Chem.-Biol. Interactions, 17 (1977) 249--255 © Elsevier/North-Holland Scientific Publishers, Ltd.
249
INDUCTION OF MIXED FUNCTION OXIDASES ON O R A L ADMINISTRATION OF D I E L D R I N
K.K. KOHLI, K.K. M A G G O N
and T.A. V E N K I T A S U B R A M A N I A N
Department of Biochemistry, Vallabhbhai Patel Chest Institute,Universityof Delhi, Delhi-110007 (India) (Received November 22nd, 1976) (Accepted February 20th, 1977)
SUMMARY
The administration of dieldrin (30 mg/kg b o d y weight) caused an increase in the liver weight of rats. The metabolism of aflatoxins B, and G, by the microsomes obtained from the liver of dieldrin-treated animals was enhanced significantly as compared to the controls showing that dieldrin increased the activity of mixed function hydroxylases. Dieldrin caused an increase in the activity of liver microsomal NADPH oxidase and a decrease in the lipid peroxidation. Dieldrin brought a b o u t an increase in the phosphatidylcholine content of rat liver.
INTRODUCTION
There are reports in the literature on induction of mixed function oxidases by chlorinated insecticides [1--3]. These have demonstrated simultaneous proliferation of smooth endoplasmic reticulum to which these enzymes are mainly confined [4]. DDT increases mixed function oxidases [5] in mice. DDT and its isomers shorten the period of hypnosis and sleeping time induced b y phenobarbital due to induction of mixed function hydroxylases [6]. However, DDT has been observed to prolong the phenobarbital induced sleeping time at a dose level of 90 mg/kg b o d y weight [7]. This has been attributed to the fact that it might inhibit the enzymes which metabolize phenobarbital. The various steps in the process of oxidative reactions have been described [8]. The first step is the combination of substrate with oxidized form of cytrochrome P 4 5 0 , thus emphasizing the role of c y t o c h r o m e P-450 in the oxidative reactions. Increased levels of cytochrome P-450 in insecticide treated animals has been reported [9--11]. The increase in c y t o c h r o m e P450 paralleled the increase in oxidative reactions. Lindane had a more pro-
250 nounced effect than DDT in increasing the activity of c y t o c h r o m e P-450 complex [11]. In view of several reports that chlorinated insecticides increase the activity of mixed function oxidases and protein synthesis [12], the effect of dieldrin on the metabolism of aflatoxins B~ and G~ by the rat liver microsomal preparation was studied. Since NADPH oxidase [ 1 3 ] , lipid peroxidation [14] and phospholipids [15] are related to mixed function oxidation reactions, these were also studied in the dieldrin-treated as well as control animals. MATERIALS AND METHODS
Chemicals Nicotinamide adenine dinucleotide phosphate (NADP), NADPH, glucose6-phosphate, nicotinamide, tris(hydroxymethylaminomethane), glucose-6phosphate dehydrogenase and thiobarbituric acid were purchased from Sigma Chemical Company, St. Louis, Missouri, U.S.A. Aflatoxins B1 and G~ were available in our laboratory. All other chemicals and solvents were purchased from BDH, Ltd., B o m b a y and were of "Analar" grade.
Animal material Wistar strain male rats 100--120 g were used in the present study. The animals weighing 60--80 g were from the stock colony of Vallabhbhai Patel Chest Institute, Delhi-7. Each animal was kept in a separate cage and fed Hind Lever rat f o o d and water ad libitum. When the animals attained the required weight, they were divided into t w o groups, control and experimental. The experimental group was fed dieldrin (30 mg/kg b o d y weight) dissolved in groundnut oil orally. The control group received only groundnut oil. After feeding, the animals were fasted and given water ad libitum. After 22--24 h of fasting the animals of both the groups were killed by decapitation and the liver removed and processed.
Preparation of microsomes Microsomes were isolated from liver obtained from control and experimental animals b y the m e t h o d of H o g e b o o m [16].
Metabolism of aflatoxins BI and GI Microsomes washed thoroughly with phosphate buffer (pH 7.4) were suspended in the same buffer. The incubation mixture contained in a total volume of 2.5 ml 140 pmoles of sodium phosphate buffer (pH 7.4), 1 pmole of NADP, 25 //moles of MgC12, 50 pmoles of nicotinamide and 200/~g of either substrate [17]. The blank consisted of boiled microsomal suspension. The incubation was carried o u t at 37°C for ten min. The reaction was stopped by addition of an equal volume of methanol. The precipitate was centrifuged down. The supernatant was extracted three times with equal volumes of chloroform. The extracts were pooled, dried over anhydrous sodium sulphate, evaporated under reduced pressure, and made u p t o a
251 known volume. The total volume was spotted on a thin layer chromatoplate coated with a 500 pm thick layer of silica gel G. The plates were developed in a solvent system, toluene : isoamylalcohol : methanol (90 : 32 : 3, v/v/v) [18]. The aflatoxin bands were located with a long wave ultraviolet lamp. The bands were scrapped and eluted three times with 3 ml of methanol. The extracts were pooled separately in each case and evaporated. The volume of each sample was noted and the optical density was measured at 363 nm in a Beckman DU Spectrophotometer. The amount of unmetabolized aflatoxin was calculated using the extinction coefficients of 22 000 for aflatoxin B1 and 18 700 for G~ [19]. The validity of this assay procedure was established in preliminary experiments, when the a m o u n t of aflatoxin B~ left unmetabolized was comparable to the values reported by Masri et al. [20].
