Tracking metabolites at single-cell resolution reveals metabolic dynamics during plant mitosis.

Analyzing only one cell allows the changes and characteristics of intracellular metabolites during the chromosome segregation process to be precisely captured and mitotic sub-phases to be dissected at the metabolite level.

Assessment of hepatic prostaglandin E2 level in carbamazepine induced liver injury.

Objective. Carbamazepine (CBZ), a widely used antiepileptic drug, is one major cause of the idiosyncratic liver injury along with immune reactions. Conversely, prostaglandin E2 (PGE2) demonstrates a hepatoprotective effect by regulating immune reactions and promoting liver repair in various types of liver injury. However, the amount of hepatic PGE2 during CBZ-induced liver injury remains elusive. In this study, we aimed to evaluate the hepatic PGE2 levels during CBZ-induced liver injury using a mouse model. Methods. Mice were orally administered with CBZ at a dose of 400 mg/kg for 4 days, and 800 mg/kg on the 5th day. Results. Plasma alanine transaminase (ALT) level increased in some of mice 24 h after the last CBZ administration. Although median value of hepatic PGE2 amount in the CBZ-treated mice showed same extent as vehicle-treated control mice, it exhibited significant elevated level in mice with severe liver injury presented by a plasma ALT level >1000 IU/L. According to these results, mice had a plasma ALT level >1000 IU/L were defined as responders and the others as non-responders in this study. Even though, the hepatic PGE2 levels increased in responders, the hepatic expression and enzyme activity related to PGE2 production were not upregulated when compared with vehicle-treated control mice. However, the hepatic 15-hydroxyprostaglandin dehydrogenase (15-PGDH) expression and activity decreased significantly in responders when compared with control mice. Conclusions. These results indicate that elevated hepatic PGE2 levels can be attributed to the downregulation of 15-PGDH expression under CBZ-induced liver injury.

Species differences in liver microsomal hydrolysis of acyl glucuronide in humans and rats.

Acyl glucuronides (AGs) are known as one of the causes of idiosyncratic drug toxicity (IDT). Although AGs can be enzymatically hydrolysed by ß-glucuronidase and esterase, much information on their characteristics and species differences is lacking. This study was aimed to clarify species differences in AG hydrolysis between human and rat liver microsomes (HLM and RLM).To evaluate the AG hydrolysis profile, and the contribution of ß-glucuronidase and esterase towards AG hydrolysis in HLM and RLM, nonsteroidal anti-inflammatory drugs (NSAIDs) were used. AGs were incubated with 0.1 M Tris-HCl buffer (pH 7.4) and 0.3 mg/mL HLM or RLM in the absence or presence of ß-glucuronidase inhibitor, D-saccharic acid 1,4-lactone (D-SL) and esterase inhibitor, phenylmethylsulfonyl fluoride (PMSF).AGs of mefenamic acid (MEF-AG) and etodolac (ETO-AG) showed significantly higher AG hydrolysis rates in RLM than in HLM. Esterases were found to serve as AG hydrolases dominantly in HLM, whereas both esterases and ß-glucuronidase equally contribute to AG hydrolysis in RLM. However, MEF-AG and ETO-AG were hydrolysed only by ß-glucuronidase.We demonstrated for the first time that the activity of AG hydrolases towards NSAID-AGs differs between humans and rats.

Rice OsHAK5 is a major potassium transporter that functions in potassium uptake with high specificity but contributes less to cesium uptake.

Cesium (Cs) in the environment is primarily absorbed by a potassium (K) transporter. OsHAK5 is a KT/HAK/KUP family K-transporter showing a high affinity for K. We created cultured rice cells whose OsHAK5 was knocked down by RNAi (named KD). In the medium containing 1.0 m m and less K, the growth of KD was significantly suppressed, suggesting that OsHAK5 greatly contributed to K absorption under limited K conditions. Although Cs suppressed the growth of KD and WT, stronger inhibition was observed on KD. Both KD and WT accumulated similar amounts of Cs when they were cultured in a medium containing Cs, whereas lower amounts of K were detected in KD. These results suggest that OsHAK5 was less involved in the absorption of Cs, although it was essential to K absorption under limited K conditions. In contrast, this means that another transporter may contribute to cesium uptake in rice.

