EFFECTS OF ORALLY ADMINISTERED POTASSIUM DICHROMATE IN REPRODUCTIVE AND NON-REPRODUCTIVE TISSUES OF FEMALE RATS

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1 Journal of Tissue Research Vol. 4 (1) (2004) ISSN: (Printed in India) Original Article EFFECTS OF ORALLY ADMINISTERED POTASSIUM DICHROMATE IN REPRODUCTIVE AND NON-REPRODUCTIVE TISSUES OF FEMALE RATS RAO, M.V., CHAWLA, S.L., DAMORE, D. AND DARJI, J. Reproductive Endocrinology and Toxicology Unit, Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad India. zooldeptgu@satyam.net.in Abstract: Effect of oral administration of potassium dichromate at 5 and 10 mg/ kg body weight daily for 30 days was investigated in reproductive organs and liver of adult female rats. The results revealed that in ovary, protein, cholesterol and ascorbic acid were decreased. Similarly in uterus, proteins and lipid levels were significantly declined with an increase in its glycogen levels. However, increased glycogen levels in the liver with a concomitant decline in phosphorylase indicated carbohydrate metabolic defect in these tissues as a result of chromium poisoning. This is further evidenced by a reduction in the activity of succinate dehydrogenase in the liver. Elevation in serum transaminase levels with a reduction in cholesterol also indicated the liver toxicity. Moreover, blood cell counts were also altered. These results clearly revealed the toxicity of potassium dichromate in reproductive organs and liver in female rats which may probably be due to the metal accumulation in these tissues. Key words: Potassium dichromate, Reproductive tissues, Liver, Blood INTRODUCTION Naturally occurring chromium compounds are generally in the trivalent state [chromium (III)] while hexavalent chromium compounds [chromium (VI)] are produced industrially by the oxidation of chromium (III) compounds and are used for chrome plating, manufacture of dye and pigments, leather tanning and wood preserving. Chromium (III) is an essential nutrient that plays a role in glucose, fat and protein metabolism by potentiating the action of insulin (ATSDR, 1998; Anderson, 1992). The hexavalent form is more toxic than the most common trivalent forms. The hexavalent chromium can enter cells via facilitated diffusion through non-specific anion channels (Levis et al., 1978). A large number of studies have demonstrated that chromium (VI) induces toxic, carcinogenic, genotoxic, dermatotoxic, nephrotoxic and neurotoxic effects in human and animals (Von Burg and Liu, 1993; Stohs and Bagchi, 1995; Barceloux, 1999; Kawanishi et al., 2002). It also induces oxidative stress, DNA damage, apoptotic cell death and altered gene expression (Bagchi et al., 2002). It has been reported that the exposure of pregnant mice to 46 mg chromium (VI)/kg/day as potassium dichromate in drinking water during gestation resulted in increased preimplantation and post implantation loss, and decreased litter size (Trivedi et al., 1989). Delayed sexual maturation was observed in female mice offsprings treated with Cr (III) and Cr (VI) compounds. Fertility was also reduced (Al-Hamood et al., 1998). Rajvanshi (2002) studied the effects of chromium salt in male mice to indicate the male reproductive toxicity. Data on female reproductive tract is scanty. Hence the present investigation was undertaken in some reproductive tissues of cyclic female rats. MATERIALS AND METHODS Healthy adult female albino rats (Rattus norvegicus) of Charles Foster strain, weighing between g were obtained from Alembic Chemicals, Baroda, India following ethical guidelines. They were housed in an air-conditioned animal house at a temperature of 26±2 C and exposed to h. of day light and were maintained on standard chow and water ad libitum. The animals were divided into three groups namely control, low (5mg/kg) and high dose (10mg/kg) treated groups. Potassium dichromate (S.D. fine chemicals Ltd, Mumbai) was dissolved in distilled water. The doses selected were based on the LD 50 values. All the treatments were given orally and daily for 30 days. animals were given distilled water. 121

