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The effect of Sudan III on drug metabolizing enzymes
Chem.-Biol. Interactions, 48 (1984) 129--143 Elsevier Scientific Publishers Ireland Ltd.
129
T H E E F F E C T O F SUDAN III ON D R UG M E T A B O L I Z I N G ENZYMES* SHOICHI FUJITAa'**, JACK PEISACHa, HIDEKO OHKAWAb, YUKO YOSHIDAb, SHIGERU ADACHIb, TAKASHI UESUGIb, MASAHIKO SUZUKIc and TOKUJI SUZUKIc aDepartment of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, N Y 10461 (U.S.A.), bDepartment of Biopharmacy, Meiji College of Pharmacy, Setagaya-Ku, Tokyo, 154 (Japan) and CDepartment of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Chiba University, Chiba, 261 (Japan)
(Received March 2nd, 1983) (Revision received July 29th, 1983) (Accepted August 4th, 1983)
SUMMARY We have examined the induction of drug metabolizing enzymes in rat liver microsomes by azo dye, 1-(p-phenylazophenylazo)-2-naphthol {Sudan III). Marked increases were observed in the levels of c y t o c h r o m e P-448 as well as in p-nitroanisole O-demethylase (p-NAD), amaranth (AR) and neoprontosil reductases (NPR) and 7 - e t h o x y c o u m a r i n O-deethylase (ECD) activities. On th e o th er hand, a m i n o p y r e n e N-demethylase activity was n o t significantly increased. Further, induced ECD activity was inhibited 90% by a specific a n t i b o d y against c y t o c h r o m e P-448 while the inhibition observed with an a n t i b o d y against c y t o c h r o m e P-450 was less than 25%. Simultaneous administration o f Sudan III and 3 - m e t h y l c h o l a n t h e n e (3-MC) induced c y t o c h r o m e P-448 up to a level brought a b o u t by either Sudan III or 3-MC t r e a t m e n t alone. In contrast, Sudan III did n o t induce c y t o c h r o m e P - 4 4 8 in the 3-MC insensitive DBA/2 mouse. Solubilized microsomes f r o m Sudan III-treated rats showed an identical sodium d o d e c y l sulfate pol yacryl am i de gel electrophoretic (SDS-PAGE) pa t t e r n with those f r om 3-MC-treated animals. It is concluded *This work was supported in part by U.S.P.H.S. Grant HL-13399 from the National Heart and Lung Institute and in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. **Current address: Department of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Chiba University, Chiba, Japan 261. tTo whom correspondence should be sent. Abbreviations: APD, aminopyrine-N-demethylase; AR, amaranth reductase; DMBA, 7,12dimethylbenzanthracene; ECD, 7-ethoxycoumarin O-deethylase; 3-MC, 3-methylcholanthrene; p-NAD, p-nitroanisole O-demethylase; NPR, neoprontosil reductase; PB, phenobarbital; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Sudan III, 1-(p-phenylazophenylazo)-2-naphthol; ZAH, zoxazolamine hydroxylase. 0009-2797/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
130 that the c y t o c h r o m e P-448 induced in liver by Sudan III is very similar to that induced by 3-MC. Sudan III also induced UDP-glucuronyltransferase activity towards 1-naphthol and estradiol. It did not induce NADPH-cytochrome c reductase, nor any of the enzymes which constitute the microsomal electron transport chain except for c y t o c h r o m e P-448.
Key words: Drug metabolism -- Induction -- Cytochrome P-450 -- Cytochrome P-448 -- Sudan III -- UDP-glucuronyltransferase INTRODUCTION
The effect of the azo dye Sudan III on hepatic drug metabolism is of special interest as there have been a number of reports implicating this comp o u n d in the prevention of chemical carcinogenesis in rats [1,2]. Similar prevention has been observed with polycyclic aromatic hydrocarbons if they are administered a few days prior to exposure to carcinogenic doses [1,3]. These observations have led to the suggestion t h a t these agents act by inducing hepatic drug-metabolizing enzymes and thus accelerate the elimination of toxic metabolites. This was borne out, for example, by Conney and Levin [3] who showed that Sudan III treatment inhibited 7,12-dimethylbenzanthracene (DMBA) carcinogenesis by inducing the metabolism of this c o m p o u n d in liver. The fact t h a t Sudan III induces P-450 type cytochromes [4,5] seems rather contradictory to the fact that it prevents chemical carcinogenesis, because c y t o c h r o m e P-448 has been shown to activate an otherwise nontoxic c o m p o u n d to an ultimate carcinogen [6]. In order to resolve this contradiction, the nature of cytochrome P-450 induction and its effect on hepatic microsomal monooxygenase activities, as well as the induction of other enzyme activities in microsomes by Sudan III were investigated. EXPERIMENTAL PROCEDURES
Chemicals NADP, NADPH, NADH and glucose 6-phosphate were obtained from K y o w a Hakko Co. (Japan), glucose-6-phosphate dehydrogenase from Sigma and 7-ethoxycoumarin and 7-hydroxycoumarin from Aldrich. Ethylisocyanide was prepared according to the m e t h o d of Jackson and McKusic [7]. Sudan III was obtained from T o k y o Kasei (Japan) and was recrystallized from benzene. 3-MC was purchased from Eastman Kodak Co. Animals Male Wistar rats weighing 100 + 10 g were divided into 6 groups, each consisting of 4 rats. They were injected intraperitoneally for 3 days with either 3-MC (20 mg/kg/day) or Sudan III (20 mg/kg/day), each dissolved in 0.5 ml of corn oil or phenobarbital (PB) (80 mg/kg/day) dissolved in 0.5 ml of 0.9%
131 NaC1. Control groups were injected with corn oil or saline. Male C57BL/6Cr and DBA/2Cr mice were injected for 4 days with the same inducers with the doses of 40 mg/kg/day. Hepatic microsomes were prepared according to the m e t h o d of Omura and Sato [8,9]. Protein concentrations were determined by the L o w r y procedure [ 10].
Spectrophotometric studies CO difference spectra of the microsomal preparations were obtained by the method of Omura and Sato [8,9] and the contents of the cytochromes were determined using a millimolar extinction coefficient of 91 for A4s0-490. Cytochrome bs was assayed by difference spectroscopy of NADH reduced minus oxidized microsomes using a millimolar extinction coefficient of 185 for A42s-410 [8]. Ethyl isocyanide difference spectra [11,12] were obtained as follows: microsomes ( 0 . 4 - 0 . 8 mg protein/roll were suspended in 1.0 M potassium phosphate buffer at various pH-values. Sodium dithionite (2 mg/ml) was added and the sample was divided in half. Ethyl isocyanide was added to one half, with the other half as reference and the spectrum was obtained with a Cary 219 spectrophotometer. Enzyme assays NAD(P)H oxidase activity was determined from the absorption change at 340 nm. Assay mixtures contained 0.05 M potassium phosphate buffer (pH 7.4) 1 mM MgC12, a b o u t 0.6 mg/ml microsomal protein and 0.1 mM NAD(P)H. NAD(P)H-cytochrome c reductase was assayed according to the prc~ cedure of Masters et al. [13]. Assay mixtures in 0.05 M potassium phosphate buffer (pH 7.4) contained 5 pm c y t o c h r o m e c, 0.1 mM EDTA, 0.1 mM KCN and microsomal protein at a final concentration 0.1 mg/ml for the NADPH-cytochrome c reductase assay and 0.01 mg/ml for the N A D H - c y t o c h r o m e c reductase assay. Reactions were started by a d d i n g NAD(P)H to a final concentration of 0.1 mM. Increase in the absorption at 550 nm was followed using a Hitachi 200-20 spectrophotometer. Aminopyrine-N-demethylase (APD) was assayed at 37°C by a modification of the method of Cochin and Axelrod [14]. The assay mixture, in 0.1 M potassium phosphate buffer (pH 7.4) contained 10 mM glucose 6-phosphate, 10 units/ml of glucose-6-phosphate dehydrogenase, 2 mM MgCI:, 7.5 mM semicarbazide-HC1 (pH adjusted to 7.4 with NaOH), 1.0 mM aminopyrine and about 1.2 mg/ml microsomal protein. The mixture was preincubated for 5 min. The reaction was initiated by the addition of 400 pM (final concentration) NADP. Oxygen gas was blown across the surface of the reaction mixture during the 15-rain incubation period. The reaction was stopped b y the addition of 1 ml of 10% ZnSO4. Five minutes later, 1 ml of 0.5 M NaOH was added to the mixture which was then stirred and centrifuged at 1000 X g for 30 rain. Formaldehyde content in the supernatant was assayed by the method of Nash [15].
