Cancer Letters, 3 (1977) 189--195 © Elsevier/North-Holland Scientific Publishers, Ltd.
189
RELATIONSHIP OF A R Y L HYDROCARBON HYDROXYLASE ACTIVITY TO BENZO(a)PYRENE-METABOLIZING ACTIVITY OF CELLS IN CULTURE
LEILA DIAMOND and WILLIAM M. BAIRD The Wistar Institute o f A n a t o m y and Biology, 36th Street at Spruce, Philadelphia, Pennsylvania 19104 (U.S.A.)
(Received 19 April 1977) (Revised version received 16 May 1977) (Accepted 25 May 1977)
SUMMARY The basal level o f aryl hydrocarbon hydroxylase (AHH) activity was high in cultures of low passage hamster e m b r y o (HE) cells but AHH inducibility by benz(a)-anthracene (BA) was low; in R72/3 rat liver cells, basal activity was low and inducibility was high. The metabolism of ~H--benzo(a)pyrene was similar in cultures of BA-induced R72/3 cells and uninduced HE cells. Thus, low AHH inducibility m a y n o t always be an indication of the cells' absolute hydrocarbonmetabolizing capacity.
INTRODUCTION It is now generally accepted t h a t polycyclic aromatic hydrocarbons require metabolic activation for carcinogenic activity and that an initial step is oxidation by the microsomal oxygenase system, AHH. Kellerman et al. [9] reported a positive correlation between high AHH inducibility of phytohemagglutininstimulated h u m a n l y m p h o c y t e s and susceptibility of the individual to bronchogenic carcinoma. However, there are suggestions that for some cells or tissues, AHH inducibility may n o t be an appropriate indicator of their hydrocarbon-metabolizing capacity. In a study of the effects of cigarette smoke inhalation on AHH activity in mouse tissues, Abramson and H u t t o n [1] noted that AHH inducibility in lung was frequently a poor indicator of an animal's Address correspondence to: Dr. Leila Diamond, The Wistar Institute of Anatomy and
Biology, 36th Street at Spruce, Philadelphia, Pennsylvania 19104, U.S.A. Abbreviations: AHH, aryl hydrocarbon hydroxylase; BA, benz(a)anthracene; BP, benzo-
(a)pyrene; DMSO, dimethyl sulfoxide; HE, hamster embryo.
190
maximal level of A H H activity. Coomes et al. [5] studied cultured human l y m p h o c y t e s from approx. 100 individuals and observed no correlation b e t w e e n basal A H H levels and A H H inducibility. To investigate the relationship b e t w e e n the A H H inducibility and actual hydrocarbon-metabolizing capacity o f a cell, we selected t w o cell lines that differ widely in inducibility and compared their capacity to metabolize the carcinogenic hydrocarbon, benzo(a)pyrene (BP). MATERIALS AND METHODS
Primary Syrian hamster e m b r y o (HE) cell cultures were prepared from 12- to 13-day-old e m b r y o s as described previously [3]. The R 7 2 / 3 cell line is derived from a t u m o r produced b y rat liver cells that had transformed spontaneously in culture [ 7 ] . The cells were grown as m o n o l a y e r cultures in Eagle's basal medium supplemented with 10% bovine fetal serum (Reheis Chemical Co. or Flow Labs. ) or bovine calf serum (Industrial Biological Labs.). To measure AHH activity and BP metabolism, the cells were seeded at a density of 5--7 • 104 cells/cm 2 in l()0-mm plastic dishes and after 24 h the cultures were refed with fresh medium. A stock solution of BA [Eastman Organic Chemicals; 3 mg BA/ml dimethyl sulfoxide (DMSO)] was diluted with medium and added to the plates to give a final concentration of 3 ~g BA/ml medium; DMSO was added to control plates at a final concentration of 0.1%. Sixteen hours later, cells from 5 control and 5 BA-treated plates were harvested by scraping and the cell pellets were stored at --70 °C for subsequent A H H assay. Preliminary experiments had indicated that with both HE and R72[3 cells A H H activity was maximal after 16 h of BA treatment. The A H H activity of the cell pellets was measured by the fluorescence assay of Nebert and Gelboin [ 11] with the modifications described by Diamond et al. [6]. One unit of AHH catalyses in 1 rain the formation of phenolic products with fluorescence' equivalent to that o f 1 pmol 3-hydroxy-BP; AHH specific activity is expressed as units/mg cell protein. At the same time that plates were harvested for A H H assay, BP metabolism was measured in three additional plates of each group. The cell monolayers were rinsed twice with 8 ml medium supplemented with serum and were refed with 15 ml fresh medium containing 0.5 nmol 3H--BP (Amersham-Searle Corp., spec. act. 3.0 Ci/mmol)/ml or 5.0 nmol 3H--BP (spec. act. 8.3 Ci/mmol)/ml. One plate containing ~H--BP b u t no cells was used as a blank. The a m o u n t of 3H--BP metabolized to water-soluble metabolites was determined by removing 0.2 ml of medium from each culture vessel and extracting with chloroform-methanol according to the procedure described by Baird and Diamond [3]. The a m o u n t of radioactivity in the organic and aqueous phases was measured by counting duplicate 0.1 ml samples in 7-ml polyethylene vials containing 4 ml scintillant {TT-21, Y o r k t o w n Res.) in a Packard Tri-Carb liquid scintillation counter and correcting for'counting efficiency by automatic external standard ratios.
