Potent Bacterial Mutagen Research Articles

Identification of mutagens in drinking water

Mutagenicity is frequently detected in drinking water, often as a product of the reaction of chlorine with naturally occurring organic substances during drinking water disinfection. The production of mutagenicity resulting from chlorination of aqueous solutions of humic and fulvic acid, phenolic compounds, protein, peptides and amino acids was investigated using the mutagenicity assay with Salmonella typhimurium. The relevance of these compounds as precursors of mutagenicity in drinking water treatment chlorination is discussed.Chlorination products were identified, including 2-phenyl-2,2-dichloroethanal, a product of the chlorination of phenylalanine. This was shown to be a bacterial mutagen. The highly potent bacterial mutagen, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), was detected in concentrated extracts of chlorinated tyrosine and UK drinking water. This compound accounted for a substantial proportion of the mutagenic activity of the extracts. Brominated analogues (BMX) of MX were also highly potent mutagens and were produced when tyrosine was chlorinated in the presence of bromide. Many chlorination products, including mutagens, were common to chlorinated humic, fulvic and amino acids and chlorinated drinking water.

Hemangioendothelial sarcoma in brown adipose tissue of mouse induced by carcinogenic heterocyclic amine, Glu-P-1.

2-Amino-6-methyldipyrido[1,2-a:3',2'-d]limidazole, a potent bacterial mutagen from glutamic acid pyrolysate, when given to male CDF1 mice at a concentration of 0.05% in the diet induced blood vessel tumors characterized histopathologically. These hemangioendothelial sarcomas showed positive alkaline phosphatase activity histochemically and Weibel-Palade bodies ultrastructurally. Neoplasms were predilective for brown adipose tissue and foci of atypical cells, not peliosis-like lesions, appear to be an early stage in tumor development.

DNA adduct formation of the carcinogen 2-amino-3-methylimidazo[4,5- f]-quinoline in target tissues of the F-344 rat

2-Amino-3-methylimidazo[4,5- f]quinoline (IQ), a potent bacterial mutagen present in broiled sardines, cooked beef and beef extract, is a carcinogen in the F-344 rat, affecting mainly the liver and small and large intestines. Using 32P-postlabeling assays, the formation of IQ-DNA adducts was examined in male F-344 rats. Twenty-four hours after i.p. doses (5–50 mg IQ/kg) the liver, small and large intestine each showed the presence of 5 adducts, the liver and large intestine having an average of 18.1 and 2.4 times as many adducts, respectively, as the small intestine. None of these adducts could be detected in vehicletreated animals. It is concluded that IQ forms specific DNA adducts in target tissues of the F-344 rat.

Mutagenicity of bi-, tri- and tetra-cyclic aromatic hydrocarbons in the “taped-plate assay” and in the conventional Salmonella mutagenicity assay

Aromatic hydrocarbons in the range of 1–4 nuclear rings were examined for mutagenicity in the so-called “taped-plate assay”. This modification of the Ames assay is particularly equipped for the detection of volatile mutagens. Of the many compounds tested only phenanthrene, pyrene, benzo[ c]phenanthrene and benzoacenaphthylene were positive in this assay. The present data underline the exceptional behaviour of fluoranthene by being a rather potent bacterial mutagen with a volatile nature (as found in a previous study).

Studies on the potent bacterial mutagen, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5 H)-furanone: aqueous stability, XAD recovery and analytical determination in drinking water and in chlorinated humic acid solutions

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5 H)-furanone (MX) was detected by gas chromatography/mass spectrometry in drinking water samples from 3 locations in the U.S.A., and also in a chlorinated humic acid solution. MX appears to account for a significant proportion of the mutagenicity of these samples, as measured in the Ames test using strain TA100 without metabolic activation. Studies on recovery of MX from spiked water samples by XAD-2/8 resin adsorption/acetone elution indicated that sample acidification prior to resin adsorption was essential to the effective recovery of MX. The stability of MX in aqueous solution was pH and temperature dependent. At 23°C the order of stability, based on persistence of mutagenic activity was found to be: pH 2 > pH 4 > pH 8 > pH 6. The half-life at pH 8 and 23°C was 4.6 days. One of the degradation products has been tentatively identified as 2-chloro-3-(dichloromethyl)-4-oxo-2-butenoic acid, an open form of MX which appears to be in the “E” configuration. Overall, these results suggest that MX is formed during water chlorination as a result of reaction of chlorine with humic substances, and that a substantial fraction of the MX formed is likely to persist throughout the distribution system.

