Acta pharmacol. et toxicol. 1977, 41, 129-140.
From the Department of Pharmacology, University of Oulu, SF-90220 Oulu 22, Finland
The Metabolism of Benzo(a)pyrene in Isolated Perfused Lungs from Variously-treated Rats BY Kirsi Vlhakangas, Kaisu Nevasaari, Olavi Pelkonen and Niilo T. Klrki (Received July 7, 1976; Accepted December 14, 1976)
Abstract: Rats were pretreated either by the injection of 3-methylcholanthrene (MC) 40 mg/kg intraperitoneally for 3 days or by giving phenobarbitone (PB) 0.5 g/l in the drinking water for 7 days. Benzo(a)pyrene (BP) and its metabolites were measured by thin-layer chromatography (TLC) and radiometry in samples of perfusion medium up to two hours. In perfusion medium the half-life of BP was about 15 min. in the perfusions of lungs from MC-pretreated rats and 60-90 min. in the perfusions of lungs from PB-pretreated and control rats. The appearance of water-soluble metabolites was most marked in the perfusions of lungs from MC-pretreated rats being at fifteen minutes 2.5-fold and a t two hours 2-fold in comparison to the controls. In the perfusions of lungs from PB-pretreated rats the accumulation was retarded perhaps indicating an inhibition of lung enzymes by PB. Water-soluble metabolites included glucuronic and sulphuric acid conjugates as well as those conjugates resistant to acid hydrolysis. Phenols were the most pronounced organic-soluble metabolites at the beginning of perfusion of lungs from MC-pretreated rats. At fifteen minutes the amount of phenols in the perfusions of lungs from PB-pretreated rats was I/, and in controls I / , of that in the perfusions of lungs from MC-pretreated rats. Towards the end of perfusion phenols decreased in the perfusions of lungs from MCpretreated rats. In other perfusions all metabolite fractions increased slowly during the perfusion. Covalently bound radioactivity and BP-hydroxylase in lung homogenates were studied after perfusions. The covalent binding of radioactivity in the lungs from MC-pretreated rats was 3.5 times higher than in control lungs and the lungs from PB-pretreated rats. There was a correlation between covalent binding and the activity of BP-hydroxylase in lung homogenates. Exposure of rats to cigarette smoke resulted in effects on the BP metabolism resembling those of MC pretreatment. Key-words: Benzo(a)pyrene metabolism - pulmonary drug metabolism perfusion - covalent binding - enzyme induction - rats.
-
lung
It has been established beyond doubt that cigarette smoking and lung cancer are causally related. Since cigarette smoke contains numerous poly-
130
KIRSI VAHAKANGAS ET AL.
cyclic aromatic hydrocarbons, which in animal experiments have been shown to be powerful carcinogens (Suss et al. 1973), one is inclined to link polycyclic aromatic hydrocarbons, for example benzo(a)pyrene, and the carcinogenic process in lungs. The development of lung tumours has been studied in vivo in different experimental animals. Other approaches have been included in the study of lung microsomal oxidative enzyme systems (CAPDEWLA et al. 1975), which are thought to be responsible for the activation oC procarcinogens to ultimate reactive forms, an example being the conversion of benzo(a)pyrene to an epoxide, which is bound covalently to cellular macromolecules and initiate the carcinogenic process (Sms & GROVER1974). Some workers have used as a model different kinds of organ and cell cultures derived from lung tissue (LEUCHTENBERGER et al. 1974). All these systems have advantages and disadvantages, and in this study we have also investigated another modd system, namely the in vitro perfusion of isolated lung. Recently, VALNIO et d. (1976) have reported a study on the fate of benzo(a)pyrene in short-term oncethrough perfusion of the isolated rat lung. We have studied short- and long-term recirculating perfusion of lungs with benzo(a)pyrene and have tried to analyze the metabolite pattern, i. e. covalent binding and other parameters as a function of time. Materials and Methods Pretreatment of animals and the drugs used. Non-fasted male rats (SpragueDawley) weighing 190-260 g were used in all experiments. One group was given phenobarbitone in the drinking water (0.5 g/l) for 7 days, controls drinking water ad