High-intensity exercise impairs intestinal barrier function by generating oxidative stress.

The intestine functions as a barrier preventing the entry of extrinsic factors into the body. This barrier function is disrupted by oxidative damage along with an impaired mucosal layer. Excessive exercise can generate oxidative stress in the intestinal tissue; however, the effect of exercise-induced oxidative stress on intestinal permeability is unclear. In this study, we examined the involvement of oxidative stress in barrier function of the ileum of mice following high-intensity exercise. Male ICR mice (12-week-old) were divided into sedentary and exercise groups. Mice in the exercise group underwent a single bout of treadmill running, and the ileum was collected for histological and biochemical analyses. Plasma fluorescence intensity level after oral administration of fluorescein isothiocyanate-dextran gradually increased until 30 min after exercise in response to intensity of exercise. Relatively high levels of oxidative proteins and low level of claudin-1, a tight-junction protein, were observed in the exercise group. Treatment with a xanthine oxidase inhibitor suppressed exercise-induced increases in intestinal permeability. Moreover, excessive exercise training for two weeks led to relatively high intestinal permeability at rest. These results suggest that high-intensity exercise increases intestinal permeability and tight junction damage, which may be mediated by oxidative stress.

Covalent adduction of serotonin-derived quinones to the SARS-CoV-2 main protease expressed in a cultured cell.

The SARS-CoV-2 main protease is an essential molecule for viral replication and is often targeted by medications to treat the infection. In this study, we investigated the possible inhibitory action of endogenous quinones on the enzyme. Recombinant SARS-CoV-2 main protease was exposed to tryptamine-4,5-dione (TD) or quinone from 5-hydroxyindoleacetic acid (Q5HIAA). As a result, the protease activity was considerably decreased in a dose-dependent manner. The IC50 values of the quinones toward the enzyme were approximately 0.28 µM (TD) and 0.49 µM (Q5HIAA). Blot analyses using specific antibodies to quinone-modified proteins revealed that quinones were adducted to the enzyme at concentrations as low as 0.12 µM. Intact mass analyses showed that one or two quinone molecules were covalently adducted onto the main protease. Chymotrypsin-digested main protease analyses revealed that the quinones bind to thiol residues at the enzyme's active site. When TD or Q5HIAA were exposed to cultured cells expressing the viral enzyme, quinone-modified enzyme was identified in the cell lysate, suggesting that even extracellularly generated quinones could react with the viral enzyme expressed in an infected cell. Thus, these endogenous quinones could act as inhibitors of the viral enzyme.

Chemical and in vitro biological formation of octahydrocurcumin stereoisomers, and in vitro activity of raceme and meso-octahydrocurcumin.

The PtO2-catalyzed hydrogenation of curcumin produced slightly predominant meso-octahydrocurcumin than raceme octahydrocurcumin. Similar result was found in the product obtained from tetrahydrocurcumin and NaBH4, whereas using palladium carbon as a catalyst increased the meso-octahydrocurcumin ratio. Compared with chemical methods, baker's yeast produced 3S,5S-octahydrocurcumin and meso-octahydrocurcumin from tetrahydrocurcumin. The different activity between raceme and meso-octahydrocurcumin was not found in our experiments.

Benzyl isothiocyanate and its metabolites inhibit cell proliferation through protein modification in mouse preosteoclast RAW264.7 cells.

