The formation of trihalomethane (THM) from 37 organohalogen compounds, disinfection by-products, was measured by the headspace-gas chromatography/mass spectrometry (GC/MS) method. Among these 37 organohalogen compounds, 10, including trichloroacetones, trihaloacetonitriles and trihaloacetic acids, produced THMs upon heating of aqueous solutions at 60°C for 30 min in a headspace sampler. The amounts and composition of THMs produced by heating depended on the position and number of the bound chlorine or bromine. In addition, the production of THMs from some of the organohalogen compounds upon heating was strongly affected by pH, temperature and heating duration. These results suggest that the headspace-GC/MS method overestimates the net concentration of THM due to positive errors.
The pollution level of the Tsurumi River flowing into Tokyo Bay was studied longitudinally using river sediment extracts in an assay system based on the arylhydrocarbon receptor (AhR)-dependent induction of 7-ethoxycoumarin O-deethylase (ECOD) activity in HepG2 cells. The sampling points of river sediment were as follows: Namamugi, Kami-Sueyoshi, Tsunashima, Shin-Yokohama A and B (downstream and upstream, respectively, from the former open-air industrial waste incineration site), Kozukue and Nakayama (Fig. 1). ECOD activity was induced to different extents in all samples tested. Namamugi, located in the middle of Keihin Industrial District, was considered to be the most polluted, followed by Shin-Yokohama A and Tsunashima. Results from these samples showed reverse U-shaped dose-response curves in terms of ECOD activity as well as 3-methylcholantrene (3-MC) although the expression of CYP1A1 mRNA in HepG2 cells remained roughly constant at higher concentrations as in the case of 3-MC, thus implying post-transcriptional suppression.
Cytidine 5′-diphospho (CDP)-choline: 1-O-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (AAG-CPT), an enzyme responsible for de novo synthesis of platelet-activating factor (PAF), was solubilized from porcine spleen microsomes using digitonin. Although the activity of the solubilized enzyme was relatively stable, further purification by sequential chromatography on Toyopearl HW-65 gel filtration and diethylaminoethyl (DEAE)-Toyopearl 650 caused a remarkable decrease in enzyme activity, which was partially recovered by the exogenously addition of phospholipids such as egg phosphatidylcholine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine, dioleoylphosphatidylglycerol, and dioleoylphosphatidylserine. In contrast, dioleoylphosphatidic acid (DOPA) showed an inhibitory effect on enzyme activity. In addition, lysophospholipids such as monooleoylphosphatidylcholine, monooleoylphosphatidylethanolamine, monooleoylphosphatidylglycerol, and monooleoylphosphatidic acid showed an inhibitory effect on the enzyme activity. When DOPA was concomitantly added with DOPC, the enzyme activity reactivated by DOPC decreased with the ratio of DOPA to DOPC. Furthermore, we examined whether phosphatidic acid (PA) or lysophospholipids had an inhibitory effect on AAG-CPT activity under more physiological conditions. Treatment of microsomes with exogenously added phospholipase D or phospholipase A2 resulted in a decrease in AAG-CPT. In addition, when endogenous phospholipase D was activated by fatty acids such as oleic acid and arachidonic acid to generate PA in the porcine microsomes, the enzyme activity was significantly inhibited. The molecular weight of the enzyme solubilized from porcine spleen microsomes was estimated to be 440 kDa based on gel-filtration column chromatography on Toyopearl HW-65, suggesting that this enzyme formed a complex with other protein molecules and membrane phospholipids, and that these phospholipids were necessary to maintain the enzyme activity. Our results indicate that environmental membrane phospholipids containing PA and/or lysophospholipids are important factors in the regulation of the enzyme for de novo synthesis of PAF.
Mutagenicity of size-fractioned airborne particles collected with a multi-stage fractioned sampler, an Andersen low pressure impactor, was measured by microsuspension assay using Salmonella typhimurium YG1024 strain (× 20 conc. bacterial solution). Fine particle samples showed mutagenicity with a good dose-response relationship under the conditions of both with and without a metabolic activation system (S9 mix). In generally, the activities without S9 mix were higher than those with S9 mix, and the highest activity per unit air volume was observed in the sample of 0.52 μm in diameter. Most of the negatives were observed in coarse particle samples. The size distribution of mutagenic activity per unit air volume was skewed to the smaller size range and had one peak at 0.52 μm, although that of mass concentration was bimodal, having two peaks bounded around 1-2 μm in diameter. The highest mutagenic activity per unit mass of particles in a series of fractioned samples was observed in the sample of less than 0.22 μm in diameter. It was suggested that ultrafine particles were more mutagenic than fine particles (PM 2.5-0.1), as if the mass concentrations of ultrafine particles were 10% or less than those of fine particles. It was also shown the Andersen low pressure impactor is very useful for studies of carcinogens and mutagens in suspended particles in ambient air.
