Glutaraldehyde has been in widespread use in hospitals to sterilize instruments. Routine exposure to glutaraldehyde is, however, known to cause adverse health effects. Authors have applied and evaluated a passive sampler (DSD-DNPH) for the determination of glutaraldehyde in air at ppb level. The sampler consists of porous polyethylene tube uniformly packed with 2,4-dinitrophenylhydrazine (DNPH) coated silica gel as a reactive adsorbent. The sampling duration of this device was designed for 8 hr to apply to field measurements in workplace. After sampling, DNPH derivatives were eluted by acetonitrile and subsequently determined by HPLC. A sampling rate of the sampler was determined by chamber experiments and resulted in 40 ml/min for glutaraldehyde. Effects of temperature and humidity on the rate were not apparent. No significant effect of exposure time, air concentration and back diffusion on the sampling performance was suggested by dynamic adsorption model. The sampling rate was then validated in field measurements comparing with a previous active sampling. The diffusion sampler was successfully used for determination of 4-180 ppb of glutaraldehyde and gave similar results to active sampling in indoor air. Limit of quantitation (LOQ) of the diffusion sampler resulted in 3.9 ppb for 8-hr exposure in air.

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We have reported the neuroprotection provided by an extract of buckwheat (BWE, Fagopyrum esculentum Moench) on neuronal damage in the repeated cerebral ischemia model and demonstrated that BWE inhibited the excess glutamate release induced by repeated cerebral ischemia, suggesting that BWE had a free radical-scavenging in addition to anti-glutamate action. In the present study, we studied the neuroprotective effects of Kudingcha extract (KDE, Ligustrum purpurascens Y. C.) on scavenging by the 2,2-diphenyl-1-picylhydrazyl (DPPH) radical in primary cultured hippocampal neurons, and on spatial memory impairment induced by scopolamine in an eight-arm radial maze in comparison with BWE. The effects of KDE (0.01-1 mg/ml) were more potent than those of BWE (0.01-1 mg/ml) in scavenging the DPPH radical (1 mM). KDE (100 μg/ml) prevented the cell damage induced by glutamate (300 μM) or kainate (1 mM), which was more potent than BWE (100 μg/ml), but BWE suppressed the cellular damage induced by β-amyloid_(25-35) (20 μM) more potently than KDE (100 μg/ml). BWE (600 mg/kg), but not KDE, significantly suppressed the increase in errors induced by scopolamine (0.5 mg/kg i.p.) in the eight-arm radial maze. The results suggest that BWE may protect against cholinergic dysfunction and that KDE protects more effectively against glutaminergic dysfunction.

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To clarify the contamination levels of surface soil with mutagens in urban areas of Aichi prefecture, Japan, and Bangkok, Thailand, 60 and 67 surface soil samples were collected in Aichi and Bangkok, respectively, and mutagenicities of the organic extracts from these soil samples were examined in the Ames assay using Salmonella typhimurium (S. typhimurium) TA98 and TA100 with and without a mammalian metabolic system (S9 mix). Most of the soil samples collected in both areas showed mutagenicity in TA98 with and without S9 mix and in TA100 with S9 mix. Thirteen (22%) soil samples collected in Aichi showed high mutagenicity (more than 1000 revertants/g of soil) in TA98 with and/or without S9 mix, and six soil samples induced more than 4000 revertants/g of soil in TA98. Mutagenic potencies of most of the soil samples from Bangkok were moderate (100-500 revertants/g of soil) or low (less than 100 revertants/g of soil) in both strains with and without S9 mix, and only one sample induced more than 1000 revertants/g of soil in both strains. In the characterization of mutagens, organic extracts of two and one soil samples from Aichi and Bangkok, respectively, were separated on an octadecylsilyl (ODS) column for HPLC, and mutagenicities of the resulting fractions were examined using S. typhimurium YG1024 without S9 mix. For the two soil samples from Aichi, retention times of the distinct mutagenic fractions were similar at 40-74 min, and especially high mutagenicities were detected in three fractions corresponding to dinitropyrene isomers. In contrast, distinct mutagenicities were detected at retention times of 6-29 min for the sample from Bangkok, and marked mutagenicity was found in the fraction corresponding to another nitroarene. These results indicate that surface soils in urban areas in Aichi and Bangkok are commonly contaminated with mutagens, but major mutagenic constituents in the surface soils are different.

