Biomedical subjects

Kazuichi Hayakawa

Publications and source records attributed to Kazuichi Hayakawa.

At least 19 recordsLinked to original sources

PubMed2006

Simultaneous determination of urinary hydroxylated metabolites of naphthalene, fluorene, phenanthrene, fluoranthene and pyrene as multiple biomarkers of exposure to polycyclic aromatic hydrocarbons.

A method is presented for determining monohydroxy polycyclic aromatic hydrocarbons (OHPAHs) having 2-, 3- and 4-rings in human urine by using high-performance liquid chromatography with fluorescence detection. A urine sample containing conjugates of OHPAHs was hydrolysed in the presence of beta-glucuronidase/aryl sulfatase and the solution was cleaned up with a solid-phase extraction (C(18) and silica). Eight OHPAHs, namely 1- and 2-hydroxynaphthalenes, 2-hydroxyfluorene, 2-, 3- and 4-hydroxyphenanthrenes, 3-hydroxyfluoranthene and 1-hydroxypyrene, were separated and 1- and 9-hydroxyphenanthrenes co-eluted on an alkylamide-type reversed-phase column with fluorimetric detection. The urinary concentrations of OHPAHs were quantified by using deuterated 1-hydoxypyrene as an internal standard. The method showed good repeatability for inter- and intra-day precisions as well as good linearity of calibration curves (r (2) ranged from 0.996 to 0.999). The limits of detection (S/N=3) were in the range from 2.3 fmol to 2.2 pmol per injection. This method was successfully applied to urine samples from non-smoking taxi drivers, traffic policemen and rural villagers of Chiang Mai, Thailand. The results showed higher urinary concentrations of OHPAHs in rural villagers, consistent with higher respiratory exposure to PAHs.

Adult↗

PubMed2006

Development of septum-free injector for gas chromatography and its application to the samples with a high boiling point.

A novel apparatus with a simple structure has been developed for introducing samples into the vaporizing chamber of a gas chromatograph. It requires no septum due to the gas sealing structure over the carrier gas supply line. The septum-free injector made it possible to use injection port temperatures as high as 450 degrees C. Repetitive injection of samples with boiling points below 300 degrees C resulted in peak areas with relative standard deviations between 1.25 and 3.28% (n=5) and good linearity (r(2)>0.9942) for the calibration curve. In the analysis of polycyclic aromatic hydrocarbons and a base oil, the peak areas of components with high boiling points increased as the injection port temperature was increased to 450 degrees C.

Calibration↗

PubMed2006

Determination of 1-nitropyrene metabolites by high-performance liquid chromatography with chemiluminescence detection.

Three metabolites of 1-nitropyrene, i.e., 3-hydoxy-1-nitropyrene (3-OHNP), 6-hydoxy-1-nitropyrene (6-OHNP) and 8-hydoxy-1-nitropyrene (8-OHNP), were sensitively and selectively detected by HPLC with peroxyoxalate chemiluminescence (CL) detection. In the system, the three OHNPs were reduced to their corresponding amino derivatives through a reduction column packed with Pt/Rh-coated alumina and then concentrated on a concentrator column. By rotating a switching valve, the analytes were eluted into a separator column (ODS), separated and then detected by the bis(2,4,6-trichlorphenyl)oxalate-H(2)O(2) CL system. The detection limits (signal-to-noise ratio=3) were in the range from 5fmol (6-OHNP and 8-OHNP) to 12fmol (3-OHNP) per injection. To demonstrate the proposed method, it was used to determine three compounds in the incubation mixture of 1-nitropyrene and rat S9 mix.

Chromatography, High Pressure Liquid↗

PubMed2006

Damage to and recovery of coastlines polluted with C-heavy oil spilled from the Nakhodka.

