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Effect of intratracheally administered lindane on aldrin and benzo(a)pyrene contents in lungs of rats
Toxicology Letters, 69 (1993) 63-67 0 1993 Elsevier Science Publishers B.V. All rights reserved 037%4274/93/$06.00
63
TOXLET 02926
Effect of intratracheally administered lindane on aldrin and benzo(a)pyrene contents in lungs of rats
Munawwar A. Khan, Mohammad A. Matin and Mirza U. Beg Industrial Toxicology Research Centre, Lucknow (India)
(Received 5 November 1992) (Accepted 20 January 1993) Key words: Lindane; Lung; Aldrin; Benzo(a)pyrene; Hydroxylase
SUMMARY Pretreatment with lindane resulted in inhibition of benzo(a)pyrene hydroxylase activity in the lungs of rats. The enzyme activity tended to return to normal 5 days after the administration of lindane. Studies with benzo(a)pyrene and aldrin indicated reduced elimination of these compounds from the lungs of lindanetreated animals, suggesting that lindane may alter the clearance of certain substances or compounds from lungs. The delayed clearance of these compounds from lungs may be an indirect effect of lindane related to inhibition of certain metabolizing enzymes.
INTRODUCTION
Lindane, the y-isomer of hexachlorocyclohexane (syn. benzene hexachloride) is an organochlorine pesticide which is extensively used in agriculture and public health programs in a number of developing countries. Lindane in high doses produces muscle fibrillation, tremors and convulsions [ 1,2]. These symptoms are accompanied by lesions or biochemical changes in liver, kidney and other tissues [3,4]. There are also reports indicating changes in the metabolism of certain xenobiotics in lungs following exposure to certain organochlorine pesticides [5]. In the present study, we investigated the effect of lindane on the metabolism of benzo(a)pyrene and aldrin in the lungs of rats.
Correspondence to and present address: Munawwar A. Khan, Ph.D., Visiting Scientist, Department Pathology, F-05, University of Texas Medical Branch, Galveston, TX 77555-0605, USA.
of
64
MATERIALS
AND METHODS
Male albino rats (Wistar strain), 150 ? 10 g, were used in experiments. The animals were maintained on a 12-h light/dark cycle and had food and water ad libitum except 12 h before experiments. The trachea was exposed by a mid-line incision after inducing light diethyl ether anaesthesia in rats; lindane was dissolved in peanut oil and injected intratracheally in a dose of 5 mg/kg in each animal. After the injection, the skin was stitched and the animals kept under observation. The intratracheal route of administration was chosen simply to achieve maximum concentration of the pesticide in the lungs. The controls were injected with the solvent by the same route. Benzo(a)pyrene (25 mg/kg) or aldrin (2.5 mg/kg) were also injected intravenously (femoral vein) in controls and lindane-treated animals 1, 3 and 5 days after the administration of lindane. The animals were decapitated 6 h after the last treatment and the lungs were quickly removed from the thoracic cavity. Aldrin was extracted with hexane and assayed by gas-liquid chromatography (GLC) according to the procedure described by Heeshane et al. [6]. The samples were analyzed on Chemito-3865 gas chromatograph, using Electron Capture (Nickel) Detectors at the following operating conditions: detector temperature 220°C; injector temperature 2 10°C; column temperature 200°C; gas flow 40 ml/min; gas pressure 4 KP/Cm; carrier gas 99% ultra pure nitrogen (IOLAR), column coiled stainless steel 6’ x l/8” packed with liquid phase 5% OV-17 on Cn W (HP) MT-300” C. Estimation of benzo(a)pyrene The operating conditions for the estimation of benzo(a)pyrene were slightly different; it was estimated on GLC using FID connected to 5% OV-17 on Cn W(HP) 6’ x l/8”, S.S. column, MT 300°C. Temperatures for injection, column and detector were kept at 350°C 280°C and 350°C respectively. A mixture of H, and 0, were used as fuel with flow rate of 30 and 300 ml/min, respectively, while N, was used as carrier gas with flow rate of 30 ml/min. Peaks were identified by comparing with those of standards. Assay of benzo(a)pyrene hydroxylase activity Benzo(a)pyrene hydroxylase was assayed by the method of Dehenen et al. [7] using benzo(a)pyrene as the substrate. The enzyme activity was assayed in microsomes prepared according to the method of Johannesen et al. [8]. Estimation of proteins The proteins were estimated according to the method of Lowry et al. [9] using bovine serum albumin as the reference. The data were analyzed statistically using Student’s ‘t’ test. Significant differences between the means calculated as P values are given in the tables.
