Modulation of superoxide production in goldfish (Carassius auratus) exposed to and recovering from sublethal copper levels

Fish & Shellfish Immunology (1998) 8, 245–259

Modulation of superoxide production in goldfish (Carassius auratus) exposed to and recovering from sublethal copper levels S. V. JACOBSON

AND

R. REIMSCHUESSEL*

University of Maryland School of Medicine, Department of Pathology, Aquatic Pathobiology Center, 10 S. Pine St. M.S.T.F. 700, Baltimore, MD 21201, U.S.A. (Received 1 September 1997, accepted in revised form 12 November 1997) The e#ects of copper and recovery from sublethal (100 ìg/l) copper exposure were studied in three groups of goldfish (Carassius auratus) (1) control (2) copper (4–25 d) and (3) recovery (4 d exposure followed by 3–21 d recovery). The mean concentration of copper measured in the liver of the continuously exposed group was significantly higher than in the control and recovery groups by 11 and 18 days of exposure, respectively. In the posterior kidney, copper accumulation in the copper group was not significantly greater than the control and recovery groups until 18 days of exposure. The liver contained 4–5-fold higher concentrations of copper than the posterior kidney in all three treatment groups. Levels of copper in the posterior kidney correlated with those of the anterior kidney. There were no measurable di#erences in liver, kidney, or serum copper levels within or between the control and recovery groups at any time point. The production of superoxide (O2
) was compared between groups by measuring the reduction of NBT in anterior kidney phagocytes. In the copper group, production of O2
increased compared to controls in response to exposure periods lasting up to 11 days. O2
production in the recovery group also increased in response to 4 days of copper exposure. However, after 3 and 7 days of recovery, O2
production decreased below then increased above the control levels, respectively. By 18 days of copper exposure or 14 days of recovery, O2
production was no longer significantly di#erent from control values. To determine if the decreased O2
production measured on recovery day 3 was mediated by a humoral factor, phagocytes from untreated fish were incubated with serum from control or 3 d recovery fish. Compared to the control serum, in vitro exposure to serum from copper-recovered animals resulted in a 29–34% decrease in O2
production. This was consistent with the 25% decrease measured in phagocytes from copper-recovered animals. It is concluded that the decrease in O2
production demonstrated in goldfish recovering 3 days from acute (4 d) sublethal copper exposure is not due to a direct e#ect of copper but to a factor(s) present in the serum of the recovering animals.  1998 Academic Press Limited

Key words:

Fish, phagocytes, recovery.

reactive

oxygen

intermediates,

copper,

*To whom correspondence should be addressed. 1050–4648/98/040245+15 $25.00/0/fi970131

245

 1998 Academic Press Limited

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S. V. JACOBSON AND R. REIMSCHUESSEL

