Genotoxic evaluation of the antimicrobial drug, trimethoprim, in cultured human lymphocytes

The antimicrobial drug, trimethoprim, was evaluated for genotoxicity in human peripheral blood lymphocyte cultures set-up from two healthy donors. Sister-chromatid exchanges (SCE) and micronuclei (MN) were scored as genetic endpoints. The treatment was done using different trimethoprim concentrations ranging from 1 to 100 μg/ml. From our results, we can conclude that this drug is able to induce both cytotoxic and moderate genotoxic effects, as revealed by the increases seen in SCE and MN frequencies in cultures from the two donors and, at least, at one of the concentrations tested.     

Different effects of chlorpromazine on bleomycin-and epirubicin induced cytotoxicity

The effect of the antiemetic chlorpromazine on bleomycin- and epirubicin cytotoxicity was testedin vitro. Chlorpromazine (0.1 or 0.01 mg/l) enhanced epirubicin-induced toxicity to cultured Chinese fibroblasts whereas 0.01 mg/l chlorpromazine inhibited the cytotoxicity of bleomycin. The results encourage further studies on the effects of commonly used antiemetics on the cytotoxicity and antitumoral effects of anticancer chemotherapeutics.     

The cytogenetic effect of bleomycin on human peripheral lymphocytes in vitro and in vivo.

The cytogenetic effect of bleomycin (BLM) in human lymphocytes was studied after exposure to different doses during the G0 and G2 phases. BLM produced a marked specific effect on the cell cycle. The main aberration types after exposure in tg0 were dicentrics and deletions; and after exposure in G2, open chromatid breaks. A linear dose--response was calculated for all these aberration types as well as for the number of aberrant cells. In the G2 experiments, partially and totally pulverized cells also increased linearly with dose. The intercellular distributions of the most frequent aberration types after exposure in G0 and G2--the dicentrics and chromatid breaks, respectively--showed over-dispersion. These results show that the cytogenetic effect of BLM may be compared with that of densely ionizing irradiation. Preliminary results of chromosome analysis of three cancer patients in the course of BLM therapy showed effects similar to those in the G0 experiments.     

Genotoxicity of cadmium chloride in human lymphocytes evaluated by the comet assay and cytogenetic tests

Peripheral blood lymphocytes were tested in vitro for genotoxic effects of cadmium chloride. Whole blood samples of four healthy, non-smoking subjects were preincubated with CdCl2 in concentrations of 10(-4), 10(-3), and 5 . 10(-3) mol/L for three hours before the cells were assessed for DNA-damage using the single cell alkaline gel electrophoresis assay (comet assay) or cultivated for chromosomal aberrations (CA), sister chromatid exchanges (SCE), and the micronucleus (MN) test. The comet assay showed notable interindividual differences. The results of the cytogenetic tests showed an increase in the frequency of CA, MN, and SCE with CdCl2 in the treated cultures, yet none was able to show a correlation between concentrations of cadmium chloride and the frequency of damages. The MN slides were stained with Giemsa and with DNA fluorochrome 4', 6'-diamidino-2-phenylindole (DAPI). The frequency of MN in slides stained with DAPI was significantly higher than in those stained with Giemsa, which might be due to an underestimation of small micronuclei in Giemsa-stained slides.     

Low dose effects of chemicals as assessed by the flow cytometric in vivo micronucleus assay

Using flow cytometric automation of the mouse in vivo, micronucleus assay increases the sensitivity of the test. This is achieved through a very large increase in the number of cells scored, by a factor of 100×, which in turn greatly reduces the sampling error. With this method, dose–response relationships of in vivo micronucleus induction for four model agents mitomycin C (MMC), diepoxybutane (DEB), cyclophosphamide (CPA), and colchicine (COL) were studied at low dose levels. For the three clastogens MMC, DEB and CPA, linear dose–response relationships were found over the dose ranges studied, even in the very low dose region (defined as the dose region where the frequency of micronucleated erythrocytes is less than twice the baseline frequency). This is consistent with the view that no threshold should exist for genotoxic agents which target DNA. For COL a dose range was found, in which the frequency of micronucleated erythrocytes did not increase with dose, possibly indicating an in vivo threshold. The flow cytometric in vivo micronucleus assay represents one possibility for in vivo low dose–response studies.     

