In vitro and in vivo chromosomal aberrations induced by megazol

With the re-emergence of Human African Trypanosomiasis (HAT) on the one hand, which are increasingly resistant to current therapies, and the stage-dependent effectiveness or even the prohibitive cost of these therapies on the other hand, megazol, a 5-nitroimidazole thiadiazole highly active against various trypanosomal species, was assessed for its genotoxic potential. Very little information has become available until now. Two batches of megazol were provided by two different suppliers: Far-Manguinhos, a part of the Fiocruz foundation, under the Brazilian Minister of Health, and Delphia, a French company. These two batches, obtained by different synthetic routes, were studied by means of the in vitro micronucleus assay on L5178Y mouse lymphoma cells, in its microscale version. Both batches of magazol displayed a strong genotoxic activity in this screening assay. A second batch from Delphia was then investigated by use of two tests, i.e. the in vitro metaphase analysis with human lymphocytes and the in vivo micronucleus test in rat bone-marrow. Megazol was shown to be a potent inducer of in vitro and in vivo chromosomal aberrations. Although megazol is a potent trypanocidal agent and is orally bio-available, its toxicity dictates that it should not be developed further for the treatment of HAT and Chagas disease. All development work has therefore been discontinued.     

In vitro and in vivo chromosomal aberrations induced by megazol

With the re-emergence of Human African Trypanosomiasis (HAT) on the one hand, which are increasingly resistant to current therapies, and the stage-dependent effectiveness or even the prohibitive cost of these therapies on the other hand, megazol, a 5-nitroimidazole thiadiazole highly active against various trypanosomal species, was assessed for its genotoxic potential. Very little information has become available until now. Two batches of megazol were provided by two different suppliers: Far-Manguinhos, a part of the Fiocruz foundation, under the Brazilian Minister of Health, and Delphia, a French company. These two batches, obtained by different synthetic routes, were studied by means of the in vitro micronucleus assay on L5178Y mouse lymphoma cells, in its microscale version. Both batches of magazol displayed a strong genotoxic activity in this screening assay. A second batch from Delphia was then investigated by use of two tests, i.e. the in vitro metaphase analysis with human lymphocytes and the in vivo micronucleus test in rat bone-marrow. Megazol was shown to be a potent inducer of in vitro and in vivo chromosomal aberrations. Although megazol is a potent trypanocidal agent and is orally bio-available, its toxicity dictates that it should not be developed further for the treatment of HAT and Chagas disease. All development work has therefore been discontinued.     

Effects of the formaldehyde releasing preservatives dimethylol urea and diazolidinyl urea in several short-term genotoxicity tests

The two formaldehyde (FA)-releasers dimethylol urea (DMU) and diazolidinyl urea (DZU) are widely used as preservatives or additives. They were tested for genotoxicity in three short-term test systems, i.e. in the Salmonella typhimurium mutagenicity assay, in the in vitro micronucleus test with V79 Chinese hamster cells and in the in vitro tubulin assembly assay using isolated tubulin from pig brains. The polymerization products obtained in the tubulin assembly assay were examined additionally by electron microscopy.In the S. typhimurium mutagenicity assay with the pre-incubation assay both FA-releasers tested show a clear and concentration-dependent increase in the number of revertants in strains TA98, TA100 and TA102 with and without metabolic activation (rat liver S9 mix). In all cases, a biologically relevant increase in the number of revertants was achieved within the concentration range tested (DZU: 0.04-1.8 micromol per plate, DMU: 0.21-8.33 micromol per plate). FA was tested at 0.06-2.5 micromol per plate and lead to similar effects.Both compounds induce the formation of micronuclei (concentration range tested: DZU: 2.5-50 micromol/l, DMU: 3.3-333 micromol/l). However, DMU shows a comparatively weaker effect exclusively in the absence of the metabolizing enzymes. By contrast, DZU yields a distinct increase of the micronucleus rate in the absence and in the presence of S9. In addition, DZU predominantly causes an increase of large micronuclei, which suggests that this compound has a marked aneugenic potential. Cytotoxic effects accompany the clastogenic effects of both DMU and DZU.The examination of DMU and DZU in view of a possible aneugenic potential in the tubulin assembly assay yielded the following results: DMU at concentrations up to 10 mmol/l did not influence the formation of microtubuli, whereas DZU inhibited this process completely at 3 mmol/l. FA at 6 mmol/l completely inhibited the tubulin assembly. These results could clearly be confirmed by electron microscopy examination. The different potential of the two compounds with respect to the inhibition of tubulin formation is apparently due to a significant difference in the degree of FA release.According to these results, both compounds have to be considered as genotoxic in vitro. On account of these data and because of the widespread use of these two compounds in various products used in daily life, a reevaluation of the risk associated with these compounds seems to be necessary.     

