Rg3-enriched Korean Red Ginseng extract inhibits blood-brain barrier disruption in an animal model of multiple sclerosis by modulating expression of NADPH oxidase 2 and 4.

BACKGROUND: Multiple sclerosis (MS) and its animal model, the experimental autoimmune encephalomyelitis (EAE), are primarily characterized as dysfunction of the blood-brain barrier (BBB). Ginsenoside-Rg3-enriched Korean Red Ginseng extract (Rg3-KRGE) is known to exert neuroprotective, anti-inflammatory, and anti-oxidative effects on neurological disorders. However, effects of Rg3-KRGE in EAE remain unclear. METHODS: Here, we investigated whether Rg3-KRGE may improve the symptoms and pathological features of myelin oligodendroglial glycoprotein (MOG)35-55 peptide - induced chronic EAE mice through improving the integrity of the BBB. RESULTS: Rg3-KRGE decreased EAE score and spinal demyelination. Rg3-KRGE inhibited Evan's blue dye leakage in spinal cord, suppressed increases of adhesion molecule platelet endothelial cell adhesion molecule-1, extracellular matrix proteins fibronection, and matrix metallopeptidase-9, and prevented decreases of tight junction proteins zonula occludens-1, claudin-3, and claudin-5 in spinal cord following EAE induction. Rg3-KRGE repressed increases of proinflammatory transcripts cyclooxygenase-2, inducible nitric oxide synthase, interleukin (IL)-1 beta, IL-6, and tumor necrosis factor-alpha, but enhanced expression levels of anti-inflammatory transcripts arginase-1 and IL-10 in the spinal cord following EAE induction. Rg3-KRGE inhibited the expression of oxidative stress markers (MitoSOX and 4-hydroxynonenal), the enhancement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) and NOX4, and NADPH activity in the spinal cord of chronic EAE mice. Furthermore, apocynin, a NOX inhibitor, mimicked beneficial effects of Rg3-KRGE in chronic EAE mice. CONCLUSION: Our findings suggest that Rg3-KRGE might alleviate behavioral symptoms and pathological features of MS by improving BBB integrity through modulation of NOX2/4 expression.

Comparative evaluation of canine cadaver embalming methods for veterinary anatomy education.

Formalin-embalmed cadavers have been extensively used to teach anatomy. Although they ensure the preservation of anatomical structures without microbial contamination, they are considerably rigid and cannot be used to study the joint and muscle movements. Moreover, formalin irritates the eyes and airways and is carcinogenic on chronic exposure. To overcome the disadvantages of formalin-fixed cadavers, we investigated the usefulness of alternative embalming methods using saturated salt solution (SS) and Thiel's solution (TS). We compared the three solutions based on the following parameters: cost of the embalming solution; preservation of anatomical structure, color, flexibility, and texture; and microbial contamination. Convenience of anatomical structure identification and preferences in anatomical laboratory practice were evaluated using questionnaires answered by veterinary undergraduate students. Cost of the embalming solution was the lowest for formalin solution (FS) and most expensive for TS. All cadavers were successfully preserved without significant putrefaction and were useful for teaching veterinary anatomy. Cadavers embalmed with SS or TS were superior in facilitating joint and muscle movement. Compared to FS, the color and texture of muscles and internal organs were similar to those of living animals and there was no irritating and offensive smell in SS and TS cadavers. Students preferred the SS and TS cadavers for their usefulness in identification of anatomical structures, highlighting their usefulness in veterinary anatomy education.

Ginseng Gintonin Attenuates Lead-Induced Rat Cerebellar Impairments during Gestation and Lactation.

