Major environmental characteristics of swine husbandry that affect exposure to dust and airborne endotoxins.

Inhalation of organic dust or endotoxin in the dust is considered a major risk factor for occupational respiratory illnesses. Eighteen environmental characteristics associated with animal husbandry were surveyed at 36 swine farms in seven provinces throughout South Korea. Association of these factors with levels of indoor inhalable or respirable dust or endotoxin in each type of dust was analyzed using backward stepwise multiple linear regression models. Mean levels of inhalable and respirable dust were 0.5 ± 0.35 and 0.13 ± 0.12 mg/m3 air, respectively, and mean endotoxin levels were 676 ± 463 and 48.4 ± 68.2 EU/m3, respectively, in each dust. Factors negatively associated with inhalable dust levels included pig age, indoor farm temperature, number of pigs in the building, hr/week of indoor farm work, and partly slatted floor. Factors positively associated with inhalable dust levels included floor cleaning by manual scraping and slurry deposit duration. Factors negatively associated with the level of endotoxin in inhalable dust included pig age, temperature, number of pigs, hr/week of indoor farm work, and partly slatted floor. Factors negatively associated with respirable dust level included area of the confinement building, whereas factors positively associated with respirable dust level included the number of pigs and stocking density. Endotoxin levels in respirable dust were negatively associated with h/week of indoor farm work and partly slatted floor. Overall, data suggest that husbandry variables may be adjusted to control dust and airborne endotoxin levels in swine farms.

Optimizing the cutoff for the identification of skin sensitizers by the HaCaSens assay: Introducing an ROC-analysis-based cutoff approach.

The worldwide restricted use of animal testing makes it challenging to identify the skin sensitizing potentials of newly manufactured products. The HaCaSens assay has shown promise as an in vitro skin sensitizing assay comparable to existing assays, and is currently under pre-validation. However, there is little agreement on how to assess the results of the assay to discriminate sensitizers from non-sensitizers as the stimulation index (SI) cutoff value was arbitrarily chosen without appropriate statistical methods. Here, we investigated the SI cutoff values in identifying sensitizers to obtain the optimal value. Sensitivities and specificities were calculated for a set of 30 test substances, and plotted in receiver operator characteristics (ROC) curves. The SI cutoff values with the highest sum of sensitivity and specificity according to LLNA data were 2.2, 1.8 and 3.0 for interleukin 1α (IL-1α), interleukin 6 (IL-6), and the combination of the two cytokines respectively. Also, the same statistical analysis of human data demonstrated optimal SI cutoff values 2.0, 2.0 and 3.2 for the same respective parameters. When considering the predictive capacity of each possible SI cutoff value determined by ROC curves, the optimal value for HaCaSens is 3.0 for the combination of IL-1α and IL-6 as it had the highest sensitivity (90.9%), specificity (75.0%) and accuracy (86.7%) based on LLNA data. Thus, we recommend the wide use of the SI cutoff value of 3.0 to ensure consistent endpoints.

Preventive Effect of Mebicar and Ginsenoside Rg1 on Neurobehavioral and Immunological Disruptions Caused by Intermittent Unpredictable Stress in Mice.

OBJECTIVES: Ginsenoside Rg1 and mebicar have been reported to have broad efficacy spectrum, including anti-anxiety and anti-stress. These drugs have been used not only for treatment but also for the purpose of increasing resistance from disease. A specific aim of this study was to investigate whether mebicar or ginsenoside Rg1 can prevent physiological changes resulting from intermittent unpredictable stress (IUS). METHODS: Seven week-old Balb/cByJ mice were administered orally with mebicar (10 mg/kg) or ginsenoside Rg1 (10 mg/kg) starting from a week before they were exposed to IUS until the end of the experiment. IUS, which consists of psychological stress and physical fatigue, was set as 3 bouts (24 h/bout) exposure in a 2-week period. RESULTS: IUS caused hyperactivity and anxiety-like behavior, which were not inhibited by mebicar or ginsenoside Rg1. IUS mice treated with mebicar or ginsenoside Rg1 recovered rapidly from anxiety-like behavior induced by the multiplexed stress compared to the mice not orally treated with mebicar or ginsenoside Rg1. Mebicar or ginsenoside Rg1 could not prevent the decrease of brain-derived neurotropic factor by IUS exposure. However, mebicar or ginsenoside Rg1 prevented elevation of serum corticosterone and secretion of proinflammatory cytokines from splenocytes due to IUS exposure. CONCLUSIONS: This study suggests that mebicar or ginsenoside Rg1 may have little preventive effect on neurobehavioral disruption by IUS exposure, but mebicar or ginsenoside Rg1 shortened the lasting duration of the anxiety caused by exposure to a novel environment. The anti-stress effect of mebicar and ginsenoside Rg1 may be restricted in peripheral stress responses.

