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Stimulation of hepatic drug-metabolizing enzymes by DDT, polycyclic hydrocarbons or phenobarbital in adrenalectomized or castrated mice
TOXICOLOGY
AND
Stimulation
APPLIED
PHARMACOLOGY
(1974)
28,465-476
of Hepatic Drug-Metabolizing Enzymes Polycyclic Hydrocarbons or Phenobarbital in Adrenalectomized or Castrated Mice
by DDT,
RAJENDRAS. CHHABRAAND JAMES R. FOUTS Pharmacology
and Toxicology Branch, National Institute of Environmental National Institutes of Health, Research Triangle Park, North Received
October
5,1973;
accepted
December
Health Sciences, Carolina
II,1973
Stimulation of Hepatic Drug-Metabolizing Enzymesby DDT, Polycyclic Hydrocarbons or Phenobarbital in Adrenalectomized or CastratedMice. CHHABRA,R. S. AND FOUTS, J. R. (1974). Toxicol. Appl. Pharmacol. 28, 465-476. Benzpyrene treatment in rats increasesthe hepatic microsomal aniline hydroxylase pH optimum from 7 to 8. In mice treated with benzpyrene, no suchshift in pH optimum of aniline hydroxylase wasobserved. Further studieson hormonal regulation of ethylmorphine-N-demethylase, aniline and benzpyrenehydroxylase in mice indicatedthat adrenalectomy, castration or adrenalectomyplus castration do not lower the basal(“uninduced”) activities of theseenzymes(in rat with similar operations, basal activities are lowered by SO-90~0depending on the enzyme). Also the magnitude of stimulation of hepatic microsomal drug metabolizing enzymes(HMDME) by pretreatmentof micewith DDT, benzpyrene,3-MC or phenobarbitalin micewithout adrenals,testesor both wasnot lessthan in sham-operated control mice(asoccursin rats), but insteadwassomewhat higher. Thesefindings suggestthat rats and mice differ in hormonal regulations of basalactivities of HMDME and in the responseof HMDME in animalspretreatedwith inducersof HMDME.
One of the factors that may alter hepatic microsomal enzymes metabolizing foreign chemicals, is the hormonal state of the animal. It has been shown by many researchers that in the absence of various endocrine glands such as testes, adrenals or pituitary there is usually a lowered rate of drug biotransformation. Kato and Gillette (1965) reported that there was a decrease in hepatic microsomal metabolism of hexobarbital and aminopyrine in adrenalectomized or castrated rats, but no alteration in aniline hydroxylase was noticed. Similarly, Castro ef al. (1970) found lower activities of ethylmorphine-N-demethylase, NADP cytochrome c reductase, Cytochrome P-450 reductase and cytochrome P-450 content in adrenalectomized as compared with intact rats. Nebert and Gelboin (1969) observed a similar pattern with benzpyrene hydroxylase in adrenalectomized and hypophysectomized as compared with intact rats. There seems to be a species variation in hormonal regulation of hepatic drug metabolizing enzymes since hexobarbital metabolism was not altered by adrenalectomy in mice and rabbits (Kato et al., 1971). Wada ef al. (1972) reported no change in aminopyrineN-demethylase activity in adrenalectomized or castrated as compared with intact mice. The presence of hormones in the rat is also not necessary for stimulation (induction) Copyright Q 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
465
466
CHHABRA AND FOUTS
of the hepatic microsomal drug metabolism by drugs and polycyclic hydrocarbons (Conney, 1967). In a recent study (Chhabra and Fouts, 1973) it was reported that benzpyrene and DDT can increase hepatic drug metabolism in mice provided benzpyrene or DDT is given for longer duration (3 days) and at higher doses than those usually administered (100 or 200 mg/kg). The inducing effect is short: seen at 24 hr, but not at 96 hr after the last injection. This kind of stimulation of hepatic drug metabolism of enzymes in mice might be due to the “stress’ produced by injection of large doses of DDT or benzpyrene. Stress-induced stimulation of hepatic drug metabolizing enzymes in rats is mediated through adrenal hormones and/or sex hormones as suggested by Furner and Stitzel (1968). The present study was, therefore, designed to determine whether DDT or benzpyrene directly stimulated the hepatic drug metabolism in mice or if stimulation was mediated through adrenal or sex hormones. METHODS Materials. For pH optima experiments, 4- to 5-week-old male, Swiss-Webster mice were obtained from Arthur Sutter Farm, Springfield, Missouri. All mice weighed between 25 and 30 g. The sham-operated, adrenalectomized, castrated, and adrenalectomized-castrated male Swiss-Webster mice of CD, strain were purchased from Charles River Laboratories, Wilmington, Massachusetts. These animals when operated on were 35-37 days old, and they were 42-44 days old when used in the experiments. For control groups, mice were sham-operated at the same time as the others were subjected to ablation of endocrine glands. The residual hormone concentrations in tissue or blood were not measured in animals subjected to adrenalectomy, castration or both. The rats used were male, CD strain weighing 175-200 g and were obtained from Charles River Laboratories. Both mice and rats were fed Wayne Lab-Blox and water ad libitum, except the adrenalectomized and adrenalectomized-castrated mice which were given 0.9 % saline for drinking. Rats were housed S/cage in stainless-steel hanging cages; mice in plastic boxes with Absorb-On@ bedding (5 mice/box). Technical DDT, 3,4-benzpyrene and 3-methylcholanthrene (3-MC) were obtained from Fisher Chemicals. Treatment. DDT, benzpyrene and 3-MC were dissolved in Mazola corn oil; phenobarbital sodium was dissolved in 0.9 % saline. All inducers were given ip once each day for 3 days. The total volume of corn oil or saline solution injected in each mouse was 0.1 ml/IO g body weight; control mice received corn oil. Animals were killed for enzyme assays 24 hr after the last treatment. Preparation of microsomesand enzyme assays. In mice, livers were excised after removal of gall bladders; 4-5 livers were pooled from each treatment group and placed in ice cold KCl-HEPES buffer, pH 7.4 (1.25 mM HEPES in 1.15 ‘A KCI). Prior to homogenization, livers were washed 2-3 times with KCI-HEPES pH 7.4. The microsomal fractions from rat or mouse livers were prepared as described previously (Chhabra and Fouts, 1973). The in vitro assay of microsomal iV-demethylation of aminopyrine, benzphetamine, 1 HEPES = N-2-hydroxyethylpiperazine-W-2 St. Louis, Missouri.