Stereoselective Covalent Adduct Formation of Acyl Glucuronide Metabolite of Nonsteroidal Anti-Inflammatory Drugs with UDP-Glucuronosyltransferase.

A reactive metabolite of nonsteroidal anti-inflammatory drugs (NSAIDs), acyl-ß-D-glucuronide (AG), covalently binds to endogenous proteins. The covalent adduct formation of NSAIDs-AG may lead to the dysfunction of target proteins. Therefore, it is important to clarify the detailed characterization of the formation of covalent protein adducts of NSAID-AG. UDP-glucuronosyltransferase (UGT) catalyzes the conversion of NSAIDs to NSAIDs-AG. The aim of this study was to perform a quantitative analysis of the covalent adduct formation of NSAIDs-AG with UGT. Diclofenac-AG and ketoprofen-AG formed covalent adducts with organelle proteins. Next, the number of covalent adducts formed between NSAIDs-AG and UGT isoforms (UGT1A1, UGT1A9, UGT2B4, and UGT2B9) was determined. The capacity of diclofenac-AG to form covalent adducts with UGT1A9 or UGT2B7 was approximately 10 times higher than that of mefenamic acid-AG. The amounts of covalent adducts of AG of propionic acid derivative NSAIDs with UGT2B were higher than those with UGT1A. Stereoselectivity was observed upon covalent binding to UGT. A significant negative correlation between the half-lives of NSAIDs-AG in phosphate buffers and the amount of covalent adduct with UGT2B7 was observed, suggesting the more labile NSAID-AG forms higher irreversible bindings to UGT. This report provides comprehensive information on the covalent adduct formation of NSAIDs-AGs with UGT.

Slc25a39 and Slc25a40 Expression in Mice with Bile Duct Ligation or Lipopolysaccharide Treatment.

SLC25A39/40, involved in mitochondrial GSH (mGSH) import from the cytoplasm, is essential for protection against oxidative stress and mitochondrial dysfunction. We examined the effects of cholestasis, through bile duct ligation (BDL) and lipopolysaccharide (LPS)-induced inflammation in mice, on Slc25a39/40 expression. Additionally, we used human clear cell renal carcinoma (KMRC-1) cells to elucidate the mechanism of regulation of SLC25A39/40 expression in the kidneys after LPS treatment. BDL resulted in a decrease in Slc25a39 mRNA in the liver and a decrease in Slc25a39/40 mRNA and protein in the kidneys. Consequently, there was a significant decrease in mGSH levels in the kidneys of BDL mice compared with those in sham mice. LPS treatment resulted in increased Slc25a40 expression in the kidneys. In KMRC-1 cells, the combination treatment of LPS-RS or FPS-ZM1 with LPS suppressed the LPS-induced increase in SLC25A40, suggesting that SLC25A40 expression could be regulated by the signaling pathway via toll-like receptor 4 and the receptor for advanced glycation end products, respectively. Our findings contribute to understanding the role of mGSH in the maintenance of the mitochondrial redox state. To the best of our knowledge, this is the first study that demonstrates the changes in Slc25a39/40 expression in mice with cholestasis-associated renal injury and LPS-induced inflammation.

Diclofenac-Induced Cytotoxicity in Direct and Indirect Co-Culture of HepG2 Cells with Differentiated THP-1 Cells.

