Development of validated sandwich ELISA for detecting peanut allergen Ara h 3 in food.

Peanut is an important food that can cause food allergies, often leading to moderate and severe allergic symptoms such as skin rashes, asthma, and even anaphylactic shock.Research indicates that Ara h 3 is one of the major peanut allergen. In order to establish a simple analytical method for detecting Ara h 3, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) with antibodies that were induced from purified Ara h 3. The experimental results showed that the purified Ara h 3 had good purity, and we successfully prepared capture and detection antibodies. The method established in this study exhibited high specificity and did not cross-react with soybeans, cashew nuts, and sesame. For validation, including precision, recovery and sensitivity were in good condition. We also detected the Ara h 3 in peanut related foods. Overall, the ELISA developed in this study is a reliable method for Ara h 3 detection.

The effect of papain hydrolysis on tropomyosin levels in shrimp.

Shrimp allergy is a serious public health problem. Epidemiological studies indicated the main cause of food allergy is the ingestion of shrimp and crab. Consequently, reducing the risk of shrimp and crab allergy is a major research focus. Few studies have demonstrated the effect of enzyme hydrolysis on reduction of shrimp allergens in food. In this study, we used papain, a commonly used enzyme in the food industry, as an ingredient to decrease levels of allergen tropomyosin in shrimp. 0-50U of papain was used to treat the shrimp meat, and then heated to measure the levels of tropomyosin, and determined the change of the protein secondary structure. The results showed that 20U of papain decrease the tropomyosin levels up to 80 %, which was further enhanced when combined with 3 min of heating. Furthermore, the result of Fourier-transform infrared spectroscopy (FTIR) showed alteration of secondary protein structure after the processing. The processing developed in this study may be an effective method used to change the levels and structure of tropomyosin.

Chronic blue light-emitting diode exposure harvests gut dysbiosis related to cholesterol dysregulation.

Night shift workers have been associated with circadian dysregulation and metabolic disorders, which are tightly coevolved with gut microbiota. The chronic impacts of light-emitting diode (LED) lighting at night on gut microbiota and serum lipids were investigated. Male C57BL/6 mice were exposed to blue or white LED lighting at Zeitgeber time 13.5-14 (ZT; ZT0 is the onset of "lights on" and ZT12 is the "lights off" onset under 12-hour light, 12-hour dark schedule). After 33 weeks, only the high irradiance (7.2 J/cm2) of blue LED light reduced the alpha diversity of gut microbiota. The high irradiance of white LED light and the low irradiance (3.6 J/cm2) of both lights did not change microbial alpha diversity. However, the low irradiance, but not the high one, of both blue and white LED illuminations significantly increased serum total cholesterol (TCHO), but not triglyceride (TG). There was no significant difference of microbial abundance between two lights. The ratio of beneficial to harmful bacteria decreased at a low irradiance but increased at a high irradiance of blue light. Notably, this ratio was negatively correlated with serum TCHO but positively correlated with bile acid biosynthesis pathway. Therefore, chronic blue LED lighting at a high irradiance may harvest gut dysbiosis in association with decreased alpha diversity and the ratio of beneficial to harmful bacteria to specifically dysregulates TCHO metabolism in mice. Night shift workers are recommended to be avoid of blue LED lighting for a long and lasting time.

Acetylation Enhances the Anticancer Activity and Oral Bioavailability of 5-Demethyltangeretin.

