Conjugations, as well as oxidations by P-450 and fp₃, are main activation mechanisms for chemical carcinogens, though the conjugation of xenobiotics has been know ordinarily to be a process to make them water-soluble for excretion. O-Sulfation of arylhydroxamic acids, hydroxylamines, and hydroxymethylarenes by sulfotransferases transform these carcinogens to highly reactive sulfate esters which act as good leaving groups with concomitant formation of carbonium and/or nitrenium ions which can react readily with DNA, RNA, and protein, and consequently exert carcinogenicity. Acetyl-CoA-dependent acetylation and N, O-transacylation also activate arylhydroxylamines and hydroxamic acids, respectively, by the same mechanism as proposed for the sulfation. Aminoacylation of arylhydroxylamines by aminoacyl-tRNA synthetases, which are not drug-metabolizing enzymes, is in the same category of activation of carcinogens by acylation. Despite of its importance in detoxifying a variety of electrophiles formed in vivo, GSH conjugation is recognized to activate a certain type of compounds, such as 1, 1-, 1, 2- di-and poly-haloalkanes, of which the 1, 2-dihaloalkanes form reactive episulfonium ions of GSH. A possible participation of reactive glucuronides and phosphoric acid esters of carcinogens are discussed in their carcinogenesis mechanisms.

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The concentration of metallothionein (MT) in the human pancreas was determined by the Sephadex G-75 method. It was higher than 51 μg/g in 15 of 18 cases, and was in the range of 218-834 μg/g in six cases. Clear peaks of zinc and cadmium in the metallothionein fraction were observed in the elution profile of the pancreas supernatant on a Sephadex G-75 column. A positive relationship was found between zinc and metallothionein concentrations in the human pancreas (r=0.439, p<0.05), but no relationship was observed between the age of patients and MT concentration. These results indicate that metallothionein in the human pancreas is zinc, cadmium-thionein, and that the metallothionein concentration is high in most human pancreases. Metallothionein may be one of the main zinc-binding components in the human pancreas.

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The complete microbial degradation of chlorhexidine is described. Two new isolates of bacteria (strains No. A-1 and No. A-3) utilized chlorhexidine as a sole nitrogen source for growth. The growth yield of both strains was about 60 g of protein per mol of nitrogen of chlorhexidine, and was similar to that in the case of ammonium ion as a nitrogen source. Different degradation patterns of chlorhexidine were observed on the high-performance liquid chromatograms (HPLC) of the culture filtrates of the strains. The strain No. A-3 (Pseudomonas sp.) was found to form transient intermediates, detectable on the HPLC, during the microbial degradation, whereas the strain No. A-1 (Flavobacterium sp.) degraded chlorhexidine without formation of such transient intermediates.

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Comparative studies on the absorption, distribution, and excretion of flame retardants, tris (2, 3-dibromopropyl) phosphate (TDBPP), bis (2, 3-dibromopropyl) phosphate (BDBPP), tris (1, 3-dichloro-2-propyl) phosphate (TDCPP), tris (1-chloro-2-propyl) phosphate (TMCPP) and tris (2-chlorethyl) phosphate (TMCEP), were carried out in rats. During 168 h after an oral administration of an equimolar dose (50 μmol/kg) of these ¹⁴C-labeled flame retardants, the decreasing order of levels of ¹⁴C-radioactivity excreted was TMCEP»TMCPP>TDBPP>BDBPP>TDCPP in urine, TDCPP>TDBPP>BDBPP>TMCPP»TMCEP in feces, and BDBPP>TDCPP>TDBPP»TMCPP>TMCEP in expired air. The amounts remaining in the carcass were in the order TDBPP=BDBPP>TDCPP>TMCEP=TMCPP. Almost 100% of the radioactivity administered was recovered, in the ranges of 43.2 to 93.4% in urine, 5.6 to 39.2% in feces, 1.7 to 19.4% in expired air (as ¹⁴CO₂), and 0.7 to 3.7% in the body. Biliary excretion of the five flame retardants ranged from 24.6 to 44.9% of the dose by 48 h. The order of biliary excretion was as follows : TMCPP>TDCPP>BDBPP=TDBPP>TMCEP. Tris- or bis- (dihalogenated alkyl) phosphates were more slowly absorbed, distributed and excreted than tris (monohalogenated alkyl) phosphates. In binding studies in vitro, the order of covalent binding to S-9 of liver and kidney was as follows : TDBPP>MDBPP>BDBPP.

