Yoshiyuki Nakamura

Phthalic acid esters in various foodstuffs and biological materials

Phthalic acid esters (PAEs) are ubiquitous environmental contaminants in Japan. Di-n-butyl phthalate (DNBP) and di-2-ethylhexyl phthalate (DEHP) are the most commonly found of nine PAEs examined. DNBP and DEHP residues in 22 kinds of commerical foodstuffs (55 examples), mostly in plastic containers, are recorded. The level is generally higher in powdered foodstuffs than in other materials and the content of PAEs increased sharply during their storage. A close correlation is found between the content of PAE residues in packing materials and those of foodstuffs. The levels of DNBP and DEHP in human blood samples are found to be much higher than those of PCBs.

Fetotoxic Effects of Mono-2-ethylhexyl Phthalate (MEHP) in Mice

Mono(2-ethylhexyl) phthalate (MEHP), one of the main metabolites of di(2-ethylhexyl) phthalate (DEHP), exerted embryo/fetotoxic effects similar to those of DEHP at lower doses. Oral administration of MEHP (1 mL/kg) to the mice of 8 days gestation resulted in less than 32% of live fetuses, all of which were deformed. When DEHP (10 mL/kg) was given to the pregnant mice of 8 days gestation, approximately 0.03% and 0.003% of the administered dose was found in fetuses as DEHP and MEHP, respectively, after 12 hr. The presence of the MEHP in fetuses is probably due to the transplacental crossing of the MEHP formed in the maternal body, since the fetuses of mice up to day 9 of pregnancy showed no hydrolytic activity of DEHP to MEHP. Crossing of MEHP through the placenta was proven by an experiment in which MEHP was administered in pregnant mice. A single injection of MEHP (25 or 50 mg/kg), but not DEHP (500 mg/kg) into pregnant mice, induced a significantly high incidence of somatic mutations in the coat hair of offspring of mice (KYG, female X PW, male; C57BL/6Crj, female X PW, male). All these data suggest that MEHP could be responsible for the embryotoxic/fetotoxic effects observed with DEHP.

Suppression of aflatoxin B1- or methyl methanesulfonate-induced chromosome aberrations in rat bone marrow cells after treatment with S-methyl methanethiosulfonate.

The suppressive effect of S-methyl methanethiosulfonate (MMTS) on aflatoxin B1 (AFB1)- or methyl methanesulfonate (MMS)-induced chromosome aberrations (CA) in rat bone marrow cells was studied. MMTS significantly suppressed CA induced by both AFB1 (an indirect-acting carcinogen) and MMS (a direct-acting carcinogen). Suppression was observed at all periods (6, 12, 18, 24 and 48 h) after AFB1 or MMS treatment and in all doses of AFB1 (5, 10 and 20 mg/kg) or MMS (50, 75 and 100 mg/kg) investigated. AFB1-induced CA was potently suppressed by MMTS given between 2 h before and 6 h after the AFB1 injection. The suppression of AFB1-induced CA by MMTS paralleled the dose of MMTS when MMTS was given in a dose range of 1-20 mg/kg body weight. MMS-induced CA was potently suppressed by MMTS given between 2 h before and 2 h after the MMS injection. The suppressive effect of MMTS on MMS-induced CA paralleled the dose of MMTS when MMTS was given in a dose range of 1-15 mg/kg body weight. Diphenyl disulfide, which modifies -SH groups in proteins like MMTS, also significantly suppressed both AFB1- and MMS-induced CA. Although other mechanisms are not excluded, the suppression of carcinogen-induced CA by MMTS may result from the ability of MMTS to modify -SH groups in proteins. The juices of cabbage and onion, which contain considerable amounts of MMTS and S-methyl-L-cysteinesulfoxide (the precursor of MMTS), also significantly suppressed AFB1- or MMS-induced CA. These results suggest that MMTS is a possible chemopreventive agent against cancer.

