Dose-effect relationship for induction of hepatic monooxygenase activity in rainbow trout and carp by Aroclor 1254

Aquatic Toxicology, 4 (1983) 51-61

51

Elsevier

DOSE-EFFECT RELATIONSHIP FOR INDUCTION OF HEPATIC MONOOXYGENASE ACTIVITY IN RAINBOW TROUT AND CARP BY AROCLOR 1254" MARK J. MELANCON and JOHN J. LECH

Department of Pharmacology and Toxicology, The Medical College of Wisconsin, P.O. Box 26509, Milwaukee, WI53226, U.S.A. (Received 4 October 1982; accepted 27 December 1982)

A range of doses of Aroclor 1254 or 3,4,3 ',4'-tetrachlorobiphenyl was administered i.p. via a single injection to rainbow trout and to carp. Hepatic microsomes were prepared and examined for monooxygenase activity. Rainbow trout hepatic microsomes were assayed for ethoxycoumarin- and ethoxyresorufin-O-deethylase activities. For both deethylase activities, peak activity was observed at < 100 mg Aroclor 1254/kg and < 1 mg 3,4,3',4'-tetrachlorobiphenyl/kg, while significantly increased deethylase activities were observed with 0.2 mg Aroclor 1254/kg and 0.01 mg 3,4,3',4'-tetrachlorobiphenyl/kg. Monooxygenase activity in carp receiving these PCBs, as monitored by ethoxyresorufin-Odeethylase activity, gave similar responses. These results suggest that in some areas environmental exposure of fish to PCBs may be sufficient to cause induction of hepatic monooxygenase activity. It also appears that the potency of planar PCB isomers as inducers in fish is sufficiently great to account for the effect o f Aroclor 1254 upon hepatic monooxygenase activity in these species. Key words: polychlorinated biphenyls; monooxygenase activity; induction; rainbow trout; carp

INTRODUCTION

Fish in the environment are exposed to a wide variety of pollutants, both directly from the water and from their food supply. The capacity of many species of fish to metabolize various types of chemicals including pesticides and industrial pollutants has been reviewed recently (Bend and James, 1979; Lech and Bend, 1980). Much of the capacity to biotransform xenobiotic chemicals resides in the monooxygenase (MO) system in liver. This enzyme system is also able to metabolize endogenous steroids in the fish (Hansson et al., 1979). A number of chemicals which are aquatic pollutants have been shown to induce (increase) the activity of these enzyme systems in many species, including fish. Polychlorinated biphenyls (PCBs) have been shown to induce hepatic MO activity in many fish species including rain* This work was supported in part by NIEHS grant No. ES01980, the Wisconsin Sea Grant College Program and NIEHS Aquatic Biomedical Center Grant No. ES01985. 0166-445X/83/$03.00 © Elsevier Science Publishers B.V.

52

bow trout (Lidman et al., 1976; Elcombe and Lech, 1978, 1979; Hansson et al., 1980). carp (Sivarajah et al., 1978; Melancon et al., 1981), brook trout (Addison et al., 1978), channel catfish (Hill et al., 1976), coho salmon (Gruger et al., 1977), sheepshead (James and Little, 1981) and mullet (Narbonne and Gallis, 1979). Early studies of the ability of PCBs to induce hepatic MO activity in fish generally have utilized Aroclor 1254, Aroclor 1242, Phenoclor DP6 or Clophen A50 at dose levels of 50 mg/kg body weight or higher. Only two studies, involving oral dosing, have utilized dosages which approached environmental exposure levels (Gruger et al., 1977; Forlin and Lidman, 1978). Because of the large number of fish which are exposed to and accumulate PCBs in the environment it is of some interest to know just how much PCBs are required to cause induction of hepatic monooxygenase activity in fish. A precise answer to this question would require knowing liver levels or circulating blood levels of such inducing agents which are effective after a given time of exposure. As a first step in examining the relationship o f PCB pollution and increased hepatic MO activity in fish in the environment, a series of experiments was initiated with rainbow trout and carp to determine hepatic MO activity after exposure to a wide range of doses o f Aroclor 1254 and of 3,4,3' ,4'-tetrachlorobiphenyl. The latter is a component of Aroclor 1254 (Albro et al., 1981) and as a planar PCB is a 3-methylcholanthrenetype inducer (Goldstein et al., 1977; Poland and Glover, 1977) which may be responsible for much of the induction of hepatic MO activity caused by Aroclor 1254 in fish. MATERIALS AND METHODS

