Induction of drug metabolizing enzyme and drug transporter expression by antifungal triazole pesticides in human HepaSH hepatocytes.

Triazole pesticides are widely used fungicides, to which humans are rather highly exposed. They are known to activate drug-sensing receptors regulating expression of hepatic drug metabolizing enzymes and drug transporters, thus suggesting that the hepatic drug detoxification system is modified by these agrochemicals. To investigate this hypothesis, the effects of 9 triazole fungicides towards expression of drug metabolizing enzymes and transporters were characterized in cultured human HepaSH cells, that are human hepatocytes deriving from chimeric humanized liver TK-NOG mice. Most of triazoles used at 10 µM were found to act as inducers of cytochrome P-450 (CYP) 1A1, CYP1A2, CYP2B6, CYP3A4 and UDP-glucuronosyltransferase 1A1 mRNA levels and of CYP3A4 protein; some triazoles also enhanced mRNA expression of the canalicular transporters P-glycoprotein/MDR1, multidrug resistance-associated protein 2 and breast cancer resistance protein. Triazoles however concomitantly inhibited CYP2B6 and CYP3A4 activities and thus appeared as dual regulators of these CYPs, being both inducers of their expression and inhibitors of their activity. The inducing effect however predominated, at least for bromuconazole, propiconazole and tebuconazole. Bromuconazole was moreover predicted to enhance CYP2B6 and CYP3A4 expression in humans exposed to this fungicide in a chronic, acute or occupational context. These data demonstrate that key-actors of the human hepatic detoxification system are impacted by triazole pesticides, which may have to be considered for the risk assessment of these agrochemicals. They additionally highlight that the use of human HepaSH cells as surrogates to primary human hepatocytes represents an attractive and promising way for studying hepatic effects of environmental chemicals.

Systemic identification of functionally conserved lncRNA metabolic regulators in human and mouse livers.

BACKGROUND & AIMS: Unlike protein-coding genes, the majority of human long non-coding RNAs (lncRNAs) lack conservation based on their sequences, posing a challenge for investigating their role in a pathophysiological context for clinical translation. This study explores the hypothesis that non-conserved lncRNAs in human and mouse livers may share similar metabolic functions, giving rise to functionally conserved lncRNA metabolic regulators (fcLMRs). METHODS: We developed a sequence-independent strategy to select putative fcLMRs, and performed extensive analysis to determine the functional similarities of putative human and mouse LMR pairs (h/mLMRs). RESULTS: We found that several pairs of putative fcLMRs share similar functions in regulating gene expression. We further demonstrated that a pair of fcLMRs, h/mLMR1, robustly regulated triglyceride levels by modulating the expression of a similar set of lipogenic genes. Mechanistically, h/mLMR1 binds to PABPC1, a regulator of protein translation, via short motifs on either lncRNA with divergent sequences but similar structures. This interaction inhibits protein translation, activating an amino acid-mTOR-SREBP1 axis to regulate lipogenic gene expression. Intriguingly, PABPC1-binding motifs on each lncRNA fully rescued the functions of their corresponding LMRs in the opposite species. Given the elevated expression of h/mLMR1 in humans and mice with hepatic steatosis, the PABPC1-binding motif on hLMR1 emerges as a potential non-conserved human drug target whose functions can be fully validated in a physiologically relevant setting before clinical studies. CONCLUSIONS: Our study supports that fcLMRs represent a novel and prevalent biological phenomenon, and deep phenotyping of genetic mLMR mouse models constitutes a powerful approach to understand the pathophysiological role of lncRNAs in the human liver.

Urinary bladder carcinogenic potential of 4,4'-methylenebis(2-chloroaniline) in humanized-liver mice.

Occupational exposure to 4,4'-methylenebis(2-chloroaniline) (MOCA) has been linked to an increased risk of bladder cancer among employees in Japanese plants, indicating its significance as a risk factor for urinary bladder cancer. To investigate the role of MOCA metabolism in bladder carcinogenesis, we administered MOCA to non-humanized (F1-TKm30 mice) and humanized-liver mice for 4 and 28 weeks. We compared MOCA-induced changes in metabolic enzyme expression, metabolite formation, and effects on the urinary bladder epithelium in the two models. At week 4, MOCA exposure induced simple hyperplasia, cell proliferation, and DNA damage in the urothelium of the humanized-liver mice, while in the non-humanized mice these effects were not observed. Notably, the concentration of 4-amino-4'-hydroxylamino-3,3'-dichlorodiphenylmethane (N-OH-MOCA) in the urine of humanized-liver mice was more than 10 times higher than that in non-humanized mice at the 4-week mark. Additionally, we observed distinct differences in the expression of cytochrome P450 isoforms between the two models. Although no bladder tumors were detected after 28 weeks of treatment in either group, these findings suggest that N-OH-MOCA significantly contributes to the carcinogenic potential of MOCA in humans.

