Natural antioxidant formula ameliorates lipopolysaccharide-induced impairment of hippocampal neurogenesis and contextual fear memory through suppression of neuroinflammation in rats.

This study investigated the ameliorating effects of a natural antioxidant formula (NAF) consisting of Ginkgo biloba leaf extract, docosahexaenoic acid/eicosapentaenoic acid, ferulic acid, flaxseed oil, vitamin E, and vitamin B12 on a lipopolysaccharide (LPS)-induced cognitive dysfunction model in rats. Six-week-old rats received a diet containing 0.5% (w/w) NAF for 38 days from Day 1, and LPS (1 mg/kg body weight) was administered intraperitoneally once daily on Days 8 and 10. On Day 11, LPS alone increased interleukin-1ß and tumor necrosis factor-α in the hippocampus and cerebral cortex and the numbers of M1-type microglia/macrophages and GFAP+ reactive astrocytes in the hilus of the hippocampal dentate gyrus. NAF treatment decreased brain proinflammatory cytokine levels and increased the number of M2-type microglia/macrophages. During Days 34-38, LPS alone impaired fear memory acquisition and the extinction learning process, and NAF facilitated fear extinction learning. On Day 38, LPS alone decreased the number of type-3 neural progenitor cells in the hippocampal neurogenic niche, and NAF restored the number of type-3 neural progenitor cells and increased the numbers of both immature granule cells in the neurogenic niche and reelin+ hilar interneurons. Thus, NAF exhibited anti-inflammatory effects and ameliorated LPS-induced adverse effects on hippocampal neurogenesis and fear memory learning, possibly through amplification of reelin signaling by hilar interneurons. These results suggest that neuroinflammation is a key factor in the development of LPS-induced impairment of fear memory learning, and supplementation with NAF in the present study helped to prevent hippocampal neurogenesis and disruptive neurobehaviors caused by neuroinflammation.

Effects of Bacillus subtilis on Production Performance, Bone Physiological Property, and Hematology Indexes in Laying Hens.

This study was to investigate the effects of Bacillus subtilis on production performance and bone pathophysiological characteristics of layers. Twenty-four 48-week-old Lohmann Pink-shell laying hens were randomly divided into two groups: a basic diet (control) and the basic diet mixed with Bacillus subtilis (0.5 g/kg) for a 60-day trial. Statistically, independent-sample t-test was used to assess the treatment differences. The results showed that Bacillus subtilis supplementation improved the percent of marketable eggs (p < 0.05) with reduced numbers of broken and soft-shelled eggs but had no effects on egg weight, height of albumen, yolk color, and Haugh unit (p > 0.05). Bacillus subtilis supplement also elevated maximum load (p = 0.06), maximum stress (p = 0.01), stiffness (p < 0.01), and Young's modulus (p < 0.01) but suppressed maximum strain (p = 0.06) in the femur. In addition, compared with control birds, phosphorous concentration (p < 0.01) was reduced in serum at day 61 but increased in the femur (p < 0.05) in Bacillus subtilis fed birds. Bacillus subtilis fed birds also had lower magnesium concentrations in both femur (p = 0.04) and feces (p = 0.09). Furthermore, Bacillus subtilis increased plasma estrogen concentration (p = 0.01) and femur TNF receptor superfamily member 11b (OPG) expression (p < 0.05) but reduced plasma IL-1 (p < 0.01) and TNF-α (p < 0.01) concentrations. These results indicate that Bacillus subtilis could be used as a health promotor to reduce overproduction-induced inflammation and associated bone damage and to increase marketable egg production. The data provide evidence for developing a management strategy to use Bacillus subtilis as a feed additive to improve marketable egg production and health and welfare status of laying hens.

Determination of lucidin-specific DNA adducts by liquid chromatography with tandem mass spectrometry in the livers and kidneys of rats given lucidin-3-O-primeveroside.

Lucidin-3-O-primeveroside (LuP) is a component of madder color (MC), a compound which is carcinogenic in the kidney and liver of rats. Since LuP is metabolized to generate genotoxic compounds such as lucidin (Luc) and rubiadin, it is likely that these play key roles in MC carcinogenesis. In fact, after incubation of Luc with calf thymus DNA, Luc-N(2)-dG and N(6)-dA adducts were reportedly formed, possibly via the sulfotransferase metabolic pathway. However, the precise extent of formation in vivo remains uncertain. In the present study, to quantitatively determine Luc-specific DNA adducts in in vivo samples, we developed an online sample purification method using column-switching and an isotope dilution LC-ESI-MS/MS technique. The limits of quantification were 0.2 and 0.04 fmol on column for Luc-N(2)-dG and N(6)-dA adducts, respectively. Using the new analytical method, we attempted to measure adduct levels in the kidneys and livers of rats treated with 0.06, 0.3, and 1.5% LuP in the diet for one week. Luc-N(2)-dG and N(6)-dA adducts in these organs were detected at ranges from 7.97 to 51.67/10(9) dG and from1.83 to 37.10/10(9) dA, respectively. Dose-dependent increases of each adduct were observed in both organs. These quantitative data obtained with our newly developed analytical method might help to improve our understanding of MC carcinogenesis.

Antioxidant enzymatically modified isoquercitrin or melatonin supplementation reduces oxidative stress-mediated hepatocellular tumor promotion of oxfendazole in rats.

