The inhibitory effect of cysteine on the mutagenic activities of several carcinogens.

The Salmonella/microsome mutagenesis assay was used to determine the effect of cysteine (alpha-amino-beta-mercaptopropionic acid) on the mutagenic actions of several carcinogens: N-methyl-N'-nitro-N-nitrosoguanidine. N-acetoxy-2-acetylaminofluorene, N-hydroxy-2-acetylaminofluorene, 4-nitroquinoline-1-oxide, methyl methanesulfonate, 5-nitro-2-furaldehyde semicarbazone, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, aflatoxin B1 and the nitrosation products of methylurea and methylguanidine. Cysteine, at non-toxic concentrations, significantly decreased the frequency of reversion to histidine prototrophy when it was added to treatment mixtures. The extent of the inhibition of mutagenic action by cysteine depended on the carcinogen studied as well as the doses of cysteine and carcinogen employed. Cysteine (2.5--10 mM) completely inhibited the mutagenic actions of N-methyl-N'-nitro-N-nitrosoguanidine and methylguanidine nitrosation products while only partially preventing the mutagenic effects of the other carcinogens assayed. Inhibition of 5-nitro-2-furaldehyde semicarbazone-induced mutagenesis occurred only with higher cysteine concentrations (20--200 mM).     

The bacterial mutagenicity of three naturally occurring indoles after reaction with nitrous acid

Three naturally occurring indoles were evaluated for potential nitrosatability using the Nitrosation Assay Procedure (NAP test) as recommended by the World Health Organisation. All three indoles i.e. tryptophan, tryptamine and 5-hydroxy-tryptamine were nitrosated to products which were directly mutagenic for S. typhimurium TA1537. In addition, the products of nitrosation of tryptamine and 5-hydroxytryptamine were also mutagenic for strains TA1538, TA98 and TA1535 without the need for metabolic activation. The sensitivities of the frameshift-detecting strains TA1537, TA1538 and TA98 were of particular interest, since nitroso compounds are characteristically base-substitution mutagens. The mutagenic effects of the products formed after nitrosation of each indole at pH 3.6, were eliminated in the presence of S9 mix. This was not the case when the nitrosation assay was carried out at pH 2.6. At this pH the mutagenicity of the nitrosated products varied in the presence of S9 mix and depended upon the nature of the indole undergoing nitrosation, and the bacterial test strain utilised for the mutagenicity assay. This indicated that more than one mutagenic product was responsible for the observed effects. As well as pH, a number of other factors influenced the formation of mutagenic nitroso products. Most notably, the concentrations of precursor compounds (sodium nitrite, and indole) present in the NAP test were of critical importance. As the sodium nitrite concentration was reduced from that recommended by the W.H.O. (40 mM), so the mutagenicity decreased. For all three compounds significant mutagenic effects were lost at sodium nitrite concentrations below 15 mM. In conclusion the data presented in this paper clearly demonstrates that individuals are chronically exposed to naturally occurring substances which readily nitrosate in excess nitrous acid and yield bacterial mutagens.     

UV-irradiation potentiates the antimutagenicity of p-aminobenzoic and p-aminosalicylic acids in Salmonella typhimurium

UV-irradiation (254 nm, 10 or 20 J/cm2) of p-aminobenzoic acid (PABA) and p-aminosalicylic acid (NaPAS) potentiated their antimutagenicity towards N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis in Salmonella typhimurium. Their inhibitory action towards the formation of the mutagen N-methyl-N-nitrosourea from the nitrosation mixture of N-methylurea and nitrite was also increased by UV-irradiation. In contrast, UV-irradiated PABA exhibited no inhibitory effects towards the mutagenicity of sodium azide or 3-azidoglycerol. Neither PABA nor NaPAS nor their UV-irradiation products were themselves mutagenic in the Ames assay.     

