EFFECT OF CHRONIC FLUORIDE INGESTION AND AGING ON DISACCHARIDASE ACTIVITIES AND INTESTINAL SOLUTE UPTAKE IN ETHANOL FED RATS

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1 69 January-March EFFECT OF CHRONIC FLUORIDE INGESTION AND AGING ON DISACCHARIDASE ACTIVITIES AND INTESTINAL SOLUTE UPTAKE IN ETHANOL FED RATS Shailender Singh Chauhan, a Akhtar Mahmood, b Nishant Kaushal, b Sudarshan Ojha b Chandigarh, India SUMMARY: The current investigation was designed to study the effect of chronic fluoride (F) ingestion and aging on disaccharidase activities and intestinal solute uptake in ethanol fed rats. Sodium fluoride (NaF, 25mg/kg) or/and 30% ethanol (EtOH, 1mL/kg) was given to 6- and 18-month-old animals daily through the intra-gastric route for,, and 90 days. Disaccharidase activities in the intestinal brush border membrane (BBM) were significantly decreased with age and the NaF or/and EtOH treatments. However, in each age group, the observed decline in enzyme activities was maximum in co-treated animals. Intestinal solute uptake in the control group showed a significant decline with age. Animals receiving F exhibited a progressive increase (6-month-old animals) and decline (18-month-old animals) in solute uptake from day to 90. Administration of EtOH alone, or together with F, showed a significant reduction in intestinal solute uptake in both age groups. These findings suggest that F and aging impair disaccharidase enzyme activities and intestinal solute uptake in a duration specific manner and these effects are aggravated with alcohol co-administration. These changes may affect the overall absorptive and transport functions in the gastrointestinal tract. Keywords: Age; Co-exposure; Disaccharidase activities; Ethanol fed rats; Fluoride ingestion; Intestinal brush border membrane; Solute uptake. INTRODUCTION Numerous studies have shown the toxic effects of excessive fluoride (F) and ethanol (EtOH) consumption on different tissues leading to serious damage and pathological changes. 1,2 Intestinal enterocytes, which play an important role in digestion and absorption mechanisms are primarily exposed to F or EtOH following oral ingestion. Humans residing in F endemic areas or consuming ethanol (alcoholics) exhibit gastric aberrations and compromised intestinal absorption. 3,4 Animals exposed to F through drinking water for a prolonged period also show gastrointestinal manifestations. 5,6 Co-exposures to F and ethanol may be common among the alcoholic populace in F endemic areas worldwide. However, little attention has been given to evaluate fluoride-alcohol interactions and their combined effects on the structure and function of the gastrointestinal tract. In our previous studies, we reported lipid peroxidation, impaired antioxidant defense systems, and mitochondrial dysfunctions in rat intestine exposed to F and/ or ethanol. 7,8 In the present investigation, we investigate the effects of chronic F ingestion and aging on disaccharidase enzyme activities and intestinal solute uptake in ethanol fed rats. a For correspondence: Dr Shailender Singh Chauhan, Senior Research Officer, WHO-Project, Advanced Pediatrics Centre, Post Graduate Institute of Medical Education and Research, Chandigarh , India; chauhan.shailender@gmail.com; b Department of Biochemistry, Panjab University, Chandigarh , India.

