Protective effects of acerola juice on genotoxicity induced by iron in vivo

Metal ions such as iron can induce DNA damage by inducing reactive oxygen species (ROS) and oxidative stress. Vitamin C is one of the most widely consumed antioxidants worldwide, present in many fruits and vegetables, especially inMalpighia glabra L., popularly known as acerola, native to Brazil. Acerola is considered a functional fruit due to its high antioxidant properties and phenolic contents, and therefore is consumed to prevent diseases or as adjuvant in treatment strategies. Here, the influence of ripe and unripe acerola juices on iron genotoxicity was analyzed in vivo using the comet assay and micronucleus test. The comet assay results showed that acerola juice exerted no genotoxic or antigenotoxic activity. Neither ripe nor unripe acerola juices were mutagenic to animals treated with juices, in micronucleus test. However, when compared to iron group, the pre-treatment with acerola juices exerted antimutagenic activity, decreasing significantly micronucleus mean values in bone marrow. Stage of ripeness did not influence the interaction of acerola compounds with DNA, and both ripe and unripe acerola juices exerted protective effect over DNA damage generated by iron.     

Juglone, a naphthoquinone from walnut, exerts cytotoxic and genotoxic effects against cultured melanoma tumor cells

This study demonstrates cytotoxic and genotoxic potential of juglone, a chief constituent of walnut, and its underlying mechanisms against melanoma cells. MTT assay and clonogenic assay were used to study cytotoxicity, micronucleus assay to assess genotoxicity, glutathione (GSH) assay and 2′,7′-dicholorofluorescein diacetate (DCFH-DA) assay to evaluate the oxidative stress induction. Apoptosis/necrosis induction was analysed by flow cytometry. We observed a concentration-dependent decrease in cell survival with a corresponding increase in the lactate dehydrogenase levels. A dose-dependent increase in the frequency of micronucleated binucleate cells indicated the potential of juglone to induce cytogenetic damage in melanoma tumor cells. Moreover, results of the micronuclei study indicated division delay in the proliferating cell population by showing decrease in the cytokinesis blocked proliferation index. Further, juglone-induced apoptosis and necrosis could be demonstrated by oligonucleosomal ladder formation, microscopic analysis, increase in the hypodiploid fraction (sub Go peak in DNA histogram), as well as an increased percentage of AnnexinV(+)/PI(+) cells detected by flow cytometry. A significant concentration-dependent decrease in the glutathione levels and increase in dichlorofluorescein (DCF) fluorescence after juglone treatment confirmed the ability of juglone to generate intracellular reactive oxygen species. The cytotoxic effect of juglone can be attributed to mechanisms including the induction of oxidative stress, cell membrane damage, and a clastogenic action leading to cell death by both apoptosis and necrosis.     

2-Aminoanthracene, diethylstilboestrol and vinblastine tested in the in vitro mammalian cell micronucleus test at British American Tobacco UK in support of the OECD draft Test Guideline 487

Reference genotoxic compounds 2-aminoanthracene, diethylstilboestrol and vinblastine were tested in the in vitro micronucleus assay using Chinese hamster V79 derived cells in the laboratories of British American Tobacco in the UK. The work was conducted in support of the cytotoxicity measures recommended in the 2007 version of the OECD Test Guideline 487. The three compounds were positive in the assay in the presence and absence of the cytokinesis blocking agent cytochalasin B at concentrations that did not exceed the recommended cytotoxic limits determined by relative population doubling, relative increase in cell counts, relative cell counts and cytokinesis block proliferation index. Consequently, this work supports the hypothesis that relative population doubling, relative increase in cell counts and relative cell counts are appropriate measures of cytotoxicity for the non-cytokinesis blocked in vitro micronucleus assay.     

