Lack of mutagenicity of ochratoxin A and B, citrinin, patulin and cnestine in Salmonella typhimurium TA102.

The Aspergillus mycotoxins ochratoxin A and B, citrinin and patulin as well as combinations of ochratoxin A and citrinin did not induce reverse mutations in Salmonella typhimurium strain TA102. Therefore there is no indication for the induction of oxidative damage or crosslinks. The same is true for cnestine, a compound extracted from the plant Cnestis glabra.

The mutagenicity of edible mushrooms in a histidine-independent bacterial test system.

In mutagenicity screening of 40 edible mushroom species, special attention was paid to the selection of the test system, since complex mixtures such as mushroom extracts may interfere with the genetic endpoint of the assay. This paper shows that the weak mutagenicity of some mushrooms in the Ames test, as reported by several authors, is actually an artefact due to the presence of free histidine in the mushroom extracts, which apparently increases the reversion of bacteria from histidine auxotrophy to prototrophy. To avoid amino-acid interaction, a combination of the forward mutation assay to 8-azaguanine resistance in Salmonella typhimurium TM677 and a liquid test was used. Out of 35 wild and commercially grown mushrooms tested, 13 species exhibited mutagenic activity. In the case of the five samples of dried mushrooms, weak mutagenicity could be detected for Auricularia sp. The presence of microsomal enzymes (S-9) reduced the mutagenic effects of all the mushrooms, with the exception of Agaricus abruptibulbus and Cantharellus cibarius, where metabolic activation enhanced the mutagenic activity.

Antimutagenic effects of mushrooms.

A heat-resistant factor in ethanol extracts of the fungus Craterellus cornucopioides completely inhibited the mutagenicity of aflatoxin B1, benzo[a]pyrene, the acridine half mustard ICR-191 and 2-nitrofluorene in a forward-mutation system using Salmonella typhimurium TM677 (screening for 8-azaguanine resistance). There was no inhibitory effect on the mutagenic activity of 4-nitroquinoline-N-oxide, methyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine. Experiments performed to elucidate the mechanism of the antimutagenic effect showed that neither an alteration of cell viability nor an interference with the excision-repair and the inducible SOS-repair system was involved. The conceivable mechanisms for the antimutagenicity of the ethanol extract include direct chemical interaction with the mutagen and/or inhibition of the activation process in the case of the promutagens. The antimutagenic activity of Craterellus cornucopioides is not unique among mushroom species. The ethanol extracts of 6 other mushrooms showed a similar antimutagenic activity.

Salmonella/mammalian microsome assay with tetranitromethane and 3-nitro-L-tyrosine.

The nitrosating agent tetranitromethane (TNM) and the nitrosation product 3-nitro-L-tyrosine (NT) were tested for mutagenic activity in the Salmonella/mammalian microsome assay. TNM showed strong genotoxic activity: it was mutagenic in all tester strains used (TA97, TA98, TA100, and TA102). The maximum mutagenic activity was reached between 16 and 32 micrograms/plate using the standard plate test; higher amounts led to distinct bactericidal effects. The mutagenicity was independent of an in vitro activation system. In the preincubation assay an increased bactericidal effect was observed. In contrast to TNM, NT, the nitrosation product, was non-mutagenic and non-toxic in the standard plate test and with the preincubation method up to 5000 micrograms/plate with and without S9 mix and with all tester strains used. Although TNM is a strong direct-acting mutagen, its nitrosating effect on proteins does lead to nongenotoxic nitro products of tyrosine in proteins.

Genotoxicity of aniline derivatives in various short-term tests.

Various substituted aniline derivatives were tested for genotoxicity in several short-term tests in order to examine the hypothesis that a substitution at both ortho positions (2,6-disubstitution) could prevent genotoxicity due to steric hindrance of an enzymatic activation to electrophilic intermediates. In the Salmonella/microsome assay, 2,6-dialkylsubstituted anilines and 2,4,6-trimethylaniline (2,4,6-TMA) were weakly mutagenic in strain TA100 when 20% S9 mix was used, although effects were small compared to those of 2,4-dimethylaniline and 2,4,5-trimethylaniline (2,4,5-TMA). In Drosophila melanogaster, however, 2,4,6-TMA and 2,4,6-trichloroaniline (TCA) were mutagenic in the wing spot test at 2-3 times lower doses than 2,4,5-TMA. In the 6-thioguanine resistance test in cultured fibroblasts, 2,4,6-TMA was again mutagenic at lower doses than 2,4,5-TMA. Two methylene-bis-aniline derivatives were also tested with the above methods: 4,4'-methylene-bis-(2-chloroaniline) (MOCA) was moderately genotoxic in all 3 test systems whereas 4,4'-methylene-bis-(2-ethyl-6-methylaniline) (MMEA) showed no genotoxicity at all. DNA binding studies in rats, however, revealed that both MOCA and MMEA produced DNA adducts in the liver at levels typically found for moderately strong genotoxic carcinogens. These results indicate that the predictive value of the in vitro test systems and particularly the Salmonella/microsome assay is inadequate to detect genotoxicity in aromatic amines. Genotoxicity seems to be a general property of aniline derivatives and does not seem to be greatly influenced by substitution at both ortho positions.