The damage to and recovery of the Japanese coastline from Suzu, Ishikawa Prefecture to Mikuni, Fukui Prefecture was investigated visually over three years after a C-heavy oil spill from the Russian tanker "Nakhodka" in the Japan Sea on January 2, 1997. The beached C-heavy oil tended to remain for a long time on coasts of bedrock and boulder/cobble/pebble but it was removed rapidly from coasts of gravel/sand and man-made structures such as concrete tetrapods. On the coasts of the latter type, wave energy appeared to be the main force removing the oil. One year after the spill, C-heavy oil tended to remain strongly on the sheltered coasts of bedrock and boulder/cobble/pebble. Even on coasts of this type, the contamination was remarkably absent by 2 years after the spill. The concentration levels of polycyclic aromatic hydrocarbons (PAHs) in oil lumps, sand and seawater were monitored during 3 years following the spill. The concentrations of PAHs having 2 or 3 rings decreased more quickly than did those of PAHs having 4 or more rings, suggesting that volatilization was the main cause of the decrease. On the other hand, the concentrations of PAHs having 4 to 6 rings did not start to decrease until 7 months after the spill. The main cause of the decrease seemed to be photolysis. The concentration of BaP in seawater off the polluted coasts was high 1 month after the spill and then decreased. Three years after the spill, the level fell to the sub ng/L level, which was as low as the level in seawater along unpolluted clean coasts in Japan. The concentration of BaP in greenling was higher than the normal level only during the first two months after the spill. These results suggest that the coastlines in Ishikawa and Fukui Prefectures that were polluted with C-heavy oil recovered in 3 years.

Animals↗

PubMed2006

Homologue and isomer distribution of dioxins observed in water samples collected from Kahokugata Lagoon and inflowing rivers, Japan.

Water samples were collected at 17 sites in Kahokugata Water Basin, a closed water basin in central Japan. We determined the concentration of dioxins of the water samples. Linear relationships between toxic equivalent (TEQ) concentrations of dioxin and concentrations of suspended solid (SS) were obtained at sites in Kahokugata Lagoon and in the rivers flowing into the lagoon. Homologue composition of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) indicated that all the water samples were still strongly influenced by chlorinated herbicides, such as chloronitrofen (CNP) and pentachlorophenol (PCP) that had been widely used in rice fields. The main isomer distributions of the PCDD homologues were not significantly different among the sampling sites, while the main isomer distributions of the PCDF homologues were considerably different among the sampling sites. At a few sampling points in the downstream part of one of the rivers, high concentrations of 1,3,6,7,8-pentachloro dibenzofuran (1,3,6,7,8-PeCDF) and its related isomers (1,3,6,8-chlorine-substituted PCDFs) were traced to a dye manufacturing plant. These non-toxic isomers are usually only minor constituents in environmental water samples and are not indicators of any known dioxin sources. The dyeing discharge was found to make a contribution only in the water samples collected near the plant and the seasonal variation of the contribution might depend on the flow rate of the river.

Dioxins↗

PubMed2006

Occurrence of polybrominated diphenyl ethers and polychlorinated biphenyls in shark liver oil supplements.

Results are reported of a pilot survey of concentrations of polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) in shark liver oil supplements. Eleven brands of dietary supplements were analysed using an isotope dilution GC/MS method. Total concentrations of 10 PBDE congeners (BDE-28, -47, -49, -66, -99, -100, -153, -154, -155 and -183) ranged from 0.1 to 53 ng g-1 oil weight and total concentrations of six PCB congeners (CB-28/31, -52, -118, -153 and -180) in the samples ranged from 16 to 340 ng g-1 oil weight (undetected values are not included). Two brands of Japanese deep-sea shark liver oil contained the highest levels of PBDEs (49-53 ng g-1 oil weight) and PCBs (290-340 ng g-1 oil weight). These results indicate that PBDEs may have entered Japanese deep-sea waters.

Animals↗

PubMed2006

A new on-line sample preparation system for the liquid chromatography/time-of-flight mass spectrometry simultaneous analysis of pesticides in river water.

