Mutagenicity and Identification of the Reaction Products of Aqueous Chlorine or Chlorine Dioxide with L-Tryptophan

Nonvolatile products generated from reactions of graded molar ratios of aqueous chlorine or chlorine dioxide with L-tryptophan (1:1, 3:1 and 7:1) were shown to be direct-acting mutagens to Salmonella typhimurium TA100 and TA98. Increasing the ratio of disinfectant relative to amino acid led to increased mutagenic activity, with mutagenicity highest at the 7:1 molar ratio. Several fluorescent bands obtained after thin layer chromatographic fractionation of the reaction mixtures were shown to be more mutagenic than the reaction mixtures. GC/MS analysis of the compounds in a highly mutagenic fraction of the aqueous chlorine reaction products identified 1,1,3-trichloropro-panonc, 1,1,3,3-tetrachloropropanone and dichloroquinoline.     

Human cell mutagens in respirable airborne particles from the northeastern United States. 2. Quantification of mutagens and other organic compounds

Few reports have characterized mutagenic compounds in respirable airborne particles (<2.5 micrometers in diameter; PM2.5) collected at different sites on a regional scale (hundreds of km). Previously, we reported differences in the human (h1A1v2) cell mutagenicity of whole and fractionated organic extracts of PM2.5 samples collected in Boston, MA, Rochester, NY, and Quabbin Reservoir, a rural site in western MA. Herein we describe the analysis of mutagens and other organic compounds in these samples. Gas chromatography-mass spectrometry (GC-MS) was used to quantify approximately 150 organic compounds, including 31 known human cell mutagens. Molecular weight (MW) 226-302 amu PAHs were the most important mutagens identified: cyclopenta[cd]pyrene accounted for 1-2% of the measured mutagenicity of the samples, MW 252 PAHs accounted for 4-6%, MW 276-278 PAHs accounted for 2-5%, and MW 302 PAHs accounted for 2-3%. 6H-benzo-[cd]pyren-6-one, a PAH ketone, accounted for 3-5% of the mutagenicity. The same compounds accounted for similar portions of the total attributed mutagenicity in each sample. Mutagen levels were similar in the Boston and Rochester samples, and both were significantly higher than the Quabbin sample. This may explain whythe mutagenicities of the Boston and Rochester samples were higher than the Quabbin sample. The levels of mutagens found in semipolar fractions, however, could not explain why the mutagenicity of semipolar fractions was 2-fold higher in the Rochester sample than in the Boston sample. Known mutagens accounted for only 16-26% of the total mutagenicity of the unfractionated extracts, and only approximately 20% of the mutagenicity of the nonpolar and semipolar fractions. The remaining mutagenicity is likely attributable to other, as-yet unknown, semipolar and polar mutagens, or to interactions among chemical constituents of the samples. These findings are consistent with similar studies performed on airborne particles from Los Angeles and Washington, DC, thus indicating that PAHs, PAH-ketones, and as-yet unidentified polar organic compounds are widely distributed airborne human cell mutagens.     

Antimutagenic Effects of Apple Juices: Interference with Heat Load Measurement by Microbiological Methods

Whole heated clear apple juice subjected to a modified Salmonella mutagenicity assay did not show any mutagenic response. However, when fractionated, one fraction obtained from gel filtration did show a dose-related mutagenic response. This suggested the presence of antimutagenic factors. The Ames standard mutagenicity test was used to investigate the antimutagenic activity of apple juice samples against the direct acting mutagens nitroquinoline-N-oxide (NQO) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). There was a dose and heat load dependent reduction in the mutagenicity of both mutagens, indicating the presence of antimutagenic factors.     