Assessment of in vitro cytotoxic and genotoxic activities of some trimethoprim conjugates

Trimethoprim, a commonly used antibacterial agent, is widely applied in the treatment of variety of infections in human. A few studies have demonstrated an extensive exposure of man to antibiotics, but there is still a lack of data for cytotoxic effects including nephrotoxicity, gastrointestinal toxicity, hematotoxicity, neurotoxicity and ototoxicity. The main purpose behind this study was to determine cytotoxic and genotoxic activities of trimethoprim (1), trimethoprim with maleic acid (2) and trimethoprim in conjugation with oxalic acid dihydrate (3). The cytotoxic effects of these three conjugates were elucidated by employing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoium bromide (MTT) assay using embryonic rat fibroblast-like cell line (F2408) and H-ras oncogene activated embryonic rat fibroblast-like cancer cell line (5RP7). Additionally, determination of genotoxic activity of these three compounds were studied by using cytokinesis blocked micronucleus assay (CBMN) in human lymphocytes. The results demonstrated that trimethoprim alone and its combination with other compounds are able to induce both cytotoxic and genotoxic damage on cultured cells (F2408, 5RP7, human lymphocytes).     

Genotoxicity of the coating lacquer on food cans, bisphenol A diglycidyl ether (BADGE), its hydrolysis products and a chlorohydrin of BADGE

The epoxy resin bisphenol A diglycidyl ether (BADGE), its hydrolysis products and a chlorohydrin of BADGE (BADGE.2HCl), were examined for their genotoxicity in the micronucleus test (MNT) with human peripheral blood lymphocytes in vitro, in presence and in absence of an exogenous metabolizing system S9 rat liver. The treatment was done using different compound concentrations up to cytotoxic doses. The concentrations tested ranged between 12.5 to 62.5microg/ml of BADGE, 12.5 to 62.5microg/ml of first BADGE hydrolysis product (BADGE.H(2)O), 25.0 to 100.0microg/ml of second BADGE hydrolysis product (BADGE.2H(2)O) and 6.25 to 50.0microg/ml of BADGE.2HCl. These compounds are able to induce both cytotoxic and genotoxic effects, as revealed by the increases observed in cytokinesis block proliferation index (CBPI) and in micronuclei (MN) frequencies, respectively.     

双歧杆菌发酵果蔬汁对小鼠免疫调节机制的研究

目的研究双歧杆菌发酵果蔬汁对小鼠免疫调节作用的机制。方法将小鼠随机分成纯净水对照组、非发酵果蔬汁组和发酵果蔬汁低、中、高三个剂量组,饮水法喂饲小鼠30d,检测小鼠脾细胞的增殖能力及T淋巴细胞亚群,ELISA法测定血清IL-6和IFN-γ水平。结果发酵果蔬汁可增强小鼠脾细胞的增殖能力,T细胞表面标志CD3、CD4、CD8表达增强,促进血清中IL-6和IFN-γ的生成。结论双歧杆菌发酵果蔬汁能提高小鼠的体液、细胞免疫和非特异性免疫功能,并呈剂量效应关系。     

Assessment of In Vivo and In Vitro Genotoxicity of Glibenclamide in Eukaryotic Cells

Glibenclamide is an oral hypoglycemic drug commonly prescribed for the treatment of type 2 diabetes mellitus, whose anti-tumor activity has been recently described in several human cancer cells. The mutagenic potential of such an antidiabetic drug and its recombinogenic activity in eukaryotic cells were evaluated, the latter for the first time. The mutagenic potential of glibenclamide in therapeutically plasma (0.6 μM) and higher concentrations (10 μM, 100 μM, 240 μM and 480 μM) was assessed by the in vitro mammalian cell micronucleus test in human lymphocytes. Since the loss of heterozygosity arising from allelic recombination is an important biologically significant consequence of oxidative damage, the glibenclamide recombinogenic activity at 1 μM, 10 μM and 100 μM concentrations was evaluated by the in vivo homozygotization assay. Glibenclamide failed to alter the frequency of micronuclei between 0.6 μM and 480 μM concentrations and the cytokinesis block proliferation index between 0.6 μM and 240 μM concentrations. On the other hand, glibenclamide changed the cell-proliferation kinetics when used at 480 μM. In the homozygotization assay, the homozygotization indices for the analyzed markers were lower than 2.0 and demonstrated the lack of recombinogenic activity of glibenclamide. Data in the current study demonstrate that glibenclamide, in current experimental conditions, is devoid of significant genotoxic effects. This fact encourages further investigations on the use of this antidiabetic agent as a chemotherapeutic drug.     