Current on-line solid-phase extraction methods combined with HPLC for shortening the clean-up operation are not suitable for simultaneously detecting compounds that have a wide variety of hydrophobicities. To solve these problems, we designed a new on-line sample preparation system. The system consists of an eluting pump, a mixing TEE connector, a 10-port 2-position valve and a solid-phase extraction precolumn. The eluate from the precolumn is diluted with a weak solvent from the HPLC at the TEE connection to load low hydrophobic compounds onto the analytical column. The proposed on-line sample preparation system was successfully applied to the simultaneous analysis of 21 pesticides in river water using LC/TOF-MS. In this method, the recoveries from river water samples were 67 to 126% (mean 83%), the reproducibility (CV%) was in the range from 1.1 to 11% (mean 5.6%), the calibration curve was linear in the range from 1 ppb to 500 ppb (r > 0.999) and the detection limits (S/N = 3) were in the range from 0.0034 ppb (daimuron) to 3.3 ppb (oxine-copper).

Automation↗

PubMed2005

Comparison of traditional cloud-point extraction and on-line flow-injection cloud-point extraction with a chemiluminescence method using benzo[a]pyrene as a marker.

In this work, using benzo(a)pyrene (BaP) as marker, the analytical merits of on-line flow-injection cloud-point extraction (FI CPE), including preconcentration factor, extraction efficiency, sample throughput, and analysis time were evaluated by use of peroxyoxalate chemiluminescence (CL) detection. Moreover, by detailed discussion of several preconcentration conditions for traditional and on-line FI CPE the advantages of on-line FI CPE became conspicuously apparent. When coupled with separation techniques such as high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE), on-line FI CPE-CL has much potential for analysis of low concentrations of polycyclic aromatic hydrocarbons (PAH) in environmental samples.

Benzo(a)pyrene↗

PubMed2005

Direct-acting mutagenicity of extracts of coal burning-derived particulates and contribution of nitropolycyclic aromatic hydrocarbons.

Benzene-ethanol extracts from particulates produced by coal burning were separated into four fractions by silica-gel column chromatography using n-hexane (240 ml), n-hexane-dichloromethane (3:1, v/v) (200 ml), dichloromethane (200 ml) and methanol (450 ml), as the corresponding eluents. The mutagenicity of each fraction was assayed by the Ames test using the Salmonellatyphimurium YG1024 strain. The nitropolycyclic aromatic hydrocarbons (NPAHs) of each fraction were assayed by high-performance liquid chromatography with chemiluminescence detection. The highest activity was observed in the n-hexane-dichloromethane fraction (Fr. 2). The mutagenic contribution of this fraction was 69.9% of the total of the four fractions. Ten of 11 NPAHs detected were in Fr. 2 and one (1-nitropyrene) was most concentrated in Fr. 3. Among the NPAHs examined, 3-nitrobenzanthrone made the largest mutagenic contribution. This is the first report of detection of 3-nitrobenzanthrone in coal burning-derived particulates.

Air Pollutants↗

PubMed2005

Chiral ligand exchange micellar electrokinetic chromatography using borate anion as a central ion.

Three compounds having 1,2-diol structure (1-phenyl-1,2-ethanediol, 3-phenoxy-1,2-propanediol, and 3-benzyloxy-1,2-propanediol) were enantioseparated by ligand exchange MEKC using (5S)-pinanediol (SPD) as a chiral selector and borate anion as a central ion together with SDS. When (S)-1,2-propanediol, (S)-1,2,4-butanetriol, or (S)-3-tert-butylamino-1,2-propanediol were used as the chiral ligand instead of SPD, these three compounds were not enantioseparated. When borate was replaced with 2-aminoethane-1-sulfonate or N-cyclohexyl-3-aminopropanesulfonate, no chiral separation was achieved. Therefore, the hydrophobic interaction between the chiral selector and the chiral analytes within the transient diastereomeric complex may play an important role in the enantioseparation achieved by the proposed method.

Borates↗

PubMed2005

Evaluation of estrogenic activities of hydroxylated polycyclic aromatic hydrocarbons in cigarette smoke condensate.

