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.     

Aspects of chloroquine mutagenicity

Using the Ames plate reversion and fluctuation tests, the mutagenic activity of chloroquine was tested in the new tester strains of Salmonella typhimurium, TA97, TA102, and Escherichia coli strains WP2, WP2hcr, WP6 and WP67. The E. coli transconjugants obtained from the mating transfer of R-plasmid(s) in strains TA97 and TA102 respectively to E. coli WP2, i.e. EE97 and EE102, were also tested. Chloroquine reverted strain TA97 from histidine dependence to independence and also reverted E. coli strains EE97 and EE102 from tryptophan dependence to independence. The E. coli strains WP2, WP2hcr; WP6 and WP67 and S. typhimurium TA102 were not affected. S. typhimurium TA97 could be reverted with 250 ng/ml of chloroquine (therapeutic blood level of chloroquine is 300 ng/ml). Reversion generally occurred optimally at the relatively lower concentrations of chloroquine i.e. 25, 50 micrograms/ml than at higher concentrations. From the properties of the reverted tester strains, the results indicated that chloroquine per se mediated frameshift reversion.     

Mutagenic activity of structurally related oxiranes and siloranes in Salmonella typhimurium

The in vitro and in vivo genotoxicity of parthenin, a sesquiterpene lactone from Parthenium hysterophorus L. with allergenic and irritant action, was assessed in three short-term tests: bacterial reversion in Salmonella typhimurium and Escherichia coli, in vitro chromosomal aberrations in peripheral blood lymphocytes and micronuclei in mouse peripheral blood. Parthenin was not mutagenic in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 but a weak response was observed in TA 102 (+S9) from 0.19 to 1.22 micromole per plate. Concentrations of 7.62 micromole per plate or higher were toxic, but the effect was reduced when S9 was present. Screening of oxidative mutagenesis with E. coli strains IC 188 and IC 203 gave negative results. Parthenin induced chromosomal aberrations, mainly chromatid breaks, in blood lymphocytes exposed to 10-60 microM during 20 h. An association was found with cytotoxicity, since concomitant nuclear alterations such as pycnosis, micronuclei and karyorrhexis were observed. Sister chromatid exchanges (SCEs) in lymphocytes were not influenced by exposure to parthenin; rather a decrease was observed at 60 microM. On the other hand, a minor increment in polyploid metaphases was found at 40 microM. When a single intraperitoneal (i.p.) dose of 4-31 mg/kg of parthenin was administered to mice, a positive increase in the micronucleated reticulocyte (RET) frequency was observed at 48 h for both sexes at the highest dose.     

Standardization of conditions for the metabolic activation of N-nitrosodiethylamine in mutagenicity tests

Like all nitrosamines, N-nitrosodiethylamine (NDEA) requires metabolic activation in order to exert its carcinogenic effects. This activation involves cytochrome P450s (CYP), which generates unstable metabolites that react with the DNA of cells in the immediate vicinity of metabolite formation. Although NDEA is carcinogenic, it has been considered a weak mutagen in classic genotoxicity assays. We used optimized Salmonella/mammalian microsome genotoxicity assays to assess the mutagenicity and toxicity of low concentrations of NDEA. Using a fixed concentration of NDEA (36.5 mg/ml), we varied the length of preincubation in the presence of different concentrations of an S9 metabolic activation mixture. Salmonella typhimurium strains TA97 and TA102 were resistant to NDEA-induced mutagenesis, even after a preincubation of up to 120 min and the use of different concentrations of the S9 mix. Strain TA98 was susceptible to mutagenesis by NDEA in the absence of the S9 mix and after preincubation with NDEA for 90 min. When bacteria of this strain were preincubated with NDEA for 60 min, mutagenesis was detected at an S9 mix concentration >9.55 mg/ml. NDEA also induced mutagenesis in strain TA100 after preincubation for 90 or 120 min, and this effect was dependent on the S9 concentration. E. coli strain BH990 also showed a concentration-dependent response, with only 60% of the cells surviving after a 120-min preincubation with NDEA in the presence of 19.1 mg S9 mix/ml.     

