Activities of pollutant metabolising and detoxication systems in the liver of the plaice, Pleuronectes platessa: Sex and seasonal variations in non-induced fish

Camp. Eiochem. Phpiol. Vol. 96C, No. I, pp. 185-192, 1990 Printed in Great Britain

0306-4492/90 $3.00 + 0.00 Q 1990 Pergamon Press plc

ACTIVITIES OF POLLUTANT METABOLISING AND DETOXICATION SYSTEMS IN THE LIVER OF THE

PLAICE, PLEURONECTES PLATESSA: SEX AND SEASONAL VARIATIONS IN NON-INDUCED FISH

STEPHEN GEORGE, PETER YOUNG, MICHAEL LEAVER and DOUGLAS CLARKE

NERC Unit of Aquatic Biochemistry, University of Stirling, Stirling FK9 4LA, Scotland. Telephone: (0786) 79692, Fax: (0786) 64994

(Received 24 January 1990)

Abstract-l. The ability of immature plaice to perform a number of representative hepatic phase I and II biotransformations was examined.

2. Activities attributable to cytochrome P-450 IA1 and phenol UDP-glucuronosyltransferase activity were markedly higher in plaice than other fish or rats. Glucuronidation of sex hormones and bilirubin, conjugation of ethacrynic acid with glutathione and the sulphation of phenols was very much lower in plaice than in rats, indicating that these are minor pathways in this fish.

3. MFO activities in sexually mature female plaice were 2(r25% of those in immature fish, whilst there were no significant differences in glutathione S-transferase or glutathione peroxidase activity.

4. Seasonal variations in activities were determined for use in biomonitoring studies. With the exception of microsomal GST, all enzymes and the liver somatic index showed a marked seasonal variation, activities were lower in summer and showed a gradual rise in autumn and winter with a peak in spring. This pattern appeared to be the inverse of the environmental temperature, indicative of a possible temperature-dependent effect; however, there was not direct proportionality and photoperiod, feeding behaviour, plasma corticosteroid and plasma glucose also show a similar cyclical variation.

5. Cytochrome P-450 dependent ethoxyresorufin 0-deethylase and cytosolic glutathione S-transferase activities in immature male fish showed a two-fold elevation over those in immature females during late winter and spring in the period when gonadal regression might be occurring and may be due to elevated 1 I-ketotestosterone levels.

INTRODUCTION

The ability of fish to metabolise various xenobiotic organic compounds is now well known, and much effort is now being placed upon obtaining an under- standing of the toxicology of aquatic pollutants and their effects upon individuals and populations. A major objective of many of these studies is to deter- mine the environmental significance of release of xenobiotics into the oceans. The first and most critical stage in metabolism of many of these non- polar lipid soluble compounds is mediated by the mixed function oxygenase (cytochrome P-450 or MFO) system, which carries out a series of oxidation reactions resulting in their conversion to more polar metabolites which may be conjugated by the phase II systems (predominantly the transferases) to form water soluble derivatives capable of being excreted in bile or urine (reviewed by Bend and James, 1978; Kleinow et al., 1987; Stegeman and Kloepper-Sams, 1987). A characteristic feature of many of these enzyme systems, particularly the cytochromes P-450, is their induced synthesis in response to many xenobiotics of environmental interest, including polycyclic aromatic hydrocarbons (James and Bend, 1980), polychlorinated and polybrominated biphenyls (Ad- dison et al., 1985; Elcombe and Lech, 1978) as well as petroleum hydrocarbons (Payne and Penrose, 1975; Walton et al., 1983). Induction of this enzyme system has therefore been proposed as a sensitive

indicator of environmental pollution (Payne et al., 1987; Addison et al., 1985; Haasch et al., 1989). Unfortunately it is also sensitive to hormonal effects which may mask or even prevent induction (Forlin and Hansson, 1982; Vodicnik and Lech, 1983). Ad- dison et ul. (1985) proposed that a benthic flatfish, the winter flounder (Pseudopleuronectes americanus) was a suitable test species for environmental monitoring due to its wide distribution, habit and lack of appreci- able migration. In Europe, the flounder (Plutichthys Jesus) is a brackish-water species, which is therefore suitable for estuarine monitoring, and a more appro- priate species for monitoring the coastal waters of the U.K. is the plaice, Pleuronectes plutessu.

Considerable interspecies differences appear to exist in metabolism and selective toxicity of many compounds, prime examples being the toxicity of TFM to lampreys which unlike insensitive species such as the trout do not rapidly clear the compound as the glucuronide (Lech and Statham, 1975), or the toxicity of Aflatoxin Bl to the trout which readily produces the carcinogenic epoxide, but is unable to detoxify this metabolite by formation of its glutathione conjugate (Loveland et al., 1984). Since the plaice is used for statutory toxicity testing, it is therefore important that the characteristics of its xenobiotic metabolising systems are determined. In previous studies we have reported some properties of plaice cytochrome P-450s (Leaver, 1988; Leaver et al., 1988), UDP-glucuronosyl transferases (Clarke et al.,

185

186 SmprmR GEORGE et al.

1988) and glutathione S-transferases (George et al., 1989) and effects of administration of a polyaromatic hydrocarbon (George and Young, 1986) and cadmium (George, 1987) on these systems. In the present study we have have examined the ability of the plaice to carry out a number of representative phase I and II biotransformations, and have investigated the seasonal and sexual variation in a number of these activities.

