Search

Occupational exposure of pharmacists to drugs during powder drug preparation in dispensing pharmacies was investigated. First, we determined frequently prescribed tipepidine hibenzate and ambroxol hydrochloride suspended in the air of the dispensing room. The median concentration of the drugs in the air was 0.01 μg/m3 and 0.02 μg/m3, respectively; these values indicate that the air in the dispensing room was contaminated with powder drug. To estimate drug exposure during powder drug preparation, drug dust was collected near the nose of workers. Analysis of the active ingredients of the drugs used in the preparation revealed that eight drugs, including bethanechol, l-carbocisteine, and zonisamide, were detected in the range of 1.5-1220 μg/m3. Assuming that the respiratory volume of an adult was 0.008 m3/min, it was estimated that 0.4-36 μg of the ingredients were exposed per prescription by multiplying concentration, respiratory volume and sampling time (3-5 min). Furthermore, the effect of wearing a medical mask on the drug powder exposure was evaluated using a self-made apparatus. When the amount of drug powder collected on filters that is either covered with or without a medical mask was compared, the covered filter exhibited reduced drug powder accumulation to less than 10% the amount collected on the uncovered filter. The present data suggested that a medical mask might decrease the drug dust allergies in dispensing pharmacists.

Context and objective: The present study is to elucidate the comparative inhibition of tetrameric carbonyl reductase (TCBR) activity by alkyl 4-pyridyl ketones, and to characterize its substrate-binding domain.Materials and methods: The inhibitory effects of alkyl 4-pyridyl ketones on the stereoselective reduction of 4-benzoylpyridine (4-BP) catalyzed by TCBR were examined in the cytosolic fraction of pig heart.Results: Of alkyl 4-pyridyl ketones, 4-hexanoylpyridine, which has a straight-chain alkyl group of five carbon atoms, inhibited most potently TCBR activity and was a competitive inhibitor. Furthermore, cyclohexyl pentyl ketone, which is substituted by cyclohexyl group instead of phenyl group of hexanophenone, had much lower ability to be reduced than hexanophenone.Discussion and conclusion: These results suggest that in addition to a hydrophobic cleft corresponding to a straight-chain alkyl group of five carbon atoms, a hydrophobic pocket with affinity for an aromatic group is located in th...

1. When benzophenone-3 (2-hydroxy-4-methoxybenzophenone; BP-3) was incubated with liver microsomes of untreated rats in the presence of NADPH, the 5-hydroxylated metabolite, 2,5-dihydroxy-4-methoxybenzophenone (5-OH-BP-3), was formed as a major novel metabolite of BP-3. The 4-desmethylated metabolite, 2,4-dihydroxybenzophenone (2,4-diOH-BP), previously reported as the major in vivo metabolite of BP-3, was also detected. However, the amount of 5-OH-BP-3 formed in vitro was about the same as that of 2,4-diOH-BP. 2. The oxidase activity affording 5-OH-BP-3 was inhibited by SKF 525-A and ketoconazole, and partly by quinidine and sulfaphenazole. The oxidase activity affording 2,4-diOH-BP was inhibited by SKF 525-A, ketoconazole and α-naphthoflavone, and partly by sulfaphenazole. 3. The oxidase activity affording 5-OH-BP-3 was enhanced in liver microsomes of dexamethasone-, phenobarbital- and 3-methylcholanthrene-treated rats. The activity affording 2,4-diOH-BP was enhanced in liver microsomes of 3-methylcholanthrene- and phenobarbital-treated rats. 4. When examined recombinant rat cytochrome P450 isoforms catalyzing the metabolism of BP-3, 5-hydroxylation was catalyzed by P450 3A2, 1A1, 2B1, 2C6 and 2D1, while 4-desmethylation was catalyzed by P450 2C6 and 1A1.

