Induction of hepatic microsomal drug metabolism by azo compounds: A structure-activity relationship

Chem.-Biol. Interactions, 52 (1984) 15--37 Elsevier Scientific Publishers Ireland Ltd.

15

I N D U C T I O N O F H E P A T I C M I C R O S O M A L D R U G M E T A B O L I S M BY AZO COMPOUNDS: A STRUCTURE-ACTIVITY RELATIONSHIP*

SHOICHI FUJITA a, MASAHIKO SUZUKIa, JACK PEISACHb and TOKUJI SUZUKIa aDepartment of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoicho, Chiba, 260 (Japan) and bDepartment of Molecular Pharmacology, Albert Einstein College of Medicine, Yeshiva University, Bronx, N Y 10461 (U.S.A.) (Received November 4th, 1983) (Revision received July 19th, 1984) (Accepted July 25th, 1984)

SUMMARY T h e s t r u c t u r e - a c t i v i t y relationship o f 40 azo c o m p o u n d s in their ability t o i n d u c e c y t o c h r o m e P - 4 4 8 and associated m o n o o x y g e n a s e activities, as well as U D P - g l u c u r o n y l t r a n s f e r a s e ( U D P G T ) activity, was investigated. Regardless o f t h e i r s t r u c t u r e , h y d r o p h i l i c azo d y e s and lipophilic a z o b e n z e n e derivatives were n o t able t o i n d u c e these e n z y m e activities. O n l y t h o s e lipophilic azo d y e s with 1 - a z o - 2 - n a p h t h o l or 1 - a z o - 2 - n a p h t h y l a m i n e m o i e t i e s were able t o i n d u c e c y t o c h r o m e P - 4 4 8 and related m o n o o x y g e n a s e activities, as well as U D P G T activity. T h e e x t e n t o f i n d u c t i o n is c o m p a r a b l e to or greater t h a n t h a t caused b y 3 - m e t h y l c h o l a n t h r e n e (3-MC). It is suggested t h a t t h o s e azo d y e s capable o f i n d u c i n g P - 4 5 0 t y p e c y t o c h r o m e s can f o r m c o p l a n a r s t r u c t u r e s with t h r e e fused, 6 - m e m b e r e d rings t h r o u g h intramolecular h y d r o g e n b o n d i n g . These s t r u c t u r e s are a n a l o g o u s t o p o l y c y c l i c arom a t i c h y d r o c a r b o n s t h a t can also induce.

K e y words: I n d u c t i o n - A z o c o m p o u n d s - - C y t o c h r o m e P - 4 5 0 - D r u g m e t a b o l i s m - - B a y region - - A h locus - S t r u c t u r e - a c t i v a t i n g relationships

*This work was supported in part by grants in aid for scientific research from the Ministry of Education, Science and Culture of Japan to S.F. and U.S.P.H.S. Grant HL13399 from the National Heart and Lung Institute to J.P. A preliminary form of this study appeared in Biochem. Pharmacol., 30 (1981 ) 1147--1150. Abbreviations: APD, aminopyrine N-demethylase; BP, benzo[a]pyrene; ECD, 7-ethoxycoumarin O-deethylase; HBH, hexobarbital hydroxylase; 3-MC, 3-methylcholanthrene; p-NAD, p-nitroanisole O-demethylase; PB, phenobarbital; PCB, polychlorinated biphenyl; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxine; UDPGT, UDP-glucuronyltransferase; ZAH, zoxazolamine hydroxylase. 0009-2797/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

16 INTRODUCTION The cytochromes P-450 and associated conjugating enzymes, play a crucial role in detoxification and activation of xenobiotics. These drug-metabolizing enzymes are generally inducible, and over 200 substances, including the prototypical inducers phenobarbital (PB) and 3-MC, are known to induce metabolism of themselves and/or other compounds [1]. PB is a general inducer of the monooxygenase activities of several cytochromes P-450 [2] and also of NADPH cytochrome c reductase [3], while 3-MC is a more specific inducer of a limited number of cytochrome P-450 isozymes, including cytochrome P-448 [4]. A recent review by Snyder and Remmer [5] as well as numerous reports on the induction of cytochrome P-448 suggest that there are several classes of inducers. One such class consists of polycyclic aromatic hydrocarbons which have a coplanar structure with more than two fused, six-membered rings, such as are found in 3-MC and benzo[a] pyrene (BP) [6]. Structurally similar fl-naphthoflavone is included in this class [7 ]. A second class includes polyhalogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzop-dioxine (TCDD) and polychlorinated biphenyls (PCBs) [8,9] *. Another class of cytochrome P-448 inducers was suggested from the experiments of Ichikawa and Yamamo [11] who reported that an azo compound, Sudan III (1-phenylazophenylazo-2-naphthol) induces cytochrome P-450 in rabbit liver. We have recently shown that Sudan III, like 3-MC, induces cytochrome P-448 [12]. Since this c o m p o u n d does not contain halogen and only possesses a fused, two-ring structure, rather than a three-ring structure, Sudan III and other azo compounds might then be classified as unique inducers of cytochrome P-448. However, as only certain azo compounds induce cytochrome P-448 [13,14], it was of interest to study the structure-activity relationship of the induction of drug metabolism by these materials. It was also of interest to compare the patterns of induction by azo compounds to those by 3-MC using several monooxygenase activities as parameters. EXPERIMENTALPROCEDURE

Azo compounds Aromatic azo compounds used in this study are listed in Table I. Compounds I, II, III, VI, VII, X, XII, XIII, XVII--XXV, XXXI and XL are commercially available. Compounds IV, V, VII, IX and XI were generous gifts from Dr. Yukio Mori of Gifu College of Pharmacy, Japan whose contribution is gratefully acknowledged. Other compounds except XXIX and XXX *A recent report [10] indicates that a few compounds with single ring structures can effectively compete with TCDD and bind to the induction specific cytosolic receptor protein. It awaits further investigations to confirm if these compounds represent another class of cytochrome P-448 inducers.

17 were synthesized in this laboratory by coupling diazotized toluidines and naphthols. Compounds XXIX and XXX were synthesized according to the methods of MiSller et al. [15] and Badger et al. [16]. Lipophilic dyes* were repeatedly recrystallized from acetone and benzene or alcohol while hydrophilic dyes were recrystallized from H20. Purity o f lipophilic dyes was assessed by thin-layer chromatography with the same solvent system as that used for recrystallization. Melting points for all compounds were compared with literature values.

