ABSTRACT
The effects of sublethal waterborne Zn2+ (150 µg l-1=2.3 µmol l-1) on the kinetics of unidirectional Ca2+ influx were studied in juvenile freshwater rainbow trout during chronic exposure (60 days) at a water [Ca2+] of 1.0 mmol l-1. An unexposed group held under identical conditions served as control. The presence of Zn2+ in the water increased the apparent Km for Ca2+ influx by up to 300 % with only a small inhibitory effect (35 % at most) on the maximum rate of uptake (Jmax). These results, in combination with earlier data showing that Ca2+ competitively inhibits Zn2+ uptake, suggest that Zn2+ and Ca2+ compete for the same uptake sites. Acute withdrawal of Zn2+ after 3 h of exposure resulted in a 23-fold reduction in Km for Ca2+, but a persistent small depression of Jmax. During prolonged exposure to Zn2+, the apparent Km for Ca2+ remained greatly elevated and Jmax remained slightly depressed. The actual Ca2+ influx in hard water ([Ca2+]=1.0 mmol l-1) decreased marginally and paralleled the small changes in Jmax. The increases in apparent Km had a negligible influence on the actual Ca2+ influx because Km values (38–230 µmol l-1), even when elevated by Zn2+, remained below the water [Ca2+] (1000 µmol l-1). Rainbow trout exposed to Zn2+ exhibited a slower rate of protein synthesis in the gills (measured on day 23) and an increased tolerance to Zn2+ challenge (measured on both days 27 and 50). Unidirectional Zn2+ influx, measured at the end of the exposure period, was significantly reduced in the Zn2+-exposed fish. There were no changes in hepatic or branchial Zn2+, Cu2+ or metallothionein concentrations. We hypothesize that, during exposure to sublethal [Zn2+] in hard water, the fish may change the Km for a mutual Ca2+/Zn2+ carrier so as to reduce markedly Zn2+ influx without greatly altering Ca2+ influx. This reduced Zn2+ influx, rather than metallothionein induction, may be the basis of adaptation to elevated concentrations of waterborne Zn2+.
Introduction
According to the present model of branchial Ca2+ uptake in freshwater fish, Ca2+ transport occurs via the mitochondria-rich ‘chloride’ cells (Perry and Wood, 1985; Flik et al. 1985; Perry and Flik, 1988). Calcium passes the apical membrane of the chloride cells through voltage-independent Ca2+ channels, driven by its electrochemical gradient. Inside the chloride cell, Ca2+ binds to proteins, such as calmodulin, thus keeping the intracellular Ca2+ activity low. Ca2+ is then transported over the basolateral membrane by a high-affinity Ca2+-ATPase.
The primary effect of an acute increase in waterborne [Zn2+] on freshwater fish is believed to be an impaired branchial Ca2+ influx that, in turn, leads to hypocalcaemia (Spry and Wood, 1985). Chemically, Zn2+ is closely related to Cd2+, which also causes hypocalcaemia, primarily by inhibiting the Ca2+-transporting ATPase in the basolateral membrane of the chloride cells (Verbost et al. 1987, 1988, 1989b; Fu et al. 1989). At present, it is not known how Zn2+ interferes with branchial Ca2+ uptake.
Fish exposed to sublethal levels of metals are often able to adapt, i.e. to make physiological adjustments which compensate for the altered environmental condition and confer increased tolerance to the metal (Chapman, 1985; McDonald and Wood, 1993). At least in freshwater fish, the mechanisms responsible for this adaptation are likely to act at the gill. The gill is the primary site of ionoregulation, acid–base balance, nitrogenous waste excretion and gas exchange; these systems, in particular ionoregulation, are especially sensitive to metal toxicity (Wood, 1992). Effects at the level of the gills may take place before the metals are distributed to other parts of the body. McDonald and Wood (1993) have proposed that metal adaptation results from a two-step process, involving initial damage to the gill epithelium followed by a period of tissue repair, during which the actual mechanisms responsible for the adaptation are expressed. Such mechanisms could include a reduced metal uptake, induction of the heavy metal binding protein metallothionein (MT), compensatory synthesis of ion-transporting ATPases and a proliferation or increased turnover of ionocytes (Hogstrand and Haux, 1991; Roesijadi, 1992; McDonald and Wood, 1993).
There is indirect evidence suggesting that induction of branchial MT could be involved in the adaptation of freshwater rainbow trout, Oncorhynchus mykiss, to Zn2+ and other metals (Kito et al. 1982; Benson and Birge, 1985; Bradley et al. 1985; Olsson et al. 1989; Norey et al. 1990). The recent development of a highly specific radioimmunoassay (RIA) system for MT in fish (Hogstrand and Haux, 1990) should facilitate future research in this area. There is direct evidence for a reduced rate of accumulation of Zn2+ in gills of adapted fish (Bradley et al. 1985). Similarly, adaptation to Cd2+ could be associated with MT induction and a reduced uptake of Cd2+ by the gills (Kito et al. 1982; Benson and Birge, 1985; Verbost et al. 1989b; Wicklund Glynn and Olsson, 1991).