132 The assay method for zoxazolamine hydroxylase (ZAH) was essentially the same as that described by Trevor [ 16 ]. The assay mixture consisting of 4 mM MgC12, 10 mM glucose 6-phosphate, 2 units/ml of glucose-6-phosphate dehydrogenase, 0.1 mg/ml zoxazolamine and about 2 mg/ml microsomal protein in oxygen-saturated 0.1 M potassium phosphate buffer (pH 7.4) in a final volume of 1 ml, was preincubated for 5 min at 37°C in a shaking water bath. The reaction was started by the addition of 10 pl of 50 mM NADP. The incubation was carried out for 15 min and the reaction was stopped by the addition of 1 ml of 1 M NaOH. Non-hydroxylated zoxazolamine in the mixture was extracted by vigorous vortex mixing into 12 ml of n-heptane containing 1.5% isoamyl alcohol. The mixture was then centrifuged at 1000 X g for 10 min. Ten milliliters of the heptane layer was transferred to a centrifuge tube containing 4 ml of 0.1 M HCI, mixed well and centrifuged as above. Optical absorptions of the HC1 layers at 278 nm were measured using a Hitachi 340 spectrophotometer. The assay m e t h o d for p-nitroanisole O-demethylase (p-NAD) described by Netter and Seidel [17] was modified as follows. The assay mixture in a total volume of 1 ml contained essentially the same ingredients as those for the zoxazolamine hydroxylase assay as described above, p-Nitroanisole, 0.6 mM, was used as a substrate. Microsomal protein c o n t e n t in the mixture was about 1 mg/ml. The reaction was started with the addition of 10 ~l of 50 mM NADP after 5 min preincubation at 37°C. The reaction mixture was then incubated for 20 min and stopped by the addition of 1 ml of 10% ZnSO4 and 2 ml of saturated Ba(OH)2. The mixture was centrifuged for 30 min at 1000 X g, and the absorption of the supernatant was measured at 420 nm. A:naranth reductase (AR) and neoprontosil reductase (NPR) activities were assayed according to the method described by Fujita and Peisach [ 18]. ECD activity was assayed according to the fluorometric m e t h o d described by Ullrich and Weber [19] using a microsomal protein concentration of 0.5 mg/ml. The production of 7-hydroxycoumarin was followed with a Hitachi 204-A fluorometer with excitation at 360 nm and emission at 460 nm. UDP-glucuronyltransferase activity was assayed according to the methods of Otani et al. [20] and Rao et al. [21]. Disk-SDS-PA GE A microsomal preparation suspended in 0.05 M potassium phosphate buffer (pH 7.4) containing 30% glycerol and 1 mM EDTA was solubilized with sodium cholate (3 mg/mg microsomal protein) at 0°C. Polyethylene glycol 6000, 50%, was added to a final concentration of 8% and the mixture was stirred for 10 min and centrifuged for 45 min at 3500 X g. The pellet was discarded. Polyethylene glycol 6000 was then added to a final concentration of 16%. The mixture was stirred for 10 min and centrifuged at 105 000 X g for 1 h. The pellet was homogenized in the buffer described above. Disc-
133 SDS-PAGE of the solubilized microsomes was carried out according to the m e t h o d of Weber and Osborn [22]. The gel contained 7.5% acrylamide and 0.1% SDS. Electrophoresis was carried out at 20°C for 7.5 h at 6 mA/gel. Malachite green, 0.005%, was used as a marker. The gels were then fixed and stained with 0.025% coomasie brilliant blue R 250 by the m e t h o d of Fazekas de St. Groth et al. [23]. As molecular weight standards, bovine serum albumin (68 000), ovalbumin (45 000) and cytochrome c (12 000) from Boehringer Mannheim were used.
Preparation of anti-PB-P-450 and anti-3-MC-P-448 antibodies Immunoglobulin fractions containing antibodies to rat liver microsomal cytochromes P-450 and P-448 prepared from immunized rabbit serum were generous gifts from Dr. Y. Okada, Kyushu University, Japan. Purified protein used for rabbit immunization were obtained according to the methods of Negishi et al. [24] for cytochrome P-450, and Hashimoto and Imai [ 25] for c y t o c h r o m e P-448. Purified cytochromes (700 Ug) were mixed with the same volume of Freund's complete adjuvant (DIFCO) and were injected subcutaneously into male white rabbits. After 3 weeks, the rabbits were injected with 2 mg of cytochrome. A week later, the serum from the blood of the immunized animals was fractionated with a m m o n i u m sulfate {0--45%). Immunoglobulin fractions were dissolved in 0.9% NaC1/10 mM sodium phosphate buffer (pH 7.5) to a final concentration of about 40 rag/ ml. Ethoxycoumarin-O-deethylase activity was assayed according to the m e t h o d described above, except in the presence of various concentrations of antibodies 2.5--20 times the concentration of microsomal protein in the reaction mixture. The activity in the presence of equal concentrations of the control immunoglobulin from non-treated animals was taken as 100% activity. RESULTS
The induction of cytochrome P-448 A comparison was made of the CO difference spectra of reduced microsomes from Sudan III treated rats with those from 3-MC or PB treated animals. A 2 nm absorption shift from 450 nm to 448 nm was observed for samples from b o t h Sudan III and 3-MC treated animals. Table I summarizes the concentrations of P-450 t y p e c y t o c h r o m e in liver m i c r a somes from rats treated with PB, Sudan III or 3-MC. Marked increases were observed in microsomes from drug treated animals. 3-MC or Sudan III alone or Sudan III injected together with 3-MC induced cytochrome P-448, while PB induced c y t o c h r o m e P-450. Simultaneous treatment with 3-MC and Sudan III did not lead to any additive induction over each c o m p o u n d alone, suggesting t h a t Sudan III and 3-MC induce the same species of cytochroms P-448.
134 TABLE I EFFECT OF PB, 3-MC AND SUDAN III TREATMENT ON LIVER MICROSOMAL P-450 CYTOCHROMES IN WISTAR RATS AND DBA/2 AND C57BL/6 MICE Groups of animals were injected i.p. for 3 days with PB dissolved in PSS, or with 3-MC or Sudan III, each dissolved in corn oil, at the doses indicated in brackets. Contents of.P-450 type cytochromes were determined from the CO difference spectra of the reduced microsomes. Standard deviations were obtained from the individual differences for 4 animals. Cytochrome contents are expressed in nmol/mg microsomal protein. Animals
Treatment (mg/kg)
~max of CO Difference (nmol]mg)
Cytochrome P-450 (nmol/mg)
Wistar
PSSa
450
0.47 +_0.01
Rats
Corn oil PB (80) 3-MC (20) 3-MC (40) Sudan III (20) 3-MC (20) + Sudan III (20)
450 450 448 448 448 440
0.53 1.57 1.19 1.25 1.10 1.27
DBA/2 Mice
Corn oil 3-MC (40} Sudan III (40)
450 450 450
0.50 +0.10 0.68 +_0.10 0.60 + 0.05
C57BL/6 Mice
Corn oil 3-MC (40) Sudan III (40)
450 448 448
0.65 _+0.03 1.48 _+0.06 1.11 +_0.07
+- 0.08 _+0.22 _+0.11 +-0.07 +_0.04 +_0.22
a Physiological saline.
T r e a t m e n t with either 3-MC or S u d a n I I I did n o t alter t h e levels o f P - 4 5 0 t y p e c y t o c h r o m e s n o r cause t h e spectral shift of 2 n m in t h e CO d i f f e r e n c e s p e c t r u m for D B A / 2 mice, the ' n o n - i n d u c i b l e ' strain (Table I). P o l a n d et al. [ 2 6 ] d e m o n s t r a t e d t h a t this m o u s e has a l o w level or a p o o r a f f i n i t y o f c y t o s o l i c r e c e p t o r p r o t e i n , the m a j o r r e g u l a t o r y gene p r o d u c t o f t h e m u r i n e Ah locus w h i c h regulates t h e i n d u c t i o n o f c y t o c h r o m e P - 4 4 8 b y p o l y c y c l i c a r o m a t i c c o m p o u n d s such as 3-MC, b e n z p y r e n e , ~-naphthoflavone or 2 , 3 , 7 , 8 - t e t r a - c h l o r o d i b e n z o - p - d i o x i n [ 2 7 ] . Thus t h e lack o f effect with S u d a n III is c o m p a r a b l e t o t h a t observed with 3-MC.
Ethyl isocyanide difference spectra A l t h o u g h t h e CO d i f f e r e n c e s p e c t r a o f m i c r o s o m e s f r o m S u d a n I I I a n d 3-MC t r e a t e d animals were a l m o s t identical, t h e p H d e p e n d e n c e of t h e e t h y l i s o c y a n i d e d i f f e r e n c e spectra was n o t t h e same. Figure 1 shows the e f f e c t o f p H o n t h e a m p l i t u d e s o f a b s o r p t i o n m a x i m a near 430 n m a n d 455 n m o f e t h y l i s o c y a n i d e d i f f e r e n c e spectra, a n d t h e A430/A4ss ratios at p H 7 are collected in Table II. F o r m i c r o s o m e s f r o m 3-MC t r e a t e d animals,
135 both maxima had equal intensity at pH 6.70, the crossover point for the titration curves. With Sudan III treatment, this occurred at pH 7.03; for animals treated with Sudan III plus 3-MC (pH 6.75). For comparison, w e show data for microsomes f~om PB treated animals where the pH crossover was at 7.5. The values reported here are within range of pH crossovers reported for microsomes after 3-MC treatment ( 6 . 4 - 6 . 7 ) and after PB treatment (7.4--7.8) [12]. Our results reflect the tendency for microsomes containing larger concentrations of cytochrome P-448 to show a lower pH intercept than those t h a t contain c y t o c h r o m e P-450.
Components o f the microsomal electron transport chain Table III shows the enzyme activities of various components of the microsomal electron transport chain and of cytochrome bs content. PB treatment [ i*-~
(a/
J05 ! 670
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80
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Fig. I. The effect of pH on the absorption maxima near 430 and 455 nm of ethylisocyanide difference spectra of rat liver microsomes. Ethylisocyanide difference spectra at various values of pH for (a) 3-MC treated animals, (b) 3-MC + Sudan III treated animals, (c) Sudan III-treated animals and (d) PB treated animals were obtained according to the method described in the text. The A,30__~s0/microsomal protein concentration (o o) and A,55_,s0/microsomal protein concentration (o o) were obtained from the spectra and were plotted against pH-values.
