Eeffects of perfluorooctane sulfonate (PFOS) on maleic dialdehyde (MDA) content, superoxide dismutase (SOD) activity and total antioxidation capability (T-AOC) were compared in mice at different postnatal developmental stages, and concentrations and distributions of PFOS in different tissues were measured simultaneously. The male and female mice at postnatal day (PD) 7, PD 14, PD 21, PD 28 and PD 35 were distributed randomly to dosage group (50 mg/kg body weight) and control group (0 mg/kg body weight). Mice were administered with PFOS by once subcutaneous injection. Subsequently, after 24 hr, MDA content, SOD activity and T-AOC in brain and liver were analyzed. The PFOS concentrations in blood, brain and liver were determined by high-performance liquid chromatography negative electrospray tandem mass spectrometry (LC-MS). PFOS induced degression of the body weights of mice evidently and increase of relative weights of liver. Meanwhile, it depressed the SOD activity and T-AOC in brain and liver. The concentrations and distribution percentages of PFOS in blood, brain and liver of mice were significantly different at various postnatal developmental stages. Achieved results in this study indicate that younger mice pups were more sensitive to PFOS exposure. In addition, significant distinctions in concentrations and distribution percentages of PFOS in various tissues were demonstrated in this study. The gender difference observed was greater in the older mice. Thus it is worth giving attention especially to adverse effects of PFOS on foetus and children.
Phthalates, such as butyl benzyl phthalate (BBP), di-n-butyl phthalate (DBP), and their metabolites (mono-buyl phthalate [MBP], mono-benzyl phthalate [MBzP] and phthalic acid [PA]), are known to obstruct normal development in mammals. BBP and DBP have been reported to have estrogenic activity, while MBP and MBzP exhibit weak or no estrogenic effects. We previously showed that BBP and DBP have inhibitory roles on nicotinic acetylcholine receptors. Nicotinic acetylcholine receptors are widely distributed and have roles in developmental processes. In this study, the effects of BBP, DBP, MBP, MBzP and PA on calcium signaling coupled to nicotinic acetylcholine receptors was investigated in bovine adrenal chromaffin cells and human neuroblastoma SH-SY5Y cells. Following an epibatidine-induced [Ca2+]c increase, the IC50s of BBP, DBP, MBzP and MBP were 3.41, 5.01, 432 and 695 microM in bovine adrenal chromaffin cells and 0.28, 0.44, 58 and 116 microM in human SH-SY5Y cells, respectively. Although PA suppressed the epibatidine-induced [Ca2+]c increase, the suppression was less than with MBP. The suppression potency of phthalates was related to their chemical structures. The suppression effects of BBP, DBP, MBP and MBzP remained similar potency under chronic treatments. This study demonstrated that MBP, MBzP and PA, the metabolites of BBP and DBP, had suppressor roles on the calcium signaling pathway coupled to nicotinic receptors.
Although phosphatidylinositol (PI) is an important component in all plants and animals, there is no toxicity report when purified PI is orally administrated to animals. As a safety evaluation of PI, acute, subchronic and genotoxicity studies were conducted with purified PI from soy lecithin (Asahi Kasei PI). Up to 2,000 mg/kg of Asahi Kasei PI was administrated once orally to male and female rats. There were no deaths or any clinical sign in any group throughout the observation period. Then, Asahi Kasei PI was repeatedly administered orally to male and female rats at daily doses of 100, 300 and 1,000 mg/kg for 13 weeks. Neither death nor any toxicological signs during the administration period and no changes related to the test substance administered were observed in any group with regard to body weight, food consumption, ophthalmoscopy, hematology, blood biochemistry, necropsy, organ weights or histopathology. Based on these results, the no-observed-adverse effect level (NOAEL) of Asahi Kasei PI was considered to be 1,000 mg/kg/day for male and female rats. Genotoxicity evaluation of Asahi Kasei PI was also carried out by the bacterial reverse mutation test (Ames test) and in vitro chromosome aberration test in compliance with the Japanese guidelines on genotoxicity testing of pharmaceuticals, the OECD guidelines for testing chemicals and guidelines for designation of food additives and for revision of standards for use of food additives. The results indicate neither increases of revertant colonies nor chromosome aberration, suggesting that Asahi Kasei PI has high safety in genotoxicity.
