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Biochemical basis for the acquisition of resistance to benzo[a]pyrene in clones of mouse liver cells in culture
Chem.-Biol. Interactions, 23 (1978) 331--344 © Elsevier/North-Holland Scientific Publishers Ltd.
331
BIOCHEMICAL BASIS FOR THE ACQUISITION OF RESISTANCE TO BENZO[a] PYRENE IN CLONES OF MOUSE LIVER CELLS IN CULTURE*
JOSEPH R. LANDOLPH, JOSEPH F. BECKER, HOWARD GAMPER, JAMES C. BARTHOLOMEW and MELVIN CALVIN
Laboratory of Chemical Biodynamics, Lawrence Berkeley Laboratory University of California, Berkeley, Calif. 94720 (U.S.A.) (Received May 1st, 1978) (Revision received July 10th, 1978) (Accepted July 15th, 1978)
SUMMARY
In a Namru mouse liver epithelial cell strain designated NMuLi, aryl hydrocarbon hydroxylase (AHH) activity peaked at 12 h post-induction with 1 ug/ml of benzo(a)pyrene (BaP) in both confluent and growing cells. Maximal levels of AHH activity were reached on day two post-plating. This induced activity was inhibited in vitro 78% by gassing the incubation mixture with carbon monoxide for 15 s, and inhibited 93% by addition of 40 pg/ml of 7,8 benzoflavone(BF). Induced AHH levels were higher in epithelial clones that were sensitive to the toxicity of BaP than in resistant clones. The survival fraction of clones from NMuLi and of subclones derived from a sensitive clone of NMuLi after BaP treatment was a negative exponential function of the maximal induced AHH activity in the clones. One of the clones, NMuLi cl 8, was extremely susceptible to the toxic effects of BaP, the +(trans)-7~, 8~-dihydroxy-7,8
*Excerpts from this work submitted in partial fulfillment of the doctoral thesis requirements for Joseph R. Landolph at the University of California, Berkeley. Abbreviations: AHH, aryl hydrocarbon hydroxylase; BaP, benzo[a ] pyrene;BF, 7,8 benzoflavone; BudR, bromodeoxyuridine; 7,8-diol, + (trans) 7a ,8~-dihydroxy-7,8-dihydro-BaP; diolepoxide, (+)-7a,8~-dihydroxy-9~,10#-epoxy-7,8,9,10-tetrahydro-BaP; 3-MC, 3-methylcholanthrene; Tetrol, 7,8,9,10-tetrahydroxy, 7,8,9,10-tetrahydro-BaP.
332 BaP and the 7,8-diol, but still extremely susceptible to the toxic effects of the diol-epoxide. The slight toxicity to BaP in this clone was inhibited by BF, b u t the toxicity of the 7,8-diol to this clone was not inhibited by BF. A typical c y t o c h r o m e P 4 5 0 inhibitor, metyrapone, had no effect on the toxicity of BaP, the 7,8-diol, or the diol-epoxide to either clone 7 or clone 8. The results suggest that these liver cells possess two enzymes that play some role in polycyclic hydrocarbon-induced toxicity. Enzyme A, a BaPinducible enzyme that is inhibitable by BF, efficiently metabolizes BaP to the 7,8-diol and the 7,8-diol to the diol-epoxide. It is responsible for most of the hydrocarbon toxicity. Enzyme B is not inhibitable by BF and metabolizes the 7,8-diol less efficiently to the diol-epoxide or efficiently to other, less toxic products.
INTRODUCTION Drug resistance and its related enzymology have been studied in many mammalian cell culture systems. In general, cells become resistant to a toxic agent by losing enzyme or protein activities that either transport the agent into the cell, metabolize it further to a more toxic form, or incorporate it into cellular components. Prominent examples are loss of an active transport function for BudR 2 in haploid frog cells resistant to this toxic agent [ 1], and loss of hypoxanthine-guanine-phosphoribosyl-transferase activity in variants resistant to 8-azaguanine [2]. Resistance to the toxicity of carcinogenic polycyclic aromatic hydrocarbons is analogous to the previous cases. Aryl hydrocarbon hydrolase (AHH) metabolizes these hydrocarbons to toxic and mutagenic derivatives [3--9] in the cell. It has been shown that the inducibility of AHH [3,4], the ability of the cell to metabolize hydrocarbons to water-soluble products [ 1 0 ] , and the binding of these hydrocarbons to nucleic acids and proteins of human and rodent cells [11] all correlated with the c y t o t o x i c i t y of these agents. Parallel to other drug resistance cases, decreases in A H H have been found to decrease polycyclic hydrocarbon toxicity [3,4]. A question of interest is w h y in many hydrocarbon toxicity studies cell lines rapidly acquire a high level of variants resistant to the hydrocarbons [4,10--14]. It has been reported that known mutagens do not increase the frequency of BaP resistance [14], and the high rate of spontaneous resistance has precluded the use of hydrocarbon resistance in somatic mutation studies up to the present. Such studies could be of theoretical interest, since AHH is expressed as a simple autosomal dominant marker in animal studies [15], and hydrocarbon resistance would be expected to have a high rate of induced mutation. An understanding of the high rates of spontaneous hydrocarbon resistance might serve as an analogue toward understanding the high rates (10-2--10 -3 ) of chemically-induced malignant transformation. Nebert and Gelboin have shown that various types of hamster fetus cells
333 lose AHH with passage [15]. AHH is now known to consist o f more than one enzyme activity [16--20], b u t the specific enzyme(s) associated with hydrocarbon toxicity is n o t known. Many of these studies were performed with fibroblasts or mixed fibroblastic-epithelial systems. In humans, 92% of cancer incidence occurs in epithelial tissues [21]. The observation of Huberman and Sachs that human embryo cultures containing greater than 29% epithelial cells metabolized 3--25 times more BaP to water- and alkali-soluble derivatives than did fibroblastic cultures from the same e m b r y o [22] indicated that epithelial cells might play a more active role in metabolizing carcinogens than fibroblastic tissues. Since metabolism of carcinogens often leads to activated derivatives, the situation could explain higher epithelial cancer rates. Liver, however, is one organ with many epithelial cells that is relatively refractory to t u m o r induction after treatment of animals with carcinogenic polycyclic aromatic hydrocarbons [23]. One possibility for this insensitivity related to other tissues is that liver possesses additional types of hydrocarbon-metabolizing enzymes that produce less damaging metabolites (in BaP metabolism, for instance, oxidation at sites other than the 7,8,9 and 10 positions which lead to the diol-epoxides) and/or higher levels of such detoxifying enzymes as epoxide hydrase and glutathione-S-epoxide-transferases. Recent advances in cell culture techniques have n o w made it possible to isolate epithelial cell strains. A previous study from this laboratory demonstrated that various rodent epithelial cell strains were extremely sensitive to the toxicity of the carcinogen BaP [24]. In the most sensitive epithelial cell strain designated NMuLi, and derived from the livers of Namru mice b y Owens [ 2 9 ] , we showed that AHH induction and cell division or some growth-associated process were necessary for BaP to cause toxicity [26]. Consequently, we extended these studies by investigating the differential toxicities of various metabolites of BaP, including the diol-epoxide and its diol precursor, in BaP-resistant and BaP-sensitive clones derived from this liver cell line. The syn- and anti-diol-epoxides are among the most mutagenic and toxic of the known BaP metabolites [5--7,9,25], initiate skin tumors in mice [ 2 7 ] , and have been suggested as proximate carcinogenic forms of BaP [5]. This study sought to define w h y liver cells in vivo are refractory to polycyclic hydrocarbon-induced tumorigenesis by examining the relative importance of the different metabolic functions of the liver and by more clearly defining the specific enzyme function lost from the AHH complex that results in loss of BaP-induced toxicity in cultured liver cells. Such knowledge could also make possible the use of hydrocarbon resistance as a marker in somatic mutation studies and lend insight as to w h y cells lose differentiated functions in culture. MATERIALS AND METHODS
Cells
The NMuLi cell strain was cultured as previously described [ 2 6 ] , except
