Xenobiotica the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Effect of Polycyclic Aromatic Hydrocarbons on Hepatic Microsomal Enzymes and Disposition of Methylnaphthalene in Rainbow Trout in vivo C. N. Statham, C. R. Elcombe, S. P. Szyjka & J. J. Lech To cite this article: C. N. Statham, C. R. Elcombe, S. P. Szyjka & J. J. Lech (1978) Effect of Polycyclic Aromatic Hydrocarbons on Hepatic Microsomal Enzymes and Disposition of Methylnaphthalene in Rainbow Trout in vivo, Xenobiotica, 8:2, 65-71 To link to this article: http://dx.doi.org/10.3109/00498257809060385
Published online: 30 Sep 2009.
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Date: 11 November 2015, At: 07:42
XENOBIOTICA,
1978, VOL. 8, NO. 2, 65-71
Effect of Polycyclic Aromatic Hydrocarbons on Hepatic Microsomal Enzymes and Disposition of Methylnaphthalene in Rainbow Trout in vivo
Downloaded by [University of California, San Diego] at 07:42 11 November 2015
C. N. STATHAM, C. R. ELCOMBE, S. P. SZYJKAand J. J. LECH* Department of Pharmacology, Medical College of Wisconsin, Milwaukee, Wisconsin 53233, U. S.A. and The Center for Great Lakes Studies, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, U.S.A. (Received 22 January 1977) 1. The effects of 3-methylcholanthrene, 2,3-benzanthracene and ,!I-naphthoflavone on xenobiotic metabolism in rainbow trout were studied. 2. These three polycyclic aromatic hydrocarbons increase hepatic arylhydrocarbon (benzo[a]pyrene) hydroxylase activity without altering glucuronyl transferase activity. 3. All three polycyclic aromatic hydrocarbons increased hepatic microsomal cytochrome P-450 levels by approximately joy,. 4. Pretreatment of trout with 2,3-benzanthracene resulted in an increase in the metabolism and biliary excretion of 2-methylnaphthalene in vivo. 5 . These studies demonstrate that the induction of mono-oxygenation by polycyclic aromatic hydrocarbons can result in significant effects upon the metabolism and excretion of xenobiotics by fish in vivo.
Introduction Many chemicals, such as phenobarbital, 3-methylcholanthrene and benzo[a]pyrene, are capable of inducing the hepatic microsomal mono-oxygenases in many species of mammals (Conney, 1967; Remmer, 1968). This increased activity has been attributed to the de novo synthesis of cytochrome P-450. Few studies have been directed towards this phenomenon in fish. Buhler and Rasmusson (1968) and Gutman and Kidron (1971) found small or variable induction of mono-oxygenation reactions in fish. However, the inducing agents studied were all of the ' barbiturate class ' (phenobarbital, phenylbutazone, DDT). More recently, it has been shown that inducers of the ' polycyclic aromatic hydrocarbon class ' (e.g. 3-methylcholanthrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin) increase mono-oxygenase reactions in certain species of marine fish (Bend et al., 1973, 1977; James et al., 1977) and freshwater fish (Ahokas et al., 1976; Lidman et al., 1976). Evidence that induction of mono-oxygenation may be relevant in the natural environment has been provided by investigations which demonstrated increased levels of benzo[a]pyrene hydroxylase activity in liver of fish sampled from oilpolluted waters (Payne & Penrose, 1975 ; Payne, 1976). This paper examines the effects of several known inducers on microsomal enzyme activities in rainbow trout, and the effects of induction on the disposition of 2-methylnaphthalene in vivo, a compound which is metabolized by the microsomal mono-oxygenase system of rainbow trout.
* USPHS Career Development Awardee #ES-00002. X.B.
