Recent progress of high performance liquid chromatography (HPLC) for drugs of abuse was reviewed on over 100 papers. First, papers concerning screening analyses for drugs of abuse using HPLC were reviewed and discussed about their usefulness. Secondly, the progress of new analytical methods for high-sensitive detection of amphetamines, morphine, codeine, 6-acetylmorphine, cannabinoids, cocaine and others were reviewed by dividing into 1. fluorometric, 2. electrochemical, and 3. chemiluminescence detections. Thirdly, new studies on the chiral assay for amphetamines using chiral derivatization or chiral column were reviewed. Furthermore, the application to discrimination between use of over-the-counter drugs and illegal amphetamines was exemplified. Fourthly, the utility of a photodiode array detector and a mass detector for the confirmation of drugs was shown by citing several papers. Moreover, it was discussed about automation of the HPLC analysis using the column switching method, capillary electrophoresis and supercritical fluid chromatography applied for the analysis of drugs of abuse.

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We summarized the present status of viruses detected in the water system in the human living environment such as sewage, reclaimed water, river water, drinking water, fish and shellfish, and swimming pool water. In addition, the effects of these viruses on human health were discussed based on previous cases of waterborne virus infection and epidemiological data. There are no marked cases of waterborne virus infection in Japan. However, as virus contamination of river water, a source of drinking water, and reclaim from an increase in sewage with advances in urbanization, surveillance of waterborne viruses is needed. We also introduced the present isolation methods of viruses in water. For surveillance of viruses in water, reliable detection methods that can be readily performed in many institutions should be established.

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The inhibitory effects of water extracts of five genera of aloe (Aloe arborescens MILLER, A. ferox M., A. barbadensis M., A. africana M., A. saponaria HAWAII) and aloe powder of Japanese Pharmacopoeia (JP Aloe powder) on histamine release from rat peritoneal mast cells induced by antigen and compound 48/80 were investigated in vitro. All the aloe extracts tested inhibited histamine release from mast cells induced by antigen and compound 48/80 in a concentration-dependent manner. The extract of A. ferox M. and JP Aloe powder paticularly strongly inhibited release by either antigen or compound 48/80 at medium inhibitory concentration (IC₅₀) of 0.16 and 0.07 mg/ml, respectively, for antigen and of 0.41 and 0.29 mg/ml for compound 48/80. These results suggest that aloes may be useful in the treatment of type I immediate allergic disorders. Barbaloin, a major constituent of aloes, also exhibited concentration-dependent inhibitory effects on histamine release induced by these two substances, with IC₅₀ being 0.02 and 0.06 mg/ml, respectively. From its yield and inhibitory effects, barbaloin is considered to account for the inhibitory action of the water extracts of A. arborescens M., A. africana M. and JP Aloe powder. Although tranilast, a current antiallergic agent, strongly inhibited antigeninduced histamine release (IC₅₀=0.07 mg/ml), it showed a weak inhibitory effect on compound 48/80-induced histamine release (IC₅₀=0.84 mg/ml). These results suggest that the mechanism of inhibitory action of barbaloin on histamine release differs from that of tranilast.

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A large quantity of discharge from sewage treatment plants flows into urban rivers. To evaluate the part this discharge plays in the organic pollution of river water, the distribution of urobilin was investigated. The amount of urobilin increased remarkably after rainfall, suggesting that the rivers are contaminated not only with discharge, but also with untreated sewage. Some rivers which run through both rural and urban areas were more polluted in their upper reaches than in their lower reaches, indicating that sewage was not treated properly in the upper reaches and flowed into the rest of the river. The contribution rate of untreated sewage, calculated from the amount of COD/urobilin of river water, was from 50 to 100% in the Kanda River in rainy weather. These investigations revealed that the main source of organic pollution of the river was sewage overflow.

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Aloenin is a major constituent of Aloe arborescens MILLER which has been utilized in Japan as a folk remedy for burns, insect bites, and skin reaction. In the present study, the effects of aloenin on sebaceous gland size, hair growth and damaged skin were investigated. Aloenin significantly promoted hair growth in depilated mice, but did not affect sebaceous gland function in the hamster ear. Aloenin also had recuperative effects on tape-stripped human skin as determined from parameters such as the shape factor of corneocytes, thick abrasion, nuclear ghosts and cellular arrangement of corneocytes. Since aloenin is effective in healing damaged skin, it may be useful for the treatment of dermatological conditions in the future.

