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Effect of certain drugs and environmental agents on microsomal enzyme systems
The Science of the Total Environment Elsevier Publishing Company, Amsterdam - - Printed in Belgium
EFFECT OF CERTAIN DRUGS AND ENVIRONMENTAL AGENTS ON MICROSOMAL ENZYME SYSTEMS
M A R J O R I E BICK* A N D L A W R E N C E FISHBEIN
National Institute of Environmental Health Sciences National Institutes of Health, Public Health Service and Department of Health, Education and Welfare Research Triangle Park, North Carolina 27709 (U. S. A.) (Received November 18th, 1971)
SUMMARY
Hepatic microsomal drug metabolizing enzymes induced in rats by p,p'-DDT reach peak activity 5 days after injection, and return to control values on the 15-19th day. Reinduction with different characteristics occurred and persisted for 50 days. Massive doses are used to produce induction in experimental animals. When smaller amounts similar to those given to man therapeutically were used, it was difficult to assess if hepatic induction occurred. There was some evidence of induction after giving diphenylhydantoin, acetylsalicylic acid, and the environmental agent pyrene. Some inhibitory effects were observed after giving phenacetin and trifluoperazine. Administration of DDT and phenobarbitone p e r o s (p.o.) increased nitroanisole demethylase activity in the small intestine. Hepatic mierosomal induction produced by phenobarbitone and DDT was independent of the route of administration, and concurrent dosage did not affect the degree of induction. The effect of induction, in relation to combined exposure to pesticides, drugs and other agents, is discussed with regard to DDT storage levels in patients on anticonvulsant therapy. INTRODUCTION
Numerous studies have shown that the metabolism of drugs by enzymes in the hepatic endoplasmic reticulum is enhanced by pretreatment of animals with chlorinated hydrocarbons 1, polycyclic hydrocarbons 2, or drugs 3. Recently, phenobarbitone and dicophane (DDT) have been used to lower serum bilirubin in certain forms of congenital unconjugated hyperbilirubinaemia in which glucuronyl transferase is reduced 4'5. Also, Davies e t al. 6 reported that blood levels of DDE were lower in a group of patients being treated with the anticonvulsant drugs phenobarbitone and/or Phenytoin (diphenylhydantoin) than the general population. There * Present address: Clinical Research Centre, Northwick Park Hospital, Harrow, Middlesex HA1 3UJ, England.
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was some evidence of an additive affect when both drugs were given simultaneously, and Phenytoin was apparently more effective than phenobarbital if the drugs were administered alone. The residue of DDE in adipose tissue of patients on similar anticonvulsant therapy was also significantly lower than that found in patients not taking anticonvulsant drugs. The mechanism of these clinical effects could be the induction of hepatic microsomal enzymes, resulting in increased conjugation and excretion of bilirubin, and enhanced metabolism of DDT to its metabolites with subsequent excretion and decreased storage in fat. Drugs, commonly used in clinical practice; and the environmental agent pyrene, have been given in this study alone, and together with DDT, to see if the substances themselves were microsomal enzyme inducers and if their administration enhanced the inductive effect of DDT. Also, the duration of microsomal enzyme induction produced by DDT has been studied. MATERIALSAND METHODS Animals
Male Spragtje Dawley rats weighing 145-250 g were used. Animals had free access to feed (commercial pellets) and water. Test compounds were given once daily for seven days, p.o. and intraperitoneally (i.p.), or by both routes simultaneously. Animals were killed by asphyxiation with carbon dioxide, between 8 and 11 a.m., twenty-four hours after the last dosage. In one experiment, to assess the duration of enzyme induction following the injection of DDT, animals were killed on thirteen selected days over a seven-week period. Tissue preparation
Microsomal pellets were prepared from liver, lung, kidney and small intestine. Preparations from liver were made on the day the animals were killed. The remaining tissues were stored frozen at - 2 5 °C. The microsomal preparations from kidney, lung and the first 35-40 cm of small intestine were made by pooling the same weight (1.0_ 0.2 g) of tissue from each animal tested. Tissues were homogenized 10% (w/v) in 0.25 M sucrose containing 3mM calcium chloride using a Potter-Elvehjem homogenizer. The homogenates were filtered through mesh and centrifuged at 9,000 g for 15 min after removal of the cellular debris by centrifugation at 600g for l0 min: Microsomes were prepared by centrifuging the 9,000g supernatant at 100,000g for 60 min in a Spinco Model L 265B, or at 20,000 g for 90 min in a Serval Model RC2-B centrifuge. All procedures were carried out at 2-4°C. The microsomes used for the eytochrome P-450 assays were resuspended in 0.15M potassium chloride and recentrifuged. Pellets for enzyme assays were washed with sucrose solution, recentrifuged and suspended in phosphate buffer, 0.1M, pH 7.4, to a suitable volume (protein content, 3-5 mg/ml). Protein content was determined by the method of Lowry et al. 7 E n z y m e assays
Demethylation of ethylmorphine was determined by measuring the amount 182
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of formaldehyde formed s according to the method of Cochin and Axelrod 9. The nitroanisole and aminopyrine demethylases were estimated by modification of the methods described by Gilbert and Golding 1°. Aminopyrine (500/~M) and p-nitroanisole (1.OmM) were used as substrates and the buffer concentration adjusted to 100 mM. Cytochrome P-450 was determined by the carbon monoxide difference spectrum after reduction with dithionite 11. Glucose-6-phosphatase (D-glucose-6phosphate hydrolase E.C. 3.1.39) was determined by the method of Nordlie and Arion 12. Compounds tested The compounds tested, and the dosages given were: acetylsalicylic acid U.S.P. (Aldrich Chemical Company) 40 mg/kg p.o., 20 mg/kg i.p.; 5,5-diphenylhydantoin sodium (Platz and Bauer, Inc.) 20 mg/kg p.o. and i.p. ; phenacetin U.S.P. powder (Merck and Company) 40 mg/kg p.o., 20 mg/kg i.p.; phenobarbital sodium U.S.P. crystalline powder (Merck and Company) 70 mg/kg p.o. and i.p.; pyrene (Eastman Organic Chemicals) 40 mg/kg p.o. and i.p.; and trifluorperazine hydrochloride (Stelazine concentrate, Smith, Kline and French Laboratories) 0.5 mg/kg p.o. and i.p. The acetylsalicylic acid, phenobarbital and trifluorperazine were dissolved or diluted with water. The phenacetin was dissolved in ethanol and the volume adjusted with water to 5% v/v ethanol in water. Diphenylhydantoin was dissolved in sodium hydroxide (0.008 M). All compounds were tested individually and in combination with p,p'-DDT (99.0% pure, Geigy Chemical Company) dissolved in Wesson oil (dosage 30 mg/kg p.o. and i.p.). The volume given to all animals was 0.2 ml per 100 g body weight, and the ratio of solvent to Wesson oil was I:1. Control animals received identical volumes by the same routes. RESULTS Duration of induction following D D T injection Maximal induction of p-nitroanisole, ethylmorphine and aminopyrine demethylases and cytochrome P-450 content was observed on the 5th day after the last i.p. injection of DDT (Fig. 1). This induction relative to the control values was 9.5 for ethylmorphine, 5.3 for p-nitroanisole, 3.1 for aminopyrine demethylases and 2.9 for cytochrome P-450 content. The induced enzyme activities subsequently decreased to control, or near control values, on the 15-19th day post treatment. A second, 4-6-fold increase in p-nitroanisole and ethylmorphine activities was found on the 29th day. A similar increase in aminopyrine demethylase activity was found after the 40th day. There was no significant reinduction of cytochrome P-450 pigment. The activity of the reinduced p-nitroanisole and ethylmorphine demethylases decreased at a slower rate than that induced in the first 10 days after injection. The ethylmorphine demethylase activity was still 4-5 times greater than the control activity on the 50th day. Effect of drugs and pyrene on hepatic demethylase activities and cytochrome P-450 content The p.o. and i.p. administration of phenobarbital caused an increase in nitroSci. Total Environ., I (1972)