I. Introduction Environmental pollutant stress is an important determining factor in various fish diseases as fish are frequently exposed to and able to accumulate potentially immunotoxic agents present in the environment (Wester et al., 1994; Zeliko# et al., 1991). Exposure of aquatic organisms to environmental contaminants has been shown to alter fish immune responses including phagocyte reactive oxygen intermediate (ROI) production (Anderson, 1994; Elsasser et al., 1986; Roszell & Anderson, 1996; Zeliko#, 1994; Zeliko# et al., 1996). If exposure to a toxicant causes a down-regulation of ROI production, it may attenuate an animal’s ability to e#ectively kill ingested pathogens, increasing the host’s susceptibility to infectious agents (Elsasser et al., 1986; Zeliko# et al., 1994). Alternatively, overproduction of ROI may overwhelm antioxidant defence mechanisms resulting in oxidant-induced injury such as induction of lipid peroxidation, membrane destabilization and/or DNA mutations (Babior, 1984). Fish may be exposed to widely distributed immunotoxicants, such as copper, in both their natural environment and in aquacultural or aquarium facilities. In addition to its industrial uses, copper is also used in algicides, fungicides, insecticides, wood preservatives, anti-foulant paints, and animal feed and fertilizers (ATSDR, 1990). Copper concentrations may fluctuate in aquatic systems depending on the level of input from wastewater disposal, aquacultural use, marina activity, accidental spills, remediation e#orts, or increased surface run-o# following storms (ATSDR, 1990; Hall et al., 1988; Parrish & Uchrin, 1990). Environmental levels of copper surface water range from 0·5 to 1000 ìg/l (median 10 ìg/l) (Bergqvist & Sundbom, 1980; Page, 1981). In Chesapeake Bay, U.S.A., an estuary with numerous harbours, marinas, and a shipping channel, dissolved copper concentrations ranged from <10 to 80 ìg/l with a mean value of 11·7 ìg/l (Hall et al., 1988). Several coppercontaining compounds are approved by the U.S. Environmental Protection Agency (USEPA) for use as algicides and herbicides in water containing food fish. Although the use of copper sulfate as a waterborne disease therapeutant for food fish has not yet been approved by the U.S. Food and Drug Administration (USFDA), recent evidence suggests such treatment does not pose a hazard to human health (Gri$n et al., 1987). Pending USFDA approval, the application of copper sulfate in aquaculture may soon be expanded. The standard dosing of copper is often based on water hardness (Avault, 1997). For example, the dose for the system used in the present study would be approximately 216 ìg/l based on a water hardness value of 85 ìg/l (as CaCO3). Copper-related immunotoxicity in fish has been demonstrated in several experimental studies (Baker et al., 1983; Ewing et al., 1982; Hetrick et al., 1979; Muhvich et al., 1995; O’Neill, 1981; Roales & Perlmutter, 1977; Rodsaether et al., 1977). There is evidence indicating acute and chronic copper exposure suppresses specific antibody responses, modulates ROI production, and increases the incidence of fish disease. Information on whether normal anti-microbial defence mechanisms are restored upon cessation of copper exposure has not yet been reported. This may be important in terms of

GOLDFISH RECOVERING FROM SUBLETHAL COPPER LEVELS

247

monitoring the health of aquatic organisms and coordinating transport or treatment with other chemotherapeutants when the immune system is not compromised. It may also be useful when assessing potential health risks associated with periodic fluctuations in copper levels that may occur from industrial discharges or remediation projects. To determine the e#ect of exposure to and recovery from sublethal copper levels on immune function, O2
production in kidney phagocytes was assayed in three groups of goldfish: (1) control (2) continuous copper exposure and (3) recovery from copper exposure. To assess humoral factors elicited by copper treatment, O2
production was also measured in phagocytes from untreated animals that were incubated with goldfish serum (GFS) from control or recovery animals. Copper accumulation was measured in the liver, kidney, and serum.

II. Materials and Methods Protocols were developed for use with goldfish phagocytes by optimizing the following parameters: cell isolation method, bu#er composition, serum concentration, cell number, and temperature. All chemicals and reagents were purchased from Sigma Chemical (St Louis, MO) unless otherwise noted.

COPPER EXPOSURE

Three groups of goldfish were exposed to sublethal levels of waterborne copper: (1) control (no added copper) (2) copper (exposed continuously to copper until sampling at 4, 7, 11, 18 or 25 d) and (3) recovery (exposed to copper for 4 days then sampled after recovering in control water for 3, 7, 14 or 21 d). Copper (nominal 100 ìg l
1) was added as an aqueous stock solution of reagent grade CuSO4·5H2O. The 10stock solution was prepared based on the proportion of copper in CuSO4·5H2O and the volume of water in the experimental tanks. The actual measured concentration of copper (79–83 ìg l
1) to which animals were exposed is slightly less than one-half the LC50 (175 ìg l
1) determined for C. auratus previously for this system (water hardness 80–85 ìg l
1 as CaCO3) (Muhvich et al., 1995). Water changes were e#ected by flow-through prior to copper exposure then by daily static renewal during exposure periods. To ensure that the amount of water and copper was replaced accurately, 10% volume increments were measured and marked externally on each tank. Static renewals were performed by removing 50% or 60% of the water (depending on the number of fish per tank) and then replenishing the tanks to the 100% mark with fresh water. Immediately afterwards, 5 or 6 ml (for a 50% or 60% renewal, respectively) of the 10xcopper stock solution was added back to each tank near the air stone to facilitate rapid mixing. The volume of water changed daily did not exceed 60% to avoid further stressing the animals. The control and recovery groups were handled in the same manner as the continuously exposed copper group throughout the experiment.

