Mutagenicity of N-arylacetohydroxamic acids and their O-glucosides derived from chlorinated 4-nitrobiphenyl ethers

The mutagenic activity of N-arylacetohydroxamic acids, their O-acetates, their O-glucosides, and N-arylhydroxylamines, derived from chlorinated 4-nitrobiphenyl ethers (CNBs), was tested in the Salmonella reversion assay. N-Arylhydroxylamines were mutagenic by themselves; however, other compounds containing an N-acetyl group showed mutagenic activity in the presence of guinea pig liver S9. The mutagenic activation of the glucosides of N-arylacetohydroxamic acids was caused by Ms but not by S10.5, whereas their aglycones, N-arylacetohydroxamic acids, were activated to mutagens by both the fractions. The mutagenic activation of these compounds was inhibited by bis(p-nitrophenyl)phosphate, which indicates that enzymatic deacetylation is a crucial step in the mutagenic activation. Analysis of metabolites of the O-glucosides of N-arylacetohydroxamic acids by h.p.l.c. indicates that the corresponding deacetylated O-glucosides are primary metabolites, which decomposed to amino and azoxy (via hydroxylamine) derivatives, and that the deacetylating activity of S9 locates exclusively in Ms.     

Mutagenic activity of chloramines

Mutagenesis by chloramines and hypochlorous acid (HOCl) was studied to determine whether these agents could contribute to the mutagenic and potentially carcinogenic activity of stimulated leukocytes and whether environmental exposure to these agents is a cause for concern. Mutagenic activity was measured using the S. typhimurium TA97a, TA100 and TA102 tester strains. Because chloramines and HOCl are bactericidal, react rapidly with cell components, and can destroy the histidine and biotin required for the mutagenesis assay, activity can't be compared directly with that of less toxic or reactive agents. Nevertheless, chloramines were mutagenic when tested under appropriate conditions. TA100 was the most sensitive strain, and the most active mutagens were lipophilic dichloramines (RNCl2) including derivatives of histamine, ethanolamine and putrescine. Lipophilic monochloramines (RNHCl) such as histamine-monochloramine and NH2Cl were less active. Hydrophilic chloramines such as taurine-chloramines had low activity, and HOCl was inactive. The metabolic state of the bacteria was critical. Chloramines were mutagenic when added to bacteria with glucose at 37 degrees C, but killing predominated when chloramines were added at 4 degrees C or 25 degrees C, or at 37 degrees C without glucose. Production of chloramines and HOCl by leukocytes in vivo could contribute to the association of chronic inflammation and cancer as a result of: (1) the entry of membrane-permeable chloramines into normal cells followed by attack on intracellular components including DNA, and (2) the production of secondary mutagens such as compounds with carbonyl groups or carbon-chlorine bonds. On the other hand, chlorination of water supplies is perhaps more likely to destroy than create mutagens, and chloramines from the environment are unlikely to penetrate the skin and mucous membranes.     

Actual problems of genetic toxicology

The review deals with current issues of genetic toxicology and aims to develop this science at the contemporary stage. We study general approaches to assessing the genotoxic and mutagenic activity of environmental factors; to constructing a regulatory system of chemical compounds that considers the mutagenic effect in Russia and abroad; and to determining modern methods for assessing the organ specificity of mutagens, alternative methods of genetic toxicology, the mutagenic action of various factors in the survey of population, and the abilities of toxicogenomics to identify the mutagenic properties of the environment.     

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.     

A contribution to the study of the structure-mutagenicity relationship for alpha-dicarbonyl compounds using the Ames test.

The mutagenicity of a series of nine alpha-dicarbonyl compounds against S. typhimurium strain TA100 was studied using the Ames test (standard plate incorporation assay) without preincubation. Acetylbenzoyl, sodium glyoxylate and camphorquinone were not mutagenic. The following sequence of activities (in revertants per mumol of free dicarbonyl added) was obtained: glyoxal greater than methylglyoxal greater than phenylglyoxal much greater than 1,2-cyclohexanedione much greater than diacetyl greater than 3,4-hexanedione. These compounds can be grouped in three series: aldehydes, ketones and enolizable ketones (1,2-cyclohexanedione). In each of the two first groups the mutagenic activity decreases when the size of the substituent increases. No relation was found between the mutagenicity and the molecular electronic and/or resonance parameters. The low or non-existent activity of some of the chemicals studied is discussed. A relation between the mutagenic activities and the polarographic reduction potentials and, consequently, the structures of the mutagens was found.     