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Morphine and codeine were analyzed in the human urine of volunteers after they had eaten one or two bean-jam buns, which are buns decorated with poppy seeds on top. Urine specimens were collected for 24 h after ingestion, and samples were hydrolyzed using enzymes and extracted with chloroform/1-butanol (9 : 1, v/v). The extracted alkaloids were converted to pentafluoropropionlyl (PFP) derivatives. The quantitation of morphine and codeine was carried out by GC/MS using deuterated analogs as internal standards. A significant amount of morphine was found in the urine of all volunteers after the intake of two buns, the highest concentration being found in specimens taken 2 or 4 h after consumption (13.6-123.6 ng/ml). In most volunteers, morphine was found in the urine as long as 24 h after the ingestion. The urinary codeine level was low in contrast to morphine ; it was also maximum at 2 or 4 h (2.6-5.2 ng/ml) in four volunteers and was negative (< 1 ng/ml) in two. When one bean-jam bun was eaten, the urinary morphine level was very low but was detectable for up to 6 h ; codeine was not observed in any urine sample. Forensic scientists should be careful in distinguishing urinary morphine and codeine levels caused by the illicit use of morphine or heroin from those due to the ingestion of foods with poppy seeds. These results should aid in developing a reasonable cut-off level in drug screening programs designed to detect street heroin or morphine use in Japan.

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本研究において, 上水に含まれる有機物の安全性を検討するために上水を粒状活性炭に通し, 吸着された有機物をクロロホルム及びエタノール等で抽出し, 回収された有機物の亜急性影響, 変異原性及び発がん性を調べた.その結果以下のことが明らかとなった.1) クロロホルム抽出物(CCE)は肝肥大を起こさせ, 肝薬物代謝酵素誘導作用をもち, 肝細胞に脂肪変性を起こす.また, 高濃度摂取では肝に壊死巣を形成し, ライソゾーム, 滑面小胞体の増加, 核内脂肪滴の出現など障害性の変化を認めた.2) また, CCE摂取による上記の生体影響は摂取中止後6日間で正常レベルまでに回復した.3) エタノール抽出物(CAE)の少量摂取では, 著名な変化は認めなかったが, 大量摂取では肝に壊死巣の形成, 細胞質の融解, ミトコンドリアの変形など強い細胞障害性の変化を起こした.4) 変異原テストでは, CCEに強い変異原性があり, 生涯飲用した場合の慢性影響としての発癌性が懸念された.5) CCEの発癌テストでは, 強い発癌性をもつ可能性は一応否定されたが, 弱い発癌性をもつ可能性については今回のテストでは結論を導き出すことはできなかった.6) このテストで半数のマウスに肺腫瘍を発生させたCCEには多くの有機ハロゲン化合物が含まれており, 発癌物質を検索して生体影響を評価していくことが必要と思われる.7) これらは上水中に微量含まれているものであり直ちに人の健康に障害を与えるような可能性は少ないと思われる.

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The results for the qualitative and quantitative detection of diacetylmorphine (heroin) and its metabolites in the urine samples of heroin abusers are presented. The heroin and its metabolites were directly extracted with chloroform-isopropanol (3 : 1, pH ≒ 9.5, 20°C) from the urine samples. The heroin and its metabolites were separated by capillary GC and identified mass spectrometrically by full mass spectra. The heroin and its metabolites levels were determined by quantitative selected ion monitoring (SIM). As the results, heroin, 6-acetylmorphine, morphine, acetylcodeine and codeine were detected by capillary GC/MS from the urine sample of heroin abuser. The above data suggest that the capillary GC/MS method is useful for the detection of heroin in the urine samples of heroin abusers as evidence for drug use.

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In the previous paper, we reported that Cu (II) chelates of imidazole and thiazole derivatives have superoxide (SO) and OH radical (·OH) scavenging activities, and that Cu (ClO₄)₂ also have the same activity. Present paper describes the scavenging mechanisms of the Cu (II) chelates and Cu (ClO₄)₂ on active oxygen species. Phenazine methosulfate-NADH system was used as a SO generation system, and the resulting H₂O₂ and ·OH were measured. Cu (ClO₄)₂ and other Cu (II) chelates, which are estimated to produce ·OH from H₂O₂ by the Fenton reaction, generated so much amount of ·OH. But, in the case of Cu (II) chelates of N-methyl-N'-[2-(imidazol-4-yl) methylthioethyl] thiourea (I-2) and 3-(2-thioureidothiazol-4-ylmethylthio) propionitrile (T-3), the amounts of H₂O₂ and ·OH produced were less than those produced in the absent of the chelates. Therefore, it seems that these chelates have not only superoxide scavenging activity but also H₂O₂ scavenging activity, which is not followed by OH radical formation. Further, Cu (II) chelates of I-2 and T-3 showed antioxidant effects on the rat liver microsomal peroxidation induced by Fe (III)-ADP-NADPH. Finally, T-3 did not inhibit the enzymatic activity of NADPH-dependent cytochrome P-450 reductase.

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Potassium bromate (KBrO₃), a food additive, has renal toxicity with the possibility of active oxygen species. It was recognized with the DTNB method that the reduced glutathione (GSH) content in the reaction mixture was correlatively decreased by the addition of KBrO₃. The peroxidation of linoleic acid micelle by the addition of GSH and KBrO₃ was measured by the ultraviolet absorption method and oxygen electrode method. Moreover, thiobarbituric acidreactive substance (TBARS) content was correlatively increased by the addition of GSH and KBrO₃ on the microsomal system in vitro. But the increase of TBARS content was inhibited by the addition of sodium azide and it was not inhibited by the addition of superoxide dismutase, catalase, mannitol, uric acid, DTPA and EHPG respectively. The addition of sodium azide correlatively inhibited the production of TBARS in the reaction mixture. These results suggested that the singlet oxygen was produced by the reaction of GSH and KBrO₃ and was related to the lipid proxidation.

