An arsenic-binding protein in rainbow trout

An arsenic-binding protein in rainbow trout

ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY An Arsenic-Binding ADEBAYO Department of Biological 9, 1-5 (1985) Protein in Rainbow Trout A. OLADIMEJ...

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ECOTOXICOLOGY

AND

ENVIRONMENTAL

SAFETY

An Arsenic-Binding ADEBAYO Department

of Biological

9, 1-5 (1985)

Protein in Rainbow

Trout

A. OLADIMEJI

Sciences,

Ahmadu

Received

August

Bell0

University,

Zaria,

Nigeria

11, I983

A pulse dose of 5 pCi 74As-labeled arsenic acid was given in diet contained in No. 4 gelatin capsules to two groups of rainbow trout (90 + 6 g). These groups consisted of the control and the treatment group which was preexposed to 22.8 mg As/kg diet for 2 weeks. An extract of cytosol prepared from the liver of both control and pretreated fish sacrificed 48 hr after the pulse do:* was found to contain an arsenic-binding protein with molecular weight of about 19,000. This protein accounted for less than 25% of the extracted cytosol in both the control and preexposed fish. Arsenic may not induce the synthesis of thionein in the liver as the molecular weight of 19,000 is outside the range previously reported for metallothionein. The binding of arsenic to this protein may not be of any adaptive significance. 65 1985 Academic Press. Inc.

INTRODUCTION

Several authors have reported adaptive responsesin aquatic species(Sinley et al., 1974; Beattie and Pascoe, 1979). Rainbow trout were reported to adapt to sublethal levels of zinc (Sinley et al., 1974) and cadmium (Beattie and Pascoe, 1979). Eels exposed to fsublethalconcentrations of mercuric chloride (HgCl,) exhibited increased resistance by surviving subsequent exposure to usually lethal concentrations (Boquegneau et al., 1975). Resistance induced by small doses of metals in fish was attributed tlo the synthesis of metal-binding proteins which detoxify metals acquired during subfsequent exposure (Brown and Parsons, 1978; Pruell and Engelhardt, 1979). Dixon (1979) and Oladimeji et al. (1982) similarly reported that pretreatment of rainbow trout with low dosesof arsenic induced some resistance to subsequent exposure. The objective of this study is to investigate whether the increased resistance to arsenic reported by Dixon (1979) could be attributed only to the enhanced excretion of arsenic as reported by Oladimeji et al. (1982) or also to the synthesis of an arsenic-binding protein. MATERIALS

AND

METHODS

Rainbow trout, Salmo gairdneri (90 + 6 g), were obtained from Goosens Trout Farm, Otterville, Ontario, Canada. Upon arrival in the laboratory, fish were held in 500-liter aquaria, equipped with cooling units (Mini-o-cool units, Toledo, Ohio). The tanks were aerated and supplied with a continuous flow of dechlorinated water having a pH of 7.2-7.4, a hardness of 45 mg liter-‘, and dissolved oxygen of 9-l 1 ppm. The t’emperature of the water was maintained at 10 + 1°C and the fish were fed Purina Trout Chow at a daily rate of 2% body wt during acclimatization as well as during the experiments. Determination of the effect of preexposure on the protein binding of arsenic was carried out using two groups of four fish each. These consisted of the control (maintained1under identical conditions in the absenceof arsenic) and the treatment 1

0147-65 13/85 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproductmn in any form rescwed