We previously identified 1,3-, 1,6-, and 1,8-dinitropyrene (DNP) isomers as major mutagens in surface soil in three metropolitan areas of Japan. In the present study, an organic extract from surface soil collected at a park in Takatsuki in Osaka Prefecture, which showed extremely high mutagenicity in Salmonella typhimurium TA98 in the absence of mammalian metabolic system (S9 mix), was investigated to identify major mutagens. A new powerful bacterial mutagen, as well as 1,6- and 1,8-DNP isomers, was isolated from the organic extract (1.8 g) of the soil sample (2.2 kg) by column chromatography. On the basis of mass spectra, the new mutagen, which accounted for 15% of the total mutagenicity of the soil extract, was thought to be a dinitrated polycyclic aromatic hydrocarbon with a molecular weight of m/z 342. The mutagen was synthesized from benzo[e]pyrene by nitration and was determined to be 3,6-dinitrobenzo[e]pyrene (DNBeP) based on its 1H NMR spectrum. The mutagenic potency of 3,6-DNBeP in the Ames/Salmonella assay was extremely high, in that it induced 285,000 revertants/nmol in TA98 and 955,000 revertants/nmol in YG1024 without S9 mix and was comparable to those of DNP isomers, which are some the most potent bacterial mutagens reported so far. In addition to the soil sample from Takatsuki, 3,6-DNBeP was also detected in surface soil samples collected at parks in four different cities, i.e., Izumiotsu and Takaishi in Osaka Prefecture and Nagoya and Hekinan in Aichi Prefecture, and accounted for 22-29% of the total mutagenicity of these soil extracts in TA98 without S9 mix. These results suggest that 3,6-DNBeP is a major mutagen in surface soil and may largely contaminate the surface soil in these two regions in Japan.

3-Nitrobenzanthrone (3-NBA) is an extremely potent direct-acting bacterial mutagen that has been detected in diesel exhaust particles, airborne particles and so forth. Recently, 3-aminobenzanthrone (3-ABA) and 3acetylaminobenzanthrone (3-AABA) were identified as metabolites of 3-NBA in mammalian cells. In this study, to clarify the genotoxicity of 3-ABA, 3-AABA and 3-NBA in vitro and in vivo, the mutagenicity and DNA-damaging activity of these chemicals were investigated by the Ames assay and by alkaline single gel electrophoresis (Comet assay), respectively. 3-ABA and 3-AABA were mutagenic toward four Salmonella typhimurium strains, i.e. TA98, TA100, YG1024 and YG1029, in the presence of a mammalian metabolic system (S9 mix). Both chemicals showed the highest mutagenicity toward YG1024, and induced 2180000 revertants/nmol of 3-ABA and 131000 revertants/ nmol of 3-AABA. 3-NBA also showed mutagenicity toward these four strains with and without S9 mix, but the potencies of 3-NBA in these strains were decreased by the addition of S9 mix. In the presence of S9 mix, the mutagenic activities of 3-NBA in four strains were comparable to or lower than those on 3-ABA. 3-ABA, 3-AABA and 3-NBA produced statistically significant DNA damage, which was detected by an increase in the DNA tail moment, in vivo 3 hr after intraperitoneal injection at 160 mg/kg body weight. 3-ABA, 3-AABA and 3-NBA induced significant DNA damage in the liver, kidney, spleen and lung, spleen only, and liver, kidney, lung and bone marrow, respectively. At a lower dose (40 mg/kg), 3-NBA produced significant DNA damage in the lung. These results indicate that 3-ABA, 3-AABA and 3-NBA are not only mutagenic in vitro in bacteria but also genotoxic in vivo in mouse.

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.

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 β-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.

