The effect of cadmium exposure on digestive enzymes in the Eastern oyster Crassostrea virginica.
Article Type: Report
Subject: Crassostrea (Health aspects)
Oysters (Health aspects)
Cadmium (Health aspects)
Digestive enzymes (Research)
Authors: Adeyemi, Joseph A.
Deaton, Lewis E.
Pub Date: 08/01/2012
Publication: Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2012 National Shellfisheries Association, Inc. ISSN: 0730-8000
Issue: Date: August, 2012 Source Volume: 31 Source Issue: 3
Topic: Event Code: 310 Science & research
Product: Product Code: 0913050 Oysters; 3339640 Cadmium NAICS Code: 114112 Shellfish Fishing; 331419 Primary Smelting and Refining of Nonferrous Metal (except Copper and Aluminum) SIC Code: 0913 Shellfish; 3339 Primary nonferrous metals, not elsewhere classified
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 303011385
Full Text: ABSTRACT The Eastern oyster Crassostrea virginica is a marine bivalve that has been used extensively in metal bioaccumulation studies. We exposed C. virginica to 0 mg/L cadmium, 0.1 mg/L cadmium, or 0.5 mg/L cadmium in seawater for 96 h and then measure the activity of enzymes (amylase, laminarinase, and protease) in the digestive gland. The levels of cadmium in the gills and digestive glands of the animals were also determined. Exposure of the animals to 0.5 mg/L cadmium resulted in a significant decrease in the activities of amylase, laminarinase, and protease enzymes compared with oysters exposed to either 0 mg/L cadmium or 0.1 mg/L cadmium. This decrease corresponds to significantly higher cadmium levels in the gills and digestive glands of oysters exposed to 0.5 mg/L cadmium. The results of this study suggest that exposure to cadmium affects the ability of the animals to process ingested food.

KEY WORDS: cadmium, amylase activity, laminarinase activity, protease activity, eastern oyster, Crassostrea virginica

INTRODUCTION

Marine environments are often exposed to various environmental contaminations, including heavy metals such as cadmium. Concentrations of cadmium range from background levels of 0.05 [micro]g/L to 10-30 [micro]g/L in polluted aquatic habitats (Crompton 2006). Cadmium is a nonessential metal that has been shown to be highly toxic to both plants and animals, even at low concentrations (Stacey et al. 1980; Das et al. 1997).

Bivalve molluscs are ecosystem engineer species of broad impact on a variety of habitats (Ruesink et al. 2005, Wall et al. 2008, Padilla 2010, Shumway 2012). As filter feeders, they represent a major route of transfer of energy and nutrients between the water column and the substrate, acting as the primary benthic pelagic couplers in coastal environments (Wong & Levinton 2006, Trottet et al. 2008, Quan et al. 2012). In coastal and estuarine habitats, the populations of bivalves such as oysters and mussels can be extremely large and have profound effects on the seston concentration, turbidity, and light penetration of the water column (Maar et al. 2007, Wall et al. 2008). Bivalves such as oysters and mussels have been used in a variety of studies on the bioaccumulation of heavy metals (Klumpp & Burdon-Jones 1982, Boening 1999, Sokolova et al. 2005, Azarbad et al. 2010).

Oysters feed by filtering unicellular algae from the water column; the gills are feeding as well as respiratory organs (Ward & Shumway 2004). The digestive gland of oysters produces a variety of catabolic enzymes including amylase, laminarinase, and protease (Langdon & Newell 1996). The objective of this study was to examine the effects of exposure of Crassostrea virginica to concentrations of cadmium representative of those found in polluted aquatic habitats on the activity of enzymes involved in the processing of the microalgae that comprise the primary food of filter-feeding bivalves. We exposed C. virginica to 0 mg/L cadmium (control), 0.1 mg/L cadmium, or 0.5 mg/L cadmium in seawater for 96 h and measured the activities of amylase, laminarinase, and protease in the digestive gland. In addition, the levels of cadmium in the gills and digestive glands were determined.

MATERIALS AND METHODS

Animal Maintenance and Cadmium Exposure

Adult oysters, Crassostrea virginica (shell length, 80-120 ram), were purchased from a local supplier and placed in glass aquaria containing aerated artificial seawater (30 ppt) at 23[degrees]C. The animals were acclimated under these conditions for at least 2 wk and were fed on alternate days with a commercial algal blend (0.5 mL/L) containing Nannochloropsis, Tetraselmis, and Isochrysis spp., ranging in size from 2-15 [micro]m (PhytoPlex).

After the acclimatization period, the oysters were divided into 3 treatment groups with 8 individuals per group. Each group of animals was exposed to either seawater (control), seawater with 0.1 mg/L cadmium, or seawater with 0.5 mg/L cadmium. Three separate aquaria containing 2-3 oysters each were used for each treatment. The exposure to cadmium followed a static renewal system and lasted for 96 h. The concentration of cadmium in each aquarium was measured with an atomic absorption spectrophotometer (Perkin-Elmer 1100B).

