Relationship between mutagenic potency in Salmonella typhimurium strains and the chemical structure of nitro biphenyls

Most of the positional isomers of mono-, di-, tri- and tetranitrobiphenyls were synthesized and assayed for their mutagenicity in Salmonella typhimurium strains TA98, TA98NR and TA98/1,8DNP6 in the absence of S9 mix. In mono- and dinitrobiphenyls, the structure requirements favoring mutagenic activity are the presence of a nitro group at the 4-position and its absence at the 2-position. TA98 and TA98/1,8DNP6 were reverted by 2-position-free 4-nitro analogues, but TA98NR was not reverted. The results suggest that direct-acting mutagenicity involves the reduction of the nitro group by bacterial nitroreductase but does not involve specific esterification enzymes. Some of the tri- and tetranitrobiphenyls e.g. 3,4,3'-, 3,4,4'-, 3,4,3',4'- and 3,4,2',4'-derivatives reverted not only TA98 and TA98/1,8DNP6 but also TA98NR. Those derivatives commonly have 2 nitro groups at an adjoining position (3,4-dinitro group), whereas 2,4,2',4'-tetranitrobiphenyl, which has strong potency not only in TA98 and TA98/1,8DNP6 but also in TA98NR, possesses 2 nitro groups at the 2-position of each benzene ring.     

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.     

Mutagenicity of polycyclic aromatic compounds (PAC) identified in source emissions and ambient air

Several polycyclic aromatic compounds (PAC) including nitrated and oxygenated derivatives of polycyclic aromatic hydrocarbons (PAH) were tested for mutagenic activity in the Salmonella/microsome assay. Among the compounds tested the isomer mix of nitro-1-hydroxypyrenes showed the highest direct mutagenic response in both the Salmonella strain TA98 and TA100 (1251 revertants/micrograms and 463 revertants/micrograms, respectively). The direct-acting mutagenicity of the nitro-1-hydroxypyrene isomer mix was dependent upon reduction of the nitro function as evidenced by the decrease in activity observed with the nitroreductase-deficient and arylhydroxylamine esterifying-deficient tester strains. The oxygenated derivatives of PAH containing aldehyde or keto groups showed weak or no mutagenic responses. In most cases addition of S9 was essential for any mutagenic activity and the strain TA100 was more sensitive than the strain TA98. Within this group, 7H-dibenzo[c,g]fluoren-7-one showed the highest mutagenic effect; 7 and 22 revertants/micrograms using the strains TA98 and TA100, respectively.     

From mutagenic to non-mutagenic nitroarenes: effect of bulky alkyl substituents on the mutagenic activity of nitroaromatics in Salmonella typhimurium. Part II. Substituents far away from the nitro group

Derivatives of 4-nitrobiphenyl, 4-nitrosobiphenyl, 2-phenyl-5-nitropyridine (2-aza-4-nitrobiphenyl) and 2-nitrofluorene, bearing various alkyl substituents far away from the nitro group (4'-position in nitrobiphenyls, 7-position in 2-nitrofluorenes) were synthesised and tested for mutagenic potency in strains TA98 and TA100 of Salmonella typhimurium. In the absence of S9 in both strains, mutagenicity of all4'-Ad (Ad=adamantyl). Changes of the molecular shape from 'planar' to non-planar caused by the bulk of the introduced substituents (without influencing the twisting of the nitro substituent or the phenyl rings in the biphenyl compounds) may be responsible for this effect by interfering with an efficient intercalation into DNA.A comparison between experimental and theoretical values as calculated from recently developed equations (QSAR) confirmed our previous results (see the preceding paper) that mutagenicity of alkyl-substituted nitroaromatics cannot be predicted by hydrophobicity and LUMO-energies alone without including steric parameters.     