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Both cytochrome P450 (CYP) 2A6 and CYP2A13 are involved in the metabolic activation of tobacco-specific N-nitrosamines (TSNAs). Some kinds of TSNAs induce lung adenocarcinoma in laboratory animals. Thus, polymorphisms of CYP2A6 and CYP2A13 genes may be important as genetic factors of lung cancer, particularly lung adenocarcinoma. We genotyped 179 patients with lung cancer and 183 controls for CYP2A6 gene deletion-type and CYP2A13 C3375T polymorphisms by polymerase chain reaction-based techniques on DNA prepared from peripheral blood. In addition, a questionnaire was used to collect demographic, lifestyle and other information from each subject. Unconditional logistic regression was used to compute the odds ratios (ORs) and their 95% confidence intervals (95% CIs), with adjustments for several covariates found to be associated with risk. Compared with one or more copies of the T allele, the CYP2A13 CC genotype was associated with a substantially increased risk for lung adenocarcinoma (OR = 2.41, 95% CI = 1.03-6.28). Smokers with the CYP2A13 CC genotype showed significantly higher risk (OR = 4.88, 95% CI = 1.35-26.53) compared to non-smokers with possession at least one copy of the CYP2A13 T allele. Although our results indicate that the CYP2A13 gene may play a role in the development of lung adenocarcinoma, the risk for those individuals with single at-risk genotype of CYP2A13 C3375T polymorphism may not be so large. In future studies, analyses based on haplotypes are recommended to confirm the role of CYP2A13 gene in the development of lung adenocarcinoma.

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To assess the safety of the food flavoring agents, methyl anthranilate (MA) and methyl N-methylanthranilate (MNMA), their relationships with metabolism and allergenicity were examined in guinea pigs. Both MA and MNMA were hydrolyzed to anthranilic acid (AA) and N-methylanthranilic acid (N-methylAA), respectively, by guinea pig liver microsomes. These hydrolytic activities at 1000 μM were 320 and 35 nmol/min/mg protein, respectively. Moreover, MNMA was N-demethylated to MA by the liver microsomes; the oxidative activity at 1000 μM of MNMA was 2.8 nmol/min/mg protein. The N-demethylase activity for N-methylAA at 1000 μM in the liver microsomes was 3.9 nmol/min/mg protein. Kinetic analysis indicated that the V_max/K_m values of MA and MNMA hydrolyses were 15- and 7.4-fold greater in guinea pig liver microsomes than in the cytosol, respectively, suggesting that these hydrolytic activities were predominantly localized in the microsomes. Liver microsomal activities for the hydrolysis of these flavoring esters were markedly inhibited by diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and bis(p-nitrophenyl)phosphate but not by physostigmine. These hydrolytic activities were suppressed by aspirin, a substrate of carboxylesterase, in a concentration-dependent manner. The N-demethylations of MNMA and N-methylAA were inhibited by SKF 525-A, a nonselective inhibitor of cytochrome P450. At the same time as the metabolic study described above, skin reactions in guinea pigs were investigated using MA, MNMA, N-methylAA, and AA. All compounds examined elicited positive skin reactions, although MNMA and AA exhibited relatively greater sensitizing properties. These results may provide useful information about metabolism in the toxicologic evaluations of MA and MNMA.

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The effects of hexavalent chromium on the cell growth of green paramecium, Paramecium bursaria (P. bursaria), were investigated in this study. Two strains (MB-1 and F₁ generation) of P. bursaria were incubated in lettuce media supplemented with different concentrations of potassium dichromate under light (L) L (24 hr light), L dark (LD) (12 hr light : 12 hr dark) and DD (24 hr dark) conditions, and the IC₅₀ values were obtained. The IC₅₀ 7-day value showed that the toxicity of potassium dichromate was light-sensitive in both strains of P. bursaria. The results of the toxic effect of chromium on the cell shape of P. bursaria (BWK-4) showed that the body ratio of P. bursaria increased, even if the cells were incubated for 24 hr with 0.5 mM potassium dichromate solution, indicating that the cell shape of P. bursaria is very sensitive to potassium dichromate. The average amount of chromium accumulated in green paramecium ranged from 1.72 to 15.5 pg Cr/cell in a time- and concentration-dependent manner. This finding indicates the possibility of the use of P. bursaria as a biomonitor and bioaccumulator for chromium contaminants in aquatic environments. The experiment with electrical stimuli into the culture of P. bursaria indicated that cells accumulated at the negative electrode, suggesting that P. bursaria carried a positive charge. Thus, because the positive charge does not cause any significant absorption of chromium into the cells, P. bursaria may develop a variety of mechanisms for chromium accumulation in the cell. Further research studies are required to elucidate the mechanism of chromium uptake in P. bursaria.

