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 Fluoride induced gestational neurotoxicity and clinoptilolite (zeolite) as therapeutic agent 

       MAHABOOB BASHA P1* AND SHABANA BEGUM2 
Department of Zoology, Bangalore University, Bangalore 560056, INDIA
Maharanis Science college for Women, Bangalore, 560001,INDIA

*Corresponding author: Dr.P.Mahaboob Basha, Associate Professor in Zoology, Bangalore University, Bangalore-560056, INDIA. Telephone: +918022961571
Email: HYPERLINK "mailto:pmbashabub@rediffmail.com"pmbashabub@rediffmail.com































Abstract:

The optimum management of soft tissue fluorosis requires a multi disciplinary approach including the use of zeolites. Zeolites are known to have ion-exchanger and adsorbent properties. Clinoptilolite, being an important zeolite, its supplementation is anticipated in having some biological significance in eliminating free radicals. In this study premated Wistar albino rats were exposed to 200 ppm fluoride in drinking water during gestation and pups born to them were used to analyze the extent of neurotoxicity imposed in discrete brain areas, and fluoride exposure has significantly enhanced the levels of malondialdehyde (P>0.05), decreased the activities of superoxide dismutase (P>0.05), catalase (P>0.05) and glutathione peroxidase (P>0.05). Alterations were region specific and oral supplementation of clinoptilolite at dose of 10(g/kgbw not only inhibited oxidative stress but also enhanced the activity of antioxidant enzymes. These results suggest the biological significance of clinoptilolite in eliminating free radicals has therapeutic value.

Key words: Fluoride toxicity, Oxidative stress, Clinoptilolite, Developing brain.


INTRODUCTION:

Fluoride is an essential trace element, plays a role in preventing dental carries and reducing tooth decay (Warren, 2005]1. It can cross the cell membranes and enter soft tissues causing functional impairment in physiological system (Teotia, 1998]2. Age, sex, calcium intake in the diet, dose and duration of fluoride intake, and renal efficiency in fluoride handling are some of the major factors that influence the fluoride toxicity in humans (Krishnamachari, 1986; Das, 1996]3-4 and bring variations in the clinical presentation (Krishnamachari, 1986)3. Reports mainly from China and India signify that fluoride in varying concentrations induce free radical toxicity in both animals and human. 

Redox homeostasis is a usual denominator of the responses to the stress which is maintained by the balance between the production of reactive oxygen species (ROS), reactive nitrogen species, and antioxidant defense system (Celi, 2010]5. Medical and scientific data reveal that the free radical imbalance and subsequent chronic oxidative stress caused by excess intake of fluoride are the basic causes in the development of neurodegeneration (Madhusudhan et al., 2010; Basha and Madhusudhan, 2011]6-7. Although the exact triggering factors involved in the molecular mechanisms are not known, increasing evidence suggests that oxidative stress plays a major role in inducing pathogenesis. 

The current approach for management of fluoride toxicity especially to soft tissues is the supplementation of dietary antioxidants. Preponderance of studies made by supplementation of antioxidants in varying doses to address the issues of fluorosis, have shown conflicting results. In true sense the optimum management of soft tissue fluorosis requires a multi disciplinary approach involving antioxidants or any agent that can safely remove the free radical burden. In the last few decades, natural zeolite, used as a feed additive into the diets of animals, has been applied with enormous success in animal breeding for many purposes. Zeolites are tectosilicates with three dimensional aluminosilicates structure that contain water molecules, alkaline, and alkali earth metals in the structural framework (Thiruvenkatachari et al., 2008)8. Currently, there are at least 50 different types of known zeolites. Clinoptilolite is the best type which is suitable for the large numbers of applications. Natural clinoptilolite contains most of the major and trace minerals which are essential for growth of animals. These minerals are in an ionic state and can be used in animal diets for improving health conditions for animals (Mumpton and Fishman, 1977)9. Clinoptilolite appears to be stable in gastrointestinal tract of animals (Colic and Poljak-Blazi, 2002))10, and as unique selective absorbers, they can absorb heavy metals, toxins, and free radicals in the body and excretes them from animal bodies (Papaioannou et al.,2005; Tiwari,2007; Tao et al.,.,2010)11-13. The absorptive characteristics of clinoptilolites are because of their high cation-exchange capacity, which affects tissue uptake and utilization of NH4 +, Cu2+, Pb2+, Cd2+, Cs+, and other cations in animals (Mumpton F, Fishman,1977; Papaioannou et al., 2005)9,11. Besides these characteristics, clinoptilolites have also shown anti-cancerous and antioxidative effects (Paveli et al.,2001; Zarcovic et al.2003; Visnja et al. 2004)14-16. Natural clinoptilolites have positive influence on the immunity and the inflammatory processes by diminishing the synthesis of nitric oxide and superoxide anions (Visnja, 2004)16. They enhance the immune activity in animals (Jung,2010)17. Zeolites possess net negative structural charge as a result of isomorphic substitution of cations in crystal lattice; this negative charge leads to the favorable ion exchange selectivity for certain cations of zeolites. However, the negative charge also leads zeolites to have little or no approximation for anions (Cruz-Guzman et al., 2006)18. Recent studies have shown that zeolites modified with some surfactants or other cationic sorbents have an approximation affinity for heavy metal anions. These surfactant modified zeolites are effective sorbents for many types of contaminants (Ghiaci, 2004; P�rez Cordoves et al., 2008)19-20. Its supplementation is anticipated to have some biological significance in eliminating free radicals, hence this study. This study aimed to address the impact of high fluoride (200ppm) induced oxidative changes in discrete brain regions of developing rats and its mitigation by use of clinoptilolite.


