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Factors affecting hepatic microsomal enzyme systems involved in drug metabolism
FACTORS
AFFECTING
ENZYME
SYSTEMS
HEPATIC INVOLVED
MICROSOMAL IN DRUG
METABOLISM JAMES R. FOUTS Department of Pharmacology, College of Medicine State University of Iowa, Iowa City
THE metabolism of drugs in the liver is catalyzed byenzymes found invarious parts of the hepatic cell. Concentrated in the endoplasmic reticulum or ergastoplasm are a variety of drug-metabolizing enzymes. Factors affecting the activity of these enzyme systems will be described in this paper. The study of drug metabolizing enzymes of the hepatic cell's ergastoplasm would be greatly helped by histochemical techniques allowing detection of these enzyme reactions in the intact cell. Such histochemical techniques could tell whether all hepatic cells contain the enzymes and whether these drug-metabolizing enzymes were uniformly distributed throughout the ergastoplasm. Unfortunately such histochemical techniques are still unborn and indirect methods must be used. It is believed by most workers that the biochemist's "microsomal" fraction contains the fragments of the ergastoplasm of the cell. Other intracellular structures are also found in this microsomal fraction--e.g, parts of the golgi apparatus and glycogen--but electron micrographs show that this pellet consists predominantly of numbers of profiles which resemble fragments of the ergastoplasm. Morphologically, the ergastoplasm of the hepatic cell has at least two subdivisionsnthis membrane system either has a rough surface or a smooth surface. The rough surface is caused by attached ribosomes. Since ribosomes can be detached from the membranes of the ergastoplasm rather easily, one question which might be asked is: does smooth surfaced ergastoplasm represent only a denuded rough-surfaced ergastoplasm ? The question becomes more important in terms of function--do these two types of ergastoplasm possess different enzymes or chemical constituents ? Our research was concerned with whether the drug-metabolizing enzymes of microsomes were found in rough-surfaced ergastoplasm, smooth-surfaced ergastoplasm or in both types. If smooth-surfaced ergastoplasm were only a denuded rough-surfaced membrane, then unequal distribution of the drug-metabolizing enzymes would be expected only if these enzymes 225
226
JAMES R. FOUTS
were in the ribosomes. Table I shows that the reverse is t r u e l d r u g metabolizing enzymes are found predominantly in smooth-surfaced microsomes, derived presumably f r o m the smooth-Surfaced ergastoplasm.O) This would seem to mean that the m e m b r a n e c o m p o n e n t of rough-surfaced ergastoplasm is not biochemically identical with that of smooth-surfaced e r g a s t o p l a s m - - t h e smooth-surfaced m e m b r a n e would not be formed normally by stripping ribosomes off the rough-surfaced membranes. TABLE 1 Drug-Metabolizing Enzyme Activities of Rabbit Liver Rough and Smooth-Surfaced Microsomes Microsomal Enzyme Activity*t Drug substrate
Chlorpromazine Acetanilid Codeine Hexobarbital Aminopyrine
Rough
Smooth
amoles/mg N 0-25 0"29 0"11 0"15 0-05
/~moles/mg N 0'68 0.86 0.40 0"63 0.28
Ratio Smooth/Rough
2"7 3"0 3"6 4"2 5"6
*Enzyme activity in/,moles drug metabolized in 2 hr per mg nitrogen. tNitrogen content determined by Kjeldahl method. Figures in the table are the average of 4 experiments, each experiment using a different animal.
R E L A T I O N BETWEEN D R U G ENZYME A C T I V I T Y AND S T R U C T U R E OF THE E R G A S T O P L A S M A relationship seems to exist between the a m o u n t o f smooth-surfaced ergastoplasm and the measurable activity of the hepatic microsomal drug metabolizing enzymes. This has been true when enzyme activity is either greater than or less than normal. Table 2 summarizes m u c h of the research done in our laboratory on conditions in which microsomal drug metabolism is abnormal. In all o f these conditions there has been a depression or loss of microsomal drug metabolizing activity as well as a decrease or loss o f the smooth-surfaced ergastoplasm in the liver cell. The same correlation of structure and enzyme activity seems to occur when drug metabolizing enzyme activity is greater than normal. A variety o f c o m p o u n d s has been described which can cause apparent adaptive increases in hepatic microsomal enzyme activity. This work has been reviewed by Conney and Burns t2) and Remmer.t 3) Phenobarbital is one such stimulator of microsomal drug metabolism with which wehave worked. We have noted a marked proliferation of smooth-surfaced ergastoplasm in
FACTORS AFFECTING HEPATIC MICROSOMAL ENZYME SYSTEMS
227
TABLE 2 Conditions Wherein Hepatic Microsomal Drug-Metabolizing Enzyme Activity is Abnormal Condition
Animal
Microsomal drug enzyme activity
References
1. Newborn or fetus
Rabbit
Negligible for all oxidative metabolisms-both fetus and newborn. Activity detectable at about 2 weeks after birth
2. Starvation (36--48 hr)
Mouse
L Obstructive jaundice
Rabbit
t. Hepatic tumors transplantable hepatomas (Novikoff, Morris 5123) or induced by feeding butter yellow or ethionine ;. Regenerating liver (Tissue left after partial hepatectomy)
Rat
Depressed for all oxidative metabolisms. Reduction of nitro groups and azo linkages not affected. Enzyme activity returns rapidly upon refeeding 1. Obstruction causing minimal cell damage produces less effect on metabolism of hexobarbital and chlorpromazine than on other drugs. 2. Obstruction causing marked cell damage produces severe depression of all oxidative enzyme activities Oxidative and reductive enzyme activities undetectable in tumor tissue. Hepatic tissue adjacent to tumor has normal enzyme activity
1. Science 129 897 (1959). 2. J.Pharmacol Exptl. Therap. 137, 103 (1962) Proc. Soc. Exptl. Biol. Med. 103, 333 (1960).
;. Animals treated with alloxan.