Non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DIC) frequently induce drug-induced liver injury (DILI). It is unclear whether macrophages such as M1 and M2 participate in NSAID-associated DILI; elucidating this relationship could lead to a better understanding of the detailed mechanism of DILI. We co-cultured human hepatoma HepG2 cells with M1 or M2 derived from human monocytic leukemia THP-1 cells to examine the roles of M1 and M2 in DIC-induced cytotoxicity. DIC was added to the direct or indirect co-cultures of HepG2 cells with M1 or M2 (HepG2/M1 or HepG2/M2, respectively) at cell ratios of (1:0, 1:0.1, 1:0.4, and 1:1). In both direct and indirect HepG2/M2 co-cultures (1:0.4), there was lower lactate dehydrogenase release compared with HepG2/M1 co-cultures. Other NSAIDs as well as DIC showed similar protective effects of DIC-induced cytotoxicity. There were only slight differences in mRNA levels of apoptosis- and endoplasmic reticulum stress-associated factors between M1 and M2 after DIC treatment, suggesting that other factors determined the protective effects of M2 on DIC-induced cytotoxicity. Levels of high mobility group box 1 (HMGB1) in the medium and the mRNA expression levels of HMGB1 receptors were different between M1 and M2 after DIC treatment. Increased HMGB1 concentrations and expression of toll-like receptor 2 mRNA in M1 were observed compared with M2 after DIC treatment. In conclusion, these results suggested that the HMGB1/TLR2 signaling axis can be suppressed in M2 but not M1, leading to the different roles of M1 and M2 in NSAID-induced cytotoxicity.

Protein Kinase N Family Negatively Regulates Constitutive Androstane Receptor-Mediated Transcriptional Induction of Cytochrome P450 2b10 in the Livers of Mice.

In receptor-type transcription factors-mediated cytochrome P450 (P450) induction, few studies have attempted to clarify the roles of protein kinase N (PKN) in the transcriptional regulation of P450s. This study aimed to examine the involvement of PKN in the transcriptional regulation of P450s by receptor-type transcription factors, including the aryl hydrocarbon receptor, constitutive androstane receptor (CAR), and pregnane X receptor. The mRNA and protein levels and metabolic activity of P450s in the livers of wild-type (WT) and double-mutant (D) mice harboring both PKN1 kinase-negative knock-in and PKN3 knockout mutations [PKN1 T778A/T778A; PKN3 -/-] were determined after treatment with activators for receptor-type transcription factors. mRNA and protein levels and metabolic activity of CYP2B10 were significantly higher in D mice treated with the CAR activator phenobarbital (PB) but not with 1,4-bis((3,5-dichloropyridin-2-yl)oxy)benzene compared with WT mice. We examined the CAR-dependent pathway regulated by PKN after PB treatment because the extent of CYP2B10 induction in WT and D mice was notably different in response to treatment with different CAR activators. The mRNA levels of Cyp2b10 in primary hepatocytes from WT and D mice treated with PB alone or in combination with Src kinase inhibitor 1 (SKI-1) or U0126 (a mitogen-activated protein kinase inhibitor) were evaluated. Treatment of hepatocytes from D mice with the combination of PB with U0126 but not SKI-1 significantly increased the mRNA levels of Cyp2b10 compared with those from the corresponding WT mice. These findings suggest that PKN may have inhibitory effects on the Src-receptor for activated C kinase 1 (RACK1) pathway in the CAR-mediated induction of Cyp2b10 in mice livers. SIGNIFICANCE STATEMENT: This is the first report of involvement of PKN in the transcriptional regulation of P450s. The elucidation of mechanisms responsible for induction of P450s could help optimize the pharmacotherapy and improve drug development. We examined whether the mRNA and protein levels and activities of P450s were altered in double-mutant mice harboring both PKN1 kinase-negative knock-in and PKN3 knockout mutations. PKN1/3 negatively regulates CAR-mediated induction of Cyp2b10 through phosphorylation of a signaling molecule in the Src-RACK1 pathway.

AtFLL2, a member of the FLO2 gene family, affects the enlargement of leaves at the vegetative stage and facilitates the regulation of carbon metabolism and flow.

Arabidopsis thaliana FLL2, a member of the FLO2 gene family, is expressed specifically in green leaves. The fll2 mutant showed significantly large rosette leaves and reduced the chlorophyll content. The sucrose content was significantly reduced. The glucose content was higher during the vegetative growth stage but decreased during the early reproductive growth stage. The amount of assimilated starch was lower than that in the wild type plant. The expression levels of genes involved in biosynthesis of sucrose and starch were largely altered. These results suggest that, in the fll2 mutant, a small amount of photosynthetic products was used for the biosynthesis of starch, and the products were supplied to promote intracellular growth of the source organs or for transport to the sink organs. These findings suggest that FLL2 is a factor affecting the expression level of genes involved in sugar metabolism, whose mutation caused a change in the assimilated products. Abbreviations : DAS: days after sowing.