Suppressing effects of S-methyl methanethiosulfonate and diphenyl disulfide on mitomycin C-induced somatic mutation and recombination in Drosophila melanogaster and micronuclei in mice

S-Methyl methanethiosulfonate (MMTS) and diphenyl disulfide (DPDS) are temporary enzyme-sulfhydryl blocking agents. They are naturally occurring phytoalexin-like and synthetic substances known to be very potent bio-antimutagens in Escherichia coli B/r WP2. In the present paper, the suppressing effects of MMTS on mitomycin C (MMC)-induced mutant wing spots in the somatic mutation and recombination test (SMART) of Drosophila melanogaster, and of MMTS and DPDS on MMC-induced micronucleated peripheral reticulocytes are described. MMTS consistently reduced the numbers of MMC-induced small single, large single and twin spots per wing at a dose of 10-1000 micrograms/vial, in a dose-dependent manner. MMTS reduced the number of twin spots per wing on the spontaneous mutation at the dose of 1000 micrograms/vial. MMTS and DPDS dose-dependently reduced the frequencies of MMC-induced micronucleated peripheral reticulocytes at a dose of 10-40, and 3-100 micrograms/kg, respectively. Our results confirmed that enzyme-sulfhydryl blocking agents, such as MMTS and DPDS, are effective antimutagens in vivo too.

Methyl cinnamate derivatives enhance UV-induced mutagenesis due to the inhibition of DNA excision repair in Escherichia coli B/r

UV-induced mutagenesis in Escherichia coli B/r WP2 was enhanced by certain derivatives of methyl cinnamate which themselves were not mutagenic. Methyl ferulate, methyl isoferulate and methyl sinapate showed this effect markedly. Such an enhancement effect was absent with the derivatives of cinnamic acid and ethyl cinnamate and was not observed in Escherichia coli WP2s uvrA. Methyl sinapate also enhanced 4NQO-induced mutation and suppressed liquid-holding recovery in the above repair-proficient strain. The presence of methyl sinapate in plating agar medium decreased the survival of UV-irradiated cells of a recombination-repair-deficient strain, CM571 recA. However, the effect was not observed with those of WP2s uvrA. In an in vitro experiment in which the removal rate of thymine dimers was measured, methyl sinapate clearly inhibited this repair event. From these results, we conclude that methyl sinapate inhibits DNA excision repair, thus enhancing UV mutagenicity.

Suppressive Effects of S‐Methyl Methanethiosulfonate on Promotion Stage of Diethylnitrosamine‐initiated and Phenobarbital‐promoted Hepatocarcinogenesis Model

Modifying effects of S‐methyl methanethiosulfonate (MMTS) on diethylnitrosamine (DEN)‐initiated and phenobarbital (PB)‐promoted hepatocarcinogenesis were examined in rats. Five‐week‐old male F344 rats were divided into 8 groups. After a week, groups 1–5 were given DEN (100 mg/kg body weight, i.p.) once a week for 3 weeks, whereas groups 6–8 received vehicle treatment. Group 2 was given 100 ppm MMTS containing diet in the initiation phase. From 4 weeks after the start of experiment, groups 3 and 5 were fed MMTS, and groups 1–3 and 7 received drinking water containing 500 ppm PB. Group 6 was given MMTS diet alone throughout the experiment (24 weeks). The incidences of hepatocellular adenoma and total liver tumors were significantly smaller in group 3 than those of group 1. The average numbers of hepatocellular adenoma, carcinoma and total tumors in group 3 were significantly smaller than in group 1. Glutathione, S‐transferase placental form‐positive foci were also significantly decreased by MMTS treatment in the promotion phase. MMTS treatment in the initiation or promotion phase reduced ornithine decarboxylase activity in the liver of rats given DEN. The antioxidant activity against lipid peroxidation of MMTS was confirmed in tests with rabbit erythrocyte membrane ghosts or rat hepatocytes. These results suggest that MMTS is a promising chemopreventive agent for liver neoplasia when concurrently administered with PB.