libitum. The second group was given 3-methylcholanthrene (Fluka AG,Switzerland) 40 mg/kg in sesame oil (1 ml/kg) intraperitoneally once a day for 3 days, while the controls received the same amount of the vehicle. The administration of phenobarbitone and methylcholanthrene was stopped 24 hours before the removal of the lungs. A third group was exposed to cigarette smoke. Rats were placed (2 at a time) into a cylinder-like Penpex-chamber (vol. 11.06 1) equipped with adjustable ventilation. With a directed air-stream one cigarette (North-State, Amer-Tupakka Oy, Finland) was burned in 2-3 min. and all the smoke blown into the chamber. Thereafter the ventilation of the chamber was adjusted to be about 1 llmin. and the rats remained in there for 10 min. The rats were exposed to cigarette smoke in this way for 2 days before the day of the perfusions, twice at an interval of 60 min. cm the first day, once on the second day. Randomly labelled *H-benzo(a)pyrene (spec. act. about 5 cilmmol) obtained from Radiochemical Center (Amersham, England) was mixed with a sufficient amount of nodabelled benzo(a)pyrene (Sigma,St. Louis Mo., U.S.A.) and used as dmcribed later. Perfusion medium. The volume of the perfusion fluid was 50 ml, containing 12 ml of fresh rat blood
BENZO(A)PYRENE IN LUNG PERFUSION
131
obtained from larger rats (anaesthetized by ether) by cardiac puncture using heparinized (250 IU) syringe; 38 ml of Krebs-Ringer phosphate solution; 1.25 g bovine serum albumin (Fraction V, Sigma Chem. Comp., St. Louis), and 50 mg D-glucose. The perfusion medium was prepared on the day of perfusion. Since the albumin was acidic, the pH had to be adjusted to about 7.2 by the addition of NaOH. Oxygen was supplied by ventilation of the lungs. During the perfusions the pH was 7.3-7.5 and the PO, between 100-130 mmHg as monitored by Combi-Analyser (L. Eschweiler & Co.,Kiel). Perfusion equipment. The perfusion apparatus consists of an organ chamber and a glass-spiral for heating the perfusion medium, surrounded by water jackets, and a perfusion medium reservoir. The temperature was maintained constant at 37.5” k 0.5” with a thermostat (Thennomix 11, B. Braun Apparate-Bau, Melsungen). From the reservoir the perfusion medium is pumped by a perfusion pump (Peristaltic Pump, Model 1217, Harvard Appar. Co.) with a constant volume of about 20 ml/min. through the heating spiral into the lungs. The pump was used with two tubes in parallel giving virtually pulse free pumping. Operation procedure. Rats were anaesthetized for lung preparation by giving an intraperitoneal injection of urethane, 1.5 g/kg. The trachea was cannulated and ventilation was started immediately with a rate of 2 ml per stroke 16 times per minute during the operation (Starling ‘LIdeal”pump, Model E9, C. F. Palmer Ltd., London). The chest was opened, a dose of 500 IU of heparin was injected into the left ventricle of the heart. The vena cava was cut off above the diaphragm in order to diminish the venous return to the lungs during the cannulation of the pulmonary artery. The heart was grasped with a clamp, and a cannula (P.E. 200-205) was placed into the pulmonary artery via the right ventricle. The left ventricle of the heart was excised, the lungs removed and placed into the perfusion chamber on a stainless steal mesh and suspended from the trachea cannula. The liver, kidneys and perfused lungs of the rat were removed, weighed and stored at -20’ for further analysis. General procedure of the perfusion. Once the perfusion was started, the ventilation pump rate was reduced to 0.5-1 ml per stroke 16 strokes per minute in order to prevent the oxygen tension in the perfusion medium from becoming too high. After an equilibration period of 5 minutes 12.5 nmol (about 15 X 1 W cpm) of JH-3,4-benzpyrene dissolved in 0.25 ml of dimethylsulphoxide was added into the perfusion medium reservoir giving a final concentration of 0.25 pM. 1 ml samples of perfusion medium were taken before sH-3,4-benzpyrene addition and 15, 30, 60,90 and 120 min. after the addition and samples were bubbled with N, for storage. After the perfusion the lungs were infused with 5 ml of physiological saline to remove the blood, and frozen for further analysis. Analysis of radioactivity.
