The effect of alkoxy substituents on the mutagenicity of some aminoazobenzene dyes and their reductive-cleavage products

15 aminoazobenzene dyes and 7 of their reductive-cleavage products were examined in the Salmonella/microsome assay with strains TA98, TA100, TA1535, TA1537 and TA1538. Dyes tested included 5 derivatives of 4-aminoazobenzene with different alkoxy substituents (-OCH3, -OCH2CH3, -OCH2CH2 CH3, -OCH2CH2CH2CH3 or -OCH2CH2OH) in the 8-position as well as the corresponding derivatives of 4-[(4-aminophenyl)azo]-N,N-diethylaniline and 4-[(4-aminophenyl)azo]-N,N-bis(2-hydroxyethyl)aniline. In general, as the size of the substituent ortho to the primary amino group of the dyes was increased, the mutagenicity decreased. A similar trend was observed for the reductive-cleavage products. The results from the latter aspect of this study suggest that the mutagenicity of aminoazobenzene dyes can not be accounted for solely from the properties of their reductive-cleavage products.     

Mutagenicity of azo dyes in the Salmonella/microsome assay using in vitro and in vivo activation

The mutagenicity of 6 azo dyes, including direct black 38 (DB38), direct black 19 (DB19), direct brown 95 (DB95), solvent yellow 3 (SY3), trypan blue (TPB), and food black 2 (FB2), was examined in the Salmonella/microsome assay. The effect of chemical azo reduction (dithionite) and in vivo metabolism on the mutagenicity of the dyes was also studied. In vivo azo-dye metabolites were isolated from the urine of rats intubated with dyes by XAD-2 column chromatography. Urinary metabolites from all the treated animals, except animals treated with FB2, induced frame-shift mutations in strains TA1538 and TA98 in the presence of liver S9 activation. The control urine did not increase the incidence of revertants in strains TA1538 and TA98. Thus, XAD-2 chromatography can be used to isolate genotoxic metabolites from the urine of animals intubated with azo dyes.     

Mutagenic activity of 27 dyes and related chemicals in the salmonella/microsome and mouse lymphoma TK+/- assays

A total of 27 dyes and related chemicals were tested for mutagenicity in both the Salmonella typhimurium plate-incorporation and FMN-modified assays as well as the mouse lymphoma TK+/- assay. Half of the compounds tested were monoazo dyes (14); the remainder consisted of disazo (3), aminotriphenylmethane derivatives (4), and other miscellaneous (6) color compounds. The results obtained in this study are compared with data from dyes of the same batch tested in other laboratories in the Salmonella plate-incorporation assay and in both in vitro and in vivo/in vitro UDS assays. Agreement of results from the various assays that could be compared (excluding results that were equivocal or indeterminate) ranged from 80 to 91%. Sufficient data were available to provide an overall index of in vitro activity for 15 chemicals; of these, 14 compounds could be compared to and agreed with reports of their carcinogenic potential in the literature.     

Evaluation of new 2,2'-dimethyl-5,5'-dipropoxybenzidine- and 3,3'-dipropoxybenzidine-based direct dye analogs for mutagenic activity by use of the Salmonella/mammalian mutagenicity assay

As part of a continuing study aimed at establishing structure-activity relationships and heuristic principles useful for the design of non-genotoxic azo dyes, a series of new direct dyes based on two non-mutagenic benzidine analogs, 2,2'-dimethyl-5,5'-dipropoxybenzidine and 3,3'-dipropoxybenzidine, were evaluated for mutagenic activity in Salmonella typhimurium strains TA98 and TA100. These strains are widely used for mutagenicity screening and have been shown to detect the mutagenic activity of benzidine analogs. While some toxicity was seen with some dyes at high doses, all of the dyes examined were judged non-mutagenic with and without metabolic activation in the standard Salmonella plate-incorporation assay. The results in the standard test are consistent with the properties of the diamines themselves. However, only one of the dyes was non-mutagenic when a reductive-metabolism pre-incubation assay was used. The results of this study suggest that although benzidine analogs are potential replacements for benzidine, there is a need to understand which mutagenic products are produced when reductive metabolism is present. There is also a need to know whether or not metal complexes of these dyes are mutagenic. Such information will allow the development of new non-mutagenic azo dyes.     

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.     

The Salmonella mutagenicity test: evaluation of 29 drug candidates

The Salmonella mutagenicity test (Ames assay) is part of the routine screening battery applied to all new drugs at The Upjohn Company. The purpose of this paper is to report results for 29 compounds. These compounds are very diverse in chemical structure and represent classes of compounds selected because of known biological activity and other reasons. None of the compounds reported here produced an increase in revertant colonies in the Salmonella strains employed (TA98, TA100, TA1535, TA1537 and TA1538) and therefore the Salmonella mutagenicity results with these materials do not suggest potential for mutagenesis or 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.     