The mutagenic and antimutagenic effects of Ecballium elaterium fruit juice in human peripheral lymphocytes

The aim of this study was to investigate the mutagenic and antimutagenic effects of Ecballium elaterium (EE) fruit juice, which has an anti-inflammatory effect, using in vitro human peripheral lymphocytes. To investigate the mutagenic effects of the EE fruit juice, human peripheral lymphocytes were treated with three doses (18, 36, and 72 μl/l) of fruit juice alone for 24 and 48 h. For investigating the antimutagenic effects of the EE fruit juice, the human lymphocytes were also treated with the mixture of the fruit juice and 0.25 μg/ml MMC. The EE fruit juice induced the percentage of total CA when used alone (especially the percentage of structural CA than the percentage of the numerical CA) and synergically induced the percentage of total CA when used as a mixture with MMC. The EE fruit juice did not affect the SCE frequency for 24 and 48 h treatment time. In contrast, EE and MMC as a mixture sinergically induced the SCE frequency at the highest concentration for 48 h treatment time only. EE alone did not decrease the RI while it decreased the MI in a dose-dependent manner. EE and MMC as a mixture have a higher cytotoxic effect than the cytotoxic effects of EE alone. As a result, it can be concluded that EE had no antimutagenic effect while EE had a mutagenic and a cytotoxic effect in human peripheral lymphocytes. This article was submitted by the authors in English.     

Use of the cytokinesis-block micronucleus method in mouse splenocytes

Mouse splenocytes have been used in the cytokinesis-block method for the evaluation of micronuclei induced by mutagenic agents in vitro as well as in vivo. Stimulation with concanavalin A for 48 h followed by 16-24-h treatment with 5 micrograms/ml cytochalasin B was found to be an optimum condition to obtain micronuclei in the binucleated splenocytes after the cells were cultured in vitro. Under the above conditions splenocytes from mice pretreated with a single i.p. injection of cyclophosphamide gave a significant increase in micronucleus production. This increase was dependent on the dose of cyclophosphamide (r = 0.99). A dose of 50 mg/kg resulted in 22% of the binucleated cells producing micronuclei, more than 20 times the level in the untreated control. The increase was also dependent on the time of cyclophosphamide injection before removal of the spleen. A duration of 4-8 h after cyclophosphamide injection gave rather sharp optimum values for the production of micronuclei. When splenocytes from non-treated mice were treated with mitomycin C together with cytochalasin B in the above in vitro condition, there was a significant increase in micronucleus production in the binucleated cells. It was also dependent on the dose of mitomycin C (r = 0.975) and a dose of 0.5 micrograms/ml resulted in a more than 20-fold increase over the untreated control. Thus, the use of mouse splenocytes in the cytokinesis-block micronucleus assay was shown to be sensitive enough for testing mutagenic agents in vivo as well as in vitro.     

Genotoxicity of the coating lacquer on food cans, bisphenol A diglycidyl ether (BADGE), its hydrolysis products and a chlorohydrin of BADGE

The epoxy resin bisphenol A diglycidyl ether (BADGE), its hydrolysis products and a chlorohydrin of BADGE (BADGE·2HCl), were examined for their genotoxicity in the micronucleus test (MNT) with human peripheral blood lymphocytes in vitro, in presence and in absence of an exogenous metabolizing system S9 rat liver. The treatment was done using different compound concentrations up to cytotoxic doses. The concentrations tested ranged between 12.5 to 62.5 μg/ml of BADGE, 12.5 to 62.5 μg/ml of first BADGE hydrolysis product (BADGE·H₂O), 25.0 to 100.0 μg/ml of second BADGE hydrolysis product (BADGE·2H₂O) and 6.25 to 50.0 μg/ml of BADGE·2HCl. These compounds are able to induce both cytotoxic and genotoxic effects, as revealed by the increases observed in cytokinesis block proliferation index (CBPI) and in micronuclei (MN) frequencies, respectively.     