Prostaglandin Transporter OATP2A1/SLCO2A1 Is Essential for Body Temperature Regulation during Fever.

Prostaglandin E2 (PGE2) in the hypothalamus is a principal mediator of the febrile response. However, the role of organic anion transporting polypeptide 2A1 (OATP2A1/SLCO2A1), a prostaglandin transporter, in facilitating this response is unknown. Here, we investigated the effect of Slco2a1 deficiency on the body core temperature (Tc) and on the PGE2 concentration in hypothalamus interstitial fluid (Cisf) and CSF (Ccsf) of lipopolysaccharide (LPS; 100 µg/kg, i.p.)-treated mice of both sexes. Slco2a1-/- mice did not develop a febrile response. Ccsf was increased in Slco2a1+/+ and Slco2a1-/- mice, and Ccsf of Slco2a1-/- mice was well maintained at 5 h after LPS injection (1160 pg/ml) compared with Slco2a1+/+ mice (316 pg/ml). A microdialysis study revealed that Cisf peaked at 2 h after LPS injection in Slco2a1+/+ mice (841 pg/ml), whereas the increase in Cisf was negligible in Slco2a1-/- mice. The PGE2 plasma concentration in Slco2a1-/- mice (201 pg/ml) was significantly higher than that in Slco2a1+/+ mice (54 pg/ml) at 1 h after LPS injection, whereas the two groups showed similar PGE2 concentrations in the hypothalamus. Strong Oatp2a1 immunoreactivity was observed in F4/80-positive microglia and perivascular cells and in brain capillary endothelial cells. The changes in Tc and Cisf seen in LPS-injected Slco2a1+/+ mice were partially attenuated in monocyte-/macrophage-specific Slco2a1-/- (Slco2a1Fl/Fl/LysMCre/+) mice. Thus, OATP2A1 facilitates the LPS-induced febrile response by maintaining a high level of Cisf, possibly by regulating PGE2 secretion from F4/80-positive glial cells and/or facilitating PGE2 transport across the blood-brain barrier. These findings suggest that OATP2A1 is a useful therapeutic target for neuroinflammation.SIGNIFICANCE STATEMENT Fever is a physiological response caused by pyrogen-induced release of prostaglandin E2 (PGE2) in the hypothalamus, which plays a central role in regulating the set-point of body temperature. However, it is unclear whether the prostaglandin transporter OATP2A1/SLCO2A1 is involved in this response. We show here that LPS-induced fever is associated with increased PGE2 concentration in hypothalamus interstitial fluid (Cisf), but not in CSF (Ccsf), by means of a microdialysis study in global Slco2a1-knock-out mice and monocyte-/macrophage-specific Slco2a1-knock-out mice. The results suggest that OATP2A1 serves as a regulator of Cisf in F4/80-positive glial cells. OATP2A1 was detected immunohistochemically in brain capillary endothelial cells and, therefore, may also play a role in PGE2 transport across the blood-brain barrier.

Decrease in Multidrug Resistance-associated Protein 2 Activities by Knockdown of Phosphatidylinositol 4-phosphate 5-kinase in Hepatocytes and Cancer Cells.