The frozen samples and the lungs were allowed to thaw at room temperature. Lungs were homogenized in 4 volumes of 0.1 M potassium-sodium phosphate buffer, pH 7.4. All samples were extracted twice with two volumes of ethyl acetate and small samples (25 pl) were taken from both aqueous and ethyl acetate phase for calculation of radioactivity. These are referred to as water-solubIe and ethyl acetate-soluble radio-
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activity. As controls we used radioactive benzo(a)pyrene mixed with lung homogenates or rat blood and extracted as experimental samples. Ethyl acetate-soluble radioactivity was further resolved by thin layer chromatography (Silica gel G, 0.25 mm, solvent system: benzene: ethanol 19:l) according to SMS (1970) and BORGENet al. (1973). Authentic reference compounds were ccu-chromatagraphed with ethyl acetate-soluble products from the lungs and the perfusate samples, fluorescent spots were identified under UV light, marked, and cut into scintillation counting vials; 5 ml of Instagel@ (Packard) was added and the samples were counted in a scintillation spectrometer (Packard Industries, Model 3320). A 0.5-ml portion of water phase was incubated with 0.1 mg of fbglucuronidase Type I (Sigma Chemical Co., St. Louis, Missouri) in 0.5 ml of phosphate buffer (0.025 M, pH 6.8) at 37” for 30 minutes. Another 0.5-ml portion of water phase was incubated with 0.1 mg of bacterial arylsulphatase Sigma Type 3 in 0.5 ml of sodium acetate buffer (0.2 M, pH 4.5) at 37” for 30 minutes. Acid hydrolysis was carried out in 2 N hydrochloric acid at 100’ for 5 minutes. In each case, controls included only buffer or water without hydrdyzing agent. After incubation, the mixture was extracted twice with ethyl acetate and the difference between treated and control samples was taken to represent the hydrolyzed metabolite. The binding of radioactivity to lung tissue was determined according to the method of Szeuvrrz (1952). The aqueous phase of the lung tissue after two ethyl acetate extractions was further extracted twice with two volumes of ethyl acetate and precipitated with TCA (10%). TCA-precipitated material was subjected to the Siekevitz wash with the modification that nucleoprotein digestion was not performed. The final precipitate was digested with Sohenem (Packard Instrument), mixed with 10 ml of Instagel@ and the radioactivity was counted. This was referred to as “covalently bound radioactivity”. The benzo(a)pyrene hydroxylase activity determinations were carried out according & G E L B O(1968). ~ The amount of tissue homoto the fluorametric method of NEBERT genate was 5 mg of lung, liver, or kidney and the incubation time 15 minutes. Statistical treatment. Student’s t-test, applied to an unpaired comparison, was used to evaluate the significance of the difference between mean values, the criterion of which was taken as P < 0.05.
Results The effects of a pretreatment with methylcholanthrene (MC) or phenobarbitone (PB) on the metabolism of SH-benzcj(a)pyrenein isolated perfused rat lungs were studied in perfusions lasting two hours. Fig. 1 shows the disappearance of SH-benz~a)pyrene from the perfusate after MC (fig. 1A) or PB pretreatment (fig. 1B) of the rats. In the MC pretreated perfused lungs, the disappearance of SH-benz~(a)pyrenefrom the perfusate was very rapid. 30 min. after the addition of BH-BPinto the perfusion medium, the amount of the sH-BP in the perfusate of MC pretreated lungs was only one fourth olf that present in the controls. On the contrary, PB pretreatment seemed to retard significantly the disappearance of 3H-BP when compared with the respective control group (fig. 1B).
BENZO(A)PYRENE IN LUNG PERFUSION
-+--
A
15 30 60 90 TIME (MINUTES)
120
6
133
15 30 60 90 TIME (MINUTES)
120
Fig. 1. A. The disappearance of SH-benzo(a)pyrene from the perfusion medium in isolated rat lung perfusions of methylcholanthrene pretreated (40 mg/kg intraperitoneally for 3 days, -0) and control ( 0 4 )rats and the appearance of water). .- and control ( . - -0 ) perfusions. soluble metabolites in MC-pretreated ( After about 5 min. equilibration period benzo(a)pyrene (15 X 108 cpm) was added into the perfusion medium (0-time in the figures and tables). Each point is the mean k S.E. of 6 perfusions. *** P 0.001, ** P 0.01,* P < 0.05 as compared to the controls by Student's t-test.