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To investigate the effects of the nitric oxide (NO) generating rate on neuritogenesis, we compared the neuritogenic activity of several NO donors with varying generating rates; NOR1 (t1/2 = 1.8 min), NOR2 (t1/2 = 28 min), NOR3 (t1/2 = 30 min), and NOR4 (t1/2 = 60 min). Each NO donor promoted neurite outgrowth in PC12h cells in a concentration-dependent manner. However, the profiles were not dependent on the NO-generating rate. The neuritogenesis promoted by NOR4 was inhibited by the NO trapping agent carboxy-2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide, sodium salt (PTIO). NOR2 and NOR3, which have similar generating rates, were more toxic than NOR1 and NOR4 in PC12h cells. NOR2 and NOR3 induced cytotoxicity at concentrations of 100 μM and above, while NOR1 and NOR4 induced cytotoxicity at concentrations of 200 μM and above. Preconditioning medium containing these NO donors in the absence of cells were less cytotoxic towards PC12h cells. Therefore, we conclude that the neuritogenic action of NO in PC12h cells is dependent on a suitable generating rate, but not dependent on the amount of NO.

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We have found that genipin, a herbal iridoid compound, can induce neurite outgrowth in rat pheochromocytoma PC12h cells. In this study, we examined the neurotrophic effects of genipin in mouse neuroblastoma Neuro2a cells. Genipin significantly induced neurite outgrowth in a concentration-dependent manner in Neuro2a cells cultured in serum-free medium supplemented with transferrin/insulin/progesterone (TIP) without cytotoxicity. Neuro2a cells cultured in serum-containing medium were mitotic, while the proliferation of these cells cultured in serum-free medium supplemented with TIP was suppressed for up to 48 hr. Serum and TIP-free medium clearly increased the number of cells stained with trypan blue compared with serum-free medium supplemented with TIP. Damage to the cells was significantly reduced by adding genipin to the cells in serum and TIP-free medium. As a positive control for a neurotrophic action, dibutyryl cAMP, a membrane-permeable cAMP analog, also induced neuritogenesis in cells cultured in serum-free medium supplemented with TIP and enhanced the survival of cells cultured in serum and TIP-free medium. These results indicate that genipin has both neuritogenic activity and a survival effect on serum-deprivation-induced cell death in Neuro2a cells. This suggests that genipin can act as a neurotrophic factor-like compound in neuronal cells.

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We describe a rapid and simple method for the analysis of the d- and l-isomers of seven methamphetamine-related compounds and the d-isomer of pseudoephedrine (pseudoEP) by capillary electrophoresis/mass spectrometry (CE/MS) with direct injection of urine. The compounds were methamphetamine (MA), amphetamine (AP), dimethylamphetamine, ephedrine, norephedrine, methylephedrine, p-hydroxymethamphetamine (pOHMA). The electrolyte was 1 M formic acid/1 M ammonium formate (10/0.2, v/v) (pH 2.0) containing 1.5 mM heptakis-(2,6-diacetyl-6-sulfato)-β-cyclodextrin. The 14 enantiomers and d-pseudoEP were completely separated within 30 min. A urine sample was mixed with an equal volume of internal standard solution, filtered with a 0.45 μm filter and then injected into the CE/MS system. In an analysis of urine sample from a healthy person spiked with racemic MA and AP, the reproducibilities (n = 6) of the migration times and peak areas after correction by an internal standard were under 0.08% and under 3.6%, respectively. The detection limits using selected ion monitoring were 0.01 μg/ml (which corresponds to 0.02 μg/ml urine) for the enantiomers of MA, AP and pOHMA. The detection yields of the enantiomers of MA, AP and pOHMA from urine were in the range of 97.7-108.8%. The proposed method was successfully used for the chiral analysis of urine samples from MA and dimethylamphetamine (DMA) addicts and patients under selegiline pharmacotherapy.

