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iodothyronine 5'-monodeiodinase (5'-D) activity in the Indian rock pigeon, Columba livia. Administration of lead acetate (6 mg/kg body weight/day) for 20 days ...
J. Biosci., Vol. 22, Number 2, March 1997, pp 247-254. © Printed in India

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Lead inhibits type-I iodothyronine 5'-monodeiodinase in the Indian rock pigeon Columba livia: A possible involvement of essential thiol groups SHYAM SUNDER CHAURASIA, SUNANDA PANDA and ANAND KAR* School of Life Sciences, Devi Ahilya University, Vigyan Bhawan, Khandwa Road, Indore 452 001 .India MS received 20 July 1996; revised 7 November 1996 Abstract. A study has been made to reveal the mode of action of lead inhibiting type-I iodothyronine 5'-monodeiodinase (5'-D) activity in the Indian rock pigeon, Columba livia. Administration of lead acetate (6 mg/kg body weight/day) for 20 days decreased 5'-D activity and glutathione content in the liver and kidney homogenates. It also reduced the serum concentration of 3, 3', 5-triiodothyronine (T3) with a marginal increase in serum thyroxine (T4). Hepatic and renal lipid peroxidative process increased significantly following lead treatment, whereas the levels of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) were decreased. The possible involvement of lipid peroxidative process in the inhibition of 5’-D activity in Columba livia is suggested. Keywords. Lead; type-I iodothyronine 5-monodeiodinase; thyroxine; 3,3', 5-triiodothyronine; glutathione; lipid peroxidation; antioxidant enzymes.

1. Introduction The type-I iodothyronine 5'-monodeiodinase (5'-D) is the principal enzyme by which thyroxine (T4) is metabolized to 3, 3', 5-triiodothyronine (T3), the most potent thyroid hormone, in the extrathyroidal tissues. Liver and kidney are the primary deiodinating sites (Kuhn et al 1990). Various thiol protecting agents including dithiothreitol (DTT) and reduced glutathione stimulate the 5'-deiodinating system (Goswami and Rosenburg 1990); additionally the sulphydryl groups of the 5'-D are essential for the enzyme to maintain its three-dimensional configuration (Ozawa et al 1982). A variety of abnormal states such as acute and chronic illness (Greenspan 1994); pharmacological agents such as dexamethasone (Decuypere et al 1983); pesticides such as fenvalerate (Maiti et al 1995), dimethoate (Maiti et al 1996) and heavy metals such as cadmium (Paier et al 1993; Gupta et al 1995), mercury (Visser et al 1976) have been reported to impair the 5'-monodeiodination process. Some preliminary investigations have demonstrated the effects of lead on the thyroid function in mammals (Robins et al 1983; Tuppurainen et al 1988; Gennart et al 1992). However, these investigations considered only the thyroidal [131I]-uptake and the serum thyroid hormone concentrations, but not the 5'-D enzyme, despite the fact that T3 is produced in peripheral tissues by the 5'-deiodinating system (Visser 1994). We have recently reported the loss *Corresponding author (Fax, 0731-472793).

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of 5'-D by lead in birds (Chaurasia et al 1995). However, the mechanism of inhibition of 5'-D activity was not determined. The present study is an attempt to determine the possible contribution of essential thiol groups to the loss of 5'-deiodinating activity in liver and kidney homogenates of Indian rock pigeon, Columba livia.

2. Materials and methods 2.1

Experimental design

Adult male pigeons, C. livia were procured from a local supplier and were acclimated for one week in a well ventilated aviary under standard laboratory conditions (14 h light: 10 h dark, 27 ± 1°C). The birds were given diet and water ad libitum and divided into two groups of seven birds in each. Group I, birds receiving 0·1 ml of normal saline, served as the control. The birds of group II were administered subcutaneously with 0·1 ml of lead acetate (6 mg/kg body weight/day) for 20 days as used previously by Chaurasia et al (1995). Drug administration was made each day between 11·00 and 11 30 h to avoid circadian interference. 2.2 Sample preparation On the last day, blood was collected from each bird by cardiac puncture, centrifuged and the serum samples were stored at — 80°C for future use. The liver and kidney were removed, cleaned and washed twice with phosphate buffered saline (pH 7·4) prior to processing for biochemical estimations. 2.3 Determination of thyroidhormone concentrations Total circulating T3 and T4 were estimated by radioimmunoassay (RIA) in the serum samples using 8-amlino-l-naphthalene sulphonic acid (ANS) following the method of Brown et al (1970) with a minor modification (Kar and Chandola-Saklani 1985). Pigeon hormone free serum (prepared by the activated charcoal method) was used for the preparation of T3 and T4 standards. T3 and T4 antibodies and labelled hormones were supplied by Bhabha Atomic Research Center, Bombay. Lower limit sensitivity was determined to be 0·072 ng/ml for T3 and 0·14 ng/ml for T4. Interassay variation was less than 5% for both the hormones. 2.4

