Extrahepatic xenobiotic metabolism in North-European freshwater fish

Comp. Biochem. Physiol. Vol. 69C, pp. 259 to 263, 1981

0306-4492/81/040259-05502.00/0 Copyright © 1981 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

EXTRAHEPATIC XENOBIOTIC METABOLISM IN NORTH-EUROPEAN FRESHWATER FISH PIRJO LINDSTROM-SEPP.~,* ULLA KOIVUSAARIand OSMO H6.NNINEN Department of Physiology, University of Kuopio, P.O.B. 138, SF-70101 KUOPIO 10, Finland

(Received 29 December 1980) Abstract--1. Cytochrome P450 and monooxygenase activity and glucuronidation were measured in the gills, intestine, heart and kidney as well as in the liver of vendaee, perch and roach and rainbow trout for comparison. 2. Cytochrome P450 content and the specific monooxygenase activities were always highest in the liver. In the rainbow trout and vendace the renal 7-ethoxycoumarin-O-deethylase and in the roach the renal 3,4-benzpyrene hydroxylase activity was about half of that in liver. The activities in gills were relatively high in roach and vendace. In rainbow trout, perch and roach the intestinal activities were easily detectable. 3. In all fish high UDPglucuronosyltransferase activities were found in the liver and intestine. In vendace and roach both the gills and the kidney showed, however, even higher specific activities. 4. The rainbow trout showed higher hepatic and intestinal biotransformation activities than the other species.

INTRODUCTION Several fish species are able to oxidize foreign substrates in the same way as mammals in their livers (DeWaide, 1971; Ahokas, 1977; Bend & James, 1978). The metabolism goes through the cytochrome P450 dependent monooxygenase system. This pathway has also been found in some extrahepatic tissues of fish (DeWaide, 1971; Bend & James, 1978; Stegeman et al., 1979). The kidney microsomes of dogfish shark have a surprisingly high level of 7-ethoxycoumarin deethylating activity (i.e. five times the activity in the liver microsomes), and the renal 3,4-benzpyrene hydroxylase activity equals to that in the liver (Pohl et al., 1974). It is possible that the relative contribution of the extrahepatic biotransformation varies considerably from species to species and from substrate to another. In this work the extrahepatic compared to hepatic biotransformation (both oxidation and glucuronidation) of selected freshwater fish species in Northern Europe has been studied. Vendace (Coregonus albula), perch (Perca fluviatilis) and roach (Rutilus rutilus) were chosen because of their great economical and ecological importance in the lake area. Cultivated rainbow trout (Salmo oairdneri) related to vendace (salmon fish) was studied for comparison. The selected fish species differ greatly from each other in their ability to survive in polluted waters. Vendace is a very sensitive fish and it disappears soon, if the lake is polluted (Hakkarainen, 1972). Rainbow trout is somewhat more durable. Perch and roach manage to live even in heavily polluted waters. MATERIALS A N D M E T H O D S

Fish species Mature fish of both sexes were collected in lakes considered clean near Kuopio, Finland: vendace by seine or * To whom all correspondence should be addressed.

net in Lake Suvasvesi by the help of commercial fishermen, perch and roach by angling and by net in Lakes Rytky and Ayskoski, rainbow trout from Nilakkalohi Fish Farm, Ayskoski, Tervo. Vendace were 10-20cm long and weighed 15-60g; perch 12.5-21 cm and 28-140g; roach 13.5-22.5 cm and 39-215g; and rainbow trout 35-47cm and 680-1270g. All the fish were caught in summer.

Tissue preparation All the fish were killed immediately after catching and the tissues were removed and put to ice-cold 0.25 M sucrose or 0.t M potassium phosphate buffer pH 7.4 containing 0.1 M KCI, 1 mM K2EDTA, 1 mM dithiotreitol and 0.1 mM phenantroline to stabilize enzymes and to avoid the autogenous destruction by proteolytic enzymes. The vendace samples were put to this special potassium phosphate buffer due to the greater lability of its monooxygenase system (not illustrated). The intestine was taken as a whole and the lumen was cleaned. The gall bladder was removed from the liver and with gills only the filaments were taken. Kidney and heart were taken as a whole, too.

