Fish Physiology and Biochemistry vol. 13 no. 4 pp 335-342 (1994) Kugler Publications, Amsterdam/New York
Distribution and induction of cytochrome P450 1A1 in the rainbow trout brain Tommy Anderssonl* and Anders Goksoyr 2 l Department of Zoophysiology, University of Goteborg, Box 25059, 400 31 Goteborg, Sweden; 2 Laboratory of Marine Molecular Biology, University of Bergen, HIB, N-5020 Bergen, Norway Accepted: May 27, 1994 Keywords: fish, rainbow trout, brain, cytochrome P450, CYPIA1, induction, subcellular fractions
Abstract Cytochrome P450 (CYP) A1 participates in the activation as well as detoxification of environmental pollutants such as aromatic hydrocarbons. This CYP form is also efficiently induced by aromatic hydrocarbons. The presence of CYP A1 in the brain might thus be of physiological and toxicological importance. In the present investigation on rainbow trout, the distribution of 7-ethoxyresorufin-O-deethylase (EROD) activity, a cytochrome CYP A1 catalyzed reaction, was measured in whole tissue homogenates from brain parts. In control fish, a relatively high activity was found in the rainbow trout olfactory bulb compared to the other brain parts. Although an EROD induction (3 to 7-fold) by jB-naphthoflavone (BNF) was recorded in all brain parts from the rainbow trout, the highest induced activity was measured in the olfactory bulbs. To ascertain the distribution of EROD activity in cells, whole brain tissue was subfractionated by differential centrifugation. The fractionation scheme separated mitochondria (P2 fraction) and microsomes (P3 fraction) as determined by marker enzymes and electron microscopy. In control rainbow trout, a low EROD activity could be measured in the P2 fraction. BNF induced the EROD activity in both P2 and P3 fractions. Western blotting showed the induction by BNF of a protein band in the P2 and P3 fractions with a molecular mass around 58,000 when highly specific anti-cod CYP AI antibodies were used. ELISA measurements confirmed the induction of CYP 1AI protein in the rainbow trout brain subcellular fractions.
Introduction The cytochrome P450 (CYP 3 ) enzyme system is involved in bioactivation and/or detoxification of many organic compounds. Much attention has been directed towards members in the CYP 1A subfamily because of their role in metabolism and acti-
vation of aromatic hydrocarbons to cytotoxic intermediates (Guengerich 1988). Xenobiotic metabolizing CYPs are most abundant in the liver but also present in many extrahepatic tissues. In mammals, the level of CYP in the brain is considerably lower than in the liver (Guengerich and Mason 1979; Marietta et al. 1979). In spite of the low cerebral
3 Abbreviations: CYP, cytochrome P450; EROD, 7-ethoxyresorufin-O-deethylase;
BNF, -naphthoflavone; ELISA, enzyme-linked immunosorbent assay; PAH, polyaromatic hydrocarbon, PCB, polychlorinated biphenyl; PCDD, polychlorinated dibenzo-p-dioxin; PCDF polychlorinated dibenzofuran; P450 c and P450 d are trivial names for CYP AI and CYP 1A2, respectively. * Correspondenceto: Tommy B. Andersson, Department of Pharmacokinetics and Drug Metabolism, Astra Hassle AB, S-431 83 M61ndal, Sweden, Fax 46 - 31 776 3701
336 CYP levels several CYP activities have been detected and immunohistochemical studies have located individual forms to specific areas and cells (Mesnil and Testa 1984; K6hler et al. 1988; Warner et al. 1988; Hansson et al. 1990). Immunohistochemical studies also suggested that CYP 1A or immunologically similar forms are constitutively expressed in rat brain (Kohler et al. 1988). Moreover, treatment of rats with polyaromatic hydrocarbon (PAH)-type inducers increased brain 7-ethoxyresorufin-Odeethylase (EROD) activity, which indicate induction of a CYP 1A form (Walther et al. 1987; Unkila et al. 1993). The brain may thus be an important target organ for environmental pollutants, contributing to neurotoxicological or endocrinological effects of such compounds. In all fish species studied only one member of the CYP 1A subfamily has been identified. So far the biochemical and regulatory properties of CYP 1A in rainbow trout (Oncorhynchus mykiss), scup (Stenotomus chrusops) and cod (Gadus morhua) indicate that this form is related to the mammalian CYP 1Al form (for review see Goksoyr and F6rlin 1992). As in mammals, the basal levels of CYP A1 in fish are often low or non-detectable. However, the expression of CYP lAl is highly inducible by several PAHs such as polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). Several studies have shown that fish living in areas polluted with PAHs have induced hepatic CYP A1 levels. Recently CYP 1AI immunoreactivity in brain vascular endothelial cells was detected in PCDF-treated scup (Smolowitz et al. 1991). The presence of CYP Al in brain endothelial cells was also demonstrated in scup from PAH-polluted areas (Stegeman et al. 1991). Furthermore, male rainbow trout pituitary CYP 1AI was found to be elevated by a PAH-type inducer (13naphthoflavone, BNF) (Andersson et al. 1993). Pituitary cells containing inducible CYP lAl were identified as gonadotrophs containing gonadotropin II, indicating one possible link between reproductive disturbances and PAH exposure. The brain of several fish species accumulates relatively high levels of PAHs (Varanasi et al. 1989). Moreover, experimental exposure of teleost fish to several aromatic hydrocarbons such as hex-
achlorbenzene, PCBs and PCDDs has shown a great species difference in their accumulation in the brain (Ingebrigtsen and Solbakken 1985; Ingebrigtsen et al. 1992; Hektoen et al. 1992). Tape section autoradiography has revealed high concentrations of these compounds in the cod brain whereas considerably lower levels were found in the rainbow trout brain. In teleosts, the toxicological significance of brain CYP could therefore be species specific. Cod and rainbow trout were recently reported to exhibit CYP-dependent 7-ethoxycoumarin-O-deethylase activity and UDP glucuronosyltransferase activity using p-nitrophenol as substrate (Ingebrigtsen et al. 1992). In this study, we have investigated the regional and subcellular distribution as well as the inducibility of CYP in the rainbow trout brain.