PURPOSE: The plasma membrane localization and transport activity of multidrug resistance- associated protein 2 (MRP2/ABCC2) and P-glycoprotein (P-gp/ABCB1) efflux transporters are governed by transporter-associated proteins. Phosphatidylinositol 4,5-bisphosphate (PIP2) formed by phosphatidylinositol 4-phosphate 5-kinase type 1 (PIP5K1) activates the linker function of radixin for efflux transporters. Radixin is involved in the plasma membrane localization of efflux transporters. We examined whether PIP5K1 could be a target for the modulation of transporter activities in hepatocytes and cancer cells. METHODS: The effects of PIP5K1 depletion by siRNA in mouse primary hepatocytes, PANC1 human pancreatic carcinoma cells, and HepG2 human hepatocellular carcinoma cells on the intracellular accumulation of MRP2 and P-gp substrates were examined. RESULTS: PIP5K1A depletion resulted in increased intracellular accumulation of carboxydichlorofluorescein, a MRP2 fluorescent substrate, in mouse primary hepatocytes, PANC1 cells, and HepG2 cells. In PANC1 and HepG2 cells, the transport activities of MRP2 were significantly decreased by PIP5K1C depletion. However, the transport activities of P-gp were unchanged by PIP5K1 depletion. PIP2 levels were unchanged between control and PIP5K1A- or PIP5K1C-depleted HepG2 cells. MRP2 mRNA levels showed few changes in HepG2 cells following PIP5K1A or PIP5K1C depletion. The expression of phosphorylated radixin was decreased by PIP5K1A and PIP5K1C depletion, although total radixin levels were unchanged. CONCLUSIONS: These data suggest that PIP5K1A and PIP5K1C could be target proteins for modulating MRP2 function, partly because of the resulting changes of the linker function of radixin.

Changes in transporters and metabolizing enzymes in the livers of rats with bile duct ligation.

PURPOSE: Bile duct ligation (BDL) in experimental animals is widely used as an animal model of liver cholestasis and fibrosis. The transcriptional process and plasma membrane localization of transporters are regulated by nuclear receptors and scaffold proteins, respectively. However, the detailed changes of these factors in the livers of BDL rats remain unclear. To clarify the effects of BDL on the levels of transporters and metabolizing enzymes, nuclear receptors, and scaffold proteins, we investigated changes in mRNA and protein levels of livers from BDL rats. METHODS: Membrane proteins and microsomes were prepared from rats with BDL. The mRNA levels of transporters and nuclear receptors in livers of control and BDL rats were examined by real-time reverse transcription polymerase chain reaction. The protein levels of transporters, metabolizing enzymes and scaffold proteins in membrane proteins and microsomes were determined by liquid chromatography-tandem mass spectrometry-based targeted proteomics. RESULTS: Mdr1a mRNA was significantly decreased at 1 and 2 weeks of BDL. The mRNA levels of MRP2 were significantly decreased. The mRNA levels of nuclear receptors were significantly decreased in livers of 1-week BDL rats. The protein levels of P-gp were significantly increased by BDL. Regarding scaffold proteins, the protein levels of ezrin, moesin and EBP50 were significantly decreased at 2 weeks of BDL. The protein levels of radixin were significantly increased at 1 week of BDL. In 1-week BDL rats, the protein levels of metabolizing enzymes such as CYP and UGT were significantly decreased. CONCLUSIONS: This study reports the comprehensive changes of transporters, metabolizing enzymes, nuclear receptors, and ezrin/radixin/moesin proteins in the livers of BDL rats. The expression levels of nuclear receptors and radixin that regulate the transcription and localization of CYP and/or transporters were decreased by BDL.

Involvement of diclofenac acyl-ß-d-glucuronide in diclofenac-induced cytotoxicity in glutathione-depleted isolated murine hepatocytes co-cultured with peritoneal macrophages.

Direct hepatotoxic effects of drugs can occur when a parent drug and/or its reactive metabolites induces the formation of reactive oxygen species. Reactive metabolites of diclofenac (DIC) such as DIC acyl-ß-d-glucuronide (DIC-AG) bind covalently to proteins, potentially decreasing protein function or inducing an immune response. However, it is unclear whether the macrophages and GSH depletion participate in DIC-induced cytotoxicity. Mouse hepatocytes (Hep) co-cultured with peritoneal macrophages (PMs) were used to clarify the effects of presence of PM with GSH depletion on DIC-induced cytotoxicity in Hep. DIC-AG but not hydroxy-DIC concentrations in medium were significantly increased in Hep co-cultured with PM with GSH depletion. Depletion of GSH resulted in significantly higher LDH leakage. Interestingly, LDH leakage in Hep/PM (1:0.4) with GSH depletion was significantly higher than in Hep/PM (1:0 and 1:0.1) with BSO. It is likely that macrophages with GSH depletion could facilitate DIC-induced cytotoxicity.

The Distinct Roles of Class I and II RPD3-Like Histone Deacetylases in Salinity Stress Response.