-ethanesulfonic
acid from
Sigma Chemical
Co.,
STIMULATION
OF DRUG
METABOLISM
IN MICE
467
p-chloro-N-methylaniline and ethylmorphine was performed by measuring the formaldehyde formed (Nash, 1953). Benzpyrene hydroxylase activity was determined as described by Hansen (1970). The metabolism of aniline was followed by the assay of its product, p-aminophenol, as reported previously (Chhabra et al., 1972). Protein estimations were carried out by the method of Lowry et al. (1951) using bovine serum albumin as the standard. The incubation mixture (total volume 2.5 ml) for enzyme assays contained glucose 6-phosphate and MgS04 12.5 ,nmol each; NADP 2.5 pmol; 0.5 ml of 0.5 M HEPES buffer (pH 7.4); yeast glucose-6-phosphate dehydrogenase, 2 units (Sigma Chemical Co., St. Louis); and microsomal protein, 1 mg/ml. Substrate concentrations for aminopyrine, benzphetamine, p-chloro-N-methylaniline and aniline were 4 mM and for benzpyrene and ethylmorphine were 0.6 and 10 mM, respectively. For pH optima studies of aniline hydroxylase activity in rats versus mice, the aniline concentrations were 0.25,0.5, and 4 mM. All incubations were carried out for 15 min under air at 37°C. RESULTS
Efect of Benzpyrene Treatment of Rats or Mice on pH Optima of Aniline Hydroxylase in Hepatic Microsomes
Figure 1 shows the pH dependence of liver aniline hydroxylase at various aniline concentrations with hepatic microsomes from corn oil- and benzpyrene-treated mice and rats. The pH optimum for the microsomal enzyme from corn oil-treated mice and rats was at pH 7 for all substrate concentrations. The administration of benzpyrene to rats shifted upward the pH optimum of aniline hydroxylase from 7 to 7.5. The apparent magnitude of induction of aniline hydroxylase by benzpyrene administered to rats seemed to be dependent on the pH of incubation mixture and on the aniline concentrations. At lower concentrations of aniline (that is, at 0.25 mM or 0.5 n-t@, the increase in enzyme activity due to benzpyrene treatment was small compared to that seen at the 4 mM substrate concentration. In contrast to rats, in mice there was no shift in pH optima of aniline hydroxylase in microsomes from benzpyrene-treated animals, but the greatest increase in enzyme activity was seen at higher (e.g., 4 mM) substrate concentrations. Effect of Animal Pretreatment with DDT or Polycyclic Hydrocarbons on Microsomal Drug Metabolism in Sham-Operated and Adrenalectomized Mice
Table 1 shows the effect of DDT treatment on microsomal N-demethylation of p-chloro-N-methylaniline, ethylmorphine, aminopyrine and benzphetamine in shamoperated and adrenalectomized mice. DDT treatment significantly increased the N-demethylation of all the substrates used both in sham-operated and adrenalectomized mice. Table 2 shows the effect of pretreating mice with DDT, benzpyrene or phenobarbital on the microsomal metabolism of aniline or benzpyrene and on cytochrome P-450 content in sham-operated and adrenalectomized mice. There was no alteration in aniline hydroxylase or cytochrome P-450 content in mice after adrenalectomy; benzpyrene hydroxylase activity was increased in adrenalectomized mice when compared with control mice.
468
CHHABRA
AND
FOUTS
TABLE
I
116.3+ 15.8 155.51: 17.7 108.6+ 10.4
Ethylmorphine Aminopyrine Benzphetamine
397.6f 67.8’ 341.3+ 6O.lb 238.3k 20.4’
DDT __208.3f 35.6b
Sham-operated
i-241.9 i-1 19.5 +119.4
f52.5
% Change
160.8f 15.1 174.9+ 37.1 136.9+ 8.1
149.2+ 7.3
Corn oil
536.6+ 8.4b 463.5+ 31.3’ 346.6f 11.8”
263.1+ 21.6b
DDT
Adrenalectomized
+233.7 f165.0 +153.2
+76.3
% Change
5 $ g : 2
5 5 Fl % $ s
3 2
M 0Mice were treateddaily ip with DDT for 3 days and sacrificed 24 hr after the last injection. b Significantly different from corresponding mean of corn oil-treated groups, p i 0.05 (Student t test). Values are mean + SD with N= 3 independent samples of pooled livers.
136.6+ 16.4
Corn oil
p-Chloro-N-methylaniline
Drug substrate
OF VARIOUS DRUG SUBSTRATES 1~ SHAM-OPERATED AND
IV-Demethylaseactivity (nmol of HCHO formed/l5 min/mg protein)
EFFECT OF DDT TREATMENT (200 mg/kg) ON HEPATIC MICROSOMAL N-DEMETHYLATION ADRENALECTOMIZED MICE”
TABLE
2
17.8+ 1.2 25.5rt 3.2” 25.7 f: 4.1e
22.2 + 5.8
18.5+ 1.5 19.5f 2.7 21.7 f 1.7
19.1f 3.4
Sham-operated Adrenalectomized
Aniline hydroxylaseb
85515152’
497 + 85 772+ 101 1032F 266’ 800 ?I 139
715k 90 880+ 124 1360_+259’
Sham-operated Adrenalectomized
Benzpyrenehydroxylase’
95f 13 130+ 3’ 114+ 8 135+- 25’
107& 8 142rt 27 132_+23 168k20’
Sham-operated Adrenalectomized
Cytochrome P-450“
a Mice were treated daily, ip with DDT, benzpyrene, and phenobarbital for 3 days and sacrificed 24 hr after the last treatment. The data are expressed as mean f SD. b nmol ofp-aminophenol formed/mg of microsomal protein/l5 min. c Relative fluorescence/O.5 mg of microsomal protein/l5 min. d d OD x 103/mg of microsomal protein (450-490 nm). e Significantly different from corresponding mean of corn oil-treated gr0ups.p < 0.05 (Dunnett test). N = 3 independent samples of pooled livers.
Corn oil DDT Benzpyrene Phenobarbital sodium
Treatment@ (100mdkg)
EFFECTS OF VARIOUS TREATMENTS ON HEPATIC MICROSOMAL DRUG METABOLISM IN SHAM-OPERATED AND ADRENALECTOMIZED MICE
P z 8 z1 8
8
STIMULATION
OF DRUG
TABLE
METABOLISM
471
IN MICE
3
EFFECT OF 3-MC TREATMENT ON HEPATIC MICROSOMAL HYDROXYLATION OF ANILINE AND BENZPYRENE IN SHAM-OPERATED AND ADRENALE~TOMIZED MICE’ Aniline
Animal Sham-operated Adrenalectomized
hydroxylase* ___--Corn oil 3-MC 16.9 f 1.3 22.0 f 1.0
Benzpyrene .-__
24.0 f 4.5’ 29.9 + 1.8*
Corn oil ---940+85 1110+26
hydroxylase’ 3-MC __..--~~. 2215 + 330d 3433 + 208”
a The mice were treated daily, ip with 3-MC (50 mg/kg) for 3 days and sacrificed 24 hr after the last injection. The data are expressed as mean f SD. * nmol ofp-aminophenol formed/mg of microsomal protein/l5 min. c Relative fluorescence/mg of microsomal protein/l5 min. * Significantly different from corresponding mean of corn oil-treated group, p ==I0.05 (Student I test), N = 3 independent samples of pooled livers.