Reversible inhibition of mammalian tubulin assembly in vitro and effects in Saccharomyces cerevisiae D61.M by mitomycin C.

Gaulden reported a novel and unexpected mitomycin C (MMC) effect, namely a pronounced retardation of very late prophase and loss of chromosome orientation in neuroblasts of the grasshopper Chortophaga viridifasciate. Because this effect may be due to interactions of MMC with non-DNA targets, MMC was tested for its interaction with porcine brain tubulin assembly in vitro and for the induction of chromosomal malsegregation in the diploid yeast Saccharomyces cerevisiae strain D61.M. A reversible dose-dependent inhibition of tubulin assembly was observed. Since no biological activation system was present in the incubation mixture this inhibition seems to result from an interaction of unactivated MMC with the assembly process. The possible chemical activation of MMC by reduction with 1,4-dithioerythritol (DTE) was investigated by omission of this compound during isolation and polymerization of tubulin. The absence of DTE resulted in a strong reduction of the net tubulin assembly. Also under these conditions MMC led to a dose-dependent inhibition of the assembly, indicating that the effect of MMC on tubulin assembly is independent of a reductive chemical modification. In S. cerevisiae D61.M, MMC did not induce chromosome loss, but induced other genetic events (possibly mutations, deletions or mitotic recombination) as was detected by an increase of the total number and of the frequency of cycloheximide-resistant colonies. This effect could be observed with and without the addition of rat liver S9 as an exogenous activation system.

The in vitro porcine brain tubulin assembly assay: effects of a genotoxic carcinogen (aflatoxin B1), eight tumor promoters and nine miscellaneous substances.

Aflatoxin B1 (AFB1) had a reversible inhibitory effect on the assembly of porcine brain tubulin in vitro. The 30%-inhibition concentration was 0.3 mM AFB1. The 8 tumor promoters showed different effects. Five of them, anthralin, cholic acid, gamma-hexachlorocyclohexane (lindane, gamma-HCH), lithocholic acid and phenobarbital (PB), enhanced the in vitro assembly. The effect was reversible in the case of PB and anthralin, only partially reversible in the case of cholic acid and gamma-HCH, whereas the stimulating effects of lithocholic acid led to an irreversible modification of the tubulin structure, as shown by the insolubility of the microtubules at 0 degrees C. This could be confirmed by an electron microscopic study. The doses necessary for a 30% enhancement of the steady-state level were 3 mM (PB), 0.2 mM (anthralin), 6 mM (cholic acid), 0.7 mM (gamma-HCH) and less than 0.2 mM (lithocholic acid). The other 3 tumor promoters tested - diethylstilbestrol (DES), 4,4'-dichloro-diphenyl-trichloro-ethane (DDT) and saccharin - inhibited the assembly. The concentrations necessary for a 30% inhibition varied within a wide range: 0.025 mM, 0.4 mM and 7.5 mM for DES, DDT and saccharin, respectively. Five of the 9 miscellaneous compounds, namely asbestos (crocidolite), bavistan, colchicine, chloropropham and ethylacetate, showed inhibitory effects, whereas Fe2+ (a constituent of asbestos) and 5-azacytidine did not influence the assembly process. The 30%-inhibition concentrations for colchicine, ethylacetate and asbestos were 10 microM, 0.153 M and 0.19 mM, respectively. For bavistan and chloropropham the 30%-inhibition values were 0.7 mM and 2.0 mM, respectively. The inhibitory effects of chloropropham and asbestos were reversible. For colchicine and bavistan the reversibility of the effects was not assayed. In agreement with published data, dimethylsulfoxide (DMSO) and acetone enhanced the in vitro assembly of porcine brain tubulin. The doses needed for a 30% enhancement by DMSO and acetone were 0.4 mM and 0.136 M, respectively. The effect of DMSO was irreversible whereas acetone led to a reversible stimulation. Some compounds were tested for their influence on preformed microtubules (interaction with the equilibrium between assembly and disassembly). Anthralin, cholic acid, PB and DMSO showed no effect on the steady-state plateau. A slight reduction was induced by DDT and bavistan, whereas DES, colchicine and chloropropham led to a pronounced reduction.