Neuroprotective Effect and Molecular Mechanism of [6]-Gingerol against Scopolamine-Induced Amnesia in C57BL/6 Mice.

We have investigated the neuroprotective and memory enhancing effect of [6]-gingerol (GIN), a pungent ingredient of ginger, using an animal model of amnesia. To determine the neuroprotective effect of GIN on cognitive dysfunction, scopolamine (SCO, 1 mg/kg, i.p.) was injected into C57BL/6 mice, and a series of behavioral tests were conducted. SCO-induced behavior changes and memory impairments, such as decreased alteration (%) in Y-maze test, increased mean escape latency in water maze test, diminished step-through latency in passive avoidance test, and shortened freezing time in fear condition test, were significantly prevented and restored by the oral administration of GIN (10 or 25 mg/kg/day). To further verify the neuroprotective mechanism of GIN, we have focused on the brain-derived neurotrophic factor (BDNF). The administration of GIN elevated the protein expression of BDNF, which was mediated via the activation of protein kinase B/Akt- and cAMP-response element binding protein (CREB) signaling pathway. These results suggest that GIN may have preventive and/or therapeutic potentials in the management of memory deficit and cognitive impairment in mice with amnesia.

Association between endotoxin levels in dust from indoor swine housing environments and the immune responses of pigs.

Indoor animal husbandry environments are inevitably contaminated with endotoxins. Endotoxin exposure is associated with various inflammatory illnesses in animals. This cross-sectional study evaluated the relationship between the degree of endotoxin exposure and the cellular and humoral immune profiles of fattening pigs. Blood samples were taken from the jugular vein of 47 pigs from ten pig farms in Korea. Whole blood cell counts and plasma immunoglobulin (Ig) classes were determined. Peripheral-blood mononuclear cells were stimulated in vitro with concanavalin A for 48 h, and cytokines released into culture supernatants were measured. The barns in which the pigs lived were assessed for endotoxin levels in the total and respirable dust by using the limulus amebocyte lysate kinetic QCL method. Low and high endotoxin exposures were defined as ≤ 30 and ＞ 30 EU/m³, respectively. Compared to pigs with low endotoxin exposure (n = 19), highly exposed pigs (n = 28) had higher circulating neutrophil and lymphocyte (particularly B cells) counts, IgG and IgE levels, interferon-gamma (IFNγ) and interleukin (IL)-4 productions, and lower IgA levels and tumor necrosis factor-alpha (TNFα) production. The IL-4, IFNγ, and TNFα levels significantly correlated with endotoxin level and/or pig age. Constant exposure of pigs to high levels of airborne endotoxins can lead to aberrant immune profiles.

A Novel Animal Model Simulating the Beginning of Combat Exposure.

OBJECTIVE: Predator stress, social defeat stress, and fear conditioning animal models have been applied to investigate combat-related posttraumatic stress disorder (PTSD). However, no animal model psychopharmacological studies have investigated prevention of somatization of increased mental stress and fatigue at the beginning of combat exposure. This study utilized a novel animal model simulating the beginning of combat exposure that aided specification of a set of biomarkers. METHODS: Psychological stress was induced by both inescapable electric foot shock and noise stimuli. Physical fatigue was induced by sleep deprivation and forced exercise in a rotating cage. A new device reflecting simultaneous psychological stress and physical fatigue was constructed. The protocol simulating combat exposure was set as 3 rounds of 24-h exposure in a 2-week period, which was specified as intermittent unpredictable stress (IUS). RESULTS: Mice exposed to IUS (IUS mice) had significantly higher serum corticosterone levels (p < 0.05), excessive locomotive activity (p < 0.001), and anxiety-like behavior (p < 0.02) compared to control mice. IUS mice also had significantly higher IFN-γ (p < 0.001) and TNF-α (p < 0.05) levels in the supernatant of splenic T-cell culture compared to control mice. Brain-derived neurotropic factor levels were significantly decreased (p < 0.04) after IUS exposure. CONCLUSION: The proposed animal model of combat exposure reflected cognitive function impairment, behavior disturbance, and altered neuroimmune interactions without any apparent histopathological changes, and this animal model may be more applicable to protective research on war syndrome than combat-related PTSD after war because the hypothalamic-pituitary-adrenal axis has not been blunted.