Increased rates of protein synthesis in the gills might well accompany such processes of damage repair and induction of specific proteins. To our knowledge, protein synthesis rates have not been measured during prolonged exposure to sublethal metal stress, though a highly sensitive technique for such measurements is now available (Houlihan et al. 1986). Whatever the mechanisms responsible for adaptation to metals, they are likely to carry a significant metabolic cost (Calow, 1991). Thus, during the adaptation process, less energy may be available for growth, locomotion, reproduction and other important variables determining the fitness of fish. For example, it has recently been shown that an increased tolerance to aluminium is accompanied by a reduced growth rate and critical swimming speed (Wilson and Wood, 1992; Wilson et al. 1993a,b).
Unidirectional flux measurements (using radiotracers) and kinetic analysis have proved to be valuable tools to characterize branchial ion-transport systems in fish (Wood, 1992). These techniques can cast light on the nature of both transport and inhibitory mechanisms. For example, linear kinetics (a proportional relationship between uptake and substrate concentration) suggest diffusive pathways, whereas Michaelis–Menten kinetics suggest carrier-or channel-mediated transport. When the latter processes apply, increases in Km (inverse of affinity) in the presence of the putative inhibitor are diagnostic of competitive inhibition, while decreases in Jmax (maximum transport rate) are indicative of non-competitive inhibition. During prolonged exposures, changes in Km may indicate adaptive alterations in the affinity of the carrier system, while changes in Jmax may signal a restoration of the absolute numbers of carrier sites. Such experiments have been used to investigate the effects of aluminium (McDonald and Milligan, 1988; McDonald et al. 1991), Cu2+ (Laurén and McDonald, 1985, 1987; Reid and McDonald, 1988), Zn2+ (Spry and Wood, 1985, 1988, 1989a; Bentley, 1992) and Cd2+ (Verbost et al. 1987, 1989b; Reid and McDonald, 1988) on branchial uptake and excretion processes. Kinetic analysis of the effects of Ca2+ on Zn2+ uptake has clearly shown that increasing concentrations of Ca2+ in the water elevate the Km for Zn2+ influx, and the inhibitor constant (Ki) for this inhibition is close to the Km for Ca2+ uptake (Spry and Wood, 1989a). These findings suggest that Ca2+ and Zn2+ may compete for the same uptake sites at the gills.
With this background in mind, the present study had three objectives. The first was to characterize the acute effect of waterborne Zn2+ on the kinetics of unidirectional Ca2+ influx (as measured with 45Ca) in juvenile freshwater rainbow trout. For this purpose, we developed a new in vivo method to measure Ca2+ influx kinetics, based on the technique described by Laurén and McDonald (1987) for the analysis of Na+ influx kinetics. The second was to see whether adaptive or non-adaptive changes in Ca2+ kinetics occurred during chronic Zn2+ exposure (60 days) and whether such changes could be correlated with the development of an increased tolerance to Zn2+. Zinc was applied at a level (150 µg l-1=2.3 µmol l-1) below the reported Km for Zn2+ uptake (240 µg l-1= 3.7 µmol l-1) by rainbow trout in identical water quality (Spry and Wood, 1989a), and at only 0.2 % of the ambient Ca2+ concentration (1.0 mmol l-1). This concentration of Zn2+ is known to be within the nutritional range for rainbow trout in the water used, stimulating growth without effects on whole-body Ca2+ and Zn2+ concentrations (Spry and Wood, 1988). An increased survival time (LT50) in a lethal concentration of Zn2+ was used as an index of adaptation to Zn2+. The third objective was to learn more about the possible costs and mechanisms of adaptation to Zn2+ by measuring protein synthesis rates and RNA concentrations in gills, liver and the whole body (Houlihan et al. 1986), levels of MT and Zn2+ in gills and liver (Hogstrand and Haux, 1990) and rates of unidirectional Zn2+ influx (using 65Zn; Spry and Wood, 1989a) in Zn2+-adapted fish.
Materials and methods
Experimental animals
Juvenile rainbow trout, Oncorhynchus mykiss (Walbaum) (4–40 g; mean 21 g) were obtained from a local hatchery (Rainbow Springs Hatchery, New Dundee, Ontario). The fish were held in two 264-l fibreglass tanks (350 per tank), each supplied at a rate of 900 ml min-1 with a flow-through of dechlorinated, aerated Hamilton city tapwater ([Na+] 0.6 mmol l-1; [Cl-] 0.7 mmol l-1; [Ca2+] 1.0 mmol l-1; [HCO3-] 1.9 mmol l-1; pH 7.9–8.2). The temperature gradually declined from 14 to 10 °C over the 2 month holding period prior to experiments. Fish were fed dry trout pellets (Martin’s Feed Mill Ltd, Ontario) at a rate of 1 % of their body mass per day.