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anisole, ethylmorphine and aminopyrine demethylascs, and in cytochrom¢ P-450 content, that was comparable to the induction produced by DDT (Table I). Inhibition rather than induction was observed following the i.p. injection of phenacetin and trifluorperazine. The nitroanisole demethylase activities were 30-55% of the control figures after i.p. phenacetin and trifluorperazin¢ injection, and the ethylmorphine demethylase activity 21% of the control value after phenacetin i.p. injection. The animals given phenacetin did not thrive well; the mean weight gains were 34 g after i.p. treatment and 45 g after oral dosage, compared to 51 g and 44 g for the controls, but they were not obviously ill. The relative liver body weight ratios were comparable to the control values. The weight gain of the animals given trifluorperazine i.p. was 55 g. A 2-3-fold increase in ethylmorphin¢ demethylase activity was found after the administration of acetylsalicylic acid p.o. and i.p. A similar increase was found in ethylmorphine and nitroanisole demethylase activities after pyren¢ was given p.o., but increases in enzyme activities were not observed after i.p. administration. Nitroanisol¢ and aminopyrine demethylase activities were increased by 80% and 250%, respectively, after p.o. diphenylhydantoin administration. Other assays showed only a 26-60% increase in cytochrome P-450 and enzyme activities, irrespective of the route of administration of the drug (Table II). Sci. Total Environ., 1 (1972)
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Cytochrome P-450 content was increased by 30--55% in animals given acetylsalicylic acid, phenacetin and trifluorperazine, p.o. or i.p. This increase was not as large as the 2-3-fold rise seen when hepatic microsomal metabolism was induced by DDT or phenobarbital. Small increases were seen in relative liver weights, except after giving trifluorperazine i.p. They were not as large as those seen after giving DDT or phenobarbital. When Wesson oil was given i.p. to control animals, alone, or with alkali (Tables I and II), the nitroanisole demethylase activity was greater than that found after oral dosage. This difference due to the route of administration was not found when DDT dissolved in Wesson oil was given, or in the other enzyme activities and cytochrome P-450 content. Effect of D D T and phenobarbitone on enzyme activities in kidney, lung, and small intestine Intraperitoneal injection of DDT and phenobarbitone did not induce increased microsomal nitroanisole and ethylmorphine demethylase activities in lung, kidney, and small intestine. Assay values were 60-80% of control figures except for ethylmorphine demethylase activity in lung after DDT injection. Glucose-6-phosphatase activity was used as an "enzyme marker" in the tissues, and the activity was unaltered by DDT but was reduced, after giving phenobarbitone in kidney, lung, and intestine, by 27, 28 and 49%, respectively. Oral administration of DDT and phenobarbitone did not induce demethylase activities in lung and kidney. After DDT treatment, the nitroanisole and demethylase activities in lung were 65 and 34% less than control values, and after phenobarbitone treatment, 28% higher and 50% less, respectively. A marked increase in nitroanisole demethylase activity, from 1.6 to 14.2 nM/mg microsomal protein/30 rain, was found in small intestine after DDT, and to 9.8 nM/mg microsomal protein/30 rain after phenobarbitone, both given p.o. This was not associated with an increase in ethylmorphine demethylase activity. Glucose-6-phosphatase activity decreased by 33% and 49% after p.o. administration of DDT and phenobarbitone, respectively. The nitroanisole demethylase activities in kidney, lung, and small intestine microsomal preparations, from animals given Wesson oil i.p., were 3-10 times greater than activities found after oral dosage. Effect of combined administration of drugs, pyrene, and DDT on hepatic demethylase activities and cytochrome P-450 content Since the route of administration of DDT in Wesson oil had no effect on the increased hepatic microsomal metabolism and cytochrome P-450 content, the values for oral and intraperitoneal injection were combined in the controls. The route of administration of the other test compounds was considered, however. When phenobarbital was given p.o. and i.p. with DDT, induction occurred but no cumulative inductive effect was observed. Values for the demethylase activities and the cytochrome P-450 content were the same, or slightly less than, those obtained with DDT only. There was a greater increase in relative liver weight in animals given DDT and phenobarbital than in animals given DDT only (Table III). Sci. Total Environ., 1 (1972)
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The values for the enzyme activities and cytoehrome P-450, with the exception of ethylmorphine demethylase (90%), were 60-80% of the DDT control values, when trifluorperazine was given with DDT p.o. or i.p. The increased ethylmorphine demethylase activity, found after giving acetylsalicylic acid, was seen also after i.p. injection with DDT. The enzyme activity was 60% greater than that seen with DDT or DDT and phenobarbital. When pyrene was given p.o. with DDT, the nitroanisole demethylase activity was similar to that obtained with DDT only, and 35% higher than the figure for oral DDT and phenobarbital. The inhibitory effect of the i.p. injection of phenacetin was also found when phenacetin was given with DDT. The enzyme activities and cytochrome P-450 content were 45-70% of DDT control figures, and only 10-30% higher than values found in animals receiving Wesson oil. Values in animals given phenacetin and DDT orally were 75-90% of DDT controls. The weight gains were 37.5 g in animals given phenacetin and DDT i.p. and 41.3 g for controls. Relative liver weights were higher, except for animals given phenacetin and DDT p.o., when drugs and pyrene were given with DDT than when DDT was given alone. DISCUSSION Duration and induction following D D T injection Kinoshito et al. la have shown in rats that maximal induced hepatic micro-
somal N-demethylase activity occurred within 7 days, and O-demethylase and aromatic side chain hydroxylating enzyme activities in 3 weeks when DDT was added to the diet. Continuous pesticide administration for 13 weeks maintained enzyme activities at a constant elevated level and gradual dose dependent decreases followed transfer to a normal diet for four weeks. The reduction in enzyme activity was~20% for O- and N-demethylase activities in animals previously fed 25 p.p.m. DDT. Also, Hart and Fouts 14"1s demonstrated enhanced drug metabolizing activity one month after long-term DDT feeding was discontinued. Since p.o. DDT in daily dosages similar to that given in this experiment is a long-lasting stimulator of microsomal enzyme activity, it is unlikely that the return to near control values seen on the 15th to the 19th day was due to the route of administration. These findings could be due to the total amount of pesticide the animal was exposed to being less than that absorbed from the gut in the long-term feeding experiments described above. The increases in the nitroanisole and ethylmorphine demethylase activities were greater, and the peak of induction in all parameters on the 5th day was earlier than has been found with p.o. intake. It is suggested that this was due to the intraperitoneal route of the administration. The reinduction found with the O- and N-demethylases, on the 29th day, could be due to exposure to an external environment, which acted as an inducer of microsomal enzyme activity, causing sudden mobilization of DDT, DDE, and/or other metabolites from fat storage by starvation14'~6 or other stimulus. All experimental animals were housed together. The mean weight gains from the 29th to the Sci. Total Environ., 1 (1972)
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43rd day were 196 g and 253 g, for the test and control animals, respectively. This seems to exclude starvation and external environment as the cause of secondary induction. Certain differences were observed between the primary and secondary induction. These included the degree of induction, the time interval required for enzyme activity to reach a maximum, and the slow decrease in activity. Ethylmorphine values were still increased 4-5 fold, 21 days after maximum secondary activities were noted. Also, reinduction did not follow the classical pattern seen with DDT, namely an increase in the activity of enzyme using Type I substrates* with a rise in cytochrome P-450 content, and an increase in total liver and microsomal liver protein. During reinduction there was no increase in cytochrome P-450 values and the difference in relative liver: body weight ratio between the animals that received D D T and the controls was negligible after the 23rd day. If induction means an increased rate of enzyme synthesis, it can be asked if these differences were due to more than one enzyme using the same substrate, variations in the respective enzymes, or alteration in rate limiting factors. It can be assumed from the increased secondary enzyme activities that the substance responsible was indeed a potent inducing agent, for it must have been present in quantities smaller than the original cumulative dose of DDT. If a metabolite of DDT, the degree of induction was greater than has been reported for oral D D D or DDE 