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Chemical leukoderma is a skin depigmentation disorder induced through contact with certain chemicals, most of which have a p-substituted phenol structure similar to the melanin precursor tyrosine. The tyrosinase-catalyzed oxidation of phenols to highly reactive o-quinone metabolites is a critical step in inducing leukoderma through the production of melanocyte-specific damage and immunological responses.Our aim was to find an effective method to evaluate the formation of o-quinone by human tyrosinase and subsequent cellular reactions.Human tyrosinase-expressing 293T cells were exposed to various phenolic compounds, after which the reactive o-quinones generated were identified as adducts of cellular thiols. We further examined whether the o-quinone formation induces reductions in cellular GSH or viability.Among the chemicals tested, all 7 leukoderma-inducing phenols/catechol (rhododendrol, raspberry ketone, monobenzone, 4-tert-butylphenol, 4-tert-butylcatechol, 4-S-cysteaminylphenol and p-cresol) were oxidized to o-quinone metabolites and were detected as adducts of cellular glutathione and cysteine, leading to cellular glutathione reduction, whereas 2-S-cysteaminylphenol and 4-n-butylresorcinol were not. In vitro analysis using a soluble variant of human tyrosinase revealed a similar substrate-specificity. Some leukoderma-inducing phenols exhibited tyrosinase-dependent cytotoxicity in this cell model and in B16BL6 melanoma cells where tyrosinase expression was effectively modulated by siRNA knockdown.We developed a cell-based metabolite analytical method to detect human tyrosinase-catalyzed formation of o-quinone from phenolic compounds by analyzing their thiol-adducts. The detailed analysis of each metabolite was superior in sensitivity and specificity compared to cytotoxicity assays for detecting known leukoderma-inducing phenols, providing an effective strategy for safety evaluation of chemicals.

Equol (7-hydroxy-3-(4′-hydroxyphenyl)-chroman, EQ), one of the major intestinally derived metabolites of daidzein, the principal isoflavane found in soybeans and most soy foods, has recently attracted increased interest as a health-beneficial compound for estrogen-dependent diseases. However, based on its structure with two p-substituted phenols, this study aimed to examine whether EQ is a substrate for tyrosinase and whether it produces o-quinone metabolites that are highly cytotoxic to melanocyte. First, the tyrosinase-catalyzed oxidation of EQ was performed, which yielded three EQ-quinones. They were identified after being reduced to their corresponding catechols with NaBH4 or L-ascorbic acid. The binding of the EQ-quinones to N-acetyl-L-cysteine (NAC), glutathione (GSH), and bovine serum albumin via their cysteine residues was then examined. NAC and GSH afforded two mono-adducts and one di-adduct, which were identified by NMR and MS analysis. It was also found that EQ was oxidized to EQ-di-quinone in cells expressing human tyrosinase. Finally, it was confirmed that the EQ-oligomer, the EQ oxidation product, exerted potent pro-oxidant activity by oxidizing GSH to the oxidized GSSG and concomitantly producing H2O2. These results suggest that EQ-quinones could be cytotoxic to melanocytes due to their binding to cellular proteins.

Antagonizing transcriptional activity of farnesoid X receptor (FXR) in the intestine has been reported as an effective means for the treatment of nonalcoholic fatty liver disease, type 2 diabetes and obesity. We describe herein that the building blocks necessary to maintain the antagonism of our chemotype were investigated in order to modulate in vivo pharmacokinetic behavior and the tissue distribution without blunting the activity against FXR. A comprehensive understanding of the structure-activity relationship led to analog 30, which is superior to 12 in terms of its pharmacokinetic profiles by oral administration and its tissue distribution toward target tissues (liver and ileum) in rats while preserving the in vitro activity of 12 against FXR. Thus, 30 should be a candidate compound to investigate the effects of inhibiting FXR activity while simultaneously improving the outcome of nonalcoholic fatty liver disease, type 2 diabetes and obesity.

A novel real-time polymerase chain reaction (PCR) method for detecting chickpea was developed. From homologous sequences of 9-cis-epoxycarotenoid dioxygenase gene (NCED) among leguminous species, chickpea’s NCED was cloned, and the Southern-blot analysis showed that NCED is a single copy gene in a haploid genome. Its coding sequences at the 5′ terminus were found unique to chickpea and conserved among various chickpea varieties. Developed real-time PCR method targeting the unique sequences was specific to chickpea and had detection limit of approximately 30 copies per a reaction, and applicable for qualitative detection of chickpea in various forms of food products including dried, powdered, retort-packed, canned, fermented and pasted. Our results showed that the developed method enables identification of chickpea in foods.

Farnesoid X receptor (FXR) plays a major role in the control of cholesterol metabolism. Antagonizing transcriptional activity of FXR is an effective means to treat the relevant metabolic syndrome. Some of antagonists so far have the charged functions; however, they may negatively affect the pharmacokinetics. We describe herein a structure–activity relationship (SAR) exploration of nonacidic FXR antagonist 6 focusing on two regions in the structure and biological evaluation of nonacidic 10 with the characteristic N-acylated piperidine group obtained from SAR studies. As the robust affinity to FXR is feasible with our nonacidic analogue, 10 is among the most promising candidates for in vivo testing.