Chronic zinc exposure
One tank was equipped with a dosing system that added Zn2+, as ZnSO47H2O (BDH Chemicals), from a stock solution. The other served as a control tank. The flow rate of the added Zn2+ stock (45.0 mg Zn2+ l-1=0.688 mmol l-1) was maintained at 3 ml min-1, by a peristaltic pump, to achieve a concentration of Zn2+ in the tank of 150 µg l-1±10 % (2.3 µmol l-1). The exposure was initiated by adding a sufficient amount of Zn2+ stock solution in the water to obtain the desired Zn2+ concentration instantly. Water flow and dosing rates were checked daily and adjusted if necessary. The exposure to Zn2+ continued for 60 days, during which the temperature was 8–10 °C. No mortality occurred during the experiment.
Water samples from the exposure tank were taken for analysis of zinc levels before and after each change of stock solution (every fourth day). Samples were acidified with HNO3 (trace metal analysis grade, BDH Chemicals, was used in all procedures), and zinc was measured with an atomic absorption spectrophotometer (Varian AA-1275), using an air/acetylene flame.
Calcium uptake kinetics
A method was developed to measure the kinetics of unidirectional Ca2+ influx in vivo in which each fish provided one data point in a terminal measurement. Ca2+ influx was calculated from the appearance of 45Ca radioactivity in the whole body of the fish over a 4 h period (cf. Perry and Wood, 1985). The flux chambers used were 5 l polypropylene bags (black) filled with 3 l of synthetic Ca2+-free water ([NaCl] 0.7 mmol l-1; [KHCO3] 1.9 mmol l-1; pH 8.0) and equipped with an airline. The bags were placed in polyvinylchloride supports in a waterbath at the same temperature (8–10 °C) as the holding tanks. In total, at each time point, 12 flux-bags were used, representing six different water Ca2+ concentrations for the Zn2+-exposed fish and six for the controls. Five minutes before the fish were introduced to the flux-bags, a sample of a 45Ca/Ca(NO3)2 stock solution [Ca2+ (52 mmol l-1); specific activity 35 kBq mol-1] was added to each of the bags. The volumes added to the bags were 3, 6, 12, 24, 48 and 96 ml, giving approximate Ca2+ concentrations of 52, 104, 207, 413, 820 and 1615 µmol l-1, respectively.
Eight fish were added to each flux-bag at the start of each flux period. Quadruplicate water samples of 5 ml were withdrawn at 0, 2 and 4 h for later analysis of 45Ca and Ca2+. After 4 h in the flux-bags, the fish were given an overdose of anaesthetic (MS 222, 1 g per flux-bag), rinsed for 1 min in 10 mmol l-1 Ca(NO3)2 to displace surface-bound 45Ca, blotted dry, weighed, and individually wrapped in aluminium foil. The fish and the water samples were frozen immediately after the experiment and kept at -20 °C until analysis.
The frozen fish were transferred to liquid nitrogen and ground to a fine powder with a mortar and pestle. The powder was weighed out in triplicate samples of 0.5 g in glass scintillation vials. Each tissue sample was digested with 2.0 ml of liquid tissue solubilizer (NCS, Amersham) for 48 h at 45 °C. The samples were then neutralized with 20 µl of glacial acetic acid, diluted with 10 ml of scintillation fluor (OCS, Amersham) and counted in a scintillation counter (LKB 1217 Rackbeta, Pharmacia-LKB AB).
Duplicate water samples were analyzed for calcium by atomic absorption spectroscopy (Varian AA 1275), using an air/acetylene flame. The remaining duplicate water samples (5 ml each) were diluted with 10 ml of scintillation fluor (ACS, Amersham) and counted for 45Ca.
Initially, Michaelis–Menten analyses of the relationships between Jin and [Ca2+] were performed using both Eadie–Hofstee and Lineweaver–Burk linear regressions. The latter yielded generally higher correlation coefficients (r) and were therefore used throughout (e.g. Fig. 1) to yield values of Jmax, Km and their S.E.M. values for the Michaelis–Menten equation (equation 2). Note that the Lineweaver–Burk transformation yields asymmetrical S.E.M. values:
Calcium uptake kinetics in the presence and absence of zinc
One day before the start of exposure to Zn2+, a control experiment with fish from control and experimental tanks was run to determine the kinetics of Ca2+ influx in the absence of Zn2+. Next, analyses of Ca2+ influx kinetics were performed on Zn2+-exposed fish (at the exposure concentration) and control fish (no Zn2+ present) after 2, 5, 9, 15 and 50 days of exposure. For the Zn2+-exposed fish, 3.0 ml of a ZnSO4 stock solution (150 mg Zn2+ l-1=2.3 mmol l-1) was added to each of the flux-bags to give a final Zn2+ concentration of 150 µg l-1 (2.3 µmol l-1).
In addition, after the first 3 h of exposure to 150 µg l-1 Zn2+, a similar Ca2+ kinetic analysis to that described above was performed, but in the absence of Zn2+. This experiment was designed to give information about any Zn2+-induced short-term alterations in the number of Ca2+ uptake sites and their affinity for Ca2+ (i.e. changes in true Km, rather than apparent Km, which is the value measured in the presence of Zn2+).
