ARSENIC and FLUORIDE INDUCED TOXICITY in GASTROCNEMIUS MUSCLE of MICE and ITS REVERSAL by THERAPEUTIC AGENTS NJ Chinoy,A SB Nair, DD Jhala Ahmedabad, India

Fluoride 2004;37(4):243–248 Research report 243

ARSENIC AND FLUORIDE INDUCED TOXICITY IN GASTROCNEMIUS MUSCLE OF MICE AND ITS REVERSAL BY THERAPEUTIC AGENTS NJ Chinoy,a SB Nair, DD Jhala Ahmedabad, India

SUMMARY: Sodium fluoride (NaF, 5 mg/kg bw) and arsenic trioxide (As2O3, 0.05 mg/ kg bw), individually or in combination, were administered orally for 30 days to Swiss strain adult female mice (Mus musculus). Alterations in the physiology of the gastrocnemius muscle occurred with a decline in total protein levels and acetylcholinesterase activity that would affect its contractile pattern. The significantly enhanced levels of glycogen with a concomitant decrease in phosphorylase activity could alter the contraction of the quick contracting white fibres, while the decrease in activities of ATPase and succinate dehydrogenase suggests disturbance in the oxidative and energy metabolisms. Withdrawal of the NaF and/or As2O3 treatment for 30 days produced incomplete recovery. However supplementation with ascorbic acid, calcium, and vitamin E, alone or in combination, during the withdrawal period, was beneficial for recovery of the muscle parameters. These findings are important in the field of muscle physiology and kinesiology. Key words: Arsenic and muscle; Calcium phosphate; Fluoride and muscle; Gastrocnemius muscle; Mice; Toxicity reversal; Vitamin C; Vitamin E. INTRODUCTION It is now well established that ingestion of fluoride affects not only teeth and bones but also many other organs in the body. Structural and biochemical changes in several soft tissues including muscle have been reported in male and female rats and mice from different doses of fluoride.1-7 Combined arsenic/fluoride poisoning also occurs, especially in India, China, and Bangladesh, where both As and F are abundant in certain drinking water sources. Studies on the individual effects of arsenic and fluoride toxicity indicate that both cause injury to several tissues.3 However, the effects of the combined treatment of fluoride and arsenic in muscle tissue have not yet been reported. There- fore, it was considered worthwhile to study this aspect on the gastrocnemius muscle of female mice and the possible reversal of the induced effects by antidotes such as vitamins C and E and supplemental calcium. MATERIALS AND METHODS Healthy, adult female albino mice (Mus musculus) of Swiss strain, weighing between 30 and 35g were divided into thirteen groups of 10 to 12 mice each and treated according to the experimental protocol described in detail in an earlier paper.8 At the end of each treatment, the animals were weighed on an Ohaus SA animal weighing balance and sacrificed by cervical dislocation. The gastrocnemius muscle was dissected out carefully, blotted free of blood, and utilized for the study.

aFor correspondence: Reproductive Endocrinology and Toxicology Unit, Department of Zoology, School of Sciences, Gujarat University, Ahmedabad 380 009, India. E-mail: [email protected]