334 that 250 units/ml of Penicillin G (Grand Island Biological Company, Grand Island, N.Y.), and 50 pg/ml of streptomycin sulfate (Mann Research Labs., New York, N.Y.) were added to the medium. NMuLi was cloned at passage 40 from 60 mm dishes having less than 5 viable colonies/dish by the glass cylinder isolation technique of Puck et al. [ 2 8 ] . NMuLi is an epithelial cell strain derived from the livers of weanling Namru mice by Owens [29]. NMuLi and clones derived from it had become malignantly transformed by passage 10, previous to the time the experiments in this paper were c o n d u c t e d (Larry Anderson and Helene Smith, pers. comm., and J. Landolph, manuscript in preparation). Toxicity assays Toxicity assays were conducted by the clonal killing m e t h o d described previously [26]. Briefly, 200 single cells were plated on a 60 mm culture dish, allowed to attach and recover for 24 h, and then treated with the various BaP derivatives in dimethyl sulfoxide (0.2% total in the medium, which caused no toxicity by itself) for 9 days. The medium was removed and the cells were washed with 0.9% saline. The colonies were then fixed in methanol for 10 min, and stained with 1% crystal violet in 25% ethanol. Each point in a Chart represents the average +- standard deviations of the values from 4 plates in each experiment. In each chart, points n o t bracketed by error bars have standard deviations less than or equal to the size of the symbol. Chemicals The racemic 7,8-diol, the anti-diol-epoxide, and the tetrol were generous gifts from Dr. K. Straub, Laboratory of Chemical Biodynamics, and their synthesis is described elsewhere [ 3 0 ] . All c o m p o u n d s had purities of 90% or better as determined by NMR, liquid chromatography, and mass spectrometry (Dr. K. Straub, pers. comm.). BaP and organic solvents were procured and purified as previously described [ 2 6 ] . Metyrapone was purchased from Aldrich Chemical Company, Milwaukee, Wisc. A H H assay A H H activity was measured fluorimetrically as previously described [ 2 4 , 2 6 ] , which is essentially the procedure of Nebert and Gelboin [31] as modified b y Nebert and Gielen [32]. For this assay, cells were seeded at 5 × 106/roller flask (109 mm × 144 mm, Coming Glass Works, Coming, N.Y.) in 100 ml of medium. 100/~l of I mg/ml solution of BaP in acetone was added to the flasks at the appropriate time for induction of A H H activity. For the preparation of cell sonicates following induction, the medium was removed, 20 ml o f ice-cold isotonic Tris buffer [26] was added, and the cells were scraped from the flask with a rubber policeman. They were processed as previously described [ 2 4 , 2 6 ] , except that cells were sonicated three times for 45 s each time at a power of 100 W on a model W185 Sonifer-Cell disruptor (Heat Systems-Ultrasonics, Inc., Plainview,
335 N.Y.). There was a 45 s period between each sonication period to allow the dissipation of heat, and the sample was cooled to 0°C by a circulating water bath. Attempts to fractionate NMuLi cells further and concentrate the AHH by spinning o u t the mitochondria were n o t successful, since half the activity came down with the mitochondria. In two experiments, sonicating and homogenizing the cells had no effect on AHH activity from NMuLi, a BaP-sensitive clone, or BaP-resistant clone, relative to untreated cell suspensions. Since the AHH activities of the clones were so low, it was decided to use whole cell suspensions rather than microsomes in much of the work. For some of the inhibition experiments, microsomes were prepared from the crude cell sonicate first by homogenizing with a glass homogenizer in 50 mM Tris--HCl, pH 7.5, in sucrose. The homogenate was then centrifuged at 600 g for 10 min and then at 7000 g for 15 min in a Sorvall RC2B centrifuge. Both pellets were discarded. The final centrifugation was performed in a Beckman/Spinco at 75 000 g for 90 min, and the pellet was resuspended in the same buffer used to suspend the crude cell sonicate. Rat liver microsomes were prepared from male Sprague--Dawley rats (Simonsen Laboratories, Gilroy, Calif.} weighing approx. 200 g following induction of AHH with 3-methylcholanthrene and processing by the methods of Meehan et al. [ 3 3 ] . The only modification was that the vehicle for the injection of 3-MC was 0.5% methylcellulose instead of corn oil. RESULTS
Characterization of AHH activity from NMuLi cells The AHH activity in the NMuLi cells was characterized b y a number of standard inhibition studies. In microsomes prepared from BaP-induced NMuLi, carbon monoxide inhibited AHH activity b y 78%, while the control treatment, bubbling with nitrogen, caused only 13% inhibition (Table I). In the same assay, carbon monoxide inhibited A H H from 3-MC-induced rat liver microsomes by 53%. In addition, 40 pg]ml of BF inhibited the AHH activity from NMuLi by 93%. A lesser concentration of BF, 4 pg/ml, inhibited both NMuLi and rat liver microsomal A H H by a b o u t 70%. The specific activity of induced microsomal AHH from NMuLi was 14 times less than that from uninduced Namru mouse liver, 73 times less than that from 3-MC-induced rat liver microsomes, and 20--40 times less than published values from fetal rat liver primary cultures [34] and from hepatoma cultures [35].
Differential inducibility of AHH during the growth of cells To determine whether the time course of induction of AHH in NMuLi was different in growing versus non-growing cultures the experiment described in Fig. 1 was carried out. In both growing and non-growing cultures the AHH activity increased, reaching a maximum around 12 h, and then declined. A comparison of the area under these induction curves indicates that the total amount of activity induced in growing cells was greater than in non-growing
336
31
~HH
olo o
~
INDUCIBILITY
10 8-. 2ffl
5
<
Fb~
1061 /
0 V 0
16
32
48 0
HOURS OF INDUCTION ©
5
r0
DAYS
Fig. 1. (left) The time course of induction of AHH. Cells were seeded at 5 × 10'/roller flask, and induced with 1 /~g/ml of BaP for the times indicated before harvesting. • Induction at day 2 post-seeding, o Induction at day 6 post-seeding. Medium was changed at days 3 and 5 post-seeding. Cell growth always plateaued by day 4 in these experiments. Fig. 2. (right) The inducibility of AHH throughout the growth curve. At each point, cells were induced with I ~g/ml of BaP for 12 h prior to harvesting. (A) induced activity; (~) basal (uninduced) AHH activity. (o) cells/roller flask. Medium was changed on days 3,5 and 7 post-seeding. cells. T h e a m o u n t o f A H H a c t i v i t y i n d u c e d f o l l o w i n g a 12 h i n d u c t i o n was also c h e c k e d on e a c h d a y d u r i n g t h e g r o w t h o f a c u l t u r e o f NMuLi. T h e results s h o w n in Fig. 2 i n d i c a t e t h a t t h e i n d u c e d specific a c t i v i t y o f A H H was r o u g h l y 3 t i m e s h i g h e r in g r o w i n g versus n o n - g r o w i n g cells, similar to earlier results o f o t h e r s in s e c o n d a r y h a m s t e r f e t u s cells [ 3 6 ] . W h e n these results w e r e c o r r e c t e d f o r t h e increased p r o t e i n c o n t e n t o f n o n - g r o w i n g c o m p a r e d t o g r o w i n g cells, t h e A H H a c t i v i t y / c e l l was a p p r o x . 1.7 t i m e s h i g h e r in g r o w i n g cultures. This e x p e r i m e n t was carried o u t 5 t i m e s a n d in all cases t h e levels o f A H H in g r o w i n g cells r e a c h e d a m a x i m u m on d a y 2, d e c r e a s e d t o a l m o s t basal levels as t h e cells r e a c h e d c o n f l u e n c e , a n d t h e n increased, p e a k e d , a n d d e c r e a s e d again. A d d i n g BaP f o r a t h r e e - d a y p e r i o d d u r i n g log p h a s e and f o r a t h r e e - d a y p e r i o d d u r i n g c o n f l u e n c e w i t h o u t m e d i u m changes gave a similar d i f f e r e n t i a l o f i n d u c t i o n {data n o t s h o w n ) , so t h e curves r e f l e c t a p h y s i o l o g i c a l ability o f t h e cells t o i n d u c e a n d i n d i c a t e t h a t t h e y w e r e n o t l i m i t e d b y BaP e x p o s u r e times. F u r t h e r , the r a t i o o f t h e i n t e g r a t e d A H H specific a c t i v i t y d u r i n g t h e log p h a s e e x p o s u r e t o t h a t in t h e c o n f l u e n t e x p o s u r e was 3.1, a p p r o x , t h e s a m e as t h a t in t h e g r o w t h c u r v e
TABLE I E F F E C T S O F C A R B O N M O N O X I D E A N D 7,8 B E N Z O F L A V O N E IN I N H I B I T I N G A HH F R O M V A R I O U S M I C R O S O M A L P R E P A R A T I O N S Specific activity o f AHH, p m o i o f 3-hydroxy-BaP/ mg p r o t e i n / r a i n
% of complete
a. Microsomes f r o m BaP t r e a t e d NMuLi Cells s Complete Complete Complete Complete b.
