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Biological Effects of a Chayotte Extract in Wistar Rats with Induced Diabetes: A Radiopharmaceutically Analysis



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

The present research evaluated the influence of a chayotte(Sechium edule) extract (macerated) on the bioavailability of 99mTcO4Na as well as in the mass of the organs. In this study, in the biodistribution analysis, the 99mTcO4Na was administrated into female Wistar rats (diabetes and no diabetes induced) which had drunk or not the extract for 7 days. After 10 min, animals were sacrificed, the organs were isolated, the radioactivity determined in a well counter and the percentages of radioactivity per gram (%ATI/g) in the organs and mass of them (g) were calculated. The analysis of the results has indicated that in the diabetes group had been an increase in the uptake of 99mTcO4Na the in pancreas as well as in the diabetes groups treated with chayotte extract. The mass of the spleen, stomach, pancreas, heart and kidney has been altered due to the comparison of the groups. It is possible to suggest that some components of chayotte extracts present an oxidant power able to alter the biodistribution of 99mTcO4Na, as a tip, we speculate that the referred extract when metabolized in the liver may produce reactive metabolites with oxidant properties linked to the stress which is generated by diabetic status, this fact could justify by the increase of %ATI/g in the pancreas which probably may be due to the producing of AGEs in diabetes status as well as by the different molecular and cellular mechanisms related to the effects of the extract and diabetes would promote differences in the mass of the organs.

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G. F. Dire, J. S. Rodrigues, J. C.S. Oliveira, S. D.D. Vasconcelos, P. R.A. Siqueira, R. M. Duarte, M. C.L. Almeida, M.L. Fernandes and M. Bernardo-Filho, 2007. Biological Effects of a Chayotte Extract in Wistar Rats with Induced Diabetes: A Radiopharmaceutically Analysis. Pakistan Journal of Biological Sciences, 10: 568-574.

DOI: 10.3923/pjbs.2007.568.574

URL: https://scialert.net/abstract/?doi=pjbs.2007.568.574

INTRODUCTION

The formation of Advanced Glycation End products (AGEs) is an important biochemical abnormality that accompanies diabetes mellitus and likely, inflammation in general. Recent studies have been indicating that the effects of AGEs on vessel wall homeostasis may account for the rapidly progressive atherosclerosis associated with diabetes mellitus. Driven by hyperglycemia and oxidant stress, AGEs form to a greatly accelerated degree in diabetes. Within the vessel wall, collagen-linked AGEs may trap plasma proteins, quench Nitric Oxide (NO) activity and interact with specific receptors to modulate a large number of cellular properties. On plasma Low Density Lipoproteins (LDL), AGEs initiate oxidative reactions that promote the formation of oxidized LDL. Interaction of AGEs with endothelial cells as well as with other cells accumulating within the atherosclerotic plaque, such as mononuclear phagocytes and smooth muscle cells, provides a mechanism to augment vascular dysfunction. Specifically, the interaction of AGEs with vessel wall components increases vascular permeability, the expression of procoagulant activity and the generation of Reactive Oxygen Species (ROS), resulting in increased endothelial expression of endothelial leukocyte adhesion molecules. AGEs potently modulate initiating steps in atherogenesis involving blood-vessel wall interactions, triggering an inflammatory-proliferative process and furthermore, critically contribute to propagation of inflammation and vascular perturbation in established disease (Basta et al., 2004).