Impact of miR-222-3p-mediated downregulation of arylacetamide deacetylase on drug hydrolysis and lipid accumulation.

Arylacetamide deacetylase (AADAC) is involved in drug hydrolysis and lipid metabolism. In 23 human liver samples, no significant correlation was observed between AADAC mRNA (19.7-fold variation) and protein levels (137.6-fold variation), suggesting a significant contribution of post-transcriptional regulation to AADAC expression. The present study investigated whether AADAC is regulated by microRNA in the human liver and elucidate its biological significance. Computational analysis predicted two potential miR-222-3p recognition elements in the 3'-untranslated region (UTR) of AADAC. Luciferase assay revealed that the miR-222-3p recognition element was functional in downregulating AADAC expression. In HEK293 cells transfected with an AADAC expression plasmid containing 3'-UTR, miR-222-3p overexpression decreased AADAC protein level and activity, whereas miR-222-3p inhibition increased them. Similar results were observed in human hepatoma-derived Huh-1 cells endogenously expressing AADAC and HepaSH cells that are hepatocytes from chimeric mice with humanized livers. In individual human liver samples, AADAC protein levels inversely correlated with miR-222-3p levels. Overexpression of miR-222-3p resulted in increased lipid accumulation in Huh-1 cells, which was reversed by AADAC overexpression. In contrast, miR-222-3p inhibition decreased lipid accumulation, which was reversed by AADAC knockdown. In conclusion, we found that hepatic AADAC was downregulated by miR-222-3p, resulting in decreased drug hydrolysis and increased lipid accumulation.

In Vivo and In Vitro Induction of Cytochrome P450 3A4 by Thalidomide in Humanized-Liver Mice and Experimental Human Hepatocyte HepaSH cells.

Autoinduction of cytochrome P450 (P450) 3A4-mediated metabolism of thalidomide was investigated in humanized-liver mice and human hepatocyte-derived HepaSH cells. The mean plasma ratios of 5-hydroxythalidomide and glutathione adducts to thalidomide were significantly induced (3.5- and 6.0-fold, respectively) by thalidomide treatment daily at 1000 mg/kg for 3 days and measured at 2 h after the fourth administration (on day 4). 5-Hydroxythalidomide was metabolically activated by P450 3A4 in HepaSH cells pretreated with 300 and 1000 µM thalidomide, and 5,6-dihydroxythalidomide was detected. Significant induction of P450 3A4 mRNA expression (4.1-fold) in the livers of thalidomide-treated mice occurred. Thalidomide exerts a variety of actions through multiple mechanisms following bioactivation by induced human P450 3A enzymes.

A mechanistic biomarker investigation of fialuridine hepatotoxicity using the chimeric TK-NOG Hu-liver mouse model and in vitro micropatterned hepatocyte cocultures.

Fialuridine (FIAU) is a nucleoside-based drug that caused liver failure and deaths in a human clinical trial that were not predicted by nonclinical safety studies. A recent report concluded that a TK-NOG humanized liver (hu-liver) mouse model detected human-specific FIAU liver toxicity, and broader use of that model could improve drug safety testing. We further evaluated this model at similar dose levels to assess FIAU sensitivity and potential mechanistic biomarkers. Although we were unable to reproduce the marked acute liver toxicity with two separate studies (including one with a "sensitized" donor), we identified molecular biomarkers reflecting the early stages of FIAU mitochondrial toxicity, which were not seen with its stereoisomer (FIRU). Dose dependent FIAU-induced changes in hu-liver mice included more pronounced reductions in mitochondrial to nuclear DNA (mtDNA/nucDNA) ratios in human hepatocytes compared to mouse hepatocytes and kidneys of the same animals. FIAU treatment also triggered a p53 transcriptional response and opposing changes in transcripts of nuclear- and mitochondrial-encoded mitochondrial proteins. The time dependent accumulation of FIAU into mtDNA is consistent with the ≥9-week latency of liver toxicity observed for FIAU in the clinic. Similar changes were observed in an in vitro micro-patterned hepatocyte coculture system. In addition, FIAU-dependent mtDNA/nucDNA ratio and transcriptional alterations, especially reductions in mitochondrially encoded transcripts, were seen in livers of non-engrafted TK-NOG and CD-1 mice dosed for a shorter period. Conclusion: These mechanistic biomarker findings can be leveraged in an in vitro model and in a more routine preclinical model (CD-1 mice) to identify nucleosides with such a FIAU-like mitochondrial toxicity mechanistic liability potential. Further optimization of the TK-NOG hu-liver mouse model is necessary before broader adoption for drug safety testing.

