Biosynthesis of prostaglandin E2 (PGE2), the most common prostanoid with potent and various biological activities, is regulated by three sequential steps of cyclooxygenase (COX) pathway. We reported the molecular identification of cytosolic prostaglandin E synthase (cPGES), a terminal enzyme of the COX-mediated PGE2 biosynthetic pathway. Of interest, it is identical to the co-chaperone p23 that binds to heat shock protein 90 (Hsp90). Incubation of recombinant cPGES/p23 and Hsp90 resulted in a remarkable increase in PGES activity in vitro. Furthermore, A23187-induced PGE2 generation in 3Y1 cells was suppressed by Hsp90 inhibitors, which destabilized the cPGES/p23-Hsp90 complex, and reduced cPGES/p23 activity and PGE2 production to basal levels. Next, we found that cPGES/p23 underwent serine phosphorylation, which was accelerated transiently after cell activation. In activated cells, cPGES/p23 phosphorylation occurred in parallel with increased cPGES/p23 enzymic activity and PGE2 production from exogenous and endogenous arachidonic acid, and these processes were facilitated by Hsp90 that formed a tertiary complex with cPGES/p23 and protein kinase CK2. Treatment of cells with inhibitors of CK2 and Hsp90 and with a dominant-negative CK2 attenuated the formation of the cPGES/p23-CK2-Hsp90 complex and attendant cPGES/p23 phosphorylation and activation. Mutations of either of two predicted CK2 phosphorylation sites on cPGES/p23 (Ser113 and Ser118) abrogated its phosphorylation and activation both in vitro and in vivo. These results provide the first evidence that the cellular function of this eicosanoid-biosynthetic enzyme is under the control of a molecular chaperone and its client protein kinase.
Dietary habits are an important risk factor for lifestyle-related diseases. To carry out a nutrition survey of fat-soluble vitamins, we developed determination methods of fat-soluble vitamins using liquid chromatography-atmospheric pressure chemical ionization/tandem mass spectrometry or high-performance liquid chromatography with fluorescence detection. In these methods, stable isotope-labeled compounds or vitamin K analogs with a saturated side-chain were used as internal standards. These methods have high sensitivity and sufficient accuracy, and we applied them in a nutrition survey about the status of fat-soluble vitamins in Japanese women. Plasma concentrations of 25-hydroxyvitamin D3 [25(OH)D3] and 25-hydroxyvitamin D2 [25(OH)D2] in healthy postmenopausal women (n=98) were 20.5±7.9 and 0.4±1.4 ng/ml, respectively. A significant negative correlation in plasma levels between 25-hydroxyvitamin D [25(OH)D] and parathyroid hormone was observed. For vitamin K homologs, plasma levels of phylloquinone (PK), menaquinone-4 (MK-4) and menaquinone-7 (MK-7) in Japanese women of various ages (n=1409) were 1.03±0.90, 0.12±0.28 and 6.71±13.6 ng/ml, respectively. The mean total vitamin K intake of Japanese young women was about 230 μg/day, and 94% of participants met the Adequate Intake of vitamin K for women aged 18-29 years in Japan, 60 μg/day. Moreover, we determined fat-soluble vitamins in breast milk collected from Japanese lactating women and revealed that the contents of all-trans-retinol, vitamin D3, 25(OH)D3, α-tocopherol, PK and MK-4 in breast milk were 0.39±0.14 μg/ml, 0.10±0.15 ng/ml, 0.08±0.04 ng/ml, 3.96±1.84 μg/ml, 3.56±2.19 and 1.77±0.68 ng/ml, respectively.
Investigation of the amounts of chemical substances excreted into environments is important in the consideration of their influence on humans and other biologics. We performed this study to investigate the excretion levels of 4-alkylphenols, represented by the endocrine disruptors 4-octylphenol and 4-nonylphenol, from chemical products and established a simultaneous analytical method in which 4-alkylphenols were trimethylsilylated and analyzed using head-space gas chromatography-mass spectrometry (HS-GC/MS). This is a simple and innovative method that proceeds to derivatization with a one-step procedure and dissolves the whole sample as well as eliminates impurities interfering with analysis. When this method was applied to synthetic resin products, 4-nonylphenol (detection limit 0.5 μg/ml) was rapidly and simply analyzed with high sensitivity.