Mutagenic activities of ten imidazole derivatives in <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

Ten imidazole derivatives were tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100 both in the absence and presence of metabolic activation by the microsomal fraction S9 mix. In a general manner, derivatives tested exhibited a greater mutagenic activity in the TA100 strain comparing to the responses in TA 98. In the standard plate incorporation assay, 8 of these substances (80%) were found to be mutagenic for at least one of the two strains in the presence or absence of metabolic activation. Two compounds showed positive results in TA98 and 6 compounds were also mutagenic in TA100 without S9. In the presence of S9 mix, all of the 10 substances were non-mutagenic in TA98, whereas 4 compounds were positive in TA100. The results suggested the mutagenic potentials of the imidazole derivatives particularly inducing the reversion of base-pair substitutions. According to the structure-activity relationships phenyl groups in position 2 with different substituents can confer the mutagenic activity of the tested compounds. Methyl groups in different positions of these phenyl substituents can cause different types of mutations. This mutagenic effect is observed more clearly when the phenyl group is inhibited with a nitro group.     

Evaluation of the mutagenic and cytostatic potential of aristolochic acid (3,4-methylenedioxy-8-methoxy-10-nitrophenanthrene-1-carboxylic acid) and several of its derivatives

Aristolochic acid (1), a constituent of Aristolochia species, has been used for medicinal purposes since the Graeco-Roman period. Following the observation that the compound was mutagenic and carcinogenic, it was removed from pharmaceutical products. Consistent with previous reports, we have found that 1 serves as a direct-acting mutagen in Salmonella typhimurium strains TA100, TA102, TA1537 and TM677, but was not active in the nitroreductase-deficient strains TA98NR and TA100NR. However, aristolic acid (2), a compound that differs in structure only by the absence of the nitro group, was also found to be a direct-acting mutagen in Salmonella strains TA98, TA100, TA102, TA1537, and TM677, as well as strains TA98NR and TA100NR. Both compounds (1 and 2) were active mutagens when evaluated with cultured Chinese hamster ovary cells. Thus, in contrast to previous suggestions, the nitro group at position 10 is not required to induce a mutagenic response. Also, a series of structural relatives (the methyl esters of 1 and 2 (3 and 4, respectively), aristolochic acid-D (5), aristolactam (6), aristolactam A-II (7), and aristolactam-N-beta-D-glucoside (8)) were evaluated for mutagenic potential with Salmonella typhimurium strain TM677 and found to be inactive. Since compounds 3 and 4 were found to be active mutagens with Salmonella typhimurium strains TA98, TA100, TA102 and TA1537 (sufficient quantities of compounds 5-8 were not available for testing), differential sensitivity of the tester strains unrelated to mutagenic potential is suggested. Further, compounds 1, 2, and 6-8 were evaluated for potential to inhibit growth with cultured KB or P388 cells. P388 cells were substantially more sensitive, and compound 1 was the most active of the materials tested (ED5 = 0.58 microM). Compound 6 also demonstrated appreciable activity (ED50 = 4.2 microM), as did compound 8 (ED50 = 6.0 microM). It therefore appears that phenanthrene-ring substituents, in addition to the nitro group at position 10, serve important roles for biological potential. In considering the carcinogenic event induced by aristolochic acid, these functionalities should also be taken into account.     

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.     

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.     

Mutagenicity of nitro derivatives induced by exposure of aromatic compounds to nitrogen dioxide

Mutagenic nitro derivatives were readily induced when 6 kinds of chemicals were exposed to 10 ppm of nitrogen dioxide (NO2). Single nitro derivatives were formed from pyrene, phenanthrene, fluorene or chrysene. Carbazole and fluoranthene each produced 2 derivatives substituted with nitro groups at different positions. The formation of nitro derivatives was enhanced by exposure of pyrene to NO2 containing nitric acid (HNO3, less than 100-fold enhancement) or sulphur dioxide (SO2, less than 15-fold enhancement). After 24 h of exposure the yields of the nitro derivative were 0.02% with 1 ppm of NO2 in air and 2.85% with NO2 (1 ppm) containing traces of HNO3. The nitro derivatives from all but phenanthrene and carbazole were chemically identified by means of gas chromatography (GC) and mass spectrometry (MS), and the mutagenicity of the 4 kinds of authentic nitro derivatives was tested by using Salmonella strains TA98 and TA1538 with or without the S9 fraction from rat liver treated with Aroclor 1254. The nitro derivative induced from pyrene was determined to be 1-nitropyrene; that of chrysene was 6-nitrochrysene; that of fluorene was 2-nitrofluorene; and those of fluoranthene were 3-nitrofluoranthene, and 8-nitrofluoranthene. Tested with strain TA98 in the absence of the S9 fraction, the first 4 of these derivatives yielded, respectively, 3050, 269, 433 and 13 400 revertants per nmole. Thus, each nitro derivative formed was potentially a direct-acting frameshift-type mutagen. Each compound exposed to NO2 showed a decreased mutagenic activity when tested in the presence of S9 mix. A possible explanation comes from experiments in which 1-nitropyrene was incubated with the S9 mix at 37 degree C for 10 min, and 1-aminopyrene was formed. The mutagenic activity of 1-aminopyrene was appreciable, but only about one-tenth of that of 1-nitropyrene in the Ames test.     