The development of diagnostic tools using radioisotopes is widely used in almost all hospitals nationwide. In nuclear medicine practice, physicians set norms for morphological or physiological function for each organ by diagnosing a large number of patients. For every procedure, there is diagnostic data for a range of normal variations familiar to physicians. Nuclear medicine practitioners interpret diseases on the basis of deviations from these limits. Radioisotopes provide vital information to help diagnosis and therapy of various medical diseases. Data on tissue shape, function and localization within the body are relayed by use of one of various radionuclides, which can either be a free chemical species or covalently bound to part of a larger organic or inorganic moiety. These images are generated by the distribution of radioactive decay of the nuclide (Saha, 1998). 99mTc-labeled human serum albumin scintigraphy is helpful to localize the protein-losing origins and surgery is an effective treatment for Cronkhite-Canada syndrome with protein-losing enteropathy (Tseng et al., 2005). Various radiochemicals have been labelled with this nuclide. 99mTc has a half-life of 6 h and emits γ-rays of 140 keV with an abundance of 90%. 99mTc is primarily obtained from 99Mo/99mTc generator and is eluted as sodium pertechnetate (Na99mTcO4). In this chemical form, it can be used to study brain and thyroid (Saha, 1998). Intravenously administered 99mTc-pertechnetate is loosely bound to plasma proteins and rapidly moves out of the intravenous compartment. The plasma half-time clearance is approximately 30 min. Approximately 30% of the administered dose is excreted within 24 h. The total urinary and fecal excretion of 99mTc activity is about 50% in 3 days and up to 70% after 8 days. 99mTc is also trapped by the thyroid gland and it passes into the small intestine. However in the brain, the blood-brain barrier prevents Na99mTcO4 from entering brain cells. The biodistribution and kinetics of radiochemicals can be altered by a variety of chemical agents, as is widely known (Sampson, 1996). However, many factors, as drug therapy, radiation therapy, dietary conditions, besides pathological process could affect the biodistribution of the different radiopharmaceuticals (Diré et al., 2001; Gomes et al., 2002; Aguiar et al., 2002) or the labeling of blood constituents (Sampson, 1996; Vidal et al., 1998; Oliveira et al., 2000, 2002, 2003; Braga et al., 2000; Nigri et al., 2002; Santos-Filho et al., 2002). This also requires the repetition of the examination procedure resulting in the unnecessary irradiation to the patient (Saha, 1998). An increasing number of people in the world are using traditional herbs medicines. Natural medicines may contain potentially toxic ingredients and contaminants such as heavy metal. Traditional Chinese Herbal Medicines (TCHM) have been reported to cause serious hematological adverse effects (Azuno et al., 1999). 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 was stopped. The medicinal use of chayotte enclose the relief of diseases related to the kidneys, circulatory system, intestinal and cutaneous inflammation and to the cauterize the sores. The infusion of the leaves which contains a substance with cardiovascular properties is indicated to the pulmonary ailment and intestinal inflammation (Jensen and Lai, 1986; Flores, 1989). Gordon (2000) described the hypotensor effect of chayotte. Diré et al. (2001) have noticed that chayotte extract (macerated) was capable of altering the morphology of red blood cells in a qualitative analysis. In a in vitro/in vivo study, Diré et al. (2002) observed that the extracts (decoct and macerated) of chayotte were not capable of altering the radiolabeling of blood elements although they were able to alter the labeling of blood constituents in the treated animals with the referred extracts. Moreno et al. (2002), demonstrated that an extract of Ginkgo biloba has been altered the radiolabeling of blood elements in an in vitro analysis as well as the biodistribution of 99mTcO4Na. Recently new radiopharmaceuticals labelled with technetium-99m as cytokines (interleukin-IL-2, IL-6, IL-8); human polyclonal immunoglobulin (HIG); radiolabelled antigranulocyte monoclonal antibodies, radiolabelled peptides and antibiotic have been developed to improve specificity in diagnosis of inflammation and infection (Comin et al., 2002). It is described that AGEs may induction the redox-sensitive transciption nuclear factor NF-kB in response to oxidative stress in turn leads to a transcriptional activation of many genes, many of which are highly relevant for inflammation, immunity and atherosclerosis. These include tumor necrosis factors (TNF-α and TNF-β), interleukins 1,6 and 8 (IL-1, IL-6 and IL-8) interferon-γ (IFN-γ) and cell adhesion molecules. The effect of diabetes associated with natural products on the biodistribution of radiochemicals has not been fully evaluated. Type-I diabetes mellitus is the consequence of pancreatic beta-cell destruction mediated by mononuclear cells (insulitis). This insulitis process starts since many years before the onset of clinical manisfestations of the disease. The presence of insulitis could be only suspected in subejects at risk to develop autoimmune diabetes (relatives of patients affected by type I diabetes, who present positivity to disease-specific autoantibodies). Nevertheles, no tool is currently available to detect such pancreatic infliutation. This could be particulary useful in order to select patients who can benefit of specific preventive immunotherapies. In a pilot study, 123I-IL2 was able to correctly identify the insulitis process in 5 subjects at risk to develop type I diabetes. All these subjects developed diabetes within 4 years from the scintigraphy. At time of diagnosis, pancreatic beta-cell inflammation is present only in a subgroup of type I diabetic patients (approximately 50%). In recent study 99mTc-IL2 scintigraphy corerctly identified this subgroup of patients. Indeed, those patients positive to IL2 scan, presented a better metabolic control after 1-year treatment with nicotinamide. These results suggest that treatment with specific immunotherapies can be useful only if a lymphocyte infiltration is still active in endocrine pancreas although it is known that some situations as oxidative stress or drugs may alter the radiolabeling efficiency and in this case a misdiagnosis would be attributed. Pharmacologic agents that specifically inhibit AGE formation have allowed the investigation of the role of AGEs in the development of diabetic complications in animal models. AGEs linked to the vascular matrix can chemically interfere with the bioavailability of NO, an important regulator of vascular tone inducing smooth muscle cells relaxation (Signore et al., 2002).