Materials and methods Animals Rainbow trout (Oncorhynchus mykiss) were obtained from a local fish farm. Fish were kept in aquaria with circulating fresh water. BNF (50 mg kg-1 body weight) was given intraperitoneally 3 times in two day intervals. Two days after the last injection the fish were killed and the brain was removed and rinsed in ice-cold 0.1 M phosphate buffer (pH 7.4).
Homogenization and subcellularfractionation The whole brain or the various parts (defined as described by Harder (1975)) were homogenized in a Potter-Elvehjem glass-teflon homogenizer by 4 strokes in 4 volumes of 0.1 M phosphate buffer containing 0.15 M potassium chloride, 0.2 mM butylhydroxytoluene, 1 mM dithiotreitol, 0.1 mM EDTA and 0.1 mM phenylmethylsulfonyl fluoride. Thereafter the homogenate was ultrasonicated for 15 s. To study the distribution of enzyme activities in subcellular fractions the following procedure was used. The tissue homogenate was centrifuged at
337 1,240 x g for 10 min. The pellet was homogenized and recentrifuged at the same force resulting in pellet 1 (P1). The pooled supernatants were centrifuged at 15,000 x g for 20 min to yield pellet 2 (P2). The resulting supernatant was centrifuged at 180,000 x g for 90 min to obtain the final pellet (P3) and a high speed supernatant.
Assays 7-Ethoxyresorufin-O-deethylase activity was measured as described by Andersson et al. (1985). NADPH-cytochrome c reductase and cytochrome oxidase was measured as described by Masters et al. (1967) and Sottocasa et al. (1967) respectively. Protein content was determined by the method of Lowry et al. (1951), using bovine serum albumin as a standard.
Immunochemical studies Western blotting was performed after electrophoresis of samples in 9% sodium dodecylsulfate polyacrylamine gelelectrophoresis (SDS-PAGE). Transfer of the protein to nitrocellulose sheets was performed in a Mini-Protean II blotting cell (BioRad) according to the manufacturer's procedures. Molecular weight of stained proteins on the Western blot were determined by comparing the relative mobilities with molecular weight standards. Rabbit anti-cod CYP A1 (cod cytochrome P450c, Goksoyr 1985) serum was purified on a protein ASuperose column. The resulting IgG fraction was further purified by affinity chromatography, using purified cod P450 1AI coupled to Tosyl-activated magnetic beads (Dynabeads M-280, Dynalas) and was used as a primary antibody in the study of antiCYP IAl-cross-reacting proteins. Indirect ELISA was performed by the method of Goksoyr (1991), employing anti-CYP A1 IgG as primary antibody. Horseradish-peroxidase conjugated goat anti-rabbit IgG (GAR-HRP) was used as secondary antibody.
Distribution of cytochrome oxidase and NADPHcytochrome c reductase activity after subcellular fractionation of the rainbow trout brain. The values are presented as DeDuve plots (DeDuve et al. 1955) and are means of relative specific activities (RSA) from three experiments containing a pool of two brains each. RSA = the percent of total activity/per cent of total protein. The fractions from left to right are the 1,240 g pellet (PI), 15,000 g pellet (P2), 18,000 g pellet (P3) and the 180,000 supernatant.
Results EROD values measured in whole tissue homogenates from the various brain parts exhibited a wide variation in activity as can be seen from the SD values (Table 1). However, the mean EROD activity in whole tissue homogenates from the control fish were found to be rather evenly distributed between the different brain parts, except for the olfactory bulb which showed a relatively high activity. Treatmen of rainbow trout with BNF induced the activity 7-fold in the olfactory bulb, telencephalonand and cerebellum, 6-fold in the hypothalamus and 3-fold in the optic tectum. To ascertain the subcellular distribution of EROD activity in the cells, whole brain tissue was subfractionated by differential centrifugation (Fig. 1). The values are presented as DeDuve plots (DeDuve et al. 1955). Cytochrome oxidase, a marker for mitochondria, was enriched 7.6-fold in the P2 pellet and NADPH cytochrome c reductase, a Table 1. EROD activities in whole tissue homogenate of different brain parts from control and BNF treated fish. Values (pmol mg protein-' min-l) are mean SD of six samples, each sample containing tissue from two fish. *p < 0.05 (Mann-Whitney U-test) Tissue
Control
BNF
Olfactory Telencephalon Optic tectum Hypothalamus Cerebellum
1.15 +2.83 0.36± 0.56 0.37 ± 0.42 0.36 ± 0.34 0.15 ±0.20
8.32 ± 2.92* 2.74± 1.15' 1.17 +0.74* 2.14 + 0.98* 1.13 ±0.19*
338 Rainbow trout
P3 fractions from BNF-treated rainbow trout whereas no staining could be seen in fractions from control fish (Fig. 4). When the anti-cod CYP IA1 antibodies were used in an indirect ELISA the absorbance readings showed that the P3 levels were as high or higher than in the P2 fractions from control fish (Fig. 5). BNF-treatment significantly increased the CYP 1Al levels in rainbow trout P2 and P3 fractions.
