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Depression of hepatic drug-metabolizing enzyme activity by B. pertussis vaccination
European Journal of Pharmacology, 43 (1977) 281--284 © Elsewer/North-Holland Bmmedmal Press
281
Short c o m m u m c a t m n DEPRESSION OF HEPATIC DRUG-METABOLIZING ENZYME ACTIVITY BY B pertussls VACCINATION JOSEPH F WILLIAMS and ANDOR SZENTIVANYI
Department of Pharmacology, College of Medtcme, Untverslty of South Florlda, 12901 N 30th Street, Tampa, Flortda 33612, US A Received 9 March 1977, accepted 11 March 1977
J F WILLIAMS and A SZENTIVANYI, Depresszon of hepatic drug-metabolzzmg enzyme actwzty by B pertussts vaccmatmn, European J Pharmacol 43 (1977) 281--284 In wtro measurements are described on N-demethylatmn of am m o p y r m e and ethylmorphme, the hydroxylatlon of amhne as well as the level and achwty of the electron transport system m hepatm mmrosomal fractmns from pertussls vaccinated mine Results mdmate that pertusms vaccmahon of mine markedly decreases the level and actlwty of the hepatm drug-metabohzmg system Pertuss~s vaccine
Mmrosomes
L~ver
Drug metabohsm
1 Introductmn Certaan stratus of mice develop an exquisite hypersensltlvaty to histamine and other naturally occurring agents following vacclnatmn wath hve or killed Bordetella pertussts cells (Szentlvanyl, 1971). This hypertenslvaty is highly remlmscent of that produced by a pharmacologically estabhshed beta adrenergac blockade (Szentlvanyl, 1971, Ortez, 1976, Krzanowslu et a l , 1976, Polson et a l , 1976). Manlfestatmns of both hypersensltlvxtles, among others, also include an increased tolerance to exogenously administered catecholamines (Szenhvanyl, 1971). The present study was 1nitrated when xt was observed (J J. Krzanowslu, personal commumcatlon) that together with this increased tolerance to catecholamlnes, the pertussxs-sensltlzed mouse also shows a reduced tolerance to theophylhne. This seeming contradiction might be explmned by the recent fmdmg that theophylhne is a substrate for the hepatm m m r o s o m a l m i x e d f u n c t m n oxldase enzyme system (Lohmann and Mmch, 1976). The role
of thxs system m determining the lntenmty and duration of action of a wide variety of drugs is well estabhshed, suggesting that the reduced tolerance to theophylhne may reflect a hitherto unreported effect of pertussls vacclnatxon on this hepatic enzyme system This communication explores, therefore, the ability of the hver to metabohze drugs following pertussls vaccmatmn
2. Materials and methods
2 1 Senszttzatton o f ammals Adult female mine (CFW or CD-1) weighing 18--20 g and fed standard laboratory dmt and water ad hbltum were used. Merthlolated suspensions of commercial Bordetella pertusszs vaccine (4 × 101° lulled organlsms/ml, Eh Lilly & Co.) were diluted 1 : 1 with sterile pyrogen-free lsotomc saline just prior to use Groups of 10 animals received an i p. mjectlon of 7 X 109 cells/mouse and controls an eqmvalent volume (0.35 ml) of lsotomc
282
J F WILLIAMS, A SZENTIVANYI
saline 5 days after vaccmatmn, the time of p e a k sensatxvaty t o h i s t a m i n e ( S z e n t a v a n y l , 1971), 5 ammals from each group were challenged wath an a p mjectmn of hastamme (70 mg/kg of the free base), and the mcadence of d e a t h , as m d m a t l v e o f t h e d e v e l o p m e n t o f hypersensatlvaty, recorded during a subseq u e n t 6 0 man p e r i o d T h e h v e r s w e r e r e m o v e d from the remaining mine of each group for study of drug metabolism