2 70 January-March MATERIALS AND METHODS Chemicals: All the chemicals used in the present investigation were of analytical grade. Sodium fluoride (NaF) and ethanol (EtOH) were procured from the Sisco Research Laboratories (SRL) Pvt Ltd, Mumbai, India and Changshu Yangyuan Chemicals, China, respectively. The glucose-oxidase-peroxidase kit was procured from Reckon Diagnostics Pvt Ltd, Baroda, India. The radiolabelled glucose/ glycine [14C-(U)-glucose/glycine] was obtained from the Radioisotope Division, Bhabha Atomic Research Centre, Bombay, India. Animals and Treatment: Six- and 18-month-old female Sprague Dawley rats, weighing 0 2 g and g respectively, were procured from the Central Animal House of Panjab University, Chandigarh, India. They were housed in propylene cages and maintained at 22±3ºC, on a 12:12 hr light-dark cycle and a minimum % relative humidity. A standard pellet diet and water were given ad libitum. After one week of acclimatization, age matched animals were separately subjected to random group division: Control (untreated); NaF treated (25 mg/kg); 30% EtOH treated (1mL/kg); NaF + EtOH co-treated (a combination of 25 mg/kg NaF and 1mL/kg 30% EtOH). All the treatments were given orally using Ryle s tube daily at 9 am for,, and 90 days. Four overnight fasted rats from each group were euthanized under light ether anesthesia. The experimental protocol was approved by the Institute s Ethical Committee. Brush border membrane (BBM) preparation and enzyme assays: For the BBM preparation, 9 intestine was removed and washed thoroughly with 0.9% NaCl and homogenized in ice-cold buffer containing 50 mm mannitol, 2 mm Tris (ph 7.2). Solid calcium chloride was added to the homogenate (final concentration, 10 mm) and allowed to stand on ice for 15 min followed by centrifugation at 3000 g for 15 min at 4ºC. The supernatant was re-centrifuged at 0 g for 30 min at 4ºC. The pellet obtained was suspended in 50 mm sodium maleate, ph 6.8. The activities of lactase, maltase, and sucrase were assayed in the BBM using the glucose-oxidase-peroxidase system as described by Dahlqvist. 10 Protein was estimated using bovine serum albumin as the standard. 11 Uptake studies: Glucose and glycine uptake was determined in vitro as described earlier. 12 Everted intestinal rings were incubated in 5 ml of oxygenated (95% O 2 5% CO 2 ) Krebs-Ringer buffer (ph 7.4) containing 5 mmol/l glucose/glycine with trace amounts of 14C-(U)-glucose/glycine (14C)-glucose/glycine at 37ºC. After 10 min, the tissues were removed, gently blotted on filter paper, weighed, digested in 10% potassium hydroxide (KOH), and radioactivity taken up determined in a Tricarb liquid scintillation counter (Packard). After correcting for extra-cellular space, the uptake rate was calculated and expressed as µmol/10 min/g wet tissue. RESULTS Changes in the enzyme activities in the intestinal BBM: The disaccharidase (lactase, maltase, and sucrase) enzyme activities in the purified intestinal BBM of the 18-month-old animals were found to be lower than in the 6-month-old animals (Tables 1-3).

3 71 January-March Table 1. Effect of chronic fluoride ingestion and aging on lactase activity in ethanol fed rats Age (months) Treatment (days) Groups Control NaF treated EtOH treated NaF+Et OH co-treated ± ± ± ± % 13% 15% 2.34± ±0.07,,,, 1.89± ± % 19% 39% 2.31± ±0.14,, 1.57±0.19, 1.22±0.16, 90 % 32% 47% ± ± ± ±0.08 % 30% 32% 1.89± ±0.14, 1.31± ±0.14, 29% 31% 39% 1.75± ±0.10,, 1.05±0.05,,,,,, 0.81± % % 54% Values are mean±sd as µmoles/min/mg protein (n = 4); p<0.05; compared to control group; compared to NaF treated group; compared to EtOH treated group; compared to -day treatment group; compared to -day treatment group; compared to 6-months-old group; % percent decreas e c ompared t o c ontrol group. Table 2. Effect of chronic fluoride ingestion and aging on maltas e activity in ethanol fed rats Age (months) Treatment (Days ) Groups Control NaF treated EtOH treated NaF+EtOH co-treated ± ± ± ±1.09 6% 13% 2% 31.07± ±0.72, 28.70±1. 59,,,, 21.50± % 7% 31% 28.77± ±1.34,, 22.90±1. 33,,,,,, 16.60± % % 42% ± ± ±0.97,,, 21.±0.29 5% 10% 13% 21.10±0.45, 18.09±0.38,, 17.90±0.64,, 11.74±0.57,,, 14% 15% 44%.±0.90, 15.69±0.64,,, 14.37±0.11,,,, 8.61±0.23,,,,, 90 23% 29% 58% Values are mean±sd as µmoles/min/mg protein (n = 4); p<0.05; compared to control group; compared to NaF treated group; compared to EtOH treated group; compared to -day treatment group; compared to -day treatment group; compared to 6-months-old group; % percent decrease compared to control group; % percent increase compared to control group.