MATERIALS AND METHODS

Chemicals

[‘HI-benzo(a)pyrene, [“‘Cl1-naphthol and [r4C]testosterone were obtained from Amersham International. NADPH was obtained from Parke Scientific (Basingstoke). Ethoxyresorufin, resorufin and phenanthrene 4,5 epoxide were a gift from Dr M. D. Burke, University of Aberdeen. Other biochemicals were obtained from Sigma Ltd (Poole) and reagent chemicals were of AnalarTM grade from BDH Ltd (Poole).

Animals

Plaice were fished by a commercial seine boat 5-8 km off the coast of Gourdon, Kincardineshire, NE Scotland. The area is considered non-polluted and the nearest site of industrial activity of any significance, Aberdeen, is 40 km north. Fish were transported to the laboratory and main- tained for up to two days before sacrifice in a flowing sea water aquarium at 10°C.

Subcellular fractionation and assays

Livers were removed, a 20% w/v homogenate was pre- pared and fractionated as described by George and Young (1986). Assays were generally performed immediately or after storage of the fractions at -80°C for up to 10 days. Assays were performed at 25°C except for glucuronosyltransferase activity with bilirubin and testosterone, which were carried out at 37°C.

Measurements of cytochrome P-450 dependent aminopy- rine N-demethylase (AP), benzo(a)pyrene hydroxylase (AHH), biphenyl 2- and 4-hydroxylases (Bp20H and Bp40H), ethoxycoumarin 0-deethylase (ECOD), 7- ethoxyresorufin 0-deethylase (EROD) were performed as described previously (George and Young, 1986; Leaver et

al., 1988). Epoxide hydratase (EH) was assayed fluorimetri- tally using phenanthrene 4,5 epoxide as substrate, as described by Leaver (1988). Glutathione S-transferase (GST), glutathione peroxidase (GSHPx) and protein determinations were also described previously (George and Young, 1986). UDP-glucuronosyltransferase activities of lubrol PX- activated microsomes were determined with bilirubin (BIIGT), I-naphthol (NAGT), 4-nitrophenol (PhGT) and testosterone (TestGT) as substrates, as described previously (Clarke et al., 1988; George and Young, 1986). NADPH cytochrome c reductase was determined spectrophotometri- tally by the reduction of ferricytochrome c as described by Mackler (1967), and NADH cytochrome b5 reductase by reduction of ferricyanide as described by Strittmatter (1967). Sulphotransferase (ST) activity was determined by HPLC analysis of post-mitochondrial supernatants incubated with 20pM I-naphthol (I-NA) as described for hepatocytes (Morrison er al., 1985).

All values represent means f SEM of duplicate measurements. Statistical analysis of results was performed by using a Student’s l-test, and Mann-Whitney, Kruskall Wallis, Kolmogorov-Smirnov tests and other standard procedures using commercial microcomputer programs. Levels of significance are given as * = 0.01 <p < 0.05, t=p <O.Ol.

RESULTS

Projle of mixed function oxygenase parameters and phase II activities

Plaice in non-spawning condition were collected from July to January during the period of sexual quiescence. There were no statistically significant differences between male and female fish in any hepatic parameters measured during this period @ > 0.05, Mann-Whitney and Student’s t-tests); the levels of mixed function oxygenase activities concerned with xenobiotic metabolism and associated microsomal electron transport components, phase II hydrolase, transferase and peroxidase activities in hepatic microsomes and the cytosol of these fish are given in Table 1. To permit comparison with other studies and fractionation procedures, the data are expressed both in the conventional manner on the

Table I. Hepatic mixed function oxygenase components, Phase I and II enzyme activities in plaice, Pleuronrcfes plafessa

Character Units Microsomal

Cytochrome P-450 NADPH cyt. c (P-450) reductase NADH cyt. h, (ferricyanide) reductase Benzo(a)pyrene hydroxylase (AHH) Ethoxyresorufin 0-deethylase (EROD) Ethoxycoumarin 0-deethylase (ECOD) Aminopyrene N-demethylase (AP) Biphenyl 2-hydroxylase (BpZOH) Biphenyl I-hydroxylase (Bp40H) Microsomal glutathione S-transferase (usGST) Epoxide hydratase (EH) Phenol UDP-glucuronosyltransferase (NAGT) Phenol UDP-glucuronosyltransferase (PhGT) Bilirubin UDP-glucuronosyhransferdse (BiIGT) Testosterone UDP-glucuronOsyltranSferdSe (TestGT) Microsomal orotein C.vosolic

Cytosolic glutathione S-transferase (cytGST) Se-dependent glutathione peroxidase (GSHPx) I-NA Sulphotransferdse (ST)

“Ill01

nmol/min pmol/min pmol/min nmol/min pmol/min nmol/min pmol/min pmol/min pmol/min nmol/min nmolimin nmol/min pmol/min pmol/min mg/g liver

pmol/min umollmin

Per mg protein

Per g liver wt

0.53 * 0.07 12.5 f I.6 0.07 * 0.004 1.69&0.11 0.51 * 0.04 13.9 + I.2 0.13 io.015 3.0 * 0.35 1.59 i 0.01 37.3 f 2.6

