Anti-genotoxic effect of Ocimum sanctum L. extract against cyproterone acetate induced genotoxic damage in cultured mammalian cells

The anti-genotoxic effect of Ocimum sanctum L. extract was studied against the genotoxic effect induced by a synthetic progestin cyproterone acetate, on human lymphocytes using chromosomal aberrations, mitotic index, sister chromatid exchanges and replication index as a parameters. About 30 microM of cyproterone acetate was treated with O. sanctum L. infusion, at dosages of 1.075 x 10(-4), 2.125 x 10(-4) and 3.15 x 10(-4) g/ml of culture medium. A clear dose-dependent decrease in the genotoxic damage of cyproterone acetate was observed, suggesting a possible modulating role of the plant infusion. The results of the present study suggest that the plant infusion per se does not have genotoxic potential, but can modulate the genotoxicity of cyproterone acetate on human lymphocytes in vitro.     

Protective effect of curcumin on gamma-radiation induced DNA damage and lipid peroxidation in cultured human lymphocytes

The present work is aimed at evaluating the radioprotective effect of curcumin, a naturally occurring phenolic compound on gamma-radiation induced toxicity. The cellular changes were estimated by using lipid peroxidative indices like thiobarbituric acid reactive substances (TBARS), the antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH). The DNA damage was analysed by using cytokinesis blocked micronucleus assay and dicentric aberration (DC). The gamma-radiation at different doses (1, 2 and 4Gy) were found to significantly increase micronuclei (MN), DC frequencies and TBARS level whereas the levels of GSH and antioxidant enzymes were significantly decreased. The maximum damage to lymphocytes was observed at 4Gy irradiation. Curcumin pretreatment (1, 5 and 10microg/ml) significantly decreased the frequency of MN and DC. The levels of TBARS decreased and activities of SOD, CAT and GPx significantly increased along with GSH levels. At 1Gy irradiation all the concentrations of curcumin (1, 5 and 10microg/ml) significantly protected the lymphocytes from radiation damage. At 2Gy irradiation, 5 and 10microg/ml of curcumin showed significant radioprotection. Since the highest damage was observed at 4Gy irradiation both 1 and 5microg/ml of curcumin pretreatment were not sufficient to protect the lymphocytes from radiation damage but 10microg/ml of curcumin significantly protected the cultured lymphocytes from radiation damage. Thus, pretreatment with curcumin gives protection to lymphocytes against gamma-radiation induced cellular damage.     

Induction of chromosomal aberrations and sister chromatid exchanges by chlormadinone acetate in human lymphocytes: a possible role of reactive oxygen species

Genotoxicity study of a synthetic progestin chlormadinone acetate (CMA) was carried out in human lymphocytes using chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) as parameter. Effect of CMA was studied at 10, 20, 30 and 40 microM. CMA was genotoxic at 30 and 40 microM. With a view to study the possible mechanism of genotoxicity of CMA, superoxide dismutase (SOD) and catalase (CAT) were used separately and in combination along with the CMA (40 microM) at different doses. SOD treatment increased CAs and SCEs at both the doses. CAT treatment decreased the frequencies of CAs and SCEs in both, separately and in combination with SOD, suggesting a possible role of reactive oxygen species for the genotoxic damage.     

Antigenotoxic effect of allicin against SCEs induced by methyl methanesulphonate in cultured mammalian cells

Allicin, one of the sulphur compounds of garlic (Allium sativum), possesses antioxidant and thiol disulphide exchange activity and is also shown to cause a variety of activities potentially useful for human health. In this investigation, the effect of 1,5,10 and 20 microM of allicin was determined for inhibiting the rate of SCE induced by 60 microM of MMS. Cultured human lymphocytes from two female donors were used for the experiment. The levels of SCEs were lowered by allicin suggesting its antigenotoxic activity in mammalian cells in vitro.     

Chromosomal damage induced by caprolactam in human lymphocytes

Caprolactam was tested in the in vitro human lymphocyte cytogenetic assay both in the presence and absence of S9 mix at dose levels up to 5500 micrograms/ml using lymphocytes obtained from a male donor and in the presence of S9 mix using lymphocytes obtained from a female donor. Statistically significant increases in chromosomal damage were observed at 5500 micrograms/ml dose level in cells from both donors. This positive response was enhanced by the inclusion of chromosomal gaps in the calculations. It was concluded that caprolactam induces chromosomal damage in human lymphocytes in vitro albeit at comparatively high dose levels.     