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Several staining methods have been developed to monitor protein fibril formation. Two widely used dyes that are now utilized in standard staining assays are Congo red and Thioflavin T (ThT). However, non-specificity, false negative results and a requirement for expensive instrumentation have precluded the use of these dyes in the characterization of amyloidogenic proteins. In this study, we developed a simple method to follow specific binding of β₂-microglobulin (β₂m) fibrils using UV-visible (Vis) spectroscopy with the Trypan blue (TB) dye. The use of UV-Vis spectroscopy as a technique for amyloid fibril demonstration serves as an advantage due to the availability of the instrument in most laboratories. Binding of β₂m fibrils was achieved by combining a solution of TB with a concentrated fibril solution followed by UV-Vis spectroscopy. Here we observed a significant shift of the λ_max towards a longer wavelength when TB specifically binds with the fibrils. Also, the observed increase in absorbance upon binding of TB was dependent on the amount of fibrils. This new and simple assay adds to the variety of staining methods which may potentially be used to analyze other protein fibrils like the Aβ in Alzheimer's disease and the prion protein in transmissible spongiform encephalopathy.

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The antidiabetic activity of Thea sinensis was investigated in KK-Ay mice, an animal model of genetically type 2 diabetes with hyperinsulinemia. The water extract of Thea sinensis (green tea cold ext) (100 mg/kg body weight) reduced the blood glucose of KK-Ay mice 4 and 8 weeks after repeated administration and tended to decrease the plasma insulin level of KK-Ay mice under similar conditions. However, green tea cold ext did not affect blood glucose levels in normal mice. Green tea cold ext decreased blood glucose levels in an insulin tolerance test. These results suggest that the antidiabetic activity of green tea cold ext is derived, at least in part, from a decrease in plasma insulin, due to decreased insulin resistance.

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The quantity of isocitrate was determined using an apparatus containing a reactor with immobilized isocitrate dehydrogenase in a flow line. NADH formed by an enzymatic reaction was fluorometrically detected. The optimal concentration of NAD⁺ in the carrier was determined. The maximum peak areas due to NADH were observed at pH 8.0 when the pH of the carrier consisting of Tris buffer ranged from 6.0 to 8.6. Various buffer types were also examined as carrier mediums at pH 8.0. In contrast to 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) and triethanolamine buffers which afforded comparable peak areas with that of Tris buffer, phosphate buffer showed a reduced peak area. This peak area decreased with an increase in the pH of the phosphate buffer from 6.5 to 8.5, suggesting the inhibitory effect of phosphate dianions upon the binding of adenosine-5′-monophosphate (AMP) to the binding site of the enzyme. When the carrier composed of Tris buffer (0.1 M, pH 8.0) was used, the calibration curve for isocitrate was linear in the range of 0.1-50 μM (r = 1.000). The detection limit (S/N = 3) was 0.07 μM. Relative standard deviations of the peak area at 1 and 10 μM were 4.0% (n = 7) and 2.8% (n = 7), respectively. Thirty samples of isocitrate (2 μM) were analyzed for 1 hr. This method was applied to the analysis of total isocitrate in several beverages. The recovery tests for the isocitrate added to samples indicatied the reliability of the present method.

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As a part of our study to clarify the interaction of food ingredients with the aryl hydrocarbon receptor (AhR), we studied the interaction of 10 constituents (phytol, p-coumaric acid, 5 flavonol glycosides, adenosine, guanosine and uridine), isolated from aqueous alcohol extracts of spinach and komatsuna, with the AhR by applying the AhR-based bioassay for dioxins, the Ah-Immunoassay and the AhR-dependent luciferase reporter gene assay. Among them, flavonol glycosides and phytol had slight inhibitory effects on AhR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at high concentrations, however the effects were not comparable with those produced by kaempferol, regarded as one of the main antagonists of AhR. Also, flavonol glycosides showed weak luciferase inductions at high concentrations.

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Zinc promotes the proliferation of bovine aortic endothelial cells depending on endogenous fibroblast growth factor-2 whose activity is modulated by proteoglycans. In this study, we investigated the effects of zinc on the synthesis of proteoglycans in proliferating cells. It was demonstrated that zinc neither influenced the core protein synthesis nor the glycosaminoglycan chain formation during the synthesis of proteoglycan molecules, suggesting that zinc stimulation of endothelial cell proliferation is independent of proteoglycan synthesis.