Treatment o f animals F o r t y four groups of rats, each group consisting of 4 animals, were injected i.p. for 4 days with azo dyes (40 mg/kg/day) or with 3-MC (40 mg/kg/ day), each dissolved in corn oil, or with PB (80 mg/kg/day) dissolved in physiological saline solution. The animals were sacrificed on the fifth day, microsomes were prepared and pooled from livers of each group of animals and cytochrome P-450 content was determined according to the m e t h o d of Omura and Sato [17]. Microsomal protein concentrations were determined by the m e t h o d of Lowry et al. [18]. Enzymatic assays UDP-glucuronyltransferase activity was measured as previously described [ 19] using p-nitrophenol as substrate. The assay m e t h o d for zoxazolamine hydroxylase was essentially the same as that described by Trevor [20]. 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 o f 1 ml, was preincubated for 5 min at 37°C in a shaker bath. The reaction was started by t h e addition o f 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 mixing with a vortex mixer into 12 ml of n-heptane containing 1.5% isoamyl alcohol. The mixture was then centrifuged at 2000 × g for 10 min. Ten milliliters of the heptane layer were transferred to a centrifuge tube containing 4 ml of 0.1 M HC1. The contents of the tube were 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 described by Netter and Seidel [21] for p-nitroanisole O-demethylase was modified as follows: the assay mixture, in a total volume o f 1 ml, contained essentially the same ingredients as those for the zoxazolamine hydroxylase assay as described above, p-Nitroanisole, 0.6 mM, was

*The lipophilic dyes used in this study had partition coefficients less than 10% in a 0.1 M sodium phosphate buffer, pH 7.4/n-octanol system.

TABLE I AZO COMPOUNDS USED IN THIS STUDY I.

II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV. XVI. XVII. XVIII. XIX. XX. XXI. XXII. XXIII. XXIV. XXV. XXVI. XXVII. XXVIII. XXIX. XXX. XXXI. XXXII. XXXIII. XXXIV. XXXV. XXXVI. XXXVII. XXXVIII. XXXIX. XL.

Azobenzene 2,2'-Dihydroxyazobenzene Phenylazoresorcinol 3,3'-Dihydroxymethylazobenzene 3,3'-Dichloromethylazobenzene 4-Aminoazobenzene 4-Dimethylaminoazobenzene (Butter yellow) 3'-Methyl-4-dimethylaminoazobenzene 3'-Hydroxymethyl-4-dimethylaminoazobenzene 2-[ (4-Dimethylamino)phenylazo ]benzoic acid (Methyl red) 3;-Chloromet hyl-4-d imet hylaminoazobenzene N-(Cyanoethyl)ethyl-4-(4'-uitro)azobenzene (Dispers orange 25) 1-Phenylazo-2-naphthol (Sudan I) 1 -o-Tolu eneazo-2-napht hol 1 -m-Tolueneazo-2-napht hol 1 -p-Tolueneazo-2-napht hol 1-[(2,4-Dimethylphenyl)azo ]-2-naphthol (Sudan II) 1-Phenylazophenylazo-2-naphthol (Sudan III) 1 -[ 2-Methyl-4-(2-methylphenylazo)phenylazo ] -2-naphthol (Sudan IV) 1-[2-Methyl-4-(2,5-dimethylphenylazo)phenylazo]-2-naphthol (Oil red EGN) 1-[ 2,5-Dimethyl-4-(2,5-dimethylphenylazo)phenylazo ] -2-naphthol (Oil red 0) 1-(2-Thiazolylazo )-2-nap ht hol 1 -(2-Pyridylazo)-2-naphthol 1-o-Tolueueazo-2-naphthylamine (Oil yellow OB) N-Ethyl-I- [ 4-(phenylazo )phenylazo ] -2 -naphthylami ne 4-o-Tolueneazo-1 -naphthol 4-m-Tolueneazo-1 -naphthol 4-p-Tolueneazo-1 -naphthol 1 -Phenylazo-2-methoxynapht hale ne 1-Phenylazo naphthalene 4-Dimethylaminoazobenzene-4'-sulphonic acid, sodium salt (Methyl orange) 4-[(2-Hydroxy-l-naphthaleneyl)azo]benzenesulphonic acid monosodium salt (Orange II) 6-Hydroxy-5- [ (4-sulphophenyl)azo ] -2-naphthalenesulphonic acid, disodium salt 7-Hydroxy-8-(phenylazo)-l,3-naphthalenedisulphonic acid disodium salt (Orange G) 4-[(2,4-Dimethylphenyl)azo ] -3-hydroxy-2,7-naphthalene-disulphonic acid, disodium salt (Ponceau MX) 4-Phenylazophenylazo-3-hydroxy-2,7 -naphthalenedisulphonic acid disodium salt (Ponceau SS) 4-[(4-Hydroxy-l-naphthalenyl)azo ] benzenesulphonic acid, monosodium salt (Orange I) 3-Hydroxy-4-[ (4-sulpho-l-naphthalenyl)azo ]-2,7-naphthalenedisulphonie acid, trisodium salt (Amaranth) 3-[(2,4-Dimet hyl-5-sulphophenyl)azo ] -4-hydroxy-1 -naphthalenesulpho nic acid, disodium salt (Ponceau SX) 3,3'-[ [ 3,3'-Dimethyl(1,1'-biphenyl)-4,4'-diol]bis(azo)]bis(5-amino-4hydroxy)-2,7-naphthalenedisulphonic acid, tetrasodium salt (Trypan blue)

