Maternal DHA intake in mice increased DHA metabolites in the pup brain and ameliorated MeHg-induced behavioral disorder.

Although pregnant women's fish consumption is beneficial for the brain development of the fetus due to the DHA in fish, seafood also contains methylmercury (MeHg), which adversely affects fetal brain development. Epidemiological studies suggest that high DHA levels in pregnant women's sera may protect the fetal brain from MeHg-induced neurotoxicity, but the underlying mechanism is unknown. Our earlier study revealed that DHA and its metabolite 19,20-dihydroxydocosapentaenoic acid (19,20-DHDP) produced by cytochrome P450s (P450s) and soluble epoxide hydrolase (sEH) can suppress MeHg-induced cytotoxicity in mouse primary neuronal cells. In the present study, DHA supplementation to pregnant mice suppressed MeHg-induced impairments of pups' body weight, grip strength, motor function, and short-term memory. DHA supplementation also suppressed MeHg-induced oxidative stress and the decrease in the number of subplate neurons in the cerebral cortex of the pups. DHA supplementation to dams significantly increased the DHA metabolites 19,20-epoxydocosapentaenoic acid (19,20-EDP) and 19,20-DHDP as well as DHA itself in the fetal and infant brains, although the expression levels of P450s and sEH were low in the fetal brain and liver. DHA metabolites were detected in the mouse breast milk and in human umbilical cord blood, indicating the active transfer of DHA metabolites from dams to pups. These results demonstrate that DHA supplementation increased DHA and its metabolites in the mouse pup brain and alleviated the effects of MeHg on fetal brain development. Pregnant women's intake of fish containing high levels of DHA (or DHA supplementation) may help prevent MeHg-induced neurotoxicity in the fetus.

Photodegradation profiling of nitrendipine: evaluation of active pharmaceutical ingredient, tablets and its altered forms.

Nitrendipine (NTR) is a dihydropyridine drug, which is well-known as a photodegradable pharmaceutical. However, the photochemical reaction of NTR has not been evaluated in detail from now. In this study, we perform the photodegradation profiling of NTR for the elucidation of its photochemical behavior. NTR amounts during ultraviolet light (UV) irradiation were monitored using high performance liquid chromatography (HPLC). NTR was photodegraded almost completely within 24 h along with the generation of some photoproducts. Structural determination of two NTR photoproducts were carried out by means of electrospray ionization liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS). Obtained results from this study clarified one novel NTR photoproduct, a nitroso pyridine analogue, in addition to a pyridine analogue. Furthermore, photodegradation pathway of NTR was speculated based on chemical structures of NTR photoproducts to clarify its photochemical behavior. It was proposed that a singlet oxygen molecule might withdraw two hydrogen radicals resulting in the form of a pyridine analogue, and the following reduction of its nitro group might produce a nitroso pyridine analogue. Finally, we evaluated the photostability of NTR tablets and its altered forms, indicating that the change of the dosage form led to a decrease of the photostability of NTR tablets. The obtained results will be helpful for the additional research to evaluate the effect of NTR photodegradation on its own biological activities.

Evaluation of photostability of azelnidipine tablets and structure determination of its photoproducts.

Photo-exposure has a crucial effect on the natures of photosensitive pharmaceuticals in addition to their contents in medicines through the photodegradation. Generated photoproducts might be more bioactive and contribute to the expression of adverse side effects. This study aimed to clarify the photochemical behavior of medicines of azelnidipine, which is a member of dihydropyridine antihypertensive drugs, by the evaluation of its photostability and the determination of chemical structures of generated photoproducts. Calblock® tablets and its altered forms (powders and suspensions) were UV-irradiated by a black light. Residual amounts of active pharmaceutical ingredients (APIs) were monitored by high-performance liquid chromatography. The chemical structures of two photoproducts were determined by electrospray ionization tandem mass spectrometry. API of Calblock® tablets was photodegraded with the generation of several photoproducts. Its photodegradability was more significant when Calblock® tablets were crushed or suspended. Structural determination revealed that two photoproducts were benzophenone and a pyridine derivative. It was speculated that these photoproducts were generated by the elimination of diphenyl methylene radical and additional chemical reaction including oxidation and hydrolysis. Azelnidipine was photosensitive and its photodegradation in Calblock® tablets was promoted by the change of the dosage form. This difference might be derived from the light emission efficiency. This study suggests that API contents of Calblock® tablets might decrease when tablets or its altered forms are exposed to sunlight irradiation with the generation of benzophenone, which is a toxicological potent.

Tributyltin activates the Keap1-Nrf2 pathway via a macroautophagy-independent reduction in Keap1.

