In patients with Alzheimer’s disease, in addition to the core symptoms, i.e., cognitive dysfunction, behavioral and psychological symptoms of dementia (BPSD) such as aggression, anxiety, and hallucinations are known to occur frequently. Because various environmental factors influence the onset and progression of Alzheimer's disease, in the present study, BPSD-like behavioral abnormality of Amyloid β (Aβ)1-42-injected mice was assessed under social isolation, which induces behavioral abnormality. Aβ protein (500 pmol) was injected into the lateral ventricle of mice, which were individually housed. Two and three weeks after injection into adult mice, the rate of mice that exhibited aggressive behavior, i.e., biting attacks and wrestling, to the total mice, was markedly increased by Aβ injection. Aβ-injected adult mice also showed anxiety-like behavior, in addition to cognitive decline. Serum corticosterone level was markedly increased by Aβ injection. When excitability of hippocampal neurons was checked using hippocampal slices, KCl-induced presynaptic activity was enhanced in hippocampal slices prepared from Aβ-injected mice. These results suggest that social isolation housing of Aβ1-42-injected adult mice induces BPSD-like behavioral abnormality in addition to cognitive decline. It is likely that behavioral abnormality of Aβ1-42-injected adult mice is associated with excitability of hippocampal glutamatergic neurons, which is associated with the elevated corticosterone level.
To date, eight next‑generation urinary protein kidney safety biomarkers have been qualified to enable monitoring for subclinical drug‑induced kidney injury (DIKI) in rat preclinical studies; however, most DIKI biomarker studies have included only male rats. The objective of this study was to assess the utility of novel DIKI biomarkers, including but not limited to urinary total protein, albumin, cystatin C and osteopontin in female Sprague‑Dawley rats (8/group) that received repeated intravenous injections of amphotericin B (AmpB, 3 mg/kg/day) or vehicle for 10 consecutive days. Serial serum/urine samples were collected on study day (D)4, D8, and D11. Surviving animals were necropsied on D11. The AmpB‑induced kidney histopathology findings were characterized by cortical and medullary tubular alterations, interstitial inflammation, intratubular granular and inflammatory cell casts, acute pelvic inflammation and tubular mineralization. Significant elevations in urinary clusterin on D4, D8 and D11 (3.5 fold, 2.2 fold and 3.3 fold respectively) were observed (versus concurrent controls) following repeated injections of AmpB. In addition, significantly elevated (fold changes) in biomarkers, neutrophil gelatinase‑associated lipocalin (14.6 fold), albumin (13.5 fold), cystatin C (13.5 fold), total protein (3.5 fold), kidney injury molecule 1 (3.0 fold) and osteopontin (2.3 fold) were detected in urine as early as D4. These findings demonstrate the value of early elevations in nephron‑specific DIKI biomarkers for detecting subclinical AmpB nephrotoxicity in female Sprague‑Dawley rats. These findings are anticipated to provide the basis for inclusion of female rats on a case‑by‑case basis in preclinical toxicology studies designed to detect DIKI.
To investigate the changes of heme oxygenase-1 (HO-1) expression and production in rats with acute liver injury induced by lipopolysaccharide (LPS), and explore the role of HO-1 in the pathogenesis of liver injury. Liver injury was assessed histologically and the serum level of alanine transaminase (ALT) and aspartate transaminase (AST) were examined. The activity of super oxide dismutase (SOD) and the content of malondialdehyde (MDA) and carbon monoxide (CO) in liver tissues were also examined at the same time. HO-1 mRNA expression was examined at different time points following LPS treatment and the expression of HO-1 protein was determined by immunohistochemical staining. Administration of LPS caused severe hepatic damage, characterized by significant elevation of serum ALT and AST levels and hepatic MDA content as well as a remarkable reduction of liver SOD activity at 24 hr as compared with those in the control group. HO-1 activity was elevated significantly after modeling, showing a time-dependent manner from 6 to 24 hr, while expression of HO-1 protein was increased remarkably from 6 to 24 hr. Endogenous CO concentration in the liver of control rats remained very low but was elevated significantly after LPS treatment (6, 12, 24 hr), which was in accordance with the changes of HO-1. HO-1 activity and protein are increased significantly in rats with acute liver injury induced by LPS, suggesting that HO-1 plays an important role in the pathogenesis of acute hepatic damage.
