Evaluation of genotoxic potential of synthetic progestin chlormadinone acetate

The genotoxicity study of a synthetic progestin chlormadinone acetate, was carried out on mouse bone marrow cells using sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) as parameter, chlormadinone acetate was studied at three different doses, i.e. 5.62, 11.25 and 22.50 mg/kg body weight and was found to be non-genotoxic at 5.62 mg/kg body weight. But at 11.25 and 22.50 mg/kg of body weight chlormadinone acetate increases SCE (P < 0.001) and CA (P < 0.01) at significant level compared to normal control. The results suggests a genotoxic and cytotoxic effect of chlormadinone acetate in mouse bone marrow cells.     

In vivo and in vitro studies on the genotoxicity of cadmium chloride in mice

The genotoxic effect of cadmium chloride was evaluated in chromosomes of experimental mice using in vivo and in vitro studies. In vivo the induction of micronuclei, sister chromatid exchange in mouse bone marrow and chromosomal aberrations in both somatic and germ cells was investigated. Doses 1.9, 5.7 and 7.6 mg kg(-1) body wt. (single i.p. treatment) induced a significant and dose-dependent increase in the percentage of polychromatic erythrocytes with micronuclei. Such a percentage reached 2.1% with the highest tested dose, compared with 0.57% for the control (non-treated) and 2.2% for mitomycin c as the positive control. The dose of 1.9 mg kg(-1) body wt. had no significant effect with respect to sister chromatid exchange (SCE) but the doses of 5.7 and 7.6 mg kg(-1)body wt. increased the frequency of SCEs significantly. The frequency of SCE reached 7.35 +/- 0.26 per cell after treatment with the highest tested dose, which is a less than twofold increase compared with the control frequency of 4.6 +/- 0.42 per cell. However mitomycin c induced a much higher effect (12.1 +/- 0.73). Cadmium chloride also induced a significant increase in the percentage of chromosomal aberrations in mouse bone marrow at the doses of 5.7 and 9.5 mg kg(-1) body wt. (single i.p. treatment). The effect is a function of cadmium chloride concentration. Moreover, cadmium chloride induced its maximum effect concerning the induction of chromosomal aberrations in mouse bone marrow cells 24 h after treatment, compared with 12 and 48 h. In germ cells, chromosomal aberrations were observed in mouse spermatocytes 12 days post-treatment with the dose of 5.7 mg kg(-1) body wt. Moreover, a pronounced reduction in the number of spermatocytes was observed after administration of cadmium chloride (0.9, 1.9 and 5.7 mg kg(-1) body wt.) In in vitro studies, the three tested concentrations of 10, 15 and 20 microgram ml(-1) cadmium chloride induced a statistically significant increase in the frequency of SCEs in cultured mouse spleen cells. The concentrations of 15 and 20 microgram ml(-1) also induced chromosomal aberrations in mouse spleen culture. The ability of vitamin C (l-ascorbic acid) to minimize the incidence of chromosomal aberrations induced by cadmium chloride in cultured mouse spleen cells was investigated. Vitamin C at the concentrations of 3 and 6 microgram ml(-1) significantly minimized the percentage of aberrant cells induced by cadmium chloride.     

Sister-chromatid exchanges induced by disulfiram in bone marrow and spermatogonial cells of mice treated in vivo.

Disulfiram is a widely used drug to treat alcoholism due to its capacity to inhibit the metabolism of acetaldehyde; however, its genotoxic potential is not well known. Thus, the aim of this investigation was to determine whether the chemical may induce sister-chromatid exchanges (SCEs) in an in vivo study using mouse bone marrow and spermatogonial cells. We used doses of 200, 400 and 800 mg/kg body weight and compared the obtained data with the values determined in a negative control group as well as with a positive control group (cyclophosphamide, 50 mg/kg). The results in both systems indicated a weak genotoxic response by the chemical. In the case of bone marrow, a significant SCE level was achieved only with the high tested dose, but in spermatogonial cells the three doses tested showed a significant difference with respect to the negative control. No significant alterations in the mitotic index or in the cell proliferation kinetics were observed in somatic cells. Concerning the effect of cyclophosphamide, an increase in the level of SCEs was observed in both types of cells, reaching more than three times the values obtained in their respective control groups.     

