Fish Physiology and Biochemistry 18: 331-342, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.
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Isolation, partial characterization and localization of integumental peroxidase, a stress-related enzyme in the skin of a teleostean fish (Cyprinus carpio L.) L.J.S. Brokken, P.M. Verbost, W. Atsma and S.E. Wendelaar Bonga Department of Animal Physiology, Faculty of Science, University of Nijmegen, 6525 ED Nijmegen, The Netherlands Accepted: August 7, 1997 Key words: common carp, Cyprinus carpio, integumental peroxidase, blood peroxidase, leucocytes, skin, stress, cadmium
Abstract Biochemical and immunological characteristics of peroxidase activity of the skin epithelium of common carp (Cyprinus carpio) were investigated and compared with peroxidase activity of blood cells. Skin as well as blood-borne peroxidases eluted from the Superdex column as a 135 kDa protein and both probably are tetrameric molecules. Skin peroxidase activity was characterized by a Vmax of 51.5 ± 1.3 U mg–1 min–1 and a KM of 1.64 ± 0.18 mM ortho-phenylenediamine (OPD), whereas blood-borne peroxidase was characterized by a 1,000 fold higher specific activity (Vmax = 30.5 · 103 ± 2.5 · 103 U mg–1 min–1) and a higher affinity (KM = 0.875 ± 0.003 mM OPD). Polyclonal antibodies were raised against concanavalin-A purified skin peroxidase as well as blood-borne peroxidase. Immunocytochemical labelling showed that peroxidase is present in mucous cells and in mucus covering the skin and gill epithelia, as well as in erythrocytes and leucocytes. We conclude that the mucous cells of the skin produce a biochemically distinct peroxidase that is released in the mucus and may contribute to the antimicrobial properties of the mucous layer covering the skin. After exposure of the fish to cadmium the kinetic characteristics of the enzyme activity, as determined in skin homogenates, changed considerably. The Vmax increased significantly to 61.9 ± 1.1 U mg–1 min–1, and the affinity for OPD increased to the value demonstrated for blood-borne peroxidase (KM = 0.888 ± 0.045 mM OPD). Increased peroxidase levels after cadmium exposure were also demonstrated immunochemically in a dotblot assay. However, no significant changes were observed when the circulatory system of the fish was perfused prior to sampling, indicating that erythrocytes are a major contributor to the increased peroxidase activity in carp skin during cadmium exposure. This likely reflects the increased vascularization of the connective tissue layer underlying the skin epithelium, which takes place when the fish are exposed to chronic stressors including cadmium. In the cadmium-exposed fish this effect prevented the biochemical detection of stressor-related changes in epithelial peroxidase reported earlier with cytochemical methods.
Introduction Fish are very vulnerable to disturbances in their environment due to the intimate contact of the skin and gills with the surrounding water. In contrast to terrestrial vertebrates, skin keratinization
is rare in fish (Whitear 1977; Mittal and Banerjee 1980). Instead, fish are protected by a mucous layer covering the skin and gill epithelium. These epithelia are biologically very active and respond to stimuli experienced by the fish as stressors (toxic substances, water acidification, transporta-
Correspondence to: Dr. S.E. Wendelaar Bonga, at the above address; Tel. 31 24 3652476; Fax 31 24 3652714; Email
[email protected]
332 tion, temperature changes) (Iger et al. 1988, 1994a, b, c, d, e; Wendelaar Bonga and Van der Meij 1989; Iger 1992; Iger and Wendelaar Bonga 1994; Balm et al. 1995; Verbost et al. 1995; Bury et al. 1997; Nolan et al. 1997). Stressor-induced changes in fish are manifold and include primary effects (rises in plasma cortisol and catecholamine levels), secondary effects (e.g., increased plasma glucose and lactate levels, stimulated respiration and oxygen uptake) and tertiary effects (decreased growth and reproduction, and immunosuppression) (e.g., Pickering 1989; Wedemeyer et al. 1990; Sumpter 1997; Wendelaar Bonga 1997). Among the secondary effects of these disturbances are also changes in the composition of the mucous layer (Iger and Wendelaar Bonga 1994; Iger et al. 1994e; Shephard 1994). Certain enzymes can appear in the mucus after fish have been exposed to a stressor. In carp (Cyprinus carpio) exposed to acid water, water