Metabolic epidemiology of large bowel cancer. Fecal mutagens in high- and low-risk population for colon cancer. A preliminary report

Because of potential significance of fecal mutagens in the pathogenesis of colon cancer, the dietary pattern and fecal mutagens of 3 populations with distinct risk for the development of colon cancer, a high-risk population in New York Metropolitan area (non-Seventh-Day Adventists), a low-risk population of vegetarian Seventh-Day Adventists in New York Metropolitan area and a low-risk population in rural Kuopio, Finland were studied. The average daily intake of protein was the same in the 3 groups, but the sources were different, a greater portion coming from meat in the New York non-Seventh-Day Adventists and from vegetables in Seventh-Day Adventists. The intake of fat was lower in Seventh-Day Adventists and higher in Kuopio and in New York non-Seventh-Day Adventists. The intake of dietary fiber was high in Kuopio compared to other groups. Fecal samples collected for 2 days were freeze-dried extracted with peroxide-free diethyl ether, partially purified on a silica-gel column and assayed for mutagenicity using the Salmonella/mammalian microsome mutagenicity test. The mutagenic activity was observed with Salmonella typhimurium tester strain TA98 without microsomal activation and with TA100 with and without microsomal activation in high-risk subjects from New York consuming a high-fat, high-meat diet. The incidence of fecal mutagen activity was higher in volunteers from New York consuming a high-fat, high-meat diet compared to low-risk rural Kuopio population. None of the vegetarian Seventh-Day Adventists showed any mutagenic activity.     

The mutagenicity of bile acids using a fluctuation test

The mutagenicity of bile acids was detected by a fluctuation test using Salmonella typhimurium TA100 and TA98 as tester strains. Cholic acid, chenodeoxycholic acid, deoxycholic acid and ursodeoxycholic acid were mutagenic in this test while lithocholic acid was not. The mutagenicity of the bile acids on a molar basis was roughly one-fourth that of methyl methanesulfonate, a moderately potent mutagen. Epidemiological studies have shown a high correlation between levels of bile acids excreted and colon cancer. However, no evidence has previously been reported showing that bile acids are mutagenic. Our results suggest that bile acids may be important in the etiology of colon cancer.     

Mutagenicity studies on fenitrothion in bacteria and mammalian cells

The mutagenicity of fenitrothion was determined in strains of Salmonella typhimurium and Escherichia coli. Fenitrothion was found to be non-mutagenic in Salmonella typhimurium strains of TA98, TA1535 and TA1537 and in Escherichia coli WP2uvrA both with and without S9 mix, while weak mutagenicity was observed only in Salmonella typhimurium TA100 and enhanced by the addition of S9 mix. The mutagenicity observed in the TA100 strain was not expressed in a nitroreductase-deficient strain, TA100 NR, and decreased in a transacetylase-deficient strain, TA100 1,8-DNP6. The mutagenicity of fenitrothion was also examined by a gene mutation assay using the gene for hypoxanthine-guanine phosphoribosyltransferase (hgprt) in V79 Chinese hamster lung cells. Fenitrothion did not induce any increment of 6-thioguanine-resistant mutant cells at doses ranging from 0.01 to 0.3 mM regardless of the presence or absence of S9 mix. These results suggest that reduction of fenitrothion by a bacterial nitroreductase of TA100 to an active form is essential for the expression of the mutagenicity of fenitrothion in TA100 and that a bacterial transacetylase of TA100 also has an important role in the process of mutagenic activation.     