cytochrome oxidase 8 6 4. 0 0
2. Pi
Q 0.
P2 P3
S
Discussions
0
u0
rome NADPH -cytochlrome c reductase
0 h..
a
4. 2-
P3
p 0
i-
0
S
P2 I
I $
50 % of total protein
!
100
Fig. 1. Distribution of cytochrome oxidase and NADPHcytochrome c reductase activity after subcellular fractionation of the rainbow trout brain. The values are presented as DeDuve plots (DeDuve et al. 1955) and are means of relative specific activities (RSA) from three experiments containing a pool of two brains each. RSA = the per cent of total activity/per cent of total protein. The fractions from left to right are the 1,240 g pellet (P1), 15,000 g pellet (P2), 18,000 g pellet (P3) and the 180,000 supernatant.
marker for endoplasmic reticulum, was enriched 4.5-fold in the P3 pellet. Very low cytochrome oxidase activity was measured in the P3 pellet and low NADPH-cytochrome c reductase activity was measured in the P2 pellet indicating that the subfractionation method efficiently separated mitochondria and microsomes. Electronmicrographs of P2 and P3 fractions from rainbow trout brain indicated that they consisted mainly of mitochondria and microsomes, respectively (Fig. 2). A low basal EROD activity was measured in the brain P2 fraction (Fig. 3) whereas no activity was detected in the P3 fraction. BNF-treatment induced the EROD activity in both P2 and P3 fractions. By Western blotting technique using ant-cod CYP lAl IgG, a protein with relative molecular mass of 58,000 Da was weakly stained in the P2 and
The present study demonstrates that rainbow trout brain contain CYP lAl or a functionally and immunochemically similar enzyme. The brain EROD activity is considerably lower than the EROD activity measured in the liver microsoma fraction reported in other studies. In control trout, a low EROD activity of 1.4 pmol/mg protein/min was measured in the P2 fraction whereas no activity was detected in the P3 fraction. This activity in rainbow trout brain P2 fraction is 14-50-fold lower than the EROD activity reported for hepatic microsomes (Goksoyr et al. 1987, 1991). In the scup, the brain microscomal benzo(a)pyrene hydroxylase activity was previously reported to be 980-fold lower than the liver microsomal activity (Stegeman et al. 1979). Mitochondrial CYP enzymes are considered to be highly substrate specific and known to participate in physiologically important hydroxylation reactions such as steroidogenesis in gonads and adrenal tissues, vitamin D3 metabolism in kidney and liver and bile acid formation in the liver, whereas xenobiotic metabolizing P450 enzymes are considered to be restricted to the microsomal fraction (Yang and Lu 1987). However, the results from the present investigation suggest that brain xenobiotic metabolizing CYP enzymes are confined both to the mitochondria and microsomal fractions. This is in accordance with the observations in mammals where xenobiotic metabolizing CYP activities in the brain can be detected in both the mitochondrial and microsomal fractions (Walther et al. 1986, 1987; Das et al. 1982; Ghersi-Egea et al. 1988; Iscan et al. 1990). Furthermore, in mammals the total content of CYP in the mitochondrial fraction
339
Fig. 2. Electron micrographs of (A) the 15,000 g pellet (P2) and (B) the 180,000 g pellet (P3) from rainbow trout brain.
8
C
E
6
o
4 E o
E
2
0
P2
P3
Rainbow trout Fig. 3. EROD activity in brain P2 and P3 fractions from control (open bars) and BNF treated (hatched bars) rainbow trout brain. Values are means _+SD of 4-5 fish.
was considerably higher than in the microsomal fraction. However, the rainbow trout brain P2 pellet was not a pure mitochondrial fraction. It is therefore possible that other cellular organelles or
Fig. 4. Western blot analysis of P4501AI in subcellular fractions from the rainbow trout brain. I and 2 designates control P2 and P3 fractions, respectively. 3 and 4 designates 0-naphthoflavone treated P2 and P3 fractions; 20 gg of protein was applied in each lane.
small cells cosedimenting with the mitochondria could contribute to the observed EROD activity. BNF induced brain EROD activities in both P2 and P3 fractions. Western blotting and ELISA
340
0.150
a)
0 c-
o
T
0.100-
0 o u~ ~D
I
0.050-
4
0.000
P2
P3
Rainbow trout Fig. 5. P4501AI level in brain P2 and P3 from control (open bars) and BNF-treated (hatched bars) rainbow trout measured as absorbance in an indirect ELISA. Values are means SD of 4-5 fish.