Rainbow trout were purchased from Hideaway Trout Farm, Kewaskum, WI and carp were obtained from the U.S. Dept. of the Interior, Lake Mills National Fish Hatchery, Lake Mills, WI. Both species of fish were maintained in flowing dechlorinated Milwaukee city water with a 12-h light: 12-h dark cycle. The fish were fed with pelleted fish chow from Glencoe Mills, Glencoe, MN. Trout were maintained at 10-12°C while carp were generally maintained at 16-18°C. Nicotinamide adenine dinucleotide phosphate reduced form (NADPH) and 7-hydroxycoumarin were purchased from Aldrich Chemical Co., St. Louis, MO. 7-Ethoxycoumarin was synthetized by the method of Ullrich and Weber (1972). 7-Ethoxyresorufin was purchased from Pierce Chemical Co., Rockford, IL and resorufin was purchased from Eastman Organic Chemicals, Rochester, NY. Aroclor 1254 was a gift from Monsanto Chemical Co., St. Louis, MO and 3,4,3',4'-tetrachlorobiphenyl was purchased from RFR Corp., Hope, RI. All other chemical reagents and solvents used in the enzyme assays were of the highest commercial quality available.

53

Treatment offish Rainbow trout and carp were injected once intraperitoneally with a solution of the compound o f interest in corn oil or with corn oil alone. In most experiments the fish weighed < 50 g and were injected at 0.4 ml/kg body wt, while in one experiment with larger trout injection was at 1.0 ml/kg body wt. Rainbow trout were injected with Aroclor 1254 (A1254) at 0.025-200 mg/kg body wt or 3,4,3',4'-tetrachlorobiphenyl (3,4,3',4'-TCB) at 0.01-25 mg/kg body wt and killed 5 days later. Carp were injected with A1254 at 0.10-100 mg/kg body wt and killed at 3, 6 or 10 days following treatment. Because of the large number of doses of PCB studied, several separate experiments were done with each species for each compound to cover the whole range of doses. In total, the A1254 experiments involved 258 trout and 135 carp while the 3 , 4 , 3 ' , 4 ' - T C B experiments involved 82 trout.

Preparation of microsomes At the indicated times the fish were killed by a blow to the back of the head and the livers were removed and placed in ice-cold 0.154 M KCI. Individual livers were minced and rinsed in the cold KCI solution to remove adhering hemoglobin. The minced tissues were homogenized in 4 volumes of 0.25 M sucrose for 5 complete strokes using a motor-driven Potter-Elvehjem glass and Teflon homogenizer. The homogenates were centrifuged at 8500 × g for 20 min using a Sorvall RC-5 centrifuge. The resulting supernatants were centrifuged at 165000 × g for 60 min. The microsome pellets were resuspended in 0.154 M KC1 and again centrifuged at 165000 × g for 60 min. The washed liver microsomal pellets from each fish were resuspended in a buffer which contained sucrose (0.25 M), E D T A (0.025 M) and Tris (0.020 M) made to pH 7.4. The volume of buffer used was 1.0 ml or 1.0 ml/g of liver, whichever was greater. All operations were performed at 0 - 4 ° C and the microsomes were frozen until the next day.

Assays Protein was measured by the method of Lowry et al. (1951) using crystalline bovine plasma albumin as standard. 7-Ethoxycoumarin-O-deethylase activity was measured by the fluorometric method of Ullrich and Weber (1972). The reaction cuvette contained 100 nmol N A D P H , 500 nmol ethoxycoumarin, 0.2-1.0 mg microsomal protein and 66 mM Tris-HCl buffer (pH 7.4) to a final volume of 1.0 ml. 7-Ethoxyresorufin-O-deethylase activity was measured by the method of Burke and Mayer (1974). The reaction cuvette contained 500 nmol N A D P H , 0.1-1.0 mg microsomal protein, 1 nmol ethoxyresorufin and 66 mM Tris-HCl buffer (pH 7.4) to a final volume of 1.0 ml.