19 used as a substrate. Microsomal protein content in the mixture was about 1 mg/ml. After 5 min preincubation at 37°C, the reaction was started by the addition of 10 pl of 50 mM NADP. The mixture was incubated for 20 min and the reaction was 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 2000 X g and the absorption of the supernatant was measured at 420 nm. Aminopyrine N~lemethylase activity was assayed by the m e t h o d of Cochin and Axelrod [22] with a slight modification. The assay mixture contained 10 mM glucose 6-phosphate, 2 units/ml of glucose-6-phosphate dehydrogenase, 2 mM MgC12, 7.5 mM semicarbazide-HC1 (pH adjusted to 7.4 with NaOH), 1.0 mM aminopyrine and about 1.2 mg/ml microsomal protein in 0.1 M potassium phosphate buffer (pH 7.4). After preincubation of a 2.5 ml reaction mixture for 5 min at 37°C, the reaction was started by the addition of 20 pl of 50 mM NADP. It was stopped with the addition of 1 ml each of 10% ZnSO4 and 0.5 M NaOH. The stirred mixture was then centrifuged at 2000 X g for 30 min. Formaldehyde content in the supernatant was determined by adding Nash reagent to the supernatant and incubating at 60°C for 30 min as described [22]. 7-Ethoxycoumarin O-deethylase and hexobarbital hydroxylase activities was assayed according to the previously described methods [ 23,24]. RESULTS Table II indicates the effects of prototypical inducers on the levels of hepatic microsomal P-450 type cytochromes and on UDPGT activity towards p-nitrophenol, enzymes of phase I and phase II reactions of microsomal metabolism, respectively. The data in Table II should be compared with those in Tables III--X which indicate the effects of various azo compounds on induction. Table IV shows the effects of lipophilic azo compounds possessing a 1-phenylazo-2-naphthol structure. With the exceptions of XIX, XX and XXI, each caused more than a 2-fold increase in the levels of P-450 type cytochromes (here c y t o c h r o m e P-448), and more than a 5-fold increase in UDPGT activity. Compounds XIX, XX and XXI did not induce cytochrome P-448, and the induction of UDPGT activity was slight. Since these compounds are considerably larger as compared to the dyes which are k n o w n to induce, we t h o u g h t that molecular size might be one of the determinants for induction. In Fig. 2 we plotted the relationship between the size of the azo dyes in Table IV, as calculated according to Arcos et al. [6], and their potential activities as inducers as determined from percentage increase in P-450 content over control values. An inverse correlation was obtained between the size of the compounds and their ability to induce (r-- 0.926). Table V shows the effects of 1-thyazolylazo-2-naphthol (XXII) and 1pyridylazo-2-naphthol (XXIII). Although these compounds possess a 1arylazo-2-naphthol structure like the compounds in Table IV, they did

20 TABLE II INDUCTION OF CYTOCHROME P-450 AND UDPGT ACTIVITY BY PB AND 3-MC Methods for pretreatment of animals and preparations of microsomes are described in the text. Treatment

UDPGT Relative activity

Cyt. P-450 km~a

Relative conc.

Saline Corn oil PB

450 450 450

100 b 100 217

100 b 100 237

3-MC

448

198

390

aWavelength in n m at t h e Soret a b s o r p t i o n m a x i m u m o f f e r r o u s c y t o c h r o m e P - 4 5 0 / C O

complex. bRelative concentrations and activities are calculated from the averages of the values obtained from 3--4 different preparations of microsomes from 4 pooled livers in each group. Values for liver microsomes from rats given physiological saline or corn oil were taken as 100% and were compared with those from PB- or 3-MC-treated rats, respectively. Control values for cytochrome P-450 contents were 0.65 *~0.10 and 0.64 ± 0.10 nmol/mg (corn oil) and those for UDPGT were 18.6 +_2.4 and 20.1 ± 3.1 nmol/mg/min, respectively. n o t i n d u c e c y t o c h r o m e P-448. H o w e v e r , t h e y did increase U D P G T activity a b o u t 3-fold. T h e inability o f these p o t e n t i a l iron c h e l a t o r s t o i n d u c e c y t o c h r o m e P - 4 5 0 d o e s n o t a p p e a r to be d u e t o t h e d e p l e t i o n o f iron required f o r P - 4 5 0 h e m e b i o s y n t h e s i s . T h e c o a d m i n i s t r a t i o n o f X X I I with X I I I resulted in a m a r k e d 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 , c o m p a r a b l e to t h e e f f e c t o f X I I I alone (Table VI). Larger doses o f X X I I caused a slight elevation of cytochrome. Table V I I shows t h e effects o f azo c o m p o u n d s possessing a 1-arylazo-2n a p h t h y l a m i n e m o i e t y . X X I V was a g o o d i n d u c e r o f b o t h c y t o c h r o m e P - 4 4 8 and U D P G T activity, b u t XXV, w h i c h has t h e largest area a m o n g t h e lipophilic azo c o m p o u n d s t e s t e d in this s t u d y , did n o t induce. Table V I I I s h o w s t h e effect o f lipophilic 4 - a r y l a z o - l - n a p h t h o l s o n levels o f P - 4 5 0 t y p e c y t o c h r o m e s and o n U D P G T activity. These azo c o m p o u n d s , a l t h o u g h t h e y are isomers o f 1 - a r y l a z o - 2 - n a p h t h o l , are n o t inducers at t h e doses e m p l o y e d , indicating t h a t t h e p o s i t i o n o f t h e h y d r o x y l g r o u p o n t h e a r o m a t i c ring p l a y s an i m p o r t a n t role in i n d u c t i o n . In o r d e r to c o n f i r m t h e i m p o r t a n c e o f a h y d r o x y l g r o u p at t h e 2 p o s i t i o n o f t h e n a p h t h a l e n e m o i e t y , we m e t h y l a t e d t h e h y d r o x y l g r o u p o f c o m p o u n d X I I I , 1 - p h e n y l a z o - 2 - n a p h t h o l [ 1 5 ] a n d also s y n t h e s i z e d 1-phenylazo-2n a p h t h a l e n e [ 1 6 ] . We c o m p a r e d t h e inductive ability o f these t w o c o m p o u n d s ( X X I X and X X X ) with c o m p o u n d X I I I (Table IX). X X I X and X X X s h o w e d a weak i n d u c t i o n . CO d i f f e r e n c e spectra o f r e d u c e d m i c r o s o m e s f r o m rats t r e a t e d with t h e s e azo d y e s s h o w e d a b s o r p t i o n m a x i m a at 4 4 9 n m . T h e c y t o c h r o m e P - 4 5 0 c o n t e n t increased a b o u t 50% over c o n t r o l levels as

21

T A B L E III EFFECTS OF AZOBENZENE DERIVATIVES ON U D P G T A C T I V I T Y IN R A T L I V E R M I C R O S O M E S

CYTOCHROME

P-450

INDUCTION

AND

M e t h o d s f o r p r e t r e a t m e n t o f a n i m a l s a n d p r e p a r a t i o n s o f m i e r o s o m e s are d e s c r i b e d in t h e t e x t .

Treatment

C

I

C o r n Oil

~

N

=

HO .-----k

II

Cyt. P-450

N

~

~

hmaxa ( n m )

C o n e . (%)

UDPGT activity b (%)

450

100

100

450

108

154

450

72

90

450

96

90

450

95

99

OH

~ C > N = N ~ OH

III

~-N=N~--OH CH2OH

IV

CH:OH

~C~N=N~__j~ CH~CI ~--~

CHIC[ .------4

V

~/~N:N~

450

88

141

VI

~N=N~NH2

450

I 11

134

V II

~k~N=N~N(CH~)

~

450

120

140

~ j ~ - - N = N - ~__/~--N(CH3)2

450

97

137

450

84

108

450

99

172

450

92

115

450

81

CH~

VIII

CH2OH

IX

~--N=N~--N(CH3)2 HOOC

X

~--N=N~/~N(CH3)2 CH:CI i----L-.

