changes in three hepatic cytochrome p450 subfamilies during a reproductive cycle in turbot...

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 277:313–325 (1997)

© 1997 WILEY-LISS, INC.

JEZ 753

Changes in Three Hepatic Cytochrome P450Subfamilies During a Reproductive Cycle in Turbot(Scophthalmus maximus L.)

AUGUSTINE ARUKWE* AND ANDERS GOKSØYRLaboratory of Marine Molecular Biology, Department of Molecular Biology,University of Bergen, N-5020 Bergen, Norway

ABSTRACT Sexual differentiation of the hepatic cytochrome P450 system was characterizedin 2-year-old farmed turbot (Scophthalmus maximus L.) during their first spawning period (Janu-ary–September). The fish were kept in tanks supplied with continuously flowing seawater (34.5‰,ppt) at a constant temperature of 16°C and natural photoperiod (60°N). Sampling of liver samples(n = 4–6) was performed once every month for 9 months. Pronounced sex differences were re-corded in the activities of 7-ethoxyresorufin O-deethylase (EROD), 7-ethoxycoumarin O-deethylase(ECOD), and NADPH-cytochrome P450 reductase during spawning (May–July). EROD activity infemale fish decreased gradually towards the onset of ovulation in May–July to rise again in thepostspawning period. The decrease correlated with increasing gonadosomatic index and estradiol-17β (E2) levels in plasma. Immunochemical detection of CYP1A (58 kDa), CYP2K-like (47 and 52kDa), and CYP3A-like (58 and 60 kDa) proteins in Western blotting, and ELISA showed higherprotein levels in male compared to female fish from April/May–June, and significant differenceswere observed in June (CYP2K-like also in April and May). Analysis of monthly variations withinsexes during the reproductive cycle shows significant monthly changes in all parameters in bothfemale and male fish. Both CYP2K- and CYP3A-like protein levels were significantly elevated inmale fish during spawning in June. To study the induction response during spawning, β-naphthoflavone (BNF) 75 mg/kg body weight) was administered intraperitoneally to both sexes inJune. BNF caused a significant increase in EROD and ECOD activities and CYP1A protein levelsbut had no effect on NADPH-cytochrome P450 reductase activity or CYP2K-like/CYP3A-likeproteins. This study documents, for the first time in any fish species or lower vertebrate, thesexual differentiation in the liver of three different CYP subfamilies during sexual matura-tion and spawning. J. Exp. Zool. 277:313–325, 1997. © 1997 Wiley-Liss, Inc.

The cytochrome P450 (CYP or P450)1-dependentmonooxygenase system plays a central role in theoxidative metabolism or biotransformation of awide range of foreign compounds (xenobiotics), in-cluding environmental pollutants, drugs, and an-tibiotics and endogenous compounds such assteroids, bile acids, fatty acids, and prostaglan-dins (reviewed by Goksøyr and Förlin, ’92; Stege-man and Hahn, ’94). In addition, certain steps inthe biotransformation pathway are responsible forthe activation of foreign compounds to the reac-tive intermediates that ultimately result in toxic-ity, carcinogenicity, and other adverse effects (seeGuengerich, ’87; Nebert and Gonzalez, ’87; Var-anasi, ’89; Stegeman and Hahn, ’94). Several fac-tors (biotic and abiotic) are known to influencethe hepatic cytochrome P450 monooxygenase sys-tem in fish. These include sex, reproductive sta-tus and steroid levels (Förlin and Hansson, ’82;Stegeman et al., ’82; Andersson, ’90; Förlin and

Haux, ’90; Larsen et al., ’92), changes in seasonand temperature (Lindström-Seppä, ’85; Snegaroffand Bach, ’90; Lange et al., ’94; Sleiderink et al.,’95), and exposure to certain types of environmen-tal pollutants (Goksøyr and Förlin, ’92; Stegemanand Hahn, ’94; Goksøyr, ’95).

In mammals and fish, several constitutive cyto-chrome P450 forms are developmentally regulatedand display sex-specific expression (Andersson,’92a). In fish species that exhibit a strong sex dif-ference, the similarities between juvenile and re-productively active males and females have led

*Correspondence to: A. Arukwe, Laboratory of Marine MolecularBiology, Department of Molecular Biology, University of Bergen, HIB,N-5020 Bergen, Norway.

Received 12 June 1996; Revision accepted 29 October 1996Parts of this work were presented at the Fifteenth Annual Confer-

ence of European Society for Comparative Physiology and Biochem-istry (ESCPB) in Genova, Italy, 20–23 September, 1994.

1The recommended nomenclature of Nebert et al. (’91) is followedin this report.

314 A. ARUKWE AND A. GOKSØYR

to suggestions that the sex differences are due toa suppression of cytochrome P450 isoenzyme ex-pression in reproductively active females (Stege-man and Hahn, ’94).