In this paper, Caenorhabditis elegans (C. elegans) is proposed as a model organism for studying chemical effects over multiple generations. We investigated whether C. elegans responds to vertebrate steroid hormones. We found that estrogenic steroids, especially estradiol (E2), have a cholesterol-like potency in supporting the reproduction of C. elegans. In contrast, testosterone (TS) and diethylstilbestrol (DES) did not display this potency. On the other hand, E2, TS and DES supressed the fecundity rate of C. elegans, when culture carried out with cholesterol. Moreover effect of TS accumulated over generation, in contrast to the other chemicals tested. These data suggested that with convenient biomarkers such as fecundity, C. elegans might be an effective model organism for studying chemical actions, including the disruption of reproduction.
Our previous study suggested that UVB irradiation of the organophosphorus pesticide, fenthion and its photodegradation product, 3-methyl-4-methylthiophenol (MMTP) yielded fenthion sulfoxide and 3-methyl-4-methylsulfinylphenol (MMSP), and that the formation mechanism was related to generation of singlet oxygen (1O2). The objective of this study was to elucidate the 1O2-triggered photooxidation mechanism of fenthion in detail. Generation of 1O2 in the photooxidative reaction was directly detected by electron spin resonance (ESR) technique with 2,2,6,6-tetramethyl-4-piperidone (TMPD) as a selective 1O2 spin trapping agent which yields 2,2,6,6-tetramethyl-4-piperidone-1-oxyl (TEMPONE). When fenthion and MMTP solutions were irradiated by UVA or UVB light, the TEMPONE signal was observed. However, no signal was detected after exposure of MMSP, dimethyl phosphorothioate or fenthion sulfoxide solutions to the UV light. The production of the signal in fenthion and MMTP solutions was more predominant under UVB irradiation than under UVA irradiation. When the signal was detected in these solutions, fenthion sulfoxide and MMSP were also formed in amounts proportional to the signal intensity. The TEMPONE signal intensity and the formation of these oxidative products were significantly inhibited by the addition of 1O2 scavenger, l-histidine or sodium azide to the reaction medium. The study provided the first direct evidence that 1O2 is generated during photolysis of fenthion and MMTP by UV irradiation. We also proposed its oxidation mechanism of fenthion and MMTP.
Zinc is an essential and multifunctional element for all cells. Metallothionein (MT) is a low molecular weight, cystein-rich, metal-binding protein that is involved in zinc homeostasis. During the acute-phase reaction, hepatic MT production is induced and hepatic zinc accumulation is stimulated. We previously reported that MT-I and II-deficient (MT-null) mice are highly sensitive to the lethal effects of lipopolysaccharide (LPS) plus D-galactosamine (GalN). The sensitization may relate to attenuatoin of α1-acid glycoprotein (AGP) expression in MT-null mice. In the present study, we hypothesized that MT-induced hepatic zinc accumulation promotes AGP expression and prevents LPS/GalN-induced lethality. To determine whether zinc reduces LPS/GalN toxicity, zinc was administered to mice. Simultaneous administration of zinc and LPS/GalN showed no effect on the lethality of LPS/GalN in mice. Zinc administration at 3 hr prior to LPS/GalN challenge reduced LPS/GalN-induced death. However, zinc pre-administration at 24 hr before LPS/GalN challenge did not reduce LPS/GalN-induced death. The expression of AGP mRNA was elevated at 3 hr after zinc administration, 24-hr pretreatment was ineffective. The protective effect of zinc was observed in both wild-type and MT-null mice. These results show that the protective effects of zinc were not caused by MT induction, but by AGP expression. We suggest that MT-induced hepatic zinc accumulation may promote AGP expression and thus prevent LPS/GalN-induced lethality.
