Virtual Reality Images of the Home Are Useful for Patients With Hospital-Based Palliative Care: Prospective Observational Study With Analysis by Text Mining.

Background: Malignancy patients who need long-term hospitalization can feel loneliness affecting their quality of life. The global COVID-19 pandemic has caused visiting restrictions that could mean patients who might be missing out on family support and palliative care, therefore, need to adapt and change. We used virtual reality (VR) technology with the aim of reducing feelings of loneliness among these patients. Objectives: In a small cohort setting, we aimed to clarify the usefulness of VR viewing for this purpose by text mining interviews with the patients in palliative care after their VR experience, and to clarify the feasibility of this program. Design and Setting/Subjects: Four consecutive Japanese patients in the palliative care unit viewed personalized familiar persons or places through VR goggles, while communicating by telephone. After the VR experience, text mining of the patients' interviews was used to extract the words for the frequency count and co-occurrence analysis. Results: Four clusters were extracted: "relief from the pain of hospitalization by feeling safe and secure with family members nearby," "using VR to regain daily life," "immersive feeling of being in the same space as family," and "loneliness due to the realistic feeling of separation from the family through VR experience." There were no cases of VR sickness. Conclusion: Our results attained by text mining suggest the promising potential of VR imaging of familiar surroundings for patients in palliative care.

Methylmercury Decreases AMPA Receptor Subunit GluA2 Levels in Cultured Rat Cortical Neurons.

Methylmercury (MeHg) is a well-known environmental pollutant that has harmful effects on the central nervous systems of humans and animals. The molecular mechanisms of MeHg-induced neurotoxicity at low concentrations are not fully understood. Here, we investigated the effects of low-concentration MeHg on the cell viability, Ca2+ homeostasis, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA2 levels, which determine Ca2+ permeability of AMPA receptors, in rat primary cortical neurons. Exposure of cortical neurons to 100 and 300 nM MeHg for 7 d resulted in a decrease in GluA2 levels, an increase in basal intracellular Ca2+ concentration, increased phosphorylation levels of extracellular signal-regulated kinase (ERK)1/2 and p38, and decreased cell viability. Moreover, glutamate stimulation exacerbated the decrease in cell viability and increased intracellular Ca2+ levels in MeHg-treated neurons compared to control neurons. MeHg-induced neuronal cell death was ameliorated by 1-naphthyl acetyl spermine, a specific antagonist of Ca2+-permeable, GluA2-lacking AMPA receptors. Our findings raise the possibility that decreased neuronal GluA2 levels and the subsequent increase in intracellular Ca2+ concentration may contribute to MeHg-induced neurotoxicity.

Xanthine oxidase and aldehyde oxidase contribute to allopurinol metabolism in rats.

BACKGROUND: Allopurinol is used to treat hyperuricemia and gout. It is metabolized to oxypurinol by xanthine oxidase (XO), and aldehyde oxidase (AO). Allopurinol and oxypurinol are potent XO inhibitors that reduce the plasma uric acid levels. Although oxypurinol levels show large inter-individual variations, high concentrations of oxypurinol can cause various adverse effects. Therefore, it is important to understand allopurinol metabolism by XO and AO. In this study we aimed to estimate the role of AO and XO in allopurinol metabolism by pre-administering Crl:CD and Jcl:SD rats, which have known strain differences in AO activity, with XO inhibitor febuxostat. METHODS: Allopurinol (30 or 100 mg/kg) was administered to Crl:CD and Jcl:SD rats with low and high AO activity, respectively, after pretreatment with or without febuxostat. The serum concentrations of allopurinol and oxypurinol were measured, and the area under the concentration-time curve (AUC) was calculated from the 48 h serum concentration-time profile. In vivo metabolic activity was measured as the ratio AUCoxypurinol /AUCallopurinol. RESULTS: Although no strain-specific differences were observed in the AUCoxypurinol/AUCallopurinol ratio in the allopurinol (30 mg/kg)-treated group, the ratio in Jcl:SD rats was higher than that in Crl:CD rats after febuxostat pretreatment. Contrastingly, the AUC ratio of allopurinol (100 mg/kg) was approximately 2-fold higher in Jcl:SD rats than that in Crl:CD rats. These findings showed that Jcl:SD rats had higher intrinsic AO activity than Crl:CD rats did. However, febuxostat pretreatment substantially decreased the activity, as measured by the AUC ratio using allopurinol (100 mg/kg), to 46 and 63% in Crl:CD rats and Jcl:SD rats, respectively, compared to the control group without febuxostat pretreatment. CONCLUSIONS: We elucidated the role of XO and AO in allopurinol metabolism in Crl:CD and Jcl:SD rats. Notably, AO can exert a proportionately greater impact on allopurinol metabolism at high allopurinol concentrations. AO's impact on allopurinol metabolism is meaningful enough that individual differences in AO may explain allopurinol toxicity events. Considering the inter-individual differences in AO activity, these findings can aid to dose adjustment of allopurinol to avoid potential adverse effects.