Rat
Rat
Oxidative and reductive enzyme activities are low during rapid regeneration. Rate of codeine dealkylation returns to normal before any other metabolism Oxidative enzyme activities depressed. Enzyme activity returned to normal by treatment of animal with insulin
J. PharmacoL Exptl. Therap. 131, 7 (1961).
Cancer Res. 21, 667 (1961).
Biochem. Pharmacol. 7, 265 (1961). J. PharmacoL ExptL Therap. 133, 7 (1961).
livers from animals treated with phenobarbital. Similar results have been reported by H. Returner of the Free Univ. of Berlin (personal communication). STIMULATION
OF MICROSOMAL
DRUG
ENZYME
ACTIVITY
Phenobarbital causes increased drug enzyme activity for a variety of metabolic pathways--it is among the least specific stimulators of drug metabolism by microsomes. Perhaps of more interest would be the effects
228
JAMES R. FOUTS
on hepatic ergastoplasm of a stimulator which affected only one or two microsomal enzyme systems. So far the only evidence seems to indicate that increases in activity of several microsomal drug enzyme systems are accompanied by the appearance o f greater quantities of the structure where these enzymes are f o u n d - - t h e smooth-surfaced ergastoplasm. A more specific stimulator of drug enzyme activity (affecting only one or two enzymes) might not cause such marked structural changes. Phenobarbital can cause increases in microsomal drug enzyme activity in livers of a variety of animals. Generally speaking, the younger the animal, the greater is the stimulation percentagewise. However, stimulation can occur in adult animals and apparently in humans. (a) Phenobarbital can stimulate drug metabolizing enzymes in the fetus and newborn animal, but the stimulation in the fetus seems to be possible only in the terminal stages of pregnancy.(4) Microsomal drug metabolism in untreated newborn or fetal animals is absent or barely detectable. Phenobarbital can stimulate microsomal enzyme activity in some other cases where enzyme activity is very low. W o r k in our laboratory has shown that the low or absent drug enzyme activity found in starved mice, liver TABLE3 Effects of Chlordane on Hepatic Microsomal Drug-Metabolizing Enzyme Activity in the Adult Rat
Drug substrate
Dose of chlordane* (given i.p.)
Peak of stimulation (days after injection)
Hexobarbital
10 mg/kg 100 mg/kg
8 days 8-15 days
Aminopyrine
10 mg/kg 100 mg/kg
8 days 8 days
Chlorpromazine
25 mg/kg 100 mg/kg
8 days 8-15 days
Metabolism of drug:~ by liver from Controlt
Chlordanetreated
tzmoles/g liver t~moles/gliver 2.77 + 0.65 3.76 + 0.41 3.78_+0.35 6.35 _+0.76 0-35 _+0.10 0.45_+0.21
0.50 +_0.05 2.88_+0.40
l-I 1 _+0-17 1.09+0.10
1.55 _+0.05 1.58+0.16
*Chlordane dissolved in corn oil and given intraperiteneally one time only. tControl values differ from one group of animals to another. Each group of chlordanetreated animals had its own set of control animals. In all cases, metabolism of drugs by liver from chlordane-treated animals was significantly greater than by liver from untreated animals (P< 0.05). All groups had at least 4 animals with values in the table representing average metabolism + standard deviation. ~tMetabolism expressed as t~moles drug metabolized per gram liver (wet weight) in 2 hr.