Influx of zinc
The unidirectional influx of Zn2+, at the Zn2+ concentration of the exposure tank (150 µg l-1=2.3 µmol l-1), was measured for Zn2+-exposed fish and for control fish at day 56. The flux chambers used were two large black polypropylene bags, each filled with 20 l of dechlorinated Hamilton tapwater (see above) with 150 µg Zn2+ l-1 (2.3 µmol l-1) added as ZnSO4. The flux-bags were equipped with an airline and placed in a tub with a flow-through of water at the same temperature as the holding tanks. Five minutes before the fish were introduced to the bags, 2.59 MBq of carrier-free 65Zn was added to each bag. Ten fish from each tank were transferred to the flux-bags and were held there for 24 h.
Quadruplicate water samples were withdrawn from the flux-bags at 0, 12, 18 and 24 h. At the end of the flux period, the fish were lightly anaesthetized (MS 222, 20 mg l-1) and transferred, one at a time for 1 min, to a beaker with tapwater containing MS 222 (1.0 g l-1). The fish were blotted dry and a terminal blood sample (100 µl) was withdrawn with a heparinized Hamilton syringe from the caudal vessels. The blood was centrifuged and the separated plasma assayed for 65Zn activity in a γ-counter (MINAXI γ Auto-Gamma 5000 Series, Canberra-Packard). Water samples were similarly counted for 65Zn activity and total zinc was measured as above by atomic absorption spectroscopy.
The calculation is not affected by the endogenous level of Zn2+ in plasma. The Zn2+ influx was only measured at one concentration of the substrate (Zn2+).
Acute toxicity tests
LT50 tests of Zn2+-exposed fish and controls were performed at days 27 and 50. On both test occasions, ten fish from each group were transferred to a 14-l black acrylic box. The test chamber was equipped with airlines and divided in the middle by a plastic mesh to allow separate exposure of the two groups to identical water quality. To ensure an efficient water turnover, the inflow and the drain were at opposite ends. The water came from the same source and had an identical temperature to that in the holding tanks. A ZnSO4 stock (350 mg Zn2+ l-1=5.35 mmol l-1) was added at 5 ml min-1 to the incoming water (500 ml min-1) by a peristaltic pump and mixing funnel, yielding a measured Zn2+ concentration in the test chamber from both tests of 3.4±0.3 mg l-1 (52±5 µmol l-1) (mean ± S.E.; N=12). Mortality was monitored throughout the first day of exposure, and four times daily thereafter for 6 days (144 h). LT50 values ±95 % confidence limits were calculated from plots of probit mortality against log time by the methods of Litchfield (1949).
Levels of metallothionein, copper and zinc
Sixty days after the start of the exposure, 10 fish from each group were sampled for analysis of branchial and hepatic levels of metallothionein (MT), copper and zinc. Copper was measured because MT binds both copper and zinc, and therefore an altered zinc content of a tissue could change the level of copper. The fish were killed by a blow to the head, and gills and liver were dissected. Soft tissue of the gill filaments was scraped from the cartilaginous tissue with two microscope slides. The samples were transferred to cryostatic vials, frozen in liquid nitrogen and then stored at -70 °C.
The frozen tissues were later weighed and homogenized individually in 50 mmol l-1 Tris–HCl, pH 8.0, at 0 °C, using a glass–Teflon homogenizer. The volumes of buffer added in the homogenization step were 1.5 ml for liver and 1.0 ml for gill. A sample of 450 µl from each homogenate was stored at -20 °C for subsequent analysis of zinc and copper. The remainder was centrifuged at 10 000 g, 4 °C, for 20 min; the supernatant was decanted, frozen in liquid nitrogen, and stored at -70 °C until used for measurement of MT. MT levels were analyzed with a double antibody radioimmunoassay (RIA), using rabbit antiserum raised against MT from perch, Perca fluviatilis, as the first antibody, 125I-labelled rainbow trout MT as tracer, and goat anti-rabbit IgG as the second antibody (Hogstrand and Haux, 1990). The MT (I and II) from rainbow trout, used as tracer, was purified according to Olsson and Haux (1985) with the modifications described by Hogstrand and Haux (1990) for perch MT. A 10 000 g supernatant prepared from the livers of Cd2+-injected rainbow trout was used as the MT standard. The MT content of the standard was calibrated against a standard curve prepared from purified rainbow trout MT (Olsson and Haux, 1985; Hogstrand and Haux, 1990). The working range of the RIA was 10–100 ng rainbow trout MT per assay tube, which corresponds to 0.6–6 µg g-1 liver wet mass.
Liver and gill homogenates were digested in acid-washed glass tubes for 1 h with 5 vols of 70 % HNO3 at 120 °C. The samples were then cooled to room temperature and 0.75 vols of H2O2 was added. The digests were evaporated to dryness at 120 °C. 5 ml of 1 % HNO3 was finally added to the digestion tubes and copper and zinc were analyzed by atomic absorption spectroscopy as described above. No solid material was present in the final digest.