Effect of fermented soybean products intake on the overall immune safety and function in mice.

Various functional activities have been reported for the fermented soybean products doenjang (DJ) and cheonggukjang (CGJ), although no systemic investigations of their immune functions have been conducted to date. We examined the effects of an experimental diet of DJ, CGJ, or a mixture of unfermented raw material for 4 weeks on overall immunity and immune safety in mice. No significant alterations were observed in peripheral or splenic immune cells among groups. Enhanced splenic natural killer cell activity was observed in the DJ and CGJ groups compared with the plain diet group. T helper type-1 (Th1)-mediated immune responses were enhanced in the DJ and CGJ groups with an upregulated production ratio of IFN-γ vs. IL-4 and IgG2a vs. IgG1 in stimulated splenic T and B cells, respectively. Resistance to Listeria monocytogenes infection was observed in the DJ and CGJ groups. Overall, the results of this study suggest that DJ and CGJ intake consolidates humoral and cellular immunity to Th1 responses.

Epizootiological characteristics of viable bacteria and fungi in indoor air from porcine, chicken, or bovine husbandry confinement buildings.

Microorganisms found in bioaerosols from animal confinement buildings not only foster the risk of spreading diseases among livestock buildings, but also pose health hazards to farm workers and nearby residents. This study identified the various microorganisms present in the air of swine, chicken, and cattle farms with different kinds of ventilation conditions in Korea. Microbial air samples were collected onto Petri dishes with bacterial or fungal growth media using a cascade impactor. Endotoxin levels in total dust were determined by the limulus amebocyte lysate kinetic QCL method. Prevalent Gram-positive bacteria were Staphylococcus (S.) lentus, S. chromogenes, Bacillus (B.) cereus, B. licheniformis, and Enterococcus faecalis, while the dominant fungi and Gram-negative bacteria were Candida albicans and Sphingomonas paucimobilis, respectively. Considering no significant relationship between the indoor dust endotoxin levels and the isolation of Gram-negative bacteria from the indoor air, monitoring the indoor airborne endotoxin level was found to be also critical for risk assessment on health for animals or workers. The present study confirms the importance of microbiological monitoring and control on animal husbandry indoor air to ensure animal and worker welfare.

Performance standard-based validation study for local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method.

Local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method (LLNA: BrdU-FCM) is a modified non-radioisotopic technique with the additional advantages of accommodating multiple endpoints with the introduction of FCM, and refinement and reduction of animal use by using a sophisticated prescreening scheme. Reliability and accuracy of the LLNA: BrdU-FCM was determined according to OECD Test Guideline (TG) No. 429 (Skin Sensitization: Local Lymph Node Assay) performance standards (PS), with the participation of four laboratories. Transferability was demonstrated through successfully producing stimulation index (SI) values for 25% hexyl cinnamic aldehyde (HCA) consistently greater than 3, a predetermined threshold, by all participating laboratories. Within- and between-laboratory reproducibility was shown using HCA and 2,4-dinitrochlorobenzene, in which EC2.7 values (the estimated concentrations eliciting an SI of 2.7, the threshold for LLNA: BrdU-FCM) fell consistently within the acceptance ranges, 0.025-0.1% and 5-20%, respectively. Predictive capacity was tested using the final protocol version 1.3 for the 18 reference chemicals listed in OECD TG 429, of which results showed 84.6% sensitivity, 100% specificity, and 88.9% accuracy compared with the original LLNA. The data presented are considered to meet the performance criteria for the PS, and its predictive capacity was also sufficiently validated.

Altered expression levels of neurodevelopmental proteins in fetal brains of BTBR T+tf/J mice with autism-like behavioral characteristics.

