A cell-based evaluation of human tyrosinase-mediated metabolic activation of leukoderma-inducing phenolic compounds.

BACKGROUND: 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. OBJECTIVE: Our aim was to find an effective method to evaluate the formation of o-quinone by human tyrosinase and subsequent cellular reactions. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

The Oxidation of Equol by Tyrosinase Produces a Unique Di-ortho-Quinone: Possible Implications for Melanocyte Toxicity.

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.

Statins repress needle-like carbon nanotube- or cholesterol crystal-stimulated IL-1ß production by inhibiting the uptake of crystals by macrophages.

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-1ß 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-1ß production in macrophages. We found that various statins, including pitavastatin, atorvastatin, fluvastatin, and lovastatin, but not squalene synthase inhibitor, repressed IL-1ß release upon MWCNT stimulation. In addition, IL-1ß production induced by cholesterol crystals and MSU crystals, but not by ATP or nigericin, was diminished. MWCNT-stimulated IL-1ß 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-1ß release, suggesting a crucial role of protein prenylation, but not cholesterol synthesis. The statin-induced repression of MWCNT-elicited IL-1ß 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-1ß precursor protein. In summary, we describe a novel anti-inflammatory mechanism through which statins repress mature IL-1ß release induced by pathogenic crystals and nanoneedles by inhibiting the internalization of crystals by macrophages.

Identification of potent farnesoid X receptor (FXR) antagonist showing favorable PK profile and distribution toward target tissues: Comprehensive understanding of structure-activity relationship of FXR antagonists.

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.

Nonacidic Chemotype Possessing N-Acylated Piperidine Moiety as Potent Farnesoid X Receptor (FXR) Antagonists.

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.

Development and Evaluation of an Enzyme-Linked Immunosorbent Assay Using a Nonpoisonous Extraction System for the Determination of Crustacean Protein in Processed Foods.

Crustacean proteins are food allergens that cause severe allergic reactions in patients with food allergies; therefore, the identification of crustaceans such as shrimp, crab, and lobster as ingredients in processed food products is mandatory in Japan. We previously developed and validated an ELISA method coupled with an extraction process using the surfactant sodium dodecyl sulfate and the reductant 2-mercaptoethanol (2-ME) to quantify crustacean protein. However, 2-ME was designated as poisonous in Japan in 2008. Therefore, in this study, we developed and evaluated an ELISA method for detecting and quantifying crustacean protein that uses sodium sulfite (Na2SO3) in place of 2-ME for extraction. The proposed ELISA method showed high sensitivity, with an LOQ of 0.66 µg protein/g food sample. Furthermore, the proposed method showed high specificity for the Decapoda order within the subphylum Crustacea, with recoveries ranging from 83.8 to 100.8% for model processed foods, as well as high reproducibility (intra- and interassay CVs of ≤8.2%) and high correlation with our previously validated ELISA method for processed foods (correlation coefficient of 0.996). The proposed ELISA method does not require the use of poisonous reagents, provides acceptable accuracy, and is useful for the routine monitoring of food products.

Molecular phylogenetic analysis of new Entoloma rhodopolium-related species in Japan and its identification method using PCR-RFLP.

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.

Discovery and optimization of benzimidazole derivatives as a novel chemotype of farnesoid X receptor (FXR) antagonists.

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.

Development and Evaluation of Event-Specific Quantitative PCR Method for Genetically Modified Soybean MON87701.

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 (Cf) required to calculate the amount of genetically modified organism (GMO) was experimentally determined for a real-time PCR instrument. The determined Cf for the real-time PCR instrument was 1.24. For the evaluation of the developed method, a blind test was carried out in an inter-laboratory trial. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSDr), respectively. The determined biases and the RSDr values were less than 30 and 13%, respectively, at all evaluated concentrations. The limit of quantitation of the method was 0.5%, and the developed method would thus be applicable for practical analyses for the detection and quantification of MON87701.

Cas9 in Genetically Modified Food Is Unlikely to Cause Food Allergy.

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.

Different Results of IgE Binding- and Crosslinking-Based Allergy Tests Caused by Allergen Immobilization.