248

S. V. JACOBSON AND R. REIMSCHUESSEL

COPPER CONCENTRATIONS

Water Water samples for analysis of dissolved copper concentrations were taken after the addition of CuSO4 at the start of the experiment, day 0. All subsequent samples were collected before static renewal according to the following protocol. Water samples were collected in a 10% HCl acid-washed 60 cc syringe (Becton Dickinson & Co., Rutherford, NJ) after rinsing the syringe twice with the water sample and filtering 10 ml of the water sample through a sterile acrodisc 0·45 ìm filter (Gelman Sciences, Ann Arbor, MI) into a 15 ìl acid-washed polypropylene tube (Corning Inc., Corning, NY). To stabilize the sample until analysis, 100 ìl of ultrex II ultrapure HNO3 (JT Baker, Inc., Phillipsburg, NJ) was added to each sample and then capped tightly and stored. The samples were analyzed by flame atomic absorption spectrophotometry (Perkin Elmer Model 5000, Norwalk, CT) using standard instrumental conditions for copper (peak wavelength of 325·7, 15 mA). The standards were made up in 0·1 M HNO3 in order to match the matrix of the samples and were measured periodically during sample readings to ensure accuracy of the results. All of the sample values were within the linear portion of the standard curve. Three readings for each sample were averaged and expressed as ìg l
1 Tissues Tissues were removed using 10% HCl acid-washed instruments, weighed, wrapped in parafilm and stored at less than 0 C. For analysis of copper concentration, tissues were transferred into etch-labelled 1275 mm acidwashed glass test tubes and wet weights recorded. The blanks used were empty identical tubes whereas standards contained approximately 0·25 mg homogenized and acetone extracted dogfish liver (DOLT-1) and muscle (DORM-1) containing 20·8 (+1·2) and 5·22 (+0·33) copper, respectively (National Research Council of Canada, Division of Chemistry, Marine Analytical Chemistry Standards Program, Ottawa, Canada). The samples were frozen for 1 h at less than 0 C, freeze-dried overnight at –100 C (DURA-DRY, FTS Systems Inc., Stoneridge, NY) and dry weights recorded. Samples were placed in a mu%e furnace (Lindberg, Watertow, WI) initially at 200 C then at 375 C for a total of 54 h. To digest the ashed tissue, the tubes were placed in a 105–110 C heating block (Multi-Blok Heater 2097-6, Labline Instruments, Melrose Park, IL) and all samples were rehydrated with 100 ìl of distilled water (dH2O). After rehydration, 100 ìl of HNO3 followed by 100 ìl H2O2 was added to all samples, standards, and blanks and heated until material was almost dry. The alternate addition of 100 ìl of HNO3 followed by 100 ìl H2O2 was continued until material in the tubes was clear. To solubilize the dry digested tissue 100 ìl of 6 N HCl was added to all tubes. The digested tissues were adequately diluted with 1 M HCl and analyzed by graphite furnace atomic absorption spectrophotometry (Perkin Elmer Model Z5000, Norwalk, CT). Results are expressed as ìg g
1 (dry weight).

GOLDFISH RECOVERING FROM SUBLETHAL COPPER LEVELS

249

Serum The serum samples (10 ml) were diluted with 1 ml 0·1 M HNO3 and analyzed by graphite furnace atomic absorption spectrophotometry. Aqueous standards in 0·1 M HNO3 were used for the standard curve. Analysis of a standard reference material (Bovine Serum SRM 1598, National Institute of Standards and Technology, Gaithersburg, MD) produced copper values within the certified range. Results are expressed as ìg/ml. ANIMALS