136 TABLE II EFFECT OF PB, 3-MC AND SUDAN HI TREATMENTS ON A4ss/A430 RATIO OF THE ETHYLISOCYANIDE DIFFERENCE SPECTRA OF RAT LIVER MICROSOMES AT pH 7.0 The treatments of animals are described in the legend to Table I. The method tlsed to obtain ethylisocyanide difference spectra is described in the text. Treatment
PSS a Corn oil PB 3-MC Sudan III 3-MC -~ Sudan III
h m a x in the 430 nm region
h m a x in the 455 nm region
hA4ss_4s 0 ~ A430_480
428 428 428 429 429 429
455 455 455 453 453 453
0.30 0.31 0.49 1.52 0.97 1.51
a Physiological saline.
c a u s e d a 2-fold increase in N A D P H o x i d a s e levels w h i c h was n o t o b s e r v e d a f t e r t r e a t m e n t w i t h either S u d a n III or 3-MC. O n t h e o t h e r hand, 3-MC t r e a t m e n t c a u s e d a small d e c r e a s e in N A D H o x i d a s e activity, while S u d a n III and PB t r e a t m e n t h a d no effect. PB i n d u c e d N A D P H - c y t o c h r o m e c r e d u c t a s e activity, c o n f i r m i n g t h e results of o t h e r s [ 2 8 , 2 9 ] , b u t slightly d e c r e a s e d t h e N A D H - c y t o c h r o m e c r e d u c t a s e activity. A slight increase in c y t o c h r o m e bs c o n t e n t was seen in r a t liver m i c r o s o m e s f r o m a n i m a l s t r e a t e d w i t h t h e inducers u s e d in t h e s e studies. It s h o u l d be n o t e d t h a t S u d a n I I I did n o t a p p r e c i a b l y a l t e r a n y of t h e e n z y m e activities o f t h e m i c r o s o m a l e l e c t r o n t r a n s p o r t s y s t e m t h a t d i r e c t e l e c t r o n s t o c y t o c h r o m e P-450, while BP a n d 3-MC t r e a t m e n t did.
Drug metabolism APD, ECD, p - N A D , Z A H , A R a n d N P R activities o f m i c r o s o m e s f r o m t h e rats t r e a t e d w i t h various inducers are s h o w n in T a b l e IV. A m a r k e d increase in A P D activity was seen in m i c r o s o m e s f r o m PB t r e a t e d animals [ 3 0 ] , b u t n o t f r o m Sudan I I I or 3-MC t r e a t e d animals. A R a n d N P R w e r e i n d u c e d b y PB, 3-MC and S u d a n I I I a n d t h e s e activities r e f l e c t t h e levels o f t o t a l P - 4 5 0 t y p e c y t o c h r o m e s in individual m i c r o s o m a l p r e p a r a t i o n s . I n d u c t i o n o f E C D a n d p - N A D activities w e r e m o s t p r o m i n e n t in liver m i c r o s o m e s f r o m rats t r e a t e d w i t h either 3-MC or S u d a n III. T h e s e s u b s t r a t e s are k n o w n to be m e t a b o l i z e d by c y t o c h r o m e P - 4 4 8 m o r e e f f i c i e n t l y t h a n P B - i n d u c e d c y t o c h r o m e P-450. 3-MC m i c r o s o m e s h a d t h e highest Z A H activity ( a n o t h e r P-448 d e p e n d e n t r e a c t i o n ) b u t S u d a n I I I m i c r o s o m e s h a d l o w e r a c t i v i t y t h a n PB m i c r o s o m e s . Z A H a c t i v i t y p e r n m o l P - 4 5 0 h o w e v e r , was higher in S u d a n III m i c r o s o m e s t h a n in PB m i c r o s o m e s (5.5 vs. 4.2 n m o l / m i n / n m o l P-450).
6.57 6.14 14.37 8.34 6.72 8.62
+0.01 _+ 1.07 -+ 1.57 _+ 1.71 _+0 . 2 4 -+0.74
NADPH oxidase nmol/mg/min
1039.3 927.4 512.4 674.2 896.8 775.5
_+ 1 5 0 . 8 _+ 40.6 _+1 0 5 . 3 _+ 1 2 9 . 0 _+ 6.1 _+ 165.4
NADH Cytocbrome c reductase (nmol/mg/min) 77.91 89.10 123.83 75.55 91.44 87.87
_+ 7.84 _+ 1 2 . 0 8 _+ 1 0 . 0 9 _+ 5.86 _+ 4 . 6 7 _+ 1 . 9 6
NADPH Cytochrome c reductase (nmol/mg/min)
0.40 0.43 0.51 0.50 0.60 0.57
_+0 . 0 6 _+ 0.02 _+0 . 0 4 _+0.02 _+0.06 _+0.10
C y t o c h r o m e b5 (nmol/mg)
a Corn oil.
Control* PB 3-MC S u d a n III
Treatment
2.51 5.77 2.26 2.68
APD
_+0.22 _+1.44 _+0.30 _+0.07
0.27 1.37 4.33 4.04
ECD _+0.015 _+0.052 _+0.139 -+0.062
E n z y m e activities ( n m o l / m g / m i n )
0.86 1.51 2.11 2.01
-+0.07 -+0.08 _+0.02 +0.06
p-NAD
4.60 6.72 8.81 6.10
ZAH -+0.08 +0.10 _+0.18 -+0.10
2.25 7.20 5.51 5.20
AR -+0.06 -+0.52 _+0.30 _+0.21
4.10 11.80 7.20 7.00
NPR -+0.12 + 0.20 +_0.31 _+0.30
T h e assay m e t h o d s are d e s c r i b e d in t h e text. A P D a c t i v i t y is e x p r e s s e d in n m o l H C H O f o r m e d / m g m i c r o s o m a l p r o t e i n / m i n . E C D activity is expressed in n m o l 7-OH c o u m a r i n f o r m e d / m g m i c r o s o m a l p r o t e i n / m i n , p - N A D [ 1 7 ] , Z A H [ 1 6 ] , A R a n d N P R [ 1 8 ] activities are expressed as before. Values are e x p r e s s e d as m e a n activity _+ S.D.
E F F E C T O F PB, 3-MC A N D S U D A N HI ON H E P A T I C D R U G M E T A B O L I Z I N G E N Z Y M E S
T A B L E IV
a Physiological saline.
9.95 7.71 9.18 5.51 8.75 5.74
PSS a Corn oil PB 3-MC S u d a n III 3 - M C + S u d a n III
_+2.98 _+ 2.07 + 2.82 _+0.12 _+ 1.14 +1.10
NADH oxidase nmol/mg/min
Pretreatment
E n z y m e assay a n d t h e d e t e r m i n a t i o n o f c y t o c h r o m e b 5 c o n t e n t were carried o u t a c c o r d i n g t o t h e m e t h o d s d e s c r i b e d in t h e t e x t . Values are expressed as m e a n values _+ S.D.
E F F E C T OF PB, 3-MC A N D S U D A N III P R E T R E A T M E N T O N H E P A T I C M I C R O S O M A L E L E C T R O N T R A N S P O R T
T A B L E HI
b-4 C~
138 The patterns of the induction of drug metabolism by Sudan III and 3-MC were very similar, and the potency of the inductive ability of these two compounds in the doses used were very similar as well.
Antibodies against cytochromes P-448 and P-450 Figure 2 shows the effects of anti-PB-P-450 and anti-3-MC-P-448 antibodies on the activity of ECD in each of the microsomal preparations. As expected, anti-PB-P-450 antibody inhibited O-deethylation in PB microsomes up to 80% (b), while inhibition was less for control microsomes (a). Considerably less inhibition was observed with microsomes from rats treated with (c} 3-MC, (d) Sudan III or (e) 3-MC + Sudan III. The anti-3-MC-P-448 antibody, on the other hand, inhibited O-deethylation by PB microsomes up to 30% and 3-MC microsomes, Sudan III microsomes, and 3-MC + Sudan III microsomes, up to 90%. It is noteworthy that both antibodies inhibited O-deethylase activity of corn oil control microsomes up to 60% and that the anti-3-MC-P-448 antibody was not more efficient than the anti-PB-P-450 antibody in inhibiting this reaction. This suggests that the mixture o f cytochromes P-450 present in control microsomes may be different from that in PB microsomes. Disc-SDS-PA GE Figure 3 shows the disk-SDS-PAGE of solubilized and polyethylene glycol 6000 fractionated microsomes from rats treated with Sudan III and 3-MC. A band at mol. wt. 56 000 is prominent and a less prominent band with a
(a) Control
°,/o
-(b) PB
(c) 3-MC
~I
I(c) 3-~c Sudan :11
] I
100
\ 5O I
\ \
!
,
-\ ",o
Ot . . . . . 0
10
\
\, ',\
\
:
\
,\
_
i R
J 2O
10
20 0
10
20 0
10 20 0 10 20 timc---~ con ot microsomal protein
Ooncentration ot Antibodies
Fig. 2. Effects of antibodies against cytochromes P-450 and P-448 on ECD activities of liver microsomes from (a) corn oil, (b) PB, (c) 3-MC, (d) S u d a n III a n d (e) 3-MC + S u d a n III treated rats. E n z y m e activities in the presence of anti-P-450 (o .o) or antiP-448 ( , . . . . , ) antibodies were compared to those in the presence of the corresponding c o n c e n t r a t i o n s of non-specific i m m u n o g l o b u l i n fractions from the serum of noni m m u n i z e d rabbits, expressed as 100%. The assay m e t h o d is described in the text.
139 mol. wt. 54 000 is also observed. Electrophoretic patterns of these two microsomal fractions were identical. There were no new identifiable species of hemoprotein with these molecular weights induced by Sudan III other than those induced with 3-MC.