Statins are 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors that lower atherogenic LDL-cholesterol levels. Statins exert clinically relevant anti-inflammatory effects; however, the underlying molecular mechanism remains unclear. Studies have shown that endogenous and exogenous pathogenic crystals, such as cholesterol and monosodium urate (MSU), and needle-like nanomaterials, such as multi-wall carbon nanotubes (MWCNT), induce the production of IL-1β and play a critical role in the development of crystal-associated sterile inflammatory pathologies. In this study, we evaluated the effect of statins on crystal-induced IL-1β production in macrophages. We found that various statins, including pitavastatin, atorvastatin, fluvastatin, and lovastatin, but not squalene synthase inhibitor, repressed IL-1β release upon MWCNT stimulation. In addition, IL-1β production induced by cholesterol crystals and MSU crystals, but not by ATP or nigericin, was diminished. MWCNT-stimulated IL-1β release was dependent on the expression of NLRP3, but not AIM2, NLRC4, or MEFV. Statin-induced repression was accompanied by reduced levels of mature caspase-1 and decreased uptake of MWCNT into cells. Supplementation of mevalonate, geranylgeranyl pyrophosphate, or farnesyl pyrophosphate prevented the reduction in IL-1β release, suggesting a crucial role of protein prenylation, but not cholesterol synthesis. The statin-induced repression of MWCNT-elicited IL-1β release was observed in THP-1-derived and mouse peritoneal macrophages, but not in bone marrow-derived macrophages where statins act in synergy with lipopolysaccharide to enhance the expression of IL-1β precursor protein. In summary, we describe a novel anti-inflammatory mechanism through which statins repress mature IL-1β release induced by pathogenic crystals and nanoneedles by inhibiting the internalization of crystals by macrophages.

Poisonous Entoloma rhodopolium and other similar species including edible E. sarcopum are morphologically diverse. People mistake poisonous species for edible species. Classification and the detection method of these species need to be defined. The morphological and phylogenetic studies have been reported in northern Europe. In Japan, the genetic study remains unsolved. Thus, phylogenetic analysis of E. rhodopolium was conducted using ITS and RPB2 sequences, and the result was compared with that of European species. Japanese E. rhodopolium was classified into three clades, none of which belonged to the true European E. rhodopolium and other known species. Three species were defined as new species. Entoloma rhodopolium clade-I (named E. lacus) was genetically close to but morphologically separated from E. majaloides. Clade-II (E. subrhodopolium) was classified to the same group as E. sinuatum and E. subsinuatum, but distinct from these species. Clade-III was segregated from known Entoloma species including E. lupinum, and named E. pseudorhodopolium. Based on the classification, a simple identification method PCR-RFLP was developed to discriminate between poisonous species and edible E. sarcopum, which is very similar in morphology. The study can help to clarify the taxonomy of complex E. rhodopolium-related species, and to prevent food poisoning.

We describe here a novel chemotype with substituted benzimidazole scaffold for nonsteroidal farnesoid X receptor (FXR) antagonists starting from the identification of a screening hit, BB-4. Structure diversity in four regions A-D of BB-4 or 1 is discussed. In particular, regions A and C had an effect on an antagonism against FXR as demonstrated by the derivatives represented by 7 and 15, respectively. Thus, compound 19 arising from the combination of regions A and C underscored an important fact on antagonism against FXR, also showing the reduced small heterodimer partner and the increased cholesterol 7α-hydroxylase expression levels.

Identification of transgenic sequences in an unknown genetically modified (GM) papaya (Carica papaya L.) by whole genome sequence analysis was demonstrated. Whole genome sequence data were generated for a GM-positive fresh papaya fruit commodity detected in monitoring using real-time polymerase chain reaction (PCR). The sequences obtained were mapped against an open database for papaya genome sequence. Transgenic construct- and event-specific sequences were identified as a GM papaya developed to resist infection from a Papaya ringspot virus. Based on the transgenic sequences, a specific real-time PCR detection method for GM papaya applicable to various food commodities was developed. Whole genome sequence analysis enabled identifying unknown transgenic construct- and event-specific sequences in GM papaya and development of a reliable method for detecting them in papaya food commodities.

A number of genetically modified (GM) maize events have been developed and approved worldwide for commercial cultivation. A screening method is needed to monitor GM maize approved for commercialization in countries that mandate the labeling of foods containing a specified threshold level of GM crops. In Japan, a screening method has been implemented to monitor approved GM maize since 2001. However, the screening method currently used in Japan is time-consuming and requires generation of a calibration curve and experimental conversion factor (C(f)) value. We developed a simple screening method that avoids the need for a calibration curve and C(f) value. In this method, ΔC(q) values between the target sequences and the endogenous gene are calculated using multiplex real-time PCR, and the ΔΔC(q) value between the analytical and control samples is used as the criterion for determining analytical samples in which the GM organism content is below the threshold level for labeling of GM crops. An interlaboratory study indicated that the method is applicable independently with at least two models of PCR instruments used in this study.