Estrogenic activities of cigarette smoke condensates obtained from the extraction of particulate matters from mainstream and sidestream cigarette smoke with benzene/ethanol were evaluated by using a yeast two-hybrid assay system expressing human estrogen receptor alpha (hERalpha). To identify the constituents of the cigarette smoke condensate which are responsible for the estrogenic activity, the condensate was fractionated into eleven fractions by liquid-liquid extractions. Among these fractions, the neutral fractions of mainstream and sidestream smoke showed the strongest estrogen receptor-mediated activity by the yeast two-hybrid assay. Then the neutral fractions were fractionated by medium-pressure liquid chromatography with silica gel column. In the fractions that showed strong estrogenic activity, 2-hydroxyfluorene (2-OHFle), 2- and 3-hydroxyphenanthrene (2- and 3-OHPhe), 1-hydroxypyrene (1-OHPyr) and n-propyl-p-hydroxybenzoate (n-PHB) were identified by LC- and GC-MS and HPLC with fluorescence detection. 2-OHFle, 2-OHPhe and n-PHB exhibited estrogenic activity, whereas weak activity was observed with 3-OHPhe and 1-OHPyr. Several other hydroxylated polycyclic aromatic hydrocarbons having no activity were also identified. This is a first study to identify estrogenic hydroxylated PAHs in cigarette smoke condensate. The present findings points out the necessity for detailed investigation of exposure to aerosols containing apparently estrogenic compounds.

Chromatography, High Pressure Liquid↗

PubMed2005

Simultaneous chiral analysis of methamphetamine and related compounds by capillary electrophoresis/mass spectrometry using anionic cyclodextrin.

A capillary electrophoresis/mass spectrometry method for the simultaneous chiral analysis of enantiomers of methamphetamine (MA), amphetamine (AP), dimethylamphetamine (DMA), ephedrine (EP), norephedrine (NE) and methylephedrine (ME) in urine has been developed. The background electrolyte was 1 M formic acid (pH 1.7). Using 0.85 mM heptakis(2,6-diacethyl-6-sulfato)-beta-cyclodextrin as the chiral selector, the 12 enantiomers were completely separated within 25 min. The detection limits were 0.01 microg mL(-1) for the enantiomers of MA, AP, DMA, EP and ME, and 0.02 microg mL(-1) for the enantiomers of NE using selected ion monitoring. The reproducibilities of within-run (n = 4) for the migration times and peak areas of the standard mixture were under 0.58% and 7.83%, respectively. The calibration curves of the peak areas of the 12 enantiomers were linear in the range of 0.05 - 10 microg mL(-1). This method was applicable to the analysis of urine samples.

Cyclodextrins↗

PubMed2004

Chiral ligand exchange capillary electrophoresis using borate anion as a central ion.

Native DL-pantothenic acid, having a 1,3-diol structure, was chirally resolved by ligand exchange capillary electrophoresis using (S)-3-amino-1,2-propanediol as a chiral selector and the borate anion as a central ion. The optimum conditions for both high resolution and short migration time of DL-pantothenic acid were found to be 200 mM (S)-3-amino-1,2-propanediol and 200 mM borate buffer (pH 9.2) containing 15% methanol with an applied voltage of +25 kV at 20 degrees C, using direct detection at 200 nm. With this system, the resolution (Rs) of racemic pantothenic acid was approximately 1.7. When (S)-1,2-propanediol, (S)-1,2,3-propanetriol, (S)-1,3-butanediol or (S)-1-amino-2-propanol were used as chiral ligand instead of (S)-3-amino-1,2-propanediol, DL-pantothenic acid was not enantioseparated. When borate was replaced with Tris or butylborate, no chiral separation was achieved. Therefore, the ionic interaction between the amino and carboxyl groups of the ternary complex may play an important role in the enantioseparation of DL-pantothenic acid by the proposed CE system.

Anions↗

PubMed2004

Antiandrogenic activity of extracts of diesel exhaust particles emitted from diesel-engine truck under different engine loads and speeds.