measurements using anti-cod CYP lAl IgG confirmed induction of CYP1Al protein in rainbow trout brain subcellular fractions. Inhibition of EROD activity with anti-cod CYP Al would be a more clear indication that this activity is of CYP lAl origin. However, this antibody was previously shown not to inhibit rainbow trout liver microsomal EROD activity (Goksoyr et al. 1991). Although an EROD induction by BNF was recorded in all brain parts from the rainbow trout, the highest absolute activity in BNF-treated fish was measured in the olfactory bulbs. The olfactory bulb may thus be especially susceptible to CYP AI inducing compounds. Several studies on mammalian brain CYP have reported that the olfactory bulb contains higher CYP-dependent activities than other brain parts (Das et al. 1982; Iscan et al. 1990).
Unkila et al. (1993) showed that the EROD activity in the rat olfactory bulbs was highly inducible by TCDD. CYP 1Al/1A2 has been located to the tufted cells in the rat olfactory bulb by immunohistochemical methods (Kohler et al. 1988). In the scup, CYP lAl immunoreactive cells in the brain were only found in the endothelial vascu-
lar cells from experimental or environmentally induced animals (Smolowitz et al. 1991; Stegeman et al. 1991). A substantial part of the CYP in the rat brain has also been reported to be confined to the microvessels (Ghersi-Egea et al. 1988). Since xenobiotics transported in the blood must cross the endothelial cells before reaching the brain the capacity of the endothelial CYP enzyme system to convert lipophilic xenobiotics to more hydrophilic products may affect the transport of these substances into the brain. In rat, CYP A-immunoreactivity has also been found in neuronal cellbodies and glial cells (Kohler et al. 1988). In rat brain preparations, the neuronal cells exhibited 3 fold higher EROD activity than the glial cells (Dhawman et al. 1990). Furthermore, EROD activity was inducible (75-120%) by 3-methylcholanthrene in neuronal and glial cell preparations. It is clearly of toxicological importance to further study the localization of CYP in fish brain. Previous studies have shown that aromatic hydrocarbons are enriched in the cod brain whereas no corresponding accumulation can be seen in the rainbow trout brain (Ingebrigtsen and Solbakken 1985; Ingebrigtsen et al. 1992; Hektoen et al. 1992). Furthermore, in cod exposed to radiolabeled 2, 4' ,5-trichlorobiphenyl the cerebrospinal fluid accumulated radioactive compounds which were analyzed as metabolites whereas no accumulation of radioactivity was seen in rainbow trout cerebrospinal fluid (Ingebrigtsen et al. 1992). The metabolites in cod cerebrospinal fluid are probable formed in the brain and then excreted into the fluid. An EROD activity has been detected in the cod brain (Andersson and Goksoyr, unpublished results). This activity was inducible by BNF. The difference in accumulation and metabolite formation between cod and rainbow trout brain might be explained on a toxicokinetic basis due to great difference in body lipid content. However, the properties and distribution of xenobiotic metabolizing enzymes in the central nervous system may also be an important factor. The induction of CYP Al is an important phenomenon which modulates the toxicity of aromatic hydrocarbons. The increased CYP AI activity leads to a higher capacity of the system to bio-
341 transform lipophilic compounds to more polar products and thus facilitate their excretion. In addition, the presence of a brain UDP glucuronosyltransferase activity was recently found int he rainbow trout (Ingebrigtsen et al. 1992). This enzyme participates in detoxication of many substances formed by CYP monoogygenations and greatly increases their polarity which facilitate their excretion into aquous solutions such as urine and bile. However, the elimination route for water soluble products formed in the fish brain is not well studied. Watersoluble xenobiotic metabolites may, at least partly, be secreted into the cerebrospinal fluid (Ingebrigtsen et al. 1992). Induction of CYP lAI could, however, have other consequences. Highly reactive intermediates with cytotoxic and mutagenic properties may be formed during CYPcatalyzed reactions (Guengerich 1988). The importance of the CYP enzyme system in degenerative diseases of the brain and xenobiotic dependent brain toxicity is well recognized in mammalian studies (Mesnil and Testa 1984). An increased level of CYP AI could also lead to an elevated metabolism of essential endogenous compounds or to the production of bioactive substances (Nebert 1991). The purified rat CYP 1AI was recently shown to be specifically active in monohydroxylating arachidonic acid in the 19 position (Falck et al. 1990). This metabolite has been shown to stimulate Na + /K + -ATPase activity in vascular smooth muscle (Escalante et al. 1990). Further exploration of the distribution of CYP A1 in various regions and cell types of the fish will be relevant for the understanding of the toxicity of organic pollutants.
Acknowledgements We are grateful to Eva Nilsson and Helen Nilsson (Dept. Zoophysiology, G6teborg), Sissel Olsen and Kjersti Helgesen (Lab. Mar. Molec. Biol., Bergen) for expert technical assistance. This study was supported by the Swedish Environmental Protection Board (T.A.) and the Norwegian Fisheries Research Council (NFFR) (A.G.)