2-(2'-Hydroxy-3',5'-di-tert-butylphenyl)benzotriazole (HDBB), the Benzotriazole UV-stabilizer (BUVSs) known as UV-320, is widely used in plastic materials for protection against UV-irradiation. Previously, we reported that oral ingestion of HDBB induce hepatotoxicity including hepatocyte hypertrophy and necrosis in rats and, males was more susceptible compared with females in young rats while no sex-related difference was observed in preweaning rats. Phenotypes observed in our previous study imply involvement of peroxisome proliferator-activated receptor (PPAR) α in HDBB hepatotoxicity, however, direct evidence that HDBB can activate PPARα has not been provided and the mechanism which underlying the gender difference of HDBB hepatotoxicity was not clearly elucidated. Here, we conduct transcriptome analysis using microarray expression profiles in the livers of rats administered HDBB. PPARα agonist activity of HDBB was elucidated by comparison with gene expression data of typical PPARα agonist, i.e. clofibrate, WY-14643, gemfibrozil, and fenofibrate, from TG GATEs database. Moreover, we analyzed for PPARα mRNA expression in the liver of developing male and female rats. PPARα mRNA expression level was higher in males than in females on postnatal days (PNDs) 28 and 35, whereas no sex-related difference was found on PNDs 7 and 22. These results suggest that HDBB exerts its hepatotoxicity through the PPARα signal pathway and the sex-related difference in PPARα expression may contribute to the sex-related difference in susceptibility to hepatotoxicity.

A series of accidents at the Fukushima Dai-ichi Nuclear Power Plant has raised concerns about the discharge of contaminated water containing tritium ((3)H) from the nuclear power plant into the environment and into foods. In this study, we explored convenient analytical methods to measure free-water (3)H in foods using a liquid scintillation counting and azeotropic distillation method. The detection limit was 10 Bq/L, corresponding to about 0.01% of 1 mSv/year. The (3)H recoveries were 85-90% in fruits, vegetables, meats and fishes, 75-85% in rice and cereal crops, and less than 50% in sweets containing little water. We found that, in the case of sweets, adding water to the sample before the azeotropic distillation increased the recovery and precision. Then, the recoveries reached more than 75% and RSD was less than 10% in all food categories (13 kinds). Considering its sensitivity, precision and simplicity, this method is practical and useful for (3)H analysis in various foods, and should be suitable for the safety assessment of foods. In addition, we examined the level of (3)H in foods on the Japanese market. No (3)H radioactivity was detected in any of 42 analyzed foods.

Farnesoid X receptor (FXR) plays a major role in the control of cholesterol metabolism. Antagonizing transcriptional activity of FXR is an effective means to treat the relevant metabolic syndrome. Some of antagonists so far have the charged functions; however, they may negatively affect the pharmacokinetics. We describe herein a structure-activity relationship (SAR) exploration of nonacidic FXR antagonist

Stacked genetically modified (GM) maize is increasingly produced; thereby, current event-specific quantitative real-time polymerase chain reaction (qPCR) methods have led to the overestimation of GM organism (GMO) content compared with the actual weight/weight percentage of GM organism in maize samples. We developed a feasible qPCR method in which the GMO content is calculated based on the quantification of two herbicide-tolerant trait genes, 5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium sp. strain CP4 (cp4epsps) and phosphinothricin N-acetyl-transferase from Streptomyces viridochromogenes (pat) to quantify the GMO content in ground grain samples containing stacked GM maize. The GMO contents of two genes were quantified using a plasmid calibrant and summed for quantification of total GMO content. The trait-specific method revealed lower biases for examination of test samples containing stacked GM maize compared with the event-specific method. Our results clearly show that the trait-specific method is not only simple and cost-effective, but also useful in quantifying the GMO content in ground grain samples containing stacked GM maize, which are expected to be major events in the near future. The developed method would be the only feasible way to conduct the quantification of GMO content in the ground maize samples containing stacked GM maize for the verification of the labeling regulation.

Because multi-wall carbon nanotubes (MWCNTs) have asbestos-like shape and size, concerns about their pathogenicity have been raised. Contaminated metals of MWCNTs may also be responsible for their toxicity. In this study, we employed high-temperature calcined fullerene nanowhiskers (HTCFNWs), which are needle-like nanofibers composed of amorphous carbon having similar sizes to MWCNTs but neither metal impurities nor tubular structures, and investigated their ability to induce production a major proinflammatory cytokine IL-1β via the Nod-like receptor pyrin domain containing 3 (NLRP3)-containing flammasome-mediated mechanism. When exposed to THP-1 macrophages, long-HTCFNW exhibited robust IL-1β production as long and needle-like MWCNTs did, but short-HTCFNW caused very small effect. IL-1β release induced by long-HTCFNW as well as by long, needle-like MWCNTs was abolished by a caspase-1 inhibitor or siRNA-knockdown of NLRP3, indicating that NLRP3-inflammasome-mediated IL-1β production by these carbon nanofibers. Our findings indicate that the needle-like shape and length, but neither metal impurities nor tubular structures of MWCNTs were critical to robust NLRP3 activation.

Many lines of genetically modified (GM) papaya (Carica papaya Linnaeus) have been developed worldwide to resist infection from various strains of papaya ringspot virus (PRSV). We found an unidentified and unauthorized GM papaya in imported processed papaya food. Transgenic vector construct that provides resistance to the PRSV strains isolated in Thailand was detected. An original and specific real-time polymerase chain reaction method was generated to qualitatively detect the PRSV-Thailand-resistant GM papaya.

