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Metabolism of foreign compounds in freshwater crayfish (Astacus astacus L.) tissues
Aquatic Toxicology, 3 (1983) 35-46
35
Elsevier Biomedical Press
METABOLISM OF FOREIGN COMPOUNDS IN FRESHWATER CRAYFISH (ASTA CUS A S T A CUS L.) TISSUES
P. LINDSTROM-SEPP,~, U. KOIVUSAARI and O. HANNINEN
Department of Physiology, University of Kuopio, P.O.Box 138, SF-70101 Kuopio 10, Finland (Received 10 February 1982; accepted 29 April 1982)
Freshwater crayfish, Astacus astacus L. were found to have considerable amounts of cytochrome P-450 in the hepatopancreas microsomes. The content was almost as high as in rat liver microsomes. The low (7-ethoxycoumarin O-deethylase and ethylmorphine demethylase) or absent (3,4-benzpyrene hydroxylase and 7-ethoxyresorufin deethylase) monooxygenase activities recorded did not, however, correlate with the high amount of cytochrome P-450. The binding of type I and II substrates to the enzyme was poorer in crayfish hepatopancreas than in rat liver microsomes. There was also less NADPH than NADH cytochrome c reductasc activity in crayfish hepatopancreas microsomes. NADH supported faster demethylation of ethylmorphine than NADPH. Studies with hepatopancreas subfractions added to rat liver microsomes showed that hepatopancreas cytosol contained heat labile monooxygenase inhibitor(s). Only traces of 7-ethoxycoumarin O-deethylase were found in total homogenates of extrahepatopancreatic tissues like gills, intestine and green glands. From conjugation activities considerable levels of glutathione S-transferase were found in all the crayfish tissues studied. UDP-glucuronosyltransferase activity was very low in microsomal and cytosol fractions of hepatopancreas. Key words: Astacus astacus; cytochrome P-450; monooxygenase; glutathione S-transferase; UDP-glucuronosyltransferase
INTRODUCTION
The freshwater crayfish Astacus astacus L. has had economic significance in the Nordic Countries. The crayfish populations have, however, diminished during the current century. One reason has been the crayfish plague fungus (Aphanomyces astact), but this does not fully explain the disappearance. Environmental pollutants may, in places, have had some role to play in the decline. A. astacus appears to be extremely sensitive to pollutants such as DDT (Airaksinen et al., 1977), and it has been reported that this species practically lacks the biotransformation enzymes (Lang et al., 1977). Several other crustacean species especially those living in the sea have, however, significant monooxygenase activities (Brodie and Maickel 1962; Kahn et al., 1972; Bend et al., 1977; James et al., 1979b). Epoxide hydratase (Bend et al., 1977) and glutathione S-transferase activities have been found, too (James et al., 1979a). Therefore one could also expect at least some biotransformation activity in A. astacus. 0166-445X/83/0000-0000/$ 03.00 © 1983 Elsevier Biomedical Press
36 The present study was performed to reinvestigate the biotransformation ability in the tissues of A. astacus. Both monooxygenation as well as conjugation activities were observed. There was a high cytochrome P-450 content in the hepatopancreas but it appeared to have distinctly different catalytic properties than the rat liver cytochrome P-450. MATERIALS AND METHODS
Chemicals 3,4-Benzpyrene, bovine serum albumin, 7-ethoxycoumarin, isocitric-acid dehydrogenase, NADP, NADPH, NADH, phenantroline and trypsin inhibitor were purchased from Sigma Chemical Co. Aprotinin was obtained from Boehringer Mannheim, dithiotreitol from Calbiochem, ethylmorphine and aniline-HCl from Yliopiston Apteekki, Helsinki, Finland and isocitric-acid from Fluka AG. 7-Ethoxyresorufin was a gift from Dr. Antti Zitting, Institute of Occupational Health, Helsinki, Finland.
Biological material Freshwater crayfish (Astacus astacus) of both sexes were collected from June to October from lakes near Kuopio. They were kept fasting in containers with air bubbling at 5°C for 2 to 6 w. The crayfish (n = 72) were 9.1 +_ 0.7 cm (:e ___ SD) long and weighed 27.2 + 7.4 g. The hepatopancreas weighed 1.64 + 0.52 g and the green glands 0.11 + 0.04 g.
Tissue preparation The crayfish were killed by destroying the central nervous system, and tissue sampling was carried out immediately afterwards. The tissues were detached and immersed in ice-cold 0.1 M potassium phosphate buffer, pH 7.4, containing 0.1 M KC1, 1 mM K2EDTA, 1 mM dithiotreitol, 0.1 mM phenantroline and trypsin inhibitor (1 mg/ml). The tissues were homogenized in four volumes of the buffer using a Potter-Elvehjem glass-Teflon homogenizer. This special buffer was used to stabilize the labile enzymes and to avoid autogenous destruction by proteolytic enzymes. Hepatopancreas, in particular, has a lot of proteolytic activity because of the presence of both liver enzymes and digestive juice secreted by the pancreatic cells. 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 a Sorvau OTD-2 ultracentrifuge. The pellet (microsomal fraction) obtained was resuspended in the 0.1 M potassium phosphate buffer with stabilizing agents and 20% glycerol was added for
37 improving the storage. The microsomes were resuspended so that 1 ml contained microsomes from 1 g of tissue. The microsomes were stored at - 8 0 ° C and the determinations were done over 1 to 4 w when no significant inactivation occurred. The cytochrome P-450 determinations were, however, done immediately after preparation. The protein contents of the samples were determined by using the Folin-Ciocalteau method as described by Lowry et al. (1951) with bovine serum albumin as the standard.