Genotoxic evaluation of the antimalarial drug,fansidar, in cultured human lymphocytes

Fansidar (pyrimethamine-sulfadoxine) has been used extensively worldwide for the treatment of chloroquine resistant Plasmodium falciparum malaria, toxoplasmosis and Pneumocystis carinii pneumonia in patients with the acquired immunodeficiency syndrome. Because of the wide usage of pyrimethamine-sulfadoxine in developing countries and the lake of information from open literature and reports from manufacturers about the genotoxicity of such antimalarial drug, the present work was suggested. The possible genetic toxicity of fansidar has been evaluated in human peripheral blood lymphocyte cultures. The frequencies of sister-chromatid exchanges (SCE) and micronuclei (MN) were scored as genetic endpoints. Both tests covering a wide range of induced genetic damage as primary DNA damage, clastogenicity and aneugenicity. Cultures were set up by using blood samples from two healthy donors and the treatment was done using different fansidar concentrations ranging from 1:20 to 10:200 g/ml. From our results, it appears that this drug is able to induce moderate genotoxic effects, as revealed by the increases found in SCE and MN frequencies in cultures from the two donors at the two highest concentrations tested (5:100 and 10:200 g/ml). In addition, cyotoxic/cytostatic effects of fansidar were revealed by a decrease in the proliferative rate index (PRI) and in the cytokinesis block proliferation index (CBPI). Our findings suggest that the use of this drug should be restricted to situations where other antimalarial drugs cannot be used. The drug should never be given to pregnant women.     

Bone marrow and lymphocyte cytogenetics of rhesus monkeys (Macaca mulatta) treated with the clastogen mitomycin C

Rhesus monkeys (Macaca mulatta) were used to determine their effectiveness as experimental animals for different cytogenetic tests with mitomycin C (MC). The micronucleus test (MNT) and/or chromosome analysis of blood and bone marrow were made before and/or after the treatment with mitomycin C. Thus, the controls data and treated data were obtained from the same animals. With the employed methology, the micronucleus test could not be performed on living animals. Less chromosomal damage was detected in the micronucleus test of post-mortem samples than in the chromosome analysis of bone marrow. No influence by the mutagen could be observed in lymphocyte chromosomes at any of the different times of analysis. In contrast to this, bone-marrow chromosomes seemed to be highly affected by mitomycin C at day 1, 2 and 3 after injection. However, before treatment and at day 14, 16 and 17 after treatment there was no visible increase in chromosomal aberration in bone marrow.     

Inhibition of chromosome repair by caffeine or isonicotinic acid hydrazide on chromosome damage induced by mitomycin C in human lymphocytes

The frequency of interchromatic exchanges induced by mitomycin C in cultured human lymphocytes was markedly lowered in the presence of caffeine or isonicotinic acid hydrazide (INH) during a post-treatment period. The autoradiographic experiment showed that the decrease in the exchange frequency did not result from delaying or cell-killing effects by the post-treatment with caffeine or INH. Therefore, it may deduced that the exchange formation closely related to a process sensitive to caffeine or INH.     

Caffeine-induced enhancement of chromosome damage in human lymphocytes treated with methyl-methanesulphonate, mitomycin C and X-rays

Two hypothese have been put forward in the literature to explain the synergistic effect of caffeine with several mutagens: (1) binding of caffeine to DNA, and (2) inhibition of DNA repair.Autoradiographic studies with ³H- and ¹⁴C-labelled caffeine did not support the binding hypothesis. Caffeine enchanced in a synergistic way the amount of chromatid breaks and exchanged induced in human lymphocytes with methyl-methanesulphonate (MMS), mitomycin C (MC) and X-rays. The results are best explained if caffiene inhibits a post-replication repair process, particularly the filling-in of gaps in the newly synthesized DNA.     