Evaluation of mutagenic activities of leachates in landfill sites by micronucleus test and comet assay using goldfish

To develop a simple system for monitoring the presence of mutagens/carcinogens in the leachates from landfill sites, we used a micronucleus test and a single cell gel electrophoresis (comet) assay originally developed for mice and rats on goldfish (Carassius auratus). The goldfish were exposed for 9 days to the leachate with chemical and biological treatment (treated leachate) or without treatment (raw leachate). The goldfish exposed to several samples died because of the high concentrations of NaCl or ammonium ion (NH4+). In the comet assay using peripheral erythrocytes, the raw leachates showed higher mutagenic activity than the treated leachates. In the micronucleus test, it was difficult to detect the micronuclei in peripheral erythrocytes. On the other hand, the frequency of micronuclei was high in gill cells of goldfish exposed to the raw leachates compared to the treated leachates. A combination of the two bioassays was shown to be useful to evaluate the mutagenic activity of the leachates. We also propose a new scoring method for determination of water quality by using acute toxicity and mutagenic activity.     

Inhibition of -tyrosine-induced micronuclei production by phenylthiourea in human melanoma cells

It was previously found that -tyrosine oxidation product(s) are cytotoxic, genotoxic and increase the sister chromatid exchange (SCE) levels in human melanoma cells. In this work, the micronucleus assay has been performed on human melanotic and amelanotic melanoma cell lines (Carl-1 MEL and AMEL) in the presence of 1.0, 0.5 and 0.1 mM -tyrosine concentrations to investigate if melanin synthesis intermediate(s) increase micronuclei production. -Tyrosine oxidation product(s) increased the frequency of micronuclei in melanoma cells; 0.1 mM phenylthiourea (PTU), an inhibitor of -tyrosine oxidation by tyrosinase, lowered the micronucleus production to the control levels. The culture of melanoma cells with high -tyrosine in the culture medium resulted in a positive response to an ELISA-based apoptotic test. For comparison the effect of -tyrosine on micronuclei production in human amelanotic melanoma cells was also investigated; the micronucleus production in the presence of 1 mM -tyrosine in the culture medium was lower than that found with melanotic melanoma cells of the same cell line. The data suggest that melanin synthesis intermediates arising from -tyrosine oxidation may cause micronuclei production in Carl-1 human melanoma cells; the addition of PTU in the presence of -tyrosine decreased the frequency of micronuclei to about the control values thus the inhibition of melanogenesis may have some clinical implication in melanotic melanoma.     

Cytotoxic, genotoxic, and mutagenic effects of diphenyl diselenide in Chinese hamster lung fibroblasts

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organic selenium compounds, and may increase the risk of human exposure to this chemical at the workplace. In a previous study, we demonstrated the pro-oxidant action and the mutagenic properties of this compound on bacteria and yeast. In the present study, we evaluated the putative cytotoxic, pro-oxidant, genotoxic, and mutagenic properties of this molecule in V79 Chinese lung fibroblast cells. When cells were treated with increasing concentrations of DPDS, its cytotoxic activity, as determined using four cell viability endpoints, occurs in doses up to 50 microM. The MTT reduction was stimulated, which may indicate reactive oxygen species (ROS) generation. Accordingly, the treatment of cells for 3h with cytotoxic doses of DPDS increased TBARS levels, and sensitized cells to oxidative challenge, indicating a pro-oxidant effect. The measurement of total, reduced, and oxidized glutathione showed that DPDS can lead to lower intracellular glutathione depletion, with no increase in the oxidation rate in a dose- and time-dependent manner. At the higher doses, DPDS generates DNA strand breaks, as observed using the comet assay. The treatment also induced an increase in the number of binucleated cells in the micronucleus test, showing mutagenic risk by this molecule at high concentrations. Finally, pre-incubation with N-acetylcysteine, which restored GSH to normal levels, annulled DPDS pro-oxidant and genotoxic effects. These findings show that DPDS-induced oxidative stress and toxicity are closely related to intracellular level of reduced glutathione. Moreover, at lower doses, this molecule has antioxidant properties, protecting the cell against oxidative damage induced by hydrogen peroxide.     