Tributyltin (TBT) is an environmental chemical, which was used as an antifouling agent for ships. Although its use has been banned, it is still persistently present in ocean sediments. Although TBT reportedly causes various toxicity in mammals, few studies on the mechanisms of biological response against TBT toxicity exist. The well-established Keap1-Nrf2 pathway is activated as a cytoprotective mechanism under stressful conditions. The relationship between TBT and the Keap1-Nrf2 pathway remains unclear. In the present study, we evaluated the effect of TBT on the Keap1-Nrf2 pathway. TBT reduced Keap1 protein expression in Neuro2a cells, a mouse neuroblastoma cell line, after 6 hr without altering mRNA expression levels. TBT also promoted the nuclear translocation of Nrf2, a transcription factor for antioxidant proteins, after 12 hr and augmented the expression of heme oxygenase 1, a downstream protein of Nrf2. Furthermore, TBT decreased Keap1 levels in mouse embryonic fibroblast (MEF) cells, with the knockout of Atg5, which is essential for macroautophagy, as well as in wild-type MEF cells. These results suggest that TBT activates the Keap1-Nrf2 pathway via the reduction in the Keap1 protein level in a macroautophagy-independent manner. The Keap1-Nrf2 pathway is activated by conformational changes in Keap1 induced by reactive oxygen species or electrophiles. Furthermore, any unutilized Keap1 protein is degraded by macroautophagy. Understanding the novel mechanism governing the macroautophagy-independent reduction in Keap1 by TBT may provide insights into the unresolved biological response mechanism against TBT toxicity and the activation mechanism of the Keap1-Nrf2 pathway.

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.

Establishment of hyperoxic cell culture system for predicting drug-induced liver injury: reducing accumulated lipids in hepatocytes derived from chimeric mice with humanized liver.

Drug-induced liver injury (DILI) is a major adverse reaction. Species-specific differences between humans and laboratory animals make it difficult to establish evaluation models that can accurately predict DILI in the preclinical phase. Chimeric mice with humanized liver are potential predictive models for understanding DILI. Chimeric mice generated by transplanting human hepatocytes into urokinase-type plasminogen activator/severe combined immunodeficient mice are known to develop fatty liver and show lipid accumulation in isolated hepatocytes. It is speculated that the lipids accumulated in hepatocytes may interfere with DILI assessment. It is known that normal 20% oxygen culture conditions do not meet oxygen demand because oxygen consumption rate is higher than the oxygen supply rate. Therefore, we predicted that hyperoxic cultures could induce hepatocyte function and reduce accumulated lipids. A culture of chimeric mouse hepatocytes in 40% oxygen showed reduced intracellular lipid and triglyceride levels compared to those cultured in 20% oxygen on days 7 and 10. In addition, fatty acid ß-oxidation (FAO) activity increased from day 7 under 40% oxygen conditions. On the other hand, FAO activity increased on day 10 under 20% conditions. Microarray and Ingenuity Pathway Analysis showed that lipid metabolism-related pathways were downregulated under 40% oxygen conditions for 7 days, suggesting the involvement of several mechanisms in decreasing lipid levels and increasing FAO. Furthermore, some pathways related to cellular function and maintenance were upregulated under 40% oxygen conditions for 7 days. In conclusion, chimeric mouse hepatocytes cultured under hyperoxic conditions may be useful for predicting DILI.

Prenatal exposure to valproic acid causes allodynia associated with spinal microglial activation.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social communication and social interaction and the presence of restricted, repetitive behaviors. Additionally, difficulties in sensory processing commonly occur in ASD. Sensory abnormalities include heightened or reduced sensitivity to pain, but the mechanism underlying sensory phenotypes in ASD remain unknown. Emerging evidence suggests that microglia play an important role in forming and refining neuronal circuitry, and thus contribute to neuronal plasticity and nociceptive signaling. In the present study, we investigated the age-dependent tactile sensitivity in an animal model of ASD induced by prenatal exposure to valproic acid (VPA) and subsequently assessed the involvement of microglia in the spinal cord in pain processing. Pregnant ICR (CD1) mice were intraperitoneally injected with either saline or VPA (500 mg/kg) on embryonic day 12.5. Male offspring of VPA-treated mothers showed mechanical allodynia at both 4 and 8 weeks of age. In the spinal cord dorsal horn in prenatally VPA-treated mice, the numbers and staining intensities of ionized calcium-binding adapter molecule 1-positive cells were increased and the cell bodies became enlarged, indicating microglial activation. Treatment with PLX3397, a colony-stimulating factor 1 receptor inhibitor, for 10 days resulted in a decreased number of spinal microglia and attenuated mechanical allodynia in adult mice prenatally exposed to VPA. Additionally, intrathecal injection of Mac-1-saporin, a saporin-conjugated anti-CD11b antibody to deplete microglia, abolished mechanical allodynia. These findings suggest that prenatal VPA treatment causes allodynia and that spinal microglia contribute to the increased nociceptive responses.