Improved Predictability of Hepatic Clearance with Optimal pH for Acyl-Glucuronidation in Liver Microsomes.

The purpose of this study was to investigate the optimal pH for acyl-glucuronidation formation with carboxylic acid-containing compounds in human and rat liver microsomes to improve the predictability of their hepatic clearance. The optimal pH for acyl-glucuronidation of all 17 compounds was around pH 6.0 in human and rat liver microsomes. Correlation analysis was done with the predicted in vitro intrinsic clearance (CLint,in vitro) and in vivo intrinsic clearance (CLint,in vivo) calculated from available reported data of total clearance (CLtot) of 11 compounds in humans. For 8 of the 11 compounds, under the pH 6.0 condition, the CLint,in vitro were within 1/3 to 3-fold error of the observed CLint,in vivo whereas, the error was within 1/3 to 3-fold of the observed CLint,in vivo for only 3 of the 11 under the pH 7.4 condition. The intracellular pH in human and rat hepatocytes decreased in the presence of a carboxylic acid-containing compound. These findings suggest that acyl-glucuronidation in liver microsomes at pH 6.0 is closer to physiological conditions in the presence of carboxylic acid compounds, and thus, use of this pH condition is important for physiological interpretation and predictability of intrinsic clearance.

Assessment of metabolic activation of felbamate in chimeric mice with humanized liver in combination with in vitro metabolic assays.

Felbamate (FBM) is an antiepileptic drug that has minimal toxicity in preclinical toxicological species but has a serious idiosyncratic drug toxicity (IDT) in humans. The formation of reactive metabolites is common among most drugs associated with IDT, and 2-phenylpropenal (2-PP) is believed to be the cause of IDT by FBM. It is important to consider the species difference in susceptibility to IDT between experimental animals and humans. In the present study, we used an in vitro and in vivo model system to reveal species difference in IDT of FBM. Human cytochrome P450 (CYP) and carboxylesterase (CES) expressing microsomes were used to clarify the isozymes involved in the metabolism of FBM. The remaining amount of FBM was significantly reduced in incubation with microsomes expressing human CYP2C8, 2C9, 2E1, and CES1c isozymes. Chimeric mice with humanized liver are expected to predict IDT in humans. Therefore, metabolite profiles in chimeric mice with humanized liver were investigated after administration of FBM. Metabolites after glutathione (GSH) conjugation of 2-phenylpropenal (2-PP), which is the reactive metabolite responsible for FBM-induced IDT, were detected in chimeric mice plasma and liver homogenate. Mass spectrometry imaging (MSI) visualizes distribution of FBM and endogenous GSH, and GSH levels in human hepatocyte were decreased after administration of FBM. In this study, we identified CYP and CES isozymes involved in the metabolism of FBM and confirmed reactive metabolite formation and subsequent decrease in GSH using humanized animal model. These results would provide useful information for the susceptibility to IDT between experimental animals and humans.