191 RESULTS
HE cell cultures grown in medium supplemented with either calf or fetal serum had high basal levels of A H H activity (Table 1). Pretreatment with BA induced a small b u t significant (P < 0.001) increase in A H H activity in cultures in m e d i u m supplemented with calf serum; the increase in cultures in medium suppIemented with fetal serum was n o t statistically significant. In contrast, R 7 2 / 3 rat liver cells had low levels of basal A H H activity in m e d i u m supplemented with calf or fetal serum and, in b o t h cases, pretreatment with BA induced a 20- to 30-fold increase in activity (Table 1). The difference b e t w e e n m a x i m u m activity induced in the 2 sera was n o t significant. Since statistically significant A H H induction occurred in both HE and 1%72/3 cells only when the medium was supplemented with calf serum, BP metabolism was c o m p a r e d in BA-treated and control cultures containing medium supplem e n t e d with calf serum. For the first 5 h, the a m o u n t of 3H--BP metabolized to water-soluble derivatives b y HE cells was greater in cultures pretreated with BA than in untreated controls (Fig. 1). This enhancement was slight (10--40%) b u t was consistently observed in cultures in which A H H activity was induced at least 2-fold b y BA-pretreatment. There was less difference b e t w e e n the amounts of BP metabolites in control and BA-treated cultures at 7 h and no detectable difference at later times (not shown). In R 7 2 / 3 cell cultures, only a small a m o u n t of BP was metabolized during the first 3 h after addition of 3H--HP to uninduced cultures (Fig. 1). However, the cumulative a m o u n t o f BP metabolized increased by larger increments at each hourly interval, suggesting that the BP-metabolizing activity o f these cells TABLE1 A R Y L H Y D R O C A R B O N H Y D R O X Y L A S E L E V E L S IN S E C O N D A R Y H A M S T E R E M B R Y O (HE) C E L L S A N D R 7 2 / 3 R A T L I V E R C E L L S A H H Specific activity ( u n i t s / m g p r o t e i n ) Fetal serum a
Calf s e r u m
8.9 + 2.5 ( 6 . 4 - - 1 1 . 6 ) b 16.1 + 4.8 ( 1 1 . 1 - - 2 1 . 9 ) 1.8 (n = 5)
8.3 ± 3.9 ( 2 , 0 - - 1 3 . 9 ) 21.0 -+ 8.9 ( 4 . 5 - - 3 4 . 0 ) 3 (n = 18)
0.6 +- 0.5 ( 0 . 1 - - 1 . 5 ) 10.5 _+ 5.2 ( 5 . 3 - - 2 0 . 1 ) 33 (n = 6)
1.0 +- 0.8 ( 0 . 3 - - 2 . 7 ) 12.5 -+ 8.6 ( 3 . 7 - - 2 9 . 5 ) 17 (n = 9)
HE Control BA-treated Inducibility ratio R72/3 Control BA-treated Inducibility ratio
a T h r e e d i f f e r e n t lots o f b o v i n e fetal s e r u m and t w o o f b o v i n e calf s e r u m w e r e used. ~b T h e A H H values are t h e m e a n s p e c i f i c activity ~_ S.D. a n d , in p a r e n t h e s i s , t h e range f r o m n n u m b e r o f e x p e r i m e n t s . T h e i n d u c i b i l i t y ratios are t h e m e a n ratios o f n e x p e r i m e n t s .