From the Department of Pharmacology, University of Oulu, SF-90220 Oulu 22, Finland
The Metabolism of Benzo(a)pyrene in Isolated Perfused Lungs from Variously-treated Rats BY Kirsi Vlhakangas, Kaisu Nevasaari, Olavi Pelkonen and Niilo T. Klrki (Received July 7, 1976; Accepted December 14, 1976)
Abstract: Rats were pretreated either by the injection of 3-methylcholanthrene (MC) 40 mg/kg intraperitoneally for 3 days or by giving phenobarbitone (PB) 0.5 g/l in the drinking water for 7 days. Benzo(a)pyrene (BP) and its metabolites were measured by thin-layer chromatography (TLC) and radiometry in samples of perfusion medium up to two hours. In perfusion medium the half-life of BP was about 15 min. in the perfusions of lungs from MC-pretreated rats and 60-90 min. in the perfusions of lungs from PB-pretreated and control rats. The appearance of water-soluble metabolites was most marked in the perfusions of lungs from MC-pretreated rats being at fifteen minutes 2.5-fold and a t two hours 2-fold in comparison to the controls. In the perfusions of lungs from PB-pretreated rats the accumulation was retarded perhaps indicating an inhibition of lung enzymes by PB. Water-soluble metabolites included glucuronic and sulphuric acid conjugates as well as those conjugates resistant to acid hydrolysis. Phenols were the most pronounced organic-soluble metabolites at the beginning of perfusion of lungs from MC-pretreated rats. At fifteen minutes the amount of phenols in the perfusions of lungs from PB-pretreated rats was I/, and in controls I / , of that in the perfusions of lungs from MC-pretreated rats. Towards the end of perfusion phenols decreased in the perfusions of lungs from MCpretreated rats. In other perfusions all metabolite fractions increased slowly during the perfusion. Covalently bound radioactivity and BP-hydroxylase in lung homogenates were studied after perfusions. The covalent binding of radioactivity in the lungs from MC-pretreated rats was 3.5 times higher than in control lungs and the lungs from PB-pretreated rats. There was a correlation between covalent binding and the activity of BP-hydroxylase in lung homogenates. Exposure of rats to cigarette smoke resulted in effects on the BP metabolism resembling those of MC pretreatment. Key-words: Benzo(a)pyrene metabolism - pulmonary drug metabolism perfusion - covalent binding - enzyme induction - rats.
-
lung
It has been established beyond doubt that cigarette smoking and lung cancer are causally related. Since cigarette smoke contains numerous poly-
130
KIRSI VAHAKANGAS ET AL.
cyclic aromatic hydrocarbons, which in animal experiments have been shown to be powerful carcinogens (Suss et al. 1973), one is inclined to link polycyclic aromatic hydrocarbons, for example benzo(a)pyrene, and the carcinogenic process in lungs. The development of lung tumours has been studied in vivo in different experimental animals. Other approaches have been included in the study of lung microsomal oxidative enzyme systems (CAPDEWLA et al. 1975), which are thought to be responsible for the activation oC procarcinogens to ultimate reactive forms, an example being the conversion of benzo(a)pyrene to an epoxide, which is bound covalently to cellular macromolecules and initiate the carcinogenic process (Sms & GROVER1974). Some workers have used as a model different kinds of organ and cell cultures derived from lung tissue (LEUCHTENBERGER et al. 1974). All these systems have advantages and disadvantages, and in this study we have also investigated another modd system, namely the in vitro perfusion of isolated lung. Recently, VALNIO et d. (1976) have reported a study on the fate of benzo(a)pyrene in short-term oncethrough perfusion of the isolated rat lung. We have studied short- and long-term recirculating perfusion of lungs with benzo(a)pyrene and have tried to analyze the metabolite pattern, i. e. covalent binding and other parameters as a function of time. Materials and Methods Pretreatment of animals and the drugs used. Non-fasted male rats (SpragueDawley) weighing 190-260 g were used in all experiments. One group was given phenobarbitone in the drinking water (0.5 g/l) for 7 days, controls drinking water ad libitum. The second group was