Mutagenic and clastogenic properties of 3-chloro-4-(dichloromethyl)-5-hydroxy-2 (5H)-furanone: a potent bacterial mutagen in drinking water.

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) was found to be a direct-acting mutagen in the Ames test for strains TA1535, TA1538, TA92, TA97, TA98, TA100 and TA102. The highest mutagenic response (approximately 13,000 revertants/nmol) was seen in strain TA100. The TA100 response was six- to tenfold higher than in TA98, TA97, and TA102, and 100- to 500-fold higher than in TA1535, TA92, and TA1538. The addition of a 9,000 x g supernatant fraction (S-9) from livers of polychlorinated biphenyl-treated rats, along with cofactors for NADPH generation, resulted in a 90% reduction in the TA100 mutagenicity. MX induced chromosomal aberrations in Chinese hamster ovary cells after 6-8 hr exposure without S-9 at a dose as low as 4 micrograms/ml, and after 2 hr exposure with S-9 at a dose of 75 micrograms/ml. The oral dose of MX lethal to 50% (LD50) in Swiss-Webster mice was determined to be 128 mg/kg. MX did not induce micronuclei in mouse bone marrow when administered by oral gavage at doses up to 70% of the LD50.

Metabolism of 1-nitrobenzo[a]pyrene by rat liver microsomes to potent mutagenic metabolites.

1-,3- and 6-Nitrobenzo[a]pyrene (nitro-BaP), which are prototypes of nitro polycyclic aromatic hydrocarbons (nitro-PAHs) derived from a carcinogenic parent PAH, benzo[a]pyrene, are environmental contaminants and potent bacterial mutagens. In this study, the aerobic and hypoxic metabolism of 1-nitro-BaP by rat liver microsomes was studied. Aerobic metabolism of 1-nitro-BaP yielded 1-nitro-BaP trans-7,8- and 9,10-dihydrodiol, while metabolism under hypoxic conditions yielded 1-amino-BaP. The metabolites formed from aerobic metabolism of 1-nitro-BaP and 1-nitro-BaP trans-9,10-dihydrodiol by liver microsomes of untreated rats and rats pretreated with 3-methylcholanthrene and phenobarbital were quantified. Comparison of these results with those obtained with BaP and BaP trans-9,10-dihydrodiol indicates that nitro substitution at the 1-position of BaP markedly affects the regioselectivity of the P-450-containing enzymes. 1-Nitro-BaP and the three metabolites were potent mutagens in Salmonella typhimurium TA98, both in the absence and in the presence of an exogenous metabolic activation system (S9). The direct and S9-mediated mutagenicities of 1-nitro-BaP and the two dihydrodiols were decreased in the nitroreductase-deficient strain TA98NR, while TA98/1,8-DNP6, an O-acetylase-deficient strain, was less sensitive to the two dihydrodiols, both with and without S9, and 1-nitro-BaP with S9. 1-Amino-BaP was equally mutagenic in all three tester strains. These observations indicate that: the metabolism of 1-nitro-BaP involves several pathways leading to mutagenic activation; the major activation pathways of 1-nitro-BaP involve nitroreduction; nitroreduction followed by O-acetylation is the major activation pathway of 1-nitro-BaP trans-7,8- and 9,10-dihydrodiol; and 1-amino-BaP is a potent direct-acting mutagen.

The disposition and in vivo covalent binding to liver DNA of the monoazodyes 6-(p-dimethylaminophenylazo)benzothiazole (6BT) and 5-(p-dimethylaminophenylazo)indazole (5I) after administration to the rat.

6-(p-Dimethylaminophenylazo)benzothiazole (6BT) and 5-(p-dimethylaminophenylazo)indazole (5I) comprise respectively a carcinogen/non-carcinogen pair of monoazo dyes related to the hepatocarcinogen, butter yellow (DAB). While both members of the pair are potent bacterial mutagens in vitro, only 6BT induces unscheduled DNA synthesis in rat liver in vivo. To investigate factors responsible for these divergent activities we have determined in rats: relative rates of uptake from the gut after direct injection of 14C-labelled compound into intestine in situ, and after administration p.o.; distribution in selected tissues and elimination in urine, faeces and bile; binding of both compounds in vivo to liver DNA. The results revealed that, although 5I was taken up from the gut to a lesser extent than 6BT, comparable labelling associated with both compounds was detected in the presumed target organ (the liver). 5I binds in vivo to DNA much less effectively than 6BT. Therefore it would seem that other factors, such as differential metabolism in vivo, are more important than differences in uptake and distribution in accounting for the divergent activities of 6BT and 5I.