Comprehensive gene profiling of the metabolic landscape of humanized livers in mice.

BACKGROUND & AIMS: The human liver transcriptome is complex and highly dynamic, e.g. one gene may produce multiple distinct transcripts, each with distinct posttranscriptional modifications. Direct knowledge of transcriptome dynamics, however, is largely obscured by the inaccessibility of the human liver to treatments and the insufficient annotation of the human liver transcriptome at transcript and RNA modification levels. METHODS: We generated mice that carry humanized livers of identical genetic background and subjected them to representative metabolic treatments. We then analyzed the humanized livers with nanopore single-molecule direct RNA sequencing to determine the expression level, m6A modification and poly(A) tail length of all RNA transcript isoforms. Our system allows for the de novo annotation of human liver transcriptomes to reflect metabolic responses and for the study of transcriptome dynamics in parallel. RESULTS: Our analysis uncovered a vast number of novel genes and transcripts. Our transcript-level analysis of human liver transcriptomes also identified a multitude of regulated metabolic pathways that were otherwise invisible using conventional short-read RNA sequencing. We revealed for the first time the dynamic changes in m6A and poly(A) tail length of human liver transcripts, many of which are transcribed from key metabolic genes. Furthermore, we performed comparative analyses of gene regulation between humans and mice, and between two individuals using the liver-specific humanized mice, revealing that transcriptome dynamics are highly species- and genetic background-dependent. CONCLUSION: Our work revealed a complex metabolic response landscape of the human liver transcriptome and provides a novel resource to understand transcriptome dynamics of the human liver in response to physiologically relevant metabolic stimuli (https://caolab.shinyapps.io/human_hepatocyte_landscape/). IMPACT AND IMPLICATIONS: Direct knowledge of the human liver transcriptome is currently very limited, hindering the overall understanding of human liver pathophysiology. We combined a liver-specific humanized mouse model and long-read direct RNA sequencing technology to establish a de novo annotation of the human liver transcriptome and identified a multitude of regulated metabolic pathways that were otherwise invisible using conventional technologies. The extensive regulatory information on human genes we provided could enable basic scientists to infer the pathological relevance of their genes of interest and physician scientists to better pinpoint the changes in metabolic networks underlying a specific pathophysiology.

Empirical scaling factor for predicting human pharmacokinetic profiles of disproportionate metabolites using the Css-MRTpo method and chimeric mice with humanised livers.

Predicting plasma concentration-time profiles of disproportionate metabolites in humans is crucial for evaluating metabolites according to the Safety Testing guidelines. We evaluated Css-MRTpo, an empirical method, using chimeric mice with humanised livers capable of generating human-disproportionate metabolites. Azilsartan and AZ-M2 were administered to humanised chimeric mice, and pharmacokinetic parameters were obtained. Pharmacokinetic data for DS-1971a and DS-M1 in humanised chimeric mice were obtained from the literature. The human plasma concentration-time profiles of these compounds were simulated using the Css-MRTpo method. Azilsartan, DS-1971a, and PF-04937319 produced human disproportionate metabolites, AZ-M2, DS-M1, and PF-M1, respectively. The predicted human pharmacokinetic profiles of PF-04937319 and PF-M1 were obtained from a previous study, and their outcomes were re-evaluated. Our findings revealed that the plasma concentrations of the three metabolites were unexpectedly underpredicted, whereas the three unchanged drugs were reasonably predicted. Further, the introduction of the empirical scaling factor of 3, obtained from six model compounds, improved the predictability of metabolites, suggesting the potential usefulness of the Css-MRTpo method in combination with humanised chimeric mice for predicting the pharmacokinetic profiles of disproportionate metabolites at the early stage of new drug development.

