Reversal by vitamin A analogues (retinoids) of hyperplasia induced by N-methyl-N'-nitro-N-nitrosoguanidine in mouse prostate organ cultures.

The antihyperplastic activity of beta-retinoic acid (RA) and nine synthetic analogues (retinoids) was examined in organ cultures of mouse prostate made hyperplastic by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). After 8 or 10 days, when most explants developed hyperplasia, the carcinogen was withdrawn and explants were incubated in control medium and medium containing different concentrations of a retinoid. The antimitotic activity of retinoids was compared with that of RA. Different retinoids produced variable degrees of mitotic inhibition in the hyperplastic prostate epithelium. The methylketo cyclopentenyl and 1-methoxyethyl cyclopentenyl analogues of RA were at least 50-fold more active than RA in reversing MNNG-induced hyperplasia. The trimethylmethoxyphenyl analogue of RA and retinyl methyl ether were significantly more active than RA. Three analogues, N-acetyiretinylamine, retinal acetyl hydrazone, and retinal oxime, were as active as RA. The chlorotrimethylphenyl analogue showed less activity than RA, and alpha-retinyl acetate was completely devoid of mitotic inhibitory activity.

DNA methylase inhibition in vitro by N-methyl-N'-nitro-N-nitrosoguanidine.

Several alkylating carcinogens were tested for their ability to inhibit DNA methylation in an in vitro assay. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) was the only carcinogen studied that altered the methylase activity. It was further demonstrated that MNNG reacts with the DNA methylase protein and produces a mixed type of inhibition. Prevention of the MNNG effect by dithiothreitol and inhibition of the DNA methylase with iodoacetamide suggest that DNA methylase is a sulfhydryl-containing enzyme and that MNNG inactivates the enzyme by reacting with sulfhydryl groups.

Mechanisms of inhibition by ascorbate of microbial mutagenesis induced by N-nitroso compounds.

Mutagenesis induced by N-methyl-N-nitrosoguanidine (MNNG) and dimethylnitrosamine (DMN) in Salmonella TA 1530 was inhibited by ascorbate. Inhibition of MNNG-induced mutagenesis resulted from a reaction between ascorbate and MNNG that led to consumption of MNNG. The rate of this reaction was considerably enhanced by catalytic amounts of Cu(II) and Fe(III). No direct reaction between DMN and ascorbate was detectable, but relatively high concentrations of Cu(II) enchanced inhibition of DMN-induced mutagenesis by ascorbate. Added protein reduced the effectiveness of Cu(II) as a catalyst of the reaction between ascorbate and MNNG, which suggested that the microsomal protein necessary to activate DMN, may reduce the concentration of free Cu(II) and thereby lower its catalytic efficiency. Mutagenesis by N-methyl-N-nitrosourea was not inhibited by ascorbate.

Inhibitory effect of dietary selenium on carcinogenesis in rat glandular stomach induced by N-methyl-N'-nitro-N-nitrosoguanidine.

The influence of dietary selenium on the incidence of stomach carcinoma induced by N-methyl-N'-nitro-N-nitrosoguanidine was studied in 108 rats that survived for over 10 wk. The incidence of glandular stomach cancer in the high-selenium (4.0 ppm) diet group (20 carcinomas in 54 rats) was lower than in the low-selenium (0.1 ppm) diet group (33 carcinomas in 54 rats). The selenium level and glutathione peroxidase activity in the blood, liver, and stomach mucosa were significantly higher in the high-selenium diet group than in the low-selenium diet group. Glutathione peroxidase activity as well as the concentration of selenium in the glandular stomach was increased significantly in the high-selenium diet group.

Enhanced induction of gastric carcinogenesis by N-methyl-N'-nitro-N-nitrosoguanidine in deoxycorticosterone acetate-NaCl hypertensive rats and its inhibition by potassium chloride.

The effect of s.c. administration of deoxycorticosterone acetate (DOCA) plus p.o. treatment with NaCl solution on gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine and the effect of p.o. potassium supplementation on the enhanced induction of gastric carcinogenesis in DOCA-NaCl rats were investigated in Wistar rats. After 25 weeks of p.o. treatment with the carcinogen, rats received s.c. injections of DOCA (50 mg/kg) twice a week and were given 1% NaCl solution with and without 1% KCl as drinking water. In Week 52, the blood pressure, the incidence of gastric cancer, and the number of cancers per rat were significantly greater in DOCA-NaCl rats than in the untreated group. Prolonged p.o. treatment of DOCA-NaCl hypertensive rats with potassium significantly reduced their blood pressure, the incidence of gastric cancers, and their number per rat. All gastric tumors were in the glandular portions of the stomach. The norepinephrine concentration in the gastric wall and the labeling indices of gastric mucosa were significantly greater in DOCA-NaCl hypertensive rats than in the untreated group, but p.o. potassium supplementation significantly reduced the norepinephrine concentration in the gastric wall and the labeling indices of the gastric mucosa in DOCA-NaCl rats. Thus, administration of DOCA and NaCl increased the norepinephrine concentration in the gastric wall and promoted gastric carcinogenesis, and p.o. potassium supplementation decreased the norepinephrine concentration in the gastric rats. Inasmuch as the norepinephrine concentration has been used as a marker of sympathetic nervous activity, these findings suggest that the sympathetic nervous system plays an important role in gastric carcinogenesis, probably associated with cell proliferation of antral epithelial cells.

