WHllIWIUHIHINIWIWWW“WWW ‘ 1O 5 THS 03 (DC!) Twigs s ‘ Date This is to certify that the thesis entitled CHARACTERISTICS OF THE HEPATIC MONOOXYGENASE SYSTEM OF THE GOLDFISH (CARASSIUS AURATUS) AND ITS INDUCTION WITH B-NAPTHOFLAVONE presented by Jay William Gooch has been accepted towards fulfillment of the requirements for Master of Science , Fisheries and Wildlife degree in Major professor May 20, 1982 0-7639 RETURNING MATERIALS: IVIESI_J Place in book drop to LJBRARJES remove this checkout from filling-III. your record. FINES will be charged if book is returned after the date stamped below. G // 7637 CHARACTERISTICS OF THE HEPATIC MONOOXYGEN ASE SYSTEM OF THE GOLDFISH (CARASSIUS AURATUS) AND ITS INDUCTION WITH E-NAPTHOFLAVON E By Jay William Gooch A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Fisheries and Wildlife 1982 ABSTRACT CHARACTERISTICS OF THE HEPATIC MON OOXYGEN ASE SYSTEM OF THE GOLDFISH (CARASSIUS AURATUS) AND ITS ‘ INDUCTION WITH B-NARTHOFLAVONE BY Jay William Gooch ‘ Treatment of goldfish with e-napthoflavone (i.p.) resulted in induction of hepatic monooxygenase activity in a dose-dependent manner. Levels of 7- ethoxyresorufin—O-deethylase and mexacarbate NADPH dependent oxidative metabolism as well as cytochrome(s) P-450 were increased. Other substrates which have been associated with '3-methylcholanthrene type induction in mammals were unchanged as was d-aminolevulinic acid synthetase. Enzyme activities were inhibited by carbon monoxide and piperonyl butoxide confirming their cytochrome P-450 nature. NADH was found to stimulate metabolism in a synergistic manner with NADPH. The pH dependent ethyl isocyanide binding characteristics were similar in control and induced preparations while mexacarbate binding to oxidized microsomes revealed a higher apparent affinity constant (Kipp) as well as greater overall binding in induced preparations. SDS- gel electrophoresis revealed an increased band with a molecular weight of approximately 56,000. To my Mother and Father ii ACKNOWLEDGEMENTS I would like to express by deepest gratitude to Professor Fumio Matsumura for taking an interest when things looked bleak and for continued encouragement and support through the course of this work and Dr. Howard Johnson for helping me put things together in the beginning. I would also like to thank Dr. Burra V. Madhukar for his expertise and help, Dr. Don Garling for allowing me to maintain my space and Ms. Alice Ellis for her professional preparation of this manuscript. May your glow box always shine! And to Ms. Cindi Garbus ‘- a heart full of love for her patience and understanding. This study was supported by the Michigan Agricultural Experiment Station and by research grant E801963 from the National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina. iii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION MATERIALS AND METHODS Materials Methods , , ' Preparation of Microsomes Enzyme Assays Binding Studies Electron Microscope RESULTS Electron Microscope Analysis of Isolated Hepatocytes Studies on the Patterns of Induction by s-napthoflavone Characterization of Induced Microsomal Monooxygenase Systems Spectroscopic Examination of Induced Cytochrome P-450 Gel ElectrOphoresis , DISCUSSION LIST OF REFERENCES iv Page vi 11 11 11 15 _ 19 22 27 32 LIST OF TABLES Table Page 1 Biotransformation enzymes demonstrated in fish. ,,,,, 2 2 The effect of B-napthoflavone pretreatment on various parameters of the hepatic monooxygenase system of the goldfish (Carassius auratus). Fish received a single 150 mg/kg dose in corn oil and were sacrificed after 96 hours. . 16 3 The effect of B-napthoflavone treatment on selected hepatic microsomal monooxygenase enzymes and on 6 - ALA-synthetase. ................... ’17 4 The effect of added cofactors and inhibitors in vitro on monooxygenase activity from control and B - napthoflavone treated goldfish. ,,,,,,,,,,,,, 18 LIST OF FIGURES Chemical structures of inducers and substrates used in this study. ...... . ................ 