Protective effect of curcumin on γ-radiation induced DNA damage and lipid peroxidation in cultured human lymphocytes

The present work is aimed at evaluating the radioprotective effect of curcumin, a naturally occurring phenolic compound on γ-radiation induced toxicity. The cellular changes were estimated by using lipid peroxidative indices like thiobarbituric acid reactive substances (TBARS), the antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH). The DNA damage was analysed by using cytokinesis blocked micronucleus assay and dicentric aberration (DC). The γ-radiation at different doses (1, 2 and 4 Gy) were found to significantly increase micronuclei (MN), DC frequencies and TBARS level whereas the levels of GSH and antioxidant enzymes were significantly decreased. The maximum damage to lymphocytes was observed at 4 Gy irradiation. Curcumin pretreatment (1, 5 and 10 μg/ml) significantly decreased the frequency of MN and DC. The levels of TBARS decreased and activities of SOD, CAT and GPx significantly increased along with GSH levels. At 1 Gy irradiation all the concentrations of curcumin (1, 5 and 10 μg/ml) significantly protected the lymphocytes from radiation damage. At 2 Gy irradiation, 5 and 10 μg/ml of curcumin showed significant radioprotection. Since the highest damage was observed at 4 Gy irradiation both 1 and 5 μg/ml of curcumin pretreatment were not sufficient to protect the lymphocytes from radiation damage but 10 μg/ml of curcumin significantly protected the cultured lymphocytes from radiation damage. Thus, pretreatment with curcumin gives protection to lymphocytes against γ-radiation induced cellular damage.     

Protective role of sinapic acid against arsenic: induced toxicity in rats

Arsenic compounds are classified as toxicants and human carcinogens. Environmental exposure to arsenic imposes a big health issue worldwide. Sinapic acid is a phenylpropanoid compound and is found in various herbal materials and high-bran cereals. It has been reported that sinapic acid has antioxidant efficacy as metal chelators due to the orientation of functional groups. However, it has not yet been examined in experimental animals. In light of this fact, the purpose of this study was to characterize the protective role of sinapic acid against arsenic induced toxicity in rats. Rats were orally treated with arsenic alone (5 mg/kg body weight (bw)/day) plus sinapic acid at different doses (10, 20 and 40 mg/kg bw/day) for 30 days. Hepatotoxicity was measured by the increased activities of serum hepatospecific enzymes namely aspartate transaminase, alanine transaminase, alkaline phosphatase, gamma glutamyl transferase, lactate dehydrogenase and total bilirubin along with increased elevation of lipid peroxidative markers, thiobarbituric acid reactive substances, lipid hydroperoxides, protein carbonyl content and conjugated dienes. The toxic effect of arsenic was also indicated by significantly decreased activities of enzymatic antioxidants like superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, glutathione reductase and glucose-6-phosphate dehydrogenase along with non-enzymatic antioxidant like reduced glutathione. Administration of sinapic acid exhibited significant reversal of arsenic induced toxicity in hepatic tissue. The effect at a dose of 40 mg/kg bw/day was more pronounced than the other two doses (10 and 20 mg/kg bw/day). All these changes were supported by reduction of arsenic concentration and histopathological observations of the liver. These results suggest that sinapic acid has a protective effect over arsenic induced toxicity in rat.     

Apoptosis induced by membrane damage in human lymphocytes; effects of arachidonic acid and its photoproducts

The effect of arachidonic acid (AA) combined with UVA irradiation was studied in a model system mimicking phototherapy PUVA (psoralen+UVA) ex vivo in vitro. The contribution of damage to the plasma membrane by PUVA was tested on human lymphocytes derived from healthy donors. The effect of arachidonic acid (AA) combined with UVA irradiation was compared with that of a psoralen photoadduct to AA added to the culture. The adduct, obtained photochemically and purified, was characterized by NMR and MS spectrometry as a cycloadduct of psoralen to the vinylene bond of the acid (AA<>PSO). The reactions of cultured cells, manifested 20 h after treatment by changes in apoptosis and mitochondrial depolarization, were monitored by flow cytometry by tagging lymphocytes with appropriate fluorescent probes. Treatment of lymphocyte suspension within AA doses from 40 to 100 microM gradually induced a shift from Anx-V(+) (single positive cells) to late apoptotic, Anx-V(+)PI(+) (double positive cells) in a dose dependent manner. The adduct, AAPSO, induced apoptotic changes at a concentration 2-3 times higher than free AA. Combination of psoralen (1 microM ) or arachidonic acid (20-120 microM) with UVA irradiation (2-6 J/cm(2)) accelerated the plasma membrane changes in a synergic way. Preliminary studies indicated that changes in the transmembrane potential of mitochondria paralleled the apoptosis when cells were treated by AA alone. Our findings showed that UVA radiation of lymphocytes in the presence of arachidonic acid, as in the presence of psoralen, enhanced apoptosis of cells in a synergic manner. Thus, PUVA-induced apoptosis may proceed in part by a still undefined signaling pathway(s) triggered in lymphocyte membranes.     