Protein synthesis
A protein synthesis experiment was run on day 23. Four days prior to this, 10 fish from the control group had been placed into a separate tank and starved. This group was used as a positive control, since starvation is known to reduce protein synthesis relative to that in fish fed daily (Houlihan et al. 1989). On the experimental day, each fish (10 starved controls, 18.12±1.98 g; 10 fed controls, 15.18±3.23 g; 10 fed Zn2+-exposed fish, 18.96±1.59 g) was quickly dried, weighed in air to the nearest 0.01 g and then injected via the caudal vessels with 150 mmol l-1 phenylalanine, pH 7.5 (1 ml 100 g-1 fish), containing 3.7 MBq ml-1 of L-2,6-[3H]phenylalanine. Following injection, the fish were put into individual 250-ml black beakers with fitting lid and airline, containing the same water as before. After 1 h, they were killed by a blow to the head, and the gills and liver were dissected out. Soft gill tissue was immediately scraped from the cartilaginous filament and frozen in liquid nitrogen. Livers and the remaining carcass were also frozen in liquid nitrogen as quickly as possible. The samples were stored at -70 °C for later analysis.
Statistical methods
Significant differences between the control and Zn2+-exposed groups for Ca2+ influx, and the Km and Jmax values for Ca2+ influx, were evaluated by Student’s t-test (two-tailed, unpaired). All statistical tests on these data were based on N=6 per treatment, rather than N=48, because the eight fish in each test concentration were not entirely independent, but had been exposed to an identical water quality in a single flux chamber. The Mann–Whitney U-test was used to detect differences between Zn2+-exposed fish and controls in tissue levels of copper, zinc, MT and total RNA, Zn2+ influx and protein synthesis rate. Groups were considered significantly different at P<0.05. In the acute toxicity tests, the groups were considered different if the 95 % confidence limits for the LT50 values did not overlap (Litchfield, 1949).
Results
Calcium influx in juvenile rainbow trout obeyed Michaelis–Menten kinetics (Fig. 1). One day before the start of Zn2+-exposure, the Km in the absence of Zn2+ was 104 µmol l-1 and the Jmax was 62 µmol kg-1 h-1 (Figs 2 and 3). During the experimental period, both the Km and Jmax values for the control group exhibited significant fluctuations. These were more pronounced for Km, which varied from 38 to 93 µmol l-1 (Fig. 2), whereas the lowest and highest values for Jmax were 40 and 69 µmol kg-1 h-1, respectively (Fig. 3).
Despite these variations in the controls over time, exposure to waterborne Zn2+ caused consistent changes in the kinetics of Ca2+ uptake. Zn2+ markedly increased the Km for Ca2+ influx (by up to 300 %), while there was only a small depressant effect (35 % at most) on Jmax (Fig. 1). With the exception of day 15, the elevations in Km were highly significant on all days relative to the simultaneous controls (Fig. 2), whereas the reduction in Jmax was significant only on days 9 and 15 (Fig. 3). These results were indicative of a large competitive component (increased Km) and a small non-competitive component (decreased Jmax) to the inhibitory effect of Zn2+ on Ca2+ uptake.
Despite this highly effective competitive inhibition by very low levels of Zn2+ (150 µg l-1=2.3 µmol l-1), the actual reductions of Ca2+ uptake experienced by the fish during the exposure were fairly modest (Fig. 4). This occurred because the Km for Ca2+ uptake (38–230 µmol l-1), even when greatly elevated by Zn2+ competition, was so far below the available concentration of Ca2+ (1.0 mmol l-1) in the holding tanks. Indeed, at this acclimation level of Ca2+ in the water, the actual Jin (Fig. 4) closely paralleled the small alterations in Jmax (cf. Fig. 3).
Three hours after the onset of Zn2+ exposure, Ca2+ influx was analyzed for both controls and exposed fish in the absence of waterborne Zn2+. A 3 h exposure to Zn2+ caused a dramatic 23-fold decrease in Km for Ca2+ (Table 1). There was also a much smaller, but still significant, decrease (20 %) in Jmax of exposed fish (Table 1) similar to that seen in the presence of Zn2+ (cf. Fig. 3). Again, the Jmax remained the dominant factor determining the actual Jin at the high water Ca2+ level to which the fish were acclimated. Zinc-exposed fish exhibited a slightly decreased influx (15 %) of Ca2+ after the removal of Zn2+ from the water (Table 1). These results indicate that the actual competitive inhibition of Ca2+ influx, at least during the early days of exposure, may have been even greater than indicated by the large increases in apparent Km shown in Fig. 2, which were determined in the presence of Zn2+. The latter were occurring against a background of a compensatory decrease in the true Km determined in the absence of Zn2+ in the experimental fish. In contrast, the small non-competitive inhibition appeared to be a relatively stable blockade or destruction of transport sites, which remained the same whether or not the Zn2+ was still present.
The Zn2+ influx at the exposure concentration (150 µg l-1=2.3 µmol l-1) was measured on day 56. The Zn2+ influx into Zn2+-exposed fish was 26.5±3.9 nmol kg-1 h-1 (mean ± S.E., N=10), which was significantly lower than the influx, 43±5.9 nmol kg-1 h-1 (mean ± S.E., N=10), measured for controls.