6 Transmission electron micrographs of isolated hepatocytes from control (a,b) and B-napthoflavone treated (c,d) goldfish. Magnification 10,000 X. ...... 12 Time course of induction of 7-ethoxyresorufin-0- deethylase activity following a single i.p. dose of e- napthoflavone (100 tug/kg). ............... 13 Dose-response curve for 7—ethoxyresorufin-0-deethylation induction. Fish received a single i.p. injection and were sacrificed 72 hours later. ............... 14 Ethyl isocyanide binding to sodium dithionite reduced goldfish microsomes as a function of pH from control (A) and B-napthoflavone (0) induced preparations. Fish received the standard 150 mg/kg i.p. dose followed by sacrifice at 96 hours. Other aspects of the assay are described in Methods. Data is expressed as mean : S.E. The variability for the treated preparation at pH 8.75 was + .003. ........................ 20 Ethyl isocyanide binding to reduced goldfish microsomes showing a gradual shift in the absorbance of the 430 nm peak with increasing pH. ................ 21 Computer-assisted representation of mexacarbate binding to oxidized goldfish liver microsomes from control (—) and B-napthoflavone induced (---) fish. [Mexacarbate] = 0.11 mM. Microsomal protein concentration was 2.8 mg/ml. ....................... 23 Double reciprocal plot of mexacarbate binding to goldfish microsomes. Binding was done in 50 mM tris buffer (pH 7.5) at a protein concentratigi of 2.9 mg/ml;5 The apparent affinity cogstants (Ka ) were 6.05 x 10 M for control and 2-6 X 10. M for indiiced preparations. ----- 24 Sodium dodecyl sulphate-polyacrylamide gel electro- phoresis of hepatic microsomes from control (b,c) and B- napthoflavone—treated (d,e) goldfish. Well (a) contained standard proteins with molecular weights of 14,400, 21,500, 31,000, 45,000 (shown), 66,200 (shown) and 92,500. Wells contained 75 -u g protein and . the resolving gel was 7-5% acrylamide. ................... vii INTRODUCTION The contamination of the aquatic environment by foreign organic chemicals is a widespread ongoing phenomenon. Between 1961 and 1975 over 465 million fish were reported killed by pollution overloads (Biernacki, 1978). The effects of this contamination range from the overt toxicity just mentioned to the bioaccumulation of harmful levels of toxic chemicals in edible species. The well known problem of the accumulation of polychlorinated biphenyls (PCB's) in Great Lakes fish species is an excellent example of the latter. Investigations into the mechanisms of biotransformation and its relationship to fate or toxicity are thus necessary to understand the behavior of chemicals in these organisms. While initial investigators (Brodie and Maickel, 1962) failed to demonstrate the presence of enzymatic oxidations in fish, subsequent research has demonstrated the existence of a wide variety of biotransformation enzymes in various fish tissues (for review see Chambers and Yarbrough, 1976; Lech and Bend, 1980; Bend et al., 1980). The ability of fish to oxidize and conjugate exogenous as well as endogenous compounds is of great importance for the maintenance of normal metabolic function. Table 1 lists examples of the many types of reactions that have been seen in different fish species. ’ Presumably in an attempt to cope with fluctuating levels of chemical exposure, some of these enzyme systems are capable of being induced into higher activity. The most notable of these is the hepatic monooxygenase system. This system, located primarily in the endoplasmic reticulum (microsomes), functions .32 .080 0:: 83.. :8... 83...... 22 :3 3 :30 3.8.30 :38... 2.2 :3 3 :86 8.38.3.0 :30 8.3.0.33... 2.2 :3 3 3:32.... 8333.80 .8... 38:30 22 :3 3 83.3.8 28.882838: 3:... 38:30 22 :3 3 833.. 283...888..z... 88.3 0:83.. 8.3.3.80-.. .32 :3 3 33:0 «83:335....333.