Inactivation mechanism of SARS-CoV-2 by ozone in aqueous and gas phases.

The comparison of the inactivation rate of SARS-CoV-2 by ozone in water with that in gas, based on data from references and experiments, has indicated the inactivation rate of the former is remarkably higher than that of the latter. To investigate the reason for this difference, we analyzed the reaction rate using a diffusional reaction model, in which ozone is carried by micro spherical viruses to inactivate the target viruses. Using this model, we can evaluate the amount of ozone required to inactivate a virus based on the ct value. We found that inactivation in gas phase requires 1014-1015 ozone molecules per virus virion, while the inactivation in aqueous phase requires 5×1010 to 5×1011 ozone molecules. This implies that the efficiency in gas phase is 200-20,000 times lower than that in aqueous phase. This is not attributed to the lower probability of collision in gas phase than in aqueous phase. Rather, it may be due to the fact that the ozone and radicals generated by ozone react and subsequently dissipate. We proposed the diffusion of ozone into a spherical virus at a steady state and the decomposition reaction model through radicals.

Effect of Citric Acid on Prolonging the Half-life of Dissolved Ozone in Water.

To elucidate the effect of citric acid on the stability of dissolved ozone, half-lives of ozone in a citric acid solution was investigated. Prolongation of the half-life of ozone was clearly shown in the presence of citric acid in ozonized water. In the presence of ethylenediaminetetraacetic acid (EDTA), the half-life of ozone was decreased. The addition of various concentrations of citric acid to the EDTA solution, however, reversed the half-life in a concentration-dependent manner. These indicate that citric acid suppresses ozone self-decomposition in water. A citric acid-mediated suppression mechanism of ozone self-decomposition involving hydroxy radical (HO•) was proposed as follows: HO• formed by the radical chain reaction process of ozone is scavenged by a way of abstracting the hydrogen atom bound to a carbon atom located α-position of a carbonyl group. The radical chain reaction of ozone is, thus, suppressed. These findings demonstrate that the addition of citric acid to ozonized water is useful for the stabilization of ozone. This ability may contribute to the application of ozone sterilization in food production processes.

Effect of Seleno-L-methionine on Oxidative Stress in the Pancreatic Islets of a Short-Term Induced Diabetic Mouse Model in Insufficient Selenium Status.

The protective effects of seleno-L-methionine (SeMet) on oxidative stress in pancreatic islets were investigated with a short-term nicotinamide (NA) and streptozotocin (STZ)-induced diabetic mouse model. ICR mice were intraperitoneally injected twice with 100 mg/kg STZ and 120 mg/kg NA at a 1-d interval and were then orally administered 158 µg Se/kg SeMet with free access to a selenium-deficient diet for 5 weeks. Administration of SeMet significantly improved the levels of glycated hemoglobin (HbA1c), non-fasting and oral glucose tolerance-tested (OGTT) blood glucose, plasma adiponectin and hepatic glycogen that deteriorated by NA/STZ treatment. However, supplementary SeMet did not restore non-fasting plasma insulin levels in NA/STZ treatment group and significantly suppressed OGTT plasma insulin levels in the control group. Although SeMet significantly suppressed 8-hydroxy-2'-deoxyguanosine density in pancreatic islets, SeMet did not restore insulin density. The hepatic and pancreatic mRNA levels of glutathione peroxidase 1 (GPX1) increased by NA/STZ treatment or SeMet administration. These results suggest that although a physiological level of SeMet improves glucose tolerance by exhibiting insulin-mimetic activity in a short-term induced diabetic mouse model under insufficient Se status, the suppression of pancreatic oxidative stress with the induction GPX1 by SeMet supplementation is unlikely to restore insulin storage and secretion.

Effects of administering sodium selenite, methylseleninic acid, and seleno-L-methionine on glucose tolerance in a streptozotocin/nicotinamide-induced diabetic mouse model.