SGX523 causes renal toxicity through aldehyde oxidase-mediated less-soluble metabolite formation in chimeric mice with humanized livers.

SGX523 is a c-Met tyrosine kinase inhibitor that failed in clinical trials because of renal toxicity caused by crystal deposits in renal tubules. SGX523 is metabolized by aldehyde oxidase (AOX) in a species-dependent manner to the considerably less soluble 2-quinolinone-SGX523, which is likely involved in the clinically observed obstructive nephropathy. This study investigated the metabolism and renal toxicity of SGX523 in chimeric mice with humanized livers (humanized-liver mice). The 2-quinolinone-SGX523 formation activity was higher in humanized-liver mouse and human hepatocytes than in mouse hepatocytes. Additionally, this activity in the liver cytosolic fraction from humanized-liver mice was inhibited by the AOX inhibitors raloxifene and hydralazine. After oral SGX523 administration, higher maximum concentrations, larger areas under the plasma concentration versus time curves, and higher urinary concentrations of 2-quinolinone-SGX523 were observed in humanized-liver mice than in non-humanized mice. Serum creatinine and blood urea nitrogen levels were elevated in humanized-liver mice following repeated oral SGX523 administration. The accumulation of amorphous material in the tubules and infiltration of inflammatory cells around tubules were observed in the kidneys of humanized-liver mice after repeated oral SGX523 administration. These findings demonstrate that humanized-liver mice are useful for understanding the metabolism and toxicity of SGX523.

Cytochrome P450 1A2 and 2C enzymes autoinduced by omeprazole in dog hepatocytes and human HepaRG and HepaSH cells are involved in omeprazole 5-hydroxylation and sulfoxidation.

The induction assay for the cytochromes P450 (P450s) is an important tool in drug discovery and development. The inductions of dog P450 1A2 and 3A12 by omeprazole and rifampicin were functionally characterised in dog hepatocytes and were compared with induction in human HepaRG and HepaSH cells.P450 1A2-dependent ethoxyresorufin O-deethylation was induced by R,S-omeprazole and P450 3 A-dependent midazolam 1'-hydroxylation was induced by rifampicin, and both reactions were significantly enhanced in cultured dog hepatocytes and human HepaRG and HepaSH cells.Recombinant dog P450 1A2 exhibited activities towards R- and S-omeprazole 5-hydroxylation with low Km values of 23-28 µM, whereas dog P450 2C21 and 3A12 efficiently mediated S-omeprazole 5-hydroxylation and sulfoxidation, respectively, with high Vmax values of 12-17 min-1.Although omeprazole 5-hydroxylation by human P450 2C19 (and sulfoxidation by P450 3A4) in human HepaSH cells were slightly (∼2-fold) induced by R,S-omeprazole, dog P450 1A2 was autoinduced by omeprazole in dog hepatocytes and showed enhanced R-omeprazole 5-hydroxylation activity (∼5-fold).These results indicate that omeprazole can be an autoinducer of P450 1A2 in hepatocytes, and this enzyme was found to be involved in omeprazole 5-hydroxylation and sulfoxidation in dog hepatocytes and human HepaRG and HepaSH cells.

Pharmacokinetic Models Scaled-up from Humanized-liver Mouse Data Can Account for Drug Monitoring Results of Atomoxetine and Its 4-Hydroxylated and N-Demethylated Metabolites i n Pediatric Patients Genotyped for Cytochrome P450 2D6.

