Genotoxicity evaluation of norethisterone acetate

Genotoxic evaluation of a commonly used progestogen, norethisterone acetate, was undertaken using a combination of short-term in vitro and in vivo assays. The clastogenic potentiality of norethisterone acetate was evident from the chromosome aberrations and sister chromatid exchanges induced both with and without S9 mix in cultured human lymphocytes and also from the increased frequency of micronuclei formation and sister chromatid exchanges in mice. However, in the Ames Salmonella assay, both with and without S9 mix and in host-mediated assay, norethisterone acetate was unable to cause any significant increase/decrease in the His⁺ revertants/plate.     

Mutagenicity and genotoxicity of dicapthon insecticide

Mutagenic and genotoxic effects of dicapthon were investigated by using the bacterial reverse mutation assay in Salmonella typhimurium TA97, TA98, TA100 and TA102 strains with or without metabolic activation system (S9 mix), and chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) tests in human peripheral blood lymphocytes in vitro. Dicapthon was dissolved in dimethyl sulfoxide for all test systems. 0.1, 1, 10 and 100 μg/plate doses of dicapthon were found to be weakly mutagenic on S. typhimurium TA 98 without S9 mix. The human peripheral lymphocytes were treated with four experimental concentrations of dicapthon (25, 50, 100, and 200 μg/mL) for 24 and 48 h. Dicapthon increased the frequency of SCE only at the 100 μg/mL concentration for the 24 and 48 h applications. Dicapthon also induced abnormal cell frequency, CA/cell ratio and frequency of MN dose dependently for 24 and 48 h. Dicapthon showed a statistically significant cytotoxic effect by decreasing the mitotic index in all concentrations and a cytostatic effect by decreasing nuclear division index in 100 and 200 μg/mL concentrations for both treatment periods when compared with both untreated and solvent controls. These values decreased also in a dose dependent manner.     

Mutagenic activity of structurally related oxiranes and siloranes in Salmonella typhimurium

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

Genotoxicity testing of fluconazole in vivo and in vitro

The genotoxic effects of the antifungal drug fluconazole (trade name triflucan) were assessed in the chromosome aberration (CA) test in mouse bone-marrow cells in vivo and in the chromosome aberration, sister chromatid exchange (SCE) and micronucleus (MN) tests in human lymphocytes. Fluconazole was used at concentrations of 12.5, 25.0 and 50.0 mg/kg for the in vivo assay and 12.5, 25.0 and 50.0 microg/ml were used for the in vitro assay. In both test systems, a negative and a positive control (MMC) were also included. Six types of structural aberration were observed: chromatid and chromosome breaks, sister chromatid union, chromatid exchange, fragments and dicentric chromosomes. Polyploidy was observed in both the in vivo and in vitro systems. In the in vivo test, fluconazole did not significantly increase the frequency of CA. In the in vitro assays, CA, SCE and MN frequencies were significantly increased in a dose-dependent manner compared with the negative control. The mitotic, replication and cytokinesis-block proliferation indices (CBPI) were not affected by treatments with fluconazole. According to these results, fluconazole is clastogenic and aneugenic in human lymphocytes, but these effects could not be observed in mice. Further studies should be conducted in other test systems to evaluate the full genotoxic potential of fluconazole.     

Mutagenic and genotoxic effects of theophylline and theobromine in Salmonella assay and in vivo sister chromatid exchanges in bone marrow cells of mice.

The mutagenic and genotoxic effects of two methylxanthines, theophylline (TH) and theobromine (TB), were assessed in the Ames mutagenicity assay (in strains TA97a, TA100, TA102 and TA104) and in vivo sister chromatid exchanges (SCEs) in bone marrow cells of mice. These are the two most commonly used nervous system stimulators throughout the world. TH is used in the long-term treatment of asthma. Bacterial mutagenicity assay showed very weak mutagenic effects of both drugs in Salmonella strains TA102 and TA104 only in certain concentrations when S9 was added to it. No mutagenic effects were observed in any other strains used in this assay either with or without metabolic activation. But results of in vivo SCE assay indicate that these two drugs can induce significant SCE in bone marrow cells of mice.     