Gintonin, a novel ginseng-derived lysophosphatidic acid receptor ligand, improves brain functions and protects neurons from oxidative stress. However, little is known about the effects of gintonin against Pb-induced brain maldevelopment. We investigated the protective effects of gintonin on the developing cerebellum after prenatal and postnatal Pb exposure. Pregnant female rats were randomly divided into three groups: control, Pb (0.3% Pb acetate in drinking water), and Pb plus gintonin (100 mg/kg, p.o.). Blood Pb was increased in dams and pups; gintonin treatment significantly decreased blood Pb. On postnatal day 21, the number of degenerating Purkinje cells was remarkably increased while the number of calbindin-, GAD67-, NMDAR1-, LPAR1-immunoreactive intact Purkinje cells, and GABA transporter 1-immunoreactive pinceau structures were significantly reduced in Pb-exposed offspring. Following Pb exposure, gintonin ameliorated cerebellar degenerative effects, restored increased pro-apoptotic Bax, and decreased anti-apoptotic Bcl2. Gintonin treatment attenuated Pb-induced accumulation of oxidative stress (Nrf2 and Mn-SOD) and inflammation (IL-1ß and TNFα,), restoring the decreased cerebellar BDNF and Sirt1. Gintonin ameliorated Pb-induced impairment of myelin basic protein-immunoreactive myelinated fibers of Purkinje cells. Gintonin attenuated Pb-induced locomotor dysfunctions. The present study revealed the ameliorating effects of gintonin against Pb, suggesting the potential use of gintonin as a preventive agent in Pb poisoning during pregnancy and lactation.

Effects of ascorbic acid treatment on developmental alterations in calcium-binding proteins and gamma-aminobutyric acid transporter 1 in the cerebellum of lead-exposed rats during pregnancy and lactation.

In the present study, we investigated the effects of lead (Pb) and ascorbic acid co-administration on rat cerebellar development. Female rats were randomly divided into the following groups: control, Pb, and Pb plus ascorbic acid (PA) groups. From one week prior to mating, female rats were administered Pb (0.3% Pb acetate in drinking water) and ascorbic acid (100 mg/kg, oral intubation). The chemical administration was stopped on postnatal day 21 when the morphology of the offspring's cerebellum is similar to that of the adult brain. The blood Pb level was significantly increased following long-term Pb exposure. Ascorbic acid reduced Pb levels in the dams and offspring. Nissl staining demonstrated that the number of Purkinje cells was significantly reduced following Pb exposure, while ascorbic acid ameliorated this effect in the cerebellum of the offspring. Calcium-binding proteins, such as calbindin, calretinin, and parvalbumin were commonly expressed in Purkinje cells, and Pb exposure and ascorbic acid treatment resulted in similar patterns of change, namely Pb-induced impairment and ascorbic acid-mediated amelioration. The gamma-aminobutyric acid transporter 1 (GABAT1) is expressed in the pinceau structure where the somata of Purkinje cells are entwined in inhibitory synapses. The number of GABAT1-immunoreactive synapses was reduced following Pb exposure, and ascorbic acid co-treatment prevented this effect in the cerebellar cortex. Therefore, it can be concluded that ascorbic acid supplementation to mothers during gestation and lactation may have potential preventive effects against Pb-induced impairments in the developing cerebellum via protection of inhibitory neurons and synapses.

Effects of Ascorbic Acid on Osteopontin Expression and Axonal Myelination in the Developing Cerebellum of Lead-Exposed Rat Pups.

Osteopontin (OPN) is a multi-functional protein that binds to integrin and calcium-binding phosphoprotein. OPN is required for normal neuronal development and its axonal myelination. We studied the combined effect of lead (Pb) and ascorbic acid treatment on OPN expression in the developing cerebellum. We randomly divided pregnant female rats into three groups: control, Pb (lead acetate, 0.3%, drinking water), and Pb plus ascorbic acid (PA; ascorbic acid, 100 mg/kg, oral intubation) groups. The blood level of Pb was significantly increased, while ascorbic acid reduced Pb levels in the dams and pups. At postnatal day (PND) 21, results from Nissl staining and OPN immunohistochemistry demonstrated that OPN was detected in the Purkinje cell layer in the cerebellum. Ascorbic acid treatment mitigated Pb exposure-induced reduction in the number of intact Purkinje cells and OPN immunoreactive Purkinje cells in the cerebellum of pups. In addition, Pb-induced reduction in the number of oligodendrocytes and myelin-associated glycoprotein is associated with the malformation of the myelin sheath. Ascorbic acid provided protection from Pb-induced impairments. Pb-induced structural deficits in the cerebellum resulted in functional deterioration observed during locomotive tests (bar holding test and wire mesh ascending test), while ascorbic acid ameliorated these harmful effects. Present results suggest that the change of OPN is associated with myelination in the developing cerebellum. The results also demonstrated that exposure to Pb is harmful, while ascorbic acid treatment is beneficial.