Isolation of microsomal fraction The tissues (pooled, n = 3-20) were homogenized in four volumes of 0.25 M sucrose or buffer (vendace) using Potter-Elvehjem glass-Teflon homogenizer. The homogenates were centrifuged at 10,000 g for 20 min at 4°C. The supernatants were centrifuged further at 105,000 g for 60 rain in Sorvall OTD-2 ultracentrifuge. The pellet (microsomal fraction) obtained was finally resuspended in 0.25 M sucrose and when potassium phosphate buffer was used (yendace) also 20% glycerol was added. The microsomes were resuspended so that I ml contained microsomes from I g of tissue. The microsomes were stored at -80°C no longer than 1 week. The protein contents of the samples were determined by using the Folin-Ciocalteau method as described by Lowry et al. (1951).

Enzyme assays The deethylation of 7-ethoxycoumarin was measured as described by Ullrich & Weber (1972) and modified by Aitio (t978) and the hydroxylation of 3,4-benzpyrene according to the method of Wattenberg et al. (1962) and modified by 259

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PIRJO LINDSTROM-SEPP.~et al.

Nebert & Gelboin (1968). UDPglucuronosyltransferase activity was measured using 4-nitrophenol as substrate according to Isselbacher (1956) and modified by H~inninen (1968). All the determinations were carried out at 18°C, near the temperatures where the fish were living in summer. Cytochrome P450 content was measured according to the method described by Johannesen & DePierre (1978) to eliminate the disturbance of hemoglobin and methemoglobin. RESULTS Cytochrome P450 content was higher in the liver than in the other tissues of all the four fish species studied (Table 1 and Fig. 1). There were also significant amounts of cytochrome P450 in the microsomal fraction of the kidney and intestine. In the gills the spectrum could easily be recorded, but only in case of roach. In the heart there was a very high disturbing peak at 420 nm, although the method of compensating hemoglobin and methemoglobin by Johannesen & DePierre (1978) was used (not illustrated). 7-Ethoxycoumarin-O-deethylase activity was found in all the fish species studied in the five tissues sampled except in the heart of perch and roach and in the

gills of perch (Table 1 and Fig. 1). Of the extrahepatic tissues the kidneys of vendace and rainbow trout showed about half of the activity in the liver in these species. In rainbow trouts the next active tissue was intestine, which also showed some activity in the roach and perch. Low enzyme activities were detected in the gills of vendace, roach and rainbow trout. In the heart the activity could be measured only from the rainbow trouts without difficulty. 3,4-Benzpyrene hydroxylase had a wider distribution than 7-ethoxycoumarin-O-deethylase in the fish tissues (Table 1 and Fig. 1). Thus it was detectable also in the heart of perch and roach and in the gills of perch. Both the specific and total extrahepatic enzyme activities were in general very low compared to the hepatic levels. The roach, in which the enzyme activity was low in the liver, the relative activities in the gills, kidney and intestine were high. The perch liver showed as high 3,4-benzpyrene hydroxylase activity as the rainbow trout liver. In the vendace the specific hepatic activity was about half and in the roach only one fifth of that in the perch and rainbow trout, respectively. All the four fish were capable of glucuronidating 4-nitrophenol in all the tissues studied (Table 1 and

Table 1. Cytochrome P450 content (measured at room temperature) and 7-ethoxycoumarin O-deethylase, 3,4 benzpyrene hydroxylase activity and UDPglucuronosyltransferase activity (determined at 18°C) measured from microsomes prepared from several organs or rainbow trout, vendace, perch and roach Tissue