NADPH oxidase and lipid peroxidation NADPH oxidase and lipid peroxidation were assayed by the m e t h o d of Hart and Fouts [13] and Ernster and Nordenbrand [21] respectively.
Protein'assays Protein was estimated by the m e t h o d of Lowry et al. [22].
Assay of phospholipids A portion of liver was dipped in chilled saline to remove blood, dried between folds of filter p a p e r , weighed, and dropped into c h l o r o f o r m : methanol (2 : 1, v/v) mixture [23] and ground with acid-washed sand. The lipids were extracted and the extracts stored at --20°C. The phospholipids were separated b y thin layer chromatography in a solvent system containing chloroform : methanol : 7 N NH4OH (230 : 90 : 15, v/v/v) [24]. The bands corresponding to phosphatidylcholine and phosphatidylethanolamine were scraped off and transferred to test tubes. Phospholipid phosphorus was estimated according to the m e t h o d of Marinetti [25]. RESULTS AND DISCUSSION Table I shows the results of the effect of dieldrin on b o d y and liver weight of rats. Dieldrin did n o t affect the b o d y weight of the animals while liver weight was significantly increased. This is in agreement with the previous reports from our laboratory [12,26] as well as those of other investigators [27--29]. Increase in liver weight has been ascribed to the increased level of triglycerides in the livers of the animals treated with dieldrin [26]. In the present investigation metabolism of aflatoxin B1 and G1 was significantly increased as compared with control animals (Table I), suggesting the stimulation of mixed function hydroxylases by dieldrin. The results obtained are in accord with earlier reports [30,31] that aflatoxin G, is less toxic than aflatoxin B~, because it is metabolized to a greater extent (Table I). DDT treatment protected rats from aflatoxin-induced liver injury [32]
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249
INDUCTION OF MIXED FUNCTION OXIDASES ON O R A L ADMINISTRATION OF D I E L D R I N
K.K. KOHLI, K.K. M A G G O N
and T.A. V E N K I T A S U B R A M A N I A N
Department of Biochemistry, Vallabhbhai Patel Chest Institute,Universityof Delhi, Delhi-110007 (India) (Received November 22nd, 1976) (Accepted February 20th, 1977)
SUMMARY
The administration of dieldrin (30 mg/kg b o d y weight) caused an increase in the liver weight of rats. The metabolism of aflatoxins B, and G, by the microsomes obtained from the liver of dieldrin-treated animals was enhanced significantly as compared to the controls showing that dieldrin increased the activity of mixed function hydroxylases. Dieldrin caused an increase in the activity of liver microsomal NADPH oxidase and a decrease in the lipid peroxidation. Dieldrin brought a b o u t an increase in the phosphatidylcholine content of rat liver.
INTRODUCTION
There are reports in the literature on induction of mixed function oxidases by chlorinated insecticides [1--3]. These have demonstrated simultaneous proliferation of smooth endoplasmic reticulum to which these enzymes are mainly confined [4]. DDT increases mixed function oxidases [5] in mice. DDT and its isomers shorten the period of hypnosis and sleeping time induced b y phenobarbital due to induction of mixed function hydroxylases [6]. However, DDT has been observed to prolong the phenobarbital induced sleeping time at a dose level of 90 mg/kg b o d y weight [7]. This has been attributed to the fact that it might inhibit the enzymes which metabolize phenobarbital. The various steps in the process of oxidative reactions have been described [8]. The first step is the combination of substrate with oxidized form of cytrochrome P 4 5 0 , thus emphasizing the role of c y t o c h r o m e P-450 in the oxidative reactions. Increased levels of cytochrome P-450 in insecticide treated animals has been reported [9--11]. The increase in c y t o c h r o m e P450 paralleled the increase in oxidative reactions. Lindane had a more pro-
250 nounced effect than DDT in increasing the activity of c y t o c h r o m e P-450 complex [11]. In view of several reports that chlorinated insecticides increase the activity of mixed function oxidases and protein synthesis [12], the effect of dieldrin on the metabolism of aflatoxins B~ and G~ by the rat liver microsomal preparation was studied. Since NADPH oxidase [ 1 3 ] , lipid peroxidation [14] and phospholipids [15] are related to mixed function oxidation reactions, these were also studied in the dieldrin-treated as well as control animals. MATERIALS AND METHODS
Chemicals Nicotinamide adenine dinucleotide phosphate (NADP), NADPH, glucose6-phosphate, nicotinamide, tris(hydroxymethylaminomethane), glucose-6phosphate dehydrogenase and thiobarbituric acid were purchased from Sigma Chemical Company, St. Louis, Missouri, U.S.A. Aflatoxins B1 and G~ were available in our laboratory. All other chemicals and solvents were purchased from BDH, Ltd., B o m b a y and were of "Analar" grade.