Measurement of cytochrome P-450 levels and enzyme activities Cytochrome P-450 content was measured by the method of Omura and Sato (1964) or as described by Johannesen and DePierre (1978). The substrate binding spectra were recorded by the method of Schenkman et al. (1967). Ethylmorphine (type I compound) or aniline-HCl (type II compound) was added to the microsomal suspension containing 1 mg protein/ml. Cytochrome c reductase activities (NADPH or NADH as electron donor) were determined by the method of Dallner et al. (1966). The deethylation of 7-ethoxycoumarin was determined as described by Ullrich and Weber (1972) and modified by Aitio (1978) and the deethylation of 7-ethoxyresorufin as described by Burke and Mayer (1974). The hydroxylation of 3,4-benzpyrene was determined according to Wattenberg et al. (1962) as modified by Nebert and Gelboin (1968). Demethylation of ethylmorphine was determined by using the method of Anders and Mannering (1966). All the enzyme determinations were made at 18°C (Lindstr6m-Sepp~i et al., 1980). An NADPH generating system or NADPH was used as electron donor in these enzyme determinations if not mentioned otherwise. The NADPH generating system was made of 1.13 mM NADP, 12.5 mM isocitric-acid, 0.19 M Tris-HC1-0.056 M KCI buffer (pH 7.4), 12.5 mM MgCI2, 12.5 mM MnCI2 and 0.6 U isocitric-acid dehydrogenase. The influence of time and storage on monooxygenase enzyme activities was studied in various buffers by keeping total homogenates of hepatopancreas for different periods of time ( 0 - 24 h) in an ice-bath. The basic buffer was 0.1 M potassium phosphate buffer (pH 7.4) containing 0.1 M KCI, 1 mM K2EDTA and 1 mM dithiotreitol. The other three buffers were made from this by adding various protease inhibitors (trypsin inhibitor 1 mg/ml, aprotinin 1 mg/ml) or stabilizing agents (0.1 mM phenantroline). The storage of the total homogenates of hepatopancreas at - 0 ° C for 24 h did not affect 7-ethoxycoumarin O-deethylase activities significantly except in the case of buffer containing phenantroline (not illustrated). In that case the monooxygenase activity measured decreased to one third of the initial state. UDP-glucuronosyltransferase activity was measured as described by Isselbacher (1956) and H/inninen (1968) using p-nitrophenol as an aglycone. Glutathione S-
38
transferase was determined by the method of Habig et al. (1974) with 1-chloro-2,4-dinitrobenzene as substrate. These conjugation activities were also measured at 18°C. RESULTS
Cytochrome P-450 and substrate binding Cytochrome P-450 was detected in the hepatopancreas of freshwater crayfish, A. astacus (Fig. 1A) in the same concentrations (0.31 + 0.07 n m o l / m g microsomal protein, Table I) as in rat liver (0.38 + 0.07 n m o l / m g protein in our own studies or 0.42 + 0.02 n m o l / m g protein, Laitinen 1976). In green glands the cytochrome P-450 level was much lower. Hepatopancreas cytochrome P-450 was converted to inactive cytochrome P-450 in the course of time in all buffers used when total homogenates were stored at - 0°C for 24 h (Fig. 1B). The inactivation was only minimal when microsomes were stored at - 8 0 ° C for several weeks. The binding of substrates to cytochrome P-450 in microsomes of freshwater crayfish hepatopancreas and rat liver was determined using ethylmorphine (type I) and aniline (type II) as models. Ethylmorphine showed lower substrate binding in freshwater crayfish hepatopancreas than in rat liver microsomes (Fig. 2A). The spectra were qualitatively different, too. There was not any clear peak in the spectra recorded f r o m crayfish hepatopancreas. The m a x i m u m in rat was in 483-485 nm. The minimum was about 415 nm in crayfish and 416-418 nm in rat.
TABLE I Biotransformation enzyme activities measured from the microsomal fraction and cytosol of the hepatopancreas of freshwater crayfish A . astacus at 18°C.
Cytochrome P-450 N A D P H cytochrome c reductase N A D H cytochrome c reductase 3,4-Benzpyrene hydroxylase 7-Ethoxycoumarin O-deethylase 7-Ethoxyresorufin deethylase Ethylmorphine demethylase UDP-glucuronosyltransferase Glutathione S-transferase
Microsomal fraction (nmol-min - ~.mg protein - 2)
n
Cytosol ( n m o l . m i n - ~-mg protein - 1)
0.31 2.23 48.81 0 0.1 0 0.07 0.01 102.31
10 5 9 7 7 13 4 9 6
0 8.73 0.43 0 0 0 0.02 0.01 394.02
± 0.07 a _+ 0.05 ± 8.14 +_ 0.00 ± 0.05 + 0.01 ± 46.94
_+ ±
0.19 0.11
_+ 0.01 + 165.33
7 2 2 7 7 8 1 8 10
a nmol • protein-2. n = Number of measurements (in each determination more than two animals); 0 = below detection limit.
39
iI
~ F~'
I
5 Onm
I
\ //
l
B
I 450
I
I
400
'
Z.O0
10 n m
ol oo u,-,iti
/ J
Fig. 1. A. CytochromeP-450 spectra from hepatopancreas microsomesof both male ( - ) and female (---) freshwater crayfish (A. astacus) by the method of Johannesen and DePierre (1978). B. Influence of time on cytochromeP-450 measured from the homogenateof crayfish hepatopancreas kept in an ice-bath ( - , measured immediately after homogenization; ---, 3 h later; .... 24 h later).
The type II binding spectrum o f aniline also differed in crayfish hepatopancreas and rat liver microsomes (Fig. 2B). The absorption maximum in crayfish hepatopancreas microsomes was 426-431 nm and in rat liver microsomes 425-427 nm; the minima were between 407-409 nm and 385-387 nm in crayfish hepatopancreas and rat liver microsomes, respectively.
N A D P H and N A D H cytochrome c reductases in crayfish hepatopancreas Distributions o f N A D H and N A D P H cytochrome c reductase during microsomal fractionation differed in the hepatopancreas o f freshwater crayfish (Fig. 3). In the green glands, however, they behaved similarly. In hepatopancreas > 9007oof N A D H cytochrome c reductase activity was found in the microsomal fraction and in the green gland about 8507o. The rest was in the supernatant fraction. Only 20% of the N A D P H cytochrome c reductase activity was in hepatopancreas microsomes, the remaining 80°7o being found in the cytosol. In the green gland the majority (80070) o f the N A D P H cytochrome c reductase activity was located in the microsomal fraction.
Microsomal monooxygenase activities 7-Ethoxycoumarin O-deethylase activities measured in the total homogenates o f
40 B
A Crayfish
,
a' Cd.~
i
I
c
I
400
I
I
500 nm
~'~------'---
I 400
I 500 nm
RoI
Fig. 2. Substrate binding spectra measured from microsomes of crayfish hepatopancreas and rat liver. A. Ethylmorphine as substrate (a = 1.04 mM, b = 3.13 mM, c = 4.17 mM, d = 7.29 mM). B. AnilineHC1 as substrate (a' = 4.17 mM, b ' = 8.33 mM, c' = 16.67 mM, d' = 25.01 mM). The amount of cytochrome P-450 was the same (0.3 nmol • mg protein- 1) in all the recordings.
Activity %
100 // //
50
// // //
/I //
I/ //
// i/
Ii
z
~
[77~
Microsomes
F~I
105.000 x g super
not ont
// // // // // // // //
// //
//
HP
GG t
NADH
I
HP
GG
*
NADPH
Fig. 3. Distribution of N A D H and N A D P H cytochrome c reductase activities in microsomes and 105 000 x g supernatant fraction in hepatopancreas (HP) and green glands (GG) of freshwater crayfish.
hepatopancreas, gills, green glands and intestine are shown in Table II. The m o n o o x y g e n a s e activity was highest in hepatopancreas and only traces were found in other tissues. The m o n o o x y g e n a s e activities measured from the microsomal and 105 000 x g supernatant fraction o f crayfish hepatopancreas are listed in Table I. 3,4-Benzpyrene hydroxylase and 7-ethoxyresorufin deethylase activities were absent, and low levels o f 7-ethoxycoumarin O-deethylase and ethylmorphine demethylase were found in microsomal fraction, although the cytochrome P-450 content was high.
41 TABLE II 7-Ethoxycoumarin O-deethylase and glutathione S-transferase activities in freshwater crayfish determined from total homogenates o f gills, hepatopancreas, intestine and green glands.