§ 30:8... .32 .85. 0:: 8830.3 380.330 :38... 22 £8380 0:: 3:93.33 5.33... .50.... 8.3.3.8... 82 .3... 0:: 5338.30 80.3.8... 3.33:... .32 :3 3 3.8:... 3883.. :30 2.2 .83.. 0:: 83.33.. 38.2.880332 .8... 38:30 2.2 :3 3 3:30 3835832 833... 230 2.3 :0. .0 00A 0:0...»233000 6:035:32 £00.00 £05500: 0030005 82 :3 3 :3... 38:88:33-3 .30.. .8... 38:30 .32 :3 3 8.3.. 38:88:83-3 .300 :38 .3035 8.3.58... .32 .83.. 0:: 83.3.6 5.3333: .8... 38:30 82 .88... 0:: :80 8333... 80:... 2.2 .83.. 0.3 83:33... 3335.... 505.33... 39.. 38:30 22 35.33 0:: 3333.. 288.9... 3.32.... .8030 3.3.3.0.... 2.2 .3338: 0:: 93...... 38893838.. 8.330 8.33.38 33...... 2.2 .08.. :8 83.... 0.... 3.38... 8.33.38 8.33... S2 .30.... 0:: 83.... 8.8 38.33 88:83.3 8.33.38 38333.0 2.2 :3 3 8.5.3.0 38822838.. 32. 38:30 . 3:05.95... 22 .83.. I....3:38.........m 39.. 38:30 8.33383 8.8.830 ~03 35:00 0:0 9.0:: 0.00 0.3000050... 20.00380 3:09.280 9.00.503 00.09502» 65020 02.0.30». 0:009:00 00.00% 003000”. in... 5 00.03.0583 0003000.. 00.358.300.505 A mamfiv 3 via NADPH, cytochrome P-450 (named for its characteristic absorption at 450 nm when reduced and complexed with carbon monoxide), NADPH cytochrome P- 450 reductase and uses oxygen to oxidize a wide variety of chemicals (for review see Kato, 1979; Parke, 1981; and Coon, 1978). Numerous studies in mammalian systems have shown that there are distinct increases in microsomal proteins and enzyme systems after chemical exposure and several scientists have made attempts to discern the complex relationship between the induction status of the system and toxicity or carcinogenesis (for review see Conney, 1967; Nebert and Jensen, 1979; Snyder and Remmer, 1979; Nebert, 1979; Parke, 1975). Initially inducers of this system were categorized into two broad types; those of the phenobarbital (PB) or barbiturate type and those of the 3- methylcholanthrene (3-M C) or polycyclic aromatic hydrocarbon type depending on the nature of the enzyme activities induced. Also noted was that the 3-MC type of inducer caused the production of a new type of cytochrome called cytochrome P-448 (Pl-450). Today, the induction phenomenon has been shown to be considerably more complex (Snyder and Remmer, 1979; Madhukar and Matsumura, 1981). While investigations into the nature of this system in fish have received much less attention, it is now well established that fish are also capable of increasing microsomal monooxygenase activities as a result of exposure to some of the xenobioties that are known to cause induction in mammals (Pederson et al., 1974; Lidman et al., 1976; Gerhart and Carlson, 1978; Vodicinik et al., 1981). As a result of these investigations, it has been proposed that fish are incapable of responding to inducers of the phenobarbital-type, while 3-methylcholanthrene (3-MC) type compounds have been shown to cause high levels of induction (Ahokas et al., 1977; Ahokas, 1979). These workers also point to the similarity of fish liver systems to that of the P1-450 system of mammals, although 4 cytochrome P—450 spectral maxima are at approximately 449-450 nm both before and after induction. James and Bend (1980) have isolated a partially purified cytochrome P-450 from the sheepshead (a marine teleost) which exhibits an absorbance maximum at 448 nm. That the system behaves in a manner analogous to the P1-450 system of mammals may have important implications concerning the carcinogenic process in these organisms (Ahokas et al., 1977; Ahokas et al., 1979; Nebert and Jensen, 1979). We have begun our studies with the goldfish for several reasons. First, the vast majority of work done on fish has been with coldwater species while there is a lack of data on warmwater species. Second, it has been shown that some of the warmwater fish are known to have relatively high metabolic capabilities. Indeed it has been generally recognized that warmwater fish are less sensitive