Anti-genotoxic effect of naringin against bleomycin-induced genomic damage in human lymphocytes in vitro.

Naringin is a flavonoid found in grapefruit and other citrus fruits that shows antioxidant activity. The aim of the present study was to determine the anti-genotoxic and protective effects of naringin on the chemotherapeutic/radiomimetic agent bleomycin (BLM) in human blood lymphocyte cultures in vitro using micronucleus test and chromosomal aberrations (CA) assay. We tested the three doses of naringin (1, 2, 3 µg/mL) and a single dose of BLM (20 µg/mL). BLM significantly increased the total CAs and micronucleus frequency at a concentration of 20 µg/mL. Naringin did not show any toxicity in doses of 1, 2, and 3 µg/mL. Combined treatments of BLM and naringin (2 and 3 µg/mL) significantly reduced micronucleus formation. Naringin dose-dependently decreased the total chromosome aberrations frequency induced by BLM. These results indicate that naringin could prevent BLM (20 µg/mL)-induced genotoxicity.

Influence of naringin on cadmium-induced genomic damage in human lymphocytes in vitro.

Cadmium is an important toxic environmental heavy metal. Generally, occupational and environmental exposures to cadmium result from heavy metal mining, metallurgy and industrial use and the manufacturing of nickel-cadmium batteries, pigments and plastic stabilizers. Cadmium induces oxidative stress and alters the antioxidant system, resulting in oxidative DNA damage and lipid peroxidation. The effect of naringin, a grapefruit flavonone, on cadmium-induced genomic damage was studied by using an in vitro system to test for chromosomal aberrations and sister chromatid exchanges. Cadmium significantly increased the total chromosomal aberrations in human lymphocytes at concentrations of 20 and 40 µM, and although naringin alone did not induce any chromosomal aberrations, it decreased those induced by cadmium. The mitotic index was not affected by either cadmium or naringin. Cadmium also induced a significant number of sister chromatid exchanges, but naringin alone did not induce sister chromatid exchanges and was unable to decrease the frequency of sister chromatid exchanges induced by cadmium. Replicative index analysis revealed that naringin and cadmium did not significantly alter replicative index frequencies. In this study, we show that plant-based flavonoids, such as naringin, may reduce the genomic damage induced by cadmium and may protect the cellular environments from free radical damage by its possible antioxidative potential.

Genotoxic effect of epirubicin in mouse bone marrow in vivo.

The genotoxic effect of epirubicin, a semisynthetic anthracycline antibiotic which has been used as an anticancer drug, was investigated in vivo on bone marrow cells of Swiss albino mice using the micronucleus test. To determine the incidence of micronuclei, mice were injected intraperitoneally with the drug at single doses of 4, 6, 8, and 10 mg/kg body weight. Then, bone marrow was sampled 18, 24, 36, and 48 h after the treatment. Polychromatic and normochromatic erythrocytes were examined for the presence of micronuclei. Epirubicin significantly increased the frequency of micronucleated polychromatic erythrocytes (MNPCEs) for all treatment periods compared with the negative control (P < 0.001). The frequency of MNPCEs increased with the dose, but at the highest dose used (which is considered to be quite toxic), the frequency of MNPCEs was rather lower. Epirubicin also decreased the ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) for all sampling intervals, which is indicative of bone marrow cytotoxicity. It can be concluded from the present study that the anticancer drug epirubicin has genotoxic effects on mouse bone marrow cells.

Evaluation of chromosome aberrations, sister chromatid exchange and micronuclei in patients with type-1 diabetes mellitus.

