In vitro Cytotoxic and Genotoxic Effects of Methanol and Aqueous Extracts of Gymnosporia Montana Plant

Introduction: Gymnosporia montana Benth. is a medicinal herb which has been valued in Ayurvedic medicine for its hepatoprotective effect. The plant has been studied for its pharmacological, antimicrobial, and antioxidant properties, but there are no reports on its genotoxicity. Aim: Hence, in the present study, two extracts of G. montana (70% methanolic and aqueous) at different concentrations were evaluated for the in vitro cytotoxicity and genotoxicity in Human peripheral blood lymphocyte cultures (PBLC) since these are well-established techniques for the analysis of the potentially mutagenic and carcinogenic chemicals. Methodology: The 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT), Mitotic index (MI), Sister-chromatid exchanges (SCEs), Cell cycle proliferative index (CCPI), Average generation time (AGT) and Population doubling time (PDT) were scored in cultures set up from 10 different healthy donors. The treatment of the cell culture was done employing different extracts of G. montana at three concentrations (1.78µg/mL, 3.57µg/mL and 7.14µg/mL) with control and positive control (Ethyl methanesulfonate [EMS (1.93 mM)]). Results: The MTT results showed the cytotoxic effect in a concentration-dependent manner in both the methanol and aqueous extract and the IC50 value of methanol and aqueous extract was found to be 2.63 µg/mL and 3.63 µg/mL respectively. The MI (p<.001) and CCPI (p<.05) in both the extracts showed significant values at higher concentration, but at lower and mid concentrations both the extracts were non-significant and the total SCEs, AGT and PDT in all the concentrations showed non-significant results when compared with the control. Conclusion: These results indicate that the G. montana plant extracts at lower two concentrations showed no cytotoxicity and genotoxicity effects in cultured human peripheral blood lymphocytes. Therefore, we suggest that the plant extract is safe for use at the lower concentrations in traditional medicine.

T. thermophilus Rhamnolipids Induce Cytogenetic Damage on Human Lymphocytes and Bind DNA in vitro.

Aims: Bacteria including Pseudomonas and T. thermophilus secretes rhamnose–containing glycolipid biosurfactants called rhamnolipids (RLs), known as bacterial virulence factors. The aim of this investigation was the evaluation of DNA damage induced on human lymphocytes by both RLs itself, secreted in a host organism by pathogens during a bacterial attack or symbiosis and in combination with the camptothecin (CPT), and on calf thymus DNA. Study Design: Human lymphocytes and calf thymus DNA were treated with isolated T. thermophilus RLs for studying DNA damage in vitro. Methodology: RLs DNA damaging action was evaluated by the Sister Chromatid Exchanges (SCEs) methodology, a method for estimating genotoxicity of human exposure to different chemicals or other mutagenic agents and by DNA electrophoretic mobility experiments. Results: RLs at concentrations of 100 and 150 μg/mL reveal significant toxicity. The highest concentration of 200 μg/mL reveals higher genotoxicity. The frequency of SCEs/cell was increased two times over the control level. When CPT, an antineoplastic drug with DNA damaging action, was tested together with RLs the genotoxic activity was reduced significantly (P<0.01) compared to the action caused by CPT itself. Sequential increase in the concentration of RLs results in the proportional reduction of Proliferation Rate Index (PRI) which is a cytostatic index. Also, Mitotic Index (MI), a cytotoxic index, was also significantly decreased at concentration of 200 μg/mL RLs. Addition of RLs in the same concentration together with CPT doesn’t affect the MI so much. Moreover, RLs are obviously capable for strong binding to plasmid or calf thymus DNA in vitro. Conclusion: RLs exert genotoxicity, cytotoxicity and cytostaticity in human lymphocytes and play probably a protective role for cells against CPT due to RLs’ detergent capability to enrobe CPT and DNA, providing a significant property that might support its possible involvement in DNA horizontal transfer phenomena.