Involvement of aldehyde oxidase in the metabolism of aromatic and aliphatic aldehyde-odorants in the mouse olfactory epithelium.

Xenobiotic-metabolizing enzymes (XMEs) expressed in the olfactory epithelium (OE) are known to metabolize odorants. Aldehyde oxidase (AOX) recognizes a wide range of substrates among which are substrates with aldehyde groups. Some of these AOX substrates are odorants, such as benzaldehyde and n-octanal. One of the mouse AOX isoforms, namely AOX2 (mAOX2), was shown to be specifically expressed in mouse OE but its role to metabolize odorants in this tissue remains unexplored. In this study, we investigated the involvement of mouse AOX isoforms in the oxidative metabolism of aldehyde-odorants in the OE. Mouse OE extracts effectively metabolized aromatic and aliphatic aldehyde-odorants. Gene expression analysis revealed that not only mAOX2 but also the mAOX3 isoform is expressed in the OE. Furthermore, evaluation of inhibitory effects using the purified recombinant enzymes led us to identify specific inhibitors of each isoform, namely chlorpromazine, 17ß-estradiol, menadione, norharmane, and raloxifene. Using these specific inhibitors, we defined the contribution of mAOX2 and mAOX3 to the metabolism of aldehyde-odorants in the mouse OE. Taken together, these findings demonstrate that mAOX2 and mAOX3 are responsible for the oxidation of aromatic and aliphatic aldehyde-odorants in the mouse OE, implying their involvement in odor perception.

Amiodarone bioconcentration and suppression of metamorphosis in Xenopus.

Trace concentrations of a number of pharmaceutically active compounds have been detected in the aquatic environment in many countries, where they are thought to have the potential to exert adverse effects on non-target organisms. Amiodarone (AMD) is one such high-risk compound commonly used in general hospitals. AMD is known to alter normal thyroid hormone (TH) function, although little information is available regarding the specific mechanism by which this disruption occurs. Anuran tadpole metamorphosis is a TH-controlled developmental process and has proven to be useful as a screening tool for environmental pollutants suspected of disrupting TH functions. In the present study, our objective was to clarify the effects of AMD on Xenopus metamorphosis as well as to assess the bioconcentration of this pharmaceutical in the liver. We found that AMD suppressed spontaneous metamorphosis, including tail regression and hindlimb elongation in pro-metamorphic stage tadpoles, which is controlled by endogenous circulating TH, indicating that AMD is a TH antagonist. In transgenic X. laevis tadpoles carrying plasmid DNA containing TH-responsive element (TRE) and a 5'-upstream promoter region of the TH receptor (TR) ßA1 gene linked to a green fluorescent protein (EGFP) gene, triiodothyronine (T3) exposure induced a strong EGFP expression in the hind limbs, whereas the addition of AMD to T3 suppressed EGFP expression, suggesting that this drug interferes with the binding of T3 to TR, leading to the inhibition of TR-mediated gene expression. We also found AMD to be highly bioconcentrated in the liver of pro-metamorphic X. tropicalis tadpoles, and we monitored hepatic accumulation of this drug using mass spectrometry imaging (MSI). Our findings suggest that AMD imposes potential risk to aquatic wildlife by disrupting TH homeostasis, with further possibility of accumulating in organisms higher up in the food chain.

Predictability of human pharmacokinetics of drugs that undergo hepatic organic anion transporting polypeptide (OATP)-mediated transport using single-species allometric scaling in chimeric mice with humanized liver: integration with hepatic drug metabolism.

