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ISSN 2455–9393

Original Article

Association of increased oxidative burden and excessive fluoride

exposure in obese children

Balvir Singh Tomar1,2 , Deepak Nathiya1,3, Dushyant Singh Chauhan1,4, Sandeep Tripathi1,4

ABSTRACT

Background: The modern scientific evidence representing that obesity associated with fluoride exposure may be a risk of reduced health quality in paediatric population. Obesity is an independent risk factor for cardiovascular diseases and markedly elevated risk of morbidity and mortality. Presence of higher levels of fluoride in drinking water (>1.5ppm) may be serious problems in health of the obese children. In the state of Rajasthan, almost all districts have high fluoride (up to 18.0 ppm) in their drinking / ground water sources. An estimated 66.6 million people (17 states in India) including 6 million less than 14 years children are at risk

Aim: The aim of the present study was to evaluate the effect of fluoride in obese child of high endemic fluoride areas.

Method: In the present study, we selected 54 obese children from the selected area of Jaipur- India, twenty seven children (n=27) from high fluoride (F > 2.5ppm) region and twenty seven (n=27) obese children (disease control) from, where fluoride content was normal (F< 1.5ppm) in their source of drinking water. Moreover, age matched healthy controls were selected from the Jaipur district where fluoride content in water was less than 1.5 ppm. After clinical examination, lipid profiles, oxidative stress parameters namely, lipid peroxide level (LPO), superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and reduced glutathione content.

Results: The concentration of fluoride in serum was significantly correlates with their water concentration. Increased LPO levels and reduced antioxidant status in obese and fluoride exposed obese. Moreover, obese and fluoride exposed obese were more comparable.

Conclusion: On the basis of the results it may conclude that fluoride enhances the severity of disease and fluoride promotes oxidative stress in obese paediatric population. However, further in depth of studies is required for the understanding of pathophysiology of child obesity those residing in endemic area of fluoride.

INTRODUCTION

Obesity can be characterized by abnormal or excessive accumulation of fat in body. The childhood obesity is multifactorial, environmental exposure, genetics, metabolism, and sociocultural factors (Claudio, 2000). Globally there are more than 300 million adults who are obese and 42 million children who are overweight (WHO 2010; Aitlhadj et al., 2011). Current evidence has implicated obesity in conferring a greater susceptibility to the adverse effects of environmental exposure (Chung and Yoon 2008; De Silva-Sanigorski et al., 2011; Scinicariello et al., 2013; Jung et al., 2014). The obesity as a

result of environmental exposure was largely overlooked because of the contamination of chemicals in water and air. It has been demonstrated that these toxins induce insulin resistance, increase glucose (blood sugar), cholesterol and fatty liver. Among the environmental chemicals, fluoride has been naturally found in our drinking water. Ground water is one of the most important sources of drinking water and fluoride contamination in ground water is increasingly becoming a matter of great concern. An estimated 66.6 million people (17 states in India) are at risk of acquiring Fluorosis. In Rajasthan, people of 22 districts (out of 32) are presently consuming

International Journal of

Gastroenterology, Hepatology,

Transplant & Nutrition

1National referral Centre for fluoride

poisoning in India, 2Department of

Paediatrics, National Institute of Medical Science & Research, 3Nims Institute of

Pharmacy, 4Institute of Advance Science &

Technology, Nims University Rajasthan, Jaipur India.