Goldfish (Carassius auratus) were acquired from commercial sources (Hunting Creek Fisheries, Thurmont, MD). Fish were acclimated for a minimum of 4 weeks and maintained at water temperatures 29 C (2) with a 16 h:8 h light:dark photoperiod. Fish were held in 132 l glass aquaria during the acclimation period and in 170 l polypropylene tanks during the experimental exposure periods. Water quality was monitored on a routine basis for temperature, pH [7·4(+0·2)], salinity [<1 g l
1], nitrites [0·20 ìg l
1] and hardness [80–85 mg l
1 as CaCO3]. The goldfish received trout grower diet (3/32 pellets, 38% protein) (Zeigler Bros., Inc., Gardners, PA) 5 days per week, including during exposure periods. Only animals that were free of external parasites and lesions and exhibited normal behaviour were used in the studies. Fish were overdosed with MS-222. Serum and tissues were removed with acid-washed instruments and processed as described below.

SERUM PREPARATION

Serum was collected from control and recovery (4 d copper/3 d recovery) animals. Approximately 0·2–0·5 ml caudal vein blood was drawn per fish (45–65 g). Blood was pooled from each treatment group and placed in a vacutainer tube. After centrifuging at 400 g for 5 min, serum was removed and used either that day or frozen in sterile microcentrifuge tubes at –70 C.

CELL ISOLATION

Anterior kidneys were pooled from fish (35–50 g) from each treatment group and immediately placed in L-15 medium containing 5 mM glucose, 1% L-glutamine, 1% penicillin (10 000 U), streptomycin (10 mg ml
1) and 10% fetal bovine serum (FBS). Tissues were kept on ice until homogenized using 10–15 small clearance strokes in a Dounce homogenizer. Homogenate was stored overnight at 4 C in 15 ml polypropylene tubes. The following day the homogenate was layered onto 5 ml Histopaque (d=1·077 g/cm3) and centrifuged at 400 g for 20 min at 15 C. The layer above the bu#y coat was removed and washed with 5 ml L-15 at 200 g for 20 min at 15 C. The pellet was resuspended in Hanks Balanced Salt Solution (HBSS) containing 5 mM glucose, 1% penicillin (10 000 U), streptomycin (10 mg ml
1) and 1% FBS. For serum experiments, 1% or 10% goldfish serum (GFS) was used instead of

250

S. V. JACOBSON AND R. REIMSCHUESSEL

FBS. Phagocytes from the enriched population of cells were counted in an hemocytometer by trypan blue exclusion (100 ìl cells:500 ìl 50% trypan blue in HBSS). NITROBLUE TETRAZOLIUM ASSAY

Intracellular O2
production was measured according to a modified method of Pick et al. (1981). The enriched population of phagocytes was adjusted to 3107 cells ml
1 with HBSS supplemented as described above. For serum experiments, the HBSS and nitroblue tetrazolium (NBT) incubation solutions were supplemented with 1% or 10% GFS instead of FBS. One hundred ìl of the adjusted cell suspension was added to wells of 96 well microtiter plates (Falcon) in replicates of four. A duplicate plate was used for protein analysis. Cells were permitted to attach to wells for 2 h at 20 C. After this period unattached cells were washed o# with HBSS at 20 C. The plates were incubated 1 h at 20 C with 100 ìl of 0·8 ìg ml
1 NBT, 37·5 ìg ml
1 superoxide dismutase (SOD) (4400 ìg mg
1s) and 0·5 ìg ml
1 phorbol myristate acetate (PMA). The supernatant was removed and wells were washed once with HBSS and three times with 70% methanol then air-dried 10 min. To solubilize formazan deposits in a homogeneous solution, 120 ìl 2 M KOH and 140 ìl DMSO were added to each well. Plates were placed on an orbital shaker for 60 s and the absorbance (A620) read with a microtiter plate reader (Dupont, Wilmington, DE). Absorbance values from the unstimulated wells were subtracted from the stimulated wells and calculated as A620 60 min
1 and adjusted for protein concentration. Results are reported as percent of control. PROTEIN ASSAY

