Mutagenic activation of xenobiotics by plant enzymes.

Genetic evidence has indicated that plants can activate certain xenobiotics to mutagens, but biochemical evidence is as yet scarce. Nevertheless, plant microsomal enzymes and peroxidases have been shown to form reactive intermediates, the best studied examples being 2-aminofluorene, benzo[a]pyrene and pentachlorophenol. The latter two xenobiotics are converted to quinoid derivatives which are, in principle, able to redox cycle and generate active oxygen species. In analogy to results obtained in mammalian systems, covalent binding of reactive intermediates to DNA as well as fragmentation of DNA, are proposed as major mechanisms of action of mutagenic plant metabolites.

The plant cell/microbe coincubation assay for the analysis of plant-activated promutagens.

The preincubation and suspension procedures of the plant cell/microbe coincubation assay are described and the activation of 2-aminofluorene and m-phenylenediamine by cultured tobacco, cotton, carrot and maize cells is compared. The assay measures the plant activation of promutagens into genotoxins detected in Salmonella typhimurium as well as toxicity in plant and microbial cells. At concentrations of 2-aminofluorene 0-0.5 mumoles/reaction tube, the rank order of the efficiency of activation by plant cells was tobacco much greater than cotton greater than carrot. Cultured maize cells did not activate 2-aminofluorene. The tobacco cell activation of 2-aminofluorene was inhibited 50% by 750 microM diethyldithiocarbamate under conditions that did not affect the cell viability. Tobacco cells were also the most efficient plant cells in activating m-phenylenediamine (0-5 mumoles/reaction tube). The 'biological affinity' of m-phenylenediamine for the activation system in tobacco cells was approximately 100 microM.

Mutagenic activity of promutagens in plants: indirect evidence of their activation.

This review summarizes data concerning mutagenic activity of promutagens in various plant in vivo assays. These data are compared with the present knowledge about the metabolism of xenobiotics and activation of promutagens in plants obtained by biochemical studies and by the separation of the activation process from the genetic endpoints assayed for the mutagenicity. The article documents a differential response of plant species in the endogenous transforming of various classes of promutagens into mutagens. Attention is devoted to the following types of promutagens: nitrosamines, polycyclic aromatic hydrocarbons and aromatic amines, aflatoxins, pyrrolizidine alkaloids, diallate, styrene, vinylchloride, ethanol, cycasin, nitrofurans, sodium azide, s-triazine herbicides, 1,2-dibromoethane and maleic hydrazide.

The plant activation of m-phenylenediamine by Tradescantia clone 03 and clone 4430 cells in liquid suspension culture.

In this study, we expanded the use of the genus Tradescantia to investigate the plant activation of promutagens and further refine the methodology of the plant cell/microbe coincubation assay. Liquid suspension cell cultures of Tradescantia clone 03 and Tradescantia clone 4430 were used to activate the promutagen m-phenylenediamine into a mutagenic compound which was detected by Salmonella typhimurium strain TA98 in the plant cell/microbe coincubation assay. Optimum treatment parameters were established for both plant cell lines. Optimum was defined as the lowest concentration or shortest time period that provided consistently positive results and high rates of revertants. Preliminary experiments with both cell lines defined 2.5 mumoles m-phenylenediamine per plate as the optimum concentration to be used in the determination of the optimal coincubation period and the optimal concentration of plant cells. These experiments also determined the optimal physiological stage at which both clones should be used in the coincubation assay. Differences were found in the optimal of coincubation (1h for clone 03, 2 h for clone 4430) and growth stage (mid-log for clone 03, mid- to late-log for clone 4430). Similar activation responses were seen for both clones when the concentration of plant cells (mg/ml) was varied. Under optimized conditions, clone 03 cells demonstrated an approximately 10% higher activation response than clone 4430.

Promutagen activation by Vicia faba: an assay based on the induction of sister-chromatid exchanges in Chinese hamster ovary cells.

