The effect of quercetin on the mutagenicity of 2-acetylaminofluorene and benzo[alpha]pyrene in Salmonella typhimurium strains

The comutagenic and desmutagenic effect of quercetin on the mutagenicity of typical mutagens e.g. 2-acetylaminofluorene (AAF), 4-nitroquinoline-1-oxide (4NQO) and benzo[alpha]pyrene (B[a]P), in Salmonella typhimurium TA98, TA100 and TA98/1,8 DNP6 were examined. In the mixed application of AAF with quercetin in the presence of mammalian metabolic activation system (S9 mix), the numbers of revertants in TA98 increased by as much 2.2-5.0-fold compared with the sum of those in the separate applications of AAF and quercetin. A 1.4-2.7-fold increase was observed in TA100. Quercetin did not affect the mutagenicity of 4NQO, and depressed that of B[a]P. Dose-response curves for mutagenicity of quercetin with or without AAF (5 micrograms/plate) were examined. The results suggest that quercetin, present in a molarity of up to 1.5 times that of AAF, is apparently effective in enhancing the mutagenicity of AAF, because a linear dose-response curve was observed in the range of 0-5 micrograms/plate quercetin with AAF although quercetin alone was not mutagenic in the same range. Dose-response curves for mutagenicity of quercetin with or without 5 micrograms/plate B[a]P did not increase compared with that for quercetin alone. The mutagenicity of the mixed application of B[a]P with quercetin was reduced to about 60% of the sum of separate application at doses ranging from 25 to 100 micrograms/plate of quercetin. Since enhancement and depression of mutagenicity by quercetin were observed for indirect mutagens, AAF and B[a]P, respectively, in the presence of S9 mix, quercetin may affect the metabolic pathway of these mutagens.     

Diesel emissions revisited: is the carcinogenicity due to a genotoxic mechanism?

The induction of recA, umuC and sfiA genes by quercetin was studied in the presence and in the absence of S9 mix. The inducing activity of quercetin is higher for sfiA than for recA and umuC genes in the absence of S9 mix. The putative genotoxic metabolites of quercetin produced by S9 mix display different inducing activities of the three SOS genes as compared to quercetin. The induction of sfiA gene is decreased by the presence of S9 mix, whereas an opposite effect was observed concerning umuC and recA. These data suggest that the error-prone repair pathway participates in mutagenesis by quercetin and its metabolites. Moreover, the type of DNA damage exerted by quercetin seems to be determined by its metabolic fate. The importance of testing for the induction of other SOS genes, together with sfiA, in the study of SOS functions as a genotoxic index is emphasized.     

Factors affecting the mutagenic activity of quercetin for Salmonella typhimurium TA98: metal ions, antioxidants and pH

The mutagenic activity of quercetin for Salmonella typhimurium TA98 was inhibited by addition of metal salts. MnCl2 was a potent inhibitor, followed by CuCl2, FeSO4, and FeCl3, the probable mechanism being facilitated catalytic oxidation of quercetin. With quercetin incorporated at a level of 100 nmoles/plate, approximate doses (nmoles/plate) to give 50% inhibition of mutagenic activity were: MnCl2 less than 10 (-S9), 18 (+S9); CuCl2 65 (-S9), greater than 100 (+S9); FeSO4 190 (-S9), greater than 300 (+S9); or FeCl3 275 (-S9), greater than 300 (+S9). Ascorbate, superoxide dismutase, and, to a lesser extent, NADH and NADPH, all enhanced the mutagenic activity of quercetin in the absence of the mammalian-microsome (S9) system, but had no significant effect in the presence of the S9 mix. The maximum enhancement of activity by ascorbate or superoxide dismutase was approximately 87% of the increase achieved by addition of the S9 mix. Tyrosinase (catechol oxidase) substantially reduced the mutagenic activity of quercetin in the absence of the S9 mix. At lower levels of tyrosinase, activity was restored by incorporation of the S9 mix. It is proposed that the S9 mix enhances the mutagenic activity of quercetin by scavenging superoxide radicals, thus inhibiting the autoxidation of quercetin, and possibly by reducing quinone oxidation products of quercetin. The mutagenic activity of quercetin increased substantially when the pH of the media was decreased. This may be due in part to a decrease in ionization of quercetin at lower pH, thereby increasing its absorption by the tester strain, to a decrease in the rate of autoxidation of quercetin at lower pH, or to a combination of these.     