Fluoride 2004;37(4) 244 Chinoy, Nair, Jhala The protein9 and glycogen10 levels, activities of succinate dehydrogenase (SDH) (E.C. 1.3.99.1),11 phosphorylase (E.C. 2.4.1.1),12 adenosine triphosphatase (ATPase) (E.C. 3.6.1.3),13 and acetylcholinesterase (E.C. 3.1.1.7)14 were determined in the gastrocnemius muscle of both control and treated animals by using the techniques cited. For all the biochemical determinations, a minimum of 8 to10 replicates were assayed for each parameter. The data were statistically analysed by Student’s t test and Analysis of Variance (ANOVA). RESULTS The protein and SDH levels in muscle showed a significant decline after the treatments for 30 days with NaF (SDH P<0.01), As2O3, and NaF + As2O3 (P<0.001) (Groups VI to VIII) (Table 1). Withdrawal of the treatment (Group IX) resulted in an insignificant recovery in both parameters. A highly significant (P<0.001) reversal of the combined toxicity was obtained, however, after treatment with vitamin C, calcium, or vitamin E as well as their combination (Groups X-XIII) as compared to Group VIII (Table 1). A very significant (P<0.001) accumulation of glycogen occurred in muscle by NaF, As2O3, and NaF + As2O3 treatments (Groups VI-VIII). Withdrawal of treatment (Group IX) revealed a significant (P<0.01) recovery in the muscle glycogen levels as compared to Group VIII. Vitamins C or E or calcium phosphate administered alone or in combination (Groups X-XIII) resulted in highly significant (P<0.001) recovery as compared to Group VIII (Table 1). The activities of phosphorylase, ATPase, and acetylcholinesterase declined significantly (P<0.001) with NaF, As2O3, and their combined treatments (Groups VI-VIII) as compared to the controls (Groups I-V). After withdrawal of treatment (Group IX) the activities of all three enzymes were significantly (P<0.01) recovered in comparison to Group VIII. Administration of vitamins C and E as well as calcium, alone or in combination (Groups X-XIII), resulted in an even more significant (P<0.001) recovery (Table 1). DISCUSSION Arsenic and fluoride, independently or in combination, are reported to alter protein levels in muscle tissue of treated rabbits and mice,3,7,15 probably by inhibit- ing its synthesis or altering its metabolism. Lack of adequate protein turnover would have an adverse effect on the receptors, structural and contractile proteins, and the activities of various enzymes in the muscle. Arsenic and fluoride individually alter carbohydrate metabolism.16 A significant accumulation of glycogen in muscle together with a significant decline in phosphorylase activity in the present study after fluoride and arsenic treatments is in agreement with the above view.16 Glycogen is considered one of the main fuels for muscle contraction. Hence its accumulation or decreased utilization under treatment would affect the normal functioning of muscle fibres, especially the glycogen-utilizing, quick-acting, white fibres. This increase in glycogen could be due to changes in the metabolism and transport of glucose leading to hyperglyce- mia in rats by fluoride administration.17

Fluoride 2004;37(4) Arsenic/fluoride toxicity in mice muscle and its reversal 245

Table 1. Protein (mg/100 mg fresh tissue wt), glycogen concentration (µg/100 mg fresh tissue wt), activities of SDH (µg formazan formed/mg protein/15 minutes), phosphorylase (µg phosphorus released/mg protein/15 minutes), ATPase (µmoles of inorganic phosphorus released/mg protein/30 minutes), and acetylcholinesterase (AChE) (AChE/mg protein) in gastrocnemius muscle of mice in Groups I-XIIIa

Group Treatment Protein Glycogen SDH Phospho- ATPase AChE rylase I Control + distilled 26.21 ± 806.53 ± 18.29 ± 10.03 ± 3.15 ± 6.31 ± water 0.59 2.79 0.35 0.44 0.14 0.21 II Control + olive oil 26.46 ± 804.98 ± 18.89 ± 9.92 ± 3.40 ± 6.26 ± 0.60 4.62 0.75 0.38 0.18 0.19 III Control + ascor- 26.16 ± 807.74 ± 19.27 ± 9.75 ± 3.36 ± 6.26 ± bic acid (AA) 0.57 8.81 0.39 0.42 0.20 0.16 IV Control + calcium 26.63 ± 807.89 ± 19.02 ± 10.18 ± 3.41 ± 6.36 ± phosphate (Ca) 0.77 8.51 0.27 0.34 0.09 0.15 V Control + vitamin 26.97 ± 803.75 ± 19.23 ± 9.44 ± 3.44 ± 6.06 ± E (Vit. E) 0.62 6.22 0.41 0.22 0.10 0.01 VI NaF 17.06 ± 1266.67 15.90 ± 7.11 ± 1.71 ± 4.20 ± 0.50§ ± 15.71§ 0.79‡ 0.20§ 0.10§ 0.02§