Humic acids inhibit the formation but not the mutagenicity of N-methyl-N-nitrosourea

Humic acids in the form of potassium humate (KH), at concentrations exerting a strong inhibitory effect on the formation of N-methyl-N-nitrosourea (MNU) when present during the nitrosation of N-methylurea (MU) at pH 3, did not reduce the mutagenicity of preformed MNU in Tradescantia, clone 4430. The inhibitory effect of 20 mg/ml KH corresponds approximately to that of 3.75 mM (0.66 mg/ml) ascorbic acid towards the formation of MNU from the mixture of 7.5 mM MU + 7.5 mM NaNO2.     

Antimutagenic and antitumorigenic activities of nordihydroguaiaretic acid.

Nordihydroguaiaretic acid (NDGA), which occurs in the resinous exudates of many plants is used as an antioxidant in fats and oils. In this study we show that NDGA inhibited the mutagenicity of methyl methanesulfonate, benzo[a]pyrene (BP), 2-aminofluorene, and aflatoxin B1 in Salmonella typhimurium strain TA100 or TA98 in the absence and presence of rat hepatic microsomal activation system. The addition of NDGA during and after nitrosation of methylurea (MU) resulted in a dose-dependent inhibition of mutagenicity induced by nitrosation products of MU. In a two-stage skin tumorigenesis protocol using 7,12-dimethylbenz[a]anthracene (DMBA) as the initiating agent followed by twice weekly applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) as tumor promoter, pretreatment of animals with NDGA prior to DMBA application, afforded significant protection against skin tumorigenicity in female SENCAR mice. In additional studies, skin application of NDGA also inhibited the binding of topically applied [3H]BP and [3H]DMBA to epidermal DNA. When assessed in the anti-tumor promotion protocol, pretreatment of animals with NDGA before each application of TPA in DMBA-initiated mouse skin, resulted in 72% decrease in the total number of tumors when compared to non-NDGA pretreated animals. The possible mechanism(s) of the antimutagenic and anti-tumorigenic activities may be due to the multiple effects of NDGA as inhibitor of the carcinogen metabolism and DNA-adduct formation, scavenger of carcinogen free radicals, and as inhibitor of TPA-induced ornithine decarboxylase activity.     

2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potentiates nitrosation of a heterocyclic amine carcinogen by nitric oxide

Although nitrosation plays an important role in initiation of carcinogenesis, the reactive nitrogen oxygen species (RNOS) mediating this reaction by multiple pathways have not been determined. The heterocyclic amine carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was used as a target to investigate RNOS and pathways for potentiation of nitric oxide (NO)-mediated nitrosation. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) oxidizes NO to NO(2)(.) and was used as a tool to investigate NO(2)(.) potentiation of nitrosation. The IQ nitrosation product, 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline ((14)C-N-NO-IQ), was monitored by HPLC. Autoxidation of NO, generated by spermine NONOate (2.4 microM NO/min) for 7.5 min, did not convert 10 microM (14)C-IQ to N-NO-IQ. However, the presence of 15 muM CPTIO resulted in 3 microM N-NO-IQ formation. Potentiation by CPTIO occurred at low and high fluxes of NO, 0.075 to 1.2 microM/min, and over a range of IQ to CPTIO ratios of 0.5 to 10. A significant portion of N-NO-IQ formation was insensitive to azide (10 mM) inhibition, suggesting oxidative nitrosylation. NADH (0.02 mM) did not alter nitrosation by autoxidation, but effectively inhibited potentiation by CPTIO. Ascorbic acid (0.2 mM) and 5,5-dimethyl-1-pyrroline N-oxide (30 mM) inhibited nitrosation with or without CPTIO, while superoxide dismutase was not inhibitory. The RNOS produced by CPTIO had a 27-fold greater affinity for IQ than those produced by autoxidation. Results are consistent with NO(2)(.) or a RNOS like NO(2)(.) potentiating IQ oxidative nitrosylation. Nitrosation occurring at both low and high fluxes of NO can contribute to carcinogenesis.     

