COPPER TOXICITY AND METALLOTHIONEIN INDUCTION IN

FISH: EFFECT OF WATER TEMPERATURE AND PH

Cleoni dos Santos CarvalhoDepartamento de Ciências Fisiológicas

Universidade Federal de São Carlose-mail: carvcleo@yahoo.com.br

Heloisa Sobreiro Selistre de AraújoDepartamento de Ciências Fisiológicas

Universidade Federal de São CarlosTel. ++55-16-260 8333, Fax ++55-16-260 8327

e-mail: hsaraujo@power.ufscar.br

Marisa Narciso FernandesDepartamento de Ciências Fisiológicas

Universidade Federal de São Carlos Rodovia Washington Luiz, km 235; Caixa Postal 676

13565-905 São Carlos, SP, BrasilTel. ++55-16-260 8314, Fax ++55-16-260 8327

e-mail: dmnf@power.ufscar.brAbstract

Copper accumulation and metallothionein (MT) concentration were determined in the liver of Prochilodus scrofa exposed to the respective 96h-LC50 at 20 and 30oC and pH 4.5 and 8.0. Copper accumulation in the liver of copper exposed fish was greater at lower temperature and pH 8.0. MT concentration in the liver was higher in fish exposed to copper at 30oC. Copper accumulation is related to copper toxicity and the water pH is a determinant factor in this process. MT concentration in the liver seems to depend on water temperature rather than copper concentration in water.

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Introduction

Ecotoxicologists have used the expression of metallothionein (MT) as a biomarker for heavy metal contamination due to its rapid increasing concentration by induction with ions such as cadmium, mercury, zinc, and copper; although, MT may be also induced by a great variety of pathophysiological factors, including restriction of food intake, bacterial infection, and exposure to physical and inflammatory stress (Kägi, 1993; Muto et al., 1999). Copper is an essential element that serves as a cofator in a number of enzyme systems for most living organisms but, at high concentration, copper become a toxic pollutant. Copper is used today a chemotherapeutic agent in aquaculture however, the increased level of copper in aquatic environments coming from industrial or agricultural wastes. The physical and chemical characteristics of water play an important role in copper toxicity for aquatic animals. Copper speciation is directly affected by water pH, and the free cupric ion concentration is higher in water with low pH, while copper hydroxide complexes prevails in water with high pH (Payle et al., 1992; Tao et al., 2001).

Water temperature acts as a regulator of the physiological processes of poikilothermal animals, such as the fish. All life processes of living organisms are physicochemical in nature therefore, temperature affect all these processes. Although the aquatic environments are in general thermally stable, seasonal variations in temperature characterize the subtropical regions. High temperatures tend the speed up the physiological processes and, as the temperature changes, the rate of various processes must be balanced and coordinated, and the organisms must compensate or minimize the changes in its body (Reynolds and Casterlin, 1980).

Therefore, changes in water temperature, as occurs in the Southeast Brazil that range from 15-20oC during the winter and 28-35oC during the summer and in pH due to episodic ecologically accidents, as most Brazilian freshwaters is ion-poor and soft having low buffering capacity, may affect profoundly copper toxicity and also the MT response in fish living in such environment. Carvalho et al. (2004) demonstrated high copper accumulation in the liver of Prochilodus scrofa with concomitant expression of MT in the liver. To evaluate the effect of temperature on copper toxicity in fish, copper accumulation and the MT

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induction in the liver of Prochilodus scrofa, we exposed fish to copper in water with 4.5 and 8.0 pH at 30ºC. P. scrofa is highly sensitive to copper (Mazon and Fernandes, 1999) and copper toxicity is strongly influenced by water pH (Takasusuki et al., 2004; Carvalho, 2003) however, this species also show fast physiological recovery from copper effects and long-term gill histological recovery after transference to water free of copper (Cerqueira and Fernandes, 2002a,b). Furthermore, this species has a high tolerance for changes in temperature (Barrionuevo and Fernandes, 1995). The thermal range for the temperature acclimation of P. scrofa goes from 15 to 35°C (Barrionuevo and Fernandes, 1995, 1998; Fernandes et al., 1995), and displays a thermal tolerance zone, determined by the critical minimum and maximum temperatures, equivalent to 1046°C2 for small fish (Barrionuevo and Fernandes, 1995).

Materials and Methods

Juvenile P. scrofa (body mass 18.5 3 g) were holding tanks at 25 ± 1°C with a continuous flow of aerated dechlorinated tap water (water composition: pH = 7.3 ± 0.2; alkalinity = 23.7 ± 1.9 mg L-1 as CaCO3; hardness = 24.5 ± 0.2 mg L-1 as CaCO3) for 30 days, after which the water temperature was lowered or increased by 1°C on alternate days until it reached 20 or 30°C which were the average lower and higher temperature of P. scrofa natural habitat The fish were kept at this temperature for a minimum of 30 days prior to the experiments.

The experiments were done in static-systems. Control groups were maintained in aquariums with copper-free water and the pH was adjusted to 4.5 and 8.0. Copper exposed groups were maintained in aquariums, which received a concentration of copper (CuSO4.5H2O) equal the nominal 96h-LC50 of copper for P. scrofa, in water with pH 4.5 (88 ± 0.8 gCu L-1) and pH 8.0 (14 ± 0.2 gCu L-1) at 30oC (Carvalho, 2003). The water temperature in each aquarium was kept stable at 30°C. All experiments were done in duplicate. Copper concentration in water was measured using Atomic Absorption Spectrophotometry.