Genotoxicity of aloeemodin in vitro and in vivo

The present in vitro and in vivo experiments were undertaken to clarify the genotoxic potential of the hydroxyanthrachinone aloeemodin which can be found in different plant derived products for therapy of constipation. The results demonstrate that aloeemodin is able to induce mutagenic effects in vitro. Positive results were obtained in the chromosomal aberration assay with CHO cells, as well as in the Salmonella reverse mutation assay (frameshift mutations in strains TA 1537, TA 1538 and TA 98). No mutagenic potential of aloeemodin, however, was observed in the gene mutation assay with mammalian cells in vitro (HPRT assay in V79 cells). Each assay was performed in the presence and absence of an extrinsic metabolic activation system (S9-mix). In in vivo studies (micronucleus assay in bone marrow cells of NMRI mice; chromosome aberration assay in bone marrow cells of Wistar rats; mouse spot test [DBA/2J × NMRI]) no indication of a mutagenic activity of aloeemodin was found. Information about a possible reaction of aloeemodin with DNA was derived from an in vivo UDS assay. Hepatocytes of aloeemodin-treated male Wistar rats did not show DNA damage via repair synthesis. All these data suggest that aloeemodin is able to interact with DNA under certain in vitro conditions. However, in vivo the results that were negative did not indicate a genotoxic potential. Therefore, it may be assumed that a genotoxic risk for man might be unlikely.     

Cytogenetic consequences of hyperbaric oxygenation in the sequence of cell cycles of human peripheral blood lymphocytes

High pressure of oxygen induces chromosomal rearrangements in metaphases of human peripherical blood cells treated in vitro. An increase in the percentage of rearrangements was detected in the third mitosis, due mainly to chromatid breaks. The data of cytogenetical analysis of lymphocytes of human blood treated in vivo at 3 ata for 30 min are presented. A differential character of distribution spectrum of chromosomal aberrations induced after in vivo and in vitro treatment was noted. Some unusual alterations in chromosome morphology are described, namely, solid, not stained clear lense-like bodies surrounded by chromatin and localised in the long arm of chromatids.     

Evaluation of the genotoxicity of cis-bis(3-aminoflavone)dichloroplatinum(II) in comparison with cis-DDP

Short-term tests that detect genetic damage have provided information needed for evaluating carcinogenic risks of chemicals to man. The mutagenicity of cis-bis(3-aminoflavone)dichloroplatinum(II) (cis-[Pt(AF)2Cl2]) in comparison with cis-diamminedichloroplatinum(II) (cis-DDP) was evaluated in the standard plate-incorporation assay in four strains of Salmonella typhimurium: TA97a, TA98, TA100 and TA102, in experiments with and without metabolic activation. It was shown that cis-[Pt(AF)2Cl2] acts directly and is mutagenic for three strains of S. typhimurium: TA97a, TA98 and TA100. In comparison with cis-DDP this compound showed a weaker genotoxicity. Contrary to cis-DDP it has not shown toxic properties in the tester bacteria. The genotoxicity of both tested compounds was evaluated using chromosomal aberration, sister chromatid exchange and micronucleus assays, without and with metabolic activation, in human lymphocytes in vitro. The inhibitory effects of both compounds on mitotic activity, cell proliferation kinetics and nuclear division index were also compared. In all test systems applied, cis-[Pt(AF)2Cl2] was a less effective clastogen and a weaker inducer of both sister chromatid exchanges and micronuclei in comparison with cis-DDP, with and without metabolic activation. cis-[Pt(AF)2Cl2] has a direct mechanism of action and is less cytostatic and cytotoxic than the other compound. These results provide important data on the genotoxicity of cis-[Pt(AF)2Cl2] and indicate its beneficial properties as a potential anticancer drug, especially in comparison with cis-DDP.     