Salmonella/mammalian microsome assay with tetranitromethane and 3-nitro-L-tyrosine

The nitrosating agent tetranitromethane (TNM) and the nitrosation product 3-nitro-L-tyrosine (NT) were tested for mutagenic activity in the Salmonella/mammalian microsome assay. TNM showed strong genotoxic activity: it was mutagenic in all tester strains used (TA97, TA98, TA100, and TA102). The maximum mutagenic activity was reached between 16 and 32 micrograms/plate using the standard plate test; higher amounts led to distinct bactericidal effects. The mutagenicity was independent of an in vitro activation system. In the preincubation assay an increased bactericidal effect was observed. In contrast to TNM, NT, the nitrosation product, was non-mutagenic and non-toxic in the standard plate test and with the preincubation method up to 5000 micrograms/plate with and without S9 mix and with all tester strains used. Although TNM is a strong direct-acting mutagen, its nitrosating effect on proteins does lead to nongenotoxic nitro products of tyrosine in proteins.     

Mutagenicity of methylisocyanate and its reaction products to cultured mammalian cells

Methylisocyanate (MIC) induced mutagenic responses in the absence of exogenous activation in the mouse lymphoma cell forward mutation assay at concentrations as low as 8-24 microM. MIC produced predominantly small mutant colonies, suggesting the possibility of clastogenic activity. The intermediate hydrolysis product, methylamine, was also mutagenic without exogenous activation but required several hundred-fold higher concentrations (ca. 3 mM). N,N'-Dimethylurea, the final product in the reaction of methylisocyanate and water, was totally refractory in either the presence or absence of S9 for concentrations up to 57 mM (5 mg/ml). The ethyl ester of N-methylcarbamic acid was also tested since it was the only available analogue to the highly reactive N-methylcarbamic acid intermediate. This compound was mutagenic only in the presence of S9 at doses exceeding 5-40 microM, which suggested the possibility that the free acid, produced by enzymatic hydrolysis, is also mutagenic. The mutagenic activity of the ester resulted solely in the production of small mutant colonies.     

DNA damage, DNA repair and chromosome aberrations of xeroderma pigmentosum cells and controls following exposure to nitrosation products of methylguanidine

Nitrosation of methylguanidine (MG) led to products that caused DNA fragmentation (shift in sedimentation profiles of velocity centrifugation through alkaline sucrose gradients), a DNA repair synthesis (unscheduled uptake of (³H]TdR), chromosome aberrations and a lethal effect of cultured human fibroblasts. The response of repair-deficient xeroderma pigmentosum cells did not differ from that of controls. The nitrosation of MG must be carried out at a pH level below 3, in order to obtain products that react with cellular DNA. The results show that a DNA repair synthesis of human fibroblasts appear to be a sensitive assay for carcinogenic and mutagenic nitrosation products which may be formed within an organism from non-carcinogenic compounds.     

Mutagenicity of Maillard browning reaction products from various nitrosated amino acid-glucose mixtures

Ten different amino acid-glucose Maillard browning products before and after reaction with nitrite were evaluated by the Ames mutagenicity assay. No mutagenic response was observed in the methylene chloride extracts of any browning products tested before nitrosation. However, mutagenicity was showed in most of the browning mixtures, e.g., glycine-glucose, lysine-glucose (I), arginine-glucose, phenylalanine-glucose (II), and methionine-glucose after nitrosation when examined by Salmonella typhimurium strains TA98 and TA100 either with or without S-9 metabolic activation. Among the browning mixtures, (I) and (II) showed the greatest mutagenic activity after reaction with nitrite. The mutagenicity of lysine-glucose with nitrite was dependent on browning intensity, nitrosation pH, nitrosation time, nitrite level and blocking agents.     