Mechanism of inhibition of N-methyl-N'-nitro-N-nitrosoguanidine-induced mutagenesis by phenolic compounds.

At non-toxic concentrations, 2 naturally occurring phenolic compounds, caffeic acid and chlorogenic acid, suppressed the mutagenic activity of the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Salmonella typhimurium strain TA1535. The inhibitory effect was observed only when the phenolic compound and the mutagen were administered concurrently. The interaction between phenolic compounds and MNNG was also studied in a cell-free system using a colorimetric method. The results are consistent with the assumption that phenolic compounds scavenge reactive electrophilic MNNG degradation products, thereby preventing their action on critical cellular targets.

Interception of some direct-acting mutagens by ergothioneine.

Ergothioneine, a novel imidazole sulfhydryl/thione compound formed in millimolar amounts by fungi, is a potentially important defense against electrophiles and free radicals. Protection may well occur both in organisms that synthesize ergothioneine and in animals including man that ingest and store ergothioneine in red blood cells, the liver, seminal fluid, and central nervous system. Ergothioneine blocks the mutagenicity for Salmonella strain TA1950 (hisG46 uvrB) of the nitrosation products of spermidine to an extent that is approximately proportional to the ergothioneine concentration. Ergothioneine also alleviates mutagenicity of cumene and t-butyl hydroperoxides but does not react with N-methyl-N'nitro-N-nitrosoguanidine as does the cysteinyl sulfhydryl compound, glutathione.

Mechanisms of chemical mediated cytotoxicity and chemoprotection in isolated rat hepatocytes.

Although N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methylmethanesulfonate (MMS) cause injury and malondialdehyde formation in rat hepatocytes, MNNG toxicity is much more sensitive to inhibition by antioxidants. In order to quantify the relationship between toxicity and antioxidation potential, we compared 14 antioxidants that protected against MNNG and MMS toxicity. Chemoprotection was quantified as the concentration that delayed by 1 h the decline in trypan blue exclusion to less than or equal to 50%. While chemoprotection against MNNG and antioxidant efficacy were directly related (R = 0.86), chemoprotection against MMS and antioxidant efficacy were unrelated (R = 0.37). Since we hypothesized that protection against MMS involved stabilization of membranes, the capacity of the 14 compounds to stabilize membranes in an unrelated system (i.e. prevention of erythrocyte osmotic rupture) was assayed. Chemoprotection against both MNNG and MMS correlated with reduced RBC fragility (R = 0.97 and 0.70, respectively). One of the better protecting compounds, 4b,5,9b,10-tetrahydroindeno[1,2-b]indole, was also protective against hepatocellular toxicity mediated by acetaminophen, carbon tetrachloride and tert-butyl hydroperoxide, suggesting a fundamental basis in the mechanism of chemoprotection. We propose that methylating agents and perhaps other chemical toxicants destabilize cellular membranes resulting in hepatocellular injury. For MNNG, radical mediated events may result in membrane destabilization; for MMS, membranes are destabilized without concurrent radical events. The current studies provide a basis for future work to determine structure-activity relationships of chemoprotective agents, examine protection mechanisms, and develop better protective compounds.

Antimutagenic effect of p-aminobenzoic acid on the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium.

p-Aminobenzoic acid (PABA) exhibited antimutagenic activity toward N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)-induced mutagenicity in the Ames assay in Salmonella typhimurium. The antimutagenic effects were associated with an increased rate of decomposition of MNNG in the presence of PABA. The participation of other mechanisms, such as the alteration of cellular processes by PABA, however, cannot be excluded.

Antimutagenicity of propionic acid bacteria.

The antimutagenic effect of dialysed cell extracts of 4 strains of propionic acid bacteria was examined against the mutagenicity of sodium azide in the TA1535 tester strain of Salmonella typhimurium using the Ames test. It was noted that dialysates of 2 strains of Propionibacterium shermanii, P. pentosaceum and P. acnes, significantly reduced sodium azide-induced revertants. The dialysate of propionic acid cocci did not show an antimutagenic effect. The inhibitory activity was enhanced if the mutagen and extract were coincubated for 20 min prior to performing the mutagenicity assay. Antimutagenicity of dialysates from P. shermanii VKM-103 against MNNG and 9-aminoacridine was shown in S. typhimurium strains TA1535 and TA97. The antimutagenic activity was found in the protein fraction of the cell extract of P. shermanii. The proteins of the dialysate of P. shermanii were separated using a Toyopearl gel column into 3 main peaks according to their molecular weights. The antimutagenic activity towards sodium azide was found in the second and the third peaks. We suggest that dialysates of the cells of propionic acid bacteria contain several kinds of antimutagenic substances with different molecular weights.