The mitomycin C (MMC)-binding protein from MMC-producing microorganisms protects from the lethal effect of bleomycin: crystallographic analysis to elucidate the binding mode of the antibiotic to the protein

Antibiotic-producing microorganisms must be protected from the lethal effect of their own antibiotic. We have previously determined the X-ray crystal structure of the bleomycin (Bm)-binding protein, designated BLMA, as a self-resistance determinant from Bm-producing Streptomyces verticillus, which suggests that the binding of the first Bm to one of two pockets formed in the BLMA homodimer induces the cooperative binding of the second Bm to the other pocket. In the present study, we noticed that the X-ray crystallographic structure of a self-resistance determinant from a mitomycin C-producing microorganism, designated MRDP, reveals similarity to the folding pattern on the BLMA, although no sequence homology exists. To clarify the hypothesis that MRDP may function as a resistance determinant to Bm, we characterized and determined the crystal structure of MRDP complexed with the Cu(II)-bound form of BmA(2) grouped into the Bm family of antibiotics. The biochemical and structural studies for Bm binding provide evidence that the first Bm binds anti-cooperatively to a pocket of MRDP with binding affinity of the nanomolar order, whereas the second Bm binds to the other pocket, which has binding affinity of the micromolar order. The invisibility of the second Bm in the structure agrees with the observation that Escherichia coli-expressing MRDP displays lower resistance to Bm than that expressing BLMA. The structure of MRDP, which is complexed with the Cu(II)-bound BmA(2), revealed that the gamma-aminopropyldimethylsulphonium moiety of the antibiotic is sandwiched between the peripheral residues of the binding pocket and that its positively charged sulphonium head is accommodated completely in the negatively charged region of the MRDP pocket. Furthermore, the Cu(II)-bound BmA(2) has a very compact structure, in which the bithiazole ring of BmA(2) is folded back to the metal-binding domain.     

Impact of radiofrequency radiation on DNA damage and antioxidants in peripheral blood lymphocytes of humans residing in the vicinity of mobile phone base stations

Radiofrequency radiations (RFRs) emitted by mobile phone base stations have raised concerns on its adverse impact on humans residing in the vicinity of mobile phone base stations. Therefore, the present study was envisaged to evaluate the effect of RFR on the DNA damage and antioxidant status in cultured human peripheral blood lymphocytes (HPBLs) of individuals residing in the vicinity of mobile phone base stations and comparing it with healthy controls. The study groups matched for various demographic data including age, gender, dietary pattern, smoking habit, alcohol consumption, duration of mobile phone use and average daily mobile phone use. The RF power density of the exposed individuals was significantly higher (p < 0.0001) when compared to the control group. The HPBLs were cultured and the DNA damage was assessed by cytokinesis blocked micronucleus (MN) assay in the binucleate lymphocytes. The analyses of data from the exposed group (n = 40), residing within a perimeter of 80 m of mobile base stations, showed significantly (p < 0.0001) higher frequency of micronuclei when compared to the control group, residing 300 m away from the mobile base station/s. The analysis of various antioxidants in the plasma of exposed individuals revealed a significant attrition in glutathione (GSH) concentration (p < 0.01), activities of catalase (CAT) (p < 0.001) and superoxide dismutase (SOD) (p < 0.001) and rise in lipid peroxidation (LOO) when compared to controls. Multiple linear regression analyses revealed a significant association among reduced GSH concentration (p < 0.05), CAT (p < 0.001) and SOD (p < 0.001) activities and elevated MN frequency (p < 0.001) and LOO (p < 0.001) with increasing RF power density.     

The effect of hydroxyurea and mitomycin C on sperm motility in mice

The mutagen, mitomycin C, and the teratogen, hydroxyurea, were found to decrease sperm motility in mice in a dose-dependent manner. Positive results with these compounds suggest that sperm motility may have been decreased through either mutations or developmental disturbances. Sperm motility can be determined quickly and may be done in conjunction with a sperm-morphology assay.     

