Some factors determining the concentration of liver proteins for optimal mutagenicity of chemicals in the Salmonella/microsome assay.

In plate assays in the presence of S. typhimurium TA100 and various amounts of liver 9000 X g supernatant (S9) from either untreated, phenobarbitone- (PB) or Aroclor-treated rats, the S9 concentration required for optimal mutagenicity of aflatoxin B1 (AFB) depended both on the source of S9 and on the concentration of the test compound. In these assays, the water-soluble procarcinogen, dimethylnitrosamine (DMN) was mutagenic in S. typhimurium TA1530 only in the presence of a 35-fold higher concentration of liver S9 from PB-treated rats than that required for AFB, a lipophilic compound. In liquid assays, a biphasic relationship was observed in the mutagenicities in S. typhimurium TA100 of benzo[a]pyrene (BP) and AFB and the concentration of liver S9. For optimal mutagenesis of BP, the concentration of liver S9 from rats treated with methylcholanthrene (MC) was 4.4% (v/v); for AFB it was 2.2% (v/v) liver S9 from either Aroclor-treated or untreated rats. At higher concentrations of S9 the mutagenicity of BP and of AFB was related inversely to the amount of S9 per assay. The effect of Aroclor treatment on the microsomemediated mutagenicity of AFB was assay-dependent: in the liquid assay, AFB mutagenicity was decreased, whereas in the plate assay it did not change or was increased. As virtually no bacteria-bound microsomes were detected by electron microscopy, after the bacteria had been incubated in a medium containing 1-34% (v/v) MC-treated rat-liver S9, it is concluded that, in mutagenicity assays, mutagenic metabolites generated by microsomal enzymes from certain pro-carcinogens have to diffuse through the assay medium before reaching the bacteria. Thus the mutagenicity of BP was dependent on both the concentration of rat-liver microsomes and that of total cytosolic proteins and other soluble nucleophiles such as glutathione. At a concentration of 4.4% (v/v) liver S9, the mutagenicity of BP was about 3.6 times higher than in assays containing a 4-fold higher concentration of cytosolic fraction. Studies on the glutathione-dependent reduction of BP mutagenicity in plate assays has shown that, in the presence of liver S9 concentrations greater than that required for optimal mutagenicity, the reduction in mutagenicity was related directly to the concentration of liver S9. Thus, in the Salmonella/microsome assay, when the concentration of rat-liver S9 was increased over and above the amount required for the optimal mutagenicity of BP, the mutagenic metabolites of BP were inactivated (by being trapped with cytosolic nucleophiles and/or by enzymic conjugation with glutathione); this effect increased more rapidly than their rate of formation. The concentration of liver S9 for optimal mutagenicity of test compounds requiring activation catalyzed by mono-oxygenases seems, therefore, to be related to the departure from linearity of the relationship between the rate of formation of mutagenic metabolites and the concentration of liver S9.     

Selenium inhibition of benzo[a]pyrene, 3-methylcholanthrene, and 3-methylcholanthrylene mutagenicity in Salmonella typhimurium strains TA98 and TA100

Selenium (Se) decreased the mutagenicity of benzo[a]pyrene (BP), 3-methylcholanthrene (3MC), and 3-methylcholanthrylene (3MCE) in Salmonella typhimurium strains TA98 and TA100. Metabolism of BP, 3MC and 3MCE to mutagens was accomplished with the liver S9 fraction from Aroclor 1254-treated male Sprague-Dawley rats. Exposure of the bacteria to 4 nmoles BP, 10 nmoles 3MC, or 10 nmoles 3MCE in the presence of S9, and up to 200 nmoles Se as Na2SeO3 resulted in decreased mutagenicities up to 39, 66 and 60% of their respective control activities without Se in TA98 and up to 46, 52 and 64% of their respective control activities without Se in TA100. Se (200 nmoles) alone was not mutagenic in strains TA98 or TA100 with or without S9. BP, 3MC and 3MCE were not mutagenic in either strain without S9. None of the tested concentrations of BP, 3MC, 3MCE and Se were cytotoxic. Assays of the aryl hydrocarbon hydroxylase (AHH) activity in the S9 preparation revealed decreased AHH activity with increase in Se concentration. The decreased mutagenicity and AHH activity were Se (as Na2SeO3) dependent and could not be duplicated by sulfur (S as Na2SO3). Inhibition of AHH activity by Se provides an explanation of the mechanism of Se inhibition of BP, 3MC and 3MCE mutagenicities in S. typhimurium TA98 and TA100.     