54

Trout microsomes were assayed at 30°C while carp microsomes were assayed at 25-27°C. Cytochrome P-450 was quantitated by the method of Omura and Sato (1964) using an Aminco DW2 UV/VIS Spectrophotometer. Tests for significance o f elevations o f enzyme activity by PCB treatment was done by Student's t test. RESULTS

Hepatic microsomes from rainbow trout which had been injected with corn oil or A1254 in corn oil were examined for MO activity by measurement of ethoxyresorufin-O-deethylase and ethoxycoumarin-O-deethylase activities, and for cytochrome P-450 content. Figures 1 and 2 contain the data on ethoxyresorufin-Odeethylase activity and ethoxycoumarin-O-deethylase activity, respectively. In both cases the deethylase activity peaked at approximately 50 mg/kg o f A1254 and the lowest dose causing a significant elevation of deethylase activity, 0.2 mg/kg, was the same for both MO activities. Cytochrome P-450 content per mg of microsomal protein (not shown) was much less responsive to A 1254 administration and was elevated significantly only at A1254 doses o f l0 mg/kg and above. Previous studies (Melancon et al., 1981) have shown that hepatic microsomal

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100

AROCLOR 1254 INJECTED(MG/KG)

Fig. 1. Hepatic microsomal ethoxyresorufin-O-deethylase activity in rainbow trout receiving graded doses of Aroclor 1254. Five days after injection of rainbow trout with corn oil or Aroclor 1254, hepatic microsomes were assayed for ethoxyresorufin-O-deethylase activity. The tops of the stippled bars represent the means for each dose of Aroclor 1254, the solid bars represent the means for corn oil-injected trout assayed on the same days as that dose of Aroclor 1254, the vertical lines represent sE and * denotes a significant difference ( P < 0 . 0 5 ) compared to corn oil-injected trout. The n values for the dose levels 0.025, 10 and 50 m g / k g are from 4 to 8 while at other doses they range from 13 to 27.

55 ethoxyresorufin-O-deethylase activity, but not ethoxycoumarin-O-deethylase activity or c y t o c h r o m e P-450 content, is highly responsive to A1254 administration to carp. Therefore, in this study, hepatic microsomes f r o m carp which had been injected with corn oil or A1254 in corn oil were examined only for ethoxyresorufin-Odeethylase activity. The data in Fig. 3 show a m a x i m u m response o f this deethylase activity at approximately 30 m g / k g o f A1254 and the lowest dose o f A1254 which caused a significant elevation o f ethoxyresorufin-O-deethylase activity was 0.1 m g / k g . Because control values were relatively constant, with means differing by less t h a n 2-fold a m o n g seven experiments, the values indicated by asterisks are significant whether c o m p a r e d to all controls or only to those assayed on the same days. It is also apparent that the lower doses were unable to cause elevations o f E R O D activity by 3 days after injection which were observable by 6 days after injection o f the A1254. W h e n a single P C B isomer, 3 , 4 , 3 ' , 4 ' - T C B was administered to rainbow trout, the results presented in Fig. 4 were obtained. A l t h o u g h it is not apparent f r o m the g r a p h because o f the size o f the symbols, every dose o f 3 , 4 , 3 ' , 4 ' - T C B utilized resulted in significant increases in ethoxyresorufin- and e t h o x y c o u m a r i n - O deethylase activities. This was true whether based o n c o m p a r i s o n to the pooled values for all corn oil controls (n = 21) or to controls only f r o m the days on which a particular dose was studied, except for ethoxycoumarin°O-deethylase activity for 0.01 m g / k g which was elevated significantly only c o m p a r e d to controls f r o m the

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T iiii i~!iii i!iiiill i!ii~!ii!~~i!i!!i!ii

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100

1254 INJECTED(MG/KG)

Fig. 2. Hepatic microsomal ethoxycoumarin-O-deethylase activity in rainbow trout receiving graded doses of Aroclor 1254. Five days after injection of rainbow trout with corn oil or Aroclor 1254, hepatic microsomes were assayed for ethoxycoumarin-O-deethylase activity. The tops of the stippled bars represent the means for each dose of Aroclor 1254, the solid bars represent the means for corn oil-injected trout assayed on the same days as that dose of Aroclor 1254, the vertical lines represent s~ and * denotes a significant difference (P< 0.05) compared to corn oil-injected trout. The n value for the dose level 0.025 mg/kg and its control are 4 while at other doses they range from 13 to 33.