Rainbow trout

Vendacea

Perch

Roach

A. Cytochrome P450 content (pmol x mg protein- 1) Liver Kidney Heart Gills Intestine

109.0 (19)c 5.1 (10) 5.7 (6) 0.0 (6) 30.5 (15)

74.2 (52) 11.7 (29) 1.9d (34) 0.0 (29) 8.2 (25)

50.6 (29) b b 0.0 (8) 6.3 (17)

89.3 (28) 9.8 (7) 0.0 (7) 27.2 (7) 20.5 (16)

B. 7-Ethoxycoumarin-O-deethylase (pmol × min-~ × mg protein-l) Liver Kidney Heart Gills Intestine

44.38 (19) 27.05 (10) 1.98 (6) 1.2 (6) 4.39 (15)

11.19 (52) 6.43 (29) 0.14d (34) 1.67 (29) 0.22d (25)

28.45 (29) b 0.0 (10) 0.0 (8) 2.15 (17)

19.15 (28) 1.37 (7) 0.0 (7) 1.35 (7) 4.01 (16)

C. 3,4-Benzpyrene hydroxylase (pmol x min- 1 x mg protein- t) Liver Kidney Heart Gills Intestine

20.29 (19) 3.04 (10) 1.70 (6) 0.35d (6) 2.60 (15)

8.42 (52) 1.84 (29) 0.28d (34) 1.03 (29) 0.37 (25)

20.13 (29) b 1.30 (10) 2.76 (8) 1.03 (17)

4.14 (28) 2.17 (7) 0.25d (7) 2.29 (7) 1.24 (16)

D. UDPglucuronosyltransferase (pmol x min-z x mg protein-1) Liver Kidney Heart Gills Intestine

383.8 (29)¢ 60.0 (10) 89.4 (6) 70.5 (6) 245.7 (15)

66.5 (52) 88.1 (29) 43.5 (34) 133.5 (29) 93.7 (25)

93.5 (29) b 20.8 (10) 73.9 (8) 67.6 (17)

108.4 (28) 158.6 (6) 36.2 (7) 235.2 (7) 75.1 (16)

a Microsomes on 0.1 M potassium phosphate buffer. b Not measured because of insufficient material. Results from pooled animal samples (number of which is given in parentheses). d The lowest activities are in the limits of detection by the methods used.

261

Extrahepatic xenobiotic metabolism in fish A. Cytochrome P 4 5 0 100%

Rainbow trout

Perch

Vendoce

e.

8 O

a)(a) I I

2

4

3

I

I 5

2

I

3

4

5

I

2

I

3

2

3

4

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B. 7 - Ethoxycoumarin-O-deethylose

100% :[ O

$ (a)

I

I

C. 3,4-Benzpyrene

I hydroxylose

Ioo% .>,

"6 (a) I

I

1

I

D. UDPglucuronosylt ronsferose 100%

Rainbow trout

Roach

Porch

Vendace

> u

0

"

i

I

2

3

4

I

5

I

2

,,

3

4

5

I

(-*' I 2

3

4

5

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2

II 3

5

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Fig. 1. Total cytochrome /)450 content and total 7-ethoxycoumarin-O-deethylase, 3,4-benzpyrene hydroxylase and UDP glucuronosyltransferase activities measured (at 18°C) from mierosomes prepared from organs of rainbow trout, vendace, perch and roach. The activities are as a percentage of the liver total activity (liver = 100%). The tissues are (1) liver, (2) kidney, (3) heart, (4) gills and (5) intestine. ((a) = Not measured because of insufficient material).