The aim of this study was to evaluate the effect of a chayotte extract and the diabetes status induced by streptozotocin in the biodistribution of sodium pertechnetate radiopharmaceutical (Na99mTcO4) as well as the influence in the mass of the organs.

MATERIALS AND METHODS

This study was developed in the labortatories of Experimental Radiopharmacy and Experimental Pathology of the Biology Institute of the Universidade do Estado do Rio Janeiro, Brazil

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 skin of chayotte were mixtured 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 insectides. 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 chayotte. 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).

Preparing of the extract: To prepare the decoct of chayotte, this vegetable (50 g) was put in an Erlenmeyer with 500 mL of water and it was boiled on slow heat for ten minutes. After that, the solution was filtered and the watery extract was obtained. The same procedure was taken with the preparing of the macerated extract. It was used the skin (50 g) of the chayote skin which were triturated in a liquidizer with 500 mL of water. The animals were treated during 7 days. In the control the animals just have received water backwards cahyotte extracts.

Radiolabeling process
Biodistribution procedures:
It was performed 4 groups in the experimental, each group with 4 animals. The chayotte extract (macerated) was administrated (replaced by water in the treated group with chayotte and in the diabetic group treated with chayotte) during 7 days. The control group has received water like to the diabetic group. After that, 99mTcO4Na (0.3 mL, 3.7 MBq) was injected by ocular plexus. The animals were sacrificed and their organs were isolated (thyroid, brain, muscle, lung, heart, spleen, kidney, stomach, intestine, liver, bone, ovary, uterus and blood) and counted in a well counter. The %ATI in each organ was determined. The statistical analysis were performed by Tukey test (p<0.05).

Diabetes induction: The injection of streptozotocin was realized in the ventral region next to the alba line with a unique dose of 30 μg kg-1 by body weight dissolved in saline solution or in a same volume of citrate (control group). In a period of 2 h after the injection the rats were maintained without water and after that it was added sugar high quantities in their drinking during 1.5 h. After 48 h of the induction it was performed the rate of sugar tests by tail puction. It was considered diabetic the rats with rate of sugar rates above 180 dg dL-1.

Fig. 1:

Effect of diabetes and chayotte extract in the mass of the organs isolated from Wistar rats. Female Wistar rats had drunk (treated group: diabetic and not diabetic) or not the extract (control group) during 7 days. The animals were sacrificed, the organs were isolated and weighed. The mass/g was determined. A statistical analysis (Tukey test, n = 4) was performed to compare the results


Table 1:

Effect of chayotte extract and diabetes in the bioavailability of sodium pertechnetate in female Wistar Rats

Female Wistar rats had drunk (treated group: diabetic and not diabetic) or not the extract (control group) during 7 days and after that 99mTcO4Na was injected by ocular plexus. The animals were sacrificed, the organs were isolated and the % ATI/g was determined. For blood 1 mL was considered to be equivalent to 1 g. A statistical analysis (Tukey test, n = 4) was performed to compare the results