Protective effects of sodium selenite on killing and mutation by N-methyl-N'-nitro-N-nitrosoguanidine in E. coli.

Sodium selenite was found to protect Escherichia coli cells against killing and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Such protective effects were not observed when cells were treated with N-methyl-N-nitrosourea (MNU). The protection by sodium selenite was not controlled by the ada gene, which is responsible for the repair of alkylated damage in DNA. A reduction of the amount of glutathione was found when cells were treated with sodium selenite, and glutathione is known to be involved in the methylation of DNA by MNNG, not by MNU. Reduced methylation by MNNG due to the reduction of the amount of glutathione caused by abundant sodium selenite was suggested to be the mechanism of protection.

Suppressive effects of coffee on the SOS responses induced by UV and chemical mutagens.

SOS-inducing activity of UV or chemical mutagens (AF-2, 4NQO and MNNG) was strongly suppressed by instant coffee in Salmonella typhimurium TA1535/pSK1002. As decaffeinated instant coffee showed a similarly strong suppressive effect, it would seem that caffeine, a known inhibitor of SOS responses, is not responsible for the effect observed. The suppression was also shown by freshly brewed coffee extracts. However, the suppression was absent in green coffee-bean extracts. These results suggest that coffee contains some substance(s) which, apart from caffeine, suppresses SOS-inducing activity of UV or chemical mutagens and that the suppressive substance(s) are produced by roasting coffee beans.

Crude tea extracts decrease the mutagenic activity of N-methyl-N'-nitro-N-nitrosoguanidine in vitro and in intragastric tract of rats.

The effects of tea extracts and their ingredients, catechins and L-ascorbic acid (AsA), on the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were examined in vitro and in the stomachs of rats using E. coli WP2 and S. typhimurium TA100. The extracts of green tea and black tea leaves decreased the mutagenic activity of MNNG to E. coli WP2 in vitro in a desmutagenic manner. Catechins such as (-)-epigallocatechin from green tea leaves and the low-molecular-weight tannin fraction isolated from black tea extract with HP-20 resin also exhibited inhibitory effects against the mutagenic activity of MNNG. A desmutagenic effect of AsA on MNNG-induced mutagenicity was observed depending on the dose, though it was complicated. The effects were also demonstrated in the stomachs of rats by assaying the bacterial mutagenic in vitro; the tea extracts previously given orally to rats reduced the mutagenic activity of MNNG remarkably, though simultaneous administration showed less effect. The effectiveness of tea extracts for the decrease of MNNG-induced mutagenesis in vitro and in vivo suggests that the habitual drinking of tea may reduce the tumor-initiating potency of MNNG-type nitrosoureido compounds if they are formed in the stomach.

Influence of ascorbic acid on the mutagenicity of N-methyl-N-nitrosoguanidine and nitrofurans studied by SOS chromotest.

Mutagenicity inhibition of MNNG*, NFAA* as well as of nitrovin by ascorbic acid was observed. The influence of ascorbic acid on these compounds was parallelly studied also by spectroscopy. Mutagenicity inhibition was characterized by SOS chromotest. Inhibition degree was evaluated quantitatively introducing the coefficient of inhibition. The influence of ascorbic acid was expressed most markedly in MNNG. It has been found that the decrease of mutagenicity should be mainly caused by acceleration of MNNG decomposition by ascorbic acid.

Cytochrome P-450 in the activation and inactivation of carcinogens.

The capacity of isolate mouse liver microsomes to alter the mutagenicity for bacteria of the primary carcinogen N-methyl-N'-nitro-N-nitrosoguanisine (MNNG) and the secondary one dimethylnitrosamine (DMN) was studied. Microsomal activation of DMN and inactivation of MNNG were decreased by protein- and protein-cholinedeficient diets and were increased by pretreatment with microsomal enzyme inducers. The decrease and increase paralleled the content of cytochrome P-450 present in the different microsomal preparations. With human liver microsomes of differing cytochrome P-450 contents similar correlation was obtained, whereas normal rat liver microsomes did not activate or inactivate DMN or MNNG. Oxidative demethylation of DMN by mouse liver microsomes and the activation of DMN to a mutagen followed similar kinetics. Both reactions were inhibited by carbon monoxide and the inhibition was maximally reversed by monochromatic light at 450 nm. These observations indicate that at least some carcinogens are activated or inactivated by the unspecific cytochrome P-450 dependent enzyme system, suggesting that the extent of this biotransformation may be one factor influencing human carcinogenesis.