14'1s, and the identity, reason for release and mechanism of same unknown. The role of microsomal enzyme induction in the interaction of drugs and the effect of environmental agents on the metabolism of normal body constituents, have been extensively studied and, recently, reviewed by Kuntzman 17. Reinduction could be of similar practical importance. It is necessary to ascertain if reinduction occurs only after exposure to agents leaving persistent residues, and not water soluble inducers such as phenobarbital, which are excreted in a matter of days or weeks. Effect of drugs and pyrene on hepatic demethylase activities and cytochrome P-450 content In this study acetylsalicylic acid, diphenylhydantoin, phenacetin, trifluorperazine and pyrene compared with phenobarbitol or D D T did not act as inducers of microsomal drug metabolizing enzyme activity. However, these compounds and the route of administration were not without effect on the individual parameters of such activity. The amounts of phenobarbital and D D T given were those generally chosen to produce maximal induction, whereas the drug dosages on a weight basis were about the same order as those given to man. The amount of acetylsalicylic acid, diphenylhydantoin, and phenacetin was not more than 3-fold, and trifluorperazine 2-fold, a reasonable therapeutic dose, whereas the phenobarbital given was about 10-15 times greater. The same dosage was given by both routes except to animals receiving phenacetin and acetylsalicylic acid, where the i.p. dose was half * See Materials and Methods section in M. Bick and L. Fishbein, Sci. Total Environ., 1 (1972) 197. 190
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that given p.o. This decrease was made as the lethal dose (50 mg/kg/day, orally) of both substances is very similar, and the animals given phenacetin did not thrive. Studies have shown that large repeated i.p. doses of diphenylhydantoin (up to 150 mg/kg/day) in rats induce hepatic microsomal enzyme activity and metabolism of diphenylhydantoin itself ls'19. However, Platt and Cockrill2°'21 found that although diphenylhydantoin given p.o. (200mg/kg/day) increased flavoprotein enzyme activity, there was no observable effect on the terminal enzymes responsible for drug substrate oxidation, as shown by an increase in the hepatic metabolism of aminopyrine and relative liver weight. Despite this, they concluded diphenylhydantoin showed an overall trend to the "barbiturate type" inductive effect. It has been suggested that this poor response was due to slow absorption of the relatively insoluble substance from the gastrointestinal tract is. However, Maynert22 has shown in rats that within 48-72 h of administration, 85% of a p.o. dose of diphenylhydantoin can be recovered in urine and feces, and only 12-14% of the amount in feces was not converted to metabolites. The moderate inductive effect seen in these experiments after giving diphenylhydantoin was of the same order, irrespective of the route of administration. The amount of diphenylhydantoin required to produce unequivocal induction of hepatic microsomal drug metabolism in the rat must be greater than that normally used in clinical medicine (300-400 mg/day), and shown to affect extra adrenal metabolism of cortisol in man 2a. Maximum absorption from the gastrointestinal tract of acetylsalicylic acid, phenacetin, and trifluorperazine occurs within 1-4 h of administration. The compounds are metabolized rapidly by the hepatic microsomal enzyme system and excreted. In man phenacetin has a half-life of 45-90 min in plasma24. The increase in ethylmorphine demethylase activity and relative liver weight after the administration (p.o. and i.p.) of acetylsalicylic acid has been interpreted as an inductive effect. This conflicts with the finding of Burns et al. 25, that chronic administration of aspirin to dogs (100 mg/kg/day) did not accelerate its own metabolism. Also, when given to man, acetylsalicylic acid did not cause any significant change in the excretion of chlorpromazine metabolites26. This should be clarified by using larger doses, but acetylsalicylic acid consistently causes gastrointestinal irritation in the rat in dosages above 64 mg/kg, irrespective of the route of administration2:. The inhibition of enzyme activity after giving phenacetin i.p. could be due to the failure of the animals to gain weight at the same rate as the controls. However, the relative liver weight and cytochrome P-450 content were slightly higher than control values. Phenacetin has not been reported as an inducer of microsomal enzyme activity, and Howes and Hunter 28 noted that phenacetin administration had no effect on the rate of hydrolysis of acetylsalicylic acid by rat liver microsomes. Phenobarbitone and phenylbutazone, known "inducers", however, reduced the hydrolysis rate. Chlorpromazine has been shown to have a stimulating effect on its own metabolism in the rat 29, and a single large dose of trifluorperazine (15 mg/kg i.p.) increased meprobamate metabolism3°. The much smaller dose (0.5 mg/kg given i.p.) in this Sci. Total Environ., 1 (1972)
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series caused some inhibition of enzyme activity, but given orally had no effect. Trifluorperazine given to rats in amounts similar to those used in man does not appear to induce microsomal drug metabolizing enzyme activity. In considering the inhibition seen after the i.p. injection of phenacetin and trifluorperazine, it must be remembered that this route of administration is not used in man. Certain polycyclic aromatic hydrocarbons including the environmental contaminant 3,4-benzpyrene and some of its substituted derivatives have been shown to induce microsomal enzyme activity in the rat and mouse 31'a2. These compounds are hydroxylated to metabolites which are excreted in urine, bile, and feces fairly rapidly; peak excretion in bile occurred within 40 h of oral administration, though some hydrocarbon persisted in fat and adrenals for up to 80 days33.The benzpyrene hydroxylase system has been found in liver and other tissues including the gastrointestinal tract, and the activity of the system in the latter can be increased by oral administration of phenothiazones and polycyclic hydrocarbons. It has been suggested that this system represents a protective effectTM. Induction was not observed following the i.p. injection of pyrene, but after p.o. administration, relative liver weights and all demethylase activities were increased. This difference cauld be explained by the absorption of an "inducing" metabolite, produced by a hydroxylase system in the gastrointestinal tract, different to that found in liver microsomes. It assumes that the metabolite has characteristics unlike those of the parent compound, as the induction produced by polycyclic aromatic hydrocarbons differs from that caused by DDT and phenobarbitone. They do not stimulate hexobarbitol metabolism and the induced heme pigment has different spectral characteristics after carbon monoxide binding and no Type I binding sitesaS. The enzymes induced in this experiment used Type I substrates. Induction in the demethylation of 3-methyl-4-amino-azobenzene was not observed after giving pyrene i.p. 36. This is a test system whose activity can be increased by many polycyelic hydrocarbons and by phenobarbitone. Effect of DDT and phenobarbitone on enzyme activities in kidney, lung, and ~mall intestine Non-hepatic demethylase activity was investigated in animals given DDT and phenobarbitone, as only these compounds induced microsomal activity in liver, and oxidative drug metabolizing enzymes have been reported to be present in low concentrations or absent in non-hepatic tissues 37. The only evidence of induction in these tissues was an increase in nitroanisole demethylase activity in the small intestine after oral D D T and phenobarbitone. The associated decrease in glucose-6phosphatase activity supports this inductive effect, for Phandi and Baum 38 found a significant decrease in the activity of this enzyme in liver microsomes following i.p. injections of phenobarbitone. Also, Platt and Cockril121 have reported similar decreases in glucose-6-phosphatase activity after oral administration of both DDT and phenobarbitone. Studies have reported a lack of stimulation of barbiturate metabolism and demethylase activity in lung, muscle, kidney, heart and spleen after treatment with phenobarbitone, although hepatic mierosomal activity was
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increased 39. Polycyclic aromatic hydrocarbons may induce microsomal activity in non-hepatic tissues more readily than DDT or phenobarbitone. This may be a question of sensitivity in detection of induction as fluorimetric, histochemical, and radioactive techniques have been used to follow benzpyrene hydroxylase activity 34. However, aminoazo dye N-demethylase activity has been induced in lung and kidney by 3-methylcholanthrene 4°.