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Inorganic arsenic undergoes extensive reduction and oxidative methylation in cells to yield a reactive metabolite of inorganic arsenic monomethylarsonous acid (MMA^III), which has a high reactivity toward vicinal thiols. Our epidemiological study in an endemic area of chronic arsenic poisoning and in experiments with rabbits exposed to arsenic revealed that arsenic exposure results in a reduction of systemic nitric oxide (NO) production. In this study, we examined the effect of MMA^III on endothelial NO synthase (eNOS) activity. With the membrane fraction of bovine aortic endothelial cells (BAEC), it was found that MMA^III with an IC₅₀ value of 2.1 μM was a potent inhibitor of eNOS, whereas inorganic arsenic and their methylated metabolites had no effect on eNOS activity. Interestingly, addition of dithiothreitol markedly blocked the MMA^III-induced inhibition of eNOS activity. This report is the first to suggest that MMA^III interacts with eNOS protein through presumably vicinal thiols, leading to decreased eNOS activity.

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We studied the effect of exercise on ovariectomized mature multiparous rats. Old retired breeder Sprague-Dawley rats were ovariectomized and divided into a sedentary and a treadmill-trained (20 weeks) groups. Exercise training decreased body weight gain, fat weight around the uterus and plasma triglyceride content significantly compared with the sedentary group, but food intake was unaffected. Extensor digitorum longus and soleus muscle weights and their glycogen content showed a significant increase in the training group. These results suggested that exercise training in ovariectomized multiparous rats is associated with decreased body weight gain and enhanced glycogen storage.

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The Shumiya cataract rat (SCR) is a hereditary cataract model in which lens opacity appears spontaneously in both the nuclear and perinuclear regions of 10-11 week old animals. The expression of drug metabolizing enzymes, including cytochrome P450 (CYP) and conjugation enzymes, was investigated in the ocular tissues of SCRs during cataractogenesis. Significantly high levels of gene expression were detected for CYP1A1, CYP2B2, CYP2C11 and CYP2E1 in normal lenses, whereas CYP1A1 and CYP2C11 were at lower levels in the cataractous lenses. The gene expression profiles of conjugation enzymes were also compared between normal and cataractous ocular tissues in which a moderate reduction in ST1A1 and ST1B1 expression, and an extensive loss of UGT1A1 expression, were observed in the SCR animals that had developed cataracts. In contrast, the elevated expression of the GSTA family of genes was evident in cataractous lenses. The reduced expression of several drug metabolizing enzymes in the cataractous ocular tissues might therefore be related to cataractogenesis via the accumulation of endogenous or exogenous toxic substances.

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The relationship between the serum β-cryptoxanthin concentration and circulating biochemical markers of bone metabolism in healthy individuals with the intake of juice prepared from Satsuma mandarin (Citrus unshiu MARC.) containing β-cryptoxanthin was investigated. Twenty volunteers (10 men and ten women) were divided into two groups of 10 volunteers (5 men and 5 women) each, and each group was sequentially given juice (192 ml) containing two different amounts of β-cryptoxanthin once a day for 56 days as follows: either regular juice with naturally occurring β-cryptoxanthin 802 μg/100 ml or reinforced juice containing β-cryptoxanthin 1500 μg/100 ml. γ-Carboxylated osteocalcin, which is a marker of bone formation, and bone tartrate-resistant acid phosphatase (TRAP), which is a marker of bone resorption, were assayed. The serum β-cryptoxanthin concentration was significantly increased with the intake of regular juice for 56 days. This increase was significantly enhanced by the intake of β-cryptoxanthin-reinforced juice. The intake of regular juice or of β-cryptoxanthin-reinforced juice for 56 days caused a significant increase in serum γ-carboxylated osteocalcin and a significant decrease in serum bone TRAP activity. A positive relationship between serum β-cryptoxanthin and circulating γ-carboxylated osteocalcin concentrations was found using the value obtained from all groups for before intake and with the intake of regular juice and β-cryptoxanthin-reinforced juice. A negative relationship between serum β-cryptoxanthin concentration and circulating TRAP activity was observed. This study shows that a relationship between serum β-cryptoxanthin and circulating bone metabolic markers is found in healthy individuals with the intake of juice containing β-cryptoxanthin.

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Toxicity and Bioaccumulation of Hexavalent Chromium in Green Paramecium, Paramecium bursaria
M. Golam Mortuza, Toshiyuki Takahashi, Tatsuya Ueki, Toshikazu Kosaka, Hitoshi Michibata and Hiroshi Hosoya.....Vol. 51 No. 6 pp. 676 Wrong:........ supplemented with different concentrations of potassium dichromate under light (L) L (24 hr light), L dark (LD) (12 hr light : 12 hr dark) and DD (24 hr dark) conditions, and ........
Right:........ supplemented with different concentrations of potassium dichromate under LL (24 hr light), LD (12 hr light : 12 hr dark) and DD (24 hr dark) conditions, and ........

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