XI

XII

~

~ N = N ~ / ~ - - N (CH3): O2N ~ /

~ f - N ~ CHzCH2-CN N=N ~ CH2CH3

89

a W a v e l e n g t h in n m at t h e S o r e t a b s o r p t i o n m a x i m u m o f t h e f e r r o u s c y t o c h r o m e P - 4 5 0 / C O c o m p l e x . b p e r c e n t a c t i v i t i e s are c a l c u l a t e d f r o m t h e a v e r a g e s of t h e values o b t a i n e d f r o m 2 d i f f e r e n t p r e p a r a t i o n s o f m i c r o s o m e s . S.E. o f t h e s a m e t r e a t m e n t g r o u p s w e r e w i t h i n 2 0 % of average xalues.

22 TABLE IV EFFECTS OF 1-PHENYLAZO-2-NAPHTHOL DERIVATIVES ON THE INDUCTION OF CYTOCHROME P-450 AND UDPGT ACTIVITY IN RAT LIVER MICROSOMES Methods for pretreatment of animals and preparations of microsomes are described in the text. Treatment

C

Cyt. /-450 h.maxa (nm)

Conc. (%)

450

100

100

~

450

117

110

~:N ~

448

242

556

448

203

520

448

252

522

Corn Oil

N

~

XIII

Oll

__£"~H~ XIV

~

XV

CH~ ~ h

UDPGT activity b (%)

/---,

NN ~

N

XVI

H,c ~

NN

448

206

582

XVII

H,C--

N=N-

448

221

392

XVII1

~N=N ~

N=N

448

177

370

XIX

~N-N

N:N

450

119

147

N:N

450

116

139

XX

~

-N N

23 TABLE IV (Contd.) Treatment

XXI

N=N -

Cyt. P-450

--N:N

)~maxa (nm)

Conc. (%)

450

129

UDPGTb acti~ity (%)

152

aWavelength in nm at the Soret absorption maximum of the ferrouscytoehromeP*450/CO complex. bperecnt activities are calculated from the averages of the values obtained from 3 different preparations of microsomes. S.E. of the same treatment groups were within 20% of average values. c o m p a r e d t o t h e effect o f X I I I , w h i c h s h o w e d a b o u t a 200% increase over controls. I n d u c t i o n o f U D P G T b y X X I X and X X X were also n o t as great as t h a t b y X I I I . T h e w e a k e r inductive c a p a c i t y o f these azo d y e s suggested t h e possible c o n v e r s i o n o f X X I X and X X X to X I I I b y O - d e m e t h y l a t i o n and 2 - h y d r o x y l a t i o n , respectively, in t h e liver. A t h i n - l a y e r c h r o m a t o g r a p h i c a l l y identical s u b s t a n c e t o c o m p o u n d X I I I was o b t a i n e d b y i n c u b a t i n g X X I X or X X X with liver m i c r o s o m e s in t h e p r e s e n c e o f a N A D P H generating system. T h e f o r m a t i o n o f f o r m a l d e h y d e was also d e t e c t e d using Nash reagent f r o m t h e m i c r o s o m a l assay m i x t u r e c o n t a i n i n g X X I X , indicating t h a t O - d e m e t h y l a t i o n h a d t a k e n place. Alternatively, t h e w e a k inductive effect o f X X X m i g h t be caused b y 1p h e n y l a z o x y n a p h t h a l e n e , an i m p u r i t y f o r m e d during t h e synthesis [ 1 6 ] . This c o m p o u n d has been s h o w n t o assume a c o p l a n a r s t r u c t u r e w h i c h consists o f t h r e e fused six m e m b e r e d a r o m a t i c rings [ 2 5 ] . This c o p l a n a r s t r u c t u r e m a y be i m p o r t a n t in 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 as discussed below. In a n y event, m a r k e d r e d u c t i o n in inductive ability with t h e r e m o v a l o f t h e h y d r o x y l g r o u p f r o m t h e 2 - n a p h t h o l m o i e t y indicated t h e i m p o r t a n c e o f t h e p r e s e n c e o f this g r o u p at t h e 2 position. Table X shows t h e effects o f h y d r o p h i l i c azo c o m p o u n d s o n i n d u c t i o n . In spite o f t h e fact t h a t m a n y o f these c o m p o u n d s c o n t a i n a 1-arylazo-2n a p h t h o l m o i e t y , t h e c o m m o n s t r u c t u r e f o u n d in azo c o m p o u n d s t h a t

~N

Azo Bay ~ region N.

1-Phenylazo-2-naphthol

Bay region

Benzo(a) pyrene

Fig. 1. Azo bay-region structure formed by intramolecular hydrogen bonding of 1arylazo-2-naphthol and a bay region structure of BP shown for comparison.

24

•

XV

150 Xlll

•

I---

z

F-Z 0 0

1 O0

XlV

2_ >.(-9

XVlll

tZ3

H

50

I---

,,=,

r=0,926

XXI x x@~ XlX@

]/

I

I

60

80

I

100

I

120

I

i

I

I_

140

160

180

200

INCUMBRANCE AREA ( ) Fig. 2. RelatiQnship between molecular size of azo compounds and their ability to induce P-450 type cytochromes in rat liver microsomes. Molecular incumbrance areas of the azo compounds listed in Table IV were calculated according to Ref. 6 and were plotted against percentage increase in cytochrome P-450 content in liver microsomes from rats treated with these dyes. i n d u c e (see Table IV), t h e y nevertheless n e i t h e r i n d u c e P - 4 5 0 t y p e c y t o c h r o m e s n o r U D P G T activity. N o n e o f t h e lipophilic a z o b e n z e n e derivatives in Table I I I had a significant e f f e c t indicating t h a t lipophilicity alone is n o t e n o u g h for i n d u c t i o n . B o t h lipophilicity a n d a 1 - a r y l a z o - 2 - n a p h t h o l o r 1 - a r y l a z o - 2 - n a p h t h y l a m i n e s t r u c t u r e , t h e n , appears t o be a r e q u i r e m e n t f o r induction. Table XI shows t h e effects o f PB, 3-MC, and s o m e azo c o m p o u n d s f r o m Tables I I I - - X on h e p a t i c m i c r o s o m a l a m i n o p y r i n e N - d e m e t h y l a s e (APD), h e x o b a r b i t a l h y d r o x y l a s e (HBH), 7 - e t h o x y c o u m a r i n O-deeth.ylase (ECD),

