Endocrine disrupting chemicals (EDCs) are widespread in the environment and suspected of interfering with endocrine homeostasis. Bisphenol A (BPA), 2,2-bis(4-hydroxyphenyl)propane, an EDC, has been widely used throughout the world as an industrial chemical. BPA is a developmental, neural, and reproductive toxicant that mimics estrogen and can interfere with growth and body function. In a recent study, a BPA-binding protein was isolated from P2 fractions of rat brain, and identified as protein disulfide isomerase (PDI). PDI has been studied extensively as a key enzyme involved in the formation of the correct pattern of disulfide bonds in proteins. PDI is also known to be a membrane-associated 3,3',5-triiodo-L-thyronine (T3)-binding protein. BPA inhibits the binding of T3 to PDI and isomerase activity of PDI. PDI has four consecutive domains, a, b, b', and a', each with a thioredoxin fold. The domains a and a' have a catalytically active Cys-Gly-His-Cys motif, while b and b' have substrate-binding sites. To address the chemical structural requirements of BPA for the inhibitory effect of PDI, several BPA analogs were tested, suggesting that the phenolic structure is important for BPA to affect PDI functions. To investigate the biological effects of BPA on PDI functions, GH3 cells were used. GH3 is a rat pituitary tumor cell line and releases growth hormone (GH) via the T3 receptor. Over-expression of PDI suppresses the T3-induced release of GH and when the cells were treated with BPA together with T3, the amount of GH released was much increased. Thus, PDI plays an important role in the hypothalamic-pituitary-thyroid axis. In this review, the possible involvement and biological significance especially in the central nervous system, of PDI as a target of phenolic environmental chemicals, are discussed.
The concentration of antibiotics in cells must be determined for effective treatment of infectious diseases caused by obligate intracellular parasites, such as Chlamydia trachomatis and Legionella pneumophila. We confirmed the usefulness of high performance liquid chromatography (HPLC), which is already commonly used in medical facilities, for the measurement of antibiotics in cells. The measurement was carried out using a post-column with tris(2,2'-bipyridine) ruthenium (III) chemiluminescence detection. Clarithromycin (CAM) was used as the model antibiotic. The retention time of CAM on the column was 7.6±0.4 min and the detection limit was 2.0×10-3 ng on column. The linearity of the calibration curve was guaranteed until 2.0 ng on column. This level of performance is comparable to that of liquid chromatography-tandem mass spectrometry. The system was able to monitor changes in the concentration of CAM in lymphocytes. These findings suggest that HPLC, a general-purpose, already widely used detection system, could also contribute to the development of effective individual antibiotic treatment regimens at a wide variety of medical facilities.
Diabetes enhances oxidative stress and exacerbates acute ischemic injury. Previous studies have demonstrated that infarct volumes were greater in diabetic animals caused by a transient cerebral ischemia as compared with non-diabetic animals. Ascorbic acid (AA) as a naturally occurring major antioxidant is reported to be low in diabetic tissues. Therefore, we examined the effects of daily supplementation of AA on lipid peroxidation and the activity of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) in streptozotocin (STZ)-induced diabetic rat brain. Additionally, we also investigated whether AA improves the exacerbation of neuronal damage induced by middle cerebral artery occlusion followed by reperfusion (MCAO/Re) in diabetic state. Type1-diabetes was induced in male Sprague Dawley rats by STZ (60 mg/kg of intraperitoneal injection). Five weeks after STZ-injection, the diabetic rats showed enhanced lipid peroxidation and impaired activity of antioxidant enzymes in the brain. Furthermore, the gene expression of glucose transporter GLUT1, which locates in the endothelial cells of the blood-brain-barrier and transports oxidized AA as the main source of AA supply to the brain, was downregulated in the diabetic brain. AA-supplemented (100 mg/kg daily, 2 weeks) diabetic rats showed normal levels of all these parameters. A diabetic state markedly aggravated MCAO/Re-induced neurological deficits and cerebral injury assessed by infarction volume. Treatment of AA remarkably improved both parameters in the diabetic rats. These results suggest that daily intake of AA relieves the exacerbation of cerebral ischemic injury in a diabetic state, which may be attributed to improvement of augmented oxidative stress.