E
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Materials and methods Materials Rainbow trout (Salmogairdneri), weighing 80-100 g, were obtained from Kettle Moraine Springs Trout Hatchery (Adell, Wisconsin) and were held in flowing, charcoal-filtered, dechlorinated water at 12" for a minimum of one week before use. A 12 h (0600-1800) light cycle was operated. NADPf, glucose-6-phosphate, glucose-6-phosphate dehydrogenase and uridine diphosphoglucuronic acid (UDPGA) were obtained from Sigma Chemical Co. (St. Louis, Mo.). Radioactive 3-triflu0romethyl-4-nitro[U-ring-~~C]phenol (sp. activity,3.7 Ci/mol)was obtained from Mallinckrodt Chemical Co. (St. Louis, Mo.) and [7,10-14C]benzo[a]pyrene(sp. activity, 51 Ci/mol) was purchased from Amersham Searle (Des Plaines, 111.). 2-Methy1[8-14C]naphthalene (sp. activity, 8.0 Ci/mol) was supplied by California Bionuclear Corp. (Sun Valley, Calif.). All other reagents were of the highest commercially available purity. Pre-treatment of fish and preparation of microsomes Polycyclic aromatic hydrocarbons were administered to trout by intraperitoneal injection as solutions in corn oil (1 mlikg). The following compounds and doses were used: B-naphthoflavone (100 mg/kg), 2,3-benzanthracene (10 mg/kg) and 3-methylcholanthrene (20 mg/kg). These doses of inducing agents resulted in maximal stimulation of arylhydrocarbon (benzo[a]pyrene) hydroxylase. Higher amounts failed to induce further. Control animals received corn oil alone. Careful injection resulted in no observable leakage of compounds from the injection site. Fish were sacrificed by a blow on the head and the gall bladders carefully removed. The livers were excised into ice-cold 1.15% (w/v) KCI, minced and washed three times in fresh 1.15% (w/v) KC1. The minced liver was homogenized in 4 volumes of 0.25 M sucrose using a motor driven Potter-Elvehjemtype glass-Teflon homogenizer and centrifuged at 8500 g (ray=8.3 cm) for 20 min using a Sorval type SM 24 rotor in a Sorval RC-5 superspeed refrigerated centrifuge. The resultant supernatant was decanted and recentrifuged at 165 000 g (r,,-5.7 cm) for 60 min using a Beckman type 65 rotor in a Beckman L5-65 ultracentrifuge. The microsomal pellet obtained was resuspended in 0.25 M sucrose to a final microsomal protein concentration equivalent to about 1 g wet wt liver/ml. All operations were performed at 0-4" and the microsomes were used on the day of preparation. Microsomal enzyme assays Arylhydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity was measured by the method of Hansen and Fouts (1972) as modified by Statham et al. (1977). Glucose-6-phosphatase activity was determined by measurement of the inorganic phosphate released during hydrolysis of glucose-6-phosphate (Hubscher & West, 1965 ; Ames, 1966). UDPGA-elucuronvl transferase was ouantified bv the method of Statham et al. (1977) using 3-triflu0romethyl-4-nitro['~C]phenolas the aglycone substrate. All assays were performed at 25". Protein was estimated by the procedure of Ross and Schatz (1973) using crystalline bovine serum albumin standards. Cytochrome P-450 content of the microsomes was determined using an Aminco DW2 spectrophotometer by the method of Raj and Estabrook (1970), thereby avoiding spectral interference by haemoglobin. This method involves gassing both cuvettes with CO and adding Na,S,04 to the sample cuvette. Studies in vivo Rainbow trout, 80-100g, were treated with either corn oil or 2,3-benzanthracene in corn oil as described above. Both groups were placed in tanks for 48 h after which the trout were exposed to 2-methyl[14C]naphthalene (0.05 mg/l) in 50 1 txiks for 6 h. The fish were returned to fresh flowing water and sampled at various time intervals for the determination of 14C-labelledmaterial in blood, muscle, liver and bile. Samples of blood and bile were placed in scintillation vials containing 15 ml of ACS (Amersham/Searle, Des Plaines, 111.) scintillation mixture. Portions of liver and muscle (5-200 mg) were added to 1 ml of NCS (Amersham/Searle, Des Plaines, Ill.) tissue solubilizer and incubated for 24 h at 48". When the samples were dissolved, 40 pl of glacial acetic acid and 15 ml of ACS were added to the vials. The radioactivity was determined in a Searle Isocap 300 scintillation counter and quench correction was applied using a computer program. Bile obtained from induced and control fish was separately pooled and applied to a XAD-2 (Rohm and Haas, Philadelphia, PA) column. The bound 2-methyl[14C]naphthalene related material was eluted with acetone and the eluate chromatographed on precoated silica gel plates (SIL G-25-UV254, Brinkmann Instrument Co., Westbury, New York) using a mobile phase obtained from the upper layer of a mixture consisting of 1butanol-ammonia soln. (sp gr. 0.88)-water (40 : 10 : 50).
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Results T h e radioactive assays developed for arylhydrocarbon (benzo[a]pyrene) hydroxylase and UDPGA-glucuronyl transferase were linear with time and protein concentration under the prescribed condition. Benzpyrene hydroxylase activity in hepatic microsomes of rainbow trout showed typical cytochrome P-450-mediated mono-oxygenase characteristics, being dependent upon a source of NADPH and also being inhibited by piperonyl butoxide, a classical inhibitor of microsomal mono-oxygenations. UDPGA-glucuronyl transferase activity was negligible in the absence of added UDPGA but was linear for up to 20 min in the presence of UDPGA. Three polycyclic aromatic hydrocarbons (P-naphthoflavone, 3-methylcholanthrene and 2,3-benzanthracene) were investigated for their abilities to induce various microsomal enzyme activities in rainbow trout. Fig. 1 demonstrates that all three polycyclic aromatic hydrocarbons caused dramatic increases in benzpyrene hydroxylase activity without affecting glucose-6-phosphatase or UDPGA-glucuronyl transferase.