given 3-methylcholanthrene (Fluka AG,Switzerland) 40 mg/kg in sesame oil (1 ml/kg) intraperitoneally once a day for 3 days, while the controls received the same amount of the vehicle. The administration of phenobarbitone and methylcholanthrene was stopped 24 hours before the removal of the lungs. A third group was exposed to cigarette smoke. Rats were placed (2 at a time) into a cylinder-like Penpex-chamber (vol. 11.06 1) equipped with adjustable ventilation. With a directed air-stream one cigarette (North-State, Amer-Tupakka Oy, Finland) was burned in 2-3 min. and all the smoke blown into the chamber. Thereafter the ventilation of the chamber was adjusted to be about 1 llmin. and the rats remained in there for 10 min. The rats were exposed to cigarette smoke in this way for 2 days before the day of the perfusions, twice at an interval of 60 min. cm the first day, once on the second day. Randomly labelled *H-benzo(a)pyrene (spec. act. about 5 cilmmol) obtained from Radiochemical Center (Amersham, England) was mixed with a sufficient amount of nodabelled benzo(a)pyrene (Sigma,St. Louis Mo., U.S.A.) and used as dmcribed later. Perfusion medium. The volume of the perfusion fluid was 50 ml, containing 12 ml of fresh rat blood
BENZO(A)PYRENE IN LUNG PERFUSION
131
obtained from larger rats (anaesthetized by ether) by cardiac puncture using heparinized (250 IU) syringe; 38 ml of Krebs-Ringer phosphate solution; 1.25 g bovine serum albumin (Fraction V, Sigma Chem. Comp., St. Louis), and 50 mg D-glucose. The perfusion medium was prepared on the day of perfusion. Since the albumin was acidic, the pH had to be adjusted to about 7.2 by the addition of NaOH. Oxygen was supplied by ventilation of the lungs. During the perfusions the pH was 7.3-7.5 and the PO, between 100-130 mmHg as monitored by Combi-Analyser (L. Eschweiler & Co.,Kiel). Perfusion equipment. The perfusion apparatus consists of an organ chamber and a glass-spiral for heating the perfusion medium, surrounded by water jackets, and a perfusion medium reservoir. The temperature was maintained constant at 37.5” k 0.5” with a thermostat (Thennomix 11, B. Braun Apparate-Bau, Melsungen). From the reservoir the perfusion medium is pumped by a perfusion pump (Peristaltic Pump, Model 1217, Harvard Appar. Co.) with a constant volume of about 20 ml/min. through the heating spiral into the lungs. The pump was used with two tubes in parallel giving virtually pulse free pumping. Operation procedure. Rats were anaesthetized for lung preparation by giving an intraperitoneal injection of urethane, 1.5 g/kg. The trachea was cannulated and ventilation was started immediately with a rate of 2 ml per stroke 16 times per minute during the operation (Starling ‘LIdeal”pump, Model E9, C. F. Palmer Ltd., London). The chest was opened, a dose of 500 IU of heparin was injected into the left ventricle of the heart. The vena cava was cut off above the diaphragm in order to diminish the venous return to the lungs during the cannulation of the pulmonary artery. The heart was grasped with a clamp, and a cannula (P.E. 200-205) was placed into the pulmonary artery via the right ventricle. The left ventricle of the heart was excised, the lungs removed and placed into the perfusion chamber on a stainless steal mesh and suspended from the trachea cannula. The liver, kidneys and perfused lungs of the rat were removed, weighed and stored at -20’ for further analysis. General procedure of the perfusion. Once the perfusion was started, the ventilation pump rate was reduced to 0.5-1 ml per stroke 16 strokes per minute in order to prevent the oxygen tension in the perfusion medium from becoming too high. After an equilibration period of 5 minutes 12.5 nmol (about 15 X 1 W cpm) of JH-3,4-benzpyrene dissolved in 0.25 ml of dimethylsulphoxide was added into the perfusion medium reservoir giving a final concentration of 0.25 pM. 1 ml samples of perfusion medium were taken before sH-3,4-benzpyrene addition and 15, 30, 60,90 and 120 min. after the addition and samples were bubbled with N, for storage. After the perfusion the lungs were infused with 5 ml of physiological saline to remove the blood, and frozen for further analysis. Analysis of radioactivity.