Evaluation of the mutagenicity of sesquiterpenic compounds and their influence on the susceptibility towards antibiotics of two clinically relevant bacterial strains

Sesquiterpenic compounds are natural chemicals present in organisms from different Phylae or Divisions, which have proved to be important bioactive products, namely in potentiating the action of antibiotics. In the first step, the mutagenicity of nine sesquiterpenic compounds (hydrocarbons and alcohols) was screened in a Salmonella typhimurium his(-)-reversion test with strains TA98 and TA100, in the presence or absence of in vitro metabolic activation. Under the test conditions, none of the compounds showed mutagenicity up to a concentration of 222μg/plate. trans-Farnesol, nerolidol, and α-bisabolol displayed cytotoxicity when tested at concentrations ranging from 14 to 222μg/plate. Then, the combined effect of antibiotic-sesquiterpenic compounds was evaluated on two clinically relevant pathogens, Escherichia coli and Staphylococcus aureus, with well-defined resistance-sensitive profiles. The agar-disc diffusion assay revealed that all the combinations of antibiotic-sesquiterpenic compounds increased the antibacterial activity of the antibiotics tested against S. aureus. For E. coli, an antagonistic effect was observed for various combinations on the growth of this bacterium.     

Urinary mutagenicity tests in lead-exposed workers

Urinary mutagenic activity detected by the bacterial fluctuation assay, using Salmonella typhimurium TA98 and Escherichia coli WP2 uvrA with and without metabolic activation (S9 mix), was studied in a group of 21 workers exposed to inorganic lead and a control group of 22 non-occupationally exposed subjects. Occupational exposure to inorganic lead had no effect on urinary mutagenicity in the strains considered, with or without metabolic activation. In smokers (exposed and non-exposed), urinary mutagenic activity appeared to increase compared to non-smokers (exposed and non-exposed), only with Salmonella typhimurium TA98 in the presence of S9 mix.     

Mutagenicity of benzo[b]phenanthro[2,3-d]thiophene (BPT) and its metabolites in TA100 and base-specific tester strains (TA7001-TA7006) of Salmonella typhimurium: evidence of multiple pathways for the bioactivation of BPT

Benzo[b]phenanthro[2,3-d]thiophene (BPT), and a number of its metabolites, including BPT-3,4-diol, BPT sulfoxide, BPT sulfone, and 3-hydroxyBPT were assessed for their mutagenic activity in Salmonella typhimurium strain TA100, and S. typhimurium base-specific strains TA7001, TA7002, TA7003, TA7004, TA7005, and TA7006. Among the compounds tested in strain TA100, BPT, BPT sulfone, and 3-hydroxyBPT did not show any significant mutagenic response in the presence of S9. In contrast BPT sulfoxide and BPT-3,4-diol (a precursor to the bay-region diol epoxide of BPT) showed significant mutagenic activity in the presence of S9. Surprisingly, BPT sulfoxide was nearly 3.3-fold more mutagenic than BPT-3,4-diol in the presence of S9. BPT sulfoxide also displayed intrinsic mutagenic activity, which was nearly 1.5-fold less than that displayed by BPT-3,4-diol in the presence of S9. In base specific tester strains, BPT sulfoxide was the most active metabolite in strains TA7002, TA7004, and TA7005 with S9 activation. In these strains, BPT-3,4-diol was 2- to 7-fold less mutagenic than BPT sulfoxide in the presence of S9. Only in strain TA7006, BPT-3,4-diol was four-fold more mutagenic than BPT sulfoxide. The fact that BPT sulfoxide is significantly more mutagenic than BPT-3,4-diol in S. typhimurium strain TA100 suggests that the formation of sulfoxide may be the principal pathway for the metabolic activation of BPT to mutagenic products. Based on the results from Tester Strain TA7005, it indicate that BPT and its most mutagenic metabolite BPT sulfoxide induce predominantly CG --> AT transversion, which is observed as the most frequent base substitution mutation of p53 tumor-suppressor gene in human lung cancer.     

Mutagenicity of beta-glucuronidase in the Ames test

The enzyme preparation beta-glucuronidase/arylsulphatase from Helix pomatia (Boehringer) caused base-pair substitutions in Salmonella typhimurium TA100 and TA1535 strains within the dose range of 0.50-50 microliter per plate. No effect was observed in the TA98 strain. The presence of S9 mix did not substantially affect the mutagenic potential of beta-G. The number of induced revertants decreased continually from experiment to experiment carried out in the course of 12 weeks.     

