Effect of tetrandrine on micronucleus formation and sister-chromatid exchange in both in vitro and in vivo assays

The genotoxicity of tetrandrine, a drug potentially useful for the treatment of silicosis, was studied using the micronucleus and the sister-chromatid exchange (SCE) assay systems. Cultured Chinese hamster lung (V79) cells were used for the in vitro micronucleus and sister-chromatid exchange studies. Mouse bone marrow was used for the in vivo micronucleus assay and mouse spleen cells for the in vivo/in vitro sister-chromatid exchange analysis. The results show that SCE levels in V79 and in spleen cells were significantly elevated by treatment with tetrandrine at doses above 0.08 mg/ml and 100 mg/kg bw, respectively. Increased tetradrine-induced SCE in vitro was metabolic activation dependent. Tetrandrine failed to induce micronuclei at any of the doses tested. A decrease of replicative index with an increase in the concentration of tetrandrine was found both in vitro and in vivo. These results indicate that tetrandrine is a weak indirect-acting genotoxicant.     

In vitro genotoxic effects of the anticancer drug gemcitabine in human lymphocytes

This research was carried out to investigate in vitro genotoxic effects of the anticancer agent gemcitabine on the induction of chromosomal aberrations and sister-chromatid exchange in human lymphocytes. Three doses of gemcitabine (0.001, 0.002 and 0.004 microg/ml) were applied to lymphocyte cultures from 15 donors. There was a significant increase in the induction of chromosome aberrations and in the occurrence of sister-chromatid exchange in these cells. In addition, gemcitabine significantly decreased the mitotic index and replicative index for all doses. Dose-response regression lines were used to compare the individual susceptibilities to gemcitabine with respect to the chromosome aberration and sister-chromatid exchange frequencies. Our results indicate that gemcitabine is able to induce both cytotoxic and genotoxic effects in human lymphocyte cultures in vitro in a dose-dependent manner.     

Mutagenicity of vinyl chloride in man: comparison of chromosome aberrations with micronucleus and sister-chromatid exchange frequencies

The mutagenic effects of vinyl chloride monomer in man were studied in the lymphocyte culture with 3 methods: the chromosome aberration assay, the micronucleus assay and the sister-chromatid exchange method. Compared with control, values obtained by these tests are increased in workers occupationally exposed to vinyl chloride. In relation to non-smokers, smokers exposed to vinyl chloride show significant increases in sister-chromatid exchange frequencies. The problem of correlating the results of the chromosome aberration assay with micronucleus and sister-chromatid exchange frequencies is discussed.     

X-rays, microwaves and vinyl chloride monomer: their clastogenic and aneugenic activity, using the micronucleus assay on human lymphocytes.

Chromosome aberration assays, sister-chromatid exchange techniques and micronucleus assays are commonly used methods for biomonitoring genetic material damaged by chemical or physical agents. On the other hand, their aneugenic activity, which can lead to hypoploidy and may also be associated with carcinogenesis, has not been thoroughly investigated. In our study we chose the micronucleus assay with a new mathematical approach to separate clastogenic from aneugenic activity of three well-known mutagens (vinyl chloride monomer, X-rays and microwaves) on the genome of human somatic cells. The comparison of frequencies of size distribution of micronuclei in the lymphocytes of humans exposed to each of these three mutagens showed that X-rays and microwaves were preferentially clastogens while vinyl chloride monomer showed aneugenic activity as well. Microwaves possess some mutagenic characteristics typical of chemical mutagens.     

Rubber solvent: a clastogenic agent that fails to induce sister-chromatid exchanges.

Rubber solvent was tested for its ability to induce chromosome aberrations and sister-chromatid exchanges in human whole blood cultures. Following exposure to relatively low rubber solvent concentrations (0.0125% and greater) significant increases in the frequencies of chromatid gaps and breaks were observed. At higher rubber-solvent concentrations (0.05% and greater) there were also significant increases in the frequency of chromosome breaks. In contrast to the increase in chromosome aberrations following rubber-solvent exposure, rubber-solvent concentrations up to the toxic level failed to produce increases in the sister-chromatid exchange frequency.     

Genotoxic effects of thiram evaluated by sister-chromatid exchanges in human lymphocytes

The purpose of this investigation was to study the genotoxic potential of thiram (CAS No. 137-26-8) using an in vitro sister-chromatid exchange (SCE) assay with human lymphocytes. The results indicate that thiram and its metabolites increase the SCE frequencies 2-fold over those observed in the negative controls. The standard inducers cyclophosphamide and ethyl methanesulfonate increased SCE frequencies 10- and 4-fold, respectively, over untreated levels.     