Polycyclic aromatic hydrocarbons (PAHs) bound in particles from automobile emissions may cause adverse human health. In this study, a 3L heavy-duty diesel engine with and without the latest aftertreatment devices were characterized for PAH emissions and toxicity using European Transient Cycle (ETC) and the European Stationary Cycle (ESC). The latest combination of aftertreatment devices including a diesel oxidation catalyst (DOC) and a catalyzed diesel particulate filter (c-DPF) were used to remove diesel exhaust particles (DEP). Particle size distribution and number concentrations were measured using a TSI Engine Exhaust Particle Sizer. Eight PAHs [benz(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, indeno(1,2,3-cd)pyrene, dibenz(a, h)anthracene, and benzo(ghi)perylene] within the DEP were analyzed by thermal desorption-gas chromatography/mass spectrometry (TD-GC/MS). Elemental carbon (EC) and organic carbon (OC) were also analyzed with a thermal/optical carbon analyzer. The results indicated that 4-ring PAH emissions are larger than 5 or 6-ring PAH emissions, with and without the aftertreatment devices in place. Number concentrations of particles, PAHs and EC mass emissions in diesel exhaust were dramatically decreased by the aftertreatment devices: >99%, >97% and >99%, respectively. To evaluate the toxicity of PAHs in the DEP, we calculated benzo(a)pyrene equivalent (BaPeq) emissions using a toxic equivalency factor (TEF). Total BaPeq emissions of eight PAHs were also substantially decreased by the aftertreatment devices (>95%). However, the decrease rate of OC was lower than EC (>72%). We demonstrated that number concentrations of DEP, PAH emissions, EC and BaPeq emissions were similarly and substantially decreased by the latest aftertreatment devices.
To test for the role of cytotoxic T-lymphocyte associated antigen 4 (CTLA4) +49A/G and tumor necrosis factor alpha (TNF-α) -308G/A polymorphisms in the development of celiac disease (CD) in Jordanian patients we investigated 85 patients, 30 CD families and 100 healthy matched controls. In this work we found that, the frequency of the CTLA4 +49GG genotype and G allele were significantly higher in patients when compared with controls. This significant increase in the frequency of both, GG genotype and G allele, is also demonstrated in CD families. No significant differences were found in TNF-α -308G/A polymorphisms in CD patients compared with control group. No significant differences in the frequency were found when the family group compared with the control group for both AA genotype and A allele. Our results demonstrated the importance of CTLA4 +49G allele in the development of CD among Jordanian CD patients and that no significant association of this disease with TNF-α -308A allele.
Edible brown alga (hijiki in Japanese, Hijikia fusiforme) contains not only a high content of inorganic arsenic (iAs) but also various arsenosugars (AsSugs) which are metabolized to dimethylarsinic acid (DMA) in mammals. Since DMA is considered to be carcinogenic in rodents, it is necessary to accurately measure the contents of AsSugs as well as iAs for the risk assessment of seaweed consumption. Seven commercially available dried-hijiki products and two raw hijiki products were analyzed. Total-As was measured by inductively coupled plasma mass spectrometry (ICP-MS) with the Dynamic Reaction Cell (DRC) mode after acid-digestion. After water-extraction, AsSugs were detected by HPLC-MS/MS with multiple reaction monitoring in the positive ion mode and speciation analysis of arsenics was performed by HPLC-ICP-MS. The ranges of total-As obtained by acid digestion (A-TAs) in nine hijiki samples were 37.1-118.6 μg As/g dry weight (dw), and those of water extracted total-As (W-TAs) were 18.4-81.0 μg As/g dw. The ratios of water extracted iAs (W-iAs) to A-TAs ranged from 24.5 to 60.1%. The major compound detected was arsenate in all samples (8.9-70.5 μg As/g dw). Dimethylarsenosugar sulfate, AsSug 408, showed the highest peak among AsSugs detected. The content ratio of water extracted AsSugs (W-AsS) to A-TAs was estimated to be from 3.7 to 27.6%. The contents of A-TAs, W-iAs and W-AsS varied depending on the hijiki product. HPLC-MS/MS detected AsSugs more sensitively than HPLC-ICP-MS. Since iAs could not be detected by HPLC-MS/MS, combined analysis consisting of HPLC-MS/MS and HPLC-ICP-MS is necessary for accurate determination of arsenic species in seaweed products and also for the toxicological evaluation of AsSugs.