Ascorbic Acid Mitigates D-galactose-Induced Brain Aging by Increasing Hippocampal Neurogenesis and Improving Memory Function.

Ascorbic acid is essential for normal brain development and homeostasis. However, the effect of ascorbic acid on adult brain aging has not been determined. Long-term treatment with high levels of D-galactose (D-gal) induces brain aging by accumulated oxidative stress. In the present study, mice were subcutaneously administered with D-gal (150 mg/kg/day) for 10 weeks; from the seventh week, ascorbic acid (150 mg/kg/day) was orally co-administered for four weeks. Although D-gal administration alone reduced hippocampal neurogenesis and cognitive functions, co-treatment of ascorbic acid with D-gal effectively prevented D-gal-induced reduced hippocampal neurogenesis through improved cellular proliferation, neuronal differentiation, and neuronal maturation. Long-term D-gal treatment also reduced expression levels of synaptic plasticity-related markers, i.e., synaptophysin and phosphorylated Ca2+/calmodulin-dependent protein kinase II, while ascorbic acid prevented the reduction in the hippocampus. Furthermore, ascorbic acid ameliorated D-gal-induced downregulation of superoxide dismutase 1 and 2, sirtuin1, caveolin-1, and brain-derived neurotrophic factor and upregulation of interleukin 1 beta and tumor necrosis factor alpha in the hippocampus. Ascorbic acid-mediated hippocampal restoration from D-gal-induced impairment was associated with an enhanced hippocampus-dependent memory function. Therefore, ascorbic acid ameliorates D-gal-induced impairments through anti-oxidative and anti-inflammatory effects, and it could be an effective dietary supplement against adult brain aging.

Ascorbic Acid Attenuates Lead-Induced Alterations in the Synapses in the Developing Rat Cerebellum.

We evaluated the effect of lead (Pb) and ascorbic acid treatment of pregnant female rats on cerebellar development in pups. Pb was administered in drinking water (0.2% Pb acetate), and ascorbic acid (100 mg/kg) was administered through oral intubation. Fifteen female rats were randomly classified into control, Pb, and Pb plus ascorbic acid (PA) groups. The treatment of Pb and ascorbic acid treatments were terminated after birth to evaluate the effects on the gestational development of the cerebellum. At postnatal day 21 (PND21), pups were sacrificed, and blood Pb level was analyzed. Blood Pb levels of pups and dams were highest in the Pb group and reduced in the PA group. Immunohistochemistry and immunoblot assays were conducted to study the cerebellar expression levels of synaptic proteins. Along with a significant reduction in Purkinje cells, the reduction in presynaptic (synaptophysin) and postsynaptic (postsynaptic density protein 95, N-methyl-D-aspartate receptor subtype 1) marker proteins was observed in Pb-exposed pups. Ascorbic acid treatment significantly prevented Pb-induced impairment in the cerebellar synaptic proteins. Hypothesizing that brain-derived neurotrophic factor (BDNF) might be affected by Pb exposure given its importance in the regulation of synaptogenesis, we observed a Pb-induced decrease and ascorbic acid-mediated increase of BDNF in the cerebellum. Luxol fast blue staining and myelin basic protein analysis suggest that ascorbic acid treatment ameliorated the Pb exposure-induced reduction in the axonal fibers in the developing cerebellum. Overall, we conclude that ascorbic acid treatment during pregnancy can prevent Pb-induced impairments in the cerebellar development in rats.