25 f 0.4 59* IO 0.98kO.14 23.1 k 3.4

4 f 0.8 94& 17 29 f 4 680 * 91

0.28 & 0.03 6.68 k 0.64 29.3 + 12.5 730 * 310 14.2 f 1.4 160&25 38.5 f 11.8 490 f 42 160+ 14 3.7 + 0.33 240 + 5 2.46 f 0.57

- 23.6 f 0.3

0.81 * 0.04 0.055 * 0.0007

0. I I i_ 0.02

(a)

62.1 + 3.7 3.33 k 0.16

7.5 It I.6

(12) (35) (35) (31) (59)

(8) (8) (8) (8)

(41) (3)

(16) (19) (27)

(5)

(81) (46) (5)

mg/g liver 68.6 * 9.0 (65)

Fish of both sexes, body weights 170-350 g, were collected from July to January and analysed as described (Materials and Methods). Values given as mean rt SEM, for n fish.

Detoxication in the liver of plaice 187

basis of protein content of the fraction as well as by liver weight.

Effect of sexual maturity on components of xenobiotic metabolism

During the entire annual survey only six sexually mature fish were obtained-all were females caught at the end of May. Comparison of the values for these fish (Table 2) showed that both EROD and AHH activities were lower in mature compared with immature fish (p < 0.01 and p < 0.05, respectively, Student’s t-test). Activities of microsomal GST, cytosolic GST and GSHPx and protein concentrations in microsomes and cytosols were not significantly different in the sexually mature fish (Student’s t, Mann-Whitney and Kruskall Wallace tests), indicating that the depression in female fish was specific to the MFO activities.

Seasonal variation

Fish were collected in weeks 3, 7, 9, 10, 23, 32-34, 37, 40, 42, 45, 47 and 49 during 1985 and 1986. The numbers of animals varied at each sampling time from 3-10, and therefore for presentation of data these weeks were grouped into 10 groups covering 5-week periods: group A (weeks 3-7 inclusive, mid. = 5), B (weeks 8-12, mid. = lo), C (weeks 13-17, mid. = 15), and so on. The seawater temperatures derived from published values for this part of the Kincardineshire coast (Tomczak and Goedecke, 1962) are plotted in Fig. l(A) and show a cyclical variation from about 3-5°C in February-March to 12-15°C in August-September. Data for hepatic parameters and enzyme activities are presented for fish ranging from 170-350 g body weight (260 + 43 g), which from the tables presented by Lamont (1967) would be expected to be between 3 and 4 years old for this population, and thus unlikely to be sexually mature. No differences were found between the same months of different years and therefore all data have been grouped together. Determinations for all parameters were not carried out for each fish, and thus the numbers of replicates varies between groups. When dissected, although gonads were evident in the majority of fish, none of these fish possessed ripe or well-developed gonads. Liver somatic indices [liver weight expressed as a percentage of the body weight (lsi)] ranged from 0.57 to 1.57 which would also be indicative of sexually immature fish (cf. lsi’s of

Table 2. Hepatic Phase I and II activities in immature and sexually

mature female plaice

Uoits/mg Immature

Character protein (n = 6) Mature (n)

EROD nmol/min I .72 f 0.48 0.39 + 0. I I (6)t AHH pmol/min 100 * 37 42+ 16 (3)’ usGST pmol/min 0.29 + 0.04 0.26 + 0.09 (4) usprotein mg/g liver 23.5 f 3.9 20.1 f 2.1 (6) cytGST pmol/min 0.62 f 0.03 0.69 f 0.07 (4) GSHPx nmol/min 38+ 18 37 * 5 (4) cyt protein mg/g liver 61 f 5.6 58.2 + 7.5 (6)

Fish were caught in weeks 25 and 27. Sexually mature fish had fully

developed ovaries and clear swollen eggs. Mean k SEM, com-

parison by Student t-test, *p < 0.05, tp < 0.01. Abbreviations as

Table I.

A. r i i A

‘cr

--I- --t- It

B C DEFGHI J Week group

- b

,.,.1.,‘,.1.1.1’1’,.,.,.,.~

ijABCDEFGHlJab Week group

Fig. I. Seasonal variation in water temperature and hepatic parameters in immature male and female plaice, Pleuronectes plutessa. (A) Water temperature, (B) liver somatic index (Isi). Values for ---- male and ----- female fish f S.E.M. Weeks grouped into five-week periods com- mencing 14 January, A = weeks 3-7, B = weeks 8-12 and

so on.

1.8-2.6 in sexually mature flounders, after Fletcher and King, 1978). The Isi data showed quite a large interanimal variation, particularly for females which when plotted on a seasonal basis (Fig. 1B) displayed an overall trend but showed no statistically significant monthly differences. For males there was less variation, and differences were significant (p < 0.05, Kruskall Wallis) with a clear pattern of constant lsi from June to November falling to a minimum in April being observed.