Induction of mitotic chromosome loss in the diploid yeast Saccharomyces cerevisiae D61.M by genotoxic carcinogens and tumor promoters.

Three genotoxic carcinogens and eight tumor promoters were tested for induction of aneuploidy, specifically chromosome loss, in Saccharomyces cerevisiae D61.M. This is a heterozygous diploid yeast strain that permits the scoring of segregants expressing three linked recessive markers (cyhR2, ade6, and leu1), two of which (ade6 and leu1) are located close to the centromere on opposite arms of chromosome VII. The centromere marker leu was routinely checked, and a positive control (bavistan) was run with every experiment. The three genotoxic carcinogens aflatoxin B1, benzo(a)pyrene, and 7,12-dimethylbenz(a)anthracene did not induce aneuploidy, independent of the presence or absence of an exogenous metabolic activation system (rat liver homogenate; S9). Four of the eight tumor promoters tested induced chromosome loss but not mitotic recombination or mutation: cholic acid, lithocholic acid, phenobarbital, and saccharin. Diethylstilbestrol (DES) led to positive as well as to negative results in several independent experiments. In the case of the positive experiment, DES also induced putative recombinants. Three tumor promoters induced neither chromosome loss nor mitotic recombination: anthralin, 4,4'-dichloro-diphenyl-ethane (DDT) and gamma-hexachlorcyclohexane (lindane). From our experiments it can be concluded that the hypothesis put forward by Parry et al. [Nature; 294:263-265], according to which tumor promoters induce chromosome loss in yeast, is not correct in a general sense. In our set of eight tumor promoters, only one half distinctly induced chromosome loss.

Trenbolone growth promotant: covalent DNA binding in rat liver and in Salmonella typhimurium, and mutagenicity in the Ames test.

DNA binding in vivo: [6,7-3H]beta-trenbolone (beta-TBOH) was administered p.o. and i.p. to rats. After 8 or 16 h, DNA was isolated from the livers and purified to constant specific radioactivity. Enzymatic digestion to deoxyribonucleotides and separation by HPLC revealed about 90% of the DNA radioactivity eluting in the form of possible TBOH-nucleotide adducts. The extent of this genotoxicity, expressed in units of the Covalent Binding Index, CBI = (mumol TBOH bound per mol nucleotide)/(mmol TBOH administered per kg body weight) spanned from 8 to 17, i.e. was in the range found with weak genotoxic carcinogens. Ames test: low doses of beta-TBOH increased the number of revertants in Salmonella strain TA100 reproducibly and in a dose-dependent manner. The mutagenic potency was 0.2 revertants per nmol after preincubation of the bacteria (20 min at 37 degrees C) with doses between 30 and 60 micrograms per plate (47 and 94 micrograms/ml preincubation mixture). Above this dose, the number of revertants decreased to control values, accompanied by a reduction in survival. The addition of rat liver S9 inhibited the mutagenicity. DNA binding in vitro: calf thymus DNA was incubated with tritiated beta-TBOH with and without rat liver S9. Highest DNA radioactivities were determined in the absence of the "activation" system. Addition of inactive S9 (without cofactors) reduced the DNA binding by a factor of up to 20. Intermediate results were found with active S9. DNA binding in Salmonella: beta-TBOH was irreversibly bound to DNA isolated from S. typhimurium TA100 after incubation of bacteria with [3H]beta-TBOH.

Investigations on DNA binding in rat liver and in Salmonella and on mutagenicity in the Ames test by emodin, a natural anthraquinone.