to chemical pollution (Johnson, 1968). For example, work from this laboratory (Hinz and Matsumura, 197 7) has demonstrated a greater capability of goldfish to degrade 2,5,2L-trichlorobiphenyl over that of rainbow trout or bullhead catfish. Third, goldfish are readily available in many locations even during the winter. In this study we have made an attempt to characterize the general nature of the goldfish monooxygenase system by using B-napthoflavone (a known 3-MC type induca) as a model inducer. Special attention has been paid to compare similarities and differences between the goldfish system to other fish systems and to the mammalian systems. MATERIALS AND METHODS Materials B-napthoflavone (5,6—benzoflavone), aminOpyrine, and p—nitroanisole were purchased from Aldrich Chemical Company, Milwaukee, Wisconsin. 3—Methyl- monomethyl aminoazobenzene (3-MMAB) was kindly supplied by Dr. J. A. Miller, McArdle Laboratories, University of Wisconsin, Madison, Wisconsin. Benzphetamine was a gift from Dr. O'Connor, Upjohn Company, Kalamazoo, Michigan. Ethylmorphine was purchased from E. Merck Company, Rahway, New R 4-dimethylamino-3,5 [14C] xylyl N- Jersey. [140] Mexacarbate (Zectran, methylcarbamate) was provided by the Dow Chemical Company, Midland, Michigan. 7-Ethoxyresorufin was purchased from Pierce Chemical Company, Rockford, Illinois. Piperonyl butoxide was purchased from ICN, K d: K Laboratories, Plainview, New York. Ethyl isocyanide was synthesized in this laboratory according to the method of Casanova et a1. (1963). All biochemicals were purchased from Sigma Chemical Company, St. Louis, Missouri. Chemical structures of the inducers and substrates used in this study are shown in Figure 1. Goldfish (Carassius M, Common Comet variety, 20-75 gm) were initially obtained from a local pet store whereas in later studies they were obtained from the Blue Ridge Fish Hatchery, Inc., Kernersville, North Carolina. Based on levels of cytochrome P-450, levels of 7-ethoxyresorufin induction and mexacarbate binding behavior, no obvious differences between groups were noted. Fish were kept at 21°C 1 1°C for at least one week prior to use. Fish were initially fed Goldfish Food Flakes (Wardley Products Company, Inc., gNHCH3 CH P -NITR)I1NIS(1E VBaco5a¢= : m we once A: 39.3 e 930:3 3320a one—>526 seucaseomoezxofioup «o 5326.: go 3.58 259. .n PSME 9:. 62:. em up we cu gun/e|o:uu ’Mgngpo esolfiqlaeg-o __1 o 9! U!61|O.Id Bun/u 11: £22 Baez a» cooctoam one? Ba .3332: .3 29% a 3239. 53 £23.65 :oflegfioooiencuzeomoahxo:“one Lou 3.50 omcoamoeuomoa .v oSmE 9:9: .300 SN 8— 2: an 1d _ -l :w/elowu. ’Aiyxgpv asoIAqleeq-o l a “i l in “i Sui/u 15 Characterization of Induced Microsomal Monooxyggnase Systems The effect of B-napthoflavone pretreatment on various electron transfer components in the liver microsomes of the goldfish was investigated and the results are shown in Table 2. Under the standard test condition the cytochrome P-450 level increased nearly two-fold, while those for cytochrome b and 5 NADPH cytochrome c reductase were unaffected. A variety of substrates which have previously been shown to indicate the characteristics of induced monooxygenase activities in higher animals has been tested on the B -naphthoflavone-induced liver microsomes of the goldfish. The results shown in Table 3 indicate that 7-ethoxyresorufin—0—deethylation activity, known as an indicator of enzyme activity for 3-MC type inducers (Burke and Mayer, 1974), was induced nearly eight-fold. Oxidative metabolism of mexacarbate, a carbamate insecticide, was also significantly increased. Metabolism of all other substrates was unaffected. The absence of induction of 37MMAB N-demethylase activity is surprising in view of the reputation of this system to be useful in recognizing 3-MC type induction in higher animals (Madhukar and Matsumura, 197 9). . Also shown in Table 3 is the effect of B-naphthoflavone