Enhancement of the mutagenicity of 2-acetylaminofluorene by flavonoids and the structural requirements

The enhancing effects of 12 kinds of flavonoids on the mutagenicity of 2-acetylaminofluorene (AAF) in Salmonella typhimurium TA98 were investigated. In the mixed applications of AAF (22.4 nmoles/plate) with flavonoids (31.4-45.0 nmoles/plate) in the presence of a mammalian metabolic activation system (S9 mix), morin, galangin, flavonol, kaempferol, quercetin and myricetin enhanced the mutagenicity of AAF by 3.3-10.2-fold. The potency of the mutagenicity enhancing effects increased in the described order. For the mutagenicity-enhancing effects of the flavonoids on AAF, the flavonol structure, including the free 3-hydroxyl group and the 2,3-double bond, were essential. In the quercetin analogues, the 5-hydroxyl group was also essential. Further, the numbers of the hydroxyl groups substituted at the 3', 4' and 5'-positions in the B-ring contributed to an increase of the enhancing effect, whereas the substitution of a hydroxyl group at the 2'-position depressed the potency of the effect.     

Mutagenicity-enhancing effect of quercetin on the active metabolites of 2-acetylaminofluorene with mammalian metabolic activation systems

The effects of quercetin on the mutagenicity of 2-acetylaminofluorene (AAF) and its 3 active metabolites, N-hydroxy-AAF (N-OH-AAF), aminofluorene (AF) and N-acetoxy-AAF(N-OAc-AAF) were investigated. The mutagenicity assays were carried out with Salmonella typhimurium TA98, and S9, microsomes and cytosol were used as metabolic activation systems. In the presence of S9, quercetin enhanced the mutagenicity of AAF, N-OH-AAF, AF and N-OAc-AAF by 6.9-, 4.3-, 3.6- and 3.9-fold, respectively. Quercetin enhanced the mutagenicity of these substrates with microsomes, whereas it depressed the mutagenicity of these substrates with cytosol. From these results, it seemed probable that quercetin promotes the N-hydroxylation and deacetylation in the microsomes, whereas it inhibits the deacetylation in the cytosol. It was shown that in the metabolism of AAF and its metabolites, quercetin modulates the balance between the mutagenicity activation and inactivation processes, which is catalysed by the enzymes in the microsomes and cytosol, and causes enhancement of the mutagenicity of AAF.     

Mutagenicity modulating effect of quercetin on aromatic amines and acetamides

The effect of quercetin on the mutagenicity of 32 kinds of aromatic amines and their acetamides were investigated using Salmonella typhimurium TA98 with a mammalian metabolic activation system (S9 mix). Quercetin enhanced the mutagenicity of the tricyclic aromatic amines (aminofluorene, aminoanthracene and aminophenanthrene) and their acetamides by 1.2-5.9-fold. Whereas, quercetin depressed the mutagenicity of aniline derivatives, biphenyl derivatives, and bi- and tetra-cyclic amino derivatives. The modulation of mutagenicity of Trp-P-1, Trp-P-2, Glu-P-1 and Glu-P-2 (heterocyclic amines) by quercetin were liable to be affected by the content of S9 in the S9 mix. It seems that quercetin does not have the same effect as norharman, because quercetin did not enhance the mutagenicity of aniline. It is suggested that the modulation of the mutagenicity of aromatic amines and acetamides is caused by the modulation of the balance between the mutagenic activation and inactivation in the metabolism of these amines and acetamides in the presence of quercetin. In this modulation, quercetin may participate through its effects on the promotion of N-hydroxylation and the inhibition of arylhydroxylation and transacylation. The presence of tricyclic aromatic rings of amines and acetamides is a structural requirement for the mutagenicity enhancement by quercetin.     

