specific and substrate-dependent differences in the inhibitory effect of AOX activity using genetically engineered HEK293 cells over-expressing human AOX1, mouse AOX1 or mouse AOX3. The IC50 values of 10 potential inhibitors of the three AOX enzymes were determined using phthalazine and O6-benzylguanine as substrates. 17ß-Estradiol, menadione, norharmane and raloxifene exhibited marked differences in inhibitory effects between the human and mouse AOX isoforms when the phthalazine substrate was used. Some of the compounds tested exhibited substrate-dependent differences in their inhibitory effects. Docking simulations with human AOX1 and mouse AOX3 were conducted for six representative inhibitors. The rank order of the minimum binding energy reflected the order of the corresponding IC50 values. We also evaluated the potential DDI between an AOX substrate (O6-benzylguanine) and an inhibitor (hydralazine) using chimeric mice with humanized livers. Pretreatment of hydralazine increased the maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve (AUC0-24) of O6-benzylguanine compared to single administration. Our in vitro data indicate species-specific and substrate-dependent differences in the inhibitory effects on AOX activity. Our in vivo data demonstrate the existence of a DDI which may be of relevance in the clinical context.

Novel robust in vitro hepatitis B virus infection model using fresh human hepatocytes isolated from humanized mice.

The molecular mechanisms underlying the hepatitis B virus (HBV) life cycle are poorly understood because of the lack of appropriate in vitro infection models. Herein, we report a highly effective in vitro HBV infection system using fresh human hepatocytes (HHs) isolated from chimeric mice with humanized livers. After the inoculation of sera collected from HBV-infected chimeric mice or patients to HHs, we measured levels of HBV DNA, mRNA, covalently closed circular DNA, and viral protein expression in HHs. We investigated the neutralization activity of hepatitis B immune globulin and the effects of siRNA against sodium taurocholate-cotransporting polypeptide and clathrin heavy chain on HBV infection. We confirmed the expression of viral antigens in HHs and the presence of extracellular HBV DNA and hepatitis B surface antigen. The maximum infection rate was approximately 80%. Lamivudine and hepatitis B immune globulin treatment reduced HBV DNA levels in a dose-dependent manner. Knockdown of sodium taurocholate-cotransporting polypeptide and clathrin heavy chain significantly reduced the levels of hepatitis B surface antigen. Infection was successfully established using different donor HHs and inocula. Elevation of extracellular HBV DNA levels and the increase of HBV-positive HHs were blocked by continuous hepatitis B immune globulin treatment, indicating virus spread in this model. Chimeric mouse-derived HHs provide a robust in vitro infection model that can completely support the HBV life cycle.

Morphological and microarray analyses of human hepatocytes from xenogeneic host livers.

We previously produced mice with human hepatocyte (h-hep) chimeric livers by transplanting h-heps into albumin enhancer/promoter-driven urokinase-type plasminogen activator-transgenic severe combined immunodeficient (SCID) mice with liver disease. The chimeric livers were constructed with h-heps, mouse hepatocytes, and mouse hepatic sinusoidal cells (m-HSCs). Here, we investigated the morphological features of the chimeric livers and the h-hep gene expression profiles in the xenogeneic animal body. To do so, we performed immunohistochemistry, morphometric analyses, and electron microscopic observations on chimeric mouse livers, and used microarray analyses to compare gene expression patterns in hepatocytes derived from chimeric mouse hepatocytes (c-heps) and h-heps. Morphometric analysis revealed that the ratio of hepatocytes to m-HSCs in the chimeric mouse livers were twofold higher than those in the SCID mouse livers, corresponding to twin-cell plates in the chimeric mouse liver. The h-heps in the chimeric mouse did not show hypoxia even in the twin-cell plate structure, probably because of low oxygen consumption by the h-heps relative to the mouse hepatocytes (m-heps). Immunohistochemical and electron microscopic examinations revealed that the sinusoids in the chimeric mouse livers were normally constructed with h-heps and m-HSCs. However, a number of microvilli projected into the intercellular clefts on the lateral aspects of the hepatocytes, features typical of a growth phase. Microarray profiles indicated that ∼82% of 16 605 probes were within a twofold range difference between h-heps and c-heps. Cluster and principal component analyses showed that the gene expression patterns of c-heps were extremely similar to those of h-heps. In conclusion, the chimeric mouse livers were normally reconstructed with h-heps and m-HSCs, and expressed most human genes at levels similar to those in human livers, although the chimeric livers showed morphological characteristics typical of growth.

Generation and characterization of severe combined immunodeficiency rats.

Severe combined immunodeficiency (SCID) mice, the most widely used animal model of DNA-PKcs (Prkdc) deficiency, have contributed enormously to our understanding of immunodeficiency, lymphocyte development, and DNA-repair mechanisms, and they are ideal hosts for allogeneic and xenogeneic tissue transplantation. Here, we use zinc-finger nucleases to generate rats that lack either the Prkdc gene (SCID) or the Prkdc and Il2rg genes (referred to as F344-scid gamma [FSG] rats). SCID rats show several phenotypic differences from SCID mice, including growth retardation, premature senescence, and a more severe immunodeficiency without "leaky" phenotypes. Double-knockout FSG rats show an even more immunocompromised phenotype, such as the abolishment of natural killer cells. Finally, xenotransplantation of human induced pluripotent stem cells, ovarian cancer cells, and hepatocytes shows that SCID and FSG rats can act as hosts for xenogeneic tissue grafts and stem cell transplantation and may be useful for preclinical testing of new drugs.