A real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event, MON87701. First, a standard plasmid for MON87701 quantification was constructed. The conversion factor (C

Biomarkers are useful tools as indicators/predictors of disease severity and drug responsiveness, and thus, are expected to make drug development more efficient and to accelerate proper use of approved drugs. Many academic achievements on biomarkers have been reported, but only several biomarkers are used in drug development and clinical settings. We first show our results on the pharmacogenomic analysis of the anti-cancer drug irinotecan and of Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). UGT1A1*6 and *28 were significantly associated with altered pharmacokinetics of an irinotecan metabolite, SN-38, and with increased frequency of severe neutropenia. HLA* 58:01 and HLA-B*15:11/HLA-A*31:01 were associated with SJS/TEN by allopurinol and carbamazepine, respectively. Our papers have been cited in the package inserts of irinotecan and allopurinol. In addition to these genomic biomarkers, metabolomic biomarkers, which can reflect the disease phenotype and drug responsiveness, have been exploring for 12 major diseases in Japan, as a part of a multi-omics team with multi-national centers. In animal models of dilated cardiomyopathy and Alzheimer's disease, we found several changes in lipid metabolite levels in the diseased tissues. Moreover, two oxidized fatty acids were correlatively changed in the brain and plasma from Alzheimer's model mice before its onset, and thus, could be candidates for predictive biomarkers. Finally, we propose/discuss several key issues for academic researches on biomarker discovery and development, especially for newly coming researchers in the field of pharmaceutical sciences. We hope that this review would help novel biomarker identification and qualification in Japan.

Genome editing has undergone rapid development during the last three years. It is anticipated that genetically modified organisms (GMOs) for food purposes will be widely produced using the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR)/Cas9 system in the near future. However, the Cas9 gene may then enter the genomes of GMOs for food if the breeding process is not strictly managed, which could lead to the Cas9 protein or associated peptides being produced within these organisms. A variety of peptides could theoretically be produced from the Cas9 gene by using open reading frames different from that of Cas9 in the GMOs. In this study, Cas9 and the peptides potentially encoded by Cas9 genes were studied regarding their immunogenicity, in terms of the digestibility of Cas9 and the homology of the peptides to food allergens. First, the digestibility and thermal stability of Cas9 were studied. Digestibility was tested with natural or heat-denatured Cas9 in simulated gastric fluid in vitro. The two types of Cas9 were digested rapidly. Cas9 was also gradually degraded during heat treatment. Second, the peptides potentially encoded by Cas9 genes were examined for their homology to food allergens. Specifically, an 8-mer exact match search and a sliding 80-mer window search were performed using allergen databases. One of the peptides was found to have homology with a food allergen.

Chemical leukoderma is a skin depigmentation disorder known to occur in manufactural workplace through contact with chemicals, such as monobenzyl ether of hydroquinone (MBEH) and 4-tert- butylphenol (4-TBP). In the skin depigmented -legions induced by these chemicals, the number of melanocyte was severely decreased. Anti-melanoma agent 4-cysteaminylphenol (4-SCAP) and its derivatives are also known to cause leukoderma. Evidence has accumulated supporting that typical class of chemicals causing leukoderma is "4-alkyl/aryl-substituted phenols/catechols", which are structurally similar to melanin precursor tyrosine. Tyrosinase-mediated oxidation of these chemicals yields toxic ortho-quinones which bind to cellular proteins and produce reactive oxygen species. Accordingly, this tyrosinase-dependent metabolic activation is thought to cause melanocyte-specific damage and subsequent immune reactions toward melanocytes. Recently, rhododendrol, an inhibitor of tyrosinase developed for so-called lightening/whitening cosmetics, was shown to cause leukoderma in the users. In this review, I document the causes of known chemical leukoderma and rhododendrol- induced leukoderma, focusing on their common mechanisms underlying melanocyte loss.

Recent progress in chemical biology of natural products has revealed the multiligandable nature, and exploration of new natural product targets often leads to the discovery of novel drug leads. We ...

Alpha‐1‐acid glycoprotein (AGP) encoded by orosomucoid genes ( ORM1 and ORM2 ) is an acute‐phase response protein and functions as a drug‐binding protein that affects pharmacokinetics (PK)/pharmacodynamics of binding drugs. To explore the effects of genetic variations of ORMs and a role of AGP on paclitaxel (PTX) therapy, we analyzed the duplication and genetic variations/haplotypes of ORMs in 165 Japanese cancer patients and then investigated their associations with serum AGP levels and the PK parameters of PTX. No effects of ORM duplications on serum AGP levels at baseline or PK of PTX were observed, but close associations of ORM1 − 559T > A with the increases of AGP levels and area under the curve (AUC) of PTX metabolites were detected. In addition, a significant correlation between the serum AGP level and the AUCs of PTX metabolites was observed, suggesting that AGP may function as a carrier of PTX from the blood into the liver via putative receptors. This study provided useful information on the possible clinical importance of ORM genetic polymorphisms and a novel role of AGP in PTX therapy. © 2011 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4546–4559, 2011

Objective— Telmisartan, an angiotensin type I receptor blocker (ARB), protects against the progression of atherosclerosis. Here, we investigated the molecular basis of the antiatherosclerotic effects of telmisartan in macrophages and apolipoprotein E–deficient mice. Methods and Results— In macrophages, telmisartan increased peroxisome proliferator-activated receptor-γ (PPARγ) activity and PPAR ligand-binding activity. In contrast, 3 other ARBs, losartan, valsartan, and olmesartan, did not affect PPARγ activity. Interestingly, high doses of telmisartan activated PPARα in macrophages. Telmisartan induced the mRNA expression of CD36 and ATP-binding cassette transporters A1 and G1 (ABCA1/G1), and these effects were abrogated by PPARγ small interfering RNA. Telmisartan, but not other ARBs, inhibited lipopolysaccharide-induced mRNA expression of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α, and these effects were abrogated by PPARγ small interfering RNA. Moreover, telmisartan suppressed oxidized low-density lipoprotein-induced macrophage proliferation through PPARγ activation. In apolipoprotein E −/− mice, telmisartan increased the mRNA expression of ABCA1 and ABCG1, decreased atherosclerotic lesion size, decreased the number of proliferative macrophages in the lesion, and suppressed MCP-1 and tumor necrosis factor-α mRNA expression in the aorta. Conclusion— Telmisartan induced ABCA1/ABCG1 expression and suppressed MCP-1 expression and macrophage proliferation by activating PPARγ. These effects may induce antiatherogenic effects in hypertensive patients.