polluted with manure, cadmium or lead, or after wounding of the skin, increased numbers of small electron-dense vesicles were produced by the pavement cells, the upper most layer of the skin epithelium (Iger and Abraham 1990; Iger 1992; Iger and Wendelaar Bonga 1994). Similar vesicles have been observed in the skin of other fish species (Wendelaar Bonga and Meis 1981). In carp these vesicles have been shown to contain peroxidase activity, which was also found in the glycocalyx covering the pavement cells. Iger and Wendelaar Bonga (1994) further demonstrated this activity in the secretory granules of the mucous cells of the skin. While present in the glycocalyx, peroxidase may be a non-specific protectant against pathogens. The appearance of peroxidase in mucus during stress has led to the idea that peroxidase could be part of the non-specific stress response in fish. The aim of the present study is to analyze the peroxidase activity in the skin of the common carp and to determine how this activity is influenced by exposing the fish to cadmium. Smith and Ramos (1976) reported the release of free haemoglobin from erythrocytes into the surrounding tissues for several teleostean fish during stress. This free haemoglobin then infiltrates through the skin into the mucus. Since cadmium exposure has been reported to increase the erythrocyte fragility (Palace et al. 1993), peroxidase present in erythrocytes might also in-
filtrate into the skin. Therefore, blood-borne peroxidase was also analyzed and compared with epithelial peroxidase.
Materials and methods Experimental setup
Common carp were obtained from our laboratory stock. Eighteen male and female fish, weighing between 30 and 90 g, were kept in two groups for a period of 7 days. Fish were maintained in 200 l aquaria filled with non-chlorinated, well aerated Nijmegen city water at pH 7.5 and 23 °C (0.8 mM Ca2+; 3 mM Na+; 0.05 mM K+). One group (n = 6) remained in this water and served as control. To the other group (n = 12) cadmium was added to a final concentration of 0.5 µM (114 µg l–1). The actual cadmium concentration was measured daily by atomic absorption spectrophotometry (PU 9200X, Philips), and adjusted if necessary.
Mucus and blood sampling Because anesthetization not only affected mucous release, but also reduced the enzyme activity in the samples, the fish were stunned by a blow on the head followed by destruction of the brain. Several techniques for successful sampling of mucus were tested. In order to obtain a reproducible enzyme source for the determination of peroxidase activity, initially mucus was scraped from the skin. However, the skin was frequently damaged by this method and only small amounts of mucus were obtained. To obtain skin mucus in sufficient quantities and, at the same time, prevent damage to the skin, two alternative methods were tested. Exposing fish to 3 M KCl, known to stimulate mucous secretion, was effective but the subsequent enzyme assays were disturbed by the presence of KCl in the samples. Exposure of the fish to 0.5% hypochlorite also resulted in a strong increase of mucous release, but destroyed the enzyme activity in the samples. Therefore, in a second approach, skin, rather than mucus, was sampled by scraping the epidermal layers of the skin on the lateral sides of the body with a micro-
333 scopic slide. This method was used in the experiments described in this report. In order to exclude blood from the skin samples, fish were also perfused prior to sampling (perfusion medium: 150 mM NaCl, 1 mM HEPES, 1 mM Tris at pH 8.0 to which 0.5% heparin (v/v) was added). This was established by inserting a canula into the bulbus arteriosis and opening of the atrium. Only fish which showed complete clearance of blood from the gills were taken for sampling. The samples were diluted in demineralized water, sonicated for 5 min at 4 °C and centrifuged for 10 min at 2,000 × g. The supernatants were used as the enzyme source. Supernatant protein content was determined by the procedure of Bradford (1976) with bovine serum albumin as a reference. Blood was drawn into heparinized 1 ml syringes and centrifuged for 10 min at 2,000 × g. Cells in the pellet were lysed by the addition of demineralized water. Lysed cells as well as plasma were used for determination of the peroxidase activity. Whole blood smears on microscope slides were used for immunocytochemical analysis.