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.

Trehalose decreases mRNA and protein expressions of c-Jun and JunB in human cervical cancer HeLa cells.

Increasing evidence suggests that trehalose, a non-reducing disaccharide, ameliorates disease phenotypes by activating autophagy in animal models of various human diseases, including neurodegenerative diseases. Multiple in vitro studies suggest that activation of transcription factor EB, a master regulator of lysosomal biogenesis and autophagy genes, is a major contributor to trehalose-induced autophagy at later stages of exposure. However, underlying causes of trehalose-induced autophagy possibly occur at the early stage of the exposure period. In this study, we investigated the effects of short-term exposure of HeLa cells to trehalose on several signal transduction pathways to elucidate the initial events involved in its beneficial effects. Phospho-protein array analysis revealed that trehalose decreases levels of phosphorylated c-Jun, a component of the transcription factor activator protein-1, after 6 h. Trehalose also rapidly reduced mRNA expression levels of c-Jun and JunB, a member of the Jun family, within 1 h, resulting in a subsequent decrease in their protein levels. Future studies, exploring the interplay between decreased c-Jun and JunB protein levels and beneficial effects of trehalose, may provide novel insights into the mechanisms of trehalose action.

Tributyltin inhibits autophagy by decreasing lysosomal acidity in SH-SY5Y cells.

Tributyltin (TBT) is an environmental pollutant that remains in marine sediments and is toxic to mammals. For example, TBT elicits neurotoxic and immunosuppressive effects on rats. However, it is not entirely understood how TBT causes toxicity. Autophagy plays a pivotal role in protein quality control and eliminates aggregated proteins and damaged organelles. We previously reported that TBT dephosphorylates mammalian target of rapamycin (mTOR), which may be involved in enhancement of autophagosome synthesis, in primary cultures of cortical neurons. Autophagosomes can accumulate due to enhancement of autophagosome synthesis or inhibition of autophagic degradation, and we did not clarify whether TBT alters autophagic flux. Here, we investigated the mechanism by which TBT causes accumulation of autophagosomes in SH-SY5Y cells. TBT inhibited autophagy without affecting autophagosome-lysosome fusion before it caused cell death. TBT dramatically decreased the acidity of lysosomes without affecting lysosomal membrane integrity. TBT decreased the mature protein level of cathepsin B, and this may be related to the decrease in lysosomal acidity. These results suggest that TBT inhibits autophagic degradation by decreasing lysosomal acidity. Autophagy impairment may be involved in the mechanism underlying neuronal death and/or T-cell-dependent thymus atrophy induced by TBT.

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.

Prediction of human pharmacokinetics for low-clearance compounds using pharmacokinetic data from chimeric mice with humanized livers.

Development of low-clearance (CL) compounds that are slowly metabolized is a major goal in the pharmaceutical industry. However, the pursuit of low intrinsic CL (CLint ) often leads to significant challenges in evaluating the pharmacokinetics of such compounds. Although in vitro-in vivo extrapolation is widely used to predict human CL, its application has been limited for low-CLint compounds because of the low turnover of parent compounds in metabolic stability assays. To address this issue, we focused on chimeric mice with humanized livers (PXB-mice), which have been increasingly reported to accurately predict human CL in recent years. The predictive accuracy for nine low-CLint compounds with no significant turnover in a human hepatocyte assay was investigated using PXB-mouse methods, such as single-species allometric scaling (PXB-SSS) approach and a novel physiologically based scaling (PXB-PBS) approach that assumes that the CLint per hepatocyte is equal between humans and PXB-mice. The percentages of compounds with predicted CL within 2- and 3-fold ranges of the observed CL for low-CLint compounds were 89% and 100%, respectively, for both PXB-SSS and PXB-PBS approaches. Moreover, the predicted CL was mostly consistent among the methods. Conversely, the percentages of compounds with predicted CL within 2- and 3-fold ranges of the observed CL for low-CLint compounds were 50% and 63%, respectively, for multispecies allometric (MA) scaling. Overall, these PXB-mouse methods were much more accurate than conventional MA scaling approaches, suggesting that PXB-mice are useful tools for predicting the human CL of low-CLint compounds that are slowly metabolized.

Treatment with Histone Deacetylase Inhibitor Attenuates Peripheral Inflammation-Induced Cognitive Dysfunction and Microglial Activation: The Effect of SAHA as a Peripheral HDAC Inhibitor.