Gills Hepatopancreas Intestine Green glands
10 10 5 5
7-Ethoxycoumarin Odeethylase (pmol-min- l.mg protein- 1)
Glutathione S-transferase (nmol.min - ~.mg protein - l)
1.0 22.9 2.6 2.3
75.1 273.1 204.9 321.4
+ + ± ±
1.7 5.3 0.8 1.1
± 30.6 + 39.9 ± 7.4 ± 68.6
Electron donors and monooxygenase activity The influence of different electron donors was determined by studying 7-ethoxycoumarin O-deethylase, 3,4-benzpyrene hydroxylase, 7-ethoxyresorufin deethylase and ethylmorphine demethylase activities. Electron donors used were isocitric acid dehydrogenase mediated NADPH generating system, NADPH, NADH and NADPH-NADH in the case of the first two enzyme reactions. NADPH and NADH were also used when the other two reactions were studied. Cumene hydroperoxide was used to bypass the electron chain to cytochrome P-450 in studies of the oxidation of 7-ethoxycoumarin and 3,4-benzpyrene (Bend et al., 1977). No 3,4-benzpyrene hydroxylase activity could be detected from the hepatopancreas microsomes with any of the physiological electron donors used (Table III). Neither did cumene hydroperoxide reveal any 3,4-benzpyrene hydroxylase activity from the hepatopancreas microsomes of the crayfish. NADPH or NADH could not support 7-ethoxyresorufin deethylase activity. 7-Ethoxycoumarin O-deethylation TABLE III The influence of different electron donors and cumene hydroperoxide on microsomal monooxygenase activities in the hepatopancreas o f freshwater crayfish. 7-Ethoxycoumarin 3,4-Benzpyrene O-deethylase hydroxylase
Ethoxyresorufin deethylase
Ethylmorphine demethylase
(pmol-min - l.mg protein 1) -
N A D P H Generating system N A D P H (10 mM) NADH (10 mM) N A D P H (10 mM) + N A D H (10 mM) Cumene hydroperoxide
4.9 + 2.5 (3) 3.5 ± 0.3 (3) 4.3 + 0.6 (3)
0 (3) 0 (3) 0 (3)
ND 0 (5) 0 (3)
ND 69.0 ± 46.7 (4) 198.0 + 43.8 (2)
3.9 + 0.6 (3) 11.3 + 0.6 (3)
0 (3) 0 (3)
ND ND
141.0 (1) ND
Number o f experiments (in each experiment more than two animals) in parentheses. ND = not determinded; 0 = below detection limit.
42
occurred in the presence o f both N A D P H and N A D H . The rate was, however, more than doubled if cumene hydroperoxide was used instead o f the physiological electron donors. Ethylmorphine demethylase activity was three times higher in the presence of N A D H , than of N A D P H , as an electron donor. When both were present intermediate activity was seen.
Effect o f crayfish hepatopancreas subfractions on monooxygenase activity in rat liver microsomes Since the cytochrome P-450 level in crayfish hepatopancreas was considerably higher than the catalytic activities, its activity could be blocked by the presence of tissue inhibitor(s). It was studied with the aid of isolated rat liver microsomes. These were incubated with the microsomal and 105 000 g supernatant fractions of the crayfish hepatopancreas. The supernatant fraction caused an inhibition o f rat liver 7-ethoxycoumarin O-deethylase and 3,4-benzpyrene hydroxylase activities (Table IV). The heated supernatant did not, however, cause any significant inhibition. Also the crayfish hepatopancreas microsomes had some inhibitory effect in the case of 7-ethoxycoumarin O-deethylase activity. In the case of 3,4-benzpyrene hydroxylase they seemed to increase the activity in the rat liver microsomes a little as did the heated supernatant fraction, too (Table IV). This apparent activation could be the cause o f the extra protein added because 3,4-benzpyrene hydroxylase activity has been shown to be stimulated when protein is added (Pelkonen et al., 1975).
Conjugation activities in crayfish tissues Glutathione S-transferase activity was highest in the green gland but almost as high activity was seen in the hepatopancreas when measured from the tntal homo-
T A B L E IV I n f l u e n c e o f the m i c r o s o m a l f r a c t i o n , n a t i v e a n d h e a t e d 105 000 g s u p e r n a t a n t o f crayfish h e p a t o p a n creas o n the rat liver m i c r o s o m a l m o n o o x y g e n a s e activities (at 37°C). 7-Ethoxycoumarin O-deethylase (o70)
3,4-Benzpyrene hydroxylase (%)
n
RLM R L M + 10/zl M R L M + 25 /zl M
100 99.2 + 1.1 79.6 + 6.5
100 125.6 ___ 6.2 131.7 + 18.3
3 2 3
R L M + 10t~l S R L M + 25 p.l S R L M + 25 /~1 H S
49.2 + 1.1 19.3 +_ 2.7 80.6 + 0.0
95.6 _+ 4.6 48.4 +_ 24.2 147.4 _+ 7.5
2 3 2
R L M = rat liver m i c r o s o m e s = 100o70; M = m i c r o s o m e s o f c r a y f i s h h e p a t o p a n c r e a s ; S = crayfish h e p a t o p a n c r e a s cytosol; H S = h e a t e d cytosol; n = n u m b e r o f d e t e r m i n a t i o n s , in each d e t e r m i n a t i o n 10 animals
43 genates of different crayfish tissues (Table II). Considerable activities were found in intestine and in gills, too. The glutathione S-transferase activity was observed both in the microsomal and cytosol fractions of crayfish hepatopancreas (Table I). The activity was four times higher in the cytosol. The high glutathione S-transferase activity seen in crayfish was still lower than activities in rat liver (not illustrated). Some UDP-glucuronosyltransferase was found in microsomal and cytosol fractions of hepatopancreas (Table I). DISCUSSION Cytochrome P-450 was found in the hepatopancreas of the freshwater crayfish (A. astacus). Some cytochrome P-450 was also detected in the green glands. There is only one study available on freshwater crayfish (Lang et al., 1977), which reports that only traces of cytochrome P-450 could be detected. The reason for the discrepancy with our work could be progress in methodology and in the use of the enzyme stabilizing buffers. The present results indicate that proper care must be taken when the xenobiotic metabolizing enzymes are studied in the crayfish hepatopancreas. Cytochrome P-450 has been reported earlier from the hepatopancreas of the lobster, (Homarus americanus), the spiny lobster, (Panulirus argus) and the blue crab, (Cailinectes sapidus) (Elmamlouk et al., 1974; Bend et al., 1977; Bend and James, 1978; James et al., 1979b). Cytochrome P-450 has also been partially purified from the lobster hepatopancreas (James and Little, 1980). The high amount of cytochrome P-450 in the A. astacus hepatopancreas did not seem to correlate with the very low monooxygenase enzyme activities detected. The substrate binding spectra showed that the affinity of different substrates for cytochrome P-450 was much lower in the case of crayfish hepatopancreas microsomes than in rat liver microsomes. Similar observations have also been made with the cytochrome P-450 of lobster hepatopancreas (Elmamlouk et al., 1974). In that case it was concluded that lobster hepatopancreas cytochrome P-450 exhibits indeed a low affinity for the substrates, or that a bound endogenous substrate limits the enzyme-substrate interactions. Because carbon monoxide (CO) is known to inhibit cytochrome P-450 dependent oxidative metabolism, it has been supposed that the presence of CO or a CO-like ligand in lobster hepatopancreas could also produce the low oxidative activities (James et al., 1979b). It has also been suggested that the primary function of cytochrome P-450 is not the oxidative metabolism of steroids and/or xenobiotics (James et al., 1979b). In the present study the substrate spectrum of the cytochrome P-450 in the hepatopancreas of the A. astacus appeared to be quite different from that in the rat liver. No hydroxylation of 3,4-benzpyrene could be observed in the presence of any electron donors used. 7-Ethoxyresorufin deethylase was also lacking. 7-Ethoxycoumarin deethylase and ethylmorphine demethylase were, however, detected. One reason for the low monooxygenase activities in vitro in the A. astacus