Zn2+-exposed fish developed increased tolerance to a potentially lethal concentration of Zn2+. In the LT50 test started on day 27, challenge with 3.4 mg Zn2+ l-1 resulted in an LT50 of 45.5, +30.5, -18.3 h (mean, +, -95 % confidence limits) for the control fish. The LT50 for Zn2+-exposed fish was greater than 144 h (the end of the test), by which time only 40 % of the fish had died. Consequently, no statistics could be carried out, but extrapolation of the probit mortality versus log time curve gave an LT50 value for exposed fish of about 230 h. A final acute toxicity test, started on day 50, yielded LT50 values for controls of 33.3, +8.0, -6.5 h and for pre-exposed fish of 49.5, +7.3, -5.9 h. These values were significantly different.
There were no differences between the groups in the levels of zinc, copper and MT in gills and liver at day 60 of the experiment (Fig. 5). The concentration of zinc in gills (about 75 µg g-1 wet mass=1.1 µmol g-1) was five times higher than in liver. In contrast, hepatic copper content was approximately 80 µg g-1 (1.2 µmol g-1), whereas the gill copper level was only about 1 µg g-1 (15 nmol g-1). The MT level was twice as high in the liver as in the gills.
Starvation of unexposed fish was employed as a positive control for the protein synthesis experiment. This treatment caused the predicted decrease in whole-body protein synthesis rate (Ks) and RNA content (Table 2). Ks also declined significantly in the gills, but not in the liver, of starved fish. Twenty-three days of exposure to Zn2+ also significantly reduced the Ks in the gills by 11 %. With the exception of this reduction, no other effects of Zn2+ exposure were observed on protein synthesis or RNA levels.
Discussion
The Km values measured in juvenile fish (average 21 g) under control conditions in the present study agreed well with previous measurements on adult trout (average 244 g) in the same water quality (Perry and Wood, 1985). However Jmax values and the actual Ca2+ influx were two-to threefold higher than previous measurements on adult trout. This difference probably reflects allometry plus the more intensive mineralization of bone that occurs in young fish. There was a considerable variation in Km and Jmax during the experimental period for both controls and exposed fish (Figs 2, 3). We have observed the same phenomenon in a separate study (C. Hogstrand, S. D. Reid and C. M. Wood, unpublished results). The Ca2+ influx in juvenile rainbow trout appears to follow a biorhythm, a phenomenon first documented by Wagner et al. (1985, 1986), who observed that there could be a fivefold cyclic variation in the uptake of Ca2+ with a periodicity of about 11 days.
The present study shows that waterborne Zn2+ competitively inhibits the uptake of Ca2+ (Fig. 1), whereas Spry and Wood (1989a) demonstrated that waterborne Ca2+ competitively inhibited the uptake of Zn2+ in rainbow trout under similar water quality conditions. This substantiates previous evidence that these two elements compete for the same uptake sites and routes (Spry and Wood, 1985, 1988, 1989a). For both elements, oesophageal ligation experiments have eliminated drinking as a significant route of influx in freshwater rainbow trout and the gills appear to be the principal site of entry for waterborne Ca2+ and Zn2+ (Perry and Wood, 1985; Spry, 1987). It remains to be established whether the competition between Zn2+ and Ca2+ takes place at the apical membrane, the basolateral membrane of the transport cells or at both sites. There is ample evidence depicting the chloride cell as the uptake site for Ca2+ (Perry and Wood, 1985; Flik et al. 1985; Perry and Flik, 1988; McCormick et al. 1992; Perry et al. 1992; Marshall et al. 1992). The competitive interaction between Zn2+ and Ca2+ suggests that the branchial uptake of Zn2+ also occurs via the chloride cell. Cadmium, which is a very potent inhibitor of Ca2+ uptake, also seems to enter the gills through apical voltage-independent Ca2+ channels of the chloride cells (Verbost et al. 1987, 1989b).
The primary toxic effect of Cd2+ is probably an inhibition of the high-affinity Ca2+-ATPase on the basolateral membrane rather than a competition at the apical channel (Verbost et al. 1987, 1988, 1989a,b). The inhibition of Ca2+ uptake is not reversed within a 12 h period if the Cd2+ is removed, which suggests that Cd2+ causes permanent damage to the Ca2+-ATPase (Reid and McDonald, 1988). In the present study, Zn2+ exerted only a small inhibitory effect on Jmax (Fig. 3), implying that the non-competitive component was minor compared with the competitive inhibition. The inhibition of Jmax could possibly reflect allosteric site interaction. This small effect persisted when the waterborne Zn2+ was acutely removed (Table 1), indicating either a strong binding of Zn2+ to non-competitive inhibitory sites or a small reduction in the number of functional transporters. The important point is that Zn2+, in contrast to Cd2+, did not appear to cause any severe damage to the Ca2+ uptake mechanism. The probable reason is that Zn2+, unlike Cd2+, is an essential micronutrient in fish, and that branchial Zn2+ uptake is a normal phenomenon which becomes especially important when the diet is deficient in Zn2+, i.e. the system is designed to take up Zn2+ in moderate quantities (Spry et al. 1988). Only when the waterborne [Zn2+] becomes unusually elevated does the potential for damage become evident. Recently, Bentley (1992) demonstrated that the presence of Cd2+ also reduces Zn2+ influx. The nature of this inhibition is unknown, although the antagonizing effects of both Ca2+ and Cd2+ on the uptake of Zn2+ fit well to the model that Zn2+ influx occurs via the Ca2+ uptake pathway. If Zn2+ moves through the gill epithelium via the Ca2+ pathway, then the blockade of the common Ca2+/Zn2+ transport mechanism by Cd2+ would inhibit Zn2+ influx.