2 2 Preparatmn of mtcrosomal fractton The pooled hvers of 5 anamals from each g r o u p w e r e r i n s e d w i t h c o l d 1 1 5 % KC1 a n d , a f t e r r e m o v a l o f c o n n e c t i v e tassue a n d gall b l a d d e r , w e r e w e x g h e d a n d h o m o g e m z e d an 4 v o l s (4 m l / g laver) o f c o l d 1 1 5 % KC1 M l c r o somes were prepared at 4°C by differential centnfugatmn of the homogenate at 10,000 g for 20 mm and centrffugatmn of the resulting s u p e r n a t a n t a t 1 0 5 , 0 0 0 g f o r 6 0 man
2 3 Enzyme assays Mlcrosomes were resuspended m cold 1 1 5 % KC1 so t h a t e a c h m l l l l h t e r w a s e q m v a lent to 250 mg of hver, wet weight Protein concentratxons were determined by the metho d o f L o w r y e t al ( 1 9 5 1 ) u s m g b o w n e s e r u m a l b u m i n as r e f e r e n c e p r o t e i n T h e m x c r o s o m a l suspension was then used to study the N
TABLE 1 Effect of Bordetella pertussts vaccine on the actlvltms and levels of the hepatm mmrosomal drug-metabohzmg enzymes Control/stram CFW Ammopyrme N-demethylase 1 Ethylmorphme N-demethylase a Anlhne hydroxylase 2
CD-1
0 668 ± 0 045 5 0 714 +_ 0 006 47 6
Pertussls-treated/strata CFW
0 820 r 0 065 0 747 ± 0 071
CD-1
0 432 ± 0 049 6 0 418 ± 0 043 6
± 0 72
55 8
± 1 23
29 2
± 0 74 6
0 554 ± 0 052 6 0 475 +_ 0 048 6 37 2
-+ 0 84 6
CytochromeP-4503
1 03
-+ 0 0 4
128
± 008
055
± 0066
071
± 0066
Cytochromebs 3
041
± 002
048
± 002
029
+- 0 0 3 6
032
± 0026
NADPH-cytochrome c reductase 4 182
± 4 58
180
± 17 3
141
-+ 8 22 7
pmoles formaldehyde/h/mg protein 2 nmoles p-ammophenol/h/mg protein 3 nmoles/mg protein 4 nmoles cytochrome c reduced/mm/mg protein s Values expressed as mean i S D of 5--7 experiments wxth pooled hvers of 5 ammals/group 6p<001 7p< 005
140
± 10 3 7
PERTUSSIS VACCINATION AND DRUG METABOLISM
estimated from the NADH-reduced spectrum, employing an e x t m c t m n coeffmmnt of 185/ mM/cm for the absorptlwty difference between 424 and 410 nm. Statistical comparisons were made by standard procedures
3. Results Both stratus o f mine vaccinated with killed B pertuss~s developed a marked histamine hypersensltlwty Values for the m wtro activation o f liver mmrosomal drug-metabohzmg enzymes, c y t o c h r o m e P-450 and c y t o c h r o m e b5 content, and N A D P H - c y t o c h r o m e c reductase actlwty are shown in table 1 E nz ym e actlwtms were depressed t o about the same e x t e n t m b o t h strmns o f vaccinated animals In contrast to the relatively equal decrease m the three enzymatm achwtles, c y t o c h r o m e P-450 concentrations were reduced to a somewhat greater degree than those of c y t o c h r o m e bs N A D P H - c y t o c h r o m e c reductase act~wty was affected to an even lesser degree Similar, but less marked, effects were seen when animals were lnnoculated with the lower dose of 3 5 × 109 cells/mouse Injection o f mice with 1 mg/kg merthiolate, the a m o u n t of preservative in the administered volume of vaccine, had no slgmfmant effect on hepatm drug metabolism. Also, m vitro addltmn of various concentrations o f the vaccine to the microsomal preparation from control animals did not affect en zy me activity
4. D i s c u s s i o n
The results indicate t hat pertussls vacclnat m n o f mine does indeed have an effect on hepatic f u n ctio n associated with the ability to m e t a b o h z e drugs. Although the levels o f cytoch r o m e bs and N A D P H - c y t o c h r o m e c reductase activity were reduced, the total decrease m drug m e t a b o h s m is probably due to the reduced levels o f c y t o c h r o m e P-450, the terminal omdase o f the mmrosomal e n z y m e system.