44
hepatopancreas microsomes could be that the NADPH cytochrome c reductase activity in the hepatopancreas microsomes was much lower than NADH cytochrome c reductase. Bend and his collaborators (Bend et al., 1977; James et al., 1979b) have shown that the reduction of cytochrome P-450 with the help of NADPH cytochrome P-450 reductase occurs at a very low rate in lobster hepatopancreas. The present results indicate that in the hepatopancreas microsomes of A . astacus there are different preferences for the source of reducing equivalents. In the case of ethylmorphine demethylase, NADH-supported activity was considerably higher than that in the presence of NADPH. The limiting role of cytochrome P-450 reduction was demonstrated by the higher monooxygenation rate when cumene hydroperoxide was used in the studies of 7-ethoxycoumarin O-deethylase. This has been found previously in spiny lobster (Bend et al., 1977). James and Little (1980) suggested that the reduction of cytochrome P-450 in spiny lobsters could also proceed by a different mechanism, although they found some opposing data. It is noteworthy, that the subfractions of crayfish hepatopancreas inhibited 7-ethoxycoumarin O-deethylation catalyzed by isolated rat liver microsomes. The inhibitory factor was thermolabile. The same effect has also been recorded with spiny lobster hepatopancreas fractions added to sheepshead and stingray hepatic microsomes (James et al., 1979b). The digestive juice from rock crab and lobster hepatopancreas when added in vitro to skate liver microsomes leads to a potent inhibition of skate liver mixed function oxidation (Pohl et al., 1974). The investigators suggested that this could explain the absence of monooxygenase activity in crab or lobster. Lobster hepatopancreas microsomes have also been reported to inhibit the NADPH cytochrome P-450 reductase activity of sheepshead hepatic microsomes (Bend et al., 1977). In the present study the metabolism of foreign compounds and the factors having effect on it was mainly examined in the crayfish hepatopancreas. It would be interesting in future to examine more thoroughly the spectrum of xenobiotic metabolism also in the other tissues with a wider variety of substrates. Considerable glutathione S-transferase activity was found in A . astacus tissues, it was, however, lower than in rat liver. In the hepatopancreas both the microsomal and cytosol fractions contained the enzyme. In lobster, spiny lobster and blue crab glutathione S-transferase activity has also been recorded (Bend et al., 1977, James et al., 1979a). Glutathione S-transferase uses e.g. epoxides as substrates. These are formed by the cytochrome P-450 cored enzyme complex. So the origin of such compounds in crayfish needs clarification. In our studies some freshwater crayfish samples contained minimal UDP-glucuronosyl-transferase activity while others had none. UDP-glucuronosyltransferase activity has not previously been found in A . astacus (Lang et al., 1977). It can be concluded that the tissues of the crayfish (A. astacus) are capable of metabolizing foreign compounds at least to some extent. The monooxygenase system of this species appears to have a substrate spectrum of its own and only a few
45
commonly used model substrates are oxidized in vitro despite the high cytochrome P-450 level. ACKNOWLEDGEMENTS
This study has been supported by grants from Finnish Research Council for Natural Sciences (Project No. 40). We wish to thank Dr. Ossi V. Lindqvist for his advice concerning crayfish physiology and ecology, Mr. Mikko Ik~iheimo for catching the material and Miss Maija-Leena Nyk~inen for her skillful technical assistance. We thank also Dr. Olavi Pelkonen for his advice and criticism.
REFERENCES Airaksinen, M., E.L. Valkama and O.V. Lindqvist, 1977. Distribution of DDT in the crayfish Astacus astacus L. in acute test. In" Freshwater crayfish 3, edited by O.V. Lindqvist. Kuopio, Finland, pp. 349-356. Aitio, A., 1978. A simple and sensitive assay of 7-ethoxycoumarin deethylation. Analyt. Biochem. 85, 488-491. Anders, M.W. and G.J. Mannering, 1966. Kinetics of the inhibition of the N-demethylation of ethylmorphine by 2-diethylaminoethyl 2,2-diphenylvalerate HCI (SKF-525 A) and related compounds. Mol. Pharmacol. 2, 319-327. Bend, J.R. and M.O. James, 1978. Xenobiotic metabolism in marine and freshwater species. In: Biochemical and biophysical perspectives in marine biology, edited by D.C. Malins and J.R. Sargent. Vol. 4, Academic Press, New York, pp. 125-188. Bend, J.R., M.O. James and P.M. Dansette, 1977. In vitro metabolism of xenobiotics in some marine animals. Ann. N.Y. Acad. Sci. 298, 505-521. Brodie, B.B. and R.P. Maickel, 1962. Comparative biochemistry of drug metabolism. In: Proceedings of the 1st Int. pharmacology meeting, Vol. 6, edited by B.B. Brodie and R.P. Maickel, Pergamon Press, pp. 299-324. Burke, M.D. and R.T. Mayer, 1974. Ethoxyresorufin: direct fluorimetric assay of microsomal O-dealkylation which is preferentially inducible by 3-methylcholanthrene. Drug Metab. Disp. 2, 6, 583-588. Dallner, G., P. Siekevitz and G.E. Palade, 1966. Biogenesis of endoplasmic reticulum membranes. 11. Synthesis of constitutive microsomal enzymes in developing rat hepatocyte. J. Cell Biol. 30, 97-117. Elmamlouk, T.H., T. Gessner and A.C. Brownie, 1974. Occurrence of cytochrome P-450 in hepatopancreas of Homarus americanus. Comp. Biochem. Physiol. 48B, 419-425. Habig, W.H., M.J. Pabst and W.B. Jakoby, 1974. Glutathione S-transferase. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130-7139. H~inninen, O., 1968. On the metabolic regulation in the glucuronic acid pathway in the rat tissues. Ann. Acad. Sci. Fenn. A2, 142, 1-96. Isselbacher, K.J., 1956. Enzymatic mechanisms of hormone metabolism. II. Mechanisms of hormonal glucuronide formation. Recent Prog. Horm. Res. Commun. 12, 134-145. James, M.O. and P.J. Little, 1980. Characterization of cytochrome P-450 dependent mixed-function oxidation in the spiny lobster, Panulirus argus. In: Biochemistry, biophysics and regulation of cytochrome P-450, edited by J . A . Gustafsson et al., Elsevier/North-Holland Biomedical Press, pp. 113-120. James, M.O., E.R. Bowen, P.M. Dansette and J.R. Bend, 1979a. Epoxide hydratase and glutathione S-
