Life Sciences, Vol. 48, pp. 1715-1719 Printed in the U.S.A.
Pergamon Press
EFFECTS OF RECOMBINANT HUMAN I N T E R F E R O N ALPHA ON ARYL HYDROCARBON HYDROXYLASE A C T I V I T Y IN CULTURED HUMAN P E R I P H E R A L LYk~HOCYTES
S.M. Moochhala and E.J.D. Lee
Dept of Pharmacology, Faculty of Medicine National University of Singapore, Kent Ridge Crescent, Singapore 0511 (Received in final form February 22, 1991)
Sunm~ary Interferon and its reducers are known to depress drug b i o t r a n s f o r m a t i o n in vlvo by d e c r e a s i n g the levels of c y t o c h r o m e P-450 (P450) monooxygenase system in the h v e r . However, very l i t t l e is known about the e f f e c t s of i n t e r f e r o n on P450 in
extrahepatlc tissues. In this study we investigated the effects of a recombinant human interferon-alpha (rhlFN-a) on aryl hydrocarbon hydroxylase (P450IAI) in cultured human peripheral lymphocytes (HPL). Non-induced and induced (3-methylcholantherene) mitogen activated lymphocytes were used throughout the study, r h l F N - a maximally depressed AHH a c t i v i t y to approximately 58% of c o n t r o l a f t e r 24 hrs of incubation in both n o n - i n d u c e d and induced lymphocytes. However, a f t e r 48 hrs of incubation with r h I F N - ~ , AHH a c t w i t y had r e c o v e r e d to 86% of c o n t r o l in induced cells and 61% in non-induced cells. r h l F N - a had no s i g n i f i c a n t e f f e c t on e i t h e r NADH c y t o c h r o m e c r e d u c t a s e a c t i v i t y or on viable lymphocyte cell count. This is the first demonstration that r h l F N - a can have a d i r e c t depressive e f f e c t on a P450 dependent monooxygenase system in HPL. It is now known that i n f e c t i o n s and immunisations are a s s o c i a t e d with impaired metabolism of drugs such as warfarin, aminopyrine, phenytoln, acetuminophen and quinine in man (1,2). All these drugs are metabolised by the h e p a t i c c y t o c h r o m e P 4 5 0 - d e p e n d e n t monooxygenase system. In animal studies, both i n t e r f e r o n - i n d u c i n g a g e n t s and i n t e r f e r o n i t s e l f have been shown to inhibit drug metabolism by lowering hepatic P450 levels. The inhibition of drug metabolism that o c c u r s with i n f e c t i o n and immunisation has t h e r e f o r e been a t t r i b u t e d to the release of endogenous i n t e r f e r o n (1,2). It has r e c e n t l y been demonstrated that the administration of recombinant human i n t e r f e r o n alpha ( r h l F N - a ) to humans results in a s i g n i f i c a n t r e d u c t i o n in the c l e a r a n c e of a n t i p y r i n e (3) and theophylline (4). H i t h e r t o , these studies have focused on the depression of h e p a t i c P450 a c t i v i t y by i n t e r f e r o n and its inducers. R e c e n t l y however, t h e r e has been an increasing number of studies c o n c e r n e d with e x t r a h e p a t i c drug metabolism. This is probably due less to a g e n e r a l b e l i e f in the importance of these enzymes for overall drug metabolism than to the p e r c e i v e d physiological, pharmacological and t o x i c o l o g i c a l implications of s p e c i f i c P450 dependent r e a c t i o n s in the individual e x t r a h e p a t i c tissue.