MATERIAL AND METHODS
Animals:
Laboratory bred pre mated albino rats Rattus norvegicus, (Wistar strain) obtained from M/S Sri Raghavendra Enterprises, Bangalore were made to acclimatize to laboratory conditions (12 hr dark/light, 25 ( 2 0C) and fed with standard rodent pellet diet. The animals were maintained in accordance with the guidelines of National Institute of Nutrition, ICMR, Hyderabad, India and approved by the Animal Ethical Committee. The animals were assorted into groups. The control group was given tap water (Less than 1ppm fluoride) and all experimental groups received 200 ppm fluoride in water during gestation and post gestation period. The pups born to experimental animals were given orally clinoptilolite 10mg daily dose/ kg body weight from postnatal day-10 till the day of autopsy.  The selection of dose and length of exposure is based on studies reported by Madhusudhan and Basha (2010)6. At the end of 45th day of antioxidant treatment the animals were sacrificed and required brain tissues viz, cerebral cortex, medulla and cerebellum were separated and tissue homogenates were used for biochemical assays.


Chemicals:
Epinephrine and DTNB (Ellman�s reagent) procured from Sigma chemicals USA, Clinoptilolite from Zeo Inc, Augusta USA, and other AR grade chemicals from Merck India Ltd. were used for the assay.




Biochemical Assays

Tissue samples were homogenized in requisite buffer for the assessment of oxidative stress indices, such as CAT, SOD, GST, GPx and GSH activity/ levels and the adopted procedures are shown below:
Lipid peroxidation (LPO) 

Lipid peroxidation (LPO) product was estimated by measurement of thiobarbituric acid reactive substances using the method of Nehius and Samuelson (1968)21.  The pink chromogen produced by the reaction of thiobarbituric acid with malondialdehyde (MDA), a secondary product of lipid peroxidation, was estimated at 535 nm.

Superoxide Dismutase (SOD, EC 1.15.1.1)

SOD activity was assayed by measuring the inhibition of epinephrine auto-oxidation as described by Misra and Fridovich (1972)22. The absorbance was recorded at 480 nm for 60 s. Results are expressed as units/mg protein.

Catalase (CAT, EC 1.11.1.6)
CAT activity was measured as described by Aebi (1984)23. The rate constant of hydrogen peroxide (H2O2) decomposition was monitored by measuring the decrease in absorbance at 240 nm for 60s. Results are expressed as nmol of H2O2 consumed/min/mg protein.

Glutathione Peroxidase Activity (GPx, EC 1.11.1.9)

GPx activity was estimated by measuring the oxidation of DTNB as described by Rotruck et al. (1973)24 and change in absorbance was measured at 420 nm. An enzyme unit represents a decrease in GSH concentration of 0.01 log unit/min, after subtraction of Non-enzyme rate. Results are presented as �mol of GSH consumed /min/mg protein.

Protein Assay

Protein content was estimated by the method of Lowry et al. (1951)25 using bovine serum albumen (BSA) as a standard.





Statistical Analysis
The results are expressed as Mean � S.E. Values in parenthesis indicate percentage change, �+� sign indicate increase, �-� sign indicate decrease over control. Data were analyzed by employing Student�s-t test and values of p<0.05 were considered statistically significant.