Manipulation of protein stability with small molecules is a challenge in the field of drug discovery. Here we show that cellular retinoic acid binding protein-II (CRABP-II) can be specifically degraded by a novel compound, SNIPER-4, consisting of (−)-N-[(2S,3R)-3-amino-2-hydroxy-4-phenyl-butyryl]-l-leucine methyl ester and all-trans retinoic acid that are ligands for cellular inhibitor of apoptosis protein 1 (cIAP1) and CRABP-II, respectively. Mechanistic analysis revealed that SNIPER-4 induces cIAP1-mediated ubiquitylation of CRABP-II, resulting in the proteasomal degradation. The protein knockdown strategy employing the structure of SNIPER-4 could be applicable to other target proteins.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT * Association of UDP-glucuronosyltransferase 1A1 (UGT1A1) genetic polymorphisms *6 and *28 with reduced clearance of SN-38 and severe neutropenia in irinotecan therapy was demonstrated in Japanese cancer patients. * The detailed gene structure of CES1 has been characterized. * Possible functional SNPs in the promoter region have been reported. WHAT THIS STUDY ADDS * Association of functional CES1 gene number with AUC ratio [(SN-38 + SN-38G)/irinotecan], an in vivo index of CES activity, was observed in patients with irinotecan monotherapy. * No significant effects of major CES1 SNPs on irinotecan PK were detected. AIMS Human carboxylesterase 1 (CES1) hydrolyzes irinotecan to produce an active metabolite SN-38 in the liver. The human CES1 gene family consists of two functional genes, CES1A1 (1A1) and CES1A2 (1A2), which are located tail-to-tail on chromosome 16q13-q22.1 (CES1A2-1A1). The pseudogene CES1A3 (1A3) and a chimeric CES1A1 variant (var1A1) are also found as polymorphic isoforms of 1A2 and 1A1, respectively. In this study, roles of CES1 genotypes and major SNPs in irinotecan pharmacokinetics were investigated in Japanese cancer patients. METHODS CES1A diplotypes [combinations of haplotypes A (1A3-1A1), B (1A2-1A1), C (1A3-var1A1) and D (1A2-var1A1)] and the major SNPs (-75T>G and -30G>A in 1A1, and -816A>C in 1A2 and 1A3) were determined in 177 Japanese cancer patients. Associations of CES1 genotypes, number of functional CES1 genes (1A1, 1A2 and var1A1) and major SNPs, with the AUC ratio of (SN-38 + SN-38G)/irinotecan, a parameter of in vivo CES activity, were analyzed for 58 patients treated with irinotecan monotherapy. RESULTS The median AUC ratio of patients having three or four functional CES1 genes (diplotypes A/B, A/D or B/C, C/D, B/B and B/D; n= 35) was 1.24-fold of that in patients with two functional CES1 genes (diplotypes A/A, A/C and C/C; n= 23) [median (25th-75th percentiles): 0.31 (0.25-0.38) vs. 0.25 (0.20-0.32), P= 0.0134]. No significant effects of var1A1 and the major SNPs examined were observed. CONCLUSION This study suggests a gene-dose effect of functional CES1A genes on SN-38 formation in irinotecan-treated Japanese cancer patients.

Fibroblast growth factor 1 (FGF-1) enhances apolipoprotein E (apoE) expression and apoE-HDL biogenesis in autocrine fashion in astrocytes (Ito, J., Y. Nagayasu, R. Lu, A. Kheirollah, M. Hayashi, and S. Yokoyama. Astrocytes produce and secrete FGF-1, which promotes the production of apoE-HDL in a manner of autocrine action. J. Lipid Res. 2005. 46: 679-686) associated with healing of brain injury (Tada,T., J-i. Ito, M. Asai, and S. Yokoyama. Fibroblast growth factor 1 is produced prior to apolipoprotein E in the astrocytes after cryo-injury of mouse brain. Neurochem. Int. 2004. 45: 23-30). FGF-1 stimulates mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) to increase cholesterol biosynthesis and phosphatidylinositol 3-OH kinase (PI3K)/Akt to enhance apoE-HDL secretion (Ito, J., Y. Nagayasu, K. Okumura-Noji, R. Lu, T. Nishida, Y. Miura, K. Asai, A. Kheirollah, S. Nakaya, and S. Yokoyama. Mechanism for FGF-1 to regulate biogenesis of apoE-HDL in astrocytes. J. Lipid Res. 2007. 48: 2020-2027). We investigated the mechanism for FGF-1 to upregulate apoE transcription. FGF-1 increased apoE and liver X receptor alpha (LXRalpha) mRNAs in rat astrocytes. Increase of LXRalpha mRNA was suppressed by inhibition of the FGF-1 receptor-1 and MEK/ERK but not by inhibition of PI3K/Akt. The increases of apoE mRNA and apoE-HDL secretion were both inhibited by downregulation or inhibition of LXRalpha, while they were partially suppressed by inhibiting cholesterol biosynthesis. We identified the liver X receptor element responsible for activation of the rat apoE promoter by FGF-1 located between -450 and -320 bp, and the direct repeat 4 (DR4) element in this region (-448 to -433 bp) was responsible for the activation. Chromatin immunoprecipitation analysis supported that FGF-1 enhanced association of LXR with the rat apoE promoter. FGF-1 partially activated the apoE promoter even in the presence of an MEK inhibitor that inhibits the FGF-1-mediated enhancement of cholesterol biosynthesis. On the other hand, FGF-1 induced production of 25-hydroxycholesterol by MEK/ERK as an sterol regulatory element-dependent reaction besides cholesterol biosynthesis. We concluded that FGF-1-induced apoE expression in astrocytes depends on LXRalpha being mediated by both LXRalpha expression and an LXRalpha ligand biosynthesis.