Biotransformation of 1-nitropyrene to 1-aminopyrene and N-formyl-1-aminopyrene by the human intestinal microbiota.

The nitropolycyclic aromatic hydrocarbon 1-nitropyrene (1-NP) is an environmental pollutant, a potent bacterial and mammalian mutagen, and a carcinogen. The metabolism of 1-NP by the human intestinal microbiota was studied using a semicontinuous culture system that simulates the colonic lumen. [3H]-1-Nitropyrene was metabolized by the intestinal microbiota to 1-aminopyrene (1-AP) and N-formyl-1-aminopyrene (FAP) as determined by high-performance liquid chromatography (HPLC) and mass spectrometry. Twenty-four hours after the addition of [3H]-1-NP, the formylated compound and 1-AP accounted for 20 and 80% of the total metabolism, respectively. This percentage increased to 66% for FAP after 24 h following 10 d of chronic exposure to unlabeled 1-NP, suggesting metabolic adaptation to 1-NP by the microbiota. Both 1-AP and FAP have been shown to be nonmutagenic towards Salmonella typhimurium TA98, which indicates that the intestinal microflora may potentially detoxify 1-NP.

Mutagenicity to Salmonella of four derivatives of the azo mutagen 5I: some implications for structure--activity databases and the evaluation of combinations of mutagens.

A structure--activity study is described in which four new derivatives of the potent bacterial mutagen 5-dimethylaminophenylazoindazole (5I) have been evaluated for mutagenicity to Salmonella. As expected, monodemethylation of the -NMe2 group of 5I increased its mutagenic potency while replacement of the -NMe2 with a cyclic amine reduced it. However, replacement of the aromatic indazole -NH group (of 5I) by an -NMe group (yielding NMe5I) dramatically attenuated mutagenic potency, a reduction which was both unexpected and not reversed in the monomethyl analogue (NMeMA5I). In competition experiments NMe5I had an inhibitory effect on the mutagenic potency of 5I itself and on that of the nonazo mutagen 2-acetylaminofluorene 2AAF. The results illustrate some of the problems associated with evaluating mixtures for mutagenicity and of assuming simple structure--activity relationships in the absence of relevant experimental data.

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

2-Amino-3-methylimidazo[4,5-ƒ]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 β-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.

Metabolism, mutagenicity, and activation of 1-nitropyrene in vivo and in vitro

The genotoxic air pollutant 1-nitropyrene (NP) labelled with 14C was administered to rats as the pure compound (6 μmole) by intraperitoneal injection, and vapor-phase-coated onto diesel particles (380 μg/g, 5 mg/rat) by oral and intratracheal instillation. In all three cases, well over 50% of the 14C label was recovered within 24 h, 20%–30% in the urine and 40–60% in the feces. Following intratracheal instillation the majority of the particles were recovered in the lung, trachea, and esophageal cavities, while after oral dosing the particles were mainly in the feces. The principal metabolite found after incubation of the urine with β-glucoronidase (15%–25% of the urinary 14C) was 6-hydrozy- N-acetyl-1-aminopyrene (6-OH-NAAP), a potent S9-dependent bacterial mutagen (∼600 rev/nmole in Salmonella TA98 | S9) identified as the major contributor to the mutagenic activity in the urine of rats dosed with NP. Studies in germ-free rats showed that activity of the gut flora was essential both for the formation of 6-OH-NAAP, for enterohepatic recirculation of metabolites excreted into the bile after first pass hepatic metabolism, and for excretion of mutagenic activity into the urine. The similarity in elimination kinetics and metabolite profile following such diverse doses and routes of administration indicates that NP is readily released from diesel particles both in the lung and in the gastrointestinal tract, and that the dose range used (10 μg to 10 mg/kg body weight) is either well above or well below the level needed to saturate metabolism and excretory mechanisms in the rat. Twenty-four h after intracheal instillation, the lung and gastrointestinal tract were the only organs in which accumulations of 14C were still detectable; these organs might therefore be particularly exposed and vulnerable to possible damage by NP.

Mutagens in cooked foods--metabolism and genetic toxicity.