2 J. Tissue Research Vaginal smears were checked daily for assessing. The animals were sacrificed by cervical dislocation after their respective treatments. Ovary, uterus and liver of all the groups of animal were dissected out carefully, blotted free of blood and used for biochemical studies. Blood from all the animals was collected by cardiac puncture, was allowed to clot and centrifuged at 1000 g for 10 minutes to obtain serum for analysis. The levels of protein in the ovary, uterus and serum of all groups of animals were estimated by the method of Lowry et al. (1951). Estimation of cholesterol in ovary and serum was done by the method of Zlatkis et al. (1953). The method of Roe and Kuether (1943) was employed to measure the total ascorbic acid in the ovary. The glycogen concentration in uterus and liver was estimated by the method of Seifter et al. (1950). The activity of phosphorylase was assayed by the method of Cori et al. (1943) and inorganic phosphate by the technique of Fiske and Subbarow (1925). Total lipids in the uterus was done by the method of Zollner and Kirsch (1962). The activity of succinate dehydrogenase (SDH) was assayed in liver by the method of Beatty et al. (1966). The photometric assays of serum glutamate pyruvate transaminase (SGPT) and serum glutamate oxaloacetate transaminase (SGOT) were carried out by the method of Reitman and Frankel (1957). The red blood cell (RBC) and white blood cell (WBC) counts from blood were carried out using the Neubaeur chamber. The haemoglobin content of the blood was estimated using standard haemoglobinometer. For all biochemical estimations a minimum of 6-8 replicates were used. The data were statistically analyzed using Student s t test. A value of p<0.05 was considered to be significant. RESULTS Gravimetric studies: The body weights of low dose K 2 treated animals were not altered as compared to control. However, high dose of chromate treatment brought about a significant (p<0.05) decrease in the body weights. No significant changes were observed in absolute weight of ovary at low dose level but the weight of the ovary declined significantly (p<0.05) in the high dose fed rats as compared to control. However, the weight of the uterus was significantly decreased (p<0.05, p<0.01) in both the chromate treated group. But no change in the liver weight was noted (Table 1). Changes in biochemical parameters: Total protein levels in ovary and uterus were significantly (p<0.01, p<0.05) declined in high dose K 2 treated group (Tables 2 and 3).The cholesterol level in the ovary of high dose treated animals also declined significantly (p<0.05) as compared to control. However, the ascorbic acid level in ovary was significantly (p<0.01), (p<0.001) altered in both treated groups as compared to control (Table 2). A significant accumulation of glycogen (p<0.001) in the uterus and liver was noted (Tables 3 and 4) But Table 1:Body and organ weights in control and experimental animals K 2 Body weight(g) 235 ± ± ± 3.73** Ovary weight (mg) ± ± ± 2.45** Uterus weight (mg) ± ± 56.91** 366 ± 30.84* Liver weight (g) 7.55 ± ± ± 0.27 Values are mean ± S.E; *p<0.01; ** p<0.05; K 2 =Potassium dichromate Table 2:Biochemical parameters in ovary of control and experimental animals K 2 Protein (mg/100mg) ± ± ± 0.64* Cholesterol (mg/100mg) 6.74 ± ± ± 0.34** Total ascorbic acid (mg/g tissue wt) 3.48 ± ± 0.17* 1.37 ± 0.10*** Values are mean ± S.E; *p<0.01; ** p<0.05; *** p<0.001; K 2 =Potassium dichromate Table 3:Biochemical parameters in uterus of control and experimental animals K 2 Phosphorylase (µ moles of inorganic phosphate released/15 minutes) ± ± ± 2.44* Glycogen (µ g/mg) ± ± 2.10*** ± 2.30*** Protein (mg/100 mg) 2.95 ± ± ± 0.22** Total lipids (mg/100mg) ± ± ± 0.44* Values are mean ± S.E; *p<0.01; ** p<0.05; *** p<0.001; K 2 =Potassium dichromate 122

3 Rao et al. phosphorylase activity decreased significantly (p<0.01) in these tissues of high dose treated groups. The activity of SDH in liver showed a significant reduction (p<0.001) in both chromate treated groups as compared to controls (Table 4). Total lipid content in uterus of high dose K 2 treated group significantly (p<0.01) decreased as compared to control (Table 3). Blood tests: The animals fed with low and high dose of K 2 had a gradual increase (p<0.001, p<0.05) in SGOT and SGPT levels as compared to control respectively. But the levels of the cholesterol in serum declined significantly (p<0.001) in both low and high dose K 2 treated groups by chromate feeding (Table 5). RBC and WBC counts were significantly (p<0.05, p<0.001) altered in the high dose treated group as compared to controls. Similarly a significant reduction in Hb percent in both treated groups was also observed (Table 6). Cyclicity: The female rats in both K 2 treated groups showed an irregular with a predominance of metaestrus as compared to control (Table 7). Table 4: Biochemical parameters in liver of control and experimental animals K 2 Phosphorylase (µ moles of inorganic phosphate released/15 minutes) ± ± ± 0.01* Glycogen (µ g/mg tissue weight) ± ± 0.51* ± 0.31** Protein (mg/100 mg tissue weight) ± ± ± 2.31 Succinate Dehydrogenase (µg formazan/15 min/100mg tissue weight) ± ± 8.52** ± 8.79** Values are mean ± S.E; *p<0.05; ** p<0.001; K 2 =Potassium dichromate Table 5: Serum Biochemical parameters in control and experimental animals K 2 Protein (mg/ml) 