Evaluation of concentration and storage effects of mitomycin C in the diagnosis of Fanconi anemia among idiopatic aplastic anemia patients

BACKGROUND:

Fanconi anemia (FA) is a rare autosomal recessive genetic disorder that shows an increased sensitivity to the intercalating agents such as mytomycin C (MMC), measured as chromosomal aberrations. This study was conducted to differentiate between FA and “idiopathic” aplastic anemia on the basis of induced chromosomal breakage study with MMC.

MATERIALS AND METHODS:

MMC stress tests in different final concentrations of 20 and 50 ng/ml of MMC were conducted on peripheral blood lymphocytes from 32 patients with aplastic anemia and 13 healthy controls. Fifty nanograms per milliliter of MMC from old, fresh and frozen stocks was used to check the sensitivity of diagnosis on FA-diagnosed patients. Statistical analysis was used for the assessment of aberrations, including chromatid and chromosome breaks and exchanges.

RESULTS:

Eight patients (25%) with a very high percentage of chromosomal breakage were diagnosed as FA on the basis of the chromosomal breakage study. Six of these patients exhibited congenital anomalies at presentation, while another two lacked such anomalies or had minor physical problems. Freshly made MMC has shown more sensitivity to detect FA patients compared with frozen or 1-week-old MMC stock.

CONCLUSIONS:

The study indicates that freshly made MMC stress test provides an unequivocal means of differentiation between FA and “idiopathic” aplastic anemia. Further, the study, the first of its kind from Iran, stresses on the need for conducting this test in all aplastic anemia cases, even those without congenital anomalies, for accurate and timely diagnosis of FA to implement appropriate therapy.     

Effects of mitomycin C on sister chromatid exchange in normal and Bloom's syndrome cells

Bloom's syndrome lymphocytes, which are characterized by a high incidence of sister chromatid exchanges (SCE: 80.6 per cell), were treated with mitomycin C (MMC) and the effect of the chemical on SCE frequency compared with that in normal cells. Raising the concentration of MMC from 1 X 10(-9) to 1 X 10(-7) g/ml led to about 10-fold increase (61.7 SCE per cell) in the SCE frequency over the base line in normal lymphocytes (6.4 SCE per cell), though chromosome aberrations remained at a relatively low frequency. MMC caused about a two-fold rise in SCE in cells of Bloom's syndrome (128.8 SCE at 10(-9) g/ml; 139.3 SCE at 10(-8) g/ml). The frequency of chromosome aberrations in Bloom's syndrome cells at concentrations of MMC of 1 X 10(-9) and 1 X 10(-8) g/ml was 0.350 and 0.825 per cell, respectively, and low when compared to the increased number of SCE. The increased frequency of SCE in normal and Bloom's syndrome cells is in contrast to the reported findings with cells from Fanconi's anemia and xeroderma pigmentosum. The distribution of SCE in MMC-treated normal cell correlates with that of spontaneous SCE in cells of Bloom's syndrome.     

Factors influencing the frequency of mitomycin C-induced sister-chromatid exchanges in 5-bromodeoxyuridine-substituted human lymphocytes in culture.

Newly developed techniques for the detection of sister-chromatid exchanges (SCE) require the substitution of 5-bromodeoxyuridine (BrdU) for thymidine in DNA. We investigated the possibility of interactions between BrdU and one mutagen--carcinogen, mitomycin C (MMC) for the induction of both chromosomal aberrations and SCE in human peripheral lymphocytes in culture. No effect on aberration yield was found. Neither comparisons between the yields of SCE by BrdU substitution and differential staining and those detected by tritiated thymidine incorporation and autoradiography nor between the yields of SCE for different levels of BrdU incorporation provided any evidence of synergism. It was found, however, that MMC persists in cultures and continues to increase SCE frequencies for about 30 h. It was also observed that some MMC-induced DNA lesions persist long enough so that some of those present prior to S phase of the first cell cycle cause additional SCE in the third cycle.     