Inhibitory effect of saliva on glutamic acid accumulation by Lactobacillus acidophilus and the role of the lactoperoxidase-thiocyanate system

Clem, W. H. (University of Washington, Seattle), and S. J. Klebanoff. Inhibitory effect of saliva on glutamic acid accumulation by Lactobacillus acidophilus and the role of the lactoperoxidase-thiocyanate system. J. Bacteriol. 91:1848-1853. 1966.-Saliva contains an antimicrobial system which inhibits the growth of Lactobacillus acidophilus, as well as a number of other organisms, in complete growth medium. This antimicrobial system consists of the salivary peroxidase (lactoperoxidase) and thiocyanate ions, and requires the presence of H(2)O(2). Saliva inhibits the accumulation of glutamic acid and certain other amino acids by resting cells. This effect of saliva is decreased by dialysis, and thiocyanate ions restore the inhibitory effect of dialyzed saliva. The inhibitory effect of saliva is decreased by heat (100 C, 10 min), and lactoperoxidase restores the inhibitory effect of heated saliva. Thus, the inhibition of glutamic acid accumulation by saliva appears to be due in part to the lactoperoxidase-thiocyanate antimicrobial system. H(2)O(2) increases the inhibitory effect of both saliva and the lactoperoxidase-thiocyanate system on glutamic acid accumulation. The inhibition of glutamic acid accumulation is not preceded by a loss in microbial viability. The glutamic acid accumulated by L. acidophilus under the conditions employed remains largely (over 90%) as free glutamic acid. This suggests that saliva and the lactoperoxidase-thiocyanate-H(2)O(2) system inhibit the net transport of glutamic acid into the cell.     

Effects of mono-, di- and tri-hydroxybenzoic acids on the nitrosation of propranolol: structure-activity relationship

Nitrosation of propranolol under standard conditions recommended by the World Health Organization (10mM propranolol hydrochloridre, 40mM sodium nitrite, pH 3.5) was performed in the absence and in the presence of benzoic acid and of twelve mono-, di- and tri-hydroxybenzoic acids, added to the nitrosation mixture in concentrations ranging from 2 to 40mM, in order to examine their effect on the nitrosation reaction. The yield of N-nitrosopropranol (NOP) was reduced by benzoic acid and, with potency decreasing in the following order, by 2,3,4-tri-hydroxybenzoic acid>/=3,4-tri-hydroxybenzoic acid>2,5-di-hydroxybenzoic acid>2,3-di-hydroxybenzoic acid>3-hydroxybenzoic acid>2-hydroxybenzoic acid>3,4,5-tri-hydroxybenzoic acid>4-hydroxybenzoic acid; their inhibiting effect was concentration-dependent. In contrast, 2,4-di-hydroxybenzoic acid, 2,6-di-hydroxybenzoic acid and 2,4,6-tri-hydroxybenzoic acid caused an increase in the yield of NOP that was inversely related to their concentration. 3,5-Di-hydroxybenzoic acid was substantially inactive. These findings indicate that, depending on the positions of carboxyl group and hydroxyl groups on the benzene ring, mono-, di- and tri-hydroxybenzoic acids may inhibit or hasten nitrosation reactions. As compared with benzenediols and benzenetriols [Mutat. Res. 398 (1998) 75], hydroxybenzoic acids inhibit the nitrosation of propranolol to a greater extent and have the advantage of being nonmutagenic and less toxic.     

Enhancement and reduction by methylated oxypurines of the frequencies of chromatid aberrations induced by camptothecin in root-tip cells of Vicia faba.

In root-tip cells of Vicia faba the frequencies of chromatid aberrations induced by 3-h treatments with 0.05 microM camptothecin were strongly modified when the treatments were carried out in the presence of caffeine at concentrations above 1 mM. Depending on the concentration of caffeine, the clastogenic effect of camptothecin was either enhanced or reduced. At concentrations between 1 and 6 mM, caffeine increased the camptothecin-induced chromosome damage, the strongest enhancement being obtained at 5 mM. A reduction of the chromosome damage was apparent at caffeine concentrations above 10 mM, and in the presence of 20 mM caffeine the clastogenic effect of camptothecin was almost completely suppressed. When present during the camptothecin treatment, theophylline, 8-chlorocaffeine and 1,3,7,9-tetramethyluric acid influenced the induced chromosome damage in a similar way as caffeine, although with varying efficiency. If the concentrations required to produce the two types of modifying effect are used as a criterion, 8-chlorocaffeine was the most effective and 1,3,7,9-tetramethyluric acid the least, whereas caffeine and theophylline were about equally effective.     