4 72 January-March Table 3. Effect of chronic fluoride ingestion and aging on s ucras e activity in ethanol fed rats Age (months) Treatment (Days ) Groups Control NaF treated EtOH treated NaF+EtOH co-treated ± ± ± ±0.43 2% 3% 4% 9.54± ± ±0.91,,, 7.24±0.49 7% 3% 24% 8.70± ±0.56,, 6.94±0.51,,,,,, 3.96± % % 54% ± ±0.11, 5.92±0.,,,, 5.26± % 17% 26% 7.06± ±0.26,, 4.25±0.12,,, 3.02±0.,,,, 41% % 57% 6.64±0.25, 3.44±0.13,,, 3.17±0.14,,,,,,, 1.85± % 52% 72% Values are mean±sd as µmoles/min/mg protein (n = 4); p<0.05; compared to control group; compared to NaF treated group; compared to EtOH treated group; compared to -day treatment group; compared to -day treatment group; compared to 6-months-old group; % percent decrease compared to control group; % percent increase compared to control group. Compared to the control animals, the administration of NaF or EtOH alone caused, for both age groups, decreased lactase activity from day onwards (Table 1) and for maltase and sucrase at day 90 (Tables 2 and 3). The NaF and EtOH co-treated animals showed reduced disaccharidase activities at days and 90 in both age groups. The observed effects on disaccharidase activities in purified intestinal BBM were maximal in the NaF and EtOH co-treated animals after and 90 days of treatment in both age groups. The changes in disaccharidase activities increased with treatment duration from to 90 days and were usually greater in the 18-month-old group than in the 6-month-old group. Changes in the intestinal solute uptake: The solute (glucose and glycine) uptake in the 18-month-old rats was significantly lower than in the 6-month-old animals (Figures 1 and 2). The treated animals in both age groups showed a progressive decline in solute uptake throughout the experimental period, except for the NaF treatment in the 6-month-old animals, which showed a continuous increase in solute uptake from day to day 90. The observed decline in intestinal solute uptake was maximum in the co-treated animals which was further intensified with greater exposure and advancing age. After 90 days, glucose uptake was reduced by 44% and 38% in the 6- and 18-month-old animals, respectively (Figure 1). Under similar experimental conditions, the observed decline in glycine uptake was 46% in the 6- and 61% in the 18-month-old animals (Figure 2).

5 73 January-March Glucose uptake µmol/10 min/g tissue weight days treatment days treatment 90 days treatment 6-month-old animals Glucose uptake µmol/10 min/g tissue weight Control NaF treatment Ethanol treatment NaF + ethanol treatment days treatment days treatment 90 days treatment 18-month-old animals Figure 1. Effect of chronic fluoride ingestion and aging on intestinal glucose uptake in ethanol fed rats. Values are mean±sd as µmol/10 min/g tissue weight (n = 4); p<0.05; compared to control group; compared to NaF treated group; compared to EtOH treated group; compared to -day treatment group; compared to -day treatment group; compared to 6-months-old group.

6 74 January-March Glycine uptake µmol/10 min/g tissue weight days treatment days treatment 90 days treatment 6-month-old-animals Glycine uptake µmol/10 min/g tissue weight Control NaF treatment Ethanol treatment NaF + ethanol treatment days treatment days treatment 90 days treatment 18-month-old animals Figure 2. Effect of chronic fluoride ingestion and aging on intestinal glycine uptake in ethanol fed rats. Values are mean±sd as µmol/10 min/g tissue weight (n = 4); p<0.05; compared to control group; compared to NaF treated group; compared to EtOH treated group; compared to -day treatment group; compared to -day treatment group; compared to 6-months-old group.

7 75 January-March DISCUSSION Transport of nutrients from the small intestine into the circulatory system is the main mechanism associated with the absorptive cells of the gastrointestinal tract. The major nutrient transport, including hydrolysis, occurs through the intestinal BBM, an important constituent of the microvillous membrane. 13 Intestinal disaccharidases are membrane glycoproteins located on or within microvilli and are essential for the appropriate digestion of carbohydrates. 14 In the present study, the activities of lactase, maltase, and sucrose enzymes (disaccharidases) in the BBM along with solutes uptake (glucose and glycine) in the intestine of the 18- month-old animals were found to significantly lower as compared to their 6- month-old counter parts. Such observations have also been taken and reported in aged rats. 15 However, another study showed that the activities of enzymes like lactase and sucrase increased significantly with increased age in rats. 16 These discrepancies may be due to factors such as age, sex, strain of animal, and the environmental conditions. Aging is associated with a higher risk for nutritional deficiencies, which could lead to adverse functional consequences. Previous studies have shown that old age is associated with impaired intestinal adaptive ability to changes in the diet. 17 Functional and anatomic changes in the gastrointestinal system could explain part of the nutritional alterations observed in the elderly. The small intestinal epithelium in mammals has a constantly renewable population of cells. The new-born absorptive cells in the intestine undergo mitosis at very short intervals and the villi of the small intestine are constantly renewed with new cells. Small intestinal development is determined by the rate of crypt cell mitosis and the life span of villous cells and, in addition, is conditioned by hormonal and dietary factors. 18 Aging results in a higher proliferative rate of the crypt cells, possibly reducing the number of transporting enterocytes and thereby reducing nutrient absorption. Exposure to NaF or/and EtOH, resulted in altered disaccharidase activities and solute uptake in the 6- and 18-month-old animals. The NaF-induced alterations in the disaccharidase activities and the solute uptake can be attributed to the fact that the hydrofluoric acid formed from ingested F in the stomach has a corrosive effect on the gastrointestinal tract and may possibly affect intestinal histological architecture and its function. 19 The observed decrease in the activities of disaccharidases in EtOH treated animals are consistent with previous findings reporting a reduction in enzyme activity in EtOH fed rats. This decline in the disaccharidase content of the mucosa may be due to a smaller intake of carbohydrates because disaccharidase activities are known to be decreased when their substrates are reduced in the animal s diet. 21 On the other hand, morphometric studies have shown that villus height and the mucosal surface area are reduced in alcoholic- as compared to non-alcoholic-patients. 22 This hypoplasia is probably also responsible for the decrease of disaccharidase activities. The decrease in solute uptake in the intestine of EtOH fed rats may be attributed to the decrease in the Na + coupled uptake of glucose and glycine. A similar decline in solute uptake has also been reported in EtOH fed rats. 23