Mutagenic 1-chloro-3-nitropyrene (1,3-CNP), 1,6-CNP and 1,8-CNP were detected in surface soil collected in Kyoto, Japan. First, 1,3-, 1,6- and 1,8-CNPs were synthesized by chlorination of 1-nitropyrene. Mutagenic activities obtained by Ames assay using Salmonella typhimurium strain TA98 under conditions without S9 mix were 1.22 revertants per nmol for 1,3-CNP, 1.14 for 1,6-CNP and 0.83 for 1,8-CNP. Miller's aryl hydrocarbon receptor (AhR) yeast reporter gene assay showed that the three CNPs had no AhR ligand activity. Surface soil was collected in Kyoto, Japan and analyzed for 1,3-, 1,6- and 1,8-CNPs by two-dimensional high-performance liquid chromatography with fluorescence detection and a zinc on-line catalytic column. 1,3-CNP, 1,6-CNP and 1,8-CNP were detected in the ranges 9.2-13.8, 3.4-4.6 and 4.3-5.0 pg per gram of soil (n = 2), respectively.

To clarify the mutagenic potential of surface soil in the Kinki region of Japan, particularly in Osaka and neighboring cities, 62 surface soil samples were collected and their organic extracts were examined by the Ames/Salmonella assay. All of the samples were mutagenic toward TA98 in both the presence and absence of a mammalian metabolic activation system (S9 mix). While all of the samples showed mutagenicity toward TA100 with S9 mix, only 45/62 (73%) were mutagenic without S9 mix. Fifty (81%) of the samples showed higher activity toward TA98 than TA100. The mean values of the mutagenicities of soil samples collected in Osaka prefecture (n=35) toward TA98 with and without S9 mix were 2315 and 1630 revertants per gram of soil, respectively, and these were 2.9 and 2.6 times as high as the values for samples from other prefectures (n=27), respectively. Three dinitropyrene (DNP) isomers, i.e. 1,3-, 1,6- and 1,8-DNP, and 3-nitrobenzanthrone (NBA) in the surface soil samples were quantified by fluorometric detection of the corresponding amino compounds, i.e. diaminopyrene isomers and 3-aminobenzanthrone, using high-performance liquid chromatography (HPLC). The three DNP isomers were detected in all of the soil samples (n=26) that were mainly collected in Osaka prefecture, and the amounts of 1,3-, 1,6- and 1,8-DNP were 6-1526, 11-1772 and 10-2092pg/g of soil, respectively. The contribution ratios of 1,3-, 1,6- and 1,8-DNP to the mutagenicity of soil extracts toward TA98 without S9 mix were 0.2-12, 0.3-12 and 0.5-27%, respectively. The amount of 3-NBA in soil samples (n=8) was 144-1158pg/g of soil, and the contribution ratio of 3-NBA to the mutagenicity of soil extracts was 2-38%. These results suggest that the surface soils in the Kinki region were highly polluted with mutagens and the pollution levels in Osaka prefecture were higher than those in other areas. DNP isomers and 3-NBA may be major mutagens that contaminate surface soil in this region.

A sensitive method for determining 3-nitrobenzanthrone in surface soil was developed. 3-Nitrobenzanthrone was reduced to 3-aminobenzanthrone by refluxing at 60 degrees C with hydrazine and Raney nickel for 20 min, and 3-aminobenzanthrone was determined by normal-phase high-performance liquid chromatography (HPLC) with fluorescence detection. We used a cyanopropyl stationary phase and an n-hexane-ethyl acetate (3:1, v/v) mobile phase, since 3-aminobenzanthrone exhibits fluorescence in a low-polarity solvent such as n-hexane or ethyl acetate, but not in a polar solvent such as water or methanol. The calibration graph showed good linearity (r2>0.9999) in the range of 0.002-2 ng, and the detection limit was 0.002 ng (S/N=3). 3-Nitrobenzanthrone in extracts from surface soil collected in the Chubu area (central area) of Japan was determined after clean-up using silica gel chromatography and high-performance liquid chromatography on a pyrenylethyl stationary phase. The concentration of 3-nitrobenzanthrone in surface soil was determined in the range of 1.2-1020 pg/g soil.

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.