The aim of this study was to characterize sexdifferences in the hepatic cytochrome P450 sys-tem of turbot during sexual maturation, spawn-ing, and postspawning periods. We also wished tocharacterize the induction response of the enzymesystem at the peak of spawning in June whenxenobiotic metabolizing enzymes are possiblyturned off or inhibited by endogenous substances.We have used immunological and catalytic assaymethods. Three different fish CYP antibodies,cross-reacting with isozymes in different CYP sub-families (CYP1A, CYP2K, CYP3A) were used si-multaneously for the first time in such a studywith fish or any lower vertebrate. Only a few stud-ies of this enzyme system have previously beenperformed in turbot. These include studies ofCYP1A induction and temperature adaptation injuvenile stages of turbot (Peters and Livingstone,’95; Skjegstad and Goksøyr, unpublished results).

The interest in turbot as an aquaculture spe-cies has progressed from research to commercialpractice throughout Europe during the past 20years. However, the problem of achieving repro-duction in captivity has been difficult with thisbatch-spawning species. This may be explainedby poor knowledge about the reproductive endo-crinology and biochemistry of this species. Under-standing the changes occurring in the sexualmaturation of turbot might point to important fac-tors in the molecular biology of turbot reproduction.The results from this study can also contribute tothe necessary pharmacological background for che-motherapeutic and antibiotic treatments againstturbot infections and for evaluation of effectivedoses at different life stages. Furthermore, the re-sults can contribute to the background knowledgeof factors influencing the CYP1A induction re-sponse when used as a biomarker in wild orfarmed turbot in the monitoring of environmen-tal pollution.

MATERIALS AND METHODS

Fish and samplingThe turbot used in this study were produced

from eggs of one female and sperm of two maleturbots that were pooled and incubated on July7, 1991, at Selvåg Fisk A/S at Os, near Bergen,Norway. The fish were brought to the IndustrialLaboratory (ILAB) at the High Technology Cen-

ter in Bergen (HIB) as juveniles on October 28,1991, and kept in indoor circular 7,000 l tanks.Genetically, they are a rather homogeneous group,being either siblings or half-siblings. A total of 110prespawning turbot from this group was used inthis study. They were supplied with continuouslyflowing seawater (34.5‰, ppt) at a temperatureof 16°C (±0.2°C). The daily photoperiod was ad-justed to follow the natural photoperiod in Ber-gen (60°N). The animals were fed commercial drypellets (Flatfiskfôr from Felleskjøpet A/S, Bergen,Norway) daily through an automatic feeder. Theexperimental fish were expected to spawn inMay–August 1993 at the age of 2 years, with nor-mally six to ten batches of eggs with intervals ofabout 70–80 h between batches. Sampling wasdone at monthly intervals from January to Sep-tember 1993 to cover the prespawning, spawning,and postspawning periods. Sampling for the wholeperiod was by simple random sampling. Ten tur-bot (n = 4–6 of each sex) were randomly selectedand anaesthetized with metacaine (50 mg in 1 lseawater) before sacrifice. This was done becauseof other studies, such as blood sampling, that wasperformed on the same fish material and partlyto allow for easy gender differentiation, especiallyduring the prespawning period. The liver and go-nad were excised and weighed in order to calculatethe liver somatic index (LSI) and gonadosomatic in-dex (GSI) (LSI and GSI = organ weight × 100/bodyweight). The liver was placed in ice-cold 0.1 M Na-phosphate buffer (pH 7.4) with 0.15 M KCl.

Chemicals7-ethoxyresorufin (7-ER), 7-ethoxycoumarin (7-

EC), 7-hydroxycoumarin (Umbelliferone), NADPH,β-naphthoflavone (BNF), o-phenylenediamine di-hydrochloride (OPD), N,N,´,N´-tetramethyl-eth-ylenediamine (TEMED), cytochrome c, and 4-chloro-1-naphthol were purchased from SigmaChemical Co. (St. Louis, MO). Resorufin wasfrom Fluka Chemika-BioChemika (Buchs, Swit-zerland). Equipment, other chemicals (goatanti-rabbit-horseradish peroxidase [GAR-HRP])for Western blotting, and ELISA were pur-chased from Bio-Rad (Hercules, CA). Microtiterplates (MaxiSorp) were purchased from Nunc(Roskilde, Denmark). All other chemicals wereof the highest commercially available grade.

InductionThe induction was performed on a single day

on June 25, 1993, and fish were killed 5 days later.The fish used were from the same group as above.

SEX DIFFERENCES IN TURBOT HEPATIC P450 ISOZYMES 315

Twenty fish were randomly selected from the ex-perimental tank (ten of each sex). Each sex groupwas further divided into two subgroups, half ofwhich was treated with BNF (a model poly-aromatic hydrocarbon (PAH)-type inducer) andhalf acting as a control group. All the subgroupswere kept in separate 150 l tanks. All environ-mental conditions remained unchanged. BNF wasdissolved and sonicated in soyabean oil (15 mgBNF/ml). Administration was by intraperitonealinjection at a single dose of 75 mg/kg body weight.The animals were starved during the 5 day ex-perimental period. The fish were sacrificed by aflow to the head, and liver was extracted immedi-ately and placed in ice-cold 0.1 M Na-phosphatebuffer (pH 7.4) with 0.15 M KCl.