Enzyme assays Peroxidase activity was determined with orthophenylenediamine (OPD) as substrate and horseradish peroxidase (HRP) as a reference. The 500 µl reaction mixture contained OPD (concentrations ranged from 0.0863 to 22 mM) dissolved in McIlvaine’s buffer (pH 5.0) containing 50 mM citric acid and 100 mM Na2HPO4 with 7.5 · 10– 3 % hydrogen peroxide and 10 µl of volume of sample. The reaction was stopped by the addition of 500 µl 1 M H2SO4 and the absorbance was recorded spectrophotometrically at 490 nm. All kinetic measurements were made at 37°C. Results are expressed as extinction (arbitrary units) per mg protein per minute (U mg–1 min–1) or, when possible, related to HRP-equivalents (ng HRPeq mg protein–1). All the kinetic measurements were performed in one assay, which makes it legitimate to compare the Vmax values expressed in arbitrary units rather than related to ng HRPeq.
Purification of peroxidase Peroxidase was purified by affinity chromatography with the lectin concanavalin-A (Con-A) from skin homogenates (carp peroxidase 1; CP1) as well as from blood (carp peroxidase 2; CP2). Con-A binds molecules that contain “α-D-mannopyranosyl or “α-D-glucopyranosyl residues. Skin samples were suspended in Con-A buffer (pH 7.4) containing 15 mM Tris, 1 mM MnCl2 · 4H2O, 1 mM MgCl2 · 6H2O, 1 mM CaCl2 · 2H2O and 1 M NaCl, and centrifuged at 4°C for 10 min at 2,000 × g. Blood was suspended in demineralized water to lyse the blood cells and centrifuged at 4°C for 10 min at 2,000 × g. The supernatant was diluted 1:1 in Con-A buffer. Glycoproteins in the supernatants were isolated in two or three rounds on a Con-A column (Pharmacia, Uppsala, Sweden). The columns were eluted with Con-A buffer containing 0.3 M αMe-D-glucoside. Fractions showing absorption at 280 nm were pooled, applied to a Sephadex G-25 M column (Pharmacia, Uppsala, Sweden) equilibrated with 0.1 M NaHCO3 (pH 9.5), and lyophilized. After resuspending the lyophilates in a small volume of demineralized water the skin fractions were chromatographed on a Superdex 75 column (Pharmacia, Uppsala, Sweden) and the blood fractions were chromatographed on a Sepharose 12 column (Pharmacia, Uppsala, Sweden). Fractions exhibiting peroxidase activity with OPD as substrate were pooled and lyophilized. Polyclonal antibodies against the Con-A purified carp peroxidases (CP1 and CP2) were commercially raised in rabbit at Eurogentec, Belgium. Resuspended fractions of rabbit-anti-carp peroxidase (RACP1 and RACP2) were analyzed by SDS-PAGE.
Gel electrophoresis and western blotting CP1, CP2, supernatants of skin homogenates and blood were electrophoresed one-dimensionally on a 12% (native) polyacrylamide gel as described by Laemmli (1970). Samples (5 µg) were dissolved in sample buffer, containing 62.5 mM Tris-HCl (pH 6.8), 0.01% bromophenol blue and 12.5% glycerol, and left to stand for 30 min at room temperature. The samples were then loaded onto a 12% polyacrylamide gel, which was
334 electrophoresed at constant voltage (200 V). The protein bands were transferred to a nitrocellulose filter by western blotting at constant voltage (100V) for 1 h at 4 °C. The filters were then blocked with 1% powdered milk in phosphatebuffered saline (PBS; 10 mM, pH 7.6) containing 10 mM K2HPO4, 10 mM KH2PO4 and 150 mM NaCl, and subsequently incubated in PBS containing 0.1% powdered milk, 1% gelatin and rabbit-anti-carp peroxidase antibody (RACP1 or RACP2) diluted 1:5,000 overnight at 4 °C. After rinsing in PBS the filters were incubated in peroxidase-conjugated goat-anti-rabbit antibody diluted 1:2,000 for 2 h in PBS containing 0.1% powdered milk