We studied the developmental toxicity and possible mechanisms of bisphenol A (BPA) damage of rat embryos in vitro. Whole-embryo culture was used to study the damage of BPA on visceral yolk sacs (VYSs) and embryos. Micromass culture was used to investigate the effects of BPA on differentiation and proliferation of embryonic midbrain cells. Neutral red staining was used to detect cell death. Immunohistochemistry was used to evaluate the expression of inducible nitric oxide synthase (iNOS) in VYSs and embryos. Our results as following, BPA could cause damage to embryonic development and morphological differentiation in a dose-dependent manner. At the highest dose, 80 and 100 mg/l BPA delayed cardiac tube growth and differentiation of VYSs, and induced various embryonic defects. BPA also induced excessive cell death and inhibited both proliferation and differentiation of rat midbrain cells. Immunohistochemical staining showed that BPA induces abnormal expression of iNOS protein in treated VYSs and embryos. Our results indicated that BPA is a potential teratogen and has toxicity on cultured rat embryo development. BPA-related developmental toxicity is caused by damage to the VYS, excessive cell death, inhibition of cell proliferation and differentiation, and abnormal expression of iNOS.
The effect of pretreatment with zinc (Zn) compounds on the mutagenicity of benzo[a]pyrene (B[a]P) was investigated using metallothionein (MT)-I/II null mice. MT-I/II null mice and wild-type mice were subcutaneously administered ZnSO4 once a day for 2 days and gavaged B[a]P at 24 hr after the last injection of ZnSO4. B[a]P-induced micronucleus frequencies were reduced by Zn pretreatment in the wild-type mice but not in the MT-I/II null mice. Zn administration significantly increased the concentration of MT in the liver and bone marrow cells of wild-type mice, but the statuses of other cellular antioxidants, such as glutathione, catalase and superoxide dismutase, were unchanged. In addition, the activity of a major B[a]P metabolic activation enzyme, cytochrome P450 1A, was unchanged by Zn treatment in both MT-I/II null mice and wild-type mice. These results suggest that Zn pretreatment protects against the mutagenicity of B[a]P through the induction of MT synthesis. The amount of MT produced in animals may determine their sensitivity to B[a]P exposure.
Poly-trans-[(2-carboxyethyl) germasesquioxane] (Ge-132) is the most common organic germanium compound. This compound has many physiological effects, which are mediated via the modulation of immune-system activation. The intake of dietary Ge-132 causes fecal color changes in humans, and we studied the mechanism of this in a rodent model. Male Wistar rats were given a diet containing 0.05% Ge-132 for two weeks and were compared with rats given a germanium free diet. The color of their feces and cecal contents changed from grayish-green to yellow in rats fed with Ge-132. The concentrations of stercobilin, a major fecal pigment, and total bile acids in cecal contents, were significantly increased by dietary Ge-132. Stercobilin is a metabolite of the bile pigment bilirubin. These results were produced by increases in bile components, such as bilirubin and bile acids, and showed that dietary Ge-132 promotes bile secretion into the intestine. Next, we administered Ge-132 (per os) to male rats at 50 mg/kg body weight per day for four days to reveal its effect on bile (and particularly on bilirubin). Bile juice samples were collected, and their bilirubin content and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity were analyzed. The bilirubin level in the bile was significantly increased by the administration of Ge-132, and the DPPH radical scavenging activity of bile was also significantly increased. As the increases in these two factors were correlated, we supposed that the anti-oxidative properties of the bile of rats fed with Ge-132 were due to bilirubin glucuronides. Oral intake of Ge-132 also increased the mRNA expression of uridine diphosphate glucuronosyltransferase 1a1 (Ugt1a1) and bile acid coenzyme A: amino acid N-acyltransferase (Baat), which encode bile component conjugating enzymes for secretion. The acceleration of bile pigment secretion into the intestine as well as increases in the anti-oxidant activity of bilirubin was induced by oral intake of dietary Ge-132. It is suggested that the antioxidative effect of bile against oxidative stress occurs through radical trapping.