Effect of glutathione and uridine 5'-diphosphoglucuronic acid on mutagenesis by benzo[a]pyrene and aflatoxin B1 in the Salmonella/microsome assay

In the Salmonella/microsome plate or liquid assay, the addition of glutathione (GSH) and uridine 5'-diphosphoglucuronic acid (UDPGA), both cofactors for GSH-S-transferases or UDPGA-transferases, altered the rat-liver microsome-mediated mutagenesis of benzo[a]pyrene (BP) and aflatoxin B1 (AFB). With either BP or AFB, an increased, unchanged or decreased number of revertant colonies of S. typhimurium was observed, depending on the substrate concentration, the source of rat-liver 9000 X g supernatant (S9), the time of incubation and the type of mutagenicity test (liquid or plate assay). Several factors responsible for quantitative changes in the pattern of BP and AFB metabolites under various assay conditions in vitro, which alter the overall mutagenic activity of the parent compound, are discussed.     

Mutagenicity studies on coffee. The influence of different factors on the mutagenic activity in the Salmonella/mammalian microsome assay

Recently, mutagenic activity on several strains of Salmonella typhimurium has been found in many heat-processed foodstuffs. The previously reported direct-acting mutagenic activity of coffee in Salmonella typhimurium TA100 (Ames assay) was confirmed in our study. In addition to TA100, a mutagenic effect of coffee was also found by using the newly developed strain TA102. The mutagenic activity was abolished by the addition of rat-liver homogenate. 10% S9 mix completely eliminated the mutagenic activity of 30 mg of coffee per plate. The addition of reduced glutathione to active S9 further decreased the mutagenic activity and also reduced the mutagenicity together with inactivated S9. The compound or compounds responsible for this inactivation are heat-labile and seem to be located in the cytosol fraction of the S9. Part of the mutagenicity of coffee was also lost spontaneously upon incubation at temperatures between 0 degrees and 50 degrees C. The loss of activity was dependent on temperature, being more pronounced at 50 degrees C compared to 0 degrees C (at 50 degrees C approximately 50% of the mutagenic activity was lost after 6 h). As anaerobic conditions prevented this loss of mutagenicity almost totally, oxidative processes are probably responsible for the inactivation. The stability of the mutagen was not influenced by incubation at low pH values (pH 1-3), with or without the addition of pepsinogen. The mutagenic properties of methylglyoxal, which to some extent could be responsible for the mutagenic activity of coffee, were compared with those of coffee. Methylglyoxal was strongly mutagenic towards Salmonella typhimurium TA100 and TA102. Its mutagenic activity was partially inactivated by the addition of 10% S9. Glyoxalase I and II together with reduced glutathione abolished the mutagenic activity of methylglyoxal but reduced the mutagenicity of coffee by only 80%. Since these enzymes occur in mammalian cells, the mutagenic compound(s) of coffee could also be degraded in vivo. This conclusion is supported by the fact that a long-term carcinogenicity study with rats was negative. These results clearly demonstrate that the effects observed in vitro do not necessarily also occur in vivo, but that in vitro experiments may contribute to the understanding of fundamental mechanisms of chemical carcinogenesis.     

Antimutagenicity studies of chlorophyllin using the Salmonella arabinose-resistant assay system

Studies with the arabinose-resistant Salmonella forward mutation assay system were performed to determine the antimutagenic activity of chlorophyllin against the mutagenic activity of aflatoxin B1 (AFB1), 2-aminoanthracene (2AA), benzo[a]pyrene (BaP), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and solvent extracts of coal dust (CD), diesel emission particles (DE), airborne particles (AP), tobacco snuff (TS), black pepper (BP) and red wine (RW). Various concentrations of each chemical and complex mixture extract were assayed for mutagenic activity with and/or without S9 in a preincubation test. One concentration of each chemical and complex mixture extract was then tested with various concentrations of chlorophyllin. Results showed that chlorophyllin, at concentrations of 2.5 mg/plate or less, completely or almost completely inhibited the mutagenicity of 2AA, AFB1, BaP, MNNG and solvent extracts of CD, DE and RW. With concentrations from 1.25 to 5 mg/plate, chlorophyllin inhibited over 50% of the mutagenicity of AP, TS and BP extracts. These results further substantiate the antimutagenic efficacy of chlorophyllin against chemicals and complex mixtures.     