RESULTS

Due to the analysis of the results it was notice that there was a decrease in the mass of the spleen by the comparison of the control group to the group treated with chayotte extract (from 0.37±0.08 to 0.25±0.02) (Fig. 1). Comparing the group treated with chayotte extract with the diabetes group it was verified an increase of the mass (from 0.25±0.02 to 0.38±0.06) the same was noticed between the diabetes groups and the diabetes group treated with chayotte extract (from 0.38±0.06 to 0.44±0.02) (Fig. 1). Towards to the analysis of the stomach it was seen that there was a decrease in the mass in the mass in the group treated with chayotte when compared with the control group (from 1.10±0.19 to 0.87±0.21) (Fig. 1). In the pancreas there was a decrease in all groups (chayotte, diabetes and diabetes treated with chayotte) when compared to the control group (from 1.21±0.19 to 0.47±0.19; to 0.37±0.10; to 0.46±0.15) (Fig. 1). In the heart was perceived a decrease in the mass in the chayotte group compared to the control (from 0.70±0.11 to 0.52±0.02) (Fig. 1). To the analysis of the kidney it was realized an increase in the mass comparing the diabetes treated with chayotte to the control (from 0.58±0.05 to 0.75±0.04) and confronting the group treated with chayotte to the diabetes group treated with chayotte (from 0.58±0.08 to 0.75±0.04) (Fig. 1). In Table 1 has shown the effect of a chayotte extract and diabetes in the uptake of radioactivity in the organs. The analysis of the results has indicated that in the diabetes group had been an increase in the uptake of the radiopharmaceutical in pancreas (from 0.03±0.01 to 0.13±0.05) as well as in the diabetes groups treated with chayotte extract (from 0.03±0.01 to 0.11±0.05). Due to the analysis to the other organs it was not noticed any alterations in the bioavailability of sodium pertechnetate.

DISCUSSION

The distribution, uptake, retention and the elimination of radiopharmaceuticals depend on several factors, such as regional blood flow tissue metabolism and the binding to the blood elements (Hladik III et al., 1987; Sampson, 1996). The labeling of blood elements with 99mTc has many applications. It is known that extracts obtained from medical plants can alter the labeling of blood elements with 99mTc as well as the morphology of red blood cells (Oliveira et al., 1997, 2000, 2002, 2003; Vidal et al., 1998, Reiniger et al., 1999; Braga et al., 2000; Lima et al., 2002 Capriles et al., 2002). The evidence that drugs can affect either the radiolabeling as the biodistribution of red blood cells or the morphology of them in the context of nuclear medicine clinic has come to light only comparatively recently and it is an important factor in the interpretation of scintigraphic images. In this work it was noticed that chayotte extract was capable of normalizing the uptake of 99mTcO4Na in the pancreas in the animals treated with the referred extract. In comparison to that in the diabetes animals was noticed that the disease status was capable to induce a decrease in uptake of the radiopharmaceutical in pancreas. A great number of workers have turned their attention to in vitro and in vivo evaluation of drugs in the process to label blood cells and in the biodistribution of radiopharceutical (Hladik III et al., 1987; Hesslewood and Leug, 1994; Owunwanne et al., 1995; Sampson, 1996). Nigri et al. (2002), analyzing concentrations levels higher than the therapeutic levels in humans it was demonstrated that antiseizure drugs like phenobarbital, clonazepam and phenytoin have the capacity of altering the radiolabeling of blood elements. Gomes et al. (2002), have demonstrated that a component of many chemotherapeutic regimens, mitomycin-C, has altered the bioavailability of technetium-99m-labelled sodium pyrophosphate in mice.

