Hitoshi Okamoto

Mutagenic activity of amino acid pyrolyzates in Salmonella typhimurium TA 98

Pyrolyzates of 25 amino acids and 5 indole derivatives were tested for mutagenicity in the histidine-requiring mutant Salmonella typhimurium TA 98. Significant mutagenic activity was detected with pyrolyzates of most of the amino acids. These pyrolyzates required a liver microsomal fraction, as representative of mammalian metabolism, to be detected as mutagens. Among the pyrolyzates tested, the highest mutagenic activity was observed with that of L-tryptophan. As little as 10 microgram of the pyrolyzate of L-tryptophan had detectable mutagenic activity toward TA 98. The optimal pyrolysis temperatures for the formation of mutagenic products were shown to be 500 degrees C for L-tryptophan and 600 degrees C for the other amino acids. The results from pyrolyses of some indole derivatives suggest that an amino group at the alpha-position to the carboxyl group of L-tryptophan plays an important role in the formation of mutagens.

Relation between chemical constituents of tobacco and mutagenic activity of cigarette smoke condensate.

Mutagenic activities of cigarette smoke condensate were assayed in the presence of S-9 Mix using Salmonella typhimurium TA 98. The results were examined in relation to chemical data of tobacco leaves. Among the nitrogenous constituents examined, the contents of total nitrogen and protein nitrogen and the soluble nitrogenous fraction were positively and significantly related to an increase in mutagenic activity of the smoke condensate, whereas nicotine and nitrate were not important in contributing to mutagenic potency of such condensates. The age of tobacco leaves influenced the mutagenic potency of the condensate, which was lowest in leaves from the lower stalk position and increased with ascending leaf position on the stalk. Smoke condensate from tobacco with higher sugar content resulted in lower mutagenic activity. The present results, together with the previous study on the mutagenicity of the amino acid pyrolyzates, suggest that potent mutagens in cigarette smoke condensate are nitrogen-containing compounds, which may be formed from proteins and amino acids during the burning of a cigarette.

Mutagenicities of the pyrolyzates of peptides and proteins

Pyrolyzates of 10 peptides, 10 proteins and 5 naturally-occurring materials were tested for mutagenicity in the histidine-requiring mutants Salmonella typhimurium TA98 and TA100. Significant mutagenic activity was detected with pyrolyzates of most of these materials. The pyrolyzates requred a liver microsomal fraction, as representative of mammalian metabolism, for their detection as mutagens. Among the pyrolyzates tested, the highest mutagenic activity was observed with that of a tryptophan-containing peptide. The pyrolyzate of protein obtained from tobacco leaf also showed mutagenicity. The higher the protein content in the leaf the higher the mutagenic activity of the pyrolyzate. Protein in a tobacco leaf may be the principal precursor of mutagens in tobacco-smoke condensate.

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced induction in Epstein-Barr virus early antigen in Raji cells

Retinol, 5 flavonoids, 3 steroids and 7 sweetening agents were studied for their effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced early antigen (EA) of Epstein-Barr virus (EBV) in Raji cells. Concomitant treatment of Raji cells with TPA and retinol showed inhibition of EA induction. Among flavonoids, quercetin resulted in effective inhibition of EA induction by TPA and alpha-naphthoflavone showed the weakly inhibitory effect. None of the other flavonoids such as rutin, catechin and beta-naphthoflavone affected the induction of EBV-EA by TPA. beta-Estradiol obviously inhibited EBV-EA induction by TPA, but hydrocortisone did not show any inhibitory effect on it. Glycyrrhetinic acid, steviol, phyllodulcin and perrillartine also showed the remarkable inhibition of EBV-EA induction. On the other hand, glycyrrhizin and stevioside, glycosides of glycyrrhetinic acid and steviol, did not inhibit the induction of EBV-EA by TPA. Some of the inhibitors reported here may be effective on the inhibition of the in vivo tumor promotion by TPA.