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ADEBAYO

A. OLADIMHI

group of four fish which was preexposed to 22.8 mg As/kg (as sodium arsenite) of diet for 14 days, during which they were fed at a daily rate of 2% body wt. Following the 1Cday preexposure of the treatment groups, both groups were starved for a day and fed a pulse dose of 5 &i 74As-labeled arsenic acid mixed with trout chow in No. 4 gelatin capsules. The four fish from each group were sacrificed 48 hr after the single dose of radioactive arsenic. The livers were excised, weighed, and homogenized with chilled isotonic saline. The homogenate was centrifuged at 35,OOOg for 30 min in a Sorval refrigerated centrifuge to separate the residue. The supernatant was withdrawn and centrifuged at 35,000g for an additional 15 min with fresh saline, and the final supematant (cytosol) of the control and preexposed fish was fractionated by gel chromatography. The final supematant was applied directly to a 2.6 X loo-cm column of Sephacryl S-200 superfine (Pharmacia Fine Chemicals, Dorval, Quebec, Canada) and eluted at 10 ml hr-’ with 0.1 it4 Tris-HCl buffer, pH 8, containing NaCl (0.5 M). One hundred 2-ml fractions of the eluate were collected. Optical absorbance of the eluate fractions was monitored with a spectrophotometer (Pye Unicam PS68-100) at 280 nm for arsenic. Eluate fractions were also subsequently assayed for arsenic (74As) using an Ortec deep-well gamma counter, consisting of a 7.6 X 7.6-cm NaI detector crystal in a lead-shielded well. Quantitative protein determination was not carried out, but the presence or absence of protein was determined by precipitation using 5% trichloroacetic acid (TCA). Precipitation on the addition of drops of TCA indicated the presence of protein. The Sephacryl S-200 column was calibrated for molecular weight determination with horseheart cytochrome c (12,500), bovine pancreas chymotripsinogen a (25,000), hen egg albumin (45,000), and bovine serum albumin (67,000) as illustrated in Fig. 2. RESULTS All fish appeared healthy during the experiment, although the livers of fish that were pretreated with arsenic for 2 weeks were pale in color. Histological studies in subsequent studies during which rainbow trout were treated with a similar dose of arsenic for the same duration resulted in necrosis of the liver. No histological studies were carried out in the present study. The elution pattern showing the uv absorbance (280 nm) and arsenic radioactivity (cpm 74As) of the fractionated cytosol of control and treated livers is shown in Fig. 1. The elution profile of 74A~ showed two peaks of arsenic, the first (Peak I) of which coincided with the tail of the protein peak eluted in fractions 40-70 as shown in Fig. 1. When the fractions corresponding to the peaks were tested for the presence of protein using trichloroacetic acid, only peak I, the fraction associated with 74As (elution fractions 40-50), formed precipitates. The second peak of arsenic eluted in fractions 75-95. The elution profile was similar for livers from both the control and pretreated fish. Molecular weight estimates based on the calibration shown in Fig. 2 indicated that the Peak I protein-arsenic complex (eluate fractions 40-50) corresponded to molecular size of 19,000; and Peak II to molecular size 3000 containing a bigger proportion (75-85%) of the arsenic applied to the column could have been elemental arsenic and arsenic bound to peptides.

ARSENIC-BINDING

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30

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Fraction

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PROTEIN

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80

90

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FIG. 1. Gel filtration chromatography of liver cytosol. (a) Control (nonpreexposed) rainbow trout sacrificed 48 hr after ingestion of 74As-labeled arsenic acid. (b) Preexposed (22.8 mg nonradioactive As/ kg diet for 2: weeks) rainbow trout sacrificed 48 hr after ingestion of a subsequent single dose of “‘Aslabeled arsenic acid.

DISCUSSION The tissue distribution data indicated that accumulation of arsenic in liver, bile with gallbladder, and kidney was influenced by preexposure (Oladimeji, 1980) suggestingsome adaptation mechanism similar to the formation of metallothionein which has been shown for some group 2B elements (e.g., Cu, Ni, Zn, and Cd) of the periodic table (Olafson and Thompson, 1974; Coombs, 1975). The detoxification of these metals has been shown to be by synthesis of metallothionein. One mechanism of metallothionein-mediated tolerance to heavy metals is the synthesis

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ADEBAYO

I

3.3

A. OLADIMEJI

I

3.5

4.0 Log ,.

molecular

I

I

4.5

5.5

weight

FIG. 2. Determination of molecular weights of arsenic-protein complex by Sephacryl S-200 gel chromatography: Relationship between elution volume V/V, and molecular weights of standard proteins (Combitek calibration proteins (Boehringer-Mannheim, West Germany). (1) Horse heart cytochrome (12,500); (2) bovine pancreas chymotrypsinogen (25,000); (3) hen egg albumin (45,000); and (4) bovine serum albumin (67,000).