The effects of administering the selenocompounds, sodium selenite, methylseleninic acid (MSA), and seleno-L-methionine (SeMet) on glucose tolerance were compared in the nicotinamide (NA) and streptozotocin (STZ)-induced diabetic mouse model. ICR mice were intraperitoneally treated twice with STZ (100 mg/kg) 15 min after an injection of NA (120 mg/kg) at a 1-d interval. Non-fasting blood glucose levels were then monitored weekly while orally administering the selenocompounds at 158 µg Se/kg body weight with free access to a selenium-deficient diet for 5 weeks. The mean body weights of NA/STZ-induced diabetic mice were partly restored by the administration of selenocompounds, while SeMet led to a higher selenium content and glutathione peroxidase 1 activity in the pancreas. Non-fasting and oral glucose tolerance-tested blood glucose levels, which were elevated by NA/STZ, were significantly suppressed by the administration of SeMet. These results suggest that SeMet may improve glucose tolerance in a NA/STZ-induced mild diabetic mouse model by increasing bioavailability in the pancreas.

Effect of selenite on T-cell mitogenesis: contribution of ROS production and apoptosis signal-regulating kinase 1.

Although supplementation with the selenocompound, sodium selenite has been shown to stimulate the concanavalin A-induced T-cell mitogenic response, the mechanisms responsible remain unclear. This study was conducted to evaluate the relationships between the induction of apoptosis, formation of tumor necrosis factor (TNF)-alpha and reactive oxygen species (ROS), activation of apoptosis signal-regulating kinase (ASK) 1 and the thioredoxin (Trx) system when mitogenesis was stimulated by selenite. TNF-alpha was dose-dependently released by mouse splenocytes treated with selenite, and apoptosis was induced when TNF-alpha was added at the indicated concentrations. However, supplementation with selenite at low concentrations inhibited the accumulation of ROS with the increased expression of Trx reductase 1 and induction of apoptosis in wild-type splenocytes, and also at high concentrations in Trx-1-transgenic mouse splenocytes. The suppression of apoptosis was accompanied by a decrease in the expression of phospho-ASK1. These results suggest that the stimulation of T-cell mitogenesis by selenite may be partly attributed to the inhibited accumulation of ROS due to a reduced Trx-1/TR1 system, the inactivation of ASK1, and the suppression of apoptosis.

Methylseleninic acid (MSA) inhibits 17ß-estradiol-induced cell growth in breast cancer T47D cells via enhancement of the antioxidative thioredoxin/ thioredoxin reductase system.

The purpose of this study was to clarify the cell growth inhibitory mechanism of human breast cancer cells caused by selenium (Se) compounds. In the presence of 17ß-estradiol (E(2)) at physiological concentrations, growth of estrogen receptor α (ERα)-positive T47D cells was markedly inhibited by 1 × 10(-6) mol/L methylseleninic acid (MSA) with no Se related toxicity.Under conditions where cell growth was inhibited, MSA decreased ERα mRNA levels and subsequent protein levels; further decreasing expression of estrogen-responsive finger protein (Efp) which is a target gene product of ERα and promotes G2/M progression of the cell cycle. Therefore, the decline in Efp expression is presumed to be involved in G2 arrest. Coincidentally, the antioxidative thioredoxin/ thioredoxin reductase (Trx/TrxR) system in cells was enhanced by the synergistic action of E(2) and MSA. It has been reported that ROS-induced oxidative stress enhanced ERα expression. E(2) increased production of intracellular ROS in T47D cells. Meanwhile, MSA significantly decreased E(2)-induced ROS accumulation. From these results, activation of the Trx/TrxR system induced by the coexistence of MSA and E(2) suppresses oxidative stress and decreases expression of ERα, and finally induces the growth arrest of T47D cells through disruption of ERα signaling.

Difference in glucose intolerance between C57BL/6J and ICR strain mice with streptozotocin/nicotinamide-induced diabetes.