The MCF-7 human breast cancer cell line has been widely used in studies in the fields of cancer biology, steroid hormone biochemistry and toxicology. MCF-7 cells were demonstrated to be enhanced cell proliferation by estrogens. The estrogen-dependent growth of MCF-7 cells is often called E-Screen, which is one of the most sensitive assays for estrogenicity assessment. On the contrary, there is a potential for this assay to identify false positives by cell mitogens and false negatives by cytotoxic or general growth inhibitors. Therefore, when we subject environmental samples to an E-screen test, we have to examine the interaction of contaminant substances in the cells. In this study, we evaluated the endocrine-disrupting activity of multiply contaminated model samples using the E-screen test. Through this study, we demonstrated that some chemicals prevent the estrogenicity of endocrine-disrupting chemicals, and furthermore, this estrogenicity is induced by other chemicals. This result indicates that the fractionation of chemicals in environmental samples is essential for evaluating their estrogenicity using a bioassay technique.

The mutagen 3-nitrobenzanthrone was determined in rainwater. Rainwater was collected at a residential area in Kyoto, Japan. Organic matter containing 3-nitrobenzanthrone in rainwater was extracted using a 3M (St. Paul, MN, U.S.A.) Empore Extraction Disk C18, and the extract was purified on a pyrenylethyl silylated silica gel column. The purified sample was then reduced using a platinum black column heated at 80°C. The reduced product of 3nitrobenzanthrone, i.e. 3-aminobenzanthrone, was analyzed by normal-phase HPLC with fluorescence detection. 3Nitrobenzanthrone in the rainwater was determined in the range of 0.07‐2.6 ng l ‐1 . The concentration was higher from May to July and lower from October to December. 1-Nitropyrene was also determined by reversed-phase HPLC with fluorescence detection after platinum black column reduction. 1-Nitropyrene was detected in the range of 0.0056‐0.19 ng l ‐1 , and this concentration range was lower than that of 3-nitrobenzanthrone.

Mutagenic 3-nitrobenzanthrone was determined in diesel exhaust particulate matter by three-dimensional high-performance liquid chromatography. Nitrophenylethyl, C18 and pyrenylethyl stationary phases were used as the first, second and third dimensions, respectively. Methanol was used as a mobile phase for the first and second dimensions, and dichloromethane was used for the third. Each column was coupled by a 6-port valve with a concentrator column. Effluent from the third dimensional column was detected by a photodiode array detector. The calibration graph showed good linearity in the range of 1-1000 ng ml(-1), and the detection limit (S/N = 3) was 1 ng ml(-1) 3-Nitrobenzanthrone could be detected within 45 min without the requirement of a clean-up procedure. 3-Nitrobenzanthrone in diesel exhaust particulate matter was detected in the range of 27-56 pg mg(-1) extract (n = 3).

In this study, airborne particulates were collected at three sites, two in a downtown area and the other in a suburban area of Kanazawa, Japan in each season for 7 years. Two polycyclic aromatic hydrocarbons (PAHs), pyrene (Py) and benzo[a]pyrene (BaP) and four nitropolycyclic aromatic hydrocarbons (NPAHs), 1-nitropyrene (NP) and 1,3-, 1,6-, and 1,8-dinitropyrenes (DNP) were determined by high-performance liquid chromatography with fluorescence and chemiluminescence detection. At the downtown sites, the mean concentration of each DNP was about two orders of magnitude lower than that of 1-NP and more than three orders of magnitude lower than those of Py and BaP. This tendency reflected the composition of PAHs and NPAHs in diesel-engine exhaust particulates. Concentrations of these PAHs and NPAHs were higher at the downtown sites than at the suburban site, suggesting the dilution of these compounds during the transportation from the downtown to the suburban area. The concentration ratios of NPAHs to PAHs were larger at the downtown sites than at the suburban site. Studies using UV light and sunlight showed that degradation of NPAHs was faster than that of PAHs. Thus, the lower concentrations of NPAHs in the suburban sites may be due to their being photodegraded faster than PAHs during the atmospheric transportation from the downtown area to the suburban area.