A possible mechanism of TPA-mediated downregulation of neurotrophin-3 gene expression in rat cultured vascular smooth muscle cells

We have previously reported that in cultured rat vascular smooth muscle cells (VSMCs), neurotrophin-3 (NT-3) gene expression was suppressed by TPA (12-O-tetradecanoyl phorbol-13-acetate), which induces an AP-1 transcription factor. In the present study, to clarify the mechanism for TPA-mediated downregulation of NT-3 gene expression, effects of cycloheximide and dexamethasone (Dex) on the TPA-mediated downregulation were examined in VSMCs. Pretreatment with cycloheximide, an inhibitor of protein synthesis, or simultaneous treatment with Dex, an inhibitor of AP-1, suppressed the TPA-mediated downregulation of NT-3 gene expression. Furthermore, co-transfection of c-fos and c-jun expression vectors into VSMCs resulted in decrease in the NT-3 gene expression. The present findings suggest that TPA-induced AP-1 de novo synthesis causes the downregulation of NT-3 gene expression in VSMCs.

Antitumor-Promoting Effects of Tea Aqueous Nondialysates in Mouse Epidermal JB6 Cells

Our attention has focused on the primary prevention of cancer using functional ingredients of edible plants. Here we report a new antitumor-promoting component, tea aqueous nondialysates (TNDs), and discuss the modes of action. TNDs were prepared from the hot water infusion of green and black tea leaves followed by extraction with CHC13, ethyl acetate, and n-butanol and finally by dialysis. The TNDs had a molecular weight of more than 12000 and consisted of a complex mixture of tannins, hydrolyzable and condensed, containing sugar(s), quinic acid, and polyphenolic compounds such as gallates and catechins. TNDs inhibit the soft agar colony induction by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) without particular cytotoxicity and the morphological changes induced by TPA in the mouse epidermal JB6 cell lines. The chemical structure of TDNs with sugar(s) and polyphenols was required for the activity because the modifications of TNDs by β-glucosidase, polyphenoloxidase, or tannase caused the loss of activity. Reversion of TPA-induced morphological alternations was associated with an increase in the cytoskeletal actin microfilaments and fiber of the extracellular matrix, fibronectin. Therefore the antitumor-promoting effect of TNDs was different from that of green tea catechins.

Mutagenicity of Nifurpirinol (P-7138) in Escherichia coli WP2 and Salmonella typhimirium TA100

Nifurpirinol, 6-hydroxymethyl-2-[2-(5-nitro-2-furyl)vinyl]pyridine, which has been widely used as an antibacterial drug against diseases of fish, was found to be a potent mutagen. The mutagenic effect was dose-related, and the potencies were calculated to be 1.49 X 10(3), 7.24 X 10(4) and 8.49 X 10(5) revertants/1 X 10(-8) survivors at the concentration of 1 microgram/ml to :Escherichia coli B/r WP2, E. coli B/r WP2 hcr- and Salmonella typhimurium TA100, respectively, without metabolic activation. Nifurpirinol is susceptible to photodegradation, and its mutagenic activity to S. typhimurium TA100 was decreased to about one thousandth when the solution of Nifurpirinol was exposed to sunlight at room temperature for 4 h.

Chemical Constituents of Mainly Active Component Fractionated from the Aqueous Tea Non-Dialysates, an Antitumor Promoter

Much attention has been focused on the primary prevention of cancer using functional ingredients of edible plants.1 The components of tea leaves (Camellia sinensis), particularly green tea catechins, are known to be potent cancer chemopreventive agents. Phase I study for their clinical application is now going on in the United States. Tea catechins represented by (−)-epigallocatechin gallate (EGCG) have potential to inhibit/suppress not only the initiation and promotion of cancer but also to cause regression of tumorigenic cells and to inhibit the metastasis of cancer cells.2–6 Here, we introduce a new antitumor promoting component, tea aqueous non-dialysates (TNDs), as a candidate for the primary prevention of cancer,7–9 although only tea catechins have been used in many studies on the anticancer effects of tea leaves. In this paper, we describe the properties of antitumor promoting effects and the identification of principally active component of TNDs and its chemical constituents.