Preparation of microsomesMicrosomes were prepared by differential ultra-

centrifugation as described by Goksøyr and Larsen(’91). The liver and gonad were extracted imme-diately after anaesthesia (general experiment) orsacrifice (induction experiment) and weighed. Twograms of liver sample was homogenized in 1:4 ra-tio of liver weight and volume of 0.1 M Na-phos-phate buffer (pH 7.4) with 0.15 M KCl andcentrifuged at 12,000g for 20 min at 4°C. Thepostmitochondrial supernatant was recentrifugedat 100,000g for 60 min at 4°C. The microsome pel-let was resuspended in 0.1 M Na-phosphate buffer(pH 7.6) with 1 mM EDTA, 1 mM DTT, and 20%glycerol and stored in aliquots at –80°C untilanalysis.

Enzyme assaysThe NADPH–cytochrome P450 reductase activ-

ity was determined with cytochrome c as a specificelectron acceptor. The activity was determined spec-trophotometrically with an Ultrospec II (LKBBiochrom, Cambridge, UK) spectrophotometer atroom temperature as described previously (Gok-søyr and Larsen, ’91). The reaction was followedas an increase in absorption per time unit at 550nm. The 7-ethoxyresorufin O-deethylase (EROD)activity was measured at room temperature witha Perkin-Elmer (Buckinghamshire, UK) LS-5 lu-minescence spectrofluorometer as described pre-viously (Larsen et al., ’92). The analysis wascalibrated with an addition of a known amount ofresorufin as the internal standard to each sample,using the extinction coefficient of 73.2 mM–1cm–1

at 572 nm (Klotz et al., ’84). The activity of 7-ethoxycoumarin O-deethylase (ECOD) was alsodetermined fluorometrically at excitation and

emission wavelengths of 400 and 460 nm, respec-tively (Goksøyr and Larsen, ’91). Umbelliferone(20 mM in methanol) was used as an internalstandard. The activity was assayed at room tem-perature with a Perkin-Elmer LS-5 luminescencespectrofluorometer. As a quality control, twoknown samples were assayed in parallel with allassay series in order to assure the consistency ofthe results obtained with unknown samples. Op-timal buffer conditions for EROD and ECOD ac-tivities were determined in prespawning turbot(January), using pH gradients of 0.1 M Tris-HClbuffer and 0.1 M Na-phosphate buffer. The opti-mal buffer conditions for NADPH–cytochromeP450 reductase, EROD, and ECOD activities weredetermined to be pH 7.5, 7.8, and 7.8, respectively,with 0.1 M Na-phosphate buffer.

Protein and immunochemical studiesThe total amount of microsomal protein was de-

termined with the method of Bradford (’76), withbovine serum albumin (BSA) as standard. Mea-surements were conducted with a Titertek Multi-scan Plus MKII (Flow Laboratories, Costa Mesa,CA) for absorbance reading. In Western blotting,turbot microsomal proteins were separated using9% polyacrylamide gels containing sodium dodecylsulfate (SDS) (Laemmli, ’70) and electroblottedfrom gels onto nitrocellulose filters as describedby Towbin et al. (’79). The indirect ELISA wasperformed essentially as described (Goksøyr, ’91)with the modifications of Husøy et al. (’94). Cross-reactions of individual hepatic microsomal pro-teins were probed with the following anti-fish CYPantibodies: polyclonal rabbit and monoclonalmouse anti-cod CYP1A-IgG (Goksøyr, ’85; Goksøyret al., ’91a; Husøy et al., ’96), rabbit anti-rainbowtrout CYPKM2-IgG2 (Andersson, ’92a), and rab-bit anti-rainbow trout P450con serum3 (Celanderet al., ’89a) in Western blotting and indirectELISA. Horseradish-peroxidase conjugated goatanti-rabbit and anti-mouse IgG (GAR- and GAM-HRP) (Bio-Rad) were used, respectively, as sec-ondary antibody in all cases.

Estradiol-17b assayEstradiol-17β was extracted from plasma by 4:1

diethylether and n-heptane and subjected to ra-

2Available evidence suggests that CYPKM2 = CYP2K and P450con= CYP3A, but none of these proteins have been sequenced so far inturbot. Given the use of CYPKM2 and P450con antibodies and theirrecognition of turbot proteins in this study, we have referred to theimmunoreactive proteins to these antibodies as CYP2K- and CYP3A-like proteins, respectively.

