Six-well plate-based colony-forming efficacy assay and Co-Culture application to assess toxicity of metal oxide nanoparticles.

The development of a universal, label-free, and reliable in vitro toxicity testing method for nanoparticles is urgent because most nanoparticles can interfere with toxicity assays. In this regard, the colony-forming efficacy (CFE) assay has been suggested as a suitable in vitro toxicity assay for testing nanoparticles without such interference. Recently, the Organisation for Economic Co-operation and Development (OECD) developed a 60 × 15 mm Petri dish-based CFE assay for testing nanoparticles in MDCK-1 cells. However, further investigations are needed, including testing with other cell types, at a smaller scale for greater efficiency, and the application of the co-culture technique. In this study, we selected TiO2, CuO, CeO2, and SiO2 as test nanoparticles and successfully developed a 6-well plate-based CFE assay using HepG2 and A549 cells and a co-culture assay for combinations of HepG2 cells and THP-1 macrophages or A549 cells and THP-1 monocytes. The results suggest that the 6-wellplate-based CFE assay for HepG2 and A549 cells can be applied to nanoparticles, but the co-culture CFE assay has limitations in that it is not different from the single culture study, and it inhibits colony-formation by A549 cells in the presence of macrophages; this warrant further study.

Effects of tianeptine on symptoms of fibromyalgia via BDNF signaling in a fibromyalgia animal model.

Previous reports have suggested that physical and psychological stresses may trigger fibromyalgia (FM). Stress is an important risk factor in the development of depression and memory impairments. Antidepressants have been used to prevent stress-induced abnormal pain sensation. Among various antidepressants, tianeptine has been reported to be able to prevent neurodegeneration due to chronic stress and reverse decreases in hippocampal volume. To assess the possible effect of tianeptine on FM symptoms, we constructed a FM animal model induced by restraint stress with intermittent cold stress. All mice underwent nociceptive assays using electronic von Frey anesthesiometer and Hargreaves equipment. To assess the relationship between tianeptine and expression levels of brain-derived neurotrophic factor (BDNF), cAMP response element-binding protein (CREB), and phosphorylated cAMP response element-binding protein (p-CREB), western blotting and immunohistochemistry analyses were performed. In behavioral analysis, nociception tests showed that pain threshold was significantly decreased in the FM group compared to that in the control group. Western blot and immunohistochemical analyses of medial prefrontal cortex (mPFC) and hippocampus showed downregulation of BDNF and p-CREB proteins in the FM group compared to the control group. However, tianeptine recovered these changes in behavioral tests and protein level. Therefore, this FM animal model might be useful for investigating mechanisms linking BDNF-CREB pathway and pain. Our results suggest that tianeptine might potentially have therapeutic efficacy for FM.

In Vivo Evaluation of the Oral Toxicity of the Chlorobutanol.

Chlorobutanol (CB) is used as a preservative in cosmetics and has antibacterial activity. This study investigated the single- and repeated-dose 28-day oral toxicity of a CB solvent in Sprague Dawley (SD) rats. For the single-dose oral toxicity study, a dose of 62.5, 125, or 250 mg per kg of body weight (mg/kg b.w.) of CB was given once orally via gavage. For the repeated-dose 28-day toxicity study, the high dose was set as 100 mg/kg b.w./day, and the middle, middle-low, and low doses were set to 50, 25, and 12.5 mg/kg b.w./day, respectively. Body weight was not significantly changed in the repeated-dose toxicity study. Relative liver and kidney weights were significantly increased in both sexes of the 100 mg/kg b.w./day treatment group. However, there were histopathological changes in liver and kidney for females and males, respectively. These data suggested that the approximate lethal dose (ALD) of CB was over 250 mg/kg b.w./day in the single-dose study, and the no adverse effect level (NOAEL) for CB was over 50 and 12.5 mg/kg b.w./day for female and male rats in the repeated-dose toxicity study.

Colony-Forming Efficiency Assay to Assess Nanotoxicity of Graphene Nanomaterials.