Methylmercury (MeHg) is gradually changed to inorganic Hg after demethylation in animal tissues, and a selective quantification of inorganic Hg in the tissues is necessary to detect the reaction. We detected inorganic Hg formation in liver and kidney of mouse as early as 24 hr after MeHg injection. As an example of biological demethylation, the cytochrome P450 (P450)-mediated N-demethylation of drugs has been well documented, and formaldehyde was detected as a reaction product. Here we incubated mouse liver homogenate with added MeHg and observed a dose-dependent production of formaldehyde, as well as inorganic Hg formation. Since the amount of formaldehyde was approx. 500 times higher than that of the inorganic Hg that formed, the formaldehyde production would be stimulated by inorganic Hg formed from MeHg. We observed that inorganic Hg caused formaldehyde production, and it was enhanced by L-methionine and sarcosine. Thus, some biomolecules with S-methyl and N-methyl groups may function as methyl donors in the reaction. Using subcellular fractions of mouse liver, we observed that microsomal P450 did not participate in the demethylation of MeHg, but the greatest activity was located in the mitochondria-rich fraction. The addition of superoxide anion in the reaction mixture significantly enhanced the formaldehyde production, whereas Mn-superoxide dismutase depressed the reaction. Our present findings demonstrated that inorganic Hg formed by MeHg demethylation in mouse liver stimulated the endogenous formaldehyde production, and we observed that MeHg demethylation could be estimated by a formaldehyde analysis. Our results also suggested that superoxide anion is involved in the reaction.
This study aimed to develop a simpler method for determining total mercury (T-Hg) and methylmercury (MeHg) in biological samples by using methyl isobutyl ketone (MIBK) in the degreasing step. The fat in the samples was extracted by MIBK to the upper phase. T-Hg transferred into the water phase. This was followed by the extraction of MeHg from the water phase using HBr, CuCl2 and toluene. The MeHg fraction was reverse-extracted into L-cysteine-sodium acetate solution from toluene. The concentrations of T-Hg and MeHg were determined by heating vaporization atomic absorption spectrometry. Certified reference materials for T-Hg and MeHg in hair and fish were accurately measured using this method. This method was then applied to determine T-Hg and MeHg concentrations in the muscle, liver and gonads of seafood for the risk assessment of MeHg exposure. The mean T-Hg and MeHg concentrations in squid eggs were 0.023 and 0.022 µg/g, and in squid nidamental glands 0.052 and 0.049 µg/g, respectively. The MeHg/T-Hg ratios in the eggs and nidamental glands of squid were 94.4% and 96.5%, respectively. The mean T-Hg and MeHg concentrations in the gonads of sea urchins were 0.043 and 0.001 µg/g, respectively, with a MeHg/T-Hg ratio of 3.5%. We developed an efficient analytical method for T-Hg and MeHg using MIBK in the degreasing step. The new information on MeHg concentration and MeHg/T-Hg ratios in the egg or nidamental glands of squid and gonads of sea urchin will also be useful for risk assessment of mercury in seafood.
Although carbon nanotubes (CNTs) are used in many fields, including energy, healthcare, environmental technology, materials, and electronics, the adverse effects of CNTs in the brain are poorly understood. In this study, we investigated the effects of CNTs on cultured microglia, as microglia are the first responders to foreign materials. We compared the effects of sonicated suspensions of 5 kinds of CNTs and their flow-through filtered with a 0.22 µm membrane filter on microglial viability. We found that sonicated suspensions caused microglial cell damage, but their flow-through did not. The number of microglial aggregates was well correlated with the extent of the damage. We also determined that the CNT agglomerates consisted of two groups: one was phagocytosed by microglia and caused microglial cell damage, and the other caused cell damage without phagocytosis. These results suggest that phagocytosis-dependent and independent mechanisms underlie the microglial cell damage caused by CNT agglomerates and it is important to conduct studies about the relationships between physical properties of nanomaterial-agglomerates and cell damage.