A kind of hydroxylated polymethoxyflavone (PMFs) existing in the citrus genus, 5-Demethyltangeretin (5-DTAN), has been reported to possess several bioactivities in vitro and in vivo. The aim of this study was to investigate whether acetylation could enhance the anticancer activity and oral bioavailability of 5-DTAN. PC-3 human prostate cancer cells were treated with tangeretin (TAN), 5-DTAN, and 5-acetylated TAN (5-ATAN), and the results showed that the cytotoxic effect 5-ATAN (IC50 value of 5.1 µM) on the cell viability of PC-3 cells was stronger than that of TAN (IC50 value of 17.2 µM) and 5-DTAN (IC50 value of 11.8 µM). Compared to 5-DTAN, 5-ATAN treatment caused a more pronounced DNA ladder, increased the sub-G1 phase population, and induced G2/M phase arrest in the cell cycle of PC-3 cells. We also found that 5-ATAN triggered the activation of caspase-3 and the progression of the intrinsic mitochondrial pathway in PC-3 cells, suggesting the induction of apoptosis. In a cell wound healing test, 5-ATAN dose-dependently reduced the cell migration, and the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) was decreased after 48 h of 5-ATAN treatment. Moreover, oral administration of 5-ATAN showed a significantly stronger inhibitory effect on tumor size and tumor weight in tumor-bearing nude mice than those of vehicle or the 5-DTAN group (p < 0.05). Furthermore, pharmacokinetic results showed that single-dose oral administration of 5-ATAN exhibited a higher maximum concentration (Cmax) and area under the curve (AUC) of 5-DTAN in plasma than that of 5-DTAN. More extensive distribution of 5-DTAN to most tissues of mice was also observed in mice treated with 5-ATAN for 7 days. In conclusion, acetylation strongly enhances the anticancer activity and oral bioavailability of 5-DTAN and could be a promising strategy to promote the potential bioactivities of natural products.

Antioxidant Activity and Inhibitory Effects of Black Rice Leaf on the Proliferation of Human Carcinoma Cells.

The leaves of black rice, well-known as postharvest agricultural waste, contain a rich source of antioxidants with multiple benefits for human health. In the present study, the ethyl acetate fraction obtained from black rice leaf was separated into five subfractions using Sephadex LH-20 column chromatography, and their antioxidant and anticancer activities were investigated. The results revealed that among all the subfractions, subfraction 5 (Sub5) showed the highest total phenolic and flavonoid values. The antioxidant activity was also superior in Sub5 (the IC50 values are 3.23, 31.95, and 72.74 µg/mL, in the DPPH, ABTS, and reducing power assays, respectively) compared to the other subfractions. All subfractions, in a time-dependent manner, inhibited the proliferation of hepatoma (HepG2), breast (MCF-7), and colorectal (Caco-2) cancer cells, especially the Sub5. Thus, Sub5 was employed to conduct the cell cycle and cell apoptosis by flow cytometry. Sub5 significantly increased the accumulation of cells at the Sub-G1 phase in HepG2 cells (44.5%, at 48 h). Furthermore, it could trigger annexin V-detected apoptosis through mitochondrial and death receptor pathways accompanied by the suppression of PI3K/Akt and Erk signaling pathways. In addition, HPLC-DAD-MS/MS was conducted to characterize the bioactive constituents in the most potent antioxidant, cytotoxic, and apoptosis-inducing subfraction. Conclusively, Sub5 may have high potential as functional dietary supplements to inhibit the development of HepG2 liver cancer.

Evaluation of Phytochemical Contents and In Vitro Antioxidant, Anti-Inflammatory, and Anticancer Activities of Black Rice Leaf (Oryza sativa L.) Extract and Its Fractions.

Black rice leaves (Oryza sativa L.) are a major part of rice straw left in open fields after rice harvest as agricultural waste. In this study, crude ethanolic extract (CEE) and various solvent fractions (hexane (Hex), ethyl acetate (EtOAc), n-butanol (n-BuOH), and aqueous fractions) of black rice leaves were investigated for their bioactive compound contents as well as antioxidant, anti-inflammatory, and anticancer activities. The results demonstrated that among all the fractions, the n-BuOH fraction presented the greatest contents of total phenolics and flavonoids, while anthocyanins were found to be abundant in the n-BuOH and aqueous fractions, which also exhibited powerful antioxidant abilities according to DPPH and ABTS radical-scavenging assays and a reducing power assay. Regarding anti-inflammatory activity, CEE and EtOAc reduced the production of NO and cytokine secretion (PGE2, IL-6, and IL-1ß) but displayed less effect on tumor necrosis factor α (TNF-α) release in lipopolysaccharide (LPS)-induced RAW 264.7 cells. They also significantly decreased iNOS and COX-2 protein expression. Additionally, the phenolics-rich ethyl acetate fraction showed the greatest activity against HepG2 liver carcinoma cells, inhibited cell growth, increased the Sub-G1 population, and induced apoptosis via mitochondrion-dependent mechanisms. In conclusion, black rice leaves, a byproduct of rice, exhibited strong antioxidant, anti-inflammatory, and anticancer capacities and might be useful for application in functional foods and the pharmaceutical industry.