Phase I, h4FO activities

EROD activity was determined for all groups and displayed a marked seasonal and sexual variation (Fig. 2A). In immature females the activity increased fourfold from June to February-March before dropping in April-May. Male EROD activities followed this increase from July to January but increased markedly above those of females to reach peak values between April and May some sevenfold greater than the summer values. Insufficient fish were assayed for AHH activity to allow monthly sex comparisons, although the trend for all fish (males plus females),

188 STEPHEN GEORGE et al.

I-

i j ABCDEFGHI Jab Week group

O~,.,.,.,‘,.,‘.‘,‘,.,‘..,.,‘.C i j ABCDEFGHI Jab

Week group

Fig. 2. Seasonal variation in hepatic detoxication activities of immature plaice, Pleuronectes platessa. (A) Hepatic cytochrome P-450 dependent ethoxyresorufin 0-deethylase (EROD) activity and (B) cytosolic glutathione S-transferase (GST) activity. Values for ---- male and ----- female fish k S.E.M. Weeks grouped into five-week periods as

Fig. 1.

plotted in Fig. 3(A), showed an essentially similar pattern to EROD activity.

Phase II activities

Cytosolic glutathione S-transferase (cytGST) activities displayed a similar seasonal trend to the MFOs with a minimum activity around July, which rose about twofold in female fish to a maximum in about March-April before dropping to the summer values (Fig. 2B). The mean winter cytGST activities in male fish were significantly greater than those in females (approximately twofold, p < 0.01, Student’s t-test). Thus the overall seasonal sex-dependent patterns for EROD and cytGST in immature plaice were the same, although the peak in male cytGST activity was reached about l-2 months before that in EROD activity. Although data sets for other phase II enzymes were more incomplete, there was sufficient data to indicate that the activity of microsomal glutathione S-transferase (usGST) remained constant throughout the year (Fig. 3B) whilst activities of Phenol UDPglucuronosyl transferase (NAGT) and glutathione peroxidase (GSHPx) (Fig. 3C, D) appeared to follow the same trends as observed for EROD and cytosolic GST activities, displaying a minimum in the period June-August.

DISCUSSION

Interspecies comparisons of detoxication activities

Most information on detoxifying activities concern studies on rats (Vlasuk et al., 1982). However, it should be noted that marked strain differences, particularly in Phase I activities are apparent and even with the same strain considerable interlabora- tory differences have been reported. For example in male Sprague-Dawley rats, values for a typical cytochrome P-450 dependent activity, EROD, reported by Burke et al. (1985) of 0.45 nmol/min/mg microsomal protein are nearly an order of magnitude greater than that of 0.055 nmol/min/mg reported by Guengerich et al. (1982). Thus it is probably wise to restrict comparisons to relative activity levels in different species measured in the same laboratory. In Table 3 we have compared activities in plaice with those of rats measured in our laboratory (this study and those of Clarke et al., 1988, and Leaver, 1988) and in rainbow trout compared with Sprague-Dawley rats reported by Gregus et al. (1983). Thus cross-species comparisons can be drawn between the two fish species, even though different assay procedures have been used.

In general it is possible to conclude that plaice have higher activities of all the enzymes concerned with metabolism and conjugation of exogenous organics than trout, whilst the phase II activities for conjuga- tions of endogenous substrates are considerably lower in plaice. Limited measurements on other flatfish, such as the European flounder (this laboratory, unpublished results), American winter flounder (Addison et al., 1985) and starry flounder (Spies et al., 1982) suggest that this situation is applicable generally for flatfish when compared with rainbow trout. The data also support the contention (Leaver et al., 1988) that there is more than one EROD catalyst in fish; compared with rats the fish have higher EROD and lower AHH activities. The activity of Bp40H, predominantly a phenobarbitol-inducible cytochrome P-450 isoform in rat (Burke et al., 1985) is an order of magnitude lower in the plaice, whilst activities of other cytochrome P-450 dependent reactions, ECOD and Bp20H, when corrected for differences in assay temperatures, are not significantly different in rat and plaice. The epoxide hydrolase and glucuronosyltransferase activity towards p-nitrophenol were comparable or greater in plaice than in the rat, whilst the trout is remarkable in the markedly elevated ability to glucuronidate testosterone and the rela- tively low glucuronosyltransferase activity towards planar phenols. The generally lower GST activities in the two fish species compared with the rat are pre- sumably due to the absence of certain of the rat-type enzymes observed in immunoblot experiments (George et al., 1989). Sulphotransferase activities of the fish are very much lower than those of the rat-indeed a previous study with isolated plaice hepatocytes that we carried out could not detect any sulphation of phenolphthalein (Morrison et al., 1985), although some sulphate conjugates of benzo- (a)pyrene metabolites were detectable (Morrison, unpublished results).

Detoxication in the liver of plaice

lo A. T F

i j ABCDEFGHI Jab

Week group

189


” D.

$ I T



Fig. 3. Seasonal variation in hepatic detoxication activities of immature plaice, Pleuronectes plutessa. (A) hepatic cytochrome P-450 dependent benzo(a)pyrene hydroxylase (AHH) activity, (B) microsomal glutathione S-transferase (usomalGST) activity, (C) phenol UDPglucuronosyltransferase activity (NAGT) and (D) cytosolic glutathione peroxidase (GSHPx) activity. Weeks grouped into five-week periods as Fig. l-fish were not classified by sex, and zero values represent weeks where data were not obtained.