65 RESULTS
Effect of lindane on benzo(a)pyrene
hydroxylase activity
The values of benzo(a)pyrene hydroxylase activity 1,3 and 5 days after the administration of lindane in rats are given in Table I. The enzyme activity was reduced by 36% 1 day after exposure to lindane. After 5 days, the inhibition of enzyme activity was only 17% (Table I). The values were significantly different from those of controls. Effect of lindane pretreatment on benzo(a)pyrene
and aldrin content of lungs
The values of benzo(a)pyrene content in the lungs of lindane treated animals are given in Table II. In animals pretreated with lindane, the benzo(a)pyrene content was higher than in controls 1, 3 and 5 days after exposure to lindane (Table II). The data for aldrin content in controls and lindane exposed animals are given in Table II. In animals pre-exposed to lindane, the aldrin content of lungs was significantly more than in controls (Table II). DISCUSSION
The results indicate changes in the activity of benzo(a)pyrene hydroxylase and the contents of aldrin and benzo(a)pyrene in the lungs of lindane-treated animals. According to our results, the benzo(a)pyrene hydroxylase activity was maximally inhibited 1 day after exposure to lindane (Table I); however, on the fifth day, there was less inhibition in the activity of enzyme which tended to return to normal (Table I). Benzo(a)pyrene hydroxylase is reponsible for the conversion of polycyclic aromatic hydrocarbons to carcinogenic and mutagenic metabolites [lO,l 11. The amount of
TABLE I EFFECT OF LINDANE ON BENZO(a)PYRENE
HYDROXYLASE
ACTIVITY OF RAT LUNGS
Benzo(a)pyrene hydroxylase activity Days: 1 Controls Lindane-treated animals % decrease
0.25 f 0.04 0.16 ?r 0.01 * (-36)
3
5
0.23 + 0.03 0.18 f 0.02 **
0.24 f. 0.05 0.20 + 0.04 **
(-22)
(-17)
The enzyme activity was determined 1, 3 and 5 days after the administration of lindane (5 mg/kg, intratracheally). Each figure represents the mean f SE of six animals. The enzyme activity is expressed as fluorescent unit/h/mg microsomal proteins. * Significantly different from controls, P < 0.01. ** Significantly different from controls, P < 0.05.
66
TABLE II EFFECT OF LINDANE LUNGS OF RATS
ON THE CONTENTS
OF BENZO(a)PYRENE
OR ALDRIN
IN THE
Benzo(a)pyrene content (mg/lung) Days: 1 Controls Lindane-treated
animals
2.2 f 0.5 3.1 f 0.4 *
Controls Lindane-treated
animals
79.5 + 2.7 209.0 f 9.5 *
3
5
2.3 ? 0.4 3.2 & 0.3 * Aldrin content @g/g lung)
2.1 * 0.2 2.8 f 0.1 **
78.3 * 1.9 127.5 + 6.3 *
73.5 * 2.2 87.6 + 7.1 **
Benzo(a)pyrene (25 mg/kg) or aldrin (2.5 mg/kg) administered intravenously 1, 3 and 5 days after the administration of lindane (5 mg/kg, intratracheally). Each group consists of six animals. The animals were sacrificed 6 h after the administration of benzo(a)pyrene or aldrin. * Significantly different from controls, P < 0.01. ** Significantly different from controls, P < 0.05.
