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Relationship of aryl hydrocarbon hydroxylase activity to benzo(a)pyrene-metabolizing activity of cells in culture
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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HOURS Fig. 1. F o r m at i o n of water-soluble BP metabolites in cultures of secondary HE cells and R72/3 liver cells. Cells were treated with BA in DMSO (3 #g BA/ml medium) and control cells were treated with DMSO alone (0.1%) for 16 h, washed twice with fresh medium and refed with medium containing 0.5 nmol 3H--BP/ml (7.5 nmol/plate). At the indicated times, samples o f m e d i u m were extracted with chloroform--methanol and the amounts of watersoluble BP metabolites present were determined. The amount of water-soluble radioactivity recovered after organic solvent extraction of samples from plates without cells (maximum value 0.04 nmol) was subtracted from the cell value. Each point represents the total amount of water-soluble BP metabolites/plate and is the mean -+ S.D. of 3 plates. The AHH specific activity (units/mg protein) at the time of adding the 3H--BP was: He--BA, 30.1; He--control, 6.9; R72/3--BA, 29.5 ; R72/3--control, 2.2.
was being induced by the 3H--BP itself. The BA-pretreated R72/3 cells metabolized significantly more BP than did controls at each time point and the a m o u n t s of metabolites formed during each hourly interval were constant (Fig. 1). During the first 3 h, 6 times more water-soluble BP metabolites were f o r m e d in the BA-treated cultures in which AHH activity had been preinduced 13-fold t h a n were formed in the control cultures which were n o t preinduced.
193
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Fig. 2. Formation of water-soluble metabol]tes in R72/3 cells treated with 5.0 nmol 3H--BP/ml medium. Except for the higher concentration of 3H--BP, the experiment was similar to that described in the legend of Fig. 1. The maximum amount of water-soluble material in the blanks was 0.02 nmol. The AHH specific activities (units/mg protein) at the time of adding the 3H--BP were: control, 1.0; BA-treated, 9.1.
With b o t h H E a n d R 7 2 / 3 cells, c o n t r o l a n d B A - t r e a t e d cultures s h o w e d t h e s a m e p r o p o r t i o n a l d i f f e r e n c e s in BP m e t a b o l i s m w h e n t h e t o t a l a m o u n t s o f BP m e t a b o l i z e d w e r e d e t e r m i n e d b y t h i n - l a y e r c h r o m a t o g r a p h y o f organic s o l v e n t e x t r a c t s [3] as w h e n o n l y t h e w a t e r - s o l u b l e m e t a b o l i t e s w e r e m e a s u r e d . T o d e t e r m i n e w h e t h e r t h e t r u e p o t e n t i a l o f R 7 2 / 3 cells to m e t a b o l i z e BP was b e i n g assessed, a parallel e x p e r i m e n t was d o n e using a 10-fold higher conc e n t r a t i o n o f 3H--BP. ( H i g h e r c o n c e n t r a t i o n s t h a n this c o u l d n o t be u s e d b e c a u s e o f possible c y t o t o x i c e f f e c t s as well as t h e d i f f i c u l t y o f m e a s u r i n g m e t a b o l i t e s f o r m e d w i t h i n a s h o r t t i m e in the p r e s e n c e o f a large excess o f u n m e t a b o l i z e d BP.) A t a c o n c e n t r a t i o n o f 5 n m o l 3 H - - B P / m l , the d i f f e r e n c e s in t h e a m o u n t s m e t a b o l i z e d in c o n t r o l a n d B A - p r e t r e a t e d cells w e r e similar t o t h e d i f f e r e n c e s o b s e r v e d w i t h t h e l o w e r c o n c e n t r a t i o n o f BP; a f t e r 3 h the cells in
194
which AHH had been preinduced 9-fold by BA had metabolized a b o u t 5 times as much BP as control cells (Fig. 2). DISCUSSION