Chromosomal aberrations in cultured human lymphocytes treated with aldicarb, a carbamate pesticide

Aldicarb was tested for its ability to induce chromosomal aberrations in human peripheral lymphocytes in vitro, in the presence of an exogenous metabolic activation system. The pesticide induced an increase in the number of chromatid and chromosome breaks. The increase was higher in the presence of S9 mix. A positive linear association between frequencies of abnormal cells and dose of aldicarb was observed.     

Combined action of gamma irradiation and 5-bromodeoxyuridine on human chromosomes

A combined effect has been studied of 5-bromo-deoxyuridine (BDU) and gamma-radiation on human lymphocytes at the G0 stage. BDU is shown to induce chromatid aberrations irrespective of its radiation. The frequency of chromatid aberrations rises with the BDU concentration. BDU modifies the effect of gamma-radiation on human lymphocytes at the G0 stage, increasing the number of chromosome aberrations. BDU slows down the lymphocyte cell cycle.     

Adaptive response in different mitotic cycles after irradiation

The frequency of cells with chromosome aberrations and the number of aberrations per cell have been studied by metaphase analysis in the nonirradiated progeny of irradiated human blood lymphocytes. DNA fragmentation (DNA double-stranded breaks) has been investigated by DNA comet assay. To study the adaptive response (AR), PHA-stimulated lymphocytes were irradiated by the adaptive dose (0.05 Gy) in 24 h and by challenge dose (1 Gy) in 48 h after stimulation. The first through fourth mitoses were identified by 5-bromodeoxyuridine. It was found that the frequency of chromosome aberrations and double-strand breaks were increased in all mitotic cycles after the challenge irradiation. In most individuals, the adaptive response is induced by adaptive and challenge irradiations in the first and the second mitotic cycles (48 and 72 h after stimulation, respectively); however, it is absent in the third and the fourth mitoses. In the first mitosis (1Gy in 48 h after stimulation), only chromatid aberrations are observed; chromosome aberrations were registered in subsequent mitoses. DNA comet assay showed that the adaptive response was obvious at 48–72 h, but not 96 h, after stimulation. It can be concluded that the nonirradiated progeny of irradiated lymphocytes have genomic instability. The adaptive response is manifested up to the third mitosis and is explained by the decreasing number of chromatid and chromosome aberrations and DNA fragmentation. We suppose that double-stranded DNA breaks may be damage signals for the induction of adaptive response.     

Ratio of the frequency of chromosome aberrations and sister chromatid exchanges to the dose of the mutagenic exposure to cyclophosphamide in vivo and in vitro

Cyclophosphamide (CP) and its metabolites were used to compare the rate of chromosomal aberrations (CA) and sister chromatid exchange (SCE) in the rabbit lymphocytes in vivo and in vitro. The dose-dependent increase of cytogenetic effects rate appeared to be of linear and exponential dependence for SCE and CA, respectively, both in vivo and in vitro. The regression equation coefficients coincided in in vivo and in vitro experiments.     

Genotoxicity evaluation of tetramethylbenzenes

The three tetramethyl isomers of benzene (prenitene, 1,2,3,4-; izodurene, 1,2,3,5-; and durene, 1,2,4,5-tetramethylben- zene) were studied using in vitro mutagenicity and in vivo genotoxicity tests. Potency of mutate induction by these solvents was evaluated in Salmonella typhimurium cells with, and without S9-mix made from Aroclor 1254-induced rat liver S9. The potency of induction of micronuclei (MN) and sister chromatid exchanges (SCEs) by solvents was evaluated in bone marrow of mice. Izodurene displayed mutagenic potency in strains TA97a, TA98 and TA100 only in the absence of the S9-mix. In MN tests, all three tetramethylbenzenes demonstrated no clastogenic activity on the bone marrow cells. All the tested solvents were active as genotoxic compounds in the SCE tests, demonstrating a dose-response relationships.     