To clarify the alteration of androgenic and antiandrogenic activities by diesel engine conditions, we collected diesel exhaust particles (DEP) samples emitted from a diesel-engine truck under different conditions of engine loads and vehicle speeds, and DEP extract (DEPE) samples were prepared from each. The androgenic and antiandrogenic activities of the DEPE samples were examined using a prostate specific antigen (PSA) promoter-luciferase reporter gene assay in PC3/AR human prostate cancer cells. While all DEPE samples did not exhibit androgenic effects, the antiandrogenic effects were enhanced by higher engine load but not by higher vehicle speed. In this study, significant correlations between antiandrogenic and aryl hydrocarbon receptor (AhR) agonistic activities were demonstrated in PC3/AR cells by 16 polycyclic aromatic compounds and beta-naphthoflavone. Yeast two-hybrid assay and cytochrome P450 (CYP) 1A1 promoter-luciferase reporter gene assay showed that the antiandrogenic constituents acting as androgen receptor (AR) antagonists and AhR agonists were increased by only the higher engine load. In conclusion, the antiandrogenic effects of DEPE samples were enhanced by a higher engine load which resulted in DEPC samples with elevated AhR agonistic and AR antagonistic activities.

Adenocarcinoma↗

PubMed2004

Hair analysis of nicotine and cotinine for evaluating tobacco smoke exposure by liquid chromatography-mass spectrometry.

A simple liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method for the determination of nicotine and cotinine in human hair was established. In the procedure, a hair sample (10 mg) was washed with dichloromethane and digested in 2.5 M sodium hydroxide. The digest was extracted with dichloromethane and then 25 mM hydrochloric acid in methanol was added to the extract, to prevent loss of analytes. The solution was evaporated and redissolved in the mobile phase, methanol/10 mM ammonium acetate (30/70, v/v). A 20 microL aliquot of redissolved solution was subjected to analysis. Nicotine and cotinine in human hair were quantified by using deuterated analytes as internal standards. The quantification limits were 8 microg/L for nicotine and 0.9 microg/L for cotinine. The proposed method was applied to measure the concentrations of nicotine and cotinine in hair of smokers and non-smokers to evaluate their self-reported smoking and exposure to environmental tobacco smoke. In both cases, the method provided good selectivity, accuracy and precision.

Chromatography, Liquid↗

PubMed2004

Determination of atmospheric nitrobenzanthrones by high-performance liquid chromatography with chemiluminescence detection.

A method using high-performance liquid chromatography with chemiluminescence detection was developed for analyzing mutagenic nitrobenzanthrone (NBA) isomers in airborne particulates. The method was a modification of our previously described method for analyzing nitropolycyclic aromatic hydrocarbons (NPAHs). The pretreatment and reducing conditions for 1-, 2-, 3- and 10-NBAs were the same as those for NPAHs. In order to separate these NBA isomers, we used a polymeric-type ODS column (Cosmosil 5C-18MS); a mixture of 40% acetonitrile and 60% 10 mM imidazole-HClO4 buffer was employed as the mobile phase at a flow rate of 1 mL/min. The isomers of 1-, 2-, 3- and 10-NBA were determined in chemiluminescence with linear calibration graphs from 0.1 to 4 pmol, from 200 to 4000 pmol, from 1 to 50 pmol and from 10 to 400 pmol, respectively. The detection limits (S/N = 3) of 1-, 2-, 3- and 10-NBA isomers were 0.02 pmol, 35 pmol, 0.3 pmol and 3 pmol, respectively. The method was used to analyze airborne particulates at a heavy traffic site in Kanazawa. 2- and 3-NBAs were detected in the extracts of the particulates, while 1-NBA and 10-NBA were not detected. The atmospheric concentrations of 2- and 3-NBAs were 1.83 pmol/m3 and 24.7 fmol/m3, respectively.

Journal Article↗

PubMed2003

Antiandrogenic activities of diesel exhaust particle extracts in PC3/AR human prostate carcinoma cells.

