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Research Article
 

Study of the Effects of a Chayotte (Sechium edule) Extract on the Plasmid Puc. 9.1 DNA



G.F. Dire, M.F.M.C. Coura, M.C.L. Almeida, S.D.D. Vasconcelos, P.R.A. Siqueira, R.M. Duarte, J.S. Rodrigues, J.C.S. Oliveira, M.L. Fernandes and M. Bernardo-Filho
 
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ABSTRACT

Stannous chloride (SnCl2) is employed as a reducing agent to obtain Technetium-99m-labelled radiopharmaceuticals in nuclear medicine kits, being inject endogenously in humans. Toxic effects of. these kits were not studied, thus making it important to evaluate their impact in humans. The use of natural extracts as medicines is growing around the world. The chayotte (Sechium edule) is a subtropical vegetable with potent diuretic action. It is used in folk medicine due its hypotensor effect. In this study, plasmid deoxyribonucleic acid (DNA) was exposed to chayotte extracts (macerated) (0.1g mL-1) in presence of stannous chloride (SnCl2). Samples of the plasmid DNA were analyzed through agarose gel electrophoresis. The results show that the chayotte extract was capable of damaging the DNA in the presence and in the absent of SnCl2.

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G.F. Dire, M.F.M.C. Coura, M.C.L. Almeida, S.D.D. Vasconcelos, P.R.A. Siqueira, R.M. Duarte, J.S. Rodrigues, J.C.S. Oliveira, M.L. Fernandes and M. Bernardo-Filho, 2007. Study of the Effects of a Chayotte (Sechium edule) Extract on the Plasmid Puc. 9.1 DNA. Plant Pathology Journal, 6: 72-76.

DOI: 10.3923/ppj.2007.72.76

URL: https://scialert.net/abstract/?doi=ppj.2007.72.76

INTRODUCTION

Stannous chloride can cause skin and mucosal irritation in humans and when this salt is injected into laboratory animals, it can produce stimulation and subsequent depression of the central nervous system (Gleason et al., 1969). It has been suggested that SnCl2 is a powerful genotoxic (McLean et al., 1983; Oliver and Marzin, 1987), mutagenic (Singh, 1983; Triphaty et al., 1990) and carcinogenic (Ashby and Tennant, 1991) compound. In nuclear medicine, SnCl2 has been employed in scintigraphic test as Technetium-99m (99mTc) reducing agent. Besides the use of SnCl2 in nuclear medicine, this salt is also used in dentistry (dentifrices) (Hallas and Cooney, 1981; McLean et al., 1983; Rader, 1991; Budavery, 1996).