Airborne particulates were collected during the same periods in downtown Kanazawa, Sapporo and Tokyo and polycyclic aromatic hydrocarbons (PAHs) and nitropolycyclic aromatic hydrocarbons (NPAHs) were determined together with several heavy metal elements and water-soluble inorganic ions. The mean concentrations of metal elements and inorganic ions were the highest in Tokyo, followed by Sapporo and the lowest in Kanazawa both in winter and summer, and were strongly dependent on the amount of airborne particulates. These tendencies were different from the cases of PAHs and NPAHs. Correlations between the organic compounds (PAHs and NPAHs) and several inorganic pollutants were strong only in winter samples of Sapporo, but not so strong in Kanazawa and Tokyo. Because PAHs and NPAHs and several combustion-origin inorganic substances are, in general, detected mainly in the fine airborne particulates, it was reasonable that the significant correlations were found only in the winter samples of Sapporo where the generation of the coarse and crustal particulates were suppressed by snow or ice. Though the atmospheric concentrations of PAHs and NPAHs in Kanazawa were much lower than in Sapporo, the correlation among the NPAH compounds and between PAHs and NPAHs were surprisingly similar in these two cities. The strong correlations indicated that the atmospheric behaviors of the PAHs and NPAHs studied were similar in the two cities and that the source of PAHs and NPAHs might be the same. On the contrary, strong correlations were not observed in Tokyo, suggesting the possibility of multiple sources or formations/degradations of PAHs and

A sensitive method for the determination of 6-nitrochrysene (NC), 2-nitrofluoranthene (NF) and 1-, 2- and 4-nitropyrenes (NP) in airborne particulates is described. After ultrasonic extraction of airborne particulates with benzene/ethanol, the extract was injected into a high-performance liquid chromatographic (HPLC) system. The system consisted of four pumps, an auto injector, two separation columns (ODS), a reduction column (zinc/glass beads), a concentration column (ODS), a switching valve, a column oven, a chemiluminescence detector and an integrator. Nitroarenes were separated on the first separation column and reduced to their corresponding aminoarenes through the reduction column. The elution bands containing the analytes were concentrated selectively onto the concentration column by increasing the water concentration of the mobile phase. By rotating the switching valve, the analytes were introduced into the second separation column, separated and then detected chemilumigenically. Detection limits were in the 0.3–5 fmol range. Utilizing the above method, atmospheric concentrations of the above five nitroarenes in Kanazawa were determined.

Aromatic hydrocarbons in the oil spilled from•gthe Nakhodka•hand in the environmental samples including air, reached oil and sea water were determined by gas chromatography/mass spectrometric detection and high-performance liquid chromatography/fluorescence detection. Aromatic hydrocarbons determined in the oil were as follows: benzene, toluene, ethylbenzene, xylenes, naphthalene, acenaphthene, fluorene, anthracene, fluoranthene, pyrene, benz [a] anthracene, chrysene, benzo [b] fluoranthene, benzo[k] fluoranthene, benzo [a] pyrene, dibenz [a, h] anthracene and benzo [ghi] perylene. Concentrations of benzene, toluene, xylenes and naphthalene in the air collected at contaminated seashores were much lower than their toxic levels. However, a model experiment suggested that their concentrations in the air might be higher just after the oil spill. Although the naphthalene concentration in the reached oil samples tended to decrease with time