Atomoxetine is a cytochrome P450 (P450) 2D6 probe substrate and an approved medicine for attention-deficit/hyperactivity disorder. In this humanized-liver mouse study, interactions between atomoxetine and the P450 2D6 probe drug paroxetine were observed. Human physiologically based pharmacokinetic (PBPK) models were established by scaling up humanized-liver mouse data obtained in the absence or presence of paroxetine. These models could explain the drug monitoring results of atomoxetine and its primary 4-hydroxylated and N-demethylated metabolites in Japanese children aged 8-14 years and could be used to help establish the correct dosage and for the evaluation of clinical outcomes. The results of simple PBPK models (using input parameters that reflected the subjects' small body size and normal or reduced P450 2D6-dependent clearance) were in general agreement with one-point measured plasma concentrations of atomoxetine and its 4-hydroxylated and N-demethylated metabolites in 13 pediatric participants. Unexpectedly high hepatic exposure, possibly in intermediate-metabolizer patients harboring CYP2D6*10 or 2D6*36 alleles, might in part explain the adverse effects of atomoxetine prescribed alone recorded in a Japanese adverse-event database. The steady-state, one-point drug monitoring data from the participants indicated extensive biotransformation of atomoxetine to 4-hydroxyatomoxetine under individually prescribed doses of atomoxetine. These results also suggest that a relatively narrow range of 4-hydroxyatomoxetine and N-desmethylatomoxetine concentration ratios in spot urine and/or plasma samples from pediatric patients could be a simple semiquantitative determinant factor for P450 2D6 intermediate metabolizers, compared with the wide range of concentrations of the two primary metabolites and substrate in extensive metabolizers. Significance Statement Validated simple pharmacokinetic models are able to predict steady-state plasma concentrations of the approved medicine atomoxetine and its primary metabolites in the majority of pediatric patients. The package insert advises careful dose escalation, especially for poor metabolizers; however, no simple way exists to determine P450 2D6 phenotypes. A relatively narrow range ratio of 4-hydroxyatomoxetine and N-desmethylatomoxetine in spot urine/plasma samples could be a simple semi-quantitative determinant factor for P450 2D6 intermediate metabolizers to optimize or confirm the correct dosage.

Drug transporter expression and activity in cryopreserved human hepatocytes isolated from chimeric TK-NOG mice with humanized livers.

Chimeric mice with humanized liver are thought to represent a sustainable source of isolated human hepatocytes for in vitro studying detoxification of drugs in humans. Because drug transporters are now recognized as key-actors of the hepatic detoxifying process, the present study was designed to characterize mRNA expression and activity of main hepatic drug transporters in cryopreserved human hepatocytes isolated from chimeric TK-NOG mice and termed HepaSH cells. Such cells after thawing were shown to exhibit a profile of hepatic solute carrier (SLC) and ATP-binding cassette (ABC) drug transporter mRNA levels well correlated to those found in cryopreserved primary human hepatocytes or human livers. HepaSH cells used either as suspensions or as 24 h-cultures additionally displayed notable activities of uptake SLCs, including organic anion transporting polypeptides (OATPs), organic anion transporter 2 (OAT2) or sodium-taurocholate co-transporting polypeptide (NTCP). SLC transporter mRNA expression, as well as SLC activities, nevertheless fell in HepaSH cells cultured for 120 h, which may reflect a partial dedifferentiation of these cells with time in culture in the conventional monolayer culture conditions used in the study. These data therefore support the use of cryopreserved HepaSH cells as either suspensions or short-term cultures for drug transport studies.

Roles of human cytochrome P450 3A4/5 in dexamethasone 6ß-hydroxylation mediated by liver microsomes and humanized liver in chimeric mice metabolically suppressed with azamulin.

The urinary metabolic ratio of 6ß-hydroxydexamethasone to dexamethasone reportedly acts as a noninvasive marker for human cytochrome P450 (P450) 3A4/5, which is induced by rifampicin in humanized-liver mice. In the current study, the pharmacokinetics of dexamethasone in humanized-liver mice after intravenous administration (10 mg/kg) were investigated using azamulin (a time-dependent P450 3A4/5 inhibitor). After intravenous dexamethasone administration, significant differences were observed in the time-dependent plasma and 24-h urinary concentrations of 6ß-hydroxydexamethasone between untreated humanized-liver mice and humanized-liver mice treated with azamulin (daily oral doses of 15 mg/kg for 3 days). The mean ratios of 6ß-hydroxydexamethasone to dexamethasone for the maximum concentrations, the areas under the plasma concentration-versus-time curves, and urinary concentrations were significantly lower in the azamulin-treated group (59%, 58%, and 41% of the untreated values, respectively). 6ß-Hydroxydexamethasone formation was suppressed by 93% by replacing control human liver microsomes with P450 3A4/5-inactivated liver microsomes. These results suggest that the oxidation of dexamethasone in humans is mediated mainly by P450 3A4/5 (which is suppressed by azamulin), and that humanized-liver mice orally treated with azamulin may constitute an in vivo model for metabolically inactivated P450 3A4/5 in human hepatocytes transplanted into chimeric mice.