on 6- aminolevulinic acid synthetase (cs-ALAS), the. rate limiting enzyme in heme biosynthesis (Shemin and Russell, 1953). Mammalian induction studies often have shown increases in this enzyme activity apparently reflecting synthesis of new monooxygenase enzymes (Greim et al., 1970). In the case of 'the goldfish system, 5-ALAS activity was unchanged by B—naphthoflavone treatment. To further examine the natm-e of the monooxygenase system of the goldfish, the effect of i_n_ _v_it_rg addition of inhibitors and cofactors on 7- ethoxyresorufin-O-deethylation and benzphetamine-N-demethylation was studied (Table 4). The 0-deethylation activity with 7-ethoxyresorufin was slightly less 16 TABLE 2. The effect of B-napthoflavone pretreatment on various parameters of the hepatic monoxoygenase system of the goldfish (Carassius auratus). Fish received a single 150 mg/kg dose in corn oil and were sacrifia after 96 hours. a b NADPH-CY'I‘ c" CYT P-450 CYT b5 REDUCTASE Control 0.23 i .09 .070 i .02 51.01 i 0.53 Treated 0.43 : .08d .074 i .06 49.86 i 3.55 anmoles P-450/mg protein. Data are expressed as mean : S.E. bnmoles b5/mg protein cnmoles cyt c reduced/min/mg protein dSignificantly greater than control (P < .05) . b 17 TABLE 3. The effect of B-napthoflavone treatment on selected hepatic microsomal monooxygenase enzymes and on G-ALA-synthetase. Enzyme Control Treated 7-ethoxyresorufin-0-deethylase 0.015 1 .006 0.224 1.092“ (nmol resorufin/min/mg protein) itroanisole-O-demethylase 0.313 1 .004 0.358 1 .054 mol p-nitrophenol/min/mg protein) 3-MMAB-N-demethylaseb 0.787 _+_ .266 0.748 1 .109 (nmol HCHO/min/mg protein) Benzphetamine-N-demethylase 0.846 1 .445 0.605 1 .135 (nmol HCHO/ min/ mg protein) Ethylmorphine-N-demethylase 0.214 1 .104 0.257 1 .126 (nmol HCHO/min/mg protein) Aminopyrine-N-demethylase 0.853 1 .265 0.831 1 .211 (nmol HCHO/min/mg protein) Mexacarbatec 4.84 1 2.29 11.20 1 1.77d 5-ALA Synthetase 12.72 1 2.80 9.98 1 2.36 (nmol G-ALA/g liver/hr) aSignificantly greater than control (P < .02). All data are expreued as mean 1 S.E. 3-methyl-4-monomethylaminoazobenzene cradioassay - 96 of metabolites detected via TLC analysis (Benezet and Matsumura, 1974). The N-demethylated product represented approximately 1.8196 and 3.2696 in the control and treated preparations respectively. dSignificantly greater than control (P < .005). 18 .m 033. 3 .83.. 83333 2.38on 635.83% home .952 mm «a wear—own an 830:8 Awe—RE an: mZum «o 35:53.: Page? 2: 09:83 End v3.3.2: 4.560502 a 32.632 5 39.883 E323 95338» $554 5 9:53:00 335:. 55.535 22286 of 3 nonamEoo 33.3% no omeuaoohoa 2: ma communes 3e 33 :ecofia§nm 05 33:8 So: 5353 omecommxoocoe :o 955 :l.. 23.9%: new Eouweumo cocoa no «coho 05. .¢ mam—<9 19 sensitive to carbon monoxide inhibition than was the benzphetamine-N- demethylase activity. 7-ethoxyresorufin activity was completely inhibited by 0.5 mM piperonyl butoxide. 0n the other hand, benzphetamine was less sensitive to the same level of inhibitor. Caution should be used in interpretation of this data since benzphetamine-N-demethylase reaction vessels contained considerably more protein (and hence P-450) and thus may require more piperonyl butoxide for complete inhibition. Addition of 1.67 mM NADH in the presence of one half of the normal NADPH generating system resulted in a significant enhancement of enzyme activities with both substrates. NADH (1.67 mM) alone was unable to support 7-ethyoxyresorufin—0—deethylase, while benzphetamine-N-demethylase was maintained at near normal levels. Spectroscopic Examination of Induced Cytochrome P-450 Figure 5 shows the spectrum of ethyl isocyanide (EtN C) binding to reduced goldfish cytochrome P-450 as a function of pH. In accordance with the observatiom made in previous studies with higher animals (Dahl and Hodgson, 1978), the change in pH brought a profound change in the peak ratio. 