benzo(a)pyrene in rat lungs 24 h after lindane treatment was 3.7 mg (Table II) which was significantly higher than that recovered from control animals (2.2 mg). However, in rats 5 days after lindane exposure, benzo(a)pyrene content was 2.8 mg which was slightly, but significantly, more than the corresponding values in controls (Table II). The data are suggestive of the retention of benzo(a)pyrene in the lungs of lindanetreated animals. It was previously reported that benzo(a)pyrene undergoes an epoxidation reaction, forming epoxides that undergo hydroxylation yielding dihydrodiols, which form conjugates with GSH and detoxified [12-141. The inhibition of benzo(a)pyrene hydroxylase by lindane treatment (Table I) may obviously retard the formation of dihydrodiol, thereby reducing the clearance of the compound from the system (Table II). Studies with aldrin also indicated reduced elimination of the compound from the lungs of lindane treated animals. The values of aldrin content in the lungs of lindanetreated animals were significantly higher than those in corresponding controls (Table II). Aldrin is converted to dieldrin by epoxidases; the accumulation of aldrin may possibly be related to inhibition of epoxidase activity in lindane-treated animals. Thus, exposure to lindane may result in changes associated with increased concentration of aldrin and benzo(a)pyrene (Table II), which is a known carcinogen [ 151rendering animals more susceptible to their toxic effects. ACKNOWLEDGEMENT
The authors are grateful to Indian Council of Medical Research (ICMR) for hnancial assistance during the course of above study.
67
REFERENCES 1 Doull, J. (1976) The treatment of insecticide poisoning. In: Wilkinson, C.F. (Ed.), Insecticide Biochemistry and Physiology. Plenum Press, New York, pp. 649-667. 2 Van Asperen, K. (1954) Interaction of the isomers of benzene hexachloride in mice. Arch. Int. Pharmacol. Ther. 99, 368-377. 3 Barros, S.B.M. and Saliba, A.M. (1978) Toxicity of hexachlorocyclohexane in rats. Toxicology 10, 271-279. 4 Ortega, P., Hayes, Jr., W.J. and Durham, W.F. (1957) Pathologic changes in the liver of rats after feeding low levels of various insecticides. Arch. Pathol. 64, 614622. 5 Narayana, S., Bajpai, A., Tyagi, S.R. and Misra, U.K. (1985) Effect of intratracheal administration of DDT and endosulfan on cytochrome P-450 and glutathione-S transferase in lungs and liver of rats. Bull. Environ. Contam. Toxicol. 34,6367. 6 Heeshane, W.S.Q., Tabor, J.A. and Suskind, R.R. (1983) Screening method for Arochlor 1254 (PCB) in whole blood. Anal. Chem. 55, 157-160. 7 Dehenen, W., Tomingas, R. and Roos, F.A. (1973) Modified method for the assay of benzo(a)pyrene hydroxylase. Anal. Biochem. 53, 373-383. 8 Johannesen, K., De Pierre, J.W., Bergstrand, A., Deillner, G., and Ernster, L. (1977) Preparation and characterization of total rough and smooth microsomes from the lungs of control and methyl cholanthrene treated rats. Biochem. Biophys. Acta 496, 115-135. 9 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193,265-275. 10 Depierre, J.W. and Ernster, L. (1978) The metabolism of polycyclic hydr~rbons and its relationship to cancer. Biochem. Biophys. Acta 473, 149-186. 11 Heidelberger, C. (1975) Chemical carcinogenesis. Annu. Rev. Biochem. 44,79-121. 12 Habig, W.J., Pabst, M.J. and Jakoby, W.B. (1974) Glutathione-S-transferases: The first step in mercapturic acid formation. J. Biol. Chem. 249, 7120.-7126. 13 Mukhtar, H. and Breshick, E. (1976) Effects of phenobarbital and 3-methylcholanthrene administration on glutathione-s-epoxide transferase activity in rats liver. Biochem. Pharmacol. 25, 1081-1084. 14 James, M.O., Fouts, J.R. and Bend, J.R. (1976) Hepatic and extra-hepatic metabolism in vitro of an epoxide [S-‘%]styrene oxide in the rabbit. Biochem. Pharmacol. 25, 187-193. 15 Cohen, G.M., Haws, SM., Moore, B.P. and Bridges, J.W. (1976) Benzo(a)pyrene-3-yl hydrogen sulphate, a major ethyl acetate extractable metabolite of benzo(a)pyrene in human, hamster and rat lung cultures. Biochem. Pharmacol. 252561-2570.