Aniline metabolism was increased both by DDT and benzpyrene treatment, but only in adrenalectomized mice. Benzpyrene hydroxylase activity was significantly increased by benzpyrene treatment both in sham-operated and adrenalectomized mice, while phenobarbital treatment increased benzpyrene metabolism only in shamoperated mice. Cytochrome P-450 content was increased after administration of phenobarbital to sham and adrenalectomized mice. DDT treatment significantly increased the cytochrome P-450 content only in adrenalectomized mice. The effect of 3-MC (50 mg/kg) treatment on aniline or benzpyrene hydroxylase activity in mice is shown in Table 3. 3-MC, a potent enzyme inducer compared to benzpyrene, increased the activities of both enzymes in sham-operated as well as in adrenalectomized mice. Efect of Animal Treatment with DDT, Polycyclic Hydrocarbons or Phenobarbital on Hepatic Microsomal Drug Metabolism in Sham-Operated and Castrated Mice
The results of this experiment are shown in Table 4. Treatment of mice with DDT or phenobarbital increased the metabolism of ethylmorphine, aniline and benzpyrene both in sham-operated and castrated mice; however in sham-operated mice, DDT treatment did not increase the aniline or benzpyrene hydroxylase activity enough to be statistically significant. Benzpyrene and 3-MC treatment increased benzpyrene hydroxylase activity both in sham-operated and castrated mice. Cytochrome P-450 content was increased by all treatments both in castrated and intact mice. Efect of Treatment with DDT, Polycyclic Hydrocarbons or Phenobarbital on Hepatic Drug Metabolism in Sham-Operatedand Adrenalectomized-Castrated Mice
The results of this experiment are shown in Table 5. DDT or phenobarbital treatments increased the activities of all the enzymes and cytochrome P-450 concentrations compared to corn oil-treated controls in both sham-operated and adrenalectomized
Treatments
460.4 IL 51.9” 490.3 + 53.6*
Phenobarbital (10 w/kg) 40.1 + 9.9f
34.9 + 5.4
34.9 + 6.5
39.7 + 3.6f
34.0 & 7.0s
33.2 i- 2.6
37.9 + 4.91
20.7 _+ 0.3
22.5 + 5.0 33.5 + 4.8
Castrated
hy Iroxyl?se’
Shamoperated
Aniline
117
1258 + 213f
1677 + 391f
1773*739f
1154 + 307
653f
Shamoperated
Benzpyrene
1468 f
2618f198f
1786f480*
36’
1250-c 174"
693-c 159
Castrated
hydroxylased
114*
108f
93f
95 *
70+
32*
21*
6f
9”
5
Shamoperated
Cytochrome
MICE
P-450”
5
16'
3'
95 + 26*
1025
83&
97 + 10”
54+
Castrated
B Mice were treated daily, ip with “inducers” for 3 days and sacrificed 24 hr after the last treatment. The data are expressed as mean + SD. b nmol of formaldehyde formed/mg of microsomal protein/l5 min. c nmol ofp-aminophenol formed/mg of microsomal protein/l5 min. d Relative fluorescence units/O.5 mg microsomal protein/l5 min. ’ A OD x 103/mg of microsomal protein (450-490 nm). f Significantly different from corresponding mean of corn oil-treated groups.p < 0.05 (Dunnett test). N= 3 independent samples of pooled livers.
141.9 + 35.4 135.8 + 23.3
3-MC (50 m/W
+ 36.4f
138.8 + 15.8
299.0 _+ 21.7'417.0
112.5 +_ 21.2
sodium
Castrated
112.6 + 24.9 142.8 + 12.8
Shamoperated
Ethylmorphine-N-demethylaseb
Benzpyrene (100 mg/k)
(200w/kg)
DDT
Corn oil
4
EFFECT OF VARIOUS TREATMENTS ON HEPATIC MICROSOMAL DRUG METABOLISM IN SHAM-OPERATED AND CASTRA~D
TABLE
Treatment”
sodium
5
438.5 + 76.3f
49.4 22.5 + 2.8
589.5 I 98.0f
144.8 + 36.6
175.9 r_ 42.0
37.7 + 2.4,’
35.2 rt 4.6’
34.5 + 4.1”
522.6 _+ 165.8.f 33.5 _+ 3.6s
159. I +
Shamoperated
Aniline
5.6
49.4 + 6.7’
34.0 +
34.8 f 2.2/
44.9 _+ 15.3f
21.5 _+ 0.5
Adrenalectomized and castrated
hydroxylase”
76
925 I 116’
1762 & 647’
1502 & 326/
743 t-
534 rt 122
Shamoperated
Benzpyrene
38s
1030 + 181,’
1825 + 665
1647 k
960 + 262
701 _+ 195
Adrenalectomized and castrated
hydroxylased
Cytochrome
10”
140 * 17,’
124 + 10f
IlO&
111 f 19f
5
Shamoperated 84+
-
0 Mice were treated daily, ip with “inducers” for 3 days and sacrificed 24 hr after the last treatment. The data are expressed as mean I SD. b nmol of formaldehyde formedjmg of microsomal protein/l5 min. c nmol of p-aminophenol formed/mg of microsomal protein/l 5 min. d Relative fluorescence units/O.5 mg of microsomal protein/l5 min. e d OD x 103/mg of microsomal protein (45&490 nm). r Significantly different from corresponding mean of corn oil-treated groups. p ..- 0.05 (Dunnett test). N = 3 independent pooled samples.
( 100 mg/kJ
134.0 + 38.2
129.0 _+ 48.5
Benzpyrene (100 m/kg)
3-MC (50 mg/kg) Phenobarbital
353.3 _+ 92.0s
128.9 + 38.4
Ethylmorphine-N-demethylaseb .__ -AdrenalShamectomized operated and castrated
(200 m/kg)
DD-T
Corn oil
TABLE
127 rf: 21s
93,15
116 f 30f
109 + 25*
72 + 14
Adrenalectomized and castrated
P-450
EFFECT OF VARIOUS TREATMENTS ON HFPATIC MICROSOMAL DRUG METABOLISM IN SHAM-OPERATED AND ADRENALECTOMIZEDCASTRATED MICE
P t; 3 2 3 5
i
3
%
2 F s 2; 8
474
CHHABRA
AND
FOUTS
DISCUSSION Remmer (1958) showed that adrenalectomy lowers the activity of rat hepatic drug metabolizing enzymes and that this decrease could be prevented by the administration of prednisolone. Kato and Gillette (1965) found similar results, but reported that only sex-dependent enzymes were lowered by adrenalectomy, since the metabolism of aniline or zoxazolamine was not impaired by adrenalectomy in rats. Kinetic studies of ethylmorphine-N-demethylase (Castro et al., 1970) revealed that adrenalectomy in rats caused both a decrease in V,,,,, and a small increase in Km, but there was no significant alteration in the apparent affinity of microsomes for ethylmorphine as measured by the spectral binding constant KS,. Kato and Onoda (1970) showed that castration of male rats decreased the activities of sex-dependent drug metabolizing enzymes and suggested that androgens increase the affinity of cytochrome P-450 for the binding of drug substrates. Further studies of Kato et al. (1971) indicated that adrenalectomy in other species, such as mice and rabbits, does not alter the hepatic drug-metabolizing enzymes, and that this is related to the fact that there are no sex differences in the enzyme activities in these animals in contrast to the rat. They suggested that higher activity of drug-metabolizing enzymes in male versus female rats is due to the interaction of androgens to increase the binding capacity of drug substrate with cytochrome P-450. In our present study we found that unlike the situation in rats, adrenalectomy, castration or adrenalectomy with castration in mice do not usually alter the activities of drug metabolizing enzymes except benzpyrene hydroxylase, which may be increased after adrenalectomy in mice. Wada et al. (1972) reported that in male mice, the activity of aminopyrine-N-demethylase was maintained if either testes or adrenals were present, but decreased in the absence of both adrenals or testes. However, we found that ethylmorphine-iV-demethylase activity was maintained in all adrenalectomized, castrated or adrenalectomized-castrated mice. It is believed that the stimulation of hepatic drug metabolism in rats by animal treatment with drugs and polycyclic hydrocarbons is not mediated through hormones (Conney, 1967). This was further supported by the work of Juchau et al. (1965), who showed induction of benzpyrene hydroxylase in isolated perfused rat liver, where the influence of adrenals or pituitary would seem minimal. Gielen and Nebert (1971) reported that phenobarbital, polycyclic hydrocarbons or DDT, when added to fetal liver in cell culture, can induce benzpyrene hydroxylase activity. Our present study shows that there is a species difference in response of hepatic microsomal drug metabolizing enzymes to adrenalectomy and/or castration in rats and mice, but that ablation of these glands in mice (like such operations in the rat) does not prevent the induction of microsomal drug-metabolizing enzymes by animal treatment with DDT, benzpyrene, 3-MC or phenobarbital. In mice without adrenals, testes, or both, the administration of DDT, polycyclic hydrocarbons or phenobarbital stimulated the drug-metabolizing enzymes in magnitude equivalent to that observed in sham-operated animals. This is in some contrast to the effects of inducers in sham-operated versus adrenalectomized, castrated, or hypophysectomized rats where endocrine ablation seems to blunt the response to inducers of several microsomal systems (Orrenius et al., 1969; Nebert and Gelboin 1969).