0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc
1716
Interferon on AHH Activity in Lymphocytes
Vol. 48, No. 18, 1991
A preliminary study has shown t h a t administration of a consensus recombinant human i n t e r f e r o n can depress P450 level as well as the a c t i v i t y of aryl h y d r o c a r b o n h y d r o x y l a s e (AHH) in hamster spleen, adrenal, lung and kidney (5). This present study was designed to examine the e f f e c t s of a clinically used recombinant human i n t e r f e r o n alpha on the a c t i v i t y of a s p e c i f i c P450, AHH (P450IAI) in m i t o g e n - a c t i v a t e d human peripheral lymphocytes (HPL). Earlier studies have suggested t h a t studies on AHH a c t i v i t y in HPL may provide indications of similar e f f e c t s on P450 a c t i v i t i e s in less accessible tissues (6). The a c t i v i t y of AHH in HPL may also be used as an index of s u s c e p t i b i l i t y to chemical c a r c i n o g e n e s i s and t o x i c i t y (6). This study is the first to d e m o n s t r a t e a d i r e c t e f f e c t of r h l F N - a on AHH a c t i v i t y in a human e x t r a h e p a t i c tissue {i.e. HPL). Methods
Subjects:
Four h e a l t h y normal male adults (age from 31 to 37) were r e c r u i t e d for this study. All were l a b o r a t o r y s t a f f of the National U n i v e r s i t y of Singapore. These subjects were non-smokers, free from medication and had no h i s t o r y of h y p e r t e n s i o n , c a r d i o v a s c u l a r diseases or r e c e n t a c u t e or chronic r e s p i r a t o r y diseases.
Lymphocyte preparation and culture:
Lymphocytes were isolated from fresh (not more than 1 hr old) whole blood using Histopaque g r a d i e n t c e n t r i f u g a t i o n (7). All p r o c e d u r e s were performed at room temperature. Cells were c u l t u r e d (8) for 48 hrs in 95% air/5% CO 2 at 37eC b e f o r e s t a r t of experiment. Prior to culture, phytohemagglutinin and ~)okeweed mitogen were added in final c o n c e n t r a t i o n s of 1% each, 8o as to ))activate" the lymphocytes. In a n o t h e r study, mitogens plus 3 - m e t h y l c h o l a n t h r e n e (MCA, 1.5 ~M) were added simultaneously prior to c u l t u r e , in order to "induce" the lymphocytes. An aliquot of cell suspension was then removed a f t e r 48 hrs for counting. Cell viability was determined to be g r e a t e r than 95% by t r y p a n blue exlusion. Both n o n - i n d u c e d as well as induced lymphocyte (3 MCA t r e a t e d ) were f u r t h e r incubated with e i t h e r r h l F N - a [2500 IU ml - t phosphate b u f f e r saline (PBS)] or PBS for a n o t h e r 48 hrs.
AHH and NADH-dependent cytochrome c reductase (cyt c) assay:
AHH, c y t c a c t i v i t i e s and vmble cell counts were determined at 0 hr, 24 hrs and 48 hrs a f t e r incubating induced and n o n - i n d u c e d HPL with PBS or r h l F N - ~ . Cells were c o l l e c t e d by c e n t r l f u g a t i o n and washed t h r e e times with PBS b e f o r e assay. The assay for AHH and c y t c a c t i v i t i e s were determined as previously described (8). A unit of AHH a c t i v i t y is defined as the f l u o r e s c e n t equivalent of 1 pmol 3-OH b e n z o ( a ) p y r e n e produced min - l at 37°C. A unit of c y t c a c t i v i t y is the r e d u c t i o n of l nmol of c y t c min - I at 37°C. Q u a d r u p h c a t e measurements were performed at each incubation time and specific a c t i v i t i e s of AHH was expressed per unit of c y t c a c t i v i t y . Cyt c a c t i v i t y was expressed in units per 106 cells.
Statistical analysis: S t u d e n t ' s paired t - t e s t was used t h r o u g h o u t the study. Results C o n s i d e r a t i o n of the following details resulted in a r e p r o d u c i b l e ( c o e f f i c i e n t of v a r i a n c e = 0.2) assay for induced and n o n - i n d u c e d AHH in HPL: (a~ s t a n d a r d i z a t i o n of the mltogen a c t i v a t i o n step by initiation of cultures at 10 o cell m1-1, (b) addition of both phytohemagglutinin and pokeweed mitogens (1% each in final incubation) in c u l t u r e s for 48 hrs, (c) induction of AHH a c t i v i t y using 48 hrs exposure to MCA and (d) p r e s e n t a t i o n of specific AHH a c t i v i t y in terms of units AHH per unit N A D H - d e p e n d e n t c y t c a c t i v i t y .