After 96h the controls and the surviving fish from groups exposed to copper were killed and the liver was dissected. A sample of liver was taken to determine the total copper, which was done using Atomic Absorption Spectrophotometry. Cytosolic proteins were isolated from the remaining liver tissue to determine MT and total protein concentrations. MT was determined in the supernatant from the centrifuged homogenate of liver, following the method described by Ahmad et al. (2000) with slight modifications. Purified protein fractions contained MT

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were obtained using a Sephadex G-75 column and DEAE-Sepharose column following by polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli (1970). The protein concentration in the liver tissue was determined by Lowry’s method (Lowry et al., 1951). The significance of the data was determined using the ANOVA analysis of variance and the Tukey test with a 95% confidence limit was applied to compare mean values whenever the data was significant.

Results and Discussion

The study reveals important aspects regarding the effect of environmental water pH on copper toxicity and MT induction in fish. The percentage of surviving fish from groups exposed to copper (96h-LC50) in water at pH 4.5 and 8.0 was 50 and 46% at 30 ºC, respectively. No fish died from the control group. As expected, the results found by Carvalho et al. (2004) in the same species exposed to the respective 96h-LC50 copper concentration in water with pH 4.5 (98 ± 0.8 gCu L-1) and 8.0 (16 ± 0.5 gCu L-1) at 20oC was also close to 50% and confirms the influence of water pH on copper toxicity evidencing no effect of temperature in copper toxicity as reported by Carvalho (2003).

The Cu2+ form of copper, considered the more toxic form of copper species, is bioavailable in waters with pH lower than 6.0 (Tao et al., 2001) and, paradoxally, copper toxicity was lower in water with pH 4.5. In this case, the H+, which concentration is high at low pH, and Cu2+competition for Ca2+ sites in the gill membrane may be the main reason for lower copper toxicity in low water pH (Meador, 1991). In waters with high pH, the reduced concentration of H+ and low levels of Ca2+ as found in the Brazilian freshwaters may favor copper binding to gill surface membrane, increasing copper uptake and, hence, its toxicity.

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Figure 1. Copper concentration in the liver of P. scrofa exposed to copper (96h-LC50) in water with pH 4.5 and 8.0 at 20 (data from Carvalho et al., 2004) 25 oC (Takasusuki et al, 2004) and 30oC (present results). Values from the control groups (0 gCuL-1; open bars) and from groups exposed to 96h-LC50 copper concentration in water with pH 4.5 and 8.0 (black bars). The bars indicate the mean values ± SEM; (*) indicates a significant difference from the respective control group (p< 0.05); and (#) indicates a significant difference in relation to copper – pH 4.5 and pH 8.0; and (+) indicates a significant difference in relation to temperature (p< 0.05).

Copper accumulation in the liver of fish exposed to copper was significantly higher (p < 0.05) than in the controls at the end of the 96 h of copper exposure fish and higher (p < 0.05) at pH 8.0. Similar results was found by Carvalho et al. (2004) in fish acclimated at 20oC. Figure 1 shows a comparison between copper concentration in liver tissue of fish copper exposed groups at 20oC (Carvalho et al., 2004), 25oC (Takasusuki et al., 2004) and 30oC (present results).

The MT-mass protein identified in the liver of P. scrofa was lower than 15 kDa as revealed by SDS-PAGE electrophoresis. Among the numerous factors stimulating MT biosynthesis, the increased of MT concentration in aquatic animals has been associated with increased levels of metals in the aquatic environment and with the length of exposure time (Olsvik et al., 2000). However, some authors have emphasized that MT production depends, at least up to a

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**

** *

#+

*#+

certain critical concentration (Roméo et al., 1997), on the effective increase in metal inside the animal, implying a response to intracellular heavy metal content (Mayer et al., 2003).

The effect of temperature seems to be evident in the MT concentration. The mean liver MT concentrations from fish exposed to copper in water with pH 4.5 and pH 8.0, are shown in the table I. MT concentration in the liver of fish exposed to copper at 20oC were not difference on both water pH (Carvalho et al., 2004). Similar results were found in the MT concentration in the liver of fish at 30oC (p>0.05).

Table I. MT concentration in the liver of P. scrofa from groups exposed to 96h-LC50 copper concentration in water with pH 4.5 and 8.0 at 20 and 30ºC. Values are means ± SEM.

Water Temperature

(oC)

Metallothionein concentration (µg.g-1 liver wet mass)

Reference

pH 4.5 pH 8.020 126.8 ± 21.5 167.4 ± 25.6 Carvalho et al, 2004

30 710 ± 70* 1010 ± 60* present study

(*) indicates a significant difference in relation to copper – pH 4.5 and pH 8.0 at 20 and 30oC (p< 0.05).

The diffusion rate and all biological processes are higher at high temperatures. The lower copper accumulation and higher MT concentrations in the liver of fish exposed to copper at 30oC in both water pHs may be due to the higher activity of metabolic processes related to copper detoxification including the excretion rate and suggests an imbalance between copper uptake and detoxification/excretion rate in neotropical fish at low temperature, seeming to favor higher copper accumulation, since the 96-LC50 of copper in water at pH 4.5 and 8.0 was the same at water temperatures of 20°C and 30ºC (Carvalho, 2003). It could be explained by an extremely slow elimination of copper in low temperature.

In conclusion, this study provides evidence that copper accumulation is related to copper toxicity and the pH of water is a determinant factor in this process. MT

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concentration in the liver seems to depend on water temperature rather than copper concentration in water.

Acknowledgements

This study was supported by grants from the Brazilian research funding institutions FAPESP and CNPq. The authors thank the Hydrobiology and Aquaculture Station of the Furnas Hydroelectric Powerplant, Furnas, Minas Gerais, Brazil for supplying the fish. C.S. Carvalho thanks FAPESP for the award of a scholarship.

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