+ + + +
15 15 40 40
s o f CO s of N 2 ug/ml of BF ~g/ml o f BF
5.5 1.2 4.8 0.4 1.4
22 87 7.3 25
403 186 353 133
47 87 33
Liver microsomes from 3-MC t r e a t e d rats b
C o m p l e t e + 15 s o f CO Complete + 15 s of N: Complete + 4.0 ~g/ml of B F
78 ± 2 (2 expts.)
c. Unindueed microsomes from N a m r u mice livers
a This experiment was performed on N M u L i cells at day 4 post-seeding, with an 11 h induction with 1 ug/ml of BaP. b See Materials and Methods for preparations of rat liver microsomes. TABLE
II
bZ-IH IN S E N S I T I V E
AND
RESISTANT
Clone No.
CLONES
OF NMuLi
AHH, p m o l 3-hydroxyBaP/mg p r o t e i n / r a i n a Complete
7, u n i n d u c e d (2 expts). induced
+4 ug/ml BF
% Inhibition
0.08 0.65
± .02 ± 0.02
0.020 • 0.006 0.33 ± 0.03
8, uninduced induced
0.40 14.8
± 0.03 ± 0.1
-4.9
± 0.4
9, passage 2 ninduced induced
0.38 1.4
± .01 ± .2
0.13 0.23
± 0.02 ± 0.03
9, passage 13 uninduced induced
1.07 3.8
± 0.20 ± 0.7
-0.9
± 0.2
19, passage 3 uninduced induced
0.14 2.2
± 0.01 -+ 0.5
.033 • 0 . 0 0 3 0.4 ± 0.01
19, passage 11 uninduced induced
0.06 0.70
± .02 ± .03
--
0.07
±.02
9 0 ± 10
12, passage 1 uninduced induced
0.13 1.0
+~ .03 ± 0.1
0.10 0.40
± .03 ~ .05
20 ± 10 60 ± 5
12, paseage 2 uninduced induced
0.036 ± 0.003 0.16 ~ 0.01
0,08 0,12
~ 0.01 ± 0.02
N M u L i , pass. 25 -40 (5 expts) uninduced induced
0.40 8.6
-1.6
± 0.1
± .06 +- 2.8
74 ± 8 49 ± 5 -66 ±
0.8
± 0.1
0 . 0 0 9 ~ .007 2
65 -+ 2 84 ± 3
-77 ± 3
76 -+ 2 84 -+ 4
--
-I00 ± 6 c 23 ± 8
-81 ±
Clone survival in BaP, Relative to u n t r e a t e d controls b
0.40
-* 0 . 0 5
0.9
± 0.1
0.66
± .03
1.0
±
0.40
± .05
0.83
± .06
0.08
± .02
0.1
5
aAl| A H H measurements were m a d e on 5 × 106 cells grown in roller flasks and induced with 1.0 #g/ml of BaP for 12 h on day t w o post-seeding. b A value of 1.0 indicates the cells were totally resistant to this 9day BaP treatment. C A 1 0 0 % increase rather than inhibition,
338 inducibility experiment (Fig. 1). The inhibition of AHH by 4 ~g/ml of BF was the same in growing and non-growing cultures (about 60%), indicating that the same type of enzyme(s) was probably induced in both situations. When the induction of AHH was measured in clones derived from NMuLi t h a t were either sensitive or resistant to BaP toxicity the same time course of induction was observed, as well as the same effect of growth on the maximal level induced (data not shown). Levels o f A H H in sensitive and resistant clones derived from N M u L i Using a 12 h induction with 1 ~g/ml of BaP and processing the cells on day 2 post-plating, we measured the AHH levels in various clones derived from NMuLi. Clone 8, a sensitive clone, possessed an induced AHH level that was at least 4 times higher than the AHH level in any resistant clone (Table II). Clone 7, which was resistant over m a n y passages, possessed the lowest basal and next to the lowest inducible AHH level among all clones studied. Except for clone 12 at passage 2, the induced AHH levels among all clones studied was inhibited at least 50% in vitro by 4 pg/ml of BF. In all clones except for clone 12 the basal levels of AHH were also inhibited at least 65% by this concentration of BF. Also, mixing homogenates from sensitive and resistant clones did n o t cause inhibition of the sensitive cells' AHH activity indicating the lack of a diffusible inhibitor in resistant cells. A semi-log plot of survival fraction versus (maximal, day 2) induced AHH activity for various clones derived from NMuLi was linear, (data from Table II) showing t h a t the toxicity of BaP to these clones is an exponential function of the maximal induced AHH activity (plot not shown). Clonal killing curves with activated derivatives o f BaP Having established a relationship between h y d r o x y l a t i o n of BaP and toxicity, we then proceeded to use various derivatives of BaP on the p a t h w a y to the proximate diol-epoxides to determine where in the pathway of metabolism of BaP the various resistant mutants were blocked. In NMuLi clone 8, 1% of the cells were resistant to the toxicity of BaP (Fig. 3). However, less than 1 in 106 were resistant to such activated derivatives of BaP as the 7,8-diol and the diol-epoxide. An inhibitor of AHH, BF, mitigated the toxicity of both BaP and the 7,8-diol but did n o t alleviate the toxicity of the diol-epoxide in clone 8. The tetrol, which would be generated in cells by the action of epoxide hydrase or by spontaneous attack of water on the diolepoxide, was not toxic to this clone, assuming the tetrol enters the cells. BF had no effect on the toxicity of the diol-epoxide. The diol-epoxide was as toxic to NMuLi clone 7, a BaP-resistant clone, (Fig. 4), as it was to the sensitive clone 8. The 7,8-diol was less toxic to clone 7 than to the BaP-sensitive clone 8. BF completely inhibited the slight toxicity that BaP exerted on the resistant cells, but did not affect the toxicity of the 7,8-diol or the diol-epoxide to clone 7 thus suggesting the existence of two enzymes. Further, the killing of clone 8 by the 7,8-diol in the presence of BF resembled the killing curve for the resistant clone 7 by
339 NMuLi
CI8
NMuLi CI7
I0
W Z
o
-J O
0
0.1
o
~ 0.1.
q
Z
g
o
c
g
cr u._
~_
><~ 0.01--
>0.01-
or"
~
O3
0.001 ~
~
Ol
20
o.ool ~0
CONC BaP DERIVATIVE, #M
I 0
l 20
I
I 40
CONC. BaP DERIVATIVE, ,uM
Fig. 3. The toxicity of derivatives of BaP to NMuLi clone 8 in the colony killing assay. The experiment was performed on passage 11 post-isolation of this clone, passage 52 post-isolation of NMuLi. No data points for survival fractions less than 0.001 are shown. Benzoflavone concentration for the inhibition experiments was 5.22 uM. There were no survivors in the 7,8-diol survival curve at higher concentrations (data points lie below 0.01), and this curve overlapped the Diol-Epoxide killing curve. (o) Tetrol, (9) BaP; (o) BaP + BF; (A) 7,8-Diol; (~) 7,8-Diol + B F ; ( Q ) Diol-Epoxide, with and without BF. Fig. 4. The toxicity of BaP derivatives to NMuLi clone 7 was performed at the same passage number as for clone 8 in Chart 3. Symbol designations are the same as in Chart 3.
the 7,8-diol alone (Figs. 3 and 4). All other BaP resistant clones tested, whether they arose spontaneously or were BaP selected and passaged free of BaP, had the same kill kinetics by activated derivatives as did clone 7 (data not shown). At later passages, the kill kinetics for clone 8 more closely resembled those of clone 7 (data n o t shown). Attempts were made to inhibit the toxicity of BaP derivatives by using a specific inhibitor of cytochrome P450, metyrapone [37]. Metyrapone itself caused no toxicity to either clone 7 or to clone 8 at concentrations ranging from 10 -2 M to 3.3 X 10 -4 M in the medium. Similarly, these concentrations of metyrapone did n o t inhibit the toxicity of 40 pM BaP or 30/~M 7,8-diol to either clone 7 or clone 8. In addition, 3.3 X 10 -4 M metyrapone did not inhibit the toxicity of any level of BaP or the 7,8-diol from 2.5/~M to 40 ~M to either clone 7 or to clone 8 in experiments performed exactly as in Charts 3 and 4 (these negative data n o t shown).