The nano-market has grown rapidly over the past decades and a wide variety of products are now being manufactured, including those for biomedical applications. Despite the widespread use of nanomaterials in various industries, safety and health effects on humans are still controversial, and testing methods for nanotoxicity have not yet been clearly established. Nanomaterials have been reported to interfere with conventional cytotoxicity tests due to their unique properties, such as light absorption or light scattering. In this regard, the colony-forming efficacy (CFE) assay has been suggested as a suitable test method for testing some nanomaterials without these color-interferences. In this study, we selected two types of GNPs (Graphene nanoplatelets) as test nanomaterials and evaluated CFE assay to assess the cytotoxicity of GNPs. Moreover, for further investigation, including expansion into other cell types, GNPs were evaluated by the conventional cytotoxicity tests including the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), Cell Counting Kit-8 (CCK-8), and Neutral red uptake (NRU) assay using MDCK, A549 and HepG2 cells. The results of CFE assay suggest that this test method for three cell lines can be applied for GNPs. In addition, the CFE assay was able to evaluate cytotoxicity regardless more accurately of color interference caused by residual nanomaterials.

Single- and repeated-dose 28-day oral toxicity study of MDM hydantoin in Sprague-Dawley rats.

1-(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione (MDM hydantoin) is a commonly used antiseptic preservative in cosmetics. However, limited toxicity information data are available for this chemical. The aim of this study was to obtain toxicity data for MDM hydantoin through single- and repeated-dose toxicity studies in Sprague-Dawley (SD) rats. In the single-dose toxicity study, MDM hydantoin was administered once orally to SD rats at four doses (5, 50, 300, and 2000 mg/kg/day). There was no significant difference in mortality, clinical signs, and body weight change for 14 days among the animals treated with the different doses in this study. Hence, the approximate lethal dose of MDM hydantoin was considered higher than 2000 mg/kg/day. Based on the results of the dose-range finding study, a 28-day repeated-dose oral toxicity study was conducted. MDM hydantoin was administered orally to SD rats at doses of 125, 250, 500, and 1000 mg/kg/day throughout an experimental period of 28 days. In the repeated-dose oral toxicity study, the adverse effects caused by MDM hydantoin were not detected in terms of body weight, clinical signs, food and water intake, hematology, organ weights, gross pathology, and histopathology. Therefore, the no-observed-adverse-effect level of MDM hydantoin was considered to be greater than 1000 mg/kg/day.

Toxicological Assessment of Bromochlorophene: Single and Repeated-Dose 28-Day Oral Toxicity, Genotoxicity, and Dermal Application in Sprague-Dawley Rats.

Bromochlorophene (BCP) has shown good properties in sterilization and antibacterial activity and is widely used as a household chemical. We evaluated the genotoxicity, single and repeated-dose 28-day oral toxicity, and dermal application of a BCP suspension in Sprague-Dawley (SD) rats. For the single-dose toxicity study, a dose of 25-1,000 mg per kg of bodyweight (mg/kg b.w.) of BCP was given once orally to SD rats. Mortality and clinical signs were observed and recorded for the first 30 min after treatment, at 4 h post-administration, and then at least once daily for 14 days after administration. For the repeated-dose 28-day toxicity study, the high dose was set at 1,000 mg/kg b.w. and the middle, middle-low, and low dose were set to 500, 250, and 125 mg/kg, respectively. Hematology and biochemistry parameters were examined. Gross pathologic and histopathologic examinations were performed on selected tissues from all animals. A bacterial reverse mutation assay, in vitro chromosomal aberration assay, and in vivo micronucleus assay were performed to assess genotoxicity-dermal application exposure assessment of BCP in rats. A high oral approximate lethal dose (ALD) of 1,000 mg/kg was observed in the single-dose toxicity test. During the repeated-dose 28-day time period, most animal deaths after administration occurred during the first 3 weeks. The 1,000 mg/kg b.w. oral dose caused the death of six male rats (6/7) and four female rats (4/7). At 500 mg/kg b.w., the female rats showed mortality (1/7). For the biochemistry assays, cholesterol was increased significantly compared to vehicle in both sexes in the 250 and 500 mg/kg groups. Histopathological changes with treatment-related findings were observed in the pancreas in female rats treated with a high dose of BCP compared with the vehicle group. BCP showed no genotoxic effect. These data suggested that the ALD of BCP, estimated as a non-genotoxic substance, was over 1,000 mg/kg b.w. in the single-dose toxicity study, and the NOAEL of BCP was considered to be 250 mg/kg b.w. for male and female rats after repeated oral administration for 28 days under the present study conditions.