The kinetics of in vivo sister chromatid exchange induction in mouse bone marrow cells by ethylnitrosourea and methylnitrosourea

Administration of ethylnitrosourea (ENU) (20, 25, 40, 50, and 60 mg/kg body weight) or methylnitrosourea (MNU) (25, 40, 50, 60, 75, and 80 mg/kg body weight) to male CD-1 mice 2 hr after sc implantation of a 5-bromo-2'-deoxyuridine (BrdUrd) pellet (55 mg) resulted in a dose-dependent increase in sister chromatid exchanges (SCE) in bone marrow cells. Treatment with ENU (50 mg/kg body weight) at several time points prior to BrdUrd implantation resulted in a multiphasic curve of SCE induction indicating at least two events that result in SCEs. Treatment with ENU at the time of BrdUrd implantation and post-BrdUrd reflected a similar mechanism apparent in the pre-BrdUrd curve. Treatment with MNU (50 mg/kg body weight) pre- and post-BrdUrd resulted in a linear monophasic curve of SCE induction in both the pre- and post-BrdUrd time periods. The overall MNU time-course curve resembled an inverted V function suggesting the mechanisms of SCE induction for ENU and MNU are different. These observations suggest that at least one explanation for the differences in the time courses for ENU and MNU SCE induction may result from a more persistent lesion being induced by ENU. In addition, these results indicate that in vivo SCE protocols which utilize a single acute chemical exposure at or near the time of BrdUrd labeling may not be useful for judging the relative activities of genotoxic agents.     

Antigenotoxic effects of ascorbic acid against megestrol acetate-induced genotoxicity in mice

The genotoxicity of megestrol acetate was studied in mouse bone marrow cells using sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) as parameters. Megestrol acetate (8.12, 16.25 and 32.50 mg/ kg of body weight) was injected intraperitoneally separately in different groups of animals. Both CAs and SCEs were statistically increased at 16.25 and 32.50 mg/kg of body weight. Our earlier in vitro studies show the generation of free oxygen radicals, by synthetic progestins responsible for the genotoxic damage. As the genotoxic effects of steroids can be reduced by natural products having antioxidant properties, and ascorbic acid possesses antioxidant activity, ascorbic acid (20, 40 or 60 mg/kg of body weight) administered together with megestrol acetate (32.50 mg/kg of body weight) significantly decreased CAs and SCEs, suggesting an antigenotoxic role of ascorbic acid against megestrol acetate-induced genotoxic damage in mice bone marrow cells. The antigenotoxic effect was clearly dose dependent. The highest protective effect was observed at 60 mg/kg body weight of ascorbic acid treated with 32.50 mg/kg body weight of megestrol acetate.     

Sister chromatid exchange induced by 'pan masala' (a betel quid ingredient) in male mice in vivo

Sister chromatid exchange (SCE) induced in vivo by 'pan masala', a betel quid ingredient, was studied in male mice. The mice were injected ip with an aqueous suspension of pan masala at doses of 5, 12.5, 25, 50, 100 or 200 mg pan masala/kg body weight. A significant dose-related increase in SCE was observed (Cochran-Armitage trend test). The minimum effective dose was 25 mg/kg. The two highest doses caused significant delays in the cell cycle. These results indicate that pan masala is a potential DNA-damaging agent and cytotoxic to bone marrow cells.     