Histone acetylation is an essential process in the epigenetic regulation of diverse biological processes, including environmental stress responses in plants. Previously, our research group identified a histone deacetylase (HDAC) inhibitor (HDI) that confers salt tolerance in Arabidopsis (Arabidopsis thaliana). In this study, we demonstrate that class I HDAC (HDA19) and class II HDACs (HDA5/14/15/18) control responses to salt stress through different pathways. The screening of 12 different selective HDIs indicated that seven newly reported HDIs enhance salt tolerance. Genetic analysis, based on a pharmacological study, identified which HDACs function in salinity stress tolerance. In the wild-type Columbia-0 background, hda19 plants exhibit tolerance to high-salinity stress, while hda5/14/15/18 plants exhibit hypersensitivity to salt stress. Transcriptome analysis revealed that the effect of HDA19 deficiency on the response to salinity stress is distinct from that of HDA5/14/15/18 deficiencies. In hda19 plants, the expression levels of stress tolerance-related genes, late embryogenesis abundant proteins that prevent protein aggregation and positive regulators such as ABI5 and NAC019 in abscisic acid signaling, were induced strongly relative to the wild type. Neither of these elements was up-regulated in the hda5/14/15/18 plants. The mutagenesis of HDA19 by genome editing in the hda5/14/15/18 plants enhanced salt tolerance, suggesting that suppression of HDA19 masks the phenotype caused by the suppression of class II HDACs in the salinity stress response. Collectively, our results demonstrate that HDIs that inhibit class I HDACs allow the rescue of plants from salinity stress regardless of their selectivity, and they provide insight into the hierarchal regulation of environmental stress responses through HDAC isoforms.

Arabidopsis thaliana FLO2 is Involved in Efficiency of Photoassimilate Translocation, Which is Associated with Leaf Growth and Aging, Yield of Seeds and Seed Quality.

FLO2, FLOURY ENDOSPERM 2, is highly conserved in higher plants, and rice FLO2 has been predicted to be involved in regulation of accumulation of storage compounds. We analyzed the function of Arabidopsis thaliana FLO2 (AtFLO2) because A. thaliana set structurally different seeds from those of rice. Although the flo2 mutant of A. thaliana showed normal germination, inflorescence and morphogenesis of flowers, peculiar phenotypes on leaves and siliques were observed, suggesting that this gene played important roles during both the vegetative and reproductive stages. The mutant leaves showed a decrease in chloroplast numbers, and increased total biomass with faster growth. When grown in high light intensity conditions, it was observed that aging events were induced. The flo2 mutant showed depressed transportation of photoassimilates into the sink organs. In the reproductive stage, the flo2 mutant had significantly smaller size siliques, causing a reduced yield of seeds. These seeds were structurally weak, and the quality of seeds was significantly lowered, with reduction of accumulation of storage compounds by seeds. A positron-emitting tracer imaging system (PETIS) analysis detected a decreased amount of photoassimilate transport in the flo2 mutant. Therefore, it was presumed that the phenotypes of the flo2 mutant were caused by reduced performance of translocation or transportation of the photoassimilates. Our observation suggests that AtFLO2 is strongly involved in regulation of translocation and transport of assimilates, and contributes greatly to quality control of the various processes involving substance supply or transfer, such as photoassimilation, leaf enlargement, yield of seeds in a silique and accumulation of seed storage compounds.

Role of OATP2A1 in PGE(2) secretion from human colorectal cancer cells via exocytosis in response to oxidative stress.