Benzyl isothiocyanate (BITC), derived from cruciferous vegetables, is an organosulfur compound exerting antiproliferative effects in several human cancer cells. In this study, we assessed BITC as a potential osteoclastogenesis inhibitor and investigated its underlying mechanism. BITC at 5 µM significantly decreased the viability of the osteoclast-like differentiating RAW264.7 cells, coinciding with the downregulation of the primary biomarkers for osteoclast differentiation, such as the tartrate-resistant acid phosphatase activity and nuclear factor of activated T-cells gene expression. Not only BITC but also its metabolites, inhibited cell proliferation in the normal RAW264.7 cells, suggesting that BITC shows an anti-osteoclastogenesis effect in vivo after its ingestion and metabolism, possibly through an antiproliferative action. Both BITC and its metabolites also enhanced the DNA fragmentation and the caspase-3 activity, whereas their higher concentrations tended to suppress these effects. BITC was intracellularly accumulated when the cells were treated with its metabolites via their degradation into the free form. A quantitative experiment using the proteolysis/high performance liquid chromatography technique showed that the amount of BITC-lysine thiourea in the cells was also increased in a time-dependent manner, suggesting that lysine modification of the cellular proteins actually took place in the cells treated by BITC. Among the cellular proteins, the cleaved caspase-3 was identified as a potential target for lysine modification by BITC. Taken together, BITC dissociated from its metabolites as well as its free form might modulate osteoclastogenesis, possibly through inhibition of cell proliferation by protein modification.

A Simple Quality Evaluation Method for Proteoglycan after Addition to Beverages.

Over the past 10 years, many products utilizing the functionality of salmon cartilage proteoglycan have come on the market, and consumer awareness of proteoglycan has increased. During this period, the biggest issue has been how to evaluate the amount and quality of proteoglycan in the cartilage extract blended in the products. In this study, we propose an immunological method that can easily evaluate the amount and quality of proteoglycan in the proteoglycan-containing compositions. By the present method, it is possible to evaluate not only the retention of the functional domains of the core protein of proteoglycan, but also that of chondroitin sulfate chains linked to the core protein. Furthermore, the binding activity of proteoglycan to hyaluronan can be evaluated if hyaluronan is used as a probe instead of an antibody. This method is expected to be useful for proteoglycan quality evaluation during the manufacturing process and product storage.

Luteolin suppresses 5-hydroxytryptamine elevation in stimulated RBL-2H3 cells and experimental colitis mice.

Increased 5-hydroxytryptamine may be associated with the development and progression of inflammatory bowel disease. In this study, we examined the suppressive effect of flavonoids on the increased intra- and extracellular 5-hydroxytryptamine levels in rat mast RBL-2H3 cells, known to produce 5-hydroxytryptamine by the phorbol 12-myristate 13-acetate stimulation. Among the flavonoids examined, luteolin and quercetin significantly reduced the cellular 5-hydroxytryptamine concentration. Gene and protein expression analyses revealed that luteolin significantly suppressed cellular tryptophan hydroxylase 1 expression induced by phorbol 12-myristate 13-acetate stimulation. Mitogen-activated protein kinase/extracellular signal-regulated kinase signaling was also suppressed by luteolin, suggesting that this pathway is one of targets of 5-hydroxytryptamine modulation by luteolin. An in vivo experimental colitis model was prepared by administering 2.5% dextran sodium sulfate in drinking water to C57BL/6 mice for seven days. The ingestion of 0.1% dietary luteolin suppressed the increasing 5-hydroxytryptamine in the colorectal mucosa. In conclusion, luteolin possesses a suppressive effect on extensive 5-hydroxytryptamine formation in both experimental RBL-2H3 cells and colitis models.

Elevation of the serotonin-derived quinone, tryptamine-4,5-dione, in the intestine of ICR mice with dextran sulfate-induced colitis.

Inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, are chronic inflammatory disorders associated with oxidative stress. The intestines produce 5-hydroxytryptamine that may negatively affect disease state under inflammatory conditions when overproduced. 5-Hydroxytryptamine is a substrate for myeloperoxidase and is converted into reactive tryptamine-4,5-dione. Here, an experimental colitis model was established through oral administration of 5% dextran sulfate sodium to ICR mice for 7 days. Furthermore, the formation of tryptamine-4,5-dione in the colorectal mucosa/submucosa and colorectal tissue was analyzed by chemical and immunochemical methodologies. First, free tryptamine-4,5-dione in the homogenate was chemically trapped by o-phenylenediamine and analyzed as the stable phenazine derivative. Tryptamine-4,5-dione localization as adducted proteins in the colorectal tissue was immunohistochemically confirmed, and as demonstrated by both methods, this resulted in the significant increase of tryptamine-4,5-dione in dextran sulfate sodium-challenged mice compared with control mice. Immunohistochemical staining confirmed tryptamine-4,5-dione-positive staining at the myeloperoxidase accumulation site in dextran sulfate sodium-challenged mice colorectal tissue. The tryptamine-4,5-dione locus in the mice was partly matched with that of a specific marker for myeloperoxidase, halogenated tyrosine. Overall, the results possibly indicate that tryptamine-4,5-dione is generated by neutrophil myeloperoxidase in inflammatory tissue and may contribute to the development of inflammatory bowel disease.