63
TOXLET 02926
Effect of intratracheally administered lindane on aldrin and benzo(a)pyrene contents in lungs of rats
Munawwar A. Khan, Mohammad A. Matin and Mirza U. Beg Industrial Toxicology Research Centre, Lucknow (India)
(Received 5 November 1992) (Accepted 20 January 1993) Key words: Lindane; Lung; Aldrin; Benzo(a)pyrene; Hydroxylase
SUMMARY Pretreatment with lindane resulted in inhibition of benzo(a)pyrene hydroxylase activity in the lungs of rats. The enzyme activity tended to return to normal 5 days after the administration of lindane. Studies with benzo(a)pyrene and aldrin indicated reduced elimination of these compounds from the lungs of lindanetreated animals, suggesting that lindane may alter the clearance of certain substances or compounds from lungs. The delayed clearance of these compounds from lungs may be an indirect effect of lindane related to inhibition of certain metabolizing enzymes.
INTRODUCTION
Lindane, the y-isomer of hexachlorocyclohexane (syn. benzene hexachloride) is an organochlorine pesticide which is extensively used in agriculture and public health programs in a number of developing countries. Lindane in high doses produces muscle fibrillation, tremors and convulsions [ 1,2]. These symptoms are accompanied by lesions or biochemical changes in liver, kidney and other tissues [3,4]. There are also reports indicating changes in the metabolism of certain xenobiotics in lungs following exposure to certain organochlorine pesticides [5]. In the present study, we investigated the effect of lindane on the metabolism of benzo(a)pyrene and aldrin in the lungs of rats.
Correspondence to and present address: Munawwar A. Khan, Ph.D., Visiting Scientist, Department Pathology, F-05, University of Texas Medical Branch, Galveston, TX 77555-0605, USA.
of
64
MATERIALS
AND METHODS
Male albino rats (Wistar strain), 150 ? 10 g, were used in experiments. The animals were maintained on a 12-h light/dark cycle and had food and water ad libitum except 12 h before experiments. The trachea was exposed by a mid-line incision after inducing light diethyl ether anaesthesia in rats; lindane was dissolved in peanut oil and injected intratracheally in a dose of 5 mg/kg in each animal. After the injection, the skin was stitched and the animals kept under observation. The intratracheal route of administration was chosen simply to achieve maximum concentration of the pesticide in the lungs. The controls were injected with the solvent by the same route. Benzo(a)pyrene (25 mg/kg) or aldrin (2.5 mg/kg) were also injected intravenously (femoral vein) in controls and lindane-treated animals 1, 3 and 5 days after the administration of lindane. The animals were decapitated 6 h after the last treatment and the lungs were quickly removed from the thoracic cavity. Aldrin was extracted with hexane and assayed by gas-liquid chromatography (GLC) according to the procedure described by Heeshane et al. [6]. The samples were analyzed on Chemito-3865 gas chromatograph, using Electron Capture (Nickel) Detectors at the following operating conditions: detector temperature 220°C; injector temperature 2 10°C; column temperature 200°C; gas flow 40 ml/min; gas pressure 4 KP/Cm; carrier gas 99% ultra pure nitrogen (IOLAR), column coiled stainless steel 6’ x l/8” packed with liquid phase 5% OV-17 on Cn W (HP) MT-300” C. Estimation of benzo(a)pyrene The operating conditions for the estimation of benzo(a)pyrene were slightly different; it was estimated on GLC using FID connected to 5% OV-17 on Cn W(HP) 6’ x l/8”, S.S. column, MT 300°C. Temperatures for injection, column and detector were kept at 350°C 280°C and 350°C respectively. A mixture of H, and 0, were used as fuel with flow rate of 30 and 300 ml/min, respectively, while N, was used as carrier gas with flow rate of 30 ml/min. Peaks were identified by comparing with those of standards. Assay of benzo(a)pyrene hydroxylase activity Benzo(a)pyrene hydroxylase was assayed by the method of Dehenen et al. [7] using benzo(a)pyrene as the substrate. The enzyme activity was assayed in microsomes prepared according to the method of Johannesen et al. [8]. Estimation of proteins The proteins were estimated according to the method of Lowry et al. [9] using bovine serum albumin as the reference. The data were analyzed statistically using Student’s ‘t’ test. Significant differences between the means calculated as P values are given in the tables.