There are other sources of SnCl2 to which human beings are exposed to such as from environmental contamination by biocide preparations containing organic compound dimethyl stannous chloride [SnCl2 (CH3)2] (Hallas and Cooney, 1981). It is hypothesized that the toxicity of SnCl2 might be mediated by generation of. reactive oxygen species (ROS) through the reaction: Sn2+ + O2 + 2H+ → Sn4+ + H2O2. The generation hydrogen peroxide undergoes by Fenton reaction to generate OH as follows: Fe2+ + H2O2 → OH- + OH (4). It was also described that SnCl2 mediates single strand breaks in plasmid DNA through ROS formation in a dose-dependent manner (Dantas et al., 1996). In addition, the mutagenic potentiality of SnCl2 was identified by supF gene mapping (Cabral et al., 1998). It was also determined that Escherichia coli (E. coli) strains proficient in DNA repair mechanisms were more resistant to SnCl2 treatment than deficient ones, suggesting that inactivation was due to DNA damage (Aherne and O’Brien, 1999). Biological effects of metals have been reported: (i) transition metals catalyze free radical production that can be related to aging processes and neurodegenerative diseases such as Alzheimer’s diseases, Parkinson’s disease and others (Stohs and Bagchi, 1995); (ii) the association between human diseases and metal ions metabolism can be also demonstrated by Huntington’s disease and amyotrophic lateral sclerosis; (iii) neurotoxic properties of aluminum and its etiopathogenetic role in Alzheimer’a disease are still controversially argued and need further elucidation (Gutteridge et al., 1985); (iv) abnormal copper metabolism detected in the brain tissue of Wilson’s and Menke’s patients remains to be fully explained (Gutteridge et al., 1985); (v) substantial levels of zinc are found in the hippocampus and its brain deficiency can be the cause of several pathological events (Bettger and O’Dell, 1981), however; (vi) magnesium is successfully used for the treatment of migraine (Stohs and Bagchi, 1995). In addition, the molecular mechanisms underlying neurotoxicity associated with mercury, tin and manganese, also need further investigation. Tin is a heavy metal which has long been regarded as a contaminant of the environment (Wood, 1974). One of its inorganic salts, stannous chloride (SnCl2), has been widely used in daily human life, to conserve soft drinks, in food manufacturing, as a result of. processing and packaging. Studies on the biological effects of SnCl2 revealed that it can generate reactive oxygen species (ROS) and breaks in deoxyribonucleic acid (DNA) (Caldeira-de-Araújo et al., 1996) and induces lethality in E. coli, whose damage recovery depends on RecA-mediated repair (Bernardo-Filho et al., 1994b). Medicinal plants, are mainly complex products with several components with different chemical and pharmacological characteristics (Moro and Basile, 2000). In addition, many of these products are also sold as dietary supplement, but, scientific information about their safe and effective use is hard to find because limited toxicological data are available on herbal remedies and support of rigorous clinical studies is lacking (Capasso et al., 2000). The use of natural products as medicines has been growing in the entire world. Because of this fact, many studies with natural products are being developed and new drugs for treatments of diseases are being discovered. In the literature, the medicinal action mechanism of several plants has been described and different compounds, with various properties, have been isolated from the crude extracts (Leite et al., 1986; Sallé, 1996). Sechium edule (Chayotte) a subtropical vegetable with potent diuretic action, is a cucurbitaceus species which is used as food or as medication in popular medicine. It was reported a case of severe hypokalemia pregnancy and that a chayotte preparation was implicated, as the potassium level returned to normal, without recurrence of hypokalemia, once the ingestion of this vegetable stopped (Jensen and Lai, 1986). Gordon (2000) described the hypotensor effect of the chayotte. Diré et al. (2002), have shown that the extracts of chayotte (macerated and infusion) were capable of altering the labeling of blood elements with technetium-99m (99mTc) in a in vivo study. In other research Diré et al. (2001), have demonstrated that a chayotte extract (macerated) was able to alter the biodistribution of 99mTc as sodium pertechnetate (NaTcO4) as well as the shape of red blood cells through a qualitative analysis. The effect of stannous ion has been abolished by extracts of some medicinal plants (Reiniger et al., 1999; Melo et al., 2001; Lima et al., 2002, Silva et al., 2002). Bernardo et al. (2002), described that the rutin, a compound isolated from Ruta graveolens, was not capable of damaging DNA, protecting DNA from the SnCl2 redox action and inactivating the Escherichia coli (E. coli AB1157) culture.

Orsolic et al. (2005) described that many dietary constituents are chemopreventive in animal models and experiments with cultured cells are revealing various potential mechanisms of action. Compounds classified as blocking agents can prevent, or greatly reduce, initiation of carcinogenesis, or suppressing agents can act on cell proliferation. Caffeic acid (CA) and caffeic acid phenethyl ester (CAPE), members of the polyphenolic compounds, are present in high concentrations in medicinal plants and propolis, a natural beehive product. Based on the findings of the referred author it was postulated that the antitumor activity of polyphenolic compounds includes direct cytotoxic effects on tumor cells.

ROS are generated during a variety of cellular events with beneficial as well as deleterious effects to the organism (Halliwell, 1994). Some plant extracts may increase the effects of the deleterious actions of ROS (Lima et al., 2001). In the present study, we have evaluated the influence of a chayotte extracts on the topology on gel electrophoretic of plasmid DNA submitted to SnCl2.

MATERIALS AND METHODS

Characterization of the chayotte sample: Chayotte was purchased from a local market in Rio de Janeiro city, RJ, Brazil. To prepare the extract, 50 g of the skin of chayotte were mixture with 500 mL of water in an electric extractor. This preparation was filtered and this extract was considered 100%.

The presence of toxic compounds was evaluated and we did not find them in the extracts of chayotte used in our experiments. The method to verify the presence of these toxic products is based on inhibition of acetylcholinesterase in the presence of the pesticides (Cunha Bastos et al., 1991). In this method, brain acethylcholinestarase is utilized as an in vitro detector of organophosphorus and carbamate insecticides. Briefly, a preparation of acetylcholinesterase was obtained after extraction of a rat brain microsomal fraction with Triton X-100 and was incubated with the extract of cauliflower. Enzyme assay was performed by a potentiometric method based on the formation of acetic acid in the incubation mixture (preparation of acetylcholinesterase and extract of chayotte).