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity in mouse epidermis by sweetening agents and related compounds

The effects of naturally occurring sweetening agents, which inhibited the induction of Epstein-Barr virus-associated early antigen (EBV-EA) induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), and related compounds on the induction of ornithine decarboxylase (ODC) by TPA is examined. Application of glycyrrhetinic acid or steviol to mouse skin 1 h before TPA treatment showed a remarkable decrease in TPA-induced ODC activity. Post-treatment with glycyrrhetinic acid or steviol 1 h after application of TPA also resulted in a considerable depression in the induction of ODC activity. Neither glycyrrhetinic acid nor steviol alone induced epidermal ODC activity. These results suggest that glycyrrhetinic acid and steviol interfere with the process of induction of epidermal ODC by TPA treatment of mouse skin. cis-Abienol, frullanolide and norambreinolide, which have a partially similar structure in the moiety with glycyrrhetinic acid or steviol, were tested. cis-Abienol and frullanolide showed an inhibitory effect when applied 1 h before TPA treatment, but norambreinolide was not effective. A relationship between suppression of ODC activity and inhibition of EBV-EA induction is discussed.

Metabolic formation of pyrenequinones as enhancing agents of mutagenicity in Salmonella

Pyrene and 1-methylpyrene in the pyrolysis product of cellulose, which enhances mutagenicity of 2-acetylaminofluorene, must be metabolized to expose enhancing activity. 1,6-Pyrenequinone and 1,8-pyrenequinone, having high enhancement activity, were first isolated and identified in the metabolites of pyrene by rat liver microsomal fraction.

Role of type II phospholipase A2 in the inflammatory process of carrageenan-induced pleurisy in rats

In order to investigate the role of type II phospholipase A2 (PLA2) in the inflammatory process, the effect of a monoclonal antibody specific to type II PLA2 on carrageenan‐induced pleurisy was studied in rats. Intravenous injection of the antibody (MB5.2), which inhibits the catalytic activity of type II PLA2, significantly reduced both the pleural exudate volume and the intrapleural leukoeyte count, while a control antibody did not have any appreciable effect. MB5.2 caused no change in the level of type II PLA2 in the pleural fluid. These results suggest that type II PLA2 generated at inflamed sites, at least in part, have a crucial role in the pathogenesis of acute inflammation.

Enhancement of mutagenicity by hydroxypyrenes in Salmonella.

1-Hydroxypyrene and 4,5-dihydro-4,5-dihydro-4,5-dihydroxypyrene were isolated and identified in the metabolites of pyrene by rat liver microsomal fraction, and the presence of 1,8- and 1,6-dihydroxypyrene in the metabolites was proposed. These hydroxypyrenes, as well as pyrenequinones reported previously, enhanced intensively the mutagenicity of 2-acetylaminofluorene (AAF).

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced induction of Epstein-Barr virus early antigen in Raji cells by some inhibitors of tumor promotion

The effects of some compounds, which have been reported to inhibit tumor promotion in vivo, on the induction of the early antigen (EA) of Epstein-Barr virus (EBV) by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells were examined. The inhibitors of the cascade process involving arachidonic acid, indomethacin, nordihydroguaiaretic acid, phenidone and p-bromophenacyl bromide, effectively inhibited EBV-EA induction by TPA. Two flavonoids, morin and kaempferol also inhibited EA induction. Among antioxidants, butylated hydroxytoluene effectively inhibited EA induction, though alpha-tocopherol did not show any inhibition of EA induction at concentrations of up to 150 micrograms/ml. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide, a calmodulin antagonist, and esculetin showed inhibitory effects on EA induction, though slight cytotoxicity was observed. L-1-p-Tosylamino-2-phenylethyl chloromethyl ketone, a protease inhibitor, showed cytotoxicity and no specific inhibition of EA induction. Five kinds of steroids, cortisone, hydrocortisone, prednisolone, dexamethasone and fluocinolone acetonide showed no inhibitory effect on EA induction at concentrations of up to 100 micrograms/ml. In addition, the relationship between the inhibition of EBV-EA induction and that of tumor promotion is discussed.