192
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189
RELATIONSHIP OF A R Y L HYDROCARBON HYDROXYLASE ACTIVITY TO BENZO(a)PYRENE-METABOLIZING ACTIVITY OF CELLS IN CULTURE
LEILA DIAMOND and WILLIAM M. BAIRD The Wistar Institute o f A n a t o m y and Biology, 36th Street at Spruce, Philadelphia, Pennsylvania 19104 (U.S.A.)
(Received 19 April 1977) (Revised version received 16 May 1977) (Accepted 25 May 1977)
SUMMARY The basal level o f aryl hydrocarbon hydroxylase (AHH) activity was high in cultures of low passage hamster e m b r y o (HE) cells but AHH inducibility by benz(a)-anthracene (BA) was low; in R72/3 rat liver cells, basal activity was low and inducibility was high. The metabolism of ~H--benzo(a)pyrene was similar in cultures of BA-induced R72/3 cells and uninduced HE cells. Thus, low AHH inducibility m a y n o t always be an indication of the cells' absolute hydrocarbonmetabolizing capacity.
INTRODUCTION It is now generally accepted t h a t polycyclic aromatic hydrocarbons require metabolic activation for carcinogenic activity and that an initial step is oxidation by the microsomal oxygenase system, AHH. Kellerman et al. [9] reported a positive correlation between high AHH inducibility of phytohemagglutininstimulated h u m a n l y m p h o c y t e s and susceptibility of the individual to bronchogenic carcinoma. However, there are suggestions that for some cells or tissues, AHH inducibility may n o t be an appropriate indicator of their hydrocarbon-metabolizing capacity. In a study of the effects of cigarette smoke inhalation on AHH activity in mouse tissues, Abramson and H u t t o n [1] noted that AHH inducibility in lung was frequently a poor indicator of an animal's Address correspondence to: Dr. Leila Diamond, The Wistar Institute of Anatomy and
Biology, 36th Street at Spruce, Philadelphia, Pennsylvania 19104, U.S.A. Abbreviations: AHH, aryl hydrocarbon hydroxylase; BA, benz(a)anthracene; BP, benzo-
(a)pyrene; DMSO, dimethyl sulfoxide; HE, hamster embryo.
190
maximal level of A H H activity. Coomes et al. [5] studied cultured human l y m p h o c y t e s from approx. 100 individuals and observed no correlation b e t w e e n basal A H H levels and A H H inducibility. To investigate the relationship b e t w e e n the A H H inducibility and actual hydrocarbon-metabolizing capacity o f a cell, we selected t w o cell lines that differ widely in inducibility and compared their capacity to metabolize the carcinogenic hydrocarbon, benzo(a)pyrene (BP). MATERIALS AND METHODS
Primary Syrian hamster e m b r y o (HE) cell cultures were prepared from 12- to 13-day-old e m b r y o s as described previously [3]. The R 7 2 / 3 cell line is derived from a t u m o r produced b y rat liver cells that had transformed spontaneously in culture [ 7 ] . The cells were grown as m o n o l a y e r cultures in Eagle's basal medium supplemented with 10% bovine fetal serum (Reheis Chemical Co. or Flow Labs. ) or bovine calf serum (Industrial Biological Labs.). To measure AHH activity and BP metabolism, the cells were seeded at a density of 5--7 • 104 cells/cm 2 in l()0-mm plastic dishes and after 24 h the cultures were refed with fresh medium. A stock solution of BA [Eastman Organic Chemicals; 3 mg BA/ml dimethyl sulfoxide (DMSO)] was diluted with medium and added to the plates to give a final concentration of 3 ~g BA/ml medium; DMSO was added to control plates at a final concentration of 0.1%. Sixteen hours later, cells from 5 control and 5 BA-treated plates were harvested by scraping and the cell pellets were stored at --70 °C for subsequent A H H assay. Preliminary experiments had indicated that with both HE and R72[3 cells A H H activity was maximal after 16 h of BA treatment. The A H H activity of the cell pellets was measured by the fluorescence assay of Nebert and Gelboin [ 11] with the modifications described by Diamond et al. [6]. One unit of AHH catalyses in 1 rain the formation of phenolic products with fluorescence' equivalent to that o f 1 pmol 3-hydroxy-BP; AHH specific activity is expressed as units/mg cell protein. At the same time that plates were harvested for A H H assay, BP metabolism was measured in three additional plates of each group. The cell monolayers were rinsed twice with 8 ml medium supplemented with serum and were refed with 15 ml fresh medium containing 0.5 nmol 3H--BP (Amersham-Searle Corp., spec. act. 3.0 Ci/mmol)/ml or 5.0 nmol 3H--BP (spec. act. 8.3 Ci/mmol)/ml. One plate containing ~H--BP b u t no cells was used as a blank. The a m o u n t of 3H--BP metabolized to water-soluble metabolites was determined by removing 0.2 ml of medium from each culture vessel and extracting with chloroform-methanol according to the procedure described by Baird and Diamond [3]. The a m o u n t of radioactivity in the organic and aqueous phases was measured by counting duplicate 0.1 ml samples in 7-ml polyethylene vials containing 4 ml scintillant {TT-21, Y o r k t o w n Res.) in a Packard Tri-Carb liquid scintillation counter and correcting for'counting efficiency by automatic external standard ratios.