Structure-mutagenicity relationship among aminoquinolines, aza-analogues of naphthylamine, and their N-acetyl derivatives

The mutagenicity of 7 positional isomers of aminoquinolines (AQ) and their N-acetyl derivatives (AcAQ) was tested in Salmonella typhimurium TA100 and TA98 in the presence and absence of S9 mix. In a series of aminoquinolines, the order of mutagenic potency in the presence of S9 mix is: 5-AQ greater than 8-AQ greater than 7-AQ greater than 3-AQ greater than 2-AQ much greater than 4-AQ, 6-AQ. The alpha-positional isomers, 5-AQ and 8-AQ, are more mutagenic than the beta-isomer, 2-, 3-, 6-, 7-AQ's. These results are in contrast to the finding that beta-naphthylamine is more mutagenic than alpha-naphthylamine. In a series of N-acetylaminoquinolines, the order of mutagenic potency in the presence of S9 mix is: 7-AcAQ greater than 6-AcAQ greater than 8-AcAQ much greater than all the others. It is suggested that the AQ and AcAQ series might exert their mutagenicity through different molecular mechanisms (i.e., metabolic activation) from each other. The rate of metabolic activation does not seem to be correlated with the mutagenic potency of the compounds. It is noteworthy that 7-AQ and 8-AQ are mutagenic in both the strains tested in the absence of S9 mix.     

Clastogenicity of 1-nitropyrene, dinitropyrenes, fluorene and mononitrofluorenes in cultured Chinese hamster cells

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on 1-nitropyrene (NP), 3 dinitropyrenes (DNPs), fluorene and 4 mononitrofluorenes with and without metabolic activation (rat S9 mix). The 3 DNPs (1,3-, 1,6- and 1,8-DNP) induced chromosomal aberrations in the absence of S9 mix. The frequencies of cells with aberrations after treatment for 48 h were 43% at 2 micrograms/ml of 1,3-DNP, 55% at 0.1 microgram/ml of 1,6-DNP and 45% at 0.025 microgram/ml of 1,8-DNP, indicating the order of clastogenic potency as 1,8- greater than 1,6- greater than 1,3-DNP. On the other hand, 1-NP, which is known to be a direct-acting mutagen in bacteria, was negative in the chromosomal aberration test without S9 mix, but clearly positive with S9 mix. This effect was dependent on the concentration of the S9 fraction in the reaction mixture. High-pressure liquid chromatography analysis showed that 1-NP was converted by S9 mix to several metabolites, including 1-aminopyrene (AP). The clastogenic activity of 1-AP, however, was equivocal without S9 mix, suggesting that active clastogens other than 1-AP exist. Fluorene induced chromosomal aberrations only in the presence of S9 mix (61.8% at 25 micrograms/ml). 1-, 2-, 3- and 4-nitrofluorene (NF) were more clastogenic in the presence of S9 mix than in the absence of S9 mix, suggesting that NFs were converted to more active clastogens by S9 mix.     

Peptization of the Gel of Konjac Mannan

The addition of 1,8-pyrenequinone into the assay system containing rat liver homogenates (S-9) caused an approximately 10-fold increase in the mutagenicity of 2-acetylaminofluorene (AAF) in the current Salmonella reversion assay system. Since no chemical reaction between 1,8-pyrenequinone and AAF was observed, the in vitro effects of 1,8-pyrenequinone on the metabolisms of AAF with S-9 mix were studied. The enhancement of mutagenicity by 1,8-pyrenequinone was not dependent on the dose of NADPH under the present assay condition. The mutagenicity of AAF was increased approximately 4-fold by the addition of 1,8-pyrenequinone into microsomes, whereas it remained at the spontaneous level in the presence of cytosol. However, by reconstituting microsomes with cytosol, the mutagenicity enhancing activity was recovered to the original level. Since 1,8-pyrenequinone inhibited the AAF hydroxylase activity, chemical analysis of the incubation mixture of AAF was tried. This indicated that a higher amount of unmetabolized AAF remained and higher amounts of 2-aminofluorene and N-hydroxy-2-acetylaminofluorene were accumulated in the presence of 1,8-pyrenequinone compared with those in the absence of 1,8-pyrenequinone. From these results, it seems probable that 1,8-pyrenequinone inhibits C-hydroxylation (the detoxifying pathway) and promotes N-hydroxylation (the activating pathway) as well as deacetylation in the AAF metabolism.     