We previously reported a prediction method for human pharmacokinetics (PK) using single species allometric scaling (SSS) and the complex Dedrick plot in chimeric mice with humanized liver to predict the total clearance (CLt), distribution volumes in steady state (Vdss) and plasma concentration-time profiles of several drugs metabolized by cytochrome P450 (P450) and non-P450 enzymes. In the present study, we examined eight compounds (bosentan, cerivastatin, fluvastatin, pitavastatin, pravastatin, repaglinide, rosuvastatin, valsartan) as typical organic anion transporting polypeptide (OATP) substrates and six compounds metabolized by P450 and non-P450 enzymes to evaluate the predictability of CLt, Vdss and plasma concentration-time profiles after intravenous administration to chimeric mice. The predicted CLt and Vdss of drugs that undergo OATP-mediated uptake and P450/non-P450-mediated metabolism reflected the observed data from humans within a threefold error range. We also examined the possibility of predicting plasma concentration-time profiles of drugs that undergo OATP-mediated uptake using the complex Dedrick plot in chimeric mice. Most profiles could be superimposed with observed profiles from humans within a two- to threefold error range. PK prediction using SSS and the complex Dedrick plot in chimeric mice can be useful for evaluating drugs that undergo both OATP-mediated uptake and P450/non-P450-mediated metabolism.

Activation of PXR, CAR and PPARα by pyrethroid pesticides and the effect of metabolism by rat liver microsomes.

In this study, we used reporter gene assays in COS-1 cells to examine the activation of rat pregnane X receptor (PXR), rat constitutive androstane receptor (CAR) and rat peroxisome-proliferator activated receptor (PPAR)α by pyrethroid pesticides, and to understand the effects of metabolic modification on their activities. All eight pyrethroids tested in this study showed rat PXR agonistic activity; deltamethrin was the most potent, followed by cis-permethrin and cypermethrin. However, when the pyrethroids were incubated with rat liver microsomes, their rat PXR activities were decreased to various extents. Cis- and trans-permethrin showed weak rat CAR agonistic activity, while the other pyrethroids were inactive. However, fenvalerate showed dose-dependent inverse agonistic activity toward rat CAR, and this activity was reduced after metabolism. None of the pyrethroids showed rat PPARα agonistic activity, but a metabolite of cis-/trans-permethrin and phenothrin, 3-phenoxybenzoic acid, activated rat PPARα. Since PXR, CAR and PPARα regulate various xenobiotic/endobiotic-metabolizing enzymes, activation of these receptors by pyrethroids may result in endocrine disruption due to changes of hormone-metabolizing activities.

Comparative study of the effect of 17 parabens on PXR-, CAR- and PPARα-mediated transcriptional activation.

Parabens are widely used as preservatives in personal care products, medicines and foods, resulting in substantial human exposures, even though some harmful effects, such as endocrine-disrupting activity, have been reported. Pregnane X receptor (PXR), constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα), which are members of the nuclear receptor superfamily, regulate the metabolism of endogenous substrates including hormones. Therefore, we hypothesized that parabens may alter hormone-metabolizing activities by acting on these receptors, and such changes could contribute to the endocrine-disrupting activity. To test this idea, we systematically examined the effects of 17 parabens on these receptors using reporter gene assays. Nine parabens significantly activated human and rat PXR. Parabens with C2-C5 (linear and branched) side chains were most active. Butylparaben and isobutylparaben also significantly activated rat CAR. We found that long-side-chain (C7-C12) parabens showed up to 2-fold activation of PPARα at 10 µM. Furthermore, pentylparaben and hexylparaben showed rat PXR antagonistic activity and rat CAR inverse agonistic activity. The activity of butylparaben towards PXR and CAR was lost after carboxylesterase-mediated metabolism. These findings confirm that parabens influence the activities of PXR, CAR and PPARα, and thus have the potential to contribute to endocrine disruption by altering hormone metabolism.

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.

Utility of Chimeric Mice with Humanized Liver for Predicting Human Pharmacokinetics in Drug Discovery: Comparison with in Vitro-in Vivo Extrapolation and Allometric Scaling.

