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.

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.

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.

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.