56 s a m e d a y . F o r b o t h d e e t h y l a s e activities m a x i m u m responses o c c u r r e d at less t h a n 1 m g / k g o f 3 , 4 , 3 ' , 4 ' - T C B . Results o f a single e x p e r i m e n t with 3 , 4 , 3 ' , 4 ' - T C B in c a r p suggested t h a t m a x i m u m r e s p o n s e o f e t h o x y r e s o r u f i n - O - d e e t h y l a s e activity also w o u l d r e q u i r e a dose o f < 1 m g / k g o f 3 , 4 , 3 ' , 4 ' - T C B . DISCUSSION B o t h r a i n b o w t r o u t a n d c a r p s h o w e d increases in h e p a t i c m i c r o s o m a l M O activity as m o n i t o r e d b y e t h o x y r e s o r u f i n - a n d / o r e t h o x y c o u m a r i n - O - d e e t h y l a s e activities d e p e n d e n t u p o n the q u a n t i t y o f A 1 2 5 4 a d m i n i s t e r e d . The lowest single dose o f A 1 2 5 4 which e l e v a t e d h e p a t i c m i c r o s o m a l M O activity significantly was 0.2 m g / k g f o r r a i n b o w t r o u t a n d 0.1 m g / k g for carp. In b o t h species m a x i m u m activity was c a u s e d at doses b e t w e e n 10 a n d 100 m g / k g . T h e s i m i l a r i t y o f r e s p o n s e s is f u r t h e r o b s e r v e d if the r a t i o s o f e t h o x y r e s o r u f i n - O - d e e t h y l a s e activity in m i c r o s o m e s f r o m fish receiving each dose o f A 1 2 5 4 to c o r n oil c o n t r o l s killed o n the s a m e d a y s are c o m p a r e d . Because t r o u t were killed 5 d a y s after i n j e c t i o n , c a r p results f r o m 6 d a y s a f t e r i n j e c t i o n a r e used for c o m p a r i s o n . T h e m a x i m u m r a t i o for t r o u t was 85 at doses o f l 0 m g / k g a n d 25 m g / k g a n d t h a t for c a r p was 60 at 30 m g / k g . O t h e r d o s e - r e s p o n s e d a t a for P C B s in fish a r e n o t a v a i l a b l e in such detail, p a r t i c u l a r l y at doses o f < 100 m g / k g a n d s i m i l a r l y little such d a t a is a v a i l a b l e in m a m m a l i a n species. Studies in which A 1 2 5 4 was a d m i n i s t e r e d to rats in t h e diet utilized

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Fig. 3. Hepatic microsomal ethoxyresorufin-O-deethylase activity in carp receiving graded doses of Aroclor 1254. Three, six or ten days after injection of carp with corn oil or Aroclor 1254, hepatic microsomes were assayed for ethoxyresorufin-O-deethylase activity. [] represents the mean for all corn oil injected, [] represents the mean for 3 days after Aroclor 1254 injection, [] represents the mean for 6 days after Aroclor 1254 injection, [] represents the mean for 10 days after Aroclor 1254 injection, vertical lines represent sE and * denotes a significant increase (P< 0.05) compared to corn oil-injected carp. There was a total of 35 corn oil injected carp and groups of carp receiving a specific dose of Aroclor 1254 a particular number of days before sacrifice ranged from 3 to 9.

