ENVIRONMENTAL RESEARCH 52, 77--82 (1990)
Influence of Some Anti-inflammatory Drugs on the Activity of Aryl Hydrocarbon Hydroxylase and the Cytochrome P450 Content M. H.
MOSTAFA,
S. A.
S H E W E I T A , AND
N. M. ABDEL-MONEAM
Institute of Graduate Studies and Research and Faculty of Science, Alexandria University, Alexandria, Egypt Received July 25, 1989 The metaboUsm of benzo[a]pyrene is mediated by the mixed function oxidase system including the cytochrome P450-dependent aryl hydrocarbon hydroxylase. The data of the present study revealed the abifity of various commonly used anti-inflammatory drugs to alter the activity of this enzyme system, where all the tested drugs, namely phenyl butazone, ketoprofen, piroxicam, and acetaminophen, caused an increase in both the activity of aryl hydrocarbon hydroxylase and the cytochrome P450 content whether administered as a single dose or as a repeated dose for 6 consecutive days. The percentage of change for all drugs except phenyl butazone was proportional to the duration of drug administration. On the other hand, pyrazole which is chemically related to phenyl butazone, had no significant effect when administered as a single dose but caused a decrease in both studied parameters when administered as a repeated dose for 6 consecutive days. The mechanisms by which these commonly used drugs modify the aryl hydrocarbon hydroxylase activity and the cytochrome p450 content are discussed in the text. © 1990AcademicPress, Inc.
INTRODUCTION
Polycyclic aromatic hydrocarbons are ubiquitous environmental pollutants and some are believed to cause cancer in man. An important and very extensively studied member of this class of compounds is benzo[a]pyrene which has been shown to cause carcinogenic, mutagenic, and cytotoxic effects in various species and tissues (Gelboin, 1980; Conney, 1982). However, in order to exert its biological effects, benzo[a]pyrene requires metabolic activation by means of the cytochrome P450-dependent monooxygenase system found in the liver microsomes, namely the aryl hydrocarbon hydroxylase system. Various xenobiotics have been reported to alter the activity of this enzyme system such as drugs, pesticides, food additives and environmental pollutants including the polycyclic aromatic hydrocarbons themselves (Beyler et al., 1988; Rodrigues et al., 1987; Gooderham and Mannering, 1985; Huang et al., 1981; Mostafa et al., 1983, 1984; Mostafa and Weisburger, 1980). Anti-inflammatory drugs, analgesics and antipyretics, are being extensively used by the general population and especially among patients suffering from rheumatic diseases, arthritis, or different kinds of fevers. Because of the uncontrolled widespread use of anti-inflammatory drugs, this study was planned to investigate the effects of some members of this particular class of drugs on microsomal cytochrome P450-dependent aryl hydrocarbon hydroxylase activity and the terminal electron acceptor, the cytochrome P450 content. Such an investigation is 77 0013-9351/90 $3.00 Copyright© 1990by AcademicPress, Inc. All fightsof reproductionin any formreserved.
78
MOSTAFA, SHEWEITA, AND ABDEL-MONEAM
important because any alterations in the activity of this enzyme system may affect the rate at which the various polycyclic aromatic hydrocarbons including benzo[a]pyrene are metabolized to yield either inert or toxicologically active metabolites. MATERIALS AND METHODS
Animals Male Swiss albino mice (Naval American Research Unit 3 [NAMRU-3] Cairo, Egypt) weighing 18-35 g were used. The animals were caged in groups of six animals per cage and were maintained on a standard laboratory diet. The animals had free access to food and water except 24 hr before decapitation.
Administration Schedules All the tested drugs were administered intraperitoneally. Each drug was administered to two groups of mice, where one group received a single dose of the drug while the second group received a repeated dose every 24 hr for 6 consecutive days. The corresponding controls for each treatment received an equal volume of vehicle. Phenylbutazone (Nile Co. for Pharmaceuticals and Chemicals Industries, Cairo, Egypt) was administered in polyethylene glycol at a dose of 100 mg/kg body wt. Pyrazole (Aldrich Chemical Co., Milwaukee, Wisconsin) was administered in distilled water at a dose of 100 mg/kg body wt. Piroxicam (Pfizer Egypt SAA, Cairo) was administered in 0.1 M potassium phosphate buffer, pH 7.4, at a dose of 4 mg/kg body wt. Ketoprofen (Specia, Paris, France) was administered in distilled water at a dose of 100 mg/kg body wt. Acetaminophen (Faculty of Pharmacy, Alexandria University, Alexandria) was administered in distilled water at a dose of 50 mg/kg body wt.
