FLUORIDE 30 (2)
1997, pp 105-110		 International Society for Fluoride Research Table of Contents
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Fluoride Vol. 30 No. 2 1997. Research Report 105

COMBINED TOXICITY OF FLUORIDE AND
BENZENE HEXACHLORIDE TO RATS

N Ramesh, E Murali Mohan, C A Y Banu Priya,
V Amalan Stanley, K S Pillai and P Balakrishna Murthy ,
Tamil Nadu, India.

SUMMARY: Sprague-Dawley rats were given various treatments of 10 ppm fluoride, 25 ppm fluoride, 25 ppm benzene hexachloride (BHC), 10 ppm fluoride + 25 ppm BHC, or 25 ppm fluoride + 25 ppm BHC for 16 weeks ad libitum. Rats showed a decrease in body weight gain, which could be attributable to BHC alone. A synergistic effect of fluoride and BHC was seen only in females given 25 ppm fluoride + 25 ppm BHC: a decrease in red blood cells (RBC) and relative weight of the ovary. Females were more susceptible to the toxicity of fluoride and BHC than males.

Key words: Benzene hexachloride (BHC), Fluorosis; Rat; Synergism.

INTRODUCTION

Several independent studies on the toxicity of fluoride (F)1-2 and benzene hexachloride (BHC)3 have been carried out in different parts of the world. However, not many attempts have been made to understand the combined toxicity of BHC and F in laboratory animals. Fluorosis, a crippling disease due to the intake of excessive fluoride in drinking water, is found in several parts of the world.4 BHC is still used extensively in third world countries, particularly in India,5 where endemic fluorosis is a major health problem.6 Studies carried out in India have shown that residues of BHC exist in adipose tissue, breast milk,7 bovine milk8 and vegetables.9

The present study aims at understanding the combined toxicity of F and BHC to rats when given for sixteen weeks in drinking water.

MATERIALS AND METHODS

Male and female Sprague-Dawley rats (80-100 g) procured from National Animal House, Central Drug Research Institute, Lucknow, India, were acclimatized to the laboratory conditions (temp. 22 ± 2°C, relative humidity 50-70%; 12 h light and 12 h dark rhythm), and divided into six groups housed in polypropylene cages, sex wise (6 rats/cage) with autoclaved paddy husk as the bedding material. Group 1, the control, received normal feed and water ad libitum. Group 2 received 10 ppm F, Group 3 25 ppm F, Group 4 25 ppm BHC, Group 5 10 ppm F and 25 ppm BHC, and Group 6 25 ppm F and 25 ppm BHC. Both F (Ranbaxy Chem, India) and BHC (BHC 50% WP, Vaigai Chem, India) were given in drinking water. Both drinking water and feed (Lipton, India) were given ad libitum. Weekly body weight gain and feed consumption were recorded. At the end of week 16, blood was drawn for haematology from the orbital sinus of anaesthetised animals. Then the animals were sacrificed under overdose of anaesthetic ether, and the organs isolated for weight determination. Haematological parameters were analysed on an Erma Particle Counter (Erma PC 605, Japan). The data were subjected to Barlett’s test, ANOVA and Student’s t test.10

RESULTS AND DISCUSSION

Both F and BHC are known to cause mortalities in laboratory animals in long term experiments. It has been stated that mortalities occurred in rats exposed to increased concentrations of fluoride.11 Dikshith et al12 reported mortality in guinea pigs treated with 500 mg/kg body weight BHC dermally for 30 days. In the present study, five mortalities were observed: in Group 3 (25 ppm F) one male died in week 13; in Group 4 (25 ppm BHC) one female died in week 12; in Group 6 (25 ppm F + 25 ppm BHC) 2 females died, in weeks 7 and 15, and one male died in week 15.

Body weight gain of groups of rats was correlated with the exposure time (weeks) by linear regression equations. From the slopes of the equations, it may be stated that body weight gain of male rats of Groups 2 (10 ppm F) and 3 (25 ppm F) was marginally higher than that of the control rats (Group 1). Male rats belonging to Groups 4 (25 ppm BHC), 5 (10 ppm F + 25 ppm BHC) and 6 (25 ppm F + 25 ppm BHC) had less body weight gain. The latter two groups had the least body weight gain compared to the other two groups (Table 1a). Body weight gain in females of Group 2 (10 ppm F) increased, whereas in other groups it decreased, being least in Groups 4 (25 ppm BHC) and 5 (10 ppm F + 25 ppm BHC), followed by Group 6 (25 ppm F + 25 ppm BHC) (Table 1b). Studies have shown that fluoride decreased body weight gain in laboratory animals.13 Decrease in body weight observed in rats treated with BHC in the present study is in contrast with that of the mice receiving BHC by oral or dermal route for 80 weeks.14

TABLE 1a. Regression equation (week vs body weight gain) of male rats

Group

 Equation r2 Standard error
of Y estimates		 Standard error
of coefficient (b)