and 1% gelatin. The immunecomplexes were visualized using 4-chloro-1naphthol as a substrate. Dotblot assay In a separate experiment, carp (n = 15) were transferred from an aquarium with city water to one containing 0.5 µM cadmium in the water. Control fish (n = 15) were transferred to normal city water. Skin samples were taken after 1 h, 2 days and 5 days of exposure. Five fish per group were sampled at each sampling point. Peroxidase levels were determined immunochemically in a dotblot assay. RACP2 (1 µl, diluted 1:20) was applied to strips (5 × 30 mm) of nitrocellulose filter, which were subsequently blocked in PBS containing 1% powdered milk. These strips were then dipped in skin homogenates taken from control and cadmium-exposed carp. After rinsing in PBS for three times 5 min the strips were incubated overnight in PBS containing 0.1% powdered milk, 1% gelatin and biotinylated RACP2 diluted 1:5,000 at 4 °C. After rinsing in PBS for three times 5 min the strips were incubated with PBS containing 0.1% powdered milk, 1% gelatin and peroxidase labeled avidin (Sigma Chemical Co., St. Louis, Missouri, USA) diluted 1:1,000 for 2 h at room temperature. Following a final rinse in PBS for three times 5 min, the immune-complexes were visualized using 4-chloro-1-naphthol as a substrate. The staining intensities of the dots were semi-quantified by ranking the intensity from 0 to 4 (where 0 corresponded with no apparent staining and 4 corresponded with the most intense staining).
Immunocytochemistry Pieces of skin (approximately 4 × 2 mm) from the lateral side of the body and gill filaments were dissected, fixed overnight in 4% paraformaldehyde at 4 °C, dehydrated and embedded in paraffin. Five µm thick sections were dewaxed in xylene and rehydrated in graded ethanols. Blood smears were fixed in Bouin’s fixative for 5 min at room temperature. After rinsing in phosphate buffered saline (PBS; 10 mM, pH 7.6) containing 0.5% Triton X-100 and 0.1% normal goat serum (PBS-TX), the sections and blood smears were preincubated in PBS-TX containing 20% normal goat serum (v/v) for 15 min at room temperature followed by an overnight incubation at room temperature in RACP1 or RACP2 antibody diluted 1:5,000 and 1:1,000 in PBS-TX, respectively. This treatment was followed by 1 h incubation at 37 °C in goat-anti-rabbit secondary antibody conjugated to FITC (GAR/FITC; Nordic Immunologic Laboratories, Tilburg, The Netherlands) diluted 1:20 in PBS. Control incubations were carried out with the omission of either the primary or the secondary antibody. Sections and blood smears were finally mounted in Citifluor for fluorescent microscopical examination.
Statistical analysis The data are expressed as means ± SEM and analyzed with the Kruskal-Wallis test; significant difference was considered at p < 0.05.
Results Control Biochemistry Kinetic analysis of the peroxidase activity in skin homogenates of control fish showed a Vmax value of 51.5 ± 1.3 U mg–1 min–1 and a KM of 1.64 ± 0.18 mM OPD (Fig. 1). Peroxidase activity in erythrocytes was characterized by a 1,000-fold higher Vmax (30.5 × 103 ± 2.5 × 103 U mg–1 min– 1 ) as well as a higher affinity for OPD (0.875 ± 0.003 mM OPD) (Fig. 2). The activity in the
335
Fig. 2. Michaelis-Menten kinetics of the peroxidase activity in blood with varying concentrations of the substrate OPD. Each value is expressed as mean ± SEM; n = 4.
Fig. 1. Michaelis-Menten kinetics of the peroxidase activities in skin homogenates from control fish, cadmium-exposed fish and cadmium-exposed fish after perfusion of the circulatory system. Each value is expressed as mean ± SEM; n = 6.