FACTORS AFFECTING HEPATIC MICROSOMAL ENZYME SYSTEMS
229
regenerating after partial hepatectomy and rats treated with alloxan can be increased or caused to appear by treating the animal with phenobarbital (unpublished observations). These increases in enzyme activity can be blocked by the amino acid antagonist, ethionine. The effects of ethionine can usually be reversed by methionine though the reversal is not always complete (unpublished observations). Such results point to a synthesis of new protein as a possible mechanism of action of phenobarbital. It is interesting however, that some other amino acid antagonists such as norleucine, p-fluorophenylalanine and methionine sulfoxide have been unable to prevent the stimulation of microsomal enzymes by phenobarbital.O) Hepatic microsomal drug metabolizing enzyme activity can also be markedly increased by the insecticide, chlordane. Chlordane, like phenobarbital, has a relatively non-specific effect on these enzyme systems, stimulating a variety of metabolic pathways (Table 3). Unlike those with most other drug enzyme stimulators however, chlordane's effects are not seen until several days after the animal is exposed to this insecticide. Most other drug enzyme stimulators cause changes in enzyme activity within 12-36 hr of their injection and this stimulation lasts only 3-5 days. With chlordane, maximum stimulation is seen 8-10 days after treatment and lasts up to 3 weeks. Such a delayed and prolonged effect on drug enzyme activity has not been reported with any other drug enzyme stimulator to our knowledge. The effects of chlordane can be blocked by ethionine. RELATION BETWEEN HEPATIC GLYCOGEN AND MICROSOMAL DRUG ENZYMES In all of the conditions wherein microsomai drug metabolizing enzyme activity has been below normal there has also been a decrease in level of hepatic glycogen. Hepatic glycogen is stored in areas containing only smooth-surfaced ergastoplasm, and the amount of smooth ergastoplasm in these areas seems to increase and decrease as glycogen stores increase and decrease. If this relationship always holds (though it is not known to do so) then the answers to the following two questions may give a clue as to whether all smooth ergastoplasm is functionally (enzymic composition) identical: (1) can hepatic glycogen levels be altered without affecting drug metabolizing enzyme activity and (2) can microsomal drug enzyme activity be changed without altering hepatic glycogen levels? If the smooth ergastoplasm associated with glycogen were the only site of the drug-metabolizing enzymes, these 2 questions should both be answered no. If drug metabolizing enzymes were only (or nearly completely) in the smooth ergastoplasm which is not associated with glycogen, the 2 questions should have a yes answer. If the smooth ergastoplasm associated with glycogen was not
230
JAMF_SR. FOUTS
different from smooth ergastoplasm elsewhere in the cell, these questions might have different answersmyes and no, depending on how much of the total smooth ergastoplasm of the cell was affected by changes in glycogen stores. A number of drugs is known which can affect hepatic glycogen levels. Effects of such drugs on microsomal drug-metabolizing enzyme activity are now being studied in search of an answer to question number 1. The results obtained with epinephrine are given in Table 4 and these data show TABLE4 Effects of Epinephrine on Hepatic MicrosomalDrug Metabolizing Enzyme Activityin the Adult Rat Time between No. injection and of animals sacrifice hr 0 (control) 1 4 8
11 4 19 14
Hepatic glycogen level % 6.7 2"9t 0 "4"I" 0-4t
Metabolism* of Chlorpromazine
Hexobarbital
~,moles/gliver 1.2
t~moles/gliver 4"0 5"4t 3"8 3-1f
1-8t
1.2 0.7"f
Epinephrine injected intraperitoneally.Dose -- 0.5 mg/kg. *Metabolismexpressedas t~molesdrug metabolizedper gram liver(wet weight)in 2 hr. ?Denotes values significantlydifferentfrom control P< 0.05. that hepatic glycogen levels can be markedly depressed without a corresponding decrease in microsomal drug metabolizing enzyme activity. Yet there may be an effect on microsomal enzyme activity since the data also show that this enzyme activity is significantly increased when glycogen levels are depressed to about 40 per cent of normal. This increase in drug enzyme activity with a moderate decrease in hepatic glycogen levels has been seen in other conditions and deserves further study. The other question--can microsomal enzyme activity be changed without altering hepatic glycogen levels?mhas been studied using a variety of inhibitors of the microsomal enzymes. We have investigated drug enzyme activity and hepatic glycogen levels at various times after the administration of SKF525A (fl-diethylaminoethyldiphenylpropyl acetate), Lilly 18947 (2,4-dichloro-6-phonylphenoxycthyl diethylamino) iproniazid, and chloramphenicol. Each of these compounds has been shown to inhibit the metabolism of drugs by hepatic microsomal enzymes both in vitro and in vivo. tS-9)
Figure 1 shows some of the data obtained with SKF525A---comparing microsomal drug metabolism and hepatic glycogen levels. Glycogen levels
FACTORS AFFECTINGHEPATIC MICRO$OMALENZYMESYSTEMS
231
are depressed by all the drug enzyme inhibitors tested so far, and in each case the first detectable changes in glycogen levels occur after the drugenzyme activity has been markedly depressed. Recovery of glycogen and drug-enzyme activity occurs at the same time--glycogen is not replaced at a faster rate than drug enzyme activity. Neither the rate of glycogen destruction nor synthesis is abnormal in livers from animals treated with SKF525A or in homogenates of liver from normal animals to which SKF525A has been added. There is as yet no explanation of how SKF525A or any other drug enzyme inhibitor causes the depression of hepatic glycogen which we have observed.
IIo~
• Code/noMetabolism
0 I 4 8 12 24 (control) Time in Hours After Treotment With SKF 5 2 5 - A
36
FIG. 1 Effect of SKF525A (Dose: 20 mg/kg intraperitoneally) on hepatic glycogen and
microsomal drug enzyme activity. The bars represent mean values of glycogen and rate of drug metabolism. The vertical lines (arrows) indicate the standard error of the mean (slain) where s -- standard deviation and n = total number of animals -- 8. The asterisks are values significantly different (P<0.0$) from control. Control values for this figure were: Glycogen: 6.2_+ 1-4 per cent Hexobarbital metabolism: 3-5_+0.27 vmoles/2 hr Codeine metabolism: 1"9+ 0-16 ~moles/2 hr All values are per gram liver (wet weight). Thus in our studies to date, we have been unable to decrease microsomal drug enzyme activity without a corresponding change in hepatic glycogen. By using various stimulators such as phenobarbital however, we have been able to markedly increase drug enzyme activity without causing a statistically sianificamt increase in hepatic glycogen. The answers now available to the two questions about the relationship o f hepatic glycogen and drug metabolizing enzyme activity allow us to
232
JA.MES R . F O U I S
say that it is very unlikely that drug metabolizing enzymes are found only in smooth ergastoplasm which is associated with glycogen, but that it is possible for these enzymes to be only in ergastoplasm not associated with glycogen or to be found in all smooth ergastoplasm. It is hoped that further morphological as well as biochemical studies will help answer these questions as well as clarify the mechanisms by which stimulators of drug enzyme activity exert their effects.