Riccardin C, a nuclear receptor LXRalpha selective agonist, is an 18-membered macrocyclic bisbibenzyl isolated from several liverworts. Synthesis of riccardin C and its seven O-methylated derivatives was accomplished. The synthetic sequence highlights an intramolecular Suzuki-Miyaura coupling in the formation of the 18-membered biaryl linkage present in riccardin C. The structure-activity relationship of these compounds suggests that all of the phenolic hydroxy groups present in riccardin C are essential for the activation of LXRalpha.

Release of cellular cholesterol by ATP-binding cassette transporter (ABC)A1 and apolipoproteins is a major source of plasma high-density lipoprotein (HDL). Expression of ABC transporter A1 (ABCA1) is directly stimulated by liver X receptor (LXR)/retinoid X receptor (RXR) activation. We evaluated the abilities of two RXR agonists, PA024 and HX630, to increase ABCA1 expression. In differentiated THP-1 cells, the two agonists efficiently enhanced ABCA1 mRNA expression and apoA-I-dependent cellular cholesterol release. However, in RAW264 cells and undifferentiated THP-1 cells, PA024 was highly effective while HX630 was inactive in increasing ABCA1 mRNA. In parallel, the two agonists had different abilities to activate ABCA1 promoter in an LXR-responsive-element (LXRE)-dependent manner and to directly stimulate LXRalpha/RXR transactivation. The ability of HX630 to enhance ABCA1 expression was correlated closely with the cellular PPARgamma mRNA level. Moreover, HX630 was able to activate PPARgamma/RXR. Transfection of PPARgamma in RAW264 cells induced HX630-mediated activation of LXRE-dependent transcription and ABCA1 promoter, suggesting the ability of HX630 to activate PPARgamma-LXR-ABCA1 pathway. We conclude that RXR agonist PA024 and HX630 have different abilities to activate LXR/RXR, and that the cell-type-dependent effect of HX630 on ABCA1 expression and HDL generation is closely associated with this defect.

Farnesoid X receptor (FXR) controls the expression of critical genes in bile acid and cholesterol homeostasis. To study FXR and to develop a regulator of cholesterol, some nonsteroidal and steroidal ligands have been found in addition to endogenous ligands for FXR. In this study, we discovered five bile acid derivatives (methyl cholate, methyl deoxycholate, 5β-cholanic acid, 5β-cholanic acid-7α,12α-diol, and NIHS700) and two natural products (marchantin A and marchantin E) that activated FXR in the reporter assay. These compounds activated FXR to a high level comparable to the most potent endogenous bile acid, chenodeoxycholic acid, although it was not predicted from their structures; five of them were similar to the lower potency bile acids, and two were structurally much different from bile acids. The elevation levels of reporter gene expression by some of the screened compounds were varied in Cos-7, HepG2, HuH-7, and Caco-2 cells. These compounds also controlled the expression of genes regulated by FXR, and some of the compounds regulated these genes in a cell-type–specific and/or gene-selective fashion. Therefore, molecular design of the compounds can cause selective modulation of the expression of FXR target genes. Keywords:: farnesoid X receptor (FXR), reporter assay, ginkgolic acid, marchantin, cell-type–specific modulation

ABC transporter A1 (ABCA1) mediates and rate-limits biogenesis of high density lipoprotein (HDL), and hepatic ABCA1 plays a major role in regulating plasma HDL levels. HDL generation is also responsible for release of cellular cholesterol. In peripheral cells ABCA1 is up-regulated by the liver X receptor (LXR) system when cell cholesterol increases. However, cholesterol feeding has failed to show a significant increase in hepatic ABCA1 gene expression, and its expression is up-regulated by statins (3-hydroy-3-methylglutaryl-CoA reductase inhibitors), suggesting distinct regulation. In this study we investigated the mechanism of regulation of the rat hepatic ABCA1 gene and identified two major ABCA1 transcripts and two corresponding promoter regions. Compactin activated the novel liver-type promoter in rat hepatoma McARH7777 cells by binding the sterol regulatory element-binding protein-2 (SREBP-2). In contrast, compactin repressed the previously identified peripheral-type promoter in an LXR-responsive element-dependent but not E-box-dependent manner. Thus, compactin increased the liver-type transcript and decreased the peripheral-type transcript. The same two transcripts were also dominant in human and mouse livers, whereas the intestine contains only the peripheral-type transcript. Treatment of rats with pravastatin and a bile acid binding resin (colestimide), which is known to activate SREBP-2 in the liver, caused a reduction in the hepatic cholesterol level and the same differential responses in vivo, leading to increases in hepatic ABCA1 mRNA and protein and plasma HDL levels. We conclude that the dual promoter system driven by SREBP-2 and LXR regulates hepatic ABCA1 expression and may mediate the unique response of hepatic ABCA1 gene expression to cellular cholesterol status.