Nucleic acid manipulations: Plasmids were diluted, dispensed into eppendorf tubes (200 ng per tube) and incubated with 200 μg mL-1 of SnCl2. To evaluate the influence of the extract of the chayotte in DNA breakage, a concentration on a par with 0.1 g mL-1 was used. In all cases, reaction mixtures were incubated at 37°C for 40 min. The analysis of the single breaks (SSB) formation was performed using 0.8% agarose gel electrophoresis in order to separate the conformations of plasmid DNA: form I supercoiled native conformation and form II open circle resulting from SSB. Aliquots from each sample (10 μL) were mixed to 2 μL of 6x concentrated loading buffer (0.25% xylene cyanol FF; 0.25% bromofenol blue; 30% glycerol) and applied in a horizontal gel electrophoresis chamber in Tris acetate-EDTA buffer at pH 8.0. After electrophoresis, the gel was stained with ethidium bromide (0.5 μg mL-1) and the DNA bands were visualized by fluorescence in an ultraviolet (UV) transiluminator system. Permanent records were performed using a polaroid MP-4+system.

RESULTS

Figure 1 shows the electrophoresis in agarose gel of pUC. 9.1 plasmid with SnCl2 and/or the extract of macerated extract.

In Fig. 1 the electrophoretic mobility of plasmidial DNA in various experimental conditions (macerated extract) is shown.

Image for - Study of the Effects of a Chayotte (Sechium edule) Extract on the Plasmid Puc. 9.1 DNA
Fig. 1: Column 1: (Control: DNA + water), Column 2: (Chayotte 100%), Column 3: (SnCl2 200 μg mL-1), Column 4: (SnCl2- 200 μg mL-1 + Chayotte 100%), Column 5: (oxidized Chayotte-10min ), Column 6: (oxidized SnCl2- 10 min), Column 7 (oxidized Chayotte+ SnCl2- 10 min) and Column 8 (marker λ hind III). Photos of the gels were scanned

DISCUSSION

Much effort has focused on the identification of phytochemicals in plants, which exert biological effects. The knowledge of these effects are worthwhile and can help to prevent possible undesirable actions of crude extracts and/or purified substances isolated from various plants. Moreover, many times the results reported in the literature are controversies. This fact could be explained by (i) various experimental conditions and models, (ii) the characteristic and the concentration of the used material (crude extract, isolated fraction, purified substance or heated extract) and (iii) the specific condition of the growth of the studied plant. Many biological effects have been associated with the flavonoids and other antioxidant molecules (Aherne and Brien, 1999). Reactive oxygen species (ROS) have been implicated as the primary destructive intermediates in a wide range of environmental conditions as well as in an increasing number of humans disorders (mutagenesis, apoptosis, aging) (Hladik et al., 1987). SnCl2 has been used as a reducing agent (Bernardo-Filho et al., 1994; Caldeira-de-Araujo et al., 1996) in medical procedures.

Cytotoxic and genotoxic SnCl2-induced damage were demonstrated in E. coli and the effects appeared to be mediated by ROS (Caldeira-de-Araújo et al., 1999; Dantas et al., 1996,1999; Felzenszwalb et al., 1998; Reiniger et al., 1999).

The treatment of the E.coli strains AB1157 with SnCl2 in presence of Peumus boldus (Reiniger et al., 1999), Cymbopogon citratus, Maytenus ilicifolia, Baccharis genistelloides (Melo et al., 2001), Rutin (Bernardo et al., 2002) and with Brassica oleracea L. var botrytis (Lima et al., 2002) induced protection of cells against the citotoxic-SnCl2 effects. It could probably be due to oxidant properties of these extracts. However, the intensity of the protective effect against the SnCl2 effects was dependent on the considered extract. Like observed in the study of the cauliflower extract (Lima et al., 2002) the extract of chayotte was capable of inducing lesion of break type in the plasmid pUC 9.1 DNA (Lima et al., 2001). In comparison with the Bernardo et al. (2002), study, it was verified that the rutin different of the chayotte extracts have not induced lesion in the DNA molecule. In this study the extracts of chayotte (macerated) in all concentrations tested have induced lesions in the DNA molecule. In the analysis of Peumus boldus extract was noticed that it has reduced or abolished the effect of SnCl2 although the lesive effect of boldine was observed when the highest concentration of this substance was used in the presence of the reducing agent despite boldine alone has not been capable of inducing alterations in the DNA. Silva et al. (2002), working with different cultures of E. coli strains also have shown a reduction of the lethal effect induced by SnCl2 on the survival of the cultures in the presence of C. citratus, B. genistelloides, M. ilicifolia and P. boldus. In the present work it was verified that the extract had induced breaks in the DNA molecule.

CONCLUSIONS

In general we can speculate that the extract of chayotte was capable of inducing damages in pUC. 9.1 DNA molecules and considering maybe due to the generation the free radicals which might induced lesions in the DNA molecule.

ACKNOWLEDGMENTS

The present study was supported by CNPq, CAPES, FAPERJ and UERJ.

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