VII As2O3 15.70 ± 1553.07 14.25 ± 4.38 ± 1.38 ± 2.46 ± 0.39§ ± 16.37§ 0.18§ 0.15§ 0.03§ 0.01§

VIII NaF + As2O3 14.39 ± 1803.36 11.27 ± 3.06 ± 1.27 ± 2.36 ± 0.41§ ± 16.65§ 0.36§ 0.25§ 0.02§ 0.05§ IX Withdrawal of 16.42 ± 1582.07 13.48 ± 5.02 ± 1.36 ± 2.98 ± Group VIII treat- 0.55 ± 15.72‡ 0.19 0.16‡ 0.02‡ 0.21‡ ment X Withdrawal of 23.77 ± 1095.03 16.94 ± 7.36 ± 2.15 ± 4.69 ± Group VIII treat- 0.55§ ± 14.45§ 0.22§ 0.16§ 0.10§ 0.23§ ment + AA XI Withdrawal of 22.53 ± 1074.54 14.22 ± 8.64 ± 1.66 ± 5.13 ± Group VIII treat- 0.25§ ± 7.46§ 0.43§ 0.19§ 0.04§ 0.10§ ment + Ca XII Withdrawal of 22.76 ± 1078.76 15.01 ± 9.09 ± 1.75 ± 5.42 ± Group VIII treat- 0.22§ ± 14.83§ 0.54§ 0.06§ 0.05§ 0.11§ ment + Vit. E XIII Withdrawal of 26.73 ± 814.88 ± 17.92 ± 10.02 ± 3.19 ± 6.38 ± Group VIII treat- 0.43§ 14.25§ 0.22§ 0.18§ 0.07§ 0.06§ ment + AA, Ca & Vit. E aData are expressed as mean ± S.E. * P<0.05; † P<0.02; ‡ P<0.01; § P<0.001; where no sign = not significant. Comparisons between: Group I and Groups VI or VII or VIII individually; Group VIII and Groups IX or X or XI or XII or XIII individually.

Fluoride 2004;37(4) 246 Chinoy, Nair, Jhala

Table 1a. ANOVA of various parameters

Parameter Source of SS df MS F-Cal F-Tab variation

Protein Between Groups 2770.8163 12 230.9014 86.6736 1.8358 Within Groups 311.6920 117 2.6640

Glycogen Between Groups 14764873.31 12 1230406.190 792.150 1.8358 Within Groups 181730.051 117 1553.2483

SDH Between Groups 822.6903 12 68.5575 35.9964 1.8358 Within Groups 222.8344 117 1.9046

Phosphorylase Between Groups 714.2425 12 59.5202 82.8145 1.8358 Within Groups 84.0899 117 0.7187

ATPase Between Groups 99.8671 12 8.3223 77.6420 1.8358 Within Groups 12.5410 117 0.1072

AChE Between Groups 280.5569 12 23.3797 116.1786 1.8358 Within Groups 23.5450 117 0.2012 SS=Sum of squares; df=degree of freedom; MSS=Mean sum of squares; F-cal=Fisher calculated; F-tab=Fisher tabulated.