of the excess of this protein when fish were preexposed. The excess of this protein is then used to bind and detoxify metals acquired during subsequent exposure (Brown and Parsons, 1978; Pruell and Engelhardt, 1979). Since the liver and kidney have been identified as the sites for the synthesis of metallothionein, binding of these metals has been associated with a corresponding increase in their concentrations in these tissues. For instance, Olson et al. (1977) indicated that the liver of rainbow trout S. gairdneri that were exposed to mercury contained the major proportion of the Hg-thionein, suggesting the detoxification of mercury by synthesis of metallothionein. Contrary to our initial suggestion (Oladimeji et al., 1979) that arsenic was bound to thionein in the liver, this study indicates that it is not. The chromatography of soluble liver fraction showed that none of the peaks corresponded to the molecular weight range of 6000-12,000 which has been reported previously in fish (Olafson and Thompson, 1974; Coombs, 1975) for the group 2B elements. One may therefore conclude that it is possible that arsenic does not induce synthesis of thionein in the liver of rainbow trout. However, the results of this study show that arsenic is bound to some protein which has a molecular weight of about 19,000. Although the rainbow trout did show some adaptation possibly by fast metabolism of the inorganic to organic form and subsequent excretion of the metabolite (Oladimeji et al., 1979; Oladimeji et al., 1982), the binding of arsenic to protein may be of little adaptive significance.

ARSENIC-BINDING

PROTEIN

ACKNOWLEDGMENTS The author thanks Dr. A. S. W. deFreitas of the Atlantic Regional Laboratory, Halifax, Nova Scotia, and Dr. S. U. Qadri, Department of Biology, University of Ottawa, for their assistance during the study.

REFERENCES BEATTIE, J. H.. AND PASCOE, D. (1979). A cadmium-binding protein in rainbow trout. Toxicol. Lett. 4, 241-246. BOQUEGNEALI,J. M., GERDAY, C., AND SISTECHE, A. (1975). Fish mercury-binding thionein related to adaptation mechanisms. FEBS Lett. 5, 173-177. BROWN, D. A., AND PARSONS,T. R. (1978). Relationship between cytoplasmic distribution of mercury and toxic effects to zooplankton and chum salmon (Onchorynchus keta) exposed to mercury in a controlled ecosystem. J. Fish. Res. Board Canad. 35, 880-884. COOMBS, T. L. (1975). The significance of multielement analyses in metal pollution studies. In Ecotoxicological

Toxicological

Research-Effects

of Heavy

Metal

and

Organohalogen

Compounds

(A. D. McIntyre and C. F. Mills, eds.). Plenum, New York. DIXON, D. G. (1979). Acclimation to toxicants by rainbow trout its potential use in predicting safe levels. Personal communication. OLADIMEJI, A. A., QADRI, S. U., TAM, G. K. H., AND DEFREITAS, A. S. W. (1979). Metabolism of inorganic arsenic to organoarsenicals in rainbow trout (Salmo gairdneri). Ecotoxicol. Environ. Saf 3, 394-400. OLADIMEJI, A. A. (1980). Bioaccumulation and metabolism of arsenic by rainbow trout, Salmo gairdneri. Ph.D. thesis. University of Ottawa. OLADIMEJI, A. A., QADRI, S. U., AND DEFREITAS, A. S. W. (1982). Effects of acclimation of rainbow trout (Salmo gairdnert] to arsenic on retention of a subsequent dose of arsenic. Ecotoxicol. Environ. SaJ: 6, 196-203.

OLAFSON, R. W., AND THOMPSON, J. A. J. (1974). Isolation of heavy metal binding proteins from marine vertebrates. Mar. Biol. 28, 83-86. OLSON, K., SQUIBB, K. S., AND COUSINS, R. J. (1977). Methylmercury binding by trout cytosol. Unpublished. PRUELL, R. J., AND ENGELHARDT, F. R. (1979). Liver cadmium uptake, catalase inhibition and cadmium thionein production in the killifish (Fund&us heteroclitus) induced by experimental cadmium exposure. Mar. Environ. Res. 3, 101-l 1 I. SINLEY, J. R., GOETL, J. P., JR., AND DAVIES, P. H. (1974). The effects of zinc on rainbow trout Salrno gairdneri, in hard and soft water. Bull. Environ. Contam. Toxicol. 12, 193-201.