Fluoranthene, a volatile mutagenic compound, present in creosote and coal tar

Creosote, a coal-tar distillation product, contains mutagens which are volatile at 37 degrees C. After distillation of creosote we found that these volatile mutagens were present in the distillation fraction with the highest boiling range (greater than 360 degrees C). The "volatile mutagenic activity" was connected with the presence of fluoranthene, a polycyclic aromatic hydrocarbon. Commercially available fluoranthene was positive in the so-called "taped-plate assay" (the test system used for the detection of volatile mutagens) towards the strains TA98 and TA100 in the presence of S9 mix. The tested creosote and coal tar contained fluoranthene in concentrations of 5.2 and 2.2%, respectively.     

Fecal mutagens from subjects consuming a mixed-western diet

Because of potential significance of fecal mutagens (presumptive carcinogens) in the pathogenesis of colon cancer, feces from 99 healthy subjects from the New York metropolitan area were studied. The diet histories indicate that all participants were consuming a mixed-western diet which is high in total fat and low in fiber. Fecal samples that were incubated under anaerobic conditions at 37 degrees C for 96 h or frozen without incubation, were extracted with hexane: peroxide-free diethyl ether (1:1), partially purified on a silica Sep-pak cartridge and assayed for mutagenicity using the Salmonella typhimurium/mammalian microsome system. Aliquots of fecal samples incubated anaerobically showed a higher frequency of mutagenic activity (per cent samples showing activity) in strains TA98 and TA100 with and without microsomal (S9) activation. In addition, the mutagens requiring S9 activation, were more frequently inactivated when the fecal samples were frozen immediately after defecation and transported to the laboratory. Compared with hexane: ether, extraction of fecal samples with acetone increased the mutagenic activity mostly with TA98 with S9 activation. The HPLC fractionation of hexane: ether extract with methanol: water gradient using reverse phase C-18 column and UV detector at 254 nm indicated that the mutagenic activity (TA98 with S9 activation) is concentrated in several peaks. This is the first demonstration of HPLC profile of fecal samples that are active in TA98 with S9 activation. HPLC profile of fecal extracts and mutagenic activity of these extracts in strains TA98 and TA100 suggest the presence of several types of mutagens in the feces of healthy subjects consuming a high-fat, low-fiber mixed-western diet.     

Mutagenic activity of amino acid pyrolyzates in Salmonella typhimurium TA 98.

Pyrolyzates of 25 amino acids and 5 indole derivatives were tested for mutagenicity in the histidine-requiring mutant Salmonella typhimurium TA 98. Significant mutagenic activity was detected with pyrolyzates of most of the amino acids. These pyrolyzates required a liver microsomal fraction, as representative of mammalian metabolism, to be detected as mutagens. Among the pyrolyzates tested, the highest mutagenic activity was observed with that of L-tryptophan. As little as 10 microgram of the pyrolyzate of L-tryptophan had detectable mutagenic activity toward TA 98. The optimal pyrolysis temperatures for the formation of mutagenic products were shown to be 500 degrees C for L-tryptophan and 600 degrees C for the other amino acids. The results from pyrolyses of some indole derivatives suggest that an amino group at the alpha-position to the carboxyl group of L-tryptophan plays an important role in the formation of mutagens.     

The mutagenicity of nitrite-treated aqueous extract of Piper betle L

Betel quid is chewed as a masticatory material by people in certain areas of Asia. The quid chewing has been related to oral cancer by epidemiological study. The mutagenic components in the aqueous extracts of betel quid ingredients were studied. Only nitrite-treated aqueous extract of Piper betle L fruits, leaves or rhizoma were demonstrated to exhibit a mutagenic response, using Salmonella typhimurium strains TA100 and TA1535 in the Ames test. When the aqueous extract of the fruit was nitrosated, the greatest number of mutagenic substances were formed at pH 3. The formation of mutagens was enhanced by increasing the temperature from 5 to 95 degrees C. Maximum production of the mutagens occurred within 15 min when nitrosation was conducted at 35 degrees C. The mutagenic components in nitrite-treated aqueous extract of Piper betle L fruit were found to be N-nitrosopiperidine, N-nitrosopyrrolidine, N-nitrosomorpholine, and other compounds, as determined by gas chromatography-thermal energy analyzer.     