A large-scale transcriptome database of rat liver (TG-GATEs) has been established by the Toxicogenomics Project in Japan. In the present study, we focused on 8 hepatotoxic compounds within TG-GATEs, i.e., clofibrate, omeprazole, ethionine, thioacetamide, benzbromarone, propylthiouracil, Wy-14,643 and amiodarone, which induced coagulation abnormalities. Aspirin was selected as a reference compound that directly causes coagulation abnormality, but not through liver toxicity. In blood chemical examinations, for all the coagulopathic compounds there was little elevation of aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT), suggesting no severe cell death by treatment with the compounds. We extracted 344 probe sets from the data for these 8 typical drugs, which induced this phenotype at any time from 3 to 28 days of repeated administration. Principal component analysis using these probe sets clearly separated dose- and time-dependent clusters of the treated groups from their controls, except aspirin and propylthiouracil, both of which were considered to cause coagulopathy not due to their hepatotoxicity but due to their direct effects on the blood coagulation system. Reviewing the extracted genes, changes in lipid metabolism were found to be dominant. Genes related to blood coagulation were generally down-regulated by these drugs except that vitamin K epoxide reductase complex subunit 1 (Vkorc1) like 1, a paralogous gene of Vkorc1, was up-regulated. As expected, expression changes of these genes were least prominent in aspirin or propylthiouracil-treated liver. We concluded that these probe sets could be a good starting point in developing mechanism-based biomarkers for diagnosis or prognosis of hepatotoxicity-related coagulation abnormalities in the early stage of drug development.
Recently, tellurium (Te), antimony (Sb) and germanium (Ge) have been used as an alloy in phase-change optical magnetic disks, such as digital versatile disk-random access memory (DVD-RAM) and DVD-recordable disk (DVD-RW). Although these metalloids, the so-called “exotic” elements, are known to be non-essential and harmful, little is known about their toxic effects and metabolism. Metalloid compounds, tellurite, antimonite and germanium dioxide, were simultaneously administered to rats. Their distributions metabolites were determined and identified by speciation. Te and Sb accumulated in red blood cells (RBCs): Te accumulated in RBCs in the dimethylated form, while Sb accumulated in the inorganic/non-methylated form. In addition, trimethyltelluronium (TMTe) was the urinary metabolite of Te, whereas Sb in urine was not methylated but oxidized. Ge was also not methylated in rats. These results suggest that each metalloid is metabolized via a unique pathway.
A chronic toxicity study of kojic acid (KA) was performed using male F344 rats by dietary administration at concentrations of 0 (control), 0.5 and 2.0% for 55 weeks. Body weight gain was suppressed in the 2.0% group. The major hematological findings were decreased red blood cell (RBC) count and hematocrit (Ht) values at both 0.5 and 2.0%. In serum biochemistry, increased aspartate transaminase (AsT), alanine transaminase (AlT), alkaline phosphatase (ALP) and γ-glutamyl transpeptidase (γ-GTP) levels were detected in the 0.5 and 2.0% groups. Histopathologically, single cell necrosis of hepatocytes and proliferation of bile ductules in both treatment groups, and hypertrophy of hepatocytes, granulomas and proliferation of bile ducts in the 2.0% group were increased in incidence, and numbers and areas of glutathione-S-transferase placental-form (GST-P) positive foci were increased in the liver of the 2.0% group. In the thyroids, diffuse follicular cell hyperplasia at 0.5 and 2.0% and focal follicular cell hyperplasia and follicular adenoma at 2.0% were increased. A thyroid follicular carcinoma was also observed at 2.0%. Additionally, increased incidences of hyaline casts and basophilic tubules in the kidneys at 2.0% and microgranulomas containing crystals in the lung in both treatment groups were noted. At 2.0%, hypertrophy of cortical cells in zona fasciculata was also increased in the adrenals. In conclusion, no observed adverse effect level of KA was below 0.5%, which is equivalent to 227 mg/kg body weight/day in male rats.
Methylmercury (MeHg) is an environmental pollutant known to cause neurobehavioral defects and is especially toxic to the developing brain. With recent studies showing that fetal exposure to low-dose MeHg causes developmental abnormalities, it is therefore important to find ways to combat its effects as well as to clarify the mechanism(s) underlying MeHg toxicity. In the present study, the effects of MeHg on cultured neural progenitor cells (NPC) derived from mouse embryonic brain were investigated. We first confirmed the vulnerability of embryonic NPC to MeHg toxicity, NPC from the telencephalon were more sensitive to MeHg compared to those from the diencephalon. Buthionine sulfoximine (BSO) which is known to inhibit glutathione synthesis accelerated MeHg toxicity. Furthermore, antioxidants such as N-acetyl cysteine and α-tocopherol dramatically rescued the NPC from MeHg's toxic effects. Interestingly, a 12 hr delay in the addition of either antioxidant was still able to prevent the cells from undergoing cell death. Although it is now difficult to avoid MeHg exposure from our environment and contaminated foods, taking anti-oxidants from foods or supplements may prevent or diminish the toxicological effects of MeHg.