Blood glucose and plasma insulin levels between C57BL/6J and ICR strain mice with nicotinamide (NA) and streptozotocin (STZ)-induced diabetes were compared to establish a suitable strain of the experimental diabetic mouse model. The mice were intraperitoneally treated twice with STZ (100 mg/kg) 15 min after injection of NA (120 mg/kg) at a 1-day interval, and non-fasting blood glucose level was then weekly monitored for 5 weeks. The blood glucose level in ICR mice gradually increased and was about 2-times higher than that in C57BL/6J mice at the end of the observation. The plasma insulin level in ICR mice was comparatively low, compared with that in C57BL/6J mice. ICR mice were also markedly glucose-intolerant when oral glucose tolerance test was performed. These results indicate that ICR strain is more sensitive than C57BL/6J strain as a mouse model with NA/STZ-induced mild diabetes.

17beta-Estradiol increases the number of effector memory CD8+ lymphocytes in mice with contact hypersensitivity and among cultured splenocytes.

To determine how estrogen exacerbates allergies, the effects of 17beta-estradiol (E2) on lymphocyte proliferation were investigated. BALB/c mice were ovariectomized, administered 3.2 microg E2, and sensitized with 50 microL 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one. After 7 days, their spleens were excised and flow cytometrically analyzed. The CD8(+)CD45RA(-)CCR7(-) cell-to-CD8(+) cell ratio in the spleen was greater in the E2-administered mice than in the controls. Splenocytes were cultured under concanavalin A stimulation, with or without E2. After 4 days, the above ratio was greater in the case of E2-treated splenocytes. E2 increases the number of effector memory CD8(+) lymphocytes during sensitization in contact hypersensitivity.

17beta-Estradiol enhances expression of inflammatory cytokines and inducible nitric oxide synthase in mouse contact hypersensitivity.

The effects of 17beta-estradiol (E2) on the expression of cytokines, inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2 in mouse contact hypersensitivity (CHS) were examined. Three week old female mice were ovariectomized, administered 3.2 microg of E2 subcutaneously, the mice sensitized by application of 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (OXA) on their backs, and CHS induced by applying OXA to the auricles. E2 significantly increased mRNA expression of interferon (IFN)-gamma and interleukin (IL)-10 in the auricle at 6 and 24 h after allergy elicitation in the ear, respectively, when compared to untreated controls. Although there was no effect of E2 on the expression of IL-4 and COX-2 at any time, the expression of iNOS mRNA was increased by E2 treatment at 48 h after elicitation. E2 also enhanced the expression of tumor necrosis factor (TNF)-alpha and IL-1beta. Histological evaluation revealed that E2 promoted edema of the auricle dermis. The hyperplasia of the epidermis was suppressed by E2 and the cell infiltration observed after elicitation was not altered by E2. These results suggest that E2 enhances the expression of IFN-gamma, TNF-alpha, and IL-1beta to augment the edema of auricle dermis in mouse CHS.

Effects of selenium status and supplementary seleno-chemical sources on mouse T-cell mitogenesis.

Although selenium is thought to be essential for various immune responses, the excess supplementation may have an adverse effect on certain immunological functions. The present study was designed to determine the effective chemical forms of selenium and their optimal levels on T-cell mitogenesis with splenic cells from mice given a selenium-deficient diet for 8 weeks to avoid effects of cellular selenium sources. Although selenium in tissues, except for spleen and thymus, was almost depleted by feeding selenium-deficient diet, the lymphoid organs still contained low levels of selenium. Both activities of cellular glutathione peroxidase (cGPx) and thioredoxin reductase (TR) in liver and splenic cells showed a tendency to decrease by selenium deficiency. However, splenic cells were tolerant against decrease of the selenoenzyme activities, and TR was also more tolerant than cGPx. T-cell proliferation of the selenium-insufficient splenic cells induced by concanavalin A was increased by addition of Na2SeO3, Na2SeO4, Na2Se, seleno-DL-cystine, seleno-L-methionine and selenocystamine. Their promoting action was observed at levels lower than 0.1 micromol/L and was completely suppressed at the highest concentration (1 micromol/L), except for selenocystamine. Na2SeO3 was one of the efficient selenocompounds for the mitogenesis, which was concomitant with the significant induction of cGPx and TR. However, recovery of cGPx activity in the selenium-insufficient cells by supplementary Na2SeO3 was only partial,while TR activity was readily recovered from selenium deficiency. These results therefore indicate that only low levels of selenium is essential for T-cell mitogenesis even in selenium-insufficient splenic cells, and TR, which is readily recovered by Na2SeO3, may be the critical enzyme.