The multidrug treatment, CDF [cyclophosphamide (CPA), doxorubicin (DXR), and 5-fluorouracil (5-FU)], is a common chemotherapy protocol for breast cancer. However, the molecular mechanisms underlying its toxicity for breast cancer cells remain unclear. As a laboratory model of breast cancer chemotherapy, the human breast cancer cell line MCF-7 was treated with CDF or the individual CDF reagents. Western blotting analysis revealed that two effector caspases (-6 and -7) were activated following the administration of DXR, 5-FU, or the CDF multi-drug. However, after treatment with CPA alone, caspase-7 was activated, but caspase-6 was not. We next used the RNA arbitrarily primed-PCR differential display (RAP-PCR DD) method, a derivative of the differential display method, to reveal changes in gene expression with the individual or multi-anticancer drug treatment. RAP-PCR DD was performed using arbitrary PCR primers. Independent cDNA bands representing at least nine mRNA species were amplified after drug treatment. Our results strongly suggested that the administration of different single and multi-anticancer drug treatments induced the expression of several different genes, whose products may be involved in the induction of cancer cell death.
Rice bran was found to effectively adsorb pesticides in artificial gastric fluid. Equilibrium adsorption isotherms conformed to the Freundlich type (log-log linear). Pretilachlor and esprocarb were successfully removed in artificial gastric fluid with an average removal efficiency of 85.5% and 95.8%, respectively after 90 min when rice bran (10 g/l) was added to samples containing from 0.05 to 5 mg/l of pesticides. The removal of pesticides by rice bran was attributed to the uptake by intracellular particles called spherosomes.
The present study investigates effect of Sida rhomboidea ROXB. (S. rhomboidea ROXB., SR) on high fat diet (HFD) induced insulin resistance in C57BL/6J mice and the results obtained have been compared with mice fed with rosiglitazone (ROS). Changes in bodyweight, food intake, fasting blood glucose, plasma insulin, plasma and hepatic triglyceride (TG), total cholesterol (TC) and free fatty acids (FFAs) have been investigated in various experimental groups. It was observed that feeding of SR extract to HFD fed mice (HFD+SR) reduced bodyweight (p<0.05), food intake (p<0.05) and feed efficiency ratio (p<0.05). Plasma and hepatic TC, TG and FFA were also significantly lowered (p<0.05) in HFD+SR groups. Efficient clearance of glucose in intraperitoneal glucose tolerance test (IPGTT), lowered area under curve (AUCglucose) values, low plasma insulin and fasting insulin resistance index (FIRI) coupled with higher KITT values were observed in HFD+SR groups. These findings were further justified by significant reduction of adipocyte diameter (p<0.05) and surface area (p<0.05) in HFD+SR groups. This study is a first scientific report on protective role of S. rhomboidea ROXB. extract against HFD induced insulin resistance in C57BL/6J mice and strengthens the folklore claim of use of SR leaves as alternative medicine against diabetes and obesity.
The subacute effects of dihydrocapsaincin (DHC) and capsaicin (CAP) (dose=3 mg/kg body weight per day for 10 days, subcutaueous) on the number and distribution of white blood cells (WBCs) were studied in male adult rats. The administration of DHC and CAP for 10 days decreased significantly the number of total WBCs, lymphocytes and monocyte, and increased significantly the number of the neutrophil and eosinophil without changing the number of basophil. The administration of DHC significantly decreased thymus weight and increased adrenal weight, showing that DHC induced thymus atrophy and adrenal hypertrophy. These results suggest that capsaicinoids induced the decrease of acquired immunity responses and these phenomena may have in part participated in capsaicinoids-induced stress-responses in rats.