Seasonal variation The seasonal trends in plaice hepatic MFO activi-

The present results show that the activities of ties, a minimum in summer with a gradual rise throughout the autumn and winter before a sudden

the major phase I and II hepatic xenobiotic trans- formation and detoxication systems of the plaice

decline in late spring/early summer observed in the

show marked seasonal variations which may be due present study follow closely those reported for two

to both exogenous (temperature) and endogenous other flatfish species, the American winter flounder,

(hormonal) factors. Pseudopleuronectes platessa (Addison et al., 1985), and the European flounder, Platichthys Jesus

Table 3. Interspecies comparison of detoxifying activities

Relative activity compared with rat = I

Rainbow Activity Plaice trout

Ethoxyresorufin 0-deethylase (EROD) IO 3 Ethoxycoumarin 0-deethylase (ECOD) 0.6 -

Benzo(a)pyrene hydroxylase (AHH) 0.3 0.05 Biphenyl 2-hydroxylase (BpZOH) 0.4 -

Biphenyl 4-hydroxylase (Bp40H) 0.04 -

Epoxide hydrolase (EH) 2’ 0.5t p-Nitrophenol-glucuronosyltransferase 1.3 0.1 I-Naphthol-glucuronosyltransferase 0.25 0.1 Bilirubin-glucuronosyltransferase 0.025 0.5 Testosterone-glUCUrOnOsyltKtnSfe~Se 0.05 5 GST (CDNB acceptor substrate) 0.3 0.6

(Ethacrynic acid acceptor substrate) 0.02 0.75 SutphotranSfemSe 0.005~ 0.085

Assays in rats were performed at 37°C and fish at 25°C. Different substrates used: lphenanthrene 4.5 oxide, tstyrene oxide, fl-naphthol, $2~naphthol.

Activities in plaice are compared with rat = I (this study and results reported by Clarke CI al.. 1988; George er al., 1989; Leaver, 1988) and those in rainbow trout compared with rat reported by Gregus ef al., 1983).


(Tarlebo et al., 1985). Tarlebo et al. (1985) reported an apparent general trend towards higher activities in male flounder during the winter and spring, and in the present study this was clearly evident with two-four fold higher EROD activities in males than in immature females during spring. Smaller fish were utilised in the present study than the flounders analysed by Addison et al. (1985) and Tarlebo et al. (1985), and although our plaice showed signs of gonadal development, they did not mature fully and thus gross effects associated with gametogenesis were avoided. In the limited number of fully mature female fish which we analysed, extremely low MFO activities were found, in accord with previous data for many fish species (Forlin and Hansson, 1982; Koivusaari et al., 1981; Stegeman and Chevion, 1980; Tarlebo et al., 1985; Walton et al., 1983). Thus, superimposed upon a sex-independent seasonal variation there is a sex- dependent elevation in EROD activity of male plaice between March and June and a decrease in EROD activity in female fish upon gonadal maturation. UDPGT activities were not measured in mature fish in this study. Reduced UDPGT activities (Koivusaari et al., 1981) and glucuronidation of phenolphthalein in z&o (Curtis, 1983) have been reported on gonadal maturation of rainbow trout.

In contrast to the report of Stegeman (1978) for the mummichog (Fundulus heteroclitus), in the present study there was no correlation between enzyme activities and the total liver or body weights of the plaice, which is in agreement with data for some other fish species, including European flounder (Tarlebo et al., 1985), cunner (Tuutogolubrus adspersus) (Walton et al., 1983) or sanddab (Citharicthys sordidus) (Spies ef al., 1982). The seasonal patterns indicated a possible relationship between liver somatic index, temperature and enzyme specific activities in the plaice. The environmental temperature of the plaice in this study varies between about 3 and 5°C in Febru- ary-March and 12-l 5°C in August-September (data derived from Tomczak and Goedecke, 1962, and plotted in Fig. lB), and there is a marked similarity between this pattern and the Isi, particularly in male fish which showed less inter-animal variation (p < 0.01, Student’s c-test). The lsi of plaice followed the same trend as in previously published work for this population of fish in the preceding year (White and Fletcher, 1985), other plaice populations (Wingfield and Grimm, 1977; Dawson and Grimm, 1980) and the winter flounder (Fletcher and King, 1978). The increase coincides with the period of intense feeding (March-October) followed by a period of sporadic feeding or starvation until after spawning (Wimpenny, 1985). During this feeding period the number of hepatocytes in plaice liver is some two-three fold greater when compared with the spawning period (Timashova, 1981). Adaptations to environmental temperature are characteristic of poik- ilothermic animals (reviewed by Hazel and Prosser, 1974), especially freshwater fish of temperate regions such as the trout (Salmo gardnieri and Salmo trutta) which have to adapt to temperatures from around 5 to 25°C; thus at low temperature an increase in relative liver biomass (lsi) (De Waide, 1970; Egaas and Varanasi, 1982) and higher specific activities of various MFOs (Blanck et al., 1989; DeWaide, 1970;