Re-examination of potassium sorbate and sodium sorbate for possible genotoxic potential

Potassium sorbate and sodium sorbate were investigated for possible genotoxic actions using the Salmonella/mammalian-microsome test, HGPRT and sister chromatid exchange (SCE) test with Chinese hamster ovary cells, the micronucleus test on bone marrow cells of mice and Chinese hamsters, and the chromosome aberration and SCE test on Chinese hamsters. In all the in vitro tests no signs of genotoxicity were detected. Whereas no in vivo mutagenicity of potassium sorbate and sodium sorbate with freshly prepared aqueous solutions and with stored potassium sorbate was found, investigations with stored sodium sorbate revealed weak clastogenic activity by increased chromosome aberrations and elevated numbers of micronuclei at doses of 200 mg/kg body weight, but no induction of SCEs.     

Sister chromatid exchanges produced by imipramine and desipramine in mouse bone marrow cells treated in vivo

Imipramine and desipramine are two widely used tricyclic antidepressants which have shown conflicting results in regard to their in vitro genotoxic evaluation. The aim of this investigation was to determine the capacity of these compounds to induce in vivo sister-chromatid exchanges (SCEs) in mouse bone marrow cells. For each compound, the animals were organized in five groups constituted by five individuals. They were intraperitoneally (ip) administered with the test substances as follows: a negative control group treated with 0.4 ml of distilled water, a positive control group administered with cyclophosphamide (70 mg/kg), three groups treated with imipramine (7, 20 and 60 mg/kg), and three other groups treated with desipramine (2, 20 and 60 mg/kg). The general procedure included the subcutaneous implantation to each mouse of a 5-bromodesoxyuridine tablet (45 mg), and 1 h later, the administration of the chemicals involved. Twenty-one hours after the tablet implantation, the mice received colchicine, and 3 h later their femoral bone marrow was obtained in KCL, fixed, and stained with the Hoechst-Giemsa method. The results showed that both compounds were SCE inducers, starting from the second tested dose. The response of these compounds was dose-dependent, and showed that the highest tested dose increased about four times the SCE control level. The cellular proliferation kinetics was not affected by the chemicals, and the mitotic indexes were slightly diminished with the highest dose. These results indicate an in vivo genotoxic potential for both chemicals, and suggest that it is pertinent to follow their evaluation in other models.     

Effects of chlorophyllin on acetaldehyde: lack of modulation of the rate of sister-chromatid exchanges in mouse bone marrow,and of complex formation in aqueous solution

Acetaldehyde (Ace) is a reactive compound widely found in natural and industrialized products. On the other hand, chlorophyllin (Chl) is a chloropyll derivative which has shown DNA modulatory effects in several models. The first aim of the present study was to determine the capacity of Ace to increase the rate of sister-chromatid exchanges (SCEs) in mouse bone marrow cells in vivo, as well as to determine its capacity to modify the mitotic index (MI) and the average generation time (AGT). For this experiment we tested four dosages of Ace by the i.p. route (0.4, 4.0, 40.0 and 400 mg/kg), and found a genotoxic effect with the two highest dosages (more than double the basal level was observed with 400 mg/kg). We also found that none of the doses tested modified the MI or the AGT. A second objective was to explore the potential of Chl to modulate the genotoxicity of Ace in the same model. We evaluated whether an oral administration of Chl (2.0, 6.0 and 10.0 mg/kg), given 1 h before an i.p. administration of Ace (100 mg/kg), could modulate the SCEs produced by the mutagen. The result showed a similar SCE rate in both, the Ace-treated mice and those administered with the two chemicals, indicating that Chl was not a modulatory chemical on the genotoxicity of Ace. No modifications were observed concerning the MI or the AGT either. A third objective was to determine whether the two compounds (Ace and Chl) may form a molecular complex in aqueous solution. In agreement with the lack of modulatory effect by Chl, a reversed HPLC and a spectrophotometric analysis showed that the two compounds were unable to form a complex. This report confirms the importance of the specificity concerning the interaction mutagen/antimutagen.     