46 transferase activities with selected alkene and arene oxides in several marine species. Chem. Biol. Interact. 25, 321-344. James, M.O., M.A.Q. Khan and J.R. Bend, 1979b. Hepatic microsomal mixed-function oxidase activities in several marine species common to coastal Florida. Comp. Biochem. Physiol. 62C, 155-164. Johannesen, K.A.M. and J.W. DePierre, 1978. Measurement of cytochrome P-450 in the presence of large amounts of hemoglobin and methemoglobin. Analyt. Biochem. 86, 725-732. Khan, M.A.Q., W. Coello, A.A. Khan and H. Pinto, 1972. Some characteristics of the microsomal mixed function oxidase in the freshwater crayfish, Camparus. Life Sci. 11,405-415. Laitinen, M., 1976. Hepatic and duodenal drug metabolism in rat during fat deficiency. Gen. Pharmacol. 7, 263-277. Lang, M., E.L. Valkama and O.V. Lindqvist, 1977. On the detoxification of foreign compounds by the crayfish Astacus astacus L. In: Freshwater crayfish 3, edited by O.V. Lindqvist. Kuopio, Finland, pp. 343-348. Lindstr6m-Sepp/i, P., U. Koivusaari and O. H~inninen, 1980. Metabolism of xenobiotics by vendace (Coregonus albula). Comp. Biochem. Physiol. 68C, 121-126. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Nebert, D.W. and H.V. Gelboin, 1968. Substrate inducible microsomal arylhydrocarbon hydroxylase. II. Cellular responses during enzyme induction. J. Biol. Chem. 244, 6242-6249. Omura, T. and R. Sato, 1964. The carbon monoxide binding pigment of liver microsomes. J. Biol. Chem. 239, 2379-2385. Pelkonen, O., E.H. Kaltiala, N.T. K~irki, K. Jalonen and K. Py6r~il~i, 1975. Properties of benzpyrene hydroxylase from human liver and comparison with the rat, rabbit and guinea-pig enzymes. Xenobiotica 5, 8, 501-509. Pohl, R.J., J.R. Bend, A.M. Guarino and J.R. Fouts, 1974. Hepatic microsomal mixed-function oxidase activity of several marine species from coastal Maine. Drug Metab. Disp. 2, 6, 545-555. Schenkman, J.B., H. Remmer and R.W. Estabrook, 1967. Spectral studies of drug interaction with hepatic microsomal cytochrome. Mol. Pharmacol. 3, 113-123. Ullrich, V. and P. Weber, 1972. The O-dealkylation of 7-ethoxycoumarin by liver microsomes. A direct fluorometric test. Hoppe-Seyler's Z. Physiol. Chem. 353, 7, 1171-1177. Wattenberg, W.L., J.L. Leong and P.J. Strand, 1962. Benzpyrene hydroxylase activity in the gastrointestinal tract. Cancer Res. 22, 1120.
35
Elsevier Biomedical Press
METABOLISM OF FOREIGN COMPOUNDS IN FRESHWATER CRAYFISH (ASTA CUS A S T A CUS L.) TISSUES
P. LINDSTROM-SEPP,~, U. KOIVUSAARI and O. HANNINEN
Department of Physiology, University of Kuopio, P.O.Box 138, SF-70101 Kuopio 10, Finland (Received 10 February 1982; accepted 29 April 1982)
Freshwater crayfish, Astacus astacus L. were found to have considerable amounts of cytochrome P-450 in the hepatopancreas microsomes. The content was almost as high as in rat liver microsomes. The low (7-ethoxycoumarin O-deethylase and ethylmorphine demethylase) or absent (3,4-benzpyrene hydroxylase and 7-ethoxyresorufin deethylase) monooxygenase activities recorded did not, however, correlate with the high amount of cytochrome P-450. The binding of type I and II substrates to the enzyme was poorer in crayfish hepatopancreas than in rat liver microsomes. There was also less NADPH than NADH cytochrome c reductasc activity in crayfish hepatopancreas microsomes. NADH supported faster demethylation of ethylmorphine than NADPH. Studies with hepatopancreas subfractions added to rat liver microsomes showed that hepatopancreas cytosol contained heat labile monooxygenase inhibitor(s). Only traces of 7-ethoxycoumarin O-deethylase were found in total homogenates of extrahepatopancreatic tissues like gills, intestine and green glands. From conjugation activities considerable levels of glutathione S-transferase were found in all the crayfish tissues studied. UDP-glucuronosyltransferase activity was very low in microsomal and cytosol fractions of hepatopancreas. Key words: Astacus astacus; cytochrome P-450; monooxygenase; glutathione S-transferase; UDP-glucuronosyltransferase
INTRODUCTION
The freshwater crayfish Astacus astacus L. has had economic significance in the Nordic Countries. The crayfish populations have, however, diminished during the current century. One reason has been the crayfish plague fungus (Aphanomyces astact), but this does not fully explain the disappearance. Environmental pollutants may, in places, have had some role to play in the decline. A. astacus appears to be extremely sensitive to pollutants such as DDT (Airaksinen et al., 1977), and it has been reported that this species practically lacks the biotransformation enzymes (Lang et al., 1977). Several other crustacean species especially those living in the sea have, however, significant monooxygenase activities (Brodie and Maickel 1962; Kahn et al., 1972; Bend et al., 1977; James et al., 1979b). Epoxide hydratase (Bend et al., 1977) and glutathione S-transferase activities have been found, too (James et al., 1979a). Therefore one could also expect at least some biotransformation activity in A. astacus. 0166-445X/83/0000-0000/$ 03.00 © 1983 Elsevier Biomedical Press
36 The present study was performed to reinvestigate the biotransformation ability in the tissues of A. astacus. Both monooxygenation as well as conjugation activities were observed. There was a high cytochrome P-450 content in the hepatopancreas but it appeared to have distinctly different catalytic properties than the rat liver cytochrome P-450. MATERIALS AND METHODS
Chemicals 3,4-Benzpyrene, bovine serum albumin, 7-ethoxycoumarin, isocitric-acid dehydrogenase, NADP, NADPH, NADH, phenantroline and trypsin inhibitor were purchased from Sigma Chemical Co. Aprotinin was obtained from Boehringer Mannheim, dithiotreitol from Calbiochem, ethylmorphine and aniline-HCl from Yliopiston Apteekki, Helsinki, Finland and isocitric-acid from Fluka AG. 7-Ethoxyresorufin was a gift from Dr. Antti Zitting, Institute of Occupational Health, Helsinki, Finland.
Biological material Freshwater crayfish (Astacus astacus) of both sexes were collected from June to October from lakes near Kuopio. They were kept fasting in containers with air bubbling at 5°C for 2 to 6 w. The crayfish (n = 72) were 9.1 +_ 0.7 cm (:e ___ SD) long and weighed 27.2 + 7.4 g. The hepatopancreas weighed 1.64 + 0.52 g and the green glands 0.11 + 0.04 g.