Preparation of Microsomes The mice were killed by decapitation 24 hr after the last injection. Each liver was excised, washed in ice-cold 0.1 M phosphate buffer, pH 7.4, homogenized separately in 3 vol (w/v) of the same buffer, and centrifuged for 20 min at 11,000g. The supernatant fraction was then centrifuged at 105,000g for 60 min and the microsomal pellet was resuspended in the 0.1 M phosphate buffer, kept in an ice bath, and used as enzyme source.
Enzyme Assays The activity of the aryl hydrocarbon hydroxylase was assayed fluorometrically by measuring the 3-hydroxy benzo[a]pyrene as described by Wiebel and Gelboin (1975). Cytochrome P450 content was measured in dithionite-reduced microsomes by the method of Omura and Sato (1964). Microsomal protein concentrations were measured by the method of Lowry et al. (1951). Student's t-test was performed on the data and probability values (P) of less than 0.05 were considered significant.
79
C Y T O C H R O M E P450 A N D A N T I - I N F L A M M A T O R Y D R U G S
RESULTS AND DISCUSSION
The metabolism of benzo[a]pyrene is mediated by the mixed-function oxidase system, which comprises a complex mixture of enzymes. These enzymes, including the cytochrome P450-dependent aryl hydrocarbon hydroxylase, are located in the membranous fractions of mammalian hepatocytes and are responsible for the detoxification of xenobiotics which sometimes lead to the formation of ultimate carcinogens. Although many xenobiotics are able to stimulate drug-metabolizing enzymes, the number of drugs which have been reported to be inducers--when administered at given doses---is extremely low (Greim, 1976). The data of the present study revealed the ability of various commonly used anti-inflammatory drugs to alter the aryl hydrocarbon hydroxylase activity and the cytochrome P450 content of mouse liver microsomes, after single- and repeated-dose treatments. As shown in Tables 1 and 2, pretreatment of mice with phenylbutazone, ketoprophen, piroxicam, and acetaminophen significantly increased the activity of aryl hydrocarbon hydroxylase and the cytochrome P450 content when the drugs were administered on a 1-day as well as on a 6-day schedule. The percentage of increase for all drugs except phenylbutazone was found to be proportional to the duration of drug administration. Contrary to these effects, pretreatment of mice with pyrazole for 6 consecutive days significantly TABLE 1 EFFECT OF SOME ANTI-INFLAMMATORY DRUGS ON THE ACTIVITY OF ARYL HYDROCARBON HYDROXYLASE AND THE CYTOCHROME P450 CONTENT AFTER A SINGLE-DOSE TREATMENT A H H activity (pmol 3-OH B(a)P/mg microsomal pr/min) a
Treatment Pyrazole, 100 mg/kg body wt ip in distilled water Phenyl butazone 100 mg/kg body wt ip in polyethylene glycol Ketoprofen, 100 mg/kg body wt ip in distilled water Piroxicam, 4 mg/kg body wt ip in 0.1 M potassium phosphate buffer, p H 7.4 Acetaminophen, 50 mg/kg body wt ip in distilled water
Control b
Cytochrome P450 (nmol/mg microsomal pr) a
Experimental
Control b
72.93
1.354
89.81
104.17
168.24 (61% increase) P < 0.01 99.67 148.1 (49% increase) P < 0.01
1.223 N o effect NS
N o effect NS 81.40 180.27 (121% increase) P < 0.01 100.80 131.18 (30% increase) P < 0.05
Experimental
1.043
1.232 N o effect NS
0.984
1.392 (41% increase) P < 0.05)
0.952
1.419 (49% increase) P < 0.001 0.667 0.852 (28% increase) P < 0.001
Note. NS, statistically nonsignificant. a Values are the means -+ SE of six mice. b Control animals received an equivalent volume of the vehicle and were assayed together with the
treated animals.
80
MOSTAFA, SHEWEITA, AND ABDEL-MONEAM
TABLE II EFFECT OF SOME ANTI-INFLAMMATORY DRUGS ON THE ACTIVITY OF ARYL HYDROCARBON HYDROXYLASE AND THE CYTOCHROME P450 CONTENT AFTER A REPEATED-DOSE TREATMENT OF 6 DAYS AHH activity (pmol 3-OH B(a)P/mg microsomal pr/min) a Treatment Pyrazole, 100 mg/kg body wt ip in distilled water Phenyl butazone 100 mg/kg body wt ip in polyethylene glycol Ketoprofen, 100 mg/kg body wt ip in distilled water Piroxicam, 4 mg/kg body wt ip in 0.1 M potassium phosphate buffer, pH 7.4 Acetaminophen, 50 mg/kg body wt ip in distilled water
Control b
Experimental
Cytochrome P450 (nmol/mg microsomal p r ) a Control b
Experimental
89.81 37.49 (58%decrease) P < 0.01 99.84 133.88 (34%increase) P
Influence of Some Anti-inflammatory Drugs on the Activity of Aryl Hydrocarbon Hydroxylase and the Cytochrome P450 Content M. H.