Comparison of the effects of 1,2-dimethylhydrazine and cyclophosphamide on micronucleus incidence in bone marrow and colon

An in vivo micronucleus assay has been developed that utilizes colonic epithelial cells. The genotoxic effects of 1,2-dimethylhydrazine (54-07-3), a colon carcinogen, and of the nitrogen mustard, cyclophosphamide (50-18-0), on the bone-marrow polychromatic erythrocytes and on colonic epithelium from mice were compared using micronucleus induction in each organ as the end point. In the bone marrow, cyclophosphamide was a potent inducer of micronuclei, while 1,2-dimethylhydrazine administration had little effect on the micronucleus incidence. In the colon, 1,2-diemthylhydrazine was an effective inducer of micronuclei. Thus, the colonic micronucleus assay appears to be a potentially useful test for the detection of colon carcinogens.     

The mouse bone marrow micronucleus test: evaluation of 21 drug candidates

The mouse bone-marrow micronucleus test is one of the most widely used genetic toxicology assays. In this report the results of testing 21 compounds in the micronucleus test are presented. Of the 21 compounds tested, 3 potential chemotherapeutic agents were identified as strongly clastogenic. In addition, one compound was identified as a weak inducer of micronuclei in the assay. Further testing of this compound in an in vivo bone marrow metaphase analysis failed to confirm this material as clastogenic. The remaining 17 compounds were classified as negative in the assay. In general the results of the micronucleus test agreed with the results of other genetic toxicology assays on this group of compounds.     

Genotoxic and cytotoxic effects in the bone marrow of rats exposed to a low dose of paraquat via the dermal route

The genotoxic effect of the herbicide paraquat was studied in rat bone-marrow by means of the micronucleus assay. Paraquat at dose levels of 6, 15 and 30 mg/kg body weight was given to rats in a single application via the dermal route. Marrow was collected at 24, 48 and 72 h after the application. The micronucleus assay was done as recommended by standard procedures. Paraquat gave rise to an increase in the number of micronuclei in a dose-dependent manner. The number of micronucleated polychromatic erythrocytes showed a maximum at 48 h and the toxicity was further prolonged, as there was no complete recovery at 72 h. These findings suggest a genotoxic effect of paraquat even after exposure via dermal application.     

Evaluation of the genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) in a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) was evaluated in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse-mutation assay (Ames test), an in vitro human lymphocyte chromosome-aberration assay, an in vivo mouse bone-marrow micronucleus assay and an in vivo rat-liver UDS assay. Maximum test concentrations in in vitro assays were determined by the TTX limit of solubility in the formulation vehicle (0.02% acetic acid solution). In the Ames test, TTX was tested at concentrations of up to 200 microg/plate. In the chromosome-aberration assay human lymphocytes were exposed to TTX at concentrations of up to 50 microg/ml for 3 and 20 h in the absence of S9, and for 3h in the presence of S9. For the in vivo assays, maximum tested dose levels were determined by the acute lethal toxicity of TTX after subcutaneous administration. In the mouse micronucleus assay TTX dose levels of 2, 4 and 8 microg/kg were administered to male and female animals, and bone-marrow samples taken 24 and 48 h (high-dose animals only) after administration. In the UDS assay, male rats were given TTX on two occasions with a 14-h interval at dose levels of 2.4 and 8 microg/kg, the last dose being administered 2h before liver perfusion and hepatocyte culturing. Relevant vehicle and positive control cultures and animals were included in all assays. TTX was clearly shown to lack in vitro or in vivo genotoxic activity in the assays conducted in this study. The results suggest that administration of TTX as a therapeutic analgesic agent would not pose a genotoxic risk to patients.     