Tissue preparation The crayfish were killed by destroying the central nervous system, and tissue sampling was carried out immediately afterwards. The tissues were detached and immersed in ice-cold 0.1 M potassium phosphate buffer, pH 7.4, containing 0.1 M KC1, 1 mM K2EDTA, 1 mM dithiotreitol, 0.1 mM phenantroline and trypsin inhibitor (1 mg/ml). The tissues were homogenized in four volumes of the buffer using a Potter-Elvehjem glass-Teflon homogenizer. This special buffer was used to stabilize the labile enzymes and to avoid autogenous destruction by proteolytic enzymes. Hepatopancreas, in particular, has a lot of proteolytic activity because of the presence of both liver enzymes and digestive juice secreted by the pancreatic cells. 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 a Sorvau OTD-2 ultracentrifuge. The pellet (microsomal fraction) obtained was resuspended in the 0.1 M potassium phosphate buffer with stabilizing agents and 20% glycerol was added for
37 improving the storage. The microsomes were resuspended so that 1 ml contained microsomes from 1 g of tissue. The microsomes were stored at - 8 0 ° C and the determinations were done over 1 to 4 w when no significant inactivation occurred. The cytochrome P-450 determinations were, however, done immediately after preparation. The protein contents of the samples were determined by using the Folin-Ciocalteau method as described by Lowry et al. (1951) with bovine serum albumin as the standard.
Measurement of cytochrome P-450 levels and enzyme activities Cytochrome P-450 content was measured by the method of Omura and Sato (1964) or as described by Johannesen and DePierre (1978). The substrate binding spectra were recorded by the method of Schenkman et al. (1967). Ethylmorphine (type I compound) or aniline-HCl (type II compound) was added to the microsomal suspension containing 1 mg protein/ml. Cytochrome c reductase activities (NADPH or NADH as electron donor) were determined by the method of Dallner et al. (1966). The deethylation of 7-ethoxycoumarin was determined as described by Ullrich and Weber (1972) and modified by Aitio (1978) and the deethylation of 7-ethoxyresorufin as described by Burke and Mayer (1974). The hydroxylation of 3,4-benzpyrene was determined according to Wattenberg et al. (1962) as modified by Nebert and Gelboin (1968). Demethylation of ethylmorphine was determined by using the method of Anders and Mannering (1966). All the enzyme determinations were made at 18°C (Lindstr6m-Sepp~i et al., 1980). An NADPH generating system or NADPH was used as electron donor in these enzyme determinations if not mentioned otherwise. The NADPH generating system was made of 1.13 mM NADP, 12.5 mM isocitric-acid, 0.19 M Tris-HC1-0.056 M KCI buffer (pH 7.4), 12.5 mM MgCI2, 12.5 mM MnCI2 and 0.6 U isocitric-acid dehydrogenase. The influence of time and storage on monooxygenase enzyme activities was studied in various buffers by keeping total homogenates of hepatopancreas for different periods of time ( 0 - 24 h) in an ice-bath. The basic buffer was 0.1 M potassium phosphate buffer (pH 7.4) containing 0.1 M KCI, 1 mM K2EDTA and 1 mM dithiotreitol. The other three buffers were made from this by adding various protease inhibitors (trypsin inhibitor 1 mg/ml, aprotinin 1 mg/ml) or stabilizing agents (0.1 mM phenantroline). The storage of the total homogenates of hepatopancreas at - 0 ° C for 24 h did not affect 7-ethoxycoumarin O-deethylase activities significantly except in the case of buffer containing phenantroline (not illustrated). In that case the monooxygenase activity measured decreased to one third of the initial state. UDP-glucuronosyltransferase activity was measured as described by Isselbacher (1956) and H/inninen (1968) using p-nitrophenol as an aglycone. Glutathione S-
38
transferase was determined by the method of Habig et al. (1974) with 1-chloro-2,4-dinitrobenzene as substrate. These conjugation activities were also measured at 18°C. RESULTS
Cytochrome P-450 and substrate binding Cytochrome P-450 was detected in the hepatopancreas of freshwater crayfish, A. astacus (Fig. 1A) in the same concentrations (0.31 + 0.07 n m o l / m g microsomal protein, Table I) as in rat liver (0.38 + 0.07 n m o l / m g protein in our own studies or 0.42 + 0.02 n m o l / m g protein, Laitinen 1976). In green glands the cytochrome P-450 level was much lower. Hepatopancreas cytochrome P-450 was converted to inactive cytochrome P-450 in the course of time in all buffers used when total homogenates were stored at - 0°C for 24 h (Fig. 1B). The inactivation was only minimal when microsomes were stored at - 8 0 ° C for several weeks. The binding of substrates to cytochrome P-450 in microsomes of freshwater crayfish hepatopancreas and rat liver was determined using ethylmorphine (type I) and aniline (type II) as models. Ethylmorphine showed lower substrate binding in freshwater crayfish hepatopancreas than in rat liver microsomes (Fig. 2A). The spectra were qualitatively different, too. There was not any clear peak in the spectra recorded f r o m crayfish hepatopancreas. The m a x i m u m in rat was in 483-485 nm. The minimum was about 415 nm in crayfish and 416-418 nm in rat.
TABLE I Biotransformation enzyme activities measured from the microsomal fraction and cytosol of the hepatopancreas of freshwater crayfish A . astacus at 18°C.
Cytochrome P-450 N A D P H cytochrome c reductase N A D H cytochrome c reductase 3,4-Benzpyrene hydroxylase 7-Ethoxycoumarin O-deethylase 7-Ethoxyresorufin deethylase Ethylmorphine demethylase UDP-glucuronosyltransferase Glutathione S-transferase
Microsomal fraction (nmol-min - ~.mg protein - 2)
n
Cytosol ( n m o l . m i n - ~-mg protein - 1)
0.31 2.23 48.81 0 0.1 0 0.07 0.01 102.31
10 5 9 7 7 13 4 9 6
0 8.73 0.43 0 0 0 0.02 0.01 394.02
± 0.07 a _+ 0.05 ± 8.14 +_ 0.00 ± 0.05 + 0.01 ± 46.94
_+ ±
0.19 0.11
_+ 0.01 + 165.33
7 2 2 7 7 8 1 8 10
a nmol • protein-2. n = Number of measurements (in each determination more than two animals); 0 = below detection limit.
39
iI
~ F~'
I
5 Onm
I
\ //
l
B
I 450
I
I
400
'
Z.O0
10 n m
ol oo u,-,iti
/ J
Fig. 1. A. CytochromeP-450 spectra from hepatopancreas microsomesof both male ( - ) and female (---) freshwater crayfish (A. astacus) by the method of Johannesen and DePierre (1978). B. Influence of time on cytochromeP-450 measured from the homogenateof crayfish hepatopancreas kept in an ice-bath ( - , measured immediately after homogenization; ---, 3 h later; .... 24 h later).
The type II binding spectrum o f aniline also differed in crayfish hepatopancreas and rat liver microsomes (Fig. 2B). The absorption maximum in crayfish hepatopancreas microsomes was 426-431 nm and in rat liver microsomes 425-427 nm; the minima were between 407-409 nm and 385-387 nm in crayfish hepatopancreas and rat liver microsomes, respectively.
