Reactive oxygen species (ROS) are produced by the normal oxygen metabolism in cells as a consequence of respiration in an oxygen atmosphere and by some kinds of environmental mutagens. ROS attack and modify cellular macromolecules including DNA. 8-Hydroxyguanine (8-OH-Gua) is one of the DNA lesions which are produced by ROS. 8-OH-Gua mainly induces G→T mutations in vitro, in E. coli and mammalian cells. Repair enzymes for 8-OH-Gua are present in cells and prevent the mutations induced by the DNA lesion. Formation of oxidative DNA lesions which are produced by ROS appears to be involved in many biological processes including carcinogenesis and ageing. Therefore, 8-OH-Gua may be a good marker of cellular production of ROS to estimate mutagenicities and carcinogenicities of chemical compounds.

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The analytical detection methods for the irradiation treatment of food are investigated recently as international projects as well as projects carried out in our country to resolve the problems on the international trade and labelling in the market. Several promising methods such as ESR, thermoluminescence, chemical luminescence, impedance, viscosity, gas-measurement, lipid-volatile hydrocarbon, lipid-volatile cyclobutanone, germination, microbiology and so on were proposed and reviewed on the international research program organized by FAO/IAEA.

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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.

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Nicotinic acid (NA) production in meat by bacterial contamination during storage at 10°C was studied. Eighty-eight isolates from 9 meat samples produced NA or consumed nicotinamide (NAA) in nutrient broth containing NAA after incubation at 35°C for 24 h. The ten strains among these isolates converted NAA to NA and another 8 strains consumed not only NAA but also NA after incubation at 10°C for 7 d. Strain B-1, which had the highest activity for converting NAA to NA, was identified as Enterobacter agglomerans by the dot blot DNA-DNA hybridization method as well as by ordinary methods ; other strains identified were Enterobacteriaceae, Pseudomonas spp., Yersinia intermedia, Serratia liquefaciens and Aeromonas sp. NAA was converted to NA with an increase in bacterial counts in sterile meat which had been inoculated with strain B-1, whereas no NA or bacterial counts in sterile meat were detectable during storage at 10°C. These results confirm that contaminating bacteria were involved in NA production in meat during storage at 10°C.

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In the previous paper, we described that acetic acid and caproic acid contained in water were decomposed very slowly by treatment of ozone alone, but they were decomposed very rapidly by simultaneous treatment of ozone and UV irradiation. In this report 5 mM aliphatic mono- and di-carboxylic acids in the 0.05 M phosphate buffer (pH 7.0) were treated by ozone + UV, and their degradation pathways were investigated by analysing their decomposition products. Dicarboxylic acids were considered to be produced from each monocarboxylic acid by oxidation at the terminal methyl group. Monocarboxylic acids with small carbon numbers were also produced by oxidative decarboxylation. The decomposition curves of each carboxylic acid indicated straight lines against decomposition time. Therefore, the decomposition reaction was considered to the apparent zero order. The decomposition rate constants of monocarboxylic acids increased with the carbon numbers, and they were decomposed more rapidly, but the rate constants of dicarboxylic acids were nearly equal. Compared to the treatment of ozone alone, the TOC (total organic carbon) removal (%) of malonic acid or adipic acid was very high by treatment of ozone + UV irradiation and very efficient decomposition to inorganic carbon dioxide was assumed. Mutagenicity of O₃ + UV treated solutions of caproic acid was assayed with Ames test, and was found to have a little mutagenicity at halfway decomposition time, but it disappeared finally.

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An induction of metallothionein by spinach extract was studied in mice. Among determined vegetables, the greatest effect was observed after injection of spinach extract. Injection of the extract induced metallothionein in a dose-dependent manner. Metallothionein was induced not only in the liver but in the kidney by injection of the extract. A fibrinogen level in the blood increased after injection of the extract. It indicates that cytokine may be involved in the induction of metallothionein by the spinach extract.