Fish exposed to Zn2+ for 3 h showed a remarkable 23-fold increase in the affinity for Ca2+ when they were returned to Zn2+-free water (i.e. true Km; Table 1). The response was not an artefact because we have confirmed its presence after 24 h of exposure in an entirely separate study at the same Zn2+ concentration (C. Hogstrand, S. D. Reid and C. M. Wood, unpublished results). Interestingly, the response was similar to that observed when rainbow trout were transferred from the present acclimation water (1000 µmol l-1) to water containing a very low Ca2+ concentration (25 µmol l-1) for 1 day (Perry and Wood, 1985). The explanation could be that the fish or the transport system ‘senses’ low [Ca2+] when Zn2+ is added to the water, because less Ca2+ is available for uptake or is bound to the gill surface. Consequently, the observed increase in affinity of the Ca2+ uptake sites could represent an attempt to restore the normal Ca2+ transporting activity of the gill. The mechanism of this response remains unknown; it could occur via a simple surface chemistry effect on the transport cells associated with the displacement of bound Ca2+, via a cellular effect (e.g. at the channel, intracellular binding protein or basolateral enzyme) and/or via a hormonal effect. It is also unknown whether this decrease in true Km persisted throughout the exposure period. At the very least, an attenuation of the effect seems likely in view of the general increase in apparent Km with time during prolonged Zn2+ exposure (Fig. 2).
It was surprising to find that Ca2+ uptake kinetics exhibited no indication of recovery over time despite the development of increased Zn2+ tolerance seen in the LT50 tests. Indeed if anything, the apparent Km for Ca2+ influx tended to increase during prolonged Zn2+ exposure (Fig. 2). This contrasts with sublethal copper or aluminium concentrations, both of which cause an initial disturbance in Na+ uptake followed by a restoration of branchial Na+ transport as increased tolerance to the metals develops (Laurén and McDonald, 1987; McDonald et al. 1991). For example, Laurén and McDonald (1987) found an immediate increase in Km and decrease in Jmax for Na+ influx in trout exposed to sublethal [Cu2+] for 24 h (i.e. mixed competitive and non-competitive inhibition). However, over 28 days of chronic exposure, Jmax recovered almost completely, while Km was only partially restored; changes in Jmax paralleled alterations in branchial Na+/K+-ATPase activity. The important difference may be that neither copper nor aluminium is taken up by the Na+ transport pathway, whereas Zn2+ seems to be taken up by the Ca2+ transport pathway. A strategy to decrease the uptake of Zn2+ might be to reduce the affinity of the Ca2+ uptake sites. Indeed, the Zn2+ influx of exposed fish, measured at day 56, was significantly decreased relative to the simultaneous control. In accord with these observations, Bradley et al. (1985) found that Zn2+-exposed rainbow trout survived longer in LT50 tests than did controls. When the pre-exposed fish finally died, their gills did not contain more Zn2+ than the controls, although the former spent more time in the test medium before they died. Similarly, the uptake rate of Cd2+ was decreased during chronic Cd2+ exposure (Wicklund Glynn and Olsson, 1991), as well as by several pharmacological treatments known to reduce the apical permeability of the gill epithelium to Ca2+ (Verbost et al. 1989b). Thus, one mechanism to alter the influx of both Zn2+ and Cd2+ could be to change the permeability of the apical membrane to Ca2+.
The actual influx of Ca2+ (Jin), at the water [Ca2+] present in the holding tanks (1.0 mmol l-1), was only moderately decreased by exposure to Zn2+ (Fig. 4). This water Ca2+ concentration gave Jin values in the region close to saturation of the Ca2+ uptake curve (cf. Fig. 1). Therefore, the Jin was largely determined by the Jmax and changes in Km only marginally affected the actual influx. The apparent Km for Zn2+ uptake, measured by Spry and Wood (1989a) in unexposed rainbow trout in the same Hamilton tapwater ([Ca2+]=1.0 mmol l-1), was 240 µg l-1 (3.7 µmol l-1). Since the water Zn2+ concentration used in the present study (2.3 µmol l-1) was below the Km for Zn2+, a change in the affinity of the binding sites for Zn2+ would probably alter the Jin for Zn2+ significantly without having much effect on the Jin for Ca2+. We suggest that, during exposure to sublethal [Zn2+], the fish may manipulate the Km for a mutual Ca2+/Zn2+ carrier so as to reduce Zn2+ influx markedly without greatly altering Ca2+ influx. Measurements of the Km for Zn2+ transport during prolonged Zn2+ exposure and/or depletion will be required to test this hypothesis.