Effect of combined administration of drugs, pyrene, and DD T on hepatic demethylase activities and cytochrome P-450 content The hepatic demethylase activities and cytochrome P-450 values obtained after the simultaneous administration of drugs and DDT, tended to be lower than those found when DDT only was given, even if the drug itself (e.g. phenobarbital) had increased hepatic microsomal metabolism. This could be due to the drugs occupying sites on the microsomal membrane and thus preventing induction by DDT. The absence of any additive effect confirmed previous observations that many drugs including barbiturates and DDT stimulate microsomal metabolism by the same mechanism. An additive effect following combined maximum stimulatory dosage is seen only when the mechanism o£ induction of each compound is different, as with phenobarbital and certain polycyclic hydrocarbons 1a'lS'al. As a cumulative effect was not seen after giving pyrene and DDT p.o., the induced enzyme activities found after giving pyrene p.o. cannot be the result of absorption from the gastrointestinal tract of the unchanged polycyclic hydrocarbon. The changes found after giving phenacetin with DDT i.p., namely an increase in relative liver weight, inhibition of induced enzyme activities, and no marked increase in cytochrome P-450 content, were considered a toxic response. The pattern was similar to that seen after p.o. administration of well established hepatotoxic agents such as carbon tetrachloride, thioacetamide, and chloroform; i.e., liver enlargement associated with inhibition of microsomal protein synthesis and reduction in some microsomal enzymes 2°'21. This toxic response differs from the liver enlargement, accompanied by increased microsomal metabolism, that is induced by drugs and DDT. Platt and Cockrill 2°'21 consider DDT to be non-hepatotoxic in the rat. The figures obtained after the combined administration of DDT with acetylsalicylic acid (i.p.) and with diphenylhydantoin supported evidence obtained from giving them alone, that they were potential inducing agents. They included increases in relative liver weight, cytochrome P-450, and ethylmorphine demethylase activity that were comparable or greater than those found when DDT or phenobarbital and DDT were given. Many substances in the environment, e.g. polycyclic aromatic hydrocarbons and certain chlorinated insecticides, as well as drugs, stimulate microsomal drug metabolizing enzymes and so may affect drug therapy. It is difficult, owing to marked species differences in the metabolism of drugs, to choose comparable and effective dosages and extrapolate pharmacological data from experimental animals to man. A similar problem exists in assessing whether the degree of exposure to environmental agents is sufficient to cause enzyme induction. Sci. Total Environ., I (1972)
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Nevertheless, the occurrence and duration of microsomal enzyme induction, and possible reinduction like that found after DDT injection, is important to man. Therefore, the potential inductive capacity of acetylsalicylic acid, and the inhibitory effect of phenacetin and trifluorperazine as indicated by changes in individual parameters of induction, should be investigated further for these drugs are used extensively. There was little evidence from this study to suggest that the reported decreased circulatory level of DDE and lower storage levels of DDE and DDT in adipose tissue of patients on long-term anticonvulsant therapy was the result of increased dechlorination of ingested DDT due to the induced hepatic microsomal enzyme activity. Massive doses of diphenylhydantoin given i.p. and phenobarbitone increase hepatic microsomal activity in experimental animals, but the smaller amount of diphenylhydantoin given in this study did not induce such activity unequivocally. Since diphenylhydantoin metabolism in man is stimulated by phenobarbitone 42, the apparent additive effect observed in patients was unlikely to be due to enhanced microsomal induction. An additive inductive effect can be expected only when the mechanism of induction is different, as with drugs or DDT and polycyclic aromatic hydrocarbons. This was not observed in this study following concurrent administration of drugs and DDT.
REFERENCES L. G. Hart and J. R. Fouts, Proc Soc. Exp. Biol. Med., 114 (1963) 388. A. H. Conney, E. C. Miller, and J. A. Miller, J. Biol. Chem., 228 (1957) 753. A. H. Conney, C. Davison, R. Gastel, and J. J. Burns, J. Pharmacol., 130 (1960) 1. S. J. Yaffe¢, G. Levy, T. Matsuzana, and T. Bailah, New Engl. J. Med., 275 (1966) 1461. R. P. H. Thompson, C. W. T. Pilcber, J. Robinson, G. M. Statbers, A. E. M. McLean, and R. Williams, Lancet, 2 (1969) 4. 6 J. E. Davies, W. F. Edmundson, C. H. Carter, and A. Barquet, Lancet, 2 (1969) 7. 7 0 . H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. BioL Chem., 193 (1951) 265. 8 T, Nash, Biochem. J., 55 (1953) 416. 9 J. Cochin and J. Axelrod, J. Pharmacol. Exp. Ther., 125 (1959) 105. 10 D. Gilbert and L. Goldberg, Food Cosmet. Toxicol., 3 (1965) 417, 11 T. Omura, R. Sato, D. Y. Cooper, O. Rosenthal, and R. W. Eastbrook, Fed. Proc., 24 (1965) 1181. 12 R. C. Nordlie and W. J. Arion, in Methods ofEnzymology, Vol. IX, Academic Press, New York, 1966, p. 619. 13 F. K. Kinoshita, J. P. Frawley, and K. P. DuBois, ToxicoL Appl. Pharmacol., 9 (1966) 505. 14 L. G. Hart and J. R. Fouts, Naunyn-Schmiedebergs Arch. Pharmakd. Exp. Pathd., 249 (1965) 486. 15 L. H. Hart and J. R. Fouts, Biochem. Pharmacol., 14 (1965) 263. 16 W. E. Dale, T. B. Gaines, and W. J. Hayes, Jr., Toxicol. Appl. Pharmacol., 4 (1962) 89. 17 R. Kunzman, Ann. Rev. Pharmacol., 9 (1969) 21. 18 T. E. Eiing, R. D. Harbison, B. A. Becket, and J. R. Fours, J. Pharmacol. Exp. Ther., 171 (1970) 127. 19 N. Gerber and K. Arnold, J. Pharmacol. Exp. Ther., 167 (1969) 77. 20 D. S. Platt and B. L. Cockrill, Biochem. Pharmacol., 18 (1969) 429. 21 D. S. Platt and B. L. Cockrill, Biochem. Pharmacol., 18 (1969) 445. 22 E. W. Maynert, J. Pharmacol. Exp. Ther., 130 (1960) 275. 23 E. E. WERK, J. MACGEE, and 1. J. Sholiton, J. Clin. Invest., 43 (1964) 1824. 24 L. F. Prescott, M. Sansur, W. Levin, and A. H. Conney, Clin. Pharmacol. Ther., 9 (1968) 605. 25 J. J. Burns, S. A. Cucinell, R. Koster, and A. H. Conney, Ann. N. Y. Acad. Sci., 123 0965) 273. 26 L. L. Huano and K. Hirano. Biochem. Pharmacol., 16 (1967) 2023. 1 2 3 4 5
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27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
D. A. Brodie and B. J. Chase, Gastroenterology, 53 (1967) 604. J. F. Howes and W. H. Hunter, J. Pharm. Pharmacol., 20 (1968) 107. J. L. Emmerson and T. S. Miya, J. Pharmacol. Exp. Ther., 137 (1962) 148. R. Kato and P. Vassarelli, Biochem. Pharmacol., 11 (1962) 779. J. O. Mullen, M. R. Juchau, and J. R. Fouts, Biochem. Pharmacol., 15 (1966) 137. F. Dewhurst and D. A. Kitchen, Biochem. Pharmacol., 18 (1969) 942. P. M. Daniel, O. E. Pratt, and M. M. L. Prichard, Nature, 215 (1967) 1142. L. W. Wattenberg, J. L. Leong, and P. J. Strand, Cancer Res., 22 (1962) 1120. G.J. Mannering, N. E. Sladek, C. J. Parli, and D. W. Shoeman, in Micromes and Drug Oxidation, Academic Press, New York (1969) 303. J. C. Arcos, A. H. Conney, and N. P. Buu-Hoi, J. Biol. Chem., 236 (1961) 1291. J. R. Gillette, B. B. Brodie, and B. N. LaDu, J. Pharmacol. Exp. Ther., 119 (1957) 532. P. N. Phandhi and H. Baum, Life Sci., 9 (1970) 87. W. Kunz, G. Schaude, W. Schmid, and M. Siess, Arch. Pharmacol. Exp. Pathol., 254 (1966)" 470. A. G. Gilman and A. H. Conney, Biochem. Pharmacol., 12 (1963) 591. N. E. Siadek and G. J. Mannering, Mol. Pharmacol., 5 (1969) 174. S. A. Cucinell, A. H. Conney, M. Sansur, and J. J. Burns, Clin. Pharmacol. Ther., 6 (1965)420.
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195