The prevention of benzene-induced genotoxicity in mice by indomethacin

Benzene is a myelotoxin which affects hemopoietic progenitor cells leading to bone-marrow depression as well as a genotoxin which causes chromosomal abnormalities including micronucleus formation. We have demonstrated previously that benzene administered to DBA/2 or C57B1/6 mice causes bone-marrow depression and increased prostaglandin E2 levels in bone marrow; both of these effects can be prevented by the coadministration of indomethacin, a selective inhibitor of prostaglandin synthase. We report, herein, that benzene (400-600 mg/kg body weight), under conditions where it depresses bone-marrow cellularity, also induces an increase in the frequency of micronucleus formation in polychromatic erythrocytes of C57B1/6 mice which is also prevented by the coadministration of indomethacin at levels that do not inhibit cytochrome P450 or myeloperoxidase. In Swiss Webster wild-type mice doses of benzene from 400 to 1000 mg/kg were without effect on marrow cellularity, but did induce the formation of micronucleated polychromatic erythrocytes which could be prevented by indomethacin. In contrast, DBA/2 mice, a strain highly sensitive to benzene, exhibited significant bone-marrow depression at a dose of benzene of 100 mg/kg body weight. Even at this low dose, benzene is too toxic toward developing erythrocytes to allow the evaluation of micronucleus formation. The frequency of induction of micronucleated polychromatic erythrocytes by benzene thus depends on the strain of mouse used. Furthermore, micronucleus formation appears to be an early and very sensitive indicator of benzene toxicity. A possible role for prostaglandin H synthase in the geno- and myelo-toxicity of benzene is discussed.     

Cytogenetic effects of methotrexate on human cells in vivo: comparison between results obtained by chromosome studies on bone-marrow cells and blood lymphocytes and by the micronucleus test

Cytogenetic studies were performed in 22 patients treated with methotrexate (MTX). In some patients, metaphases from both bone-marrow cells and peripheral blood cells were studied. In the bone-marrow preparations an increased number of structural chromosomal aberrations was present, whereas abnormalities were not observed in the peripheral blood cells. An examination of the bone-marrow chromosomes must therefore be included in the study of the possible chromosome-breaking effect of chemical agents. The results obtained with the micronucleus test and chromosome studies were compared in 10 patients treated with MTX. The micronucleus test was more sensitive than the chromosome analysis as regards the clastogenic effect of MTX.     

Influence of vegetable oil vehicles on bone-marrow proliferation in the mouse micronucleus test

The detection of genotoxins in the mouse bone-marrow micronucleus (MN) test is sensitive to factors which may inhibit bone-marrow proliferation. We have shown that three commercially available cooking oils (olive, peanut and sunflower seed oils), commonly used as vehicles in toxicological tests, were able to induce a cytotoxic effect in mouse bone marrow. The effects observed were reversible and the magnitude of the responses varied with the oil administered. The results suggest a need to examine vehicle effects when conducting the MN test.     

Malaria infection modulates effects of genotoxic chemicals in the mouse bone-marrow micronucleus test

Malaria has been reported to modulate the activity of cytochrome-P450 enzymes (CYP). Since CYPs are involved both in the activation and detoxication of xenobiotics, we investigated whether malaria would modify the effects of chemical carcinogens in the bone-marrow micronucleus assay. Female C57BL6 mice were infected with Plasmodium berghei (ANKA) and treated (ip route) with cyclophosphamide (CPA, 25 mg/kg body weight), 7,12-dimethylbenz[a]anthracene (DMBA, 50mg/kg body weight) or ethyl methanesulfonate (EMS, 150 mg/kg body weight), on post-infection days 9-12 when parasitemia was > or =9% of RBC. Controls were age-paired non-infected mice. Bone marrows were sampled at 24 and 48 h (CPA), 24 h (EMS) or 48 h (DMBA) after treatment. The background incidence of polychromatic erythrocytes with micronuclei (MN-PCE) in malaria-infected mice was approximately twofold the background incidence in non-infected controls. Effects of indirect clastogens (CPA and DMBA) in the micronucleus assay were attenuated while the effect of EMS, a direct clastogen, was enhanced by infection. In a separate experiment, malaria was shown to decrease activities of ethoxy-(EROD, a marker for CYP1A) and benzyloxy-(BROD, CYP2B) resorufin-O-dealkylases in liver microsomes. The foregoing findings are consistent with the hypothesis that malaria-caused attenuation of genotoxicity arose from a down modulation of CYP isoforms that convert CPA (CYP2B) and DMBA (CYP1A) into their active metabolites.     