Distribution of allergens may change according to characteristics of regions. Measurements of total immunoglobulin E (TIE) and specific IgE antibodies are used to diagnose allergic diseases. In this study, we investigated sensitivity of TIE and its consistency with allergic symptoms, and compared the appropriateness of some allergy panels with features of Malatya, south-eastern Turkey province. Sera of 233 allergic patients of various age groups were tested for TIE. The specific IgE's were worked with 529 sera for food panel 5 (FP5) and one inhalant panel by using chemiluminesence technique. The sixty of inhalant panel positive sera were tested with specific IgE against house dust mites, Dermatophagoides pteronyssinus and Dermatophagoides farinae. The specific IgE against egg white, milk, wheat, corn, tomato, beef, strawberry and banana were investigated in sixty of FP5 positive sera by enzyme immunoassay method. The sensitivity of TIE was found to be 85%. We concluded that TIE can be used as a scanning test in children aged 5-18 years (69% positive) but it is not useful for 0-5 age group nor in adults (38%, 53% positive, respectively). The allergen scanning test panels should be designed according to custom of society and characteristics of the region.
Dalton's lymphoma ascites (DLA) cells were used as a model cell line to evaluate the cytotoxic concentration and anti-tumor activity of 5-fluorouracil (5-FU) through transdermal drug delivery (TDD) for solid tumors. Cytotoxicity was measured by exposing cell suspensions to increased concentrations of drug from 10-50 μg/ml and then the viable cells count was measured by the tryphan blue exclusion method. The results confirmed that 50 μg/ml of 5-FU was cytotoxic to DLA cells. The tumor volume was 0.23 cm2 and the increase in life span (ILS) was 81.8% with a maximum survival time of 39.5±1.87 days for 5-FU monolithic matrix transdermal patch in mice with solid tumors induced by DLA. The results were significantly different (p<0.05) compared to the untreated control, which had a maximum survival time of 19±1 days. The anti-tumor activity was very effective compared to intravenous therapy (ILS, 25.58%, maximum survival time, 24±2.7 days). No signs of erythema, vesiculations or bullous reaction were observed with the patches. The mean cumulative skin irritation and adherence scores for both mice and humans were zero and less than one, proving there was no irritation or sensitization. The patches adhered completely to the skin, without leaving any adhesive on skin (score=0 in human subjects). The transdermal patches had 100% flatness, a thickness of 150±0.03 mm, good content uniformity, folding endurance (>500 foldings), elegance, smoothness, transparency, and flexibility. The Velcro protection jackets were suitable for the study and prevented the mice from licking, scratching, and rubbing the patches.
Multiple drug resistant protein 1 (MDR1), P-glycoprotein, plays a role in the blood-brain barrier, preventing drug distribution into the brain, and in cancer chemotherapy. MDR1 is composed of two repeated fragments and there are six transmembrane domain (TMD) on the N-terminal of each repeat and a nucleotide-binding domain (NBD) on the C-terminal. We reported that sera from autoimmune hepatitis patients well reacted with MDR1 by enzyme-linked immuno-sorbent assay (ELISA) [Shinoda et al., (2004) Autoimmunity, 37, 473-480]. In this study, to determine antigenic sites, peptides on the extra-cellular loop (ECL), TMD and NBD of MDR1 were applied to ELISA with sera from 4 autoimmune hepatitis (AIH) patients and 4 normal individuals. The results showed that serum from Patient 3 reacted well with peptide 314-328 and weakly with peptide 957-971. Meanwhile, serum from Patient 4 reacted well with peptide 850-857 and weakly with peptide 741-755 and 957-971. All the five peptides reacted with sera from Patients 3 and 4 were located on ECL. Normal sera did not react with those peptides and the reactions of sera from Patients 1 and 2 were marginal. Sera from 4 patients and normal individuals did not react with peptides of TMD and NBD. These results suggest that some ECL on MDR1 play a role of antigenic determinants, and TMD and NBD do not. Personal specificity and diversity of antibodies from the AIH patients (such as Patients 3 and 4) against antigenic determinant were found.
The presence of eight kinds of persistent organic pollutants (POPs) such as DDT and its metabolites (DDTs), hexachlorocyclohexanes (HCHs), chlordane compounds (CHLs), drin compounds (Drins), heptachlor, hexachlorobenzene (HCB), heptachlor-epoxide, polychlorinated biphenyls (PCBs) and sixty-four polycyclic aromatic hydrocarbon compounds (PAHs) was identified using high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS) to investigate their distribution in surface sediment from Hanoi, Hue, and Ho Chi Minh in Vietnam. A survey of sediment samples from Osaka was conducted for comparison. The concentrations of ΣDDTs, ΣCHLs, ΣPCBs and ΣPAHs in Vietnam were 0.19-140, N.D.-9.0, 0.11-110, and 30-5500 ng/g-dry, respectively. Concentrations of these compounds in urban areas were higher than those in other areas. In addition, the ΣDDT concentrations in Vietnamese urban areas were higher than those in Osaka. These results suggest that most DDTs would be used as insecticides for the purpose of health services rather than as agricultural chemicals. PAH pollution in urban areas and suburbs is caused mainly by runoff of petrol, whereas in rural areas, the combustion of fossil fuels and biomass is the major pollutant source.