0n the other hand, no clear-cut difference in the pH-induced spectral changes was observed between the treated and control cytochromes. It should be noted that with the methods employed here (described in Methods) a marked instability in the ratio of the two peaks was observed as time progressed. It was also noted that pHs above 7.5 caused a shift in the position of the 430 nm peak to the extent that at a pH of 8.0 the peak was shifted to approximately 436 nm. This phenomenon is demonstrated graphically in Figure 6. In order to maintain consistency the absorbance at 430 nm was adopted throughout in plotting the data for Figure 5. Since no discernable differences were observed in binding to the heme iron moiety (as exemplified by EtNC), a suitable type I substrate was chosen to 430 - 49G nm) E I O a (a) a A Absorba nce (455-490 nm 4: Figure 5. 20 6.50 7.00 7.50 8.00 8.50 9.00 pH Ethyl isocyanide binding to sodium dithionite reduced goldfish microsomes as a function of pH from control (A) and B- napthoflavone (0) induced preparations. Fish received the standard 150 mg/kg i.p. dose followed by sacrifice at 96 hours. Other aspects of the assay are described in Methods. Data is expressed as mean 1 S.E. The variability for the treated preparation at pH 8.75 was 1 .003. pH=6.90 0.01 A pH=7.25 pH =7.45 12;;483 pH=8.00 Illllllllllllllllllll oo 4 400 WAVELE NGTHmm Figure 6. Ethyl isocyanide binding to reduced goldfish microsomes showing a gradual shift in the absorbance of the 430 nm peak with increasing pH. 22 examine the effect of B-naphthoflavone induction on binding to the protein moiety of cytochrome P-450. Preliminary tests established that mexacarbate is a suitable monooxygenase substrate for the goldfish system. This substrate has been previously shown to bind in a type I manner (Kulkarni et al., 197 5). Figure 7 demonstrates a typical type I spectrum obtained upon addition of mexacarbate to oxidized goldfish liver microsomes. This binding spectrum may be characterized by a fairly narrow absorption minimum at 420 nm followed by a broad maximum around 387 nm. A double reciprocal plot of the magnitude of the absorbance change as the microsomes were titrated with increasing amounts of mexacarbate is shown in Figure 8. An approximately 2.4-fold increase in the apparent affinity comtant (K299) over the control is evident. It should also be noted that the maximal (i.e., the point at which further addition of substrate does not increase the absorbance change) spectrum from the control was approximately one half that of the treated microsomes. Gel-electrophoresis Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is a valuable tool for examining the constitutive proteins from complex mixtures. Figure 9 shows the electrophoretic pattern of B-naphthoflavone-induced and non- induced goldfish liver microsomes. The appearance of an increased protein band at apparent molecular weight of 56,000 daltons is noted in the treated preparation. This is the general region where hemoprotein(s) P-450 are known to migrate and is also approximately the same molecular weight as the enhanced protein found in rainbow trout after B-napthoflavone induction (Elcombe and Lech, 197 9). An effort to demonstrate the presence of heme in this band region by using peroxidase staining failed. Simultaneous electrophoresis of microsomes from rat liver, however, did reveal peroxidase) activity indicating the marked species difference in this regard. Apparently all of the heme from the goldfish 0.015 HBSORBRNCE 0.005 -00005 23 0.015 Figure 7. 490 j l I I 470 450 480 410 390 370 NRVELENGTHINM] Computer-assisted representation of mexacarbate binding to oxidized goldfish liver microsomes from control (—) and B-napthoflavone induced (---) fish. [Mexacarbate] = 0.11 mM. Microsomal protein concentration was 2.8 mg/ml. 214 6:53.883 8265 :8 2 :3 x m; EB 33:8 :8 E :3 x 8.: 303 A my: 3:338 >552: 3:338 ofi. ._E\mE mfi 3w_533:8:8 530.3 a 3 m3. :3 :33: 5.. 