Inhibition of L-tyrosine-induced micronuclei production by phenylthiourea in human melanoma cells

It was previously found that L-tyrosine oxidation product(s) are cytotoxic, genotoxic and increase the sister chromatid exchange (SCE) levels in human melanoma cells. In this work, the micronucleus assay has been performed on human melanotic and amelanotic melanoma cell lines (Carl-1 MEL and AMEL) in the presence of 1.0, 0.5 and 0.1 mM L-tyrosine concentrations to investigate if melanin synthesis intermediate(s) increase micronuclei production. L-Tyrosine oxidation product(s) increased the frequency of micronuclei in melanoma cells; 0.1 mM phenylthiourea (PTU), an inhibitor of L-tyrosine oxidation by tyrosinase, lowered the micronucleus production to the control levels. The culture of melanoma cells with high L-tyrosine in the culture medium resulted in a positive response to an ELISA-based apoptotic test. For comparison the effect of L-tyrosine on micronuclei production in human amelanotic melanoma cells was also investigated; the micronucleus production in the presence of 1 mM L-tyrosine in the culture medium was lower than that found with melanotic melanoma cells of the same cell line. The data suggest that melanin synthesis intermediates arising from L-tyrosine oxidation may cause micronuclei production in Carl-1 human melanoma cells; the addition of PTU in the presence of L-tyrosine decreased the frequency of micronuclei to about the control values thus the inhibition of melanogenesis may have some clinical implication in melanotic melanoma.     

The mutagenic and anti-mutagenic effects of Ecballium elaterium fruit juice in human peripheral lymphocytes

The aim of this study was to investigate the mutagenic and anti-mutagenic effects of Ecballium elaterium (EE) fruit juice which has an anti inflammatory effect using in vitro human peripheral lymphocytes. For the investigating the mutagenic effects of EE fruit juice, human peripheral lymphocytes was treated with three doses (18, 36 and 72 microl/1) of fruit juice alone for 24 and 48 h. For the investigating the anti-mutagenic effects of the EE fruit juice, the human lymphocytes also treated with the mixture of the fruit juice and 0.25 microg/ml MMC. EE fruit juice induced the percentage of total CA when used alone (especially the percentage of structural CA than the percentage of the numerical CA) and synergically induced the percentage of total CA when used as a mixture with MMC. EE fruit juice did not affect the SCE frequency for 24 and 48 h treatment time. In contrast, EE and MMC as a mixture, sinergically induced the SCE frequency at the highest concentration for 48 h treatment time only. EE alone did not decrease the RI while it decreased the MI as a dose dependent manner. EE and MMC as a mixture have the higher cytotoxic effect than the cytotoxic effects of EE alone. As a result, it can be concluded that, EE had no anti-mutagenic effect while EE had a mutagenic and a cytotoxic effect in human peripheral lymphocytes.     

Phytochemical composition and in vitro biological activities of Morinda citrifolia fruit juice