Mutagenicity of 2,6-dinitrotoluene and its metabolites, and their related compounds in Salmonella typhimurium

The mutagenic activities of 2,6-dinitrotoluene (2,6-DNT) and its 6 metabolites, and their 8 related compounds were examined using Salmonella typhimurium strains TA98 and TA100 in the absence or presence of S9 mix. 2,6-DNT itself showed no mutagenicity toward either strain, but 2,6-dinitrobenzaldehyde (2,6-DNBAl), one of the metabolites of 2,6-DNT, showed the highest mutagenic activity in strain TA100. 2,6-DNBAl was a direct-acting mutagen, not requiring metabolic activation. The other compounds containing nitro groups showed weak or no mutagenic activity. This result suggests that the direct-acting mutagenicity of 2,6-DNBAl is mainly due to the aldehyde group of the 2,6-DNBAl molecule.     

Mutagenic substances in red and white wine in Chile, a high risk area for gastric cancer

Chilean home-made and commercial wines were analyzed for the presence of mutagenic substances using the Salmonella mutagenicity test with preincubation. Strains TA98 and TA100 were used in the absence and in the presence of S9 mix. 90% of red wines from a total of 30 samples and 54% of white wines from a total of 22 were found to be mutagenic. In all cases, S9 mix did not affect the mutagenicity of the samples. At least in one case, more than one mutagen was present, since the mutagenicity with TA98 could be selectively inactivated without affecting that with TA100. This study supports the hypothesis that wine consumption may be an important risk factor for upper gastrointestinal cancer, particularly for adenocarcinoma of the stomach, which is highly prevalent in Chile.     

Biological monitoring of bidi rollers with respect to genotoxic hazards of occupational tobacco exposure

Smokeless tobacco habits are associated with a high incidence of oropharyngeal cancer in India. Hence, the biological effects of occupational exposure to smokeless tobacco used for making bidis (the Indian version of cigarettes) were studied in 2 groups of bidi rollers designated BR-K and BR-S and in control subjects with no tobacco habits. Specific tobacco exposure and the electrophilic burden were determined by estimating urinary cotinine and thioethers respectively. Urine mutagenicity was tested with the Ames assay using Salmonella typhimurium strains TA98 and TA100. While cotinine was not detected in control samples, the mean cotinine levels (mmole/mole creatinine) in the BR-K and BR-S groups were 0.79 +/- 0.30 and 0.09 +/- 0.03 respectively. Urinary thioether excretion (mmole/mole creatinine) was significantly elevated in the BR-S group 4.59 +/- 0.52; p less than 0.001) but it was lower in the BR-K group (0.54 +/- 0.08; p less than 0.001) compared to the control (1.83 +/- 0.34). Furthermore, beta-glucuronidase-treated samples from both groups of bidi rollers exhibited increased mutagenicity to TA98 compared to the control group; in addition, BR-S samples exhibited direct mutagenicity to TA98. The results show that occupational tobacco exposure modulates the glutathione conjugation pathway and increases the mutagenic burden of bidi rollers.     

Metabolic epidemiology of colon cancer: fecal mutagens in healthy subjects from rural Kuopio and urban Helsinki, Finland

Fecal mutagenic activity and dietary pattern of rural and urban Finnish population groups with distinct risk for the development of colon cancer were studied in a low-risk population in rural Kuopio and an intermediate-risk population in urban Helsinki. The average daily intake of protein and fat was the same in the two groups but the frequency of consumption of whole-grain cereals and whole-grain bread, as well as the amount of fiber from the bread were higher in Kuopio as compared to Helsinki. Fecal samples collected for 2 days were incubated under anaerobic conditions at 37 degrees C for 96 h, extracted with hexane: peroxide-free diethyl ether, partially purified on a silica Sep-Pak cartridge, and assayed for mutagenic activity using the Salmonella typhimurium/mammalian microsome system. The fecal mutagenic activity was observed with the tester strains TA98 and TA100 with and without microsomal activation in both the population groups. The percentage of samples showing fecal mutagenic ratio greater than 3 with TA98 and TA100 with microsomal activation, was higher in volunteers from Helsinki than in Kuopio.     