The serum concentration of valproic acid (VPA) in epilepsy patients decreased by the administration of carbapenem antibiotics, such as meropenem, panipenem or imipenem, to a sub-therapeutic level. Studies to explain the decrease were carried out using almost rats by the following steps: absorption of VPA in the intestine, glucuronidation in the liver, disposition in blood and renal excretion. It is difficult to consider the inhibition of intestinal absorption, because carbapenem antibiotics are intravenously administered and do not reach the intestine at an effective concentration. The liver is the key organ for the decrease of VPA concentration by carbapenem antibiotics, because it has been reported that no decrease of the VPA level by carbapenem was found in hepatectomized rats. The most likely mechanism in liver is the activation of UDP-glucuronosyltransferase by carbapenem antibiotics. We found a 35% increase of VPA-glucuronidation activity by the pre-incubation of human liver microsomes with meropenem. We estimated that this increase fully compensates for the decrease of serum VPA level by carbapenem antibiotics in patients. Meanwhile, we could not find the in vitro activation of CYP2A6, CYP3A4, P-glycoprotein (P-gp, MDR1) and Multidrug resistance-associated protein 2 by pre-incubation with carbapenem antibiotics. Thus, we considered that the activation of VPA glucuronidation by carbapenem antibiotics is a key point in the decrease of plasma VPA level.
We developed an analytical method for volatile organic compounds (VOCs) used as film-forming agents or anti-freeze agents in water-based paints by flame ionization detection (FID-GC) and gas chromatography mass spectrometry (GC/MS). Twelve VOC components were separated using 2 types of column that differ in polarity. In addition, a pretreatment method by ethyl acetate-water partition was evaluated, and a survey of the use of VOCs in commercially available water-based paints was performed. For indoor paints, the anti-VOC measures included changes from VOC solvents to semi-VOC (SVOC) solvents such as those from 2,2,4-trimethyl-1,3-pentanediol-monoisobutyrate (TMPMIB) to 2,2,4-trimethyl-1,3-pentanediol diisobutyrate and from ethylene glycol (EG) to triethylene glycol. However, in multipurpose paints for both indoor and outdoor use, VOCs such as EG, TMPMIB, diethyleneglycol dibutyl ether, and diethyleneglycol monobutyl ether were detected. Since these products may be also used for indoor painting, indications of VOC components and caution for use on products are necessary.
Fluorinated derivatives of 4,10-diazachrysene (DAC) were tested for their mutagenicity in Salmonella typhimurium TA100 in the presence of rat liver S9 to investigate the metabolic activation pathway of DAC. We have previously reported that metabolism at the pyridine moiety of quinoline was related to its mutagenicity and that quinoline was deprived of its genotoxicity by fluorine-substitution at the position 3 in the pyridine moiety. DAC, an aza-analog of chrysene consisting of two quinoline moieties, has two pyridine and two benzene rings as metabolically susceptible sites in the molecule. The mutagenicity of DAC was only decreased by fluorine-substitution on both pyridine moieties. On the other hand, the mutagenicity of DAC was neither decreased by fluorine-substitution on just one pyridine moiety nor by fluorine-substitution on both benzene moieties. These results suggest that metabolic activation occurs on both pyridine moieties in DAC like in quinoline.
Quinoline and four monofluorinated derivatives of quinoline (FQ's) were tested for their clastogenicity in a Chinese hamster lung (CHL) cell line using chromosomal aberration (CA) and micronucleus (MN) tests. Quinoline and all the fluoroquinolines, 3-, 5-, 6-, and 8-FQ, induced CA in the presence of the metabolic activation system. However, the clastogenic property was altered by fluorine-substitution. 3-FQ showed reduced cytotoxicity and clastogenicity. It was positive only at a higher dose than the other compounds. 6-FQ was as cytotoxic and clastogenic as quinoline when tested in the lower dose range (less than 0.075 mg/ml). 5-FQ and 8-FQ were only moderately clastogenic in the CA test although their toxicity was similar to that of quinoline. The MN test showed almost the same tendency in clastogenicity as the CA test, except that 8-FQ showed a negative result. These results demonstrate that fluorine-substitution can modify the clastogenicity of quinoline, probably through interference of the metabolic activation.