STIMULATION OF DRUG METABOLISMIN MICE
475
Rickert and Fouts (1970) reported that pretreatment of rats with benzpyrene shifted the pH optimum of hepatic microsomal aniline hydroxylase from 7 to 8. Moreover, benzpyrene-induced increase in aniline metabolism might not become evident until the pH of incubation system was increased from that used with control preparations. Similar results were obtained by Hansen and Fouts (1971b) in rats, but not in mice, since they did not see any increase in the hepatic microsomal metabolism of aniline in benzpyrene treated mice. We have recently (Chhabra and Fouts, 1973) shown that benzpyrene treatment of mice increased the hepatic metabolism of various drug substrates inciuding aniline, provided the mice are treated with a very high dose of benzpyrene (100 or 200 mg/kg), for longer duration (3 days) and the animals are sacrificed for enzyme assays at or before 24 hr after the last injection. Since we could not induce aniline hydroxylase with low doses of benzpyrene in mice, the present study also reinvestigated whether there was any shift in pH optima of aniline hydroxylase in mice after effective benzpyrene pretreatment. We again found that benzpyrene administration shifted the pH optima of aniline hydroxylase in rat liver from 7 to 7.5, but this did not occur in mice. The shift in pH optima in rats was dependent on substrate concentration and seemed to be more pronounced at higher substrate concentrations. These results agreed with the earlier findings of Hansen and Fouts (1971a). Hansen and Fouts (1971a) have suggested that the shift of aniline hydroxylase pH optima in rats could be due to the synthesis of a new enzyme, or synthesis of a cytochrome P-450 with a defective Type 1 binding site, or a Type 1 binding site occupied by highly bound substrates (endogenous or exogenous). Our present study suggests that in mouse liver microsomes there may be only a single enzyme which catalyzes the metabolism of aniline, and this is not changed except in amount by benzpyrene treatment. There seems to be no formation of cytochrome P-448 since we did not observe any shift in cytochrome P-450 absorption peak from 450 to 448 nm (Chhabra and Fouts, 197333. Finally, the response of mouse liver enzymes to DDT and benzpyrene treatment 01 mice would not seem to require androgens or adrenal hormones since induction still occurs in adrenalectomized and/or castrated mice.
REFERENCES
CASTRO,J. A., GREENE, F. E., GIGON,P., SASAME,H. ANDGILLETTE,J. R. (1970). Effect of
adrenalectomy and cortisone administration on components of the liver microsomal mixed function oxygenase system of male rats which catalyses ethylmorphine metabolism. Biochem. Pharmacol.
19,2461-2467.
CHHABRA,R. S.ANDFOUTS,J. R. (1973).Stimulation of hepaticmicrosomaldrug metabolizing enzymes in mice by l,l,l-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) and 3,4-benz-
pyrene. Toxicol. Appl. Pharmacol.
25,6@-70.
CHHABRA, R. S., GRAM,T. E. ANDFOUTS,J. R. (1972).A comparativestudy of two procedures used in the determination of hepatic microsomal aniline hydroxylation. Toxicol. Appl. Pharmacol.
22,5Q-58.
C~NNEY,A. H. (1967). Pharmacological implications of microsomal enzyme induction. Pharmacol.
Rev. 19, 317-366.
DUNNETT,C. W. (1955).A multiple comparisonprocedurefor comparingseveraltreatments with a control. J. Amer. Stat. Ass. 50, 1096-l 121. FURNER,R. L. AND STITZEL,R. E. (1968). Stress-inducedalterations in microsomaldrug metabolismin the adrenalectomizedrat. Biochem. Pharmacol. 17, 121-.127.
476
CHHABRA AND FOUTS
GIELEN, J. E. AND NEBERT, D. W. (1971).Microsomalhydroxylase induction in liver cell culture
by phenobarbital,polycyclic hydrocarbonsandp,p-DDT. Science172,167-l 69. HANSEN, A. R. (1970).The effect of 3,4benzpyrene administration on the characteristicsof
hepatic microsomalmixed function oxidases.A comparative study with rats and mice. Ph.D. Thesis,Univ. of Iowa, Aug. 1970,pp. 25-27. HANSEN, A. R. AND FOUTS,J. R. (1971a).Studieson the shift in the pH optimum of hepatic microsomal aniline p-hydroxylation following treatment of rats with 3,4benzpyrene. Chem.Biol. Interactions3, 123-129. HANSEN, A. R. AND FOUTS, J. R. (1971b).A comparisonof the effectsof benzpyreneadministration on somehepatic microsomalmixed function oxidasesof rats and mice. Biochem. Pharmacol.20,3 125-3143. JUCHAU, M. R., CRAM, R. L., PLAA, G. L. AND FOUTS, J. R. (1965).The induction of benzpyrenehydroxylase in the isolatedperfusedrat liver. Biochem.Pharmacol.14,473482. KATO, R. AND GILLETTE, J. R. (1965).Sexdifferencesin the effectsof abnormal physiological stateson the metabolismof drugs by rat liver microsomes.J. Pharmacol.Exp. Ther. 150, 285-291. KATO, R. AND ONODA, K. (1970).Studieson the regulation of the activity of drug oxidation in rat liver microsomesby androgenand estrogen.Biochem.Pharmacol.19,1649-1660. KATO, R., ONODA, K. AND TAKANAKA, A. (1971).Speciesdifferencesin the effect of morphine
administration or adrenalectomyon the substrateinteractionswith cytochrome P-450and drug oxidations by liver microsomes.Biochem.Pharmacol.20,1093-1099. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. AND RANDALL, R. J. (1951).Protein measurement with the Folin phenol reagent.J. Biol. Chem.193,265-275. NASH, T. (1953). The calorimetric estimation of formaldehyde by meansof the Hantzsch reaction. Biochem.J. 55,416-421. NEBERT, D. W. AND GELBOIN, H. V. (1969).The in vivo andin vitro induction of arylhydrocarbon hydroxylase in mammaliancells of different species,tissues,strains, and developmental and hormonal states.Arch. Biochem.Biophys. 134, 76-80. ORRENIUS, S., DAS, M. AND GNOSSPELIUS, Y. (1969). Overall biochemicaleffects of drug induction on liver microsomes.In MicrosomesandDrug Oxidation (Gillette, J. R. ; CONNEY, A. H.; COSMIDES, G. J.; ESTABROOK, R. W.; Fours, J. R. AND MANNERING, G. J., eds.) pp. 251-277.Academic Press,New York. REMMER, H. (1958).Die Wirkung der Nebennierenrindeauf den Abbau von Pharmaka in den Lebermikrosomen.Naturwissenschaften 21, 522-523. RICKERT, D. E. AND FOUTS, J. R. (1970). Benzpyrenepretreatment changesthe kinetics and pH optimum for aniline hydroxylation in vitro, but not thosefor benzphetaminedemethylation in vitro by rat liver microsomes.Biochem.Pharmacol.19,381-390. WADA, F., YOSHIDA, M. AND ISOHASHI, F. (1972).Effects of adrenalectomyand castration on enzymesmetabolizingdrugsin mouseliver. J. Biochem.71,343-345.