In the labeling process of blood constituents with 99mTc is needed a reducing agent and probably the stannous ion would be oxidized in vitro studies was verified that extracts of Thuya ocidentallis (Oliveira et al., 1997), Nicotiana tabacum (Vidal et al., 1998), Maytenus ilicifolia (Oliveira et al., 2000), Syzygium jambolanum (Santos et al., 2002), Stryphnodendron adstringens (Mart.) Coville (Costa et al., 2002) and Ginkgo biloba (Moreno et al., 2002), possibly, would have oxidants compounds and the labeling of blood elements decrease in the presence of these extracts. In a research was verified that Paullinia cupana extract was capable of altering the radiolabeling of blood elements as well as to alter quantitatively the shape of red blood cells (Oliveira et al., 2002). In other in vitro study with Fucus vesiculosus extract was noticed that the referred extract has induced a qualitative alterations on the morphology of red blood cells together with alterations on the labeling of blood elements with 99mTc (Oliveira et al., 2003). In a in vivo studies Diré et al. (2002), have demonstrated that the chayotte extracts (macerated and decoct) were capable of altering the radiolabeling of blood elements. Similar results were observed with an extract of Solanun melongena (eggplant) which was capable of altering radiolabeling of blood elements with 99mTc as well as the bioavailability of NaTcO4 (Capriles et al., 2002). Moreno et al. (2002), eyed that in a in vitro study the extract of Ginkgo biloba altered the morphology of red blood cells together with the radiolabeling of blood elements, the opposite, was observed in a in vivo study which this fact may be explained by the generate of metabolites in vivo without direct action on the morphology of red blood cells despite the referred extract had been altered the biodistribution of 99mTcO4Na. Santos-Filho et al. (2002), reported that the extracts of Mentha crispa L. (mint) and Piper methysticum (Kava Kava) were capable of altering the morphology of red blood cells notwithstanding mint extract has also altered the radiolabeling process. Braga et al. (2000), in a in vitro study demonstrated that Peumus boldus did not alter the labeling of blood elements with 99mTc, in this same study it was observed that the extracts of T. ocidentallis and N. tabacum have altered the radiolabeling of blood elements as well as the morphology of red blood cells. Lima et al. (2002) in a in vivo study have shown that an extract of cauliflower (leaf) was not capable of altering the labeling of blood elements with technetium-99m. the referred author has demonstrated that cauliflower extract was not able to alter the biodistribution of 99mTcO4Na. Diré et al. (2001), in a qualitative analysis in vivo, have eyed that a chayotte extract (macerated) has induced alteration on the shape of red blood cells. We can speculate like observed by Mongelli et al. (1997), in a study with Bolax gummifera extract, that the chayotte extract when administrated to the animals due to their possible metabolization may generate reactive metabolites with oxidant properties which may be able to alter the active of cell membrane which can modify the uptake of 99mTcO4Na in pancreas as it was noticed together with the decrease of mass. Although the non alteration of the bioavailability in the other organs it was verified a variety in the mass to spleen, stomach, heart and kidney comparing the groups to the control and between them. Maybe the variation in the mass could be due to different mechanisms of action of the compounds present in the extract, by the effect of the many circumstances related to the diabetes status or by the effects of the extract associated with the ones of the diabetes which would be related to molecular and cellular pathways which would be linked to the alteration of the %ATI/g as well as to the mass by the alterations of the structure of the extra cellular matrix and the morphphysiological status of the cell. The results may be support by the study in which to dissect the contribution of RAGE-ligand interaction in the pathogenesis of diabetic vasculopathy, an acute animal model of diabetes-associated hyperpermeability was tested first, using reagents blocking the receptor itself or blocking the access of ligants to RAGE, by administering the decoy protein soluble RAGEs. Rats rendered diabetic with streptozotocin, after 9-11 weeks of diabetes shoed increased vascular permeability in multiple organs, especially the intestine, the skin and the kidney (Basta et al., 2004).

CONCLUSIONS

Due to the results obtained in this study we can speculate that Sechium edule extract and diabetes status were capable of altering the biodistribution of 99mTcO4Na in pancreas as well as the mass in many organs. This fact could be related to the presence of compounds with oxidant properties which could be produced by the metabolization of the extract and by the generation of AGEs in diabetes. Moreover, although our results were obtained with animals, we suggest paying attention with examination in nuclear medicine in patients under the treatment referred to popular medicine who is drinking chayotte extract therapeutically.

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