Mutagenicity and identification of mutagenic compounds of fumes obtained from heating peanut oil

Since the fume of cooking oil has been reported to increase the risk of lung cancer, the objectives of this study were to evaluate the mutagenicity and to find the mutagens in the fumes of peanut oil heated to the smoke point. Peanut oil prepared from roasted peanut kernel showed a lower smoke point, less unsaturated fatty acids, more fume formation, and stronger mutagenicity than that from unroasted kernel. Further investigation of mutagenic compounds was performed by the Ames test and gas chromatography/mass spectrometry analysis. Among the 12 compounds identified from the neutral fraction of methanol extract, four compounds at a dose of 10 microg per plate were mutagenic to Salmonella Typhimurium TA98 and TA100 in the order of trans-trans-2,4-decadienal > trans-trans-2,4-nonadienal > trans-2-decenal > trans-2-undecenal. Results report the enal compounds formed as the mutagens in the fumes of peanut oil and indicate that inhaling cooking fumes might cause carcinogenic risk.     

Preventive effect of adding antioxidants on mutagenic compound formation in fumes of cooking oil

The preventive effect of various antioxidants on the formation of mutagenic compounds such as trans,trans‐2,4‐decadienal in degummed peanut oil (DPO) fumes was investigated. The mutagenicity of the DPO fumes was significantly reduced by antioxidants added before heating. The addition of antioxidants increased the smoke point and oxidative stability of DPO and decreased the yield of oil fumes and the amount of mutagens. Butylated hydroxyanisole and butylated hydroxytoluene were more effective than natural antioxidants in reducing the amount of four enal compounds in fumes from DPO. Thus edible cooking oil with a high smoke point, less fume and lower mutagenicity might be developed with an appropriate antioxidant. Copyright © 2004 Society of Chemical Industry     

Class separation of mutagenic polycyclic organic material in grilled and smoked foods

A method for class separation of mutagenic polycyclic organic material in grilled and smoked foods is described. The procedure involves an initial extraction with acetone, removal of fat and proteins by precipitation at -55 degrees C, and an acid-base extraction. Further fractionation was carried out by gel filtration and silica gel chromatography. In four samples of grilled sausages, 80%-90% of the extracted mutagenicity (TA98 + S9) was contained in the basic fractions. Flame-grilled sausages showed higher mutagenicity than charcoal-grilled ones. In a smoked fish sample, the mutagenicity was low and evenly distributed between the basic and the neutral/acidic fractions. A few samples showed a weak direct-acting mutagenicity in the neutral/acidic fractions. The presence of nitrite in grilled sausages did not influence the mutagenicity markedly. Gas chromatography-selected ion monitoring was used to successfully identify a number of polycyclic aromatic hydrocarbons (PAHs) and tentatively identify several nitro-PAHs and oxygenated compounds. However, the identification of mutagens in the basic fractions was complicated by peak tailing and the presence of co-eluting material.     

Examination of Chili Pepper and Nutmeg Oleoresins Using the Salmonella/Mammalian Microsome Mutagenicity Assay

Past investigations have suggested that various arylalkene spice compounds warrant further study as possible naturally occurring mutagens and/or carcinogens. The present study carried out a detailed examination of nutmeg oleoresin, myristicin, chili pepper oleoresin, capsaicin, and vanillylamine for in vitro mutagenicity using the Salmonella/mammalian microsome mutagenicity assay. None of the materials tested displayed significant mutagenicity over a wide range of concentrations.     

MUTAGENIC ACTIVITY IN HEAT-PROCESSED FOODS AS DETERMINED BY THE AMES SALMONELLA/MUTAGENICITY ASSAY

Different concentrations of six commercially available heat-processed foods were screened for mutagenic activity using the Ames Salmonella mutagenicity assay. Bacterial strain TA 98 which detects frame shift mutations was used with S-9 mix prepared from livers of Aroclor 1254 induced rats for metabolic activation. Cornflake cereal, branflake cereal, and a breakfast replacement product were not mutagenic at 90 g concentrations or less. At concentrations of 20 g or above, liquid smoke was toxic to the bacteria. At lower concentrations of 1 and 5 g the bacterial colony survival rate was low, therefore no conclusions were made regarding its mutagenic behavior. A canned brown gravy was not mutagenic at concentrations of 75 g or less. A canned au jus gravy displayed moderately high levels of mutagenic activity of 100 g concentrations or less. The contribution of manufacturers'processing techniques to the formation of chemical mutagens in foods is discussed.     