UDP-glucuronyltransferase activity Table V shows the effect of inducers of cytochrome P-450 and P-448 on hepatic microsomal UDP-glucuronyltransferase activity. Sudan III induced UDP-glucuronyltransferase activity towards 1-naphthol and estradiol. 3-MC, on the other hand, induced activity only towards 1-naphthol, a typical substrate of so-called 'late fetal type' UDP-glucuronyltransferase. Estradiol, on the other hand, is a typical substrate of 'neonatal type' UDP-
PSS
PB
S III
MC
--68
000
~45
000
C
Fig. 3. Disk-SDS-PAGE of solubilized liver mierosomes f r o m rats treated with PB, 3-MC, and S u d a n III. Microsomes, suspended in 0.1 mM E D T A , 30% glycerol, were solubilized with s o d i u m cholate (3 m g / m g m i c r o s o m a l protein). Electrophoresis was carried o u t f r o m top to b o t t o m in the presence of SDS as described in the text. Molecular weight standards are indicated o n the right of the figure. PSS, solubilized microsomes f r o m the livers of physiological saline solution treated control rats; PB, f r o m PB-treated rats; MC, f r o m 3-MC-treated rats; SIII, f r o m S u d a n III-treated rats; C, from corn oil-treated control rats.
140 TABLE V E F F E C T O F PB, 3-MC A N D S U D A N HI ON U D P - G L U C U R O N Y L T R A N S F E R A S E A C T I V I T I E S IN R A T L I V E R M I C R O S O M E S P r e t r e a t m e n t s of animals and the assay m e t h o d s are described in E x p e r i m e n t a l Procedures. C y t o c h r o m e P-450 c o n t e n t is expressed in n m o l / m g microsomal protein. UDP-glucuronyltransferase activity is expressed in nmol glucuronide formed/rag m i c r o s o m a l protein/rain. Values are expressed as m e a n +_S.D. Treatments
PSS a Corn oil PB 3-MC Sudan III
P-450 contents
0.47 0.45 1.00 1.18 0.94
+_0.01 +_0.03 +0.12 +_0.20 +_0.03
UDP-glucuronyltransferase activity towards 1-Naphthol
Estradiol
7.6 7.5 10.9 20.0 19.6
0.14 0.13 0.26 0.16 0.24
+_0.70 +_0.78 +_0.56 +_0.92 +_0.44
+_0.015 +_0.013 +0.004 +_0.010 +_0.013
a Physiological saline.
glucuronyltransferase [ 31 ]. These activities of different types were reported to be differentially induced by 3-MC and PB respectively [32] in agreement with our results. DISCUSSION
In the present study, we show that Sudan III is an inducer of c y t o c h r o m e P-448" with a potency similar to that of 3-MC for ECD, p-nitroanisole O-demethylation, and amaranth and neoprontosil reduction**. On the other hand, Sudan III is not an inducer of APD demethylase (Table IV), an activity associated with c y t o c h r o m e P-450 [30]. The pH intercepts of the absorption maxima near 430 nm and 455 nm in the ethylisocyanide difference spectra (Fig. 1) for 3-MC and Sudan III microsomes are lower in value than the intercepts for PB and control microsomes [ 12 ]. Antibodies against purified liver microsomal c y t o c h r o m e P-448 induced b y c-naphthoflavone inhibit the ECD activity of liver microsomes from either Sudan IH or 3-MC treated rats while the antibody against purified c y t o c h r o m e P-450 is n o t as effective {Fig. 2). Disk-SDS-PAGE of microsomes from rats treated with Sudan III or 3-MC showed identical patterns (Fig. 3). All of these observations strongly suggest that liver microsomal cytochromes P°448 from 3-MC- and Sudan IIItreated animals are similar. *The terms 'cytochrome P - 4 5 0 ' and ' c y t o c h r o m e - P - 4 4 8 ' are operational definitions referring to the m i x t u r e o f m i c r o s o m a l P-450 t y p e c y t o c h r o m e s that give rise to absorptions at 450 nm and 448 nm, respectively, in the CO-difference spectrum. **These m i c r o s o m a l azoreductase activities are greater for PB-induced animals than for either 3-MC- or Sudan III-induced animals since t h e y depend on the level of total P-450 t y p e c y t o c h r o m e s which is greatest in PB-induced animals [ 18 ].
141 It is of interest to note that the mechanism o f induction of c y t o c h r o m e P-448 with Sudan III appears to be similar to the induction with 3-MC. The simultaneous administration of 3-MC and Sudan III does not lead to additive induction, as takes place after simultaneous PB and b e n z o [ a ] p y r e n e treatm e n t [33]. The DBA/2 strain of mice which is non-responsive to 3-MC treatment in the induction of cytochrome P-448 is also not responsive to Sudan III treatment. Poland et al. [26] showed that this non-responsiveness is due to the genetic defect of a polycyclic hydrocarbon inducer binding protein which is present in the cytosol. Since Sudan III failed to induce c y t o c h r o m e P-448 in this strain of mice, it is likely that the induction by Sudan III is also mediated by this protein. It is curious, though, that a structurally different c o m p o u n d like Sudan III may be able to bind to this same protein in a way similar to that of a polycyclic aromatic hydrocarbon. Other than c y t o c h r o m e P-448, Sudan III does not alter any o f the enzyme activities of the microsomal electron transport chain. Thus with Sudan III, the induction of monooxygenase activity is largely due to increased cytochrome P-448 levels. It has been known that treatment by Sudan III, and other aromatic compounds, prevents adrenal injury and mammary cancer that it caused by chemical carcinogens, such as DMBA [1,2]. Conney and Levin [3] reported that the metabolism of this polyaromatic c o m p o u n d in the liver is induced by Sudan III. It seems contradictory that an inducer of c y t o c h r o m e P-448 prevents chemical carcinogenesis if given prior to exposure to a known aromatic hydrocarbon carcinogen, since these are usually activated by c y t o c h r o m e P-448 to produce ultimate carcinogens [6 ]. It is possible that Sudan III induces enzymatic activities other than those catalyzed by c y t o c h r o m e P-448, which affect the lifetime of active intermediates. Indeed, Sudan III was shown to induce UDP-glucuronyltransferase activities towards both 1-naphthol and estradiol. The enhancement of activity by Sudan III suggests that its role in the prevention of carcinogenesis m a y be via this mechanism. In conclusion, Sudan III causes the enhancement o f drug metabolism by inducing c y t o c h r o m e P-448, an enzyme of the phase I drug-metabolizing reactivity, and UDP-glucuronyltransferase an enzyme of phase II reactivity. This Sudan III mediated induction of activating and inactivating steps of drug metabolism may be involved in the prevention of chemical carcinogene~ sis by this dye. Regardless of its usefulness as an anti-cancer agent, Sudan III is an interesting c o m p o u n d for the studies of mechanisms of induction of phase I and phase II drug metabolizing enzymes and may be a possible link between them. Finally, since Sudan III is not a carcinogen, it may be a safer c o m p o u n d to use in the laboratory for the study of c y t o c h r o m e P-448 induction than the highly carcinogenic compound, 3-MC. REFERENCES
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142 2 C.BI Huggins, N. Ueda and A. Russo, Azo dyes prevent hydrocarbon-induced leukemia in the rat, Proc. Natl. Acad. Sci., U.S.A., 75 (1978) 4524. 3 A.H. Conney and W. Levin, Induction of hepatic 7,12-dimethylbenz(a)anthracene metabolism by polycyclic aromatic hydrocarbons and aromatic azo derivatives, Life Sci., 5 (1966) 465. 4 Y. Ichikawa, B. Hagihara and T. Yamano, Magnetic and spectrophotometric properties of the microsomal carbon monoxide binding pigment, Arch. Biochem. Biophys., 120 (1967) 204. 5 S. Fujita, Microsomal Electron Transport and Azoreductase Activity, Ph.D. Thesis, Yeshiva University, New York, 1979. 6 D.W. Nebert and J.S. Felton, Evidence for the activation of 3-methylcholanthrene as a carcinogen in vivo and in vitro by P,-450 from inbred strains of mice, in: D.Y. Cooper, O. Rosenthal, R. Snyder and C. Witmer (Eds.), Cytochromes P-450 and bs, Plenum Press, New York, 1975, pp. 127--149. 7 H.L. Jackson and B.C. McKusic, Ethylisocyanide, in: Organic Synthesis Coll. Vol. IV, John Wiley and Sons, Inc., New York, 1963, pp. 438--440. 8 T. Omura and R. Sato, A new cytochrome in liver microsomes, J. Biol. Chem., 237
(1962) 1375. 9 T. Omura and R. Sato, Carbon monoxide-binding pigment of liver microsomes, J. Biol. Chem., 239 (1964) 2370. 10 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 11 Y. Imai and R. Sato, Evidence for two forms of P-450 hemoprotein in microsomal membranes, Biochem. Biophys. Res. Commun., 23 (1966) 5. 12 J. Peisach and J. Mannering, A re-evaluation of the optical titrations of the 430 and 455 nm chromophores of ethylisocyanide complexes of mammarian hepatic cytochrome P-450, Mol. Pharmacol., 11 (1975) 818. 13 B.S.S. Masters, C.,H. Williams, Jr. and H. Kamin, The preparation and properties of microsomal TPNH cytochrome c reductase from pig liver, Methods Enzymol., 10 (1967) 565. 14 J. Cochin and J. Axelrod, Biochemical and pharmacological changes in the rat following chronic administration of morphine, nalorphine and normorphine, J. Pharmacol. Exp. Thex., 125 (1959) 105. 15 T. Nash, Colorimetric estimation of formaldehyde by means of the Huntzsch reaction, Biochem. J., 55 (1953) 416. 16 A. Trevor: Correlation of drug disposition with pharmacologic actions, in: B.N. LaDu, H.G. Mandel and E.L. Way (Eds.), Fundamentals of Drug Metabolism and Drug Disposition, Williams and Wilkins, Baltimore, 1971, pp. 591--599. 17 K.L. Netter and G. Seidel, An adaptively stimulated O-demethylating system in rat liver microsomes and its kinetic properties, J. Pharmacol. Exp. Ther., 146 (1964) 61. 18 S. Fujita and J. Peisach, Liver microsomal cytochromes P-450 and azoreductase activity, J. Biol. Chem., 253 (1978) 4512. 19 V. Ullrich and P. Weber, The O-dealkylation of 7-ethoxycoumarin by liver microsomes. A direct fluorometric test, Hoppe-Seyler's Z. Physiol. Chem., 353 (1972) 1171. 20 G. Otani, M.M. Abou-E1-Makarem and K.W. Bock, UDP-glucuronyltransferase in perfused rat liver and in microsomes. III. Effects of galactosamine and carbon tetrachloride on the glucuronidation of 1-naphthol and bilirubin, Biochem. Pharmacol., 25 (1976) 1293. 21 G.S. Rao, G. Haueter, M.L. Rao and H. Breuer, An improved assay for steroid glucuronyltransferase in rat liver microsomes, Anal. Biochem., 74 (1976) 35. 22 K. Weber and M. Osborn, The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis, J. Biol. Chem., 244 (1969) 4406.