EFFECT OF CERTAIN DRUGS AND ENVIRONMENTAL AGENTS ON MICROSOMAL ENZYME SYSTEMS
M A R J O R I E BICK* A N D L A W R E N C E FISHBEIN
National Institute of Environmental Health Sciences National Institutes of Health, Public Health Service and Department of Health, Education and Welfare Research Triangle Park, North Carolina 27709 (U. S. A.) (Received November 18th, 1971)
SUMMARY
Hepatic microsomal drug metabolizing enzymes induced in rats by p,p'-DDT reach peak activity 5 days after injection, and return to control values on the 15-19th day. Reinduction with different characteristics occurred and persisted for 50 days. Massive doses are used to produce induction in experimental animals. When smaller amounts similar to those given to man therapeutically were used, it was difficult to assess if hepatic induction occurred. There was some evidence of induction after giving diphenylhydantoin, acetylsalicylic acid, and the environmental agent pyrene. Some inhibitory effects were observed after giving phenacetin and trifluoperazine. Administration of DDT and phenobarbitone p e r o s (p.o.) increased nitroanisole demethylase activity in the small intestine. Hepatic mierosomal induction produced by phenobarbitone and DDT was independent of the route of administration, and concurrent dosage did not affect the degree of induction. The effect of induction, in relation to combined exposure to pesticides, drugs and other agents, is discussed with regard to DDT storage levels in patients on anticonvulsant therapy. INTRODUCTION
Numerous studies have shown that the metabolism of drugs by enzymes in the hepatic endoplasmic reticulum is enhanced by pretreatment of animals with chlorinated hydrocarbons 1, polycyclic hydrocarbons 2, or drugs 3. Recently, phenobarbitone and dicophane (DDT) have been used to lower serum bilirubin in certain forms of congenital unconjugated hyperbilirubinaemia in which glucuronyl transferase is reduced 4'5. Also, Davies e t al. 6 reported that blood levels of DDE were lower in a group of patients being treated with the anticonvulsant drugs phenobarbitone and/or Phenytoin (diphenylhydantoin) than the general population. There * Present address: Clinical Research Centre, Northwick Park Hospital, Harrow, Middlesex HA1 3UJ, England.
Sci. Total Environ., 1 (1972)
181
was some evidence of an additive affect when both drugs were given simultaneously, and Phenytoin was apparently more effective than phenobarbital if the drugs were administered alone. The residue of DDE in adipose tissue of patients on similar anticonvulsant therapy was also significantly lower than that found in patients not taking anticonvulsant drugs. The mechanism of these clinical effects could be the induction of hepatic microsomal enzymes, resulting in increased conjugation and excretion of bilirubin, and enhanced metabolism of DDT to its metabolites with subsequent excretion and decreased storage in fat. Drugs, commonly used in clinical practice; and the environmental agent pyrene, have been given in this study alone, and together with DDT, to see if the substances themselves were microsomal enzyme inducers and if their administration enhanced the inductive effect of DDT. Also, the duration of microsomal enzyme induction produced by DDT has been studied. MATERIALSAND METHODS Animals
Male Spragtje Dawley rats weighing 145-250 g were used. Animals had free access to feed (commercial pellets) and water. Test compounds were given once daily for seven days, p.o. and intraperitoneally (i.p.), or by both routes simultaneously. Animals were killed by asphyxiation with carbon dioxide, between 8 and 11 a.m., twenty-four hours after the last dosage. In one experiment, to assess the duration of enzyme induction following the injection of DDT, animals were killed on thirteen selected days over a seven-week period. Tissue preparation
Microsomal pellets were prepared from liver, lung, kidney and small intestine. Preparations from liver were made on the day the animals were killed. The remaining tissues were stored frozen at - 2 5 °C. The microsomal preparations from kidney, lung and the first 35-40 cm of small intestine were made by pooling the same weight (1.0_ 0.2 g) of tissue from each animal tested. Tissues were homogenized 10% (w/v) in 0.25 M sucrose containing 3mM calcium chloride using a Potter-Elvehjem homogenizer. The homogenates were filtered through mesh and centrifuged at 9,000 g for 15 min after removal of the cellular debris by centrifugation at 600g for l0 min: Microsomes were prepared by centrifuging the 9,000g supernatant at 100,000g for 60 min in a Spinco Model L 265B, or at 20,000 g for 90 min in a Serval Model RC2-B centrifuge. All procedures were carried out at 2-4°C. The microsomes used for the eytochrome P-450 assays were resuspended in 0.15M potassium chloride and recentrifuged. Pellets for enzyme assays were washed with sucrose solution, recentrifuged and suspended in phosphate buffer, 0.1M, pH 7.4, to a suitable volume (protein content, 3-5 mg/ml). Protein content was determined by the method of Lowry et al. 7 E n z y m e assays
Demethylation of ethylmorphine was determined by measuring the amount 182
Sci. Total Environ., 1 (1972)
of formaldehyde formed s according to the method of Cochin and Axelrod 9. The nitroanisole and aminopyrine demethylases were estimated by modification of the methods described by Gilbert and Golding 1°. Aminopyrine (500/~M) and p-nitroanisole (1.OmM) were used as substrates and the buffer concentration adjusted to 100 mM. Cytochrome P-450 was determined by the carbon monoxide difference spectrum after reduction with dithionite 11. Glucose-6-phosphatase (D-glucose-6phosphate hydrolase E.C. 3.1.39) was determined by the method of Nordlie and Arion 12. Compounds tested The compounds tested, and the dosages given were: acetylsalicylic acid U.S.P. (Aldrich Chemical Company) 40 mg/kg p.o., 20 mg/kg i.p.; 5,5-diphenylhydantoin sodium (Platz and Bauer, Inc.) 20 mg/kg p.o. and i.p. ; phenacetin U.S.P. powder (Merck and Company) 40 mg/kg p.o., 20 mg/kg i.p.; phenobarbital sodium U.S.P. crystalline powder (Merck and Company) 70 mg/kg p.o. and i.p.; pyrene (Eastman Organic Chemicals) 40 mg/kg p.o. and i.p.; and trifluorperazine hydrochloride (Stelazine concentrate, Smith, Kline and French Laboratories) 0.5 mg/kg p.o. and i.p. The acetylsalicylic acid, phenobarbital and trifluorperazine were dissolved or diluted with water. The phenacetin was dissolved in ethanol and the volume adjusted with water to 5% v/v ethanol in water. Diphenylhydantoin was dissolved in sodium hydroxide (0.008 M). All compounds were tested individually and in combination with p,p'-DDT (99.0% pure, Geigy Chemical Company) dissolved in Wesson oil (dosage 30 mg/kg p.o. and i.p.). The volume given to all animals was 0.2 ml per 100 g body weight, and the ratio of solvent to Wesson oil was I:1. Control animals received identical volumes by the same routes. RESULTS Duration of induction following D D T injection Maximal induction of p-nitroanisole, ethylmorphine and aminopyrine demethylases and cytochrome P-450 content was observed on the 5th day after the last i.p. injection of DDT (Fig. 1). This induction relative to the control values was 9.5 for ethylmorphine, 5.3 for p-nitroanisole, 3.1 for aminopyrine demethylases and 2.9 for cytochrome P-450 content. The induced enzyme activities subsequently decreased to control, or near control values, on the 15-19th day post treatment. A second, 4-6-fold increase in p-nitroanisole and ethylmorphine activities was found on the 29th day. A similar increase in aminopyrine demethylase activity was found after the 40th day. There was no significant reinduction of cytochrome P-450 pigment. The activity of the reinduced p-nitroanisole and ethylmorphine demethylases decreased at a slower rate than that induced in the first 10 days after injection. The ethylmorphine demethylase activity was still 4-5 times greater than the control activity on the 50th day. Effect of drugs and pyrene on hepatic demethylase activities and cytochrome P-450 content The p.o. and i.p. administration of phenobarbital caused an increase in nitroSci. Total Environ., I (1972)