Grape seed extract (GSE) is one of Japan's natural food additives, and is recommended for use as an antioxidant. The component of interest is polyphenol, mainly proanthocyanidins, which are condensed tannins. A vanillin-hydrochloric acid (HCl) assay is specific for flavan-3-ol. The sensitivity of GSE components to the vanillin-HCl assay differed according to the chemical structure. We applied this assay to the determination of proanthocyanidin content in GSEs, health foods containing GSEs and grape seed oils. The concentration of proanthocyanidins in GSEs and health foods was overestimated in the vanillin-HCl assay when (+)-catechin was used as a standard. However, the concentration of proanthocyanidins in health foods with high protein or a high colored substance content was underestimated. No proanthocyanidins were detected in grape seed oil. Furthermore, we examined the content of low molecular weight compounds in GSE by reversed-phase HPLC. The percentage of low molecular weight compounds, including gallic acid, (+)-catechin, (-)-epicatechin, procyanidins B1, B2 and C was estimated to be 5.5-12.2%(w/w).
Simultaneous determination of ethanol and acetaldehyde was performed with an apparatus consisting of two enzyme reactors placed either side of an octadecylsilica column in a single flow line. The enzymes used were alcohol dehydrogenase for ethanol analysis, and aldehyde dehydrogenase for acetaldehyde analysis. The most favorable concentration of NAD+ in the carrier for the simultaneous determination of ethanol and acetaldehyde was studied in wine or sake, in which the ethanol concentration was much higher that that of acetaldehyde. An NAD+ concentration of 0.1 mM was adopted. To distinguish between NADH formed due to ethanol and that formed due to acetaldehyde, several buffers (pH 7.8) were also examined for use as the carrier medium, and triethanolamine buffer was found to be the most favorable. The calibration curve for acetaldehyde was linear (r2 = 1.000) in the range of 0.2-100 μM. With respect to ethanol, plotting the logarithm of the peak area versus that of the concentration gave a linear relationship (r2 = 0.997) in the range of 0.04-100 mM. This method was applied to the simultaneous analysis of ethanol and acetaldehyde in several types of liquor. The results were similar to those obtained using a commercially available test-kit method, suggesting the reliability and practicality of this method in analyzing real samples.
The response of environmental pollutants can be detected bioanalytically focusing on the source and matrices of concern. Cell culture bioassays are rapid and inexpensive and thus have great potential for routine monitoring of aquatic resources. Such novel in vitro assays are a new tool to investigate lipophilic and low volatile compounds and are a powerful complement to instrumental analysis. The ethoxyresorufin O-deethylase (EROD) microbioassay was conducted to determine cytochrome P4501A (CYP1A) activity in environmental samples, and the calculated 3-methylchloranthrene (3-MC) equivalent concentration (MEQ) was introduced as a new quantitative water quality parameter. The chemical MEQ was calculated by multiplying induction equivalency factor (IEF) to GC-MS analysis data. And biological (bio-) MEQ was calculated by comparing the concentration response curve of the sample with those of the 3-MC calibration curve. Therefore, chemical MEQ is an estimation of the toxic effects of polycyclic aromatic hydrocarbons (PAHs) and bio-MEQ is the total toxic effects of various CYP1A-inducing chemicals in water samples. In this study, bio-MEQ values of water samples were higher than chemical MEQ values and total PAH concentrations, indicating that there must be other compounds in the water sample effective as inducers of EROD and that the biological activities of mixture compounds are mainly due to additive effects. There was a good correlation between bio-MEQ and total PAH concentration. The difference between bio-MEQ and total PAH concentration was high in downstream areas, i.e., polluted sites. Bio-MEQ calculated based on the enzyme-inducing effect of water samples could give information about the biological potency of water samples caused by PAH-like compounds.
Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-chlorotriazine), one of the most widely used herbicides, is classified as a possible human carcinogen by the US Environmental Protection Agency and is a known endocrine disruptor. Recent research has indicated that the estrogenic and antiestrogenic effects elicited by this chemical are not mediated by the estrogen receptor. In this study, we also observed that not only atrazine but also its metabolites did not affect basal or 17β-estradiol induced MCF7-BUS cell proliferation. Therefore, to explain the apparent endocrine disrupting effects of atrazine in vivo, an estrogen-independent mechanism and its metabolites were studied. We examined the alteration in aromatase (CYP19) activity in human choriocarcinoma JEG-3 cells by atrazine and its major metabolites (hydroxyatrazine, diaminochlorotriazine), the conversion of estradiol to hydroxylated metabolites by these compounds associated with cytochrome P4501A. The common metabolite of atrazine, diaminochlorotriazine inhibited the aromatase activity responsible for estrogen synthesis in JEG-3 cells. Atrazine and its metabolites induced 7-ethoxyresorufin-O-deethylase activity and catalyzed estrogen metabolism in MCF-7 cells. These results suggest that the antiestrogenic effects of atrazine may relate to a decrement in estrogen levels caused by aromatase inhibition and estrogen metabolism stimulation by atrazine and its metabolites.