Egaas and Varanasi, 1982; Koivusaari, 1983) have been observed. It has been postulated that the latter may be attributable to changes in membrane phos- pholipid content since the difference in MFO activities was only apparent when a constant assay temperature was used-when assayed at the acclimation (environmental) temperatures of 5 and 20°C they were identical (Koivusaari, 1983; Blanck et al., 1989). The lipid analyses upon which this postulate is based (Hazel, 1979) are open to doubt (Sargent, personal communication); moreover, it would be expected that gross changes in membrane phospholipids would also affect other transmembrane enzyme activities such as UDPglucuronosyltransferase in a similar manner. Koivusaari (1983) did not observe similar adaptive changes in UDPglucuronosyl transferase activity of trout-when activity was measured at 18°C it was identical in August when fish were acclimated to 20°C and in November when they were acclimated to 2°C. It should also be a generalised phenomenon for a number of fish species; whilst similar effects on MFO activity were reported for the bluegill (Leptomis macrochirus) by Karr et aI. (1985), it was not observed in mature rainbow trout by Forlin et al., 1984, or in other species including the roach (Leusiscus rutilus) (DeWaide and Henderson, 1970) sheepshead (Archosargus probatocephalus) (James and Bend, 1980) or sanddab (Citharicthys sordidus) (Spies et al., 1982). It therefore requires further experimentation to explain the changes in MFO activities. In the present study enzymes activities were measured at constant temperatures, and therefore mathematical correction from the temperature-activity curves of the enzymes might indicate whether a temperature- dependent adaptive response was occurring. Whilst some smoothing of the data occurred, the temperature-activity maxima appeared to be out of phase and variations were still evident, indicating that other factors are operative.

Superimposed upon this seasonal variation in activities there are sex-dependent variations. In male fish we observed dramatic rises in MFO and GST activities in what would be the post-spawning period in mature fish between March and May. In mammals MFO activities in mature males are induced by testosterone (Waxman et al., 1985). Published data for mature plaice have shown that plasma testosterone levels are highest in female fish just before spawning (Wingfield and Grimm, 1977), and that in male fish 1 I-ketotestosterone levels rise dramatically near the time of spawning (Campbell et al., 1976), whilst in immature fish, such as used in this study, both testosterone and oestrogen levels are low throughout the year. The marked induction of EROD activity we observed in these immature males cannot therefore be attributed to induction by testosterone; however, it is presumed that any partially developed gametes would be resorbed during this period and it is possible that this might be under the control of 11-ketotestosterone, which might also in- duce hepatic detoxication activities. Previously published data for plaice (Wingfield and Grimm, 1977; White and Fletcher, 1985) and winter flounder (Campbell et al., 1976) have shown that glucorticoids are lower in winter when fish are not feeding and highest immediately post-spawning, thus following a

Detoxication in the liver of plaice 191

similar seasonal pattern to the lsi. Again cortisol levels are higher in females than males post-spawning (Campbell et al., 1976) indicating that whilst the overall pattern of changes in Isi and detoxication activities may be under glucocorticoid control, the sex-specific increase in male fish cannot be due to these hormones. In contrast, the extremely low levels of EROD activity observed in the few sexually mature females are in agreement with numerous reports for other species (Koivusaari and Andersson, 1984; Stegeman and Chevion, 1980; Williams et al., 1986) and are probably related to high oestrogen levels in these fish. Reduction of hepatic MFO levels and cytochrome P-450 content after oestradiol injection has been reported by Forlin and Hansson (1982).

The present study serves to provide baseline data on the scope of organic metabolism and detoxication activities in this flatfish species, and on seasonal variations which are relevant for the use of the species for environmental pollutant monitoring. Further studies of the controlling factors for these variations are clearly necessary to establish their mechanistic basis, and we are currently attempting to utilise cultured fish cells for this purpose.

REFERENCES

Addison R. F., Edwards A. and Renton K. (1985) Hepatic mixed function oxidases in winter flounder (Pseudo- pleuronectes americanus): seasonal variation and response to PCB feeding. Mar. Environ. Res. 17, 150-151.

Bend J. R. and James M. 0. (1978) Xenobiotic metabolism in marine and freshwater species. In Biochemical and Biophysical Perspectives in Marine Biology (Vol. 4), Ma- lins D. C. and Sargent J. R. (eds), pp. 126-188. Academic Press, New York.

Blanck J., Lindstrom-Seppa P., Agren J. J., Hanninen O., Rein H. and Ruckpaul K. (1989) Temperature compensation of hepatic microsomal cytochrome P-450 activity in rainbow trout. I. Thermodynamic regulation during water cooling in autumn. Comp. Biochem. Physiol. 93C, 55-60.

Burke M. D., Thompson S., Elcombe C. R., Halpert J., Haaparanta T. and Mayer R. T. (1985) Ethoxy-, pentoxy- and benzoyloxyphenoxazones and homologues: a series of substrates to distinguish different induced cytochromes P-450. Biochem. Pharmacol. 34, 583-588.

Campbell C. M., Walsh J. M. and Idler D. R. (1976) Steroids in the plasma of the winter flounder (Pseudo- pleuronectes americanus Walbaum): a seasonal study and investigation of steroid involvement in oocyte maturation. Gen. Camp. Endocrinol. 29, 14-20.

Clarke D. J., Burchell B. and George S. G. (1988) Charac- terisation and molecular analysis of hepatic microsomal UDP-glucuronosyltransferases in the marine fish, Pleuronectes platessa. Mar. Environ. Res. 24, 105-109.

Curtis L. R. (1983) Glucuronidation and biliary excretion of phenolphthalein in temperature acclimated steelhead trout (Salmo gairdneri). Comp. Biochem. Physiol. 76C, 107-111.

Dawson A. S. and Grimm A. S. (1980) Quantitative seasonal changes in the protein, lipid and energy content of the carcass, ovaries and liver of adult female plaice, Pleuronectes platessa L. J. Fish Biol. 16, 493-504.