W
co a
*
0-81 BENZOPYRENE HYDROXYLASE
C
T_, , C
T ,
,C
-T
I
\. BENZAMHRACENE BENZAMHD*rZLIC 3-METHYLCHOLANTHRENE 3 . METHYLCHOLANTHRENE p-NAPHTHOFLNONE
Fig. 1. The effect of various inducing agents on selected microsomal enzyme activities. Animals were pre-treated as described in Methods. Hepatic microsomes were prepared from individual fish 48 h after dosing. Each bar is the mean+ S.E. (n=3-5). * Indicates induced activity (T) significantly different from control (C) activity ( P < 0.05).
Time course studies for the induction of benzpyrene hydroxylase by the polycyclic aromatic compounds showed a typical time-dependent increase in mono-oxygenase activity (Fig. 2). Benzpyrene hydroxylase activity reached a maximum at around 48 h after injection of 3-methylcholanthrene and 2,3benzanthracene, while with P-naphthoflavone as the inducer, the benzpyrene hydroxylase activity was still increasing at 96 h after injection. These differences may be explained by differences in the rates of absorption of the various compounds from the interperitoneal corn oil depot into the vascular system of the fish. Similar observations by Boobis et al. (1977) have been explained on a similar basis. However, in mice, these workers found that benzpyrene hydroxylase activity returned to basal levels faster after P-naphthoflavone pre-treatment than after injections of 3-methylcholanthrene.
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1.2
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1.0
c
1 0
24
48
72
96
Tlme a f t e r injection [ h )
Fig. 2.
Time course for znduction of arylhydrocarbon (benzo[a]pyrene) hydroxylase by various polycyclic hydrocarbons. Each point is the mean of 2 separate experiments each using the pooled livers from 2 fish. 0 - 0 , corn oil control; A--A, 2,3-benzanthracene; 0-0,3-methylcholanthrene; 0- 0, 13-naphthoflavone.
T h e induction of benzpyrene hydroxylase by 3-methylcholanthrene, pnaphthoflavone and 2,3-benzanthracene was accompanied by an increase in hepatic microsomal cytochrome P-450 levels (Table 1). The P-450 haemoprotein concentrations of the induced microsomes were approximately 5076 higher than those of the control microsomes. No distinctive alterations in the position of the Soret peak of the CO complex of the Na,S,O,-reduced haemoprotein was noted after induction by the polycyclic hydrocarbons. This is in contrast with the situation in rats and other mammals, where at shift from 450 to 448 nm is seen (Sladek & Mannering, 1966). Further evidence for a true induction, rather than an activation of existing enzymes was found in vitro. T h e inclusion of /I-naphthoflavone in the benzpyrene hydroxylase assay or the preincubation of control microsomes with P-naphthoflavone and NADPH before the assay of benzpyrene hydroxylase did not increase the hydroxylation of benzo[a]pyrene. T h e data in Table 2 demonstrate that p-naphthoflavone was actually inhibitory in the benzpyrene hydroxylase assay. Table 1. CytochromeP-450 content of variously induced trout hepatic microsomes Pre-treatment of fish -
-_________
Corn oil (control) P-Naphthoflavone 3-Methylcholanthrene 2,3-Benzanthracene
Dose - _~
Cytochrome P-450 (nmol/mg protein)"
1 ml/kg 100 mg/kg 20 mg/kg 10 mg/kg
-
-
0.21 ; 0.22 0.33; 0.35 0.31 ; 0.34 0.34; 0.28
*Values obtained from pooled microsomes (6-8 fish per group) in two separate experiments. Microsomes were prepared 48 h after treatment of fish.
Polycyclic Aromatic Hydrocarbons in Rainbow Trout
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Table 2. Effect of /3-naphthoflavoneon arylhydrocarbon(benzo [a] pyrene) hydroxylase activity of control trout hepatic microsomes in vitro Addition to assay
Renzo[a]pyrene hydroxsse activity"
(70) 100
None Dimethylformamide (5 pl) /3-Naphthoflavone 10 p~ 100 ,!AM 500 p M
98.3 65.3 46.3 22.6
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P-Naphthoflavone was added dissolved in 5 111 of N,N-dimethylformamide. " nmol polar metabolites of benzo[a]pyrene produced/min/mg microsomal protein. 50
L
0
1
BiLE
-*
*
T
3'0L
2.5
0
3
12
6
Washout
Fig. 3.
*
T
time l h l
Uptake and elimination of 2-methyl['4C]napht/zalene-derived material in rainbow trout. Each point is the mean k S.E. ( n = 3 in two separate experiments). - - _ _ Control; -benzanthracene-treated. * P < 0.05. Exposure was to 0.05 mg 2-methyl[14C]naphthalene/1.