3See footnote 2.


dioimmunoassay (RIA) measurement. The assaywas performed using specific antibodies. Antise-rum and isotopes were obtained from ICN Im-muno Biologicals (Lilse, IL) and New EnglandNuclear (Boston, MA) respectively. The cross-re-activity of the RIA with other steroids is reportedby ICN as follows: estrone, 4%; estriol, 0.3%; es-tradiol-17α, 5%; 6α-hydroxy-estradiol-17β, 100%;6-keto-estradiol-17β, 100%; 17α-ethynyl-estradiol-17β, 0.3%; equilenin, 1%; testosterone, <0.1%;progesterone, <0.1% and equilin, 1%.

Statistical methodsAll data were log-transformed before statis-

tical analysis to achieve variance homogeneity(Zar, ’84). The monthly sex differences wereanalyzed with one-way analysis of variance(one-way ANOVA), and monthly variationswithin sexes were analyzed using the Tukey-Kramer test. The effects of BNF treatment wereanalyzed by a standard Student’s t-test betweencorresponding control and BNF groups. Corre-lations between variables were analyzed foreach sex group using linear regression onscatterplots. For all the tests, the level of signifi-cance was set at P ≤ 0.05 unless otherwise stated.Statistical analyses were performed using theJMP software (version 3.1.6) for Statistical Visu-alization (SAS Institute, Cary, NC) on an AppleMacintosh.

RESULTSDuring the prespawning period (January, Feb-

ruary, and March), the LSI was higher in malefish compared with female fish, with a fallingtrend through spawning (May–June) to the post-spawning period (August–September) (Fig. 1A).The female fish, in contrast, had a lower LSI inthe prespawning period, which increased slightlyand peaked in May (early spawning period) withabout 2.8% of total body weight. After this peak,the female fish LSI value fell to less than 2%from June to September. A gradual increase wasobserved in the GSI of female fish from Janu-ary to June, when the highest value of morethan 10% of total body weight was recorded(Fig. 1B). After this peak, the GSI value fellsignificantly to less than 5% of total bodyweight from July to the end of the samplingperiod in September (postspawning period). TheGSI in male fish, in contrast, did not increase sig-nificantly but showed high individual variationsfrom May–August, as evidenced with high stan-dard deviations (Fig. 1C). ANOVA analysis of

pooled LSI and GSI data shows significant sexdifferences in both parameters during the repro-ductive period (P < 0.01; results not shown). Analy-sis of female fish GSI and LSI shows significant(P < 0.001) monthly variations during the repro-ductive cycle. Pair-wise comparison shows thatJune GSI in female fish is significantly different(P < 0.001) from the rest of the months (exceptMay). No such differences were observed in malefish GSI.

The monthly variations in the yield of hepaticmicrosomal protein after subcellular fractionationindicate higher microsomal protein synthesis infemale turbot compared with male turbot through-out the reproductive period, with a distinct peakat the start of spawning (June) (Fig. 2A). WithNADPH–cytochrome P450 reductase, a higher ac-tivity was observed in male fish compared withthe female fish, with a maximum activity in Au-gust (Fig. 2B). There was a significant sex differ-ence from the last part of the prespawning period(April/May), through spawning (June/July) to thebeginning of the postspawning period (August).Activities tended to rise in female fish during thepostspawning period, contrary to the male fish,which showed a falling trend. There was pro-nounced monthly variation in the EROD activi-ties of male and female turbot (Fig. 2C). In femalefish, maximum activity was observed in January,falling below the lowest detectable limit of 0.5pmol/min/mg protein in some individual fish inJune (spawning period), and rising again fromJuly–September (postspawning period). Plasmalevels of estradiol-17β (E2) of female turbot in-creased gradually from January towards spawn-ing in June, when the maximum level wasrecorded (data from G.L. Grung presented inFig. 2E). Thereafter, a fall in mean plasma E2levels of female fish was observed during thepostspawning period. Female fish EROD activ-ity correlated negatively with GSI and plasmaE2 levels in individual fish (r = –0.7 and –0.8,respectively; P < 0.001) (Table 1).

There was a significant difference between fe-male and male fish EROD activity during thespawning period (May–July), with males showinghigher EROD activity than females (see Table 1for comparisons of P values and correlation coef-ficients of male and female turbot parameters dur-ing the reproductive cycle). EROD activity showeda high range of individual variation during thereproductive period (female plasma E2 levels inJune), as evidenced by relatively high standarddeviations. The pattern in ECOD activity showed


Fig. 1. Changes in different biological parameters of tur-bot during a reproductive cycle. Data are given as mean val-ues ± SD. **Female fish (2) significantly different from malefish (7) (P < 0.01). *Female fish significantly different from

male fish (P < 0.05). A: Liver somatic index (LSI). B:Gonadosomatic index (GSI). C: GSI of individual male turbotduring the reproductive cycle.

a higher activity in male fish, with a peak in Janu-ary and September, compared with female fish,where the activity was below the lowest detect-able limit of 0.5 pmol/min/mg protein in someindividual fish, especially in June (Fig. 2D). Asignificant sex difference in ECOD activity