65 RESULTS
Effect of lindane on benzo(a)pyrene
hydroxylase activity
The values of benzo(a)pyrene hydroxylase activity 1,3 and 5 days after the administration of lindane in rats are given in Table I. The enzyme activity was reduced by 36% 1 day after exposure to lindane. After 5 days, the inhibition of enzyme activity was only 17% (Table I). The values were significantly different from those of controls. Effect of lindane pretreatment on benzo(a)pyrene
and aldrin content of lungs
The values of benzo(a)pyrene content in the lungs of lindane treated animals are given in Table II. In animals pretreated with lindane, the benzo(a)pyrene content was higher than in controls 1, 3 and 5 days after exposure to lindane (Table II). The data for aldrin content in controls and lindane exposed animals are given in Table II. In animals pre-exposed to lindane, the aldrin content of lungs was significantly more than in controls (Table II). DISCUSSION
The results indicate changes in the activity of benzo(a)pyrene hydroxylase and the contents of aldrin and benzo(a)pyrene in the lungs of lindane-treated animals. According to our results, the benzo(a)pyrene hydroxylase activity was maximally inhibited 1 day after exposure to lindane (Table I); however, on the fifth day, there was less inhibition in the activity of enzyme which tended to return to normal (Table I). Benzo(a)pyrene hydroxylase is reponsible for the conversion of polycyclic aromatic hydrocarbons to carcinogenic and mutagenic metabolites [lO,l 11. The amount of
TABLE I EFFECT OF LINDANE ON BENZO(a)PYRENE
HYDROXYLASE
ACTIVITY OF RAT LUNGS
Benzo(a)pyrene hydroxylase activity Days: 1 Controls Lindane-treated animals % decrease
0.25 f 0.04 0.16 ?r 0.01 * (-36)
3
5
0.23 + 0.03 0.18 f 0.02 **
0.24 f. 0.05 0.20 + 0.04 **
(-22)
(-17)
The enzyme activity was determined 1, 3 and 5 days after the administration of lindane (5 mg/kg, intratracheally). Each figure represents the mean f SE of six animals. The enzyme activity is expressed as fluorescent unit/h/mg microsomal proteins. * Significantly different from controls, P < 0.01. ** Significantly different from controls, P < 0.05.
66
TABLE II EFFECT OF LINDANE LUNGS OF RATS
ON THE CONTENTS
OF BENZO(a)PYRENE
OR ALDRIN
IN THE
Benzo(a)pyrene content (mg/lung) Days: 1 Controls Lindane-treated
animals
2.2 f 0.5 3.1 f 0.4 *
Controls Lindane-treated
animals
79.5 + 2.7 209.0 f 9.5 *
3
5
2.3 ? 0.4 3.2 & 0.3 * Aldrin content @g/g lung)
2.1 * 0.2 2.8 f 0.1 **
78.3 * 1.9 127.5 + 6.3 *
73.5 * 2.2 87.6 + 7.1 **
Benzo(a)pyrene (25 mg/kg) or aldrin (2.5 mg/kg) administered intravenously 1, 3 and 5 days after the administration of lindane (5 mg/kg, intratracheally). Each group consists of six animals. The animals were sacrificed 6 h after the administration of benzo(a)pyrene or aldrin. * Significantly different from controls, P < 0.01. ** Significantly different from controls, P < 0.05.