Increased rates of metabolism and biliary excretion of 2-methyl[14C]naphthalene in nivo were also observed after pretreatment of rainbow trout with 2,3benzanthracene. Fig. 3 demonstrates that the rate of appearance of radioactive material in bile was dramatically increased by 2,3-benzanthracene pretreatment. It is interesting to note that the initial levels of I4C were higher in livers of induced trout and appeared to be retained longer during the wash-out period. Pre-treatment with 2,3-benzanthracene did not appear to significantly affect the rate of disappearance of radioactive material from blood or muscle. Thin-layer chromatography of extracts of bile indicated a greater proportion of 2-methylnaphthalene related polar radioactive materials and less parent compound in bile after induction of mono-oxygenation by 2,3-benzanthracene (Fig. 4). illthough the polar metabolites have not yet been characterized, there appear to be several different compounds in the polar fraction.
C . N . Statham et al.
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a
2 - METHYLNAPHTHALENE
-
10
* n
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L
Distance f r o m origin (cm)
Fig. 4.
Thin-layer radiochromatographic profile of 2-methylnaphthalene metabolites in trout bile Solvent: 1-butanol-ammonia soln. (sp. gr. 0.88)-water (40 : 10 : 50). The shaded spots indicate the mobility of 2-methylnaphthalene.
Discussion Polycyclic aromatic hydrocarbon inducing agents induce benzolulpyrene hydroxylase activity in several marine species (Bend et al., 1973 ; James et al., 1977), but no concomitant increase in cytochrome P-450 levels were observed. In the present studies using rainbow trout, an increase in cytochrome P-450 concentration in response to P-naphthoflavone, 3-methylcholanthrene and 2,3-benzanthracene was found. This may reflect differences in the control mechanisms of P-450 synthesis of marine and freshwater species of fish. T h e observation that P-naphthoflavone, added in vitro, inhibited the benzo[alpyrene hydroxylase activity of control microsomes suggests that the endogenous cytochrome P-450 of trout hepatic microsomes may be of the P,-450 type; because in mammalian systems P-naphthoflavone actually stimulates P-450mediated benzpyrene hydroxylase while inhibiting P1-450 benzpyrene hydroxylase activity (Wiebel et al., 1971). Furthermore, no blue shift in the absorbance maximum of the carbon monoxide complex of the ferrocytochrome was seen after induction, suggesting that the induced and endogenous cytochrome(s) are similar. T h e metabolism of 2-methyl[14C]naphthalene in vivo can be altered by pretreatment of rainbow trout with 2,3-benzanthracene. An increased biliary elimination of 14C-labelled materials in bile and a corresponding increase in polar metabolites of 2-methylnaphthalene were observed after induction of monooxygenation by 2,3-benzanthracene. Greater retention of 14C in livers from induced trout may be due to the presence of a greater quantity of polar metabolites in these livers, since it has been reported that polar metabolites of naphthalene are retained longer than naphthalene in tissues of naphthalene-treated spot shrimp (Pandalus plutycens) (Sanborn & Malins, 1977). T h e lack of effect of 2,3-benzanthracene induction upon elimination of 2-methylnaphthalene-derived radioactivity from muscle and blood suggests that the rate-limiting step in elimination from these tissues is not hepatic metabolism even though the biliary excretion of polar metabolites was increased. Factors such as transfer rates from specific tissue compartments and other routes of elimination must be considered when dealing with the overall elimination of any specific chemical.
Polycyclic Aromatic Hydrocarbons in Rainbow Trout
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Acknowledgments We would like to thank Dr. J. G. Ghazarian for the use of his Aminco D W 2 spectrophotometer. This research was supported by the National Institutes of Health Grant ES 01080, Grant R803971010 from the Environmental Protection Agency and by the Sea Grant Programme of the University of Wisconsin.
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References AHOKAS, J. T., PELKONEN, 0. & K ~ ~ R K N.I ,T. (1976). Acta Pharmac. Tax., 38, 440. AMES,B. N. (1966). In Methods in Enzymology. Editors: Colowick, P. and Kaplan, N. 0. vol. 8, p. 115. New York: Academic Press. BEND,J. R., POHL,R. J. & FOUTS,J. R. (1973). Bull. M t . Desert IsIand Biol. Lab., 13, 9. BEND,J. R., POHL,R. J., ARINC,E. & PHILPOT,R. M. (1977). In Proceedings, Third International Symposium on Microsomes and Drug Oxidations. Oxford : Pergamon Press. 0. W. & FELTON, J. S. (1977). Molec. Pharmac., 13, 259. BOOBIS, A. R., NEBERT, BUHLER,D. R. & RASMUSSON, M. E. (1968). Comp. Biochem. Physiol., 25, 223. A. H. (1967). Pharmac. Rev., 19, 317. CONNEY, GUTMAN, Y. & KIDRON, M. (1971). Biochem. Pharmac., 20, 3547. J. R. (1972). Chem. Bid. Interactions, 5, 167. HANSEN, A. R. & FOUTS, H~~BSCHER, G. & WEST,G. R. (1965). Nature, 205, 799. JAMES,M. O., FOUTS, J. R. & BEND,J. R. (1977). In Pesticides in the Aquatic Environment. Symposium at the 15th International Cong. of the Entomology SOC. LIDMAN, U., FORLIN,L., MOLANDER, 0. & AXELSON, G. (1976). Acta Pharmac. Tax., 39, 262. J. F. & PENROSE, W. R. (1975). Bull. Environ. Contam. Toxic., 14, 112. PAYNE, PAYNE, J. F. (1976). Science, 191, 945. RAJ,P. P. & ESTABROOK, R. W. (1970). Pharmacologist, 12, 261. REMMER,H. (1968). German Med. Month., 13, 53. ROSS, E. & SCHATZ, G. (1973). Analyt. Biochem., 54, 304. SANBORN, H. R. & MALINS,D. C. (1977). Pvoc. Sac. exp. Biol. Med., 154, 151. S. P., MENAHAN, L. A. & LECH,J. J. (1977). Biochem. Pharmac., STATHAM, C. N., SZYJKA, 26, 1395. N. E. & MANNERING, G. J. (1966). Biochem. Biophys. Res. Commun., 24, 668. SLADEK, WIEBEL,F. J., LEUTZ,J. C., DIAMOND, L. & GELBOIN, H. V. (1971). Arch. Biochem. Biophys., 144, 78.