Germ-cell mutagenicity of etoposide: induction of meiotic micronuclei in cultured rat seminiferous tubules

Mutagen effects on male germ cells can be quantified by meiotic micronucleus induction in vitro. Late pachytene and diakinetic primary spermatocytes are able to differentiate through meiotic divisions in vitro and develop to round spermatids. In the presence of mutagens micronucleus induction reflects the potential of the chemical to induce chromosome breakage or uneven chromosome distribution. In this study we have investigated the mutagenicity of etoposide (VP-16) and its ability to induce micronuclei S-independently in meiosis by the meiotic micronucleus method in vitro. Our results indicate that etoposide is able to cause a statistically significant increase in the frequency of micronuclei at a concentration range as low as 0.5–8 μmole/1. The meiotic micronucleus method in vitro seems to be a feasible and sensitive test system of male germ-cell mutagenesis.     

Influence of dietary carrot on cytostatic drug activity of cyclophosphamide and its main directly acting metabolite: induction of sister-chromatid exchanges in normal human lymphocytes, Chinese hamster ovary cells, and their DNA repair-deficient cell lines

We have utilized an in vivo drug metabolism technique (i.e. injecting the chemical into rat and isolating plasma with metabolites from blood) for detecting the genotoxicity of indirectly acting cyclophosphamide and its directly acting metabolite phosphoramide mustard in cultures of human peripheral blood lymphocytes of normal individuals, Fanconi's anaemia (FA) and aplastic anaemia (AA) patients, wild-type Chinese hamster ovary cells (CHO) and its DNA repair-deficient mutant 43-3B cells. In addition, the influence of dietary carrot on the clastogenic activity of these 2 chemicals in all the different cell types was studied. The genotoxicity was assessed by the ability of the metabolites of these agents to induce sister-chromatid exchanges in the treated cells. A dose-dependent increase in the frequencies of sister-chromatid exchanges was observed in all cell strains following treatment with activated metabolites of cyclophosphamide or phosphoramide mustard. The sensitivity of lymphocytes from normal donors, FA and AA patients to these 2 chemicals was similar. In CHO cell lines the induced frequency of sister-chromatid exchanges was slightly higher after treatment with the metabolites of cyclophosphamide than with phosphoramide mustard. The mutant 43-3B cells responded with higher frequencies of SCEs when compared to the wild-type CHO cells, about 1.5-2-fold, at low doses. Pretreating of rats with fresh carrot juice effectively inhibited the increase in the frequencies of sister-chromatid exchanges induced by cyclophosphamide in wild-type and mutant CHO cells (P less than 0.01), and to a lesser extent in human lymphocytes (p less than 0.05). In contrast, no inhibitory effect was observed in any of these cell types in combination of dietary carrot for direct acting phosphoramide mustard on the frequency of induced sister-chromatid exchanges. The possibility that dietary carrot exerts its antimutagenic effect by affecting the processes of enzymatic activation of cyclophosphamide is discussed.     

In vivo cytogenetic studies on mice exposed to ethylene dibromide

The pesticide, ethylene dibromide (EDB), was evaluated with in vivo cytogenetic assays to determine its genotoxicity. CD1 male mice were exposed to EDB through intraperitoneal injections. Bone marrow cells isolated from femora were analyzed for sister-chromatid exchange (SCE), chromosome aberration and micronucleus formation. The results showed that only certain concentrations of EDB tested caused a slight but significant increase in SCEs and chromosome aberrations. However, these increases were not dose-related. No increase in the polychromatic erythrocytes with micronuclei was observed following EDB exposure. Also, EDB did not cause cell-cycle delay in comparison with controls. Thus, it appears that EDB is not an effective genotoxic agent in vivo in mice.     

The persistence of sister-chromatid exchange frequencies in men occupationally exposed to vinyl chloride monomer.

The persistence of sister-chromatid exchange frequencies in a population occupationally exposed to the well known chemical mutagen vinyl chloride monomer was studied. It was shown that increased values of sister-chromatid exchange frequencies were still present in the lymphocytes of workers who had not been exposed for 8-120 days and retired persons for 5-10 years after exposure. The possible ability of vinyl chloride monomer alkylating metabolites to cause long-lasting damage of the DNA molecule is discussed.     