Vol. 48, No. 18, 1991
Interferon on AHH Activity in Lymphocytes
1717
AFIH and c y t c a c t i v i t y were not d e t e c t a b l e in freshly p r e p a r e d lymphocytes. Culturing HPL with mitogens resulted in a t w o - f o l d increase in lymphocyte cell count and allowed d e t e c t i o n of AHFI and c y t c a c t i v i t y . Induction with MCA (1.5 ~M) i n c r e a s e d AHH a c t i v i t y in mitogen a c t i v a t e d HPL by 2.5 fold without any s i g n i f i c a n t change in c y t c a c t i v i t y (Table 1). In n o n - i n d u c e d lymphocyte c u l t u r e s (Table 1), lymphocyte count c o n t i n u e d to i n c r e a s e for additional 48 hrs whilst specific AHFI and c y t c a c t i v i t i e s e i t h e r remained unchanged or d e c r e a s e d with time. Similar t i m e - c o u r s e loss in P450 dependent enzyme a c t i v i t i e s have been r e p o r t e d in n o n - i n d u c e d h e p a t o c y t e cultures. Perhaps, like in c u l t u r e d h e p a t o c y t e s , this loss in a c t i v i t y with time may be due to increase in turnover of P450 in HPL. In the induced lymphocyte c u l t u r e s however, lymphocyte counts, AHFI and c y t c a c t i v i t y remained unchanged t h r o u g h o u t the 48 hrs post incubation period, r h I F N - a (2500 IU m1-1) s i g n i f i c a n t l y depressed AHH a c t i v i t y in FIPL in both n o n - i n d u c e d and induced HPL (Table 1). r h l F N - ~ maximally depressed AHFI a c t i v i t y to approximately 58% of c o n t r o l a f t e r 24 hrs of i n c u b a t i o n in both n o n - i n d u c e d and induced FIPL. However, a f t e r 48 hrs of incubation with r h I F N - a , AFIFI a c t i v i t y had r e c o v e r e d to 86% and 61% of c o n t r o l in induced and n o n - i n d u c e d cells r e s p e c t i v e l y . Similarly, when n o n - r e d u c e d and induced FIPL were exposed to 5000 IU m1-1 of r h l F N - a , AFIFI a c t i v i t y in both p r e p a r a t i o n of HPL was depressed to 61% of c o n t r o l a f t e r 24 hrs of incubation; and 63% and 91% of c o n t r o l r e s p e c t i v e l y a f t e r 48 hrs of incubation, r h l F N - a had no s i g n i f i c a n t e f f e c t on e i t h e r c y t c a c t i v i t y or on lymphocyte cell count. At 48 hrs, AHFI a c t i v i t y a f t e r incubation with r h I F N - ~ was still higher in induced HPL than that of n o n - i n d u c e d HPL. TABLE 1 E f f e c t s Of r h l F N In HPL.
On L y m p h o c y t e Count, AHH And NADH Cyt c R e d u c t a s e
Activty .
.
.
.
.
.
Time (hrs) .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
cyt~c ( u n i t s 10u c e l l s ) Control rhlFN- Q .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
AHH ( u n i t s per u n i t c y t c) Control rhlFN- a .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Lymphocyte count
( l x l 0 ~ c e l l s ml - | ) Control rhlFN- a .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
N O N - I NDUCED 0
26.0±7.6
-
0.36±0.07
24
24.5±2.0 (100)
21.3±3.2 (87)
0.35±0.05 (100)
48
21.2±0.3
22.3±2.2 (105)
0.18±0.02 (I00)
(100)
2.1±0.3
-
0.20¢0.02" (57)
4.5±0.2 (ZOO)
4.7+0.4 (104)
0.11 a0.01* (61)
6.2±0.8 (100)
5.9+0.6
(95)
INDUCED 0
2 9 . 9 + 1.1
24
22.9±1.0 (100)
28.7±2.0 (125)
1.24±0.17 (100)
0.72±0.05* (58)
2.2±0.13 (100)
2.1±1.0
31.9±2.7 (100)
27.7±3.6 (87)
0.80±0.17 (100)
0.69±0. I0 (86)
2.3±0.15 (100)
2.5±0.04 (109)
48
0.93+0.21
-
2.1+1.0
(95)