Total protein per well was measured using bicinchoninic acid (BCA) (Pierce, Rockford, IL) according to the manufacturer’s protocol against a standard curve for bovine serum albumin (BSA) (Pierce). Results were averaged from four wells and expressed as mg protein. STATISTICAL ANALYSIS

Statistical significance (P<0·05) was determined by one-way analysis of variance (ANOVA) and LSD(T) comparison of means were performed on an IBM PC using SAS for the PC software package (SAS Institute, Cary, NC) and Statistix for Windows (Analytical Software, Tallahassee, FL). III. Results COPPER CONCENTRATIONS

Water The mean (and range) measured concentration of dissolved copper in the control group was 3·5 ìg l
1 (0–7 ìg l
1); copper group, 82·6 ìg l
1

GOLDFISH RECOVERING FROM SUBLETHAL COPPER LEVELS

251

Table 1. Liver and posterior kidney mean§ copper concentrations expressed as ìg copper g
1 dry weight of tissue in goldfish exposed to sublethal copper concentrations (nominal 100 ìg l
1) for various lengths of time 4 days

7 days

11 days

18 days

Liver control copper recovery

60·6 97·7 83·8

58·3 71·6 72·6

55·9 123·1* 79·6

64·6 165·0*†‡ 91·4

Posterior kidney control copper recovery

11·7 17·9 20·4

12·2 17·9 12·8

12·0 23·1 15·2

13·4 41·5*‡ 16·7

*Significantly (Pc0·05) di#erent from controls. †Significantly (Pc0·05) di#erent from recovery group. ‡Significantly (Pc0·05) di#erent from 4 d and 7 d copper group. §Mean of data from 2 experiments, n=5 fish per treatment group per experiment. Statistical significance determined by one-way analysis of variance (ANOVA) and LSD(T) comparison of means using Statistix for Windows.

(61–108 ìg l
1); and recovery group, 78·8 ìg l
1 (66–100 ìg l
1) during the 4 d exposure then 4·8 ìg l
1 (0–12 ìg l
1) during the recovery period. Tissues The mean copper concentrations in the liver and posterior kidney are reported in Table 1. Accumulation of copper in the liver was significantly (P<0·05) greater by day 11 in the copper group compared to the controls. By day 18, accumulation in the copper group was significantly greater than both the control and recovery groups and also greater than day 4 and 7 in the copper group. There was not a significant di#erence in copper levels measured in or between the control or recovery groups. Compared to the posterior kidney, mean copper levels in the liver were 4·6–5·2, 4·0–5·5, and 4·1–5·7 fold higher in the control, copper, and recovery groups, respectively. In the posterior kidney, copper concentrations were not significantly greater in the copper group compared to the control and recovery groups until day 18 and recovery day 14, respectively. The copper levels in the posterior kidney of the recovery group were not significantly di#erent from the controls at any time point. Copper concentrations in the posterior kidney correlated with the anterior kidney on all days sampled (data not shown). Serum Copper levels were measured in GFS collected from control (n=2 groups), recovery (n=3 groups), or 4 d copper (n=1 group) fish. The mean (and range) copper levels were 1·06 ìg ml
1 (0·96–1·16 ìg ml
1) in control GFS,

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S. V. JACOBSON AND R. REIMSCHUESSEL

200

Percent of control

copper recovery control

*

180 160

*

140 120

* *

*

100 80 60

** 4 0

7 3

11 7

18 14

25 Copper 21 Recovery

Days


Fig. 1. O2 production in phagocytes from goldfish exposed in vivo to copper. The data are expressed as percent of control based on absorbance values (A620 60 min
1 adjusted for protein) from the stimulated wells minus the unstimulated wells. Bars represent standard error. *Significantly (P<0·05) di#erent from the control group. **Significantly (P<0·05) di#erent from both the control and copper group.