AND
Stimulation
APPLIED
PHARMACOLOGY
(1974)
28,465-476
of Hepatic Drug-Metabolizing Enzymes Polycyclic Hydrocarbons or Phenobarbital in Adrenalectomized or Castrated Mice
by DDT,
RAJENDRAS. CHHABRAAND JAMES R. FOUTS Pharmacology
and Toxicology Branch, National Institute of Environmental National Institutes of Health, Research Triangle Park, North Received
October
5,1973;
accepted
December
Health Sciences, Carolina
II,1973
Stimulation of Hepatic Drug-Metabolizing Enzymesby DDT, Polycyclic Hydrocarbons or Phenobarbital in Adrenalectomized or CastratedMice. CHHABRA,R. S. AND FOUTS, J. R. (1974). Toxicol. Appl. Pharmacol. 28, 465-476. Benzpyrene treatment in rats increasesthe hepatic microsomal aniline hydroxylase pH optimum from 7 to 8. In mice treated with benzpyrene, no suchshift in pH optimum of aniline hydroxylase wasobserved. Further studieson hormonal regulation of ethylmorphine-N-demethylase, aniline and benzpyrenehydroxylase in mice indicatedthat adrenalectomy, castration or adrenalectomyplus castration do not lower the basal(“uninduced”) activities of theseenzymes(in rat with similar operations, basal activities are lowered by SO-90~0depending on the enzyme). Also the magnitude of stimulation of hepatic microsomal drug metabolizing enzymes(HMDME) by pretreatmentof micewith DDT, benzpyrene,3-MC or phenobarbitalin micewithout adrenals,testesor both wasnot lessthan in sham-operated control mice(asoccursin rats), but insteadwassomewhat higher. Thesefindings suggestthat rats and mice differ in hormonal regulations of basalactivities of HMDME and in the responseof HMDME in animalspretreatedwith inducersof HMDME.
One of the factors that may alter hepatic microsomal enzymes metabolizing foreign chemicals, is the hormonal state of the animal. It has been shown by many researchers that in the absence of various endocrine glands such as testes, adrenals or pituitary there is usually a lowered rate of drug biotransformation. Kato and Gillette (1965) reported that there was a decrease in hepatic microsomal metabolism of hexobarbital and aminopyrine in adrenalectomized or castrated rats, but no alteration in aniline hydroxylase was noticed. Similarly, Castro ef al. (1970) found lower activities of ethylmorphine-N-demethylase, NADP cytochrome c reductase, Cytochrome P-450 reductase and cytochrome P-450 content in adrenalectomized as compared with intact rats. Nebert and Gelboin (1969) observed a similar pattern with benzpyrene hydroxylase in adrenalectomized and hypophysectomized as compared with intact rats. There seems to be a species variation in hormonal regulation of hepatic drug metabolizing enzymes since hexobarbital metabolism was not altered by adrenalectomy in mice and rabbits (Kato et al., 1971). Wada ef al. (1972) reported no change in aminopyrineN-demethylase activity in adrenalectomized or castrated as compared with intact mice. The presence of hormones in the rat is also not necessary for stimulation (induction) Copyright Q 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
465
466
CHHABRA AND FOUTS
of the hepatic microsomal drug metabolism by drugs and polycyclic hydrocarbons (Conney, 1967). In a recent study (Chhabra and Fouts, 1973) it was reported that benzpyrene and DDT can increase hepatic drug metabolism in mice provided benzpyrene or DDT is given for longer duration (3 days) and at higher doses than those usually administered (100 or 200 mg/kg). The inducing effect is short: seen at 24 hr, but not at 96 hr after the last injection. This kind of stimulation of hepatic drug metabolism of enzymes in mice might be due to the “stress’ produced by injection of large doses of DDT or benzpyrene. Stress-induced stimulation of hepatic drug metabolizing enzymes in rats is mediated through adrenal hormones and/or sex hormones as suggested by Furner and Stitzel (1968). The present study was, therefore, designed to determine whether DDT or benzpyrene directly stimulated the hepatic drug metabolism in mice or if stimulation was mediated through adrenal or sex hormones. METHODS Materials. For pH optima experiments, 4- to 5-week-old male, Swiss-Webster mice were obtained from Arthur Sutter Farm, Springfield, Missouri. All mice weighed between 25 and 30 g. The sham-operated, adrenalectomized, castrated, and adrenalectomized-castrated male Swiss-Webster mice of CD, strain were purchased from Charles River Laboratories, Wilmington, Massachusetts. These animals when operated on were 35-37 days old, and they were 42-44 days old when used in the experiments. For control groups, mice were sham-operated at the same time as the others were subjected to ablation of endocrine glands. The residual hormone concentrations in tissue or blood were not measured in animals subjected to adrenalectomy, castration or both. The rats used were male, CD strain weighing 175-200 g and were obtained from Charles River Laboratories. Both mice and rats were fed Wayne Lab-Blox and water ad libitum, except the adrenalectomized and adrenalectomized-castrated mice which were given 0.9 % saline for drinking. Rats were housed S/cage in stainless-steel hanging cages; mice in plastic boxes with Absorb-On@ bedding (5 mice/box). Technical DDT, 3,4-benzpyrene and 3-methylcholanthrene (3-MC) were obtained from Fisher Chemicals. Treatment. DDT, benzpyrene and 3-MC were dissolved in Mazola corn oil; phenobarbital sodium was dissolved in 0.9 % saline. All inducers were given ip once each day for 3 days. The total volume of corn oil or saline solution injected in each mouse was 0.1 ml/IO g body weight; control mice received corn oil. Animals were killed for enzyme assays 24 hr after the last treatment. Preparation of microsomesand enzyme assays. In mice, livers were excised after removal of gall bladders; 4-5 livers were pooled from each treatment group and placed in ice cold KCl-HEPES buffer, pH 7.4 (1.25 mM HEPES in 1.15 ‘A KCI). Prior to homogenization, livers were washed 2-3 times with KCI-HEPES pH 7.4. The microsomal fractions from rat or mouse livers were prepared as described previously (Chhabra and Fouts, 1973). The in vitro assay of microsomal iV-demethylation of aminopyrine, benzphetamine, 1 HEPES = N-2-hydroxyethylpiperazine-W-2 St. Louis, Missouri.
