Brief Definitive Report
ON THE O C C U R R E N C E OF CYTOCHROME P-450 A N D ARYL H Y D R O C A R B O N H Y D R O X Y L A S E ACTIVITY IN RAT BRAIN* BY J O N A T H A N A. C O H N , , ALVITO P. ALVARES,§ A~m ATTALLAH KAPPAS
(From the Rockefeller University, New York 10021)
Liver microsomes contain a mixed function oxidase system capable of metabolizing a broad range of biologically active substances, including drugs, steroids, and environmental chemicals such as polycyclic hydrocarbons. Certain hemoproteins, collectively referred to as cytochrome P-450, function as the terminal oxidase in this system. The fact that this hepatic mixed function oxidase system is sensitive to induction and inhibition by many of its substrates and other agents has provided an approach to understanding a major mechanism by which endogenously- and environmentally-derived substances may affect each otheFs disposition and biological impact (1). Mixed function oxidase activities which depend on cytochrome P-450 are present in many tissues other than liver, including such endocrine organs as adrenal, testes, and placenta, such sites of direct xenobiotic exposure as lung, intestine, and skin, and such sites of drug action as heart and kidney. Cytochrome P-450 has also been detected in each of these tissues. Aryl hydrocarbon hydroxylase (AHH) is an example of a mixed function oxidase involving cytochrome P-450 whose distribution and properties have been widely studied. Benzo[a]pyrene (BP), which is a carcinogenic constituent of cigarette smoke and charcoal-broiled meat, is both a substrate and an inducer of this enzyme activity. Because many of the substrates of the hepatic cytochrome P-450 system have major actions on the central nervous system, this study was undertaken to determine whether this cytochrome, and the related AHH activity, could be detected in brain. M a t e r i a l s and Methods Microsomes were prepared from rat brains as follows: male, Sprague-Dawley, 125-150-g rats were decapitated and the brains were immediately removed and transferred to cold saline. Large blood vessels and clots were excised and the pooled brains were homogenized in 4 vol of KC1 (1.15%), by using a glass Teflon homogenizer. This homogenate was centrifuged for 20 m i n at ll,000g; the supernate was t h e n centrifuged for 60 min at 105,000g; the resulting microsome pellet was washed in KC1 and t h e n suspended in buffer (0.1 M potassium phosphate, pH 7.4). A H H activity was determined by using BP as the substrate by the method of Nebert and Gelboin (2), as modified by Alvares et al. (3). Brain microsomal suspensions were incubated for 30 * Supported by U. S. Public Health Service g r a n t ES-01055 and the Scaife Family Trust. ;t Biomedical Fellow at the Rockefeller University Cornell University Medical College; and received partial support from the Pharmaceutical Manufacturers Association. § Recipient of a Research Career Development Award 1 K04 ES 00010-02 from the National Institutes of Health. THE JOURNAL OF EXPERIMENTAL MEDICINE • VOLUME
145, 1977
1607
1608
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BRIEF DEFINITIVE
REPORT
min at 37°C in 1.05 ml buffer containing 100 nmol BP, 1 ~mol NADPH, 3 ~mol MgC12,and 1 mg bovine serum albumin. In other experiments, homogenate equivalent to 100 mg brain wet weight was also incubated as above, except that albumin was omitted. The reaction was stopped by adding 1 ml cold acetone, either before (for blank determinations) or after incubation. BP metabolites were then extracted from the mixture, first into hexane, and then into 1 N NaOH, in which the concentration of hydroxylated (OH-) BP was determined by measuring the fluorescence at 522 nm during excitation at 396 nm. Hydroxy-BP standards were prepared by adding genuine 8OH-BP