MOSTAFA,
S. A.
S H E W E I T A , AND
N. M. ABDEL-MONEAM
Institute of Graduate Studies and Research and Faculty of Science, Alexandria University, Alexandria, Egypt Received July 25, 1989 The metaboUsm of benzo[a]pyrene is mediated by the mixed function oxidase system including the cytochrome P450-dependent aryl hydrocarbon hydroxylase. The data of the present study revealed the abifity of various commonly used anti-inflammatory drugs to alter the activity of this enzyme system, where all the tested drugs, namely phenyl butazone, ketoprofen, piroxicam, and acetaminophen, caused an increase in both the activity of aryl hydrocarbon hydroxylase and the cytochrome P450 content whether administered as a single dose or as a repeated dose for 6 consecutive days. The percentage of change for all drugs except phenyl butazone was proportional to the duration of drug administration. On the other hand, pyrazole which is chemically related to phenyl butazone, had no significant effect when administered as a single dose but caused a decrease in both studied parameters when administered as a repeated dose for 6 consecutive days. The mechanisms by which these commonly used drugs modify the aryl hydrocarbon hydroxylase activity and the cytochrome p450 content are discussed in the text. © 1990AcademicPress, Inc.
INTRODUCTION
Polycyclic aromatic hydrocarbons are ubiquitous environmental pollutants and some are believed to cause cancer in man. An important and very extensively studied member of this class of compounds is benzo[a]pyrene which has been shown to cause carcinogenic, mutagenic, and cytotoxic effects in various species and tissues (Gelboin, 1980; Conney, 1982). However, in order to exert its biological effects, benzo[a]pyrene requires metabolic activation by means of the cytochrome P450-dependent monooxygenase system found in the liver microsomes, namely the aryl hydrocarbon hydroxylase system. Various xenobiotics have been reported to alter the activity of this enzyme system such as drugs, pesticides, food additives and environmental pollutants including the polycyclic aromatic hydrocarbons themselves (Beyler et al., 1988; Rodrigues et al., 1987; Gooderham and Mannering, 1985; Huang et al., 1981; Mostafa et al., 1983, 1984; Mostafa and Weisburger, 1980). Anti-inflammatory drugs, analgesics and antipyretics, are being extensively used by the general population and especially among patients suffering from rheumatic diseases, arthritis, or different kinds of fevers. Because of the uncontrolled widespread use of anti-inflammatory drugs, this study was planned to investigate the effects of some members of this particular class of drugs on microsomal cytochrome P450-dependent aryl hydrocarbon hydroxylase activity and the terminal electron acceptor, the cytochrome P450 content. Such an investigation is 77 0013-9351/90 $3.00 Copyright© 1990by AcademicPress, Inc. All fightsof reproductionin any formreserved.
78
MOSTAFA, SHEWEITA, AND ABDEL-MONEAM
important because any alterations in the activity of this enzyme system may affect the rate at which the various polycyclic aromatic hydrocarbons including benzo[a]pyrene are metabolized to yield either inert or toxicologically active metabolites. MATERIALS AND METHODS
Animals Male Swiss albino mice (Naval American Research Unit 3 [NAMRU-3] Cairo, Egypt) weighing 18-35 g were used. The animals were caged in groups of six animals per cage and were maintained on a standard laboratory diet. The animals had free access to food and water except 24 hr before decapitation.
Administration Schedules All the tested drugs were administered intraperitoneally. Each drug was administered to two groups of mice, where one group received a single dose of the drug while the second group received a repeated dose every 24 hr for 6 consecutive days. The corresponding controls for each treatment received an equal volume of vehicle. Phenylbutazone (Nile Co. for Pharmaceuticals and Chemicals Industries, Cairo, Egypt) was administered in polyethylene glycol at a dose of 100 mg/kg body wt. Pyrazole (Aldrich Chemical Co., Milwaukee, Wisconsin) was administered in distilled water at a dose of 100 mg/kg body wt. Piroxicam (Pfizer Egypt SAA, Cairo) was administered in 0.1 M potassium phosphate buffer, pH 7.4, at a dose of 4 mg/kg body wt. Ketoprofen (Specia, Paris, France) was administered in distilled water at a dose of 100 mg/kg body wt. Acetaminophen (Faculty of Pharmacy, Alexandria University, Alexandria) was administered in distilled water at a dose of 50 mg/kg body wt.