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Distribution and induction of cytochrome P450 1A1 in the rainbow trout brain Tommy Anderssonl* and Anders Goksoyr 2 l Department of Zoophysiology, University of Goteborg, Box 25059, 400 31 Goteborg, Sweden; 2 Laboratory of Marine Molecular Biology, University of Bergen, HIB, N-5020 Bergen, Norway Accepted: May 27, 1994 Keywords: fish, rainbow trout, brain, cytochrome P450, CYPIA1, induction, subcellular fractions
Abstract Cytochrome P450 (CYP) A1 participates in the activation as well as detoxification of environmental pollutants such as aromatic hydrocarbons. This CYP form is also efficiently induced by aromatic hydrocarbons. The presence of CYP A1 in the brain might thus be of physiological and toxicological importance. In the present investigation on rainbow trout, the distribution of 7-ethoxyresorufin-O-deethylase (EROD) activity, a cytochrome CYP A1 catalyzed reaction, was measured in whole tissue homogenates from brain parts. In control fish, a relatively high activity was found in the rainbow trout olfactory bulb compared to the other brain parts. Although an EROD induction (3 to 7-fold) by jB-naphthoflavone (BNF) was recorded in all brain parts from the rainbow trout, the highest induced activity was measured in the olfactory bulbs. To ascertain the distribution of EROD activity in cells, whole brain tissue was subfractionated by differential centrifugation. The fractionation scheme separated mitochondria (P2 fraction) and microsomes (P3 fraction) as determined by marker enzymes and electron microscopy. In control rainbow trout, a low EROD activity could be measured in the P2 fraction. BNF induced the EROD activity in both P2 and P3 fractions. Western blotting showed the induction by BNF of a protein band in the P2 and P3 fractions with a molecular mass around 58,000 when highly specific anti-cod CYP AI antibodies were used. ELISA measurements confirmed the induction of CYP 1AI protein in the rainbow trout brain subcellular fractions.
Introduction The cytochrome P450 (CYP 3 ) enzyme system is involved in bioactivation and/or detoxification of many organic compounds. Much attention has been directed towards members in the CYP 1A subfamily because of their role in metabolism and acti-
vation of aromatic hydrocarbons to cytotoxic intermediates (Guengerich 1988). Xenobiotic metabolizing CYPs are most abundant in the liver but also present in many extrahepatic tissues. In mammals, the level of CYP in the brain is considerably lower than in the liver (Guengerich and Mason 1979; Marietta et al. 1979). In spite of the low cerebral
3 Abbreviations: CYP, cytochrome P450; EROD, 7-ethoxyresorufin-O-deethylase;
BNF, -naphthoflavone; ELISA, enzyme-linked immunosorbent assay; PAH, polyaromatic hydrocarbon, PCB, polychlorinated biphenyl; PCDD, polychlorinated dibenzo-p-dioxin; PCDF polychlorinated dibenzofuran; P450 c and P450 d are trivial names for CYP AI and CYP 1A2, respectively. * Correspondenceto: Tommy B. Andersson, Department of Pharmacokinetics and Drug Metabolism, Astra Hassle AB, S-431 83 M61ndal, Sweden, Fax 46 - 31 776 3701
336 CYP levels several CYP activities have been detected and immunohistochemical studies have located individual forms to specific areas and cells (Mesnil and Testa 1984; K6hler et al. 1988; Warner et al. 1988; Hansson et al. 1990). Immunohistochemical studies also suggested that CYP 1A or immunologically similar forms are constitutively expressed in rat brain (Kohler et al. 1988). Moreover, treatment of rats with polyaromatic hydrocarbon (PAH)-type inducers increased brain 7-ethoxyresorufin-Odeethylase (EROD) activity, which indicate induction of a CYP 1A form (Walther et al. 1987; Unkila et al. 1993). The brain may thus be an important target organ for environmental pollutants, contributing to neurotoxicological or endocrinological effects of such compounds. In all fish species studied only one member of the CYP 1A subfamily has been identified. So far the biochemical and regulatory properties of CYP 1A in rainbow trout (Oncorhynchus mykiss), scup (Stenotomus chrusops) and cod (Gadus morhua) indicate that this form is related to the mammalian CYP 1Al form (for review see Goksoyr and F6rlin 1992). As in mammals, the basal levels of CYP A1 in fish are often low or non-detectable. However, the expression of CYP lAl is highly inducible by several PAHs such as polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). Several studies have shown that fish living in areas polluted with PAHs have induced hepatic CYP A1 levels. Recently CYP 1AI immunoreactivity in brain vascular endothelial cells was detected in PCDF-treated scup (Smolowitz et al. 1991). The presence of CYP Al in brain endothelial cells was also demonstrated in scup from PAH-polluted areas (Stegeman et al. 1991). Furthermore, male rainbow trout pituitary CYP 1AI was found to be elevated by a PAH-type inducer (13naphthoflavone, BNF) (Andersson et al. 1993). Pituitary cells containing inducible CYP lAl were identified as gonadotrophs containing gonadotropin II, indicating one possible link between reproductive disturbances and PAH exposure. The brain of several fish species accumulates relatively high levels of PAHs (Varanasi et al. 1989). Moreover, experimental exposure of teleost fish to several aromatic hydrocarbons such as hex-
achlorbenzene, PCBs and PCDDs has shown a great species difference in their accumulation in the brain (Ingebrigtsen and Solbakken 1985; Ingebrigtsen et al. 1992; Hektoen et al. 1992). Tape section autoradiography has revealed high concentrations of these compounds in the cod brain whereas considerably lower levels were found in the rainbow trout brain. In teleosts, the toxicological significance of brain CYP could therefore be species specific. Cod and rainbow trout were recently reported to exhibit CYP-dependent 7-ethoxycoumarin-O-deethylase activity and UDP glucuronosyltransferase activity using p-nitrophenol as substrate (Ingebrigtsen et al. 1992). In this study, we have investigated the regional and subcellular distribution as well as the inducibility of CYP in the rainbow trout brain.