The above parameters were determined in quadruplicate for each sample. The sean ± SE are shown. The values in the parenthesis indicates percent change from corresponding control (vehicle) cultures. * denotes s i s n i f i c a n t differences from vehicle (P
Pergamon Press
EFFECTS OF RECOMBINANT HUMAN I N T E R F E R O N ALPHA ON ARYL HYDROCARBON HYDROXYLASE A C T I V I T Y IN CULTURED HUMAN P E R I P H E R A L LYk~HOCYTES
S.M. Moochhala and E.J.D. Lee
Dept of Pharmacology, Faculty of Medicine National University of Singapore, Kent Ridge Crescent, Singapore 0511 (Received in final form February 22, 1991)
Sunm~ary Interferon and its reducers are known to depress drug b i o t r a n s f o r m a t i o n in vlvo by d e c r e a s i n g the levels of c y t o c h r o m e P-450 (P450) monooxygenase system in the h v e r . However, very l i t t l e is known about the e f f e c t s of i n t e r f e r o n on P450 in
extrahepatlc tissues. In this study we investigated the effects of a recombinant human interferon-alpha (rhlFN-a) on aryl hydrocarbon hydroxylase (P450IAI) in cultured human peripheral lymphocytes (HPL). Non-induced and induced (3-methylcholantherene) mitogen activated lymphocytes were used throughout the study, r h l F N - a maximally depressed AHH a c t i v i t y to approximately 58% of c o n t r o l a f t e r 24 hrs of incubation in both n o n - i n d u c e d and induced lymphocytes. However, a f t e r 48 hrs of incubation with r h I F N - ~ , AHH a c t w i t y had r e c o v e r e d to 86% of c o n t r o l in induced cells and 61% in non-induced cells. r h l F N - a had no s i g n i f i c a n t e f f e c t on e i t h e r NADH c y t o c h r o m e c r e d u c t a s e a c t i v i t y or on viable lymphocyte cell count. This is the first demonstration that r h l F N - a can have a d i r e c t depressive e f f e c t on a P450 dependent monooxygenase system in HPL. It is now known that i n f e c t i o n s and immunisations are a s s o c i a t e d with impaired metabolism of drugs such as warfarin, aminopyrine, phenytoln, acetuminophen and quinine in man (1,2). All these drugs are metabolised by the h e p a t i c c y t o c h r o m e P 4 5 0 - d e p e n d e n t monooxygenase system. In animal studies, both i n t e r f e r o n - i n d u c i n g a g e n t s and i n t e r f e r o n i t s e l f have been shown to inhibit drug metabolism by lowering hepatic P450 levels. The inhibition of drug metabolism that o c c u r s with i n f e c t i o n and immunisation has t h e r e f o r e been a t t r i b u t e d to the release of endogenous i n t e r f e r o n (1,2). It has r e c e n t l y been demonstrated that the administration of recombinant human i n t e r f e r o n alpha ( r h l F N - a ) to humans results in a s i g n i f i c a n t r e d u c t i o n in the c l e a r a n c e of a n t i p y r i n e (3) and theophylline (4). H i t h e r t o , these studies have focused on the depression of h e p a t i c P450 a c t i v i t y by i n t e r f e r o n and its inducers. R e c e n t l y however, t h e r e has been an increasing number of studies c o n c e r n e d with e x t r a h e p a t i c drug metabolism. This is probably due less to a g e n e r a l b e l i e f in the importance of these enzymes for overall drug metabolism than to the p e r c e i v e d physiological, pharmacological and t o x i c o l o g i c a l implications of s p e c i f i c P450 dependent r e a c t i o n s in the individual e x t r a h e p a t i c tissue.