(males higher than females) was recorded dur-ing the spawning and the beginning of thepostspawning periods (June–August). However,there were pronounced individual variations(more so in males than in females), as evidencedby the high standard deviations. An ANOVA test


Fig. 2. Changes in hepatic microsomal protein yield,NADPH–cytochrome P450 reductase, and different P450 ac-tivities during the reproductive cycle of turbot. Data are givenas mean values ± SD. Hepatic microsomal protein yield pergram of liver (A), NADPH–cytochrome P450 reductase activ-ity (nmol/min/mg protein) (B), 7-ethoxyresorufin O-deethylase(EROD activity, pmol/min/mg protein) (C), 7-ethoxycoumarinO-deethylase (ECOD activity, pmol/min/mg protein) (D), andEstradiol-17β (E2, ng/ml [data supplied by G.L. Grung]) (E)levels in plasma of individual female turbot during the re-productive cycle (for explanation of symbols, see legend toFig. 1). #, samples were lost accidentally.


of pooled NADPH–cytochrome P450 reductase,EROD, and ECOD data from the whole study pe-riod showed significant sex differences in all pa-rameters (P < 0.01; results not shown) at allsampling points during the annual cycle. Analy-sis of monthly variations within sexes during thereproductive cycle show significant monthlychanges (P < 0.001) in EROD, ECOD, and reduc-tase activities in both female and male fish. Gen-erally, these activities were significantly reducedin female fish during spawning (May–June) andthe early postspawning period (July–August).

The levels of the three P450 isozymes (i.e., theCYP1A, CYP2K-like, and CYP3A-like immunore-active proteins) as determined by indirect ELISAwere higher in males compared with females dur-ing the prespawning/spawning (April/May–June)period for all isozymes (Fig. 3A,B,C, respectively).Sex differences were recorded in all three param-eters during spawning in June, with males show-ing higher absorbance values than females. InWestern blotting, the CYPKM2 antibody cross-re-acted with a double protein band of 47 and 52kDa in turbot liver microsomes (Fig. 4A), whilethe P450con antibody cross-reacted with twoprotein bands of 58 and 60 kDa (Fig. 4B). Thecross-reacting CYP2K- and CYP3A-like proteinsshowed higher protein levels in males comparedto females in June (Fig. 4A,B), thus comple-menting the indirect ELISA results. Analysisof monthly variations within sexes during thereproductive cycle shows significant monthlychanges (P < 0.001) in both female and malefish. Both CYP2K- and CYP3A-like protein lev-els were significantly elevated in male fish dur-ing spawning in June.

Response to b-naphthoflavone (BNF)treatment

Treatment of spawning female and male turbotwith the model PAH-type CYP1A inducer, BNF,did not cause any change in the electron trans-port system of turbot as determined by NADPH–cytochrome P450 reductase. EROD and ECODcatalytic activities, however, showed a significantincrease after BNF treatment. The EROD activ-ity increased 29- and 44-fold in male and femaleturbot, respectively, while the ECOD activity in-creased three- and fourfold in male and femaleturbot, respectively. In all cases activity was com-pared with the corresponding control group (Table2). In Western blotting, monoclonal anti-codCYP1A-IgG (MAb-NP7) (Husøy et al., ’96) wasused to demonstrate the presence of immuno-reacting protein (Mr = 58 kDa) in both male andfemale BNF-treated turbot (Fig. 5). The 58 kDaprotein band was not detectable in the corre-sponding control group. Analysis of induction re-sponse using indirect ELISA with a polyclonalantibody showed an absorbance increase of three-and twofold in male and female turbot, respec-tively, when compared with the correspondingcontrol group.

DISCUSSION

Marked sex differences in all the parametersstudied during spawning in June demonstratethat CYP isoenzyme patterns differ between maleand female turbot. The results also revealed astrong induction response to BNF treatment atthe peak of maturation (June) in both male andfemale turbot despite the sex differences observed

TABLE 1. Results from regression analyses of different parameters (log-transformed) measured in individual male andfemale turbot during the reproductive cycle1

Correlation coefficient (P value)Male Female

EROD vs. GSI –0.3 (ns) –0.7 (<0.001)ECOD vs. GSI –0.3 (ns) –0.06 (<0.001)EROD vs. estradiol-17β2 — –0.8 (<0.001)ECOD vs. estradiol-17β — –0.5 (<0.001)Estradiol-17β vs. GSI — 0.6 (<0.001)EROD vs. CYP1A-ELISA 0.1 (ns) 0.4 (<0.01)EROD vs. NADPH-reductase 0.4(<0.01) 0.6 (<0.001)Estradiol-17β vs. CYP3A-ELISA — –0.6 (<0.001)Estradiol-17β vs. CYP2K-ELISA — –0.4 (<0.01)GSI vs. CYP2K-ELISA 0.1 (ns) –0.5 (<0.001)GSI vs. CYP3A-ELISA –0.1 (ns) –0.6 (<0.001)Prot./g liver vs. CYP3A-ELISA 0.5(<0.001) –0.3 (<0.01)1NADPH-reductase, NADPH-cytochrome P450 reductase, ns; not significant.2Data from G.L. Grung et al. (manuscript in preparation).