Predicting human pharmacokinetics (PK) such as clearance (CL) and volume of distribution (Vd) is a critical component of drug discovery. These predictions are mainly performed by in vitro-in vivo extrapolation (IVIVE) using human biological samples, such as hepatic microsomes and hepatocytes. However, some issues with this process have arisen, such as inconsistencies between in vitro and in vivo findings; the integration of predicted CYP, non-CYP and transporter-mediated human PK; and the difficulty of evaluating very metabolically stable compounds. Various approaches to solving these issues have been reported. Allometric scaling using experimental animals has also often been used. However, this method has also shown many problems due to interspecies differences, albeit that various correction methods have been proposed. Another approach involves the production of chimeric mice with humanized liver via the transplantation of human hepatocytes into mice. The livers of these mice are repopulated mostly with human hepatocytes and express human drug-metabolizing enzymes and drug transporters, suggesting that these mice are useful for solving the issues of IVIVE and allometric scaling, and more reliably predicting human PK. In this review, we summarize human PK prediction methods using IVIVE, allometric scaling and chimeric mice with humanized liver, and discuss the utility of predicting human PK in drug discovery by comparing these chimeric mice with IVIVE and allometric scaling.

Coordinated cytochrome P450 expression in mouse liver and intestine under different dietary conditions during liver regeneration after partial hepatectomy.

Liver resection is performed to remove tumors in patients with liver cancer, but the procedure's suitability depends on the regenerative ability of the liver. It is important to consider the effects of exogenous factors, such as diets, on liver regeneration for the recovery of function. The evaluation of drug metabolism during liver regeneration is also necessary because liver dysfunction is generally observed after the operation. Here, we investigated the influence of a purified diet (AIN-93G) on liver regeneration and changes in the mRNA expression of several cytochrome P450 (CYP) isoforms in the liver and small intestine using a two-thirds partial hepatectomy (PH) mouse model fed with a standard diet (MF) and a purified diet. Liver regeneration was significantly delayed in the purified diet group relative to that in the standard diet group. The liver Cyp2c55 and Cyp3a11 expression was increased at 3 day after PH especially in the purified diet group. Bile acid may partly cause the differences in liver regeneration and CYP expression between two types of diets. On the other hand, Cyp3a13 expression in the small intestine was transiently increased at day 1 after PH in both diet groups. The findings suggest that compensatory induction of the CYP expression occurred in the small intestine after attenuation of drug metabolism potential in the liver. The present results highlight the importance of the relationship between liver regeneration, drug metabolism, and exogenous factors for the effective treatment, including surgery and medication, in patients after liver resection or transplantation.

Inhibition of cytochrome P450 3A protein degradation and subsequent increase in enzymatic activity through p38 MAPK activation by acetaminophen and salicylate derivatives.

Cytochrome P450 (CYP) 3A4 plays an important role in drug metabolism. Although transcriptional regulation of CYP3A expression by chemicals has been comprehensively studied, its post-translational regulation is not fully understood. We previously reported that acetaminophen (APAP) caused accumulation of functional CYP3A protein via inhibition of CYP3A protein degradation through reduction of glycoprotein 78 (gp78), an E3 ligase of the ubiquitin proteasome system. Furthermore, N-acetyl-m-aminophenol, a regioisomer of APAP causes CYP3A protein accumulation, whereas p-acetamidobezoic acid, in which a hydroxy group of APAP was substituted for a carboxy group, did not lead to the same effects. However, the mechanism underlying the reduction of gp78 protein expression by APAP has not yet been elucidated. In this study, we selected 32 compounds including a phenolic hydroxyl group such as APAP and explored the compounds that increased CYP3A enzyme activity to analyze their common mechanism. Four compounds, including salicylate, increased CYP3A enzyme activity and led to the accumulation of functional CYP3A protein similarly to APAP. APAP and salicylate activate p38 mitogen-activated protein kinase (p38 MAPK). gp78 is known to be phosphorylated by p38 MAPK; so, we investigated the relationship between p38 MAPK and CYP3A. APAP activated p38 MAPK, decreased gp78 protein expression, and subsequently induced CYP3A protein expression in a time-dependent manner. When SB203580, a p38 MAPK inhibitor, was co-administered with APAP, the inhibitory effects of APAP on CYP3A protein degradation were suppressed. In this study, we demonstrated the involvement of the p38 MAPK-gp78 pathway in suppressing CYP3A protein degradation by APAP. Salicylate derivatives may also suppress the CYP3A protein degradation.