0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc
1716
Interferon on AHH Activity in Lymphocytes
Vol. 48, No. 18, 1991
A preliminary study has shown t h a t administration of a consensus recombinant human i n t e r f e r o n can depress P450 level as well as the a c t i v i t y of aryl h y d r o c a r b o n h y d r o x y l a s e (AHH) in hamster spleen, adrenal, lung and kidney (5). This present study was designed to examine the e f f e c t s of a clinically used recombinant human i n t e r f e r o n alpha on the a c t i v i t y of a s p e c i f i c P450, AHH (P450IAI) in m i t o g e n - a c t i v a t e d human peripheral lymphocytes (HPL). Earlier studies have suggested t h a t studies on AHH a c t i v i t y in HPL may provide indications of similar e f f e c t s on P450 a c t i v i t i e s in less accessible tissues (6). The a c t i v i t y of AHH in HPL may also be used as an index of s u s c e p t i b i l i t y to chemical c a r c i n o g e n e s i s and t o x i c i t y (6). This study is the first to d e m o n s t r a t e a d i r e c t e f f e c t of r h l F N - a on AHH a c t i v i t y in a human e x t r a h e p a t i c tissue {i.e. HPL). Methods
Subjects:
Four h e a l t h y normal male adults (age from 31 to 37) were r e c r u i t e d for this study. All were l a b o r a t o r y s t a f f of the National U n i v e r s i t y of Singapore. These subjects were non-smokers, free from medication and had no h i s t o r y of h y p e r t e n s i o n , c a r d i o v a s c u l a r diseases or r e c e n t a c u t e or chronic r e s p i r a t o r y diseases.
Lymphocyte preparation and culture:
Lymphocytes were isolated from fresh (not more than 1 hr old) whole blood using Histopaque g r a d i e n t c e n t r i f u g a t i o n (7). All p r o c e d u r e s were performed at room temperature. Cells were c u l t u r e d (8) for 48 hrs in 95% air/5% CO 2 at 37eC b e f o r e s t a r t of experiment. Prior to culture, phytohemagglutinin and ~)okeweed mitogen were added in final c o n c e n t r a t i o n s of 1% each, 8o as to ))activate" the lymphocytes. In a n o t h e r study, mitogens plus 3 - m e t h y l c h o l a n t h r e n e (MCA, 1.5 ~M) were added simultaneously prior to c u l t u r e , in order to "induce" the lymphocytes. An aliquot of cell suspension was then removed a f t e r 48 hrs for counting. Cell viability was determined to be g r e a t e r than 95% by t r y p a n blue exlusion. Both n o n - i n d u c e d as well as induced lymphocyte (3 MCA t r e a t e d ) were f u r t h e r incubated with e i t h e r r h l F N - a [2500 IU ml - t phosphate b u f f e r saline (PBS)] or PBS for a n o t h e r 48 hrs.
AHH and NADH-dependent cytochrome c reductase (cyt c) assay:
AHH, c y t c a c t i v i t i e s and vmble cell counts were determined at 0 hr, 24 hrs and 48 hrs a f t e r incubating induced and n o n - i n d u c e d HPL with PBS or r h l F N - ~ . Cells were c o l l e c t e d by c e n t r l f u g a t i o n and washed t h r e e times with PBS b e f o r e assay. The assay for AHH and c y t c a c t i v i t i e s were determined as previously described (8). A unit of AHH a c t i v i t y is defined as the f l u o r e s c e n t equivalent of 1 pmol 3-OH b e n z o ( a ) p y r e n e produced min - l at 37°C. A unit of c y t c a c t i v i t y is the r e d u c t i o n of l nmol of c y t c min - I at 37°C. Q u a d r u p h c a t e measurements were performed at each incubation time and specific a c t i v i t i e s of AHH was expressed per unit of c y t c a c t i v i t y . Cyt c a c t i v i t y was expressed in units per 106 cells.
Statistical analysis: S t u d e n t ' s paired t - t e s t was used t h r o u g h o u t the study. Results C o n s i d e r a t i o n of the following details resulted in a r e p r o d u c i b l e ( c o e f f i c i e n t of v a r i a n c e = 0.2) assay for induced and n o n - i n d u c e d AHH in HPL: (a~ s t a n d a r d i z a t i o n of the mltogen a c t i v a t i o n step by initiation of cultures at 10 o cell m1-1, (b) addition of both phytohemagglutinin and pokeweed mitogens (1% each in final incubation) in c u l t u r e s for 48 hrs, (c) induction of AHH a c t i v i t y using 48 hrs exposure to MCA and (d) p r e s e n t a t i o n of specific AHH a c t i v i t y in terms of units AHH per unit N A D H - d e p e n d e n t c y t c a c t i v i t y .