The mutagenic potencies of plant extracts containing quercetin in Salmonella typhimurium TA98 and TA100

Four commercial ethanolic plant extracts, Tinctura Alchemillae, Extractum Crataegi, Extractum Myrtilli and Tinctura Hyperici, were tested for their mutagenicity in Salmonella typhimurium TA98 and TA100 with and without S9 mix obtained from rats pretreated with phenobarbital. The extracts studied differed greatly in their mutagenic potencies but exhibited a very similar mutation pattern in which the strongest effect was always seen in tester strain TA98 with S9 mix. Simultaneously we investigated the extracts for the presence of quercetin and kaempferol. Only quercetin was detected in small amounts by thin-layer chromatography (TLC). The fractions containing quercetin were separated and collected using a Sephadex LH-20 column. Two different methods were employed to estimate the amount of quercetin in the extracts: a colorimetric assay developed by Christ and Müller, and a complexometric method by Belikov. The quercetin concentrations ranged between 2 mg (Tinctura Alchemilla) and 89 mg (Tinctura Hyperici) per 100 g of extract. We suggest that the mutagenicity of the 4 plant extracts is mainly due to the presence of quercetin for the following reasons: (1) all the plant extracts exhibit a mutation pattern which is very similar to that of quercetin, (2) the mutagenic potential of the extracts correlates well with their quercetin content, considering the fact that plant extracts are very complex mixtures often containing toxic or antimutagenic compounds.     

The genotoxic and anti-genotoxic effects of <Emphasis Type="Italic">Stachys petrokosmos</Emphasis> leaf extract in human lymphocytes using microsomal fractions

The genotoxic and anti-genotoxic effects of Stachys petrokosmos leaf extracts (Sp) were investigated in human lymphocytes. The cells were treated with 1.5, 3.0 and 6.0 μL/mL concentrations of Sp leaf extracts for 24 and 48 h treatment periods in the absence and presence of metabolic activator (S9mix). In the absence of S9mix, Sp alone did not induce chromosome aberrations and formation of micronucleus while inducing the mean sister chromatid exchange at the highest concentration. In addition, Sp decreased the mutagenic effect of mitomycin-c. Sp alone showed a cytotoxic effect determined by a decrease in the proliferation index, mitotic index and nuclear division index. On the other hand a mixture of Sp and mitomycin-c resulted in a higher cytotoxic effect especially for 48 h treatment period. In the presence of S9mix, Sp was not genotoxic and cytotoxic however, it showed an anti-genotoxic effect by decreasing the effects of cyclophosphamide.     

Analysis of the cytogenetic effect in human lymphocytes induced by metabolically activated 1- and 2-methylnaphthalene

Chromosome analyses were carried out in human lymphocytes treated in vitro with 1- and 2-methylnaphthalene (1-MN, 2-MN) in the presence and absence of the mammalian metabolic activation system, S9 mix. Without S9 mix there was no indication of induction of any significant cytogenetic effect by either compound. With S9 mix a weak clastogenic effect was apparent at 4 mM 2-MN only and sister-chromatid exchange frequencies were significantly increased at each dose of 1- and 2-MN, yet always less than twice the control level. The present observations do not indicate that 1- and 2-MN must be classified as potential genotoxic substances.     

Analysis of cytogenetic effect in human lymphocytes induced by metabolically activated 2-nitropropane

Chromosome analyses were carried out in human lymphocytes treated in vitro with 2-nitropropane (2-NP) in the presence and absence of the mammalian metabolic activation system, S9 mix. Without S9 mix, only the frequency of gaps was significantly increased at 80 mM 2-NP as compared to controls. With S9 mix, the incidences of gaps and chromatid-type aberrations were significantly increased at 60 mM and 80 mM. Sister-chromatid exchanges (SCE) have been induced at concentrations as low as 7.5 mM. The present findings demonstrate that in human lymphocytes, 2-NP requires metabolic activation to express clastogenicity and SCEs.     