Emodin (1,6,8-trihydroxy-3-methylanthraquinone), an important aglycone found in natural anthraquinone glycosides frequently used in laxative drugs, was mutagenic in the Salmonella/mammalian microsome assay (Ames test) with a specificity for strain TA1537. The mutagenic activity was activation-dependent with an optimal amount of S9 from Aroclor 1254-treated male Sprague-Dawley rats of 20% in the S9 mix (v/v) for 10 micrograms emodin per plate. Heat inactivation of the S9 for 30 min at 60 degrees C prevented mutagenicity. The addition of the cytochrome P-448 inhibitor 7,8-benzoflavone (18.5 nmoles per plate) reduced the mutagenic activity of 5.0 micrograms emodin per plate to about one third, whereas the P-450 inhibitor metyrapone (up to 1850 nmoles per plate) was without effect. To test whether a metabolite binds covalently to Salmonella DNA, [10-(14)C]emodin was radiosynthesized, large batches of bacteria were incubated with [10-(14)C]emodin and DNA was isolated. [G-3H]Aflatoxin B1 (AFB1) was used as a positive control mutagen known to act via DNA binding. DNA obtained after aflatoxin treatment could be purified to constant specific activity. With emodin, the specific activity of DNA did not remain constant after repeated precipitations so that it is unlikely that the mutagenicity of emodin is due to covalent interaction of a metabolite with DNA. The antioxidants vitamin C and E or glutathione did not reduce the mutagenicity. Emodin was also negative with strain TA102. Thus, oxygen radicals are probably not involved. When emodin was incubated with S9 alone for up to 50 h before heat-inactivation of the enzymes and addition of bacteria, the mutagenic activity did not decrease. It is concluded that the mutagenicity of emodin is due to a chemically stable, oxidized metabolite forming physico-chemical associations with DNA, possibly of the intercalative type. In order to check whether an intact mammalian organism might be able to activate emodin to a DNA-binding metabolite, radiolabelled emodin was administered by oral gavage to male SD rats and liver DNA was isolated after 72 h. Very little radioactivity was associated with the DNA. Considering that DNA radioactivity could also be due to sources other than covalent interactions, an upper limit for the covalent binding index, CBI = (mumoles chemical bound per moles DNA nucleotides)/(mmoles chemical administered per kg body weight) of 0.5 is deduced. This is 10(4) times below the CBI of AFB1.(ABSTRACT TRUNCATED AT 400 WORDS)

Endogenous promutagen activation in the yeast Saccharomyces cerevisiae: factors influencing aflatoxin B1 mutagenicity.

The formation of convertants, revertants and other types of mitotic segregants was induced in Saccharomyces cerevisiae D7 upon incubation with aflatoxin B1 (AFB1). The most distinct effects were observed for gene conversion to tryptophan prototrophy. The fact that different cytochrome P-450 inhibitors (ellipticine, penconazole and propiconazole as yeast-specific P-450 inhibitors) abolished the AFB1-induced mutagenicity indicates that activation of the promutagen AFB1 depends on the cytochrome P-450-catalyzed electron-transfer reactions. This hypothesis is further supported by the observation that the cytochrome P-450 content of yeast cells harvested at different phases during growth is directly correlated with their sensitivity for AFB1-induced tryptophan conversion.

The mutagenic activity of agaritine--a constituent of the cultivated mushroom Agaricus bisporus--and its derivatives detected with the Salmonella/mammalian microsome assay (Ames Test).

Purified agaritine (N'-(gamma-L(+)-glutamyl)-p-hydroxymethylphenylhydrazine) isolated from Agaricus bisporus, p-hydrazinobenzoic acid (its presumptive precursor) and some agaritine-degradation products were tested for mutagenic activity with the Salmonella/mammalian microsome assay (Ames test). Consistent with the literature, agaritine showed a distinct direct-acting mutagenicity with the strain TA1537 (30 revertants/mumol) and with TA97. Incubation of agaritine at alkaline pH increased the mutagenic effect. Pre-incubation of agaritine with gamma-glutamyl transferase (GT) during 10 h at room temperature (pH 8.2) even enhanced the mutagenicity by a factor of 8 to 16 depending on the strain. In accordance with this finding, synthetic p-hydroxymethylphenylhydrazine (the presumptive product of the GT catalyzed degradation) showed also a distinct direct-acting mutagenicity, but the increase was only about 3- to 6- times compared with agaritine. The hypothetical ultimate mutagenic metabolite of agaritine, the p-hydroxymethylbenzenediazonium ion, a compound occurring naturally in A. bisporus, showed the highest mutagenic activity (with TA1537 approximately 300 to 1,000 revertants/mumol).

Limitations of the salmonella/mammalian microsome assay (Ames test) to determine occupational exposure to cytostatic drugs.