Morinda citrifolia is a plant with broad nutraceutical and therapeutic effects and used in the traditional treatment of several ailments. The objective of this work is to investigate the phytochemistry of the fruit juice of M. citrifolia on one hand and on other hand to evaluate its antiradical and antibacterial activity. The phytochemical investigation was carried out by tube staining tests of the extract of two types of fruit juice of M. citrifolia. The antioxidant activity of these juices was evaluated by reducing the DPPH radical method. Concerning the antibacterial activity, it was tested on the in vitro growth of 10 reference bacterial strains using the well diffusion method. Qualitative phytochemistry of M. citrifolia fruit juices revealed the presence of large groups of secondary metabolites including polyphenols, reducing compounds, mucilage and terpernoids. The antioxidant activity of M. citrifolia fruit juices is dose-dependent and higher than that of ascorbic acid. Antimicrobial activity on other hand revealed that fruit juices inhibit growth inhibitory activity of Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, S. epidermidis, Proteus vulgaris, Streptococcus oralis, Enterococcus faecalis and Escherichia coli. This observed difference is significant for each juices on the strains (p < 0.001). These results support the use of M. citrifolia in traditional medicine and are the starting points for the development of a new drug to combat both dietary conditions and chronic conditions associated with oxidative stress.     

Cytotoxic and genotoxic effects of tambjamine D, an alkaloid isolated from the nudibranch Tambja eliora, on Chinese hamster lung fibroblasts

Marine organisms have been shown to be potential sources of bioactive compounds with pharmaceutical applications. Previous chemical investigation of the nudibranch Tambja eliora led to the isolation of the alkaloid tambjamine D. Tambjamines have been isolated from marine sources and belong to the family of 4-methoxypyrrolic-derived natural products, which display promising immunosuppressive and cytotoxic properties. Their ability to intercalate DNA and their pro-oxidant activity may be related to some of the biological effects of the 4-methoxypyrrolic alkaloids. The aim of the present investigation was to determine the cytotoxic, pro-oxidant and genotoxic properties of tambjamine D in V79 Chinese hamster lung fibroblast cells. Tambjamine D displayed a potent cytotoxic effect in V79 cells (IC50 1.2 microg/mL) evaluated by the MTT assay. Based on the MTT result, V79 cells were treated with different concentrations of tambjamine D (0.6, 1.2, 2.4 and 4.8 microg/mL). After 24h, tambjamine D reduced the number of viable cells in a concentration-dependent way at all concentrations tested, assessed by the trypan blue dye exclusion test. The hemolytic assay showed that the cytotoxic activity of tambjamine D was not related to membrane disruption (EC50>100 microg/mL). Tambjamine D increased the number of apoptotic cells in a concentration-dependent manner at all concentrations tested according to acridine orange/ethidium bromide staining, showing that the alkaloid cytotoxic effect was related to the induction of apoptosis. MTT reduction was stimulated by tambjamine D, which may indicate the generation of reactive oxygen species. Accordingly, treatment of cells with tambjamine D increased nitrite/nitrate at all concentrations and TBARS production starting at the concentration corresponding to the IC50. Tambjamine D, also, induced DNA strand breaks and increased the micronucleus cell frequency as evaluated by comet and micronucleus tests, respectively, at all concentrations evaluated, showing a genotoxic risk induced by tambjamine D.     

Spontaneous and mitomycin-C-induced micronuclei in human lymphocytes exposed to extremely low frequency pulsed magnetic fields

The cytokinesis block micronucleus method, a very sensitive cytogenetic assay, was used to ascertain the possible genotoxic effects of extremely low frequency pulsed magnetic fields in phytohemagglutinin-stimulated human lymphocytes cultures from 16 healthy donors. Four conditions were studied: i) lymphocytes not exposed to the field (control cultures); ii) lymphocytes exposed to the field; iii) lymphocytes treated with mitomycin-C and not exposed to the field; iv) lymphocytes treated with mitomycin-C and exposed to the field. Mitomycin-C-treated cultures were used as control for the micronucleus method, because it is known that mitomycin-C is a potent genotoxic agent, capable of inducing micronuclei. The frequency of micronuclei in field-exposed cultures was similar to the spontaneous frequency observed in control unexposed-cultures. Moreover, the exposure to pulsed magnetic fields did not affect the frequency of micronuclei induced by mitomycin-C, suggesting that, in the experimental conditions used, this kind of field neither affected the integrity of chromosomes nor interfered with the genotoxic activity of mitomycin-C.