340 DISCUSSION
The literature is increasingly filled with reports of multiple forms of P 4 5 0 [16,17,19,20] and multiple forms of A H H [18,34,37--30]. Huang et al. [17] purified phenobarbital-induced P450-type oxidase from mouse liver microsomes into four distinct bands with differing substrate specificities and Thomas et al. [20] have demonstrated the existence of 6 distinct forms of c y t o c h r o m e P 4 5 0 in purified rat liver microsomes. Guengerich has also resolved rabbit liver microsomes into 6 different bands all of which metabolize BaP to different extents [ 1 2 ] . Our results with different liver cell variants suggest that at least two of these P 4 5 0 - t y p e oxidases are probably involved in mediating the cytotoxicity of BaP in cultured cells. One of these enzyme activities, which we call enzyme A (see Fig. 5) is inducible b y BaP and inhibitable by BF. The loss of this enzyme in cultured cells parallels the loss of cellular sensitivity to efficient BaP-induced toxicity and efficient 7,8-diol-induced toxicity. There appears to be at least a second enzyme in these cells because even in the presence of BF, which inhibits enzyme A, both BaP-sensitive and BaPresistant clones are still sensitive to the toxic effects of the 7,8~diol, although much less so than is the BaP-sensitive clone in the absence of BF. This second enzyme would appear to metabolize the 7,8,diol either inefficiently to the diol-epoxide or else to some other product which is not as toxic as the diol-epoxide. Although it is possible that the 7,8-'diol may be generally toxic without further metabolism to the diol-epoxide, we conclude that the lack of toxicity of the tetrol, a structurally similar analogue, argues against this explanation. BoP METABOLISM SCHEME GLUTATHIONE CONJUGATES OF EPOXIDES
G L U C O S ECONJUGATES OF DIOLS
GLUTATHIONE S-EPOXIDE TRANSFERASE
BaP
~
# I
HYDRASE
7,8 OXIDE + (TOXIC)
#H
I
7,8- (TRANS) DIHYDRODIOL | +
OTHER EPOXII)ES
I A ~B
(TOXIC) Oi ~
ISOMERIZE PHENOLS
UDP GLUCURONOSYLTRANSFERA SE
OTHER . TRANSFERASES GLUCOSE AND SULFATE CONJUGATES OF PHENOLS
OTHER DIHYDRODIOLS
o DIOL-EPOXIDE (TOXIC)
Fig. 5. General scheme for the metabolism of BaP in tissue culture cells. This scheme is a modification of the one outlined for benz[a]anthracene by Heidelberger [47]. *Other transferases indicate UDP glucuronosyltransferase and phenoltranssulfonase.
341 Atlas et al. have separated rabbit liver microsomes into a number of bands, one of which has BaP-hydroxylase activity, a molecular weight of 57 000, and is inhibited b y BF [41]. Our enzyme A function is probably analogous to this band. Goujon et al. have shown that metyrapone is effective at inhibiting AHH from control preparations of rat liver microsomes b u t not from 3-MC-induced microsomes [16] and that BF is effective at inhibiting A H H from 3-MC b u t n o t from control mouse liver microsomes [37]. In our system, the inability of metyrapone to inhibit the cytotoxicity of BaP or the 7,8 diol in either the sensitive clone 8 or the resistant clone 7 implied that there was little or none of the basal in vivo enzyme in our cells. Consistent with this was the fact that the control enzyme in these cells was inhibitable by BF, similar to an earlier report [ 3 4 ] . Since liver is n o t very susceptible to malignant transformation b y polycyclic aromatic hydrocarbons in vivo [ 2 0 ] , it is possible that the control enzyme lacking in cultured cells is a factor that protects liver from the deleterious carcinogenic effects of polycyclic aromatic hydrocarbons in vivo. We would speculate that the control enzyme does n o t cause much hydroxylation in the 7,8,9,10 positions of BaP to produce the toxic diol-epoxides, b u t predominantly hydroxylates (epoxidates) the other positions o f BaP. Supporting this hypothesis, Wang and Rasmussen have shown that BaP, 3-MC and benz(a)anthracene-induced rat liver microsomes metabolize BaP proportionately more to the 7,8 and 9,10-diols than to the 4,5-diol, whereas the phenobarbital-induced rat liver microsomes metabolize BaP to the 4,5-diol and 3-hydroxy BaP more efficiently than to the 7,8 and 9,10-diols. In addition, these workers found that lung microsomes were highly inducible b y BaP and 3-MC, b u t n o t by phenobarbital, indicating that the control, phenobarbitalinducible enzyme is probably not present in lung [ 4 2 ] . In this connection, it is interesting that lung is a target for BaP-induced carcinogenesis, whereas liver apparently is not. Others have shown that rodent liver microsomes can metabolize BaP to the 4,5~1iol among other products, whereas e m b r y o cells from the same species are n o t very effective in producing the 4,5<1iol [43]. In addition, it has been demonstrated that the dioNepoxides are relatively poor substrates for the detoxifying enzyme epoxide hydrase, whereas the 4,5 oxide is a good substrate for epoxide hydrase [7]. In an analogous situation, it has been shown the BaP hydroxylase of trout is n o t inhibited by metyrapone b u t is inhibited by BF, and pointed o u t that trout develop hepatomas readily on exposure to such carcinogens as ariatoxins [ 4 4 ] . Given such a metabolism scheme, attempts to inhibit enzyme A and its deleterious metabolism and to drive the metabolism through the basal enzyme in vivo in tissues possessing it would seem reasonable. This scheme is complicated b y the fact that there are many toxic metabolites of BaP other than the ones considered, and because enzymes A and B b o t h might perform the epoxidation of the 7,8-diol to the diol~epoxide. For example, phenols are toxic [4], and are also good substrates for UDP glucuronosyltransferase [45] and for phenoltranssulfonase [ 4 6 ] .