Induction of SCEs and DNA fragmentation in bovine peripheral lymphocytes by in vitro exposure to tolylfluanid-based fungicide

The potential for genotoxic and cytotoxic effects of tolylfluanid-based fungicide (50 percent active agent) was evaluated using sister chromatid exchange (SCE) and proliferation indices (PI) in cultured bovine peripheral lymphocytes. For the detection of possible genetic damage, DNA fragmentation assay was also applied. Bovine lymphocytes cultured for 72 h were treated with the fungicide at the final concentrations of 1.75, 3.5, 8.75, and 17.5 µg/mL for the last 24 and 48 h of culture without S9 metabolic activation, and during the last 2 h of culture with S9 metabolic activation. In the SCE assays no evidence for genotoxic activity of the fungicide was found in treatments of 24 h without and 2 h with S9. After the 24 h exposure to tolylfluanid, a weak decrease in the PI was observed. With the prolonged exposure time (48 h), dose dependence in the increase of SCE frequencies was observed. Moreover, after 48 h exposure slight fragmentation of DNA at the concentrations of 3.5 and 8.75 µg/mL was demonstrated. SCE quantification is the most widely used approach for the assessment of genotoxic/cytogenetic effects of chemical compounds. Positive results in the assay at 48 h exposure indicated a potential of the fungicide to increase frequency of chromosomal damage (replication injuries) that is the confirmation of early effect of exposure.

Modulatory effects of garlic extract against the cyclophosphamide induced genotoxicity in human lymphocytes in vitro.

Cyclophosphamide (CP) is a commonly used chemotherapeutic and immunosuppressive agent which is used in the treatment of wide range of cancers and autoimmune diseases. Besides that it is a well known carcinogen. In this study by using chromosomal aberrations (CA) and sister chromatid exchanges (SCE) assays method, the modulatory effects exerted by the extract of garlic against the CP induced genotoxicity in the human lymphocyte cultures in vitro were tested. Three different doses of garlic extract were tested for their modulatory capacity on the mutagenecity exerted by 100 µg ml-1 of CP. The results indicate a significant decrease in the frequency of CA and SCE suggesting that the garlic extract modulates the CP induced genotoxicity in a dose dependent manner. These findings provide the future directions for the research on design and development of possible modulatory drugs containing garlic extract.

Concentration of Urinary Cotinine and Frequency of Sister Chromatid Exchange in Lymphocytes among Male Adolescents / 예방의학회지

OBJECTIVES: To evaluate the internal burden and hazardous effects associated with smoking in middle and high school students. METHODS: We analysed urinary cotinine (U-cotinine) concentrations and the frequency of Sister Chromatid Exchanges (SCE). A comparison was done of U-cotinine concentrations and the frequency of SCE in peripheral lymphocytes across school levels (middle vs. high) and smoking types (direct: daily & occasional smoking, indirect: usual indirect & non-smoking), in 122 males. RESULTS: The middle school student group comprised 6.8% daily smokers, 15.9% occasional smokers, 40.9% daily indirect smokers, and 35.4% nonsmokers, while the high school student group comprised 18.0%, 20.5%,39.7%, and 21.8%, respectively. The U-cotinine concentration and the frequency of SCE among the middle school students were 79.11 microgram/literand 2.0 per cell, respectively, which were significantly lower than the 146.85 microgram/liter (p=0.078) and 2.6 per cell (p=0.005) of the high school students. Among the 40 direct smokers, these two biomarkers were 235.66 microgram/literand 2.59 per cell, significantly higher than the 67.33 microgram/liter (p=0.0001) and2.1 per cell (p=0.003) among indirect smoking groups. The variation in individual U-cotinine concentration ranged widely in both the indirect and direct smoking groups. CONCLUSION: Urinary cotinine concentrations and the frequency of Sister Chromatid Exchange seem to objectively and effectively evaluate student exposure whether it was direct or indirect smoking. Consequently, these biomarkers may be useful in monitoring the objective efficacy of anti-smoking programs in adolescent populations.