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Effect of Polycyclic Aromatic Hydrocarbons on Hepatic Microsomal Enzymes and Disposition of Methylnaphthalene in Rainbow Trout in vivo C. N. Statham, C. R. Elcombe, S. P. Szyjka & J. J. Lech To cite this article: C. N. Statham, C. R. Elcombe, S. P. Szyjka & J. J. Lech (1978) Effect of Polycyclic Aromatic Hydrocarbons on Hepatic Microsomal Enzymes and Disposition of Methylnaphthalene in Rainbow Trout in vivo, Xenobiotica, 8:2, 65-71 To link to this article: http://dx.doi.org/10.3109/00498257809060385
Published online: 30 Sep 2009.
Submit your article to this journal
Article views: 8
View related articles
Citing articles: 3 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ixen20 Download by: [University of California, San Diego]
Date: 11 November 2015, At: 07:42
XENOBIOTICA,
1978, VOL. 8, NO. 2, 65-71
Effect of Polycyclic Aromatic Hydrocarbons on Hepatic Microsomal Enzymes and Disposition of Methylnaphthalene in Rainbow Trout in vivo
Downloaded by [University of California, San Diego] at 07:42 11 November 2015
C. N. STATHAM, C. R. ELCOMBE, S. P. SZYJKAand J. J. LECH* Department of Pharmacology, Medical College of Wisconsin, Milwaukee, Wisconsin 53233, U. S.A. and The Center for Great Lakes Studies, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, U.S.A. (Received 22 January 1977) 1. The effects of 3-methylcholanthrene, 2,3-benzanthracene and ,!I-naphthoflavone on xenobiotic metabolism in rainbow trout were studied. 2. These three polycyclic aromatic hydrocarbons increase hepatic arylhydrocarbon (benzo[a]pyrene) hydroxylase activity without altering glucuronyl transferase activity. 3. All three polycyclic aromatic hydrocarbons increased hepatic microsomal cytochrome P-450 levels by approximately joy,. 4. Pretreatment of trout with 2,3-benzanthracene resulted in an increase in the metabolism and biliary excretion of 2-methylnaphthalene in vivo. 5 . These studies demonstrate that the induction of mono-oxygenation by polycyclic aromatic hydrocarbons can result in significant effects upon the metabolism and excretion of xenobiotics by fish in vivo.
Introduction Many chemicals, such as phenobarbital, 3-methylcholanthrene and benzo[a]pyrene, are capable of inducing the hepatic microsomal mono-oxygenases in many species of mammals (Conney, 1967; Remmer, 1968). This increased activity has been attributed to the de novo synthesis of cytochrome P-450. Few studies have been directed towards this phenomenon in fish. Buhler and Rasmusson (1968) and Gutman and Kidron (1971) found small or variable induction of mono-oxygenation reactions in fish. However, the inducing agents studied were all of the ' barbiturate class ' (phenobarbital, phenylbutazone, DDT). More recently, it has been shown that inducers of the ' polycyclic aromatic hydrocarbon class ' (e.g. 3-methylcholanthrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin) increase mono-oxygenase reactions in certain species of marine fish (Bend et al., 1973, 1977; James et al., 1977) and freshwater fish (Ahokas et al., 1976; Lidman et al., 1976). Evidence that induction of mono-oxygenation may be relevant in the natural environment has been provided by investigations which demonstrated increased levels of benzo[a]pyrene hydroxylase activity in liver of fish sampled from oilpolluted waters (Payne & Penrose, 1975 ; Payne, 1976). This paper examines the effects of several known inducers on microsomal enzyme activities in rainbow trout, and the effects of induction on the disposition of 2-methylnaphthalene in vivo, a compound which is metabolized by the microsomal mono-oxygenase system of rainbow trout.