342 It is still an open question as to whether in our resistant cells there was complete loss of enzyme A and/or an increase in the levels of conjugating enzymes such as glutathione-S-epoxide transferase, epoxide hydrase, and UDP-glucuronosyl transferase. Diamond asked this question earlier and found that both alternatives appeared to contribute when divergent cell lines were compared [10]. Preliminary data from this lab indicate that in clone 7, using a radioactive assay, levels of total water-soluble BaP conjugates are decreased relative to clone 8, implying that the mechanism of resistance is simply a loss of enzyme A (Landolph, Becker and Bartholomew, unpublished results). It should be possible to answer on a somatic cell genetic level why this loss of enzyme A is so facile leading to such high rates of BaP resistance. In conclusion, NMuLi has proved to be an effective system for studying toxicity of BaP to epithelial cells, and parallels between different functions of AHH and toxicity response. We have demonstrated that in the absence of basal metyrapone-inhibitable AHH, high enzyme A levels are responsible for the observed toxicity of BaP to these epithelial cells, and that a loss of enzyme A in subsequent passages may be responsible for the acquisition of resistance to BaP in cultured cells. ACKNOWLEDGEMENTS
We would like to thank Dr. Thomas Meehan, Dr. Kenneth Straub and Dr. Gordon Parry for extremely helpful discussions and for criticizing the manuscript. In addition, we would especially like to thank Dr. Kenneth Straub for providing us with samples of the various derivatives of BaP used in these experiments. This study was supported by the National Cancer Institute, DHEW Contract YO1-CP-50203 and Grant CA05573-02, and the Division of Biomedical and Environmental Research of the U.S. Energy Research and Development Administration. REFERENCES 1 L. Mezger-Freed, Effect of ploidy and mutagens on bromodeoxyuridine resistance in haploid and diploid frog cells, Nature New Biol., 235 (1972) 245. 2 A.A. Van Zeeland and J.W.I.M. Simons, Ploidy level and mutation to hypoxanthineguanine-phosphoribosyl-transferase (HGPRT) deficiency in Chinese hamster cells, Mutat. Res., 28 (1975) 239. 3 W.F. Benedict, J.E. Gielen and D.W. Nebert, Polycyclic hydrocarbon-produced toxicity, transformation, and chromosomal aberrations as a function of aryl hydrocarbon hydroxylase activity in cell cultures, Int. J. Cancer, 9 (1972) 435. 4 H.V. Gelboin, E. Huberman and L. Sachs, Enzymatic hydroxylation of benzopyrene and its relationship to cytotoxicity, Proc. Nail. Acad. Sci. U.S.A., 64 (1969) 1188. 5 E. Huberman, L. Sachs, S.K. Yang and H.V. Gelboin, Identification of mutagenic metabolites o f b e n z o [ a ] p y r e n e in mammalian cells, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 606. 6 D.R. Thakker, H. Yagi, H. Akogi, M, Koreeda, A.Y.H. Lu, W. Levin, A.W. Wood, A.H. Conney and D.M. Jerina, Metabolism of b e n z o [ a ] p y r e n e VI. Stereoselective
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331
BIOCHEMICAL BASIS FOR THE ACQUISITION OF RESISTANCE TO BENZO[a] PYRENE IN CLONES OF MOUSE LIVER CELLS IN CULTURE*
JOSEPH R. LANDOLPH, JOSEPH F. BECKER, HOWARD GAMPER, JAMES C. BARTHOLOMEW and MELVIN CALVIN
Laboratory of Chemical Biodynamics, Lawrence Berkeley Laboratory University of California, Berkeley, Calif. 94720 (U.S.A.) (Received May 1st, 1978) (Revision received July 10th, 1978) (Accepted July 15th, 1978)
SUMMARY
In a Namru mouse liver epithelial cell strain designated NMuLi, aryl hydrocarbon hydroxylase (AHH) activity peaked at 12 h post-induction with 1 ug/ml of benzo(a)pyrene (BaP) in both confluent and growing cells. Maximal levels of AHH activity were reached on day two post-plating. This induced activity was inhibited in vitro 78% by gassing the incubation mixture with carbon monoxide for 15 s, and inhibited 93% by addition of 40 pg/ml of 7,8 benzoflavone(BF). Induced AHH levels were higher in epithelial clones that were sensitive to the toxicity of BaP than in resistant clones. The survival fraction of clones from NMuLi and of subclones derived from a sensitive clone of NMuLi after BaP treatment was a negative exponential function of the maximal induced AHH activity in the clones. One of the clones, NMuLi cl 8, was extremely susceptible to the toxic effects of BaP, the +(trans)-7~, 8~-dihydroxy-7,8
*Excerpts from this work submitted in partial fulfillment of the doctoral thesis requirements for Joseph R. Landolph at the University of California, Berkeley. Abbreviations: AHH, aryl hydrocarbon hydroxylase; BaP, benzo[a ] pyrene;BF, 7,8 benzoflavone; BudR, bromodeoxyuridine; 7,8-diol, + (trans) 7a ,8~-dihydroxy-7,8-dihydro-BaP; diolepoxide, (+)-7a,8~-dihydroxy-9~,10#-epoxy-7,8,9,10-tetrahydro-BaP; 3-MC, 3-methylcholanthrene; Tetrol, 7,8,9,10-tetrahydroxy, 7,8,9,10-tetrahydro-BaP.
332 BaP and the 7,8-diol, but still extremely susceptible to the toxic effects of the diol-epoxide. The slight toxicity to BaP in this clone was inhibited by BF, b u t the toxicity of the 7,8-diol to this clone was not inhibited by BF. A typical c y t o c h r o m e P 4 5 0 inhibitor, metyrapone, had no effect on the toxicity of BaP, the 7,8-diol, or the diol-epoxide to either clone 7 or clone 8. The results suggest that these liver cells possess two enzymes that play some role in polycyclic hydrocarbon-induced toxicity. Enzyme A, a BaPinducible enzyme that is inhibitable by BF, efficiently metabolizes BaP to the 7,8-diol and the 7,8-diol to the diol-epoxide. It is responsible for most of the hydrocarbon toxicity. Enzyme B is not inhibitable by BF and metabolizes the 7,8-diol less efficiently to the diol-epoxide or efficiently to other, less toxic products.
INTRODUCTION Drug resistance and its related enzymology have been studied in many mammalian cell culture systems. In general, cells become resistant to a toxic agent by losing enzyme or protein activities that either transport the agent into the cell, metabolize it further to a more toxic form, or incorporate it into cellular components. Prominent examples are loss of an active transport function for BudR 2 in haploid frog cells resistant to this toxic agent [ 1], and loss of hypoxanthine-guanine-phosphoribosyl-transferase activity in variants resistant to 8-azaguanine [2]. Resistance to the toxicity of carcinogenic polycyclic aromatic hydrocarbons is analogous to the previous cases. Aryl hydrocarbon hydrolase (AHH) metabolizes these hydrocarbons to toxic and mutagenic derivatives [3--9] in the cell. It has been shown that the inducibility of AHH [3,4], the ability of the cell to metabolize hydrocarbons to water-soluble products [ 1 0 ] , and the binding of these hydrocarbons to nucleic acids and proteins of human and rodent cells [11] all correlated with the c y t o t o x i c i t y of these agents. Parallel to other drug resistance cases, decreases in A H H have been found to decrease polycyclic hydrocarbon toxicity [3,4]. A question of interest is w h y in many hydrocarbon toxicity studies cell lines rapidly acquire a high level of variants resistant to the hydrocarbons [4,10--14]. It has been reported that known mutagens do not increase the frequency of BaP resistance [14], and the high rate of spontaneous resistance has precluded the use of hydrocarbon resistance in somatic mutation studies up to the present. Such studies could be of theoretical interest, since AHH is expressed as a simple autosomal dominant marker in animal studies [15], and hydrocarbon resistance would be expected to have a high rate of induced mutation. An understanding of the high rates of spontaneous hydrocarbon resistance might serve as an analogue toward understanding the high rates (10-2--10 -3 ) of chemically-induced malignant transformation. Nebert and Gelboin have shown that various types of hamster fetus cells
333 lose AHH with passage [15]. AHH is now known to consist o f more than one enzyme activity [16--20], b u t the specific enzyme(s) associated with hydrocarbon toxicity is n o t known. Many of these studies were performed with fibroblasts or mixed fibroblastic-epithelial systems. In humans, 92% of cancer incidence occurs in epithelial tissues [21]. The observation of Huberman and Sachs that human embryo cultures containing greater than 29% epithelial cells metabolized 3--25 times more BaP to water- and alkali-soluble derivatives than did fibroblastic cultures from the same e m b r y o [22] indicated that epithelial cells might play a more active role in metabolizing carcinogens than fibroblastic tissues. Since metabolism of carcinogens often leads to activated derivatives, the situation could explain higher epithelial cancer rates. Liver, however, is one organ with many epithelial cells that is relatively refractory to t u m o r induction after treatment of animals with carcinogenic polycyclic aromatic hydrocarbons [23]. One possibility for this insensitivity related to other tissues is that liver possesses additional types of hydrocarbon-metabolizing enzymes that produce less damaging metabolites (in BaP metabolism, for instance, oxidation at sites other than the 7,8,9 and 10 positions which lead to the diol-epoxides) and/or higher levels of such detoxifying enzymes as epoxide hydrase and glutathione-S-epoxide-transferases. Recent advances in cell culture techniques have n o w made it possible to isolate epithelial cell strains. A previous study from this laboratory demonstrated that various rodent epithelial cell strains were extremely sensitive to the toxicity of the carcinogen BaP [24]. In the most sensitive epithelial cell strain designated NMuLi, and derived from the livers of Namru mice b y Owens [ 2 9 ] , we showed that AHH induction and cell division or some growth-associated process were necessary for BaP to cause toxicity [26]. Consequently, we extended these studies by investigating the differential toxicities of various metabolites of BaP, including the diol-epoxide and its diol precursor, in BaP-resistant and BaP-sensitive clones derived from this liver cell line. The syn- and anti-diol-epoxides are among the most mutagenic and toxic of the known BaP metabolites [5--7,9,25], initiate skin tumors in mice [ 2 7 ] , and have been suggested as proximate carcinogenic forms of BaP [5]. This study sought to define w h y liver cells in vivo are refractory to polycyclic hydrocarbon-induced tumorigenesis by examining the relative importance of the different metabolic functions of the liver and by more clearly defining the specific enzyme function lost from the AHH complex that results in loss of BaP-induced toxicity in cultured liver cells. Such knowledge could also make possible the use of hydrocarbon resistance as a marker in somatic mutation studies and lend insight as to w h y cells lose differentiated functions in culture. MATERIALS AND METHODS