Plant activation of promutagens was studied using Vicia faba S10 (in vitro activation) and the extracts prepared from promutagen-treated roots of Vicia faba (in vivo activation). The induction of sister-chromatid exchanges in Chinese hamster ovary cells was used as an endpoint to evaluate the cytogenetic effects of promutagens activated by Vicia faba. Cyclophosphamide and ethyl alcohol were activated both by Vicia S10 and by the Vicia extracts, and their activation resulted in an increase in SCEs. Benzo[a]pyrene, 2-aminofluorene, and maleic hydrazide were not activated. Aniline was activated, but without effect on the induction of SCEs. The activation capacity in vitro and in vivo of Vicia faba was not very pronounced, except for the activation of ethyl alcohol, when compared with that of rat-liver S9, and showed differences in activation for the 6 chemical agents tested.

Assessment of the mutagenicity of fractions from s-triazine-treated Zea mays.

A water-soluble extract from maize plants exposed to 3 s-triazine herbicides (atrazine, simazine and cyanazine) has been shown to be mutagenic in strain TA100 of Salmonella. No mutagenic activity was observed in any control plant extracts using either water or a variety of organic solvents. Gel permeation studies of the extracts suggest that the mutagen(s) are small molecules (less than 1000 MW). HPLC fractionation suggests that the mutagens formed from each of the 3 herbicides are similar in polarity and water solubility, eluting in a 50/50 water:methanol fraction. Approximately 89% of 14C-labeled HPLC chromatographable metabolites of atrazine were also associated with this fraction, suggesting a close chemical link between a labeled but unidentified metabolite and the mutagenic activity.

N-nitrosamines: bacterial mutagenesis and in vitro metabolism.

Many nitrosamines are potent mutagens. The rate-limiting step in their in vitro metabolism to mutagens is usually a single enzymatic reaction catalyzed by one or more of the many cytochrome P-450-dependent mixed-function oxidases present in the microsomal cell fraction. Current evidence indicates that this reaction activates nitrosamines to alpha-hydroxynitrosamines, which have half-lives on the order of seconds. This product decomposes to an aldehyde and a much shorter-lived ultimate metabolite which is probably an alkyl diazonium ion or an alkyl carbocation. This may react with DNA leading to premutagenic adducts. Such adducts represent a very small fraction of the ultimate mutagen, with the rest reacting with water to yield the corresponding alcohol. Evidence for this pathway includes (1) the observation of deuterium isotope effects in metabolism and mutagenesis, (2) products (aldehydes, alcohols, and N2) consistent with this pathway, (3) studies on metabolism of nitrosamines using purified cytochrome P-450, (4) formation of DNA adducts such as O6-alkylguanines which are consistent with those expected from the ultimate mutagen, (5) expected products and genotoxic effects of other sources of activated nitrosamines, e.g., alpha-acetoxynitrosamines, alkanediazotates and related compounds. Hydroxylation of nitrosamines at other positions also occurs in vitro (usually to a lesser extent), but these products are generally stable and must be further metabolized to exert mutagenic effects (with the exception of N-nitrosoalkyl(formylmethyl)amines, which are direct-acting mutagens). Because only low percentages of nitrosamines are metabolized in vitro, the contribution to mutagenesis by secondary metabolism is small. In this respect, in vitro metabolism can differ significantly from in vivo metabolism. Bacterial mutagenesis by nitrosamines has most often been studied in Salmonella typhimurium and to a lesser extent E. coli. Mutagenesis by nitrosamines generally requires a source of microsomes (a 9000 X g supernatant fraction is often used), and NADPH. Liver fractions from Aroclor-1254- or PB-induced rodents have been most frequently employed but liver fractions from untreated animals, and homogenates of other organs (lung, kidney, nasal mucosa, and pancreas) have also been utilized. Liver homogenates from humans are generally similar to those from untreated rats in metabolizing nitrosamines to mutagens but large interindividual variations are observed. Mutagenesis is often most effective using a liquid preincubation, a slightly acidic incubation mixture and hamster liver fractions.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolic conversion of IQ and MeIQ to bacterial mutagens.