* USPHS Career Development Awardee #ES-00002. X.B.
E
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Materials and methods Materials Rainbow trout (Salmogairdneri), weighing 80-100 g, were obtained from Kettle Moraine Springs Trout Hatchery (Adell, Wisconsin) and were held in flowing, charcoal-filtered, dechlorinated water at 12" for a minimum of one week before use. A 12 h (0600-1800) light cycle was operated. NADPf, glucose-6-phosphate, glucose-6-phosphate dehydrogenase and uridine diphosphoglucuronic acid (UDPGA) were obtained from Sigma Chemical Co. (St. Louis, Mo.). Radioactive 3-triflu0romethyl-4-nitro[U-ring-~~C]phenol (sp. activity,3.7 Ci/mol)was obtained from Mallinckrodt Chemical Co. (St. Louis, Mo.) and [7,10-14C]benzo[a]pyrene(sp. activity, 51 Ci/mol) was purchased from Amersham Searle (Des Plaines, 111.). 2-Methy1[8-14C]naphthalene (sp. activity, 8.0 Ci/mol) was supplied by California Bionuclear Corp. (Sun Valley, Calif.). All other reagents were of the highest commercially available purity. Pre-treatment of fish and preparation of microsomes Polycyclic aromatic hydrocarbons were administered to trout by intraperitoneal injection as solutions in corn oil (1 mlikg). The following compounds and doses were used: B-naphthoflavone (100 mg/kg), 2,3-benzanthracene (10 mg/kg) and 3-methylcholanthrene (20 mg/kg). These doses of inducing agents resulted in maximal stimulation of arylhydrocarbon (benzo[a]pyrene) hydroxylase. Higher amounts failed to induce further. Control animals received corn oil alone. Careful injection resulted in no observable leakage of compounds from the injection site. Fish were sacrificed by a blow on the head and the gall bladders carefully removed. The livers were excised into ice-cold 1.15% (w/v) KCI, minced and washed three times in fresh 1.15% (w/v) KC1. The minced liver was homogenized in 4 volumes of 0.25 M sucrose using a motor driven Potter-Elvehjemtype glass-Teflon homogenizer and centrifuged at 8500 g (ray=8.3 cm) for 20 min using a Sorval type SM 24 rotor in a Sorval RC-5 superspeed refrigerated centrifuge. The resultant supernatant was decanted and recentrifuged at 165 000 g (r,,-5.7 cm) for 60 min using a Beckman type 65 rotor in a Beckman L5-65 ultracentrifuge. The microsomal pellet obtained was resuspended in 0.25 M sucrose to a final microsomal protein concentration equivalent to about 1 g wet wt liver/ml. All operations were performed at 0-4" and the microsomes were used on the day of preparation. Microsomal enzyme assays Arylhydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity was measured by the method of Hansen and Fouts (1972) as modified by Statham et al. (1977). Glucose-6-phosphatase activity was determined by measurement of the inorganic phosphate released during hydrolysis of glucose-6-phosphate (Hubscher & West, 1965 ; Ames, 1966). UDPGA-elucuronvl transferase was ouantified bv the method of Statham et al. (1977) using 3-triflu0romethyl-4-nitro['~C]phenolas the aglycone substrate. All assays were performed at 25". Protein was estimated by the procedure of Ross and Schatz (1973) using crystalline bovine serum albumin standards. Cytochrome P-450 content of the microsomes was determined using an Aminco DW2 spectrophotometer by the method of Raj and Estabrook (1970), thereby avoiding spectral interference by haemoglobin. This method involves gassing both cuvettes with CO and adding Na,S,04 to the sample cuvette. Studies in vivo Rainbow trout, 80-100g, were treated with either corn oil or 2,3-benzanthracene in corn oil as described above. Both groups were placed in tanks for 48 h after which the trout were exposed to 2-methyl[14C]naphthalene (0.05 mg/l) in 50 1 txiks for 6 h. The fish were returned to fresh flowing water and sampled at various time intervals for the determination of 14C-labelledmaterial in blood, muscle, liver and bile. Samples of blood and bile were placed in scintillation vials containing 15 ml of ACS (Amersham/Searle, Des Plaines, 111.) scintillation mixture. Portions of liver and muscle (5-200 mg) were added to 1 ml of NCS (Amersham/Searle, Des Plaines, Ill.) tissue solubilizer and incubated for 24 h at 48". When the samples were dissolved, 40 pl of glacial acetic acid and 15 ml of ACS were added to the vials. The radioactivity was determined in a Searle Isocap 300 scintillation counter and quench correction was applied using a computer program. Bile obtained from induced and control fish was separately pooled and applied to a XAD-2 (Rohm and Haas, Philadelphia, PA) column. The bound 2-methyl[14C]naphthalene related material was eluted with acetone and the eluate chromatographed on precoated silica gel plates (SIL G-25-UV254, Brinkmann Instrument Co., Westbury, New York) using a mobile phase obtained from the upper layer of a mixture consisting of 1butanol-ammonia soln. (sp gr. 0.88)-water (40 : 10 : 50).