SUMMARY The activity of certain hepatic microsomal enzymes which metabolize drugs is not constant. These enzyme activities can be affected by drugs, disease states and certain alterations in cellular environment which need not lead to recognizable hepatic pathology. Microsomal drug metabolizing enzyme activity can be increased above normal levels by treatment of animals with a variety of drugs (e.g. phenobarbital, chlordan) which appear to stimulate the synthesis of new enzyme protein. Absence or abnormally low levels of microsomal drug metabolizing enzyme activity are seen in fetal and newborn animals, in mice starved 48-72 hr, in rabbits suffering obstructive jaundice, in rats treated 48-72 hr previously with alloxan, in hepatic tissue remaining after partial hepatectomy, and in a variety of drug-induced and/or transplantable hepatomas (e.g. Novikoff, Morris 5123). Changes which often accompany these alterations in the rate of microsornal drug metabolism include, (1) loss of hepatic glycogen when drug metabolism slows down and increased glycogen levels when drug metabolism speeds up, and (2) loss or decrease in amount of smooth-surfaced ergastoplasm (SSE) when drug metabolism is depressed and proliferation of SSE when metabolism is increased. Other studies have shown that several microsomal drug-metabolizing enzymes are localized predominantly in smooth-surfaced microsomes which are derived presumably from SSE. Predictions of the level of microsomal drug-metabolizing enzyme activity are best made from the amount of SSE in the hepatic parenchymal cell since hepatic glycogen levels can be changed (e.g. by epinephrine injections) without changing microsomal enzyme activity.
ACKNOWLEDGMENTS The author wishes to acknowledge the efforts of D r . R. H. Adamson, Mr. R. L. Dixon, Mr. L. G. Hart, Mr. R. W. Shultice, Mr. E. F. McLuen, and Mr. L. A. Rogers. As graduate students, these individuals performed much of the research cited in this paper. The author also wishes to thank
FACTORS.AFFECTINGHEPATICMICROSOMALENZYME SYSTEMS
233
Mrs. Anne Trankle, Mrs. D o n n a Goodwin, Mr. John Mullen, and Mr. Arnold Balanoff for their excellent technical assistance in these studies. The studies reported in this paper were supported by grants (GM-06034 and CA-05648) from the U.S. Public Health Service and the American Cancer Society (P-247 A). REFERENCES 1. JAMESR. FOUTS,The metabolism of drugs by subfractions of hepatic microsomes, Biochem. Biophys. Res. Communs. 6, 373-378 0961). 2. A. H. CONNEYand J. J. BURNS,Factors influencing drug metabolism, in Advances in Pharmacology (Ed. by S. Garattini and P. A. Shore), vol. 1, Academic Press, New York, (1961), p. 31-58. 3. H. REMMER,Drug Tolerance, in Ciba Foundation Symposium on Enzymes and Drug Action (Ed. by J. L. Mongar and A. V. S. de Reuck), Little, Brown, Boston, (1962), p. 276-298. 4. LARRYG. HART, RICHARDH. ADAMSON,ROBERTC. DIXONand JAMF~R. Fours, Stimulation of hepatic microsomal drug metabolism in the newborn and fetal rabbit, J. PharmaeoL Exptl. Therap. 137, 103-106 (1962). 5. R. KATO,E. CHIESARAand P. VASSANEtJ.I,Factors influencing induction of hepatic microsomal drug-metabolizing enzymes, Bioehem. Pharmacol. 11,211-220 (1962). 6. JULIUSAXELROD,JULESR~ICMENTHALand B~RN~J~ B. BRODIE,Mechanism of the potentiating action of ~-diethylaminoethyl diphenylpropylacetate, J. PharmacoL ExptL Therap. 112, 49-54 (1954). JACKR. COOrER,JULIUSAXEIJtODand BERNARD B. BRODXE,Inhibitory effects of /3-diethylaminoethyl diphenylpropylacetate on a variety of drug metabolic pathways in vitro, J. Pharmacol. Exptl. Therap. 112, 55-63 (1954). 7. J.R. Fotrrs and BERNARDB. BRODIE,Inhibition of drug metabolic pathways by the potentiating agent 2,4-dichloro-6-phenylphenoxyethyl diethylamine, J. Pharmacol. ExptL Therap. 115, 68-73 (1955). 8. J. R. Fou~s and BERNARDB. BRODIE,On the mechanism of drug potentiation by iproniazid (2-isopropyl-l-isonicotinyl hydrazine), J. PharmacoL ExptL Therap. 116, 480-485 (1956). 9. ROeERTL. DIXONand JAMESR. FOUTS, Inhibition of microsomal drug metabolic pathways by chloramphenicol, Biochem. Pharmacol. 11, 715-720 (1962).