283
Whether the decrease in the actlwty o f the mmrosomal mixed function oxldase system is a reflection o f general t oxi ci t y, and thus a non-specific l m pm rm ent o f liver function in general, is as y e t u n d e t e r m i n e d However, m the present study no indications of a debilitated condition of the animals were observed Also, no slgmfmant difference m b o d y weight, m hver weight, or mlcrosomal protein content/g hver between control and pertussls vaccinated mine was found. Furt herm ore, ot her observations showed t hat the capacity of the liver at least to synthesize and degrade glycogen, incorporate radloact~ve amino acids into total hepatm protein, and to synthesize cyclic AMP is unlmpmred under condltmns of pertussls vaccination (Ortez, 1976, Szentlvanyl et al., 1977) Such altered m et abohsm could conceivably influence the toxmlty, half-life, and pharmacological actlwty o f substances administered m the course of studies utilizing these ammals as experimental models (Szentlvanyl, 1971, Szentlvanyl et a l , 1977) Moreover, it has been recently reported that pertussls vaccination of children increased their histamine sensltlwty, and produced o t h e r effects reminiscent o f those observed m vaccinated mine (Sen et a l , 1974) However, the relationship between these observations on children and the findings of the present c o m m u n l c a t m n is not known and requires further study
References Imal, Y , A I t o and R Sato, 1966, Evidence for blochemmally different types of vesmles in the hepatic mmrosomal fraction, J Blochem 60, 417 Krzanowskl, J J , J B Polson and A Szenttvanyl, 1976, Pulmonary patterns of adenosme-3',5'-cychc monophosphate accumulatmns m response to adrenergm or histamine shmulatlon m Bordetella pertusszs-sensltlzed mine, Blochem Pharmacol 25, 1631 Lohmann, S M and R P Mlech, 1976, Theophylllne metabohsm by the rat liver mmrosomal system, J Pharmacol Exptl Therap 196, 213 Lowry, O H , N J Rosebrough, A L Farr and R J Randall, 1951, Protein measurement with the Fohn phenol reagent, J Blol Chem 193, 265
284 Ortez, R A , 1976, E f f e c t o f h i s t a m i n e o n lung c y c h c AMP levels m n o r m a l a n d pe~tussls-vaccmated mine, B l o c h e m P h a r m a c o l 25, 523 Polson, J B , J J K r z a n o w s k l a n d A S z e n t l v a n y l , 1976, E f f e c t s o f m e t h a c h o h n e , h i s t a m i n e a n d a t r o p i n e on p u l m o n a r y g u a n o s m e - 3 ' , 5 ' - m o n o p h o s p h a t e levels m h y p e r s e n s i t i v e mice, N a u n y n S c h m m d e b A r c h P h a r m a c o l 295, 27 Sen, D K , S Aroma, S G u p t a a n d R K Sanyal, 1974, S t u d m s o n a d r e n e r g m m e c h a m s m s m r e l a t m n to histamine sensltlwty m children lmmumzed with Bordetella pertussts vaccine, J Allergy C h n I m m u n o l 54, 25 S z e n t l v a n y l , A , 1971, E f f e c t o f bacterial p r o d u c t s and a d r e n e r g m b l o c k i n g agents o n allergm reac-
J F WILLIAMS, A SZENTIVANYI tlons, m T e x t b o o k o f I m m u n o l o g i c a l Diseases, ed M S a m t e r (L,ttle, B r o w n & Co , B o s t o n , Mass ) p 356 Szentlvanyl, A, J B Polson and J J Krzanowskl, 1977, T h e altered r e a c t i v i t y o f t h e e f f e c t o r cells t o a n t l g e m c and p h a r m a c o l o g m a l i n f l u e n c e s and its r e l a t i o n to c:ychc n u c l e o t l d e s I E f f e c t o r reactlvltins m t h e a f f e r e n t l o o p o f t h e i m m u n e response, m P a t h o m e c h a m s m u s u n d P a t h o g e n e s e allerglscher R e a c k t l o n e n , ed G F l h p p (Werk-Verlag, Dr E d m u n d B a n a s c h e w s k l , G l a f e l f m g bel M u n c h e n ) ( m press) Williams, J F a n d R Karler, 1971, E f f e c t of cycloh e x l m l d e o n t h e m i x e d - f u n c t i o n oxldase s y s t e m of rat hver, Mol P h a r m a c o l 7, 269