Food phytochemicals, epigallocatechin gallate and myricetin, covalently bind to the active site of the coronavirus main protease in vitro.

SARS-CoV-2 main protease is a possible target for protection against viral infection. This study examined the inhibitory effect of food phytochemicals on the main protease of SARS-CoV-2 by determining a cleaved product after chromatographic separation. First, 37 phytochemicals, including glycosides and metabolites, were screened at 20 µM; epigallocatechin gallate, myricetin, theaflavin, herbacetin, piceatannol, myricitrin, and isothiocyanates inhibited the enzyme in varying degrees. The IC50 values were estimated from 0.4 to 33.3 µM against the 0.5-µM enzyme. The dose-dependent adduction of epigallocatechin gallate and myricetin was confirmed by quinone staining of protein blotted onto a membrane. The enzyme activity was decreased by increasing the concentration of the two phytochemicals, accompanied by increasing the respective adducted molecule estimated by intact mass spectrometry. Reduced glutathione canceled the formation of conjugate and the inhibitory effect of epigallocatechin gallate or myricetin on the enzyme, suggesting that the formation of the quinone moiety in the phytochemicals is critical for the inhibition. The covalent binding of epigallocatechin gallate or myricetin to the cysteine residue at the active site was confirmed by analyzing peptides from the chymotrypsin-digested main protease.

Methylglyoxal binds to amines in honey matrix and 2'-methoxyacetophenone is released in gaseous form into the headspace on the heating of manuka honey.

Reports on the thermal stability of manuka honey in terms of food processing have been few. This study investigated changes in nine characteristic chemicals of manuka honey during heating. Among these, methylglyoxal (MGO) and 2'-methoxyacetophenone (MAP) were significantly decreased by heating at 90 °C. To elucidate the mechanism for this decrease, artificial honey was prepared from sugars and water with MAP or MGO and then heated. The decrease of MGO was enhanced with l-proline, lysine, or arginine derivatives, accompanied by formation of 2-acetyl-1-pyrroline, MGO-derived lysine dimer, or argpyrimidine, respectively, suggesting that an amino-carbonyl reaction is one pathway for the loss of MGO. The decrease of MAP in the artificial honey depended on the volume of headspace in a vessel. MAP from heated manuka honey was also detected in the gas phase, indicating that MAP was vaporized. Heating could thus reduce the beneficial and/or signature molecules in honey.

Effects of dietary fiber on vascular calcification by repetitive diet-induced fluctuations in plasma phosphorus in early-stage chronic kidney disease rats.

Vascular calcification progresses under hyperphosphatemia, and represents a risk factor for cardiovascular disease in chronic kidney disease (CKD) patients. We recently indicated that phosphorus (P) fluctuations also exacerbated vascular calcification in early-stage CKD rats. Dietary fiber intake is reportedly associated with cardiovascular risk. This study investigated the effects of dietary fiber on vascular calcification by repeated P fluctuations in early-stage CKD rats. Unilateral nephrectomy rats were used as an early-stage CKD model. For 36 days, a P fluctuation (LH) group was fed low-P (0.02% P) and high-P (1.2% P) diets alternating every 2 days, and a P fluctuation with dietary fiber intake (LH + F) group was fed low-P and high-P diets containing dietary fiber alternating every 2 days. The effect on vascular calcification was measured calcium content. Effects on uremic toxin were measured levels of indoxyl sulfate (IS) and investigated gut microbiota. The LH + F group showed significantly reduced vessel calcium content compared to the LH group. Further, dietary fiber inhibited increases in blood levels of IS after intake of high-P diet, and decreased uremic toxin-producing intestinal bacteria. Dietary fiber may help suppress progression of vascular calcification due to repeated P fluctuations in early-stage CKD rats by decreasing uremic toxin-producing intestinal bacteria.