Oxidative stress-induced DNA damage seems to play a role in the pathogenesis of type-1 diabetes mellitus and its complications. Several in vitro assays have been used to measure the DNA damage produced by oxidative stress. In the present study, we aimed to investigate the frequency of sister chromatid exchange (SCE), chromosomal aberrations (CA) and micronuclei (MN) in type-1 diabetes mellitus patients compared with healthy controls. SCE, CA and MN tests were carried out with the blood-cell cultures from 35 type-1 diabetic patients and 15 healthy, age- and sex-matched control subjects. The mean age of the type-1 diabetic patients was 31.89 +/- 10.01 years, with a mean duration of the diabetes of 7.8 +/- 6.02 years. The mean level of HbA1c of the type-1 diabetic patients was 8.37+/-1.36%. Only three (8.5%) patients with type-1 diabetes mellitus had an HbA1c level below 7%. Patients with type-1 diabetes mellitus showed a higher frequency of SCE compared with controls (5.44 +/- 1.47 and 2.54 +/- 0.82, respectively, p < 0.001), but there was no significant correlation between the duration of diabetes, HbA1c and SCE. No significant difference was found in CA or MN frequency in type-1 diabetic patients compared with controls. In conclusion, these results suggest that type-1 diabetes mellitus is a condition with genomic instability characterized by an increased level of SCE. Hyperglycemia-induced oxidative stress may be the underlying factor of the increased SCE frequency.

A biomonitoring study of genotoxic risk to workers of transformers and distribution line stations.

A cytogenetic monitoring study was carried out on a group of workers from transformer and distribution line stations in the Bursa province of Turkey, to investigate the genotoxic risk of occupational exposure to extremely low frequency electric (ELF) and magnetic fields (EMF). Cytogenetic analysis, namely chromosomal aberrations (CAs) and micronucleus (MN) tests were performed on a strictly selected group of 55 workers and compared to 17 controls. CA and MN frequencies in electrical workers appeared significantly higher than in controls (p < 0.001, 0.05, respectively). The frequency of CA in exposed groups were significantly enhanced with the years of exposure (p < 0.01). The effect of smoking on the level of CA and MN was not significant in the control and exposure groups. The results of this study demonstrated that a significant induction of cytogenetic damage in peripheral lymphocytes of workers engaged to occupational exposure to ELMF in electric transformer and distribution stations.

In vivo evaluation of the toxic effects of pyrimethamine on spermatogenesis in male mice.

Pyrimethamine is known to have antimalarial activities and used clinically in the therapy of toxoplasmosis and human immunodeficiency virus-associated pneumonia. In this study we aimed to test the effects of pyrimethamine on spermatogenesis in mice. For this aim, animals were given pyrimethamine as a single application and the doses were 5, 10, 20, and 40 mg/kg. For the spermatogenic effects, the sperm shape abnormality, epididymal sperm counts, and testes weights were evaluated at the end of days 7, 14, 21, 28, and 35 after single pyrimethamine injection at the first day. Pyrimethamine increased the frequency of abnormal sperm shape for all studied weeks except the first week and its germ cell stage-specific effects correspond to spermatozoa, spermatids, and spermatocytes. It also decreased the epididymal sperm counts at the end of days 28 and 35, which corresponds to the spermatocyte stage of mouse spermatogenesis.

In vitro antigenotoxicity of Ulva rigida C. Agardh (Chlorophyceae) extract against induction of chromosome aberration, sister chromatid exchange and micronuclei by mutagenic agent MMC.

OBJECTIVE: To determine the in vitro possible clastogenic and cytotoxic activities of Ulva rigida crude extracts (URE), and identify their antigenotoxic and protective effects on chemotherapeutic agent mitomycine-C (MMC). METHODS: Anti-clastogenic and anti-genotoxic activities of Ulva rigida crude extracts (URE) were studied using chromosome aberration (CA), sister chromatid exchange (SCE), and micronuclei (MN) tests in human lymphocytes cultured in vitro. RESULTS: The chromosome aberration, sister chromatid exchange or micronuclei tests showed that URE at concentrations of 10, 20, and 40 microg/mL had no clastogenic activity in human lymphocyte cell culture. Three doses of URE significantly decreased the number of chromosomal aberrations and the frequencies of SCE and MN when compared with the culture treated with MMC (P < 0.0001). CONCLUSION: Although URE itself is not a clastogenic or cytotoxic substance, it possesses strong antigenotoxic, anti-clastogenic, and protective effects on MMC in vitro.