instead of BP to incubation mixtures and extracting as for blank determinations. The activation and fluorescence spectra of the extracted metabolites after incubation of brain microsomes or homogenates with BP, and the spectra of 8-OH-BP, added to and immediately extracted from unincubated mixtures, were identical. Protein was determined by using bovine serum albumin as the standard (4). The carbon monoxide (CO) complex of reduced cytochrome P-450 strongly adsorbs light at 450 nm. Because both the reduced cytochrome, alone, and the CO-complex of the oxidized cytochrome do not strongly absorb light of this wavelength, difference spectra permit detection of the cytochrome in turbid suspensions (5-7). Results and Discussion In Fig. 1 A, the difference s p e c t r u m b e t w e e n the CO-complex of dithionitereduced b r a i n m i c r o s o m e s and t h e CO-complex of oxidized m i c r o s o m e s is depicted. T h e a b s o r b a n c e m a x i m u m (425 nm) and m i n i m u m (408 nm) in this s p e c t r u m correspond to t h e oxidized-minus-reduced s p e c t r u m of cytochrome bs, which h a s b e e n p r e v i o u s l y detected in b r a i n m i c r o s o m e s (8). C y t o c h r o m e b5 does not bind CO. H o w e v e r , the deflection in a b s o r b a n c e n e a r 450 n m is only pronounced in the presence of CO, as in Fig. 1 A, a n d is not c h a r a c t e r i s t i c of cytochrome bs. The effect of N A D H on t h e dithionite-difference s p e c t r u m of COb o u n d b r a i n m i c r o s o m e s supports the idea t h a t this deflection is due to cytochrome P-450. N A D H t r a n s f e r s its r e d u c i n g e q u i v a l e n t s directly to c y t e c h r o m e b5 v i a the flavoprotein, cytochrome b5 r e d u c t a s e (E.C. 1.6.2.2.), which h a s b e e n detected in b r a i n m i c r o s o m e s (8). C y t o c h r o m e P-450 differs f r o m cytochrome b5 in t h a t it c a n n o t be directly reduced by N A D H aerobically (5). For t h i s reason, the addition of N A D H to b o t h the s a m p l e a n d reference cuvettes which were c o m p a r e d in Fig. 1 A should r e s u l t in a d i m i n i s h e d c o n t r i b u t i o n to the absorbance difference b y c y t o c h r o m e b5 (which would become reduced in t h e reference as well as the s a m p l e cuvette), a n d a n unaffected contribution b y c y t o c h r o m e P450 (which would r e m a i n oxidized in t h e reference cuvette). Fig. 1 B depicts t h e difference s p e c t r u m a f t e r adding N A D H to the c u v e t t e s c o m p a r e d in Fig. 1 A. As expected, the addition of N A D H r e s u l t e d in a difference s p e c t r u m w i t h a d i m i n i s h e d m i n i m u m at 408 n m a n d a d i m i n i s h e d m a x i m u m at 425 n m , yielding resolution of t h e a b s o r b a n c e at 450 n m as a distinct p e a k . A s p e c t r u m indistinguishable f r o m t h a t in Fig. 1 B was o b t a i n e d e i t h e r w h e n m o r e N A D H w a s added (to a final concentration of 1 mM) or w h e n N A D H w a s added to the m i c r o s o m e s before exposure to CO, a d j u s t m e n t of t h e b a s e line, a n d addition of dithionite to t h e s a m p l e c u v e t t e only. The CO-difference s p e c t r u m of dithionite-reduced b r a i n m i c r o s o m e s provided direct evidence t h a t cytochrome P-450 w a s p r e s e n t in t h e s e suspensions. Such a s p e c t r u m is p r e s e n t e d in Fig. 2 A. T h e absorbance m a x i m u m at 454 n m reflects the a b s o r b a n c e difference b e t w e e n t h e CO-complex of reduced c y t o c h r o m e P-450 a n d the reduced cytochrome, alone. The other a b s o r b a n c e m a x i m u m (420 nm) a n d the m i n i m u m (438 nm) in this s p e c t r u m m a y be due to c y t o c h r o m e P-420 or
COHN
ET AL.