AFFECTING
ENZYME
SYSTEMS
HEPATIC INVOLVED
MICROSOMAL IN DRUG
METABOLISM JAMES R. FOUTS Department of Pharmacology, College of Medicine State University of Iowa, Iowa City
THE metabolism of drugs in the liver is catalyzed byenzymes found invarious parts of the hepatic cell. Concentrated in the endoplasmic reticulum or ergastoplasm are a variety of drug-metabolizing enzymes. Factors affecting the activity of these enzyme systems will be described in this paper. The study of drug metabolizing enzymes of the hepatic cell's ergastoplasm would be greatly helped by histochemical techniques allowing detection of these enzyme reactions in the intact cell. Such histochemical techniques could tell whether all hepatic cells contain the enzymes and whether these drug-metabolizing enzymes were uniformly distributed throughout the ergastoplasm. Unfortunately such histochemical techniques are still unborn and indirect methods must be used. It is believed by most workers that the biochemist's "microsomal" fraction contains the fragments of the ergastoplasm of the cell. Other intracellular structures are also found in this microsomal fraction--e.g, parts of the golgi apparatus and glycogen--but electron micrographs show that this pellet consists predominantly of numbers of profiles which resemble fragments of the ergastoplasm. Morphologically, the ergastoplasm of the hepatic cell has at least two subdivisionsnthis membrane system either has a rough surface or a smooth surface. The rough surface is caused by attached ribosomes. Since ribosomes can be detached from the membranes of the ergastoplasm rather easily, one question which might be asked is: does smooth surfaced ergastoplasm represent only a denuded rough-surfaced ergastoplasm ? The question becomes more important in terms of function--do these two types of ergastoplasm possess different enzymes or chemical constituents ? Our research was concerned with whether the drug-metabolizing enzymes of microsomes were found in rough-surfaced ergastoplasm, smooth-surfaced ergastoplasm or in both types. If smooth-surfaced ergastoplasm were only a denuded rough-surfaced membrane, then unequal distribution of the drug-metabolizing enzymes would be expected only if these enzymes 225
226
JAMES R. FOUTS
were in the ribosomes. Table I shows that the reverse is t r u e l d r u g metabolizing enzymes are found predominantly in smooth-surfaced microsomes, derived presumably f r o m the smooth-Surfaced ergastoplasm.O) This would seem to mean that the m e m b r a n e c o m p o n e n t of rough-surfaced ergastoplasm is not biochemically identical with that of smooth-surfaced e r g a s t o p l a s m - - t h e smooth-surfaced m e m b r a n e would not be formed normally by stripping ribosomes off the rough-surfaced membranes. TABLE 1 Drug-Metabolizing Enzyme Activities of Rabbit Liver Rough and Smooth-Surfaced Microsomes Microsomal Enzyme Activity*t Drug substrate
Chlorpromazine Acetanilid Codeine Hexobarbital Aminopyrine
Rough
Smooth
amoles/mg N 0-25 0"29 0"11 0"15 0-05
/~moles/mg N 0'68 0.86 0.40 0"63 0.28
Ratio Smooth/Rough
2"7 3"0 3"6 4"2 5"6
*Enzyme activity in/,moles drug metabolized in 2 hr per mg nitrogen. tNitrogen content determined by Kjeldahl method. Figures in the table are the average of 4 experiments, each experiment using a different animal.
R E L A T I O N BETWEEN D R U G ENZYME A C T I V I T Y AND S T R U C T U R E OF THE E R G A S T O P L A S M A relationship seems to exist between the a m o u n t o f smooth-surfaced ergastoplasm and the measurable activity of the hepatic microsomal drug metabolizing enzymes. This has been true when enzyme activity is either greater than or less than normal. Table 2 summarizes m u c h of the research done in our laboratory on conditions in which microsomal drug metabolism is abnormal. In all o f these conditions there has been a depression or loss of microsomal drug metabolizing activity as well as a decrease or loss o f the smooth-surfaced ergastoplasm in the liver cell. The same correlation of structure and enzyme activity seems to occur when drug metabolizing enzyme activity is greater than normal. A variety o f c o m p o u n d s has been described which can cause apparent adaptive increases in hepatic microsomal enzyme activity. This work has been reviewed by Conney and Burns t2) and Remmer.t 3) Phenobarbital is one such stimulator of microsomal drug metabolism with which wehave worked. We have noted a marked proliferation of smooth-surfaced ergastoplasm in
FACTORS AFFECTING HEPATIC MICROSOMAL ENZYME SYSTEMS
227
TABLE 2 Conditions Wherein Hepatic Microsomal Drug-Metabolizing Enzyme Activity is Abnormal Condition
Animal
Microsomal drug enzyme activity
References
1. Newborn or fetus
Rabbit
Negligible for all oxidative metabolisms-both fetus and newborn. Activity detectable at about 2 weeks after birth
2. Starvation (36--48 hr)
Mouse
L Obstructive jaundice
Rabbit
t. Hepatic tumors transplantable hepatomas (Novikoff, Morris 5123) or induced by feeding butter yellow or ethionine ;. Regenerating liver (Tissue left after partial hepatectomy)
Rat
Depressed for all oxidative metabolisms. Reduction of nitro groups and azo linkages not affected. Enzyme activity returns rapidly upon refeeding 1. Obstruction causing minimal cell damage produces less effect on metabolism of hexobarbital and chlorpromazine than on other drugs. 2. Obstruction causing marked cell damage produces severe depression of all oxidative enzyme activities Oxidative and reductive enzyme activities undetectable in tumor tissue. Hepatic tissue adjacent to tumor has normal enzyme activity
1. Science 129 897 (1959). 2. J.Pharmacol Exptl. Therap. 137, 103 (1962) Proc. Soc. Exptl. Biol. Med. 103, 333 (1960).
;. Animals treated with alloxan.