Effect of various alkyl and unsaturated substituents on the mutagenicity of some nitrophenyl thioethers.

A variety of nitro-substituted phenyl alkyl/aryl thioethers and nitroso-substituted phenyl alkyl/aryl thioethers have been synthesized and tested for their mutagenicity towards Salmonella typhimurium strain TA100, TA98, TA98NR and TA98/1,8-DNP(6) in the absence of S9 mix. The relative order of mutagenicity in TA98 and TA100 among p-nitrophenyl thioethers having alkyl or aryl substituents is allyl>phenyl>benzyl>butyl>propyl>ethyl>methyl. Compounds having an alkyl chain C(6) to C(12) were found to be non-mutagenic. Among the various positional isomers (ortho, meta and para) of nitro-substituted diphenyl thioethers only the compounds having the -NO(2) function at the para position is mutagenic, whereas compounds having a -NO(2) function at ortho and meta are non-mutagenic. However, the reduced intermediate, ortho-nitroso derivative was found to be mutagenic in all the four strains but the meta-nitroso derivative was found to be non-mutagenic. All mutagens were found to be non-mutagenic when tested in nitroreductase deficient strain TA98NR, whereas their nitroso intermediates are found to be mutagenic. A substantial fall in the mutagenic activity is observed when some mutagens are tested in O-acetyltransferase deficient strain TA98/1,8-DNP(6).     

Formation of mutagens by heating creatine and glucose

The mutagenic activity toward $\text{Salmonella typhimurium}$ TA 98 and TA 100 was investigated by heat treatment at temperatures up to 200°C of meat with identified components such as protein, adenine, creatine and a mixture of each of the 17 amino acids or glucose. Mutagenicity of these nitrogenous compounds was detected at the temperature of 150°C by adding glucose, consequently the yield of mutagenic activity by heating creatine and glucose was remarkably high. It is assumed that mutagens would be formed by the reaction of creatine and sugars during cooking of meat.     

Formation of mutagens by amino-carbonyl reactions

The formation of mutagens by amino-carbonyl reactions of 20 kinds of amino acid and sugars after heating at 100 degrees C for 10 h was examined by the Ames test. The browned solutions of Gly, Ala, Val, Leu, Ile, Ser, Thr, Gln, Lys X HCl, Arg, Phe, Cys, Met and Pro with Glc caused mutation of Salmonella typhimurium TA100 and/or TA98 with or without S9 mix. The presence of S9 mix increased the mutagenic activity of the browned solutions of Cys and Phe with Glc on TA100 and of those of Gly, Ala, Val, Ile and Cys on TA98, but decreased the activity of other solutions. No revertants of Salmonella were induced by the browned solutions of Trp, Tyr, Asp, Asn, Glu and (Cys)2 with Glc. Among positive browned solutions, Cys, Lys, Arg and Phe had the stronger activity, but their activity was weak compared with that of pyrolysates or chemical mutagens such as Trp-P-1, Trp-P-2 and 4-nitroquinoline-N-oxide. The mutagenic activity of the browned solutions increased with prolongation of heating time and varied with the pH of the reaction mixture. Fru, Gal, Ara, Xyl, Man, Lac and Suc also had the ability to form mutagens in the browning reactions with amino acids.     

Formation of new heterocyclic amine mutagens by heating creatinine, alanine, threonine and glucose.

A mixture of alanine, threonine, creatinine and glucose was heated in diethylene glycol and water (5:1, v/v) for 15 min at 200 degrees C. The mutagens formed were purified by high-performance liquid chromatography using the Ames/Salmonella mutagenic activity to guide the purification. The structures of the purified mutagens were determined using UV absorption, mass and NMR spectrometry. A new mutagenic compound with a mass number of 217 was found and its mass spectrum did not correspond to any known mutagen derived from food. This new compound accounted for 4% of the total mutagenic activity. Other mutagenic compounds were identified as MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline), 4,8-DiMeIQx (2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline), and a new mutagen 4,7,8-TriMeIQx (2-amino-3,4,7,8-tetramethylimidazo[4,5-f]quinoxaline) with a mutagenic activity of 73,000 TA98 revertants per microgram. The percentage of the mutagenic activity attributable to MeIQx, 4,8-DiMeIQx and 4,7,8-TriMeIQx was 10%, 70% and 3%, respectively. The yield of MeIQx, 4,8-DiMeIQx and 4,7,8-TriMeIQx was 10, 36 and 6 nmole/mmole creatinine. The formation of TriMeIQx from natural meat components suggests that this new quinoxaline mutagen may be present in cooked foods.     