Table 1. Analysis of peroxidase activity in carp skin homogenates and blood. Skin homogenates were collected from control fish, cadmium-exposed fish and cadmium-exposed fish after perfusion of the vascular system.
supernatant was negligible compared with that of the erythrocyte fraction (Table 1). Immunochemistry Figure 3a shows the elution pattern of the Con-Apurified blood samples. Fractions 8, 9, 10 and 11 exhibited peroxidase activity, and these fractions were pooled and lyophilized. Subsequent chromatography of the resuspended lyophilate on a Superose 12 column revealed one major peak corresponding with the fractions exhibiting peroxidase activity (Fig. 3b). Con-A-purified carp peroxidases from skin homogenates (CP1) and blood (CP2) were analyzed by polyacrylamide gel a
Skin homogenates Control Cadmium Cadmium + perfusion Blood Plasma Pellet
Vmax (U mg–1 min–1)
KM (mM OPD)
51.5 ± 1.3 61.9 ± 1.1*
1.64 ± 0.18 0.888 ± 0.045**
53.6 ± 1.8
1.54 ± 0.22
30.1 ± 2.0 30.5 103 ± 2.5 103
·
·
nd 0.875 ± 0.003
Each value is expressed as mean ± SEM; skin: n = 6; blood: n = 4. nd, not determined. *, p < 0.05; **, p < 0.01 by KruskalWallis’ test as compared with control values. b
Fig. 3. Superose 12 chromatography of the Con-A purified blood sample (a). When the fractions exhibiting peroxidase activity were pooled the active fractions eluted as a single glycoprotein peak from the Superose 12 column (b).
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Fig. 4A. Paraffin section of control carp skin fixed in 4% paraformaldehyde and stained with RACP1. Immunofluorescent staining is clearly located in the mucous cells (m) and the mucous layer covering the epidermis (arrows). The club cells (c) are negative. The pavement cells (p) and filament cells (f) show the same low immune-reactivity as in the controls. (× 500) Fig. 4B. Paraffin section of a control filament tip fixed in 4% paraformaldehyde and stained with RACP1. Immunofluorescent staining is located in the mucous cells (arrows). (× 320) Fig. 4C. Blood smear from carp fixed in Bouin’s fixative and stained with RACP2. Immunofluorescence is concentrated in the leucocytes (l). The erythrocytes (e) are lightly stained. Control incubations (i.e., with the omission of the first or second antibody) were negative. (× 870) →
337 electrophoresis, western blotting and subsequent labelling with the polyclonal RACP1 or RACP2 antisera. In both cases, this resulted in a clear band of 64 kDa, as well as a smear between 100 and 200 kDa. A similar pattern was produced when whole skin homogenates were analyzed. In blood, an additional band of 70 kDa was detected. No peroxidase activity could be detected biochemically once the enzyme was blotted onto nitrocellulose filter. However, enzyme activity was detected in the 135 kDa fraction which was initially collected from the Superdex column. Immunocytochemistry Labelling of skin and gill sections with either the RACP1 or RACP2 antiserum resulted in staining of mucus covering the skin and gill epithelia as well as the mucus present in the mucous cells in these epithelia (Fig. 4A, B). However, in the gills only mucous cells located in the filament tips were stained. Labelling with RACP1 resulted in more pronounced aspecific staining throughout the epidermis compared with RACP2. In the latter case, aspecific staining was virtually absent. When blood cells were labelled in Bouin-fixed blood smears with either RACP1 or RACP2, both the leucocytes and the erythrocytes were stained (Fig. 4C). However, the labelling of leucocytes was more intense with RACP2 than with RACP1. Control incubations (i.e., when either the primary or the secondary antibody was omitted) did not show any staining.
Cadmium exposure Biochemistry After carp had been exposed to a sublethal dose of cadmium for 7 days Vmax of the peroxidase
activity in the skin significantly (p < 0.05) increased compared with control values. However, when the cadmium-exposed carp were perfused before sampling no difference in Vmax compared with controls was observed (Fig. 1). The affinity of the peroxidase for OPD increased significantly (p < 0.01) after cadmium exposure. After perfusion however, again no difference in peroxidase affinity between this group and the control group was apparent (Table 1). Dotblot assay Skin samples from carp exposed to a sublethal cadmium concentration in the water were also analyzed immunochemically by dotblot assay. No difference was found after 1 h of exposure compared with controls. However, after 2 and 5 days, dotblots from skin samples of the cadmium-exposed fish were stained more intensely than those of control fish (Fig. 5). The same samples were also analyzed biochemically. After 1 h of exposure both the control and the exposed group showed an increase in peroxidase activity as compared with control values at the beginning of the experiment. However, after 2 and 5 days the cadmium-exposed group exhibited significantly higher (p < 0.001) peroxidase activities compared with the controls (Fig. 6). Immunocytochemistry After cadmium exposure the staining pattern did not differ from that described for control tissues. Again, mucous cells and the mucous layer covering the skin and gill epithelia were stained. Leucocytes present in the cadmium-exposed skin did not stain when RACP1 was employed (Fig. 4D), but did stain with RACP2 (Fig. 4E, F). These leucocytes were absent in skin from control fish.