8 76 January-March In conclusion, the present results indicate that disaccharidase activities and solute uptake are affected during chronic F ingestion and aging in the intestine of ethanol fed rats. The significance of these findings is that the alcoholic populace residing in F endemic areas may have a increased susceptibility to age associated gastrointestinal abnormalities. ACKNOWLEDGMENTS The authors are grateful to the University Grants Commission, New Delhi, India, for financial assistance [Grant No. F14-2/07(SA.III)]. REFERENCES 1 Barbier O, Arreola-Mendoza L, Del Razo LM. Molecular mechanisms of fluoride toxicity. Chem Biol Interact 10;188(2): Jung MK, Callaci JJ, Lauing KL, Otis JS, Radek KA, Jones MK, Kovacs EJ. Alcohol exposure and mechanisms of tissue injury and repair. Alcohol Clin Exp Res 11;35(3): Susheela AK, Kumar A, Bhatnagar M, Bahadur R. Prevalence of endemic fluorosis with gastrointestinal manifestations in people living in some North-Indian villages. Fluoride 1993;26(2): Bode C, Bode JC. Alcohol s role in gastrointestinal tract disorders. Alcohol Health Res World 1997;21(1): Choubisa SL. Status of fluorosis in animals. Proc Natl Acad Sci India Sect B Biol Sci 12;82(3): Choubisa SL. Fluoride toxicosis in immature herbivorous domestic animals living in low fluoride water endemic areas of Rajasthan, India: an observational survey. Fluoride 13;46(1): Chauhan SS, Ojha S, Mahmood A. Modulation of lipid peroxidation and antioxidant defense systems in rat intestine by sub-chronic fluoride and ethanol administration. Alcohol 11;45(7): Chauhan SS, Mahmood A, Ojha S. Ethanol and age enhances fluoride toxicity through oxidative stress and mitochondrial dysfunctions in rat intestine. Mol Cell Biochem 13;384: Kessler M, Acuto O, Storelli C, Murer H, Müller M, Semenza G. A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes: their use in investigating some properties of D-glucose and choline transport systems. Biochem Biophys Acta 1978;506(1): Dahlqvist A. Method for the assay of intestinal disaccharidase. Anal Biochem 1964;7(1): Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem1951;193: Bhalla S, Mahmood S, Mahmood A. Effect of prenatal exposure to ethanol on postnatal development of intestinal transport functions in rats. Eur J Nutr 04;43: Ferraris RP, Villenas SA, Diamond J. Regulation of brush-border enzyme activities and enterocyte migration rates in mouse small intestine. Am J Physiol. Gastrointestinal and Liver Physiology 1992;262(6 Pt 1): Robayo-Torres CC, Quezada-Calvillo R, Nichols BL. Disaccharide digestion: clinical and molecular aspects. Clin Gastroenterol Hepatol 06;4(3): Lee MF, Russell RM, Montgomery RK, Krasinski SD. Total intestinal lactase and sucrase activities are reduced in aged rats. J Nutr 1997;127(7): Raul F, Gosse F, Doffoel M, Darmenton P, Wessely JY. Age related increase of brush border enzyme activities along the small intestine. Gut 1988;29(11): Ferraris RP, Vinnakota RR. Regulation of intestinal nutrient transport is impaired in aged mice. J Nutr 1993;123(3):

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