Cells
The NMuLi cell strain was cultured as previously described [ 2 6 ] , except
334 that 250 units/ml of Penicillin G (Grand Island Biological Company, Grand Island, N.Y.), and 50 pg/ml of streptomycin sulfate (Mann Research Labs., New York, N.Y.) were added to the medium. NMuLi was cloned at passage 40 from 60 mm dishes having less than 5 viable colonies/dish by the glass cylinder isolation technique of Puck et al. [ 2 8 ] . NMuLi is an epithelial cell strain derived from the livers of weanling Namru mice by Owens [29]. NMuLi and clones derived from it had become malignantly transformed by passage 10, previous to the time the experiments in this paper were c o n d u c t e d (Larry Anderson and Helene Smith, pers. comm., and J. Landolph, manuscript in preparation). Toxicity assays Toxicity assays were conducted by the clonal killing m e t h o d described previously [26]. Briefly, 200 single cells were plated on a 60 mm culture dish, allowed to attach and recover for 24 h, and then treated with the various BaP derivatives in dimethyl sulfoxide (0.2% total in the medium, which caused no toxicity by itself) for 9 days. The medium was removed and the cells were washed with 0.9% saline. The colonies were then fixed in methanol for 10 min, and stained with 1% crystal violet in 25% ethanol. Each point in a Chart represents the average +- standard deviations of the values from 4 plates in each experiment. In each chart, points n o t bracketed by error bars have standard deviations less than or equal to the size of the symbol. Chemicals The racemic 7,8-diol, the anti-diol-epoxide, and the tetrol were generous gifts from Dr. K. Straub, Laboratory of Chemical Biodynamics, and their synthesis is described elsewhere [ 3 0 ] . All c o m p o u n d s had purities of 90% or better as determined by NMR, liquid chromatography, and mass spectrometry (Dr. K. Straub, pers. comm.). BaP and organic solvents were procured and purified as previously described [ 2 6 ] . Metyrapone was purchased from Aldrich Chemical Company, Milwaukee, Wisc. A H H assay A H H activity was measured fluorimetrically as previously described [ 2 4 , 2 6 ] , which is essentially the procedure of Nebert and Gelboin [31] as modified b y Nebert and Gielen [32]. For this assay, cells were seeded at 5 × 106/roller flask (109 mm × 144 mm, Coming Glass Works, Coming, N.Y.) in 100 ml of medium. 100/~l of I mg/ml solution of BaP in acetone was added to the flasks at the appropriate time for induction of A H H activity. For the preparation of cell sonicates following induction, the medium was removed, 20 ml o f ice-cold isotonic Tris buffer [26] was added, and the cells were scraped from the flask with a rubber policeman. They were processed as previously described [ 2 4 , 2 6 ] , except that cells were sonicated three times for 45 s each time at a power of 100 W on a model W185 Sonifer-Cell disruptor (Heat Systems-Ultrasonics, Inc., Plainview,
335 N.Y.). There was a 45 s period between each sonication period to allow the dissipation of heat, and the sample was cooled to 0°C by a circulating water bath. Attempts to fractionate NMuLi cells further and concentrate the AHH by spinning o u t the mitochondria were n o t successful, since half the activity came down with the mitochondria. In two experiments, sonicating and homogenizing the cells had no effect on AHH activity from NMuLi, a BaP-sensitive clone, or BaP-resistant clone, relative to untreated cell suspensions. Since the AHH activities of the clones were so low, it was decided to use whole cell suspensions rather than microsomes in much of the work. For some of the inhibition experiments, microsomes were prepared from the crude cell sonicate first by homogenizing with a glass homogenizer in 50 mM Tris--HCl, pH 7.5, in sucrose. The homogenate was then centrifuged at 600 g for 10 min and then at 7000 g for 15 min in a Sorvall RC2B centrifuge. Both pellets were discarded. The final centrifugation was performed in a Beckman/Spinco at 75 000 g for 90 min, and the pellet was resuspended in the same buffer used to suspend the crude cell sonicate. Rat liver microsomes were prepared from male Sprague--Dawley rats (Simonsen Laboratories, Gilroy, Calif.} weighing approx. 200 g following induction of AHH with 3-methylcholanthrene and processing by the methods of Meehan et al. [ 3 3 ] . The only modification was that the vehicle for the injection of 3-MC was 0.5% methylcellulose instead of corn oil. RESULTS
Characterization of AHH activity from NMuLi cells The AHH activity in the NMuLi cells was characterized b y a number of standard inhibition studies. In microsomes prepared from BaP-induced NMuLi, carbon monoxide inhibited AHH activity b y 78%, while the control treatment, bubbling with nitrogen, caused only 13% inhibition (Table I). In the same assay, carbon monoxide inhibited A H H from 3-MC-induced rat liver microsomes by 53%. In addition, 40 pg]ml of BF inhibited the AHH activity from NMuLi by 93%. A lesser concentration of BF, 4 pg/ml, inhibited both NMuLi and rat liver microsomal A H H by a b o u t 70%. The specific activity of induced microsomal AHH from NMuLi was 14 times less than that from uninduced Namru mouse liver, 73 times less than that from 3-MC-induced rat liver microsomes, and 20--40 times less than published values from fetal rat liver primary cultures [34] and from hepatoma cultures [35].
Differential inducibility of AHH during the growth of cells To determine whether the time course of induction of AHH in NMuLi was different in growing versus non-growing cultures the experiment described in Fig. 1 was carried out. In both growing and non-growing cultures the AHH activity increased, reaching a maximum around 12 h, and then declined. A comparison of the area under these induction curves indicates that the total amount of activity induced in growing cells was greater than in non-growing
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r0
DAYS
Fig. 1. (left) The time course of induction of AHH. Cells were seeded at 5 × 10'/roller flask, and induced with 1 /~g/ml of BaP for the times indicated before harvesting. • Induction at day 2 post-seeding, o Induction at day 6 post-seeding. Medium was changed at days 3 and 5 post-seeding. Cell growth always plateaued by day 4 in these experiments. Fig. 2. (right) The inducibility of AHH throughout the growth curve. At each point, cells were induced with I ~g/ml of BaP for 12 h prior to harvesting. (A) induced activity; (~) basal (uninduced) AHH activity. (o) cells/roller flask. Medium was changed on days 3,5 and 7 post-seeding. cells. T h e a m o u n t o f A H H a c t i v i t y i n d u c e d f o l l o w i n g a 12 h i n d u c t i o n was also c h e c k e d on e a c h d a y d u r i n g t h e g r o w t h o f a c u l t u r e o f NMuLi. T h e results s h o w n in Fig. 2 i n d i c a t e t h a t t h e i n d u c e d specific a c t i v i t y o f A H H was r o u g h l y 3 t i m e s h i g h e r in g r o w i n g versus n o n - g r o w i n g cells, similar to earlier results o f o t h e r s in s e c o n d a r y h a m s t e r f e t u s cells [ 3 6 ] . W h e n these results w e r e c o r r e c t e d f o r t h e increased p r o t e i n c o n t e n t o f n o n - g r o w i n g c o m p a r e d t o g r o w i n g cells, t h e A H H a c t i v i t y / c e l l was a p p r o x . 1.7 t i m e s h i g h e r in g r o w i n g cultures. This e x p e r i m e n t was carried o u t 5 t i m e s a n d in all cases t h e levels o f A H H in g r o w i n g cells r e a c h e d a m a x i m u m on d a y 2, d e c r e a s e d t o a l m o s t basal levels as t h e cells r e a c h e d c o n f l u e n c e , a n d t h e n increased, p e a k e d , a n d d e c r e a s e d again. A d d i n g BaP f o r a t h r e e - d a y p e r i o d d u r i n g log p h a s e and f o r a t h r e e - d a y p e r i o d d u r i n g c o n f l u e n c e w i t h o u t m e d i u m changes gave a similar d i f f e r e n t i a l o f i n d u c t i o n {data n o t s h o w n ) , so t h e curves r e f l e c t a p h y s i o l o g i c a l ability o f t h e cells t o i n d u c e a n d i n d i c a t e t h a t t h e y w e r e n o t l i m i t e d b y BaP e x p o s u r e times. F u r t h e r , the r a t i o o f t h e i n t e g r a t e d A H H specific a c t i v i t y d u r i n g t h e log p h a s e e x p o s u r e t o t h a t in t h e c o n f l u e n t e x p o s u r e was 3.1, a p p r o x , t h e s a m e as t h a t in t h e g r o w t h c u r v e
TABLE I E F F E C T S O F C A R B O N M O N O X I D E A N D 7,8 B E N Z O F L A V O N E IN I N H I B I T I N G A HH F R O M V A R I O U S M I C R O S O M A L P R E P A R A T I O N S Specific activity o f AHH, p m o i o f 3-hydroxy-BaP/ mg p r o t e i n / r a i n
% of complete
a. Microsomes f r o m BaP t r e a t e d NMuLi Cells s Complete Complete Complete Complete b.