Ascorbic Acid Supplementation Prevents the Detrimental Effects of Prenatal and Postnatal Lead Exposure on the Purkinje Cell and Related Proteins in the Cerebellum of Developing Rats.

We investigated the effects of lead (Pb) and ascorbic acid co-administration on rat cerebellar development. Prior to mating, rats were randomly divided into control, Pb, and Pb plus ascorbic acid (PA) groups. Pregnant rats were administered Pb in drinking water (0.3% Pb acetate), and ascorbic acid (100 mg/kg) via oral intubation until the end of the experiment. Offspring were sacrificed at postnatal day 21, the age at which the morphology of the cerebellar cortex in developing pups is similar to that of the adult brain. In the cerebellum, Pb exposure significantly reduced Purkinje cells and ascorbic acid prevented their reduction. Along with the change of the Purkinje cells, long-term Pb exposure significantly reduced the expression of the synaptic marker (synaptophysin), γ-aminobutyric acid (GABA)-synthesizing enzyme (glutamic acid decarboxylase 67), and axonal myelin basic protein while ascorbic acid co-treatment attenuated Pb-mediated reduction of these proteins in the cerebellum of pups. However, glutamatergic N-methyl-D-aspartate receptor subtype 1 (NMDAR1), anchoring postsynaptic density protein 95 (PSD95), and antioxidant superoxide dismutases (SODs) were adversely changed; Pb exposure increased the expression of NMDAR1, PSD95, and SODs while ascorbic acid co-administration attenuated Pb-mediated induction. Although further studies are required about the neurotoxicity of the Pb exposure, the results presented here suggest that developmental Pb exposure disrupted normal development of Purkinje cells by increasing glutamatergic and oxidative stress in the cerebellum. Additionally, ascorbic acid co-treatment is beneficial in attenuating prenatal and postnatal Pb exposure-induced maldevelopment of Purkinje cells in the developing cerebellum.

Korean Red Ginseng mitigates spinal demyelination in a model of acute multiple sclerosis by downregulating p38 mitogen-activated protein kinase and nuclear factor-κB signaling pathways.

BACKGROUND: The potential therapeutic values of Korean Red Ginseng extract (KRGE) in autoimmune disorders of nervous system have not been fully investigated. METHODS: We used an acute experimental autoimmune encephalomyelitis animal model of multiple sclerosis and determined the effects and mechanism of KRGE on spinal myelination. RESULTS: Pretreatment with KRGE (100 mg/kg, orally) for 10 days before immunization with myelin basic protein (MBP)68-82 peptide exerted a protective effect against demyelination in the spinal cord, with inhibited recruitment and activation of immune cells including microglia, decreased mRNA expression of detrimental inflammatory mediators (interleukin-6, interferon-γ, and cyclooxygenase-2), but increased mRNA expression of protective inflammatory mediators (insulin-like growth factor ß1, transforming growth factor ß, and vascular endothelial growth factor-1). These results were associated with significant downregulation of p38 mitogen-activated protein kinase and nuclear factor-κB signaling pathways in microglia/macrophages, T cells, and astrocytes. CONCLUSION: Our findings suggest that KRGE alleviates spinal demyelination in acute experimental autoimmune encephalomyelitis through inhibiting the activation of the p38 mitogen-activated protein kinase/nuclear factor-κB signaling pathway. Therefore, KRGE might be used as a new therapeutic for autoimmune disorders such as multiple sclerosis, although further investigation is needed.

Neonatal influenza virus infection affects myelination in influenza-recovered mouse brain.