Chronic inflammation induced by reactive oxygen species is associated with increased risk of developing colorectal cancer (CRC), and prostaglandin E2 (PGE2), which serves as a key mediator of inflammatory responses, plays an important role in CRC initiation and progression. Therefore, in the present study, we aimed to investigate the role of prostaglandin transporter OATP2A1/SLCO2A1 in the changes of PGE2 disposition in CRC cells in response to oxidative stress. H2O2 induced translocation of cytoplasmic OATP2A1 to plasma membranes in LoVo and COLO 320DM cells, but not in Caco-2 cells. The shift of subcellular OATP2A1 was abolished in the presence of anti-oxidant N-acetyl-L-cysteine or an inhibitor of protein kinase C, which evokes exocytosis. Exposure of LoVo cells to H2O2 caused an increase in the amount of extracellular PGE2 without changing the sum of intra- and extracellular PGE2. OATP2A1 knockdown decreased extracellular PGE2 in LoVo cells. In addition, extracellular PGE2 was significantly reduced by exocytosis inhibitor cytochalasin D, suggesting that H2O2-induced PGE2 release occurs in an exocytotic manner. Furthermore, mRNA expression of vascular endothelial growth factor (VEGF) was significantly reduced in LoVo cells by knockdown of OATP2A1. These results suggest that cytoplasmic OATP2A1 likely facilitates PGE2 loading into suitable intracellular compartment(s) for efficient exocytotic PGE2 release from CRC cells exposed to oxidative stress.

Inhibition of Methotrexate Uptake via Organic Anion Transporters OAT1 and OAT3 by Glucuronides of Nonsteroidal Anti-inflammatory Drugs.

Combination therapy of non-steroidal anti-inflammatory drugs (NSAIDs) and methotrexate (MTX) sometimes triggers adverse effects, such as liver injury, renal failure, gastrointestinal disorders, and myelosuppression, owing to the reduction of MTX clearance. Previous reports have suggested that NSAIDs inhibit renal MTX uptake via organic anion transporters (OATs) and reduced folate transporter (RFC)-1 and efflux via multidrug resistance-associated proteins (MRPs). Recently, our laboratory found inhibitory effects of NSAIDs-glucuronide (NSAIDs-Glu), a major metabolite of NSAIDs, on MRP-mediated MTX transport as a new site of interaction between MTX and NSAIDs. However, it remains unclear that whether NSAIDs-Glu inhibit renal uptake of MTX. Therefore, the present study aimed to evaluate inhibitory effects of several NSAIDs-Glu (diclofenac, R- and S-ibuprofen, R- and S-flurbiprofen, and R- and S-naproxen) on human OAT1 and OAT3-mediated MTX transport. In this study, [3H]MTX uptake was observed by using human OAT1 and OAT3-overexpressing HEK293 cells in the presence or absence of NSAIDs-Glu. All examined NSAIDs-Glu exhibited concentration-dependent inhibitory effects on MTX uptake via OAT1 and OAT3. Our results indicated that NSAIDs-Glu are more potent (5- to 15-fold) inhibitors of OAT3 than OAT1. Moreover, stereoselective inhibitory effects of NSAIDs-Glu on OATs-mediated MTX uptake were not observed, unlike on MRPs-mediated transport. These findings suggest that inhibition of OAT1 and OAT3-mediated renal uptake of MTX by plasma NSAIDs-Glu may be one of the competitive sites underlying complex drug interaction between MTX and NSAIDs.

Involvement of Reactive Metabolites of Diclofenac in Cytotoxicity in Sandwich-Cultured Rat Hepatocytes.

BACKGROUND AND OBJECTIVES: Diclofenac (DIC) is metabolized to reactive metabolites such as diclofenac acyl-ß-d-glucuronide (DIC-AG). It is possible that such reactive metabolites could cause tissue damage by formation of covalent protein adducts and other modification of cellular proteins or by induction of immune responses against its covalent protein adducts. However, the detailed mechanisms of idiosyncratic drug-induced liver injury (DILI) have been unclear. The objective is to clarify the involvement of DIC-AG and 4'hydroxydiclofenac (4'OH-DIC) in acute DILI. METHODS: We examined the effects of inhibiting DIC-AG and 4'OH-DIC production on covalent protein adduct formation and lactate dehydrogenase leakage using sandwich-cultured rat hepatocytes (SCRHs). RESULTS: After pretreatment of SCRH with (-)-borneol (BOR, a uridine diphosphate (UDP)-glucuronosyltransferase inhibitor) or sulfaphenazole (SUL, a cytochrome P450 2C9 inhibitor) for 30 minutes, intracellular concentrations of DIC, DIC-AG, and 4'OH-DIC were determined after further treating cells with 300 µM DIC for 3 hours. The decreased levels of reactive metabolites caused by BOR or SUL pretreatment resulted in decreased lactate dehydrogenase leakage from SCRH, although the formation of covalent protein adducts was not affected. CONCLUSION: These results suggested that both DIC-AG and 4'OH-DIC may be involved in acute cytotoxicity by DIC.