With most cells, one effect of treatment with a h y d r o c a r b o n is the induction o f the hydrocarbon-oxidizing enzyme system, AHH, and attempts have been made to relate the magnitude of this induction to the biological response of the cells to the h y d r o c a r b o n (see ref. 8). The results presented here demonstrate the difficulties inherent in this approach. N o t only do the particular sera and culture conditions affect AHH inducibility [ 2 , 4 ] , b u t the inducibility ratios m a y n o t reflect the true BP-metabolizing capacity of the cells. Although HE cells had low A H H inducibility and R 7 2 / 3 cells had high, the metabolism of BP in uninduced HE cells and induced R 7 2 / 3 cells was similar. The fact that BP metabolism in b o t h control and BA-pretreated HE cells resembled BP metabolism in BA-pretreated R 7 2 / 3 cells suggests that untreated HE cells were expressing essentially their maximal BP-metabolizing capacity. Kouri et al. [10] measured AHH levels and inducibility and the conversion of 3H--BP to aqueous-acetone-soluble metabolites in a n u m b e r of different cell lines. Their data show that in control cells exposed to 2 pg 3H--BP/ml, a concentration of BP sufficient to induce AHH activity, the amounts of 3H--BP metabolized in 24 h were directly correlated with the levels o f BA-induced AHH, even in cells in which the control and induced A H H levels were t o o low to calculate inducibility ratios. Thus, their results and those reported here show that absolute levels of induced A H H are a measure of the hydrocarbonmetabolizing capacity of cells, while the absolute levels of uninduced A H H are a measure of the initial metabolizing activity after exposure to the hydrocarbon. However, as shown in this paper, inducibility need n o t correspond to the cells' m a x i m u m hydrocarbon-metabolizing capacity. ACKNOWLEDGEMENTS
This study was supported in part by USPHS grants CA-08936, CA-19948 and CA-10815 from the National Cancer Institute. Ms. Mary Geisz provided excellent technical assistance. REFERENCES 1 Abramson, R.K. and Hutton, J.J. (1975) Effects of cigarette smoking on aryl hydrocarbon hydroxylase activity in lungs and tissues o f inbred mice. Cancer Res., 35, 23--29. 2 Atlas, S.A., Vesell, E.S. and Nebert, D.W. (1976) Genetic control o f interindividual variations in the inducibility o f aryl h y d r o c a r b o n hydroxylase in cultured human lymphocytes. Cancer Res., 36, 4619--4630. 3 Baird, W.M. and Diamond, L. (1976) Effect o f 7,8-benzoflavone on the formation of benzo [a ] pyrene-DNA-bound products in hamster e m b r y o cells. Chem.-Biol. InCeeract., 13, 67--75.
195
4 Bast, R.C., Jr., Okuda, T., Plotkin, E., Tarone, R., Rapp, H.J. and Gelboin, H.V. (1976) Development of an assay for aryl hydrocarbon [benzo(a)pyrene ] hydroxylase in human peripheral blood monocytes. Cancer Res., 36, 1967--1974. 5 Coomes, M.L., Mason, W.A., Muijsson, I.E., Cantrell, E.T., Anderson, D.E. and Busbee, D.L. (1976) Aryl hydrocarbon hydroxylase and 16~-hydroxylase in cultured human lymphocytes. Biochem. Genet., 14,671--685. 6 Diamond, L., McFall, R., Miller, J. and Gelboin, H.V. (1972) The effects of two isomeric benzoflavones on aryl hydrocarbon hydroxylase and the toxicity and carcinogenicity of polycyclic hydrocarbons. Cancer Res., 32, 731--736. 7 Diamond, L., McFall, R., Tashiro, Y. and Sabatini, D. (1973) The WIRL-3 rat liver cell lines and their transformed derivatives. Cancer Res., 33, 2627--2636. 8 Freudenthal, R. and Jones, P.W. (Editors) (1976) Carcinogenesis: A Comprehensive Survey (Vol. 1). Polynuclear Aromatic Hydrocarbons: Chemistry, Metabolism, and Carcinogenesis. Raven Press, New York. 9 Kellermann, G., Shaw, C.R. and Luyten-Kellermann, M. (1973) Aryl hydrocarbon hydroxylase inducibility and bronchogenic carcinoma. N. Engl. J. Med., 289,934--937. 10 Kouri, R.E., Kiefer, R. and Zimme~man, E.M. (1974) Hydrocarbon-metabolizing activity of various mammalian cells in culture. In Vitro, 10, 18--25. 11 Nebert, D.W. and Gelboin, H.V. (1968) Substrate-inducible microsomal aryl hydrocarbon hydroxylase in mammalian cell culture. I. Assay and properties of induced enzyme. J. Biol. Chem., 243, 6242--6249.