Multiple-Reaction Monitoring Tandem Mass Method for Determination of Phenolics and Water-Soluble Vitamins in Eccoilopus formosanus.

This study established a validated method for the quantitative and qualitative determination of eight signature compounds in Eccoilopus formosanus. We used multiple-reaction monitoring scanning for quantification, and switched the electrospray ion source polarity between positive and negative modes in a single chromatographic run. The precursor-to-product ion transitions were m/z 355/163, m/z 181/163, m/z 265/122, m/z 269/117, m/z 170/152, m/z 377.2/180.7, m/z 169/124.8 and m/z 193/134 for chlorogenic acid, caffeic acid, thiamine, apigenin, pyridoxamin, riboflavin, gallic acid and ferulic acid, respectively. The developed method was also validated for accuracy, precision and limit of quantification. In this method, eight compounds were quantified with correlation coefficients of greater than 0.995. A high recovery (81.5-94.1%) and good reproducibility was obtained for five phenolics and three vitamins with the relative standard deviation, ranging from 1.2 to 3.5%. This method may be applied to the determination of both phenolics and water-soluble vitamins in cereal grain. The results may suggest that the extract of E. formosanus could be a good source of bioactive phytochemicals.

HPLC-DAD-ESI-MS Analysis for Simultaneous Quantitation of Phenolics in Taiwan Elderberry and Its Anti-Glycation Activity.

Sambucus formosana is most commonly used as a traditional herb medicine in Taiwan. In this study, high performance liquid chromatography equipped with photodiode array detection-mass (HPLC-DAD-ESI-MS) method was developed for the identification and quantification of bioactive phenolics. The developed method was also validated for accuracy, precision, limit of detection, and quantification. In this method, chlorogenic acid, rutin, isoquercetrin, nictoflorin, astragalin, and quercetin were quantified in linearity range of 10-100 (µg/mL) with a correlation coefficient of greater than 0.996. High recovery (86.5-93.1%) and good reproducibility were obtained for six phenolics with the relative standard deviation ranging from 1.7-3.1%. Therefore, the proposed method for simultaneous quantification of six bioactive phenolics in the extract and fractions of S. formosana using HPLC-DAD-ESI-MS detection under the optimized conditions is accurate and validated. Among the results, methanol extract showed the greatest values of total phenolic content (93.1 mg gallic acid equivalent/g). Additionally, the methanol extract revealed best antioxidant capacity based on the DPPH scavenging activity and anti-glycation activity (IC50 was observed at 97.1 and 77.9 µg/mL, respectively).

Identification of pyruvate kinase 2 as a possible crab allergen and analysis of allergenic proteins in crabs consumed in Taiwan.

In Taiwan, crab is one of the main causes for food allergy. Several proteins are recognized as crustacean allergens, and tropomyosin is known to be the major one. However, sensitization patterns of Taiwanese patients to crustacean allergens remain unclear. Therefore, we analyzed the specific-IgE binding ability of crucifix crab (Charybdis feriatus) allergens by western blot using patients' sera. In particular, we found a 56 kDa protein in crucifix crab reacted with specific-IgEs in patients' sera, and we further identified the protein as a novel crab allergen pyruvate kinase 2. Additionally, little is known about tropomyosin contents in crabs consumed in Taiwan. Thus, we also quantified the levels of tropomyosin by using enzyme-linked immunosorbent assay (ELISA) among raw and cooked crab species. Our results showed tropomyosin levels varied depending on crab species. In summary, these findings improve the understanding of crustacean allergens and contribute to the clinical diagnosis of crustacean allergies.