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A simple and rapid biodegradation test method (cultivation method) was developed for the screening of chemicals which were hard to be degradable by microorganisms in the natural water. Namely, a water, acetone or dimethyl-sulfoxide solution (0.1 ml) of the test chemicals was added to a mixture of river or sea water (4.9 ml) from unpolluted area and an autoclaved solution (5.0 ml) of 0.2% peptone (added by 3% NaCl for sea water) in a sterile test tube (25×200 mm) with a tight plug. After sealed with film and fixed at angle 30° in a dark box, test tubes were incubated at 30°C and shaked at 120 rpm. At appropriate times during cultivation, the remaining chemicals were determined for obtaining the degradation percentage by growing microorgaisms, which were examined by the measurement of turbidity (OD at 610 nm) of the mixture. This cultivation method gave considerably constant results on many chemicals tested, when degradability was judged from degradation percentage after 3 d-incubation as follows : 100-50% to be easy-, 50-15% to be moderate-, and 15-0% to be hard-of-degradability.

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Phosphate on the surface of vegetables was determined by the spectrometric detection of the molybdophosphate-Malachite Green aggregate after preconcentration with a membrane filter. Vegetables obtained commercially were washed by an adequate volume of water, and the Mo-MG reagent (mixed solution of ammonium molybdate and Malachite Green) was added to the washings. The molybdophosphate-Malachite Green aggregate formed was selectively collected on a nitrocellulose membrane filter (3 μm pore size) and dissolved in methylcellosolve together with the membrane filter. The absorbance at 627 nm was proportional to the concentration of phosphate in the washings with the molar absorptivity of 2.7×10⁵ M^-1 cm^-1. The method was applied to the determination of phosphate on the surface of vegetable samples ; soybean sprouts, edible burdock roots and young taroes. In the case of soybean sprouts, the amounts of phosphate (20-37 μg P/g sample) for the white samples (8 samples) were higher than those for natural color samples (12 samples). For the samples of edible burdock roots packed in vinylresine bags, the amount of phosphate was about 170 mg P/g sample which was about two-fold larger than the natural one. Phosphate was also detected for young taroes (peeled and washed at the market) with an amount about twenty-fold larger than the natural one (1 μg P/cm² surface). Phosphate used for the surface treatment did not penetrate taroes deeper than 1 mm.

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A simple and rapid method for the determination of tylosin in chicken, pork and beef has been developed by high-performance liquid chromatography (HPLC). The drug was extracted from meat with 0.5% metaphosphoric acid-methanol (7 : 3), and followed by liquid-liquid partition for clean-up procedure. The HPLC separation was carried out on an Inertsil ODS column (25 cm×4.6 mm i.d.) using 0.05 M sodium dihydrogenphosphate (pH 2.4)-acetonitrile (65 : 35) as a mobile phase, and detected at 287 nm with 0.04 a.u.f.s. Although tylosin consists of four major components such as tylosin A, B, C and D, their molecular extinction coefficients at 287 nm maximum were virtually identical. Therefore, the method of calculating the contents of tylosin was carried out by total peak area normalization of peaks of tylosin A, B, C and D observed on the chromatogram. The calibration graph for tylosin was rectilinear from 10 to 200 ng. The average recoveries of tylosin added to chicken, pork and beef at the level 1.0 μg/g were 88.5, 88.4 and 84.5%, and the coefficients of variation were 2.5, 3.4 and 4.1%, respectively. The limits of detection of tylosin in chicken, pork and beef were 0.05μg/g. The accuracy and reproducibility of the present method were enough for residual analysis, and an excellent correlation (r=0.99) between the HPLC method and the bioassay method was obtained.