Mutagenicity studies of human fibroblast interferon (HuIFN-beta)

Human fibroblast interferon (HuIFN-beta) was studied for mutagenicity using the Ames method and in vitro cytogenetics. HuIFN-beta had no mutagenic effect on S. typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) and E. coli (WP2 uvrA) at concentrations of 3, 30, 300, 3000, 30 000 or 300 000 IU/plate. In the cytogenetic study, HuIFN-beta had no clastogenic effect on human peripheral blood lymphocytes at concentrations of 3, 30, 300, 3000, or 30 000 IU/ml. These results suggest that HuIFN-beta has no mutagenic potential.     

Genotoxicity assay of oil dispersants in bacteria (mutation, differential lethality, SOS DNA-repair) and yeast (mitotic crossing-over)

5 oil dispersants and a sample of paraffin were devoid of mutagenic activity in the Ames reversion test, with and without S9 mix, using 7 his- S. typhimurium strains (TA1535, TA1537, TA1538, TA97, TA98, TA100, TA102). However, 3 dispersants produced direct DNA damage in E. coli WP2, which was not repairable in repair-deficient strains (WP2uvrA, CM871, TM1080), as shown by two different DNA-repair test procedures. The uvrA excision-repair system was in all cases the most important mechanism involved in repairing the DNA damage produced by oil dispersants, while the combination of uvrA with other genetic defects (polA, recA, lexA) decreased the efficiency of the system. The observed genotoxic effects were considerably lowered in the presence of S9 mix containing liver S9 fractions from Aroclor-treated rats. The sample of oil dispersant yielding the most pronounced DNA damage in repair-deficient E. coli failed to induce gene sfiA in E. coli (strain PQ37), using the SOS chromotest, or mitotic crossing-over in Saccharomyces cerevisiae (strain D5). The direct toxicity of the oil dispersant to both bacterial and yeast cells was markedly decreased in the presence of rat-liver preparations. These two short-term tests were effective in detecting the genotoxicity of both direct-acting compounds (such as 4-nitroquinoline N-oxide and methyl methanesulfonate) and procarcinogens (such as cyclophosphamide, 2-aminoanthracene and 2-aminofluorene). Moreover, the SOS chromotest was successfully applied to discriminate the activity of chromium compounds as related to their valence (i.e. Cr(VI) genotoxic and Cr(III) inactive). Combination of oil dispersants with Cr(VI) compounds did not affect the direct mutagenicity to S. typhimurium (TA102) of a soluble salt (sodium dichromate) nor did it result in any release of a water-soluble salt (lead chromate), as also confirmed by analytical methods. On the other hand, exposure to sunlight tended to decrease, to a slow rate, the direct genotoxicity of an oil dispersant in the bacterial DNA-repair test.     

Mutagenic and genotoxic evaluation of bisphenol F diglycidyl ether (BFDGE) in prokaryotic and eukaryotic systems

The epoxy resin bisphenol F diglycidyl ether (BFDGE), was examined for its mutagenicity in prokaryotic assays (Salmonella typhimurium His(-) and Escherichia coli Trp(-) tests) and its genotoxicity in eukaryotic systems (sister chromatid exchange (SCE) and micronucleus tests in human lymphocytes), in the presence or absence of an exogenous metabolizing system (S9 from rat liver). In the prokaryotic tests, the concentrations of BFDGE ranged between 100 and 5000 micro g per plate, and in the eukaryotic assays from 12.5 to 62.5 micro g/ml. The compound is able to induce mutagenic effects in bacterial strains TA100, TA1535, WP2uvrA and IC3327, as revealed by the increase observed in the number of induced revertants. With respect to the genotoxicity assays, BFDGE induces an increase in the frequency of sister chromatid exchanges and micronuclei in human peripheral blood lymphocytes.     