Thirty-one endocrine disruptors, including alkylphenols, phthalates and styrene oligomers, in diesel and gasoline exhaust particulate matter were determined simultaneously by gas chromatography/mass spectrometry (GC/MS). When particulate matter was subjected to Soxhlet extraction and Kuderna-Danish concentration, four phthalates, an adipate and five styrene trimers were found to exist as contaminants from the ambient air. Regarding the other 21 compounds, only benzo[a]pyrene and n-pentylphenol were detected in the range of 0.5-11 and 0.1-1.1 μg/km driving distance, respectively, from both diesel and gasoline exhaust particulate matter. When particulate matter was subjected to only ultrasonic extraction, the above phthalates, adipate and styrene trimers were not detected as contaminants from the ambient air. Using this treatment method, the above phthalates, adipate and styrene trimers were not detected in either of the two types of exhaust particulate matter. The above results revealed that the presence of such endocrine disruptors as benzo[a]pyrene and n-pentylphenol in diesel and gasoline exhaust particulate matter must be investigated in greater detail. Furthermore, it becomes clear that prevention of contamination during sample pretreatment is necessary.
Enzymatic hydrolysis of phthalate esters in human saliva was investigated to characterize salivary esterase in the formation of monoesters from their diesters. The monoesters formed were analyzed by GC/MS after incubation of phthalate diesters in the saliva. Hydrolytic activity in the supernatant obtained by centrifugation of the saliva at 1350 × g was equivalent to that in whole saliva, and the activity was inhibited by the addition of denaturing protein. The hydrolytic activity was dependent on the protein concentration in the supernatant. The optimum temperature and pH of the hydrolysis was 50°C and 8.2, respectively. In addition, the 80% acetone powder of the supernatant showed high substrate specificity for straight-chain alkyl group of phthalate diesters, especially the butyl group, whereas almost no specificity was seen for the 2-ethylhexyl and benzyl groups. These results indicate that not the oral flora but salivary esterase, such as lingual lipase, is involved in phthalate monoester formation from the diesters in human saliva, and do not act on the hydrolysis of monoester.
The tumor-promoting activity of four phthalate diesters and three monoesters were tested in vitro in a screening assay using a transformed and non transformed BALB/c3T3 cell mixture and in a transformation assay using Bhas cells (v-Ha-ras-transfected BALB/3T3 cells). Di-2-ethylhexyl phthalate (DEHP), di-n-butyl phthalate (DBP), and their monoesters increased cell proliferation in the screening assay. Mono-n-butyl phthalate (MBuP) and mono-2-ethylhexyl phthalate (MEHP) promoted Bhas cell transformation, whereas the activities of DBP and DEHP were weak. The activity of MEHP was about 1/500 that of 12-O-tetra-decanoyl-phorbol-13-acetate. MBuP and MEHP therefore showed tumor-promoting activity, whereas all the other phthalate esters tested, including di-iso-nonyl phthalate, had little or no such activity. In addition, MBuP and MEHP were formed by hydrolysis in Bhas cells exposed to DBP and DEHP for 4 days. We postulate that MEHP plays a role in the slight ability of DEHP to promote cell proliferation.
After adding the silylating agents N, O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) or N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA) to the indirubin solution and leaving for 1 hr at 60°C, the color remained red. Indirubin could be measured by GC/MS after replacing the active hydrogen on the amino group with -Si(CH3)3 or -Si(CH3)2C2H5 groups. However, peak tailing was observed and the quantitative and detection limits were not sensitive enough for practical use. Indirubin silylated at four sites was observed under the reaction conditions as follows; solvent dichloromethane: acetone (8 : 2), 90°C reaction temperature, 1 hr reaction time, BSTFA derivative, pyridine catalyst. The color of the solution changed from red to colorless. Retention time appeared to be faster and the peak shape improved. Under these conditions, the quantitative and detection limits of indirubin were 5 ppb and 0.1 ppb, respectively.