DNA damage and repair induced in vitro by nitrilotriacetic acid (NTA) in human lymphocytes

In cultured human lymphocytes we determined the ability of nitrilotriacetic acid (NTA) to inhibit DNA replication and to stimulate DNA repair synthesis (UDS), as well as to influence the UDS induced by UV irradiation. In phytohemagglutinin-stimulated lymphocytes a strong inhibition of DNA replication was induced by NTA concentrations above 10(-3) M, which was accompanied by a marked cell lethality, whereas at lower doses the incorporation of tritiated thymidine (3H-TdR) into DNA or treated cells was slightly increased in comparison to untreated cells. When, after NTA pretreatment, UDS was determined by scintillation spectrometry or autoradiography in unstimulated G0 lymphocytes, UV-irradiated or unirradiated, an increased incorporation of 3H-TdR was observed, positively correlated with the NTA doses. This effect was only partially due to the expansion of the intracellular TdR pool as a consequence of the stimulation of 3H-TdR uptake by NTA. Even after normalization of the scintillometric data by the radioactivities of the soluble nucleotide fraction, significant increase of DNA repair synthesis was detected after treatment with 7.5 x 10(-3)-10(-2) M NTA.     

Evidence against an intermediary role of abscisic acid in stomatal closure induced by phenylmercuric acetate and faresol

The hypothesis was tested that stomatal closure induced by the antitranspirants phenylmercuric acetate and farnesol is mediated by an increase in abscisic acid content. Isolated leaves of Spinacia oleracea L. cv. Müma, Tradescantia X andersoniana Ludw. et Rohw. and Commelina communis L. were incubated in solutions of phenylmercuric acetate (10⁻³ M ) and of farnesol (2 × 10⁻³ M ). In all three species, a reduction in stomatal aperture was observed. The extent and velocity of the reaction differed between the species and also between the two compounds applied. The abscisic acid content of the leaves was determined after stomatal closure had been achieved. No significant increase in abscisic acid level was found in treated leaves of Spinacia and Commelina . In Tradescantia , on the contrary, a reduction to about 50% was observed after 120 min treatment. Visible damage of the treated leaves and membrane alterations observable by electron microscopy occurred, especially after treatment with farnesol. These changes in membrane structure suggest a connection with the reduction in stomatal aperture.     

Cytogenetic damage induced in human lymphocytes by sodium bisulfite.

The frequencies of chromosomal aberrations (CA), sister-chromatid exchanges (SCE), and micronuclei (MN) in human blood lymphocytes exposed to sodium bisulfite (sulfur dioxide) at various concentrations ranging from 5 x 10(-5) M to 2 x 10(-3) M in vitro were studied. It was shown that sodium bisulfite (NaHSO3 and Na2SO3, 1:3 M/M) caused an increase in SCE and MN in human blood lymphocytes in a dose-dependent manner, and also induced mitotic delays and decreased mitotic index. For CA, our results indicated that sodium bisulfite induced an increase of chromatid-type aberrations in lymphocytes from three of four donors in a dose-dependent manner. The chemical at low concentrations induced chromatid-type aberrations, but not chromosome-type aberrations; high concentrations induced both chromatid- and chromosome-type aberrations. No cytogenetic damage in human lymphocytes was induced by sodium sulfate. The results have confirmed that sulfur dioxide is a clastogenic and genotoxic agent.     