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A simple and rapid method for high-performance liquid chromatographic determination of riboflavin sodium phosphate (FMN) based on its conversion to riboflavin (FR) with acid phosphatase was developed. A mixture for the conversion of FMN to FR contained, in a final volume of 4.0 ml, 1.0 ml of 0.8 M sodium acetate buffer (pH 4.8), 20.0 units of acid phosphatase (Type I : from wheat germ, Sigma Chemical Co.) and less than 0.5 mg of FMN. After incubation for 30 min at 37°C in an amber test tube with a stopper, the reaction mixture was filtered through a 0.45 μm membrane filter. The filtrate was injected to reversed-phase high performance liquid chromatography equipped with an octadecylsilyl column (L-column ODS, 4.6 mm i.d.×250 mm) and detected for absorbance at 270 nm. FR in the acidic filtrate was stable for at least 24 h. The recoveries of FMN in drugs and soft drinks by this method were 97.7-100.8% with the coefficients of variation of 0.4-2.8%. Impurity FR was detected in all samples under the same conditions without enzymatic hydrolysis. This method could be applied to the separative determination of FR and FMN.

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A simple and rapid method for the determination of methyl mercury in fish and shellfish using a gas chromatograph with electron capture detection (GC/ECD) was developed. Lipids and other organic interference in the test materials were eliminated by pretreatment with acetone and benzene, and then the methyl mercury extracted with the acidified benzene was analyzed by GC/ECD. Good recoveries (tuna, 90.3±2.3% ; shark, 105.5±3.2% ; sail-fish, 87.5±2.7% ; and shrimp meat, 88.4±2.6%) were obtained from samples spiked with 1 ppm of methyl mercury. The detection limit of methyl mercury was approximately 0.0016 ppm.

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A simple method by high performance liquid chromatography (HPLC) was developed for the simultaneous determination of 4 preservatives in cosmetics, chlorhexidine gluconate (GCH), benzalkoniumchloride (BzAC), benzethonium chloride (BzEC) and cetylpyridinium chloride (CPC). A sample of a cosmetic containing GCH, BzAC, BzEC and CPC was dissolved in tetrahydrofuran (THF) or methanol (MeOH). For the elution of BzAC, BzEC and CPC, the sample was passed through a Bondelute SCX cartridge. After washing the cartridge with MeOH. BzAC, BzEC and CPC were eluted with 0.1 M NaClO₄/MeOH. On the other hand, for the elution of GCH, the sample was passed through Bondelute CBA cartrige. After washing the cartridge with MeOH, GCH was eluted with a solution of 0.2 M KH₂PO₄-CH₃CN (1 : 1). The optimum condition for the separation by HPLC of 4 preservatives in cosmetics was as follows : column, TSK gel ODS 80 TM (4.6 mm i.d.×150 mm) ; mobile phase, CH₃CN-H₂O-THF-acetic acid (40 : 40 : 20 : 0.2) containing 0.2% sodium lauryl sulfate, 1.2 ml/min ; column temperature, 50°C ; detection wavelength, 263 nm. Recovery of 4 preservatives in 10 samples of cosmetics (6 different types of cosmetics) was almost more than 95%. Detection limits of GCH, BzAC, BzEC and CPC were 0.00003%, 0.001%, 0.0002%, 0.00005%, respectively (S/N ratio=10, 10 μl, 0.005 a.u.f.s, λ_max)