Induction of chromosome aberrations in lymphocytes of mice after subchronic exposure to benzene

The induction of chromosome aberrations in lymphocytes of mice after subchronic exposure to benzene was investigated. 4 groups of 5 Swiss (ICR) male mice were given orally a solution of benzene every day for 14 days except days 5 and 10. The daily doses were 0, 36.6, 73.2 and 146.4 mg/kg. Mice were sacrificed on day 15, lymphocytes were obtained by perfusion of the spleen and the cells were cultured in RPMI 1640 medium. After 48 h of culture, cells were harvested for cytogenetic analysis. A significant dose-dependent increase in the frequency of cells with chromatid aberrations were found (p less than 0.001). A significant increase in polyploid cells were also observed (p less than or equal to 0.05). This study represents the first report on the induction of chromosome aberrations and polyploid cells in lymphocytes of mice after subchronic exposure to benzene. Such dual activity of benzene suggests that benzene may be responsible for more human health problems than currently estimated.     

Genotoxicity assessment of ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN)

Ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN) are relatively insensitive explosive compounds that are being explored as safe alternatives to other more sensitive compounds. When used in combination with other high explosives they are an improvement and may provide additional safety during storage and use. The genetic toxicity of these compounds was evaluated to predict the potential adverse human health effects from exposure by using a standard genetic toxicity test battery which included: a gene mutation test in bacteria (Ames), an in vitro Chinese Hamster Ovary (CHO) cell chromosome aberration test and an in vivo mouse micronucleus test. The results of the Ames test showed that EDDN increased the mean number of revertants per plate with strain TA100, without activation, at 5000μg/plate compared to the solvent control, which indicated a positive result. No positive results were observed with the other tester strains with or without activation in Salmonella typhimurium strains TA98, TA1535, TA1537, and Escherichia coli strain WP2 uvrA. DETN was negative for all Salmonella tester strains and E. coli up to 5000μg/plate both with and without metabolic activation. The CHO cell chromosome aberration assay was performed using EDDN and DETN at concentrations up to 5000μg/mL. The results indicate that these compounds did not induce structural chromosomal aberrations at all tested concentrations in CHO cells, with or without metabolic activation. EDDN and DETN, when tested in vivo in the CD-1 mouse at doses up to 2000mg/kg, did not induce any significant increase in the number of micronuclei in bone marrow erythrocytes. These studies demonstrate that EDDN is mutagenic in one strain of Salmonella (TA100) but was negative in other strains, for in vitro induction of chromosomal aberrations in CHO cells, and for micronuclei in the in vivo mouse micronucleus assay. DETN was not genotoxic in all in vitro and in vivo tests. These results show the in vitro and in vivo genotoxicity potential of these chemicals.     

Cytogenetic analyses of the in vitro and in vivo responses of murine cells to peroxyacetyl nitrate (PAN)

Peroxyacetyl nitrate (PAN) is one of a class of common air pollutant formed by the action of sunlight on volatile organic compounds and nitrogen oxides. PAN has been shown to be a bacterial mutagen. To determine if PAN can cause DNA damage in mammalian cells, we exposed murine peripheral blood lymphocytes (PBLs) to various volumes of PAN in vitro and analyzed the cells for chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and DNA damage using the single cell gel (SCG) assay. At in vitro concentrations of PAN that were cytotoxic (inhibited cell division), an increase in DNA damage was noted in the SCG assay. At lower exposure levels that permitted cell division, no increases in SCEs, CAs, or DNA damage were evident. For in vivo studies, male mice were exposed nose-only by inhalation for 1 h to 0, 15, 39 or 78 ppm PAN, and their lung cells removed and cultured for the scoring of SCEs and CAs. In addition, PBLs and lung cells were analyzed by the SCG assay. No dose-related effects were found in any of the assays. These data indicate that PAN does not appear to be a potent clastogen or DNA damaging agent in mammalian cells in vivo or in vitro.     