Demonstration of a powerful mutagenic dinitropyrene in airborne particulate matter

Of the many nitroarenes, dinitropyrenes (DNPs) have the potential to revert Salmonella typhimurium his- mutants. This study was conducted to investigate the potential mutagens present in airborne particulate matter collected in Santiago, Chile. 5 organic substances extracted with dichloromethane showed mutagenic rates of from 38.9 to 287 revertants per m3 of air for S. typhimurium his- strain TA98 without S9 mix. 4 of the samples had greatly reduced mutagenicity for strain TA98/1,8DNP6 but not for strain TA98NR. The 1-nitropyrene (1-NP) content accounted for 0.06-0.15 microgram per g of particulate, as determined by high-performance liquid chromatography (HPLC), but the contribution of the compound to mutagenicity was less than 1% of the total activity. On the other hand, by using two columns in the HPLC, DNPs of 1,6- and 1,8-isomers were detected in the samples pooled after the determination of 1-NP, and the amount of the derivatives was about 0.2 microgram per g of particulate matter.     

The basis of the insensitivity of Salmonella typhimurium strain TA98/1,8-DNP6 to the mutagenic action of nitroarenes

Salmonella typhimurium tester strain TA98/1,8-DNP6 is resistant to the mutagenicity of 1,8-dinitropyrene because it lacks an esterification enzyme which is needed for the formation of the ultimate mutagen, presumably the corresponding hydroxamic acid ester. This enzyme does not appear to be required for the activation of all nitroarenes and arylamines, as some of these are fully active in TA98/1,8-DNP. It is suggested that these form electrophilic arylnitrenium ions nonenzymatically from nitroso- and N-hydroxylamino-arenes intermediates. The esterification enzyme appears to be a transacetylase. An assay using 2-aminofluorene as the acetyl acceptor is described. Derivatives of S. typhimurium TA100 also lacking this enzyme were obtained by Tn5-mediated mutagenesis.     

Host-mediated mutagenicity experiments with benzo[a]pyrene and two of its metabolites

Benzo[a]pyrene (BP) and two of its major metabolites, the ultimate mutagen BP-4,5-oxide and the proximate mutagen trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (BP-7,8-diol) were investigated for mutagenicity in Salmonella typhimurium TA1538, TA98 and TA100 using an intrasanguineous host-mediated assay. BP and BP-4,5-oxide were not mutagenic under any experimental conditions. BP-7,8-diol was inactive with the strain TA1538 but was mutagenic with the strains TA98 and TA100. The effect was potentiated by pretreatment of the host mice with the cytochrome P-450 inducer 5,6-benzoflavone. We conclude: (i) one of the reasons for the observed insensitivity of the intrasanguineous host-mediated assay towards BP is that BP-4,5-oxide, which contributes to the microsome-mediated mutagenicity of BP, is inactive in the host-mediated assay; (ii) the finding that BP-7,8-diol is mutagenic in the host-mediated assay demonstrates that the lack of mutagenicity of BP is not intrinsic; (iii) the potentiated mutagenicity after treatment of the hosts with 5,6-benzoflavone suggests that cytochrome P-450 is more important in the activation of BP-7,8-diol in this system than other enzymes (e.g. prostaglandin synthase) that can also activate this compound in vitro.     

Enhancing effect of saccharides on the mutagenicity of 2-chloro-4-methylthiobutanoic acid

The mutagenicity of 2-chloro-4-methylthiobutanoic acid (CMBA), a nitrite-treated Sanma fish mutagen, in Salmonella typhimurium TA100 was enhanced by addition of D-glucose during the CMBA-treatment. Several other monosaccharides also enhanced the mutagenicity of CMBA, and the order of the enhancing potency was found to be D-mannose, D-glucose>D-fructose, D-ribose, D-galactose. A disaccharide, maltose, showed only little enhancement. No enhancement was found with L-glucose. We investigated whether saccharides affect uptake of [methyl-14C]CMBA into S. typhimurium TA100. Saccharides which enhanced CMBA-induced mutagenesis increased the uptake. L-Glucose did not enhance the uptake. There was good correlation between the enhanced mutagenesis and increased radioactivity in Salmonella, suggesting that the enhancing effect of monosaccharide on the CMBA-induced mutagenesis results from the enhanced uptake of the mutagen into bacteria.     