Influenza virus infection is a zoonosis that has great socioeconomic effects worldwide. Influenza infection induces respiratory symptoms, while the influenza virus can infect brain and leave central nervous system sequelae. As children are more vulnerable to infection, they are at risk of long-term neurological effects once their brains are infected. We previously demonstrated that functional changes in hippocampal neurons were observed in mice recovered from neonatal influenza infection. In this study, we investigated changes in myelination properties that could affect neural dysfunction. Mice were infected with the influenza virus on postnatal day 5. Tissues were harvested from recovered mice 21-days post-infection. The expression levels for myelin basic protein (MBP) were determined, and immunohistochemical staining and transmission electron microscopy were performed. Real-time polymerase chain reaction and Western blot analyses showed that mRNA and protein expressions increased in the hippocampus and cerebellum of recovered mice. Increased MBP-staining signal was observed in the recovered mouse brain. By calculating the relative thickness of myelin sheath in relation to nerve fiber diameter (G-ratio) from electron photomicrographs, an increased G-ratio was observed in both the hippocampus and cerebellum of recovered mice. Influenza infection in oligodendrocyte-enriched primary brain cell cultures showed that proinflammatory cytokines may induce MBP upregulation. These results suggested that increased MBP expression could be a compensatory change related to hypomyelination, which may underlie neural dysfunction in recovered mice. In summary, the present results demonstrate that influenza infection during the neonatal period affects myelination and further induces functional changes in influenza-recovered mouse brain.

Comparative Study on the Epidemiological Trends and Aspects of Murine Typhus in Korea in the Last Decade (2006-2015).

INTRODUCTION: In this study, we undertook a retrospective analysis of the epidemiological aspects and risk factors of murine typhus (MT) in Korea in the last decade (2006-2015). MATERIALS AND METHODS: MT infections in Korea using a total of 411 cases obtained from the Diseases Web Statistical System of the Korea Center for Disease Control and Prevention. RESULTS: In a total of 411 MT infection cases, the cumulative incidence rate was 0.08/100,000 populations. Men were slightly more infected by MT as compared to women (51.3% vs. 48.7%), and a higher incidence of MT was observed in people aged over 40 years (93.4%; P < 0.01). The seasonal pattern of outbreaks revealed that most infections occurred from October to November (69.1% of the total cases) (P < 0.01). Significantly more outbreaks occurred in the southern part (53.5%) of the Korean peninsula as compared to its northern (33.3%) and central (10.7%) parts, as well as the Jeju Island (1.0%) (P < 0.01). In addition, the number of MT infections was significantly higher in rural and sea-village (87.6%) than in urban areas (12.4%; P < 0.01). CONCLUSION: In conclusion, the rapid reemergence of MT outbreaks can be minimized through health education, and a strong enforcement of control measures against rats and their ectoparasites could markedly reduce the transmission of this infection to humans in high-risk areas.

Gintonin Attenuates D-Galactose-Induced Hippocampal Senescence by Improving Long-Term Hippocampal Potentiation, Neurogenesis, and Cognitive Functions.

BACKGROUND: Ginseng has been used to improve brain function and increase longevity. However, little is known about the ingredients of ginseng and molecular mechanisms of its anti-brain aging effects. Gintonin is a novel exogenous ginseng-derived lysophosphatidic acid (LPA) receptor ligand; LPA and LPA1 receptors are involved in adult hippocampal neurogenesis. D-galactose (D-gal) is used to induce brain -aging in animal models because long-term treatment with D-gal facilitates hippocampal aging in experimental adult animals by decreasing hippocampal neurogenesis and inducing learning and memory dysfunction. OBJECTIVE: To investigate the protective effects of gintonin on D-gal-induced hippocampal senescence, impairment of long-term potentiation (LTP), and memory dysfunction. METHODS: Brain hippocampal aging was induced by D-gal administration (150 mg/kg/day, s.c.; 10 weeks). From the 7th week, gintonin (50 or 100 mg/kg/day, per os) was co-administered with D-gal for 4 weeks. We performed histological analyses, LTP measurements, and object location test. RESULTS: Co-administration of gintonin ameliorated D-gal-induced reductions in hippocampal Ki67-immunoreactive proliferating cells, doublecortin-immunoreactive neuroblasts, 5-bromo-2'-deoxyuridine-incorporating NeuN-immunoreactive mature neurons, and LPA1 receptor expression. Co-administration of gintonin in D-gal-treated mice increased the expression of phosphorylated cyclic adenosine monophosphate response element binding protein in the hippocampal dentate gyrus. In addition, co-administration of gintonin in D-gal-treated mice enhanced LTP and restored the cognitive functions compared with those in mice treated with D-gal only. CONCLUSION: These results show that gintonin administration restores D-gal-induced memory deficits by enhancing hippocampal LPA1 receptor expression, LTP, and neurogenesis. Finally, the present study shows that gintonin exerts anti-brain aging effects that are responsible for alleviating brain aging-related dysfunction.