191 RESULTS
HE cell cultures grown in medium supplemented with either calf or fetal serum had high basal levels of A H H activity (Table 1). Pretreatment with BA induced a small b u t significant (P < 0.001) increase in A H H activity in cultures in m e d i u m supplemented with calf serum; the increase in cultures in medium suppIemented with fetal serum was n o t statistically significant. In contrast, R 7 2 / 3 rat liver cells had low levels of basal A H H activity in m e d i u m supplemented with calf or fetal serum and, in b o t h cases, pretreatment with BA induced a 20- to 30-fold increase in activity (Table 1). The difference b e t w e e n m a x i m u m activity induced in the 2 sera was n o t significant. Since statistically significant A H H induction occurred in both HE and 1%72/3 cells only when the medium was supplemented with calf serum, BP metabolism was c o m p a r e d in BA-treated and control cultures containing medium supplem e n t e d with calf serum. For the first 5 h, the a m o u n t of 3H--BP metabolized to water-soluble derivatives b y HE cells was greater in cultures pretreated with BA than in untreated controls (Fig. 1). This enhancement was slight (10--40%) b u t was consistently observed in cultures in which A H H activity was induced at least 2-fold b y BA-pretreatment. There was less difference b e t w e e n the amounts of BP metabolites in control and BA-treated cultures at 7 h and no detectable difference at later times (not shown). In R 7 2 / 3 cell cultures, only a small a m o u n t of BP was metabolized during the first 3 h after addition of 3H--HP to uninduced cultures (Fig. 1). However, the cumulative a m o u n t o f BP metabolized increased by larger increments at each hourly interval, suggesting that the BP-metabolizing activity o f these cells TABLE1 A R Y L H Y D R O C A R B O N H Y D R O X Y L A S E L E V E L S IN S E C O N D A R Y H A M S T E R E M B R Y O (HE) C E L L S A N D R 7 2 / 3 R A T L I V E R C E L L S A H H Specific activity ( u n i t s / m g p r o t e i n ) Fetal serum a
Calf s e r u m
8.9 + 2.5 ( 6 . 4 - - 1 1 . 6 ) b 16.1 + 4.8 ( 1 1 . 1 - - 2 1 . 9 ) 1.8 (n = 5)
8.3 ± 3.9 ( 2 , 0 - - 1 3 . 9 ) 21.0 -+ 8.9 ( 4 . 5 - - 3 4 . 0 ) 3 (n = 18)
0.6 +- 0.5 ( 0 . 1 - - 1 . 5 ) 10.5 _+ 5.2 ( 5 . 3 - - 2 0 . 1 ) 33 (n = 6)
1.0 +- 0.8 ( 0 . 3 - - 2 . 7 ) 12.5 -+ 8.6 ( 3 . 7 - - 2 9 . 5 ) 17 (n = 9)
HE Control BA-treated Inducibility ratio R72/3 Control BA-treated Inducibility ratio
a T h r e e d i f f e r e n t lots o f b o v i n e fetal s e r u m and t w o o f b o v i n e calf s e r u m w e r e used. ~b T h e A H H values are t h e m e a n s p e c i f i c activity ~_ S.D. a n d , in p a r e n t h e s i s , t h e range f r o m n n u m b e r o f e x p e r i m e n t s . T h e i n d u c i b i l i t y ratios are t h e m e a n ratios o f n e x p e r i m e n t s .
192
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