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Ooysoffer Lost Injection DDT Fig. 1. Effect of time on demethylas¢ activities and cytochrome P-450 content in rat liver microsomes following i.p. injection of DDT. © O, p-nitroanisole; • • , ethylmorphine; A A, aminopyrine; [-] [] cytochrome P-450. The values represent the average from two animals except on the eighth day (four animals). The control activity is the mean value for each assay over the whole time period.
anisole, ethylmorphine and aminopyrine demethylascs, and in cytochrom¢ P-450 content, that was comparable to the induction produced by DDT (Table I). Inhibition rather than induction was observed following the i.p. injection of phenacetin and trifluorperazine. The nitroanisole demethylase activities were 30-55% of the control figures after i.p. phenacetin and trifluorperazin¢ injection, and the ethylmorphine demethylase activity 21% of the control value after phenacetin i.p. injection. The animals given phenacetin did not thrive well; the mean weight gains were 34 g after i.p. treatment and 45 g after oral dosage, compared to 51 g and 44 g for the controls, but they were not obviously ill. The relative liver body weight ratios were comparable to the control values. The weight gain of the animals given trifluorperazine i.p. was 55 g. A 2-3-fold increase in ethylmorphin¢ demethylase activity was found after the administration of acetylsalicylic acid p.o. and i.p. A similar increase was found in ethylmorphine and nitroanisole demethylase activities after pyren¢ was given p.o., but increases in enzyme activities were not observed after i.p. administration. Nitroanisol¢ and aminopyrine demethylase activities were increased by 80% and 250%, respectively, after p.o. diphenylhydantoin administration. Other assays showed only a 26-60% increase in cytochrome P-450 and enzyme activities, irrespective of the route of administration of the drug (Table II). Sci. Total Environ., 1 (1972)
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Cytochrome P-450 content was increased by 30--55% in animals given acetylsalicylic acid, phenacetin and trifluorperazine, p.o. or i.p. This increase was not as large as the 2-3-fold rise seen when hepatic microsomal metabolism was induced by DDT or phenobarbital. Small increases were seen in relative liver weights, except after giving trifluorperazine i.p. They were not as large as those seen after giving DDT or phenobarbital. When Wesson oil was given i.p. to control animals, alone, or with alkali (Tables I and II), the nitroanisole demethylase activity was greater than that found after oral dosage. This difference due to the route of administration was not found when DDT dissolved in Wesson oil was given, or in the other enzyme activities and cytochrome P-450 content. Effect of D D T and phenobarbitone on enzyme activities in kidney, lung, and small intestine Intraperitoneal injection of DDT and phenobarbitone did not induce increased microsomal nitroanisole and ethylmorphine demethylase activities in lung, kidney, and small intestine. Assay values were 60-80% of control figures except for ethylmorphine demethylase activity in lung after DDT injection. Glucose-6-phosphatase activity was used as an "enzyme marker" in the tissues, and the activity was unaltered by DDT but was reduced, after giving phenobarbitone in kidney, lung, and intestine, by 27, 28 and 49%, respectively. Oral administration of DDT and phenobarbitone did not induce demethylase activities in lung and kidney. After DDT treatment, the nitroanisole and demethylase activities in lung were 65 and 34% less than control values, and after phenobarbitone treatment, 28% higher and 50% less, respectively. A marked increase in nitroanisole demethylase activity, from 1.6 to 14.2 nM/mg microsomal protein/30 rain, was found in small intestine after DDT, and to 9.8 nM/mg microsomal protein/30 rain after phenobarbitone, both given p.o. This was not associated with an increase in ethylmorphine demethylase activity. Glucose-6-phosphatase activity decreased by 33% and 49% after p.o. administration of DDT and phenobarbitone, respectively. The nitroanisole demethylase activities in kidney, lung, and small intestine microsomal preparations, from animals given Wesson oil i.p., were 3-10 times greater than activities found after oral dosage. Effect of combined administration of drugs, pyrene, and DDT on hepatic demethylase activities and cytochrome P-450 content Since the route of administration of DDT in Wesson oil had no effect on the increased hepatic microsomal metabolism and cytochrome P-450 content, the values for oral and intraperitoneal injection were combined in the controls. The route of administration of the other test compounds was considered, however. When phenobarbital was given p.o. and i.p. with DDT, induction occurred but no cumulative inductive effect was observed. Values for the demethylase activities and the cytochrome P-450 content were the same, or slightly less than, those obtained with DDT only. There was a greater increase in relative liver weight in animals given DDT and phenobarbital than in animals given DDT only (Table III). Sci. Total Environ., 1 (1972)
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The values for the enzyme activities and cytoehrome P-450, with the exception of ethylmorphine demethylase (90%), were 60-80% of the DDT control values, when trifluorperazine was given with DDT p.o. or i.p. The increased ethylmorphine demethylase activity, found after giving acetylsalicylic acid, was seen also after i.p. injection with DDT. The enzyme activity was 60% greater than that seen with DDT or DDT and phenobarbital. When pyrene was given p.o. with DDT, the nitroanisole demethylase activity was similar to that obtained with DDT only, and 35% higher than the figure for oral DDT and phenobarbital. The inhibitory effect of the i.p. injection of phenacetin was also found when phenacetin was given with DDT. The enzyme activities and cytochrome P-450 content were 45-70% of DDT control figures, and only 10-30% higher than values found in animals receiving Wesson oil. Values in animals given phenacetin and DDT orally were 75-90% of DDT controls. The weight gains were 37.5 g in animals given phenacetin and DDT i.p. and 41.3 g for controls. Relative liver weights were higher, except for animals given phenacetin and DDT p.o., when drugs and pyrene were given with DDT than when DDT was given alone. DISCUSSION Duration and induction following D D T injection Kinoshito et al. la have shown in rats that maximal induced hepatic micro-
somal N-demethylase activity occurred within 7 days, and O-demethylase and aromatic side chain hydroxylating enzyme activities in 3 weeks when DDT was added to the diet. Continuous pesticide administration for 13 weeks maintained enzyme activities at a constant elevated level and gradual dose dependent decreases followed transfer to a normal diet for four weeks. The reduction in enzyme activity was~20% for O- and N-demethylase activities in animals previously fed 25 p.p.m. DDT. Also, Hart and Fouts 14"1s demonstrated enhanced drug metabolizing activity one month after long-term DDT feeding was discontinued. Since p.o. DDT in daily dosages similar to that given in this experiment is a long-lasting stimulator of microsomal enzyme activity, it is unlikely that the return to near control values seen on the 15th to the 19th day was due to the route of administration. These findings could be due to the total amount of pesticide the animal was exposed to being less than that absorbed from the gut in the long-term feeding experiments described above. The increases in the nitroanisole and ethylmorphine demethylase activities were greater, and the peak of induction in all parameters on the 5th day was earlier than has been found with p.o. intake. It is suggested that this was due to the intraperitoneal route of the administration. The reinduction found with the O- and N-demethylases, on the 29th day, could be due to exposure to an external environment, which acted as an inducer of microsomal enzyme activity, causing sudden mobilization of DDT, DDE, and/or other metabolites from fat storage by starvation14'~6 or other stimulus. All experimental animals were housed together. The mean weight gains from the 29th to the Sci. Total Environ., 1 (1972)