We collected diesel exhaust particles (DEPs) emitted from three diesel-engine vehicles--a car, a bus, and a truck--in daily use, and prepared DEP extracts (DEPEs), designated as EC, EB, or ET, respectively. The androgenic and antiandrogenic effects of the DEPE samples were examined by a luciferase reporter assay in human prostate carcinoma PC3/AR cells transiently transfected with a prostate specific antigen gene promoter-driven luciferase expression vector pGLPSA5.8. PC3/AR is a subline of human prostate carcinoma PC3 transformed to stably express wild-type human androgen receptor (AR). While DEPE samples did not exhibit any androgenic effect, they exerted antiandrogenic effect, inhibiting dihydrotestosterone (10 pM) -induced luciferase activity by 24 to 52% at an extract concentration of 10 microg/ml. The antiandrogenic effect was greater in the following order: ET > EB > EC. Co-treatment of PC3/AR cells with SKF-525A, a nonselective inhibitor of cytochrome P450 (CYP) enzymes, enhanced the antiandrogenic effect, indicating that the antiandrogenic effect is caused by intact species of DEPE constituents. The antiandrogenic effect of DEPE samples was reversed by alpha-naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist. The antiandrogenic activity of a DEPE sample correlated with its AhR agonist activity assayed in PC3/AR cells transiently transfected with CYP1A1 gene promoter-driven luciferase expression vector pLUC1A1. Equimolar mixtures of ten polycyclic aromatic hydrocarbons (PAHs) having four or more rings, structures found in the DEPEs, showed significant antiandrogenic effects and AhR agonist activity at concentrations equivalent to those found in DEPE samples. Further, DEPE samples elicited only antiandrogenic effects in recombinant yeast cells, which express beta-galactosidase in response to androgen. A competitive AR binding assay showed that AR-binding constituents exist in DEPE samples, indicating that greater part of AR-binding constituents in DEPEs are AR antagonists. All these findings show that DEPE samples exhibit significant antiandrogenic effect in cell-based transcription assay and that this effect is due in part to the constituents with AhR agonist activity including PAHs and to the constituents with AR antagonist activity.

Adenocarcinoma↗

PubMed2003

A role of aryl hydrocarbon receptor in the antiandrogenic effects of polycyclic aromatic hydrocarbons in LNCaP human prostate carcinoma cells.

The role of aryl hydrocarbon receptor (AhR) on the antiandrogenic effects of polycyclic aromatic hydrocarbons (PAHs) was studied in LNCaP cells. The PAHs used in this study were chrysene (Chr), benzo[ k]fluoranthene (BkF), benzo[ a]pyrene (BaP), anthracene (Ant) and pyrene (Pyr). Chr, BkF and BaP acted as AhR agonists in LNCaP cells, while Ant and Pyr did not. The antiandrogenic effects of the PAHs were evaluated on the basis of regulation of prostate-specific antigen (PSA) mRNA and protein levels by 5alpha-dihydrotestosterone (DHT). Chr, BkF and BaP exhibited an antiandrogenic effect, but Ant and Pyr did not. alpha-Naphthoflavone (alpha-NF), an AhR antagonist, reversed the antiandrogen action of Chr, BkF and BaP, suggesting a requirement for activated AhR. The antiandrogenic PAHs did not significantly decrease androgen receptor (AR) levels or cellular DHT concentrations. Gel mobility shift assays revealed that Chr, BkF and BaP inhibited the binding of AR in nuclear extracts to oligonucleotide probes containing the AR-responsive element (ARE), whereas Ant and Pyr had no effect. The antiandrogenic PAHs elevated mRNA levels of c-fos and c-jun. Since activator protein-1 (AP-1), a heterodimer of c-jun and c-fos proteins, is known to inhibit binding of AR to ARE by protein-protein interaction with AR, the findings in the present study suggest a possible involvement of AP-1 in the antiandrogenic effects of PAHs acting as AhR agonists. These results suggest that AhR can stimulate AP-1 expression resulting in inhibition of the binding of AR to ARE in the transcription regulatory region of target genes such as PSA.

Androgen Antagonists↗