Perfluorinated compounds are widespread in the environment, animals, and humans, and have been proposed to be included in the list of persistent organic pollutants that have the potential to cause a global pollution problem similar to that caused by existing persistent organic pollutants. Daphnia magna was used to evaluate the chronic effects of the ammonium salt of pentadecafluorooctanoic acid (PFOA) and the potassium salt of perfluorooctane sulfonate (PFOS) on the reproduction and biochemical responses of aquatic animals. Reproduction of Daphnia magna was a more sensitive response than survival or enzyme activities in daphnia exposed to PFOA or PFOS. No observed effect concentrations (NOECs) of daphnia reproduction were 1 and 10 mg/l after 21 days of exposure to PFOA and PFOS, respectively. The median lethal concentration (LC50) and survival NOEC values of PFOA for Daphnia magna were all greater than 100 mg/l after 21 days of exposure. The LC50 and survival NOEC values of PFOS were 9.1 mg/l and 5 mg/l after 21 days exposure. No significant changes in cholinesterase, catalase and heme peroxidase activities were observed between controls and exposure to PFOA or PFOS. This suggests that these enzyme activities are not sensitive biomarkers of exposure to these two chemicals or their effects in daphnids after chronic treatment. Based on the results of this study and other findings published in the literature, it is suggested that current PFOS and PFOA levels in freshwater may have no harmful impact on the aquatic environment. However, more information on the long-term ecological effects of PFOS and PFOA on diverse aquatic species is still needed to provide important information for adequately assessing the ecological risks of PFOS and PFOA.
A simple, rapid and inexpensive sample preparation method using extraction by shaking and sulfuric acid treatment was developed for determining eight organochlorine pesticides in kampo products by gas chromatography/mass spectrometry with negative chemical ionization. The recoveries were between 71% and 117%, and most relative standard deviations were less than 10%. The proposed method was applied to twenty-three samples of kampo formulae, and no samples were contaminated by eight organochlorine pesticides over the quantification limit.
We measured the amount of formaldehyde (FA) in cosmetics containing FA-donor preservatives [imidazolidinyl urea (IU), dimethyloldimethyl hydantoin (DM), diazolidinyl urea (DU), quaternium-15 (QU), and bronopol (BP)] and explored the factors affecting FA release. FA was detected in all the 89 cosmetic products tested. FA concentrations of cosmetics declared to contain DM and DU were significantly higher than those of cosmetics declared to contain IU and BP. Detected FA concentration of samples produced in the U.S.A. was significantly higher than that of samples produced in European countries. A weak proportional relationship was observed between the pH value and the released FA concentration of the cosmetic products containing DM and DU. There were no significant differences in the FA concentrations of various categories of cosmetics (lotion, gel, conditioner, shampoo, body wash, and others). Cosmetics containing a blend of amines, amides, or hydrolyzed proteins together with FA-donor preservatives had a lower FA concentration than the others.
In order to clarify the physiological roles played by MerP, MerT and MerE in Gram-negative bacteria, we constructed the plasmids pTP4 and pTPE21, which contained the genes merR, merT and merP, from the Pseudomonas K-62 plasmid pMR26, or the same genes with the merE gene of Tn21 from the Shigella flexneri plasmid NR1 (R100), respectively. Cells containing pTP4 showed increased hypersensitivity to Hg(II), but maintained a normal sensitivity to CH3Hg(I). However, cells with pTPE21 exhibited increased hypersensitivity to Hg(II) and CH3Hg(I). Cells with pTP4 accumulated appreciably more Hg(II) than control cells, but no significant difference was observed in their uptake of 14CH3Hg(I). In contrast, the cells containing pTPE21 accumulated significantly larger amounts of Hg(II) and 14CH3Hg(I) than either control cells or cells with pTP4. These results suggest that the mer operons have evolved to redirect uptake of mercurials into dedicated, specific and relatively high-affinity transport systems comprising the small periplasmic protein MerP and two inner membrane proteins, MerT and MerE.