Animal material Wistar strain male rats 100--120 g were used in the present study. The animals weighing 60--80 g were from the stock colony of Vallabhbhai Patel Chest Institute, Delhi-7. Each animal was kept in a separate cage and fed Hind Lever rat f o o d and water ad libitum. When the animals attained the required weight, they were divided into t w o groups, control and experimental. The experimental group was fed dieldrin (30 mg/kg b o d y weight) dissolved in groundnut oil orally. The control group received only groundnut oil. After feeding, the animals were fasted and given water ad libitum. After 22--24 h of fasting the animals of both the groups were killed by decapitation and the liver removed and processed.
Preparation of microsomes Microsomes were isolated from liver obtained from control and experimental animals b y the m e t h o d of H o g e b o o m [16].
Metabolism of aflatoxins BI and GI Microsomes washed thoroughly with phosphate buffer (pH 7.4) were suspended in the same buffer. The incubation mixture contained in a total volume of 2.5 ml 140 pmoles of sodium phosphate buffer (pH 7.4), 1 pmole of NADP, 25 //moles of MgC12, 50 pmoles of nicotinamide and 200/~g of either substrate [17]. The blank consisted of boiled microsomal suspension. The incubation was carried o u t at 37°C for ten min. The reaction was stopped by addition of an equal volume of methanol. The precipitate was centrifuged down. The supernatant was extracted three times with equal volumes of chloroform. The extracts were pooled, dried over anhydrous sodium sulphate, evaporated under reduced pressure, and made u p t o a
251 known volume. The total volume was spotted on a thin layer chromatoplate coated with a 500 pm thick layer of silica gel G. The plates were developed in a solvent system, toluene : isoamylalcohol : methanol (90 : 32 : 3, v/v/v) [18]. The aflatoxin bands were located with a long wave ultraviolet lamp. The bands were scrapped and eluted three times with 3 ml of methanol. The extracts were pooled separately in each case and evaporated. The volume of each sample was noted and the optical density was measured at 363 nm in a Beckman DU Spectrophotometer. The amount of unmetabolized aflatoxin was calculated using the extinction coefficients of 22 000 for aflatoxin B1 and 18 700 for G~ [19]. The validity of this assay procedure was established in preliminary experiments, when the a m o u n t of aflatoxin B~ left unmetabolized was comparable to the values reported by Masri et al. [20].
NADPH oxidase and lipid peroxidation NADPH oxidase and lipid peroxidation were assayed by the m e t h o d of Hart and Fouts [13] and Ernster and Nordenbrand [21] respectively.
Protein'assays Protein was estimated by the m e t h o d of Lowry et al. [22].
Assay of phospholipids A portion of liver was dipped in chilled saline to remove blood, dried between folds of filter p a p e r , weighed, and dropped into c h l o r o f o r m : methanol (2 : 1, v/v) mixture [23] and ground with acid-washed sand. The lipids were extracted and the extracts stored at --20°C. The phospholipids were separated b y thin layer chromatography in a solvent system containing chloroform : methanol : 7 N NH4OH (230 : 90 : 15, v/v/v) [24]. The bands corresponding to phosphatidylcholine and phosphatidylethanolamine were scraped off and transferred to test tubes. Phospholipid phosphorus was estimated according to the m e t h o d of Marinetti [25]. RESULTS AND DISCUSSION Table I shows the results of the effect of dieldrin on b o d y and liver weight of rats. Dieldrin did n o t affect the b o d y weight of the animals while liver weight was significantly increased. This is in agreement with the previous reports from our laboratory [12,26] as well as those of other investigators [27--29]. Increase in liver weight has been ascribed to the increased level of triglycerides in the livers of the animals treated with dieldrin [26]. In the present investigation metabolism of aflatoxin B1 and G1 was significantly increased as compared with control animals (Table I), suggesting the stimulation of mixed function hydroxylases by dieldrin. The results obtained are in accord with earlier reports [30,31] that aflatoxin G, is less toxic than aflatoxin B~, because it is metabolized to a greater extent (Table I). DDT treatment protected rats from aflatoxin-induced liver injury [32]
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