2-Hydroxyemodin, an active metabolite of emodin in the hepatic microsomes of rats

The hepatic microsomes derived from rats transformed emodin (1,3,8-trihydroxy-6-methyl-anthraquinone), an anthraquinone present in fungal metabolites and constituent of rhubarb, into at least 10 anthraquinoid metabolites. Metabolite d proved to be mutagenic to Salmonella typhimurium TA1537 in the absence of activation system. MS, NMR, UV and mutagenicity test analysis revealed that metabolite d was 2-hydroxyemodin (1,2,3,8-tetrahydroxy-6-methyl-anthraquinone) and exhibited mutagenicity in doses of 2-20 micrograms/plate. In addition to this active metabolite, TLC analysis revealed the formation of 4-hydroxyemodin (metabolite a), 5-hydroxyemodin (metabolite b), 7-hydroxyemodin (metabolite d') and others. No mutagenicity of these monohydroxyemodins was demonstrated in the absence of activation system.     

Nucleic acid binding and mutagenicity of active metabolites of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a potent mutagen and carcinogen present in heated foodstuffs. The covalent binding of MeIQx to calf thymus DNA and calf liver RNA with microsomal activation was demonstrated. A major metabolite which exerts a direct mutagenic effect on S. typhimurium TA98 was found by HPLC analysis after incubation of MeIQx with rat liver microsomal fraction. The metabolite was identified as 2-hydroxyamino-3,8-dimethylimidazo[4,5-f]quinoxaline (N-OH-MeIQx). Synthetic N-OH-MeIQx was found to bind non-enzymatically to DNA and RNA at neutral pH even at 0 degrees C. Addition of acetic anhydride increased the binding of N-OH-MeIQx to DNA 10 times. These results suggest that MeIQx is metabolized to N-OH-MeIQx by microsomal cytochrome P-450 and further activated to an acetylated form that binds efficiently to nucleic acids in rat liver. Preferential modification of polyguanylic acid suggests that guanine residues of DNA are mainly modified with MeIQx. Synthetic N-OH-MeIQx exerted direct mutagenic activity on S. typhimurium TA98 inducing 150,000 rev/micrograms. Pentachlorophenol (PCP) caused a dose-dependent inhibition of this mutagenic effect, but 2,6-dichloro-4-nitrophenol (DCNP) did not. Thus the acetyltransferase of S. typhimurium seems to be important for the high mutagenicity of MeIQx after its microsomal activation.     

Metabolic activation of emodin in the reconstituted cytochrome P-450 system of the hepatic microsomes of rats

Studies were undertaken to elucidate further the mechanism by which emodin, an anthraquinoid mycotoxin and constituent of rhubarb, was converted into a direct-acting mutagen to Salmonella typhimurium TA1537 by the hepatic microsomes and the reconstituted cytochrome P-450 system. Emodin was activated into a mutagenic principle(s) in the reconstituted cytochrome P-450 system, and its mutagenicity was significantly higher with the fraction II (P-448 type) than the fraction I (P-450 type) derived from the hepatic microsomes of PCB-induced rats. Thin-layer chromatographic analysis revealed that the purified cytochrome II-a (maximal CO-differential spectrum at 448.0 nm and high-spin form) activity converted emodin into 2-hydroxy-emodin, a direct-acting mutagen.     

ω-Hydroxymodin, a major hepatic metabolite of emodin in various animals and its mutagenic activity

The hepatic microsomes derived from various animal species transformed emodin (1,3,8-trihydroxy-6-methylanthraquinone), and anthraquinoid pigment present in fungal metabolites and a constituent of plant medicines, into an unidentified anthraquinone h, along with 2-hydroxy-, 4-hydroxy- and 7-hydroxyemodins. TLC, UV, MS and NMR clarified this unidentified major metabolite as ω-hydroxy-emodin (1,3,8-trihydroxy-6-hydroxymethylanthraquinone). Among 7 animal species, the highest activity to produce this ω-hydroxyemodin was observed in the hepatic microsomes of guinea pig and rat, followed by mouse and rabbit. The microsomal activity to convert emodin into ω-hydroxyemodin was accelerated by the pretreatment of animals with phenobarbital, and inhibited by SKF 525A. The microsomal hydroxylation reactions of the methyl residue and the anthraquinoid nucleus of emodin were presumed to be catalyzed regiospecifically by multiple forms of cytochrome P-450.