Effects of Panax ginseng C.A. Meyer extract on the offspring of adult mice with maternal immune activation.

To understand maternal immune activation (MIA) during prenatal development, the synthetic double­stranded RNA polyriboinosinic­polyribocytidylic acid [poly(I:C)] has been widely used in animal models to induce behavioral deficits similar to those in schizophrenia and other psychotic disorders. Panax ginseng C.A. Meyer (PG) extract is widely used to treat various kinds of nervous system disorders in Asia particularly China and Korea. The present study aimed to examine the effects of PG extract on MIA offspring using behavioral activity tests and protein expression analyses. Pregnant mice were exposed to poly(I:C) (5 mg/kg) or vehicle treatment on gestation day 9, and the resulting MIA offspring were subjected to vehicle or PG (300 mg/kg) treatment. In the acoustic startle response test, MIA­induced sensorimotor gating deficit was ameliorated by PG. The majority of behavioral parameters measured in the social interaction (non­aggressive or/and aggressive pattern), open field (number/duration of behavior) and forced swimming test (immobility behavior) were significantly altered in the MIA offspring. Western blot and immunohistochemical analyses of the medial prefrontal cortex indicated that the expression levels of certain neurodevelopmental proteins, including dihydropyrimidinase­related 2, LIM and SH3 domain 1, neurofilament medium, and discs large homolog 4, were decreased in the untreated MIA offspring, whereas PG treatment improved behavioral impairments and increased neurodevelopmental protein expression in MIA offspring. These results suggested that PG may be useful in neurodevelopmental disorder therapy, including psychiatric disorders such as schizophrenia, owing to its antipsychotic effects.

Antinociceptive effect of Valeriana fauriei regulates BDNF signaling in an animal model of fibromyalgia.

The genus Valeriana has been widely used in popular medicine for centuries, to treat sleep disorders, anxiety, epilepsy and insomnia. Recent studies have focused on the novel pharmacological effects of Valeriana fauriei Briq. (VF) species. Previous studies have attempted to determine the pharmacological functions of Valeriana in various human diseases, particularly with regards to its neuroprotective effects, and its ability to reduce pain and stress. The present study constructed an animal model of fibromyalgia (FM), which was induced by intermittent cold stress with slight modification. Subsequently, the study aimed to determine whether VF exerts antinociceptive effects on the FM­like model following oral administration of VF extracts. The effects of VF extracts on the FM model were investigated by analyzing behavioral activity, including pain, and detecting protein expression. In the behavioral analysis, the results of a nociception assay indicated that the pain threshold was significantly decreased in the FM group. Subsequently, western blotting and immunohistochemical analyses of the hippocampus demonstrated that the protein expression levels of brain­derived neurotrophic factor (BDNF) and phosphorylated­cAMP response element­binding protein were downregulated in the FM group. Conversely, VF restored these levels. These results suggested that the effects of VF extract on a model of FM may be associated with its modulatory effects on the BDNF signaling pathway in the hippocampus and medial prefrontal cortex. In conclusion, the mechanism underlying the protective effects of VF as a therapeutic agent against FM may involve the BDNF signaling pathway.

Association between endothelin­1 and fibromyalgia syndrome.

Fibromyalgia syndrome (FMS) is characterized by widespread chronic musculoskeletal pain, stiffness and pressure hyperalgesia at soft tissue tender points. Patients with FMS may exhibit a tendency towards cold extremities and cold­induced vasospasm. Endothelin­1 (EDN1) is a potent vasoconstrictor that is mainly produced by endothelial cells. The present study aimed to determine whether plasma expression levels avvnd single­nucleotide polymorphism (SNP; rs1800541) of the EDN1 gene were associated with FMS and/or any of its clinical variables. Plasma EDN1 levels were assessed by ELISA, and SNP genotypes were determined using polymerase chain reaction­high­resolution melting curve analysis. Patients with the TG genotype and the G allele may have an elevated risk of FMS. In addition, patients with FMS with the TG genotype and/or T allele exhibited higher plasma EDN1 levels compared with healthy controls. EDN1 levels increased significantly in patients with FMS compared with normal controls. In addition, EDN1 SNP was found to be associated with susceptibility to FMS.