Mutagenicity of methyl isocyanate in the modified test conditions of Ames Salmonella/microsome liquid-preincubation procedure

Methyl isocyanate (MIC) was tested for mutagenicity using the Ames Salmonella/microsome liquid-preincubation procedure with slight modification of test conditions. In the modification the preincubation mixture was incubated at 10 degrees C for 60 min. MIC was assayed both in the presence and absence of Aroclor-1254-induced S9, using 5 tester strains of Salmonella typhimurium, TA97a, TA98, TA100, TA102 and TA104. MIC induced mutagenic response in two base-pair substitution strains, TA100 and TA104, in the presence and absence of S9. However, mutagenic response in the presence of S9 was low as compared to that in the absence of S9. In the comparative mutagenic activity at 3 different preincubation test conditions (37 degrees C for 20 min, 20 degrees C for 40 min and 10 degrees C for 60 min), optimum mutagenic response was observed at 10 degrees C for the 60-min test condition. However, no mutagenic response was observed at 37 degrees C for the 20-min test condition.     

Mutagenicity and co-mutagenicity of catechol on Salmonella

Catechol was not mutagenic for Salmonella typhimurium TA98, TA100 or TA1537 in the presence or absence of S9 mix. At the lower level of S9 in the Ames method, the mutagenic activity of benzo[a]pyrene decreased with the increased addition of catechol. When catechol was added to the pre-incubation mixture at a higher concentration than in the conventional Ames method, the mutagenic activity of benzo[a]pyrene increased with the increased addition of catechol. Catechol is believed to be a co-mutagen for benzo[a]pyrene in the presence of a sufficient amount of S9 in the incubation mixture.     

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.     

Genotoxicity of apomorphine and various catecholamines in the Salmonella mutagenicity test (Ames test) and in tests for primary DNA damage using DNA repair-deficient B. subtilis strains (rec assay)

Apomorphine, N-nor-N-propyl-apomorphine, dopamine, L-DOPA, 6-hydroxydopamine and adrenaline were evaluated for genotoxicity using the Ames test and DNA repair-deficient and DNA repair-proficient Bacillus subtilis strains (rec assay, H17/M45; HLL3g/HJ-15). In the absence of an S9 liver homogenate, apomorphine induced frame-shift mutations in Salmonella typhimurium, mainly in strain TA1537; no indication of DNA-damaging effects in B. subtilis was observed. N-Nor-N-propyl-apomorphine was tested using strain TA1537 only and found to be mutagenic. Dopamine, L-DOPA, 6-hydroxydopamine and adrenaline were non-mutagenic when tested without S9, whereas they were all more toxic for DNA repair-deficient than for DNA repair-proficient B. subtilis strains, indicating a DNA-damaging potential. In a second set of experiments the mode of action of apomorphine and the relevance of the positive Ames test data were investigated. Glutathione in physiological concentrations reduced the mutagenic effect of apomorphine in a dose-dependent way, both in the presence and the absence of S9. S9 also reduced the mutagenicity of apomorphine. By comparing the effects of a complete S9 mix with those of a preparation without glucose-6-phosphate and NADP, it became clear that S9 also had an activating effect, overshadowed under standard conditions by its deactivating activity. Apomorphine was not mutagenic under anaerobic conditions. Superoxide dismutase and catalase reduced the mutagenic effect of apomorphine. All test conditions which reduced the mutagenic effect also inhibited the dark discoloration of the tester plates, indicating a retardation of apomorphine oxidation. It can, therefore, be concluded that oxidation of apomorphine leads to mutagenic products which induce frame-shift mutations in Salmonella typhimurium. This oxidation was prevented both by glutathione in concentrations well below physiological levels and/or by catalase and superoxide dismutase. Under these conditions, apomorphine was non-mutagenic in therapeutic concentrations as well as at higher dose levels. The possibility of genotoxic side effects occurring in patients treated with apomorphine as an emetic drug is therefore considered to be very unlikely.     