Address for Correspondence: Prof. (Dr.) Balvir Singh Tomar

Email:chairman@nimsuniversity.org

Access this article online QR Code

Website: www.journal.pghtn.com

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ISSN 2455–9393

fluoride (Pushpendra et al 2012; Chauhan et al 2013) greater

than permissible limit. High groundwater fluoride

concentrations have been reported in India, China, Spain, and Mexico, where levels are higher than 1.5 ppm (Wang et al., 2007). An estimated 66.6 million people (17 states in India) including 6 million less than 14 years children are at risk of acquiring fluorosis (ICMR 2004; Sigh et al., 2013)). In Rajasthan, people of 22 districts (out of 32) are presently consuming fluoride greater than permissible limit (Sigh et al., 2013). It is reported that fluoride is one of the factor that may reduce IQ level and behavioral impairment (Verma et al., 2017). In the light of new scientific evidence demonstrating that fluoride may be associated with child obesity and increase risk mortality and morbidity. The mechanisms by which fluoride produce such effects are still not clear. Animal studies demonstrated that fluoride generates reacting oxygen species (ROS) (Chauhan et al. 2013; Rao et al. 2006). Different enzymatic and non-enzymatic antioxidants regulated production of ROS by normal physiological processes but excessive production of ROS may leads to oxidative insult. Interactions between fluoride and free-radical reactions have been studied in various biological systems. However, the relationship between fluoride toxicity, oxidative stress and obesity is still not clear. Oxidative stress is a condition that indicates the imbalance between the pro-oxidants and antioxidants leading to the oxidative damage to lipids, proteins and DNA. It is reported that fluoride also affects the renal tubules evidenced by ultrastructural changes (Quadri et., 2017). Chinoy et al (2004) reported that antioxidant supplementation with diet reverse the toxic effects of fluoride in the body. Beltowski (2000) has been reported that obesity leads to oxidative stress which can contribute obesity and associated diseases (Sfar et al., 2013; Dennis et al., 2013). Fluoride is one of the factors associated with pathophysiology of child obesity (Sakeenabi et al., 2012).

Keeping in view the paucity of information in relation to high fluoride exposure in obese pediatric population residing in endemic areas and its impact on severity of disease, the present study was undertaken.

MATERIAL & METHODS

Study Design:

Thirty seven obese children (male, age- 7 to 13 years) were selected from the high fluoride region of the eastern regions (Dousa district) in Rajasthan India where fluoride content in water is more than 1.5 ppm. Thirty five obese children selected as positive control from the non fluorotic region. For the comparison of obesity, thirty five apparently non obese healthy children were also included in a group. The subjects and control were investigated clinically. The subjects were similar living conditions and differ minimally in terms of lifestyle, parental education level, socioeconomic status, and medical care. Informed consent for participating in the study was obtained from the parents. Children with endocrine disorder such as diabetes, hypothyroidism, Cushing’s syndrome, other metabolic disorder, renal and liver disease, active infection, on medication

are excluded in this study. The study proposal was approved by the Institutional ethical committee.

Blood Collection and fluoride estimation:

A venous blood sample (3 ml) was collected at 9:00 a.m. from each child after an overnight fast of not more than 12 hours. The concentration of fluoride in the sources of drinking water of each family and serum of were investigated using fluoride selective electrode (¬¬¬¬Thermo Fischer, Singapore).

Biochemical studies:

Lipid profile were estimated using commercially available kit with enzymatic procedures for the measurement of cholesterol, triglycerides and high density lipoprotein (HDL). The low density lipoprotein (LDL) cholesterol level was calculated by using the formula [LDL = TC - (HDL + TG/5)]. Blood lysate was prepared to estimate oxidative stress parameter. The protein content was measured (Lowry et al, 1952) using bovine serum albumin (BSA) as standard. The lipid peroxide (LPx) levels were measured (Okhawa et al (1979) using thiobarbituric acid

reacting substances (TBARS), estimated by

spectrophotometrically at 532 nm and expressed as nmole of MDA /mg protein. The superoxide dismutase (SOD EC 1: 15.1.1) activity was determined from its ability to inhibit the reduction of NBT in presence of PMS (McChord and Fridovich 1969). The reaction was monitored spectrophotometrically at 560nm. The SOD activity was expressed as U/mg protein (1 unit is the amount of enzyme that inhibit the reduction of NBT by one half in above reaction mixture). Catalase (CAT, EC 1.11.1.6) activity was assayed as per the previously described method (Aebi et al (1974) using hydrogen peroxide as substrate; the decomposition of H2O2 was followed at 240nm on spectrophotometer. The CAT activity was expressed as U/mg protein. The glutathione peroxidase (GSHPx, EC 1.11.1.0) was assayed using GSH, NADPH and H2O2 as reactants (Pagila and Valentine (1967). The oxidation of GSH into GSSG was measured in terms of oxidation of NADPH to NADP+ and assayed as decrease in the absorbance of reaction mixture at 340 nm on spectrophotometer. The activity of GSHPx was expressed as n moles of NADPH oxidized / min / mg protein. Reduced glutathione was measured in deprotonized supernatent in lysate with tetrachloroacetic acid, centrifuged and supernatant was used for the estimation of reduced glutathione (GSH) by the use of Ellman reagent (5, 5’ dithiobis (2-nitro benzoic acid) (Ellman et al, 1959). The optical density of the pale colour was measured on the spectrophotometer on 412 nm. An appropriate standard (pure GSH) was run simultaneously. The level of GSH was expressed as µmole GSH / mg protein