N A D P H and N A D H cytochrome c reductases in crayfish hepatopancreas Distributions o f N A D H and N A D P H cytochrome c reductase during microsomal fractionation differed in the hepatopancreas o f freshwater crayfish (Fig. 3). In the green glands, however, they behaved similarly. In hepatopancreas > 9007oof N A D H cytochrome c reductase activity was found in the microsomal fraction and in the green gland about 8507o. The rest was in the supernatant fraction. Only 20% of the N A D P H cytochrome c reductase activity was in hepatopancreas microsomes, the remaining 80°7o being found in the cytosol. In the green gland the majority (80070) o f the N A D P H cytochrome c reductase activity was located in the microsomal fraction.
Microsomal monooxygenase activities 7-Ethoxycoumarin O-deethylase activities measured in the total homogenates o f
40 B
A Crayfish
,
a' Cd.~
i
I
c
I
400
I
I
500 nm
~'~------'---
I 400
I 500 nm
RoI
Fig. 2. Substrate binding spectra measured from microsomes of crayfish hepatopancreas and rat liver. A. Ethylmorphine as substrate (a = 1.04 mM, b = 3.13 mM, c = 4.17 mM, d = 7.29 mM). B. AnilineHC1 as substrate (a' = 4.17 mM, b ' = 8.33 mM, c' = 16.67 mM, d' = 25.01 mM). The amount of cytochrome P-450 was the same (0.3 nmol • mg protein- 1) in all the recordings.
Activity %
100 // //
50
// // //
/I //
I/ //
// i/
Ii
z
~
[77~
Microsomes
F~I
105.000 x g super
not ont
// // // // // // // //
// //
//
HP
GG t
NADH
I
HP
GG
*
NADPH
Fig. 3. Distribution of N A D H and N A D P H cytochrome c reductase activities in microsomes and 105 000 x g supernatant fraction in hepatopancreas (HP) and green glands (GG) of freshwater crayfish.
hepatopancreas, gills, green glands and intestine are shown in Table II. The m o n o o x y g e n a s e activity was highest in hepatopancreas and only traces were found in other tissues. The m o n o o x y g e n a s e activities measured from the microsomal and 105 000 x g supernatant fraction o f crayfish hepatopancreas are listed in Table I. 3,4-Benzpyrene hydroxylase and 7-ethoxyresorufin deethylase activities were absent, and low levels o f 7-ethoxycoumarin O-deethylase and ethylmorphine demethylase were found in microsomal fraction, although the cytochrome P-450 content was high.
41 TABLE II 7-Ethoxycoumarin O-deethylase and glutathione S-transferase activities in freshwater crayfish determined from total homogenates o f gills, hepatopancreas, intestine and green glands.
Gills Hepatopancreas Intestine Green glands
10 10 5 5
7-Ethoxycoumarin Odeethylase (pmol-min- l.mg protein- 1)
Glutathione S-transferase (nmol.min - ~.mg protein - l)
1.0 22.9 2.6 2.3
75.1 273.1 204.9 321.4
+ + ± ±
1.7 5.3 0.8 1.1
± 30.6 + 39.9 ± 7.4 ± 68.6
Electron donors and monooxygenase activity The influence of different electron donors was determined by studying 7-ethoxycoumarin O-deethylase, 3,4-benzpyrene hydroxylase, 7-ethoxyresorufin deethylase and ethylmorphine demethylase activities. Electron donors used were isocitric acid dehydrogenase mediated NADPH generating system, NADPH, NADH and NADPH-NADH in the case of the first two enzyme reactions. NADPH and NADH were also used when the other two reactions were studied. Cumene hydroperoxide was used to bypass the electron chain to cytochrome P-450 in studies of the oxidation of 7-ethoxycoumarin and 3,4-benzpyrene (Bend et al., 1977). No 3,4-benzpyrene hydroxylase activity could be detected from the hepatopancreas microsomes with any of the physiological electron donors used (Table III). Neither did cumene hydroperoxide reveal any 3,4-benzpyrene hydroxylase activity from the hepatopancreas microsomes of the crayfish. NADPH or NADH could not support 7-ethoxyresorufin deethylase activity. 7-Ethoxycoumarin O-deethylation TABLE III The influence of different electron donors and cumene hydroperoxide on microsomal monooxygenase activities in the hepatopancreas o f freshwater crayfish. 7-Ethoxycoumarin 3,4-Benzpyrene O-deethylase hydroxylase
Ethoxyresorufin deethylase
Ethylmorphine demethylase
(pmol-min - l.mg protein 1) -
N A D P H Generating system N A D P H (10 mM) NADH (10 mM) N A D P H (10 mM) + N A D H (10 mM) Cumene hydroperoxide
4.9 + 2.5 (3) 3.5 ± 0.3 (3) 4.3 + 0.6 (3)
0 (3) 0 (3) 0 (3)
ND 0 (5) 0 (3)
ND 69.0 ± 46.7 (4) 198.0 + 43.8 (2)
3.9 + 0.6 (3) 11.3 + 0.6 (3)
0 (3) 0 (3)
ND ND
141.0 (1) ND
Number o f experiments (in each experiment more than two animals) in parentheses. ND = not determinded; 0 = below detection limit.
42
occurred in the presence o f both N A D P H and N A D H . The rate was, however, more than doubled if cumene hydroperoxide was used instead o f the physiological electron donors. Ethylmorphine demethylase activity was three times higher in the presence of N A D H , than of N A D P H , as an electron donor. When both were present intermediate activity was seen.
Effect o f crayfish hepatopancreas subfractions on monooxygenase activity in rat liver microsomes Since the cytochrome P-450 level in crayfish hepatopancreas was considerably higher than the catalytic activities, its activity could be blocked by the presence of tissue inhibitor(s). It was studied with the aid of isolated rat liver microsomes. These were incubated with the microsomal and 105 000 g supernatant fractions of the crayfish hepatopancreas. The supernatant fraction caused an inhibition o f rat liver 7-ethoxycoumarin O-deethylase and 3,4-benzpyrene hydroxylase activities (Table IV). The heated supernatant did not, however, cause any significant inhibition. Also the crayfish hepatopancreas microsomes had some inhibitory effect in the case of 7-ethoxycoumarin O-deethylase activity. In the case of 3,4-benzpyrene hydroxylase they seemed to increase the activity in the rat liver microsomes a little as did the heated supernatant fraction, too (Table IV). This apparent activation could be the cause o f the extra protein added because 3,4-benzpyrene hydroxylase activity has been shown to be stimulated when protein is added (Pelkonen et al., 1975).