0·71 ìg ml
1 (0·64–0·74 ìg ml
1) in recovery GFS, and 0·95 ìg ml
1 (no range) in 4 d copper GFS. Levels were compared in the control and recovery groups; there were no significant di#erences measured. NBT REDUCTION ASSAYS

In vivo copper exposure Acute exposure of goldfish to sublethal copper concentrations increased (129–154% of controls) PMA-stimulated O2
production and was significantly (P<0·05) di#erent from controls on days 4, 7, and 11 (Fig. 1). The O2
response was also significantly (P<0·05) enhanced (132% of controls) after 4 days of copper exposure in the recovery group. However, after 3 days of recovery in control water, O2
production decreased to 75% of controls and was significantly (P<0·05) di#erent from both the control and copper groups. By 7 days of recovery, O2
production exceeded both the control (184% of controls) and copper group but was significantly (P<0·05) di#erent only from the controls. By 18 days of copper exposure or 14 days of recovery, values returned to within 12% of controls and were no longer significantly di#erent. In vitro exposure Phagocytes from untreated fish were incubated with GFS (1% or 10%) collected from control or 3 d recovery fish. O2
production in cells exposed to 1% or 10% recovery GFS was compared to cells exposed to 1% or 10% control GFS, respectively (Fig. 2). A significant (GFS 1% P=0·0278; GFS 10% P=0·0003) decrease, 71% or 66% of controls, was observed in cells treated with

GOLDFISH RECOVERING FROM SUBLETHAL COPPER LEVELS

253

125

*

75

*

Percent of control

100

50

25

0 control 1% recovery 1%

control 10% recovery 10%

Fig. 2. O2
production in control goldfish phagocytes exposed in vitro to GFS (1% or 10%) from control or 3 d recovery fish. The data are expressed as percent of control based on absorbance values (A620 60 min
1 adjusted for protein) from the stimulated wells minus the unstimulated wells. Bars represent standard error. *Significantly (P<0·05) di#erent from controls.

1% or 10% recovery GFS, respectively. There was no significant di#erence in O2
production between the 1% and 10% GFS treated cells. IV. Discussion The average sublethal copper dose to which the fish were exposed did not produce any mortalities during the course of the experiment, although during the copper exposure animals exhibited behavioural changes (lethargy, decreased appetites, and laboured respiration) reported to occur with low doses of copper. The e#ects on the immune system reported here occurred at concentrations comparable to those found in the environment or used in aquaculture. The mean concentrations of copper in the livers of control and copperexposed animals in the present study were intermediate of those reported elsewhere. In brown bullheads (Ictalurus nebulosus) exposed 6 or 30 days to copper (104 ìg l
1), mean copper levels were 33 ìg g
1 or 116 ìg g
1, dry weight, respectively, whereas levels in the controls were 24 and 23 ìg g
1, dry weight, respectively (Brungs et al., 1973). In coho salmon (Oncorhynchus kisutch) exposed 14 days to 0, 70 or 140 ìg l
1 copper, mean levels of copper in the liver were approximately 75, 100, or 175 ìg g
1 dry weight, respectively (Buckley et al., 1982). The copper concentrations measured in the posterior kidney of control and copper-exposed fish in the present experiment were also comparable to levels reported elsewhere. In the studies using brown bullheads and coho salmon referred to above, copper levels in the posterior kidney ranged from 6–9 ìg g
1 (dry weight) in the controls and 5–45 ìg g
1 (dry

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S. V. JACOBSON AND R. REIMSCHUESSEL