[Contribution of chimeric mice with a humanized liver to the evaluation of pharmacology, toxicity, and pharmacokinetics in drug discovery and development].

To develop new drugs with high efficacy and safety, it is important to predict the pharmacological, toxicological, and pharmacokinetic profiles of drug candidates in humans. Chimeric mice with a humanized liver are mice in which human hepatocytes have been transplanted, such that mouse liver cells are replaced with human hepatocytes; these mice have been used as prediction models. Studies performed thus far indicate that chimeric mice with a humanized liver can be used for the prediction of human-specific metabolite formation and plasma concentration-time curves for several drugs. Furthermore, studies advocate the utility of chimeric mice with a humanized liver for modelling drug-induced hepatotoxicity and disease such as hepatitis virus infection in safety and pharmacological evaluations respectively. Taken together, these findings indicate that chimeric mice with a humanized liver can be used to evaluate the relationship between pharmacokinetics, toxicity, and efficacy; the contribution by active metabolites may also be assessed. In recent years, new and improved animal models have been developed to overcome the disadvantages of chimeric mice with a humanized liver. It is expected that their usefulness for optimization of drug candidates and translational research in drug discovery and development will further increase.

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.

Comparison of Drug Metabolism and Its Related Hepatotoxic Effects in HepaRG, Cryopreserved Human Hepatocytes, and HepG2 Cell Cultures.

Differentiated HepaRG cells maintain liver-specific functions such as drug-metabolizing enzymes. In this study, the feasibility of HepaRG cells as a human hepatocyte model for in vitro toxicity assessment was examined using selected hepatotoxic compounds. First, basal drug-metabolizing enzyme activities (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, uridine 5'-diphospho-glucuronosyltransferase [UGT], and sulfotransferases [SULT]) were measured in HepaRG, human hepatocytes, and HepG2 cells. Enzyme activities in differentiated HepaRG cells were comparable to those in human hepatocytes and much higher than those in HepG2 cells, except for SULT activity. Second, we examined the cytotoxicity of hepatotoxic compounds, acetaminophen (APAP), aflatoxin B1 (AFB1), cyclophosphamide (CPA), tamoxifen (TAM), and troglitazone (TGZ) in HepaRG cells and human hepatocytes. AFB1- and CPA-induced cytotoxicities against HepaRG cells were comparable to those against human hepatocytes. Furthermore, the cytotoxicities of these compounds were inhibited by 1-aminobenzotriazole (ABT), a broad CYP inhibitor, in both cells and were likely mediated by metabolic activation by CYP. Finally, toxicogenomics analysis of HepG2 and HepaRG cells after exposure to AFB1 and CPA revealed that numerous p53-related genes were upregulated- and the expression of these genes was greater in HepaRG than in HepG2 cells. These results suggest that gene expression profiles of HepaRG cells were affected more considerably by the toxic mechanisms of AFB1 and CPA than the profiles of HepG2 cells were. Therefore, our investigation shows that HepaRG cells could be useful human hepatic cellular models for toxicity studies.

Tributyltin induces epigenetic changes and decreases the expression of nuclear respiratory factor-1.