17beta-estradiol enhances contact hypersensitivity in a hair cycle-dependent manner and retards thymic atrophy with age.

The effect of 17beta-estradiol (E2) on murine contact hypersensitivity (CHS), thymic atrophy, and hair-cycle change related to growth was investigated. Female mice were ovariectomized. E2 (3.2 microg) was injected subcutaneously along with the sensitizer 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (OXA), and a hypersensitive reaction was elicited with OXA on the ear in mice of various ages. E2 enhanced allergy only in 3- and 7-week-old mice, just prior to hair regrowth. Age-related thymus atrophy was repressed in the E2-treated mice compared with the control mice. E2 alone did not cause thymus involution, but it did inhibit thymus involution and regeneration after CHS.

Contribution of thioredoxin reductase to T-cell mitogenesis and NF-kappaB DNA-binding promoted by selenite.

Although the essential role of selenium for cellular immune responses is obvious, delineation of the functions is lacking because selenium can either promote or inhibit cell growth, cytokine production, and activation of transcription factor nuclear factor-kappaB (NF-kappaB). Studies with human thioredoxin-1 (Trx-1)-transgenic (Tg) mice were conducted to evaluate the relationship between stimulation of T-cell mitogenic response by sodium selenite and the intracellular Trx-1 levels, and the activities of selenoenzymes and NF-kappaB-DNA binding. Concanavalin A-induced mitogenesis of wild-type mouse splenic cells was stimulated by exposure to low levels of selenite (0.02-0.1 microM), with augmentation of NF-kappaB-DNA binding activity. Treatment with NF-kappaB nuclear translocation inhibitor SN50 or thioredoxin reductase (TR) inhibitor aurothioglucose depressed this stimulatory action. The mitogenic response of Trx-1-Tg mouse splenic cells was enhanced by exposure to relatively high levels of selenite (> or = 0.05 microM), compared with the wild-type mouse. Selenite also augmented TR activity but not cellular glutathione peroxidase activity in the Trx-1-overexpressed cells. These results suggest that the stimulation of T-cell mitogenic response by the physiological levels of selenite is predominantly caused by increased TR activity, which may lead to reduction of Trx-1 dependent on the intracellular expression level and promotion of DNA binding of NF-kappaB.

17beta-estradiol enhances contact hypersensitivity and IFN-gamma expression in inflamed skin of BALB/c mice.

The effects of 17beta-estradiol (E(2)) on mouse contact hypersensitivity (CHS) elicited at the ears by 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (OXA) were examined. Male and female BALB/c mice were sham-treated or gonadectomized, and then subcutaneously injected with E(2) twice a week for 4 weeks. The mice were sensitized by OXA application to their back and CHS was elicited at the ears. E(2) enhanced the ear swelling of all groups at 6h after the elicitation. E(2) had no effect on the mitogenesis of splenic lymphocytes or nitric oxide synthesis by peritoneal macrophages. E(2) increased the number of thymic cells in female mice, but not male mice, and had no effect on the splenic cells of either female or male mice. Evaluation of the cytokine expressions in the inflamed skin revealed that E(2) enhanced the expression of interferon-gamma, but had no effect on the expression of interleukin-4. These results suggest that E(2) affects the thymus and enhances the production of interferon-gamma in skin to augment the skin swelling in CHS elicited by OXA.

Cystathionine gamma-lyase contributes to selenomethionine detoxification and cytosolic glutathione peroxidase biosynthesis in mouse liver.