Genotoxicity of selected pesticides in the mouse bone-marrow micronucleus test and in the sister-chromatid exchange test with human lymphocytes in vitro

Selected pesticides, (aldicarb, 1,3-dichloropropene, methidathion, parathion, triadimefon, vinclozolin) were tested for their clastogenic and aneugenic activities in the mouse bone-marrow micronucleus (MN) test in vivo and for their sister-chromatid exchange-inducing activities in human lymphocytes in vitro in the presence and absence of an exogenous metabolizing system from rat-liver S9. 1,3-Dichloropropene significantly increased the frequencies of micronucleated polychromatic erythrocytes (PCE) in bone-marrow cells of female mice from 3.3 MN/1000 PCE to 15.3 MN/1000 PCE (187 mg per kg body weight). 1,3-Dichloropropene (100 μM) induced 16.0 SCE/metaphase after 24 h of incubation as compared with the basal rate of 11.2 SCE/metaphase (− S9) and of 15.4 SCE/metaphase as compared with 10.5 SCE/metaphase of the control (+ S9). These values were statistically significantly different from each other. The other pesticides tested did neither increase the rate of micronuclei significantly in polychromatic erythrocytes in male nor in female animals. Aldicarb and methidathion induced a significant increase in SCEs in human lymphocytes in vitro only without the metabolic activating system: aldicarb, 5 μm, 24 h incubation: 15.5 SCE/metaphase; control: 12.6 SCE/metaphase; methidathion, 100 μM, 24 h incubation: 15.8 SCE/metaphase, control: 11.1 SCE/metaphase. Parathion, triadimefon and vinclozolin did not have any SCE-inducing effects.     

Induction of sister chromatid exchanges by 2,4-dichlorophenoxyacetic acid in somatic and germ cells of mice exposed in vivo.

2,4-dichlorophenoxyacetic acid (2,4-D) is one of the most widely used selective herbicides throughout the world; however, the studies that have been conducted to establish its genotoxic potential have given conflicting results. The aim of this investigation was to determine whether the herbicide increases the frequency of sister chromatid exchanges (SCEs) in bone marrow and spermatogonial cells of mice exposed in vivo. The experiment included an oral administration of 2,4-D to three groups of mice (50,100 and 200 mg/kg), as well as to a control group of animals administered with distilled water, pH 10.5 and another group injected with cyclophosphamide (50 mg/kg). In somatic cells, the results showed a significant SCE increase with the two high doses tested, a response that was manifested in a dose-dependent manner. With regard to the mitotic index and the cell proliferation kinetics, there were no modifications exerted by 2,4-D; however, cyclophosphamide induced cytotoxic damage and a cell-cycle delay. With respect to the germ cells, the genotoxic results were similar to those described earlier; that is, there was a significant SCE increase induced by the two high 2,4-D doses tested and a higher genotoxic damage was observed in the animals treated with cyclophosphamide. Our investigation established that 2,4-D is a moderate genotoxicant in mice treated in vivo with high doses, and suggests a minor hazard for humans in the present conditions of its use.     

Inhibition of benzo[a]pyrene-induced bone marrow micronuclei formation by diallyl thioethers in mice.

Diallyl thioethers (DATEs), naturally occurring compounds present in garlic, were investigated for their putative ability to inhibit benzo[a]pyrene-induced genotoxicity in ICR and C3H strains of mice. The mouse bone marrow micronucleus assay was used as an indicator of in vivo genotoxicity. A dose of 0.67 mmol total DATEs/kg body weight inhibited formation of micronucleated polychromatic erythrocytes (MPCEs) by 24%, and 0.33 mmol DATEs inhibited formation of MPCEs by 45%. Possibly the toxicity of DATEs accounted for less inhibition with the higher dose. Formation of MPCEs were inhibited only slightly by DATEs in C3H mice. These results indicate that the mouse bone marrow micronucleus assay can be used to identify organosulfur components of garlic that inhibit genotoxicity.     

Antigenotoxic effect of apigenin against mitomycin C induced genotoxic damage in mice bone marrow cells

The antigenotoxic effect of apigenin was studied against the genotoxic damage induced by mitomycin C on mouse bone marrow cells using sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) as parameters. Apigenin was studied at three different doses i.e. 10, 20 and 40 mg/kg b.w. and was found to be non-genotoxic at all the above three doses. Mitomycin C at 2 mg/kg b.w. was given along with the 10, 20 and 40 mg/kg bw of apigenin. A significant decrease in SCEs and CAs was observed, suggesting a protective role of apigenin against the genotoxicity of mitomycin C on mice bone marrow cells.     