Absence of mutagenic activity of ticlopidine in the Ames Salmonella/microsome test

Ticlopidine hydrochloride, 5-(o-chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydrochloride, a platelet aggregation inhibitor, was tested for mutagenic activity in the Ames Salmonella/mammalian microsome test. Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were employed. Two of these strains (TA1535 and TA100) are sensitive to base-pair substitution mutagens, and the remaining 3 are sensitive to frame-shift mutagens. There was no evidence that ticlopidine hydrochloride had any mutagenic activity either in the presence or absence of a liver microsomal supplement.     

Enhancement of the mutagenicity of IQ and MeIQ by nitrite in the Salmonella system.

The fried food mutagens IQ, MeIQ, Glu-P-1 and Trp-P-2 were treated with nitrite at pH 3.0 for 1 h at 37 degrees C. The resulting reaction mixtures were tested for mutagenicity towards Salmonella typhimurium TA97, TA98, TA100 and TA1535. Glu-P-1 and Trp-P-2 were readily converted to weak or non-mutagenic deaminated compounds, whereas IQ and MeIQ were converted to extremely strong mutagenic derivatives in both the presence and the absence of rat liver S9 mix. The mutagenicity of MeIQ in TA98 was enhanced by nitrite up to 3-fold, while that of nitrosated MeIQ was further enhanced by S9 mix up to 15-fold. The nitrosation products of MeIQ were resolved into 7 bands by TLC on silica gel plate. Bands I, III, V and VI were highly mutagenic to both TA98 and TA100. The experimental results suggest that the non-enzymatic formation of direct-acting mutagens from indirect-acting mutagens such as IQ or MeIQ might be physiologically important, especially with regard to the etiology of human gastrointestinal tract tumors.     

Fate of the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in Sprague-Dawley rats. I. Mutagens in the urine

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is a potent bacterial mutagen formed during cooking of beef. IQ was administered intravenously to Sprague-Dawley rats at concentrations ranging from 7.5-50 mg/kg body weight. Urine was collected and analyzed for mutagenicity. Urinary mutagens were found which required activation by S9 mix, and reverted Ames test strains TA98 and TA100, but not TA1535 or TA1537. The amount of urinary mutagen(s) were related to IQ dose administered and were excreted within 48 h. Additional mutagenic activity was not released after incubation with beta-glucuronidase or aryl sulfatase. Analysis of urinary mutagens by HPLC indicates that the majority of mutagenic activity is due to unchanged IQ, but a small peak of mutagenic activity may correspond to N-acetyl or 3-N-demethylated metabolite. Since only 1% of the administered mutagenic activity is recovered in the urine, IQ may be readily detoxified in vivo.     

Mutagenic activity of pyrolysates of cyanocobalamin and some other water-soluble vitamins in the model system with the Salmonella/mammalian microsomes

Pyrolysates of cyanocobalamin, thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, and ascorbic acid were tested for mutagenicity in the histidine-requiring mutants Salmonella typhimurium TA98 and TA100. Each vitamin was sealed in a glass tube and heated at 100-600 degrees C in a muffle furnace. Methanol-chloroform extracts of the pyrolysate of each vitamin tested did not show any mutagenicity in either TA98 or TA100 without rat liver 9000 x g supernatant fraction (S9) added. In the presence of S9, the B-group vitamins (cyanocobalamin, thiamine hydrochloride, riboflavin, and pyridoxine hydrochloride) were all mutagenic in TA98 and TA100, with the highest activity among the vitamins tested found in the pyrolysate of cyanocobalamin. The pyrolysate of 0.25 mumole cyanocobalamin produced 3200 revertants, while the pyrolysates of 0.25 mumole thiamine hydrochloride and riboflavin produced only 910 revertants, and the pyrolysate of pyridoxine hydrochloride did not show any mutagenicity at that amount. The mutagenicity was generally more active to TA98 than to TA100, indicating that frameshift-type mutagens were contained in the pyrolysates. The pyrolysate of ascorbic acid did not show any mutagenic activity in either TA98 or TA100 under the present experimental conditions.     