Evaluation of the genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) in a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) was evaluated in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse-mutation assay (Ames test), an in vitro human lymphocyte chromosome-aberration assay, an in vivo mouse bone-marrow micronucleus assay and an in vivo rat-liver UDS assay.Maximum test concentrations in in vitro assays were determined by the TTX limit of solubility in the formulation vehicle (0.02% acetic acid solution). In the Ames test, TTX was tested at concentrations of up to 200 μg/plate. In the chromosome-aberration assay human lymphocytes were exposed to TTX at concentrations of up to 50 μg/ml for 3 and 20 h in the absence of S9, and for 3 h in the presence of S9. For the in vivo assays, maximum tested dose levels were determined by the acute lethal toxicity of TTX after subcutaneous administration. In the mouse micronucleus assay TTX dose levels of 2, 4 and 8 μg/kg were administered to male and female animals, and bone-marrow samples taken 24 and 48 h (high-dose animals only) after administration. In the UDS assay, male rats were given TTX on two occasions with a 14-h interval at dose levels of 2.4 and 8 μg/kg, the last dose being administered 2 h before liver perfusion and hepatocyte culturing. Relevant vehicle and positive control cultures and animals were included in all assays.TTX was clearly shown to lack in vitro or in vivo genotoxic activity in the assays conducted in this study. The results suggest that administration of TTX as a therapeutic analgesic agent would not pose a genotoxic risk to patients.     

Evaluation of the micronucleus assay in bone marrow and peripheral blood of rats for the determination of cigarette mainstream-smoke activity

The mammalian in vivo micronucleus assay is widely used as part of the genotoxicity testing battery required during the development of new drugs. As such, the in vivo micronucleus assay has been used in a battery of assays for the assessment of cigarette ingredients or design modifications to help ensure that there is no increase in risk or any new risk introduced by these additions or modifications. The present series of studies was conducted to optimize and evaluate this assay for the assessment of the effects of mainstream smoke on the micronucleus frequency in the bone marrow and peripheral blood of rats. In a first experiment, the optimal conditions for performing the micronucleus assay in these tissues were determined. This was done by use of two compounds known for their micronucleus-inducing activity, i.e., the clastogen cyclophosphamide and the aneugen colchicine. In a second experiment, the effects of tube restraint on untreated control rats were investigated. In a third experiment, the optimal conditions were used to assess the clastogenic/aneugenic activity of cigarette smoke in Sprague-Dawley rats. The rat micronucleus assay in both bone marrow and peripheral blood is able to detect clastogenic and aneugenic activity. The flow cytometric determination of micronucleated cells in rat blood is at least as sensitive as determinations in bone marrow. No statistically significant differences were observed in micronucleus frequencies between rats with and without the additional stress of tube restraint; however, the cautious approach would be to use a fresh-air-exposed group (with tube restraint) as the negative control in inhalation experiments. Using the conditions identified as optimal in the above-mentioned experiments, the micronucleus assay was not able to detect effects induced by smoke from conventional cigarettes. Nevertheless, the micronucleus assay will remain a valuable tool as part of a testing battery used to investigate possible adverse effects related to product modifications.     