Evaluation of chemical-specific IgG antibodies in male workers from a urethane foam factory.

BACKGROUND: Plastic resins are complex chemicals that contain toluene diisocyanate (TDI) and/or trimellitic anhydride (TMA), which cause occupational allergies (OA), including respiratory allergies. Serum IgGs against TDI and TMA have been suggested as potential markers of the exposure status and as exploring cause of OA. Although TDI-specific IgG has been examined for suspected OA, TMA-specific IgG is not commonly evaluated in a urethane foam factory. This study therefore investigated both TDI- and TMA-specific IgGs in suspected OA patients and to evaluate the usefulness of the measurement of multiple chemical-specific IgG measurement for practical monitoring. METHODS: Blood samples were collected from two male workers who developed respiratory allergies supposedly caused by occupational exposure to TDI and/or TMA for the presence of TDI- and TMA-specific IgGs. In addition, blood samples from 75 male workers from a urethane foam factory, along with 87 male control subjects, were collected in 2014 and tested for the same IgGs in 2014. The presence and levels of TDI- and TMA-specific serum IgGs were measured using dot blot assays. RESULTS: We found that controls had mean concentrations of TDI- and TMA-specific IgGs of 0.98 and 2.10 µg/mL, respectively. In the two workers with respiratory allergies, the TDI-specific IgG concentrations were 15.6 and 9.51 µg/mL, and TMA-specific IgG concentrations were 4.56 and 14.4 µg/mL, which are clearly higher than those in controls. Mean concentrations of TDI- and TMA-specific IgGs in the factory workers were 1.89 and 2.41 µg/mL, respectively, and are significantly higher than those of the controls (P < 0.001 and P < 0.026 for TDI- and TMA-specific IgGs, respectively). CONCLUSION: The workers suspected of OA showed an evidently high level of TDI- and TMA-specific IgG, and these levels in workers at the urethane foam factory were also significantly higher than those in controls. In conclusion, the measurement of TDI- and TMA-specific IgG among workers using plastic resins is helpful to monitor their exposure status.

Identification of pyruvate kinase as a novel allergen in whiteleg shrimp (Litopenaeus vannamei) by specific-IgE present in patients with shrimp allergy.

Food allergy is one of the most important health issues worldwide. In Taiwan, current literature suggests shrimps and crabs are the most common causes of food allergy, and are frequently associated with acute allergic reactions such as urticaria, atopic dermatitis, and asthma. However, knowledge regarding the shrimp allergens remains limited. Thus, there is an urgent need to establish comprehensive information for elucidating underlying triggers for food allergy. In this study, whiteleg shrimp (Litopenaeus vannamei) was used to evaluate the IgE-binding properties of various shrimp proteins to 7 allergic patients' sera by western blot. A 63 kDa protein was found in raw and cooked shrimp bound to specific-IgEs in 7 and 4 patients' sera, respectively. This protein was further identified as pyruvate kinase based on the proteomic mass spectrometry. This study identifies an important shrimp allergen unique to Taiwan and further testing and prevention measures might be implemented in the allergen analysis.

Lifetime Increased Risk of Adult Onset Atopic Dermatitis in Adolescent and Adult Patients with Food Allergy.

Food allergy can result in life-threatening anaphylaxis. Atopic dermatitis (AD) causes intense itching and impaired quality of life. Previous studies have shown that patients with classical early-onset AD tend to develop food allergy and that 10% of adults with food allergies have concomitant AD. However, it is not known whether late-onset food allergy leads to adult-onset AD, a recently recognized disease entity. Using an initial cohort of one-million subjects, this study retrospectively followed-up 2851 patients with food allergy (age > 12 years) for 14 years and compared them with 11,404 matched controls. While 2.8% (81) of the 2851 food allergy patients developed AD, only 2.0% (227) of the 11,404 controls developed AD. Multivariate regression analysis showed that food allergy patients were more likely to develop AD (adjusted hazard ratio = 2.49, p < 0.0001). Controls had a 1.99% risk of developing AD, while food allergy patients had a significantly higher risk (7.18% and 3.46% for patients with ≥3 and <3 food allergy claims, respectively) of developing adult-onset AD. This is the first study to describe the chronological and dose-dependent associations between food allergy in adolescence and the development of adult-onset AD.