Anticholinesterases, such as organophosphorus pesticides and warfare nerve agents, present a significant health threat. Onset of symptoms after exposure can be rapid, requiring quick-acting, efficacious therapy to mitigate the effects. The goal of the current study was to identify the safest antidote with the highest therapeutic index (TI = oxime 24-hr LD50/oxime ED50) from a panel of four oximes deemed most efficacious in a previous study. The oximes tested were pralidoxime chloride (2-PAM Cl), MMB4 DMS, HLö-7 DMS, and obidoxime Cl2. The 24-hr median lethal dose (LD50) for the four by intramuscular (IM) injection and the median effective dose (ED50) were determined. In the ED50 study, male guinea pigs clipped of hair received 2x LD50 topical challenges of undiluted Russian VX (VR), VX, or phorate oxon (PHO) and, at the onset of cholinergic signs, IM therapy of atropine (0.4 mg/kg) and varying levels of oxime. Survival was assessed at 3 hr after onset clinical signs. The 3-hr 90th percentile dose (ED90) for each oxime was compared to the guinea pig pre-hospital human-equivalent dose of 2-PAM Cl, 149 µmol/kg. The TI was calculated for each OP/oxime combination. Against VR, MMB4 DMS had a higher TI than HLö-7 DMS, whereas 2-PAM Cl and obidoxime Cl2 were ineffective. Against VX, MMB4 DMS > HLö-7 DMS > 2-PAM Cl > obidoxime Cl2. Against PHO, all performed better than 2-PAM Cl. MMB4 DMS was the most effective oxime as it was the only oxime with ED90 < 149 µmol/kg against all three topical OPs tested.
After the life-threatening cytokine release syndrome (CRS) occurred in the clinical study of the anti-CD28 monoclonal antibody (mAb) TGN1412, in vitro cytokine release assays using human blood cells have been proposed for non-clinical evaluation of the potential risk of CRS. Two basic assay formats are frequently used: human peripheral blood mononuclear cells (PBMC) with immobilized mAbs, and whole blood with aqueous mAbs. However, the suitability of the whole blood cytokine assay (WBCA) has been questioned, because an unrealistically large sample size would be required to detect the potential risk of CRS induced by TGN1412, which has low sensitivity. We performed a WBCA using peripheral blood obtained from 68 healthy volunteers to compare two high risk mAbs, the TGN1412 analogue anti-CD28 superagonistic mAb (CD28SA) and the FcγR-mediated alemutuzumab, with a low risk mAb, panitumumab. Based on the cytokine measurements in this study, the sample size required to detect a statistically significant increase in cytokines with 90% power and 5% significance was determined to be n = 9 for CD28SA and n = 5 for alemtuzumab. The most sensitive marker was IL-8. The results suggest that WBCA is a practical test design that can warn of the potential risk of FcγR-mediated alemtuzumab and T-cell activating CD28SA but, because there was apparently a lower response to CD28SA, it cannot be used as a risk-ranking tool. WBCA is suggested to be a helpful tool for identifying potential FcγR-mediated hazards, but further mechanistic understanding of the response to CD28SA is necessary before applying it to T cell-stimulating mAbs.
It has been recognized that the use of nanoparticles (NPs) in the cosmetic industry results in products with better efficacy and functionality. However, recent advances in molecular toxicology have revealed that NP exposure can promote cytotoxicity and oxidative damage, which has raised health concerns in the use of NPs in personal care products. Nevertheless, the mechanistic basis for the toxicity and safety of cosmetic NPs is poorly understood. The goal of the study was to determine the cytotoxicity and intracellular distribution of titanium dioxide (TiO2) NPs containing fatty acid composites (palmitoleic acid, palmitic acid, stearic acid and oleic acid) commonly used in cosmetic products. Two types of cells, human fibroblast skin cells and adenocarcinoma lung cells, were exposed to either bare TiO2 NPs or TiO2 NPs mixed with fatty acids for up to 48 hr. NMR analysis confirmed that the fatty acid composites remained in the NPs after wash. The cytotoxicity of TiO2 NPs was determined by cell viability measurement using quantitative confocal microscopy, and the localization of two different forms of TiO2 NPs were assessed using electron spectroscopic imaging with transmission electron microscopy. TiO2 NPs containing fatty acids posed significantly reduced cytotoxicity (80-88% decreases) than bare NPs in both cell types. Furthermore, there was less intracellular penetration of the NPs containing fatty acid composites compared with bare NPs. These results provide important insights into the role of fatty acids in protecting the cells from possible toxicity caused by NPs used in the production of cosmetic products.