Reduction in Ames Salmonella mutagenicity of mainstream cigarette smoke condensate by tobacco protein removal

The mutagenic activity of cigarette smoke condensates (CSC) made from tobacco before and after removal of protein was assessed by the Ames Salmonella assay in bacterial strains TA98 and TA100. Removal of protein and peptides from flue-cured tobacco via water extraction followed by protease digestion reduced the mutagenicity of the resultant CSC by 80% in the TA98 strain and 50% in the TA100 strain. Similarly, reductions of 81% in TA98 and 54% in TA100 were seen following water extraction and protease digestion of burley tobacco. The significant reductions in Ames mutagenicity following protein removal suggest that protein pyrolysis products are a principal contributor to the genotoxicity of CSC as measured in this assay.     

Mutagenicity of derivatives and metabolites of 1-nitropyrene: activation by rat liver S9 and bacterial enzymes

The mutagenicity and activation requirements of purified synthetic derivatives and potential metabolites of 1-nitropyrene have been characterized in the Ames plate incorporation assay with the Salmonella tester strains TA98, TA98NR and TA98/1,8-DNP6, in the presence or absence of exogenous metabolic activation provided by Aroclor-induced rat liver S9. All the compounds tested (1-aminopyrene, N-acetyl-1-aminopyrene, N-hydroxy-N-acetyl-1-aminopyrene, 3-hydroxy-1-nitropyrene, 6-hydroxy-1-nitropyrene, and 8-hydroxy-1-nitropyrene) exhibited mutagenic activity under one or more assay conditions. 1-Nitropyrene was metabolized to 3-hydroxy-1-nitropyrene, 6- or 8-hydroxy-1-nitropyrene, 1-aminopyrene, N-acetyl-1-aminopyrene and other unidentified products (including some bound to protein) by an S9 preparation analogous to that used for exogenous metabolic activation in the Ames assay. 1-Nitropyrene and 3-hydroxy-1-nitropyrene were activated primarily by the 'classical' nitroreductase, while the other compounds, particularly in the presence of S9 metabolic activation, were dependent on transesterification for expression of their mutagenicity.     

A comparative study of mutagenic and SOS-inducing activity of biphenyls, phenanthrenequinones and fluorenones

A total of 23 chemicals--biphenyls, phenanthrenequinones and fluorenones--were tested for mutagenicity towards Salmonella typhimurium strains TA1538, TA1535 and TA98. SOS-inducing activity of the same chemicals was studied in terms of the SOS-inducing potency in Escherichia coli PQ37, using an automated instrument controlled by a dedicated computer program for the SOS Chromotest. Of the 23 chemicals studied 14 induced His+ revertants in S. typhimurium TA1538 hisD305 (-1 frameshift); none induced His+ reversions in TA1535 (base-pair substitution). The mutagenicity of the chemicals in S. typhimurium TA98 (pKM 101) was lower than in TA1538. There was a close correlation between mutagenicity and SOS-inducing activity of fluorenones and phenanthrenequinones. None of the biphenyls tested induced SOS response and this property does not depend upon the mutagenic activity of the chemicals. SOS Chromotest is particularly valid in detecting chemicals which give rise to base-pair substitutions through SOS induction. If positive results are obtained, the Salmonella assay may be omitted. However, this test cannot replace the Ames test especially for the primary screening of mutagenicity of chemicals with unknown structure.     

Developing azo and formazan dyes based on environmental considerations: Salmonella mutagenicity

In previous papers, the synthesis and chemical properties of iron-complexed azo and formazan dyes were reported. It was shown that in certain cases iron could be substituted for the traditionally used metals such as chromium and cobalt, without having an adverse effect on dye stability. While these results suggested that the iron analogs were potential replacements for the commercially used chromium and cobalt prototypes, characterization of potentially adverse environmental effects of the new dyes was deemed an essential step in their further development. The present paper provides results from using the Salmonella/mammalian microsome assay to determine the mutagenicity of some important commercial metal complexed dyes, their unmetallized forms, and the corresponding iron-complexed analogs. The study compared the mutagenic properties of six unmetallized azo dyes, six commercial cobalt- or chromium-complexed azo dyes, six iron-complexed azo dyes, six unmetallized formazan dyes, and six iron-complexed formazan dyes. The results of this study suggest that the mutagenicity of the unmetallized dye precursors plays a role in determining the mutagenicity of the iron-complexes. For the monoazo dye containing a nitro group, metal complex formation using iron or chromium decreased or removed mutagenicity in TA100; however, little reduction in mutagenicity was noted in TA98. For the formazan dye containing a nitro group, metal-complex formation using iron increased mutagenicity. Results varied for metal-complexes of azo and formazan dyes without nitro groups, but in general, the metal-complexed dyes based on mutagenic ligands were also mutagenic, while those dyes based on nonmutagenic ligands were nonmutagenic.     