Exposure of Vicia faba and Pisum sativum to copper-induced genotoxicity

The potential genotoxicity of Cu(2+) was investigated in Vicia faba and Pisum sativum seedlings in hydroponic culture conditions. Cu(2+) caused a dose-dependent increase in micronuclei frequencies in both plant models. Cytological analysis of root tips cells showed clastogenic and aneugenic effects of this heavy metal on V. faba root meristems. Cu(2+) induced chromosomal alterations at the lowest concentration used (2.5 mM) when incubated for 42 h, indicating the potent mutagenic effect of this ion. A spectrum of chromosomal abnormalities was observed in V. faba root meristems, illustrating the genotoxic events leading to micronuclei formation.     

Laser pyrolysis products-genotoxic, clastogenic and mutagenic effects of the particulate aerosol fractions

Laser therapy has gained wide acceptance and application in many medical disciplines. Nevertheless, during surgical procedures, the thermal destruction of tissue creates a smoke plume. Recent research data indicate that pyrolysates liberated during vaporisation of tissue induce DNA damage. However, assessing potential health hazards during medical laser treatment requires comprehensive insight into the cytotoxic, genotoxic, clastogenic and mutagenic capacity of laser pyrolysis products (LPP). Therefore, the aim of this study was to evaluate the cytotoxic, genotoxic, clastogenic and mutagenic potential of substances resulting from laser irradiation. Four different types of porcine tissues were irradiated with a surgical CO2 laser, the aerosols were sampled under defined conditions and subjected to the SCE test, micronucleus test and the HPRT test. The results showed that the pyrolysis products are strong inducers of cytotoxic effects. The pyrolysis products induced positive effects in the SCE test, micronucleus test and the HPRT test. The ability and extent to induce genotoxic and mutagenic effects turned out to be dependent on the type of tissue that had been irradiated. In general, the effects were most pronounced with liver pyrolysate. In all test systems, a clear dose relationship could be established. In conclusion, we were able to prove that the particulate fraction of laser pyrolysis aerosols originating from biological tissues undoubtedly have to be classified as cytotoxic, genotoxic, clastogenic and mutagenic. Therefore, they could be potential health hazards for humans.     

The effect of humic acids,fractionated according to molecular mass,on the formation and mutagenicity of N-methyl-N-nitrosourea

Fractions of humic acids (HA-K⁺) of molecular mass between 500 and 300 000 and exceeding 300 000 showed a very high nitrite depleting ability, whereas the fraction of HA-K⁺ with molecular mass lower than 500 had little or no such effect. Autoclaving HA-K⁺ (121 °C, 20 min) decreased the nitrite-depleting ability to about a half. This observation correlates with the results of mutagenic studies in Arabidopsis thaliana, demonstrating the inhibitory effect of HA-K⁺ and its fractions on the formation of mutagenic N-methyl-N-nitrosourea (MNU) from the nitrosation mixture of N-methylurea and nitrite. Nonfractionated HA-K⁺ had no inhibitory effect towards the mutagenic activity of preformed direct acting MNU or towards the activation of the promutagen N-nitrosodimethylamine to a mutagenic product.     

Mutagenic effects of nitrogen dioxide combined with methylurea and ethylurea in Drosophila melanogaster

The standard sex-linked recessive lethal test was used to test whether NO2 induces lethal mutations in male germ cells of Drosophila in the presence or absence of alkylureas. Methylurea, ethylurea and NO2 alone did not enhance the mutation frequency significantly. However, highly significant enhancement in the mutation frequency was observed when adult flies were exposed to NO2 (150--280 ppm) for 3 h after ingestion of methylurea (0.1 M) or ethylurea (0.1 M) for 2 days. Oral administration of ethylnitrosourea and also of methylurea or ethylurea that had been exposed to NO2 in vitro were more effective in increasing the mutation frequency than methylurea or ethylurea combined in vivo with NO2. These results suggest that ingested alkylurea is converted in vivo by inhaled NO2 to highly mutagenic nitrosoalkylurea and/or other mutagens. No significant enhancement of the mutation frequency was observed when flies were fed on methylurea solution after they had been exposed to NO2.     