TABLE V EFFECTS OF 1-THIAZOLYLAZO-2-NAPHTHOL AND 1-PYRIDYLAZO-1-NAPHTHOL ON THE L E V E L S O F C Y T O C H R O M E P - 4 5 0 A N D U D P G T A C T I V I T Y IN R A T L I V E R M I C R O S O M E S M e t h o d s f o r p r e t r e a t m e n t o f a n i m a l s a n d p r e p a r a t i o n s o f m i c r o s o m e s are d e s c r i b e d in t h e t e x t . Treatment

C

Cyt. P-450

C o r n Oil

UDPGT a c t i v i t y b (%)

)~max a ( n m )

Cone. (%)

450

100

100

450

90

329

450

97

293

OH XXII

~

~-~=N S

OH XXIII

N=N

a W a v e l e n g t h in n m at t h e S o r e t a b s o r p t i o n m a x i m u m o f t h e f e r r o u s c y t o c h r o m e P - 4 5 0 / C O c o m p l e x . b p e r c e n t a c t i v i t i e s are c a l c u l a t e d f r o m t h e a v e r a g e s o f t h e v a l u e s o b t a i n e d f r o m 3 d i f f e r e n t p r e p a r a t i o n s o f m i c r o s o m e s . S.E. o f t h e s a m e t r e a t m e n t g r o u p s w e r e w i t h i n 2 0 % o f a v e r a g e values. T A B L E VI DIFFERENTIAL EFFECTS OF AROMATIC MOIETIES LINKED TO AZO NITROGEN OF 1-AZO2 - N A P H T H O L O N 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 5 0 IN L I V E R M I C R O S O M E S F R O M RATS TREATED WITH THESE COMPOUNDS Treatment

Cyt. P-450

Azo dyes

mg/kg

kmax a (nm)

Content (nmol/mg prot.)

450

0 . 6 6 -+ 0 . 1 0

40

448

1.91 + 0 . 1 5

C o r n Oil L HO ~ XIII

~N:N--~_~

HO

~ XXIII

XIII + XXIII b

N=N

HO

20

450

0,73 ± 0.05

40

450

0.79 ± 0.05

80

450

0 . 8 9 _+0 . 1 3

40 + 40

448

1 . 5 1 +- 0 . 2 0

a ve l e n g t h m n m a t t h e S o r e t a b s o r p t i o n m a x i m u m o f t h e f e r r o u s c y t o c h r o m e P - 4 5 0 / C O c o m p l e x . bWa Simultaneous administration of 1-(2-pyridylazo)-2-naphthol and 1-phenylazo-2-naphthol (40 mg/kg each) for 3 days following 1-day treatment with 40 mg/kg of 1-(2-pyridylazo)-2-naphthol alone.

26 TABLE VII E F F E C T S OF 1 - A R Y L A Z O - 2 - N A P H T H Y L A M I N E D E R I V A T I V E S ON T H E C Y T O C H R O M E P - 4 5 0 A N D U D P G T A C T I V I T Y IN R A T L I V E R M I C R O S O M E S

INDUCTION

OF

M e t h o d s for p r e t r e a t m e n t o f a n i m a l s a n d p r e p a r a t i o n s of m i c r o s o m e s are d e s c r i b e d in t h e t e x t . Treatment

C

C o r n Oil

~

XXIV

XXV

Cyt. P - 4 5 0

CH,

?,.maxa ( n m )

Cone. (%)

450

100

100

448

210

376

450

102

NH~

-N=N~

N=N--~ N

UDPGT a c t i v i t y b (%)

HNCH~CH~

N~

81

a W a v e l e n g t h in n m at t h e S o r e t a b s o r p t i o n m a x i m u m o f t h e f e r r o u s c y t o c h r o m e P-4501CO c o m p l e x . b p e r c e n t activities gre c a l c u l a t e d f r o m t h e a v e r a g e s o f t h e v a l u e s o b t a i n e d f r o m 3 d i f f e r e n t p r e p a r a t i o n s of m i c r o s o m e s . S.E. of t h e s a m e t r e a t m e n t g r o u p s w e r e w i t h i n 20% o f a v e r a g e values.

TABLE VIII E F F E C T S O F 4 - P H E N Y L A Z O - 1 - N A P H T H O L D E R I V A T I V E S ON 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 5 0 A N D U D P G T A C T I V I T Y IN R A T L I V E R M I C R O S O M E S M e t h o d s for p r e t r e a t m e n t o f a n i m a l s a n d P r e p a r a t i o n s of m i c r o s o m e s are d e s c r i b e d in t h e t e x t . Cyt. P-450

Treatment

C

Corn Oil

XXVI

xxvI I

XXVIII

N=N--

n

~N:N-~__~)~--

H~C ( r ~

N=N ~ X X f O H

~.max a ( n m )

Conc. (%)

UDPGT activityb (%)

450

100

100

450

110

134

450

t 02

116

450

107

131

a w a v e l e n g t h in n m at t h e S o r e t a b s o r p t i o n m a x i m u m of t h e f e r r o u s c y t o c h r o m e P-4501CO c o m p l e x . b p e r c e n t acti~4ties are c a l c u l a t e d f r o m t h e a v e r a g e s o f t h e v a l u e s o b t a i n e d f r o m 3 d i f f e r e n t p r e p a r a t i o n s of m i c r o s o m e s . S.E. o f t h e s a m e t r e a t m e n t g r o u p s w e r e w i t h i n 20% of a v e r a g e values.