Recently developed in our laboratories is an efficient extraction procedure incorporating XAD resin adsorption which yields from 200 degrees C grilled ground beef an extract containing 230 Salmonella TA1538 revertants per g fresh weight of original ground beef. These mutagenic components are specific for frameshift-sensitive Salmonella strains and have an absolute requirement for metabolic activation. S9 activation by cytochrome P-448 inducers, Aroclor 1254 (PCB), 3-methylcholanthrene (3-MC) and B-naphthoflavone (BNF), resulted in the largest mutagenic response. Phenobarbital induction gave 20% of the PCB response and Pregnenolone-16a-carbonitrile and corn oil were inactive. Human liver microsomes and BNF-induced rodent intestinal S9 were also active metabolizing fractions. Normal-phase HPLC separation of methanol-extractable metabolites generated from reaction of 2-amino-3-methylimidazo [4,5-f]quinoline (IQ), a mutagenic component of broiled food, rat liver microsomes and cofactors resulted in one direct-acting mutagenic peak and a second more polar peak still requiring metabolic activation. Two potent thermally-produced bacterial mutagens, Trp-P-2 and IQ, were examined in mammalian cells. In excision repair-deficient CHO cells, Trp-P-2 exposure caused cytotoxicity, mutagenicity (thioguanine and azaadenine resistances), sister chromatid exchange, and chromosomal aberrations at concentrations more than 30-fold lower than those for IQ. In normal repair-proficient CHO cells Trp-P-2 was one-half as active and IQ was inactive. Relative to Trp-P-2, IQ is much more potent in the Salmonella bacterial system than in mammalian CHO cells.

Activation of 2-amino-3-methylimidazo[4,5-ƒ]quinoline and 2-aminofluorene for bacterial mutagenesis by primary monolayer cultures of adult rat hepatocytes

Primary monolayer cultures of adult rat hepatocytes readily activate 2-amino-3-methylimidazo[4,5-ƒ]quinoline (IQ), a potent bacterial mutagen (Ames test) present in some cooked foods. In a co-culture system consisting of intact hepatocytes, bacteria, and IQ, the number of revertant colonies produced increased proportionately with hepatocyte density to a maximum at about 6 × 10 5 hepatocytes. When the density exceeded 6 × 10 5 hepatocytes per dish, the number of revertant colonies markedly declined. The decline was not due to bacterial death. In standard Ames tests disrupted hepatocytes prepared from similarly handled monolayer cultures or from hepatocytes just after isolation (uncultured), or rat-liver S9, were only about 1 10 th as efficient as the co-cultured intact hepatocytes (at optimum density) in activating IQ for bacterial mutagenesis. In contrast, 2-aminofluorene was poorly activated by intact hepatocytes. Disrupted hepatocytes and rat-liver S9 in standard Ames tests were much more effective than co-cultured intact hepatocytes in activating 2-aminofluorene for bacterial mutagenesis. For intact hepatocytes an inhibitor of cytochrome P-450, 2-[(2,4-dichloro-6-phenyl)-phenoxyl]ethylamine, inhibited IQ activation by 94–98%, and AF activation by 64–70%.

Mutagenicity and genotoxicity of nitroarenes: All nitro-containing chemicals were not created equal

The nitrated polycyclic aromatic hydrocarbons constitute a group of chemicals of environmental concern which display a broad spectrum of mutagenic, genotoxic and carcinogenic properties. Some members of the group are the most potent direct-acting bacterial mutagens while others exhibit low levels of potencies which require metabolic activation mixtures. Bacterial mutagenicity is dependent upon reduction of the nitro function. In mammalian cell systems the genetic and genotoxic effects of these nitrated chemicals include the induction of unscheduled DNA synthesis, sister-chromatid exchanges, chromosomal aberrations, gene mutations and cell transformation. The qualitative as well as quantitative expression of these effects is dependent upon the species and tissue of origin as well as culture history of the cell which in turn determine their enzymic capabilities and the conversion of these nitroarenes to ultimate mutagens and genotoxicants. In eukaryotic cells the following bioactivation pathways have been recognized: (a) reduction of the nitro moiety, (b) ring oxidation (the nature of which is influenced by the nitro function) followed by reduction of the nitro group, and (c) ring oxidation without concomitant reduction of the nitro moiety.