Rhodobacter capsulatus OlsA is a bifunctional enzyme active in both ornithine lipid and phosphatidic acid biosynthesis.

The Rhodobacter capsulatus genome contains three genes (olsA [plsC138], plsC316, and plsC3498) that are annotated as lysophosphatidic acid (1-acyl-sn-glycerol-3-phosphate) acyltransferase (AGPAT). Of these genes, olsA was previously shown to be an O-acyltransferase in the second step of ornithine lipid biosynthesis, which is important for optimal steady-state levels of c-type cytochromes (S. Aygun-Sunar, S. Mandaci, H.-G. Koch, I. V. J. Murray, H. Goldfine, and F. Daldal. Mol. Microbiol. 61:418-435, 2006). The roles of the remaining plsC316 and plsC3498 genes remained unknown. In this work, these genes were cloned, and chromosomal insertion-deletion mutations inactivating them were obtained to define their function. Characterization of these mutants indicated that, unlike the Escherichia coli plsC, neither plsC316 nor plsC3498 was essential in R. capsulatus. In contrast, no plsC316 olsA double mutant could be isolated, indicating that an intact copy of either olsA or plsC316 was required for R. capsulatus growth under the conditions tested. Compared to OlsA null mutants, PlsC316 null mutants contained ornithine lipid and had no c-type cytochrome-related phenotype. However, they exhibited slight growth impairment and highly altered total fatty acid and phospholipid profiles. Heterologous expression in an E. coli plsC(Ts) mutant of either R. capsulatus plsC316 or olsA gene products supported growth at a nonpermissive temperature, exhibited AGPAT activity in vitro, and restored phosphatidic acid biosynthesis. The more vigorous AGPAT activity displayed by PlsC316 suggested that plsC316 encodes the main AGPAT required for glycerophospholipid synthesis in R. capsulatus, while olsA acts as an alternative AGPAT that is specific for ornithine lipid synthesis. This study therefore revealed for the first time that some OlsA enzymes, like the enzyme of R. capsulatus, are bifunctional and involved in both membrane ornithine lipid and glycerophospholipid biosynthesis.

Low frequency of p53 and k-ras codon 12 mutations in non-small cell lung carcinoma (NSCLC) tumors and surgical margins.

AIMS AND BACKGROUND: Lung cancer is one of the most common cancers and has became a predominant cause of cancer-related death throughout the world. The k-ras codon 12 mutation, which is the most common lung cancer mutation, is found in 15 to 30% of all lung cancers. Furthermore, the p53 gene has a very important role in the biological properties of tumor cells, and it is mutated in about 50% of non-small cell lung cancers. Residual tumor cells remain in surgical margins diagnosed as tumor free by histopathological techniques, and they can play a role in forming any local recurrence. Molecular methods may be exploited that are sensitive enough to detect small numbers of tumor cells. METHODS: In the present study, we examined p53 gene mutations and k-ras codon 12 mutations from the tumor samples and surgical margins of 34 non-small-cell lung cancer patients. P53 gene mutations were analyzed by single strand conformational polymorphism analysis, heterodublex analysis and DNA sequencing. K-ras codon 12 mutations were analyzed by the mutagenic PCR-restricted fragment length polymorphism method. RESULTS: A p53 mutation was detected only in primary tumors of 3 out of 34 patients (8.82%). These mutations were clustered in exon 5. Moreover, a k-ras codon 12 mutation was detected in both the primary tumor and the surgical margin tissues of 2 out of 34 patients (5.88%). CONCLUSIONS: The detected mutation rate was low, in the range given in the literature. We think that different mechanisms related to other genes and individual genetic differences might play a role in cancer formation in our study group. We believe that molecular studies are necessary to identify biomarkers and to determine genetic alterations in histopathologically normal surgical margins.

A comparative study on the genotoxic effect of pyrimethamine in bone marrow and spermatogonial mice cells.