Induction of micronuclei by vinyl acetate in mouse bone marrow cells and cultured human lymphocytes

A dose-dependent increase in micronucleated polychromatic erythrocytes was observed in the bone marrow of male C57B1/6 mice 30 h after a single intraperitoneal injection of vinyl acetate (250, 500, 1000 or 2000 mg/kg b.wt.; (9-14 animals per group). The effect was statistically significant at 1000 mg/kg (1.33 +/- 0.29% vs. 0.6 +/- 0.10% in olive oil-treated controls) and at 2000 mg/kg (1.57 +/- 0.19%) of vinyl acetate. These doses were fatal to 6 (1000 mg/kg) and 8 (2000 mg/kg) out of 14 animals in both groups. The ratio of polychromatic to normochromatic cells decreased as a function of vinyl acetate dose. Cyclophosphamide (20 mg/kg), used as a positive control chemical, induced a clear increase in micronucleated polychromatic erythrocytes (2.07 +/- 0.20%). None of the treatments affected the number of micronuclei in normochromatic erythrocytes. In human whole-blood lymphocyte cultures, micronucleus induction by a 48-h treatment with vinyl acetate (0.125, 0.25, 0.5, 1 and 2 mM; 24 h after culture initiation) was studied in lymphocytes with preserved cytoplasm from smear slides prepared by a method involving the removal of erythrocytes at harvest by sodium cyanide treatment to improve preparation quality. The frequency of micronucleated lymphocytes reached a peak at 0.5 mM (3.2 +/- 1.0% vs. 0.9 +/- 0.1% in control cultures) and 1 mM (3.1 +/- 0.7%), with a decline at 2 mM probably because of a toxic effect resulting in mitotic inhibition.     

Genotoxic potential of medroxyprogesterone acetate in cultured human peripheral blood lymphocytes

Medroxyprogesterone acetate was studied at three different concentrations (1, 5 and 10 microM), for its genotoxic effects in human peripheral blood lymphocyte culture using chromosomal aberrations and sister chromatid exchanges as parameters. Duplicate peripheral blood cultures were treated with three different concentrations (1, 5 and 10 microM) of medroxyprogesterone acetate. The study was carried out both in the absence as well as in the presence of metabolic activation (S9 mix) with and without NADP. Medroxyprogesterone acetate was found genotoxic at 5 and 10 microM in the presence of S9 mix with NADP. To study the possible mechanism of the genotoxicity of medroxyprogesterone acetate, superoxide dismutase and catalase at different doses were used separately and in combination with 10 microM of medroxyprogesterone at different doses in the presence of S9 mix with NADP. Superoxide dismutase treatment results in an increase of the genotoxic damage but catalase treatment reduce the genotoxic damage of medroxyprogesterone acetate. Catalase treatment in combination with superoxide dismutase also results in the further reduction of the genotoxic damage. The results of the present study reveal that medroxyprogesterone acetate is genotoxic only in the presence of metabolic activation (S9 mix) with NADP. Treatments with superoxide dismutase and catalase suggests the possible generation of reactive oxygen species by redox cycling of various forms of quinones, similar to estrogens, that are the results of aromatic hydroxylation by cytochrome P450s.     

Caffeic acid protects human peripheral blood lymphocytes against gamma radiation-induced cellular damage

In the present study, we investigated in vitro radioprotective potential of caffeic acid (CA), a naturally occurring catecholic acid against gamma radiation-induced cellular changes. Different concentrations of CA (5.5, 11, 22, 44, 66, and 88 microM) were incubated with lymphocytes for 30 min prior to gamma-irradiation, and micronuclei (MN) scoring and comet assay were performed to fix the effective concentration of CA against gamma-irradiation. Among all concentrations, 66 microM of CA showed the optimum protection by effectively decreasing the MN frequencies and comet attributes. From the above-mentioned results, 66 microM of CA was selected as the effective concentration and was further used to investigate its radioprotective efficacy. For that purpose, a separate experiment was carried out on the lymphocytes in which lymphocytes were preincubated with CA (66 microM) and were exposed to different doses of radiation (1, 2, 3, and 4 Gy). Genetic damage (MN, dicentric aberration, and comet attributes) and biochemical changes were measured. Gamma-irradiated lymphocytes showed a dose-dependent increase in the genetic damage and thiobarbituric acid reactive substances, accompanied by the significant decrease in the antioxidant status, whereas CA pretreatment positively modulated all the radiation-induced changes through its antioxidant potential. The current study demonstrates that CA is effective in protecting lymphocytes against radiation-induced toxicity and encourages further in vivo study to evaluate radioprotective efficacy of CA.     

Prion protein protects against DNA damage induced by paraquat in cultured cells

Exposure of cells to paraquat leads to production of superoxide anion (O2*-). This reacts with hydrogen peroxide to give the hydroxyl radical (*OH), leading to lipid peroxidation and cell death. In this study, we investigated the effects of cellular prion protein (PrPC) overexpression on paraquat-induced toxicity by using an established model system, rabbit kidney epithelial A74 cells, which express a doxycycline-inducible murine PrPC gene. PrPC overexpression was found to significantly reduce paraquat-induced cell toxicity, DNA damage, and malondialdehyde acid levels. Superoxide dismutase (total SOD and CuZn-SOD) and glutathione peroxidase activities were higher in doxycycline-stimulated cells. Our findings clearly show that PrPC overexpression plays a protective role against paraquat toxicity, probably by virtue of its superoxide dismutase-like activity.     