The herbicide 2,4-Dichlorophenoxyacetic acid (2,4-D) is globally used in agriculture and has been linked to human sperm abnormalities in vivo. However, its effects on ejaculated human spermatozoa in vitro have not been characterized. Therefore, we examined the effects of 2,4-D on the functions of ejaculated human spermatozoa in vitro, including: sperm motility, the ability to move through a viscous medium, capacitation, and the acrosome reaction. Different doses of 2,4-D (10 nM, 100 nM, 1 µM, 10 µM, 100 µM, and 200 µM) were applied to human spermatozoa prepared from normal fresh semen samples. The results indicated that 2,4-D did not affect the viability, capacitation, or spontaneous acrosome reactions of human spermatozoa, but it dose-dependently inhibited the total motility, progressive motility, ability to penetrate viscous medium, and progesterone-induced capacitation and acrosome reaction rates. These results suggest that exposure to 2,4-D and its accumulation in the seminal plasma and follicular fluid might increase the risk of infertility. Our findings provide new insights for understanding the male reproductive toxicity of 2,4-D.
The in vitro cytochrome P450 (CYP)-inhibitory effects of 11 parabens and 7 phthalates used in consumer products, as well as their hydrolytic metabolites, were investigated, using rat liver microsomes as an enzyme source. The effects on individual CYP isozymes were evaluated by assaying inhibition of activities towards specific substrates, i.e., ethoxyresorufin O-dealkylase (EROD), methoxyresorufin O-dealkylase (MROD), pentoxyresorufin O-dealkylase (PROD), 7-benzyloxy-4-trifluoromethylcoumarin dealkylase (BFCD), 7-methoxy-4-trifluoromethylcoumarin dealkylase (MFCD) and 7-ethoxy-4-trifluoromethylcoumarin dealkylase (EFCD) activities. These activities were dose-dependently inhibited, most potently by medium-side-chain parabens (C6-9) and phthalates (C4-6), and less potently by shorter- and longer-side-chain esters. The hydrolytic product of parabens, 4-hydroxybenzoic acid, was not inhibitory, while those of phthalates, phthalic acid monoesters, showed lower inhibitory activity than the parent phthalates. Parabens showed relatively potent inhibition of MFCD activity, considered to be mainly due to CYP2C, and phthalates showed relatively potent inhibition of PROD activity, considered to be mainly due to CYP2B.
The classic toxicity of carbon tetrachloride (CCl4) is to induce liver lesion and liver fibrosis. Liver fibrosis is a consequence of chronic liver lesion, which can progress into liver cirrhosis even hepatocarcinoma. However, the toxicological mechanisms of CCl4-induced liver fibrosis remain not fully understood. We combined transcriptomic and proteomic analysis and biological network technology, predicted toxicological targets and regulatory networks of CCl4 inliver fibrosis. Wistar rats were treated with CCl4 for 9 weeks. Histopathological changes, hydroxyproline (Hyp) contents, serum ALT and AST in the CCl4-treated group were significantly higher than that of CCl4-untreated group. CCl4-treated and -untreated liver tissues were examined by microarray and iTRAQ. The results showed that 3535 genes (fold change ≥ 1.5, P < 0.05) and 1412 proteins (fold change ≥ 1.2, P < 0.05) were differentially expressed. Moreover, the integrative analysis of transcriptomics and proteomics data showed 523 overlapped proteins, enriched in 182 GO terms including oxidation reduction, response to oxidative stress, inflammatory response, extracellular matrix organization, etc. Furthermore, KEGG pathway analysis showed that 36 pathways including retinol metabolism, PPAR signaling pathway, glycolysis/gluconeogenesis, arachidonic acid metabolism, metabolism of xenobiotics by cytochrome P450 and drug metabolism. Network of protein-protein interaction (PPI) and key function with their related targets were performed and the degree of network was calculated with Cytoscape. The expression of key targets such as CYP4A3, ALDH2 and ALDH7A1 decreased after CCl4 treatment. Therefore, the toxicological mechanisms of CCl4-induced liver fibrosis may be related with multi biological process, pathway and targets which may provide potential protection reaction mechanism for CCl4 detoxication in the liver.
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