[Occupational Exposure of Pharmacists to Drugs during the Preparation of Powder Drugs in Dispensing Pharmacies].

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.

Comparative inhibition of tetrameric carbonyl reductase activity in pig heart cytosol by alkyl 4-pyridyl ketones.

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 the substrate-binding domain of TCBR.

2,5-Dihydroxy-4-methoxybenzophenone: a novel major in vitro metabolite of benzophenone-3 formed by rat and human liver microsomes.

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.

Intramolecular rearrangement of linolenate peroxyl radicals in lipoxygenase reactions at lower oxygen content.

Variation of tissue oxygen content is thought to be a possible factor in determining the structural diversity of hydroperoxy fatty acids. In the present study, we evaluated the structural diversity of intermediate carbon-centered radicals at lower oxygen content. When the buffered solution (pH 7.4) containing 1.0 mM alpha-linolenic acid, 1.0 muM soybean 15-lipoxygenase, and 1.0 mM nitroxyl radical [3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmDeltaP)], which selectively traps carbon-centered radicals, was incubated in a sealed vial, the generation of linolenate hydroperoxide was completed within 1 min. In the subsequent reaction at lower oxygen content, the production of the [LnA-H+O(2)].-CmDeltaP adduct was ascertained by liquid chromatography tandem mass spectrometry with precursor ion scanning. Furthermore, HPLC analysis with photodiode array detection showed that the adduct exhibits an absorption maximum at 278 nm, indicating a conjugated triene moiety. On the basis of these facts, the structure of the adduct was speculated to be C(2)H(5)-CH(CmDeltaP)-CH = CH-CH = CH-CH = CH-CH(OOH) -C(7)H(14)-COOH. We proposed a possible reaction pathway as follows: a linolenate 9-peroxyl radical generated in the lipoxygenase reaction might be converted into C(2)H(5)-.CH-CH = CH-CH = CH-CH = CH-CH(OOH) -C(7)H(14)-COOH through an intramolecular rearrangement. This intermediate radical may give rise to hydroperoxy fatty acids with structural diversity.

Detection of a novel mutagen, 3,6-dinitrobenzo[e]pyrene, as a major contaminant in surface soil in Osaka and Aichi Prefectures, Japan.

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.

Detection of genistein as an estrogenic contaminant of river water in Osaka.

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.

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.

Comprehensive two-dimensional high-performance liquid chromatography for the separation of polycyclic aromatic hydrocarbons.

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.

Mutagenic activity and quantification of nitroarenes in surface soil in the Kinki region of Japan.

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.

Determination of 3-nitrobenzanthrone in surface soil by normal-phase high-performance liquid chromatography with fluorescence detection.

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.

Determination of mutagenic 3-nitrobenzanthrone in diesel exhaust particulate matter by three-dimensional high-performance liquid chromatography.

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).

An automatic method for the determination of carcinogenic 1-nitropyrene in extracts from automobile exhaust particulate matter.

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.