The effect of ethanol on the formation of N2-ethylidene-dG adducts in mice: implications for alcohol-related carcinogenicity of the oral cavity and esophagus.

The present study aimed to experimentally confirm that long-term alcohol drinking causes a high risk of oral and esophageal cancer in aldehyde dehydrogenase 2 (ALDH2)-deficient individuals. Aldh2 knockout mice, an animal model of ALDH2-deficiency, were treated with 8% ethanol for 14 months. Levels of acetaldehyde-derived DNA adducts were increased in esophagus, tongue and submandibular gland. Our finding that a lack of Aldh2 leads to more DNA damage after chronic ethanol treatment in mice supports epidemiological findings on the carcinogenicity of alcohol in ALDH2-deficient individuals who drink chronically.

Historical review on development of environmental quality standards and guideline values for air pollutants in Japan.

Environmental quality standards (EQSs) have been established as desirable levels to be maintained for protection of human health and the conservation of the living environment by Basic Environment Law. EQSs in ambient air had been set for 10 substances (sulfur dioxide (SO(2)), carbon monoxide (CO), suspended particulate matter (SPM), nitrogen dioxide (NO(2)) and photochemical oxidants (Ox), benzene, tetrachloroethylene, trichloroethylene, dioxins and dichloromethane) and guideline values for 7 (acrylonitorile, vinyl chloride monomer, mercury, nickel compounds, 1,3-butadiene, chloroform and 1,2-dichloromethane) in Japan by 2009. EQSs for the classical (or traditional) air pollutants, SO(2), CO, SPM, NO(2) and Ox, were set according to the minimal requirement to protect human health, based on evidence from epidemiological studies conducted before the 1970s. In 1996, the Central Environment Council designated substances which may be hazardous air pollutants and substances requiring priority action, and adopted the concept of risk assessment to set EQSs and guideline values. A life-long risk level (virtually safe dose) of 10(-5) was used to set EQS for benzene, and guideline values for vinyl chloride monomer, nickel compounds, and 1,3-butadiene. EQSs for trichloroethylene, tetrachloroethylene and dichloromethane, and guideline values for acrylonitorile and mercury were set using uncertain factors and lowest observed adverse effect (LOAEL)/no observed adverse effect level (NOAEL). The results of animal experiments were utilized to set guideline values for chloroform and 1,2-dichloroethane. The benchmark approach and human equivalent concentration (HEC) were adopted for 1,2-dichloroethane. The history of setting EQSs and guideline values for hazardous air pollutants is one of adopting new concepts into risk assessment.

Formation of acetaldehyde-derived DNA adducts due to alcohol exposure.