The Zn2+-exposed fish in the present study displayed an increased tolerance to a lethal concentration of Zn2+ from the first test at day 27. Since plasma Ca2+ was not analyzed, it is not known whether the fish also showed a physiological recovery in terms of normalized plasma [Ca2+]. During chronic exposure of tilapia to Cd2+, the plasma [Ca2+] was restored, a response which appeared to be correlated with increased prolactin cell activity, increased plasma cortisol concentration and a proliferation of chloride cells, though it was not clear whether unidirectional Ca2+ influx, efflux or both were corrected (Fu et al. 1989, 1990). In the present study on rainbow trout, the Jmax of both controls and exposed fish declined during the experimental period. Thus, there was no evidence for an increased number of Ca2+ uptake sites associated with the Zn2+ adaptation.
Induction of metallothionein has often been depicted as a possible mechanism responsible for adaptation and development of increased tolerance to metals (Chapman, 1985; Hamilton and Mehrle, 1986; Roesijadi, 1992; McDonald and Wood, 1993). While there is convincing evidence that MT protects cells from metal toxicity, there are few experimental data directly supporting the idea that MT is the dominant factor for the development of increased tolerance to metals in fish (see Hogstrand and Haux, 1991). One of the few is the work of Bradley et al. (1985), who exposed rainbow trout to a much higher, but still sublethal, level of waterborne Zn2+ (670 µg l-1=10.2 µmol l-1) and noted a direct correlation between the development of increased tolerance and the induction of a heat-stable sulphydryl-rich protein (‘putative MT’) in gills and liver. Spry and Wood (1989b) also found induction of a heat-stable metal-binding protein in the gills of trout exposed to 148 µg Zn2+ l-1 (2.3 µmol l-1) for 16 weeks, but concluded that the protein was not MT. In the present study, MT was measured directly by a highly specific RIA (Hogstrand and Haux, 1990). The fish in our study exhibited an enhanced tolerance to Zn2+ without any increase in branchial or hepatic MT (Fig. 5). Moreover, even if all of the MT in the gill (20.7 µg g-1≈3.2 nmol g-1) bound zinc only (i.e. 7 atoms of zinc per MT molecule; Olsson and Haux, 1985), the total zinc-binding capacity of the branchial MT would still be no more than 1.9 % of the zinc present in the gill (1.2 µmol g-1). Even more interestingly, after 60 days of exposure, there was no accumulation of zinc in liver or gill tissue (Fig. 5). With no increased zinc content in these tissues, elevated levels of MT would not be predicted. Bradley et al. (1985), using a much higher exposure level of waterborne Zn2+, found only very modest levels of gill and liver Zn2+ build-up, but apparently this was enough to induce ‘MT’ synthesis. The lack of zinc accumulation and MT induction, found in the present study, suggests that the Zn2+ concentration used was within the nutritional range. If MT is involved in the process of Zn2+ adaptation, it would be as a second line of defence when the level of Zn2+ exposure is high enough to cause a net accumulation in tissues.
As the Zn2+ adaptation develops and the mechanisms responsible for this are expressed, an enhanced metabolic activity could be predicted. An increase in metabolic activity would be reflected in elevated levels of RNA and an increased protein synthesis rate. However, the only significant change found in these variables in Zn2+-exposed fish was a moderate decrease in the rate of protein synthesis in gill tissue. The lack of increased protein synthesis rates in response to the treatment again points towards the conclusion that the Zn2+ exposure was below the toxic range.
As predicted, in starved fish there was a reduction in the RNA content of muscle and in the rate of protein synthesis in the gills and muscle. However, the protein synthesis rate of the liver remained unchanged. The latter observation, though somewhat surprising, agrees with the data of McMillan and Houlihan (1988), who found that protein synthesis rates were similar in the livers of fasted or fully fed rainbow trout.
In summary, the present study supports previous evidence that Zn2+ and Ca2+ compete directly for a common uptake route at the gills. The Km for Ca2+ uptake is far below the normal water [Ca2+], so Jin normally occurs at close to the maximum rate set by Jmax. There is only a small non-competitive component (i.e. decrease in Jmax), but a large competitive component (increase in Km), to the inhibitory effect of Zn2+ on Ca2+ transport. During chronic sublethal Zn2+ exposure, a significantly increased tolerance to Zn2+ can develop without the induction of MT in gills or liver. During this period, the apparent affinity of the transport mechanism for Ca2+ is greatly decreased, which may be a way to minimize Zn2+ uptake via the same route without greatly affecting Ca2+ uptake. This is supported by the findings that the Zn2+ influx was decreased in Zn2+-adapted fish and that there was no zinc build-up in gills or liver.
ACKNOWLEDGEMENTS
This study was supported by an NSERC Strategic Grant in Environmental Quality and a DFO/NSERC Science Subvention Program Grant to CMW, grants from Längmanska kulturfonden and Kungliga Hvitfeldtska Stipendieinrättningen to C.H., and a PDF from SJFR to C.H. We are grateful to Dr D. Houlihan for his instructions in the protein synthesis assay.