281
Short c o m m u m c a t m n DEPRESSION OF HEPATIC DRUG-METABOLIZING ENZYME ACTIVITY BY B pertussls VACCINATION JOSEPH F WILLIAMS and ANDOR SZENTIVANYI
Department of Pharmacology, College of Medtcme, Untverslty of South Florlda, 12901 N 30th Street, Tampa, Flortda 33612, US A Received 9 March 1977, accepted 11 March 1977
J F WILLIAMS and A SZENTIVANYI, Depresszon of hepatic drug-metabolzzmg enzyme actwzty by B pertussts vaccmatmn, European J Pharmacol 43 (1977) 281--284 In wtro measurements are described on N-demethylatmn of am m o p y r m e and ethylmorphme, the hydroxylatlon of amhne as well as the level and achwty of the electron transport system m hepatm mmrosomal fractmns from pertussls vaccinated mine Results mdmate that pertusms vaccmahon of mine markedly decreases the level and actlwty of the hepatm drug-metabohzmg system Pertuss~s vaccine
Mmrosomes
L~ver
Drug metabohsm
1 Introductmn Certaan stratus of mice develop an exquisite hypersensltlvaty to histamine and other naturally occurring agents following vacclnatmn wath hve or killed Bordetella pertussts cells (Szentlvanyl, 1971). This hypertenslvaty is highly remlmscent of that produced by a pharmacologically estabhshed beta adrenergac blockade (Szentlvanyl, 1971, Ortez, 1976, Krzanowslu et a l , 1976, Polson et a l , 1976). Manlfestatmns of both hypersensltlvxtles, among others, also include an increased tolerance to exogenously administered catecholamines (Szenhvanyl, 1971). The present study was 1nitrated when xt was observed (J J. Krzanowslu, personal commumcatlon) that together with this increased tolerance to catecholamlnes, the pertussxs-sensltlzed mouse also shows a reduced tolerance to theophylhne. This seeming contradiction might be explmned by the recent fmdmg that theophylhne is a substrate for the hepatm m m r o s o m a l m i x e d f u n c t m n oxldase enzyme system (Lohmann and Mmch, 1976). The role
of thxs system m determining the lntenmty and duration of action of a wide variety of drugs is well estabhshed, suggesting that the reduced tolerance to theophylhne may reflect a hitherto unreported effect of pertussls vacclnatxon on this hepatic enzyme system This communication explores, therefore, the ability of the hver to metabohze drugs following pertussls vaccmatmn
2. Materials and methods
2 1 Senszttzatton o f ammals Adult female mine (CFW or CD-1) weighing 18--20 g and fed standard laboratory dmt and water ad hbltum were used. Merthlolated suspensions of commercial Bordetella pertusszs vaccine (4 × 101° lulled organlsms/ml, Eh Lilly & Co.) were diluted 1 : 1 with sterile pyrogen-free lsotomc saline just prior to use Groups of 10 animals received an i p. mjectlon of 7 X 109 cells/mouse and controls an eqmvalent volume (0.35 ml) of lsotomc
282
J F WILLIAMS, A SZENTIVANYI
saline 5 days after vaccmatmn, the time of p e a k sensatxvaty t o h i s t a m i n e ( S z e n t a v a n y l , 1971), 5 ammals from each group were challenged wath an a p mjectmn of hastamme (70 mg/kg of the free base), and the mcadence of d e a t h , as m d m a t l v e o f t h e d e v e l o p m e n t o f hypersensatlvaty, recorded during a subseq u e n t 6 0 man p e r i o d T h e h v e r s w e r e r e m o v e d from the remaining mine of each group for study of drug metabolism