-ethanesulfonic
acid from
Sigma Chemical
Co.,
STIMULATION
OF DRUG
METABOLISM
IN MICE
467
p-chloro-N-methylaniline and ethylmorphine was performed by measuring the formaldehyde formed (Nash, 1953). Benzpyrene hydroxylase activity was determined as described by Hansen (1970). The metabolism of aniline was followed by the assay of its product, p-aminophenol, as reported previously (Chhabra et al., 1972). Protein estimations were carried out by the method of Lowry et al. (1951) using bovine serum albumin as the standard. The incubation mixture (total volume 2.5 ml) for enzyme assays contained glucose 6-phosphate and MgS04 12.5 ,nmol each; NADP 2.5 pmol; 0.5 ml of 0.5 M HEPES buffer (pH 7.4); yeast glucose-6-phosphate dehydrogenase, 2 units (Sigma Chemical Co., St. Louis); and microsomal protein, 1 mg/ml. Substrate concentrations for aminopyrine, benzphetamine, p-chloro-N-methylaniline and aniline were 4 mM and for benzpyrene and ethylmorphine were 0.6 and 10 mM, respectively. For pH optima studies of aniline hydroxylase activity in rats versus mice, the aniline concentrations were 0.25,0.5, and 4 mM. All incubations were carried out for 15 min under air at 37°C. RESULTS
Efect of Benzpyrene Treatment of Rats or Mice on pH Optima of Aniline Hydroxylase in Hepatic Microsomes
Figure 1 shows the pH dependence of liver aniline hydroxylase at various aniline concentrations with hepatic microsomes from corn oil- and benzpyrene-treated mice and rats. The pH optimum for the microsomal enzyme from corn oil-treated mice and rats was at pH 7 for all substrate concentrations. The administration of benzpyrene to rats shifted upward the pH optimum of aniline hydroxylase from 7 to 7.5. The apparent magnitude of induction of aniline hydroxylase by benzpyrene administered to rats seemed to be dependent on the pH of incubation mixture and on the aniline concentrations. At lower concentrations of aniline (that is, at 0.25 mM or 0.5 n-t@, the increase in enzyme activity due to benzpyrene treatment was small compared to that seen at the 4 mM substrate concentration. In contrast to rats, in mice there was no shift in pH optima of aniline hydroxylase in microsomes from benzpyrene-treated animals, but the greatest increase in enzyme activity was seen at higher (e.g., 4 mM) substrate concentrations. Effect of Animal Pretreatment with DDT or Polycyclic Hydrocarbons on Microsomal Drug Metabolism in Sham-Operated and Adrenalectomized Mice
Table 1 shows the effect of DDT treatment on microsomal N-demethylation of p-chloro-N-methylaniline, ethylmorphine, aminopyrine and benzphetamine in shamoperated and adrenalectomized mice. DDT treatment significantly increased the N-demethylation of all the substrates used both in sham-operated and adrenalectomized mice. Table 2 shows the effect of pretreating mice with DDT, benzpyrene or phenobarbital on the microsomal metabolism of aniline or benzpyrene and on cytochrome P-450 content in sham-operated and adrenalectomized mice. There was no alteration in aniline hydroxylase or cytochrome P-450 content in mice after adrenalectomy; benzpyrene hydroxylase activity was increased in adrenalectomized mice when compared with control mice.
468
CHHABRA
AND
FOUTS
TABLE
I
116.3+ 15.8 155.51: 17.7 108.6+ 10.4
Ethylmorphine Aminopyrine Benzphetamine
397.6f 67.8’ 341.3+ 6O.lb 238.3k 20.4’
DDT __208.3f 35.6b
Sham-operated
i-241.9 i-1 19.5 +119.4
f52.5
% Change
160.8f 15.1 174.9+ 37.1 136.9+ 8.1
149.2+ 7.3
Corn oil
536.6+ 8.4b 463.5+ 31.3’ 346.6f 11.8”
263.1+ 21.6b
DDT
Adrenalectomized
+233.7 f165.0 +153.2
+76.3
% Change
5 $ g : 2
5 5 Fl % $ s
3 2
M 0Mice were treateddaily ip with DDT for 3 days and sacrificed 24 hr after the last injection. b Significantly different from corresponding mean of corn oil-treated groups, p i 0.05 (Student t test). Values are mean + SD with N= 3 independent samples of pooled livers.
136.6+ 16.4
Corn oil
p-Chloro-N-methylaniline
Drug substrate
OF VARIOUS DRUG SUBSTRATES 1~ SHAM-OPERATED AND
IV-Demethylaseactivity (nmol of HCHO formed/l5 min/mg protein)
EFFECT OF DDT TREATMENT (200 mg/kg) ON HEPATIC MICROSOMAL N-DEMETHYLATION ADRENALECTOMIZED MICE”
TABLE
2
17.8+ 1.2 25.5rt 3.2” 25.7 f: 4.1e
22.2 + 5.8
18.5+ 1.5 19.5f 2.7 21.7 f 1.7
19.1f 3.4
Sham-operated Adrenalectomized
Aniline hydroxylaseb
85515152’
497 + 85 772+ 101 1032F 266’ 800 ?I 139
715k 90 880+ 124 1360_+259’
Sham-operated Adrenalectomized
Benzpyrenehydroxylase’
95f 13 130+ 3’ 114+ 8 135+- 25’
107& 8 142rt 27 132_+23 168k20’
Sham-operated Adrenalectomized
Cytochrome P-450“
a Mice were treated daily, ip with DDT, benzpyrene, and phenobarbital for 3 days and sacrificed 24 hr after the last treatment. The data are expressed as mean f SD. b nmol ofp-aminophenol formed/mg of microsomal protein/l5 min. c Relative fluorescence/O.5 mg of microsomal protein/l5 min. d d OD x 103/mg of microsomal protein (450-490 nm). e Significantly different from corresponding mean of corn oil-treated gr0ups.p < 0.05 (Dunnett test). N = 3 independent samples of pooled livers.
Corn oil DDT Benzpyrene Phenobarbital sodium
Treatment@ (100mdkg)
EFFECTS OF VARIOUS TREATMENTS ON HEPATIC MICROSOMAL DRUG METABOLISM IN SHAM-OPERATED AND ADRENALECTOMIZED MICE
P z 8 z1 8
8
STIMULATION
OF DRUG
TABLE
METABOLISM
471
IN MICE
3
EFFECT OF 3-MC TREATMENT ON HEPATIC MICROSOMAL HYDROXYLATION OF ANILINE AND BENZPYRENE IN SHAM-OPERATED AND ADRENALE~TOMIZED MICE’ Aniline
Animal Sham-operated Adrenalectomized
hydroxylase* ___--Corn oil 3-MC 16.9 f 1.3 22.0 f 1.0
Benzpyrene .-__
24.0 f 4.5’ 29.9 + 1.8*
Corn oil ---940+85 1110+26
hydroxylase’ 3-MC __..--~~. 2215 + 330d 3433 + 208”
a The mice were treated daily, ip with 3-MC (50 mg/kg) for 3 days and sacrificed 24 hr after the last injection. The data are expressed as mean f SD. * nmol ofp-aminophenol formed/mg of microsomal protein/l5 min. c Relative fluorescence/mg of microsomal protein/l5 min. * Significantly different from corresponding mean of corn oil-treated group, p ==I0.05 (Student I test), N = 3 independent samples of pooled livers.