Ames Test for Mutagenicity on Pacific Whiting Treated with Hydrogen Peroxide

Raw Pacific whiting (Merluccius productus) treated with hydrogen peroxide or potassium bromate was tested for mutagenicity by the Ames Salmonella/microsome assay with strains TA 98 and TA 100 without and with S-9 activation. To examine the effects of long-term exposure, cooked whiting treated with hydrogen peroxide and stored up to 6 months at -26°C was also tested. In contrast to the raw-treated fish, the cooked sample contained 78% catalasereactive peroxide up to 6 months later. For testing, acidic, neutral and basic fractions were obtained by modifying the procedure of Felton et al. (Mutat. Res., 1981) to reduce emulsion formation. No extract from either potassium bromate or hydrogen peroxide treatment produced mutagens.     

Antimutagenicity of curcumin and related compounds against genotoxic heterocyclic amines from cooked food: The structural requirement

Curcumin (a major constituent of widely-used spice and colouring agent, turmeric) was found to be very effective in antagonising the S9-mediated mutagenicity of several food-derived heterocyclic amines. In order to understand the chemical basis of antimutagenic properties of curcumin against these mutagens, we have studied the structure–activity relationship between curcumin and its naturally-occurring derivatives, namely demethoxycurcumin and bisdemethoxycurcumin, and other structurally-related natural and synthetic analogues of curcumin, namely tetrahydrocurcumin, dibenzoylmethane, dibenzoylpropane, vanillin, ferulic acid, isoferulic acid and caffeic acid, using Ames Salmonella/reversion assay, against different classes of cooked food mutagens. We conclude that unsaturation in the side chain, a methoxy group on the benzene ring and a central β-diketone moiety in the curcumin molecule are the important structural requirements responsible for high antimutagenic potential of curcumin against cooked food heterocyclic amines.     

Possible mechanisms of antimutagenicity in fermented soymilk prepared with a coculture of Streptococcus infantis and Bifidobacterium infantis

The possible mechanisms of antimutagenicity against 4-nitroquinoline-N-oxide (4-NQO; a direct mutagen) and 3,2'-dimethyl-4-amino-biphenyl (DMAB; an indirect mutagen) were examined in fermented soymilk prepared with a coculture of Streptococcus thermophilus and Bifidobacterium infantis. The antimutagenicity in the fermented soymilk was not due to the bioantimutagenic effect of modulation of DNA repair processes. The mutagenicity of DMAB decreased with increased preincubation of fermented soymilk and the DMAB metabolite but not with intact DMAB or an S9 mixture. Mutagenicity of 4-NQO was not affected by preincubation of fermented soymilk with this mutagen. Mutagenicity of both 4-NQO and DMAB was reduced when Salmonella Typhimurium TA 100 was pretreated with fermented soymilk, indicating that fermented soymilk affected the function of the bacterial cell, which might also lead to reduced mutagenicity of the tested mutagens. Desmutagenic and blocking effects were the main mechanisms of antimutagenicity in the fermented soymilk against DMBA. In contrast, the antimutagenic effect of the fermented soymilk on 4-NQO was primarily due to a blocking effect.     

Reactions of Aqueous Chlorine and Chlorine Dioxide with Tryptophan, N-Methyltryptophan, and 3-Indolelactic Acid: Kinetic and Mutagenicity Studies

Reactions of tryptophan, N-methyltryptophan and 3-indolelactic acid with aqueous chlorine or chlorine dioxide (ClO₂) in 0.1M potassium phosphate buffer, pH 6.0, were investigated to determine any structural relationships with regards to kinetics and mutagenicity. The reaction with ClO₂ followed pseudo-first order kinetics, with the half-life of the respective compounds being 36, 22, and 8 milliseconds. The formation of a dark precipitate in the reaction of tryptophan with HOCl precluded any kinetic comparison. The reaction products of tryptophan with hypochlorous acid (HOCl) or ClO₂ were mutagenic to Salmonella typhimurium TA98 and TA100; while those of N-methyltryptophan with HOCl and ClO₂ were more mutagenic toward TA98. Higher recoveries of the reaction products were achieved by passing the acidified (pH 2.5) mixture through an XAD-8/XAD-2 resin column.     