2 2 Preparatmn of mtcrosomal fractton The pooled hvers of 5 anamals from each g r o u p w e r e r i n s e d w i t h c o l d 1 1 5 % KC1 a n d , a f t e r r e m o v a l o f c o n n e c t i v e tassue a n d gall b l a d d e r , w e r e w e x g h e d a n d h o m o g e m z e d an 4 v o l s (4 m l / g laver) o f c o l d 1 1 5 % KC1 M l c r o somes were prepared at 4°C by differential centnfugatmn of the homogenate at 10,000 g for 20 mm and centrffugatmn of the resulting s u p e r n a t a n t a t 1 0 5 , 0 0 0 g f o r 6 0 man
2 3 Enzyme assays Mlcrosomes were resuspended m cold 1 1 5 % KC1 so t h a t e a c h m l l l l h t e r w a s e q m v a lent to 250 mg of hver, wet weight Protein concentratxons were determined by the metho d o f L o w r y e t al ( 1 9 5 1 ) u s m g b o w n e s e r u m a l b u m i n as r e f e r e n c e p r o t e i n T h e m x c r o s o m a l suspension was then used to study the N
TABLE 1 Effect of Bordetella pertussts vaccine on the actlvltms and levels of the hepatm mmrosomal drug-metabohzmg enzymes Control/stram CFW Ammopyrme N-demethylase 1 Ethylmorphme N-demethylase a Anlhne hydroxylase 2
CD-1
0 668 ± 0 045 5 0 714 +_ 0 006 47 6
Pertussls-treated/strata CFW
0 820 r 0 065 0 747 ± 0 071
CD-1
0 432 ± 0 049 6 0 418 ± 0 043 6
± 0 72
55 8
± 1 23
29 2
± 0 74 6
0 554 ± 0 052 6 0 475 +_ 0 048 6 37 2
-+ 0 84 6
CytochromeP-4503
1 03
-+ 0 0 4
128
± 008
055
± 0066
071
± 0066
Cytochromebs 3
041
± 002
048
± 002
029
+- 0 0 3 6
032
± 0026
NADPH-cytochrome c reductase 4 182
± 4 58
180
± 17 3
141
-+ 8 22 7
pmoles formaldehyde/h/mg protein 2 nmoles p-ammophenol/h/mg protein 3 nmoles/mg protein 4 nmoles cytochrome c reduced/mm/mg protein s Values expressed as mean i S D of 5--7 experiments wxth pooled hvers of 5 ammals/group 6p<001 7p< 005
140
± 10 3 7
PERTUSSIS VACCINATION AND DRUG METABOLISM
estimated from the NADH-reduced spectrum, employing an e x t m c t m n coeffmmnt of 185/ mM/cm for the absorptlwty difference between 424 and 410 nm. Statistical comparisons were made by standard procedures
3. Results Both stratus o f mine vaccinated with killed B pertuss~s developed a marked histamine hypersensltlwty Values for the m wtro activation o f liver mmrosomal drug-metabohzmg enzymes, c y t o c h r o m e P-450 and c y t o c h r o m e b5 content, and N A D P H - c y t o c h r o m e c reductase actlwty are shown in table 1 E nz ym e actlwtms were depressed t o about the same e x t e n t m b o t h strmns o f vaccinated animals In contrast to the relatively equal decrease m the three enzymatm achwtles, c y t o c h r o m e P-450 concentrations were reduced to a somewhat greater degree than those of c y t o c h r o m e bs N A D P H - c y t o c h r o m e c reductase act~wty was affected to an even lesser degree Similar, but less marked, effects were seen when animals were lnnoculated with the lower dose of 3 5 × 109 cells/mouse Injection o f mice with 1 mg/kg merthiolate, the a m o u n t of preservative in the administered volume of vaccine, had no slgmfmant effect on hepatm drug metabolism. Also, m vitro addltmn of various concentrations o f the vaccine to the microsomal preparation from control animals did not affect en zy me activity
4. D i s c u s s i o n
The results indicate t hat pertussls vacclnat m n o f mine does indeed have an effect on hepatic f u n ctio n associated with the ability to m e t a b o h z e drugs. Although the levels o f cytoch r o m e bs and N A D P H - c y t o c h r o m e c reductase activity were reduced, the total decrease m drug m e t a b o h s m is probably due to the reduced levels o f c y t o c h r o m e P-450, the terminal omdase o f the mmrosomal e n z y m e system.