3.76 ± ± ± 0.29 Cholesterol (mg/ml) 6.14 ± ± 0.63** 4.23 ± 0.22** SGOT (miu/ml) 14.0 ± ± 0.07** 18.0 ± 0.66** SGPT (miu/ml) 10.5 ± ± ± 0.33* Values are mean ± S.E; *p<0.05; ** p<0.001; K 2 =Potassium dichromate SGOT=Serum glutamate oxaloacetate transaminase; SGPT=Serum glutamate pyruvate transaminases Table 6: Haematological parameters in control and experimental animals K 2 Hb(gm%) ± ± 0.74* ± 1.52* RBC count (Million/cubic mm) 5.21 ± ± ± 0.41* WBC count (Thousand/cubic mm) 9200 ± ± 0.02** 5400 ± 0.08** Values are mean ± S.E; *p<0.05; ** p<0.001; K 2 =Potassium dichromate; RBC=Red blood cells; WBC=White blood cells. Table 7: Cyclicity in control and experimental animals Days 1 P 2 E 3 M 4 M 5 D 6 LP 7 LE 8 M 9 M 10 LD 30 Regular Irregular With predominance of metaestrus K 2 Irregular With predominance of metaestrus DISCUSSION The results of the present study revealed a significant decline in body as well as tissues (ovary and uterine) weights with high doses of Cr (VI). Elbetieha and Al- Hamood (1997) and Al-Hamood et al. (1998) also observed a decline in ovarian and uterine weights in Cr (III) exposed females. In a recent study Chundawat and Sood (2003) also reported decrease in food and water intake in Cr (VI) intoxicated developing chick leading to a significant loss of body and tissues (liver, kidney and muscles) weights and reduction in various biochemical parameters. Thus the reduction in the weight of reproductive tissue could be due to alteration in food intake as well as the physiological changes resulting due to Cr (VI) toxicity as also evident in present study. P=Proestrus; E=Estrus; M=Metaestrus; D=Diestrus; LP=Late proestrus; LD=Late diestrus; LE=Late estrrus Proteins level in the ovary is significantly decreased indicating a probable fall in protein synthesis as pro- 123

4 J. Tissue Research tein synthesizing machinery is badly damaged during Cr (IV) intoxication (Chowdhuri et al., 2001). It is well known that heavy metals bind with SH groups of proteins rendering them inactive and/or inhibit their synthesis (De Bruin, 1976; Rao, 1997). The reduction in the cholesterol in ovary and serum could be attributed to the alterations in its synthesis, conversion and transport as a result of chromium feeding. Ascorbic acid is known to be antistress factor and is involved in several oxido-reduction reaction in tissues and cells. Moreover its involvement in ovarian steroidogenesis with cholesterol in gonad is well documented by several investigators (Rajvanshi et al., 2001). Its protective action on chromium toxicity is well documented via its free radical mechanisms and detoxification effect (Karimov, 1988). In our study the was affected indicating chromium interference with the ovarian hormonal milieu that regulates estrus cycle. Most of the animals had irregular cycles with prolongation of metaestrus stage, leading to probable induction of infertility. The level of total lipids in uterus was reduced significantly by chromate treatment. Abraham et al. (1982) have reported that in rabbits fed a high cholesterol diet, chromium not only decreased cholesterol accumulation but also increased the removal rate of cholesterol and lipids that have already been deposited in the aorta. Further, chromium supplementation to normal and diabetic adult men increases high density lipoprotein cholesterol and decreases triglycerides and total cholesterol (Anderson, 1986). A significant accumulation of glycogen with a concomitant decline in phosphorylase activity in uterus and liver as evident in the present study indicates a carbohydrate metabolic change in these tissues. In any instance of poisoning, the change in the glycogen storage is found to be correlated with dysfunction of the liver (De Bruin, 1976). The reduction in the level of SDH activity in liver is an indication of chromium toxicity as SDH is a mitochondrial enzyme and a decrease in its activity might cause a block in the Krebs cycle kinetics. Liver mitochondria has been reported to accumulate Cr (IV) (Langard, 1979), which affected mitochondrial enzymes. Additionally the activities of serum transaminases which are known to be markers of liver function were significantly increased. Thus it is evident that Cr (IV) is highly toxic to liver. Das Neves et al. (2002) also reported liver toxicity after single administration of Cr (VI) or [Cr (V)-BT] (2) to mice. Hematological parameters like haemoglobin and cell counts exhibited alterations in the K 2 treated groups in the present study. Studies by NTP (1996) have reported a decline in mean cell volume (MCV) and mean cell haemoglobin (MCH) values at doses of 8.4 and 9.8 mg Cr (VI)/kg/day in male and female rats. Lewalter et al. 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