-
%
,
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Polycyclic Aromatic Hydrocarbons in Rainbow Trout
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Results T h e radioactive assays developed for arylhydrocarbon (benzo[a]pyrene) hydroxylase and UDPGA-glucuronyl transferase were linear with time and protein concentration under the prescribed condition. Benzpyrene hydroxylase activity in hepatic microsomes of rainbow trout showed typical cytochrome P-450-mediated mono-oxygenase characteristics, being dependent upon a source of NADPH and also being inhibited by piperonyl butoxide, a classical inhibitor of microsomal mono-oxygenations. UDPGA-glucuronyl transferase activity was negligible in the absence of added UDPGA but was linear for up to 20 min in the presence of UDPGA. Three polycyclic aromatic hydrocarbons (P-naphthoflavone, 3-methylcholanthrene and 2,3-benzanthracene) were investigated for their abilities to induce various microsomal enzyme activities in rainbow trout. Fig. 1 demonstrates that all three polycyclic aromatic hydrocarbons caused dramatic increases in benzpyrene hydroxylase activity without affecting glucose-6-phosphatase or UDPGA-glucuronyl transferase.
W
co a
*
0-81 BENZOPYRENE HYDROXYLASE
C
T_, , C
T ,
,C
-T
I
\. BENZAMHRACENE BENZAMHD*rZLIC 3-METHYLCHOLANTHRENE 3 . METHYLCHOLANTHRENE p-NAPHTHOFLNONE
Fig. 1. The effect of various inducing agents on selected microsomal enzyme activities. Animals were pre-treated as described in Methods. Hepatic microsomes were prepared from individual fish 48 h after dosing. Each bar is the mean+ S.E. (n=3-5). * Indicates induced activity (T) significantly different from control (C) activity ( P < 0.05).
Time course studies for the induction of benzpyrene hydroxylase by the polycyclic aromatic compounds showed a typical time-dependent increase in mono-oxygenase activity (Fig. 2). Benzpyrene hydroxylase activity reached a maximum at around 48 h after injection of 3-methylcholanthrene and 2,3benzanthracene, while with P-naphthoflavone as the inducer, the benzpyrene hydroxylase activity was still increasing at 96 h after injection. These differences may be explained by differences in the rates of absorption of the various compounds from the interperitoneal corn oil depot into the vascular system of the fish. Similar observations by Boobis et al. (1977) have been explained on a similar basis. However, in mice, these workers found that benzpyrene hydroxylase activity returned to basal levels faster after P-naphthoflavone pre-treatment than after injections of 3-methylcholanthrene.
C. N . Statham et al.
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1.2
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1.0
c
1 0
24
48
72
96
Tlme a f t e r injection [ h )
Fig. 2.
Time course for znduction of arylhydrocarbon (benzo[a]pyrene) hydroxylase by various polycyclic hydrocarbons. Each point is the mean of 2 separate experiments each using the pooled livers from 2 fish. 0 - 0 , corn oil control; A--A, 2,3-benzanthracene; 0-0,3-methylcholanthrene; 0- 0, 13-naphthoflavone.
T h e induction of benzpyrene hydroxylase by 3-methylcholanthrene, pnaphthoflavone and 2,3-benzanthracene was accompanied by an increase in hepatic microsomal cytochrome P-450 levels (Table 1). The P-450 haemoprotein concentrations of the induced microsomes were approximately 5076 higher than those of the control microsomes. No distinctive alterations in the position of the Soret peak of the CO complex of the Na,S,O,-reduced haemoprotein was noted after induction by the polycyclic hydrocarbons. This is in contrast with the situation in rats and other mammals, where at shift from 450 to 448 nm is seen (Sladek & Mannering, 1966). Further evidence for a true induction, rather than an activation of existing enzymes was found in vitro. T h e inclusion of /I-naphthoflavone in the benzpyrene hydroxylase assay or the preincubation of control microsomes with P-naphthoflavone and NADPH before the assay of benzpyrene hydroxylase did not increase the hydroxylation of benzo[a]pyrene. T h e data in Table 2 demonstrate that p-naphthoflavone was actually inhibitory in the benzpyrene hydroxylase assay. Table 1. CytochromeP-450 content of variously induced trout hepatic microsomes Pre-treatment of fish -
-_________
Corn oil (control) P-Naphthoflavone 3-Methylcholanthrene 2,3-Benzanthracene
Dose - _~
Cytochrome P-450 (nmol/mg protein)"
1 ml/kg 100 mg/kg 20 mg/kg 10 mg/kg
-
-
0.21 ; 0.22 0.33; 0.35 0.31 ; 0.34 0.34; 0.28
*Values obtained from pooled microsomes (6-8 fish per group) in two separate experiments. Microsomes were prepared 48 h after treatment of fish.