benzo(a)pyrene in rat lungs 24 h after lindane treatment was 3.7 mg (Table II) which was significantly higher than that recovered from control animals (2.2 mg). However, in rats 5 days after lindane exposure, benzo(a)pyrene content was 2.8 mg which was slightly, but significantly, more than the corresponding values in controls (Table II). The data are suggestive of the retention of benzo(a)pyrene in the lungs of lindanetreated animals. It was previously reported that benzo(a)pyrene undergoes an epoxidation reaction, forming epoxides that undergo hydroxylation yielding dihydrodiols, which form conjugates with GSH and detoxified [12-141. The inhibition of benzo(a)pyrene hydroxylase by lindane treatment (Table I) may obviously retard the formation of dihydrodiol, thereby reducing the clearance of the compound from the system (Table II). Studies with aldrin also indicated reduced elimination of the compound from the lungs of lindane treated animals. The values of aldrin content in the lungs of lindanetreated animals were significantly higher than those in corresponding controls (Table II). Aldrin is converted to dieldrin by epoxidases; the accumulation of aldrin may possibly be related to inhibition of epoxidase activity in lindane-treated animals. Thus, exposure to lindane may result in changes associated with increased concentration of aldrin and benzo(a)pyrene (Table II), which is a known carcinogen [ 151rendering animals more susceptible to their toxic effects. ACKNOWLEDGEMENT
The authors are grateful to Indian Council of Medical Research (ICMR) for hnancial assistance during the course of above study.
67
REFERENCES 1 Doull, J. (1976) The treatment of insecticide poisoning. In: Wilkinson, C.F. (Ed.), Insecticide Biochemistry and Physiology. Plenum Press, New York, pp. 649-667. 2 Van Asperen, K. (1954) Interaction of the isomers of benzene hexachloride in mice. Arch. Int. Pharmacol. Ther. 99, 368-377. 3 Barros, S.B.M. and Saliba, A.M. (1978) Toxicity of hexachlorocyclohexane in rats. Toxicology 10, 271-279. 4 Ortega, P., Hayes, Jr., W.J. and Durham, W.F. (1957) Pathologic changes in the liver of rats after feeding low levels of various insecticides. Arch. Pathol. 64, 614622. 5 Narayana, S., Bajpai, A., Tyagi, S.R. and Misra, U.K. (1985) Effect of intratracheal administration of DDT and endosulfan on cytochrome P-450 and glutathione-S transferase in lungs and liver of rats. Bull. Environ. Contam. Toxicol. 34,6367. 6 Heeshane, W.S.Q., Tabor, J.A. and Suskind, R.R. (1983) Screening method for Arochlor 1254 (PCB) in whole blood. Anal. Chem. 55, 157-160. 7 Dehenen, W., Tomingas, R. and Roos, F.A. (1973) Modified method for the assay of benzo(a)pyrene hydroxylase. Anal. Biochem. 53, 373-383. 8 Johannesen, K., De Pierre, J.W., Bergstrand, A., Deillner, G., and Ernster, L. (1977) Preparation and characterization of total rough and smooth microsomes from the lungs of control and methyl cholanthrene treated rats. Biochem. Biophys. Acta 496, 115-135. 9 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193,265-275. 10 Depierre, J.W. and Ernster, L. (1978) The metabolism of polycyclic hydr~rbons and its relationship to cancer. Biochem. Biophys. Acta 473, 149-186. 11 Heidelberger, C. (1975) Chemical carcinogenesis. Annu. Rev. Biochem. 44,79-121. 12 Habig, W.J., Pabst, M.J. and Jakoby, W.B. (1974) Glutathione-S-transferases: The first step in mercapturic acid formation. J. Biol. Chem. 249, 7120.-7126. 13 Mukhtar, H. and Breshick, E. (1976) Effects of phenobarbital and 3-methylcholanthrene administration on glutathione-s-epoxide transferase activity in rats liver. Biochem. Pharmacol. 25, 1081-1084. 14 James, M.O., Fouts, J.R. and Bend, J.R. (1976) Hepatic and extra-hepatic metabolism in vitro of an epoxide [S-‘%]styrene oxide in the rabbit. Biochem. Pharmacol. 25, 187-193. 15 Cohen, G.M., Haws, SM., Moore, B.P. and Bridges, J.W. (1976) Benzo(a)pyrene-3-yl hydrogen sulphate, a major ethyl acetate extractable metabolite of benzo(a)pyrene in human, hamster and rat lung cultures. Biochem. Pharmacol. 252561-2570.