Biochemical analysis of a new sugary-type rice mutant, Hemisugary1, carrying a novel allele of the sugary-1 gene.

MAIN CONCLUSION: A novel allele of the sugary-1 rice mutant was isolated. The single amino acid change led to isoamylase activity reduction and accumulation of high-molecular-weight phytoglycogen in seeds. A new sugary rice variety with an improved seed appearance has been isolated and designated Hemisugary1. This mutant, which was derived from Japonica-type cultivar Tsugaruroman treated with sodium azide, has about half the isoamylase activity of seeds in the original Tsugaruroman. The mutant also accumulates significant phytoglycogen, albeit approximately 40% of the total phytoglycogen in the existing sugary cultivar Ayunohikari which is defective in its most isoamylase activity. The site of mutation was identified using a re-sequence of the whole genome and a cleaved amplified polymorphic sequence (CAPS) marker. The hemisugary phenotypes of the F2 progeny were entirely consistent with the results of genotyping using the CAPS marker. Segregation analysis of the F2 population showed that the hemisugary phenotype was controlled by a single recessive gene, which was produced by a G → A single nucleotide polymorphism in the sugary-1 gene, resulting in a missense mutation from glycine to aspartic acid at amino acid position 333. Zymogram showed that this amino acid replacement resulted in a decrease in isoamylase activity with a concomitant reduction in the formation of isoamylase complexes. Phytoglycogen molecules from Hemisugary1 seeds were 3.5 times larger and contained more short glucan chains than did Ayunohikari seeds. Our data provide new insights into the relationship between isoamylase structure and phytoglycogen formation.

Dynamics of the Cellular Metabolism of Leptosperin Found in Manuka Honey.

Leptosperin (methyl syringate ß-d-gentiobioside) is abundantly found in manuka honey, which is widely used because of its antibacterial and possible anti-inflammatory activities. The aim of this study was to examine the molecular mechanism underlying the metabolism of leptosperin. Five phytochemicals (leptosperin, methyl syringate (MSYR), glucuronate conjugate of MSYR (MSYR-GA), sulfonate conjugate of MSYR (MSYR-S), and syringic acid (SYR)) were separately incubated with HepG2 and Caco-2 cells. After incubation, we found that the concentration of MSYR decreased, whereas the concentrations of SYR, MSYR-GA, and MSYR-S increased. By profiling with inhibitors and carboxylesterases (CES1, 2), we found that the conversion from MSYR to SYR was mediated by CES1. Lipopolysaccharide-stimulated RAW264.7 cells restored MSYR-GA to MSYR possibly by the secreted ß-glucuronidase. All of the mice administered with leptosperin, MSYR, or manuka honey showed higher MSYR (13.84 ± 11.51, 14.29 ± 9.19, or 6.66 ± 2.30 nM) and SYR (1.85 ± 0.66, 6.01 ± 1.20, or 8.16 ± 3.10 nM) levels in the plasma compared with that of the vehicle controls (3.33 ± 1.45 (MSYR) and 1.85 ± 0.66 (SYR) nM). The findings of our study indicate that the unique metabolic pathways of these compounds may account for possible functionalities of manuka honey.

Covalent Modification of Phosphatidylethanolamine by Benzyl Isothiocyanate and the Resultant Generation of Ethanolamine Adduct as Its Metabolite.