Materials and methods Animals Rainbow trout (Oncorhynchus mykiss) were obtained from a local fish farm. Fish were kept in aquaria with circulating fresh water. BNF (50 mg kg-1 body weight) was given intraperitoneally 3 times in two day intervals. Two days after the last injection the fish were killed and the brain was removed and rinsed in ice-cold 0.1 M phosphate buffer (pH 7.4).
Homogenization and subcellularfractionation The whole brain or the various parts (defined as described by Harder (1975)) were homogenized in a Potter-Elvehjem glass-teflon homogenizer by 4 strokes in 4 volumes of 0.1 M phosphate buffer containing 0.15 M potassium chloride, 0.2 mM butylhydroxytoluene, 1 mM dithiotreitol, 0.1 mM EDTA and 0.1 mM phenylmethylsulfonyl fluoride. Thereafter the homogenate was ultrasonicated for 15 s. To study the distribution of enzyme activities in subcellular fractions the following procedure was used. The tissue homogenate was centrifuged at
337 1,240 x g for 10 min. The pellet was homogenized and recentrifuged at the same force resulting in pellet 1 (P1). The pooled supernatants were centrifuged at 15,000 x g for 20 min to yield pellet 2 (P2). The resulting supernatant was centrifuged at 180,000 x g for 90 min to obtain the final pellet (P3) and a high speed supernatant.
Assays 7-Ethoxyresorufin-O-deethylase activity was measured as described by Andersson et al. (1985). NADPH-cytochrome c reductase and cytochrome oxidase was measured as described by Masters et al. (1967) and Sottocasa et al. (1967) respectively. Protein content was determined by the method of Lowry et al. (1951), using bovine serum albumin as a standard.
Immunochemical studies Western blotting was performed after electrophoresis of samples in 9% sodium dodecylsulfate polyacrylamine gelelectrophoresis (SDS-PAGE). Transfer of the protein to nitrocellulose sheets was performed in a Mini-Protean II blotting cell (BioRad) according to the manufacturer's procedures. Molecular weight of stained proteins on the Western blot were determined by comparing the relative mobilities with molecular weight standards. Rabbit anti-cod CYP A1 (cod cytochrome P450c, Goksoyr 1985) serum was purified on a protein ASuperose column. The resulting IgG fraction was further purified by affinity chromatography, using purified cod P450 1AI coupled to Tosyl-activated magnetic beads (Dynabeads M-280, Dynalas) and was used as a primary antibody in the study of antiCYP IAl-cross-reacting proteins. Indirect ELISA was performed by the method of Goksoyr (1991), employing anti-CYP A1 IgG as primary antibody. Horseradish-peroxidase conjugated goat anti-rabbit IgG (GAR-HRP) was used as secondary antibody.
Distribution of cytochrome oxidase and NADPHcytochrome c reductase activity after subcellular fractionation of the rainbow trout brain. The values are presented as DeDuve plots (DeDuve et al. 1955) and are means of relative specific activities (RSA) from three experiments containing a pool of two brains each. RSA = the percent of total activity/per cent of total protein. The fractions from left to right are the 1,240 g pellet (PI), 15,000 g pellet (P2), 18,000 g pellet (P3) and the 180,000 supernatant.
Results EROD values measured in whole tissue homogenates from the various brain parts exhibited a wide variation in activity as can be seen from the SD values (Table 1). However, the mean EROD activity in whole tissue homogenates from the control fish were found to be rather evenly distributed between the different brain parts, except for the olfactory bulb which showed a relatively high activity. Treatmen of rainbow trout with BNF induced the activity 7-fold in the olfactory bulb, telencephalonand and cerebellum, 6-fold in the hypothalamus and 3-fold in the optic tectum. To ascertain the subcellular distribution of EROD activity in the cells, whole brain tissue was subfractionated by differential centrifugation (Fig. 1). The values are presented as DeDuve plots (DeDuve et al. 1955). Cytochrome oxidase, a marker for mitochondria, was enriched 7.6-fold in the P2 pellet and NADPH cytochrome c reductase, a Table 1. EROD activities in whole tissue homogenate of different brain parts from control and BNF treated fish. Values (pmol mg protein-' min-l) are mean SD of six samples, each sample containing tissue from two fish. *p < 0.05 (Mann-Whitney U-test) Tissue
Control
BNF
Olfactory Telencephalon Optic tectum Hypothalamus Cerebellum
1.15 +2.83 0.36± 0.56 0.37 ± 0.42 0.36 ± 0.34 0.15 ±0.20
8.32 ± 2.92* 2.74± 1.15' 1.17 +0.74* 2.14 + 0.98* 1.13 ±0.19*
338 Rainbow trout
P3 fractions from BNF-treated rainbow trout whereas no staining could be seen in fractions from control fish (Fig. 4). When the anti-cod CYP IA1 antibodies were used in an indirect ELISA the absorbance readings showed that the P3 levels were as high or higher than in the P2 fractions from control fish (Fig. 5). BNF-treatment significantly increased the CYP 1Al levels in rainbow trout P2 and P3 fractions.