Mutagenicity of 8-ethoxycaffeine in vitro : Induction of point mutations in the salmonella/microsome test and of sister-chromatid exchanges as well as chromosomal aberrations in Chinese hamster ovary cells (CHO line)

The caffeine derivative 8-ethoxycaffeine (EOC) was tested in 3 different test systems in vitro. Each experiment was carried out with and without S9 mix. Incubation temperatures were 20 and 37 degrees C. (1) In the Salmonella/microsome test, EOC behaved as a pro-mutagen in the Salmonella typhimurium strain TA1535. No mutagenic activity was found in experiments without S9 mix. The influence of temperature was negligible. The mutagenic activity of EOC depended mainly on the mammals used to prepare the S9 fraction and on the agents given to them to induce liver enzymes. (2) EOC did not induce sister-chromatid exchanges in cell cultures, either at 20 or at 37 degrees C. (3) On the other hand, EOC induced chromosomal aberrations when the cells were incubated at 37 degrees C without S9 mix.     

Screening complex hazardous wastes for mutagenic activity using a modified version of the TLC/Salmonella assay

10 complex hazardous wastes were tested for mutagenic activity using a modified version of the TLC/Salmonella assay developed by Bj?rseth et al. (1982). This fractionation/bioassay scheme couples thin-layer chromatography (TLC) with the Salmonella/mammalian-microsome (Ames) assay for the detection of mutagenic constituents in complex mixtures. Crude (unadulterated) hazardous wastes and selected hazardous waste extracts were fractionated on commercially available cellulose TLC plates. Mutagenicity testing was performed in situ by applying a single overlay of minimal growth agar, tester strain TA98 or TA100, and the optional metabolic activation system directly onto the developed chromatogram. A mutagenic effect was indicated either by the appearance of localized clusters of revertant colonies or by an increase in total revertant growth vis-à-vis control plates. 7 of 10 hazardous wastes (including tars, emulsions, sludges, and spent acids and caustics) demonstrated mutagenic activity when tested by this method. To assess the sensitivity of the modified TLC/Salmonella assay, 14 Salmonella mutagens from a wide range of chemical classes and polarities were tested. Selected compounds included heterocyclics, aromatic amines, alkylating agents, antitumor agents, a nitrosamine and a nitroaromatic. 11 of the 14 mutagens were positive in this test system. The 3 compounds refractory to analysis included a polycyclic aromatic hydrocarbon and two volatiles.     

Comutagenic effects exerted by N-nitroso compounds.

Mutagenesis induced by dimethylnitrosamine (DMN) and N-methyl-N-nitrosourea (NMU) in Salmonella typhimurium TA100 and TA1530 is characterized by biphasic dose and time response curves. At low doses or short incubation times mutagenic response is minimal, but increases rapidly when an apparent threshold dose or threshold incubation time is exceeded. Bacteria pretreated with subthreshold doses of DMN or NMU were many times more sensitive to the mutagenic effects of methylating and ethylating N-nitroso compounds than were untreated bacteria. The growth phase of the bacteria had little effect on the percentage enhancement of mutagenesis caused by pretreatment with NMU although exponentially growing cells were more sensitive to mutagenesis induced by NMU or diethylnitrosamine. Mutagenesis induced by methylmethanesulfonate and N-propyl-N'-nitro-N-nitrosoguanidine was not significantly enhanced by pretreatment of bacteria with NMU or NEU suggesting that the former mutagens act by different mechanisms than NMU or NEU.     

Kinetic analysis of the role of histidine chloramines in hypochlorous acid mediated protein oxidation

Hypochlorous acid (HOCl) is a powerful oxidant generated from H(2)O(2) and chloride ions by the heme enzyme myeloperoxidase (MPO) released from activated leukocytes. In addition to its potent antibacterial effects, excessive HOCl production can lead to host tissue damage, with this implicated in human diseases such as atherosclerosis, cystic fibrosis, and arthritis. HOCl reacts rapidly with biological materials, with proteins being major targets. Chlorinated amines (chloramines) formed from Lys and His side chains and alpha-amino groups on proteins are major products of these reactions; these materials are however also oxidants and can undergo further reactions. In this study, the kinetics of reaction of His side-chain chloramines with other protein components have been investigated by UV/visible spectroscopy and stopped flow methods at pH 7.4 and 22 degrees C, using the chloramines of the model compound 4-imidazoleacetic acid and N-alpha-acetyl-histidine. The second-order rate constants decrease in a similar order (Cys > Met > disulfide bonds > Trp approximately alpha-amino > Lys > Tyr > backbone amides > Arg) to the corresponding reactions of HOCl, but are typically 5-25 times slower. These rate constants are consistent with His side-chain chloramines being important secondary oxidants in HOCl-mediated damage. These studies suggest that formation and subsequent reactions of His side-chain chloramines may be responsible for the targeted secondary modification of selected protein residues by HOCl that has previously been observed experimentally and highlight the importance of chloramine structure on their subsequent reactivity.     