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Many artificial or natural occurring compounds are used as food additives. Although they are very useful in food distribution, the addition of some of them to food is strictly regulated by the law because of their toxicities or mutagenicities. We have investigated the effect of xenobiotics on rabbit platelet functions by means of thromboxane B₂ (TXB₂) synthesis which was determined by the EIA methods. The present study has been carried out to investigate the effect of five antioxidants ; butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), dl-α-tocopherol (VE), l-ascorbyl palmitate (AP), and propyl gallate (PG) ; used as food additives on rabbit platelet functions both in vitro and ex vivo. Among five antioxidants used, BHT, BHA, AP and PG inhibited A-23187-induced TXB₂ synthesis at the concentration of 10^-5M when added simultaneously in vitro. On the other hand, all compounds revealed significant inhibitory effects against thrombin-induced TXB₂ synthesis at 10^-7M and over. Pretreatment of platelet with these compounds also inhibited both agonist-induced TXB₂ syntheses, and this inhibitory effect was reduced partly by removal of BHA and PG when platelet was stimulated by A-23187, but no reduction was observed when platelet was stimulated by thrombin. Both agonist-induced activations of platelet from rabbit fed ADI levels of each compound with diet for 5 d were inhibited significantly. These results reveal that the ADI levels of antioxidants used as food additives inhibit the agonist-induced TXB₂ synthesis in rabbit platelet.

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A systematic analytical procedure for dichlorvos (DDVP) and trichlorfon (DEP), organophosphorus pesticides, and furthermore for desmethyl dichlorvos (DM-DDVP), desmethyl trichlorfon (DM-DEP), dimethyl phosphate (DMP) and monomethyl phosphate (MMP), their metabolites and degradation products, was investigated. From the environmental water, drinks and human urine, after salting-out, only DDVP was first extracted with n-hexane and then the others were extracted with acetonitrile-diethylether (1 : 1, v/v) at about 1N by HCl. TLC was performed on silica-gel plate using acetonitrile-water (85 : 15, v/v) and isopropanol-water-28% ammonium hydroxide (75 : 24 : 1, v/v) as development systems. By colorations with 4-(p-nitrobenzyl) pyridine (NBP) and o-tolidine, they were relatively well detected. It was possible to determine DEP, DM-DDVP, DM-DEP, DMP and MMP by FID-GC and GC-MS after trimethylsilyl derivatization of them with N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA). The detection limits of their aqueous solution were 0.1-3 μg/ml by FID-GC and 0.2-10 μg/ml by scanning mode GC-MS. The coefficients of variation for their spiked urine sample by GC analysis in this way were 2.7-7.6% at 50μg/ml, respectively.

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A highly sensitive detection method of PCDDs in environmental water was developed by the concentration method using polyamide columns. PCDDs in water samples were adsorbed first to a polyamide column to which 2-chlorodibenzo-p-dioxin (MCDD) and octachlorodibenzo-p-dioxin (OCDD) were shown to be adsorbed quantitatively. The PCDDs, then, were eluted from the column by ethanol and dichloromethane containing 1% acetic acid. The eluted MCDD and OCDD were analyzed and quantified by GC-MS-SIM with 97% and 98% of recovery, respectively. By this method, the levels of PCDDs were determined in environmental water taken at four points of Tokyo Bay and four points of river flue into Tokyo Bay. The highest PCDDs levels were obtained from the sample of Arakawa River with a level of about 400 pg/liter. The concentration method using polyamide column could be used in routine work since this method enables to quantify efficiently PCDDs even at ppq levels.

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Concentrations of four volatile chlorinated hydrocarbons (VCHs), i.e. 1, 1, 1-trichloroethane (MCF), trichloroethylene (TCE), tetrachloroethylene (PCE) and tetrachloromethane (PCM), in the ambient air at a rural site and four industrial sites were monitored. One week measurements of the VCHs were carried out in recent 3 years (April, 1989 to March, 1992). Mean concentrations at all sites of MCF, TCE, PCE and PCM were 1.3 ppb (0.25 to 3.2 ppb), 0.97 ppb (0.053 to 4.6 ppb), 0.29 ppb (0.039 to 0.84 ppb) and 0.12 ppb (0.097 to 0.24 ppb), respectively. The concentration of VCH used at the industrial site was most elevated compared with the other sites. PCM concentrations at all sites except that located near chemical plants were close to the background concentrations. MCF, TCE and PCE concentrations at the rural site were higher in winter season than in summer season ; this seems to be caused by meteorological factors. Some of those at the industrial sites showed individual variations, caused by both meteorological factors and operation condition of the industry.

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