Fig. 1). UDPglucuronosyltransferase activity differed in distribution from the cytochrome P450 content and the monooxygenase activity. The gills showed the highest specific activities in the roach and vendace. In these species also the renal activities were higher than the levels in the liver. In the perch the UDPglucuronosyltransferase activities in the gills and intestine were close to the hepatic level, although the activity was highest in the liver. In the rainbow trout the liver showed higher glucuronidating activity than any other tissue. The next active organ was the intestine in this species. The activities in the heart, gills and kidney were about the same and approximately one fourth or sixth of that in the liver. Of the different species the rainbow trout appeared to be superior in

glucuronidation capacity. The specific UDPglucuronosyltransferase activity in the liver was higher than in any other species (3.5 and 5.8 times the activity in the roach and vendace liver, respectively). DISCUSSION The results obtained indicate that in the extrahepatic tissues like in the gills and intestine, through which the adsorption of xenobiotics occurs, there is measurable biotransformation activity in several fish species. Although cytochrome P450 could not necessarily be recorded from the microsomal fractions of the gills, this tissue almost consistently showed some monooxygenase activity in the fish species studied. In

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PmJo LINDSTRoM-SEPP,~et al.

spite of the low activity the monooxygenase system in location of the monooxygenase system also in fish the gills may have some physiological significance, (DeWaide, 1971; Bend & James, 1978; Stegeman et since xenobiotics can penetrate their membranes. The al., 1979). On the other hand the contribution of at glucurononidation capacity of the gills is most prob- least the intestine to the total glucuronidating caably of physiological significance, since in two species pacity is significant at least in vendace and rainbow studied (roach and vendace) the highest specific trout. Cytochrome P450 content compared to the UDPglucuronosyltransferase activities were seen in this organ. Also in the two other species the enzyme monooxygenase activities appeared to differ in distribution. There were tissues from where cytochrome activity was in gills easily detectable. The intestine is subjected to the load of xenobiotics, P450 could not be directly recorded, although which occur in the feed. Cytochrome P450 as well as 3,4-benzpyrene hydroxylase activity was present. In the two monooxygenase activities could consistently the measurement of cytochrome P450 is not necessbe measured from the intestinal samples. Although arily, however, easy, when the levels are low. the monooxygenase activities were only a fraction of Monooxygenase activities measured as 7-ethoxycouthat seen in the liver, they may have physiological marin-O-deethylase and 3,4-benzpyrene hydroxylase significance in metabolizing the compounds during showed both similarities and dissimilarities in their adsorption. The intestinal UDPglucuronosyltransfer- pattern of distribution in this study. These suggest ase activities were in all species studied either close to differences in the relative occurrence of cytochrome the hepatic levels or even higher. At least those com- P450 isoenzymes in the different tissues. pounds having proper functional groups may be effecOur results show wide existence of UDPglucurotively conjugated during the passage through the nosyltransferase in the tissues of the freshwater fish. It mucosa. When the activity values reported here are appears that the distribution of this enzyme is not compared with the figures obtained from the liver, it paralled to that of the monooxygenase system, since should be noted that the intestinal samples contained the relative activities vary independently. In this reall the gut layers. It is known from the mammals that spect the fish resemble mammals (Aitio, 1973). the muscular layers are practically devoid of UDPgluAt least in marine species some other conjugacuronosyltransferase activity (e.g. Toivonen et al., tion reactions also occur. James et al. (1979) have found 1973). If this is true also in the fish, the intestinal that glutathione S-transferase exists in the liver and mucosa may be the most active tissue in fish in glumany extrahepatic organs studied (gills, intestine, curonidating xenobiotics. gonadal tissues) in several marine species. Bend et al. Of the inner organs the kidney in addition to the (1977) have found a wide distribution of glutathione liver showed considerable monooxygenase and glu- S-transferase in organs of the little skate, too. curonidation activities as is known to be the case also in mammals (Aitio, 1973). It has been reported that Acknowledgements--This study has been supported by dogfish shark has as high or higher monooxygenase grants from Finnish Research Council for Natural Sciences activity (3,4-benzpyrene hydroxylase and ethoxycou- (Project No. 40). We thank Mr Mikko Ik~iheimo for his marin-O-deethylase) in the kidney than in the liver help in catching the fish. microsomes (Pohl et al., 1974). 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