Effect of Valeriana fauriei extract on the offspring of adult rats exposed to prenatal stress.

Exposing a pregnant female to stress is a risk factor for the development of psychiatric disorders in the offspring. In the present study, we examined the effects of an extract of Valeriana fauriei (VF) root (100 mg/kg/day, administered on postnatal days 35-56) on behavioral patterns as well as protein expression in the prefrontal cortex of the offspring of prenatally-stressed rats. Modified behavioral tests, including the forced swim test, the open field test, a social interaction test and the prepulse inhibition test were performed and many of the parameters were found to decrease in the offspring of the rats exposed to PNS compared with the offspring of the non-stressed rats. Western blot and immunohistochemical analyses of the prefrontal cortex revealed that the downregulation of several neurodevelopmental proteins in the offspring of rats dams exposed to PNS was reversed after treatment with VF extract. These findings demonstrate that the downregulation of several proteins in the prefrontal cortex of the offspring of prenatally­stressed rats may be associated with subsequent behavioral changes, and that these phenomena recovered following VF treatment. Our results suggest that VF decreases the incidence of prenatal stress related-psychiatric disorders, such as depression and schizophrenia.

15-hydroxyprostaglandin dehydrogenase is upregulated by hydroxychloroquine in rheumatoid arthritis fibroblast-like synoviocytes.

15-Hydroxyprostaglandin dehydrogenase (HPGD) is the key enzyme responsible for the metabolic inactivation of prostaglandin E2 (PGE2) catabolism. PGE2 is one of the predominant catabolic factors involved in rheumatoid arthritis (RA). However, the expression and regulation of HPGD in RA fibroblast­like synoviocyte (FLS) remain to be elucidated. Disease­modifying anti­rheumatic drugs (DMARDs) are the most important anti­arthritic drugs, which reduce the effect of joint injury. The aim of the present study was to assess the expression of HPGD in RA tissues and cells, and normal synovial tissues and cells. The effect of the most popular DMARDs, hydroxychloroquine, on the expression of HPGD in RA­FLS was also investigated. Western blotting and immunohistochemical analysis demonstrated that the expression levels of HPGD in human synovium were lower in RA synovium compared with the normal and OA synovium. In RA­FLS, the expression of HPGD was increased following treatment with several DMARDs, including sulfasalazine, methotrexate, and hydroxychloroquine. Hydroxychloroquine (10 µM) treatment induced the phosphorylation of ERK, SAPK/JNK and p38. Hydroxychloroquine induced a decrease in the release of PGE2, which was restored by mitogen­activated protein (MAP) kinase pathway inhibitors. Hydroxychloroquine may therefore, affect the pathogenesis of RA through the MAP kinase pathway by regulating the expression of HPGD.

Influence of Panax ginseng on the offspring of adult rats exposed to prenatal stress.

The exposure of pregnant females to stress during a critical period of fetal brain development is an environmental risk factor for the development of schizophrenia in adult offspring. Schizophrenia is a group of common mental disorders of unclear origin, affecting approximately 1% of the global population, showing a generally young age at onset. In the present study, a repeated variable stress paradigm was applied to pregnant rats during the final week of gestation. The effects of an extract of Panax ginseng C.A. Meyer (PG) on rats exposed to prenatal stress (PNS) were investigated in terms of behavioral activity and protein expression analyses. In the behavioral tests, grooming behavior in a social interaction test, line-crossing behavior in an open-field test and swimming activity in a forced-swim test were decreased in the rats exposed to PNS compared with the non-stressed offspring; the changes in behavioral activity were reversed upon oral treatment with PG (300 mg/kg). Subsequently, western blot analysis and immunohistochemical analyses of the prefrontal cortex and hippocampus revealed that the downregulation of several neurodevelopmental genes which occurred following exposure to PNS was reversed upon treatment with PG. The current findings demonstrate that the downregulation of several genes following exposure to PNS may affect subsequent behavioral changes, and that these phenomena are reversed following treatment with PG during pregnancy. Our results suggest that oral treatment with PG reduces the incidence of psychiatric disorders, such as schizophrenia.