ω-Hydroxyemodin was not mutagenic to Salmonella typhimurium in the absence of S9, but exhibited mutagenicity in the presence of an activating system. This genotoxic potential was comparable to 2-hydroxyemodin, a direct-acting mutagen.     

The effects of pretreatment with cytochrome P-450 inducers and preincubation with a cytochrome P-450 effector on the mutagenicity of genotoxic carcinogens mediated by hepatic and renal S9 from two species of marine fish

A series of experiments was designed to characterize the cytochrome P-450-dependent activation of 7 genotoxic carcinogens in the Salmonella preincubation assay by hepatic postmitochondrial fractions (S9) from the oyster toadfish and the Americal eel and by renal S9 from the toadfish. Significant S9-dependent mutagenicity was observed for benzo[a]pyrene (BAP), 2-aminoanthracene (2AA), aflatoxin B1 (AFB1), 7,12-dimethylbenz[a]anthracene (DMBA) and cyclophosphamide (CP) with hepatic S9 from untreated fish (UI S9) of both species and with renal S9 from untreated toadfish, although renal UI S9 was only marginally effective for activating AFB1. Neither UI S9 from toadfish liver or kidney nor that from eel liver consistently affected the direct mutagenicity of ethylene dibromide (EDB) or substantially activated dimethylnitrosamine (DMN). Pretreatment of toadfish with 3-methylcholanthrene (MC) decreased the mutagenicity of 2AA and increased the mutagenicities of BAP, AFB1 and DMBA, whereas, pretreatment of eels with MC increased the mutagenicities of BAP, 2AA and AFB1. Pretreatment of toadfish with Aroclor 1254 (AC) decreased the mutagenicity of AFB1 and increased the mutagenicity of 2AA, whereas, pretreatment of eels with AC increased the mutagenicities of BAP and DMBA. Pretreatment of toadfish with beta-napthoflavone (BNF) effected changes similar to those by pretreatment with MC except that the mutagenicity of AFB1 was not increased. Coincubation with 10(-4) M alpha-napthoflavone (ANF) decreased the mutagenicity of BAP mediated by toadfish MC and BNF S9 and eel AC S9 and decreased the mutagenicity of AFB1 mediated by toadfish MC and BNF S9 and by eel MC S9. Coincubation with ANF increased the mutagenicity of AFB1 mediated by toadfish and eel AC S9 and increased the mutagenicity of 2AA mediated by eel AC S9. Pretreatment of toadfish with MC, BNF and AC decreased the mutagenicity of 2AA mediated by renal S9 and ANF decreased the mutagenicity of 2AA mediated by renal UI and BNF S9. MC pretreatment of toadfish and eels and BNF pretreatment of toadfish induced BAP monooxygenase activity in hepatic microsomes. ANF (10(-4) M) inhibited the BAP monooxygenase activity of MC microsomes from toadfish and eels and of BNF microsomes from toadfish. The conjugation effectors diethyl maleate and salicylamide alone or combined had little or no effect on the mutagenicities of BAP and 2AA mediated by toadfish and eel UI and MC S9.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evaluation of mutagenic and antimutagenic activities of alpha-bisabolol in the Salmonella/microsome assay