Cytogenetic studies on 1,1-dichloroethylene and its two isomers in mammalian cells in vitro and in vivo

Chromosomal aberration and sister-chromatid exchange (SCE) tests in vitro on 1,1-dichloroethylene (1,1-DCE), its two isomers, cis- and trans-1,2-DCE, and two possible metabolites of 1,1-DCE, chloroacetyl chloride and chloroacetic acid, were carried out using a Chinese hamster cell line, CHL. 1,1-DCE induced chromosomal aberrations in the presence of S9 mix prepared from the rat liver, but not in the absence of S9 mix. SCEs were also slightly induced by 1,1-DCE only in the presence of S9 mix. On the other hand, two isomers and two metabolites of 1,1-DCE induced neither chromosomal aberrations nor SCEs with and without S9 mix. 1,1-DCE, however, was negative even at a sublethal dose in the micronucleus test using mouse bone marrow, fetal liver and blood.     

Use of rat primary lung cells for studying genotoxicity with the sister-chromatid exchange and micronucleus assays

Primary culture of lung cells from CD rats was established for pulmonary genotoxicity studies using two genetic endpoints, sister-chromatid exchange (SCE) and micronucleus formation (MN). In the cell isolation study, a combined enzyme separation of rat lungs with trypsin (1.3 mg/ml) plus collagenase (50 U/ml) gave the highest yield of viable and colony-forming cells. For the MN assay, the cytokinesis block induced by cytochalasin B (CYB) was employed to enumerate MN in binucleated (BN) cells. Treatment of primary lung cells with 2 micrograms CYB/ml for two days appeared to be optimal for scoring micronuclei in CYB-induced BN cells. By this procedure, mitomycin C (MMC), triethylenemelamine, and benzo[a]pyrene caused a dose-related increase in micronucleated BN cells in vitro without metabolic activation. In the SCE assay, maximum second-division metaphases were obtained after cells were incubated with bromodeoxyuridine for 48-54 h. After this incubation time, high frequencies of SCE induced by MMC and 3-methylcholanthrene after in vitro exposure (without S9 activation) or in vivo exposure were observed. The results indicate that rat primary lung cells can metabolize polycyclic aromatic hydrocarbons and that this lung cell system is potentially useful for the detection of pulmonary genotoxicants.     

Mutagenicity of methyl 2-benzimidazolecarbamate, diethylstilbestrol and estradiol: structural chromosomal aberrations, sister-chromatid exchanges, C-mitoses, polyploidies and micronuclei

Methyl 2-benzimidazolecarbamate (MBC), diethylstilbestrol (DES) and estradiol were tested with regard to their ability to induce C-mitoses, polyploidies, micronuclei, structural chromosomal aberrations and sister-chromatid exchanges (SCE) in human peripheral lymphocytes in vitro. The compounds did not induce structural chromosomal aberrations either in the presence or absence of metabolic activation. MBC and estradiol were negative in the SCE test. DES induced SCE rates which were not even twice the control level and which were independent of dose and of metabolic activation. All compounds induced C-mitoses, polyploidies and micronuclei. The micronuclei are interpreted as resulting from errors in the anaphase distribution of chromosomes by spindle disturbances rather than from structural chromosomal aberrations.     

The assessment of genotoxic effects in lymphocyte cultures of infants treated with chloral hydrate

Chloral hydrate is a sedative commonly used in pediatric medicine. It was evaluated for genotoxicity in cultured peripheral blood lymphocytes of infants who were given chloral hydrate for sedation. Sister chromatid exchange and micronucleus frequencies were determined before and after chloral hydrate administration. After treatment, the frequencies of sister chromatid exchange and micronuclei were significantly increased, suggesting that chloral hydrate has moderate genotoxic potential in infants.     

Radiation induction of micronuclei in human lymphocytes

An examination of the cytokinesis-blocked micronucleus technique confirmed its potential usefulness as a method of biological dosimetry for radiation accidents. Several advantages and disadvantages of the system are discussed. It has been demonstrated that under these conditions of these experiments, the blocking agent, cytochalasin B does not induce micronuclei or unstable chromosome aberrations. The induction of sister-chromatid exchanges proved just significant.Analysis of the dose response for 250 kVp X-rays indicates that although the Y = αD + βD² model fits the data, the relationship does not correspond to that for total aberration induction as might have been expected. The background frequency of micronuclei and the value of the α coefficient are higher than for total aberrations and the β term is lower. This indicates that simple incorporation of acentric chromosome fragments into micronuclei may not wholly account for the phenomenon.     

Genotoxicity studies on the antimicrobial drug sulfamethoxazole in cultured human lymphocytes

The genotoxicity of the antimicrobial drug sulfamethoxazole was evaluated in cultured human peripheral blood lymphocytes. The frequencies of sister-chromatid exchange (SCE) and micronuclei (MN) were scored as genetic endpoints. Both tests cover a wide range of induced genetic damage such as primary DNA damage, clastogenicity and aneugenicity. Cultures were set up with blood samples from two healthy donors and the treatment was done with different sulfamethoxazole concentrations ranging from 10 to 500 microg/ml. From the results obtained it appears that this drug is able to induce weak genotoxic effects, as revealed by the slight increase in the SCE and MN frequencies, at least at one of the two highest concentrations tested. However, the results of the SCE assay should be interpreted with caution because the increase is just significant. In addition, cyotoxic/cytostatic effects of sulfamethoxazole were revealed by a decrease in the proliferative rate index (PRI) and in the cytokinesis block proliferation index (CBPI).     