Control

 Y = 17.05 + 5.29 X 0.92 7.16 0.43

10 ppm fluoride

 Y = 18.02 + 5.97 X 0.92 7.92 0.47

25 ppm fluoride

 Y = 18.09 + 5.86 X 0.93 7.24 0.43

25 ppm BHC

 Y = 18.18 + 5.21 X 0.96 5.05 0.30

10 ppm fluoride +
25 ppm BHC

 Y = 17.55 + 4.99 X 0.93 6.57 0.39

25 ppm fluoride +
25 ppm BHC

 Y = 19.05 + 5.07 X 0.93 6.23 0.37
  Y = a + bX, where Y = Body weight gain; X = week (1-16); a = intercept; b = slope


TABLE 1b. Regression equation (week vs body weight gain) of female rats
Group Equation r2 Standard error
of Y estimates		 Standard error
of coefficient (b)

Control

 Y = 8.52 + 2.53 X 0.96 2.30 0.14

10 ppm fluoride

 Y = 8.04 + 2.67 X 0.95 2.81 0.17

25 ppm fluoride

 Y = 8.55 + 2.46 X 0.95 2.65 0.16

25 ppm BHC

 Y = 6.58 + 1.74 X 0.86 2.92 0.17

10 ppm fluoride +
25 ppm BHC

 Y = 5.84 + 1 76 X 0.94 2.09 0.13

25 ppm fluoride +
25 ppm BHC

 Y = 11.86 + 2.10 X 0.94 2.54 0.15

Y = a + bX, where Y = Body weight gain; X = week (1-16); a = intercept; b = slope


Males of Group 6 (25 ppm F + 25 ppm BHC) showed a decrease in weight of brain (Table 2a) whereas females of Group 4 (25 ppm BHC) showed an increase (Table 2b). Abalis et al 15 showed that BHC has an important effect particularly on the central nervous system. Lu et al 16 have stated that fluoride adversely affects various central nervous system agents in rats. Males and females of Group 3 (25 ppm F) and females of Groups 2 (10 ppm F) and 5 (10 ppm F + 25 ppm BHC) showed a decrease in the heart weight. Caruso et al 17 showed various cardiovascular defects, including hypertrophy of the ventricles and vasodilation, upon fluoride ingestion. Females of Group 4 (25 ppm BHC) showed an increase in the weight of the kidney. Philip et al 18 showed histopathological changes in kidney of Mus booduga following BHC treatment. Females of Group 6 (25 ppm F + 25 ppm BHC) showed a significant decrease in the weight of the ovaries. Even though fluoride and BHC per se failed to produce any change, the combination of both affected the weight of the ovaries.


TABLE 2a. Relative organ weight (%) of male rats given
fluoride or BHC or combination of fluoride and BHC

Groups

 Brain Heart Kidney Ovaries
Control 0.55 ± 0.05 0.32 ± 0.014 0.31 ± 0.03 0.45 ± 0.03
10 ppm F 0.52 ± 0.03 0.33 ± 0.003 0.35 ± 0.006 0.51*± 0.02
25 ppm F 0.50 ± 0.06 0.28*± 0.03 0.33 ± 0.05 0.50 ± 0.05
25 ppm BHC 0.53 ± 0.05 0.31 ± 0.006 0.32 ± 0.03 0.47 ± 0.07
10 ppm F +
25 ppm BHC		 0.49 ± 0.03 0.30 ± 0.039 0.33 ± 0.04 0.43 ± 0.06
25 ppm F +
25 ppm BHC		 0.48*± 0.08 0.33 ± 0.037 0.36 ± 0.03 0.46 ± 0.04
  Values are expressed as Mean ± Standard Deviation (n = 6)
  * Significant (P < 0.05)


TABLE 2b. Relative organ weight (%) of female rats given
fluoride or BHC or combination of fluoride and BHC
Groups Brain Heart Kidney Ovaries
Control 0.74 ± 0.11 0.39 ± 0.05 0.33 ± 0.02 0.05 ± 0.03
10 ppm F 0.69 ± 0.11 0.30* ± 0.01 0.30 ± 0.05 0.05 ± 0.006
25 ppm F 0.67 ± 0.11 0.30*± 0.01 0.30 ± 0.05 0.05 ± 0.02
25 ppm BHC 0.87*± 0.12 0.40 ± 0.05 0.37*± 0.01 0.03 ± 0.02
10 ppm F +
25 ppm BHC		 0.75 ± 0.04 0.34*± 0.03 0.34 ± 0.05 0.03 ± 0.015
25 ppm F +
25 ppm BHC		 0.75 ± 0.06 0.38 ± 0.06 0.35 ± 0.01 0.03*± 0.009
 Values are expressed as Mean ± Standard Deviation (n = 6)
 * Significant (P < 0.05)


None of the haematological parameters (WBC, RBC, HGB, HCT, MCV, MCH and MCHC) of males evaluated in the study showed a change due to the treatment of F, BHC or combinations of F and BHC.

Decrease in white blood cells (WBC) was evident in females of all the groups, except Group 5 (10 ppm F + 25 ppm BHC). It is apparent that a marginal decrease in WBC occurred in this group also. A decrease in WBC was reported in rabbits given 20 mg/kg body weight of F orally for 30 days.19 No significant change in WBC was observed in a study conducted in rats to evaluate the toxicity of BHC.20 Therefore it may be assumed that fluoride was solely responsible for decreasing WBC in female rats.