Evaluation of the micronucleus assay in bone marrow and peripheral blood of rats for the determination of cigarette mainstream-smoke activity

The mammalian in vivo micronucleus assay is widely used as part of the genotoxicity testing battery required during the development of new drugs. As such, the in vivo micronucleus assay has been used in a battery of assays for the assessment of cigarette ingredients or design modifications to help ensure that there is no increase in risk or any new risk introduced by these additions or modifications. The present series of studies was conducted to optimize and evaluate this assay for the assessment of the effects of mainstream smoke on the micronucleus frequency in the bone marrow and peripheral blood of rats. In a first experiment, the optimal conditions for performing the micronucleus assay in these tissues were determined. This was done by use of two compounds known for their micronucleus-inducing activity, i.e., the clastogen cyclophosphamide and the aneugen colchicine. In a second experiment, the effects of tube restraint on untreated control rats were investigated. In a third experiment, the optimal conditions were used to assess the clastogenic/aneugenic activity of cigarette smoke in Sprague-Dawley rats. The rat micronucleus assay in both bone marrow and peripheral blood is able to detect clastogenic and aneugenic activity. The flow cytometric determination of micronucleated cells in rat blood is at least as sensitive as determinations in bone marrow. No statistically significant differences were observed in micronucleus frequencies between rats with and without the additional stress of tube restraint; however, the cautious approach would be to use a fresh-air-exposed group (with tube restraint) as the negative control in inhalation experiments. Using the conditions identified as optimal in the above-mentioned experiments, the micronucleus assay was not able to detect effects induced by smoke from conventional cigarettes. Nevertheless, the micronucleus assay will remain a valuable tool as part of a testing battery used to investigate possible adverse effects related to product modifications.     

Assessment of the genotoxicity of imidacloprid and metalaxyl in cultured human lymphocytes and rat bone-marrow

Imidacloprid and metalaxyl are two pesticides that are widely used in agriculture, either separately, or in combination. These agents were studied for their possible genotoxic effects with respect to the following cytogenetic end-points: (1) in vitro micronucleus (MN) formation and sister-chromatid exchange (SCE) induction in human lymphocytes and (2) in vivo micronucleus induction in polychromatic erythrocytes (PCEs) of the rat bone-marrow. The results of the MN analysis indicate that MN frequencies after treatment with both pesticides, separately or as a mixture, do not significantly differ from those in the controls except after treatment with metalaxyl alone at 50 microg/ml (p<0.05). The results of the SCE analysis show that SCE frequencies after treatment with imidacloprid do not differ significantly from those in the controls. A statistically significant increase (p<0.05) in SCE frequency resulted from treatments with metalaxyl at 5, 10 and 100 microg/ml and with the combination of imidacloprid and metalaxyl at 100 and 200 microg/ml. Finally, the in vivo micronucleus assay with rat bone-marrow polychromatic erythrocytes showed a statistically significant effect upon separate treatments with imidacloprid and metalaxyl at doses of 300 mg/kg body weight (b.w.) (p<0.01) or upon combined treatment with 200 mg/Kg b.w. (p<0.001) and 400 mg/kg b.w. (p<0.05).     

Relative distribution of mature erythrocytes, polychromatic erythrocytes (PE) and micronucleated PE on mouse bone-marrow smears: control observations

Maps are presented showing the distribution of normal erythrocytes (NE), polychromatic erythrocytes (PE) and micronucleated PE (MPE) on 3 mouse bone-marrow smears. Adjacent areas of each slide were assessed along their whole length, and it was found that both the incidence of MPE and the ratio of PE:NE varied between different regions of the slide. These two variables were essentially independent of each other. The maps give the impression of clusters or islands of MPE on the slide, but these become less significant when the number of erythrocytes in those areas is taken into account. The present data confirm our previous conclusion that the resolving power of the bone-marrow micronucleus assay is related to the number of PE assessed for MPE per slide. Our data also illustrate that in instances where small departures from control values are observed, clarification/confirmation of the result can be obtained by extending the assessment of slides for MPE. It is proposed that the present recognition of the inherent sensitivity contained within this assay necessitates careful examination of the criteria for a positive response. The selection of PE suitable for assessment is discussed, and our recent 24-h oral corn oil control data are analysed.     