283
Whether the decrease in the actlwty o f the mmrosomal mixed function oxldase system is a reflection o f general t oxi ci t y, and thus a non-specific l m pm rm ent o f liver function in general, is as y e t u n d e t e r m i n e d However, m the present study no indications of a debilitated condition of the animals were observed Also, no slgmfmant difference m b o d y weight, m hver weight, or mlcrosomal protein content/g hver between control and pertussls vaccinated mine was found. Furt herm ore, ot her observations showed t hat the capacity of the liver at least to synthesize and degrade glycogen, incorporate radloact~ve amino acids into total hepatm protein, and to synthesize cyclic AMP is unlmpmred under condltmns of pertussls vaccination (Ortez, 1976, Szentlvanyl et al., 1977) Such altered m et abohsm could conceivably influence the toxmlty, half-life, and pharmacological actlwty o f substances administered m the course of studies utilizing these ammals as experimental models (Szentlvanyl, 1971, Szentlvanyl et a l , 1977) Moreover, it has been recently reported that pertussls vaccination of children increased their histamine sensltlwty, and produced o t h e r effects reminiscent o f those observed m vaccinated mine (Sen et a l , 1974) However, the relationship between these observations on children and the findings of the present c o m m u n l c a t m n is not known and requires further study
References Imal, Y , A I t o and R Sato, 1966, Evidence for blochemmally different types of vesmles in the hepatic mmrosomal fraction, J Blochem 60, 417 Krzanowskl, J J , J B Polson and A Szenttvanyl, 1976, Pulmonary patterns of adenosme-3',5'-cychc monophosphate accumulatmns m response to adrenergm or histamine shmulatlon m Bordetella pertusszs-sensltlzed mine, Blochem Pharmacol 25, 1631 Lohmann, S M and R P Mlech, 1976, Theophylllne metabohsm by the rat liver mmrosomal system, J Pharmacol Exptl Therap 196, 213 Lowry, O H , N J Rosebrough, A L Farr and R J Randall, 1951, Protein measurement with the Fohn phenol reagent, J Blol Chem 193, 265
284 Ortez, R A , 1976, E f f e c t o f h i s t a m i n e o n lung c y c h c AMP levels m n o r m a l a n d pe~tussls-vaccmated mine, B l o c h e m P h a r m a c o l 25, 523 Polson, J B , J J K r z a n o w s k l a n d A S z e n t l v a n y l , 1976, E f f e c t s o f m e t h a c h o h n e , h i s t a m i n e a n d a t r o p i n e on p u l m o n a r y g u a n o s m e - 3 ' , 5 ' - m o n o p h o s p h a t e levels m h y p e r s e n s i t i v e mice, N a u n y n S c h m m d e b A r c h P h a r m a c o l 295, 27 Sen, D K , S Aroma, S G u p t a a n d R K Sanyal, 1974, S t u d m s o n a d r e n e r g m m e c h a m s m s m r e l a t m n to histamine sensltlwty m children lmmumzed with Bordetella pertussts vaccine, J Allergy C h n I m m u n o l 54, 25 S z e n t l v a n y l , A , 1971, E f f e c t o f bacterial p r o d u c t s and a d r e n e r g m b l o c k i n g agents o n allergm reac-
J F WILLIAMS, A SZENTIVANYI tlons, m T e x t b o o k o f I m m u n o l o g i c a l Diseases, ed M S a m t e r (L,ttle, B r o w n & Co , B o s t o n , Mass ) p 356 Szentlvanyl, A, J B Polson and J J Krzanowskl, 1977, T h e altered r e a c t i v i t y o f t h e e f f e c t o r cells t o a n t l g e m c and p h a r m a c o l o g m a l i n f l u e n c e s and its r e l a t i o n to c:ychc n u c l e o t l d e s I E f f e c t o r reactlvltins m t h e a f f e r e n t l o o p o f t h e i m m u n e response, m P a t h o m e c h a m s m u s u n d P a t h o g e n e s e allerglscher R e a c k t l o n e n , ed G F l h p p (Werk-Verlag, Dr E d m u n d B a n a s c h e w s k l , G l a f e l f m g bel M u n c h e n ) ( m press) Williams, J F a n d R Karler, 1971, E f f e c t of cycloh e x l m l d e o n t h e m i x e d - f u n c t i o n oxldase s y s t e m of rat hver, Mol P h a r m a c o l 7, 269