Correlations between 15 polycyclic aromatic hydrocarbons (PAH) and the mutagenicity of the total PAH fraction in ambient air particles in La Spezia (Italy)

Airborne particulate matter has been monitored 4 times a month for 1 year (1988) in the city of La Spezia (Italy). The polycyclic aromatic hydrocarbon (PAH) fractions were extracted, purified and characterized for the content of 15 individual PAH. In general when concentrations of individual PAH were compared statistical correlation was obtained. Mutagenicity studies were performed by the use of the Ames plate test with the Salmonella strains TA98, TA100, TA98NR and TA98DNP6 with and without metabolic activation (S9 mix). The TA98 strain was by far the most responsive and the S9 mix was absolutely required as expected when PAH are assayed. Besides mutagenicity, toxicity was also considered and it proved to be correlated with mutagenicity in TA98, +S9. The TA98NR and TA98DNP6 strains showed no appreciable differences from the parental strain TA98 indicating the absence of significant amounts of direct-acting nitro derivatives in our PAH samples. Of the 15 PAH considered in this study the amounts of cyclopental[c,d]pyrene (CPP) correlated best with mutagenicity. The role of CPP in contributing to the indirect mutagenicity of urban air PAH samples is discussed.     

Structural specificity of aromatic compounds with special reference to mutagenic activity in Salmonella typhimurium--a series of chloro- or fluoro-nitrobenzene derivatives

The mutagenicity of 21 chloro- or fluoronitrobenzene compounds and 9 chloro- or fluorobenzene compounds in Salmonella typhimurium (strains TA98, TA1538, TA1537, TA100 and TA1535) was examined. The tests were carried out under the conditions of absence and presence of liver microsomal activation. 15 nitro-group compounds had mutagenic activity; above all, compounds of fluoronitrobenzene were mutagenic for both types of strain. On the other hand, chloronitrobenzene compounds were mutagenic for base-pair substitution strains only. Mutagenic activity was exhibited by all compounds having a chloro or fluoro substituent at the para and ortho position in the nitrobenzene nucleus. All compounds without a nitro substituent showed no mutagenic activity.     

From mutagenic to non-mutagenic nitroarenes: effect of bulky alkyl substituents on the mutagenic activity of 4-nitrobiphenyl in Salmonella typhimurium. Part I. Substituents ortho to the nitro group and in 2'-position

Eleven alkyl substituted derivatives of 4-nitrobiphenyl (4NBp) and two corresponding nitroso compounds were synthesised and tested for mutagenic potency in strains TA98 and TA100 of Salmonella typhimurium. The mutagenicity of compounds substituted ortho to the nitro group (3-methyl-, 3-ethyl-, 3-isopropyl-, 3-tertbutyl-, 3, 5-diethyl-, 3,5-diisopropyl-, and 3,5-ditertbutyl-4NBp) decreased with growing steric demand of the alkyl substituents in both tester strains. The most sterically hindered compounds were non-mutagenic even at highest concentrations. This reduction of mutagenicity is correlated with deviations from the coplanar orientation of the nitro group relative to the aromatic plane. Since a comparable decrease of mutagenicity for the nitroso compounds (4NOBp and 3-tertbutyl-4NOBp) was not observed, the first reduction step of non-planar nitro groups must be inhibited.Alkyl groups in the 2'-position of 4NBp (2'-methyl-, 2'-ethyl-, 2'-isopropyl-, and 2',4', 6'-trimethyl-4NBp) also reduced mutagenic activity with increasing size and removed it completely for the most sterically hindered species (2'-isopropyl-, 2',4',6'-trimethyl-4NBp). In this case, the co-planarity of the nitro group is not affected but the twisting of the two aromatic rings, which is associated with a less effective charge delocalisation of the nitrenium ion.The experimental mutagenicities of all nitro compounds were compared to predicted values, that are based on recently developed empirical equations. While there was reasonable correspondence for the parent compound (4NBp), its ortho methylated derivative (3-methyl-4NBp) and two highly hydrophobic dialkylated species (3,5-diisopropyl- and 3, 5-ditertbutyl-4NBp), predictions for all other alkyl substituted compounds were too high. Thus, steric parameters should be included to improve the general validity of predictions by means of quantitative structure-activity relationships (QSAR).     