Succinate dehydrogenase (SDH), a mitochondrial enzyme, catalyses the oxida- tion of succinate to fumarate in the Krebs cycle. Its decrease after treatment implies a block in the cycle with a probable accumulation of succinate. The reduction in activity of SDH in gastrocnemius muscle of fluoride-treated mice is in agreement with the data of others,18,19 and the results have been correlated with alterations in the structure of mitochondria and skeletal muscle fibres.20 The decrease in activities of adenosine triphosphatase (ATPase) after fluoride treatment in the present study, and as reported by others,18,20,21 suggests that the energy metabolism is disturbed probably due to structural and functional changes in muscle. Likewise, the reduced acetylcholinesterase (AChE) activity in muscle after treatment indicates an effect on transmission of nerve impulses at the neuro- muscular junctions.18 In the present study, the combined treatment with NaF + As2O3 caused more toxicity than the individual treatments in all muscle parameters studied. An and co-workers22 have also reported synergism between arsenic and fluoride. Increase in serum creatinine23 in fluorotic humans and changes in connective tissue24 in rats might also be responsible for the structural and physiological manifestations in the muscle of fluorotic mice. As noted in the results, upon withdrawal of the combined treatment, some recovery occurred in muscle, but a significant recovery in gastrocnemius muscle

Fluoride 2004;37(4) Arsenic/fluoride toxicity in mice muscle and its reversal 247 was observed by the administration of vitamins C or E or calcium phosphate either alone or in combination. Ascorbic acid (vitamin C) is an antioxidant with detoxification properties.25 It activates adenyl cyclase but inhibits phosphodiesterase (PDE), similar to the action of calcium, which will result in an increase in c-AMP levels and cause activation of several enzymes.26 Supplementation with calcium also brought about significant recovery in numerous enzyme activities in fluoride or arsenic intoxicated mice, rats, rabbits, and guinea pigs.1-,3,6,7 Calcium phosphate ingestion might have helped in overcoming the hypocalcemia induced by fluoride/ arsenic treatment,27,28 and it may also have acted synergistically with vitamin C.5,7,29 Vitamin E (α-tocopherol), a potent antioxidant, exerts its protective effect pri- marily through destruction of cell damaging free radical oxygen species. In the present study, vitamin E also helped to reverse NaF + As2O3 induced toxicity in gastrocnemius muscle. Together with vitamin C, vitamin E provides an additional antioxidant effect, since vitamin E interacts non-enzymatically with ascorbic acid which enhances its radical scavenging effects.30 Hence it is clear that vitamin C and vitamin E as well as calcium have an additive or synergistic effect. Thus the interaction of all three antidotes resulted in the reversal of the NaF and As2O3 induced toxicity in gastrocnemius muscle of female mice. This is an important finding in muscle physiology and has potentially valuable implications in the field of kinesiology. This paper was presented at the XXIV Conference of the International Society for Fluoride Research, Otsu, Shiga, Japan, 4–7 September 2001. REFERENCES 1 Chinoy NJ. Effects of fluoride on physiology of some animals and human beings. Indian J Environ Toxicol 1991;1(1):17-32. 2 Chinoy NJ. Effects of fluoride on some organs of rats and their reversal. Proc Zool Soc Calcutta. 1991;44(1):11-5. 3 Chinoy NJ. Studies on fluoride, aluminium and arsenic toxicity in mammals and amelioration by some antidotes. In: Tripathi G, editor. Modern Trends in Environmental Biology. New Delhi: CBS Publishers; 2002. p. 164-93. 4 Chinoy NJ, Patel TN. Reversible toxicity of fluoride and aluminium in liver and gastrocnemius muscle of female mice. Fluoride 1999;32(4):215-29. 5 Chinoy NJ, Memon MR. Beneficial effects of some vitamins and calcium on fluoride and aluminium toxicity on gastrocnemius muscle and liver of male mice. Fluoride 2001;34(1):21-33. 6 Chinoy NJ, Joseph R, Sequeira E, Narayana MV. Effects of sodium fluoride on the muscle and liver of albino rats. Indian J Environ Toxicol 1991;1(2):129-34. 7 Chinoy NJ, Sharma M, Mathews Michael. Beneficial effects of ascorbic acid and calcium on reversal of fluoride toxicity in male rats. Fluoride 1993;26(1):45-56. 8 Chinoy NJ, Tewari K, Jhala DD. Fluoride and/or arsenic toxicity in mice testis with formation of giant cells and subsequent recovery by some antidotes. Fluoride 2004;37(3):172-84. 9 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with Folin phenol reagent. J Biol Chem 1951;193:26575. 10 Seifter S, Dayton S, Novic B, Muntwyler E. The estimation of glycogen with anthrone reagent. Arch Biochem 1950;25:191200. 11 Beatty CH, Basinger GM, Dully CC, Bocek RM. Comparision of red and white voluntary skeletal muscle of several species of primates. J Histochem Cytochem 1966;14:590600.