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Coloring is added to many foods and beverages to compensate for the loss of color during processing and to enhance their visual appeal. We have been studying the effect of food additives around the ADI level on cellular functions using washed rabbit platelets. The present study was carried out to investigate the effect of natural red colors on rabbit platelet functions both in vitro and ex vivo. Among the 7 natural red colors used (red cabbage, elderberry, purple corn, lac, cochineal, monascus and beet red colors), red cabbage, elderberry and purple corn had an inhibitory effect on A-23187-induced thromboxane B₂ (TXB₂) synthesis. On the other hand, all colors affected thrombin-induced TXB₂ syntheses at concentrations up to 0.2% : red cabbage stimulated, cochineal stimulated or inhibited depending on its concentration, and others inhibited. In the pretreatment experiment, monascus, beet red and cochineal inhibited A-23187-induced TXB₂ synthesis, and the effects of the former two colors were irreversible. Thrombin-induced TXB₂ syntheses were inhibited by pretreatment with all colors except red cabbage which had a stimulatory effect. These effects were irreversible except for purple corn. Thrombin-induced TXB₂ synthesis in the platelet from rabbits, after administering drinking water to which the recommended highest level of each color was added for 5 d, were affected by 3 colors. Namely, elderberry and beet red inhibited and purple corn stimulated. These results indicate that the amount of these compounds added to foods should be reduced to the minimum necessary to lower the risk of their potential hazard to humans.

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The determination of formaldehyde (HCHO) by the 4-amino-3-hydrazino-5-mercapto-1, 2, 4-triazole (AHMT) method was disturbed by cupric ion Cu²⁺. In the presence of Cu²⁺, the absorbance of the HCHO-AHMT reaction product decreased rapidly, and at the same time the absorption bands shifted to shorter wavelengths. Cobaltous ion Co²⁺ showed similar interference to Cu²⁺ in the determination of HCHO. Addition of EDTA markedly inhibited the interference of Cu²⁺, which enables us to determine HCHO in the presence of Cu²⁺.

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The toxicities on mice and Escherichia coli were examined on the lipid extracts from oyster digestive glands treated with 4% acetic acid or PBS at 37°C for 3 h and their lipid fractions (Frs. 1-4) obtained by TLC after the acid treatment. Almost all mice that had been administered intraperitoneally the lipid extracts were alive for 24 h after the injection. The mice which had been given Fr. 2 mainly consisting of free fatty acids, the oxides and monoglycerides were killed within 5 h after the injection. Histopathological observation were injury of villi and hemorrhage in the upper intestine, as well as hemorrhage in the kidneys. Fraction 2 markedly inhibited the growth of E. coli. Among the standard fatty acids, polyunsaturated fatty acids (linolic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA)) and oxidized EPA or DHA also inhibited the growth at the concentrations of 4.4-5.5×10^-3 mM.

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Analytical methods for the determination of imazalil, thiabendazole (TBZ), diphenyl (DP) and o-phenylphenol (OPP) in citrus fruits and for the determination of TBZ and imazalil in banana were described. The four fungicides were simultaneously extracted with ethylacetate, and distributed into two fractions, namely, the imazalil and TBZ fraction and the DP and OPP fraction, by a liquid-liquid partitioning process for cleanup. The fungicides in each fraction were chromatographed on a J'sphere ODS-M80 column with acetonitrile-methanol-water mixture (47 : 13 : 40) adjusted pH to 2.4 containing 0.01 M sodium dodecyl sulfate as mobile phase. Detection of DP, OPP and TBZ derived from citrus fruits were carried out by fluorescence detector set at Ex. 285 nm and Em. 340 nm, and imazalil derived from citrus fruits was detected by UV spectrophotometric detector set at 220 nm. Detection of TBZ and imazalil derived from banana, the former was detected at Ex. 305 nm and Em. 350 nm and the latter was detected at UV 220 nm. Recoveries of DP, OPP, TBZ and imazalil from citrus fruits spiked at 20 μg/g of DP and 2.0 μg/g of others were 77.3-81.9%, 84.8-91.8%, 80.1-85.9% and 79.2-81.3%, respectively, and recoveries of TBZ and imazalil from banana spiked at 2.0 μg/g were 80.2% and 78.7%, respectively. The detection limits of DP, OPP, TBZ and imazalil in citrus fruits were 0.5, 0.05, 0.02 and 0.05 μg/g, respectively, and the detection limits of TBZ and imazalil in banana were 0.005 and 0.02 μg/g, respectively.

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