Sericoside is a traditional herbal saponin from Terminalia sericea (Family: Combretaceae). We studied the anti-inflammatory effect of sericoside on acute inflammatory colitis induced by ethanolic 2,4,6-trinitrobenzene sulfonic acid in male rats. Intestinal mucosa lesions were judged by their macroscopic damage score and measuring by myeloperoxidase (MPO) activity. Pretreatment of sericoside (30 mg/kg daily by gavage for 10 days) significantly reduced the macroscopic damage score and inhibited MPO activity. These results showed that sericoside attenuated the inflammatory effect and suggested its possible role in treating acute inflammatory diseases such as inflammatory bowel disease.
The pharmacokinetics and utilization (flavocoenzyme formation) of an intravenously administered flavin mononucleotide (FMN) or riboflavin (RF) in rats were assessed using ethylenediamine-N, N, N', N'-tetraacetic acid (EDTA)-treated plasma. Serial blood samples were collected via a syringe containing EDTA, used to prevent flavin adenin dinucleotide (FAD) hydrolysis, from the left jugular vein and plasma samples were analyzed for FAD, FMN and RF levels using a reversed-phase HPLC method. After the intravenous bolus administration of a single dose of FMN (500 nmol/kg), FAD was identified in plasma. However, increased levels of FAD in plasma were much less pronounced than that of RF levels. In contrast, after the intravenous bolus administration of single dose of RF (500 nmol/kg), both FAD and FMN levels were not different from the endogenous levels. Evidence is given in this paper that intravenously administered FMN affects the plasma levels of FAD in rats, whereas insensible changes in FMN and FAD levels were not confirmed by intravenously administered RF. The plasma kinetics of total flavin after intravenous bolus administration of RF, FMN or FAD to rats was also estimated. Statistical analysis of individual data revealed a high correlation between the total flavin level obtained by summation of each flavin level and that obtained with the lumiflavin (LF) method. Since this method is highly sensitive, it could be used to give precise values for the pharmacokinetic parameters of flavins.
We investigated the acute effect of a single dose of dried-bonito broth (DBB) on peripheral blood flow using a laser Doppler blood flow meter. In a randomized, double-blind, crossover, placebo-controlled study, 19 healthy female subjects ingested DBB (4900 mg) or placebo. The peripheral blood flow was measured before ingestion and at 5, 10, 15, 20, 25, 30, 45, and 60 min after ingestion of the test diet. Blood flow significantly increased after DBB ingestion, and the area under the blood flow-time curves calculated up to 60 min (AUC0-60) for the ingestion with DBB was significantly higher than that for the placebo ingestion (p<0.001). Following consumption of lower dose of DBB (2450 mg), an increase in peripheral blood flow was observed and that the AUC0-60 after subjects consumed DBB was significantly higher than that after they consumed the placebo (p<0.01). The mean AUC0-60 for the treatment with 4900 mg DBB was about 2 times that for 2450 mg DBB, suggesting that an orally administered single dose of DBB might have an acute dose-dependent effect on peripheral blood flow.
High levels of arsenic (As) contamination are found in the groundwater of Vietnam. To determine the distribution of arsenic and other metal contamination in the groundwater of the Mekong River Delta, we examined the contamination status of As and other metals in two regions, Tien Giang Province and Dong Thap Province. The concentration of total As in the groundwater, which is used for the drinking water supply, ranged from 0.9 μg/l to 321 μg/l, and 27% of the shallow-well water samples exceeded the World Health Organization (WHO) provisional guideline of 10 μg/l. Also, 91% and 27% of shallow-well water samples had higher concentrations of manganese (Mn) and barium (Ba) than those stated in the WHO drinking water guidelines, respectively. On the other hand, such contamination was not found in the deep-well water samples examined. These results suggest that pollution by As, Mn, and Ba is widely distributed in the shallow aquifer of the Mekong River Delta and thus the health of the people consuming shallow-well water in both provinces might be at considerable risk.
A single injection of an overdose of selenite is known to induce cataracts in young rat eyes. In our current study, we have investigated the gene expression profiles of the cytochrome P450s (CYPs) in the ocular tissues of rats developing such selenite-induced cataracts. Seven days after the injection of selenite, the expression levels of the CYP1A1 and CYP2E1 genes in the lenses of these animals increased and a slight reduction in the expression of the CYP3A gene family could be observed in the extralenticular tissues. When pantethine, an antioxidant, was administered 30 min before selenite injection, the extent of the CYP gene expression changes diminished. These results suggest that selenite-induced oxidative stress may play a role in both the induction and downregulation of CYP genes. In addition, the elevated expression of CYP1A1 and CYP2E1 in the lens may underlie the enhancement of cataract development induced by selenite.