Mutagenicity studies of p-substituted benzyl derivatives in the Ames Salmonella plate-incorporation assay

The mutagenicity of 24 benzyl derivatives, containing a variety of substituents and leaving groups, were assayed in strain TA100 using the Ames plate-incorporation assay. p-Nitrobenzyl chloride (12 000 revertants/mumole), p-nitrobenzyl tosylate (6100 revertants/mumole), and p-acetoxybenzyl chloride (100 revertants/mumole) were mutagenic; none of the remaining 21 compounds were mutagenic. p-Nitrobenzyl chloride was also found to be mutagenic in strain TA98 (700 revertants/mumole), but not in strain TA98NR (a strain deficient in nitro reductase activity). p-Acetoxybenzyl chloride was nonenzymatically hydrolyzed to p-hydroxybenzyl alcohol and p-acetoxybenzyl alcohol. These findings suggest that nitrobenzyl derivatives were mutagenic due to nitro reductive metabolism and that p-acetoxybenzyl chloride was mutagenic due to the intermediate formation of p-hydroxybenzyl chloride during the hydrolysis of p-acetoxybenzyl chloride.     

Further examination of the effects of nitrosylation on Alternaria alternata mycotoxin mutagenicity in vitro

Previously, Alternaria extract and metabolite mutagenicities+/-nitrosylation were characterized using Ames Salmonella strains TA98 and TA100, which are both reverted at GC sites. To examine other targets for mutation, the metabolites Altertoxin I (ATX I), Altenuene (ALT), Alternariol (AOH), Alternariol monomethyl ether (AME), Tentoxin (TENT), Tenuazonic acid (TA) and Radicinin (RAD) were reexamined+/-nitrosylation, using Ames Salmonella strain TA97, sensitive to frameshift mutations at a run of C's, as well as strains TA102 and TA104, reverted by base pair mutations at AT sites and more sensitive to oxidative damage. ATX I was also assessed for mammalian mutagenicity at the Hprt gene locus in Chinese hamster V79 lung fibroblasts and rat hepatoma H4IIE cells. When tested from 1 to 100 microg/plate without nitrosylation, ATX I was mutagenic in TA102+/-rat liver S9 for activation and weakly mutagenic in TA104+/-S9, demonstrating direct-acting AT base pair mutagenicity. AOH was also directly mutagenic at AT sites in TA102+/-S9 while AME was weakly mutagenic in TA102+/-S9 and TA104+S9. Nitrosylation of ATX I enhanced mutagenicity at AT sites in TA104+/-S9 but produced little change in TA102+/-S9 compared to native ATX I. However, nitrosylated ATX I generated a potent direct-acting frameshift mutagen at C sites in TA97+/-S9. While ATX I was not mutagenic in either V79 cells or H4IIE cells, 5 and 10 microg/ml nitrosylated ATX I produced a doubling of 6-thioguanine resistant V79 colonies and 0.5 and 1 microg/ml were mutagenic to H4IIE cells, becoming toxic at higher concentrations. These results suggest ATX I, AME and AOH induce mutations at AT sites, possibly through oxidative damage, with nitrosylation enhancing ATX I frameshift mutagenicity at runs of C's. Nitrosylated ATX I was also directly mutagenic in mammalian test systems.     

Mutagenicity of some commercially available nitro compounds for Salmonella typhimurium.