Type -I 5'-D assay

Hepatic and renal 5'-D activity were estimated by the method of Decuypere et al (1983) with a minor modification (Chaurasia et al 1995). In brief, liver and kidney homogenates were homogenized in ice cold phosphate buffer (1:4 weight/volume, 0·15 M, pH 6·5 with 0·25 Μ sucrose and 5 mM ethylene diamine tetraacetic acid). The homogenates were centrifuged at 2000 g for 30 min at 4°C. The supernatant collected was used for subsequent assays. To 100 µl supernatant 200 µl of DTT (4 mM) and 10 µl T4(4 µΜ) were added; this served as the test sample. In control tube, T4 was replaced by equivalent amount of buffer. The tubes were incubated at 37°C for 1 h. The enzymatic

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reaction was stopped by adding 1 ml of 95% ethanol, vortexed and kept overnight at — 80°C. Next day, the samples were centrifuged at 3000 g for 40 min at 4°C and the amount of T3 formed was estimated by RIA. Samples were estimated in duplicates and standards were prepared in 100 µ1 ethanol extracts. Results are expressed as ng of T3 generated per hour of incubation per mg protein. 2.5 Determination of lipid peroxidation and antioxidant enzymes For this, liver and kidney tissues were homogenized in 10% ice cold phosphate buffer (0·1 M, pH 7·4). The homogenates were centrifuged at 15,000 g for 30 min at 4°C and the supernatant was used for further assays. Lipid peroxidation (LPO) was determined by thiobarbituric acid (TBA) reaction with malondialdehyde (MDA), a product formed due to the peroxidation of lipids (Ohkawa et al 1979). The absorbance was measured at 532 nm using a Shimadzu UV- 160A spectrophotometer (Japan). The LPO was expressed as nM of MDA formed per hour per mg protein. The hepatic and renal superoxide dismutase (SOD) activity was assayed according to the method of Marklund and Marklund (1974) and catalase activity following the method of Aebi (1983). 2.6 Glutathione determination Reduced sulphydryl groups (GSH) were assayed using Ellman's reagent (Ellman 1959). 2.7 Protein determination The protein content was measured in 10 µ1 supernatant by the method of Lowry et al (1951) with bovine serum albumin as the standard. 2.8 Statistical analysis The data were expressed as mean ± SE and the results were compared by Student's t test.

3. Results Birds of both the control and treated groups were found to be healthy throughout the experiment. Lead inhibited the thyroid function in male pigeons significantly decreasing the serum T3 concentration (P < 0·001), while the serum T4 concentration exhibited a marginal increase (figure la). The 5'-D activity also decreased significantly in liver (P < 0·001) and kidney (P < 0· 01) homogenates as shown in figure lb. On the other hand, a significant increase in the lipid peroxide level in liver (P < 0·01) and kidney (P < 0 ·01) tissues, was observed following lead treatment (figure 2a) with a concomitant decrease in the GSH content (figure 2b; Ρ < 0·001 for both the tissues). Lead also caused a significant reduction in the activities of tissue antioxidant enzymes (figure 3), such as SOD (P < 0·00l for both the tissues) and catalase (Ρ < 0·001 for liver and Ρ < 0·01 for kidney).

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Figure 1. Effects of lead acetate (6 mg/kg body weight) on (a) circulating thyroid hormone concentrations (ng/ml) and (b) type-I iodothyronine 5'-monodeiodinase activity (ng T3 generated/h/mgprotein) ofliver and kidney homogenatesin Indian rock pigeon, C. livia. Each bar represents the mean ± SEM for seven birds. Significant differences from the control; *P< 0·01, **P< 0·001.

4. Discussion Decreased serum T3 and a marginal increase in serum T4 concentrations following metal treatment corroborate earlier reports (Tuppurainen et al 1988; Gennart et al 1992). However, these two studies have ignored the importance of type-I 5'-D enzyme, which is primarily involved in the production of T3. We have recently reported a depressed 5'-D activity in bird and fish following lead exposure, with concomitant

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Figure 2. Effects of lead acetate (6 mg/kg body weight) on (a) lipid peroxide level (nM of MDA formed/h/mg protein) and (b) glutathione content (µΜ GSH/mgprotein) of hepatic and renal tissues in Indian rock pigeon, C. livia. Each bar represents the mean ± SEM for seven birds. Significant differences from the control; *P < 0 · 01, * * P < 0 001.

decrease in serum T3 concentration (Chaurasia et al 1995, 1996). In the present study, one of the possible mechanisms of reduction of 5'-monodeiodination has been elucidated. Type-I 5'-D enzyme is believed to have a pivotal role in maintaining circulating concentrations of T3 as extrathyroidal conversion accounts for approximately 80% of the total T 3 production (Chopra et al 1978). Low concentration of serum T3 and a marginal increase in serum T4 in lead treated pigeon may be due to the decreased formation of T3 following the inhibition of type-I 5'-D activity in liver and kidney homogenates as observed in the present study. However, the increase in T4 was not

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Figure 3. Efects of lead acetate (6 mg/kg body weight) on (a) superoxide dismutase (SOD) activity (units/mg protein; one unit of the SOD is defined as the enzyme activity which inhibits autoxidation of pyrogallol by 50%) and catalase activity (µΜ of H2O2 decomposed/min/mg protein) of liver and kidney homogenates in Indian rock pigeon, C. livia. Each bar represents the mean ± SEM for seven birds. Significant differences from the control: * Ρ < 0·01, **P