Lack of activity of the bacterial mutagen emodin in HGPRT and SCE assay with V79 Chinese hamster cells

Genotoxicity of emodin was studied in the Salmonella/microsome assay, the sister-chromatid exchange (SCE) assay and the hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) forward mutation assay with V79 Chinese hamster cells. In the Salmonella/microsome assay, emodin was found to be positive in TA97, TA100 and TA1537 in the presence of liver homogenate. In TA1537 a weak direct mutagenicity was also observed. In both mammalian test systems, no genotoxicity was found either with or without metabolic activation.     

Mutagenicity of Maillard browning reaction products from various nitrosated amino acid-glucose mixtures

Ten different amino acid-glucose Maillard browning products before and after reaction with nitrite were evaluated by the Ames mutagenicity assay. No mutagenic response was observed in the methylene chloride extracts of any browning products tested before nitrosation. However, mutagenicity was showed in most of the browning mixtures, e.g., glycine-glucose, lysine-glucose (I), arginine-glucose, phenylalanine-glucose (II), and methionine-glucose after nitrosation when examined by Salmonella typhimurium strains TA98 and TA100 either with or without S-9 metabolic activation. Among the browning mixtures, (I) and (II) showed the greatest mutagenic activity after reaction with nitrite. The mutagenicity of lysine-glucose with nitrite was dependent on browning intensity, nitrosation pH, nitrosation time, nitrite level and blocking agents.     

Mutagenicity of products formed by ozonation of naphthoresorcinol in aqueous solutions

The mutagenicity of products formed by ozonation of naphthoresorcinol in aqueous solution was assayed with Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA104 in the presence and absence of S9 mix from phenobarbital- and 5,6-benzoflavone-induced rat liver. Ozonated naphthoresorcinol was mutagenic in TA97, TA98, TA100 and TA104 without S9 mix. By the addition of S9 mix, the mutagenic activity of ozonated naphthoresorcinol was markedly suppressed in TA98 and TA100, but became positive in TA102. High-performance liquid chromatography (HPLC) after derivatization to 2,4-dinitrophenylhydrazones demonstrated the formation of glyoxal as an ozonation product of naphthoresorcinol. Ion chromatographic technique also demonstrated the formation of o-phthalic acid, muconic acid, maleic acid, mesoxalic acid, glyoxylic acid and oxalic acid as ozonation products. The mutagenicity assays of these identified products with five Salmonella showed that glyoxal and glyoxylic acid were directly mutagenic; the former in TA100, TA102 and TA104, the latter in TA97, TA100 and TA104. In the presence of S9 mix, glyoxylic acid gave a positive response of mutagenicity for TA102. The experimental evidence supported that glyoxal and glyoxylic acid may contribute to the mutagenicity of ozonated naphthoresorcinol.     

Mutagenicity of selected sulfonated azo dyes in the Salmonella/microsome assay: use of aerobic and anaerobic activation procedures

A selection of 16 sulfonated azo dyes of both the monoazo type and diazo dyes based on benzidine, o-tolidine and o-dianisidine were assayed for mutagenicity in Salmonella typhimurium strains TA98 and TA100 employing both aerobic and anaerobic preincubation procedures. 3 food dyes, FD & C Red No. 40 and Yellows No. 5 and No. 6 were non-mutagenic in all tests. 5 dyes were mutagenic with aerobic treatment (trypan blue, Pontacyl Sky Blue 4BX, Congo Red, Eriochrome Blue Black B, dimethylaminoazobenzene) and 6 were mutagenic aerobically with riboflavin and cofactors (Deltapurpurin, trypan blue, Pontacyl Sky Blue 4BX, Congo Red, methyl orange, Ponceau 3R). Anaerobic preincubation involving enzymatic reduction of the dyes led to a different pattern of mutagenicity, with trypan blue giving much enhanced mutagenicity; Eriochrome Blue Black B, Pontacyl Sky Blue 4BX, Deltapurpurin and Congo Red exhibiting similar activity to aerobic preincubation; and methyl orange and Ponceau 3R yielding no mutagenicity. The results are interpreted with respect to an hypothesis involving partial reduction of the azo bond under differing degrees of aerobiosis via azo-anion radicals and hydrazo intermediates.     

Mutagenic and genotoxic effects of theophylline and theobromine in Salmonella assay and in vivo sister chromatid exchanges in bone marrow cells of mice.

The mutagenic and genotoxic effects of two methylxanthines, theophylline (TH) and theobromine (TB), were assessed in the Ames mutagenicity assay (in strains TA97a, TA100, TA102 and TA104) and in vivo sister chromatid exchanges (SCEs) in bone marrow cells of mice. These are the two most commonly used nervous system stimulators throughout the world. TH is used in the long-term treatment of asthma. Bacterial mutagenicity assay showed very weak mutagenic effects of both drugs in Salmonella strains TA102 and TA104 only in certain concentrations when S9 was added to it. No mutagenic effects were observed in any other strains used in this assay either with or without metabolic activation. But results of in vivo SCE assay indicate that these two drugs can induce significant SCE in bone marrow cells of mice.     