cytochrome oxidase 8 6 4. 0 0
2. Pi
Q 0.
P2 P3
S
Discussions
0
u0
rome NADPH -cytochlrome c reductase
0 h..
a
4. 2-
P3
p 0
i-
0
S
P2 I
I $
50 % of total protein
!
100
Fig. 1. Distribution of cytochrome oxidase and NADPHcytochrome c reductase activity after subcellular fractionation of the rainbow trout brain. The values are presented as DeDuve plots (DeDuve et al. 1955) and are means of relative specific activities (RSA) from three experiments containing a pool of two brains each. RSA = the per cent of total activity/per cent of total protein. The fractions from left to right are the 1,240 g pellet (P1), 15,000 g pellet (P2), 18,000 g pellet (P3) and the 180,000 supernatant.
marker for endoplasmic reticulum, was enriched 4.5-fold in the P3 pellet. Very low cytochrome oxidase activity was measured in the P3 pellet and low NADPH-cytochrome c reductase activity was measured in the P2 pellet indicating that the subfractionation method efficiently separated mitochondria and microsomes. Electronmicrographs of P2 and P3 fractions from rainbow trout brain indicated that they consisted mainly of mitochondria and microsomes, respectively (Fig. 2). A low basal EROD activity was measured in the brain P2 fraction (Fig. 3) whereas no activity was detected in the P3 fraction. BNF-treatment induced the EROD activity in both P2 and P3 fractions. By Western blotting technique using ant-cod CYP lAl IgG, a protein with relative molecular mass of 58,000 Da was weakly stained in the P2 and
The present study demonstrates that rainbow trout brain contain CYP lAl or a functionally and immunochemically similar enzyme. The brain EROD activity is considerably lower than the EROD activity measured in the liver microsoma fraction reported in other studies. In control trout, a low EROD activity of 1.4 pmol/mg protein/min was measured in the P2 fraction whereas no activity was detected in the P3 fraction. This activity in rainbow trout brain P2 fraction is 14-50-fold lower than the EROD activity reported for hepatic microsomes (Goksoyr et al. 1987, 1991). In the scup, the brain microscomal benzo(a)pyrene hydroxylase activity was previously reported to be 980-fold lower than the liver microsomal activity (Stegeman et al. 1979). Mitochondrial CYP enzymes are considered to be highly substrate specific and known to participate in physiologically important hydroxylation reactions such as steroidogenesis in gonads and adrenal tissues, vitamin D3 metabolism in kidney and liver and bile acid formation in the liver, whereas xenobiotic metabolizing P450 enzymes are considered to be restricted to the microsomal fraction (Yang and Lu 1987). However, the results from the present investigation suggest that brain xenobiotic metabolizing CYP enzymes are confined both to the mitochondria and microsomal fractions. This is in accordance with the observations in mammals where xenobiotic metabolizing CYP activities in the brain can be detected in both the mitochondrial and microsomal fractions (Walther et al. 1986, 1987; Das et al. 1982; Ghersi-Egea et al. 1988; Iscan et al. 1990). Furthermore, in mammals the total content of CYP in the mitochondrial fraction
339
Fig. 2. Electron micrographs of (A) the 15,000 g pellet (P2) and (B) the 180,000 g pellet (P3) from rainbow trout brain.
8
C
E
6
o
4 E o
E
2
0
P2
P3
Rainbow trout Fig. 3. EROD activity in brain P2 and P3 fractions from control (open bars) and BNF treated (hatched bars) rainbow trout brain. Values are means _+SD of 4-5 fish.
was considerably higher than in the microsomal fraction. However, the rainbow trout brain P2 pellet was not a pure mitochondrial fraction. It is therefore possible that other cellular organelles or
Fig. 4. Western blot analysis of P4501AI in subcellular fractions from the rainbow trout brain. I and 2 designates control P2 and P3 fractions, respectively. 3 and 4 designates 0-naphthoflavone treated P2 and P3 fractions; 20 gg of protein was applied in each lane.
small cells cosedimenting with the mitochondria could contribute to the observed EROD activity. BNF induced brain EROD activities in both P2 and P3 fractions. Western blotting and ELISA
340
0.150
a)
0 c-
o
T
0.100-
0 o u~ ~D
I
0.050-
4
0.000
P2
P3
Rainbow trout Fig. 5. P4501AI level in brain P2 and P3 from control (open bars) and BNF-treated (hatched bars) rainbow trout measured as absorbance in an indirect ELISA. Values are means SD of 4-5 fish.