Benzoyl chloride and 53 commercially available aromatic heterocyclic and aliphatic nitro compounds were tested for mutagenicity in Salmonella typhimurium TA98 and TA100. 34 of 53 nitro compounds (64%) were mutagenic, 4 in TA100 only, 15 in TA98 only, and 15 in both strains. 13 of the heterocyclic derivatives of pyridine, indole, indazole, quinoline, and benzimidazole were mutagenic. 21 of 34 mutagenic nitro compounds were bactericidal. Nitromethane was the only aliphatic tested and was not mutagenic. Benzoyl chloride, a human carcinogen, was mutagenic for TA98.     

Relationships among direct-acting mutagenicity, nitro group orientation and polarographic reduction potential of 6-nitrobenzo[a]pyrene, 7-nitrobenz[a]anthracene and their derivatives

The direct-acting mutagenicity in Salmonella typhimurium strains TA98 and TA100 and the half-wave reduction potentials of 6-nitrobenzo[a]pyrene (6-nitro-BaP), 7-nitrobenz[a]anthracene (7-nitro-BA), and a series of their derivatives were compared. The common structural feature of these compounds is that their nitro substituents, in order to minimize steric hindrance, preferentially adopt an orientation perpendicular or nearly perpendicular to the aromatic rings. All of the compounds, except 7-hydroxy-6-nitro-BaP and 7-acetoxy-6-nitro-BaP, were found to exhibit very weak or no direct-acting mutagenicity. 7-Acetoxy-6-nitro-BaP and 7-hydroxy-6-nitro-BaP were also found to have the lowest reduction potentials among the tested compounds. The results suggest that a combination of the orientation of the nitro substituent and the first half-wave reduction potential of the compound may correlate with the direct-acting bacterial mutagenicity of nitro-polycyclic aromatic hydrocarbons.     

Dependence on Salmonella typhimurium enzymes of mutagenicities of nitrobenzene and its derivatives in the presence of rat-liver S9 and norharman

Dependence on S. typhimurium enzymes of mutagenicities of nitrobenzene (NB) and o-, p-chloronitrobenzenes (o-, p-CNBs), which are only mutagenic in the presence of S9 and norharman (NOH), was investigated using a nitroreductase-deficient strain TA98NR and an esterifying enzyme-deficient strain TA98/1,8-DNP6. NB exhibited mutagenicity towards TA98 but did not towards TA98NR strain in spite of the presence of S9 in the assay system. The mutagenicity of o-CNB towards TA98NR was significantly lower than that of o-CNB towards TA98. In contrast to NB and o-CNB, synthesized phenylhydroxylamine (PHA) and o-chlorophenylhydroxylamine (o-CPHA) exhibited approximately the same mutagenicity towards both tester strains. These results indicate that the nitroreduction required for the appearance of mutagenicity of the nitrobenzene derivatives in the presence of S9 and NOH is dependent on the nitroreductase of the tester strain. In addition, the mutagenicities of PHA and p-CPHA were significantly higher towards TA98/1,8-DNP6 than towards TA98, suggesting that the esterification of their hydroxylamines produced inactivation rather than activation. From these results, it was concluded that S9 and NOH play a role in metabolic activation other than the reduction of the nitro group to hydroxylamine and subsequent esterification for the mutagenesis of NB and its derivatives.     

Review of the Salmonella typhimurium mutagenicity of benzidine, benzidine analogues, and benzidine-based dyes

We have reviewed the mutagenicity of benzidine analogues (including benzidine-based dyes), with a primary emphasis on evaluating results of the Salmonella/microsome mutagenicity assay. Many of these amines are mutagenic in tester strains TA98 and TA100 but require exogenous mammalian activation (S9) for activity. A few amines with halogen or nitro-groups in the structure are direct-acting mutagens. The addition of a sulfonic acid moiety to the molecule of benzidine reduced the mutagenicity of benzidine; whereas, methoxy, chloro, or methyl group additions did not. Complexation with a metal ion also decreased the mutagenicity. A substitution of an alkyl group on the ortho position next to an amine group also influenced the mutagenicity. Most carcinogenic benzidine analogues are mutagenic, and their metabolism to electrophiles that interact with DNA, leading to mutations, plays a central role in their carcinogenesis.     

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.