Subsequent products after antioxidant actions of beta-carotene and alpha-tocopherol have no Salmonella mutagenicity

Beta-carotene and alpha-tocopherol are important antioxidants biologically, but whether their oxidized products are toxic or not remains to be discovered. Here, we chromatographically separated 5 pure products or isomeric mixtures from reaction mixtures of beta-carotene and reactive oxygens, and 17 lipid-radical scavenging products of alpha-tocopherol. The products were tested for mutagenicity using Salmonella typhimurium TA98, TA100, TA102, and TA104, in the presence and absence of S9. None showed mutagenicity against any of the four strains, or cytotoxicity that influenced the survival of the bacteria. Lipid-peroxides have been known to increase the formation of mutagens from dietary procarcinogens such as heterocyclic amines. So, we also measured the activity to increase 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) mutagenicity. The products from beta-carotene and alpha-tocopherol did not increase, but rather several of them suppressed, the mutagenicity of Trp-P-2. Thus, the products of beta-carotene and alpha-tocopherol formed after the antioxidant actions were not genotoxic.     

Host-mediated mutagenicity experiments with benzo[a]pyrene and two of its metabolites

Benzo[a]pyrene (BP) and two of its major metabolites, the ultimate mutagen BP-4,5-oxide and the proximate mutagen trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (BP-7,8-diol) were investigated for mutagenicity in Salmonella typhimurium TA1538, TA98 and TA100 using an intrasanguineous host-mediated assay. BP and BP-4,5-oxide were not mutagenic under any experimental conditions. BP-7,8-diol was inactive with the strain TA1538 but was mutagenic with the strains TA98 and TA100. The effect was potentiated by pretreatment of the host mice with the cytochrome P-450 inducer 5,6-benzoflavone. We conclude: (i) one of the reasons for the observed insensitivity of the intrasanguineous host-mediated assay towards BP is that BP-4,5-oxide, which contributes to the microsome-mediated mutagenicity of BP, is inactive in the host-mediated assay; (ii) the finding that BP-7,8-diol is mutagenic in the host-mediated assay demonstrates that the lack of mutagenicity of BP is not intrinsic; (iii) the potentiated mutagenicity after treatment of the hosts with 5,6-benzoflavone suggests that cytochrome P-450 is more important in the activation of BP-7,8-diol in this system than other enzymes (e.g. prostaglandin synthase) that can also activate this compound in vitro.     

Role of bacterial nitroreductase and O-acetyltransferase in urine mutagenicity assay of rats exposed to 2,4,6-trinitrotoluene (TNT)

The urine mutagenicity of rats exposed to 2,4,6-trinitrotoluene (TNT) by i.p. injection was studied in the Salmonella assay using indicator strains with various levels of 'classical' nitroreductase or acetyl-CoA:N-hydroxylarylamine O-acetyltransferase activity. The strains used were the conventional Salmonella typhimurium TA98, nitroreductase-deficient TA98NR and -overproducing YG1021, and O-acetyltransferase-deficient TA98/1,8-DNP6 and -overproducing YG1024. TA98, YG1021 and YG1024 clearly detected the increase of direct urine mutagenicity. A slight increase of mutagenicity was also detected with metabolic activation in YG1021 and YG1024. High levels of both nitroreductase and O-acetyltransferase significantly increased the sensitivity of the indicator strain to the mutagenicity of urine caused by TNT exposure, while the nitroreductase- or O-acetyltransferase-deficient strains gave negative responses.     

Testing of some azo dyes and their reduction products for mutagenicity using Salmonella typhimurium TA 1538

A series of ten azo dyes as well as various single ring aromatic amines substituted on the benzene ring were tested for bacterial mutagenicity with Salmonella typhimurium TA 1538 using a soft-agar overlay method. Two dyes, sudan 2 and chrysoidin induced mutation but only in the presence of a rat liver preparation. Chrysoidin was the more active. Testing of its reduction products, aniline and 1,2,4-triaminobenzene showed a liver metabolite of the latter compound could be responsible for the mutagenic effect, having a comparable mutagenicity with 1,2-diamino-4-nitro-benzene, one of the mutagenic constituents of hair dyes. Structure-activity studies on a series of ring-substituted anilines indicated that mutagenic activity required at least two positions to be substituted with either amino or nitro groups, or one of each. The bacteria as well as the liver enzyme preparation may partake in the activation of these chemicals. The correlation between mutagenicity and carcinogenicity for this group of compounds is discussed.