Structure-activity relationships of nitro and methyl-nitro derivatives of indoline, indole, indazole and benzimidazole in Salmonella typhimurium

The mutagenic activities of eleven nitro derivatives and eleven N-methyl-nitro derivatives of indoline, indole, indazole and benzimidazole were investigated in Salmonella TA98 and TA100. The presence of a nitro group at C4 or C7 resulted in only weakly or nonmutagenic compounds, while a nitro group at C2, C5 or C6 usually resulted in measurable mutagenic activity in the non-N-methylated compounds. Methylation of a ring nitrogen usually reduced the mutagenic activity of these nitroheterocyclics except 2-nitro-benzimidazole, which resulted in a better than 300-fold increase in mutagenic activity. A proposed mechanism for the increased mutagenic activity obtained by methylation of imidazole nitrogens may provide insights into the reasons for the potent mutagenicities observed for several similarly methylated cooked-food mutagens.     

Mutagenicity of adsorbates to a copper-phthalocyanine derivative recovered from municipal river water

Blue cotton, bearing a covalently bound copper-phthalocyanine derivative capable of adsorbing polycyclic aromatic hydrocarbons (PAHs) over 3 rings, was applied to recover mutagens from the Katsura River which is a tributary of the Yodo River. The Ames Salmonella/microsome assay with TA98 and TA100 of the blue cotton concentrate recovered from the river water demonstrated indirect mutagenicity toward TA98. The subfractions separated by Sephadex G-25 gel chromatography also showed direct mutagenicity in strains YG1021 and YG1024, the nitroreductase- and O-acetyltransferase-overproducing derivatives of TA98; this activity was greatly increased by the addition of S9 mix, especially in YG1024. However, these subfractions were less mutagenic with TA98NR or TA98/1,8-DNP6, regardless of whether S9 mix was present or not. The behaviors of these mutagenic activities therefore suggested that frameshift mutagens of both directly mutagenic nitroarenes and indirectly mutagenic aminoarenes were present in the blue cotton concentrate from the river water.     

Mutagenesis by photoaffinity labeling using selected azidofluorenes

Several 2-azidofluorenes have been synthesized for use as photoaffinity labels inside bacteria. In the dark they were not mutagenic for any Salmonella typhimurium tested. When photolyzed inside the bacteria, all were mutagenic for strain TA1538 to varying degrees, and were considerably less mutagenic in the corresponding repair positive TA1978. None were mutagenic for strain TA1535 or TA1537, although most compounds were toxic for those strains when photolyzed.     

The effect of temperature on the formation of mutagens in heated beef stock and cooked ground beef

The microsome-activatable mutagens (chromatographically distinguishable from benzo[a]pyrene and from the mutagens produced from pyrolysed amino acids and proteins) previously found in beef extract and in bacterial nutrients which contain beef extract are produced when beef stock is heated. Reflux boiling of beef stock at 100°C results in a linear increase in mutagenic activity toward Salmonella strain TA1538. The rate of production of mutagenic activity at temperatures between 68°C and 98°C conforms closely to the Arrhenius equation, yielding an activation energy of 23 738 calories per mole. Extrapolation from these data predicts a sharp rise in the rate of mutagen formation between 140 and 180°C. This expectation is confirmed when ground beef patties (hamburgers) are prepared in various conventional electrically-heated appliances which operate at different cooking temperatures within this range. The mutagenic activity of hamburger cooked at high temperatures is limited to the surface layers; the temperature of the inside of the hamburger does not exceed 100°C during cooking. No mutagenic activity is found in comparable samples of uncooked meat. The results indicated that the mutagens may be formed as a result of the temperatures encountered in certain conventional cooking procedures.