N-nitrosamine and mutagenicity formation in Chinese salted fish after digestion.

Salted and dried fish (Nemipterus virgatus), acquired from Hong Kong, was treated with 0.43-110 mM nitrite during in vitro digestion using gastric enzymes and the volatile N-nitrosamine content and mutagenicity on Salmonella typhimurium TA100 assayed without concentration. N-Nitrosodimethylamine (NDMA; the only nitrosamine detected) formation was second order in nitrite concentration. When 10 g of fish was treated with 6.96 mM nitrite, 394 nM NDMA was formed. Thiocyanate was catalytic for NDMA formation at nitrite concentration greater than 0.87 mM and when the ratio of thiocyanate to nitrite was greater than 1. Approximately a 50% inhibition in NDMA formation by ascorbic acid was seen when the ratio of ascorbate to nitrite was approximately 2 or greater and the nitrite concentration was 1.74 mM. Mutagenicity increased with increasing nitrite concentration but the addition of thiocyanate did not increase mutagenicity over nitrite alone. Ascorbate increased mutagenicity even though NDMA formation was inhibited. Even at nitrite concentrations greater than 100-fold higher than expected in vivo, there was insufficient NDMA formed to account for the observed mutagenicity. These data do not exclude the possibility that the observed mutagenicity was due to non-volatile N-nitroso compounds, however, this possibility seems unlikely given the effects of ascorbate and thiocyanate which would be expected to inhibit and enhance non-volatile N-nitroso compound formation.     

Inhibition of cooked food-induced mutagenesis by dietary constituents: Comparison of two natural isothiocyanates

Sulforaphane(1-isothiocyanato-(4R)-(methylsulphinyl)butane), a major constituent of broccoli (Brassica oleracea, var. italica) and a structurally related natural aliphatic isothiocyanate, sulforaphen (4-isothiocyanato-(1R)-(methylsulphinyl)-1-(E)-butene), found in radish (Raphanus sativus L., Cruciferae) were investigated for their antimutagenic potential against different classes of cooked food mutagens (heterocyclic amines) in the Ames assay using Salmonella typhimurium TA98 and TA100 strains in the presence of Aroclor 1254-induced rat liver S9. Results of the in vitro antimutagenicity studies in the TA100 strain strongly suggest that both isothiocyanates were potent inhibitors of the mutagenicity induced by all the tested mutagens. Sulforaphen, possessing unsaturation in the alkyl chain of its structure, was, however, found to be 1.3–1.5 times more active than sulforaphane. These studies strongly warrant further investigations of sulforaphen for its potential as a chemopreventive agent.     

Mutagenic and carcinogenic hazards of settled house dust. II: Salmonella mutagenicity

Settled house dust (SHD) is a complex mixture that contains numerous chemical contaminants. Very little is known about the hazards of SHD as compared to other complex matrices such as air and soil. In this study, the mutagenic hazards associated with the extracts of sieved dust from 52 homes were examined using the Salmonella Mutagenicity Test. All of the SHD samples displayed mutagenic activity and the mean mutagenic potencies ranged from 2300to 23 600 revertants per gram. Testing with various Salmonella strains revealed a predominance of frameshift mutagens in the dust samples. Analyses showed that polycyclic aromatic hydrocarbons (PAHs) were likely responsible for a quarter of the mutagenic activity of the SHD samples. In an effort to identify factors that influenced dust mutagenicity, the relationships between SHD mutagenicity and household activities were investigated. Mutagenicity was positively correlated with parameters such as the time since last vacuuming (r2 = 0.11, p < 0.05) and the number of people living in the home (r2 = 0.11-0.43, p < 0.05). However, the causative factors responsible for these relationships remain unclear.     