The frozen samples and the lungs were allowed to thaw at room temperature. Lungs were homogenized in 4 volumes of 0.1 M potassium-sodium phosphate buffer, pH 7.4. All samples were extracted twice with two volumes of ethyl acetate and small samples (25 pl) were taken from both aqueous and ethyl acetate phase for calculation of radioactivity. These are referred to as water-solubIe and ethyl acetate-soluble radio-
132
KIRSI VWHAKANGAS ET AL.
activity. As controls we used radioactive benzo(a)pyrene mixed with lung homogenates or rat blood and extracted as experimental samples. Ethyl acetate-soluble radioactivity was further resolved by thin layer chromatography (Silica gel G, 0.25 mm, solvent system: benzene: ethanol 19:l) according to SMS (1970) and BORGENet al. (1973). Authentic reference compounds were ccu-chromatagraphed with ethyl acetate-soluble products from the lungs and the perfusate samples, fluorescent spots were identified under UV light, marked, and cut into scintillation counting vials; 5 ml of Instagel@ (Packard) was added and the samples were counted in a scintillation spectrometer (Packard Industries, Model 3320). A 0.5-ml portion of water phase was incubated with 0.1 mg of fbglucuronidase Type I (Sigma Chemical Co., St. Louis, Missouri) in 0.5 ml of phosphate buffer (0.025 M, pH 6.8) at 37” for 30 minutes. Another 0.5-ml portion of water phase was incubated with 0.1 mg of bacterial arylsulphatase Sigma Type 3 in 0.5 ml of sodium acetate buffer (0.2 M, pH 4.5) at 37” for 30 minutes. Acid hydrolysis was carried out in 2 N hydrochloric acid at 100’ for 5 minutes. In each case, controls included only buffer or water without hydrdyzing agent. After incubation, the mixture was extracted twice with ethyl acetate and the difference between treated and control samples was taken to represent the hydrolyzed metabolite. The binding of radioactivity to lung tissue was determined according to the method of Szeuvrrz (1952). The aqueous phase of the lung tissue after two ethyl acetate extractions was further extracted twice with two volumes of ethyl acetate and precipitated with TCA (10%). TCA-precipitated material was subjected to the Siekevitz wash with the modification that nucleoprotein digestion was not performed. The final precipitate was digested with Sohenem (Packard Instrument), mixed with 10 ml of Instagel@ and the radioactivity was counted. This was referred to as “covalently bound radioactivity”. The benzo(a)pyrene hydroxylase activity determinations were carried out according & G E L B O(1968). ~ The amount of tissue homoto the fluorametric method of NEBERT genate was 5 mg of lung, liver, or kidney and the incubation time 15 minutes. Statistical treatment. Student’s t-test, applied to an unpaired comparison, was used to evaluate the significance of the difference between mean values, the criterion of which was taken as P < 0.05.
Results The effects of a pretreatment with methylcholanthrene (MC) or phenobarbitone (PB) on the metabolism of SH-benzcj(a)pyrenein isolated perfused rat lungs were studied in perfusions lasting two hours. Fig. 1 shows the disappearance of SH-benz~a)pyrene from the perfusate after MC (fig. 1A) or PB pretreatment (fig. 1B) of the rats. In the MC pretreated perfused lungs, the disappearance of SH-benz~(a)pyrenefrom the perfusate was very rapid. 30 min. after the addition of BH-BPinto the perfusion medium, the amount of the sH-BP in the perfusate of MC pretreated lungs was only one fourth olf that present in the controls. On the contrary, PB pretreatment seemed to retard significantly the disappearance of 3H-BP when compared with the respective control group (fig. 1B).
BENZO(A)PYRENE IN LUNG PERFUSION
-+--
A
15 30 60 90 TIME (MINUTES)
120
6
133
15 30 60 90 TIME (MINUTES)
120
Fig. 1. A. The disappearance of SH-benzo(a)pyrene from the perfusion medium in isolated rat lung perfusions of methylcholanthrene pretreated (40 mg/kg intraperitoneally for 3 days, -0) and control ( 0 4 )rats and the appearance of water). .- and control ( . - -0 ) perfusions. soluble metabolites in MC-pretreated ( After about 5 min. equilibration period benzo(a)pyrene (15 X 108 cpm) was added into the perfusion medium (0-time in the figures and tables). Each point is the mean k S.E. of 6 perfusions. *** P 0.001, ** P 0.01,* P < 0.05 as compared to the controls by Student's t-test.