Autism is a brain developmental disorder with characteristics of social interaction defects, language and communication dysfunction, and repetitive behavior. Occurrence of autism is continuously increasing, but the cause of autism is not clearly defined. Genetic linkage or environmental factors were proposed as sources for pathogenesis of autism. BTBR T+tf/J (BTBR) mice were reported as an appropriate animal model for autism investigation because of their similarities in behavioral abnormalities with human autistic subjects. The aim of this study was to evaluate expression levels of proteins involved with brain development at fetal stage of BTBR mice. FVB/NJ mice were used as a control strain because of their social behaviors. Level of fetal brain immunoglobulin (Ig) G deposit was also evaluated. Fetal brains were obtained at d 18 of gestational period. Thirty-one and 27 fetuses were obtained from 3 pregnant BTBR and FVB dams, respectively. The level of glial fibrillary acidic protein expression was significantly lower in fetal brains of BTBR than FVB/NJ mice. Expression of brain-derived neurotrophic factor and myelin basic protein was significantly more upregulated in BTBR than in FVB/NJ mice. No significant difference was obtained for nerve growth factor between the two strains. Levels of IgG isotypes deposited in fetal brain of BTBR mice were significantly higher than in FVB mice except for IgG1. Overall, these results suggest that prenatal alterations in expression of various fetal brain proteins may be implicated in aberrant behavioral characteristics of BTBR mice.

Protective Effect of Arabinoxylan against Scopolamine-Induced Learning and Memory Impairment.

The purpose of this study is to investigate the memory enhancing effect and underlying molecular mechanism of arabinoxylan (AX), a major component of dietary fiber in wheat against scopolamine (SCO)-induced amnesia in Sprague-Dawley (SD) rats. Diverse behavior tests including Y-maze, Morris water maze, and passive avoidance tests were performed to measure cognitive functions. SCO significantly decreased the spontaneous alterations in Y-maze test and step-through latency in passive avoidance test, whereas increased time spent to find the hidden platform in Morris water maze test compared with the sham control group. In contrast, oral administration of AX (25 mg/kg and 50 mg/kg) effectively reversed the SCO-induced cognitive impairments in SD rats. Furthermore, AX treatment up-regulated the expression of brain-derived neurotrophic factor (BDNF) in the cortex and hippo-campus via promoting activation of cAMP response element binding protein (CREB). Therefore, our findings suggest that AX can improve SCO-induced learning and memory impairment possibly through activation of CREB and up-regulation of BDNF levels, thereby exhibiting a cognition-enhancing potential.

Decaffeinated coffee prevents scopolamine-induced memory impairment in rats.

INTRODUCTION: Several human studies have reported that coffee consumption improves cognitive performance. In the present study, we investigated whether instant decaffeinated coffee also ameliorates cognitive performance and attenuates the detrimental effects of scopolamine on memory. METHODS: Memory performance was evaluated in Morris water maze test and passive avoidance test. Instant decaffeinated coffee (p.o.) at 120 or 240 mg/kg in Sprague-Dawley rats, which is equivalent to approximately three or six cups of coffee, respectively, in a 60 kg human, was administered for two weeks. RESULTS: Oral gavage administration of instant decaffeinated coffee inhibited scopolamine-induced memory impairment, which was measured by Morris water maze test and passive avoidance test. Instant decaffeinated coffee suppressed scopolamine-mediated elevation of tumor necrosis factor-α (TNF-α) and stimulation of nuclear factor-κB (NF-κB) pathway (i.e., phosphorylation of IκBα and p65) in the rat hippocampus. DISCUSSION: These findings suggest that caffeine-free decaffeinated coffee may prevent memory impairment in human through the inhibition of NF-κB activation and subsequent TNF-α production.

[6]-Gingerol attenuates ß-amyloid-induced oxidative cell death via fortifying cellular antioxidant defense system.