A validation study of an in vitro skin irritation testing method using a reconstructed human skin model has been conducted by the European Centre for the Validation of Alternative Methods (ECVAM), and a protocol using EpiSkin (SkinEthic, France) has been approved. The structural and performance criteria of skin models for testing are defined in the ECVAM Performance Standards announced along with the approval. We have performed several evaluations of the new reconstructed human epidermal model LabCyte EPI-MODEL, and confirmed that it is applicable to skin irritation testing as defined in the ECVAM Performance Standards. We selected 19 materials (nine irritants and ten non-irritants) available in Japan as test chemicals among the 20 reference chemicals described in the ECVAM Performance Standard. A test chemical was applied to the surface of the LabCyte EPI-MODEL for 15 min, after which it was completely removed and the model then post-incubated for 42 hr. Cell v iability was measured by MTT assay and skin irritancy of the test chemical evaluated. In addition, interleukin-1 alpha (IL-1α) concentration in the culture supernatant after post-incubation was measured to provide a complementary evaluation of skin irritation. Evaluation of the 19 test chemicals resulted in 79% accuracy, 78% sensitivity and 80% specificity, confirming that the in vitro skin irritancy of the LabCyte EPI-MODEL correlates highly with in vivo skin irritation. These results suggest that LabCyte EPI-MODEL is applicable to the skin irritation testing protocol set out in the ECVAM Performance Standards.
Amount of dietary protein is known to influence blood pressure in humans and animal models. However, contradictory reports are available on the influence of source of dietary protein and soy isoflavones on blood pressure. Information on potential effect of anthocyanins, potent flavonoid antioxidants widely distributed in fruits and vegetables, on hypertension is also limited. Therefore, this study was conducted to examine whether source of dietary protein (casein vs. soybean protein, washed by alcohol to remove most isoflavones), dietary extracted isoflavones and anthocyanins modulate the lifespan of Stroke-prone Spontaneously Hypertensive (SHRSP) rats, one of the most suitable models for hemorrhagic stroke. Body weight and systolic blood-pressure matched groups of 47 day-old SHRSP rats (n = 16) received semi-purified diets containing 200 g/kg protein (casein or soybean) supplemented with 0 or 500 mg/kg isoflavones (NOVASOYTM, a commercial soy isoflavones supplement extracted from soybean), and 0 or 500 mg/kg anthocyanins (extracted from elderberry). The drinking water contained 10 g/l sodium chloride to induce early hypertension. Survival times and survival rates of rats were determined. The survival rates were determined for each group and expressed as a percentage of the original number of rats still alive on a given day. The survival times and survival rates of animals fed casein and soybean protein diets were not different (P > 0.05). However, there was a significant effect of supplementation with isoflavones or anthocyanins on survival times and survival rates. Death occurred significantly earlier (P < 0.05) in the isoflavones- or anthocyanins-supplemented groups.
Blood circulation and the route of nutritional supply both change dramatically in the immediate neonatal period. These systemic shifts lead to adjustment of metabolic patterns in the neonate, with alterations in the spectrum of metabolites in body fluids. We have investigated whether 1H-NMR-based metabolic profiling (NMR-MP) with principal component analysis (PCA) can be used to evaluate metabolite profiles in highly-diluted samples of individual neonatal mouse urine. We report that a 60-fold dilution of urine gave a reproducible NMR-MP analysis. Here the NMR spectral patterns and PCA score plot clearly delineated the developmental changes in urine metabolites in the immediate neonatal period. These results suggest that NMR-MP may offer a powerful method for assessing the physiology and toxicity of chemicals in neonatal periods of experimental animals.
Genotoxicity of superparamagnetic iron-platinum (FePt) nanoparticles (NPs) capped with 2-aminoethanethiol (AET) was evaluated using the bacterial reverse mutation assay (Ames test) and in vitro chromosomal aberration test. Mutagenicity of AET-capped FePt NPs was found to be negative in the Ames test, while clastogenicity of FePt NPs seemed to be false-positive in the in vitro chromosomal aberration test using Chinese hamster lung fibroblast cells. However, further detailed in vitro genotoxicity tests, such as DNA adduct studies, are necessary to conclude that a positive aberration result is irrelevant.