Pyrimethamine is an antimalarial agent widely used in clinical therapy. We aimed to compare its mutagenic potential in mammalian spermatogonial and bone marrow cells. For studying chromosomal aberrations mice were treated acutely (single treatment) with 4 dose levels of pyrimethamine (5, 10, 20 and 40 mg/kg). Pyrimethamine was found to produce a significant increase in structural chromosomal aberrations after acute treatment in bone marrow cells of mice (p < 0.001). It also induced chromosome abnormalities in spermatogonial cells (p < 0.05) at the highest dose.

Germination responses to GA3 and stratification of threatened Festuca L. species from Eastern Mediterranean.

The seed germination characteristics of three threatened Festuca sp. [F. punctoria Sm., F. cyllenica Boiss. et Heldr. subsp. uluana Markgr.-Dannenb., F. paphlagonica (St.-Yves) Markgr.-Dannenb. subsp. paphlagonica] were investigated. These species are endemic and spread on alpine belt. The study was carried out with wet-cold and dry-cold stratification throughout 15 days, different doses of GA3 (50, 100 and 150 ppm) and hormone-stratification combined treatments, and non-treatment series. We found that the germination rates of three fescue seeds for various treatment series were different. The mean germination percentage of F. cyllenica was higher (80%) than that of F. punctoria and F. paphlagonica which were fairly low (50-60%). Germination rates increased by wet-stratification treatment in F. punctoria and also increased with 100 ppm GA3 application to the seeds of F. paphlagonica. When taken into consideration the germination percentages of all fescue species, the seeds of F. punctoria and F. paphlagonica can be dormant, but the seeds of F. cyllenica are non-dormant.

Genotoxic effects induced by fotemustine and vinorelbine in human lymphocytes.

The aim of this study was to investigate the in vitro genotoxic effects of the anticancer drugs fotemustine and vinorelbine on human lymphocytes and to determine individual and sex-related responses to these drugs. Fotemustine is a DNA-alkylating drug while vinorelbine is a semi-synthetic Vinca alkaloid. The study was carried out with twenty independent healthy donors for each drug. We have tested the ability of these drugs to induce chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) as well as effect on the mitotic index (MI) in cultured human lymphocytes. Fotemustine was shown to induce CAs and SCEs at all concentrations tested (2, 4 and 8 microg/ml) in a dose-dependent manner. Additionally it also decreased the mitotic index in a similar dose-dependent manner. Vinorelbine had no effect on structural CAs, but it significantly increased the numerical CAs at all doses tested (0.5, 1 and 2 microg/ml). Vinorelbine also induced SCE events and increased the MI values. Two-way analyses of variance were used to compare the individual and gender-related susceptibilities to fotemustine and vinorelbine with respect to the CA, SCE and MI values. The results indicated that individuals in fotemustine treatment groups showed different genotoxic responses with respect to CA and SCE induction and additional findings indicated a gender-specific response in this group. Individuals in the vinorelbine test group also exhibited statistically significant numerical CA, SCE and MI responses to vinorelbine. A statistically significant gender-related SCE response to this drug was also evident. This study indicates that these drugs have potentially harmful effects on human health.

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.

The investigation of the genotoxic effects of fenarimol and propamocarb in mouse bone marrow in vivo.

In this study, we aimed to evaluate the genotoxic effects of fungicides fenarimol and propamocarb which are used to protect crops from fungi. For this reason, bone-marrow micronucleus and chromosome aberration tests were carried out in Swiss albino mice. Mice were injected with four different doses of fenarimol and propamocarb intraperitoneally; 50, 100, 200 and 400 mg/kg b.w. Fenarimol did not induce any significant increase in micronucleated erythrocytes after 24, 36, and 48 h treatment but it decreased the ratio of polychromatic/normochromatic erythrocytes at all dose groups and sampling intervals. Fenarimol did not increase the number of chromosome aberrations significantly, but it reduced the mitotic index at the higher doses (P < 0.05). Propamocarb did not increase the frequency of micronucleated erythrocytes, but decreased the polychromatic/normochromatic erythrocytes ratio at all sampling intervals. Propamocarb increased only gaps in total chromosome aberrations, but when gaps were excluded, there were no significant differences in total aberrations between the control and dose groups (P > 0.05). Propamocarb also reduced the mitotic index compared with the negative control group (P < 0.001). Contributing these results, we can suggest that fenarimol and propamocarb are non-genotoxic in mouse bone marrow in vivo but have cytotoxic effects.