Rat
Rat
Oxidative and reductive enzyme activities are low during rapid regeneration. Rate of codeine dealkylation returns to normal before any other metabolism Oxidative enzyme activities depressed. Enzyme activity returned to normal by treatment of animal with insulin
J. PharmacoL Exptl. Therap. 131, 7 (1961).
Cancer Res. 21, 667 (1961).
Biochem. Pharmacol. 7, 265 (1961). J. PharmacoL ExptL Therap. 133, 7 (1961).
livers from animals treated with phenobarbital. Similar results have been reported by H. Returner of the Free Univ. of Berlin (personal communication). STIMULATION
OF MICROSOMAL
DRUG
ENZYME
ACTIVITY
Phenobarbital causes increased drug enzyme activity for a variety of metabolic pathways--it is among the least specific stimulators of drug metabolism by microsomes. Perhaps of more interest would be the effects
228
JAMES R. FOUTS
on hepatic ergastoplasm of a stimulator which affected only one or two microsomal enzyme systems. So far the only evidence seems to indicate that increases in activity of several microsomal drug enzyme systems are accompanied by the appearance o f greater quantities of the structure where these enzymes are f o u n d - - t h e smooth-surfaced ergastoplasm. A more specific stimulator of drug enzyme activity (affecting only one or two enzymes) might not cause such marked structural changes. Phenobarbital can cause increases in microsomal drug enzyme activity in livers of a variety of animals. Generally speaking, the younger the animal, the greater is the stimulation percentagewise. However, stimulation can occur in adult animals and apparently in humans. (a) Phenobarbital can stimulate drug metabolizing enzymes in the fetus and newborn animal, but the stimulation in the fetus seems to be possible only in the terminal stages of pregnancy.(4) Microsomal drug metabolism in untreated newborn or fetal animals is absent or barely detectable. Phenobarbital can stimulate microsomal enzyme activity in some other cases where enzyme activity is very low. W o r k in our laboratory has shown that the low or absent drug enzyme activity found in starved mice, liver TABLE3 Effects of Chlordane on Hepatic Microsomal Drug-Metabolizing Enzyme Activity in the Adult Rat
Drug substrate
Dose of chlordane* (given i.p.)
Peak of stimulation (days after injection)
Hexobarbital
10 mg/kg 100 mg/kg
8 days 8-15 days
Aminopyrine
10 mg/kg 100 mg/kg
8 days 8 days
Chlorpromazine
25 mg/kg 100 mg/kg
8 days 8-15 days
Metabolism of drug:~ by liver from Controlt
Chlordanetreated
tzmoles/g liver t~moles/gliver 2.77 + 0.65 3.76 + 0.41 3.78_+0.35 6.35 _+0.76 0-35 _+0.10 0.45_+0.21
0.50 +_0.05 2.88_+0.40
l-I 1 _+0-17 1.09+0.10
1.55 _+0.05 1.58+0.16
*Chlordane dissolved in corn oil and given intraperiteneally one time only. tControl values differ from one group of animals to another. Each group of chlordanetreated animals had its own set of control animals. In all cases, metabolism of drugs by liver from chlordane-treated animals was significantly greater than by liver from untreated animals (P< 0.05). All groups had at least 4 animals with values in the table representing average metabolism + standard deviation. ~tMetabolism expressed as t~moles drug metabolized per gram liver (wet weight) in 2 hr.