Ultrastructural Changes and Expression of PCNA and RPE65 in Sodium Iodate-Induced Acute Retinal Pigment Epithelium Degeneration Model.

Alteration in retinal pigment epithelium (RPE) results in the visual dysfunction and blindness of retinal degenerative diseases. Injection of sodium iodate (NaIO3) generates degeneration of RPE. We analyzed the sequential ultrastructure and expression of proliferating cell nuclear antigen (PCNA) and retina-specific RPE65 in NaIO3-induced retinal degeneration model. Adult male rats were injected 1% NaIO3 (50 mg/kg) and eyes were enucleated at 1, 3, 5, 7 and 14 days post-injection (DPI), fixed, and processed for histological analysis. NaIO3-induced retinal degeneration was successfully established. At 1 DPI, most RPE cells were degenerated and replaced by a few proliferating RPE cells in the peripheral area. At 3 DPI, the RPE and photoreceptor out segments (POS) underwent a marked morphological change, including POS disruption, accumulation of residual bodies in RPE and POS, and hyperplasia of the RPE cell. At 5 DPI, POS showed a maximum increase in the outer segment debris and the retina showed partial detachment. These abnormal morphological changes gradually decreased by day 7. At 14 DPI, the damaged RPE and POS were partially regenerated from the peripheral to the central region. Expression of PCNA and RPE65 increased from day 3 onward. The damaged RPE showed earlier expression of PCNA and RPE65 than POS. The RPE damaged by NaIO3 rapidly proliferated to put down roots on Bruch's membrane from the peripheral retina and proliferation and hyperplasia of the RPE had a regular direction of progress. Therefore, NaIO3-induced acute changes in retina mimic the patho-morphologic features of RPE-related diseases.

Ascorbic acid ameliorates lead-induced apoptosis in the cerebellar cortex of developing rats.

We investigated the effects of the gestational administration of lead (Pb) and ascorbic acid on cerebellar development. Pregnant female rats were randomly assigned to the control, Pb, or Pb plus ascorbic acid (PA) groups; six offspring per cage were randomly selected for analysis. Compared to the control group, fewer Purkinje cells were observed in the Pb-exposed pups at postnatal day 21. However, co-administrating Pb and ascorbic acid inhibited the Pb-induced reduction in Purkinje cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining, which detected DNA fragmentation in the dying cells, showed more TUNEL-positive cells in the Pb group, while co-treatment with Pb and ascorbic acid mitigated the Pb-induced cellular degeneration. Using immunohistochemistry and immunoblotting, we additionally found that Pb exposure induced a rise in the apoptotic factor Bax in the cerebellum, while Pb plus ascorbic acid treatment ameliorated this Bax induction. Since, Pb competes with the iron in the cell and the accumulation of free iron triggers oxidative stress, we performed iron staining, which revealed that ascorbic acid prevented the Pb-induced rises in iron-reactive cells and iron-reactivity. The anti-oxidant enzyme manganese-dependent superoxide dismutase showed change patterns that were similar to those of iron in the cerebellum. Finally, the pups' blood Pb levels were highest in the Pb group but were reduced in the PA group. Our findings suggest that ascorbic acid effectively ameliorates Pb-induced apoptosis and oxidative stress in the cerebellum. The present results imply that ascorbic acid treatment during pregnancy may protect against Pb-mediated developmental impairments in the cerebellum.

Dynamic residual pattern of azoxystrobin in Swiss chard with contribution to safety evaluation.