27 T A B L E IX E F F E C T S O F 1 - P H E N Y L A Z O - 2 - N A P H T H A L E N E D E R I V A T I V E S ON T H E L E V E L S O F C Y T O C H R O M E P-450 A N D U D P G T A C T I V I T Y IN R A T L I V E R M I C R O S O M E S M e t h o d s for p r e t r e a t m e n t o f a n i m a l s and p r e p a r a t i o n s of m i c r o s o m e s are d e s c r i b e d in t h e t e x t . C y t o c h r o m e P - 4 5 0 c o n c e n t r a t i o n s in liver m i c r o s o m e s f r o m c o r n oil t r e a t e d c o n t r o l rats w e r e 0 . 6 4 * 0 . 0 0 7 n m o l / m g m i c r o s o m a l p r o t e i n . U D P G T activities of t h e s a m e m i c r o s o m e s were 25.3 ± 2.8 n m o l / m i n / m g m i c r o s o m a l p r o t e i n . T h e s e v a l u e s w e r e t a k e n as 100%. Treatment

Cyt. / ' - 4 5 0 Cbnc. (%)

UDPGT activity b (%)

. 450

100

100

448

309

766

kmax a (nm) C

C o r n Oil

XlII

~-N=N

~_~.~,CH,

a w a v e l e n g t h in n m at t h e Sorer a b s o r p t i o n m a x i m u m of t h e f e r r o u s c y t o c h r o m e P - 4 5 0 / C O c o m p l e x . b p e r c e n t activities are c a l c u l a t e d f r o m t h e a v e r a g e s o f t h e values o b t a i n e d f r o m 3 d i f f e r e n t p r e p a r a t i o n s o f m i c r o s o m e s . S.E. o f t h e s a m e t r e a t m e n t g r o u p s w e r e w i t h i n 20% of a v e r a g e values. TABLE X E F F E C T S OF H Y D R O P H I L I C AZO COMPOUNDS ON T H E I N D U C T I O N OF C Y T O C H R O M E P - 4 5 0 A N D U D P G T A C T I V I T Y IN R A T L I V E R M I C R O S O M E S M e t h o d s for p r e t r e a t m e n t of a n i m a l s a n d p r e p a r a t i o n s o f m i c r o s o m e s are d e s c r i b e d in t h e t e x t . Treatment

Cyt. P - 4 5 0 hmax a (nm)

Cone. (%)

UDPGT activity b (%)

PSS

Physiological Saline Solution

450

100

100

XXXI

NaO3S--~--N=N--~--N(CH,)2

450

82

113

450

89

121

450

96

121

OH

XXXII NaOaS--~N=N~ OH A---, X X i II I

NaO3S--~--N=N~

k----../.~O3Na

28 TABLE X (Corltd,} Treatment

Cyt. P-450

UDPGT activity b

k

a

Cone,

(%)

OH XXXIV

@~__j~-N:N-~__4)

450

110

116

450

95

14~

451)

72

77

450

92

82

450

83

120

,oo

,2,

NaO~S ~ g O ~ N a CH3 XXXV

IItC ~

HH~O3SO3Na

-N=N--~ M-----/~O3Na OH SO~Na

XXXV|

~

N=N ~ - - N = N

~ ~SO3Na

XXXVII

NaO,S ~ - - N = N

~--OH

H

()~Na

XXXVIII O~Na NaO D~__.N ~"

CH3

N1

O 11 H 1

[

OH

S

V SO,Na

H,~' ~I

~ I

bn, :

14(~ N

' :

aWavelength in nm at the Soret absorption maximum of the ferrouscytochromeP-450/CO c o m p l e x . bpercent activities are calculated from the averages of the values obtained from 3 different preparations of mierosomes. S.E. of the same treatment groups were within 20% of average values.

zoxazolamine hydroxylase (ZAH) and p-nitroanisole O-demethylase (p-NAD) activities. Activities of APD and HBH were significantly induced by PB, but were not induced by either 3-MC or by azo compounds. ECD, ZAH and p-NAD activities were induced by both PB and 3-MC, and by lipophilic azo compounds containing a 1-phenylazo-2-naphthol moiety. The patterns of induction of monooxygenase and UDPGT activities by certain azo compounds parallels that by 3-MC, suggesting that these compounds are 3-MC-type inducers.

29 TABLE

XI

EFFECTS

OF

VARIOUS

ACTIVITIES

AS

AZ0

COMPARED

COMPOUNDS WITH

THE

ON EFFECTS

HEPATIC OF

MICROSOMAL

THE

PROTOTYPE

MONOOXYGENASE INDUCERS.

PB

AND

3-MC Methods

for

preparations

of

microsmncs

and

monooxygenase

assas

methods

are

described

text. Monooxygrnasr

Treatment

ECD

100

100 (1.2)

100

(3.0) 100

PB

(0.24) 207

(3.1) 163

(1.1) 137

3-MC

1181

223

200

A.Ais

011

@-@J

0 0

-N=N

6 0 i:,

6 -N-N 6

100

HBH 100 (0.21)

APD 100

100

(7.7) 100

(0.22)

(7.2)

1221

172

84

94

96

63

89

88

Y6

82

87

88

1153

168

150

92

72

1177

195

150

98

80

210

215

124

121

215

209

89

lo8

112

\‘I1

xv

p-NAD

100

Corn

XIII

ZAH

(70)a

(o.281b

Saline

Physiological

1

activities

0

bn

OH

XVI

XVIII

(,>,_N-@N-

0

837

80

XXIV

0“ly_y ; (.r8 771

205

221

122

91

256

131

126

124

94

330

103

113

91

91

0

XXII

[)-,=,-

6 8 & 8 0

XXIII

06

-_N+-

0

in the

30 T A B L E X I (Contd.) Treatment

M o n o o x y g e n a s e activities (%)a ECD

((~x)

XXVII

N=N_( ( ~ O

XXVIII

H

ZAH

p-NAD

HBH

APD

86

136

78

99

124

115

105

105

138

109

97

101

98

133

100

OH XXXII1

a p e r c e n t activities are c a l c u l a t e d f r o m t h e a v e r a g e s o f v a l u e s o b t a i n e d f r o m 3 d i f f e r e n t p r e p a r a t i o n s o f m i c r o s o m e s f r o m 4 p o o l e d livers in each. S t a n d a r d e r r o r s of t h e s a m e t r e a t m e n t g r o u p s w e r e w i t h i n 15% of t h e a v e r a g e values. b A v e r a g e c o n t r o l m o n o o x y g e n a s e activities, e x p r e s s e d in n m o l / m i n / m g m i c r o s o m a l p r o t e i n , are indic a t e d in p a r e n t h e s e s .