143 23 S. Fazekas de St. Groth, R.G. Weber and A. Datyner, Two new staining procedures for quantitative estimation of proteins on electrophoretic strips, Biochim. Biophys. Acta: 71 (1963) 377. 24 M. Negishi, Y. Fujii-Kuriyama, Y. Ashiro and Y. Imai, Site of phenobarbital treated rats, Biochem. Biophys. Res. Commun., 71 (1976) 1153. 25 C. Hashimoto and Y. Imai, Purification of a substrate complex of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-treated rabbits, Biochem. Biophys. Res. Commun., 68 (1976) 821. 26 A. Poland, E. Glover and A~S. Kende, Stereospecific high affinity binding of 2,3,7,8 tetrachlorodibenzo-p-dioxin by hepatic cytosoL Evidence that the binding species is a receptor for induction of arylhydrocarbon hydroxylase, J. Biol. Chem., 251 (1976) 4936. 27 A.B. Okey, G.P. Bondy, M.E. Mason, G.F. Kahl, H.J. Eisen, T.M. Guenthner and D.W. Nebert, Regulatory gene product of the Ah locus, J. Biol. Chem., 254 (1979) 11636. 28 C. Ionannides and D.V. Parke, Mechanism of induction of hepatic microsomal drug metabolizing enzymes by a series of barbiturates, J. Pharm. Pharmacol., 27 (1975) 739. 29 L. Ernster and S. Orrenius, Sustrate induced synthesis of the hydroxylating enzyme system of liver microsomes, Fed. Proc., 24 (1965) 1190. 30 T. Matsubara, A. Touchi and Y. Tochino, Hepatic aminopyrine N-demethylase system: Further studies of assay procedure, Jap. J. Pharmacol., 27 (1977) 127. 31 G.J. Wishart and M.T. Campbell, Demonstration of two functionally heterogenous groups within the activities of UDP-glucuronyltransferase towards a series of 4 alkylsubstituted phenols, Biochem. J., 178 (1979) 443. 32 K.W. Bock, W. Frohling, H. Kemmer and B. Rexer, Effects of phenobarbital and 3-methylcholanthrene on substrate specificity of rat liver microsomal UDP-glucuronyltransferase, Biochim. Biophys. Acta, 327 (1973) 46. 33 J.R. Gillette, Factors that effect the stimulation of the microsomal drug enzymes induced by foreign compounds, Adv. Enzyme Reg., 1 (1963) 215.
129
T H E E F F E C T O F SUDAN III ON D R UG M E T A B O L I Z I N G ENZYMES* SHOICHI FUJITAa'**, JACK PEISACHa, HIDEKO OHKAWAb, YUKO YOSHIDAb, SHIGERU ADACHIb, TAKASHI UESUGIb, MASAHIKO SUZUKIc and TOKUJI SUZUKIc aDepartment of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, N Y 10461 (U.S.A.), bDepartment of Biopharmacy, Meiji College of Pharmacy, Setagaya-Ku, Tokyo, 154 (Japan) and CDepartment of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Chiba University, Chiba, 261 (Japan)
(Received March 2nd, 1983) (Revision received July 29th, 1983) (Accepted August 4th, 1983)
SUMMARY We have examined the induction of drug metabolizing enzymes in rat liver microsomes by azo dye, 1-(p-phenylazophenylazo)-2-naphthol {Sudan III). Marked increases were observed in the levels of c y t o c h r o m e P-448 as well as in p-nitroanisole O-demethylase (p-NAD), amaranth (AR) and neoprontosil reductases (NPR) and 7 - e t h o x y c o u m a r i n O-deethylase (ECD) activities. On th e o th er hand, a m i n o p y r e n e N-demethylase activity was n o t significantly increased. Further, induced ECD activity was inhibited 90% by a specific a n t i b o d y against c y t o c h r o m e P-448 while the inhibition observed with an a n t i b o d y against c y t o c h r o m e P-450 was less than 25%. Simultaneous administration o f Sudan III and 3 - m e t h y l c h o l a n t h e n e (3-MC) induced c y t o c h r o m e P-448 up to a level brought a b o u t by either Sudan III or 3-MC t r e a t m e n t alone. In contrast, Sudan III did n o t induce c y t o c h r o m e P - 4 4 8 in the 3-MC insensitive DBA/2 mouse. Solubilized microsomes f r o m Sudan III-treated rats showed an identical sodium d o d e c y l sulfate pol yacryl am i de gel electrophoretic (SDS-PAGE) pa t t e r n with those f r om 3-MC-treated animals. It is concluded *This work was supported in part by U.S.P.H.S. Grant HL-13399 from the National Heart and Lung Institute and in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. **Current address: Department of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Chiba University, Chiba, Japan 261. tTo whom correspondence should be sent. Abbreviations: APD, aminopyrine-N-demethylase; AR, amaranth reductase; DMBA, 7,12dimethylbenzanthracene; ECD, 7-ethoxycoumarin O-deethylase; 3-MC, 3-methylcholanthrene; p-NAD, p-nitroanisole O-demethylase; NPR, neoprontosil reductase; PB, phenobarbital; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Sudan III, 1-(p-phenylazophenylazo)-2-naphthol; ZAH, zoxazolamine hydroxylase. 0009-2797/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
130 that the c y t o c h r o m e P-448 induced in liver by Sudan III is very similar to that induced by 3-MC. Sudan III also induced UDP-glucuronyltransferase activity towards 1-naphthol and estradiol. It did not induce NADPH-cytochrome c reductase, nor any of the enzymes which constitute the microsomal electron transport chain except for c y t o c h r o m e P-448.
Key words: Drug metabolism -- Induction -- Cytochrome P-450 -- Cytochrome P-448 -- Sudan III -- UDP-glucuronyltransferase INTRODUCTION
The effect of the azo dye Sudan III on hepatic drug metabolism is of special interest as there have been a number of reports implicating this comp o u n d in the prevention of chemical carcinogenesis in rats [1,2]. Similar prevention has been observed with polycyclic aromatic hydrocarbons if they are administered a few days prior to exposure to carcinogenic doses [1,3]. These observations have led to the suggestion t h a t these agents act by inducing hepatic drug-metabolizing enzymes and thus accelerate the elimination of toxic metabolites. This was borne out, for example, by Conney and Levin [3] who showed that Sudan III treatment inhibited 7,12-dimethylbenzanthracene (DMBA) carcinogenesis by inducing the metabolism of this c o m p o u n d in liver. The fact t h a t Sudan III induces P-450 type cytochromes [4,5] seems rather contradictory to the fact that it prevents chemical carcinogenesis, because c y t o c h r o m e P-448 has been shown to activate an otherwise nontoxic c o m p o u n d to an ultimate carcinogen [6]. In order to resolve this contradiction, the nature of cytochrome P-450 induction and its effect on hepatic microsomal monooxygenase activities, as well as the induction of other enzyme activities in microsomes by Sudan III were investigated. EXPERIMENTAL PROCEDURES
Chemicals NADP, NADPH, NADH and glucose 6-phosphate were obtained from K y o w a Hakko Co. (Japan), glucose-6-phosphate dehydrogenase from Sigma and 7-ethoxycoumarin and 7-hydroxycoumarin from Aldrich. Ethylisocyanide was prepared according to the m e t h o d of Jackson and McKusic [7]. Sudan III was obtained from T o k y o Kasei (Japan) and was recrystallized from benzene. 3-MC was purchased from Eastman Kodak Co. Animals Male Wistar rats weighing 100 + 10 g were divided into 6 groups, each consisting of 4 rats. They were injected intraperitoneally for 3 days with either 3-MC (20 mg/kg/day) or Sudan III (20 mg/kg/day), each dissolved in 0.5 ml of corn oil or phenobarbital (PB) (80 mg/kg/day) dissolved in 0.5 ml of 0.9%
131 NaC1. Control groups were injected with corn oil or saline. Male C57BL/6Cr and DBA/2Cr mice were injected for 4 days with the same inducers with the doses of 40 mg/kg/day. Hepatic microsomes were prepared according to the m e t h o d of Omura and Sato [8,9]. Protein concentrations were determined by the L o w r y procedure [ 10].
