Shigenobu Mizusaki

Mutagenicity of polycyclic aromatic hydrocarbons and quinones on Salmonella typhimurium TA97.

18 polycyclic aromatic hydrocarbons (PAHs) and 7 quinones were tested for mutagenicity using Salmonella typhimurium TA97, TA98 and TA100 with or without metabolic activation. In the presence of metabolic activation, TA97 was more susceptible to mutation than either TA98 or TA100 by many of PAHs tested. PAHs such as 1-methylphenanthrene, fluoranthene, pyrene, benzo[a]pyrene, benzo[e]pyrene and perylene had high mutagenic effects on TA97 in the presence of metabolic activation. 1,6- and 1,8-pyrenequinones were also highly mutagenic on TA97 in the presence or absence of metabolic activation. It appears that pyrene is mutagenic through its metabolic conversion to pyrenequinones.

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.

Factors affecting mutagenic activity of cigarette smoke condensate in Salmonella typhimurium TA 1538

Smoke condensates from Burley tobacco, bright-type tobacco and various brands of commercial cigarettes were tested for mutagenicity by using a microsomal test system with Salmonella typhimurium TA 1538. Smoke condensate from Burley tobacco had much higher mutagenic activity than that from bright-type tobacco. Increased mutagenic activity was observed with smoke condensates from Burley tobacco grown with increasing amounts of nitrogen fertilizer, and from commercial cigarettes blended with Burley tobacco. There was a significant correlation between nitrate content of cigarette and mutagenic activity of the resulting smoke condensate. The results suggest that nitrate in cigarettes may influence the formation of potential mutagens during the burning of a cigarette.

N-methylputrescine oxidase from tobacco roots

Abstract N -Methylputrescine oxidase, catalyzing the oxidative deamination of the primary amino group of N -methylputrescine, was demonstrated in roots of tobacco plants, and purified 150-fold. N -Methylpyrrolinium salt was identified as the reaction product by comparison with the authentic compound. The enzyme had a pH optimum at 8·0 and the K m value for N -methylputrescine was 4·5 × 10 −4 M. Putrescine and cadaverine were also oxidized. Activity of the enzyme was strongly inhibited by carbonyl reagents, thiol inhibitors and diethyldithiocarbamate, but hydrazine derivatives were without significant effect on the enzyme activity. The enzyme was localized exclusively in the roots and its activity markedly increased on decapitation of the shoots. The demonstration of N -methylputrescine oxidase, in addition to putrescine N -methyltransferase, provides evidence that the biosynthetic route of nicotine involves N -methylputrescine and 4-methylaminobutanal ( N -methylpyrrolinium salt) as intermediates.

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.

Metabolism of nicotinic acid in tobacco plants

Abstract 14C from nicotinic acid-6-14C which was administered to tobacco plants was incorporated into nine pyridine compounds during 3 hr incubation; two of these were identified as nicotinic acid-N-glucoside and 6-hydroxynicotinic acid. Nicotinic acid was only incorporated into intermediates of the pyridine nucleotide cycle to a limited extent the main product being the glucoside. Nicotinic acid glucoside was incorporated into nicotine with the same efficiency as nicotinic acid, but the rate of the incorporation was markedly reduced when unlabelled nicotinic acid was also fed, suggesting that the glucoside is not involved in the direct route of nicotine biosynthesis.

Phytochemical studies on tobacco alkaloids—XI: A new alkaloid inNicotiana tabacum roots

Abstract A new alkaloid has been isolated from the roots of Nicotiana tabacum . Its chemical structure was confirmed as 2,4-di(3-pyridyl)piperidine ( anatalline ) by elemental analyses, u.v., i.r. and NMR and mass spectroscopy and finally by its dehydrogenation to nicotelline. The biosynthetic route for this compound may be similar to that of anatabine, but it is not formed in a manner similar to anabasine from lysine.

Inhibition of 12-O-Tetradecanoylphorbol-13-Acetate-Stimulated 32Pi Incorporation into Phospholipids and Protein Phosphorylation by 2,7, 11-Cembratriene-4,6-Diol, an Antitumor-Promoting Agent

A possible mechanism of antitumor-promoting activity of alpha-2,7,11-cembratriene-4,6-diol (alpha-CBT) was studied. alpha-CBT inhibited the 32Pi incorporation into phospholipids of HeLa cells stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast to the property to interact with calmodulin of many other antitumor-promoting agents, which were proved to inhibit TPA-stimulated 32Pi incorporation into phospholipids, alpha-CBT did not show any interaction with calmodulin; i.e., the fluorescence of N-phenyl-1-naphthylamine enhanced by binding with Ca2+-calmodulin was not influenced by treatment with alpha-CBT. TPA-stimulated phosphorylation of 47-kilodalton protein, which is phosphorylated by protein kinase C in human platelets, was found to be inhibited by alpha-CBT. However, the specific binding of 3H-TPA to mouse epidermal particulate fraction was not inhibited by treatment with alpha-CBT. These results suggest that alpha-CBT inhibits the activity of protein kinase C by another mode of action rather than the effect on its receptor site, and that this action and calmodulin-independent inhibitory effect on phospholipid metabolism of alpha-CBT may play a certain role in its antitumor-promoting activity in vivo.