RESULTS

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be significantly (p<0.05) increased in fluoride exposed obese groups when compared with the control and disease control groups. In addition, hart rate were also markedly increased in fluoride exposed obese group than that of healthy control. The concentration of cholesterol, triglycerides, HDL and LDL were found to significantly (p<0.05) increased in both disease control and fluoride exposed obese groups when compared with the controls (table-1). The concentration of fluoride in drinking water and serum of fluoride exposed group were found to be markedly (p<0.001) elevated in fluoride exposed group as comparison with control and diseased control group (table-1). The fluoride level in serum was directly proportional to the concentration of fluoride in drinking water.

Table-1 Characteristics of Children including this study

Data are presented as mean ±SD. The superscript indicates significance level (p<0.05) between control vs obese (*) and obese vs fluoride exposed obese (#).

The mean concentration of oxidative stress markers namely, LPO, SOD, CAT, GPx, GR and GSH in obese and fluoride exposed obese are compared with control in the figure-1 and respective percentage change are presented in table-2.

Table-2 Percentage change in between groups

The lipid peroxide levels were found to be markedly increased by 35% (p<0.01) in obese and 73% (p<0.001) in fluoride exposed obese subjects when compared with the controls. On the other hand, 28% (p<0.01) increment was observed in fluoride exposed group as compared to obese. The activity of SOD was found to be significantly reduced by 17% (p<0.05) in obese and 30% (p<0.001) in fluoride exposed obese subjects when compared with the controls, while, 15% (p<0.05)

reduction was observed in fluoride exposed group as compared to obese. The activity of catalase was significantly reduced by 23% (p<0.05) in obese and 32% (p<0.001) in fluoride exposed obese subjects when compared with their age matched controls. The activity of GPx and GR were found to be significantly (p<0.05) reduced by 12% and 11% in obese as compared to their controls respectively. Moreover, fluoride exposed group exhibited significant (p<0.001) reduction by 22% of GPx and 25% of GR when compared with the controls respectively. The fluoride exposed group indicated 11% (p<0.05) of GPx and 16% (p<0.05) of GR reduction in compared with disease control- obese group. The concentration of glutathione was found to be significantly increased by 17% (p<0.01) in obese and 28% (p<0.001) in fluoride exposed obese subjects when compared with the controls. On the other hand, 14% (p<0.05) increment was observed in fluoride exposed group as compared to obese.

Figure-1 The mean concentration of oxidative stress markers namely, LPO, SOD, CAT, GPx, GR and GSH in obese and fluoride exposed obese

DISCUSSION

The association of obesity and fluoride toxicity in children are great concern of the research (Yen and Hu, 2013; Sakeenabi et al., 2012). In the present study we observed additional significant changes in fluoride exposed children with obesity. It is most comparable to disease control group. The elevated concentration of serum fluoride has been recognized as a consistent marker of fluoride exposure and can also be used as one of the biomarkers to assess the effect of endemic fluorosis. The large difference between fluoride concentrations in serum of control and subjects correlated with concentration of fluoride