T A B L E XIi E F F E C T S O F PB, 3-MC A N D A Z O C O M P O U N D S 1 - P H E N Y L A Z O P H E N Y L A Z O - 2 - N A P H T H O L ( S U D A N Ill) A N D 1 - m - T O L U E N E A Z O - 2 - N A P H T H O L ON C Y T O C H R O M E P-450 L E V E L S IN L I V E R M I C R O S O M E S O F C 5 7 B L / 6 A N D D B A / 2 MICE Values are e x p r e s s e d as m e a n ± S.D. f r o m 5 d i f f e r e n t p r e p a r a t i o n s of m i c r o s o m e s in each t r e a t m e n t group. Treatment

C 5 7 B L / 6 Cr

D B A / 2 Cr

Cyt. Po450

Cyt. P - 4 5 0

kmaxa

Content

kma a

Content

(nm)

(nmol/mg)

(m,n)

(nmol/mg)

PSS

450

0 . 5 0 -+ 0 . 0 3

450

0 . 6 5 -+ 0.01

C

450

0 . 6 0 +- 0 . 0 5

450

0.59 +- 0 . 0 5

PB

450

1.24 *_ 0 . 0 8

450

1.52 _+0 . 0 3

448

1.48 ± 0 . 0 6

450

0 . 6 7 +_0 . 0 1

448

1.11 +- 0.07

450

0.54 + 0.02

3-MC

~ H~C/

/ HO XVIII

~/~N=N

~--N:N--

~

31 T A B L E X I l (Contd.) Treatment

H~C XV

C 5 7 B L / 6 Cr

D B A / 2 Cr

Cyt. P - 4 5 0

Cyt. P-450

kmaxa (nm)

Content (nmol/mg)

hmaxa (nm)

Content

448

1.52 -+ 0.09

449

0 . 9 8 -* 0 . 0 6

(nmol/mg)

HO

~ N = N _ / ( ~ _ . .~ z - . . -x

a W a v e l e n g t h in n m at t h e Soret a b s o r p t i o n m a x i m u m of t h e ferrous c y t o c h r o m e P-450/C0 c o m p l e x .

Compounds XXII and XXIII show unusual patterns of induction. As can be seen (Table IV), these azo compounds did not induce cytochrome P-450 nor cause a hypsochromic shift in the CO difference spectrum of reduced microsomes. They did not induce ZAH or p-NAD activities but caused more than a 2-fold increase in ECD activity. These results suggest that the c y t o c h r o m e P-450 species engaging in ZAH and p-NAD activities may be different from those associated with ECD activities. In order to investigate whether those azo compounds that induce cytochrome P-448 do so by binding to the same receptor protein as does 3-MC, two of the azo compounds that induce, XVIII and XV, were tested with the DBA/2 strain of mice in which the receptor protein does not bind 3-MC due to a genetic defect in the A h locus [26]. As indicated in Table XII, 3-MC, XVIII, and XV were able to induce cytochrome P-448 by more than 2-fold in normal, C57BL/6J mice. Compound XV was the most potent of the three. In contrast, 3-MC and XVIII did not induce c y t o c h r o m e P-448 in genetically defective DBA/2 mice, while XV did induce cytochrome P-448 about 1.5-fold over controls and also caused a 1-nm hypsochromic shift in the CO difference spectrum of dithionite reduced microsomes. DISCUSSION In spite of the fact that m a n y azo compounds are used to color food, cosmetics, fabrics and paints, and that many azo dyes are known carcinogens [27], little, if any, information is available on the effect of azo dyes on liver enzymes. We therefore, surveyed a number of azo compounds in order to study their effects on liver microsomal drug metabolizing enzymes, with particular attention to the P-450 t y p e cytochromes. As can be seen in Tables IV and VII, those lipophilic azo dyes which possess 2naphthol or 2-naphthylamine moieties, including Sudan I (XIII), Sudan II (XVII), Sudan III (XVIII), 1-o-tolueneazo-2-naphthol (XIV), 1-m-tolueneazo2-naphthol (XV), 1-p-tolueneazo-2-naphthol (XVI) and oil yellow OB (XXIV) are p o t e n t inducers. They induce cytochrome levels more than 2-fold, and

32 UDPGT activity, more t h a n 4-fold over controls, and are therefore as potent inducers as 3-MC (Table II). As indicated in Table XI, various monooxygenase activities are also induced by azo compounds, and the pattern of induction resembles that of 3-MC. Those azo compounds that induce ECD, p-NAD and ZAH activities do not induce APD and HBH activities. These results suggest that those azo compounds that do induce are all 3-MC type inducers and differ from PCB inducers as classified by Alvares et al. [28]. Arochlor 1254, a commercial mixture of PCB isomers, produces a complex pattern of induction of microsomal monooxygenase activity in rat liver which is comparable to that produced by the combined administration of PB and 3-MC, i.e., it induces ethy~norphine N
33 p o u n d s and even their weak inductive ability seems to depend on the conversion o f t he naphthalene or m e t h o x y n a p t h a l e n e moieties to 2-naphthol, again indicating t he i m por t ance o f t he presence of OH group. Water soluble azo c o m p o u n d s , such as the ones listed in Table X, do n o t induce even when t h e y do contain the 1-phenylazo-2-naphthol m o i e t y in their structure. Thus, lipophilicity is a n o t h e r requi rem ent for activity. Among lipophilic azo c o m p o u n d s , those which do not contain napht hol moieties i.e., t he azobenzenes listed in Table III, do not induce, indicating that lipophilicity and an azo structure is not enough for induction. A 2n a p h t h o l structure is needed. It should be n o t e d t hat 2-naphthol itself does n o t induce indicating t ha t the azo linkage is required. Therefore, the smallest effective unit for induction is 1-arylazo-2-naphthol. The h y d r o x y l group on the 2-naphthol m o i e t y can be replaced by an amine {Table VII). Why are certain azo c o m p o u n d s then able to induce and others not? With structures differing from those of carcinogenic polycyclic aromatic hydrocarbons, like 3-MC, or polyhalogenated aromatics, like TCDD, it was t h o u g h t that azo c o m p o u n d s f r om a new class of c y t o c h r o m e P-448 inducers. However, as indicated in Fig. 2, 1-arylazo-2-naphthol has been shown by infrared [29,30] and NMR [31] spectroscopic analysis to form an intramolecular h y d r o g en bond be t w een t he phenolic h y d r o x y l and one o f t he azo nitrogens, producing a six-membered ring with a phenanthrene-like structure. Although the third ring thus f or m e d is heterocyclic, t he overall shape of the planar structure with three six-membered rings resembles the bay-region structure o f carcinogenic polycyclic aromatic h y d r o c a r b o n s [ 3 2 ] . It is interesting to n o te that only those azo c o m p o u n d s capable of forming this t y p e of azo bay-region structure can induce c y t o c h r o m e P-448". The bay-region structure has been shown to be important in chemical carcinogenesis [ 3 2 ] . The activated forms o f polycyclic aromatic hydrocarbons possessing t he bay-region structure interact strongly with cellular macromolecules resulting in t he alterations of gene expression and carcinogenic t r a n s f o r m a t i o n of cells [ 3 3 ] . The apparently different p h e n o m e n o n , induction, involves a process in c o m m o n with chemical carcinogenesis, i.e., t h e interaction of a c o m p o u n d with cellular macromolecules and subsequent gene expression [ 3 4 - - 3 6 ] . The fact that these polycyclic aromatic hydrocarbon carcinogens are know n inducers of c y t o c h r o m e P-448, suggests that the bay region structure is required for the induction process. If th e 'bay-region hypothesis of induction' is indeed correct, then t he f o r m a t i o n of a coplanar, third, six-membered ring is an absolute requirement for an azo c o m p o u n d to induce c y t o c h r o m e P-448 and UDPGT activity. *Although 1-phenylazo-2-naphthol is reported to be extensively metabolized [33 ], it may be argued that intramolecular hydrogen bonding serves to neutralize the polarizing effects of the hydroxyl group and thereby slowing metabolic conjugation and elimination. However, removal of the hydroxyl group from this structure to form less polar compounds, as in 1-phenylazo naphthalene (XXX) or substitution of a methyoxyl group (XXIX), markedly reduces the inductive potency of the 2-naphthol (Table IX).