Study on mutagenicity and antimutagenicity of BHT and its derivatives in a bacterial assay

The mutagenicity of butylated hydroxytoluene (BHT) and its derivatives was investigated by the Ames method using Salmonella typhimurium TA98 and TA100 with or without S9 mix. The compounds were not mutagenic in either indicator strain at concentrations ranging from 50 to 330 micrograms/plate (SQ: 3,5,3',5'-tetra-tert-butylstilbenequinone; VI-III: unidentified), 500 micrograms/plate (BE: 3,5,3',5'-tetra-tert-4,4'-dihydroxy-1,2-diphenylethylene; VI: 2,6-di-tert-butyl-4-methyl-4-tert-butylperoxy-2,5-cyclohexadienone ; VI-I: unidentified; VI-II: 3-acetyl-2,5-di-tert-cyclopenta-2,4-dienone) and 1000 micrograms/plate (BHT). The antimutagenic effects of BHT and its derivatives on mutagenesis by chemical agents were investigated in Salmonella typhimurium TA98 and TA100 and Escherichia coli WP-2 hcr-. VI-II suppressed the mutagenesis induced in TA98 and TA100 by 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) and that induced in WP-2 hcr- by 4-nitroquinoline-1-oxide (4NQO) without decreasing cell viability. In WP-2 hcr-, the mutagenesis induced by AF-2 and ethyl methanesulfonate was also suppressed significantly. Mutations induced by methyl methanesulfonate were slightly inhibited. However, VI-II had no effect on the mutagenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine.     

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.     

Soil mutagens are airborne mutagens: variation of mutagenic activities induced in Salmonella typhimurium TA 98 and TA 100 by organic extracts of agricultural and forest soils in dependence on location and season

As our hypothesis was that soil mutagens are airborne mutagens, possibly modified by soil microorganisms, we checked solvent extracts from agricultural and forest soils collected during late summer in the environment of Mainz, a region highly charged by anthropogenic air pollution, or near Bayreuth, a rural low charged region of Germany, or in a remote region of western Corsica without anthropogenic air pollution for the presence of mutagenicity in Salmonella typhimurium. Levels of mutagenic activities were quantified by calculation of revertants/g from the initial slope of dose-response curves applying tester strains S. typhimurium TA 98 and TA 100 in the absence and presence of an activation system from rat liver (S9). Three soils from Corsica did not induce mutagenicity under any test condition. However, most soils from Germany exhibited mutagenic activities, though preferentially in strain TA 98, but no statistically significant differences could be detected between 27 soils from the Mainz and nine soils from the Bayreuth regions. On the other hand, no correlation could be detected between the levels of mutagenic activities at any test condition and agricultural practice - rye growing, viniculture, fruit growing, meadow, and fallow - texture of soils - % composition of clay, slit, and sand - or the contents of organic matter. The only significant difference of mutagenicity was, however, found with S. typhimurium TA 98-S9 between forest soils of pH approximately 4.0 as compared with agricultural soils of pH approximately 7.0. The presence of antimutagens in soil as demonstrated by the course of dose-response curves of the three soils from Corsica may be another possible confounder. Calculation of mean values of mutagenic activities for all soils from Germany gave the following results: S. typhimurium TA 98: 69.7+/-153.2 (-S9); 63.0+/-176.3 (+S9); S. typhimurium TA 100:-144.7+/-399.4 (-S9); 43.3+/-172.0 (+S9) revertants/g of dry soil. In another series of experiments, soil mutagenicity in 10 rye fields near Mainz was monitored for 1 year. It became evident that low levels of mutagenic activities in late summer increased during autumn, reached a peak in late winter, and subsequently, decreased during spring and summer. These results agree with the hypothesis of an airborne origin of soil mutagens, deposition, and an adjacent transformation to non-mutagenic compounds by soil microorganisms.     

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.     

Bacterial reversion assay and micronucleus test carried out on hydrogenated glucose syrups 'Malti-Towa' (powder) and maltitol crystal

Two preparations of maltitol (4-O-alpha-D-glucopyranosyl-D-sorbitol), hydrogenated glucose syrups and maltitol crystal, were examined for genotoxic potential by a battery of short-term tests. In the bacterial reversion assay, maltitol induced no detectable revertants in any of the tester strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538, or Escherichia coli WP2/pKM101 at doses of 0.5-50 mg per plate with and without rat liver S9 mix. In the micronucleus test, no significant increase in the frequency of micronucleated erythrocytes was observed in bone marrow of mice after administration of the two preparations at 3.75-30 g per kg by gastric intubation.