A sensitive determination method of 1 3-dinitropyrene (1,3-DNP), 1,6-DNP, 1,8-DNP, and 1-nitropyrene (1-NP) in airborne particulates has been developed. After adding 2-fluoro-7-nitrofluorene as an internal standard, these compounds were extracted with benzene-ethanol, cleaned, and reduced chemically to their corresponding amino derivatives. They were separated on a reversed-phase column with an acetonitrile-imidazole buffer and chemilumigenically detected using bis(2,4,6-trichlorophenyl)oxalate and hydrogen peroxide as postcolumn reagents. Their concentrations in urban air showed two peaks, in the morning and evening, and were higher in autumn and winter than in spring and summer. Mean concentrations were as follows : 1,3-DNP, 2.19 ± 0.81 fmol/m 3 ; 1,6-DNP, 4.03 ± 1.52 fmol/m 3 ; 1,8-DNP, 3.63 ± 1.40 fmol/m 3 ; 1-NP, 0.70 ± 0.28 pmol/m 3 . The mutagenic contribution of each DNP at the above level in air might be comparable to that of 1-NP or more.

Direct-acting mutagenic 1, 3-dinitropyrene (1, 3-DNP), 1, 6-DNP, 1, 8-DNP and 1-nitropyrene (1-NP) and indirect-acting mutagenic benzo [a] pyrene (BaP) in airborne particulates collected by the side of a busy intersection in downtown Kanazawa were determined by high-performance liquid chromatography (HPLC) with chemiluminescence and fluorescence detection. Time courses of their concentrations were high in the morning (8 : 00-10 : 00) and evening (16 : 00-20 : 00) and low from the midnight until early morning (0 : 00-6 : 00). Levels of each DNP (in the range of fmol/m3) and 1-NP (in the range of sub pmol/m3) were, respectively, more than three and about one order of magnitude lower than that of BaP. Large correlation coefficients (0.85-0.91) between their concentrations, traffic volume, and carbon monoxide and nitrogen monoxide concentrations suggested that the main source was vehicles. Utilizing the concentration ratios, ([1, 3-DNP]+[1, 6-DNP]+[1, 8-DNP])/[1-NP], in airborne particulates (0.014), gasoline particulates (0.56) and diesel particulates (0.013), contributions (%) of dieselengine vehicles to the three DNPs and 1-NP in the air were estimated to be 94.3% and 99.8%, respectively.

A simultaneous determination method for polycyclic aromatic hydrocarbons (PAHs)and nitro-PAHs (NPAHs) in airborne particulates was developed. Airborne particulates were extracted with benzene/ethanol, cleaned up by liquid-liquid partition, refluxed in the presence of NaSH and then the resultant solution was injected into an HPLC system. The system was hybridized into two systems with different optimum conditions for PAHsand NPAHs by introducing a switching valve and a mini separation column. Mobil ephases were acetonitrile/water (75 : 25) for PAHs, and acetonitrile/imidazole HClO4 buffer (1:1) for NPAHs and the postcolumn reagent solution for NPAHs was an acetonitrile solution containing bis(2, 4, 6-trichlorophenyl)oxalate and H2O2. By this method, four NPAHs (1, 3-, 1, 6-, 1, 8-dinitropyrenes and 1-nitropyrene) and eight PAHs (fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a] pyrene and coronene) in sub-milligram quantities of airborne particulates were de-termined chemilumigenically and fluorogenically, respectively using 2-fluoro-7-nitrofluorene as an internal standard within sixty minutes.

The estrogenic activity in water at various localities on Lake Biwa-Yodo River, a representative watershed in Japan, was measured using a recombinant yeast that expresses the human estrogen receptor. The yeast bioassay revealed that the activities of 13 water samples had an average value of 14 pmol/L (3.8 ng/L) (17beta-estradiol equivalent) with a very wide range from 0 to 72 pmol/L (0-19.6 ng/ L), and two of the samples had prominent levels of activity (72 pmol/L (19.6 ng/L) and 56 pmol/L (15.2 ng/L)). We analyzed these two samples with instrumental approaches. A high-performance liquid chromatogram profile showed that the strong activity in one sample, which was collected just downstream of a sewage-treatment plant, would be due to 17beta-estradiol and estrone, whose source is considered to be human urine contained in the effluent of the plant. The activity in the other sample, which was obtained from a tributary river in a primarily residential area with some industrial development (i.e., Osaka City), however, did not correspond to 17beta-estradiol, estrone, or synthetic chemicals known as estrogenic. Analysis of a fraction with estrogenic activity by liquid chromatography-mass spectrometry (LC-MS) provided evidence that the activity in the water sample resulted from the presence of genistein, an isoflavone compound of plant origin.