34

This can explain why an h y d r o x y l group at the 2-position of the naphthalene moiety is critical for induction. If this group were at any other position, the azo c o m p o u n d would not be able to form a bay-region structure, and therefore would be unable to induce (see, for example, compounds XXVI, XXVII, and XXVIII in Table VIII). Exceptions to this hypothesis are XXII and XXIII which do not induce cytochrome P-448 in spite of the fact that t h e y possess a 1-arylazo-2-naphthol structure (and their molecular size is smaller than 150 A2). Both these compounds however, are p o t e n t metal chelators and as such, are not capable of forming an azo bay-region structure [37] (Fig. 3B). It is suggested that the resulting compounds do not have the ability to induce cytochrome P-448. Even if 1-phenyl azo-2-naphthol, which has the ability to induce, were to form a metal complex, the azo bay-region structure would still be retained (Ref. 38 and Fig. 3A). It is of interest to note that the induction of c y t o c h r o m e P-448 coincides with that of UDPGT in most cases, except for XXII and XXIII. The possible link between genes for these enzymes has been suggested [4]. Our results with XXII and XXIII seem to contradict this view. There is a 2--3-fold increase of UDPGT activity, yet c y t o c h r o m e P-450 levels were unaffected. This result demonstrates the possibility of separate induction of a detoxifying enzyme without induction o f an activating enzyme. Interestingly enough, azo compounds do n o t have to possess the bayregion like structure to be carcinogenic, while t h e y do have to possess the bay-region like structure to be inducers of cytochrome P-448. Conversely, azo compounds do n o t have to be able to induce c y t o c h r o m e P-448 in order to be carcinogenic. Many of the azo compounds in Table III are known carcinogens, but do not form bay-region structures and do not induce cytochrome P-448, while Sudan III (XVIII) a noncarcinogen, forms a bayregion structure and induces the cytochrome [12]. A reason why those azo compounds that induce cytochrome P-448 are not potent carcinogens, in spite of the fact that t h e y form the bay-region like structure, may be due to their ability to induce cytochrome P-448 which is capable of detoxifying the azo linkage in a reductive process [39]. It is suggested from our studies that the bay-region structure may be required for the interaction of azo c o m p o u n d inducers with the inductionspecific receptor protein. In order to obtain indirect evidence for this, we

(A)

(B)

Fig. 3. Metal complexes of 1-phenylazoi2-naphthol (A)and li(2-thiazo|y|azo)-2-naphthol (B) [37,38].

35 co mp ar ed t he effect of 1-m-tolueneazo-2-naphthol (XV), the most p o t e n t P-448 inducer among the azo c o m p o u n d s tested, and Sudan III (XVIII), one o f the less p o t e n t inducers, on t he levels of liver microsomal P-450 t y p e c y t o c h r o m e s in genetically defective DBA/2 and normal C57BL/6J mice. F r o m the results indicated in Table XII, XVIII does not induce c y t o c h r o m e P-448 in DBA/2 mice, while it causes a 2-fold increase in c y t o c h r o m e P-448 levels in C57BL/6 mice, comparable to the effect of 3-MC. The more p o t e n t inducer, XV, causes a 3-fold increase in the levels o f c y t o c h r o m e P-448 in C57BL/6 mice, and a 1.5-fold increase in DBA/2 mice, the latter accompanied by a 1-nm h y p s o c h r o m i c shift in t he absorption peak wavelength of th e CO difference spectrum. It is suggested that these azo c o m p o u n d s induce c y t o c h r o m e P-448 via t he same mechanism as 3-MC, i.e., t h e y bind to the induction specific r e c e p t o r protein, t he p r o d u c t o f t he A h locus. C o m p o u n d XV would t h e n have a higher affinity for t he r e c e p t o r protein than either XVIII or 3-MC, as it induces c y t o c h r o m e P-448 in DBA/2 mice, although to a limited extent. Since polycylic aromatic h y d r o c a r b o n s such as 3-MC and those azo c o m p o u n d s which induce c y t o c h r o m e P-448 have t he bay-region structure in c o m m o n , it is suggested that for polycyclic aromatic c o m p o u n d s including azo dyes, this structure m ay be suited for t he interaction with the r e c e p t o r protein. Needless to say, for polyhalogenated c o m p o u n d s and for single ring aromatics, o t h e r structural properties must be required for the binding. In summary, only lipophilic azo c o m p o u n d s which are capable o f forming an azo bay-region structure by intramolecular h y d r o g e n bonding and with a molecular incumbrance area less than 150 A2 can induce hepatic microsomal c y t o c h r o m e P-448 and associated m o n o o x y g e n a s e activities and UDPGT activity. It is suggested t hat these azo c o m p o u n d s interact with t he induction specific cytosolic r e c e p t o r protein, t he regulator gene p r o d u c t of t he A h locus. The i m por t ance of azo c o m p o u n d s in c y t o c h r o m e P-448 induction is 2-fold: their structure-activity relationship for induction is suggestive of a mo r e general structure-activity relationship inclusive o f t he polycyclic aromatic hydr oc a r bons , i.e., the 'bay-region hypothesis o f induction' and t h e y represent a new set of p o t e n t inducers of c y t o c h r o m e P-448 and associated drug metabolism. REFERENCES

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