189
43rd day were 196 g and 253 g, for the test and control animals, respectively. This seems to exclude starvation and external environment as the cause of secondary induction. Certain differences were observed between the primary and secondary induction. These included the degree of induction, the time interval required for enzyme activity to reach a maximum, and the slow decrease in activity. Ethylmorphine values were still increased 4-5 fold, 21 days after maximum secondary activities were noted. Also, reinduction did not follow the classical pattern seen with DDT, namely an increase in the activity of enzyme using Type I substrates* with a rise in cytochrome P-450 content, and an increase in total liver and microsomal liver protein. During reinduction there was no increase in cytochrome P-450 values and the difference in relative liver: body weight ratio between the animals that received D D T and the controls was negligible after the 23rd day. If induction means an increased rate of enzyme synthesis, it can be asked if these differences were due to more than one enzyme using the same substrate, variations in the respective enzymes, or alteration in rate limiting factors. It can be assumed from the increased secondary enzyme activities that the substance responsible was indeed a potent inducing agent, for it must have been present in quantities smaller than the original cumulative dose of DDT. If a metabolite of DDT, the degree of induction was greater than has been reported for oral D D D or DDE 14'1s, and the identity, reason for release and mechanism of same unknown. The role of microsomal enzyme induction in the interaction of drugs and the effect of environmental agents on the metabolism of normal body constituents, have been extensively studied and, recently, reviewed by Kuntzman 17. Reinduction could be of similar practical importance. It is necessary to ascertain if reinduction occurs only after exposure to agents leaving persistent residues, and not water soluble inducers such as phenobarbital, which are excreted in a matter of days or weeks. Effect of drugs and pyrene on hepatic demethylase activities and cytochrome P-450 content In this study acetylsalicylic acid, diphenylhydantoin, phenacetin, trifluorperazine and pyrene compared with phenobarbitol or D D T did not act as inducers of microsomal drug metabolizing enzyme activity. However, these compounds and the route of administration were not without effect on the individual parameters of such activity. The amounts of phenobarbital and D D T given were those generally chosen to produce maximal induction, whereas the drug dosages on a weight basis were about the same order as those given to man. The amount of acetylsalicylic acid, diphenylhydantoin, and phenacetin was not more than 3-fold, and trifluorperazine 2-fold, a reasonable therapeutic dose, whereas the phenobarbital given was about 10-15 times greater. The same dosage was given by both routes except to animals receiving phenacetin and acetylsalicylic acid, where the i.p. dose was half * See Materials and Methods section in M. Bick and L. Fishbein, Sci. Total Environ., 1 (1972) 197. 190
Sci. Total Environ., 1 (1972)
that given p.o. This decrease was made as the lethal dose (50 mg/kg/day, orally) of both substances is very similar, and the animals given phenacetin did not thrive. Studies have shown that large repeated i.p. doses of diphenylhydantoin (up to 150 mg/kg/day) in rats induce hepatic microsomal enzyme activity and metabolism of diphenylhydantoin itself ls'19. However, Platt and Cockrill2°'21 found that although diphenylhydantoin given p.o. (200mg/kg/day) increased flavoprotein enzyme activity, there was no observable effect on the terminal enzymes responsible for drug substrate oxidation, as shown by an increase in the hepatic metabolism of aminopyrine and relative liver weight. Despite this, they concluded diphenylhydantoin showed an overall trend to the "barbiturate type" inductive effect. It has been suggested that this poor response was due to slow absorption of the relatively insoluble substance from the gastrointestinal tract is. However, Maynert22 has shown in rats that within 48-72 h of administration, 85% of a p.o. dose of diphenylhydantoin can be recovered in urine and feces, and only 12-14% of the amount in feces was not converted to metabolites. The moderate inductive effect seen in these experiments after giving diphenylhydantoin was of the same order, irrespective of the route of administration. The amount of diphenylhydantoin required to produce unequivocal induction of hepatic microsomal drug metabolism in the rat must be greater than that normally used in clinical medicine (300-400 mg/day), and shown to affect extra adrenal metabolism of cortisol in man 2a. Maximum absorption from the gastrointestinal tract of acetylsalicylic acid, phenacetin, and trifluorperazine occurs within 1-4 h of administration. The compounds are metabolized rapidly by the hepatic microsomal enzyme system and excreted. In man phenacetin has a half-life of 45-90 min in plasma24. The increase in ethylmorphine demethylase activity and relative liver weight after the administration (p.o. and i.p.) of acetylsalicylic acid has been interpreted as an inductive effect. This conflicts with the finding of Burns et al. 25, that chronic administration of aspirin to dogs (100 mg/kg/day) did not accelerate its own metabolism. Also, when given to man, acetylsalicylic acid did not cause any significant change in the excretion of chlorpromazine metabolites26. This should be clarified by using larger doses, but acetylsalicylic acid consistently causes gastrointestinal irritation in the rat in dosages above 64 mg/kg, irrespective of the route of administration2:. The inhibition of enzyme activity after giving phenacetin i.p. could be due to the failure of the animals to gain weight at the same rate as the controls. However, the relative liver weight and cytochrome P-450 content were slightly higher than control values. Phenacetin has not been reported as an inducer of microsomal enzyme activity, and Howes and Hunter 28 noted that phenacetin administration had no effect on the rate of hydrolysis of acetylsalicylic acid by rat liver microsomes. Phenobarbitone and phenylbutazone, known "inducers", however, reduced the hydrolysis rate. Chlorpromazine has been shown to have a stimulating effect on its own metabolism in the rat 29, and a single large dose of trifluorperazine (15 mg/kg i.p.) increased meprobamate metabolism3°. The much smaller dose (0.5 mg/kg given i.p.) in this Sci. Total Environ., 1 (1972)