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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HOURS Fig. 1. F o r m at i o n of water-soluble BP metabolites in cultures of secondary HE cells and R72/3 liver cells. Cells were treated with BA in DMSO (3 #g BA/ml medium) and control cells were treated with DMSO alone (0.1%) for 16 h, washed twice with fresh medium and refed with medium containing 0.5 nmol 3H--BP/ml (7.5 nmol/plate). At the indicated times, samples o f m e d i u m were extracted with chloroform--methanol and the amounts of watersoluble BP metabolites present were determined. The amount of water-soluble radioactivity recovered after organic solvent extraction of samples from plates without cells (maximum value 0.04 nmol) was subtracted from the cell value. Each point represents the total amount of water-soluble BP metabolites/plate and is the mean -+ S.D. of 3 plates. The AHH specific activity (units/mg protein) at the time of adding the 3H--BP was: He--BA, 30.1; He--control, 6.9; R72/3--BA, 29.5 ; R72/3--control, 2.2.
was being induced by the 3H--BP itself. The BA-pretreated R72/3 cells metabolized significantly more BP than did controls at each time point and the a m o u n t s of metabolites formed during each hourly interval were constant (Fig. 1). During the first 3 h, 6 times more water-soluble BP metabolites were f o r m e d in the BA-treated cultures in which AHH activity had been preinduced 13-fold t h a n were formed in the control cultures which were n o t preinduced.
193
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HOURS
Fig. 2. Formation of water-soluble metabol]tes in R72/3 cells treated with 5.0 nmol 3H--BP/ml medium. Except for the higher concentration of 3H--BP, the experiment was similar to that described in the legend of Fig. 1. The maximum amount of water-soluble material in the blanks was 0.02 nmol. The AHH specific activities (units/mg protein) at the time of adding the 3H--BP were: control, 1.0; BA-treated, 9.1.
With b o t h H E a n d R 7 2 / 3 cells, c o n t r o l a n d B A - t r e a t e d cultures s h o w e d t h e s a m e p r o p o r t i o n a l d i f f e r e n c e s in BP m e t a b o l i s m w h e n t h e t o t a l a m o u n t s o f BP m e t a b o l i z e d w e r e d e t e r m i n e d b y t h i n - l a y e r c h r o m a t o g r a p h y o f organic s o l v e n t e x t r a c t s [3] as w h e n o n l y t h e w a t e r - s o l u b l e m e t a b o l i t e s w e r e m e a s u r e d . T o d e t e r m i n e w h e t h e r t h e t r u e p o t e n t i a l o f R 7 2 / 3 cells to m e t a b o l i z e BP was b e i n g assessed, a parallel e x p e r i m e n t was d o n e using a 10-fold higher conc e n t r a t i o n o f 3H--BP. ( H i g h e r c o n c e n t r a t i o n s t h a n this c o u l d n o t be u s e d b e c a u s e o f possible c y t o t o x i c e f f e c t s as well as t h e d i f f i c u l t y o f m e a s u r i n g m e t a b o l i t e s f o r m e d w i t h i n a s h o r t t i m e in the p r e s e n c e o f a large excess o f u n m e t a b o l i z e d BP.) A t a c o n c e n t r a t i o n o f 5 n m o l 3 H - - B P / m l , the d i f f e r e n c e s in t h e a m o u n t s m e t a b o l i z e d in c o n t r o l a n d B A - p r e t r e a t e d cells w e r e similar t o t h e d i f f e r e n c e s o b s e r v e d w i t h t h e l o w e r c o n c e n t r a t i o n o f BP; a f t e r 3 h the cells in
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which AHH had been preinduced 9-fold by BA had metabolized a b o u t 5 times as much BP as control cells (Fig. 2). DISCUSSION