HepaSH cells: Experimental human hepatocytes with lesser inter-individual variation and more sustainable availability than primary human hepatocytes.

Primary human hepatocytes (PHHs) have been commonly used as the gold standard in many drug metabolism studies, regardless of having large inter-individual variation. These inter-individual variations in PHHs arise primarily from genetic polymorphisms, as well as from donor health conditions and storage conditions prior to cell processing. To equalize the effects of the latter two factors, PHHs were transplanted to quality-controlled mice providing human hepatocyte proliferation niches, and engrafted livers were generated. Cells that were harvested from engrafted livers, call this as experimental human hepatocytes (EHH; termed HepaSH cells), were stably and reproducibly produced from 1014 chimeric mice produced by using 17 different PHHs. Expression levels of acute phase reactant (APR) genes as indicators of a systemic reaction to the environmental/inflammatory insults of liver donors varied widely among PHHs. In contrast to PHHs, the expression of APR genes in HepaSH cells was found to converge within a narrower range than in donor PHHs. Further, large individual differences in the expression levels of drug metabolism-related genes (28 genes) observed in PHHs were greatly reduced among HepaSH cells produced in a unified in vivo environment, and none deviated from the range of gene expression levels in the PHHs. The HepaSH cells displayed a similar level of drug-metabolizing enzyme activity and gene expression as the average PHHs but retained their characteristics for drug-metabolizing enzyme gene polymorphisms. Furthermore, long-term 2D culture was possible and HBV infection was confirmed. These results suggest that the stably and reproducibly providable HepaSH cells with lesser inter-individual differences in drug-metabolizing properties, may have a potential to substitution for PHH as practical standardized human hepatocytes in drug discovery research.

o-Toluidine metabolism and effects in the urinary bladder of humanized-liver mice.

Occupational exposure to aromatic amines is one of the most important risk factors for urinary bladder cancer. When considering the carcinogenesis of aromatic amines, metabolism of aromatic amines in the liver is an important factor. In the present study, we administered ortho-toluidine (OTD) in the diet to mice for 4 weeks. We used NOG-TKm30 mice (control) and humanized-liver mice, established via human hepatocyte transplantation, to compare differences in OTD-induced expression of metabolic enzymes in human and mouse liver cells. We also investigated OTD-urinary metabolites and proliferative effects on the urinary bladder epithelium. RNA and immunohistochemical analyses revealed that expression of N-acetyltransferases mRNA in the liver tended to be lower than that of the P450 enzymes, and that OTD administration had little effect on N-acetyltransferase mRNA expression levels. However, expression of CYP3A4 was increased in the livers of humanized-liver mice, and expression of Cyp2c29 (human CYP2C9/19) was increased in the livers of NOG-TKm30 mice. OTD metabolites in the urine and cell proliferation activities in the bladder urothelium of NOG-TKm30 and humanized-liver mice were similar. However, the concentration of OTD in the urine of NOG-TKm30 mice was markedly higher than in the urine of humanized-liver mice. These data demonstrate differences in hepatic metabolic enzyme expression induced by OTD in human and mouse liver cells, and consequent differences in the metabolism of OTD by human and mouse liver cells. This type of difference could have a profound impact on the carcinogenicity of compounds that are metabolized by the liver, and consequently, would be important in the extrapolation of data from animals to humans.

The Unique Human N10-Glucuronidated Metabolite Formation from Olanzapine in Chimeric NOG-TKm30 Mice with Humanized Livers.