2:. on 5 0:8 83 m5o5m £338.82: .533.» 3 «:35: 33.88me .3 33 38.333 03on .m 0.53.: 3.330985. 25: 8 m. o m? 8.. a... - u u q - -8 2. w 1100 m. 830...... m L W. 3380.4 L8 w a m ,.. -8. m U m -8. 25 Figure 9. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of hepatic microsomes from control (b,c) and B—napthoflavone—treated (d,e) goldfish. Well (a) contained standard proteins with molecular weights of 14,400, 21,500, 31,000, 45,000 (shown), 66,200 (shown) and 92,500. Wells contained 75 u g protein and the resolving gel was 7.596 acrylamide. 26 preparations had dissociated from the microsomes quite easily as peroxidase activity was noted at the migration front of the gel. DISCUSSION The results of this study indicate that goldfish possess a B-napthoflavone inducible monooxygenase system as has been seen in other marine and freshwater species (Elcombe and Lech, 1979; James and Bend, 1980). Dose response studies show a general linear relationship within the dose range tested, while time course experiments demonstrate maximal induction four to five days post- treatment. Cytochrome P-450 levels of the uninduced goldfish were similar to those reported for rainbow trout (Elcombe and Lech, 1979), sheepshead (James and Bend, 1980), lake trout (Ahokas et al., 1977) and the common carp (Guiney et al., 1980). Significant induction, while variable, was readily achieved. Cytochrome b5 levels were found to be generally greater than those reported for rainbow trout or brook trout (Stegeman and Chevion, 1980). Levels of NADPH cytochrome-c—reductase were also uninduced and compare favorably with other species of fish tested (James and Bend, 1980; Stanton and Khan, 1975; James et al., 1979). This enzyme, however, appears to be less active than in most mammalian systems (reviewed by Kato, 1979). The substrates chosen for examination of inductive effects were meant to cover a variety of the types of cytochrome(s) P—450 found in mammalian systems (e.g., Madhukar and Matsumura, 1981). Some interesting differences were noted between goldfish and mammals, particularly with respect to 3-MMAB-N- demethylase and 6-ALA-synthetase. Both of these activities have been found to be inducible by 3-MC type inducers in mammalian systems (Madhukar and 27 28 Matsumura, 1981), while they are not induced in goldfish. Indeed, Madhukar and Matsumura (1979) showed nearly two-fold induction of 3-MMAB-N-demethylase activity following phenobarbital treatment of rats demonstrating that induction of 3-M MAB metabolism is not specific to 3-MC type induction. Curiously, mexacarbate oxidative metabolism, which proceeds primarily through a N- demethylation in our system (see footnote, Table 2), is apparently stimulated by B-napthoflavone induction. This is supported by the increase in the apparent affinity constant (K299) based on mexacarbate binding to the microsomal preparation. Mexacarbate may prove to be a useful substrate when examining the induction of monooxygenase activity in fish. 6 -ALA-synthetase did not increase after B-napthoflavone induction even though SDS - PAGE indicates formation of an increased protein band in the hemoprotein region. There are at least two possible explanations for this: (1) there is a high enough titer of the porphyrine-heme pool to supply the new protein synthesized in the goldfish liver or (2) the increased band in the electrophoresis gel is not of a hemoprotein nature since it was not possible to detect peroxidase activity in this or any other region of the gel. Based on the increases in enzyme activity, binding of substrate and the work of others (Elcombe and Lech, 197 9; Vodicinik et al., 1981), the former explanation appears to be more plausible. The results in Table 4 apparently represent observations previously not investigated in fish liver monooxygenase systems. The synergism seen when NADH is added with NADPH for both of the substrates