The metabolic conversion of 2-amino-3-methyl- and 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline (IQ and MeIQ respectively) to bacterial mutagens was studied using a bacterial mutation assay. Studies were performed using S9 fractions derived from either corn oil (uninduced) or Aroclor-1254-treated Sprague-Dawley rats. Aroclor 1254 treatment lowered the S9 protein concentration required for optimum levels of mutagenesis, enhanced the numbers of mutants observed and altered the effects of metabolic inhibitors and cofactors added to the assay. Studies with uninduced preparations revealed that IQ and MeIQ exhibited similar responses to the effects of metabolic inhibitors and cofactors involved in detoxication reactions. Both IQ and MeIQ activation appeared to be inhibited by the biogenic amines tryptamine and tyramine and inactivated by conjugation with either acetyl coenzyme A or glutathione.

Activation of carcinogenic N-nitrosopropylamines to mutagens by lung and pancreas S9 fractions from various animal species and man.

The mutagenic potential of 7 carcinogenic N-nitrosopropylamines was examined by the Ames liquid incubation assay, using lung and pancreas 9000 X g supernatant (S9) fractions from rats, hamsters, mice, rabbits, monkeys and humans for metabolic activation. N-Nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP), N-nitrosobis(2-oxopropyl)amine (BOP) and N-nitrosomethyl(2-oxopropyl)amine (MOP) showed positive mutagenicity in strain TA100 in the presence of lung S9 from each of the uninduced animals and humans. Besides the 3 N-nitrosopropylamines, N-nitrosomethyl(2-hydroxypropyl)amine (MHP) was also positive in the presence of lung S9 from polychlorinated biphenyl (PCB)-induced rats, hamsters and mice. On the other hand, in the presence of pancreas S9 from uninduced or PCB-induced animals, only HPOP and BOP showed positive mutagenicity. In contrast, N-nitrosobis(2-hydroxypropyl)amine (BHP), N-nitrosobis(2-acetoxypropyl)amine (BAP) and N-nitroso-2,6-dimethylmorpholine (NDMM) showed negative mutagenicity in the presence of lung and pancreas S9 from either uninduced or PCB-induced animals and humans. HPOP was a direct-acting mutagen, and lung and pancreas S9 from 5 animal species and man did not affect the activity. BOP was mutagenic even in the presence of bovine serum albumin. The mutagenic activation of MHP by lung S9 from PCB-induced rats, hamsters and mice was completely inhibited by preincubation in an atmosphere of carbon monoxide or by addition of cytochrome c or metyrapone to the S9 mixture, whereas 7,8-benzoflavone totally lacked this effect. However, that of MOP was insensitive to these inhibitors. These results of mutagenicity assay indicate that only the methyl derivatives of N-nitrosopropylamines, MHP and MOP are activated by the lung from 5 animal species and man, whereas the pancreas from all the tested animals did not activate the 7 N-nitrosopropylamines to mutagens, and that the phenobarbital-inducible major cytochrome P-450 in the lung of rodents is involved in the mutagenic activation of MHP.

Formation of bacterial mutagens from the reaction of chewing tobacco with nitrite.

Using the Salmonella/microsome assay system, the mutagenicity of chewing tobacco extracts (CTE) treated with and without sodium nitrite under acidic conditions was examined. Mutagenic activity was found only for nitrite-treated CTE in both tester strains, TA98 and TA100, and was independent of metabolic activation. Formation of mutagenic substances from CTE by nitrite was dependent on acidic pHs (the highest at pH 2) and could be inhibited by ascorbate. The mutagenic potency of CTE plus nitrite was proportional to the content of nitroso compounds generated in the reaction mixture, indicating that the nitrosation process was involved. The possible in vivo nitrosation and the potential health effect are discussed.

Evaluation of the mutagenic activity of some aza-aromatic hydrocarbons.

The mutagenic activity of 7 aza-aromatic hydrocarbons, which are suspected of being environmental pollutants, was assessed using the Salmonella assay. The compounds tested were: 1-azachrysene, 2-azachrysene, 4-azachrysene, 1-azabenz[a]anthracene, 2-azabenz[a]anthracene, 9-azabenz[a]anthracene, and 12-benzo[a]pyrene. None of the compounds was mutagenic in the absence of S9, but all were mutagenic in the presence of S9.