Formation of a diazoquinone-type mutagen from acetaminophen treated with nitrite under acidic conditions

Acetaminophen (paracetamol) showed mutagenicity to Salmonella typhimurium TA100 and TA98 either with or without S9 mix when treated with excess nitrite in acidic solution. The mutagen was isolated and identified as 4-acetylamino-6-diazo-2,4-cyclohexadienone. Mutation tests and product analysis by high-performance liquid chromatography, however, suggested that this type of mutagen was hardly formed in the digestive tract of normal subjects.     

Mutagenicity tests on anthelmintics: microsomal activation of viprynium embonate to a mutagen.

Eight anthelmintic preparations readily available in Australia were tested for mutagenicity in the Salmonella typhimurium test system. A slightly modified version of the procedure recommended by Ames et al. [2] was adopted, in that the test samples were placed in "wells" cut out of the agar of a plate previously seeded with the appropriate tester strain. Addition of a mixture of rat liver microsomal enzymes and appropriate co-factors ("S-9 mix") to one of the two wells on a single plate allowed a possible requirement for metabolic activation to be recognised. Using this procedure, viprynium embonate was found to be non-mutagenic. It was however, activated by the rat liver microsome preparation to a mutagen capable of causing both base-pair substitution (detected with strain TA100) and frameshift (detected with strain TA98) mutations. The other seven compounds tested all gave negative results in this system.     

Formation of mutagens during the frying of Hawaiian fish: correlation with creatine and creatinine content

Compounds mutagenic toward Salmonella typhimurium strain TA98 in the presence of rat-liver homogenates (S9) were formed when fish flesh was fried at 199 degrees C. Three species of Hawaiian fish commonly consumed in Hawaii (skipjack tuna, Katsuwonus pelamis; yellowfin tuna, Neothunnus macropterus; and milkfish, Chanos chanos) were cooked in an electric skillet, along with samples of sole (Microstomus pacificus). Organic extracts of the fish were tested in the Ames Salmonella mutagenic assay using tester strain TA98 and S9. Basic organic extracts of fried, but not raw, samples exhibited significant mutagenicity. The levels of mutagenicity were also higher among the red flesh Hawaiian fish ('ahi, aku and awa) than with the white flesh sole. Creatine and creatinine contents were highest in the Hawaiian fish and lower in the sole. Creatine levels in the fish were 50-100 times greater than the creatinine content and varied from a high of 645 mg/100 g wet weight of fish for yellowfin tuna to a low value of 251 mg/100 g for sole. Mutagen levels are only approximately related to creatine/creatinine levels suggesting that other components contained in these fish may be as important as the guanidines in determining the levels of mutagen in the cooked fish.     

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.     

Sesamol exhibits antimutagenic activity against oxygen species mediated mutagenicity

The effects of sesamol, a phenolic compound responsible for the high resistance of sesame oil to oxidative deterioration as compared with other vegetable oils, have been investigated after mutagen treatment in various strains of Salmonella typhimurium. Sesamol was shown to exhibit strong antimutagenic effects in the Ames tester strains TA100 and TA102. The TA102 strain has been shown to be highly sensitive to reactive oxygen species. Mutagenicity was induced by the generation of oxygen radicals by tert-butylhydroperoxide (t-BOOH) or hydrogen peroxide (H(2)O(2)); therefore, the antimutagenic property of sesamol was attributed to its antioxidant properties. The superoxide and hydroxyl radical scavenging capabilities have further been elucidated using in vitro test systems. It was further shown to have a desmutagenic effect on t-BOOH-induced mutagenesis in TA102 strain. Sesamol also inhibited the mutagenicity of sodium azide (Na-azide) in TA100 tester strain while it had no effect on nitroquinoline-N-oxide (NQNO)-induced mutagenesis in TA98 strain of Salmonella typhimurium. Since active oxygen species are involved in multiple stage processes of carcinogenicity, this compound may also exhibit anticarcinogenic properties.     