The physicochemical nature of allergen molecules differ from the liquid phase to the solid phase. However, conventional allergy tests are based on the detection of immunoglobulin (Ig)E binding to immobilized allergens. We recently developed an in vitro allergy testing method using a luciferase-reporting humanized rat mast cell line to detect IgE crosslinking-induced luciferase expression (EXiLE test). The aim of the present study was to evaluate the effects of antigen immobilization on the results of different in vitro allergy tests using two anti-ovalbumin (OVA) antibodies (Abs), E-C1 and E-G5, with different properties in the OVA-induced allergic reaction. Both Abs showed clear binding to OVA with an enzyme-linked immunosorbent assay and by BIAcore analysis. However, only E-C1 potentiated EXiLE response for the liquid-phase OVA. On the other hand, OVA immobilized on solid-phase induced EXiLE responses in both E-C1 Ab- and E-G5 Ab-sensitized mast cells. Western blotting of OVA indicated that E-C1 Ab binds both to OVA monomers and dimers, unlike E-G5 Ab, which probably binds only to the OVA dimer. These results suggest that antigen immobilization enhanced IgE crosslinking ability through multimerization of allergen molecules in the solid phase, resulting in an increase in false positives in IgE binding-based conventional in vitro allergy tests. These findings shed light on the physicochemical nature of antigens as an important factor for the development and evaluation of in vitro allergy tests and suggest that mast cell activation-based allergy testing with liquid-phase allergens is a promising strategy to evaluate the physiological interactions of IgE and allergens.

Transcriptome analyses demonstrate that Peroxisome Proliferator-Activated Receptor α (PPARα) activity of an ultraviolet absorber, 2-(2'-hydroxy-3',5'-di-tert-butylphenyl)benzotriazole, as possible mechanism of their toxicity and the gender differences.

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.

An Analytical Method to Measure Free-Water Tritium in Foods using Azeotropic Distillation.

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.

Interlaboratory validation data on real-time polymerase chain reaction detection for unauthorized genetically modified papaya line PRSV-YK.

This article is referred to research article entitled "Whole genome sequence analysis of unidentified genetically modified papaya for development of a specific detection method" (Nakamura et al., 2016) [1]. Real-time polymerase chain reaction (PCR) detection method for unauthorized genetically modified (GM) papaya (Carica papaya L.) line PRSV-YK (PRSV-YK detection method) was developed using whole genome sequence data (DDBJ Sequenced Read Archive under accession No. PRJDB3976). Interlaboratory validation datasets for PRSV-YK detection method were provided. Data indicating homogeneity of samples prepared for interlaboratory validation were included. Specificity and sensitivity test data for PRSV-YK detection method were also provided.

Development and Interlaboratory Validation of a Simple Screening Method for Genetically Modified Maize Using a ΔΔC(q)-Based Multiplex Real-Time PCR Assay.

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.

Whole genome sequence analysis of unidentified genetically modified papaya for development of a specific detection method.

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.

Differential analyses of major allergen proteins in wild-type rice and rice producing a fragment of anti-rotavirus antibody.

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.

Characterization of hepatic lipid profiles in a mouse model with nonalcoholic steatohepatitis and subsequent fibrosis.

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.

Oral administration of apple condensed tannins delays rheumatoid arthritis development in mice via downregulation of T helper 17 (Th17) cell responses.

Apples are known to contain high concentrations of phenolic compounds such as condensed tannins. Consumption of condensed tannins has been reported to reduce the risk of many types of chronic diseases including allergies. However, their therapeutic effectiveness and potential in treating autoimmune disease remain controversial. Here, the effect of oral administration of apple condensed tannins (ACT) prepared from apples (Malus pumila cv. Fuji) on bovine type II collagen (CII)-induced arthritis in DBA1/J mice, a well-established murine model of human rheumatoid arthritis (RA), was evaluated. As compared to the control (without ACT administration) group, RA development was delayed and a significant reduction in the RA clinical score was observed in the ACT-administered group. Using cultured splenocytes isolated from CII-immunized mice, ACT-administration was shown to decrease the CII-induced increases in IL-17 expression and production in vitro. We propose that downregulation of T helper (Th) 17 cells is responsible for the ACT-induced RA suppression.

[Leukoderma caused by chemicals: mechanisms underlying 4-alkyl/aryl-substituted phenols- and rhododendrol-induced melanocyte loss].

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.