Fig. 4D. Paraffin section of cadmium-exposed carp skin fixed in 4% paraformaldehyde and stained with RACP1. Immunofluorescent staining is concentrated in the mucous cells (m) and the mucous layer covering the epidermis (arrows). No immunereactive leucocytes can be detected in the epidermis (compare with Fig. 4E). The pavement cells and the filament cells show the same low immune-reactivity as in the controls (not shown). The club cells (c) are negative. (× 500) Fig. 4E. Paraffin section of cadmium-exposed carp skin fixed in 4% paraformaldehyde and stained with RACP2. Immunofluorescent staining is located in the mucous cells (m). Note the presence of immune-reactive leucocytes infiltrating the epidermis (arrows). The pavement cells, filament cells and club cells are negative. (× 500) Fig. 4F. Paraffin section of cadmium-exposed carp skin fixed in 4% paraformaldehyde and stained with RACP2. Immunofluorescent staining is located in huge numbers of leucocytes infiltrating the epidermis (arrows). (× 500)
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Fig. 5. Peroxidase levels in skin homogenates collected from control fish and fish exposed to cadmium for different time periods. Peroxidase levels are expressed as relative staining intensities; n = 5.
Fig. 6. Peroxidase activity in skin homogenates collected from control fish and fish exposed to cadmium for different time periods. Peroxidase activities are expressed as mean ± SEM; n = 5. ***, significantly different (p < 0.001) from control values.
Discussion
lected from the Superdex column. Analyzing this fraction by gel electrophoresis and western blotting probably denatured the enzyme in such a way that no enzyme activity could be detected anymore. With antibodies raised against this fraction a clear 64 kDa band was detected on western blots. Occasionally, an immune reactive 32 kDa band also was formed, suggesting that the 64 kDa band represents the denatured, dimeric state of the enzyme, which appears to be active only in a tetrameric 135 kDa conformation. The smear between 100 and 200 kDa and the additional 70 kDa band in blood suggest that the antisera crossreact with other, yet unknown, antigens. Immunohistochemical labelling of gill and skin sections showed that the peroxidase is present in mucus covering the skin and gill epithelia, as well as the mucous cells in these tissues. Apparently the peroxidase is produced by the mucocytes and co-released with the mucus. It may function as a non-specific protective mechanism against invading pathogens. It should be noted that fixing the tissue in 4% paraformaldehyde resulted in poor preservation of the mucous layer covering the epithelia. Although Iger et al. (1994e) reported peroxidase activity in electron-dense vesicles in the pavement cells of carp exposed to cadmium, no immune reactivity was observed in these cells. Since these vesicles were very small (150–200 nm in diameter) and usually rather limited in number, they may escape detection using the light
In this report the purification, partial characterization and localization of peroxidase from skin and blood cells of the common carp are described. Scraping the epidermal cell layers of the skin with a microscopic slide resulted in a reproducible sampling method of the peroxidase source in the skin, while circumventing problems caused by exposing the fish to either KCl or hypochlorite. The biochemical characteristics of carp skin peroxidase and blood-borne peroxidase differed significantly. Peroxidase in blood had a higher Vmax as well as a higher affinity for OPD than in skin homogenates. We presented the first biochemical characterization of a specific type of peroxidase in fish skin. Cytochemical evidence for peroxidase activity in the skin of carp has been reported earlier by our group (Iger and Wendelaar Bonga 1994). Peroxidase activity can therefore be added to the growing number of enzyme activities that have been detected in teleostean fish skin, which includes lysozyme (Fletcher and Grant 1968; Fletcher and White 1973), trypsin (Hjelmeland et al. 1983; Brown et al. 1990), carbonic anhydrase (Wright et al. 1986; Rahim et al. 1988) and alkaline phosphatase (Iger and Abraham 1990). When skin homogenates were purified, enzyme activity was detected in a 135 kDa fraction col-