Spectrophotometric studies CO difference spectra of the microsomal preparations were obtained by the method of Omura and Sato [8,9] and the contents of the cytochromes were determined using a millimolar extinction coefficient of 91 for A4s0-490. Cytochrome bs was assayed by difference spectroscopy of NADH reduced minus oxidized microsomes using a millimolar extinction coefficient of 185 for A42s-410 [8]. Ethyl isocyanide difference spectra [11,12] were obtained as follows: microsomes ( 0 . 4 - 0 . 8 mg protein/roll were suspended in 1.0 M potassium phosphate buffer at various pH-values. Sodium dithionite (2 mg/ml) was added and the sample was divided in half. Ethyl isocyanide was added to one half, with the other half as reference and the spectrum was obtained with a Cary 219 spectrophotometer. Enzyme assays NAD(P)H oxidase activity was determined from the absorption change at 340 nm. Assay mixtures contained 0.05 M potassium phosphate buffer (pH 7.4) 1 mM MgC12, a b o u t 0.6 mg/ml microsomal protein and 0.1 mM NAD(P)H. NAD(P)H-cytochrome c reductase was assayed according to the prc~ cedure of Masters et al. [13]. Assay mixtures in 0.05 M potassium phosphate buffer (pH 7.4) contained 5 pm c y t o c h r o m e c, 0.1 mM EDTA, 0.1 mM KCN and microsomal protein at a final concentration 0.1 mg/ml for the NADPH-cytochrome c reductase assay and 0.01 mg/ml for the N A D H - c y t o c h r o m e c reductase assay. Reactions were started by a d d i n g NAD(P)H to a final concentration of 0.1 mM. Increase in the absorption at 550 nm was followed using a Hitachi 200-20 spectrophotometer. Aminopyrine-N-demethylase (APD) was assayed at 37°C by a modification of the method of Cochin and Axelrod [14]. The assay mixture, in 0.1 M potassium phosphate buffer (pH 7.4) contained 10 mM glucose 6-phosphate, 10 units/ml of glucose-6-phosphate dehydrogenase, 2 mM MgCI:, 7.5 mM semicarbazide-HC1 (pH adjusted to 7.4 with NaOH), 1.0 mM aminopyrine and about 1.2 mg/ml microsomal protein. The mixture was preincubated for 5 min. The reaction was initiated by the addition of 400 pM (final concentration) NADP. Oxygen gas was blown across the surface of the reaction mixture during the 15-rain incubation period. The reaction was stopped b y the addition of 1 ml of 10% ZnSO4. Five minutes later, 1 ml of 0.5 M NaOH was added to the mixture which was then stirred and centrifuged at 1000 X g for 30 rain. Formaldehyde content in the supernatant was assayed by the method of Nash [15].
132 The assay method for zoxazolamine hydroxylase (ZAH) was essentially the same as that described by Trevor [ 16 ]. The assay mixture consisting of 4 mM MgC12, 10 mM glucose 6-phosphate, 2 units/ml of glucose-6-phosphate dehydrogenase, 0.1 mg/ml zoxazolamine and about 2 mg/ml microsomal protein in oxygen-saturated 0.1 M potassium phosphate buffer (pH 7.4) in a final volume of 1 ml, was preincubated for 5 min at 37°C in a shaking water bath. The reaction was started by the addition of 10 pl of 50 mM NADP. The incubation was carried out for 15 min and the reaction was stopped by the addition of 1 ml of 1 M NaOH. Non-hydroxylated zoxazolamine in the mixture was extracted by vigorous vortex mixing into 12 ml of n-heptane containing 1.5% isoamyl alcohol. The mixture was then centrifuged at 1000 X g for 10 min. Ten milliliters of the heptane layer was transferred to a centrifuge tube containing 4 ml of 0.1 M HCI, mixed well and centrifuged as above. Optical absorptions of the HC1 layers at 278 nm were measured using a Hitachi 340 spectrophotometer. The assay m e t h o d for p-nitroanisole O-demethylase (p-NAD) described by Netter and Seidel [17] was modified as follows. The assay mixture in a total volume of 1 ml contained essentially the same ingredients as those for the zoxazolamine hydroxylase assay as described above, p-Nitroanisole, 0.6 mM, was used as a substrate. Microsomal protein c o n t e n t in the mixture was about 1 mg/ml. The reaction was started with the addition of 10 ~l of 50 mM NADP after 5 min preincubation at 37°C. The reaction mixture was then incubated for 20 min and stopped by the addition of 1 ml of 10% ZnSO4 and 2 ml of saturated Ba(OH)2. The mixture was centrifuged for 30 min at 1000 X g, and the absorption of the supernatant was measured at 420 nm. A:naranth reductase (AR) and neoprontosil reductase (NPR) activities were assayed according to the method described by Fujita and Peisach [ 18]. ECD activity was assayed according to the fluorometric m e t h o d described by Ullrich and Weber [19] using a microsomal protein concentration of 0.5 mg/ml. The production of 7-hydroxycoumarin was followed with a Hitachi 204-A fluorometer with excitation at 360 nm and emission at 460 nm. UDP-glucuronyltransferase activity was assayed according to the methods of Otani et al. [20] and Rao et al. [21]. Disk-SDS-PA GE A microsomal preparation suspended in 0.05 M potassium phosphate buffer (pH 7.4) containing 30% glycerol and 1 mM EDTA was solubilized with sodium cholate (3 mg/mg microsomal protein) at 0°C. Polyethylene glycol 6000, 50%, was added to a final concentration of 8% and the mixture was stirred for 10 min and centrifuged for 45 min at 3500 X g. The pellet was discarded. Polyethylene glycol 6000 was then added to a final concentration of 16%. The mixture was stirred for 10 min and centrifuged at 105 000 X g for 1 h. The pellet was homogenized in the buffer described above. Disc-
133 SDS-PAGE of the solubilized microsomes was carried out according to the m e t h o d of Weber and Osborn [22]. The gel contained 7.5% acrylamide and 0.1% SDS. Electrophoresis was carried out at 20°C for 7.5 h at 6 mA/gel. Malachite green, 0.005%, was used as a marker. The gels were then fixed and stained with 0.025% coomasie brilliant blue R 250 by the m e t h o d of Fazekas de St. Groth et al. [23]. As molecular weight standards, bovine serum albumin (68 000), ovalbumin (45 000) and cytochrome c (12 000) from Boehringer Mannheim were used.
Preparation of anti-PB-P-450 and anti-3-MC-P-448 antibodies Immunoglobulin fractions containing antibodies to rat liver microsomal cytochromes P-450 and P-448 prepared from immunized rabbit serum were generous gifts from Dr. Y. Okada, Kyushu University, Japan. Purified protein used for rabbit immunization were obtained according to the methods of Negishi et al. [24] for cytochrome P-450, and Hashimoto and Imai [ 25] for c y t o c h r o m e P-448. Purified cytochromes (700 Ug) were mixed with the same volume of Freund's complete adjuvant (DIFCO) and were injected subcutaneously into male white rabbits. After 3 weeks, the rabbits were injected with 2 mg of cytochrome. A week later, the serum from the blood of the immunized animals was fractionated with a m m o n i u m sulfate {0--45%). Immunoglobulin fractions were dissolved in 0.9% NaC1/10 mM sodium phosphate buffer (pH 7.5) to a final concentration of about 40 rag/ ml. Ethoxycoumarin-O-deethylase activity was assayed according to the m e t h o d described above, except in the presence of various concentrations of antibodies 2.5--20 times the concentration of microsomal protein in the reaction mixture. The activity in the presence of equal concentrations of the control immunoglobulin from non-treated animals was taken as 100% activity. RESULTS
The induction of cytochrome P-448 A comparison was made of the CO difference spectra of reduced microsomes from Sudan III treated rats with those from 3-MC or PB treated animals. A 2 nm absorption shift from 450 nm to 448 nm was observed for samples from b o t h Sudan III and 3-MC treated animals. Table I summarizes the concentrations of P-450 t y p e c y t o c h r o m e in liver m i c r a somes from rats treated with PB, Sudan III or 3-MC. Marked increases were observed in microsomes from drug treated animals. 3-MC or Sudan III alone or Sudan III injected together with 3-MC induced cytochrome P-448, while PB induced c y t o c h r o m e P-450. Simultaneous treatment with 3-MC and Sudan III did not lead to any additive induction over each c o m p o u n d alone, suggesting t h a t Sudan III and 3-MC induce the same species of cytochroms P-448.
134 TABLE I EFFECT OF PB, 3-MC AND SUDAN III TREATMENT ON LIVER MICROSOMAL P-450 CYTOCHROMES IN WISTAR RATS AND DBA/2 AND C57BL/6 MICE Groups of animals were injected i.p. for 3 days with PB dissolved in PSS, or with 3-MC or Sudan III, each dissolved in corn oil, at the doses indicated in brackets. Contents of.P-450 type cytochromes were determined from the CO difference spectra of the reduced microsomes. Standard deviations were obtained from the individual differences for 4 animals. Cytochrome contents are expressed in nmol/mg microsomal protein. Animals
Treatment (mg/kg)
~max of CO Difference (nmol]mg)
Cytochrome P-450 (nmol/mg)
Wistar
PSSa
450
0.47 +_0.01
Rats
Corn oil PB (80) 3-MC (20) 3-MC (40) Sudan III (20) 3-MC (20) + Sudan III (20)
450 450 448 448 448 440
0.53 1.57 1.19 1.25 1.10 1.27
DBA/2 Mice
Corn oil 3-MC (40} Sudan III (40)
450 450 450
0.50 +0.10 0.68 +_0.10 0.60 + 0.05
C57BL/6 Mice
Corn oil 3-MC (40) Sudan III (40)
450 448 448
0.65 _+0.03 1.48 _+0.06 1.11 +_0.07
+- 0.08 _+0.22 _+0.11 +-0.07 +_0.04 +_0.22
a Physiological saline.
T r e a t m e n t with either 3-MC or S u d a n I I I did n o t alter t h e levels o f P - 4 5 0 t y p e c y t o c h r o m e s n o r cause t h e spectral shift of 2 n m in t h e CO d i f f e r e n c e s p e c t r u m for D B A / 2 mice, the ' n o n - i n d u c i b l e ' strain (Table I). P o l a n d et al. [ 2 6 ] d e m o n s t r a t e d t h a t this m o u s e has a l o w level or a p o o r a f f i n i t y o f c y t o s o l i c r e c e p t o r p r o t e i n , the m a j o r r e g u l a t o r y gene p r o d u c t o f t h e m u r i n e Ah locus w h i c h regulates t h e i n d u c t i o n o f c y t o c h r o m e P - 4 4 8 b y p o l y c y c l i c a r o m a t i c c o m p o u n d s such as 3-MC, b e n z p y r e n e , ~-naphthoflavone or 2 , 3 , 7 , 8 - t e t r a - c h l o r o d i b e n z o - p - d i o x i n [ 2 7 ] . Thus t h e lack o f effect with S u d a n III is c o m p a r a b l e t o t h a t observed with 3-MC.