To clarify whether enzymatically modified isoquercitrin (EMIQ) or melatonin (MLT) supplementation reduces oxidative stress-mediated hepatocellular tumor-promoting effect of oxfendazole (OX), a benzimidazole anthelmintic, male rats were administered a single intraperitoneal injection of N-diethylnitrosamine (DEN) and were fed a diet containing OX (500 ppm) for 10 weeks with or without EMIQ (2,000 ppm) or MLT (100 ppm) in the drinking water after DEN initiation. One week after the commencement of the administration of OX, rats were subjected to two-thirds of partial hepatectomy. The number of GST-P-positive foci promoted by OX was significantly inhibited by the combined antioxidant EMIQ or MLT administration, and the area of GST-P-positive foci was inhibited by the administration of MLT. Real-time RT-PCR analysis revealed decreases in mRNA expression levels of cytochrome P450, family 2, subfamily b, polypeptide 2 (Cyp2b2) and malic enzyme 1 (Me1) in the DEN-OX-EMIQ and DEN-OX-MLT groups and decreases in mRNA expression levels of Cyp1a1 and aldo-keto reductase family 7, member A3 (Akr7a3) in the DEN-OX-MLT group compared to those in the DEN-OX group. In in vitro ROS production assay, inhibited production of NADPH-dependent ROS was observed by the treatment with EMIQ or MLT. These results suggest that coadministration of EMIQ or MLT suppresses the hepatocellular tumor-promoting activity of OX in rats through the decrease in ROS production by the activation of CYPs.

Suppressive effect of Siraitia grosvenorii extract on dicyclanil-promoted hepatocellular proliferative lesions in male mice.

Dicyclanil (DC) generates reactive oxygen species (ROS) due to Cyp1a1 induction, and DNA damage caused by oxidative stress is probably involved in hepatocarcinogenesis in mice. To clarify the modifying effect of the Siraitia grosvenorii extract (SGE), which has antioxidative properties, we employed a 2-stage liver carcinogenesis model in partially hepatectomized male ICR mice. Mice maintained on diet containing DC at a concentration of 1,500 ppm for 9 weeks after a single intraperitoneal injection of diethylnitrosamine (DEN) at a dose of 30 mg/kg and they were given water containing 2,500 ppm of SGE for 11 weeks including 2 weeks as pre-administration on DC. SGE inhibited the induction of gamma-glutamyltranspeptidase-positive hepatocytes, lipid peroxidation, and gene expression of Cyp1a1, all of which were caused by DC. To examine whether SGE indirectly inhibits Cyp1a1 expression induced by inhibition of aryl hydrocarbon receptor (Ahr)-mediated signal transduction caused by DC, mice with high (C57BL/6J mice) and low affinities (DBA/2J mice) to Ahr were given DC-containing diet and/or SGE-containing tap water for 2 weeks. Cyp1a1 gene expression was significantly lower in C57BL/6J mice administered DC + SGE than in C57BL/6J mice administered DC alone; there was no difference in the Cyp1a1 expression between DBA/2J mice administered DC + SGE and DC alone. These results suggest that SGE suppresses the induction of Cyp1a1, leading to inhibition of ROS generation and consequently inhibited hepatocarcinogenesis, probably due to suppression of Ahr activity.

Thirteen-week repeated dose toxicity of Siraitia grosvenori extract in Wistar Hannover (GALAS) rats.

Siraitia grosvenori extract has been used as a food additive. As a part of the safety assessment of the extracts, a 13-week repeated dose toxicity study was performed in Wistar Hannover (GALAS) rats. Male and female rats were divided into five groups consisting of eight animals each and given diet containing 0%, 0.04%, 0.2%, 1%, and 5% of S. grosvenori extract for 13 weeks. During the experiment, no deaths were observed in any groups, and there were no remarkable changes in general appearance, body weight, food and water consumption, hematological and serum biochemical parameters, organ weight and histopathological findings between the control and treated groups. On the basis of these data, the no-observed-adverse effect level (NOAEL) of S. grosvenori extract in Wistar Hannover rats was considered to be 5% (2520 mg/kg/day in males and 3200 mg/kg/day in females) or more.

Molecular pathological analysis for determining the possible mechanism of piperonyl butoxide-induced hepatocarcinogenesis in mice.

Piperonyl butoxide (PBO), alpha-[2-(2-butoxyethoxy)ethoxy]-4,5-methylene-dioxy-2-propyltoluene, is widely used as a synergist for pyrethrins. In order to clarify the possible mechanism of non-genotoxic hepatocarcinogenesis induced by PBO, molecular pathological analyses consisting of low-density microarray analysis and real-time reverse transcriptase (RT)-PCR were performed in male ICR mice fed a basal powdered diet containing 6000 or 0 ppm PBO for 1, 4, or 8 weeks. The animals were sacrificed at weeks 1, 4, and 8, and the livers were histopathologically examined and analyzed for gene expression using the microarray at weeks 1 and 4 followed by real-time RT-PCR at each time point. Reactive oxygen species (ROS) products were also measured using liver microsomes. At each time point, the hepatocytes of PBO-treated mice showed centrilobular hypertrophy and increased lipofuscin deposition in Schmorl staining. The ROS products were significantly increased in the liver microsomes of PBO-treated mice. In the microarray analysis, the expression of oxidative and metabolic stress-related genes--cytochrome P450 (Cyp) 1A1, Cyp2A5 (week 1 only), Cyp2B9, Cyp2B10, and NADPH-cytochrome P450 oxidoreductase (Por) was over-expressed in mice given PBO at weeks 1 and 4. Fluctuations of these genes were confirmed by real-time RT-PCR in PBO-treated mice at each time point. In additional real-time RT-PCR, the expression of Cyclin D1 gene, key regulator of cell-cycle progression, and Xrcc5 gene, DNA damage repair-related gene, was significantly increased at each time point and at week 8, respectively. These results suggest the possibility that PBO has the potential to generate ROS via the metabolic pathway and to induce oxidative stress, including oxidative DNA damage, resulting in the induction of hepatocellular tumors in mice.