Various metal powders were batch screened as possible on-line reducers for nitropolycyclic aromatic hydrocarbons (NPAHs), in particular 1,3-dinitropyrene (1,3-DNP), 1,6-DNP, 1,8-DNP and 1-nitropyrene (1-NP) and its metabolites. Zinc was found to be an excellent reducer in chemiluminescent compatible mobile phases without any associated disposal problems. Zinc reducer column parameters, namely pH, buffer concentration, packing composition (zinc:glass beads or zinc:silica gel) and column length were all optimized for the HPLC determination system with chemiluminescence detection, named NPAH analyser. The system consisted of three pumps, sample injector, reducer column packed with zinc:glass beads, switching valve with a cutting loop, separation column (ODS), chemiluminescence detector and integrator. The mobile phase for the separation column and chemiluminescence reagent solution were respectively acetonitrile:imidazole–HClO4 and acetonitrile containing bis(2,4,6-trichlorophenyl)oxalate and H2O2. When the reducer column was introduced before the separation column, 1,3-, 1,6-, 1,8-DNPs and 1-NP in benzene:ethanol extracts from airborne particulates were separately determined with detection limits of 2–50 fmol, respectively, in a 100 μL sample. When the reducer column was introduced just after the separation column, 1-NP, 1-nitrosopyrene and 1-aminopyrene in the incubation mixture of 1-NP and the Salmonella typhimurium YG1021 strain were separately determined with detection limits as low as sub fmol levels. 1,8-DNP and its metabolite were also separately determined in the incubation mixture of 1,8-DNP and the S. typhimurium TA98 strain.

Distributions of 1,3-dinitropyrene (1,3-DNP), 1,6-DNP, 1,8-DNP, 1-nitropyrene (1-NP) and mutagenicity in airborne particulates collected in downtown Kanazawa, Japan with an Andersen high-volume air sampler were examined. Mutagenicities of benzene-ethanol extract from particulates were determined by the Ames test using S. typhimurium strains with S9 mix, while concentrations of DNPs and 1-NP were determined by high-performance liquid chromatography (HPLC) using chemiluminescence detection. In the finest particulate fraction (smaller than 1.1 μm), 68% and 75% of the total mutagenicities were observed in TA98 and YG1024 strains, respectively. In the same fraction, 65–82% of three DNPs as well as 84% of 1-NP were observed. Mutagenic contributions of 1,3-DNP, 1,6-DNP, 1,8-DNP and 1-NP in the extract were respectively 0.6, 1.2, 1.8 and 1.6% in the TA98 stain, and 2.5, 5, 9 and 2.1% in the YG1024 strain.

A comprehensive two-dimensional HPLC system for the separation of polycyclic aromatic hydrocarbons was developed using a pentabromobenzyl column as the first dimension and two short monolithic C18 columns as the second dimension. The primary column and two secondary columns were coupled by a 10-port 2-position valve. The effluent from the first dimension was repetitively injected into the second dimension every 12 s. Due to its resolution, this technique is a powerful tool for the separation of polycyclic aromatic hydrocarbons in a complex matrix such as environmental samples.

An automatic method for the determination of carcinogenic 1-nitropyrene in extracts from automobile exhaust particulate matter has been developed. Organic matter was extracted from particulate matter and the extract was concentrated. Then the extract was injected into a two-dimensional (2D) high-performance liquid chromatograph (HPLC) with a fluorescence detection system. To improve the sensitivity a large sample volume (300 microl) was applied to the HPLC system using the double on-column focusing technique. To achieve full automation and high selectivity a reduction column packed with platinum black was introduced after the 2D HPLC system using a C18 column as the primary column and a pentabromobenzyl column as the secondary column. This HPLC system could determine 0.01-3,000 ng ml(-1) of 1-nitropyrene in an extract from diesel and gasoline exhaust particulate matter at 40 min intervals.