In Vitro Binding of the Food Mutagen 2-Amino-3-methylimidazo[4,5-<italic>f</italic>]quinoline to Dietary Fibers<xref ref-type="fn" rid="fn2">2</xref>

The ability of three kinds of fiber to bind to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a potent bacterial mutagen formed during the cooking of meat, was investigated to determine interactions among important food components. At pH 6.5 corn bran, wheat bran, and alfalfa meal each bound approximately 50% of the available IQ after incubation for 1 hour. Binding was pH-dependent, occurred optimally between pH 4 and 6, and was probably due to a cation-exchange mechanism. The pH at which IQ is efficiently bound to fiber overlaps the range of pH in the human gastrointestinal tract.

Apparent absence of recombinogenic activity of nitropyrenes for yeast

Nitropyrenes have been shown to be potent bacterial and mammalian mutagens. However, they failed to induce any recombinogenic activity in Saccharomyces cerevisiae D4 even at elevated concentrations and following extended periods of exposure. A plausible explanation for this lack of activity is the absence or the lack of activation of the enzyme required for the activation of nitropyrenes in this test system under the experimental (aerobic) conditions employed.

Comparative mutagenicity of a coal combustion fly ash extract in salmonella typhimurium and chinese hamster ovary cells

The dichloromethane extract of a coal combustion fly ash sample obtained from an experimental fluidized bed coal combustor was tested for mutagenicity in Salmonella typhimurium and cultured Chinese hamster ovary (CHO) cells. The extract was directly mutagenic in S typhimurium strain TA98 and the nitroreductase deficient strains TA98NR and TA98/1,8DNP6. The mutagenicity observed in TA98NR and TA98/1,8DNP6 was lower than that in TA98. Addition of exogenous Aroclor 1254-induced rat liver supernatant (liver S9) decreased the bacterial mutagenicity of the extract. A different mutagenic response was observed in CHO cells. In the absence of liver S9, although the extract was cytotoxic to CHO cells, no significant mutagenicity was observed. Addition of exogenous liver S9 decreased the cytotoxicity and increased the mutagenicity at both Na+-K+-ATPase and hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene loci in CHO cells. Using gas chromatography/mass spectrometry (GC/MS) and tandem quadruple mass spectrometry, a number of polynuclear aromatic hydrocarbons (PAHs) and nitrated PAHs (nitro-PAHs) were tentatively identified and quantitated. A possible explanation of the difference in bacterial and mammalian mutagenicity of the extract is that the bacterial mutagenicity was induced by the nitro-PAHs that are potent bacterial mutagens and mammalian mutagenicity was induced by both PAHs and nitro-PAHs that are promutagens.

Nitrated polycyclic aromatic hydrocarbons: potent bacterial mutagens and stimulators of DNA repair synthesis in cultured human cells.

Ten polycyclic aromatic hydrocarbons (PAHs), viz. anthracene pyrene, chrysene, perylene, fluoranthene, benzo[a]pyrene, benzo[a]pyrene, benz[a]anthracene, benzo[ghi]perylene, benzo[k]fluoranthene, have been nitrated using concentrated nitric acid and the crude nitrated mixture examined for biological activity. All the nitro PAHs examined were mutagenic to Salmonella typhimurium in the absence of a rat liver preparation. Addition of Aroclor-1254 induced liver had little effect on mutagenicity. Mutagenic potency differed for the various nitrated mixtures with nitrated pyrene and nitrated fluoranthene the most potent and nitrated anthracene the least potent. Both frame-shift and base-substitution mutations were induced by the nitrated PAHs. The nitrated PAHs were also able to induce DNA repair synthesis in cultured HeLa cells in the absence of liver, indicating that these cells have the necessary enzymes to activate nitro PAHs. Potency again varied from compound to compound with nitrated pyrene appearing to be the most active. Isolation of individual components from the crude nitrated mixtures has not been carried out in this study. In view of the possible wide-spread distribution of nitrated PAHs in the environment further work is required to assess the carcinogenic potency of these compounds which possibly pose a risk to man.

ChemInform Abstract: SYNTHESIS OF CYCLOPENTA(CD)PYRENE 3,4‐EPOXIDE, THE ULTIMATE MUTAGENIC METABOLITE OF THE ENVIRONMENTAL CARCINOGEN, CYCLOPENTA(CD)PYRENE

AbstractDas Cyclopentapyren (I) ergibt mit N‐Bromsuccinimid das Bromhydrin (II), das zum Epoxid (III) führt.