The assessment of micronucleated polychromatic erythrocytes in rat bone marrow. Technical and statistical considerations

Traditionally, the mouse is the species of choice for the rodent bone marrow micronucleus assay (MN). However, the rat is used for most other toxicological studies. The suitability of the rat as a test species for the MN was therefore investigated. In this paper, the methodological aspects of the assay have been considered. The distribution and incidence of micronucleated polychromatic erythrocytes (MPEs) on bone marrow slides prepared by two techniques, the conventional smear and the paint-brush technique, were assessed in control and cyclophosphamide-dosed male and females rats. MPEs were shown to be homogeneously distributed when assessed over a large number of PEs on slides prepared by both techniques, but when viewed over a few hundred PEs (less than 500 PEs), the incidence of MPEs on the same slides was seen to vary considerably (0-10 MPEs/500 PEs). Variability was within acceptable limits when at least 1000 PEs/animal were analysed. The spontaneous incidence of MPEs in the AP rat is low (0-2 MPEs/1000 PEs). Cyclophosphamide increased the incidence markedly and there was a wide inter-animal variability in the response (10-40 MPEs/1000 PEs). The paint-brush technique is considered technically simpler and recommended over the smear technique. This study shows that MPEs can be accurately scored in the bone-marrow of the rate provided due consideration is given to staining and sample size of PEs analysed per animal.     

Comparative analysis of cytotoxic, genotoxic and antioxidant effects of 2,2,4,7-tetramethyl-1,2,3,4-tetrahydroquinoline and ethoxyquin on human lymphocytes

2,2,4,7-Tetramethyl-1,2,3,4-tetrahydroquinoline (THQ) is a new synthetic compound with potential antioxidant activity. In this study, cytotoxic, genotoxic and antioxidant activities of THQ were studied on human lymphocytes with the use of the trypan blue exclusion assay, the TUNEL method, the comet assay and the micronucleus test. The activities of THQ were compared with those of a structurally similar compound-ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline, EQ), which is used in animal feeds as a preservative. Cytotoxic effects of THQ were observed after 1-h treatment at the concentration of 500 microM and after 24-h treatments at the concentrations of 250-500 microM. Although the micronucleus test did not reveal a genotoxic effect of THQ, in the comet assay the statistically significant increase in DNA damage was observed as compared with the control. On the other hand, the protection of human lymphocytes against DNA damage induced by hydrogen peroxide suggests an antioxidant activity of THQ. The comparative analysis of THQ and EQ activities performed in these studies revealed that THQ was less cytotoxic and less genotoxic than EQ. Slightly lower antioxidant activity of THQ was also shown in the comet assay when it was used at the lower studied doses (1-5 microM), but for the highest one (10 microM) its efficiency was similar to that of EQ. In the micronucleus assay THQ was more effective than EQ in protecting the cultured lymphocytes from clastogenicity of H2O2. We believe that THQ is worthy of further detailed studies on its antioxidant properties to confirm its usefulness as a preservative.     

Micronucleus induction in bone-marrow cells following consumption of cooked beef in mice. Preliminary investigations.

The bone-marrow micronucleus assay was used to investigate whether the consumption of cooked meat could induce chromosome damage. There was no difference in the micronucleus frequency of mice on normal diet (cereal-based, non-purified diet) and mice on normal diet supplemented with microwaved meat. However, supplementing the normal diet with well-done pan-fried meat or rare charcoal-barbecued meat or well-done charcoal-barbecued meat produced significant increments in the micronucleus frequency of polychromatic erythrocytes--the increments were of the order of 73% (p = 0.045), 90% (p = 0.047) and 136% (p = 0.001) respectively and they were observed after a 21-day feeding trial. These results suggest that ingestion of well-done pan-fried or barbecued meat may increase genetic damage, however, the accompanying decreased intake of vegetable constituents may have also contributed to the observed changes.     

Evaluation of the mutagenic potential of the hair dye N-methyl-amino-2-nitro-4-N',N'-bis-(2-hydroxyethyl)-aminobenzene in a battery consisting of Drosophila and mammalian cytogenetic assays

The compound N-methyl-amino-2-nitro-4-N', N'-bis(2-hydroxyethyl)-aminobenzene was tested for mutagenic activity in the sex-linked recessive lethal test with Drosophila melanogaster, the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) with Chinese hamster ovary (CHO) cells in vitro, and the micronucleus test with mouse bone-marrow cells in vivo. Consistently negative results were obtained with the 3 tests. The SCE tests gave positive results with prolonged treatments. It is concluded that reliable decisions about mutagenic activity cannot be based on the induction, in vitro, of SCEs alone.