339 microscope. Another possibility is that the cytochemical method used by Iger and Wendelaar Bonga (1994), based on the conventional diaminibenzidine (DAB) technique, is represented by a type of peroxidase activity that was not detected by our antibodies. According to Vacca et al. (1978) the DAB technique also detects pseudoperoxidase activity (i.e., non-enzymatic peroxidase activity) when employed at pH 7.2–7.6. This pseudoperoxidase activity can, however, be suppressed selectively by lowering the pH of the DAB solution to pH 5.5. After cadmium exposure the skin was invaded with leucocytes. This has also been observed by Iger et al. (1994e). In paraformaldehyde-fixed skin tissue, these leucocytes were positively stained only by the RACP2 antiserum, indicating that both antisera were directed against different antigens. This was supported by the observation that RACP1 resulted in a higher background staining throughout the epidermis than RACP2 and by the fact that in blood smears fixed with Bouin’s, immunereactivity could be detected in the leucocytes with RACP1. However, the staining was less intense than when RACP2 was used. This study further demonstrated that the peroxidase activity in the skin increased significantly when carp were exposed to a sublethal cadmium concentration in the water for 7 days. No increase in enzyme activity was found when the fish were perfused prior to sampling. This suggests that the increase in activity is not, or not noticeably, caused by increased peroxidase activity produced by the mucous cells of the skin epithelium, but due to blood-borne peroxidases. The result that the affinity of peroxidase for ortho-phenylenediamine (OPD) in stressed carp skin was as high as that in blood supports this view. Only a small amount of blood would suffice to increase the peroxidase activity in skin samples significantly due to the extremely high peroxidase activity of erythrocytes and leucocytes. Immunocytochemical labelling of blood smears with RACP1 or RACP2 resulted in stained erythrocytes as well as leucocytes. When analyzed by SDS-PAGE, the antibody recognizes similar bands in blood as in skin homogenates. High peroxidase levels in erythrocytes and leucocytes have been demonstrated for several species including carp (Catton 1951; Conroy 1972; Blaxhall and Daisley 1973;
Bielek 1981). According to Malkovics et al. (1977) who compared peroxidase levels in carp, pigeon and pig erythrocytes, fish have one of the highest values described in vertebrates. In mammals the peroxidase of leucocytes participates in microbicidal activity (Ellis 1977). During stress large numbers of leucocytes infiltrate the skin epithelium (Iger and Abraham 1990; Iger 1992; Iger and Wendelaar Bonga 1994; Iger et al. 1994e), which could increase the peroxidase activity. However, as these are unlikely to be removed from the skin epithelium by perfusion of the circulatory system, we conclude that the increase in peroxidase activity after cadmium exposure as measured in our experiment was not caused by peroxidases derived from leucocytes and that the erythrocytes were the major source responsible for this increase. This is unlikely to have been brought about by an increased haematocrit, as cadmium exposure has been reported to result in depressed rather than increased erythrocyte numbers in a variety of teleostean fishes (Larsson, 1975; Johansson-Sjobeck and Larsson 1978; Gill and Pant 1985; Karlsson-Norrgen et al. 1985; Gill and Epple 1993; Kaviraj and Das 1995). The mechanism of cadmium-induced anemia is not clear, although reduced intestinal iron uptake (Freeland and Cousins 1973) and shortened life span of erythrocytes (Berlin and Friberg 1960) have been suggested as contributory causes. How then can the increase in skin peroxidase activity in the cadmium-exposed fish be explained? We suggest that the vascularization of the skin increases during cadmium exposure. It is known that the distance between capillaries and basal lamina in the skin of carp decreases during exposure to cadmium (Iger et al. 1994e), copper (Iger et al. 1994d), lead (Iger 1992) and acidified water (Iger and Wendelaar Bonga 1994) as a result of outgrowth of blood capillaries (angiogenesis). Similar responses have been reported for carp skin after wounding (Iger and Abraham 1990) and in the esophageal epithelium of tilapia (Oreochromis mossambicus) during adaptation to salinity changes (Cataldi et al. 1988). This indicates that angiogenesis in epithelial tissue may be part of a general response to stressors in fish. In skin, this response could be connected with the increased metabolic requirements of the epidermis as a result of the enhanced cellular activity