FACTORS AFFECTING HEPATIC MICROSOMAL ENZYME SYSTEMS
229
regenerating after partial hepatectomy and rats treated with alloxan can be increased or caused to appear by treating the animal with phenobarbital (unpublished observations). These increases in enzyme activity can be blocked by the amino acid antagonist, ethionine. The effects of ethionine can usually be reversed by methionine though the reversal is not always complete (unpublished observations). Such results point to a synthesis of new protein as a possible mechanism of action of phenobarbital. It is interesting however, that some other amino acid antagonists such as norleucine, p-fluorophenylalanine and methionine sulfoxide have been unable to prevent the stimulation of microsomal enzymes by phenobarbital.O) Hepatic microsomal drug metabolizing enzyme activity can also be markedly increased by the insecticide, chlordane. Chlordane, like phenobarbital, has a relatively non-specific effect on these enzyme systems, stimulating a variety of metabolic pathways (Table 3). Unlike those with most other drug enzyme stimulators however, chlordane's effects are not seen until several days after the animal is exposed to this insecticide. Most other drug enzyme stimulators cause changes in enzyme activity within 12-36 hr of their injection and this stimulation lasts only 3-5 days. With chlordane, maximum stimulation is seen 8-10 days after treatment and lasts up to 3 weeks. Such a delayed and prolonged effect on drug enzyme activity has not been reported with any other drug enzyme stimulator to our knowledge. The effects of chlordane can be blocked by ethionine. RELATION BETWEEN HEPATIC GLYCOGEN AND MICROSOMAL DRUG ENZYMES In all of the conditions wherein microsomai drug metabolizing enzyme activity has been below normal there has also been a decrease in level of hepatic glycogen. Hepatic glycogen is stored in areas containing only smooth-surfaced ergastoplasm, and the amount of smooth ergastoplasm in these areas seems to increase and decrease as glycogen stores increase and decrease. If this relationship always holds (though it is not known to do so) then the answers to the following two questions may give a clue as to whether all smooth ergastoplasm is functionally (enzymic composition) identical: (1) can hepatic glycogen levels be altered without affecting drug metabolizing enzyme activity and (2) can microsomal drug enzyme activity be changed without altering hepatic glycogen levels? If the smooth ergastoplasm associated with glycogen were the only site of the drug-metabolizing enzymes, these 2 questions should both be answered no. If drug metabolizing enzymes were only (or nearly completely) in the smooth ergastoplasm which is not associated with glycogen, the 2 questions should have a yes answer. If the smooth ergastoplasm associated with glycogen was not
230
JAMF_SR. FOUTS
different from smooth ergastoplasm elsewhere in the cell, these questions might have different answersmyes and no, depending on how much of the total smooth ergastoplasm of the cell was affected by changes in glycogen stores. A number of drugs is known which can affect hepatic glycogen levels. Effects of such drugs on microsomal drug-metabolizing enzyme activity are now being studied in search of an answer to question number 1. The results obtained with epinephrine are given in Table 4 and these data show TABLE4 Effects of Epinephrine on Hepatic MicrosomalDrug Metabolizing Enzyme Activityin the Adult Rat Time between No. injection and of animals sacrifice hr 0 (control) 1 4 8
11 4 19 14
Hepatic glycogen level % 6.7 2"9t 0 "4"I" 0-4t
Metabolism* of Chlorpromazine
Hexobarbital
~,moles/gliver 1.2
t~moles/gliver 4"0 5"4t 3"8 3-1f
1-8t
1.2 0.7"f
Epinephrine injected intraperitoneally.Dose -- 0.5 mg/kg. *Metabolismexpressedas t~molesdrug metabolizedper gram liver(wet weight)in 2 hr. ?Denotes values significantlydifferentfrom control P< 0.05. that hepatic glycogen levels can be markedly depressed without a corresponding decrease in microsomal drug metabolizing enzyme activity. Yet there may be an effect on microsomal enzyme activity since the data also show that this enzyme activity is significantly increased when glycogen levels are depressed to about 40 per cent of normal. This increase in drug enzyme activity with a moderate decrease in hepatic glycogen levels has been seen in other conditions and deserves further study. The other question--can microsomal enzyme activity be changed without altering hepatic glycogen levels?mhas been studied using a variety of inhibitors of the microsomal enzymes. We have investigated drug enzyme activity and hepatic glycogen levels at various times after the administration of SKF525A (fl-diethylaminoethyldiphenylpropyl acetate), Lilly 18947 (2,4-dichloro-6-phonylphenoxycthyl diethylamino) iproniazid, and chloramphenicol. Each of these compounds has been shown to inhibit the metabolism of drugs by hepatic microsomal enzymes both in vitro and in vivo. tS-9)
Figure 1 shows some of the data obtained with SKF525A---comparing microsomal drug metabolism and hepatic glycogen levels. Glycogen levels
FACTORS AFFECTINGHEPATIC MICRO$OMALENZYMESYSTEMS
231
are depressed by all the drug enzyme inhibitors tested so far, and in each case the first detectable changes in glycogen levels occur after the drugenzyme activity has been markedly depressed. Recovery of glycogen and drug-enzyme activity occurs at the same time--glycogen is not replaced at a faster rate than drug enzyme activity. Neither the rate of glycogen destruction nor synthesis is abnormal in livers from animals treated with SKF525A or in homogenates of liver from normal animals to which SKF525A has been added. There is as yet no explanation of how SKF525A or any other drug enzyme inhibitor causes the depression of hepatic glycogen which we have observed.
IIo~
• Code/noMetabolism
0 I 4 8 12 24 (control) Time in Hours After Treotment With SKF 5 2 5 - A
36
FIG. 1 Effect of SKF525A (Dose: 20 mg/kg intraperitoneally) on hepatic glycogen and
microsomal drug enzyme activity. The bars represent mean values of glycogen and rate of drug metabolism. The vertical lines (arrows) indicate the standard error of the mean (slain) where s -- standard deviation and n = total number of animals -- 8. The asterisks are values significantly different (P<0.0$) from control. Control values for this figure were: Glycogen: 6.2_+ 1-4 per cent Hexobarbital metabolism: 3-5_+0.27 vmoles/2 hr Codeine metabolism: 1"9+ 0-16 ~moles/2 hr All values are per gram liver (wet weight). Thus in our studies to date, we have been unable to decrease microsomal drug enzyme activity without a corresponding change in hepatic glycogen. By using various stimulators such as phenobarbital however, we have been able to markedly increase drug enzyme activity without causing a statistically sianificamt increase in hepatic glycogen. The answers now available to the two questions about the relationship o f hepatic glycogen and drug metabolizing enzyme activity allow us to
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JA.MES R . F O U I S
say that it is very unlikely that drug metabolizing enzymes are found only in smooth ergastoplasm which is associated with glycogen, but that it is possible for these enzymes to be only in ergastoplasm not associated with glycogen or to be found in all smooth ergastoplasm. It is hoped that further morphological as well as biochemical studies will help answer these questions as well as clarify the mechanisms by which stimulators of drug enzyme activity exert their effects.