Fluoride 2004;37(4) 248 Chinoy, Nair, Jhala 12 Cori CF, Cori GT, Green A. Crystalline muscle phosphorylase III. kinetics. J Biol Chem 1943;151:39-55. 13 Quinn PJ, White IG. Distribution of adenosine triphosphatase activity in ram and bull spermatozoa. J Reprod Fert 1968;15:44952. 14 de la Huerga J, Yesinick C, Popper H. Colorimetric method for the determination of serum cho- linesterase. Am J Clin Pathol 1952;22:1126-33. 15 Shashi A, Singh JP, Thapar SP. Protein degradation in skeletal muscle of rabbit during experimental fluorosis. Fluoride 1992;25(3):155-8. 16 de Bruin A. Carbohydrates and carbohydrate metabolism. In: Biochemical Toxicology of Environ- mental Agents. Amsterdam: Elsevier/North Holland Biomedical Press; 1976; p 471-525. 17 Sakurai T, Suzuki K, Taki T, Suketa Y. The mechanism of changes in metabolism and transport of glucose caused by fluoride administration to rats [abstract]. Fluoride 1993;26(3):210. 18 Lakshmi VM, Reddy KP. Effects of fluoride accumulation on some enzymes of brain and gastrocnemius muscle of mice. Fluoride 2000;33(1):17-26. 19 Pang YX, Guo YQ, Zhu P, Fu KW, Sun YF, Tang RQ. The effects of fluoride, alone and in combination with selenium, on the morphology and histochemistry of skeletal muscle. Fluoride 1996;29(2):59-62. 20 Shashi A. Fluoride toxicity and muscular manifestations: histopathological effects in rabbit. Fluo- ride 1989;22(2):72-7. 21 Park S, Ajtai K, Burghardt TP. Inhibition of myosin ATPase by metal fluoride complexes. Biochim et Biophys Acta 1999;1430(1):127-140. 22 An D, He YG, Hu QX. Poisoning by coal smoke containing arsenic and fluoride. Fluoride 1997;30(1):29-32. 23 Wang J, Zheng ZA, Zhang LS, Cao DM, Chen KZ, Lu D. An experimental study for early diagnos- tic feature in fluorosis. Fluoride 1993;26(1):61-5. 24 Stanbury JB, Embery G. The excretion of urinary glycosaminoglycans by fluorotic rats. Fluoride 1993;26(4):247-56. 25 Chinoy NJ. Ascorbic acid turnover in animal and human tissues. J Anim Morphol Physiol 1978; Silver Jubilee Volume:68-85. 26 Pasternak CA. An Introduction to Human Biochemistry. New York: Oxford University Press; 1979. p 199-219. 27 Boink AB, Wemer J, Meulenbelt J, Vaessen HA, de Wildt DJ. The mechanism of fluoride-induced hypocalcemia. Hum Exp Toxicol 1994;13(3):149-55. 28 Verma RJ, Sherlin DM. Hypocalcaemia in parental and F1 generation rats treated with sodium fluoride. Food Chem Toxicol 2002;40(4):551-4. 29 Chinoy NJ, Memon MR, Shah SD. Beneficial effects of vitamins C, E and calcium on nucleic acids and protein in gastrocnemius muscle and liver of fluoride and/or aluminium intoxicated mice. Indian J Environ Toxicol. In press 2004. 30 Bender DA. Nutritional Biochemistry of the Vitamins. New York: Cambridge University Press; 1992. p. 87-105.

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