weight) in the copper-exposed fish. Hepatic copper levels are generally greater than renal levels following aqueous copper exposure (Buckley et al., 1982). This was also true in the present study. Copper levels were 4–5-fold greater in the liver than in the posterior kidney in all of the treatment groups at every time point. The concentration of copper in the posterior kidney correlated with the anterior kidney on all days sampled. Copper accumulation in the posterior kidney of the copper group was not significantly di#erent from the control and recovery groups except on exposure day 18. Although there may have been a significant trend of accumulation discernible with a greater number of samples, one was not observed by Buckley et al. (1982) either. In the liver, accumulation in the copper group was significantly greater by day 11 compared with the controls. Within the copper group, livers from animals exposed 18 days had significantly greater copper levels than those exposed for 4 or 7 days but not 11 days. These data may suggest that compared to controls, copper accumulates in the liver as a function of time then plateaus when the animal has adapted to the copper exposure. Other laboratory studies have also reported a time-dependent accumulation of copper in the liver that reaches equilibrium within 1 month of exposure. In studies that exposed fish up to 20 months, copper concentrations peaked by 30 days of exposure and remained relatively constant after this time (Brungs et al., 1973; Buckley et al., 1982). In a di#erent study in which channel catfish (Ictalurus punctatus) were exposed to copper for 10 weeks, copper also accumulated in the liver in a timeand dose-dependent manner up to 4 weeks but then decreased over the next 6 weeks of exposure (Gri$n et al., 1997). As discussed by Buckley et al. (1982), adaptation may occur after 4 weeks of copper exposure by increased synthesis of copper-binding metallothioneines to sequester additional copper and/or by regulation of copper uptake or excretion in an e#ort to prevent excessive copper accumulation in the liver. Whether an animal maintains a constant level of copper in the liver or depurates it after 4 weeks of exposure may depend on which of the adaptive mechanisms is more predominant. The present study was designed to investigate the response of animals recovering from copper exposure, therefore, animals were not exposed to copper for longer than 25 days. Based on the results reported here and those reported by other researchers, it is reasonable to state that copper accumulates in the liver as a function of time for up to 30 days. In the recovery and control groups, there were no significant di#erences in copper levels in the liver, kidney, or serum within or between the two groups at any time during the experiment. Therefore it is concluded that the following changes in phagocyte function observed in copper-recovered animals are not a direct e#ect of copper. The production of O2
was enhanced in response to acute copper exposure. Exposure to low concentrations of metals has previously been shown to enhance fish immune responses. In medaka (Oryzias latipes), production of PMA-stimulated O2
increased in response to 5 days of exposure to equimolar concentrations (6 ìg l
1) of cadmium, inorganic mercury and nickel (Zeliko# et al., 1996). In the present study, O2
production in goldfish recovering in control water 3 or 7 days following 4 days of copper exposure decreased below

GOLDFISH RECOVERING FROM SUBLETHAL COPPER LEVELS

255

then rebounded above, respectively, the levels of control and continuously exposed groups. Production of O2
in medaka recovering in control water for 3 or 10 days following cadmium (60 ìg l
1) exposure decreased to control levels or 40% below control levels, respectively (Zeliko# et al., 1996). The changes observed during the recovery period correlated with changes in macrophage activation. The results of the cadmium study di#er from the present study in that the suppression below control values occurred on recovery day 10 instead of day 4 and no further time point was investigated to determine if O2
production may have rebounded above or returned to control values. A similar pattern of respiratory burst modulation may exist but is delayed due to di#erences in cadmium versus copper metabolism or species variations. The mechanism by which modulation of O2
production in response to copper exposure occurs is not presently understood. It is possible that these changes are due to the actions of cytokines. Resting macrophages are activated by cytokines such as gamma interferon (IFN-ã), tumour necrosis factor (TNF), and interleukin 1 (IL-1) so that they become potent e#ector cells e$cient in the degradation of phagocytosed particles (Abbas et al., 1994; Nathan et al., 1983). Priming of the respiratory burst response also occurs in fish by the actions of macrophage activating factors (MAF) present in the supernatant harvested from mitogen-stimulated head kidney leukocytes (Graham & Secombes, 1988; Neumann & Belosevic, 1996). These factors are believed to share structural and functional similarities with mammalian INF-ã, TNF, and IL-1 (Graham & Secombes, 1990; Jang et al., 1995; Neumann & Belosevic, 1996; Secombes et al., 1996b; Verburg-van Kemenade et al., 1995). Down-regulation of the mammalian respiratory burst may be mediated by several di#erent mechanisms (Bogdan & Nathan, 1993), e.g., suppression of IFN-ã, downregulation of cytokine receptors, or by factors such as TGF-â and IL-10 (Chao et al., 1991; Tsunawaki et al., 1988). In fish, factors that have been shown to down-regulate the respiratory burst include crowding stress and humoral factors, e.g. cortisol, eicosanoids (prostaglandin), and TGF-â1 (Secombes et al., 1996b). The significant decrease (29% or 34%) in O2
production observed in the present study of untreated goldfish phagocytes incubated with 1% or 10% serum from copper-recovered fish, respectively, and its correlation with O2
suppression (25%) in phagocytes of copper-recovered fish, suggests the decrease in O2
response on recovery day 3 is also mediated by a humoral substance. Preliminary experiments using a human rTGF-â1 immunoassay kit to measure activated TGF-â1 in serum samples collected from control, 4 d copper, and recovery fish produced no significant results. It should be mentioned that the analysis of goldfish TGF-B1 using a human rTGF-â1 immunoassay has not yet been validated. It is possible that the assay was not sensitive enough for use with goldfish or that the cross reactivity measured was not TGF-â1. Suppression of MAF or enhancement of other negative regulators of immunity such as interleukin-4 (IL-4), interleukin-10 (IL-10), prostaglandins and/or cortisol (Bogdan & Nathan, 1993; Secombes et al., 1996b) may also be responsible for the reduction in O2
production. Corticosteroids have been demonstrated to suppress the chemiluminescent