Protective effect of Raphanus sativus on H2O2 induced oxidative damage in human lymphocytes

Raphanus sativus, a common cruciferous vegetable has been attributed to possess a number of pharmacological and therapeutic properties. This present study evaluated the protective effect of different parts of R. sativus such as root, stem and leaf obtained with a variety of extraction solvents against cell death and oxidative DNA damage induced by hydrogen peroxide (H₂O₂) in normal human lymphocytes. R. sativus extracts as such showed no cytotoxicity and genotoxicity to the lymphocytes at the tested concentrations. Of the different extracts, hexane extract of root and methanolic extract of stem and leaf showed significant protective effect against oxidative damage induced by 200 μM H₂O₂ in a dose dependent manner, as compared to cells exposed only to H₂O₂. Our results suggest that the protective effect afforded by R. sativus extract could be related to the presence of isothiocyanates and polyphenolics, as they possess significant capacity to remove reactive species by virtue of their ability to scavenge free radicals and induce antioxidant enzyme system in the cells.     

Sister-chromatid exchanges (SCE) induced by p-dichlorobenzene in cultured human lymphocytes

We have investigated the ability of p-dichlorobenzene (p-DCB) to induce sister-chromatid exchanges (SCE) in cultured human lymphocytes. From our results we can conclude that p-DCB was able to induce a cytotoxic effect, measured as a decrease in third and second metaphases, together with an increase of SCEs.     

Chromosomal damage induced in human lymphocytes by low doses of D-T neutrons

Unstable chromosome aberrations induced by in vitro irradiation with zero plus seven low doses of 14.8 MeV D-T neutrons in the range 3.55-244 mGy have been analysed in human peripheral blood lymphocytes. In order to obtain the required large numbers of scored cells for such low doses, fourteen laboratories participated in the experiment. The dose responses for dicentrics, excess acentrics and total aberrations, fitted well to the Y = alpha D model. The alpha coefficient of yield for dicentrics, 1.60 +/- 0.07 X 10(-2) Gy-1, compares well with the values obtained in previous studies with D-T neutrons at somewhat higher doses. Results from a previous collaborative study using 250 kVp X-rays over a comparable dose range indicated the possible existence of a threshold below 50 mGy. In the present study there is no clear evidence for neutrons for such a threshold. However, the data were insufficient to permit the rejection of a possible threshold below approximately 10 mGy.     

Protective effect of apigenin on radiation-induced chromosomal damage in human lymphocytes

The potential use of flavonoids as a radioprotector is of increasing interest because of their high antioxidant activity and abundance in the diet. The aim of this study is to examine genotoxic and radioprotective effects of one of the most common flavonoids, apigenin, on radiation-induced chromosome aberrations in human lymphocytes. The cytokinesis-block micronucleus (CBMN) assay was used to evaluate such effects of apigenin. Blood samples were collected from two non-smoking healthy male volunteers who had no history of previous exposure to other clastogenic agents. Isolated lymphocytes were cultured. There were two tubes per concentration for all treatments. To evaluate the genotoxicity of apigenin, cells were first treated with different concentrations of apigenin (0, 2.5, 5, 10 and 25 microg/mL) at 24 h after culture initiation, followed by cytochalasin-B (Cyt-B) treatment (3 microg/mL) and cell harvest at 44 and 72 h, respectively. Secondly, to investigate the radioprotective effect, cell cultures were exposed to different concentrations of apigenin as described above for 30 min before being irradiated to 2 Gy of 137Cs gamma rays (at a dose rate of 0.75 Gy/min). In all instances, the frequency of MN was scored in binucleated (BN) cells. The nuclear proliferation index also was calculated. We did not detect an increase in the frequency of MN in non-irradiated human lymphocyte cultures treated with 2.5, 5.0 or 10 microg/mL apigenin; although, we did observe an increase in cultures treated with 25 microg/mL apigenin (the highest concentration of apigenin used in our study). We also observed a significant increase in the frequency of MN in irradiated cells overall; however, the frequency was decreased as the concentration of apigenin increased, suggesting a radioprotective effect. These findings provide a basis for additional studies to help clarify the potential use and benefit of apigenin as a radioprotector.