Urine samples of nursing personnel working in medical oncology divisions of several Swiss hospitals were examined for mutagenic activity. Urine samples were concentrated 100 times following XAD-2 chromatography and mutagenicity was determined using the Salmonella/mammalian microsome assay (Ames test). Apart from the urine samples of patients treated with cytostatic drugs and urine samples of nurses who are cigarette smokers, no mutagenic activity could be found. Also following exposure to an increased and defined quantity of cytostatic drugs no mutagenicity could be recovered from the urine. Four different nurses worked with cyclophosphamide, methotrexate, 5-fluorouracil, adriamycin and cis-platinum for 3-4 hr without using any protection such as gloves, masks or a vertical laminar airflow hood. Aqueous extracts of filters, through which air was pumped during the whole experiment (a personal air-sampler was fixed near the face of the test persons), were non-mutagenic. Parallel to the mutagenicity test chemical analyses were also done. The methotrexate content was determined in serum samples and the aqueous filter extracts and urine samples were examined for cis-platinum. All chemical determinations were negative. With the aid of urine concentrates of a patient treated with sub-therapeutic doses of cyclophosphamide as well as with normal urine to which single small amounts of different cytostatics (adriamycin, cyclophosphamide, cis-platinum) were added, the detection limits for the corresponding cytostatic drugs were determined and found to be in the range of 2-10 mg for cyclophosphamide and approx. 10 micrograms for adriamycin. Cis-platinum was lost during the passage through the XAD-2 columns. With these results at hand the sensitivity of the hitherto preferably used method (Ames test) for the monitoring of exposure to cytostatic drugs must be seriously questioned.

Phenobarbital induces aneuploidy in Saccharomyces cerevisiae and stimulates the assembly of porcine brain tubulin.

Phenobarbital (PB) specifically induces mitotic chromosomal malsegregation in the diploid Saccharomyces cerevisiae strain D61.M but no other genetic events such as mitotic recombination or point mutations. In accordance with the hypothesis that PB exerts its genotoxic activity by an interaction with tubulin, it stimulates the GTP-promoted assembly of porcine brain tubulin in vitro. This process is reversible thus excluding an unspecific denaturation of the tubulin protein by PB.

The influence of roasting procedure on the formation of mutagenic compounds in coffee.

Mutagenic products can be formed during the processing of food and especially as a result of heat treatment. Direct acting mutagenic activity was found in extracts of instant coffee and roasted coffee beans using Salmonella typhimurium TA100 in vitro. The mutagenic activities of the four pure coffee varieties examined (Coffea arabica Santos, Coffea arabica Columbia, Coffee robusta Indonesia, Coffee robusta Camerun) were within the same range. Twenty milligrams per plate freeze-dried powder prepared from aqueous roast coffee extracts induced between six and ten times the number of revertants found in the negative controls. Green coffee beans had no mutagenic activity. Mutagenicity increased with roasting time up to 4 min in the Probat drum roaster and then remained constant (i.e. no further increase after 8 min, the time normally used to roast coffee). The genotoxic compounds were quickly formed at temperatures below 220 degrees C (in normally roasted coffee the beans must reach a temperature of 220 degrees C). Mutagenic activity was independent of the roasting procedure (Jetzon procedure v. Probat drum roaster).

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.

Goitrin--a nitrosatable constitutent of plant foodstuffs.

N-nitrosamides are known as direct-acting carcinogens at the site of their formation; they do not need any metabolic activation in vivo. The conditions leading to their formation in the stomach, and also their genotoxicity, have been thoroughly studied with some model compounds. Several reports link this type of compound to the induction of gastric cancer in human. However, only limited data are presently available about possible precursors of N-nitrosamides in foods. In the present study we found that goitrin --a naturally occurring compound in cruciferous vegetables and rape--could be easily nitrosated by treatment with nitrite under stomach conditions, yielding with loss of sulfur the N-nitroso- oxazolidone 4 (fig.). This product has a mutagenicity pattern and potency similar to that of N-nitroso-N-methyl-N'- nitroguanidine (MNNG) in the Ames Salmonella/mammalian microsome test.

The Salmonella/mammalian-microsome assay: variations of the test protocol; results of a questionnaire returned by 87 laboratories.

The answers to a questionnaire sent to 147 laboratories in Europe using the Salmonella/mammalian-microsome assay are presented. In mutagenicity testing, where research projects in environmental mutagenesis predominate, the Salmonella/mammalian-microsome assay is the most often used test. Of all laboratories that answered the questionnaire, 80% are using the test with occasional modifications (preincubation, fluctuation test). Most of the differences concern the activation system used. This questionnaire is part of a European collaborative study which aims to improve the standardization of the Salmonella/mammalian-microsome assay.

Criteria for the standardization of Salmonella mutagenicity tests: results of a collaborative study. III. The influence of the composition and preparation of the minimal medium in the Salmonella mutagenicity test.

The influence of factors connected with the preparation of the minimal medium for the Ames test has been investigated. Faulty sterilization procedures can lead to the generation of toxic and/or mutagenic by-products in the minimal medium. Changes in histidine concentration affect not only the number of spontaneously arising colonies on the plate, but also the number of induced mutants. Although the number of spontaneous mutants increases slightly with increasing histidine concentration, the influence on the number of induced mutants depends on the nature of the mutagen tested.