Suppressive effect of aspirin on chromosome aberration induced by mitomycin C in mice

Chromosome aberrations induced by mitomycin C (MMC) were suppressed by aspirin in a mouse micronucleus test with peripheral blood and bone marrow cells. Aspirin at doses of 0.5, 5, and 50 mg/kg was injected intraperitoneally or per administered orally 0.5, 6, or 24 h after administration of MMC and then peripheral blood and/or bone marrow cells were sampled 48 h after administration of MMC. The suppressive effect of aspirin was more pronounced in the aspirin-treated groups 24 h than 0.5 and 6 h after administration of MMC. In the aspirin-treated group at 24 h, the frequency of polychromatic erythrocytes with micronuclei was decreased by about 60-80% after intraperitoneal injection and by about 40-70% after oral administration. It is suggested that aspirin may directly act on MMC metabolites, but not on MMC itself.     

Increased sister-chromatid exchange frequency in preimplantation mouse embryos after maternal cyclophosphamide treatment before implantation

The effect of a toxic agent in vivo on sister-chromatid exchange (SCE) frequency of preimplantation mouse embryos and bone marrow cells was determined using combined in vivo treatment and in vitro culture in the presence of 5-bromo-2-desoxyuridine (BrdU) for differential staining of the chromatids. In mice exposed to cyclophosphamide (CPA) on day 2 of pregnancy SCE frequency was increased dose-dependently both in embryos and bone marrow cells 1 h after treatment. It returned to control values in bone marrow cells obtained 24 h after exposure but was still significantly increased in the embryos. A closer time-related evaluation of SCE on day 2 of gestation showed a significant increase in SCE in bone marrow cells and in embryos obtained 20-60 min after CPA treatment. Furthermore, SCE frequency was the most sensitive toxicological endpoint to detect embryotoxic effects of CPA treatment before implantation, since it was significantly increased in embryos exposed to 5 mg/kg CPA on day 2 of pregnancy while embryolethality at term and both cytogenetical (structural chromosomal aberrations, micronuclei) and developmental parameters (cell number, differentiation in culture) before implantation did not indicate any toxic effect.     

Further evidence that dichloromethane does not induce chromosome damage

Dichloromethane (DCM) is a widely used industrial solvent that has been determined to be a carcinogen in rats and mice. In vitro and in vivo analyses of chromosome damage induced by this agent have provided conflicting results. In order to further investigate the clastogenic potential of DCM in vivo, we analyzed sister chromatid exchanges (SCEs) and chromosome aberrations (CAs) in mouse bone marrow cells following intraperitoneal exposures of 100-2000 mg kg-1 DCM. Dichloromethane failed to increase the frequencies of either SCEs or CAs.     

In vivo cytogenetic studies on mice exposed to Orange G, a food colourant

Orange G, a monoazo dye, used as a food colourant, was evaluated with in vivo cytogenetic assays to determine its genotoxicity. Swiss albino male mice were exposed to Orange G through intraperitoneal injections. Bone marrow cells isolated from femora were analyzed for sister chromatid exchanges (SCE) and chromosome aberrations. The results showed that the incidence of SCEs and chromosome aberrations were significantly higher than controls at certain concentrations. 25 mg/kg of Orange G was found to be the minimum effective dose for the induction of both SCEs and chromosome aberrations. Orange G is thus found to be clastogenic and genotoxic in vivo in mice.     

Anti-genotoxicity and glutathione S-transferase activity in mice pretreated with caffeinated and decaffeinated coffee.