PURIFICATION AND PROPERTIES OF DESMUTAGENIC FACTOR FROM BROCCOLI (BRASSICA OLERANCEA VAR. ITALICA PLENCK) FOR MUTAGENIC PRINCIPLE OF TRYPTOPHAN PYROLYSATE

A desmutagenic factor which inhibited the mutagenicity of the mutagens, Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole), Trp-P-2 (3-amino-1-methyl)-5H-pyrido[4,3-b]indole), ethidiumbromide and 2-aminoanthracene, was purified from broccoli (Brassica olerancea var. italica plenck). The factor was not sedimented by ultra-centrifugation at 200,000 xg for 2 h. It adsorbed to a DEAE-cellulose column and was eluted with low concentration of potassium chloride. The purified factor exhibited a heme-like protein absorption spectrum with a Soret band at 403 nm and α and β bands at 640 and 504 nm, respectively. The molecualr weight was estimated to be approximately 53,000 by SDS-gel electrophoresis.     

Reduction of Heterocyclic Aromatic Amine Formation and Overall Mutagenicity in Fried Ground Beef Patties by Organosulfur Compounds

ABSTRACT: The effects of several organosulfur compounds on heterocyclic aromatic amine (HAA) formation and overall mutagenicity in fried ground beef patties were evaluated. The greatest inhibition of total HAA formation was achieved with diallyl disulfide (78%) and dipropyl disulfide (70%); these compounds also reduced overall mutagenicity by 75 and 65%, respectively. The addition of diallyl sulfide, allyl methyl sulfide, and allyl mercaptan also significantly ( p < 0.05) reduced mutagenicity, with reductions of 56, 43, and 30%, respectively. The addition of cysteine and cystine, however, did not reduce the mutagenicity of cooked meat, an observation confirmed by the relatively small reductions in HAA concentrations.     

Mutagen Formation in Beefburgers Processed by Frying or Microwave with use of Flavoring and Browning Agents

Traditional frying produced mutagens in a dose-dependent manner (0.5–4.0g equivalents of beefburger extract) as detected by Salmonella typhimutium strains TA97 and TA98 (sensitive to frame-shift mutagens). Mutagenicity was associated with beefburger crusts. In beef burgers cooked by microwave, in presence or absence of browning agent and flavoring, bacterial mutagens were not detected at equivalent dose concentrations. In addition to reduction of mutagen formation, microwave cooking produced an equally acceptable product as judged by limited flavor testing.     

In vivo breakdown of dietary [methyl−14C] and [uronate-6−14C]pectin-labelled spinach cell walls by rat intestinal microorganisms and incorporation of 14C into host tissues

The metabolism of dietary plant cell wall (PCW) pectin was followed in the rat using PCW isolated from spinach cell cultures ¹⁴C-labelled in the galacturonic acid residues or pectic methyl ester groups. ¹⁴C-PCW were rapidly broken down in the caecum and colon of the rat and generated several groups of soluble products. [¹⁴C]Methanol was released from [methyl^?14C]pectin-labelled PCW and some of the methanol was converted to ¹⁴C-labelled volatile acids. ¹⁴C-Labelled volatile acids were also generated from [uronate-6-¹⁴C]pectin-labelled PCW along with soluble but non-volatile material. ¹⁴C derived from both cell wall types was taken up by the caecal and colonic mucosa and transported to the liver, pelt and other body tissues. Some ¹⁴C was excreted as ¹⁴CO₂ and in urine.     

Effect of Pectin and Lignin on Mutagenicity in the Ames Assay

The effect of incorporating varying concentrations of pectin and lignin into the Ames assay with the known mutagens, benzo(a) pyrene, 3-methyl cholanthrene, and N-methyl-N-nitro-N-nitrosoguanidine, on mutagenic activity was investigated. The addition of 50 μg, 150 μg, and 300 μg concentrations of pectin and lignin failed to block the mutagenicity of benzo-pyrene and 3-methyl cholanthrene in the Ames/mammalian microsome assay using Salmonella typhimurium TA 98 and TA 100 with and without preincubation. The incorporation of 50 μg, 150 μg, and 300 μg of pectin and lignin did not block the mutagenic activity of MNNG in the Ames Spot test with Salmonella typhimurium TA 100.