This study aimed at quantifying the residual amount of azoxystrobin in Swiss chard samples grown under greenhouse conditions at two different locations (Gwangju and Naju, Republic of Korea). Samples were extracted with acetonitrile, separated by salting out, and subjected to purification by using solid-phase extraction. The analyte was identified using liquid chromatography-ultraviolet detection. The linearity of the calibration range was excellent with coefficient of determination 1.00. Recovery at three different spiking levels (0.1, 0.5, and 4 mg/kg) ranged between 82.89 and 109.46% with relative standard deviation <3. The limit of quantification, 0.01 mg/kg, was considerably much lower than the maximum residue limit (50 mg/kg) set by the Korean Ministry of Food and Drug Safety. The developed methodology was successfully used for field-treated leaves, which were collected randomly at 0-14 days following azoxystrobin application. The rate of disappearance in/on Swiss chard was ascribed to first-order kinetics with a half-life of 8 and 5 days, in leaves grown in Gwangju and Naju greenhouses, respectively. Risk assessments revealed that the acceptable daily intake percentage is substantially below the risk level of consumption at day 0 (in both areas), thus encouraging its safe consumption.

A quick and effective methodology for analyzing dinotefuran and its highly polar metabolites in plum using liquid chromatography-tandem mass spectrometry.

A simple and effective method was developed for analyzing dinotefuran and its three metabolites (MNG, UF, and DN) in plum using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Due to the polarity and high water miscibility, dinotefuran and some of its metabolites (especially DN) have some limitations to be extracted with acetonitrile and salt following the "QuEChERS" sample preparation methodology. Alternatively, the samples were extracted with methanol, and purified with dispersive-solid phase extraction procedure (d-SPE) using primary secondary amine (PSA) and C18 sorbents after filtration, and mass up. Due to the suppression effect originated from plum matrix, matrix-matched calibration curves, which provided good linearity with coefficient of determination (R2)≥0.998, were used for quantification of all analytes. Blank plum samples fortified with 2 spiking levels (10×LOQ and 50×LOQ) yielded satisfactory recoveries for all tested analytes in the range of 83.01 to 110.18% with relative standard deviation (RSD)≤8.91. The method was successfully applied to field-incurred plum samples and dinotefuran and all metabolites were positively detected and quantified. In conclusion, we suggest that the method can be expanded to polar compounds having solvent and partitioning problems in any of the versions of QuEChERS.

Ascorbic Acid Ameliorates Gestational Lead Exposure-Induced Developmental Alteration in GAD67 and c-Kit Expression in the Rat Cerebellar Cortex.

In the present study, we investigated the effects of ascorbic acid on lead-exposed developing cerebellum. Female rats were divided into the following three groups: control (distilled water), lead (0.2% lead acetate), and lead plus ascorbic acid (100 mg/kg/day, 10% solution). To evaluate the effect of lead exposure and ascorbic acid treatment accurately on the cerebellar development for the gestational period, we halted further treatment with lead and ascorbic acid in the dams after delivery of the pups. Although the ascorbic acid slightly decreased the lead level in pups, lead level was still high in the group treated with lead plus ascorbic acid group compared with the control group. The blood lead levels indicated that the ascorbic acid could facilitate both the excretion and transfer of lead from a dam to its pups via milk. At postnatal day 21, lead exposure significantly reduced the number of Purkinje cells in the cerebellar cortex of pups. Additionally, lead treatment induced degenerative changes such as reduction of glutamic acid decarboxylase (GAD67) and c-kit expressions are observed in the developing cerebellar cortex. In the cerebellum of the pups from the lead plus ascorbic acid group, reduction of the number of Purkinje cells, GAD67 expression, and c-kit immunopositivity were remarkably restored compared with the lead group. Our present results suggested that ascorbic acid treatment to lead-exposed dam exerted protective effects on the developing cerebellum against lead-induced neurotoxicity.

Residual dynamic and risk assessment of dimethomorph in Swiss chard grown at two different sites.