alpha-Bisabolol (BISA) is a sesquiterpene alcohol found in the oils of chamomile (Matricaria chamomilla) and other plants. BISA has been widely used in dermatological and cosmetic formulations. This study was undertaken to investigate the mutagenicity and antimutagenicity of BISA in the Salmonella/microsome assay. Mutagenicity of BISA was evaluated with TA100, TA98, TA97a and TA1535 Salmonella typhimurium strains, without and with addition of S9 mixture. No increase in the number of his+ revertant colonies over the negative (solvent) control values was observed with any of the four tester strains. In the antimutagenicity assays, BISA was tested up to the highest nontoxic dose (i.e. 50 and 150 microg/plate, with and without S9 mix, respectively) against direct-acting (sodium azide, SA; 4-nitroquinoline-N-oxide, 4-NQNO; 2-nitrofluorene, 2-NF; and nitro-o-phenylenediamine, NPD) as well as indirect-acting (cyclophosphamide, CP; benzo[a]pyrene, B[a]P; aflatoxin B1, AFB1; 2-aminoanthracene, 2-AA; and 2-aminofluorene, 2-AF) mutagens. BISA did not alter mutagenic activity of SA and of NPD, and showed only a weak inhibitory effect on the mutagenicity induced by 4-NQNO and 2-NF. The mutagenic effects of AFB1, CP, B[a]P, 2-AA and 2-AF, on the other hand, were all markedly and dose-dependently reduced by BISA. It was also found that BISA inhibited pentoxyresorufin-o-depentylase (PROD, IC50 2.76 microM) and ethoxyresorufin-o-deethylase (EROD, 33.67 microM), which are markers for cytochromes CYP2B1 and 1A1 in rat liver microsomes. Since CYP2B1 converts AFB1 and CP into mutagenic metabolites, and CYP1A1 activates B[a]P, 2-AA and 2-AF, results suggest that BISA-induced antimutagenicity could be mediated by an inhibitory effect on the metabolic activation of these promutagens.     

Mutagenic properties of 2-amino-N6-hydroxyadenine in Salmonella and in Chinese hamster lung cells in culture

The mutagenicity of the base analogue, 2-amino-N6-hydroxyadenine (AHA), was tested in Salmonella typhimurium TA100 and TA98 and in Chinese hamster lung (CHL) cells. AHA showed very potent mutagenicity in TA100 without S9 mix, inducing 25,000 revertants/micrograms. The mutagenicity increased about 2-fold upon addition of S9 mix containing 10 microliters S9. AHA was found to be one of the strongest mutagens for TA100. Addition of S9 mix containing 100 microliters S9 induced no significant increase of revertants with AHA at amounts up to 50 ng per plate. AHA was also mutagenic for the frameshift mutant, TA98, without S9 mix, the mutagenicity for TA98 being about 1/1000 of that for TA100. When the mutagenicity of AHA was tested in CHL cells, with diphtheria toxin resistance (DTr) as a selective marker in the absence of S9 mix with a 3-h treatment of cells, DTr mutants increased dose-dependently at concentrations of 2.5-15 micrograms/ml. When cells were incubated with AHA for 24 h, a 200-fold increase in the number of DTr mutants was observed; the mutagenicity was 500-fold higher than that of ethyl methanesulfonate. This marked increase of mutagenicity by prolonged incubation may indicate that AHA induces mutations mainly after incorporation into DNA. The addition of a small amount of S9 increased the mutagenicity obtained with a 3-h treatment 2-fold, but a larger amount of S9 decreased the mutagenicity as was found with S. typhimurium TA100.     

Effects of norharman on the mutagenicity of chlorophenylhydroxylamine and its metabolism with rat liver S9

o,p-Chlorophenylhydroxylamines (CPHAs) (10468-16-3, 823-86-9) only demonstrated mutagenicity in the presence of S9 mix and norharman (NOH) (244-63-3), as well as chloronitrobenzenes. The mutagenic activity of o-CPHA was 30 times higher than that of p-CPHA. When o-CPHA was preincubated with S9 mix without NOH, the mutagenic activity disappeared rapidly. The decrease in activity during the preincubation was suppressed by addition of NOH. HPLC analysis revealed that o-CPHA was metabolized to o-chloroaniline (o-CA) (95-51-2) and that the metabolic reduction was inhibited by NOH. When microsomes containing NADPH were used instead of S9 mix, o-CPHA exhibited only very weak mutagenicity. The activity in the microsome system, however, was greatly enhanced by adding cytosol or ascorbic acid (50-81-7). These phenomena were only observed in the conventional plate incorporation method. In the case of the liquid incubation assay, in which test compound metabolism and tester strain mutation only occur in the liquid incubation medium, the mutagenic activity of o-CPHA in the microsome system with NOH was comparable to that in the S9 system, indicating that o-CPHA was activated by an enzyme in microsomes in the presence of NOH. Consequently, it was concluded that NOH not only affects the metabolic inactivation of o-CPHA to o-CA by S9, but also the metabolic activation of o-CPHA by microsomes. No appreciable enhancing effects of cytosol and ascorbic acid were observed in the liquid incubation assay, suggesting that these factors affect the stability of CPHA or an active metabolite. The microsome activation of o-CPHA was dependent on NADPH and oxygen; SKF-525A (62-68-0), metyrapone (54-36-4) and alpha-naphthoflavone (604-59-1) inhibited the mutagenic activity by about 50%, suggesting that cytochrome P-450 was involved in the metabolic activation.     