To develop oral antibody therapy against rotavirus infection, we previously produced a recombinant fragment of llama heavy-chain antibody to rotavirus (ARP1) in rice seeds (MucoRice-ARP1). We intend to use a purification-free rice powder for clinical application but needed to check whether MucoRice-ARP1 had increased levels of known allergen proteins. For this purpose, we used two-dimensional fluorescence difference gel electrophoresis to compare the allergen protein levels in MucoRice-ARP1 and wild-type rice. We detected no notable differences, except in the levels of α-amylase/trypsin inhibitor-like family proteins. Because by this approach we could not completely separate ARP1 from the proteins of this family, we confirmed the absence of changes in the levels of these allergens by using shotgun mass spectrometry as well as immunoblot. By using immunoelectron microscopy, we also showed that RAG2, a member of the α-amylase/trypsin inhibitor-like protein family, was relocated from protein bodies II to the plasma membrane or cell wall in MucoRice-ARP1 seed. The relocation did not affect the level of RAG2. We demonstrated that most of the known rice allergens were not considerably upregulated by the genetic modification in MucoRice-ARP1. Our data suggest that MucoRice-ARP1 is a potentially safe oral antibody for clinical application.

Nonalcoholic steatohepatitis (NASH) is a major health problem since it often leads to hepatocellular carcinoma. However, the underlying mechanisms of NASH development and subsequent fibrosis have yet to be clarified. We compared comprehensive lipidomic profiles between mice with high fat diet (HFD)-induced steatosis and STAM mice with NASH and subsequent fibrosis. The STAM mouse is a model that demonstrates NASH progression resembling the disease in humans: STAM mice manifest NASH at 8 weeks, which progresses to fibrosis at 12 weeks and finally develop hepatocellular carcinoma. Overall, 250 lipid molecules were detected in the liver using liquid chromatography-mass spectrometry. We found that STAM mice with NASH presented a significantly higher abundance of sphingolipids and lower levels of triacylglycerols than the HFD-fed control mice. The abundance of certain fatty acids in phospholipid side chains was also significantly different between STAM and control mice, although global levels of phosphatidylcholines and phosphatidylethanolamines were comparable. Finally, increase in levels of acylcarnitines and some diacylglycerols was observed in STAM mice toward the fibrosis stage, but not in age-matched control mice. Our study provides insights into the lipid status of the steatotic, NASH and fibrotic liver that would help elucidate the molecular pathophysiology of NASH progression.

Hydrolysed wheat protein (HWP), which has high emulsifying property and water retentivity, is added to various foods and cosmetics to improve their texture. Recently, several studies showed that a specific HWP formula could induce IgE-mediated hypersensitivity by skin contact and/or food ingestion. In Japan, a number of HWP-caused systemic wheat allergy cases were reported, including wheat-dependent exercise-induced anaphylaxis after sensitisation to acid-hydrolysed gluten additive [Glupearl 19S® (Glp19S)] in a cosmetic soap. These allergic manifestations are believed to result from deamidated peptides in high-molecular weight fractions of Glp19S. However, relationships between the properties of HWP and its allergenicity remain unknown. Here, we experimentally assessed the cross-reactivity of IgE from Glp19S-sensitised patients' sera by examining the IgE-binding capacity to various HWPs generated under different conditions and the ability to elicit mast cell activation. We prepared three different HWP types by acidic hydrolysis (0.1M HCl treatment at 100°C), alkaline hydrolysis (1M NaOH treatment at 100°C) and enzymatic hydrolysis (Neutrase® digestion at 50°C) for 0.5, 1, 3, 6, 9, 12 and 24 h, respectively. We examined the capacity of IgE binding to various acid-HWPs and native gluten using Western blotting. After 0.5-1 h of acid hydrolysis, IgE immunoblotting with a patient's serum showed smears at around 40-70-kDa proteins. However, after 3 h of acid hydrolysis, IgE binding to 40-70-kDa proteins time-dependently decreased. Regarding mast cell activation by acid-HWPs, a 0.5-h hydrolysis sample showed dramatically increased mast cell activation, whereas native gluten had little effect on it. Even after prolonged hydrolysis, acid-HWPs still retained the ability to activate mast cells, with only slightly decreased activity levels. The alkaline and enzymatic hydrolysis samples are currently under investigation to assess cross-reactivity. In conclusion, we showed that allergenic HWP had specific molecular properties given by hydrolysis. These results showed that it may be possible to reduce the risk of HWP-caused allergy by the regulation of hydrolysis procedures.

The farnesoid X receptor (FXR) is a bile acid/alcohol-activated nuclear receptor that regulates lipid homeostasis. Unlike other steroid receptors, FXR binds bile acids in an orientation that allows the steroid nucleus A ring to face helix 12 in the receptor, a crucial domain for coactivator-recruitment. Because most naturally occurring bile acids and alcohols contain a cis-oriented A ring, which is distinct from that of other steroids and cholesterol metabolites, we investigated the role of this 5beta-configuration in FXR activation. The results showed that the 5beta-(A/B cis) bile alcohols 5beta-cyprinol and bufol are potent FXR agonists, whereas their 5alpha-(A/B trans) counterparts antagonize FXR transactivation and target gene expression. Both isomers bound to FXR, but their ability to induce coactivator-recruitment and thereby induce transactivation differed. These findings suggest a critical role for the A-ring orientation of bile salts in agonist/antagonist function.