The role of bacterial metabolism in transformation of non-mutagenic compounds into mutagens. I. Participation of Escherichia coli nitroreductase in creation of mutagens from non-mutagenic new pesticides]

Investigations concerned Escherichia coli nitroreductase in creation of mutagens from non-mutagenic pesticides-derivatives of urea. Three new compounds were studied: N-phenyl-N'-methylurea (IPO 4328), N-methyl,N-(2-hydroxyethyl)-N'phenylurea (IPO 2363), N-(2-hydroxyethyl), N-methyl-N'-(3,4 dichloroethyl) urea, and diurone-3-(3,4 dichlorophenyl)-1,1 dimethylurea. These compounds were incubated in anaerobic conditions with cells of E. coli K-12 (KF) strain and nitrate or nitrite. Using Ames test, mutagenicity of resulting metabolites was investigated. It was found that during incubation of herbicide IPO 4328 with cells of E. coli K-12 (KF) and nitrate, mutagenic product for strain of S. typhimurium TA 1537 is created. Very weak mutagenic metabolite for the same strain was appearing during incubation of herbicide IPO 2363 with cells of E. coli K-12 (KF) in presence of nitrite. Incubation of investigated compounds with E. coli K-12 (KF) cells alone did not result in appearance of mutagenic substances. Thus, role of Escherichia coli in creation of mutagenic compounds from non-mutagenic derivatives of urea consisted of nitrite from nitrate production with participation of nitroreductase, which afterwards in absence of bacteria or action of their enzymes reacted with investigated pesticides.     

Application of Salmonella strains with altered nitroreductase and O-acetyltransferase activities to the evaluation of the mutagenicity of airborne particles

The Ames test was applied to evaluation of the mutagenicity of month's samples of airborne particles from the center of Wroc?aw (SW Poland) collected in August and December 1997. The strains used for the study were TA 98, TA 100 and their derivatives: TA 98 NR, YG 1021, YG 1024, YG 1026, YG 1029, YG 1041, YG 1042. Both studied samples were mutagenic for almost all tested strains, with the exception of the August sample which did not influence the strain TA 100 without the metabolic activation with the S9 fraction. The December sample exhibited higher genotoxic activity than the August sample. Mutagenicity ratios of the strains with reduced nitroreductase and O-acetyltransferase activities were higher, and of the strain without the nitroreductase--lower than those of the parent strains. This indicates that nitro and amino derivatives of PAHs are responsible for the significant proportion of total mutagenicity of the studied samples of particulates. Metabolic activation with the S9 fraction caused the increase of the mutagenic activity of the samples, which indicates the presence of promutagens. The GC-MS analysis revealed the presence of known indirect mutagens from the PAHs group.     

Formation of mutagens following chlorination of humic acid. A model for mutagen formation during drinking water treatment

Aqueous chlorination of humic acids results in the formation of compounds with direct-acting mutagenic activity in the Ames/Salmonella plate assay for tester strains TA98, TA100, TA1535, TA1537 and TA1538. The addition of a rat-liver microsomal fraction (S9) plus cofactors causes a substantial decrease of activity, the extent of which is tester strain dependent. The non-chlorinated humic acids are not mutagenic either in the presence or absence of S9. Formation of mutagenic activity and of total organic halogen (TOX) is linearly related to humic concentration in the range of 0.2-1.6 mg/ml total organic carbon (TOC), and to chlorine concentration in the range of 0.1-1.0 chlorine equivalents per mole of carbon. The mutagenic activity is due predominantly to non-volatile compounds. Mutagenic activity is also detectable, after sample concentration by lyophilization, upon chlorination at a humic acid level of 0.02 mg/ml TOC. The specific mutagenic activities (per mg TOX), and also the degree of chlorine incorporation into humic acid, at 0.02 mg/ml TOC are similar to those present after chlorination at 1 mg/ml TOC. Production of mutagens is greatly dependent on the chlorination pH, with a pattern of decreasing mutagenic activity with increasing pH. This order of activity can be at least partially explained by the alkali liability of the compounds. Chlorination of commercial humic acids is proposed as a model for examination of mutagen formation during water chlorination.     