Vol. 48, No. 18, 1991
Interferon on AHH Activity in Lymphocytes
1717
AFIH and c y t c a c t i v i t y were not d e t e c t a b l e in freshly p r e p a r e d lymphocytes. Culturing HPL with mitogens resulted in a t w o - f o l d increase in lymphocyte cell count and allowed d e t e c t i o n of AHFI and c y t c a c t i v i t y . Induction with MCA (1.5 ~M) i n c r e a s e d AHH a c t i v i t y in mitogen a c t i v a t e d HPL by 2.5 fold without any s i g n i f i c a n t change in c y t c a c t i v i t y (Table 1). In n o n - i n d u c e d lymphocyte c u l t u r e s (Table 1), lymphocyte count c o n t i n u e d to i n c r e a s e for additional 48 hrs whilst specific AHFI and c y t c a c t i v i t i e s e i t h e r remained unchanged or d e c r e a s e d with time. Similar t i m e - c o u r s e loss in P450 dependent enzyme a c t i v i t i e s have been r e p o r t e d in n o n - i n d u c e d h e p a t o c y t e cultures. Perhaps, like in c u l t u r e d h e p a t o c y t e s , this loss in a c t i v i t y with time may be due to increase in turnover of P450 in HPL. In the induced lymphocyte c u l t u r e s however, lymphocyte counts, AHFI and c y t c a c t i v i t y remained unchanged t h r o u g h o u t the 48 hrs post incubation period, r h I F N - a (2500 IU m1-1) s i g n i f i c a n t l y depressed AHH a c t i v i t y in FIPL in both n o n - i n d u c e d and induced HPL (Table 1). r h l F N - ~ maximally depressed AHFI a c t i v i t y to approximately 58% of c o n t r o l a f t e r 24 hrs of i n c u b a t i o n in both n o n - i n d u c e d and induced FIPL. However, a f t e r 48 hrs of incubation with r h I F N - a , AFIFI a c t i v i t y had r e c o v e r e d to 86% and 61% of c o n t r o l in induced and n o n - i n d u c e d cells r e s p e c t i v e l y . Similarly, when n o n - r e d u c e d and induced FIPL were exposed to 5000 IU m1-1 of r h l F N - a , AFIFI a c t i v i t y in both p r e p a r a t i o n of HPL was depressed to 61% of c o n t r o l a f t e r 24 hrs of incubation; and 63% and 91% of c o n t r o l r e s p e c t i v e l y a f t e r 48 hrs of incubation, r h l F N - a had no s i g n i f i c a n t e f f e c t on e i t h e r c y t c a c t i v i t y or on lymphocyte cell count. At 48 hrs, AHFI a c t i v i t y a f t e r incubation with r h I F N - ~ was still higher in induced HPL than that of n o n - i n d u c e d HPL. TABLE 1 E f f e c t s Of r h l F N In HPL.
On L y m p h o c y t e Count, AHH And NADH Cyt c R e d u c t a s e
Activty .
.
.
.
.
.
Time (hrs) .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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cyt~c ( u n i t s 10u c e l l s ) Control rhlFN- Q .
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AHH ( u n i t s per u n i t c y t c) Control rhlFN- a .
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Lymphocyte count
( l x l 0 ~ c e l l s ml - | ) Control rhlFN- a .
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N O N - I NDUCED 0
26.0±7.6
-
0.36±0.07
24
24.5±2.0 (100)
21.3±3.2 (87)
0.35±0.05 (100)
48
21.2±0.3
22.3±2.2 (105)
0.18±0.02 (I00)
(100)
2.1±0.3
-
0.20¢0.02" (57)
4.5±0.2 (ZOO)
4.7+0.4 (104)
0.11 a0.01* (61)
6.2±0.8 (100)
5.9+0.6
(95)
INDUCED 0
2 9 . 9 + 1.1
24
22.9±1.0 (100)
28.7±2.0 (125)
1.24±0.17 (100)
0.72±0.05* (58)
2.2±0.13 (100)
2.1±1.0
31.9±2.7 (100)
27.7±3.6 (87)
0.80±0.17 (100)
0.69±0. I0 (86)
2.3±0.15 (100)
2.5±0.04 (109)
48
0.93+0.21
-
2.1+1.0
(95)
The above parameters were determined in quadruplicate for each sample. The sean ± SE are shown. The values in the parenthesis indicates percent change from corresponding control (vehicle) cultures. * denotes s i s n i f i c a n t differences from vehicle (P