256

S. V. JACOBSON AND R. REIMSCHUESSEL

response in fish phagocytes (Stave & Roberson, 1985) and are known to increase in response to sublethal copper exposure (Schreck & Lorz, 1978). In yearling coho salmon (Oncorhynchus kisutch) expose to 60 and 90 ìg l
1 Cu (as CuCl2), cortisol levels immediately increased in a dose-dependent manner after exposure began, briefly decreased toward basal levels by 24 h, then increased significantly again and remained elevated for the duration of the 170 h experiment. Indirect mechanisms of copper toxicity, such as enhanced cortisol production or decreased caloric intake, cannot be excluded as the mechanism by which copper exposure modulates O2
production. Attributing changes in O2
production to one factor may not be possible as several di#erent factors may be involved and their actions may be synergistic or antagonistic and may also depend on the levels produced, duration of exposure, and source as well as the activation state of the target phagocytes (Abbas et al., 1994; Bogdan & Nathan, 1993; Jang et al., 1995; Novoa et al., 1996; Secombes et al., 1996a). For example, addition of TGF-â1 to rainbow trout (Oncorhynchus mykiss) macrophages, activated with supernatant harvested from mitogen-stimulated head kidney leukocytes, inhibited (up to 61%) the respiratory burst although it had a stimulatory e#ect on non-activated macrophages (Secombes et al., 1996b). The changes in the oxidative response in the present study were significant only up to 11 days of copper exposure. After this time the animals may be able to adapt to the copper exposure in an e#ort to prevent further ROIinduced injury of host tissues Neumann & Belosevic (1996) have suggested that macrophages may become anergic to additional stimulation by MAF. From their research it was concluded that the primed state of a goldfish macrophage cell line (GMCL) 24–48 h after an initial stimulation with MAFcontaining supernatant, could not be restored with a second exposure. Deactivation of the primed response may have been the result of intracellular events. In conclusion, O2
production in goldfish phagocytes was significantly increased in response to acute sublethal copper exposure. In animals recovering 3 days from acute exposure, O2
production was significantly decreased to levels below that of control animals. This decrease was not a direct e#ect of accumulated copper in the liver, kidney or serum and appeared to be mediated by a humoral substance. By 7 days of recovery, a compensatory reaction occurred in that O2
production increased from below control levels to that which was significantly greater than the controls. The e#ects of copper on phagocyte function were reversible and possibly act to prevent oxidative tissue injury in the host. Although the mechanism of copper-mediated modulation of ROI production is not yet known, these findings may be important in terms of monitoring fish health and risk assessment during periods of fluctuating copper levels. The authors would like to express their gratitude to the University of Maryland School of Medicine, Pathology Department and Program of Toxicology for their financial support. We would also like to thank Robert S. Anderson, Marianne W. Curry, and Kristine Y. Patterson for their assistance with various parts of this manuscript.

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