Aniline metabolism was increased both by DDT and benzpyrene treatment, but only in adrenalectomized mice. Benzpyrene hydroxylase activity was significantly increased by benzpyrene treatment both in sham-operated and adrenalectomized mice, while phenobarbital treatment increased benzpyrene metabolism only in shamoperated mice. Cytochrome P-450 content was increased after administration of phenobarbital to sham and adrenalectomized mice. DDT treatment significantly increased the cytochrome P-450 content only in adrenalectomized mice. The effect of 3-MC (50 mg/kg) treatment on aniline or benzpyrene hydroxylase activity in mice is shown in Table 3. 3-MC, a potent enzyme inducer compared to benzpyrene, increased the activities of both enzymes in sham-operated as well as in adrenalectomized mice. Efect of Animal Treatment with DDT, Polycyclic Hydrocarbons or Phenobarbital on Hepatic Microsomal Drug Metabolism in Sham-Operated and Castrated Mice
The results of this experiment are shown in Table 4. Treatment of mice with DDT or phenobarbital increased the metabolism of ethylmorphine, aniline and benzpyrene both in sham-operated and castrated mice; however in sham-operated mice, DDT treatment did not increase the aniline or benzpyrene hydroxylase activity enough to be statistically significant. Benzpyrene and 3-MC treatment increased benzpyrene hydroxylase activity both in sham-operated and castrated mice. Cytochrome P-450 content was increased by all treatments both in castrated and intact mice. Efect of Treatment with DDT, Polycyclic Hydrocarbons or Phenobarbital on Hepatic Drug Metabolism in Sham-Operatedand Adrenalectomized-Castrated Mice
The results of this experiment are shown in Table 5. DDT or phenobarbital treatments increased the activities of all the enzymes and cytochrome P-450 concentrations compared to corn oil-treated controls in both sham-operated and adrenalectomized
Treatments
460.4 IL 51.9” 490.3 + 53.6*
Phenobarbital (10 w/kg) 40.1 + 9.9f
34.9 + 5.4
34.9 + 6.5
39.7 + 3.6f
34.0 & 7.0s
33.2 i- 2.6
37.9 + 4.91
20.7 _+ 0.3
22.5 + 5.0 33.5 + 4.8
Castrated
hy Iroxyl?se’
Shamoperated
Aniline
117
1258 + 213f
1677 + 391f
1773*739f
1154 + 307
653f
Shamoperated
Benzpyrene
1468 f
2618f198f
1786f480*
36’
1250-c 174"
693-c 159
Castrated
hydroxylased
114*
108f
93f
95 *
70+
32*
21*
6f
9”
5
Shamoperated
Cytochrome
MICE
P-450”
5
16'
3'
95 + 26*
1025
83&
97 + 10”
54+
Castrated
B Mice were treated daily, ip with “inducers” for 3 days and sacrificed 24 hr after the last treatment. The data are expressed as mean + SD. b nmol of formaldehyde formed/mg of microsomal protein/l5 min. c nmol ofp-aminophenol formed/mg of microsomal protein/l5 min. d Relative fluorescence units/O.5 mg microsomal protein/l5 min. ’ A OD x 103/mg of microsomal protein (450-490 nm). f Significantly different from corresponding mean of corn oil-treated groups.p < 0.05 (Dunnett test). N= 3 independent samples of pooled livers.
141.9 + 35.4 135.8 + 23.3
3-MC (50 m/W
+ 36.4f
138.8 + 15.8
299.0 _+ 21.7'417.0
112.5 +_ 21.2
sodium
Castrated
112.6 + 24.9 142.8 + 12.8
Shamoperated
Ethylmorphine-N-demethylaseb
Benzpyrene (100 mg/k)
(200w/kg)
DDT
Corn oil
4
EFFECT OF VARIOUS TREATMENTS ON HEPATIC MICROSOMAL DRUG METABOLISM IN SHAM-OPERATED AND CASTRA~D
TABLE
Treatment”
sodium
5
438.5 + 76.3f
49.4 22.5 + 2.8
589.5 I 98.0f
144.8 + 36.6
175.9 r_ 42.0
37.7 + 2.4,’
35.2 rt 4.6’
34.5 + 4.1”
522.6 _+ 165.8.f 33.5 _+ 3.6s
159. I +
Shamoperated
Aniline
5.6
49.4 + 6.7’
34.0 +
34.8 f 2.2/
44.9 _+ 15.3f
21.5 _+ 0.5
Adrenalectomized and castrated
hydroxylase”
76
925 I 116’
1762 & 647’
1502 & 326/
743 t-
534 rt 122
Shamoperated
Benzpyrene
38s
1030 + 181,’
1825 + 665
1647 k
960 + 262
701 _+ 195
Adrenalectomized and castrated
hydroxylased
Cytochrome
10”
140 * 17,’
124 + 10f
IlO&
111 f 19f
5
Shamoperated 84+
-
0 Mice were treated daily, ip with “inducers” for 3 days and sacrificed 24 hr after the last treatment. The data are expressed as mean I SD. b nmol of formaldehyde formedjmg of microsomal protein/l5 min. c nmol of p-aminophenol formed/mg of microsomal protein/l 5 min. d Relative fluorescence units/O.5 mg of microsomal protein/l5 min. e d OD x 103/mg of microsomal protein (45&490 nm). r Significantly different from corresponding mean of corn oil-treated groups. p ..- 0.05 (Dunnett test). N = 3 independent pooled samples.
( 100 mg/kJ
134.0 + 38.2
129.0 _+ 48.5
Benzpyrene (100 m/kg)
3-MC (50 mg/kg) Phenobarbital
353.3 _+ 92.0s
128.9 + 38.4
Ethylmorphine-N-demethylaseb .__ -AdrenalShamectomized operated and castrated
(200 m/kg)
DD-T
Corn oil
TABLE
127 rf: 21s
93,15
116 f 30f
109 + 25*
72 + 14
Adrenalectomized and castrated
P-450
EFFECT OF VARIOUS TREATMENTS ON HFPATIC MICROSOMAL DRUG METABOLISM IN SHAM-OPERATED AND ADRENALECTOMIZEDCASTRATED MICE
P t; 3 2 3 5
i
3
%
2 F s 2; 8
474
CHHABRA
AND
FOUTS
DISCUSSION Remmer (1958) showed that adrenalectomy lowers the activity of rat hepatic drug metabolizing enzymes and that this decrease could be prevented by the administration of prednisolone. Kato and Gillette (1965) found similar results, but reported that only sex-dependent enzymes were lowered by adrenalectomy, since the metabolism of aniline or zoxazolamine was not impaired by adrenalectomy in rats. Kinetic studies of ethylmorphine-N-demethylase (Castro et al., 1970) revealed that adrenalectomy in rats caused both a decrease in V,,,,, and a small increase in Km, but there was no significant alteration in the apparent affinity of microsomes for ethylmorphine as measured by the spectral binding constant KS,. Kato and Onoda (1970) showed that castration of male rats decreased the activities of sex-dependent drug metabolizing enzymes and suggested that androgens increase the affinity of cytochrome P-450 for the binding of drug substrates. Further studies of Kato et al. (1971) indicated that adrenalectomy in other species, such as mice and rabbits, does not alter the hepatic drug-metabolizing enzymes, and that this is related to the fact that there are no sex differences in the enzyme activities in these animals in contrast to the rat. They suggested that higher activity of drug-metabolizing enzymes in male versus female rats is due to the interaction of androgens to increase the binding capacity of drug substrate with cytochrome P-450. In our present study we found that unlike the situation in rats, adrenalectomy, castration or adrenalectomy with castration in mice do not usually alter the activities of drug metabolizing enzymes except benzpyrene hydroxylase, which may be increased after adrenalectomy in mice. Wada et al. (1972) reported that in male mice, the activity of aminopyrine-N-demethylase was maintained if either testes or adrenals were present, but decreased in the absence of both adrenals or testes. However, we found that ethylmorphine-iV-demethylase activity was maintained in all adrenalectomized, castrated or adrenalectomized-castrated mice. It is believed that the stimulation of hepatic drug metabolism in rats by animal treatment with drugs and polycyclic hydrocarbons is not mediated through hormones (Conney, 1967). This was further supported by the work of Juchau et al. (1965), who showed induction of benzpyrene hydroxylase in isolated perfused rat liver, where the influence of adrenals or pituitary would seem minimal. Gielen and Nebert (1971) reported that phenobarbital, polycyclic hydrocarbons or DDT, when added to fetal liver in cell culture, can induce benzpyrene hydroxylase activity. Our present study shows that there is a species difference in response of hepatic microsomal drug metabolizing enzymes to adrenalectomy and/or castration in rats and mice, but that ablation of these glands in mice (like such operations in the rat) does not prevent the induction of microsomal drug-metabolizing enzymes by animal treatment with DDT, benzpyrene, 3-MC or phenobarbital. In mice without adrenals, testes, or both, the administration of DDT, polycyclic hydrocarbons or phenobarbital stimulated the drug-metabolizing enzymes in magnitude equivalent to that observed in sham-operated animals. This is in some contrast to the effects of inducers in sham-operated versus adrenalectomized, castrated, or hypophysectomized rats where endocrine ablation seems to blunt the response to inducers of several microsomal systems (Orrenius et al., 1969; Nebert and Gelboin 1969).