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In the chlorination of miscellaneous nitrogenous compounds contained in water, N-chloro compounds are formed from amine or imine in the compounds. Although the chlorine in N-chloro compounds has property of combined available chlorine, reactivity of each N-chloro compound is different each other. In the fractional determination of available chlorines by the N, N-diethyl-p-phenylene diamine (DPD) method or amperometric titration etc. different amounts of potassium iodide are added to differentiate each class of N-chloro compound. The velocity of color reaction of DPD depends mainly on the concentration of residual available chlorine. Consequently, the color development can be approximated by a first-order reaction. From the recorded curve of color development at 550 nm, periods to reach 85% and 95% value of the maximum absorbance are measured respectively. The rate constants of the first-order reaction K are calculated from these values. The rate constants increase in proportion to KI concentrations, and all experimental data points are essentially on a straight line. The time required to finish DPD color development and reciprocal of KI concentration are also correlated. In the case of added 30 μM KI, the DPD color development is completed within a period from 19 to 38 s, except chloropiperazine (over 80 s). The relationship between rate constant K and pKa of each N-compound has a good correlation.

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改良ランキン装置を用いた通気蒸留法による簡便かつ高感度のシアン定量法を開発した.この方法は, 粉末試料0.2gを用い, 0℃で20分更に50℃で2時間通気蒸留して, 遊離型シアンと結合型(配糖体)シアンを分別捕集し, クロルシアンを生成させ, ヘッドスペース法によりECD-GLCで定量するものである.この方法は従来の方法に比べて, 遊離シアンと結合型(配糖体)シアンが同時に測定可能であり, 少量の試料を用いるだけですみ, 操作が簡便で分析時間を短縮することができ, 微量定量が可能である, という利点がある.ヘッドスペース法によるシアンの定量限界は, 1試験管あたり0.1μgであった.また, 市販のアーモンドプードル, 小豆及び輸入サルタニ豆の粉末各0.2gを用いて遊離シアン, アミグダリン及びリナマリンの添加回収実験を行ったところ, 以下のような結果を得た.遊離シアンの回収率は, 酵素添加の有無にかかわらず92.0-101.5%であった.アーモンドプードルでのアミグダリンの回収率は, 酵素添加時は90.0%, 酵素無添加時は16.1%であった.小豆粉末でのリナマリンの回収率は酵素添加時は89.7%, 酵素無添加時は13.2%であった.一方, サルタニ豆でのリナマリンの回収率は酵素添加の有無にかかわらず87.5-90.0%であった.したがって, サルタニ豆のような輸入雑豆の場合は酵素添加は不要と考えられた.本法を用いて, 輸入ベビーライマ豆, バター豆, サルタニ豆のシアン含有量を分別定量した.ベビーライマ豆の総シアン含有量は45.0-76.9ppm, 遊離シアン含有量は0.5-3.1ppm, バター豆の総シアン含有量は241.6-326.4ppm, 遊離シアン含有量は0.1-6.4ppm, サルタニ豆の総シアン含有量は327.2ppm, 遊離シアン含有量は3.3ppmであった.これらの定量値は, 以前に報告されているものとほぽ同じであり, 本法は実際の輸入豆のシアン含有量の測定に十分用いうることが示された.

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We analyzed stimulants in the urine by automated head-space gas chromatography. A 6-ml of urine-containing stimulants was pipetted into a 20 ml autosampler vial together with 0.5 ml of 20% sodium hydroxide and 3.5 g of potassium carbonate. After the vial was sealed and heated for 20 min at 80°C, 0.8 ml of the head space gas was injected into the gas chromatograph equipped with a FID and fusedsilica capillary column (DB-17 : 30 m×0.32 mm i.d., film thickness 0.5 μm), by using Hamilton gas tight syringe. Addition of potassium carbonate to the urine increased the detection limit 10 to 100 times. The calibration curves showed a linearity in the range of 0.1 to 15 μg/ml for methamphetamine and amphetamine. The relative standard deviations were 4.8% for methamphetamine and 4.1% for amphetamine when the stimulants concentration was 10 μg/ml. Detection limits of methamphetamine and amphetamine were 0.02 μg/ml and 0.05 μg/ml, respectively.