measurements using anti-cod CYP lAl IgG confirmed induction of CYP1Al protein in rainbow trout brain subcellular fractions. Inhibition of EROD activity with anti-cod CYP Al would be a more clear indication that this activity is of CYP lAl origin. However, this antibody was previously shown not to inhibit rainbow trout liver microsomal EROD activity (Goksoyr et al. 1991). Although an EROD induction by BNF was recorded in all brain parts from the rainbow trout, the highest absolute activity in BNF-treated fish was measured in the olfactory bulbs. The olfactory bulb may thus be especially susceptible to CYP AI inducing compounds. Several studies on mammalian brain CYP have reported that the olfactory bulb contains higher CYP-dependent activities than other brain parts (Das et al. 1982; Iscan et al. 1990).
Unkila et al. (1993) showed that the EROD activity in the rat olfactory bulbs was highly inducible by TCDD. CYP 1Al/1A2 has been located to the tufted cells in the rat olfactory bulb by immunohistochemical methods (Kohler et al. 1988). In the scup, CYP lAl immunoreactive cells in the brain were only found in the endothelial vascu-
lar cells from experimental or environmentally induced animals (Smolowitz et al. 1991; Stegeman et al. 1991). A substantial part of the CYP in the rat brain has also been reported to be confined to the microvessels (Ghersi-Egea et al. 1988). Since xenobiotics transported in the blood must cross the endothelial cells before reaching the brain the capacity of the endothelial CYP enzyme system to convert lipophilic xenobiotics to more hydrophilic products may affect the transport of these substances into the brain. In rat, CYP A-immunoreactivity has also been found in neuronal cellbodies and glial cells (Kohler et al. 1988). In rat brain preparations, the neuronal cells exhibited 3 fold higher EROD activity than the glial cells (Dhawman et al. 1990). Furthermore, EROD activity was inducible (75-120%) by 3-methylcholanthrene in neuronal and glial cell preparations. It is clearly of toxicological importance to further study the localization of CYP in fish brain. Previous studies have shown that aromatic hydrocarbons are enriched in the cod brain whereas no corresponding accumulation can be seen in the rainbow trout brain (Ingebrigtsen and Solbakken 1985; Ingebrigtsen et al. 1992; Hektoen et al. 1992). Furthermore, in cod exposed to radiolabeled 2, 4' ,5-trichlorobiphenyl the cerebrospinal fluid accumulated radioactive compounds which were analyzed as metabolites whereas no accumulation of radioactivity was seen in rainbow trout cerebrospinal fluid (Ingebrigtsen et al. 1992). The metabolites in cod cerebrospinal fluid are probable formed in the brain and then excreted into the fluid. An EROD activity has been detected in the cod brain (Andersson and Goksoyr, unpublished results). This activity was inducible by BNF. The difference in accumulation and metabolite formation between cod and rainbow trout brain might be explained on a toxicokinetic basis due to great difference in body lipid content. However, the properties and distribution of xenobiotic metabolizing enzymes in the central nervous system may also be an important factor. The induction of CYP Al is an important phenomenon which modulates the toxicity of aromatic hydrocarbons. The increased CYP AI activity leads to a higher capacity of the system to bio-
341 transform lipophilic compounds to more polar products and thus facilitate their excretion. In addition, the presence of a brain UDP glucuronosyltransferase activity was recently found int he rainbow trout (Ingebrigtsen et al. 1992). This enzyme participates in detoxication of many substances formed by CYP monoogygenations and greatly increases their polarity which facilitate their excretion into aquous solutions such as urine and bile. However, the elimination route for water soluble products formed in the fish brain is not well studied. Watersoluble xenobiotic metabolites may, at least partly, be secreted into the cerebrospinal fluid (Ingebrigtsen et al. 1992). Induction of CYP lAI could, however, have other consequences. Highly reactive intermediates with cytotoxic and mutagenic properties may be formed during CYPcatalyzed reactions (Guengerich 1988). The importance of the CYP enzyme system in degenerative diseases of the brain and xenobiotic dependent brain toxicity is well recognized in mammalian studies (Mesnil and Testa 1984). An increased level of CYP AI could also lead to an elevated metabolism of essential endogenous compounds or to the production of bioactive substances (Nebert 1991). The purified rat CYP 1AI was recently shown to be specifically active in monohydroxylating arachidonic acid in the 19 position (Falck et al. 1990). This metabolite has been shown to stimulate Na + /K + -ATPase activity in vascular smooth muscle (Escalante et al. 1990). Further exploration of the distribution of CYP A1 in various regions and cell types of the fish will be relevant for the understanding of the toxicity of organic pollutants.
Acknowledgements We are grateful to Eva Nilsson and Helen Nilsson (Dept. Zoophysiology, G6teborg), Sissel Olsen and Kjersti Helgesen (Lab. Mar. Molec. Biol., Bergen) for expert technical assistance. This study was supported by the Swedish Environmental Protection Board (T.A.) and the Norwegian Fisheries Research Council (NFFR) (A.G.)
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