Data and Statistical Analysis:
The results expressed as mean � S. D. (% change) of different groups. The data were examined for statistical significance employing paired Student�s �t� test. 
The percentage recovery was calculated by using the formula � 
% Recovery =  EMBED Equation.3  
Where A - % change noticed in fluoride exposed group and B - % change noticed in fluoride and antioxidants exposed group 

RESULTS: 
The results of the present experiment are depicted in the table (1-2).  It is evident from data depicted in table (1) that fluoride intoxication has considerably augmented the MDA levels indicating increased lipid peroxidation followed by a marked decrease in the activity level of catalase, SOD, and GSH-Px enzymes in discrete regions of CNS studied. The apparent alterations brought perturbations in the antioxidant homeostasis and among the discrete regions, cortex and medulla found to be highly vulnerable to fluoride toxicity. Further supplementation of clinoptilolite for 15days found beneficial and protected brain regions by elevating the antioxidant levels to safeguarding the developing nervous system. 

It is evident from the data depicted in table (2) that clinoptilolite at dose of 10mg/kgbw for 15days offered amelioration by elevating the MDA levels to 83.60%, 50.00% and 25.03% in cortex, cerebellum and medulla regions respectively. Similar rate of  amelioration was observed with  regard to other antioxidant enzymes (viz., SOD 47.39%, 46.59% and 51.03; catalase 50.79%, 32.97% and 46.82%; GPx 38.94%, 54.52%, and 64.03% in cortex, cerebellum and medulla regions respectively).
DISCUSSION:

Life is characterized by a steady generation of reactive oxygen species balanced by a similar rate of their consumption by antioxidants. To maintain homeostasis, a continuous regeneration of antioxidant production is required, when this is not met, oxidative stress occurs resulting in pathophysiological events. The antioxidants may prevent these damages induced by oxidation of protein and lipid. The antioxidant enzymes include GSH-Px, SOD, and CAT. These are the three main antioxidant enzymes in the body, which remove unwanted " O2 ", ROOH, hydrogen peroxide (H2O2) and produced by free radicals. Superoxide dismutase catalyzes superoxide radical dismutation; GSH concentration plays an important role in protecting cells against oxidative stress (Ermak, 2002; Sies, 1999)26-27. Glutathione peroxidase decomposes H2O2, GSH and SOD protect cells against toxic compounds and oxygen radicals (Nita,2001)28. Decreased activity of these enzymes will induce increased free radicals and then lead to damage of the corresponding tissue. Malondialdehyde is an ending product of lipid peroxidation, so the amounts of MDA could be used to assess the extent of lipid peroxidation (Satoshi, 1989)29. In the present study, the malondialdehyde (MDA) level which is the marker of lipid peroxidation was found to increase significantly in discrete brain regions of rats treated with fluoride. Reaction of oxygen free radicals with the methylene groups of polyunsaturated fatty acids initiates the peroxidation of lipids present in biological membranes (O�Donnell and Lynch, 1998)30, hence fluoride exposure might have generated reactive oxygen species which in turn would have caused lipid peroxidation as evidenced by an increase in malondialdehyde levels over subsequent generations. Moreover the brain tissue is deficient in oxidative defense mechanisms and therefore is at a greater risk of damage mediated by ROS resulting in molecular and cellular dysfunction (Ogawa, 1994)31. In addition, brain has a high ratio of membrane surface area to cytoplasmic ratio, extended axonal morphology prone to injury, and neuronal cells are non-replacing. 

High lipid content in the brain may be responsible for the vulnerability of the discrete brain regions to oxidative stress. The increased oxidative stress observed in fluoride toxicosis indicates the sensitivity of the developing nervous system and prone to lipid peroxidation and oxidative damage. In this study the activity levels of GSH-Px found decreased in all the regions of CNS upon fluoride intoxication. This could be due to increased utilization of glutathione in curbing the free radical mediated toxic actions. When the tissues are exposed to oxidative stress they increase the activity and expression of antioxidant enzymes as a compensatory mechanism against free radical-mediated damage. Nevertheless, the alterations observed in the level of antioxidant enzymes may be inadequate to counteract the potential damage in many conditions of oxidative stress. Since the oxidative stress observed in the present study is the key factor in altering the antioxidant homeostasis, regardless of etiology and is associated with decreased SOD concentrations in nervous tissue and decreased SOD activity may lead to decreased production of hydrogen peroxide that could reduce the oxidation of glutathione by glutathione peroxidase. The findings of Reddy et al., (2009)32 and Hassan and Aziz (2010)33 showed similar results and corroborate the present study findings.  