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Alkylating activities of various alkylating agents were examined by using nicotinamide. One μmol of alkylating agent was incubated with nicotinamide (25-200 μmol) in a phosphate buffer (pH 7.4) at 37°C for 24 h. The resulting N¹-alkylnicotinamide was converted to 2-alkyl-6-phenyl-2, 8-dihydro-8-oxo-2, 7-naphthyridine and analyzed by high-performance liquid chromatography with a fluorescent detector. Alkylating activity was expressed as percentage of the formed N¹-alkylnicotinamide against 1 μmol of the standard N¹-alkylnicotinamide. In methylation and ethylation, the alkylating activity was in the order of methyl methanesulfonate (100%), dimethyl sulfate (68%), methyl iodide (21%) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (20%), and ethyl methanesulfonate (93%), diethyl sulfate (68%), ethyl iodide (3%) on addition of 200 μmol nicotinamide. MNNG known as mutagen and carcinogen, have not so high activity. Propylene oxide and 1, 2-butylene oxide show 100% alkylating activities under the same reaction conditions as described above on addition of 200 μmol nicotinamide.

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The adsorption behavior of an ammonia, trimethylamine gas mixture on plasma-treated activated carbons (PT-ACs) was investigated by using adsorption isotherms. The adsorption capacity of the PT-AC was 1.2-1.3 times larger than that of corresponding raw activated carbons (R-ACs) for a 1 : 1 gas mixture. The experimental adsorption behavior of the mixture coincided well with the theoretical adsorption on the PT-ACs, while a substantial discrepancy was observed between them on the R-ACs. This may be because the competitive adsorption of individual components was less on the PT-ACs than on the R-ACs. Adsorption of the ammonia, trimethylamine mixture seemed to be enhanced at low concentrations on PT-ACs by the acid functional groups formed by oxygenplasma treatment. The PT-ACs, which show high adsorption capacity compared to the R-ACs, seem to be promising adsorbents for ammonia, trimethylamine gas mixtures at low concentration levels.

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The aim of the present study was to confirm putrefactive formation of 3-methylbutyrylurea (3 M) from α-bromoisovalerylurea (BV). A commercial minced pork sample was spiked with BV and incubated at room temperature (above 25°C) for 1 month in a closed vessel. After the incubation, BV and 3 M were determined by thin-layer chromatography using guaiac gum reagent combined with treatment of the plate with chlorine gas and potassium iodide reagent. A spot of 3 M was detected and increased with the time of putrefaction, accompanied with a decrease of the BV spot. This finding suggested that 3 M was formed from BV in a putrefying environment.

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Gas chromatographic methods with ⁶³Ni electron capture detection (GC-ECD) or mass spectrometry (selected ion monitoring, SIM) was investigated for the determination of tributyltin compounds in fish. Tributyltin compounds in a homogenized flesh sample of fish were extracted as tributyltin chloride (TBTC) with a hydrochloric acid-methanol mixture and transferred to a hexane solution. The extract was charged to an alumina column and eluted stepwise with ethyl acetate followed by hexane-ethanol (4 : 1, v/v) mixture. TBTC eluted with the latter solvent was transferred to a hexane layer by liquid-liquid extraction, and the hexane layer was concentrated by a rotary evaporator. The concentrate was subjected to gas chromatographic separation on a 10% KOCL-Sn column. The calibration curves were linear in the range of 0.1-2 μg/ml with GC-ECD and in the range of 0.05-1 μg/ml with SIM, respectively. The detection limit of TBTC was 0.005 μg/g in sample by the SIM method. Eleven samples of fish were analyzed by the GC-ECD and SIM methods, and the determined values by both the methods were in good agreement.

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Toxicity of phenol and 7 chlorophenols from mono-Cl to penta-Cl on 3 species of daphnia, Daphnia magna, Daphnia carinata and Daphnia pulex has been determined according to OECD guideline for testing of chemicals. The responses of each daphnia to chemicals were measured by acute immobilization of young daphnia less than 24 h old (24 h-EC₅₀) and reproduction inhibition during 14 d (14 d-Maximum Acceptable Toxicity Concentration, MATC). The 24 h-EC₅₀ values were correlated with 6 physico-chemical parameters of chlorophenols. The parameters employed in this quantitative structure-activity relationship (QSAR) analyses were : n-octanol/water partition coefficient, log P_OW ; dissociation constant, pK_a ; Hammett Σσ constant ; index of valence molecular connectivity, ¹χ^V ; perimeter of the efficient cross section of molecule, ΣD ; melting point, mp. In immobilization studies, 24 h-EC₅₀ values of each daphnia decreases as substituted chlorine atom increases and QSAR study shows that log P_OW, a parameter of hydrophobicity, gives the best correlation. Toxic responses of 3 kinds of daphnia are almost the same each other and 24 h-EC₅₀ value of each chlorophenol is almost the same as 96 h-LC₅₀ in fish, P. reticulata. On the other hand, in the reproduction inhibition study using Daphnia magna, MATC values of mono-, di-, tri-, tetra- and pentachlorophenol are almost the same each other and no relationship was found between MATC values and substituted chlorine numbers.