We earlier found that seleno-l-methionine (L-SeMet) as a food source of selenium (Se) is directly converted to methylselenol (CH3SeH), alpha-ketobutyrate, and ammonia by the mouse hepatic cystathionine gamma-lyase. The purpose of this study was to clarify the biological role of cystathionine gamma-lyase in Se detoxification and cytosolic glutathione peroxidase (cGPx) biosynthesis because another metabolic pathway to CH3SeH via seleno-l-cystathionine and seleno-l-cysteine (l-SeCyH) from l-SeMet has been shown by several enzymatic reactions. When mice were treated with either toxic doses of l-SeMet or a Se-deficient diet, the cystathionine gamma-lyase activity for l-SeMet was invariable, suggesting that this enzyme was effective in both detoxification and biotransformation of Se. Concerning Se biotransformation into cGPx, production of H2Se as the possible precursor was not observed by the in vitro reaction of the liver cytosol with CH3SeH. When l-SeMet was administered at the nutritional dose to mice fed a Se-deficient diet, levels of both cGPx mRNA and cGPx protein were significantly restored. This recovery was not comparatively suppressed by coadministration of periodate-oxidized adenosine, an inhibitor of S-adenosylhomocysteinase, where the conversion of l-SeMet to l-SeCyH is inhibited. However, the recovery was strongly suppressed when propargylglycine, an inhibitor of cystathionine gamma-lyase that catalyzes the alpha,gamma-elimination reaction of both l-SeMet and seleno-l-cystathionine, was treated. These results suggest that cystathionine gamma-lyase is a notable enzyme in SeMet metabolism and that CH3SeH produced by the enzymatic reaction is utilized for cGPx biosynthesis.

Identification of mouse selenomethionine alpha,gamma-elimination enzyme: cystathionine gamma-lyase catalyzes its reaction to generate methylselenol.

The purpose of this study was to identify the seleno-L-methionine (L-SeMet) alpha,gamma-elimination enzyme that catalyzes L-SeMet to generate methylselenol (CH3SeH), a notable intermediate for the metabolism of selenium compounds, in mammalian tissues. The enzyme purified from ICR mouse liver was separated by one-dimensional gel electrophoresis, and the specific band was subjected to in-gel trypsin digestion followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric analysis. In the peptide mass fingerprinting search, the mass numbers of 14 peptides produced by tryptic digestion of the enzyme were consistent with the theoretical mass numbers calculated from the amino acid sequence of murine cystathionine gamma-lyase (E.C. 4.4.1.1). The peptide sequence tags search was also performed to obtain the amino acid sequence data of five tryptic peptides. These peptides were significantly identical to the partial amino acid sequences of cystathionine gamma-lyase. This enzyme was clearly shown to catalyze the alpha,gamma-elimination reaction of L-cystathionine by the enzymological research. The Km value for the catalysis of L-cystathionine was 0.81 mM and Vmax was 0.0013 unit/mg protein. These results suggested that cystathionine gamma-lyase catalyzes L-SeMet to generate CH3SeH by its alpha,gamma-elimination reaction.

Purification and characterization of mouse hepatic enzyme that converts selenomethionine to methylselenol by its alpha,gamma-elimination.