Tributyltin (TBT), a common organotin environmental pollutant, has been widely used as a component of marine antifouling paints. We previously reported that exposure to TBT inhibits the expression and DNA binding of nuclear respiratory factor-1 (NRF-1) and causes neurotoxicity. In the present study, we focused on the epigenetic effects of TBT and investigated whether TBT decreases NRF-1 expression via epigenetic modifications in SH-SY5Y human neuroblastoma cells. First, we found that exposure to 300 nM TBT decreases NRF-1 expression. We examined epigenetic changes induced by TBT, and showed that TBT causes hypermethylation of the NRF-1 promoter region, increases the amount of methyl-CpG-binding protein 2 (MeCP2) bound to the NRF-1 promoter, and alters the expression of DNA methyltransferases and ten-eleven translocation (TET) demethylation enzymes. These results suggest that epigenetic changes play an important role in regulation of NRF-1 expression. Next, we investigated effect of NRF-1 expression decrease on cells, and TBT reduces mitochondrial membrane potential and overexpression of NRF-1 rescued this reduction in membrane potential. Thus, we suggested that NRF-1 is important for maintaining mitochondrial membrane potential. Our study indicates that TBT causes epigenetic changes such as hypermethylation, which increases recruitment of MeCP2 to the NRF-1 promoter and probably lead to decreased of NRF-1 expression and mitochondrial membrane potential. Therefore, this research provides new evidence of the epigenetic action caused by organotin.

Chimeric mice with humanized liver: Application in drug metabolism and pharmacokinetics studies for drug discovery.

Predicting human drug metabolism and pharmacokinetics (PK) is key to drug discovery. In particular, it is important to predict human PK, metabolite profiles and drug-drug interactions (DDIs). Various methods have been used for such predictions, including in vitro metabolic studies using human biological samples, such as hepatic microsomes and hepatocytes, and in vivo studies using experimental animals. However, prediction studies using these methods are often inconclusive due to discrepancies between in vitro and in vivo results, and interspecies differences in drug metabolism. Further, the prediction methods have changed from qualitative to quantitative to solve these issues. Chimeric mice with humanized liver have been developed, in which mouse liver cells are mostly replaced with human hepatocytes. Since human drug metabolizing enzymes are expressed in the liver of these mice, they are regarded as suitable models for mimicking the drug metabolism and PK observed in humans; therefore, these mice are useful for predicting human drug metabolism and PK. In this review, we discuss the current state, issues, and future directions of predicting human drug metabolism and PK using chimeric mice with humanized liver in drug discovery.

Carbofuran causes neuronal vulnerability to glutamate by decreasing GluA2 protein levels in rat primary cortical neurons.

Glutamate receptor 2 (GluA2/GluR2) is one of the four subunits of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR); an increase in GluA2-lacking AMPARs contributes to neuronal vulnerability to excitotoxicity because of the receptor's high Ca2+ permeability. Carbofuran is a carbamate pesticide used in agricultural areas to increase crop productivity. Due to its broad-spectrum action, carbofuran has also been used as an insecticide, nematicide, and acaricide. In this study, we investigated the effect of carbofuran on GluA2 protein expression. The 9-day treatment of rat primary cortical neurons with 1 µM and 10 µM carbofuran decreased GluA2 protein expression, but not that of GluA1, GluA3, or GluA4 (i.e., other AMPAR subunits). Decreased GluA2 protein expression was also observed on the cell surface membrane of 10 µM carbofuran-treated neurons, and these neurons showed an increase in 25 µM glutamate-triggered Ca2+ influx. Treatment with 50 µM glutamate, which did not affect the viability of control neurons, significantly decreased the viability of 10 µM carbofuran-treated neurons, and this effect was abolished by pre-treatment with 300 µM 1-naphthylacetylspermine, an antagonist of GluA2-lacking AMPAR. At a concentration of 100 µM, but not 1 or 10 µM, carbofuran significantly decreased acetylcholine esterase activity, a well-known target of this chemical. These results suggest that carbofuran decreases GluA2 protein expression and increases neuronal vulnerability to glutamate toxicity at concentrations that do not affect acetylcholine esterase activity.