DeWaide J. H. (1970) Species differences in hepatic drug oxidation in mammals and fishes in relation to thermal acclimation. Camp. Gen. Pharmacol. 1, 375-384.

DeWaide J. H. and Henderson P. Th. 11970) Seasonal variation of hepatic drug metabolism in the roach Leusis- cus rutilis L. Camp. Biochem. Physiol. 32, 487497.

Egaas E. and Varanasi U. (1982) Effects of polychlorinated biphenyls and environmental temperature on in vitro formation of henzo(a)pyrene metabolites by the liver of trout (Salmo gairdneri). Biochem. Pharmacol. 31, 561-566.

Elcombe C. R. and Lech J. J. (1978) Induction of mono- oxygenation in rainbow trout by polybrominated biphenyls: a comparative study. Environ. Health perspect. 23, 309-3 14.

Fletcher G. L. and King M. J. (1978) Seasonal dynamics of Cu2+, Zn*+, Ca*+ and Mg*+ in gonads of winter flounder (Pseudopleuronectes americanus): evidence for summer storage of Zn*+ for winter gonad development in females. Can. J. Zoo. 56, 284-290.

Forlin L., Andersson T., Koivusaari U. and Hansson T. (1984) Influence of biological and environmental factors on hepatic steroid and xenobiotic metabolism in fish: interaction with PCB and /I-naphthoflavone. Mar. Enui- ron. Res. 14, 47-58.

Forlin L. and Hansson T. (1982) Effects of oestradiol-17/I and hypophysectomy on hepatic mixed function oxidases in rainbow trout. J. Endocrinol. 95, 245-252.

George S. G. (1989) Cadmium effects on plaice liver zeno- biotic and metal detoxication systems: dose response. Aquatic Toxicology 15, 303-3 10.

George S., Buchanan G., Nimmo I. and Hayes J. (1989) Fish and mammalian liver cytosolic glutathione S-transferases: substrate specificities and immunological comparison. Mar. Environ. Res. 28, 4146.

George S. G. and Young P. (1986) The time course of effects of cadmium and 3-methylcholanthrene on activities of enzymes of xenobiotic metabolism and metallothionein levels in the plaice, Pleuronectes platessa. Comp. Biochem. Physiol. 83C, 374.

Gregus Z., Watkins J. B., Thompson T. N., Harvey M. J., Rozman K. and Klaassen C. D. (1983) Hepatic phase I and phase II biotransformations in quail and trout: comparison to other species commonly used in toxicity testing. Toxicol. appl. Pharmacol. 67, 430-44 I.

Guengerich F. P., Dannan G. A., Wright S. T., Martin M. V. and Karminsky L. S. (1982) Purification and characterisation of liver microsomal cytochromes P-450: electrophoretic, spectral, catalytic and immunochemical properties and inducibility of eight isozymes isolated from rats treated with phenobarbital or /I-naphthoflavone. Biochemistry 21, 60196030.

Haasch M. L., Wejksnora P. J., Stegeman J. J. and Lech J. J. (1989) Cloned rainbow trout liver P, 450 complimentary DNA as a potential environmental monitor. Toxic. appl. Pharmacol. 98, 362-368.

Hazel J. R. (1979) Influence of thermal acclimation on membrane lipid composition of rainbow trout liver. Am. J. Physiol. 236, R9l-RIOI.

Hazel J. R. and Prosser C. L. (1974) Molecular mechanism of temperature compensation in poikilotherms. Physiol. Rev. 54, 620-677.

James M. 0. and Bend J. R. (1980) Polycyclic aromatic hydrocarbon induction of cytochrome P-450 dependent mixed-function oxidases of marine fish. Toxic. appl. Phar- macol. 54, 117.-133.

Karr S. W., Reinert R. E. and Wade A. E. (1985) The effects of temperature on the cytochrome P-450 system of ther- mally acclimated bluegill. Camp. Biochem. Physiol. 8OC, 135-139.

Kleinow K. M., Melancon M. and Lech J. J. (1987) Biotransformation and induction: implications for toxicity, bioaccumulation and monitoring of environmental xenobiotics in fish. Environ. Health Perspect. 71, 105-l 19.

Koivusaari U. (1983) Thermal acclimation of hepatic poly- substrate monooxygenase and UDPglucuronosyltrans- ferase of mature rainbow trout (Salmogairdneri).~J. Exp. Zool. 227, 3542.


Koivusaari U. and Andersson T. (1984) Partial temperature compensation of hepatic biotransformation enzymes in juvenile rainbow trout (Sabno gairdneri) during water warming in spring. Camp. Biochem. Physiol. 78B, 223-226.

Koivusaari U., Harri M. and Hanninen 0. (1981) Seasonal variation of hepatic biotransformation in female and male rainbow trout (Salmo gairdneri). Cornp. Biochem. Physiol. 7oc, 1499157.

Lamont J. M. (1967) Plaice investigations in Scottish waters. Mar. Res. 3, 1-51.

Leaver M. J. (1988) Studies on the effects of oil drill cuttings on marine sediments and on hepatic cytochrome P-450 monooxygenases of plaice. Ph.D. thesis, University of Aberdeen, Scotland.