ß-Amyloid (Aß) is involved in the formation of senile plaques, the typical neuropathological marker for Alzheimer's disease (AD) and has been reported to cause apoptosis in neurons via oxidative and/or nitrosative stress. In this study, we have investigated the neuroprotective effect and molecular mechanism of [6]-gingerol, a pungent ingredient of ginger against Αß(25-35)-induced oxidative and/or nitrosative cell death in SH-SY5Y cells. [6]-Gingerol pretreatment protected against Aß(25-35)-induced cytotoxicity and apoptotic cell death such as DNA fragmentation, disruption of mitochondrial membrane potential, elevated Bax/Bcl-2 ratio, and activation of caspase-3. To elucidate the neuroprotective mechanism of [6]-gingerol, we have examined Aß(25-35)-induced oxidative and/or nitrosative stress and cellular antioxidant defense system against them. [6]-Gingerol effectively suppressed Aß(25-35)-induced intracellular accumulation of reactive oxygen and/or nitrogen species and restored Aß(25-35)-depleted endogenous antioxidant glutathione levels. Furthermore, [6]-gingerol treatment up-regulated the mRNA and protein expression of antioxidant enzymes such as γ-glutamylcysteine ligase (GCL) and heme oxygenase-1 (HO-1), the rate limiting enzymes in the glutathione biosynthesis and the degradation of heme, respectively. The expression of aforementioned antioxidant enzymes seemed to be mediated by activation of NF-E2-related factor 2 (Nrf2). These results suggest that [6]-gingerol exhibits preventive and/or therapeutic potential for the management of AD via augmentation of antioxidant capacity.

Neuroprotective effects of Triticum aestivum L. against beta-amyloid-induced cell death and memory impairments.

beta-Amyloid (A beta) is a key component of senile plaques, neuropathological hallmarks of Alzheimer's disease (AD) and has been reported to induce cell death via oxidative stress. This study investigated the protective effects of Triticum aestivum L. (TAL) on A beta-induced apoptosis in SH-SY5Y cells and cognitive dysfunctions in Sprague-Dawley (SD) rats. Cells treated with A beta exhibited decreased viability and apoptotic features, such as DNA fragmentation, alterations in mitochondria and an increased Bax/Bcl-2 ratio, which were attenuated by TAL extract (TALE) pretreatment. To elucidate the neuroprotective mechanisms of TALE, the study examined A beta-induced oxidative stress and cellular defense. TALE pretreatment suppressed A beta-increased intracellular accumulation of reactive oxygen species (ROS) via up-regulation of glutathione, an essential endogenous antioxidant. To further verify the effect of TALE on memory impairments, A beta or scopolamine was injected in SD rats and a water maze task conducted as a spatial memory test. A beta or scopolamine treatment increased the time taken to find the platform during training trials, which was decreased by TALE pretreatment. Furthermore, one of the active components of TALE, total dietary fiber also effectively inhibited A beta-induced cytotoxicity and scopolamine-caused memory deficits. These results suggest that TALE may have preventive and/or therapeutic potential in the management of AD.

Neuroprotective effect of epigallocatechin-3-gallate against beta-amyloid-induced oxidative and nitrosative cell death via augmentation of antioxidant defense capacity.

beta-Amyloid (Abeta) peptide, a major component of senile plaques has been regarded to play a crucial role in the development and neuropathogenesis of Alzheimer's disease (AD). Increasing data from in vitro and in vivo studies indicate that Abeta-induced damages in neurons and glia are mediated via nitrosative as well as oxidative stress. Therefore, recent researches have been focused on searching for dietary and herbal manipulations to protect against the Abeta-induced oxidative and/or nitrosative cell death. Epigallocatechin-3-gallate (EGCG), one of these candidates is a major polyphenolic compound present in green tea and has been reported to exhibit potent antioxidant and anti-inflammatory properties. In the present study, we have investigated the effect of EGCG against Abeta-induced oxidative and/or nitrosative cell death in BV2 microglia. Abeta treatment led to apoptosis in BV2 cells as revealed by DNA fragmentation, perturbation of mitochondrial transmembrane potential, and alterations in the expression of apoptosis-regulator Bcl-2 family proteins. EGCG pretreatment effectively ameliorated Abeta-induced cytotoxicity and manifestation of proapoptotic signals. Furthermore, BV2 cells exposed to Abeta underwent nitrosative stress as shown by the increased expression of inducible nitric oxide synthase (iNOS) and subsequent production of nitric oxide (NO) and peroxynitrite, which were effectively suppressed by EGCG pretreatment. To elucidate a molecular mechanism underlying the neuroprotective effect of EGCG, we have examined the cellular metabolism of reduced glutathione (GSH) with antioxidant properties. EGCG treatment fortified cellular GSH pool through elevated mRNA expression of gamma-glutamylcysteine ligase (GCL), the rate limiting enzyme in the glutathione biosynthesis. These results suggest that EGCG may have preventive and/or therapeutic potential in AD patients by augmenting cellular antioxidant defense capacity and attenuating Abeta-mediated oxidative and/or nitrosative cell death.