Restoring or maintaining oxidative buffering capacity therefore represents one useful therapeutic approach to minimize neuro-degeneration thereby beneficial effects of clinoptilolite was assessed. Zeolites are hydrated synthetic micro porous crystals with well-defined structures containing AlO4 and SiO4 linked through the common oxygen atoms (Thiruvenkatachari et al., 2008)8. Clinoptilolite being a natural zeolite acts as an antioxidant, chelator and helps in the removal of heavy metals from the body (Colic and Poljak-Blazi, 2002)10. It is suggested that the adding of clinoptilolite may support antioxidant system (Wang, 2011)34. Wang et al.(2011)34 found that the supplementation with zinc-bearing clinoptilolite increased SOD enzyme activity in ileal mucosa and decreased intestinal MDA concentrations, thus reducing the oxidative stress in chicken. In the present study the supplementation of clinoptilolite at a dose of 10mg/kgbw for 35-days potentially minimized the toxic burden by increasing the antioxidant defense in discrete regions of brain tissue studied suggesting the prominent role of clinoptilolite in the management of soft tissue fluorosis. The antioxidant responsiveness of clinoptilolite supplementation have biological significance in eliminating reactive free radicals that may otherwise affect the normal cell functioning. 

In conclusion, fluoride in high doses duly proves to cause oxidative stress, and perturb the antioxidant homeostasis. The supplementation of clinoptilolite at a dose of 10mg/kgbw for 15-days offers a promising result in minimizing toxicity and can use as therapeutic agent. 

Acknowledgements:
Authors are thankful to University Grants Commission, New Delhi, India for research grants.
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Table 1: Fluoride (200ppm) induced changes in the level of antioxidant enzymes and their mitigation by Zeolite�clinoptilolite supplementation in discrete brain regions of developing rats
_______________________________________________________________________

Antioxidant Enzyme	   Cortex__________Cerebellum________Medulla_____________

LPO (nmoles of MDA/mg tissue)

Control               		 2.93 � 0.02                     	 1.61 � 0.02                       1.94 � 0.05                     
200ppm F            	 	6.71 � 0.02 (+129.01)*      	2.33 � 0.06 (+44.72)*         2.46 � 0.06 (+26.80)*
200ppm F + Clinoptilolite 	2.31 � 0.03 (+21.16)*�    	1.25 � 0.06 (-22.36)*         1.29 � 0.04 (-33.51)*

______________________________________________________________________________________

CATALASE (� moles/min/mg protein)

Control 			125.2 � 4.69 		119.2 � 5.63 		125.1 � 2.14   	
200ppm F		086.4 � 4.24 (-30.99)* 	072.2 � 2.83 (-39.43)* 	073.4 � 2.60 (-41.33)*
200ppm F + Clinoptilolite	106.1 � 4.67 (-15.25)	 087.7 � 1.60 (-26.43)* 	097.6 � 2.54 (-21.98)*   
_______________________________________________________________________________________

SOD (� moles/min/mg protein)

Control 			4.66 � 0.14 		3.56 � 0.12 		3.31 � 0.04
200ppm F 		2.93 � 0.09 (-37.12)* 	2.53 � 0.04 (-28.93)* 	2.33 � 0.03 (-29.61)*
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___________________________________________________________________________________________________
GPX (�g of GSH consumed/min/mg protein)

Control 			3.43 � 0.04 		2.55 � 0.06		 2.46 � 0.04
200ppm F	 	2.12 � 0.02 (-38.19)* 	1.78 � 0.05 (-30.17)* 	1.32 � 0.03 (-46.34)*
200ppm F + Clinoptilolite	2.63 � 0.04 (-23.32)*� 	2.20 � 0.03 (-13.72)*� 	2.05 � 0.02 (-16.67)*�

____________________________________________________________________________________________________

Values - Mean activity � S.E.M. of  6 observations, Values in the parenthesis indicate %change,
�-� sign indicate decrease, �+� sign indicate increase over controls
* P<0.05 in comparison to control group, � P < 0.05 in comparison to respective fluoride treated group.


Table 2: Percent (%) Recovery in the levels of antioxidant variables indicated as a functional consequence of clinoptilolite supplementation on fluoride exposure in different regions of developing brain.

Antioxidant treated  Biochemical variables studiedBrain regionsCerebral cortexCerebellumMedulla(Value indicate % recovery over Fluoride toxicity)Clinoptilolite (10mg/ kgbw)LPO -83.60-50.00-25.03SOD
Catalase+47.39
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