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.     

Activation of mutagens by hepatocytes and liver 9000 X g supernatant from human origin in the Salmonella typhimurium mutagenicity assay. Comparison with rat liver preparations

The mutagenicity of 10 known genotoxic compounds, of several chemical classes, was measured in Salmonella typhimurium mutagenicity assays comprising isolated human hepatocytes or human liver 9000 X g supernatant (S9) from 4 different individuals, as activating system. The mutagenic activity of several compounds as determined with the Salmonella/hepatocyte suspension assay showed obvious differences when compared with the values obtained in the Salmonella/S9 plate assay. For instance, the mutagenic activity of BZ, DMN and DEN appeared to be much higher in the hepatocyte assay than in the S9 assay. However, 2-AF and 2-AAF were activated more effectively into mutagens in the S9 assay than in the hepatocyte assay. 2-AF was slightly more mutagenic than 2-AAF in the hepatocyte assay, whereas it was far more mutagenic than 2-AAF in the S9 assay. DMN was found more mutagenic than DEN in the hepatocyte assay, whereas in the S9 assay DEN appeared to be slightly more mutagenic. Furthermore, great interindividual differences in the metabolic activation of certain compounds, e.g. BZ and DMN, were observed in the hepatocyte suspension assay, whereas these variations were less evident in the S9 plate assay. Comparison of the mutagenicity data obtained with the human liver preparations, with those obtained with rat liver preparations, showed great interspecies differences in the capacity to activate certain chemicals into mutagens. The use of human liver preparations, in particular isolated human hepatocytes, may be of great value in studies on inter- and intraspecies variations in metabolic activation of genotoxic agents.     

Mutagenicity monitoring of airborne particulate matter (PM10) in the Czech Republic.

The mutagenic activities associated with inhalable airborne particulate matter (PM10) collected over a year in four towns (Czech Republic) have been determined. The dichloromethane extracts were tested for mutagenicity using the Ames plate incorporation test and the Kado microsuspension test both with Salmonella typhimurium TA98 and its derivative YG1041 tester strains in the presence and absence of S9 mixture. The aim of this study was to assess the suitability of both bacterial mutagenicity tests and to choose the appropriate indicator strain for monitoring purposes. To elucidate the correlation between mutagenicity and polycyclic aromatic hydrocarbons (PAHs), the concentration of PAHs in the air samples were determined by GC/MS. In general, the significant mutagenicity was obtained in organic extracts of all samples, but differences according to the method and tester strain used were observed. In both mutagenicity tests, the extractable organic mass (EOM) exhibited higher mutagenicity in the YG1041 strain (up to 97 rev/microg in the plate incorporation and 568 rev/microg in the microsuspension tests) than those in TA98 (up to 2.2 rev/microg in the plate incorporation and 14.5 rev/microg in the microsuspension tests). In the plate incorporation test, the direct mutagenic activity in YG1041 was on average 60-fold higher and in microsuspension assay 45-fold higher with respect to strain TA98. In the presence of S9 mix, the mutagenic potency in YG1041 declined (P<0.001) in summer, but increased in TA98 (P<0.05) in samples collected during the winter season. The microsuspension assay provided higher mutagenic responses in both tester strains, but in both strains a significant decrease of mutagenic potency was observed in the presence of S9 mix (P<0.001 for YG1041, P<0.05 for TA98 in winter). The mutagenic potencies detected with both indicator strains correlated well (r=0.54 to 0.87) within each mutagenicity test used but not (for TA98) or moderately (r=0.44 to 0. 66 for YG1041) between both of the tests. The mutagenic activity (in rev/m(3)) likewise the concentration of benzo[a]pyrene and sum of carcinogenic PAHs showed seasonal variation with distinctly higher values during winter season. A correlation between the PAH concentrations and the mutagenicity results for the plate incorporation, but not for the microsuspension tests was found. In samples from higher industrial areas, the higher mutagenicity values were obtained in plate incorporation test with TA98 and in both tests with YG1041 in summer season (P<0.05). According to our results, plate incorporation test seems to be more informative than microsuspension assay. For routine ambient air mutagenicity monitoring, the use of YG1041 tester strain without metabolic activation and the plate incorporation test are to be recommended.     