Curcumins as inhibitors of nitrosation in vitro

The effects of turmeric extract and its pure yellow pigments curcumin I, II and III were tested on the nitrosation of methylurea by sodium nitrite at pH 3.6 and 30 degrees C. The nitrosomethylurea formed was monitored by checking the mutagenicity in S. typhimurium strains TA1535 and TA100 without metabolic activation. Turmeric extract as well as curcumins exhibit dose-dependent decreases of nitrosation. Curcumin III was the most effective nitrosation inhibitor among the compounds tested. The simultaneous treatment of inhibitor with nitrosation precursors was essential and pre- or post-treatment of inhibitor had no effect on the mutagenicity of nitrosomethylurea. The binding of nitrite with the inhibitors was studied at pH 3.6 and 30 degrees C. Curcumin I shows a dose-dependent depletion of nitrite ions thus making nitrite non-available for nitrosation. Curcumin I and III when tested also showed a time-dependent depletion of nitrite ions at pH 3.6 and 30 degrees C. Curcumin III has a higher affinity for nitrite ions than curcumin I.     

Evaluation of clastogenicity of formic acid, acetic acid and lactic acid on cultured mammalian cells

Using Chinese hamster ovary K1 cells, chromosomal aberration tests were carried out with formic acid, acetic acid and lactic acid, and the relationship between the pH of the medium and the clastogenic activity was examined. The medium used was Ham's F12 supplemented with 17 mM NaHCO3 and 10% fetal calf serum. All of these acids induced chromosomal aberrations at the initial pH of ca. 6.0 or below (about 10-14 mM of each acid) both with and without S9 mix. Exposure of cells to about pH 5.7 or below (about 12-16 mM of each acid) was found to be toxic. When the culture medium was first acidified with each of these acids and then neutralized to pH 6.4 or pH 7.2 with NaOH, no clastogenic activity was observed. Using F12 medium supplemented with 34 mM NaHCO3 as a buffer, no clastogenic activity was observed at doses up to 25 mM of these acids (initial pH 5.8-6.0). However, it was found that about 10% of the cells had aberrations at pH 5.7 or below (27.5-32.5 mM of each acid). Furthermore, when 30 mM HEPES was used as a buffer, chromosomal aberrations were not induced at doses up to 20 mM formic acid and acetic acid (initial pH 7.0-7.1), and at doses up to 30 mM lactic acid (initial pH 6.6). In the initial pH range of 6.4-6.7 (25-32.5 mM of each acid), chromosomal aberrations were observed. The above results show that these acids themselves are non-clastogenic, and the pseudo-positive reactions attributable to non-physiological pH could be eliminated by either neutralization of the treatment medium or enhancement of the buffering ability.     

Generation of an endogenous DNA-methylating agent by nitrosation in Escherichia coli.

Escherichia coli ada ogt mutants, which are totally deficient in O6-methylguanine-DNA methyltransferases, have an increased spontaneous mutation rate. This phenotype is particularly evident in starving cells and suggests the generation of an endogenous DNA alkylating agent under this growth condition. We have found that in wild-type cells, the level of the inducible Ada protein is 20-fold higher in stationary-phase and starving cells than in rapidly growing cells, thus enhancing the defense of these cells against DNA damage. The increased level of Ada in stationary cells is dependent on RpoS, a stationary-phase-specific sigma subunit of RNA polymerase. We have also identified a potential source of the mutagenic agent. Nitrosation of amides and related compounds can generate directly acting methylating agents and can be catalyzed by bacteria] enzymes. E. coli moa mutants, which are defective in the synthesis of a molybdopterin cofactor required by several reductases, are deficient in nitrosation activity. It is reported here that a moa mutant shows reduced generation of a mutagenic methylating agent from methylamine (or methylurea) and nitrite added to agar plates. Moreover, a moa mutation eliminates much of the spontaneous mutagenesis in ada ogt mutants. These observations indicate that the major endogenous mutagen is not S-adenosylmethionine but arises by bacterially catalyzed nitrosation.