In vivo anti-genotoxic effects of caffeinated and decaffeinated instant coffee were compared in mice after pretreatment either by gavage for 10 consecutive days or in the drinking water for 2 weeks. Changes in hepatic sulfhydryl (-SH) content and glutathione S-transferase (GST) activity were evaluated in pretreated animals. Both caffeinated and decaffeinated instant coffee induced a moderate increase in -SH content and GST activity following pretreatment (with 70, 140 and 280 mg/kg body weight) by gavage for 10 days. This enhancement was not always dose dependent. The maximum effect on GST activity was observed at a dose of 140 mg/kg body weight/day. However, such an effect was not observed after administration of drinking water containing 2% caffeinated/decaffeinated instant coffee for 2 weeks. Results of the bone marrow micronucleus test for evaluating genotoxic effects revealed that both caffeinated and decaffeinated instant coffee (140 mg/kg body weight/day) could exert significant anti-genotoxic effects against ip injected benzo[a]pyrene (BP), cyclophosphamide (CPH), 7,12-dimethylbenz[a]anthracene (DMBA), mitomycin C (MMC) and procarbazine (PCB) in animals pretreated by gavage. Anti-genotoxic effects against BP, DMBA and urethane (URE) were evaluated in animals that received drinking water containing 2% caffeinated/decaffeinated instant coffee for 2 weeks. With the exception of the anti-genotoxic effect of decaffeinated coffee against DMBA, there was no significant change in genotoxicity after the above pretreatment. From this work, there is no evidence for any significant difference in the in vivo anti-genotoxicity of caffeinated and decaffeinated instant coffee.     

Genotoxic effects of silver nanoparticles with/without coating in human liver HepG2 cells and in mice

With the rapid expansion of human exposure to silver nanoparticles (AgNPs), the genotoxicity screening is critical to the biosafety evaluation of nanosilver. This study assessed DNA damage and chromosomal aberration in the human hepatoma cell line (HepG2) as well as the effects on the micronucleus of bone marrow in mice induced by 20 nm polyvinylpyrrolidone‐coated nanosilver (PVP‐AgNPs) and 20 nm bare nanosilver (AgNPs). Our results showed that the two types of AgNPs, in doses of 20‐160 μg/mL, could cause genetic toxicological changes on HepG2 cells. The DNA damage degree of HepG2 cells in 20 nm AgNPs was higher than that in 20 nm PVP‐AgNPs, while the 20 nm PVP‐AgNPs caused more serious chromosomal aberration than 20 nm AgNPs. Both kinds of AgNPs caused genetic toxicity in a dose‐dependent manner in HepG2 cells. In the micronucleus test on mouse bone marrow cells, in doses of 10, 50 and 250 mg/kg body weight administered orally for 28 days once a day, the two kinds of AgNPs have no obvious inhibitory effect on the mouse bone marrow cells, and the effect of chromosome aberration could be documented at the high dose of 250 mg/kg. These results suggest that AgNPs have genotoxic effects in HepG2 cells and limited effects on bone marrow in mice; both in vitro and in vivo tests could be of great importance on the evaluation of genotoxicity of nanosilver. These findings can provide useful toxicological information that can help to assess genetic toxicity of nanosilver in vitro and in vivo.     

Effect of chlorophyllin on mercuric chloride-induced clastogenicity in mice.

The effect of chlorophyllin (1.5 mg/kg body weight) on the clastogenicity of mercuric chloride (HgCl2) was studied in vivo in mouse bone marrow cells. HgCl2 (3.0, 6.0 and 12.0 mg/kg body weight) administered by gavage induced chromosomal aberrations at frequencies directly proportional to the dose. Chlorophyllin was not clastogenic, and significantly reduced the mitotic index when given alone. Chlorophyllin administered simultaneously with HgCl2 significantly reduced the frequencies of chromosomal aberrations in a dose-dependent manner. When given simultaneously with the lowest HgCl2 concentration tested (3.0 mg/kg body weight), chlorophyllin provided total protection. A lower degree of protection was given by chlorophyllin administered 2 hr before HgCl2. The data demonstrate the potential of green plant components to modify the genotoxic activity of HgCl2 when administered orally.