Olanzapine is an antipsychotic agent with species-dependent pharmacokinetic profiles in both humans and animals. In the present study, the metabolic profiles of olanzapine in vitro and in vivo were compared in non-transplanted immunodeficient NOG-TKm30 mice and chimeric mice with humanized livers (hereafter humanized-liver mice). Hepatic microsomal fractions prepared from humanized-liver mice and humans mediated olanzapine N10-glucuronidation, whereas fractions from cynomolgus monkeys, marmosets, minipigs, dogs, rabbits, guinea pigs, rats, CD1 mice, and NOG-TKm30 mice did not. The olanzapine N10-glucuronidation activity in liver microsomes from humanized-liver mice was inhibited by hecogenin, a human UDP-glucuronosyltransferase (UGT) 1A4 inhibitor. In addition, hepatocytes from humanized-liver mice suggest that olanzapine N10-glucuronidation was a major metabolic pathway in the livers of humanized-liver mice. After a single oral dose of olanzapine (10 mg/kg body weight) to humanized-liver mice and control NOG-TKm30 mice, olanzapine N10-glucuronide isomers and olanzapine N4'-glucuronide were detected only in the plasma of humanized-liver mice. In contrast, the area under the curve for N4'-demethylolanzapine, 2-hydroxymethylolanzapine, and 7-hydroxyolanzapine glucuronide was higher in NOG-TKm30 mice than that in humanized-liver mice. The cumulative excreted amounts of olanzapine N10-glucuronide isomers were high in the urine and feces from humanized-liver mice, whereas the cumulative excreted amounts of 2-hydroxymethylolanzapine were higher in NOG-TKm30 mice than in humanized-liver mice. Thus, production of human-specific olanzapine N10-glucuronide was observed in humanized-liver mice, which was consistent with the in vitro glucuronidation data. These results suggest that humanized-liver mice are useful for studying drug oxidation and conjugation of olanzapine in humans. SIGNIFICANCE STATEMENT: Human-specific olanzapine N10-glucuronide isomers were generated in chimeric NOG-TKm30 mice with humanized livers (humanized-liver mice), and high UGT1A4-dependent N10-glucuronidation was observed in the liver microsomes from humanized-liver mice. Hence, humanized-liver mice may be a suitable model for studying UGT1A4-dependent biotransformation of drugs in humans.

TNF receptor-related factor 3 inactivation promotes the development of intrahepatic cholangiocarcinoma through NF-κB-inducing kinase-mediated hepatocyte transdifferentiation.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is a deadly but poorly understood disease, and its treatment options are very limited. The aim of this study was to identify the molecular drivers of ICC and search for therapeutic targets. APPROACH AND RESULTS: We performed a Sleeping Beauty transposon-based in vivo insertional mutagenesis screen in liver-specific Pten -deficient mice and identified TNF receptor-related factor 3 ( Traf3 ) as the most significantly mutated gene in murine ICCs in a loss-of-function manner. Liver-specific Traf3 deletion caused marked cholangiocyte overgrowth and spontaneous development of ICC in Pten knockout and KrasG12D mutant mice. Hepatocyte-specific, but not cholangiocyte-specific, Traf3 -deficient and Pten -deficient mice recapitulated these phenotypes. Lineage tracing and single-cell RNA sequencing suggested that these ICCs were derived from hepatocytes through transdifferentiation. TRAF3 and PTEN inhibition induced a transdifferentiation-like phenotype of hepatocyte-lineage cells into proliferative cholangiocytes through NF-κB-inducing kinase (NIK) up-regulation in vitro. Intrahepatic NIK levels were elevated in liver-specific Traf3 -deficient and Pten -deficient mice, and NIK inhibition alleviated cholangiocyte overgrowth. In human ICCs, we identified an inverse correlation between TRAF3 and NIK expression, with low TRAF3 or high NIK expression associated with poor prognosis. Finally, we showed that NIK inhibition by a small molecule inhibitor or gene silencing suppressed the growth of multiple human ICC cells in vitro and ICC xenografts in vivo. CONCLUSIONS: TRAF3 inactivation promotes ICC development through NIK-mediated hepatocyte transdifferentiation. The oncogenic TRAF3-NIK axis may be a potential therapeutic target for ICC.

Forward and reverse dosimetry for aniline and 2,6-dimethylaniline in humans extrapolated from humanized-liver mouse data using simplified physiologically based pharmacokinetic models.

Although human urinary aniline and 2,6-dimethylaniline were unexpectedly detected in biomonitoring data, little is known about the daily intake doses of aniline and 2,6-dimethylaniline in the living environment or their relation to tolerable daily intake (TDI) values in humans. In the current study, to evaluate the daily oral intake of aniline and 2,6-dimethylaniline in humans, forward and reverse dosimetry was carried out using simplified in silico physiologically based pharmacokinetic (PBPK) modeling established using in vivo experimental pharmacokinetic data. These data were from humanized-liver mice after single oral doses of 100 mg/kg aniline (previously determined) and 116 mg/kg 2,6-dimethylanine (currently investigated). The in vivo elimination rates of 2,6-dimethylaniline from plasma in humanized-liver mice were generally slow compared with those of aniline. Faster in vitro metabolic elimination rates of aniline mediated by liver 9000 × g supernatant fractions from rats than those from humans may suggest the existence of higher first-pass effects in rats than in humanized-liver mice. In silico aniline and 2,6-dimethylaniline concentration curves in human urine after virtual oral administrations were estimated by human PBPK models created with data from humanized-liver mice. Reverse dosimetry analysis using human PBPK models estimated the daily intake of aniline, based on reported human urinary concentrations in biomonitoring data, to be roughly similar to the aniline TDI level. These results suggest that forward and reverse dosimetry using simplified human PBPK models founded on data from humanized-liver mice can be used to evaluate possible higher than expected exposures of aniline and 2,6-dimethylaniline in humans.