The objective of this study was to purify and characterize a mouse hepatic enzyme that directly generates CH3SeH from seleno-l-methionine (l-SeMet) by the alpha,gamma-elimination reaction. The l-SeMet alpha,gamma-elimination enzyme was ubiquitous in tissues from ICR mice and the activity was relatively high in the large intestine, brain, and muscle, as well as the liver. Aging and sex of the mice did not have any significant influence on the activity in the liver. The enzyme was purified from the mouse liver by ammonium sulfate precipitation and four kinds of column chromatography. These procedures yielded a homogeneous enzyme, which was purified approx 1000-fold relative to mouse liver extract. Overall recovery was approx 8%. The purified enzyme had a molecular mass of approx 160 kDa with four identical subunits. The Km value of the enzyme for the catalysis of l-SeMet was 15.5 mM, and the Vmax was 0.29 units/mg protein. Pyridoxal 5'-phosphate (pyridoxal-P) was required as a cofactor because the holoenzyme could be resolved to the apoenzyme by incubation with hydroxylamine and reconstituted by addition of pyridoxal-P. The enzyme showed the optimum activity at around pH 8.0 and the highest activity at 50 degrees C; it catalyzed the alpha,gamma-elimination reactions of several analogs such as d,l-homocysteine and l-homoserine in addition to l-SeMet. This enzyme also catalyzed the alpha,beta-elimination reaction of Se-methylseleno-l-cysteine. However, l-methionine was inert. Therefore, the purified enzyme was different from the bacterial l-methionine gamma-lyase that metabolizes l-SeMet to CH3SeH, in terms of the substrate specificity. These results were the first identification of a mammalian enzyme that specifically catalyzes the alpha,gamma-elimination reaction of l-SeMet and immediately converts it to CH3SeH, an important metabolite of Se.

Aqueous photodegradation of fenthion by ultraviolet B irradiation: contribution of singlet oxygen in photodegradation and photochemical hydrolysis.

The objective of this study was to evaluate the photodegradation of the organophosphorus pesticide fenthion in the environment from a human health effect viewpoint. The major photodegradation products of fenthion in an aqueous solution under UVB irradiation (280-320nm radiation) were identified as fenthion sulfoxide, 3-methyl-4-methylthiophenol (MMTP), dimethyl phosphorothioate and 3-methyl-4-methylsulfinylphenol (MMSP). MMTP, dimethyl phosphorothioate and MMSP were discovered as novel photodegradation products of fenthion. Kinetic analysis of these products showed the formation of MMTP and dimethyl phosphorothioate by the photochemical hydrolysis of fenthion, which was accelerated under alkaline conditions. The former was further oxidized to MMSP. Fenthion sulfoxide was directly produced by the oxidative reaction of fenthion. Contribution of dissolved oxygen in this photooxidation was observed by replacing the air with nitrogen gas in the reaction system, which prevented oxidative formation of fenthion sulfoxide from fenthion and MMSP from MMTP. These oxidative compounds were also formed from fenthion in the presence of singlet oxygen (1O2) generated by the visible light irradiation of rose bengal solution, while 1O2 scavengers, L-histidine and sodium azide (NaN3) inhibited this reaction. The aqueous photolysis mechanisms of fenthion were proposed from a kinetic photolysis experiment study as follows: there were two kinds of UVB photodegradation pathways of fenthion, one being photochemical hydrolysis of the phosphorus-O-phenyl ester to form MMTP and dimethyl phosphorothioate, and the other oxygenation triggered by 1O2 and producing fenthion sulfoxide and MMSP. Therefore, the steady photodegradation products of fenthion in the water environment may be fenthion sulfoxide and MMSP.

Estrogen receptor alpha in mouse splenic lymphocytes: possible involvement in immunity.

To elucidate relevance of estrogens to immune responses, we investigated whether estrogen receptor alpha (ERalpha) exists in mouse splenic B cells and T cells and the effect of 17beta-estradiol and endocrine disrupting chemicals (EDCs) on lymphocyte mitogenesis. ERalpha was identified in both male and female mouse splenic cells using RT-PCR. Crude splenic cells were stained with anti-ER antibody, and the distribution of ERalpha in the splenic B cells and part of the splenic T cells was confirmed by flow cytometry. 17beta-Estradiol inhibited B cell mitogenesis at the concentration of 10(-8) M and T cell mitogenesis at the concentration of 10(-6) M. Some EDCs, diethylstilbestrol, bisphenol A, p-nonylphenol and di-2-ethylhexylphthalate, suppressed lymphocyte mitogenesis at the concentration of 10(-6)-10(-5) M. We therefore suggest that estrogen may suppress lymphocyte mitogenesis through ERalpha in B and T cells.