+ + + +
15 15 40 40
s o f CO s of N 2 ug/ml of BF ~g/ml o f BF
5.5 1.2 4.8 0.4 1.4
22 87 7.3 25
403 186 353 133
47 87 33
Liver microsomes from 3-MC t r e a t e d rats b
C o m p l e t e + 15 s o f CO Complete + 15 s of N: Complete + 4.0 ~g/ml of B F
78 ± 2 (2 expts.)
c. Unindueed microsomes from N a m r u mice livers
a This experiment was performed on N M u L i cells at day 4 post-seeding, with an 11 h induction with 1 ug/ml of BaP. b See Materials and Methods for preparations of rat liver microsomes. TABLE
II
bZ-IH IN S E N S I T I V E
AND
RESISTANT
Clone No.
CLONES
OF NMuLi
AHH, p m o l 3-hydroxyBaP/mg p r o t e i n / r a i n a Complete
7, u n i n d u c e d (2 expts). induced
+4 ug/ml BF
% Inhibition
0.08 0.65
± .02 ± 0.02
0.020 • 0.006 0.33 ± 0.03
8, uninduced induced
0.40 14.8
± 0.03 ± 0.1
-4.9
± 0.4
9, passage 2 ninduced induced
0.38 1.4
± .01 ± .2
0.13 0.23
± 0.02 ± 0.03
9, passage 13 uninduced induced
1.07 3.8
± 0.20 ± 0.7
-0.9
± 0.2
19, passage 3 uninduced induced
0.14 2.2
± 0.01 -+ 0.5
.033 • 0 . 0 0 3 0.4 ± 0.01
19, passage 11 uninduced induced
0.06 0.70
± .02 ± .03
--
0.07
±.02
9 0 ± 10
12, passage 1 uninduced induced
0.13 1.0
+~ .03 ± 0.1
0.10 0.40
± .03 ~ .05
20 ± 10 60 ± 5
12, paseage 2 uninduced induced
0.036 ± 0.003 0.16 ~ 0.01
0,08 0,12
~ 0.01 ± 0.02
N M u L i , pass. 25 -40 (5 expts) uninduced induced
0.40 8.6
-1.6
± 0.1
± .06 +- 2.8
74 ± 8 49 ± 5 -66 ±
0.8
± 0.1
0 . 0 0 9 ~ .007 2
65 -+ 2 84 ± 3
-77 ± 3
76 -+ 2 84 -+ 4
--
-I00 ± 6 c 23 ± 8
-81 ±
Clone survival in BaP, Relative to u n t r e a t e d controls b
0.40
-* 0 . 0 5
0.9
± 0.1
0.66
± .03
1.0
±
0.40
± .05
0.83
± .06
0.08
± .02
0.1
5
aAl| A H H measurements were m a d e on 5 × 106 cells grown in roller flasks and induced with 1.0 #g/ml of BaP for 12 h on day t w o post-seeding. b A value of 1.0 indicates the cells were totally resistant to this 9day BaP treatment. C A 1 0 0 % increase rather than inhibition,
338 inducibility experiment (Fig. 1). The inhibition of AHH by 4 ~g/ml of BF was the same in growing and non-growing cultures (about 60%), indicating that the same type of enzyme(s) was probably induced in both situations. When the induction of AHH was measured in clones derived from NMuLi t h a t were either sensitive or resistant to BaP toxicity the same time course of induction was observed, as well as the same effect of growth on the maximal level induced (data not shown). Levels o f A H H in sensitive and resistant clones derived from N M u L i Using a 12 h induction with 1 ~g/ml of BaP and processing the cells on day 2 post-plating, we measured the AHH levels in various clones derived from NMuLi. Clone 8, a sensitive clone, possessed an induced AHH level that was at least 4 times higher than the AHH level in any resistant clone (Table II). Clone 7, which was resistant over m a n y passages, possessed the lowest basal and next to the lowest inducible AHH level among all clones studied. Except for clone 12 at passage 2, the induced AHH levels among all clones studied was inhibited at least 50% in vitro by 4 pg/ml of BF. In all clones except for clone 12 the basal levels of AHH were also inhibited at least 65% by this concentration of BF. Also, mixing homogenates from sensitive and resistant clones did n o t cause inhibition of the sensitive cells' AHH activity indicating the lack of a diffusible inhibitor in resistant cells. A semi-log plot of survival fraction versus (maximal, day 2) induced AHH activity for various clones derived from NMuLi was linear, (data from Table II) showing t h a t the toxicity of BaP to these clones is an exponential function of the maximal induced AHH activity (plot not shown). Clonal killing curves with activated derivatives o f BaP Having established a relationship between h y d r o x y l a t i o n of BaP and toxicity, we then proceeded to use various derivatives of BaP on the p a t h w a y to the proximate diol-epoxides to determine where in the pathway of metabolism of BaP the various resistant mutants were blocked. In NMuLi clone 8, 1% of the cells were resistant to the toxicity of BaP (Fig. 3). However, less than 1 in 106 were resistant to such activated derivatives of BaP as the 7,8-diol and the diol-epoxide. An inhibitor of AHH, BF, mitigated the toxicity of both BaP and the 7,8-diol but did n o t alleviate the toxicity of the diol-epoxide in clone 8. The tetrol, which would be generated in cells by the action of epoxide hydrase or by spontaneous attack of water on the diolepoxide, was not toxic to this clone, assuming the tetrol enters the cells. BF had no effect on the toxicity of the diol-epoxide. The diol-epoxide was as toxic to NMuLi clone 7, a BaP-resistant clone, (Fig. 4), as it was to the sensitive clone 8. The 7,8-diol was less toxic to clone 7 than to the BaP-sensitive clone 8. BF completely inhibited the slight toxicity that BaP exerted on the resistant cells, but did not affect the toxicity of the 7,8-diol or the diol-epoxide to clone 7 thus suggesting the existence of two enzymes. Further, the killing of clone 8 by the 7,8-diol in the presence of BF resembled the killing curve for the resistant clone 7 by
339 NMuLi
CI8
NMuLi CI7
I0
W Z
o
-J O
0
0.1
o
~ 0.1.
q
Z
g
o
c
g
cr u._
~_
><~ 0.01--
>0.01-
or"
~
O3
0.001 ~
~
Ol
20
o.ool ~0
CONC BaP DERIVATIVE, #M
I 0
l 20
I
I 40
CONC. BaP DERIVATIVE, ,uM
Fig. 3. The toxicity of derivatives of BaP to NMuLi clone 8 in the colony killing assay. The experiment was performed on passage 11 post-isolation of this clone, passage 52 post-isolation of NMuLi. No data points for survival fractions less than 0.001 are shown. Benzoflavone concentration for the inhibition experiments was 5.22 uM. There were no survivors in the 7,8-diol survival curve at higher concentrations (data points lie below 0.01), and this curve overlapped the Diol-Epoxide killing curve. (o) Tetrol, (9) BaP; (o) BaP + BF; (A) 7,8-Diol; (~) 7,8-Diol + B F ; ( Q ) Diol-Epoxide, with and without BF. Fig. 4. The toxicity of BaP derivatives to NMuLi clone 7 was performed at the same passage number as for clone 8 in Chart 3. Symbol designations are the same as in Chart 3.
the 7,8-diol alone (Figs. 3 and 4). All other BaP resistant clones tested, whether they arose spontaneously or were BaP selected and passaged free of BaP, had the same kill kinetics by activated derivatives as did clone 7 (data not shown). At later passages, the kill kinetics for clone 8 more closely resembled those of clone 7 (data n o t shown). Attempts were made to inhibit the toxicity of BaP derivatives by using a specific inhibitor of cytochrome P450, metyrapone [37]. Metyrapone itself caused no toxicity to either clone 7 or to clone 8 at concentrations ranging from 10 -2 M to 3.3 X 10 -4 M in the medium. Similarly, these concentrations of metyrapone did n o t inhibit the toxicity of 40 pM BaP or 30/~M 7,8-diol to either clone 7 or clone 8. In addition, 3.3 X 10 -4 M metyrapone did not inhibit the toxicity of any level of BaP or the 7,8-diol from 2.5/~M to 40 ~M to either clone 7 or to clone 8 in experiments performed exactly as in Charts 3 and 4 (these negative data n o t shown).