Genotoxicity of two anticancer drugs, gemcitabine and topotecan, in mouse bone marrow in vivo.

In this study, the genotoxic effects of gemcitabine and topotecan were investigated in mouse bone marrow cells using the micronucleus and chromosomal aberration test systems. Gemcitabine increased the frequency of micronuclei, particularly at the median dose for the 24-, 36-, and 48-h sampling intervals. It had cytotoxic effects on the bone marrow and decreased the polychromatic/normochromatic erythrocyte ratio dose-dependently for all sampling intervals. Gemcitabine significantly decreased the mitotic index at the 24-h time point. It increased the number of abnormal cells and induced a significant increase in total chromosomal aberrations. For the 6-h sampling time, gemcitabine neither induced chromosomal aberrations nor reduced the mitotic index. Topotecan also induced high levels of micronuclei, particularly for the 24- and 36-h sampling times and it decreased the polychromatic/normochromatic erythrocyte ratio for all sampling intervals, which is indicative of bone marrow cytotoxicity. The bone marrow metaphase analysis showed that topotecan significantly elevated the number of abnormal metaphases and total chromosomal aberrations at 6 and 24h, in a dose-dependent manner. It also decreased the mitotic index for both sampling intervals. In conclusion, the results of this study indicate that the two chemotherapeutics gemcitabine and topotecan have cytotoxic and genotoxic effects in mouse bone marrow.

Radio-protective effect of cinnamic acid, a phenolic phytochemical, on genomic instability induced by X-rays in human blood lymphocytes in vitro.

The present study was designed to determine the protective activity of cinnamic acid against induction by X-rays of genomic instability in normal human blood lymphocytes. This radio-protective activity was assessed by use of the cytokinesis-block micronucleus test and the alkaline comet assay, with human blood lymphocytes isolated from two healthy donors. A Siemens Mevatron MD2 (Siemens AG, USA, 1994) linear accelerator was used for the irradiation with 1 or 2 Gy. Treatment of the lymphocytes with cinnamic acid prior to irradiation reduced the number of micronuclei when compared with that in control samples. Treatment with cinnamic acid without irradiation did not increase the number of micronuclei and did not show a cytostatic effect in the lymphocytes. The results of the alkaline comet assay revealed that cinnamic acid reduces the DNA damage induced by X-rays, showing a significant radio-protective effect. Cinnamic acid decreased the frequency of irradiation-induced micronuclei by 16-55% and reduced DNA breakage by 17-50%, as determined by the alkaline comet assay. Cinnamic acid may thus act as a radio-protective compound, and future studies may focus on elucidating the mechanism by which cinnamic acid offers radioprotection.

Radioprotection by two phenolic compounds: chlorogenic and quinic acid, on X-ray induced DNA damage in human blood lymphocytes in vitro.

The present study was designed to determine the radioprotective effect of two phytochemicals, namely, quinic acid and chlorogenic acid, against X-ray irradiation-induced genomic instability in non-tumorigenic human blood lymphocytes. The protective ability of two phenolic acids against radiation-induced DNA damage was assessed using the alkaline comet assay in human blood lymphocytes isolated from two healthy human donors. A Siemens Mevatron MD2 (Siemens AG, USA, 1994) linear accelerator was used for irradiation. The results of the alkaline comet assay revealed that quinic acid and chlorogenic acid decreased the DNA damage induced by X-ray irradiation and provided a significant radioprotective effect. Quinic acid decreased the presence of irradiation-induced DNA damage by 5.99-53.57% and chlorogenic acid by 4.49-48.15%, as determined by the alkaline comet assay. The results show that quinic acid and chlorogenic acid may act as radioprotective compounds. Future studies should focus on determining the mechanism by which these phenolic acids provide radioprotection.