It has been demonstrated that peripheral inflammation induces cognitive dysfunction. Several histone deacetylase (HDAC) inhibitors ameliorate cognitive dysfunction in animal models of not only peripheral inflammation but also Alzheimer's disease. However, it is not clear which HDAC expressed in the central nervous system or peripheral tissues is involved in the therapeutic effect of HDAC inhibition on cognitive dysfunction. Hence, the present study investigated the effect of peripheral HDAC inhibition on peripheral inflammation-induced cognitive dysfunction. Suberoylanilide hydroxamic acid (SAHA), a pan-HDAC inhibitor that is mainly distributed in peripheral tissues after intraperitoneal administration, was found to prevent peripheral inflammation-induced cognitive dysfunction. Moreover, pretreatment with SAHA dramatically increased mRNA expression of interleukin-10, an anti-inflammatory cytokine, in peripheral and central tissues and attenuated peripheral inflammation-induced microglial activation in the CA3 region of the hippocampus. Minocycline, a macrophage/microglia inhibitor, also ameliorated cognitive dysfunction. Furthermore, as a result of treatment with liposomal clodronate, depletion of peripheral macrophages partially ameliorated the peripheral inflammation-evoked cognitive dysfunction. Taken together, these findings demonstrate that inhibition of peripheral HDAC plays a critical role in preventing cognitive dysfunction induced by peripheral inflammation via the regulation of anti-inflammatory cytokine production and the inhibition of microglial functions in the hippocampus. Thus, these findings could provide support for inhibition of peripheral HDAC as a novel therapeutic strategy for inflammation-induced cognitive dysfunction.

MiT/TFE family members suppress L-leucyl-L-leucine methyl ester-induced cell death.

Lysosomes are degradative organelles essential for cell homeostasis. However, various internal and external stimuli, including L-leucyl-L-leucine methyl ester (LLOMe), which is one of the common lysosomotropic agents, permeabilize the lysosomal membrane, leading to lysosome-dependent cell death because of leakage of lysosomal contents to the cytosol. The microphthalmia/transcription factor E (MiT/TFE) family members, which include transcription factor EB (TFEB), transcription factor E3 (TFE3), and microphthalmia-associated transcription factor (MITF), are master regulators of lysosomal biogenesis and are known to be involved in the lysosomal stress response. However, their protective effects against cell death associated with lysosomal-membrane damage are still poorly understood. In this study, we confirmed that LLOMe-induced lysosomal damage triggered nuclear translocation of TFEB/TFE3/MITF and increased the mRNA levels of their target genes encoding lysosomal hydrolases and lysosomal membrane proteins in HeLa cells. Furthermore, we revealed that TFEB/TFE3/MITF knockdown exacerbated LLOMe-induced cell death. However, TFEB overexpression only slightly attenuated LLOMe-induced cell death, despite enhanced LLOMe-induced increase in CTSD mRNA levels, implying that the endogenous levels of MiT/TFE family members might be sufficient to promote lysosomal biogenesis in response to lysosomal-membrane damage. Our results suggest that MiT/TFE family members suppress the cell death associated with lysosomal-membrane damage.

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.

CYP1A2 Downregulation by Obeticholic Acid: Usefulness as a Positive Control for the In Vitro Evaluation of Drug-Drug Interactions.

Cytochrome P450 (CYP) downregulation is a mechanism of drug-drug interaction encountered in pharmaceutical development which is difficult to evaluate in vitro because of the scarcity of evidence. A previous clinical study of obeticholic acid (OCA) with caffeine suggested that OCA may be a useful positive control to establish a method to evaluate CYP1A2 downregulation and to investigate the mechanism of its downregulation. In the present study, we investigated the ability of OCA to downregulate CYP1A2 in human hepatocytes. OCA suppressed CYP1A2 mRNA expression and CYP1A2 enzyme activity without causing direct inhibition of CYP1A2 or cytotoxicity, suggesting that OCA downregulates CYP1A2 in vitro. OCA significantly suppressed the induction of CYP1A2 mRNA expression by omeprazole in a concentration-dependent manner, suggesting that a combination of inducers and new chemical entities would be helpful to investigate the mechanism of CYP1A2 downregulation and to evaluate the potential of new chemical entities for downregulation and investigate their downregulation mechanism. We also showed that CYP1A2 was transcriptionally downregulated by OCA and that a reduction in aryl hydrocarbon receptor mRNA expression is a possible mechanism of CYP1A2 downregulation by OCA. These results indicate that OCA would be a suitable positive control for studies of CYP1A2 downregulation.

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.

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.