Ethyl isocyanide difference spectra A l t h o u g h t h e CO d i f f e r e n c e s p e c t r a o f m i c r o s o m e s f r o m S u d a n I I I a n d 3-MC t r e a t e d animals were a l m o s t identical, t h e p H d e p e n d e n c e of t h e e t h y l i s o c y a n i d e d i f f e r e n c e spectra was n o t t h e same. Figure 1 shows the e f f e c t o f p H o n t h e a m p l i t u d e s o f a b s o r p t i o n m a x i m a near 430 n m a n d 455 n m o f e t h y l i s o c y a n i d e d i f f e r e n c e spectra, a n d t h e A430/A4ss ratios at p H 7 are collected in Table II. F o r m i c r o s o m e s f r o m 3-MC t r e a t e d animals,
135 both maxima had equal intensity at pH 6.70, the crossover point for the titration curves. With Sudan III treatment, this occurred at pH 7.03; for animals treated with Sudan III plus 3-MC (pH 6.75). For comparison, w e show data for microsomes f~om PB treated animals where the pH crossover was at 7.5. The values reported here are within range of pH crossovers reported for microsomes after 3-MC treatment ( 6 . 4 - 6 . 7 ) and after PB treatment (7.4--7.8) [12]. Our results reflect the tendency for microsomes containing larger concentrations of cytochrome P-448 to show a lower pH intercept than those t h a t contain c y t o c h r o m e P-450.
Components o f the microsomal electron transport chain Table III shows the enzyme activities of various components of the microsomal electron transport chain and of cytochrome bs content. PB treatment [ i*-~
(a/
J05 ! 670
0b
n
I
q
~
I
-
-
~
005 ~
;
-
(b)
-b
~d ~
u
8 V
005
(c)
703
£
" ~ .
o
~
i
(d)
F
•
i
7so
~
o,o
"
55
60
65
I
70
'5
80
85
pH
Fig. I. The effect of pH on the absorption maxima near 430 and 455 nm of ethylisocyanide difference spectra of rat liver microsomes. Ethylisocyanide difference spectra at various values of pH for (a) 3-MC treated animals, (b) 3-MC + Sudan III treated animals, (c) Sudan III-treated animals and (d) PB treated animals were obtained according to the method described in the text. The A,30__~s0/microsomal protein concentration (o o) and A,55_,s0/microsomal protein concentration (o o) were obtained from the spectra and were plotted against pH-values.
136 TABLE II EFFECT OF PB, 3-MC AND SUDAN HI TREATMENTS ON A4ss/A430 RATIO OF THE ETHYLISOCYANIDE DIFFERENCE SPECTRA OF RAT LIVER MICROSOMES AT pH 7.0 The treatments of animals are described in the legend to Table I. The method tlsed to obtain ethylisocyanide difference spectra is described in the text. Treatment
PSS a Corn oil PB 3-MC Sudan III 3-MC -~ Sudan III
h m a x in the 430 nm region
h m a x in the 455 nm region
hA4ss_4s 0 ~ A430_480
428 428 428 429 429 429
455 455 455 453 453 453
0.30 0.31 0.49 1.52 0.97 1.51
a Physiological saline.
c a u s e d a 2-fold increase in N A D P H o x i d a s e levels w h i c h was n o t o b s e r v e d a f t e r t r e a t m e n t w i t h either S u d a n III or 3-MC. O n t h e o t h e r hand, 3-MC t r e a t m e n t c a u s e d a small d e c r e a s e in N A D H o x i d a s e activity, while S u d a n III and PB t r e a t m e n t h a d no effect. PB i n d u c e d N A D P H - c y t o c h r o m e c r e d u c t a s e activity, c o n f i r m i n g t h e results of o t h e r s [ 2 8 , 2 9 ] , b u t slightly d e c r e a s e d t h e N A D H - c y t o c h r o m e c r e d u c t a s e activity. A slight increase in c y t o c h r o m e bs c o n t e n t was seen in r a t liver m i c r o s o m e s f r o m a n i m a l s t r e a t e d w i t h t h e inducers u s e d in t h e s e studies. It s h o u l d be n o t e d t h a t S u d a n I I I did n o t a p p r e c i a b l y a l t e r a n y of t h e e n z y m e activities o f t h e m i c r o s o m a l e l e c t r o n t r a n s p o r t s y s t e m t h a t d i r e c t e l e c t r o n s t o c y t o c h r o m e P-450, while BP a n d 3-MC t r e a t m e n t did.
Drug metabolism APD, ECD, p - N A D , Z A H , A R a n d N P R activities o f m i c r o s o m e s f r o m t h e rats t r e a t e d w i t h various inducers are s h o w n in T a b l e IV. A m a r k e d increase in A P D activity was seen in m i c r o s o m e s f r o m PB t r e a t e d animals [ 3 0 ] , b u t n o t f r o m Sudan I I I or 3-MC t r e a t e d animals. A R a n d N P R w e r e i n d u c e d b y PB, 3-MC and S u d a n I I I a n d t h e s e activities r e f l e c t t h e levels o f t o t a l P - 4 5 0 t y p e c y t o c h r o m e s in individual m i c r o s o m a l p r e p a r a t i o n s . I n d u c t i o n o f E C D a n d p - N A D activities w e r e m o s t p r o m i n e n t in liver m i c r o s o m e s f r o m rats t r e a t e d w i t h either 3-MC or S u d a n III. T h e s e s u b s t r a t e s are k n o w n to be m e t a b o l i z e d by c y t o c h r o m e P - 4 4 8 m o r e e f f i c i e n t l y t h a n P B - i n d u c e d c y t o c h r o m e P-450. 3-MC m i c r o s o m e s h a d t h e highest Z A H activity ( a n o t h e r P-448 d e p e n d e n t r e a c t i o n ) b u t S u d a n I I I m i c r o s o m e s h a d l o w e r a c t i v i t y t h a n PB m i c r o s o m e s . Z A H a c t i v i t y p e r n m o l P - 4 5 0 h o w e v e r , was higher in S u d a n III m i c r o s o m e s t h a n in PB m i c r o s o m e s (5.5 vs. 4.2 n m o l / m i n / n m o l P-450).
6.57 6.14 14.37 8.34 6.72 8.62
+0.01 _+ 1.07 -+ 1.57 _+ 1.71 _+0 . 2 4 -+0.74
NADPH oxidase nmol/mg/min
1039.3 927.4 512.4 674.2 896.8 775.5
_+ 1 5 0 . 8 _+ 40.6 _+1 0 5 . 3 _+ 1 2 9 . 0 _+ 6.1 _+ 165.4
NADH Cytocbrome c reductase (nmol/mg/min) 77.91 89.10 123.83 75.55 91.44 87.87
_+ 7.84 _+ 1 2 . 0 8 _+ 1 0 . 0 9 _+ 5.86 _+ 4 . 6 7 _+ 1 . 9 6
NADPH Cytochrome c reductase (nmol/mg/min)
0.40 0.43 0.51 0.50 0.60 0.57
_+0 . 0 6 _+ 0.02 _+0 . 0 4 _+0.02 _+0.06 _+0.10
C y t o c h r o m e b5 (nmol/mg)
a Corn oil.
Control* PB 3-MC S u d a n III
Treatment
2.51 5.77 2.26 2.68
APD
_+0.22 _+1.44 _+0.30 _+0.07
0.27 1.37 4.33 4.04
ECD _+0.015 _+0.052 _+0.139 -+0.062
E n z y m e activities ( n m o l / m g / m i n )
0.86 1.51 2.11 2.01
-+0.07 -+0.08 _+0.02 +0.06
p-NAD
4.60 6.72 8.81 6.10
ZAH -+0.08 +0.10 _+0.18 -+0.10
2.25 7.20 5.51 5.20
AR -+0.06 -+0.52 _+0.30 _+0.21
4.10 11.80 7.20 7.00
NPR -+0.12 + 0.20 +_0.31 _+0.30
T h e assay m e t h o d s are d e s c r i b e d in t h e text. A P D a c t i v i t y is e x p r e s s e d in n m o l H C H O f o r m e d / m g m i c r o s o m a l p r o t e i n / m i n . E C D activity is expressed in n m o l 7-OH c o u m a r i n f o r m e d / m g m i c r o s o m a l p r o t e i n / m i n , p - N A D [ 1 7 ] , Z A H [ 1 6 ] , A R a n d N P R [ 1 8 ] activities are expressed as before. Values are e x p r e s s e d as m e a n activity _+ S.D.
E F F E C T O F PB, 3-MC A N D S U D A N HI ON H E P A T I C D R U G M E T A B O L I Z I N G E N Z Y M E S
T A B L E IV
a Physiological saline.
9.95 7.71 9.18 5.51 8.75 5.74
PSS a Corn oil PB 3-MC S u d a n III 3 - M C + S u d a n III
_+2.98 _+ 2.07 + 2.82 _+0.12 _+ 1.14 +1.10
NADH oxidase nmol/mg/min
Pretreatment
E n z y m e assay a n d t h e d e t e r m i n a t i o n o f c y t o c h r o m e b 5 c o n t e n t were carried o u t a c c o r d i n g t o t h e m e t h o d s d e s c r i b e d in t h e t e x t . Values are expressed as m e a n values _+ S.D.