In vitro analyses of the anti-fibrotic effect of SPARC silencing in human Tenon's fibroblasts: comparisons with mitomycin C

Failure of glaucoma filtration surgery (GFS) is commonly attributed to scarring at the surgical site. The human Tenon's fibroblasts (HTFs) are considered the major cell type contributing to the fibrotic response. We previously showed that SPARC (secreted protein, acidic, rich in cysteine) knockout mice had improved surgical success in a murine model of GFS. To understand the mechanisms of SPARC deficiency in delaying subconjunctival fibrosis, we used the gene silencing approach to reduce SPARC expression in HTFs and examined parameters important for wound repair and fibrosis. Mitomycin C-treated HTFs were used for comparison. We demonstrate that SPARC-silenced HTFs showed normal proliferation and negligible cellular necrosis but were impaired in motility and collagen gel contraction. The expression of pro-fibrotic genes including collagen I, MMP-2, MMP-9, MMP-14, IL-8, MCP-1 and TGF-β(2) were also reduced. Importantly, TGF-β(2) failed to induce significant collagen I and fibronectin expressions in the SPARC-silenced HTFs. Together, these data demonstrate that SPARC knockdown in HTFs modulates fibroblast functions important for wound fibrosis and is therefore a promising strategy in the development of anti-scarring therapeutics.     

Effects of caffeine on chromosome aberrations and sister-chromatid exchanges induced by mitomycin C in BrdU-labeled human chromosomes.

The BrdU-Hoechst staining technique has been used in analyzing the effect of caffeine (CAF) on chromosome aberrations and sister-chromatid exchanges (SCEs) induced by mitomycin C (MC). CAF increased the frequency of SCE in MC-treated chromosomes in all specimens. The combination of MC and CAF caused a remarkable increase in all types of chromosome aberrations, but the most startling effect was the appearance of many cells with multiple aberrations (shattered chromosomes). The BrdU-Hoechst technique showed that the shattered chromosomes did not appear in cells that had replicated only once, but did occur in cells which replicated twice in the presence of MC and CAF. The large majority of chromatid breaks observed did not involve areas common to SCE; and the SCE frequency significantly increased in spite of the existence of multiple breaks. This indicates that very few of the breaks are incomplete exchanges and that the mechanism for formation of SCE might be different from that of chromosome breaks. In another experiment, monofunctional-MC (M-MC) had a small effect on SCE rates, though it induced shattered chromosomes with CAF post-treatment. Possible differences in the mechanisms leading to SCE and chromosome breaks are discussed.     

The effect of mitomycin C treatment of infected erythrocytes on infection with Babesia microti and Babesia rodhaini in mice

In vitro treatment of Babesia microti infected erythrocytes with mitomycin C before their injection into mice prolonged the prepatent period of infection, reduced the levels of the infection in the ‘breakthrough’ parasitaemia and induced protection against reinfection. Treatment of B. microti with mitomycin C at a concentration of 25 μg ml^?1 resulted in a mean peak parasitaemia of 6.2% in the infected mice compared with 46.5% in control mice injected with untreated B. microti parasites. In addition, mice survived a normally fatal B. rodhaini infection if injected with 6.2 × 10⁷ infected erythrocytes treated with 25 μg ml^?1 mitomycin C and four of five mice survived infection with 6.2 × 10⁵ similarly treated infected erythrocytes. However, the degree of protection against B. rodhaini was dependent on the concentration of mitomycin C used to treat the parasites and treatment of 5 × 10⁷ infected erythrocytes with 50 μg ml^?1 resulted in survival of only four of the five infected mice. In addition, when 100 μg ml^?1 of mitomycin C was used to treat B. rodhaini parasites, the course of infection, although delayed, was indistinguishable from that seen in the control mice and all the mice died. The latter results and the lack of efficacy of comparable numbers of heat killed parasites suggested the necessity for sufficient, non-replicating, mitomycin C treated parasites to metabolize and produce and/or present protective antigens to the host.