Mutagenicities of nitrosated carboline derivatives

Food-borne amines have been considered as the potential precursors of endogenous carcinogenic N-nitroso compounds in humans. A compound which yields a direct mutagen after nitrite treatment was isolated from soy sauce and was identified as 1-methyl-1,2,3,4-tetrahydro-2-carboline-3-carboxylic acid (MTCA) (Wakabayashi, et al., 1983). The mutagenicities of other carboline derivatives such as harman, norharman, harmaline, harmalol, harmine, and harmol were studied. Like MTCA, the nitrosated carboline derivatives showed higher mutagenic activity as compared to their corresponding parent compounds. The demethylated analogue of MTCA, 1,2,3,4-tetrahydro-2-carboline-3-carboxylic acid was synthesized and its nitrosated products were shown to be mutagenic to Salmonella typhimurium TA 100 and TA 98. The potent mutagen Trp-P-2 is a typical 3-carboline derivative. The mutagenicity of Trp-P-2 was suppressed remarkably after nitrosation. Several 3-carboline derivatives also showed the similar property. Nitrosation of MTCA gave several derivatives which were isolated and showed direct mutagenicity to Salmonella typhimurium TA 98. Further characterization of these new carboline derivatives is in progress.     

Mutagenicity of aliphatic epoxides.

The mutagenicity of 17 aliphatic epoxides was determined using the specially constructed mutants of Salmonella typhimurium developed by Ames. The activity of these epoxides together with those reported in the literature as mutagens in strains TA100 and TA1535 depended on the degree of substitution around the oxirane ring. Monosubstituted oxiranes were the most potent mutagens in both strains. 1,1-Disubstitution resulted in the complete loss or reduction of mutagenicity, trans-1,2-Disubstituted, and tetrasubstituted oxiranes all lacked mutagenicity, while the cis-1,2-disubstituted oxiranes tested were weakly mutagenic in strain TA100 only. For the monosubstituted compounds the presence of electron-withdrawing substituents increased mutagenicity.     

The mutagenicity of natural products from marine algae.

5 polyhalogenated hydrocarbon natural products isolated from the marine red alga Plocamium spp. were tested for mutagenicity in the Ames reversion assay. All 5 of the compounds induced revertants in Salmonella typhimurium strains TA100 and TA1535, indicating the mutational events involved base substitutions. One of the compounds, designated cross-conjugated ketone, was shown to be almost 200 times more effective as a mutagen than was ethyl methanesulfonate.     

Preliminary studies on the ability of Drosophila microsomal preparations to activate mutagens and carcinogens

Subcellular fractions from Drosophila melanogaster, known to have several xenobiotic-metabolizing enzymatic activities, were investigated with respect to their ability to biotransform compounds that require metabolic activation before exerting mutagenic effects. Nitrofurazone, dimethylnitrosamine, cyclophosphamide and 2-acetylaminofluorene were activated to mutagens upon incubation with Drosophila microsomes or 20000 x g supernatant: mutagenicity was observed in Chinese hamster ovary cells, Escherichia coli strains 343/113/R-9 and 343/113/uvrB, and Salmonella typhimurium TA1538. Under the conditions used, microsomal preparations of Drosophila were not able to activate benzo[a]pyrene to a mutagen for Salmonella typhimurium TA98. The spectrum of mutagenic effects observed shows some correlation with the known mutagenicity of these compounds in vivo in Drosophila melanogaster. Drosophila microsomes appeared to be at least as active as rat-liver microsomes when compared in this type of mutagenicity testing.