Liver X receptors (LXRs) alpha and beta share considerable sequence homology and several functions, respond to the same endogenous and synthetic ligands, and play critical roles in maintaining lipid homeostasis. In this study, liverwort-derived riccardin C (RC) and F (RF) were identified as an LXRalpha agonist/LXRbeta antagonist and an LXRalpha antagonist, respectively. RC and RF bound to LXRs, but had different abilities to recruit a coactivator and thereby induce transactivation. Despite its unique subtype-selective activity, RC enhanced ABCA1 and ABCG1 expression and cellular cholesterol efflux in THP-1 cells. RC may provide a novel tool for identifying subtype-function and drug development.

Objective— Fibrates are widely used drugs to reduce plasma triglyceride and increase high-density lipoprotein. Their active forms, fibric acids, are peroxisome proliferator-activated receptor-α activators, but no direct evidence has been demonstrated for their activation of ATP-binding cassette transporter A1 (ABCA1) in relation to clinically used fibrates. We investigated the reaction of fenofibric acid in this regard. Methods and Results— Fenofibric acid was examined for the effect of increase of ABCA1 activity. It enhanced ABCA1 gene transcription and its protein level in macrophage cell line cells and fibroblasts and increased apolipoprotein A-I–mediated cellular lipid release, all in a dose-dependent manner. Enhancement of the gene transcription was examined by using a reporter assay system for liver X receptor responsive element (LXRE) and its inactive mutant. The results demonstrated that the effect of fenofibric acid is dependent on active LXRE. Conclusions— Fenofibric acid increased transcription of ABCA1 gene in a liver X receptor–dependent manner.

HX531 is a retinoid X receptor (RXR) antagonist that inhibits 9-cis retinoic acid-induced neutrophilic differentiation of HL-60 cells. In order to elucidate the inhibitory mechanism of HX531, we have developed a novel ligand sensor assay for RXR in which the receptor-coactivator interaction is directly monitored using surface plasmon resonance (SPR) biosensor technology. A 20-mer peptide from steroid receptor coactivator-1 (SRC-1), containing nuclear receptor interaction motif LXXLL was immobilized on the surface of a BIAcore sensor chip. Injection of human recombinant RXR with or without 9-cis retinoic acid resulted in ligand-dependent interaction with the SRC-1 peptide. Kinetic analysis revealed dissociation constants (KD) of 9-cis RA-preincubated RXR to SRC-1 was 5.92 x 10(-8)M. Using this technique, we found that 1 microM HX531 reduced the ka value of liganded-RXR with SRC-1, suggesting that HX531 reduced the affinity of RXR to SRC-1. This SPR assay system was applied to obtain quantitative kinetic data of RXR ligand binding to the SRC-1 peptide and the alteration of these data by antagonists.

Objective—Release of cellular cholesterol and phospholipid mediated by helical apolipoprotein and ATP-binding cassette transporter (ABC) A1 is a major source of plasma HDL. We investigated the effect of calcium channel blockers on this reaction.Methods and Results—Expression of ABCA1, apoA-I–mediated cellular lipid release, and HDL production were enhanced in cAMP analogue-treated RAW264 cells by verapamil, and similar effects were also observed with other calcium channel blockers. The verapamil treatment resulted in rapid increase in ABCA1 protein and its mRNA, but not the ABCG1 mRNA, another target gene product of the nuclear receptor liver X receptor (LXR). By using the cells transfected with a mouse ABCA1 promoter–luciferase construct (−1238 to +57bp), verapamil was shown to enhance the transcriptional activity. However, it did not increase transcription of LXR response element-driven luciferase vector.Conclusions—The data demonstrated that verapamil increases ABCA1 expression through LXR-independent mechanism and thereby increases apoA-I–mediated cellular lipid release and production of HDL.

Ligand binding to nuclear receptors leads to a conformational change that increases the affinity of the receptors to coactivator proteins. We have developed a ligand sensor assay for farnesoid X receptor (FXR) in which the receptor-coactivator interaction can be directly monitored using surface plasmon resonance biosensor technology. A 25-mer peptide from coactivator SRC1 containing the LXXLL nuclear receptor interaction motif was immobilized on the surface of a BIAcore sensor chip. Injection of the FXR ligand binding domain (FXRLBD) with or without the most potent natural ligand, chenodeoxycholic acid (CDCA), over the surface of the chip resulted in a ligand- and LXXLL motif-dependent interaction. Kinetic analysis revealed that CDCA and its conjugates decreased the equilibrium dissociation constant (K(d)) by 8-11-fold, indicating an increased affinity. Using this technique, we found that a synthetic bile acid sulfonate, 3alpha,7alpha-dihydroxy-5beta-cholane-24-sulfonate, which was inactive in a FXR response element-driven luciferase assay using CV-1 cells, caused the most potent interaction, comparable to the reaction produced by CDCA. This method provides a rapid and reliable in vitro ligand assay for FXR. This kinetic analysis-featured technique may be applicable to mechanistic studies.