Chromosome damage induced in human lymphocytes by 5-methoxypsoralen and 8-methoxypsoralen plus UV-A

The induction of structural chromosome aberrations and sister chromatid exchanges (SCE) was studied in human lymphocytes in vitro after treatment with the two bifunctional furocoumarins 5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MOP) in the presence of UV-A. The results show that both psoralens induce a dose-dependent increase in the SCE rate as well as in structural chromosome aberrations. 5-MOP was 2.0-2.5 times more effective for the induction of chromosome breaks and had a slightly stronger effect with respect to SCE induction. A significant influence on proliferation kinetics could be observed only with 5-MOP plus UV-A.     

Cytogenetic studies of three triazine herbicides. II. In vivo micronucleus studies in mouse bone marrow

Atrazine, simazine, and cyanazine are widely used preemergence and postemergence triazine herbicides that have made their way into the potable water supply of many agricultural communities. Although there are several contradictory genotoxicity studies in the literature, our previous in vitro studies with human lymphocytes showed that atrazine, simazine, and cyanazine did not induce sister chromatid exchanges (SCEs) or chromosome aberrations (CAs) up to the limits of solubility in aqueous medium using 0.5% dimethyl sulfoxide. To expand upon these results and to ensure that our in vitro findings could be replicated in an in vivo system, mice were treated with each triazine by two intraperitoneal injections, 24h apart. The animals were sacrificed and the bone marrow removed for micronucleus (MN) analysis, 24h after the last injection. Two to four independent trials were performed for MN analysis in polychromatic erythrocytes, and in some trials the spleen was removed, cultured, and analyzed for SCEs and CAs. None of the triazines investigated induced MN in the bone marrow, even at doses that caused significant bone marrow suppression and/or death. These results indicate that atrazine, simazine, and cyanazine are not genotoxic as measured by the bone marrow MN assay in mice following high dose exposures.     

Evaluation of the cytogenetic effect of the herbicide treflan and of a number of its metabolites on mammalian somatic cells

Investigation of the cytogenetic effect of two samples of trifluralin, a herbicide, (chemically pure active ingredient and 25% emulsion concentrate) and its eight metabolites in the culture of human peripheral lymphocytes in vitro and in the bone marrow cells of mice in vivo has revealed that trifluralin itself does not induce chromosome aberrations both in vitro and in vivo, but some of its derivatives possess moderate (metabolites III and VII) or weak (metabolites II and XI) clastogenic activity. The results obtained should be taken into account for the hygienic regulation of trifluralin use allowing for the reality of entrance of its mutagenic derivatives into the human organism and for the degree of its genetic danger.     

Genotoxic evaluation of Ara-C by multiple parameters

The cytogenetic effects of the antimetabolite, cytosine arabinoside (Ara-C) are evaluated using in vivo and in vitro test systems and applying multiple parameters. The in vivo assay was carried out on 8-10-week-old inbred Swiss albino male mice using bone marrow as the somatic test system and the cells of testis as the meiotic test system. In vitro human leukocyte cultures were also employed. In vivo experimental doses were computed on surface area basis within the therapeutic dose range and injected intraperitoneally and for in vitro they were calculated on blood volume basis. Evaluation of somatic chromosome mutations included conventional screening for chromosome aberrations, variations in mitotic index and sister-chromatid exchanges (SCEs) by in vivo and in vitro methods besides studies on meiotic test systems using conventional screening for chromosome and sperm-head abnormalities. The quantitative data were subjected to statistical analysis by applying appropriate tests to evaluate their significance. The results of in vivo and in vitro experiments reveal the chromosome mutational activity of the compound. This is further supported by data on SCEs from both systems. However, a comparison of both demonstrated a differential mutagenic response of the drug, more in vivo than in vitro. This is also true for SCEs. Even though the mechanisms involved in causing chromosome aberrations and SCEs are different, the data on both corroborate each other on induction of chromosome mutations.