Approaches to genetic control for the use of pesticides

Analysis of data available in literature has shown that 65% of 400 pesticides studied for their mutagenicity exert a mutagenic effect on any test-object. The number of revealed mutagens approaches 100% when four or more test-objects are used. Recommendations for quantitative estimation of genetic risk worked out under conditions of model experiments with standard mutagens are not available for pesticides since they are slight mutagens. The necessity for genetic stage-by-stage monitoring of pesticide use is substantiated. This monitoring should be carried out at the stage of experimental studies by means of classifications by the degree of potential mutagenic danger (the method is described) and at the stage of ecological and genetic investigations--by means of the regulation for application with due regard for the summary mutagenic background.     

Cytogenetic activity of various organophosphorus insecticides

The action of certain insecticides belonging to the group of organophosphorous compounds (chlorophos, metaphos, etaphos, phosphamid, phosalone, carbophos, selecron) has been studied by the test of chromosome aberrations in Crepis capillaris cells. It is shown that these insecticides possess mutagenic activity in all phases of the cell cycle and are mutagens of the undelayed action.     

Studies on the mutagenicity and tumor-initiating activity of methylated fluorenes

Several methylated analogs of fluorene were evaluated as mutagens in Salmonella typhimurium TA98 and TA100 in the presence and absence of microsomal activation. Among the methylated derivatives of fluorene assayed were 9-methylfluorene, 2-fluoro- and 2,7-difluoro-9-methylfluorene, 1,9-, 2,9-, 3,9- and 4,9-dimethylfluorene, 2,3,9-trimethylfluorene and 2,7,9-trimethylfluorene. Mutagenic activity was observed for several of these fluorene derivatives in the presence of rat liver homogenate. The data support a previous observation that a single methyl substituent in the 9-position of fluorene is associated with mutagenic activity within this series of compounds. Substitution with fluorine at both the 2- and 7-positions of 9-methylfluorene was not associated with a loss of mutagenic activity as evidenced by the similar mutagenic activity of 2,7-difluoro-9-methylfluorene and 9-methylfluorene. However, 2,7,9-trimethylfluorene was not mutagenic under these assay conditions. 9-Methylfluorene, 1,9-, 2,9-, 3,9- and 4,9-dimethylfluorene and 2,3,9-trimethylfluorene were active as mutagens in the presence of rat liver homogenate, but were inactive as tumor initiators when assayed on mouse skin.     

Studies on the effect of ascorbic acid and selenium on the genotoxicity of nitrofurans: nitrofurazone and furazolidone

The genotoxic properties of nitrofurazone and furazolidone were studied using the Ames test and SOS-chromotest. Both compounds were found to act as strong mutagens on the TA97 and TA102 strains of S. typhimurium and to induce the SOS-repair system in the PQ37 strain of E. coli. A good concordance was found between the mutagenic activity and the ability to induce the SOS system. Ascorbic acid and sodium selenite only very slightly lowered the genotoxic effect of the 2 nitrofurans studied both in the Ames test and in the SOS-chromotest.     

Mutagenic potency of some conjugated nitroaromatic compounds and its relationship to structure

The mutagenicities of 12 conjugated non-fused nitroaromatic compounds and 1 amino analogue were determined in strains TA100 and TA98 of Salmonella typhimurium. Reversions by p-nitroaromatics increased in the order of the acetophenone, benzaldehyde, styrene, chalcone, cinnamic acid and stilbene indicating the importance for mutagenic potency of extended conjugation to the p-nitrophenyl substituent. Highest mutagenicity was found with alpha-substituted 4-nitrostyryl derivatives of which the phenyl derivative (31 revertants per nmole in TA100) was the most active. Generally, the TA100 strain was more sensitive than TA98 to these mutagens and S9 treatment was unnecessary for activity, although 4-nitrochalcone required S9 activation. Para-nitro isomers of the cinnamic acids and chalcones were much more active than the corresponding ortho and meta isomers. The 4-aminocinnamic acid analogue was inactive suggesting that complete reduction in Salmonella of 4-nitrocinnamic acid to an active amino derivative is not response for the high mutagenicity of the former. Mutagenicity of these p-nitrostyryl compounds may be explained by the covalent interaction of the electrophilic benzylic carbon with Salmonella DNA.     