Polycyclic Aromatic Hydrocarbons in Rainbow Trout
69
Table 2. Effect of /3-naphthoflavoneon arylhydrocarbon(benzo [a] pyrene) hydroxylase activity of control trout hepatic microsomes in vitro Addition to assay
Renzo[a]pyrene hydroxsse activity"
(70) 100
None Dimethylformamide (5 pl) /3-Naphthoflavone 10 p~ 100 ,!AM 500 p M
98.3 65.3 46.3 22.6
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P-Naphthoflavone was added dissolved in 5 111 of N,N-dimethylformamide. " nmol polar metabolites of benzo[a]pyrene produced/min/mg microsomal protein. 50
L
0
1
BiLE
-*
*
T
3'0L
2.5
0
3
12
6
Washout
Fig. 3.
*
T
time l h l
Uptake and elimination of 2-methyl['4C]napht/zalene-derived material in rainbow trout. Each point is the mean k S.E. ( n = 3 in two separate experiments). - - _ _ Control; -benzanthracene-treated. * P < 0.05. Exposure was to 0.05 mg 2-methyl[14C]naphthalene/1.
Increased rates of metabolism and biliary excretion of 2-methyl[14C]naphthalene in nivo were also observed after pretreatment of rainbow trout with 2,3benzanthracene. Fig. 3 demonstrates that the rate of appearance of radioactive material in bile was dramatically increased by 2,3-benzanthracene pretreatment. It is interesting to note that the initial levels of I4C were higher in livers of induced trout and appeared to be retained longer during the wash-out period. Pre-treatment with 2,3-benzanthracene did not appear to significantly affect the rate of disappearance of radioactive material from blood or muscle. Thin-layer chromatography of extracts of bile indicated a greater proportion of 2-methylnaphthalene related polar radioactive materials and less parent compound in bile after induction of mono-oxygenation by 2,3-benzanthracene (Fig. 4). illthough the polar metabolites have not yet been characterized, there appear to be several different compounds in the polar fraction.
C . N . Statham et al.
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a
2 - METHYLNAPHTHALENE
-
10
* n
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L
Distance f r o m origin (cm)
Fig. 4.
Thin-layer radiochromatographic profile of 2-methylnaphthalene metabolites in trout bile Solvent: 1-butanol-ammonia soln. (sp. gr. 0.88)-water (40 : 10 : 50). The shaded spots indicate the mobility of 2-methylnaphthalene.
Discussion Polycyclic aromatic hydrocarbon inducing agents induce benzolulpyrene hydroxylase activity in several marine species (Bend et al., 1973 ; James et al., 1977), but no concomitant increase in cytochrome P-450 levels were observed. In the present studies using rainbow trout, an increase in cytochrome P-450 concentration in response to P-naphthoflavone, 3-methylcholanthrene and 2,3-benzanthracene was found. This may reflect differences in the control mechanisms of P-450 synthesis of marine and freshwater species of fish. T h e observation that P-naphthoflavone, added in vitro, inhibited the benzo[alpyrene hydroxylase activity of control microsomes suggests that the endogenous cytochrome P-450 of trout hepatic microsomes may be of the P,-450 type; because in mammalian systems P-naphthoflavone actually stimulates P-450mediated benzpyrene hydroxylase while inhibiting P1-450 benzpyrene hydroxylase activity (Wiebel et al., 1971). Furthermore, no blue shift in the absorbance maximum of the carbon monoxide complex of the ferrocytochrome was seen after induction, suggesting that the induced and endogenous cytochrome(s) are similar. T h e metabolism of 2-methyl[14C]naphthalene in vivo can be altered by pretreatment of rainbow trout with 2,3-benzanthracene. An increased biliary elimination of 14C-labelled materials in bile and a corresponding increase in polar metabolites of 2-methylnaphthalene were observed after induction of monooxygenation by 2,3-benzanthracene. Greater retention of 14C in livers from induced trout may be due to the presence of a greater quantity of polar metabolites in these livers, since it has been reported that polar metabolites of naphthalene are retained longer than naphthalene in tissues of naphthalene-treated spot shrimp (Pandalus plutycens) (Sanborn & Malins, 1977). T h e lack of effect of 2,3-benzanthracene induction upon elimination of 2-methylnaphthalene-derived radioactivity from muscle and blood suggests that the rate-limiting step in elimination from these tissues is not hepatic metabolism even though the biliary excretion of polar metabolites was increased. Factors such as transfer rates from specific tissue compartments and other routes of elimination must be considered when dealing with the overall elimination of any specific chemical.
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Acknowledgments We would like to thank Dr. J. G. Ghazarian for the use of his Aminco D W 2 spectrophotometer. This research was supported by the National Institutes of Health Grant ES 01080, Grant R803971010 from the Environmental Protection Agency and by the Sea Grant Programme of the University of Wisconsin.
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