Vitamin C and cancer. How convincing a connection?

More research is needed to draw definitive conclusions on the relationship between vitamin C intake and cancer, but the following general statements can be made. The effectiveness of megadoses (greater than 1 gm/day) of vitamin C for the cure or prevention of cancer is still unproven; in fact, the safety of megadoses is still in question. Therefore, they are not recommended for the general public at present and, if used at all, should be used under medical supervision. Epidemiologic evidence suggests that vitamin C-rich foods may be beneficial in preventing cancer, and their consumption should be encouraged as a measure to reduce the incidence of cancer. Well-controlled studies should be undertaken to elucidate the relationship between vitamin C and cancer, using both vitamin-containing foods and vitamin C supplements at different intake levels.

Rutin-induced beta-glucosidase activity in Streptococcus faecium VGH-1 and Streptococcus sp. strain FRP-17 isolated from human feces: formation of the mutagen, quercetin, from rutin.

A fecal isolate, Streptococcus sp. strain FRP-17, and strain VGH-1 of Streptococcus faecium were shown to contain beta-glucosidases which converted rutin (quercetin-3-O-beta-D-glucose-alpha-L-rhamnose) to quercetin and were active against o-nitrophenyl-beta-D-glucose. The activity against rutin could be measured by increased mutagenicity in the Ames assay or visualized on thin-layer chromatography plates. In both organisms, the beta-glucosidase activities were inducible by the addition of rutin to the growth media. Several closely related strains of Streptococcus spp. lacked any beta-glucosidase activity. In cell preparations of the active organisms, activities with rutin and o-nitrophenyl-beta-D-glucose were optimal at pH 6.8 and could be enhanced by increasing the ionic strength of the assay system. At low ionic strengths, both quercetin and a new product (intermediate between the polarities of rutin and quercetin) were formed by the incubation of rutin with cell preparations of either active organism. This product disappeared with increased ionic strength, suggesting that it may be a reaction intermediate, quercetin-3-O-beta-D-glucose. These results suggest that the beta-glucosidase active against rutin and that active against o-nitrophenyl-beta-D-glucose are the same.

[Cooking meat in microwave ovens does not cause formation of mutagenic substances]. / La cottura di carne con forni a microonde non determina la formazione di sostanze mutagene.

UNLABELLED: During the cooking process of meat, mutagenic and/or carcinogenic substances can be formed that can induce tumours of the gastro-intestinal tract or of other organs in the rat. The formation of these substances is proportionate to the cooking time, the cooking surface and the quantity of fats contained in meat. A comparison is made between beef cooked on a grid where the temperature reaches 200 degrees C, and cooked in a microwave oven (Cuocorapido Candy 500 CL, frequency 2450 Mhz) where the temperature does not exceed 100 degrees C. Mutagenic substances were extracted by the Commoner technique and mutagenic activity was assayed with the Ames test. RESULTS: no mutagenic activity was demonstrated in the extracts of meat cooked in microwave ovens, while mutagenic activity was clearly demonstrated in the extracts of meat cooked on a grid.

In vitro production of human fecal mutagen.

The feces of some normal humans were previously shown to be mutagenic by the Salmonella mutagenicity assay with strain TA100. In the present study, the mutagenicity of feces of certain donors can be increased by anaerobic incubation for 96 h. The increase in mutagenicity did not occur upon incubation in the cold or in air, in the presence of antimicrobial agents or if the feces were sterilized by heat. On thin-layer chromatographs, the relative mobility of fecal mutagen for all donors after incubation was the same in any one of 4 different solvent systems. The major mutagenicity appears to be due to a single type of compound which may be produced by anaerobic bacteria.

Mutagenicity of 5,6-dimethoxysterigmatocystin, a metabolite from Aspergillus multicolor, in the Salmonella/microsome system.

The natural sterigmatocystin derivative, 5,6-dimethoxysterigmatocystin, was found to be a mutagen for Salmonella typhimurium strains TA98 and TA100 after metabolic activation in a mammalian microsome system.