57

dietary levels of 0.1, 1 and 10 ppm (Turner and Green, 1974) and I, 5 and 25 ppm (Bruckner et al., 1977). In both studies the presence of PCBs in the diet at 1 ppm or less had little effect on hepatic microsomal enzyme activity, but neither of these studies gave complete dose-response curves. The latter study showed that ingestion of just over 1 mg/kg of A1254 did not increase aminopyrine-N-demethylase activity but did increase acetanilide hydroxylase activity slightly. A more complete dose-response study was done in rat by injection of pure PCB isomers (Goldstein et al., 1977). In this study aryl hydrocarbon hydroxylase activity was increased significantly by 4 daily doses of 3,4,5,3',4',5'-hexachlorobiphenyl at 0.08 mg/kg but not at 0.02 mg/kg and maximum induction appeared to have resulted from 4 daily doses at 2 mg/kg. It may be that parameters known to affect MO activity and induction such as composition of diet (Mehrle et al., 1974) and sexual maturation (Stegeman and Chevion, 1980) affect the response of hepatic MO induction in fish more than differences in fish species. Other likely factors are temperature and quantities of depot fat. Previous studies which we have done with PCBs and other lipophilic pollutants show relatively rapid appearance of the lipophilic chemical in depot fat (Guiney et al., 1977; Melancon and Lech, 1978; Glickman et al., 1977). Substantialdifferences in depot fat could significantly affect the amount of a lipophilic chemical available to the remainder of a fish.

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3,4,3~4'-TETRACHLOROBIPHENYL INJECTED (MG/KG) Fig. 4. Hepatic microsomal ethoxyresorufin-O-deethylase and ethoxycoumarin-O-deethylase activities in rainbow trout receiving graded doses of 3,4,3' ,4'-tetrachlorobiphenyl. Five days after injection of rainbow trout with corn oil or 3 , 4 , 3 ' , 4 ' - T C B , hepatic microsomes were assayed for ethoxycoumarin-Odeethylase activity (*) and ethoxyresorufin-O-deethylase activity ( • ). The open symbols represent the mean value for all corn oil injected carp. The vertical lines represent SE and the numbers in parenthesis above each dose level represent n.

58 Body b u r d e n s o f PCBs c o n s i d e r a b l y higher t h a n the doses o f PCBs required for i n d u c t i o n o f hepatic m i c r o s o m a l M O activity in r a i n b o w t r o u t a n d carp have been o b s e r v e d in b o t h species in the e n v i r o n m e n t , p a r t i c u l a r l y in the G r e a t Lakes. In m a n y cases these b o d y b u r d e n s o f P C B s are so great t h a t it seems likely that these fish possess elevated levels of hepatic M O activity. This does n o t however permit the c o n c l u s i o n t h a t a p a r t i c u l a r b o d y b u r d e n of P C B s , for example 5 m g / k g (5 p p m ) is e q u i v a l e n t to a single dose of 5 m g / k g o f PCBs. I n a fish in the e n v i r o n m e n t which has b e e n receiving small c o n t i n u o u s doses of PCBs t h r o u g h the water a n d / o r diet m u c h o f the b o d y b u r d e n of P C B s m a y be in relatively stable depots a n d the q u a n t i ty available to cause i n d u c t i o n o f hepatic M O activity m a y well be less t h a n that available following a single dose o f PCBs equal to the b o d y b u r d e n . Similarly the same dose o f P C B s acquired via diet or by water exposure m a y result in differing m a g n i t u d e s o f i n d u c t i o n o f hepatic M O activity. Studies i n t o the i n t e r r e l a t i o n s h i p s b e t w e e n dose, r o u t e o f a d m i n i s t r a t i o n , d u r a t i o n o f a d m i n i s t r a t i o n , time after adm i n i s t r a t i o n , tissue levels o f PCBs a n d extent o f i n d u c t i o n o f hepatic M O activity are necessary before accurate e s t i m a t i o n s can be m a d e as to the extent o f i n d u c t i o n in fish caused by exposure to P C B s in the e n v i r o n m e n t . N u m e r o u s studies o n the characteristics o f c o m m e r c i a l mixtures o f PCBs, such

TABLE I Hepatic microsomal monooxygenase activity in fish receiving various PCBsa PCB

Corn oil

PCB

PCB/

(mg/kg body wt) (nmol/min per mg microsomal protein)

Corn oil

0.2 0.5 0.2 0.6

0.092 0.060 0.051 0.014

_+ 0.012 (16)c +_ 0.011 (27) + 0.09 (21) + 0.004 (8)

0.212 _+ 0.059 (14) 0.409 + 0.115 (27) 2.81 + 0.96 (13) 0.356 + 0.051 (8)

2 7 55 25

0.2 0.5 0.2 0.6

0.101 0.077 0.054 0.091

+ + + +

0.220 +__0.032 (14) 0.209 +_ 0.048 (26) 0.399 + 0.379 (13) 0.647 _+ 0.078 (8)