Prostaglandin transporter (OATP2A1/SLCO2A1) contributes to local disposition of eicosapentaenoic acid-derived PGE3.

Eicosapentaenoic acid (EPA)-derived prostaglandin E3 (PGE3) possesses an anti-inflammatory effect; however, information for transporters that regulate its peri-cellular concentration is limited. The present study, therefore, aimed to clarify transporters involved in local disposition of PGE3. PGE3 uptake was assessed in HEK293 cells transfected with OATP2A1/SLCO2A1, OATP1B1/SLCO1B1, OATP2B1/SLCO2B1, OAT1/SLC22A6, OCT1/SLC22A1 or OCT2/SLC22A2 genes, compared with HEK293 cells transfected with plasmid vector alone (Mock). PGE3 uptake by OATP2A1-expressing HEK293 cells (HEK/2A1) was the highest and followed by HEK/1B1, while no significantly higher uptake of PGE3 than Mock cells was detected by other transporters. Saturation kinetics in PGE3 uptake by HEK/2A1 estimated the Km as 7.202 ± 0.595 µM, which was 22 times higher than that of PGE2 (Km=0.331 ± 0.131 µM). Furthermore, tissue disposition of PGE3 was examined in wild-type (WT) and Slco2a1-deficient (Slco2a1(-/-)) mice after oral administration of EPA ethyl ester (EPA-E) when they underwent intraperitoneal injection of endotoxin (e.g., lipopolysaccharide). PGE3 concentration was significantly higher in the lung, and tended to increase in the colon, stomach, and kidney of Slco2a1(-/-), compared to WT mice. Ratio of PGE2 metabolite 15-keto PGE2 over PGE2 concentration was significantly lower in the lung and colon of Slco2a1(-/-) than that of WT mice, suggesting that PGE3 metabolism is downregulated in Slco2a1(-/-) mice. In conclusion, PGE3 was found to be a substrate of OATP2A1, and local disposition of PGE3 could be regulated by OATP2A1 at least in the lung.

High-resolution genetic mapping and physical map construction for the fertility restorer Rfm1 locus in barley.

KEY MESSAGE: High-resolution genetic linkage mapping and BAC physical mapping narrowed the fertility restorer locus Rfm1 in barley to a sub-centimorgan genetic interval and a 208-kb physical interval. Rfm1 restores the fertility of msm1 and msm2 male-sterile cytoplasms in barley. The fertility restoration gene is located on the short arm of chromosome 6H (6HS), and we pursued a positional cloning of this gene. Starting from a previous result that has delimited Rfm1 within a 10.8 cM region on 6HS, we developed novel CAPS and SSR markers tightly linked to the gene in barley using the sequence information from the syntenic region of rice and barley genome assemblies. Next, we performed fine mapping of the Rfm1 locus. To isolate recombinants, we surveyed 3,638 F2 plants derived from a cross between the CMS strain and the Rf strain with adjacent markers (NAS2090 and NAS1080). This analysis identified 175 recombinant plants from the F2 population to build a high-resolution map with nine markers tightly linked to the Rfm1 locus. Rfm1 was located within the 0.14 cM region delimited by two markers (NAS9113 and NAS9200). Using these flanking markers as well as marker cosegregating with Rfm1 (NAS9133), we screened the BAC libraries of the cultivar Morex, an rfm1 carrier. We isolated 11 BAC clones and constructed a BAC physical map using their fingerprints. Finally, we delimited the Rfm1 locus encompassing the rfm1 allele on a 208-kb contig composed of three minimally overlapping BAC clones. This precise localization of the Rfm1 locus in the barley genome is expected to greatly accelerate the future map-based cloning of the Rfm1 gene by sequence analysis and its genetic transformation for the complementation of cytoplasmic male-sterile plants.