A decrease in RBC was observed in females of Group 6. Pillai et al 21 observed a decrease in RBC in mice given a single dose of 17.3 mg F/kg body weight. Rats given 150 ppm F in drinking water for 75 days showed a decrease in red cell count.22 Similarly a decrease in erythrocyte count was observed in rats treated with BHC.20 As no change in RBC occurred in other groups, it may be assumed that a synergistic effect of fluoride and BHC on RBC took place at the higher dose level (25 ppm).

From the marked decrease in slopes of regression equations established between body weight gain and exposure weeks, it is presumed that the females are more vulnerable to the toxicity of fluoride and BHC than males. Females showed changes in relative weight of a greater number of organs than males. BHC was responsible for decreasing the body weight of rats. Other than decrease in RBC and relative weight of the ovary of females of Group 6 (25 ppm F + 25 ppm BHC), the present findings do not confirm a synergistic interaction between F and BHC

Acknowledgment: The authors are grateful to the management of the Fredrick Institute of Plant Protection and Toxicology for the facilities provided to carry out the work. The authors also thank Mr A. Selvam for maintaining the animals during the course of the study.

REFERENCES

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  2. Shupe JL., Olson AE, Peterson HB, Low JB. Fluoride toxicosis in wild ungulates. Journal of the American Veterinary Medical Association 185 (11) 1295-1300 1984.
  3. Shrivastava SC, Dhavan S. Study of pesticide usage by farmers around Lucknow and gas liquid chromatographic analysis of gammaxene residues present in pond and well water of the related village. Indian Journal of Biological Research 5 34-37 1987.
  4. Allcroft R, Burns KN, Nancy HC. Fluorosis in cattle development and alleviation: Experimental studies. Animal Disease Surveys. Report No.2 Part II. Her Majesty’s Stationary Office, London 1965 pp 1-58.
  5. Pillai MKK. Pesticide pollution of soil, water and air in Delhi area. Indian Journal of Science and Total Environment 55 321-327 1986.
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  10. Anderson D, Conning DM. Experimental Toxicology. The basic issue. Royal Society of Chemistry, Cambridge 1993 pp 405-440.
  11. National Toxicology Program Technical Report on the Toxicology and Carcinogenesis Studies of Sodium Fluoride in F344/N Rats and B6C3F1 Mice (Series No. 393). US Department of Health and Human Services, 1990.
  12. Dikshith TS, Datta KK, Kushwat HS, Raizada RB. Histopathological and biochemical changes in guinea pigs after repeated dermal exposure to benzene hexachloride. Toxicology 10 (1) 55-66 1978.
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  14. Kashyap SK, Nigam SK, Gupta RC et al. Carcinogenicity of hexachlorocyclohexane in pure inbred Swiss mice. Journal of Environmental Science and Health. 14 (3) 305-318 1979.
  15. Abalis IM, Eldefrawi ME, Eldefrawi AT. Effects of insecticides of gamma amino butyric acid-induced chloride influx into rat brain microsacs. Journal of Toxicology and Environmental Health 18 (1) 13-23 1986.
  16. Lu FC, Mazurkiewiez IM, Grewal RS et al. The effect of sodium fluoride on response to various central nervous system agents in rats. Toxicology and Applied Pharmacology 3 31-38 1961.
  17. Caruso PS, Maynard EA, Distefano V. Pharmacology of sodium fluoride In: Smith FA (Ed). Handbook of Experimental Pharmacology. VoI. 20 Part ll Springer Verlag, New York 1970 pp 144-165.
  18. Philip GH, Sriraman PK, Ramamurthi R. Histopathological changes in liver and kidney of Mus booduga following oral benzene hexachloride feeding. Bulletin of Environmental Contamination and Toxicology 42 (4) 499-502 1989.
  19. Soldatovic D, Nadeljkovic TM. Influence of different concentrations of sodium fluoride and sodium fluorosilicate - in cases of chronic intoxication - on the number of erythrocytes and leukocytes and on the haemoglobin content and iron content of rabbit blood. Acta Pharmaceutica 21 181-186 1971.
  20. Pesticides Residues in Food - 1989 (Food and Agricultural Organization Plant Production and Protection Paper (100/2). Part II. ToxicoIogy). World Health Organization, Geneva 1989 pp 141-155.
  21. Pillai KS, Mathai AT, Deshmukh PB. Effect of fluoride on reproduction in mice. Fluoride 22 (4) 165-168 1989.
  22. Kahl S, Wojcik K, Ewy Z. Effect of fluoride on some haematological indices and iron distribution in the blood and iron - storing tissues of rats. Bulletin Academie Polonaise des Sciences. Serie des Sciences Biologique 21 389-393 1973.

__________________________________

Department of Toxicology, Fredrick Institute of Plant Protection and Toxicology,
Padappai 601 301, Chengai Anna District, Tamil Nadu, India
106 N Ramesh, E Murali Mohan, C A Y Banu Priya et al.

FLUORIDE 30 (2)
1997, pp 105-110		 International Society for Fluoride Research
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