2 3 7 7

0.3 0.2

0.063 _+ 0.011 (4) 0.041 _+ 0.022 (3)

0.113 _+ 0.024 (4) 0.830 _+ 0.224 (6)

2 20

Trout

ERODb Aroclor 1254 Aroclor 1254 3,4,3',4'-TCB 3,4,5,3',4'-PeCBd ECOD Aroclor 1254 Aroclor 1254 3,4,3',4'-TCB 3,4,5,3',4'-PeCB Carp EROD Aroclor 1254 3,4,3',4'-TCB

0.011 (16) 0.009 (27) 0.007 (21) 0.010 (8)

aData ~vas obtained as described in 'Materials and Methods'. The data from 3,4,5,3',4'-PeCB was provided by Dr. M.J. Vodicnik with chemicals supplied by Dr. H. Yushimura. hEROD = ethoxyresorufin-O-deethylase; ECOD = ethoxycoumarin-O-deethylase. CMean +_ SE, value in parenthesis is number of fish used for individual hepatic microsome preparations. d3,4,5,3',4'-PeCB = 3,4,5,3',4'-pentachlorobiphenyl.

59 as the Aroclors, as inducing agents have shown that they induce both the phenobarbital-inducible and 3-methylcholanthrene-inducible microsomal enzyme activities. It has been shown that the phenobarbital-like inducing characteristic resides in non-planar PCBs (those with chlorine at the 2 position) and the 3-methylcholanthrene-like inducing characteristic resides in planar PCBs (those with no chlorine at the 2 position) (Poland and Glover, 1977; Goldstein et al., 1977). While hepatic microsomal MO activity is inducible in fish by 3-methylcholanthrenetype agents, it is nonresponsive to phenobarbital and similar inducing agents (for reviews, see Bend and James, 1979, Lech et al., 1982). One would expect then in fish that planar PCB isomers would be effective inducers of MO activity while nonplanar isomers would not be. Recent studies in this laboratory with pure PCB isomers and polybrominated biphenyl isomers have been consistent with this (Elcombe et al., 1979; Melancon et al., 1981; Lech et al., 1982). 2,3,7,8-Tetrachlorodibenzodioxin which may be present in commercial PCB mixtures is also an inducer of MO activity in fish (Vodicnik et al., 1981). If planar PCBs such as 3,4,3',4'-TCB and 3,4,5,3',4'-pentachlorobiphenyl, which are present in A1254 at low levels, are responsible for the 3-methylcholanthrene-like inducing properties of A1254 in fish, they must be much more potent than A1254. To facilitate comparison of the potencies of A1254 and these planar PCBs to induce MO activity in rainbow trout and carp a listing has been made (Table I) of the induction resulting from treatment of fish with these chemicals at similar doses (0.2-0.6 mg/kg) for similar periods (5 or 6 days before sacrifice). In addition to the data described in 'Results', this table includes some data obtained by Dr. N.J. Vodicnik of The Medical College of Wisconsin using 3,4,5,3' ,4'-pentachlorobiphenyl supplied by Dr. H. Yashimura of Kyushu University, Japan. It is obvious that both of these planar isomers are more potent inducers than A1254 in these studies. As mentioned earlier, the response to 3,4,3',4'-tetrachlorobiphenyl appeared to peak around a dose of 0.2 mg/kg. Although 3,4,5,3',4'-pentachlorobiphenyl exhibited greater potency than A1254, it is likely that this isomer has such high potency that a dose of 0.6 mg/kg may already be beyond the maximum of the dose-response curve. The greater potecy of 3,4,3',4'-tetrachlorobiphenyl and 3,4,5,3',4' -pentachlorobipheny than A 1254 is consistent with the small amounts o f planar PCB isomers in A1254 being responsible for its inducing ability in fish. Studies are continuing to relate the dose of PCB administered, the route of administration, the level of PCB in selected tissues and the hepatic microsomal NO activity. ACKNOWLEDGEMENTS The authors wish to acknowledge the excellent technical assistance of Ms. Sue Swartz, Ms. Sue Bunke and Mr. Steven Yeo.

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