Fig. 3. Immunochemical analysis of turbot hepatic CYPprotein levels during the reproductive cycle, using indirectELISA. Data are presented as in previous figures. ELISA ab-

sorbances (492 nm) of CYP1A (A), CYP2K-like (B), andCYP3A-like (C) immunoreactive protein levels.

during spawning. In addition, monthly variationswithin sexes in all parameters during the repro-ductive cycle show significant changes (P < 0.001)in all parameters in female and in most male fishparameters. The CYP isozyme patterns deter-mined immunochemically showed significant el-

evation of CYP2K- and CYP3A-like proteins inmale fish during spawning in June. Generally,these results are in accordance with the demon-strations of others that sex-dependent changes oc-cur in fish cytochrome P450 system during sexualmaturation and spawning (e.g., Förlin and Haux,


’90; Pajor et al., ’91; Larsen et al., ’92). However,this is the first time this concept is validated im-munochemically for isozymes in three differentCYP subfamilies simultaneously in fish during aspawning cycle.

The female fish LSI reached a maximum in May.The rise and fall of the female fish LSI is sug-gested to be a result of increased hepatic synthe-sis of vitellogenin (reviewed by Mommsen andWalsh, ’88; Norberg, ’89) and zona radiata pro-teins (zrp) (Oppen-Berntsen et al., ’92, ’94), al-though these parameters were not studied inthe present investigation. The growth of the oo-cytes is manifested in the strong increase of thegonadosomatic index, which reached a maximumin June with values ranging from 8–25% of totalbody weight. After this maximum, the exact go-

nadal weight was difficult to obtain, partly due tobatch ovulation. However, the rather large stan-dard deviations from late May to late August seemto suggest that most of the females started ovula-tion in May, with some differences in the timingof ovulation cycles. Nevertheless, it should benoted that a constant temperature of 16°C maybe suboptimal for turbot during the spawning sea-son and might disturb the individual ovulatorycycles (Howell and Scott, ’88). The male GSI wasmuch smaller than the female, with a maximumof 1.3% of total body weight.

The observed variations in NADPH–cytochromeP450 reductase activity correspond with whatLarsen et al. (’92) reported in salmon, where theNADPH-reductase activity decreased from theprespawning (sexual maturation) to spawningperiod. The variations in the female NADPH-reductase activity correlates positively with thevariations in female EROD activity (r = 0.6; P< 0.001). Greater contents of liver microsomalP450, b5, and NADPH–P450 reductase activi-ties in mature males compared to females haveearlier been reported in a number of species(Stegeman and Chevion, ’80; Koivusaari et al., ’81;Mckee et al., ’83; Stegeman and Woodin, ’84;Lindström-Seppä, ’85; Larsen et al., ’92).

EROD activity showed a dramatic fall in femaleturbot during the spawning period, displaying asignificant sex difference from May to July. Themean value for the EROD activity was very low(2.1 pmol/min/mg protein) in female turbot in Mayand June. Plasma levels of the single sex steroidanalysed in this study in female fish, estradiol-17β (E2), peaked during spawning in June (lowEROD). Marked decreases in monooxygenase ac-tivities and cytochrome P450 contents of juvenilebrook trout (Salvelinus fontinalis) and rainbowtrout (Salmo gairdnerii) treated with estradiolhave earlier been reported (Stegeman et al., ’82;Hansson et al., ’82). Our results suggest that theindividual variation and, in general, the monthlyfluctuations in the EROD values during the

Fig. 4. Western blots of turbot hepatic microsomes probedwith anti-rainbow trout CYPKM2 (CYP2K-like) IgG (A) oranti-rainbow trout P450con (CYP3A-like) serum (B). Individu-als are sampled in June (representative for the spawning pe-riod). Proteins were separated using 9% sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) be-fore blotting. Lanes 1–4: Females. Lanes 5–10: Males. Therelative molecular mass of cross-reacting proteins (47 and 52and 58 and 60 kDa) are indicated in panels A and B, respec-tively. Microsomal protein (20 µg) was applied in each well.std, = prestained molecular weight standards (Bio-Rad).