Diethylstilbestrol and 11 derivatives: a mutagenicity study with Salmonella typhimurium.

Diethylstilbestrol was tested for mutagenicity with his- S. typhimurium strains under 10 different matabolic situations (no exogenous metabolizing system; S9 mix from liver homogenate of rats induced with Aroclor 1254, with or without inhibition of epoxide hydratase; liver and/or kidney S9 mix from control or hamsters treated with Aroclor 1254; horse-radish peroxidase + H2O2). Under none of these conditions did diethylstilbestrol give any indication of a mutagenic effect. Furthermore, 11 metabolites and other derivatives of diethylstilbestrol, 2 of them potent inducers of sister-chromatid exchange in cultured fibroblasts, were not mutagenic with any of the 4 tester strains (S. typhimurium TA100, TA98, TA1537, TA1535) in the presence or absence of S9 mix from liver homogenate of rats induced with Aroclor 1254. Thus, one of the few known human carcinogens is very resistant to detection by the mammalian enzyme-mediated Salmonella typhimurium mutagenicity test (Ames test). This is especially remarkable since the metabolizing systems used included: (1) some of very high metabolic activity (S9 mix from liver homogenate of rats and hamsters induced with Aroclor 1254); (2) metabolizing systems from organs susceptible to the carcinogenic activity of diethylstilbestrol (hamster kidney); as well as (3) a mixture of (1) and (2) in case both activities are required for the carcinogenic effect in the whole animal.     

Influence of S9 mix on the expression of mutants in the L-arabinose resistance test of Salmonella typhimurium

S9 mix produces an effect similar to that of D-glucose in the L-arabinose resistance test of Salmonella typhimurium, releasing the growth inhibition exerted by L-arabinose. Two elements are responsible for this effect: the glucose-6-phosphate present in the cofactors of the S9 mix and the S9 fraction itself. UV light was used as a mutagen to compare the efficiency of S9 mix and D-glucose in allowing phenotypic expression of mutants in selective plates with L-arabinose; 0.5 ml of S9 mix per plate showed and efficacy similar to that of 0.5 mg of D-glucose per plate. To verify that the S9 mix is equivalent to D-glucose traces in selective plates with respect to the number of induced mutants in compounds requiring metabolic activation, we utilized 2 direct-acting nitrofurans. Our conclusion is that activation of agents could be erroneously attributed to the S9 mix, when plates with 0.5 mg of D-glucose are compared to plates with 0.5 ml of S9 mix plus 0.5 mg of D-glucose. Our results suggest that D-glucose traces be omitted in experiments requiring the presence of the S9 mixture.     

Hypersensitive character of Bloom syndrome B-lymphoblastoid cell lines usable for sensitive carcinogen detection

Various carcinogens were tested with regard to the induction of sister-chromatid exchanges (SCEs) and chromosome aberrations using 3 types of Bloom syndrome (BS) B-lymphoblastoid cell lines (LCLs) (type I with normal frequency of SCEs and normal karyotype; type II with high frequency of SCEs and normal karyotype; type III with high frequency of SCEs and abnormal karyotypes) in the presence and absence of S9 mix. Three types of BS B-LCLs and normal cells showed different responses to the various carcinogens in the level of SCE induction. BS type I cells had the same SCE response as normal cells to carcinogens. Some carcinogens that require metabolic activation (S9 mix) had little effect on type II cells without S9 mix but had high SCE levels with S9 mix. BS type III cells were highly susceptible to both direct and indirect carcinogens with respect to high SCE increase without S9 mix (ca. 140 SCEs/cell), though some carcinogens produced SCEs rated in the medium (ca. 120 SCEs/cell) range, and had a high rate (more than 10%) of centromere spreading (CS), in addition to quadriradials. Therefore BS type III is a unique cell line which can be used to detect carcinogens.     