340 and increased cellular turnover that has been reported for this tissue during metal and low pH exposure (Iger and Wendelaar Bonga 1994; Iger et al. 1994d, e). Since the skin samples collected in this study contained some subepithelial tissue, stimulated angiogenesis during cadmium exposure might explain why skin samples taken from stressed fish contain more blood peroxidase than skin samples from control fish. Peroxidase levels were also determined immunochemically in a dotblot assay. We expected that this approach would be more sensitive than the biochemical assay, since loss of enzyme activity (e.g. due to storage) is circumvented. After 1 h no difference between samples from control fish and from cadmium-exposed fish was evident. Probably, the control fish as well as the cadmium-exposed fish experienced an initial handling stress due to transferring both groups to a new tank, as indicated by the biochemical data. Both groups had significantly elevated peroxidase activities compared with a control group that had not been transferred. After 2 and 5 days of exposure however, the cadmium-exposed fish exhibited higher peroxidase levels in the skin than the controls did. At these time points the increased peroxidase levels in the control fish had returned to prestress levels. The same result was achieved when these samples were analyzed biochemically, indicating that the dotblot approach is a reliable immunochemical method to determine peroxidase levels in skin homogenates in carp. However, this method does not seem to be more sensitive than the biochemical approach. Heavy metal exposure induces a variety of changes in blood parameters in fish, such as a decrease of haematocrit and leucocrit values (Tort and Torres 1988), reduction of plasma electrolytes (Pratap et al. 1989) and elevation of plasma cortisol and glucose levels (Fu et al. 1990; Pratap and Wendelaar Bonga 1990). Many authors have reported histopathological changes in the skin and branchial epithelium after exposure to cadmium (Oronsaye and Brafield 1984; Mallatt 1985; Karlsson-Norrgen et al. 1985; Pratap and Wendelaar Bonga 1993; Iger et al. 1994d, e). Cadmium exposure in carp has been shown to result in increased plasma cortisol levels (Iger et al. 1994e), which is known to promote processes essential for adaptation to stressors, e.g., energy mobiliza-
tion, osmoregulation or synthesis of metallothioneins (Fu et al. 1990; Barton and Iwama 1991; Redding et al. 1991). Consequently, plasma cortisol levels have been considered a useful stress indicator. Since electron microscopical observations indicated the appearance of peroxidase in mucus during exposure to a variety of stressful stimuli that evoked a rise in plasma cortisol (Wendelaar Bonga and Meis 1981; Iger and Abraham 1990; Iger 1992; Iger and Wendelaar Bonga 1994), we hypothesized that the peroxidase level in mucus might be a reliable, easy and non-invasive way to determine stress in fish. Skin samples taken by scraping the epidermal cells contained sufficient amounts of peroxidase to detect biochemically as well as immunochemically. However, although electron microscopical observations indicated an increase in peroxidase levels in skin of stressed fish (Iger and Abraham 1990; Iger 1992; Iger and Wendelaar Bonga 1994), the present results on cadmium-exposed fish show that this increase is too small to be detected biochemically, and it appeared to be obscured completely by the high peroxidase activity produced by the blood cells penetrating the skin of stressed fish. Peroxidase present in the skin epithelium differs in the affinity for OPD from that present in blood, but no distinction can be made between the activity of epithelial peroxidase and of blood peroxidase in a biochemical assay. Therefore increased skin peroxidase levels are not considered to be useful as a stress indicator when measured biochemically.
Acknowledgements This research was financially supported by the Technology Foundation (STW; NBI22.2832).
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