340 DISCUSSION
The literature is increasingly filled with reports of multiple forms of P 4 5 0 [16,17,19,20] and multiple forms of A H H [18,34,37--30]. Huang et al. [17] purified phenobarbital-induced P450-type oxidase from mouse liver microsomes into four distinct bands with differing substrate specificities and Thomas et al. [20] have demonstrated the existence of 6 distinct forms of c y t o c h r o m e P 4 5 0 in purified rat liver microsomes. Guengerich has also resolved rabbit liver microsomes into 6 different bands all of which metabolize BaP to different extents [ 1 2 ] . Our results with different liver cell variants suggest that at least two of these P 4 5 0 - t y p e oxidases are probably involved in mediating the cytotoxicity of BaP in cultured cells. One of these enzyme activities, which we call enzyme A (see Fig. 5) is inducible b y BaP and inhibitable by BF. The loss of this enzyme in cultured cells parallels the loss of cellular sensitivity to efficient BaP-induced toxicity and efficient 7,8-diol-induced toxicity. There appears to be at least a second enzyme in these cells because even in the presence of BF, which inhibits enzyme A, both BaP-sensitive and BaPresistant clones are still sensitive to the toxic effects of the 7,8~diol, although much less so than is the BaP-sensitive clone in the absence of BF. This second enzyme would appear to metabolize the 7,8,diol either inefficiently to the diol-epoxide or else to some other product which is not as toxic as the diol-epoxide. Although it is possible that the 7,8-'diol may be generally toxic without further metabolism to the diol-epoxide, we conclude that the lack of toxicity of the tetrol, a structurally similar analogue, argues against this explanation. BoP METABOLISM SCHEME GLUTATHIONE CONJUGATES OF EPOXIDES
G L U C O S ECONJUGATES OF DIOLS
GLUTATHIONE S-EPOXIDE TRANSFERASE
BaP
~
# I
HYDRASE
7,8 OXIDE + (TOXIC)
#H
I
7,8- (TRANS) DIHYDRODIOL | +
OTHER EPOXII)ES
I A ~B
(TOXIC) Oi ~
ISOMERIZE PHENOLS
UDP GLUCURONOSYLTRANSFERA SE
OTHER . TRANSFERASES GLUCOSE AND SULFATE CONJUGATES OF PHENOLS
OTHER DIHYDRODIOLS
o DIOL-EPOXIDE (TOXIC)
Fig. 5. General scheme for the metabolism of BaP in tissue culture cells. This scheme is a modification of the one outlined for benz[a]anthracene by Heidelberger [47]. *Other transferases indicate UDP glucuronosyltransferase and phenoltranssulfonase.
341 Atlas et al. have separated rabbit liver microsomes into a number of bands, one of which has BaP-hydroxylase activity, a molecular weight of 57 000, and is inhibited b y BF [41]. Our enzyme A function is probably analogous to this band. Goujon et al. have shown that metyrapone is effective at inhibiting AHH from control preparations of rat liver microsomes b u t not from 3-MC-induced microsomes [16] and that BF is effective at inhibiting A H H from 3-MC b u t n o t from control mouse liver microsomes [37]. In our system, the inability of metyrapone to inhibit the cytotoxicity of BaP or the 7,8 diol in either the sensitive clone 8 or the resistant clone 7 implied that there was little or none of the basal in vivo enzyme in our cells. Consistent with this was the fact that the control enzyme in these cells was inhibitable by BF, similar to an earlier report [ 3 4 ] . Since liver is n o t very susceptible to malignant transformation b y polycyclic aromatic hydrocarbons in vivo [ 2 0 ] , it is possible that the control enzyme lacking in cultured cells is a factor that protects liver from the deleterious carcinogenic effects of polycyclic aromatic hydrocarbons in vivo. We would speculate that the control enzyme does n o t cause much hydroxylation in the 7,8,9,10 positions of BaP to produce the toxic diol-epoxides, b u t predominantly hydroxylates (epoxidates) the other positions o f BaP. Supporting this hypothesis, Wang and Rasmussen have shown that BaP, 3-MC and benz(a)anthracene-induced rat liver microsomes metabolize BaP proportionately more to the 7,8 and 9,10-diols than to the 4,5-diol, whereas the phenobarbital-induced rat liver microsomes metabolize BaP to the 4,5-diol and 3-hydroxy BaP more efficiently than to the 7,8 and 9,10-diols. In addition, these workers found that lung microsomes were highly inducible b y BaP and 3-MC, b u t n o t by phenobarbital, indicating that the control, phenobarbitalinducible enzyme is probably not present in lung [ 4 2 ] . In this connection, it is interesting that lung is a target for BaP-induced carcinogenesis, whereas liver apparently is not. Others have shown that rodent liver microsomes can metabolize BaP to the 4,5~1iol among other products, whereas e m b r y o cells from the same species are n o t very effective in producing the 4,5<1iol [43]. In addition, it has been demonstrated that the dioNepoxides are relatively poor substrates for the detoxifying enzyme epoxide hydrase, whereas the 4,5 oxide is a good substrate for epoxide hydrase [7]. In an analogous situation, it has been shown the BaP hydroxylase of trout is n o t inhibited by metyrapone b u t is inhibited by BF, and pointed o u t that trout develop hepatomas readily on exposure to such carcinogens as ariatoxins [ 4 4 ] . Given such a metabolism scheme, attempts to inhibit enzyme A and its deleterious metabolism and to drive the metabolism through the basal enzyme in vivo in tissues possessing it would seem reasonable. This scheme is complicated b y the fact that there are many toxic metabolites of BaP other than the ones considered, and because enzymes A and B b o t h might perform the epoxidation of the 7,8-diol to the diol~epoxide. For example, phenols are toxic [4], and are also good substrates for UDP glucuronosyltransferase [45] and for phenoltranssulfonase [ 4 6 ] .
342 It is still an open question as to whether in our resistant cells there was complete loss of enzyme A and/or an increase in the levels of conjugating enzymes such as glutathione-S-epoxide transferase, epoxide hydrase, and UDP-glucuronosyl transferase. Diamond asked this question earlier and found that both alternatives appeared to contribute when divergent cell lines were compared [10]. Preliminary data from this lab indicate that in clone 7, using a radioactive assay, levels of total water-soluble BaP conjugates are decreased relative to clone 8, implying that the mechanism of resistance is simply a loss of enzyme A (Landolph, Becker and Bartholomew, unpublished results). It should be possible to answer on a somatic cell genetic level why this loss of enzyme A is so facile leading to such high rates of BaP resistance. In conclusion, NMuLi has proved to be an effective system for studying toxicity of BaP to epithelial cells, and parallels between different functions of AHH and toxicity response. We have demonstrated that in the absence of basal metyrapone-inhibitable AHH, high enzyme A levels are responsible for the observed toxicity of BaP to these epithelial cells, and that a loss of enzyme A in subsequent passages may be responsible for the acquisition of resistance to BaP in cultured cells. ACKNOWLEDGEMENTS
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28 29 30 31
32 33
34 35 36 37 38 39
40 41 42 43
44 45 46 47
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