Treatment with hepatocyte transplantation in a novel mouse model of persistent liver failure.

Background and Aim: Cell-based transplantation therapy using hepatocytes, hepatic stem cells, hepatocyte-like cells induced from stem cells, etc. is thought to be an attractive alternative to liver transplantation, and have been studied to date. For its clinical application, however, it is extremely important to develop a model that reproduces the pathological conditions with indication for treatment and enables the study for the ideal treatment strategy. Methods: The transgenic mice which express the thymidine kinase (TK) gene of human herpes simplex virus (HSV) in their hepatocytes with normal immunity has been developed (designated as HSVtk). After ganciclovir (GCV) administration which injure TK-expressing hepatocytes, the primary hepatocytes (PHs) isolated from green fluorescent protein (GFP) transgenic mouse (GFP-tg) were transplanted to HSVtk intrasplenically, and replacement index (RI) with transplanted PHs in the liver, liver histology, and mRNA expressions in the liver were analyzed up to 8 weeks after transplantation. Results: HSVtk without PH transplantation after GCV administration developed persistent liver failure with degenerated hepatocytes, persistent elevation of ALT and hepatic p16 mRNA levels, suggesting the existence of cellular senescence in the base of the disease. When autologous GFP-PHs were transplanted to HSVtk, the transplanted cells were successfully engrafted in the liver. Eight weeks after transplantation, serum ALT levels and liver histology were almost normalized, while RIs varied from 19.8 to 73.8%. Since the hepatic p16 mRNA levels were decreased significantly in these mice, the senescence of hepatocytes associated with liver injury was thought to be resolved. On the other hand, allogenic GFP-PHs transplanted to HSVtk were eliminated as early as 1 week after transplantation. In these mice, hepatic p16 mRNA levels were significantly increased at 8 weeks after transplantation, suggesting the aggravation of hepatocyte senescence. FK506 administration to HSVtk protected the transplanted hepatocytes with allogenic background from rejection at 2 weeks after transplantation, but the condition of mice and the senescent status in the liver seemed worsened. Conclusions: The mouse model with HSVtk/GCV system was useful for studying the mechanism of liver regeneration and the immune rejection responses in the hepatocyte transplantation treatment. It may also be utilized to develop the effective remedies to avoid immune rejection.

Formation of reactive metabolites of benzbromarone in humanized-liver mice.

Benzbromarone, a uricosuric drug, has the potential to cause serious hepatotoxicity. Several studies have shown the formation of reactive metabolites of benzbromarone and their association with hepatotoxicity in mice. However, it is unknown whether those reactive metabolites are generated in humans in vivo. In the present study, we firstly investigated the pharmacokinetic profiles of benzbromarone in chimeric TK-NOG mice transplanted with human hepatocytes (humanized-liver mice) and then investigated whether reactive metabolites could be generated. The area under the plasma concentration-time curve ratio of benzbromarone and its major metabolites (benzbromarone: 1'-hydroxy benzbromarone: 6-hydroxy benzbromarone) in humanized-liver mice was 1: 1.2: 0.7, which was similar to that reported in humans. In addition, glutathione conjugates and their further metabolites derived from the epoxidation of the benzofuran ring and 1',6-dihydroxylation of benzbromarone were detected in the livers, urine and plasma. Furthermore, their peak intensities in mass spectrometry showed markedly higher levels compared with those of TK-NOG mice. These results suggested that the metabolic profiles of benzbromarone in humanized-liver mice were similar to those in humans and that the reactive metabolites detected in humanized-liver mice could be generated and are associated with the benzbromarone-induced hepatotoxicity in humans.