Diesel-engine exhaust and airborne particulates were collected in downtown and suburban areas and five mutagenic nitroarenes (2-nitrofluoranthene (NF), 1-, 2- and 4-nitropyrenes (NPs) and 6-nitrochrysene (NC) in benzene-ethanol extracts were determined by high-performance liquid chromatography with chemiluminescence detection. 1- and 4-NPs and 6-NC were found in diesel-engine exhaust particulates, however, 2-NF and 2-NP were not detected. On the other hand, all five nitroarenes were found in airborne particulates. Diurnal concentration patterns of 4-NP and 6-NC in the downtown area were similar to the pattern of 1-NP, but the patterns of 2-NF and 2-NP were different from the pattern of 1-NP. These results strongly suggested that 2-NF and 2-NP are formed in the atmosphere. Of the five nitroarenes, 2-NF had the largest suburban / downtown concentration ratio. 2- and 4-NPs and 6-NC all showed larger ratios than did 1-NP. These results showed that 2-NF and 2-NP were formed in the atmosphere, but that the secondary formation of 4-NP and 6-NC were not known. Taking mutagenic activity and the atmospheric concentration into consideration, the mutagenic contribution of 2-NF was estimated to be comparable with that of 1-NP in the downtown area, and more than that of 1-NP in the suburban area. This result suggested that the mutagenic contribution of the minor nitroarenes formed in urban air might not be negligible.

Air samples were collected at the sides of busy traffic roads in downtown Kanazawa, Sapporo and Tokyo during the same periods both in the daytime and in the night, in winter and summer. We determined the levels of polycyclic aromatic hydrocarbons (PAHs) and nitropolycyclic aromatic hydrocarbons (NPAHs), which are known to be carcinogenic and/or mutagenic. PAHs and NPAHs were analyzed simultaneously with an HPLC system with fluorescence detection for PAHs and another HPLC system with chemiluminescence detection for NPAHs, respectively. The amounts of airborne particulates were larger in the daytime than in the night in all three cities. This result was thought to reflect the diurnal change of such urban activities as traffic volume. Many of the PAH and NPAH compounds showed higher concentrations in the daytime than in the night, and also showed higher concentrations in winter than in summer in the three cities. This result coincided with the trends observed in our continuous surveys in downtown Kanazawa for several years. However, 5-ring PAHs, such as benzo [a] pyrene, and dinitropyrenes in Tokyo did not show such diurnal or seasonal differences. The composition ratio of NPAHs and the contribution of diesel-engine vehicles to the atmospheric concentrations of NPAHs were calculated from all the data of this survey. The concentration ratio of dinitropyrenes to 1-nitropyrene became larger with an increase in the ratio of diesel-engine vehicles to the total vehicles registered, suggesting a large contribution of diesel-engine vehicles to the concentrations of these NPAHs in urban air.

Polycyclic aromatic hydrocarbons (PAH) and nitropolycyclic aromatic hydrocarbons (NPAH) in airborne particulates collected simultaneously at downtown and suburban sites and in exhaust particulates from diesel and gasoline engine vehicles were determined. The mean concentrations of all compounds in the atmosphere were lower at the suburban site. The differences between the two sites were smaller in airborne particulates than in the atmosphere. However, the difference in NPAH concentrations in particulates between the two sites was greater than the difference in PAH concentrations, suggesting that the NPAH were less stable than the PAH. The concentrations of 1-nitropyrene and 1,3-, 1,6- and 1,8-dinitropyrenes were generally much higher in automobile exhaust particulates than in airborne particulates because the former were collected directly. However, 2-nitrofluoranthene and 2-nitropyrene were observed in airborne particulates but not in automobile exhaust particulates. The diurnal concentrations s...