E F F E C T OF PB, 3-MC A N D S U D A N III P R E T R E A T M E N T O N H E P A T I C M I C R O S O M A L E L E C T R O N T R A N S P O R T
T A B L E HI
b-4 C~
138 The patterns of the induction of drug metabolism by Sudan III and 3-MC were very similar, and the potency of the inductive ability of these two compounds in the doses used were very similar as well.
Antibodies against cytochromes P-448 and P-450 Figure 2 shows the effects of anti-PB-P-450 and anti-3-MC-P-448 antibodies on the activity of ECD in each of the microsomal preparations. As expected, anti-PB-P-450 antibody inhibited O-deethylation in PB microsomes up to 80% (b), while inhibition was less for control microsomes (a). Considerably less inhibition was observed with microsomes from rats treated with (c} 3-MC, (d) Sudan III or (e) 3-MC + Sudan III. The anti-3-MC-P-448 antibody, on the other hand, inhibited O-deethylation by PB microsomes up to 30% and 3-MC microsomes, Sudan III microsomes, and 3-MC + Sudan III microsomes, up to 90%. It is noteworthy that both antibodies inhibited O-deethylase activity of corn oil control microsomes up to 60% and that the anti-3-MC-P-448 antibody was not more efficient than the anti-PB-P-450 antibody in inhibiting this reaction. This suggests that the mixture o f cytochromes P-450 present in control microsomes may be different from that in PB microsomes. Disc-SDS-PA GE Figure 3 shows the disk-SDS-PAGE of solubilized and polyethylene glycol 6000 fractionated microsomes from rats treated with Sudan III and 3-MC. A band at mol. wt. 56 000 is prominent and a less prominent band with a
(a) Control
°,/o
-(b) PB
(c) 3-MC
~I
I(c) 3-~c Sudan :11
] I
100
\ 5O I
\ \
!
,
-\ ",o
Ot . . . . . 0
10
\
\, ',\
\
:
\
,\
_
i R
J 2O
10
20 0
10
20 0
10 20 0 10 20 timc---~ con ot microsomal protein
Ooncentration ot Antibodies
Fig. 2. Effects of antibodies against cytochromes P-450 and P-448 on ECD activities of liver microsomes from (a) corn oil, (b) PB, (c) 3-MC, (d) S u d a n III a n d (e) 3-MC + S u d a n III treated rats. E n z y m e activities in the presence of anti-P-450 (o .o) or antiP-448 ( , . . . . , ) antibodies were compared to those in the presence of the corresponding c o n c e n t r a t i o n s of non-specific i m m u n o g l o b u l i n fractions from the serum of noni m m u n i z e d rabbits, expressed as 100%. The assay m e t h o d is described in the text.
139 mol. wt. 54 000 is also observed. Electrophoretic patterns of these two microsomal fractions were identical. There were no new identifiable species of hemoprotein with these molecular weights induced by Sudan III other than those induced with 3-MC.
UDP-glucuronyltransferase activity Table V shows the effect of inducers of cytochrome P-450 and P-448 on hepatic microsomal UDP-glucuronyltransferase activity. Sudan III induced UDP-glucuronyltransferase activity towards 1-naphthol and estradiol. 3-MC, on the other hand, induced activity only towards 1-naphthol, a typical substrate of so-called 'late fetal type' UDP-glucuronyltransferase. Estradiol, on the other hand, is a typical substrate of 'neonatal type' UDP-
PSS
PB
S III
MC
--68
000
~45
000
C
Fig. 3. Disk-SDS-PAGE of solubilized liver mierosomes f r o m rats treated with PB, 3-MC, and S u d a n III. Microsomes, suspended in 0.1 mM E D T A , 30% glycerol, were solubilized with s o d i u m cholate (3 m g / m g m i c r o s o m a l protein). Electrophoresis was carried o u t f r o m top to b o t t o m in the presence of SDS as described in the text. Molecular weight standards are indicated o n the right of the figure. PSS, solubilized microsomes f r o m the livers of physiological saline solution treated control rats; PB, f r o m PB-treated rats; MC, f r o m 3-MC-treated rats; SIII, f r o m S u d a n III-treated rats; C, from corn oil-treated control rats.
140 TABLE V E F F E C T O F PB, 3-MC A N D S U D A N HI ON U D P - G L U C U R O N Y L T R A N S F E R A S E A C T I V I T I E S IN R A T L I V E R M I C R O S O M E S P r e t r e a t m e n t s of animals and the assay m e t h o d s are described in E x p e r i m e n t a l Procedures. C y t o c h r o m e P-450 c o n t e n t is expressed in n m o l / m g microsomal protein. UDP-glucuronyltransferase activity is expressed in nmol glucuronide formed/rag m i c r o s o m a l protein/rain. Values are expressed as m e a n +_S.D. Treatments
PSS a Corn oil PB 3-MC Sudan III
P-450 contents
0.47 0.45 1.00 1.18 0.94
+_0.01 +_0.03 +0.12 +_0.20 +_0.03
UDP-glucuronyltransferase activity towards 1-Naphthol
Estradiol
7.6 7.5 10.9 20.0 19.6
0.14 0.13 0.26 0.16 0.24
+_0.70 +_0.78 +_0.56 +_0.92 +_0.44
+_0.015 +_0.013 +0.004 +_0.010 +_0.013
a Physiological saline.
glucuronyltransferase [ 31 ]. These activities of different types were reported to be differentially induced by 3-MC and PB respectively [32] in agreement with our results. DISCUSSION
In the present study, we show that Sudan III is an inducer of c y t o c h r o m e P-448" with a potency similar to that of 3-MC for ECD, p-nitroanisole O-demethylation, and amaranth and neoprontosil reduction**. On the other hand, Sudan III is not an inducer of APD demethylase (Table IV), an activity associated with c y t o c h r o m e P-450 [30]. The pH intercepts of the absorption maxima near 430 nm and 455 nm in the ethylisocyanide difference spectra (Fig. 1) for 3-MC and Sudan III microsomes are lower in value than the intercepts for PB and control microsomes [ 12 ]. Antibodies against purified liver microsomal c y t o c h r o m e P-448 induced b y c-naphthoflavone inhibit the ECD activity of liver microsomes from either Sudan IH or 3-MC treated rats while the antibody against purified c y t o c h r o m e P-450 is n o t as effective {Fig. 2). Disk-SDS-PAGE of microsomes from rats treated with Sudan III or 3-MC showed identical patterns (Fig. 3). All of these observations strongly suggest that liver microsomal cytochromes P°448 from 3-MC- and Sudan IIItreated animals are similar. *The terms 'cytochrome P - 4 5 0 ' and ' c y t o c h r o m e - P - 4 4 8 ' are operational definitions referring to the m i x t u r e o f m i c r o s o m a l P-450 t y p e c y t o c h r o m e s that give rise to absorptions at 450 nm and 448 nm, respectively, in the CO-difference spectrum. **These m i c r o s o m a l azoreductase activities are greater for PB-induced animals than for either 3-MC- or Sudan III-induced animals since t h e y depend on the level of total P-450 t y p e c y t o c h r o m e s which is greatest in PB-induced animals [ 18 ].
141 It is of interest to note that the mechanism o f induction of c y t o c h r o m e P-448 with Sudan III appears to be similar to the induction with 3-MC. The simultaneous administration of 3-MC and Sudan III does not lead to additive induction, as takes place after simultaneous PB and b e n z o [ a ] p y r e n e treatm e n t [33]. The DBA/2 strain of mice which is non-responsive to 3-MC treatment in the induction of cytochrome P-448 is also not responsive to Sudan III treatment. Poland et al. [26] showed that this non-responsiveness is due to the genetic defect of a polycyclic hydrocarbon inducer binding protein which is present in the cytosol. Since Sudan III failed to induce c y t o c h r o m e P-448 in this strain of mice, it is likely that the induction by Sudan III is also mediated by this protein. It is curious, though, that a structurally different c o m p o u n d like Sudan III may be able to bind to this same protein in a way similar to that of a polycyclic aromatic hydrocarbon. Other than c y t o c h r o m e P-448, Sudan III does not alter any o f the enzyme activities of the microsomal electron transport chain. Thus with Sudan III, the induction of monooxygenase activity is largely due to increased cytochrome P-448 levels. It has been known that treatment by Sudan III, and other aromatic compounds, prevents adrenal injury and mammary cancer that it caused by chemical carcinogens, such as DMBA [1,2]. Conney and Levin [3] reported that the metabolism of this polyaromatic c o m p o u n d in the liver is induced by Sudan III. It seems contradictory that an inducer of c y t o c h r o m e P-448 prevents chemical carcinogenesis if given prior to exposure to a known aromatic hydrocarbon carcinogen, since these are usually activated by c y t o c h r o m e P-448 to produce ultimate carcinogens [6 ]. It is possible that Sudan III induces enzymatic activities other than those catalyzed by c y t o c h r o m e P-448, which affect the lifetime of active intermediates. Indeed, Sudan III was shown to induce UDP-glucuronyltransferase activities towards both 1-naphthol and estradiol. The enhancement of activity by Sudan III suggests that its role in the prevention of carcinogenesis m a y be via this mechanism. In conclusion, Sudan III causes the enhancement o f drug metabolism by inducing c y t o c h r o m e P-448, an enzyme of the phase I drug-metabolizing reactivity, and UDP-glucuronyltransferase an enzyme of phase II reactivity. This Sudan III mediated induction of activating and inactivating steps of drug metabolism may be involved in the prevention of chemical carcinogene~ sis by this dye. Regardless of its usefulness as an anti-cancer agent, Sudan III is an interesting c o m p o u n d for the studies of mechanisms of induction of phase I and phase II drug metabolizing enzymes and may be a possible link between them. Finally, since Sudan III is not a carcinogen, it may be a safer c o m p o u n d to use in the laboratory for the study of c y t o c h r o m e P-448 induction than the highly carcinogenic compound, 3-MC. REFERENCES
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