tested is quite similar to the phenomenon seen in rabbits (Cohen and Estabrook 1971). It is also interesting to note that benzphetamine-N-demethylation is supported by NADH nearly as well as with NADPH. Whether or not these effects are mediated through cytochrome b in fish as they are in mammals (Staudt et al., 1974; 5 29 Kamataki and Kitagawa, 1977) remains to be seen. However, based on other qualitative similarities betwen the two systems, cytochrome b5 involvement would appear to be likely. Ethyl isocyanide binding to reduced microsomes has been used in the past (Sladek and Mannering, 1966; Imai and Siekevitz, 1971) to demonstrate differences in the binding behavior of microsomal preparations before and after induction. Generally an increase in the ratio of the absorbance at 455 and 430 nanometers has been observed after treatment with 3-MC type inducers. This effect has not been observed in rainbow trout (Elcombe and Lech, 1979). In a similar manner, induced rat liver microsomes show a shift in the pH dependent crossover point (the point at which the absorbance of the 455 nm and 430 nm peaks is equal) when compared to uninduced preparations. In contrast, we were unable to demonstrate any significant differences in the nature of the pH dependent binding between control and induced preparations. This observation is consistent with other observations in goldfish liver systems that the characteristies of control and induced preparations are qualitatively very similar. The gradual shift in the absorbance of the 430 nm peak to 436 nm at higher pI-l's is an observation we have not seen described by any other authors. This could be due to a conversion of a portion of the cytochrome(s) P-450 to P-420 which is known to exhibit a single absorbance at 433 nm when complexed with ethyl isocyanide (Imai and Sato, 1967). However, the fact that the shift appears to be gradual as the pH is changed would seem to indicate that other phenomena are involved. Also, Imai and Sato (1967) report that the absorbance of the P- 420—ethyl isocyanide complex is insensitive to changes in pH. Substrate-induced spectral interactions with oxidized microsomes have been seen in virtually every system investigated (reviewed by Schenkman et al., 30 1981). Those substrates that elicit a type I binding spectrum are believed to bind to the apoprotein moiety of the cytochrome P-450 enzyme. In this system the qualitative nature of the mexacarbate-induced type I difference spectrum was similar with both control and treated preparations. The fact that titration with substrate reveals differences in apparent affinity and also in the maximum inducible spectrum could be due to the synthesis of a different form of cytochrome P-450 capable of undergoing a low to high spin transition of the heme iron (Cinti et al., 197 9) at lower substrate concentrations. Schenkman et al. (1981) suggest that this process could lead to an enhanced rate of cytochrome P-450 reduction which in turn would lead to increased rates of metabolism. Alternatively, it could be postulated that a change had occurred in the protein environment of a preexisting cytochrome P-450 enzyme which would affect the nature of the spin equilibrium surrounding that particular cytochrome P—450 enabling the low to high spin transition to occur more readily. Both of these possibilities could manifest themselves as the observed phenomenon. Although SDS - PAGE shows an increased protein band of approximately 56,000 MW in microsomes from B-napthoflavone-treated fish, this alone is not sufficient to distinguish between the two possibilities. Other investigations (Vodicinik et al., 1981; Bend et al., 1973; Addison et al., 197 7) have demonstrated that fish are apparently refractive to inducers other than the 3-MC type. Apparently goldfish are also incapable of responding to mirex at 100 mg/kg. 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