With most cells, one effect of treatment with a h y d r o c a r b o n is the induction o f the hydrocarbon-oxidizing enzyme system, AHH, and attempts have been made to relate the magnitude of this induction to the biological response of the cells to the h y d r o c a r b o n (see ref. 8). The results presented here demonstrate the difficulties inherent in this approach. N o t only do the particular sera and culture conditions affect AHH inducibility [ 2 , 4 ] , b u t the inducibility ratios m a y n o t reflect the true BP-metabolizing capacity of the cells. Although HE cells had low A H H inducibility and R 7 2 / 3 cells had high, the metabolism of BP in uninduced HE cells and induced R 7 2 / 3 cells was similar. The fact that BP metabolism in b o t h control and BA-pretreated HE cells resembled BP metabolism in BA-pretreated R 7 2 / 3 cells suggests that untreated HE cells were expressing essentially their maximal BP-metabolizing capacity. Kouri et al. [10] measured AHH levels and inducibility and the conversion of 3H--BP to aqueous-acetone-soluble metabolites in a n u m b e r of different cell lines. Their data show that in control cells exposed to 2 pg 3H--BP/ml, a concentration of BP sufficient to induce AHH activity, the amounts of 3H--BP metabolized in 24 h were directly correlated with the levels o f BA-induced AHH, even in cells in which the control and induced A H H levels were t o o low to calculate inducibility ratios. Thus, their results and those reported here show that absolute levels of induced A H H are a measure of the hydrocarbonmetabolizing capacity of cells, while the absolute levels of uninduced A H H are a measure of the initial metabolizing activity after exposure to the hydrocarbon. However, as shown in this paper, inducibility need n o t correspond to the cells' m a x i m u m hydrocarbon-metabolizing capacity. ACKNOWLEDGEMENTS
This study was supported in part by USPHS grants CA-08936, CA-19948 and CA-10815 from the National Cancer Institute. Ms. Mary Geisz provided excellent technical assistance. REFERENCES 1 Abramson, R.K. and Hutton, J.J. (1975) Effects of cigarette smoking on aryl hydrocarbon hydroxylase activity in lungs and tissues o f inbred mice. Cancer Res., 35, 23--29. 2 Atlas, S.A., Vesell, E.S. and Nebert, D.W. (1976) Genetic control o f interindividual variations in the inducibility o f aryl h y d r o c a r b o n hydroxylase in cultured human lymphocytes. Cancer Res., 36, 4619--4630. 3 Baird, W.M. and Diamond, L. (1976) Effect o f 7,8-benzoflavone on the formation of benzo [a ] pyrene-DNA-bound products in hamster e m b r y o cells. Chem.-Biol. InCeeract., 13, 67--75.
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4 Bast, R.C., Jr., Okuda, T., Plotkin, E., Tarone, R., Rapp, H.J. and Gelboin, H.V. (1976) Development of an assay for aryl hydrocarbon [benzo(a)pyrene ] hydroxylase in human peripheral blood monocytes. Cancer Res., 36, 1967--1974. 5 Coomes, M.L., Mason, W.A., Muijsson, I.E., Cantrell, E.T., Anderson, D.E. and Busbee, D.L. (1976) Aryl hydrocarbon hydroxylase and 16~-hydroxylase in cultured human lymphocytes. Biochem. Genet., 14,671--685. 6 Diamond, L., McFall, R., Miller, J. and Gelboin, H.V. (1972) The effects of two isomeric benzoflavones on aryl hydrocarbon hydroxylase and the toxicity and carcinogenicity of polycyclic hydrocarbons. Cancer Res., 32, 731--736. 7 Diamond, L., McFall, R., Tashiro, Y. and Sabatini, D. (1973) The WIRL-3 rat liver cell lines and their transformed derivatives. Cancer Res., 33, 2627--2636. 8 Freudenthal, R. and Jones, P.W. (Editors) (1976) Carcinogenesis: A Comprehensive Survey (Vol. 1). Polynuclear Aromatic Hydrocarbons: Chemistry, Metabolism, and Carcinogenesis. Raven Press, New York. 9 Kellermann, G., Shaw, C.R. and Luyten-Kellermann, M. (1973) Aryl hydrocarbon hydroxylase inducibility and bronchogenic carcinoma. N. Engl. J. Med., 289,934--937. 10 Kouri, R.E., Kiefer, R. and Zimme~man, E.M. (1974) Hydrocarbon-metabolizing activity of various mammalian cells in culture. In Vitro, 10, 18--25. 11 Nebert, D.W. and Gelboin, H.V. (1968) Substrate-inducible microsomal aryl hydrocarbon hydroxylase in mammalian cell culture. I. Assay and properties of induced enzyme. J. Biol. Chem., 243, 6242--6249.