SUMMARY The activity of certain hepatic microsomal enzymes which metabolize drugs is not constant. These enzyme activities can be affected by drugs, disease states and certain alterations in cellular environment which need not lead to recognizable hepatic pathology. Microsomal drug metabolizing enzyme activity can be increased above normal levels by treatment of animals with a variety of drugs (e.g. phenobarbital, chlordan) which appear to stimulate the synthesis of new enzyme protein. Absence or abnormally low levels of microsomal drug metabolizing enzyme activity are seen in fetal and newborn animals, in mice starved 48-72 hr, in rabbits suffering obstructive jaundice, in rats treated 48-72 hr previously with alloxan, in hepatic tissue remaining after partial hepatectomy, and in a variety of drug-induced and/or transplantable hepatomas (e.g. Novikoff, Morris 5123). Changes which often accompany these alterations in the rate of microsornal drug metabolism include, (1) loss of hepatic glycogen when drug metabolism slows down and increased glycogen levels when drug metabolism speeds up, and (2) loss or decrease in amount of smooth-surfaced ergastoplasm (SSE) when drug metabolism is depressed and proliferation of SSE when metabolism is increased. Other studies have shown that several microsomal drug-metabolizing enzymes are localized predominantly in smooth-surfaced microsomes which are derived presumably from SSE. Predictions of the level of microsomal drug-metabolizing enzyme activity are best made from the amount of SSE in the hepatic parenchymal cell since hepatic glycogen levels can be changed (e.g. by epinephrine injections) without changing microsomal enzyme activity.
ACKNOWLEDGMENTS The author wishes to acknowledge the efforts of D r . R. H. Adamson, Mr. R. L. Dixon, Mr. L. G. Hart, Mr. R. W. Shultice, Mr. E. F. McLuen, and Mr. L. A. Rogers. As graduate students, these individuals performed much of the research cited in this paper. The author also wishes to thank
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Mrs. Anne Trankle, Mrs. D o n n a Goodwin, Mr. John Mullen, and Mr. Arnold Balanoff for their excellent technical assistance in these studies. The studies reported in this paper were supported by grants (GM-06034 and CA-05648) from the U.S. Public Health Service and the American Cancer Society (P-247 A). REFERENCES 1. JAMESR. FOUTS,The metabolism of drugs by subfractions of hepatic microsomes, Biochem. Biophys. Res. Communs. 6, 373-378 0961). 2. A. H. CONNEYand J. J. BURNS,Factors influencing drug metabolism, in Advances in Pharmacology (Ed. by S. Garattini and P. A. Shore), vol. 1, Academic Press, New York, (1961), p. 31-58. 3. H. REMMER,Drug Tolerance, in Ciba Foundation Symposium on Enzymes and Drug Action (Ed. by J. L. Mongar and A. V. S. de Reuck), Little, Brown, Boston, (1962), p. 276-298. 4. LARRYG. HART, RICHARDH. ADAMSON,ROBERTC. DIXONand JAMF~R. Fours, Stimulation of hepatic microsomal drug metabolism in the newborn and fetal rabbit, J. PharmaeoL Exptl. Therap. 137, 103-106 (1962). 5. R. KATO,E. CHIESARAand P. VASSANEtJ.I,Factors influencing induction of hepatic microsomal drug-metabolizing enzymes, Bioehem. Pharmacol. 11,211-220 (1962). 6. JULIUSAXELROD,JULESR~ICMENTHALand B~RN~J~ B. BRODIE,Mechanism of the potentiating action of ~-diethylaminoethyl diphenylpropylacetate, J. PharmacoL ExptL Therap. 112, 49-54 (1954). JACKR. COOrER,JULIUSAXEIJtODand BERNARD B. BRODXE,Inhibitory effects of /3-diethylaminoethyl diphenylpropylacetate on a variety of drug metabolic pathways in vitro, J. Pharmacol. Exptl. Therap. 112, 55-63 (1954). 7. J.R. Fotrrs and BERNARDB. BRODIE,Inhibition of drug metabolic pathways by the potentiating agent 2,4-dichloro-6-phenylphenoxyethyl diethylamine, J. Pharmacol. ExptL Therap. 115, 68-73 (1955). 8. J. R. Fou~s and BERNARDB. BRODIE,On the mechanism of drug potentiation by iproniazid (2-isopropyl-l-isonicotinyl hydrazine), J. PharmacoL ExptL Therap. 116, 480-485 (1956). 9. ROeERTL. DIXONand JAMESR. FOUTS, Inhibition of microsomal drug metabolic pathways by chloramphenicol, Biochem. Pharmacol. 11, 715-720 (1962).