Epidemiological studies have identified chronic alcohol consumption as a significant risk factor for cancers of the upper aerodigestive tract, including the oral cavity, pharynx, larynx and esophagus, and for cancer of the liver. Ingested ethanol is mainly oxidized by the enzymes alcohol dehydrogenase (ADH), cytochrome P-450 2E1 (CYP2E1), and catalase to form acetaldehyde, which is subsequently oxidized by aldehyde dehydrogenase 2 (ALDH2) to produce acetate. Polymorphisms of the genes which encode enzymes for ethanol metabolism affect the ethanol/acetaldehyde oxidizing capacity. ADH1B*2 allele (ADH1B, one of the enzyme in ADH family) is commonly observed in Asian population, has much higher enzymatic activity than ADH1B*1 allele. Otherwise, approximately 40% of Japanese have single nucleotide polymorphisms (SNPs) of the ALDH2 gene. The ALDH2 *2 allele encodes a protein with an amino acid change from glutamate to lysine (derived from the ALDH2*1 allele) and devoid of enzymatic activity. Neither the homozygote (ALDH2*2/*2) nor heterozygote (ALDH2*1/*2) is able to metabolize acetaldehyde promptly. Acetaldehyde is a genotoxic compound that reacts with DNA to form primarily a Schiff base N(2)-ethylidene-2'-deoxyguanosine (N(2)-ethylidene-dG) adduct, which may be converted by reducing agents to N(2)-ethyl-2'-deoxyguanosine (N(2)-ethyl-dG) in vivo, and strongly blocked translesion DNA synthesis. Several studies have demonstrated a relationship between ALDH2 genotypes and the development of certain types of cancer. On the other hand, the drinking of alcohol induces the expression of CYP2E1, resulting in an increase in reactive oxygen species (ROS) and oxidative DNA damage. This review covers the combined effects of alcohol and ALDH2 polymorphisms on cancer risk. Studies show that ALDH2*1/*2 heterozygotes who habitually consume alcohol have higher rates of cancer than ALDH2*1/*1 homozygotes. Moreover, they support that chronic alcohol consumption contributes to formation of various DNA adducts. Although some DNA adducts formation is demonstrated to be an initiation step of carcinogenesis, it is still unclear that whether these alcohol-related DNA adducts are true factors or initiators of cancer. Future studies are needed to better characterize and to validate the roles of these DNA adducts in human study.

Effects of acetaldehyde inhalation in mitochondrial aldehyde dehydrogenase deficient mice (Aldh2-/-).

Human body might be exposed to acetaldehyde from smoking or occupational environment, which is known to be associated with cancer through the formation of DNA adducts, in particular, N2-ethylidene-2'- deoxyguanosine (N2-ethylidene-dG). Aldehyde dehydrogenase 2 (ALDH2) is the major enzyme that contribute to the detoxification of acetaldehyde in human body. In this study, wild type (Aldh2+/+) and Aldh2KO (Aldh2-/-) mice were exposed to the air containing 0, 125, 500 ppm acetaldehyde for 2 weeks. After inhalation, levels of N2- ethylidene-dG in the chromosomal DNA were analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). N2-ethylidene-dG levels in livers of Aldh2-/- mice were always lower than those of Aldh2+/+ mice, suggesting that Aldh2 deficiency might cause the induction of acetaldehyde metabolizing enzymes in the liver such as P450s. The differences between Aldh2-/- and Aldh2+/+ mice were greater in the order of nasal epithelium > lung > dorsal skin, suggesting that nasal epithelium and lung are the major target sites for acetaldehyde. Acetaldehyde inhalation may cause a high risk in nasal epithelium and lung cancers for individuals with inactive ALDH2.

Characteristics of aldehyde dehydrogenase 2 (Aldh2) knockout mice.

Acetaldehyde is an intermediate of ethanol oxidation. It covalently binds to DNA, and is known as a carcinogen. Aldehyde dehydrogenase 2 (ALDH2) is an important enzyme that oxidizes acetaldehyde. Approximately 45% of Chinese and Japanese individuals have the inactive ALDH2 genotypes (ALDH2*2/*2 and ALDH2*1/*2), and Aldh2 knockout mice appear to be a valid animal model for humans with inactive ALDH2. This review gives an overview of published studies on Aldh2 knockout mice, which were treated with ethanol or acetaldehyde. According to these studies, it was found that Aldh2 -/- mice (Aldh2 knockout mice) are more susceptible to ethanol and acetaldehyde-induced toxicity than Aldh2 +/+ mice (wild type mice). When mice were fed with ethanol, the mortality was increased. When they were exposed to atmospheres containing acetaldehyde, the Aldh2 -/- mice showed more severe toxic symptoms, like weight loss and higher blood acetaldehyde levels, as compared with the Aldh2 +/+ mice. Thus, ethanol and acetaldehyde treatment affects Aldh2 knockout mice more than wild type mice. Based on these findings, it is suggested that ethanol consumption and acetaldehyde inhalation are inferred to pose a higher risk to ALDH2-inactive humans. These results also support that ALDH2-deficient humans who habitually consume alcohol have a higher rate of cancer than humans with functional ALDH2.