TABLE 2. Effects of β-naphthoflavone (BNF) treatment on the hepatic cytochrome P450 system of spawning turbot1

Control BNF-TreatedMale Female Male Female

EROD 21 ± 15 9 ± 6 606 ± 96* 476 ± 227*ECOD 22 ± 7 12 ± 5 59 ± 10* 54 ± 25*NADPH-reductase 28 ± 3 20 ± 5 24 ± 6 21 ± 6CYP1A-ELISA 0.2 ± 0.02 0.3 ± 0.1 0.6 ± 0.1* 0.4 ± 0.1*1For explanation of abbreviations, see Table 1. EROD and ECOD activities (pmol/min/mg protein), NADPH-reductase activity (nmol/min/mgprotein), CYP1A = ELISA (absorbance at 492 nm).*BNF = treated significantly different from corresponding control group (p < 0.001, n = 5 in all cases).


spawning period reflect the fluctuations in plasmasteroid (E2) levels of batch-spawning turbot. Onthe contrary, other species (single spawners) showa continuous fall in EROD activity as steroid lev-els increase prior to spawning (e.g., Larsen et al.,’92; Förlin and Haux, ’90). The basal levels of tur-bot hepatic microsomal EROD activity were verymuch lower than those reported in salmon (Larsenet al., ’92). The CYP1A protein levels determinedby ELISA indicate that the protein was presentalso in female turbot when EROD activity wasabsent. The CYP1A-ELISA absorbance has a lowcorrelation with EROD activity in male but cor-relates fairly well in female fish (see Table 1). Thismight be explained as a result of lower individualvariations (low standard deviations) found inCYP1A absorbance compared with EROD activ-ity. Furthermore, catalytic activities can be inhib-ited by sex steroids (E2) (Gray et al., ’91) or otherendogenous and exogenous inhibitors of enzymeactivity. Our results are consistent with the re-sults of Larsen et al. (’92), who showed that ERODactivity was inhibited during spawning in salmon,while CYP1A-ELISA absorbance revealed thepresence of CYP1A proteins.

There was a wide range of individual variationin the male ECOD activity, as evidenced by thelarge standard deviations. The observed ECODactivity corresponds to the finding of Koivusaariet al. (’81), where the activities of ECOD werehigher in male rainbow trout than female butfailed to reach a statistical significance until closeto spawning time in March. However, the modestvariation and high standard deviation observedin the ECOD activity during the reproductive pe-

Fig. 5. Effects of BNF treatment on CYP1A protein ex-pression in sexually mature turbot liver microsomes probedwith monoclonal anti-cod CYP1A-IgG (MAb-NP7). Proteinswere separated using 9% sodium dodecyl sulfate–polyacryla-mide gel electrophoresis (SDS-PAGE) before blotting. Lane1: BNF-treated cod. Lane 2: Control cod. Lanes 3–4: Con-trol female turbot. Lanes 5–7: BNF-treated female turbot.Lanes 8, 9: Control male turbot. Lanes 10–12: BNF-treatedmale turbot. Microsomal protein (20 µg) in 10 µl was appliedper well. Note that the BNF-treated individual in lane 5 dis-played lower EROD activity than the other BNF-treated fish(89 pmol/min/mg protein vs. 460–749 pmol/min/mg protein),as reflected in the staining intensity.

riod might be explained as a result of the earlierobservation that ECOD activity exhibits a bipha-sic kinetics in liver microsomes from untreatedrainbow trout (Celander et al., ’89b). In mammals(Ullrich and Weber, ’72; Greenlee and Poland, ’78;Guengerich, ’78; Snegaroff, ’82; Boobis et al., ’86)and fish (Klotz et al., ’86), this is believed to bedue to the presence of more than one form of cy-tochrome P450 that catalyzes the O-deethylationof 7-ethoxycoumarin.

There are some controversial results availableon the sex differences in the levels of cytochromeP450–dependent monoxygenase activities in fish.In some studies no such differences have been re-ported (Buhler and Rasmusson, ’68; Dewaide andHenderson, ’70), while a number of more recentreports did find higher levels of cytochrome P450and higher activities of several monoxygenase re-actions in males of different fish species (Förlin,’80; Stegeman and Chevion, ’80; Hansson et al.,’82; Stegeman et al., ’82; Lindström-Seppä, ’85;Williams et al., ’86; Förlin and Haux, ’90; Larsenet al., ’92; Elskus et al., ’92; George et al., ’90;Tarlebø et al., ’85; Addison and Willis, ’82). Theresults from this study support the concept thata sexual differentiation of the hepatic microsomalcytochrome P450 monooxygenase activities occursin late stages of sexual maturation in fish. In ad-dition, our results also indicate that estradiol-17β(E2) or other sex steroids may affect the catalyticactivity of P450 isoenzymes directly. In our labo-ratory, it has been shown that estradiol is a di-rect inhibitor of microsomal EROD activity in vitro(S.O. Olsen, unpublished results). It is most likelythat such in situ regulation of enzyme activitymay be of critical importance in the signal trans-duction processes that regulate maturation andspawning in fish.