Nitroarenes in Suimon River sediment

Mutagenic activity was observed in sediments of the Suimon River bed with and without S9 mix. The direct-acting mutagens in the sediment were investigated. The sediment was extracted with methanol and fractionated on a Silica gel column. The benzene fraction from the Silica gel column exhibited mutagenic activity without S9 mix in strain TA98, while it failed to show mutagenic activity in nitroreductase-deficient strain TA98NR. This observation led to the suspicion that nitro compounds were the direct-acting mutagens of these samples. The benzene fraction was treated by heptafluorobutyric anhydride (HFBA) and investigated with gas chromatography equipped with an electron capture detector (GC-ECD). 2-Nitrofluorene, 4,4'-dinitrobiphenyl, 2,7-dinitrofluorene and 1-nitropyrene were detected and measured quantitatively. The mutagenic activity of a mixture of these compounds was compared with that of the original fraction and the direct-acting mutagenicity of Suimon River sediment can be explained by these nitroarenes, especially 1-nitropyrene.     

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.     

Nitrosoguanidine-induced mutations inStreptomyces indicus

The mutagenic activities of five 1-alkyl derivatives of 3-nitro-l-nitrosoguanidine,viz. methyl, ethyl, propyl, isopentyl and octyl, were tested onStreptomyces indicus. All the compounds were found to be mutagenic. The methyl and ethyl derivatives were most effective regarding mutational frequency. The effectiveness decreased with increasing number of C-atoms in the alkyl group of the mutagens. The compounds tested had a much higher mutagenic efficiency than UV-rays. A marked variation existed in antibiotic activity among the biochemical mutants: increase, decrease or complete absence of such activity were seen.     

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.     

Determination of the activity of 16 hydrazine derivatives in the bioluminescence test for genotoxic agents

The activity of 16 hydrazine derivatives was determined in the bioluminescence test for genotoxic agents (BLT). Hydrazine compounds that were shown to exert mutagenic activity in the Ames test were also active in the BLT. Isoniazid and p-tolylhydrazine which reacted as weak mutagens in the Ames test were highly active in the BLT.     

The mutagenicity of halogenated alkanols and their phosphoric acid esters for Salmonella typhimurium.

9 halogenated alkanols, 9 corresponding tris (haloalkyl)phosphates, and 2 bis-(2,3-dibromopropyl)phosphate salts were evaluated for mutagenicity against Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538, with and without rat liver in vitro metabolic activation system (S9 mix). Most of the test samples showed mutagenic activity in the strains TA100 and TA1535, but not in the strains TA98, TA1537 and TA1538. In general, the mutagenic activities of the phosphates obtained with S9 mix were greater than the activities obtained without S9 mix. Among the phosphates, several structure--activity relationships were found; i.e., (i) the bromoalkyl derivatives were more mutagenic than the corresponding chloroalkyl derivatives, (ii) the beta-haloethyl derivatives were more mutagenic than the gamma-halopropyl derivatives, (iii) the phosphates having adjacent beta and gamma halogen atoms in the alkyl moiety, e.g., tris-(2,3-dibromopropyl)phosphate, were particularly potent mutagens, (iv) the branched carbon chain reduced the mutagenic activities in spite of the presence of beta-halogen atoms, e.g., tris(1-bromomethyl-2-bromoethyl)phosphate. However, such relations did not necessarily apply to the halogenated alkanols. It is concluded that the metabolic activation pathway via haloalkanols to mutagens must not be in common with all tris-BP-like phosphates.