B R I E F D E F I N I T I V E REPORT
1609
31C
c~
o x 2
2 cn £}
< I
ON THE O C C U R R E N C E OF CYTOCHROME P-450 A N D ARYL H Y D R O C A R B O N H Y D R O X Y L A S E ACTIVITY IN RAT BRAIN* BY J O N A T H A N A. C O H N , , ALVITO P. ALVARES,§ A~m ATTALLAH KAPPAS
(From the Rockefeller University, New York 10021)
Liver microsomes contain a mixed function oxidase system capable of metabolizing a broad range of biologically active substances, including drugs, steroids, and environmental chemicals such as polycyclic hydrocarbons. Certain hemoproteins, collectively referred to as cytochrome P-450, function as the terminal oxidase in this system. The fact that this hepatic mixed function oxidase system is sensitive to induction and inhibition by many of its substrates and other agents has provided an approach to understanding a major mechanism by which endogenously- and environmentally-derived substances may affect each otheFs disposition and biological impact (1). Mixed function oxidase activities which depend on cytochrome P-450 are present in many tissues other than liver, including such endocrine organs as adrenal, testes, and placenta, such sites of direct xenobiotic exposure as lung, intestine, and skin, and such sites of drug action as heart and kidney. Cytochrome P-450 has also been detected in each of these tissues. Aryl hydrocarbon hydroxylase (AHH) is an example of a mixed function oxidase involving cytochrome P-450 whose distribution and properties have been widely studied. Benzo[a]pyrene (BP), which is a carcinogenic constituent of cigarette smoke and charcoal-broiled meat, is both a substrate and an inducer of this enzyme activity. Because many of the substrates of the hepatic cytochrome P-450 system have major actions on the central nervous system, this study was undertaken to determine whether this cytochrome, and the related AHH activity, could be detected in brain. M a t e r i a l s and Methods Microsomes were prepared from rat brains as follows: male, Sprague-Dawley, 125-150-g rats were decapitated and the brains were immediately removed and transferred to cold saline. Large blood vessels and clots were excised and the pooled brains were homogenized in 4 vol of KC1 (1.15%), by using a glass Teflon homogenizer. This homogenate was centrifuged for 20 m i n at ll,000g; the supernate was t h e n centrifuged for 60 min at 105,000g; the resulting microsome pellet was washed in KC1 and t h e n suspended in buffer (0.1 M potassium phosphate, pH 7.4). A H H activity was determined by using BP as the substrate by the method of Nebert and Gelboin (2), as modified by Alvares et al. (3). Brain microsomal suspensions were incubated for 30 * Supported by U. S. Public Health Service g r a n t ES-01055 and the Scaife Family Trust. ;t Biomedical Fellow at the Rockefeller University Cornell University Medical College; and received partial support from the Pharmaceutical Manufacturers Association. § Recipient of a Research Career Development Award 1 K04 ES 00010-02 from the National Institutes of Health. THE JOURNAL OF EXPERIMENTAL MEDICINE • VOLUME
145, 1977
1607
1608
COHN
E T AL.