The changes in male CYP2K-like cross-react-ing proteins observed in this study in both ELISAand Western blotting are in accordance with theresults of Andersson (’92b), which demonstratedthat 11-ketotestosterone (one of the spermatogen-esis regulatory hormones) induces CYP2K-likeproteins in juvenile rainbow trout trunk kidney.There was a negative correlation between GSIand CYP2K-like protein levels in individual fe-male turbot during the reproductive cycle (r =–0.5; P < 0.001) (Table 1). In males, a nonsig-nificant positive correlation between GSI andCYP2K-like protein levels was observed (r = 0.1)(Table 1). The CYP2K-like cross-reacting proteinsin Western blotting showed a continuous doubleprotein band pattern (47 and 52 kDa) in both


sexes from April to July. These two protein bandswere much stronger in male than in female tur-bot in May and June compared with the rest ofthe period. It has been suggested that CYPLMC2(LM2) and KM2 could be similar proteins bothbelonging to the CYP2K subfamily because oftheir metabolism of progesterone at the 16α po-sition (Stegeman, ’93).

The CYP3A-like protein levels determined withELISA showed that the protein was present athigher levels in male than in female turbot dur-ing spawning in June, where a significant sex dif-ference was observed. The variations in femaleturbot CYP3A-like cross-reacting protein levelscorrelated negatively with GSI during the repro-ductive cycle (r = –0.6; P < 0.001). Larsen et al.(’92), in accordance with this result, recorded afall in female salmon CYP3A-like protein levelsdetermined by ELISA during an annual reproduc-tive cycle. In males, a nonsignificant negative cor-relation between GSI and CYP3A-like proteinlevels was observed (r = 0.1) during the repro-ductive cycle. Cross-reactivity of turbot liver mi-crosomes to anti-rainbow trout CYP3A-likeprotein showed several protein bands of differ-ent molecular weight in Western blotting, but twoof the bands (58 and 60 kDa) were persistent inall samples. In June, there was a sex differencein these two protein bands, with stronger inten-sity in males than in females, thus demonstrat-ing the differentiated regulation of CYP3A-likeproteins during the late stages of the reproduc-tive cycle. The sex differences observed in theCYP3A-like proteins in this study are consistentwith earlier description of the fish CYP3A-like pro-teins as the major catalyst of 6β-hydroxylation ofsteroid hormones such as progesterone and tes-tosterone (Klotz et al., ’86; Miranda et al., ’89, ’91).This suggests that the 6β-hydroxylation of thesesteroid hormones by CYP3A may occur generallyin fish (Pajor et al., ’91) but is more pronouncedin spawning males. The remarkable sex differ-ences observed in the CYP2K-like and CYP3A-like proteins in June suggest also that fishprobably follow the general vertebrate pattern oftestosterone and progesterone metabolism by cy-tochrome P450 isoenzymes (Cheng and Schenk-man, ’83), but further studies will be needed toidentify the specific roles of these isoenzymes inturbot sexual maturation.

The immunochemical data from this study dem-onstrate that, at least in turbot, some of the sexdifferences observed during spawning are due toincreased levels of CYP isozymes in males rather

than suppression of the levels in females, as haspreviously been the accepted concept (Stegemanand Hahn, ’94).

In this study, induction of CYP1A protein wasapparent even at the peak of sexual maturation,when the isozyme levels were at the lowest de-tectable basal levels. Immunochemical detectiondemonstrated that female and male turbot expressa 58 kDa CYP1A protein. EROD activity of BNF-treated fish did not correlate well with CYP1AELISA of the same samples (result not shown),although there is a general agreement of theCYP1A isoenzyme as the predominant ERODcatalyst in fish (e.g., Goksøyr et al., ’91b, ’94;Goksøyr and Förlin, ’92). There was no evidenceof sex differences in the induction response.

Since the P450 system metabolizes both endog-enous and exogenous substances, interactions be-tween foreign chemicals and physiological processesare possible. In this respect, the relationships be-tween induction of biotransformation enzymes infish liver and altered steroid metabolism in vitroand in vivo deserves more attention. In severalinvestigations a relationship between elevatedP450 activities and disturbed physiological endo-crine functions, essential for successful reproduc-tion, has been found (Spies et al., ’85; Anderssonet al., ’88; Sivarajah et al., ’78; Thomas, ’90). Al-though no links between the induction of P450and impaired reproductive functions have yetbeen established, it is nevertheless importantthat the mechanism by which potential P450inducers may affect sexual development and fer-tility is elucidated.

ACKNOWLEDGMENTSThis study was supported by the Norwegian Re-

search Council (NFR). We want to thank the man-agement of the Industrial Laboratories (ILAB,High Technology Centre in Bergen [HIB]) andGuri Lerøy Grung for supplying estradiol-17βdata. The help of Sissel O. Olsen and Kjersti A.Helgesen during the sampling and analysis peri-ods is gratefully appreciated. We are also gratefulto Malin Celander and Tommy Andersson for pro-viding the rainbow trout P450con and P450KM2antibodies respectively. We will also like to thankMalin Celander for comments on the manuscript.

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changes in three hepatic cytochrome p450 subfamilies during a reproductive cycle in turbot...

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