Residue analysis of dimethomorph in Swiss chard cultivated at two different locations under greenhouse conditions was conducted using high-performance liquid chromatography-ultraviolet detection and confirmed by tandem mass spectrometry. The randomly collected samples (over 14 days) were extracted with acetonitrile and purified using a Florisil solid-phase extraction cartridge. Linearity over a concentration range of 0.05-50.0 mg/L had an excellent coefficient of determination of 0.9996. Recovery rate ranged from 82.98 to 95.43% with relative standard deviations ≤5.12% and limits of detection and quantification of 0.003 and 0.01 mg/kg, respectively. The initial deposits [day 0 (2 h post-application)] were considerably lower (7.57 and 8.55 mg/kg for sites 1 and 2, respectively) than the maximum residue limit (30 mg/kg) set by the Korean Ministry of Food and Drug Safety. The dissipation half-life was approximately the same, being 5.0 and 5.1 days for sites 1 and 2, respectively. Risk assessment estimated as acceptable daily intake revealed a value of 0.084 or 0.094% (day 0) and 0.014% (10 days post-application), for sites 1 and 2, respectively. The values indicated that dimethomorph can be safely used on Swiss chard, with no hazardous effects expected for Korean consumers.

Bithionol residue analysis in animal-derived food products by an effective and rugged extraction method coupled with liquid chromatography-tandem mass spectrometry.

Herein, we developed a simple analytical procedure for the quantitation of bithionol residues in animal-derived food products such as porcine muscle, eggs, milk, eel, flatfish, and shrimp using a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method coupled with liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI+/MS-MS). Samples were extracted with 0.1% solution of formic acid in acetonitrile and the extract was purified using a C18 sorbent. Separation was performed on a Waters XBridge™ C18 reversed-phase analytical column using 0.1% solution of formic acid/acetonitrile as the mobile phase. Six-point matrix-matched calibration indicated good linearity, with the calculated coefficients of determination (R2) being≥0.9813. Intra- and inter-day recoveries (determined at spiking levels equivalent to 1×and 2×the limit of quantitation (0.25µg/kg)) ranged between 80.0 and 94.0%, with the corresponding relative standard deviations (RSDs) being≤8.2%. The developed experimental protocol was applied to different samples purchased from local markets in Seoul, which were tested negative for bithionol residues. In conclusion, the proposed method proved to be versatile and precise, being ideally suited for the routine detection of bithionol residues in animal-derived food products with various protein and fat contents.

Quantification of bupivacaine hydrochloride and isoflupredone acetate residues in porcine muscle, beef, milk, egg, shrimp, flatfish, and eel using a simplified extraction method coupled with liquid chromatography-triple quadrupole tandem mass spectrometry.

In this study, a simple analytical approach has been developed and validated for the determination of bupivacaine hydrochloride and isoflupredone acetate residues in porcine muscle, beef, milk, egg, shrimp, flatfish, and eel using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A 0.1% solution of acetic acid in acetonitrile combined with n-hexane was used for deproteinization and defatting of all tested matrices and the target drugs were well separated on a Waters Xbridge™ C18 analytical column using a mobile phase consisting of 0.1% acetic acid (A) and 0.1% solution of acetic acid in methanol (B). The linearity estimated from six-point matrix-matched calibrations was good, with coefficients of determination ≥0.9873. The limits of quantification (LOQs) for bupivacaine hydrochloride and isoflupredone acetate were 1 and 2ngg-1, respectively. Recovery percentages in the ranges of 72.51-112.39% (bupivacaine hydrochloride) and 72.58-114.56% (isoflupredone acetate) were obtained from three different fortification concentrations with relative standard deviations (RSDs) of <15.14%. All samples for the experimental work and method application were collected from the local markets in Seoul, Republic of Korea, and none of them tested positive for the target drugs. In conclusion, a simple method using a 0.1% solution of acetic acid in acetonitrile and n-hexane followed by LC-MS/MS could effectively extract bupivacaine hydrochloride and isoflupredone acetate from porcine muscle, beef, milk, egg, shrimp, flatfish, and eel samples.