BRIEF DEFINITIVE
REPORT
min at 37°C in 1.05 ml buffer containing 100 nmol BP, 1 ~mol NADPH, 3 ~mol MgC12,and 1 mg bovine serum albumin. In other experiments, homogenate equivalent to 100 mg brain wet weight was also incubated as above, except that albumin was omitted. The reaction was stopped by adding 1 ml cold acetone, either before (for blank determinations) or after incubation. BP metabolites were then extracted from the mixture, first into hexane, and then into 1 N NaOH, in which the concentration of hydroxylated (OH-) BP was determined by measuring the fluorescence at 522 nm during excitation at 396 nm. Hydroxy-BP standards were prepared by adding genuine 8OH-BP instead of BP to incubation mixtures and extracting as for blank determinations. The activation and fluorescence spectra of the extracted metabolites after incubation of brain microsomes or homogenates with BP, and the spectra of 8-OH-BP, added to and immediately extracted from unincubated mixtures, were identical. Protein was determined by using bovine serum albumin as the standard (4). The carbon monoxide (CO) complex of reduced cytochrome P-450 strongly adsorbs light at 450 nm. Because both the reduced cytochrome, alone, and the CO-complex of the oxidized cytochrome do not strongly absorb light of this wavelength, difference spectra permit detection of the cytochrome in turbid suspensions (5-7). Results and Discussion In Fig. 1 A, the difference s p e c t r u m b e t w e e n the CO-complex of dithionitereduced b r a i n m i c r o s o m e s and t h e CO-complex of oxidized m i c r o s o m e s is depicted. T h e a b s o r b a n c e m a x i m u m (425 nm) and m i n i m u m (408 nm) in this s p e c t r u m correspond to t h e oxidized-minus-reduced s p e c t r u m of cytochrome bs, which h a s b e e n p r e v i o u s l y detected in b r a i n m i c r o s o m e s (8). C y t o c h r o m e b5 does not bind CO. H o w e v e r , the deflection in a b s o r b a n c e n e a r 450 n m is only pronounced in the presence of CO, as in Fig. 1 A, a n d is not c h a r a c t e r i s t i c of cytochrome bs. The effect of N A D H on t h e dithionite-difference s p e c t r u m of COb o u n d b r a i n m i c r o s o m e s supports the idea t h a t this deflection is due to cytochrome P-450. N A D H t r a n s f e r s its r e d u c i n g e q u i v a l e n t s directly to c y t e c h r o m e b5 v i a the flavoprotein, cytochrome b5 r e d u c t a s e (E.C. 1.6.2.2.), which h a s b e e n detected in b r a i n m i c r o s o m e s (8). C y t o c h r o m e P-450 differs f r o m cytochrome b5 in t h a t it c a n n o t be directly reduced by N A D H aerobically (5). For t h i s reason, the addition of N A D H to b o t h the s a m p l e a n d reference cuvettes which were c o m p a r e d in Fig. 1 A should r e s u l t in a d i m i n i s h e d c o n t r i b u t i o n to the absorbance difference b y c y t o c h r o m e b5 (which would become reduced in t h e reference as well as the s a m p l e cuvette), a n d a n unaffected contribution b y c y t o c h r o m e P450 (which would r e m a i n oxidized in t h e reference cuvette). Fig. 1 B depicts t h e difference s p e c t r u m a f t e r adding N A D H to the c u v e t t e s c o m p a r e d in Fig. 1 A. As expected, the addition of N A D H r e s u l t e d in a difference s p e c t r u m w i t h a d i m i n i s h e d m i n i m u m at 408 n m a n d a d i m i n i s h e d m a x i m u m at 425 n m , yielding resolution of t h e a b s o r b a n c e at 450 n m as a distinct p e a k . A s p e c t r u m indistinguishable f r o m t h a t in Fig. 1 B was o b t a i n e d e i t h e r w h e n m o r e N A D H w a s added (to a final concentration of 1 mM) or w h e n N A D H w a s added to the m i c r o s o m e s before exposure to CO, a d j u s t m e n t of t h e b a s e line, a n d addition of dithionite to t h e s a m p l e c u v e t t e only. The CO-difference s p e c t r u m of dithionite-reduced b r a i n m i c r o s o m e s provided direct evidence t h a t cytochrome P-450 w a s p r e s e n t in t h e s e suspensions. Such a s p e c t r u m is p r e s e n t e d in Fig. 2 A. T h e absorbance m a x i m u m at 454 n m reflects the a b s o r b a n c e difference b e t w e e n t h e CO-complex of reduced c y t o c h r o m e P-450 a n d the reduced cytochrome, alone. The other a b s o r b a n c e m a x i m u m (420 nm) a n d the m i n i m u m (438 nm) in this s p e c t r u m m a y be due to c y t o c h r o m e P-420 or
COHN
ET AL.
B R I E F D E F I N I T I V E REPORT
1609
31C
c~
o x 2
2 cn £}
< I