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series caused some inhibition of enzyme activity, but given orally had no effect. Trifluorperazine given to rats in amounts similar to those used in man does not appear to induce microsomal drug metabolizing enzyme activity. In considering the inhibition seen after the i.p. injection of phenacetin and trifluorperazine, it must be remembered that this route of administration is not used in man. Certain polycyclic aromatic hydrocarbons including the environmental contaminant 3,4-benzpyrene and some of its substituted derivatives have been shown to induce microsomal enzyme activity in the rat and mouse 31'a2. These compounds are hydroxylated to metabolites which are excreted in urine, bile, and feces fairly rapidly; peak excretion in bile occurred within 40 h of oral administration, though some hydrocarbon persisted in fat and adrenals for up to 80 days33.The benzpyrene hydroxylase system has been found in liver and other tissues including the gastrointestinal tract, and the activity of the system in the latter can be increased by oral administration of phenothiazones and polycyclic hydrocarbons. It has been suggested that this system represents a protective effectTM. Induction was not observed following the i.p. injection of pyrene, but after p.o. administration, relative liver weights and all demethylase activities were increased. This difference cauld be explained by the absorption of an "inducing" metabolite, produced by a hydroxylase system in the gastrointestinal tract, different to that found in liver microsomes. It assumes that the metabolite has characteristics unlike those of the parent compound, as the induction produced by polycyclic aromatic hydrocarbons differs from that caused by DDT and phenobarbitone. They do not stimulate hexobarbitol metabolism and the induced heme pigment has different spectral characteristics after carbon monoxide binding and no Type I binding sitesaS. The enzymes induced in this experiment used Type I substrates. Induction in the demethylation of 3-methyl-4-amino-azobenzene was not observed after giving pyrene i.p. 36. This is a test system whose activity can be increased by many polycyelic hydrocarbons and by phenobarbitone. Effect of DDT and phenobarbitone on enzyme activities in kidney, lung, and ~mall intestine Non-hepatic demethylase activity was investigated in animals given DDT and phenobarbitone, as only these compounds induced microsomal activity in liver, and oxidative drug metabolizing enzymes have been reported to be present in low concentrations or absent in non-hepatic tissues 37. The only evidence of induction in these tissues was an increase in nitroanisole demethylase activity in the small intestine after oral D D T and phenobarbitone. The associated decrease in glucose-6phosphatase activity supports this inductive effect, for Phandi and Baum 38 found a significant decrease in the activity of this enzyme in liver microsomes following i.p. injections of phenobarbitone. Also, Platt and Cockril121 have reported similar decreases in glucose-6-phosphatase activity after oral administration of both DDT and phenobarbitone. Studies have reported a lack of stimulation of barbiturate metabolism and demethylase activity in lung, muscle, kidney, heart and spleen after treatment with phenobarbitone, although hepatic mierosomal activity was
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increased 39. Polycyclic aromatic hydrocarbons may induce microsomal activity in non-hepatic tissues more readily than DDT or phenobarbitone. This may be a question of sensitivity in detection of induction as fluorimetric, histochemical, and radioactive techniques have been used to follow benzpyrene hydroxylase activity 34. However, aminoazo dye N-demethylase activity has been induced in lung and kidney by 3-methylcholanthrene 4°.
Effect of combined administration of drugs, pyrene, and DD T on hepatic demethylase activities and cytochrome P-450 content The hepatic demethylase activities and cytochrome P-450 values obtained after the simultaneous administration of drugs and DDT, tended to be lower than those found when DDT only was given, even if the drug itself (e.g. phenobarbital) had increased hepatic microsomal metabolism. This could be due to the drugs occupying sites on the microsomal membrane and thus preventing induction by DDT. The absence of any additive effect confirmed previous observations that many drugs including barbiturates and DDT stimulate microsomal metabolism by the same mechanism. An additive effect following combined maximum stimulatory dosage is seen only when the mechanism o£ induction of each compound is different, as with phenobarbital and certain polycyclic hydrocarbons 1a'lS'al. As a cumulative effect was not seen after giving pyrene and DDT p.o., the induced enzyme activities found after giving pyrene p.o. cannot be the result of absorption from the gastrointestinal tract of the unchanged polycyclic hydrocarbon. The changes found after giving phenacetin with DDT i.p., namely an increase in relative liver weight, inhibition of induced enzyme activities, and no marked increase in cytochrome P-450 content, were considered a toxic response. The pattern was similar to that seen after p.o. administration of well established hepatotoxic agents such as carbon tetrachloride, thioacetamide, and chloroform; i.e., liver enlargement associated with inhibition of microsomal protein synthesis and reduction in some microsomal enzymes 2°'21. This toxic response differs from the liver enlargement, accompanied by increased microsomal metabolism, that is induced by drugs and DDT. Platt and Cockrill 2°'21 consider DDT to be non-hepatotoxic in the rat. The figures obtained after the combined administration of DDT with acetylsalicylic acid (i.p.) and with diphenylhydantoin supported evidence obtained from giving them alone, that they were potential inducing agents. They included increases in relative liver weight, cytochrome P-450, and ethylmorphine demethylase activity that were comparable or greater than those found when DDT or phenobarbital and DDT were given. Many substances in the environment, e.g. polycyclic aromatic hydrocarbons and certain chlorinated insecticides, as well as drugs, stimulate microsomal drug metabolizing enzymes and so may affect drug therapy. It is difficult, owing to marked species differences in the metabolism of drugs, to choose comparable and effective dosages and extrapolate pharmacological data from experimental animals to man. A similar problem exists in assessing whether the degree of exposure to environmental agents is sufficient to cause enzyme induction. Sci. Total Environ., I (1972)
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Nevertheless, the occurrence and duration of microsomal enzyme induction, and possible reinduction like that found after DDT injection, is important to man. Therefore, the potential inductive capacity of acetylsalicylic acid, and the inhibitory effect of phenacetin and trifluorperazine as indicated by changes in individual parameters of induction, should be investigated further for these drugs are used extensively. There was little evidence from this study to suggest that the reported decreased circulatory level of DDE and lower storage levels of DDE and DDT in adipose tissue of patients on long-term anticonvulsant therapy was the result of increased dechlorination of ingested DDT due to the induced hepatic microsomal enzyme activity. Massive doses of diphenylhydantoin given i.p. and phenobarbitone increase hepatic microsomal activity in experimental animals, but the smaller amount of diphenylhydantoin given in this study did not induce such activity unequivocally. Since diphenylhydantoin metabolism in man is stimulated by phenobarbitone 42, the apparent additive effect observed in patients was unlikely to be due to enhanced microsomal induction. An additive inductive effect can be expected only when the mechanism of induction is different, as with drugs or DDT and polycyclic aromatic hydrocarbons. This was not observed in this study following concurrent administration of drugs and DDT.
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