Control Obese Obese + F

Age in years 10.4 ± 2.3 11.3 ± 2.1ns 10.9 ± 1.9 ns

BMI (kg/m2) 15.4 ±1.3 22.3 ± 2.2 ns 23.1 ± 2.3 ns

SBP (mmHg) 104.4 ± 9.3 114.1 ± 9.2 ns 121.3 ± 10.5*#

DSP (mmHg) 56.4 ± 7.5 58.9 ± 6.7 ns 65.5 ± 10.3 *#

HR (bpm) 86.9 ±7.9 88.5 ± 8.3 ns 87.8 ± 9.2*

Cholesterol (mg /dl) 144.2 ± 14.8 157.6 ± 12.9* 156.8 ± 11.6*

Triglycerides (mg /dl) 73.7 ± 10.7 86.7 ± 9.3* 96.9 ± 11.4*#

HDL (mg /dl) 49.4 ± 6.1 42.6 ± 4.9* 38.5 ± 4.5*

LDL(mg /dl) 77.9 ± 11.9 86.7 ± 9.7* 89.8 ± 10.9*

Fluoride in water (ppm) 1.03 ± 0.15 1.07 ± 0.16 ns 5.7 ± 1.6*#

Fluoride in serum 0.023 ±

0.012

0.021 ± 0.013 ns 0.197 ± 0.08*#

CT Vs Obes CT Vs Obes + F Obese Vs Obes +

F

LPO 35.18** 72.77*** 27.81**

SOD -16.67* -29.54*** -15.45*

Catalase -22.63* -31.89*** -11.96ns

GPx -12.33* -22.14*** -11.19*

GR -10.57* -24.83*** -15.94**

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in drinking water of controls and subjects. It is suggestive that fluoride directly incorporated into the blood and it may deposit in different body organs, bones and teeth.

It is well known that obesity may induce systemic oxidative stress. It is reported that oxidative stress is responsible for an irregular production of adipokines, which may contributes to the development of the metabolic syndrome (Ihab et al. 2007). In the present study, we observed elevated levels of lipid peroxidation and reduced antioxidant status in obese and fluoride exposed obese children as comparison with their age matched controls (figure-1). The most interestingly, fluoride increases lipid peroxide levels of higher than that of disease control. In this study, activity of antioxidant enzymes namely SOD, GPx, GR and glutathione content markedly reduced in fluoride exposed population than in healthy control and disease control. Increased lipid peroxidation is the potent biomarker of oxidative stress. It has also often been associated with reduced

antioxidant status. ROS occurs under different

pathophysiological conditions in many diseases. Increase rate of free radical generation causes dysfunction and promotes oxidative damage to tissue and cells. Moreover, it is more associated with their pathological effects that ultimately lead to protein and cellular damage as well as cell death (Belle 2011; Li and Engelhardt 2006; Jenner 1998). Chronic fluoride can severely damage human health, but its pathogenesis is poorly understood. As in the case of many acute and chronic degenerative diseases, increased rate of ROS productions and lipid peroxide levels have even been considered to play an important role in the pathogenesis of chronic fluoride toxicity (Gazzano et al., 2010; Hanaa et al., 2009). The fluoride toxicity and its association with obesity

It has been reported that excessive fluoride exposure can damage the redox balance of the cells in tissues, decrease antioxidant defense capacity (Bharti and R. S. Srivastava 2009; Bouaziz, et al., 2007; Shivarajashankara et al., 2001). As evident by our study, we observed significant increment of lipid peroxidation and reduced antioxidant status. Present finding are similar with Shivarajashankara et al., 2001 and Saralakumari and Rao 1991. They are reported a close association between chronic fluoride toxicity and increased oxidative stress in humans and in experimental animals. Vani et al (2000) has also reported that fluoride induces inhibition of antioxidant enzymes associated with free radical metabolism. Increased oxidative stress in obesity with fluoride exhibiting weakening of antioxidant defense status is concordant with the study of Armutcu et al., 2008. Moreover, Roberts et al., 2006 also showed that reduced antioxidant in obese animal models. In addition, vitamin E and β-carotene were also found to be reduced in obese children (Molnar et al., 2004).

CONCLUSION

On the basis of the results it may conclude that fluoride exposure promote oxidative stress in obese children. These alterations may induce excess pathophysiological changes or severity of disease due to lack of proper drinking water source.

However, further in depth studies is required for the understanding of pathophysiology of obesity associated with fluoride exposure.

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