Genotoxic and anti-genotoxic effects of vanillic acid against mitomycin C-induced genomic damage in human lymphocytes in vitro.

Vanillic acid, a vegetable phenolic compound, is a strong antioxidant. The aim of the present study was to determine its effects on mitomycin C-induced DNA damage in human blood lymphocyte cultures in vitro, both alone and in combination with mitomycin C (MMC). The cytokinesis block micronucleus test and alkaline comet assay were used to determine genotoxic damage and anti-genotoxic effects of vanillic acid at the DNA and chromosome levels. MMC induced genotoxicity at a dose of 0.25 µg/ml. Vanillic acid (1 µg/ml) significantly reduced both the rates of DNA damaged cells and the frequency of micronucleated cells. A high dose of vanillic acid (2 µg/ml) itself had genotoxic effects on DNA. In addition, both test systems showed similar results when tested with the negative control, consisting of dimethyl sulfoxide (DMSO) in combination with vanillic acid (1 µg/ml) +MMC. In conclusion, vanillic acid could prevent oxidative damage to DNA and chromosomes when used at an appropriately low dose.

Evaluation of genotoxicity from Nilufer Stream (Bursa/Turkey) water using piscine micronucleus test.

In the present study, the in vivo micronucleus (MN) test in fish erythrocytes was used to evaluate the genotoxic potentials of water samples collected from four different sites along the Nilufer Stream which empties into the Marmara Sea on the north-west of Turkey. Nilufer Stream receives discharges of industrial and domestic wastes resulting from industrialization and urbanization activities in Bursa city. Nile tilapias (Oreochromis niloticus) were exposed to collected water samples under laboratory conditions for 3 and 6 days. Micronuclei analyses were carried out in peripheral blood erythrocytes. In addition to micronuclei, other nuclear abnormalities (NAs) such as bi-nucleated cells and binuclei with nucleoplasmic bridge and cells with blebbed, notched and lobbed nuclei, were assessed in the erythrocytes. Chemical analyses were also carried out in the water samples to assess the presence of major pollutants. MN and NA frequencies were significantly elevated in fishes exposed to water from polluted areas compared to those exposed to clean water sample. The results of this study indicate that Nilufer Stream is contaminated with potential genotoxic chemicals and the genotoxicity is possibly related with the industrial, agricultural and domestic activities.

Evaluation of anti-oxidative, genotoxic and antigenotoxic potency of Codium tomentosum Stackhouse ethanolic extract in human lymphocytes in vitro.

The genome is constantly exposed to agents, both exogenous and endogenous, that damage DNA. Consequently, it is very important that determination of this agents and the protective agents. In this work, we evaluated the antigenotoxic/antimutagenic activity of the crude ethanolic extracts of Codium tomentosum Stackhouse (Chlorophyceae) (CTE), collected from The Coast of South East Marmara Sea, in human lymphocytes culture in vitro against genotoxic/mutagenic agents MMC, EMS and H(2)O(2) by using chromosome aberration (CA), sister chromatid exchange (SCE) and micronuclei (MN) assays as experimental endpoints. Also, in the present study, we determined total phenolic content and total antioxidant capacity (in soluble lipid and water). In addition, total protein, total carbohydrate, vitamins (A, C and E) and pigments (chlorophyll a, chlorophyll b and carotene) contents were also determined. Results of CA, SCE and MN tests show that CTEs have not shown genotoxic effect. In CTE plus MMC-, EMS- or H(2)O(2)- treated cultures, CA, SCE and MN frequency which induced by MMC, EMS or H(2)O(2) has been decreased significantly (p<0.05-0.001). This is the first report on genotoxicity/antigenotoxicity and anti-oxidative capacity of Codium tomentosum. Our results have clearly shown that CTE has strong anti-oxidative and antigenotoxic effect.