Conjugation activities in crayfish tissues Glutathione S-transferase activity was highest in the green gland but almost as high activity was seen in the hepatopancreas when measured from the tntal homo-
T A B L E IV I n f l u e n c e o f the m i c r o s o m a l f r a c t i o n , n a t i v e a n d h e a t e d 105 000 g s u p e r n a t a n t o f crayfish h e p a t o p a n creas o n the rat liver m i c r o s o m a l m o n o o x y g e n a s e activities (at 37°C). 7-Ethoxycoumarin O-deethylase (o70)
3,4-Benzpyrene hydroxylase (%)
n
RLM R L M + 10/zl M R L M + 25 /zl M
100 99.2 + 1.1 79.6 + 6.5
100 125.6 ___ 6.2 131.7 + 18.3
3 2 3
R L M + 10t~l S R L M + 25 p.l S R L M + 25 /~1 H S
49.2 + 1.1 19.3 +_ 2.7 80.6 + 0.0
95.6 _+ 4.6 48.4 +_ 24.2 147.4 _+ 7.5
2 3 2
R L M = rat liver m i c r o s o m e s = 100o70; M = m i c r o s o m e s o f c r a y f i s h h e p a t o p a n c r e a s ; S = crayfish h e p a t o p a n c r e a s cytosol; H S = h e a t e d cytosol; n = n u m b e r o f d e t e r m i n a t i o n s , in each d e t e r m i n a t i o n 10 animals
43 genates of different crayfish tissues (Table II). Considerable activities were found in intestine and in gills, too. The glutathione S-transferase activity was observed both in the microsomal and cytosol fractions of crayfish hepatopancreas (Table I). The activity was four times higher in the cytosol. The high glutathione S-transferase activity seen in crayfish was still lower than activities in rat liver (not illustrated). Some UDP-glucuronosyltransferase was found in microsomal and cytosol fractions of hepatopancreas (Table I). DISCUSSION Cytochrome P-450 was found in the hepatopancreas of the freshwater crayfish (A. astacus). Some cytochrome P-450 was also detected in the green glands. There is only one study available on freshwater crayfish (Lang et al., 1977), which reports that only traces of cytochrome P-450 could be detected. The reason for the discrepancy with our work could be progress in methodology and in the use of the enzyme stabilizing buffers. The present results indicate that proper care must be taken when the xenobiotic metabolizing enzymes are studied in the crayfish hepatopancreas. Cytochrome P-450 has been reported earlier from the hepatopancreas of the lobster, (Homarus americanus), the spiny lobster, (Panulirus argus) and the blue crab, (Cailinectes sapidus) (Elmamlouk et al., 1974; Bend et al., 1977; Bend and James, 1978; James et al., 1979b). Cytochrome P-450 has also been partially purified from the lobster hepatopancreas (James and Little, 1980). The high amount of cytochrome P-450 in the A. astacus hepatopancreas did not seem to correlate with the very low monooxygenase enzyme activities detected. The substrate binding spectra showed that the affinity of different substrates for cytochrome P-450 was much lower in the case of crayfish hepatopancreas microsomes than in rat liver microsomes. Similar observations have also been made with the cytochrome P-450 of lobster hepatopancreas (Elmamlouk et al., 1974). In that case it was concluded that lobster hepatopancreas cytochrome P-450 exhibits indeed a low affinity for the substrates, or that a bound endogenous substrate limits the enzyme-substrate interactions. Because carbon monoxide (CO) is known to inhibit cytochrome P-450 dependent oxidative metabolism, it has been supposed that the presence of CO or a CO-like ligand in lobster hepatopancreas could also produce the low oxidative activities (James et al., 1979b). It has also been suggested that the primary function of cytochrome P-450 is not the oxidative metabolism of steroids and/or xenobiotics (James et al., 1979b). In the present study the substrate spectrum of the cytochrome P-450 in the hepatopancreas of the A. astacus appeared to be quite different from that in the rat liver. No hydroxylation of 3,4-benzpyrene could be observed in the presence of any electron donors used. 7-Ethoxyresorufin deethylase was also lacking. 7-Ethoxycoumarin deethylase and ethylmorphine demethylase were, however, detected. One reason for the low monooxygenase activities in vitro in the A. astacus
44
hepatopancreas microsomes could be that the NADPH cytochrome c reductase activity in the hepatopancreas microsomes was much lower than NADH cytochrome c reductase. Bend and his collaborators (Bend et al., 1977; James et al., 1979b) have shown that the reduction of cytochrome P-450 with the help of NADPH cytochrome P-450 reductase occurs at a very low rate in lobster hepatopancreas. The present results indicate that in the hepatopancreas microsomes of A . astacus there are different preferences for the source of reducing equivalents. In the case of ethylmorphine demethylase, NADH-supported activity was considerably higher than that in the presence of NADPH. The limiting role of cytochrome P-450 reduction was demonstrated by the higher monooxygenation rate when cumene hydroperoxide was used in the studies of 7-ethoxycoumarin O-deethylase. This has been found previously in spiny lobster (Bend et al., 1977). James and Little (1980) suggested that the reduction of cytochrome P-450 in spiny lobsters could also proceed by a different mechanism, although they found some opposing data. It is noteworthy, that the subfractions of crayfish hepatopancreas inhibited 7-ethoxycoumarin O-deethylation catalyzed by isolated rat liver microsomes. The inhibitory factor was thermolabile. The same effect has also been recorded with spiny lobster hepatopancreas fractions added to sheepshead and stingray hepatic microsomes (James et al., 1979b). The digestive juice from rock crab and lobster hepatopancreas when added in vitro to skate liver microsomes leads to a potent inhibition of skate liver mixed function oxidation (Pohl et al., 1974). The investigators suggested that this could explain the absence of monooxygenase activity in crab or lobster. Lobster hepatopancreas microsomes have also been reported to inhibit the NADPH cytochrome P-450 reductase activity of sheepshead hepatic microsomes (Bend et al., 1977). In the present study the metabolism of foreign compounds and the factors having effect on it was mainly examined in the crayfish hepatopancreas. It would be interesting in future to examine more thoroughly the spectrum of xenobiotic metabolism also in the other tissues with a wider variety of substrates. Considerable glutathione S-transferase activity was found in A . astacus tissues, it was, however, lower than in rat liver. In the hepatopancreas both the microsomal and cytosol fractions contained the enzyme. In lobster, spiny lobster and blue crab glutathione S-transferase activity has also been recorded (Bend et al., 1977, James et al., 1979a). Glutathione S-transferase uses e.g. epoxides as substrates. These are formed by the cytochrome P-450 cored enzyme complex. So the origin of such compounds in crayfish needs clarification. In our studies some freshwater crayfish samples contained minimal UDP-glucuronosyl-transferase activity while others had none. UDP-glucuronosyltransferase activity has not previously been found in A . astacus (Lang et al., 1977). It can be concluded that the tissues of the crayfish (A. astacus) are capable of metabolizing foreign compounds at least to some extent. The monooxygenase system of this species appears to have a substrate spectrum of its own and only a few
45
commonly used model substrates are oxidized in vitro despite the high cytochrome P-450 level. ACKNOWLEDGEMENTS
This study has been supported by grants from Finnish Research Council for Natural Sciences (Project No. 40). We wish to thank Dr. Ossi V. Lindqvist for his advice concerning crayfish physiology and ecology, Mr. Mikko Ik~iheimo for catching the material and Miss Maija-Leena Nyk~inen for her skillful technical assistance. We thank also Dr. Olavi Pelkonen for his advice and criticism.
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