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Rapid analysis of propylene glycol (PG) in fatty foods was developed by using kieselguhr column chromatography. 2-5 g of sample suspended in hot water (70-80°C) was applied to the Extrelut 20 column. After standing for 15 min, 70 ml of hexane was put on the column to remove fat. PG retained in the column was eluted with distilled water. 50 ml of aqueous eluate was collected. PG was quantitated by gas chromatography with trimetylene glycol as an intenal standard. Recoveries of PG added to margarines and mayonnaises were from 98% to 105%. Since proposed purification process is very rapid, many samples can be treated at the same time. Among commercial margarines (25 samples), shortenings (3 samples) and mayonnaises (3 samples), PG was detected in eight margarines (range 37-261 ppm).

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The present study was focused on the preparation of a homogeneous sample solution for the determination of chloroform in the tissue of brain or lung of a rabbit. Into a pair of vial (7 ml) both of 200 mg of brain sample and 0.4 ml of 10% (v/v) Triton X-100 in water were taken, and adjusted to a total volume of 0.8 ml with water. Each vial was sealed with a screw-cap with a septum lid. The sample mixture was irradiated by ultrasonic wave (20 KC, 100 W) for 5 min under cooling on running water. Into the resulting duplicate tissue homogenate samples was injected 200 μl of either one of standard sample solutions, (a) or (b) through the septum lid : (a), 20% methanol solution (200 μl) containing 30 μg of CHCl₃ and 20 μg of CH₃CCl₃ (internal standard) ; (b), the same solution containing 15 μg of CHCl₃ and 20 μg of CH₃CCl₃. The both vials were allowed to stand for 15 min at 65°C on a heating block. A portion (400 μl) of the headspace gas was analyzed by GC with a flame ionization detector. GC conditions used were as follows : Glass column (1.6 m×3 mm i.d.) packed with Chromosorb 102 (80-100 mesh) ; Column oven, 170°C ; Detector and injector temperatures, 220°C ; Carrier gas, N₂ 50 ml/min. In the case of lung tissue sample, the mixture was incubated at 35°C for 2 h in the presence of collagenase in the sealed vial prior to the sonication. The successive experimental mannuals were mostly the same as those of the brain. The present headspace GC was found to be reliable for the determination of CHCl₃ in a range of 2 to 40 μg/ml in the homogeneous sample solution.

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Monochlorodibenzofuran, which is considered to be formed by the reaction of dibenzofuran with chlorine, has been recently detected in tap water in some region of Japan. The mutagenicities of 1-, 2-, 3-, and 4-chlorodibenzofurans were tested to Salmonella typhimurium TA 98 and TA 100 in the absence or presence of rat liver S 9. 1-Chlorodibenzofuran and 4-chlorodibenzofuran were practically non-mutagenic, while 2-chlorodibenzofuran was weakly mutagenic to TA 98. Unlike these three isomers, 3-chlorodibenzofuran was strongly mutagenic to both TA 98 and TA 100. The mutagenic activity was about one fifth and one twentieth of that of benzo [α] pyrene to TA 98 and TA 100 respectively. Its mutagenic activity was more potent to TA 98 than to TA 100. Chlorinated aromatic compounds usually show no mutagenic activity in Salmonella assay system. The result of this study suggests that the number and the position of the chlorine substitution on the benzene ring play an important role in the manifestation of mutagenicity as well as the carcinogenicity in polychlorinated aromatic compounds.

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Biodegradability of organic compounds in river and sea water was tested according to the simple and rapid method, the cultivation method, which was reported in our previous report. Out of 170 chemicals tested, 103 compounds were judged to be hard-, 43 to be moderate-, and 24 to be easy-of-degradability. In comparison with the MITI method, our method was applicable to wider range of compounds with less expensive equipments and gave more strict marks in 3 d, indicating that our method is a useful screening test method for biodegradability of chemicals in environmental water.

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