STIMULATION OF DRUG METABOLISMIN MICE
475
Rickert and Fouts (1970) reported that pretreatment of rats with benzpyrene shifted the pH optimum of hepatic microsomal aniline hydroxylase from 7 to 8. Moreover, benzpyrene-induced increase in aniline metabolism might not become evident until the pH of incubation system was increased from that used with control preparations. Similar results were obtained by Hansen and Fouts (1971b) in rats, but not in mice, since they did not see any increase in the hepatic microsomal metabolism of aniline in benzpyrene treated mice. We have recently (Chhabra and Fouts, 1973) shown that benzpyrene treatment of mice increased the hepatic metabolism of various drug substrates inciuding aniline, provided the mice are treated with a very high dose of benzpyrene (100 or 200 mg/kg), for longer duration (3 days) and the animals are sacrificed for enzyme assays at or before 24 hr after the last injection. Since we could not induce aniline hydroxylase with low doses of benzpyrene in mice, the present study also reinvestigated whether there was any shift in pH optima of aniline hydroxylase in mice after effective benzpyrene pretreatment. We again found that benzpyrene administration shifted the pH optima of aniline hydroxylase in rat liver from 7 to 7.5, but this did not occur in mice. The shift in pH optima in rats was dependent on substrate concentration and seemed to be more pronounced at higher substrate concentrations. These results agreed with the earlier findings of Hansen and Fouts (1971a). Hansen and Fouts (1971a) have suggested that the shift of aniline hydroxylase pH optima in rats could be due to the synthesis of a new enzyme, or synthesis of a cytochrome P-450 with a defective Type 1 binding site, or a Type 1 binding site occupied by highly bound substrates (endogenous or exogenous). Our present study suggests that in mouse liver microsomes there may be only a single enzyme which catalyzes the metabolism of aniline, and this is not changed except in amount by benzpyrene treatment. There seems to be no formation of cytochrome P-448 since we did not observe any shift in cytochrome P-450 absorption peak from 450 to 448 nm (Chhabra and Fouts, 197333. Finally, the response of mouse liver enzymes to DDT and benzpyrene treatment 01 mice would not seem to require androgens or adrenal hormones since induction still occurs in adrenalectomized and/or castrated mice.
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adrenalectomy and cortisone administration on components of the liver microsomal mixed function oxygenase system of male rats which catalyses ethylmorphine metabolism. Biochem. Pharmacol.
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CHHABRA,R. S.ANDFOUTS,J. R. (1973).Stimulation of hepaticmicrosomaldrug metabolizing enzymes in mice by l,l,l-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) and 3,4-benz-
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25,6@-70.
CHHABRA, R. S., GRAM,T. E. ANDFOUTS,J. R. (1972).A comparativestudy of two procedures used in the determination of hepatic microsomal aniline hydroxylation. Toxicol. Appl. Pharmacol.
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C~NNEY,A. H. (1967). Pharmacological implications of microsomal enzyme induction. Pharmacol.
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DUNNETT,C. W. (1955).A multiple comparisonprocedurefor comparingseveraltreatments with a control. J. Amer. Stat. Ass. 50, 1096-l 121. FURNER,R. L. AND STITZEL,R. E. (1968). Stress-inducedalterations in microsomaldrug metabolismin the adrenalectomizedrat. Biochem. Pharmacol. 17, 121-.127.
476
CHHABRA AND FOUTS
GIELEN, J. E. AND NEBERT, D. W. (1971).Microsomalhydroxylase induction in liver cell culture
by phenobarbital,polycyclic hydrocarbonsandp,p-DDT. Science172,167-l 69. HANSEN, A. R. (1970).The effect of 3,4benzpyrene administration on the characteristicsof
hepatic microsomalmixed function oxidases.A comparative study with rats and mice. Ph.D. Thesis,Univ. of Iowa, Aug. 1970,pp. 25-27. HANSEN, A. R. AND FOUTS,J. R. (1971a).Studieson the shift in the pH optimum of hepatic microsomal aniline p-hydroxylation following treatment of rats with 3,4benzpyrene. Chem.Biol. Interactions3, 123-129. HANSEN, A. R. AND FOUTS, J. R. (1971b).A comparisonof the effectsof benzpyreneadministration on somehepatic microsomalmixed function oxidasesof rats and mice. Biochem. Pharmacol.20,3 125-3143. JUCHAU, M. R., CRAM, R. L., PLAA, G. L. AND FOUTS, J. R. (1965).The induction of benzpyrenehydroxylase in the isolatedperfusedrat liver. Biochem.Pharmacol.14,473482. KATO, R. AND GILLETTE, J. R. (1965).Sexdifferencesin the effectsof abnormal physiological stateson the metabolismof drugs by rat liver microsomes.J. Pharmacol.Exp. Ther. 150, 285-291. KATO, R. AND ONODA, K. (1970).Studieson the regulation of the activity of drug oxidation in rat liver microsomesby androgenand estrogen.Biochem.Pharmacol.19,1649-1660. KATO, R., ONODA, K. AND TAKANAKA, A. (1971).Speciesdifferencesin the effect of morphine
administration or adrenalectomyon the substrateinteractionswith cytochrome P-450and drug oxidations by liver microsomes.Biochem.Pharmacol.20,1093-1099. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. AND RANDALL, R. J. (1951).Protein measurement with the Folin phenol reagent.J. Biol. Chem.193,265-275. NASH, T. (1953). The calorimetric estimation of formaldehyde by meansof the Hantzsch reaction. Biochem.J. 55,416-421. NEBERT, D. W. AND GELBOIN, H. V. (1969).The in vivo andin vitro induction of arylhydrocarbon hydroxylase in mammaliancells of different species,tissues,strains, and developmental and hormonal states.Arch. Biochem.Biophys. 134, 76-80. ORRENIUS, S., DAS, M. AND GNOSSPELIUS, Y. (1969). Overall biochemicaleffects of drug induction on liver microsomes.In MicrosomesandDrug Oxidation (Gillette, J. R. ; CONNEY, A. H.; COSMIDES, G. J.; ESTABROOK, R. W.; Fours, J. R. AND MANNERING, G. J., eds.) pp. 251-277.Academic Press,New York. REMMER, H. (1958).Die Wirkung der Nebennierenrindeauf den Abbau von Pharmaka in den Lebermikrosomen.Naturwissenschaften 21, 522-523. RICKERT, D. E. AND FOUTS, J. R. (1970). Benzpyrenepretreatment changesthe kinetics and pH optimum for aniline hydroxylation in vitro, but not thosefor benzphetaminedemethylation in vitro by rat liver microsomes.Biochem.Pharmacol.19,381-390. WADA, F., YOSHIDA, M. AND ISOHASHI, F. (1972).Effects of adrenalectomyand castration on enzymesmetabolizingdrugsin mouseliver. J. Biochem.71,343-345.