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.     

Evaluation of azo food dyes for mutagenicity and inhibition of mutagenicity by methods using Salmonella typhimurium

The mutagenicity of 4 azo dyes (FD&C Yellow No. 5, FD&C Yellow No. 6, FD&C Red No. 40 and amaranth) that are widely used to color food has been evaluated. 4 different methods were used: (1) the standard Ames plate-incorporation assay performed directly on the dyes in the absence of S9 and in the presence of rat- or hamster-liver S9; (2) application of the standard plate assay to ether extracts of aqueous solutions of the dyes; (3) a variant of the standard assay, using hamster liver S9, preincubation, flavin mononucleotide (FMN) and other modifications designed to facilitate azo reduction; and (4) reduction of the dyes with sodium dithionite, followed by ether extraction and the standard plate assay. Assays that include chemical reduction (methods 3 and 4) were included because azo compounds ingested orally are reduced in the intestine with the release of free aromatic amines. No mutagenic activity was seen for any of the azo dyes tested by using the standard Ames plate assay (method 1). Ether extracts of some samples of FD&C Yellow No. 6, FD&C Red No. 40 and amaranth were active (method 2), but only at high doses, generally 250 mg-equivalents or more per plate. These results indicate the presence of low levels of ether-extractable mutagenic impurities. The FMN preincubation assay (method 3) gave negative results for all dye samples tested. Most batches of FD&C Red No. 40 tested had mutagenic activity that was detectable when the ether extract of less than 1 mg of dithionite-reduced dye was plated in the presence of S9 (method 4). This finding implies that an impurity in these samples of FD&C Red No. 40 can be reduced to yield an ether-extractable mutagen. Dithionite-reduced samples of FD&C Yellow No. 6 and amaranth showed ether-extractable mutagenic activity only at much higher doses than those at which activity was seen with most dithionite-reduced samples of FD&C Red No. 40 (method 4). FD&C Yellow No. 5 showed no mutagenic activity with this method. Mutagenic activity was not detected when FD&C Red No. 40 was tested by using the azo reduction preincubation assay with FMN (method 3).(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Structures of mutagens produced by the co-mutagen norharman with o- and m-toluidine isomers

Norharman, abundantly present in cigarette smoke and cooked foods, is not mutagenic to Salmonella typhimurium strains. However, norharman shows mutagenicity to S. typhimurium TA98 and YG1024 in the presence of S9 mix when coexisting with aromatic amines, including aniline, o- and m-toluidines. We previously reported that the mutagenicity from norharman and aniline in the presence of S9 mix was due to the formation of a mutagenic compound, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman). In the present study, we analyzed the mutagens produced by norharman with o- or m-toluidine in the presence of S9 mix. When norharman and o-toluidine were reacted at 37 degrees C for 20 min, two mutagenic compounds, which were mutagenic with and without S9 mix, respectively, were produced, and these were isolated by HPLC. The former mutagen was deduced to be 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-3'-methylphenylnorharman) on the basis of various spectral data, and this new heterocyclic amine was confirmed by its chemical synthesis. The latter mutagen was identified to be the hydroxyamino derivative. Amino-3'-methylphenylnorharman induced 41,000 revertants of TA98, and 698,000 revertants of YG1024 per microg with S9 mix. Formation of the same DNA adducts was observed in YG1024 when amino-3'-methylphenylnorharman or a mixture of norharman plus o-toluidine was incubated with S9 mix. These observations suggest that norharman reacts with o-toluidine in the presence of S9 mix to produce amino-3'-methylphenylnorharman, and this compound is metabolically activated to yield its hydroxyamino derivative. After activation by O-acetyltransferase, it might bind to DNA and exert mutagenicity in S. typhimurium TA98 and YG1024. When norharman and m-toluidine were reacted in the presence of S9 mix, 9-(4'-amino-2'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-2'-methylphenylnorharman) was identified as a mutagen. Thus, the mutagenicity of norharman with m-toluidine may follow a mechanism similar to that with o-toluidine.