Leaver M. J., Burke M. D., George S. G., Davies J. M. and Rafaelli D. (1988) Induction of cytochrome P-450 monoxygenase activities in plaice by ‘model’ inducers and drilling muds. Mar. Environ. Res. 24, 27-30.

Lech J. J. and Statham C. N. (1975) Role of glucuronide formation in the selective toxicity of 3-trifluoromethyl- 4-nitrophenol (TFM) for the sea lamprey: comparative aspects of TFM uptake and conjugation in sea lamprey and rainbow trout. Toxicol. appl. Pharmac. 31, 150-158.

Loveland P. M., Nixon J. E. and Bailey G. S. (1984) Glucuronides in bile of rainbow trout (Salmo gairdneri) injected with [‘HI aflatoxin Bl and the effects of dietary b-naphthoflavone. Camp. Biochem. Physiol. 78C, 13-19.

Mackler B. (1967) Microsomal DPNH-cytochrome c reductase. Meths. Enzymol. 10, 551-553.

Morrison H., Young P. and George S. G. (1985) Conjuga- tion of organic compounds in isolated hepatocytes from a marine fish, the plaice, Pleuronectes plafessa. Biochem. pharmacol. 34, 3933-3938.

Payne J. F., Fancey L. L., Rahimtula A. D. and Porter E. L. (1987) Review and perspective on the use of mixed function oxygenase enzymes in biological monitoring. Comp. Biochem. Physiol. 86C, 233-245.

Payne J. F. and Penrose W. R. (1975) Induction of arylhydrocarbon (benzo(a)pyrene) hydroxylase in fish by petroleum. Bull. environ. contam. Toxicol. 14, 112-116.

Spies R. B., Felton J. S. and Dillard L. (1982) Hepatic mixed function oxidases in California flatfishes are increased in contaminated environments by oil and PCB ingestion. Mar. Biol. 70, 117-127.

Stegeman J. J. (1978) Influence of environmental contami- nation on cytochrome P-450 mixed-function oxygenase in fish: implications for recovery in the Wild Harbour Marsh. J. Fish. res. Bd. Can. 35, 668674.

Stegeman J. J. and Chevion M. (1980) Sex differences in cytochrome P-450 and mixed function oxygenase activity in gonadally mature trout. Biochem. Pharmacol. 31, 553-558.

Stegeman J. J. and Kloepper-Sams P. (1987) Cytochrome P-450 isozymes and monooxygenase activity in aquatic animals. Env. Health Perspect. 71, 87-95.

Strittmatter P. (1967) NADH-cytochrome b, reductase. Meths. Enzymol. 10, 561-565.

Tarlebo J., Solbakken J. E. and Palmork K. H. (1985) Variation in hepatic arylhydrocarbon hydroxylase activity in flounder, Platichthys Jesus: a baseline study. Helgolander Meeres 39, 187-199.

Timashova L. V. (1981) Seasonal changes in the structure of the liver of the plaice, Pleuronectesplatessa. J. Icthyol. 21, 145-151.

Tomczak G. and Goedecke E. (1962) Monatskarten der temperatur der Nordsee, dargestellt fur verschiedene Tiefenhorizonte. Erganzungsh Dtsch Hydrogr. Z. 7, 16.

Vlasuk G. P., Ghrayeb J., Ryan D. E., Reik L., Thomas P. E., Levin W. and Walz F. G. Jr (1982) Multiplicity, strain differences and topology of phenobarbital-induced cytochromes P-450 in rat liver microsomes. Biochemistry 21, 789-798.

Vodicnik M. J. and Lech J. J. (1983) The effect of sex steroids and pregnenolone-l6-a-carbonitrile on the hepatic microsomal monooxygenase system of rainbow trout (Salmo gairdneri). J. Steroid Biochem. 18, 323-328.

Walton D.-G., Fancy L. L., Green J. M., Kiceniuk J. W. and Penrose W. R. (1983) Seasonal changes in arvl hvdro- . I -

carbon hydroxylase activity of a ma&e fish Tautogo- labrus aakpersus (Walbaum) with and without petroleum exposure. Camp. Biochem. Physiol. 76C, 247-253.

Waxman D. J., Dannan G. A. and Guengerich F. P. (1985) Regulation of rat hepatic cytochrome P-450: age-dependent expression, hormonal imprinting and xenobiotic inducibility of sex-specific isoenzymes. Biochemistry 24, 44094417.

White A. and Fletcher T. C. (1985) Seasonal changes in serum glucose and condition of the plaice, Pleuronectes platessa L. J. Fish Biol. 26, 7555764.

Williams D. E., Masters B. S. S., Lech J. J. and Buhler D. R. (1986) Sex differences in cytochrome P-450 isozyme composition and activity in kidney microsomes of mature rainbow trout. Biochem. Pharmacol. 35. 2017-2023.

Wimpenny R. S. (1953) The Plaice (Buckland lectures for 1949). Arnold and Co., London.

Winefield J. C. and Grimm A. S. (1977) Seasonal changes in-plasma cortisol, testosterone and oestradiol-17/I in ihe plaice, Pleuronectes platessa L. Gen. camp. Endocr. 31, l-11.

Documents

Activities of pollutant metabolising and detoxication systems in the liver of the plaice, Pleuronectes platessa: Sex and seasonal variations in non-induced fish