Mutagenicity of N-acetyl and N,N'-diacetyl derivatives of 3 aromatic amines used as epoxy-resin hardeners

3 epoxy-resin hardeners, 4,4'-diaminodiphenyl ether (DDE), 4,4'-diaminodiphenylmethane (DDM), and 4,4'-diaminodiphenylsulfone (DDS), and their N-acetyl and N,N'-diacetyl derivatives were examined for their mutagenicity using Salmonella typhimurium TA98 and TA100 as the tester stains and an S9 mix containing a rat-liver 9000 X g supernatant fraction as the metabolic activation system. DDE and DDM were mutagenic towards TA98 and TA100 in the presence of S9 mix while DDS exhibited no significant mutagenic activity towards these tester strains. These epoxy-resin hardeners were metabolized in vivo and their N-acetyl and N,N'-diacetyl metabolites were found in the urine. Among these acetyl metabolites, only N-acetyl-DDE was found to be mutagenic towards TA98 and TA100 in the presence of S9 mix. None of these acetyl metabolites exhibited significant mutagenic activity towards these tester strains in the absence of S9 mix.     

Induction of chromosome aberrations and sister-chromatid exchanges by caprolactam in vitro

Caprolactam (CAP) induced chromosome aberrations in whole-blood cultures of human lymphocytes at 50 mM without metabolic activation (24-h treatment) and at 200 mM in the presence of rat liver S9 mix (1-h treatment). CAP also produced a dose-dependent increase in polyploid cells, the effect being statistically significant at 25 and 50 mM without S9 mix and at 100 and 200 mM with S9 mix. Without metabolic activation, there was an increase in hypodiploid cells at 50 mM and hyperdiploid cells at 12.5 mM. In Chinese hamster ovary cells, CAP produced a marginal elevation of sister-chromatid exchanges at 125 mM in the presence of S9 mix (4-h treatment). The results show that CAP is able to induce cytogenetic changes in vitro at very high toxic concentrations.     

Rec effect of certain textile dyes in Bacillus subtilis

A large number of compounds are toxic, genotoxic, mutagenic, teratogenic and/or carcinogenic. The genotoxicity of four textile dyes commonly used in India namely Sulphur Red Brown 360 (SRB), Jade Green 2G (JG), Reactofix Turquoise Blue 5GFL (RTB) and Direct Scarlet 4BS (DS) was determined by Bacillus subtilis spore Rec assay, both in the presence and absence of metabolizing activation mixture (S9 mix). Each dye was toxic at higher dose levels. A dose-dependent increase in the depth of growth inhibition zones was observed for all dyes. Zones of inhibition were usually clearer at higher doses of the dyes and with Rec- bacteria, but were translucent with Rec+ bacteria. SRB and DS were toxic to Rec+ and Rec- bacteria. JG was less genotoxic in the absence of S9 mix, however, its genotoxic potential increased in the presence of S9 mix. Reactofix T blue was more genotoxic in the absence of S9 mixture.     

Mutagenicity testing of seminal fluid: seminal fluid increases the mutagenicity of the precursor mutagen benzo[a]pyrene in the presence of S9 mix

Small amounts of seminal fluid strongly enhanced the mutagenicity of the precursor mutagen benzo[a]pyrene (BP) in the Salmonella/microsome test. This previously unreported effect was found only in the presence of S9 mix for metabolic activation. The increase far exceeded the additive effect expected from experiments where seminal fluid and BP were tested separately with S9 mix. Testing of the direct-acting mutagen 4-nitro-o-phenylene-diamine (NPD) together with seminal fluid resulted in a lower mutagenic activity than that of NPD alone. Seminal fluid had a bactericidal effect on the Salmonella bacteria, thus only volumes up to 40 microliter could be used per plate. The mutagenic effect of only seminal fluid and S9 mix was slightly increased over controls in a standard Ames test, but was equal to the spontaneous mutation rate with a preincubation test modified according to Kado and coworkers. There were no significant differences between seminal plasma from smokers and non-smokers in any experimental series. Seminal fluid concentrated 20-fold by extraction with the mutagen-removing adsorbant Mutasorb did not have any enhancing effect on the mutagenicity of BP, nor did it exhibit any mutagenic activity in itself with or without S9 mix.