Benzyl isothiocyanate (BITC), a dietary isothiocyanate (ITC) derived from cruciferous vegetables, has anticancer properties. It is believed that the ITC moiety (-N═C═S) that reacts predominantly with thiol compounds plays a central role in triggering the activities resulting from these properties. Recent studies have demonstrated that ITCs also covalently modify amino moieties in a protein. In this study, we examined the chemical reaction between BITC and the aminophospholipid, phosphatidylethanolamine (PE), in the cell membrane or lipoprotein particle. To detect the BITC-modified PE, the bond between ethanolamine (EA) and phosphatidic acid in PE was cleaved using phospholipase D to form the BITC-EA adduct, which was then measured. BITC-EA was detected from the BITC-treated unilamellar liposome and low-density lipoprotein even with only a few micromoles of BITC treatment, suggesting that BITC might react with not only a thiol/amino group of a protein but also an amino moiety of an aminophospholipid. Moreover, after incorporating BITC-PE included in the liposomes into the cultured cells or after direct exposure of BITC to the cells, free BITC-EA was excreted and accumulated in the medium in a time-dependent manner. It indicates that an intracellular enzyme catalyzes the cleavage of BITC-PE to produce BITC-EA. Because the ITC-amine adduct is stable, the ITC-EA adduct could be a promising indicator of ITC exposure in vivo.

Covalent adduction of endogenous and food-derived quinones to a protein: its biological significance.

There are many chemically reactive compounds, including quinone, in living systems and also food. Even after the ingestion of food polyphenols, quinones derived from catechol moieties could form endogenously in the body. Dopaquinone, dopamine quinone, estrogen-derived quinones, tryptamine-4,5-dione, and ubiquinone are examples of an endogenous quinone. These indicate that quinone is ubiquitously formed or present in living systems and food. Quinones can induce a variety of hazardous effects and also could have beneficial physiological effects. This review focuses on the chemical reactivity of quinone toward a biomolecule and its biological action.

Lymphatic metabolites of quercetin after intestinal administration of quercetin-3-glucoside and its aglycone in rats.

Quercetin is a major flavonoid, present as its glycosidic forms in plant foods. In this study, quercetin-3-glucoside (Q3G) was administered intraduodenally to thoracic lymph-cannulated rats, and its lymphatic transport was investigated. The resulting lymphatic and plasma metabolites were identified with LC-MS/MS and compared with those after administration of quercetin aglycone. The total concentration of quercetin metabolites in the lymph was about four times lower than that in the plasma, and quercetin and its methylated form isorhamnetin were detected as their glucuronides, sulfates and diglucuronides both in the lymph and the plasma after Q3G and quercetin administrations. The lymph levels of the glucuronides after Q3G administration were lower than those after quercetin administration, whereas those in the plasma showed the opposite pattern. Both the lymph and plasma levels of the sulfates after Q3G administration were lower than those after quercetin administration. Some of the intestinal metabolites like quercetin monoglucuronides were transported directly into the lymph and the hepatic metabolites like the diglucuronides were eventually transferred from the plasma into the lymph. These results indicate that the absorbed Q3G is partly transported into the intestinal lymph as quercetin metabolites. Deglycosylation in the enterocyte is also suggested to affect the subsequent metabolic pathways.

Radioprotective activity of blackcurrant extract evaluated by in vitro micronucleus and gene mutation assays in TK6 human lymphoblastoid cells.

INTRODUCTION: Blackcurrant (Ribs nigrum L.) is a classical fruit that has long been used to prepare juice, jam, liqueur, and sometimes medicines in Europe. Previously, we reported a genome defense effect by the antioxidative activity of several types of blackcurrant extracts (BCEs) in yeast and human cell gene mutation assays. In this study, we determined if BCE exerted radioprotective activity against DNA damage, chromosomal aberration, and gene mutations in the TK6 human lymphoblastoid cell line. We prepared aqueous BCE extracted from mature fruits cultivated in the Aomori Prefecture, Japan. FINDINGS: In the micronucleus test and TK gene mutation assay, TK6 cells were irradiated with 0, 0.125, 0.250, 0.500, and 1.000 Gy with or without 1.0 mg/mL BCE. Intracellular hydrogen peroxide (H2O2) was measured using the fluorescent probe BES-H2O2-Ac. Induction of micronuclei and gene mutations by γ-irradiation exposure was suppressed in combination with BCE. In addition, BCE reduced intracellular H2O2 levels caused by γ-irradiation. CONCLUSIONS: Our findings clearly support the genome defense potential of blackcurrant against γ-induced DNA damage. We postulate that these genome defense activities are related to the antioxidant compounds in blackcurrant.