Preparation of Microsomes The mice were killed by decapitation 24 hr after the last injection. Each liver was excised, washed in ice-cold 0.1 M phosphate buffer, pH 7.4, homogenized separately in 3 vol (w/v) of the same buffer, and centrifuged for 20 min at 11,000g. The supernatant fraction was then centrifuged at 105,000g for 60 min and the microsomal pellet was resuspended in the 0.1 M phosphate buffer, kept in an ice bath, and used as enzyme source.
Enzyme Assays The activity of the aryl hydrocarbon hydroxylase was assayed fluorometrically by measuring the 3-hydroxy benzo[a]pyrene as described by Wiebel and Gelboin (1975). Cytochrome P450 content was measured in dithionite-reduced microsomes by the method of Omura and Sato (1964). Microsomal protein concentrations were measured by the method of Lowry et al. (1951). Student's t-test was performed on the data and probability values (P) of less than 0.05 were considered significant.
79
C Y T O C H R O M E P450 A N D A N T I - I N F L A M M A T O R Y D R U G S
RESULTS AND DISCUSSION
The metabolism of benzo[a]pyrene is mediated by the mixed-function oxidase system, which comprises a complex mixture of enzymes. These enzymes, including the cytochrome P450-dependent aryl hydrocarbon hydroxylase, are located in the membranous fractions of mammalian hepatocytes and are responsible for the detoxification of xenobiotics which sometimes lead to the formation of ultimate carcinogens. Although many xenobiotics are able to stimulate drug-metabolizing enzymes, the number of drugs which have been reported to be inducers--when administered at given doses---is extremely low (Greim, 1976). The data of the present study revealed the ability of various commonly used anti-inflammatory drugs to alter the aryl hydrocarbon hydroxylase activity and the cytochrome P450 content of mouse liver microsomes, after single- and repeated-dose treatments. As shown in Tables 1 and 2, pretreatment of mice with phenylbutazone, ketoprophen, piroxicam, and acetaminophen significantly increased the activity of aryl hydrocarbon hydroxylase and the cytochrome P450 content when the drugs were administered on a 1-day as well as on a 6-day schedule. The percentage of increase for all drugs except phenylbutazone was found to be proportional to the duration of drug administration. Contrary to these effects, pretreatment of mice with pyrazole for 6 consecutive days significantly TABLE 1 EFFECT OF SOME ANTI-INFLAMMATORY DRUGS ON THE ACTIVITY OF ARYL HYDROCARBON HYDROXYLASE AND THE CYTOCHROME P450 CONTENT AFTER A SINGLE-DOSE TREATMENT A H H activity (pmol 3-OH B(a)P/mg microsomal pr/min) a
Treatment Pyrazole, 100 mg/kg body wt ip in distilled water Phenyl butazone 100 mg/kg body wt ip in polyethylene glycol Ketoprofen, 100 mg/kg body wt ip in distilled water Piroxicam, 4 mg/kg body wt ip in 0.1 M potassium phosphate buffer, p H 7.4 Acetaminophen, 50 mg/kg body wt ip in distilled water
Control b
Cytochrome P450 (nmol/mg microsomal pr) a
Experimental
Control b
72.93
1.354
89.81
104.17
168.24 (61% increase) P < 0.01 99.67 148.1 (49% increase) P < 0.01
1.223 N o effect NS
N o effect NS 81.40 180.27 (121% increase) P < 0.01 100.80 131.18 (30% increase) P < 0.05
Experimental
1.043
1.232 N o effect NS
0.984
1.392 (41% increase) P < 0.05)
0.952
1.419 (49% increase) P < 0.001 0.667 0.852 (28% increase) P < 0.001
Note. NS, statistically nonsignificant. a Values are the means -+ SE of six mice. b Control animals received an equivalent volume of the vehicle and were assayed together with the
treated animals.
80
MOSTAFA, SHEWEITA, AND ABDEL-MONEAM
TABLE II EFFECT OF SOME ANTI-INFLAMMATORY DRUGS ON THE ACTIVITY OF ARYL HYDROCARBON HYDROXYLASE AND THE CYTOCHROME P450 CONTENT AFTER A REPEATED-DOSE TREATMENT OF 6 DAYS AHH activity (pmol 3-OH B(a)P/mg microsomal pr/min) a Treatment Pyrazole, 100 mg/kg body wt ip in distilled water Phenyl butazone 100 mg/kg body wt ip in polyethylene glycol Ketoprofen, 100 mg/kg body wt ip in distilled water Piroxicam, 4 mg/kg body wt ip in 0.1 M potassium phosphate buffer, pH 7.4 Acetaminophen, 50 mg/kg body wt ip in distilled water
Control b
Experimental
Cytochrome P450 (nmol/mg microsomal p r ) a Control b
Experimental
89.81 37.49 (58%decrease) P < 0.01 99.84 133.88 (34%increase) P