Immunofluorescent staining of kinetochores in micronuclei: a new assay for the detection of aneuploidy

The immunofluorescent staining of kinetochores in micronuclei with antikinetochore antibodies was used to develop an in vitro assay for aneuploidy-inducing agents. The results show that about 80% of micronuclei induced by either colchicine or chloral hydrate contained kinetochores; only 9% of X-ray-induced micronuclei reacted positively to the antibody. These findings indicate that the in vitro micronucleus assay coupled with immunofluorescent staining of kinetochores can be a useful method for assessing the ability of chemicals to induce aneuploidy and/or chromosome aberrations.     

An in vitro micronucleus assay for determining the radiosensitivity of hepatocytes

An in vitro micronucleus assay was developed for primary cultures of rat hepatocytes and utilized to determine the oxygen enhancement ratio (OER). Freshly isolated Fischer 344 female rat hepatocytes were irradiated in suspension either in air or in anoxia, cultured for 60 h to allow for the maximum expression of micronuclei, fixed with methanol-glacial acetic acid, stained with the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI), and scored with a fluorescent microscope for the presence of micronuclei. The percentage of cells with micronuclei increased linearly with dose, and the slopes of the relationships were 0.044 +/- 0.001 Gy-1 and 0.015 +/- 0.001 Gy-1 in air and anoxia, respectively. The calculated OER of 2.9 +/- 0.5 is similar to that previously obtained for hepatocyte cell survival. Our data demonstrate that this in vitro hepatocyte micronucleus assay is a rapid and sensitive method to further investigate those factors which influence the radiosensitivity of hepatocytes.     

Sister-chromatid exchanges in beta-thalassaemic patients under conditions of in vivo and in vitro depletion of folic acid.

In order to investigate the effect of folate depletion, lymphocyte sister-chromatid exchange (SCE) rates were compared among homozygous beta-thalassaemic patients with low folic acid levels, heterozygous beta-thalassaemic patients with normal folate levels and healthy persons with normal haemoglobin, in cultures with both normal and depleted folate conditions. Significantly higher SCE rates were found in homozygous patients in all assays, but the in vitro folate depletion did not induce an increase in SCE frequency in any group.     

In vitro genotoxicity of para-phenylenediamine and its N-monoacetyl or N,N'-diacetyl metabolites

para-Phenylenediamine (PPD), a widely used ingredient of oxidative hair dyes, is converted by human hepatocytes and in the human epidermis, or after topical application to rats, to its N-monoacetylated (MAPPD) and/or N,N'-diacetylated (DAPPD) derivatives. We investigated in vitro genotoxic properties of PPD, MAPPD and DAPPD in the Ames test, the micronucleus test (MNT) in human lymphocytes and the mouse lymphoma assay (Hprt locus, PPD only). Given that MAPPD and DAPPD are actual human skin and hepatic metabolites of PPD and represent the substances to which humans are systemically exposed, they were tested in the absence of metabolic activation. In the Ames test, PPD was slightly mutagenic in Salmonella typhimurium strain TA98 in the presence of a rat liver metabolic activation system (S-9), whereas MAPPD and DAPPD were negative in all strains. When tested up to toxic doses, PPD did not induce mutation at the Hprt locus of L5178Y mouse lymphoma cells in two independent experiments, either in the absence or presence of S-9, suggesting that PPD is non-mutagenic in mammalian cells. In the in vitro micronucleus test, PPD induced micronuclei (MN) in cultured human peripheral blood lymphocytes (HL) in the presence of S-9, when tested following 24-h PHA stimulation. No increases in MN frequency were observed in the absence of S-9, when tested following 24-h PHA stimulation. However, PPD induced MN both in the absence and presence of metabolic activation, when tested following 48-h PHA stimulation. In contrast, MAPPD and DAPPD did not induce MN in HL when tested up to 10mM concentrations or to their limit of solubility, respectively, after either 24- or 48-h stimulation. In conclusion, the results of the Ames and MN tests confirm that PPD has a slight genotoxic potential in vitro, although it was non-mutagenic in mammalian cells. Given that MAPPD and DAPPD were negative in the Ames and the MN tests, these acetylated conversion products are considered to be detoxified metabolites that are biologically less reactive than the parent molecule PPD.