Cytotoxic and cytoprotective effects of tryptamine-4,5-dione on neuronal cells: a double-edged sword.

Serotonin (5-hydroxytryptamine) is a putative substrate for myeloperoxidase, which may convert it into the reactive quinone tryptamine-4,5-dione (TD). In this study, we found that the viability of human SH-SY5Y neuroblastoma cells treated with 25 µM TD was increased to approximately 117%. On the other hand, the cell viability was significantly decreased by exposure to TD (150-200 µM), with an increase in intracellular reactive oxygen species (ROS). Interestingly, pre-treatment of SH-SY5Y cells with 100 µM TD prevented cell death and suppressed intracellular ROS generation evoked by the addition of hydrogen peroxide (H2O2). Expression of the phase-II antioxidant enzyme NAD(P)H: quinone oxidoreductase 1 and haem oxygenase 1 were upregulated by TD at a concentration of 50-100 µM. Nuclear factor erythroid 2-related factor 2 (Nrf2), the regulator of these enzyme, was translocated from the cytosol to the nucleus by 100 µM TD. In summary, moderate concentrations of TD may increase the self-defence capacity of neuronal cells against oxidative stress.

In vivo absorption and metabolism of leptosperin and methyl syringate, abundantly present in manuka honey.

SCOPE: Manuka honey, which shows strong nonperoxide-dependent antibacterial activity, contains unique components, such as methyl syringate 4-O-ß-D-gentiobioside (leptosperin) and its aglycone, methyl syringate (MSYR). To determine the potential for biological activity evoked by the ingestion of leptosperin and MSYR, we investigated the absorption and metabolism of these components in manuka honey. METHODS AND RESULTS: The incubation of MSYR with liver microsomes or S9 fractions in vitro resulted in the formation of MSYR-glucuronide (MSYR-GA), MSYR-sulfate (MSYR-S), and syringic acid as metabolites. Then, manuka honey (15 g) was fed to healthy human volunteers. MSYR-GA, MSYR-S, and MSYR were detected in both plasma and urine. Within plasma, their levels were highest within 0.5 h to 1 h post-ingestion, and most metabolites disappeared within 3 h. In conjunction with the disappearances, a significant amount of metabolites along with trace leptosperin was excreted in urine within 4 h. To elucidate the detailed metabolisms of leptosperin and MSYR, each compound was separately administered to mice. In each case, MSYR-GA, MSYR-S, and MSYR were detected in both plasma and urine. CONCLUSION: This study shows the major molecular pathway for leptosperin and MSYR metabolism and could facilitate an understanding of biological functions of manuka honey post ingestion.

Oxidation of proline decreases immunoreactivity and alters structure of barley prolamin.

Elimination of celiac-toxic prolamin peptides and proteins is essential for Triticeae products to be gluten-free. Instead of enzymatic hydrolysis, in this study we investigated metal-catalyzed oxidation of two model peptides, QQPFP, and PQPQLPY, together with a hordein isolate from barley (Hordeum vulgare L.). We established a multiple reaction monitoring (MRM) LC-MS method to detect and quantify proline oxidation fragments. In addition to fragmentation, aggregation and side chain modifications were identified, including free thiol loss, carbonyl formation, and dityrosine formation. The immunoreactivity of the oxidized hordein isolate was considerably decreased in all metal-catalyzed oxidation systems. Cleavage of peptides or protein fragments at the numerous proline residues partially accounts for the decrease. Metal-catalyzed oxidation can thus be used in the modification and elimination of celiac-toxic peptides and proteins.