Enzyme Assays

After the 96-h period of cadmium exposure, the animals were opened by cutting the adductor muscles, and digestive gland tissue was removed and homogenized in ice-cold buffer consisting of 50 mM N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid) (HEPES) and 500 mM NaCl, with a of pH 7.6. The homogenates were centrifuged (Beckman microfuge E), and the supernatant fluid was removed and stored on ice until they were assayed for enzyme activities. The protein content of aliquots of the tissue extracts was determined by a modified Lowry assay (Miller 1951) using bovine serum albumin as a standard.

Amylase activity in the digestive gland extracts was determined by measuring the glucose released from starch by the enzyme with 4-hydroxybenzhydrazide (PAHBAH). The PAHBAH assay solution was prepared fresh each day by mixing 10 mL PAHBAH stock (10 g PAHBAH and 10 mL concentrated HCl diluted to a volume of 200 mL) with 90 mL of a diluent solution. The diluent was prepared by dissolving 24.9 g trisodium citrate in 500 mL deionized [H.sub.2]O, adding 2.2 g Ca[Cl.sub.2] and 40 g NaOH, and diluting this mixture to 2 L total volume. The assay mixture for amylase activity consisted of 400 [micro]L HEPES buffer and 200 [micro]L potato starch solution (10 mg/mL deionized [H.sub.2]O) in a 1.5-mL microcentrifuge tube. To start the reaction, 400 [micro]L of the tissue extract was added to the assay mixture. After a 4-h incubation at room temperature, 1 mL PAHBAH assay solution was added to the assay tubes. The tubes were heated in a boiling water bath for 10 min, then cooled to room temperature, and the absorbance of the solution was measured at a wavelength of 410 nm in a spectrophotometer (Perkin-Elmer lambda 3). To control for absorbance resulting from the presence of glycosylated proteins in the tissue extracts, tubes containing 600 [micro]L HEPES buffer and 400 [micro]L extract were assayed as well. A standard curve obtained by assaying tubes with a range of 0-1,000 nmol glucose was used for quantification. Laminarinase activity in the tissue extracts was measured similarly, with 10 mg/mL laminarin substituted for the starch in the assay mixture. Enzyme activities were expressed as nanomoles of glucose per milligram protein.

Protease activity was measured using azocasein as a substrate (Deaton 1987). The assay tubes contained 100 [micro]L azocasein (5 mg/mL), 400 [micro]L imidazole buffer, and 500 [micro]L of the digestive gland extract. After 4 h of incubation, 500 [micro]L 10% trichloroacetic acid was added to the assay tubes and they were centrifuged. A 1-mL aliquot of the supernatant was removed and mixed with 2 mL deionized water. The absorbance of this mixture was measured at a wavelength of 440 nm. The enzyme activity was expressed as units per milligram protein.

Tissue Cadmium Accumulation

The concentrations of cadmium in the gill and digestive glands from animals from each treatment group were determined. Tissue samples were dissected from the oysters and rinsed with double-deionized water to remove the externally adsorbed cadmium. The tissues were transferred individually into 2-mL tubes and were stored at -70[degrees]C for later analysis. The tissue samples were later thawed on ice, then dried in an oven at 60[degrees]C for 48 h to obtain the dry weight. The tissues were then digested in concentrated nitric acid (1 mL) for 24 h inside the microcentrifuge tubes. The concentration of cadmium in the tissue digests was measured by atomic absorption spectrometry (Perkin-Elmer 1100B).

Statistical Analyses

In all cases, data were first analyzed using a 2-way mixed-model analysis of variance, with factors being the treatment group and aquaria (because multiple aquaria were used per group). Because the aquaria effect was not significant, this factor was eliminated from the model. The amylase activity, laminarinase activity, protease activity, and tissue cadmium data were analyzed with 1-way analysis of variance, to detect the differences among the means of the different treatment groups. This was followed by Tukey's multiple comparison tests whenever there was a significant difference. We performed Student's t-test to show the difference between the gill and digestive gland cadmium concentrations for each of the treatment groups. All statistics were performed using JMP version 9.0 software (SAS Inc.). For reporting purposes, data were expressed as mean [+ or -] SD, and statistical significance was assumed at P [less than or equal to] 0.05.

[FIGURE 1 OMITTED]

RESULTS

Amylase, Laminarinase, and Protease Activity

Exposure to the highest concentration of cadmium (0.5 mg/L) resulted in a reduction in amylase activity ([F.sub.2,21] : 7.2464, P = 0.0040; Fig. 1), laminarinase activity ([F.sub.2,21] : 38.5571, P < 0.0001; Fig. 2), and protease activity ([F.sub.2,21] = 8.4635, P = 0.0021; Fig. 3). There were no differences in amylase, laminarinase, and protease activity between the control oysters and those exposed to 0.1 mg/L cadmium.

Tissue Cadmium Levels

There was a correlation between the levels of cadmium in both the gills and the digestive glands and the exposure concentrations (Fig. 4). There was a statistically significant difference in cadmium levels in the gill ([F.sub.2,21] = 237.9222, P < 0.0001; Fig. 4) and digestive glands ([F.sub.2,21] = 22.9462, P < 0.0001; Fig. 4) among the treatment groups. The accumulation of cadmium was higher in the digestive gland more than in the gill for the control (T = 6.9142, P = 0.0002, df = 14; Fig. 4) and the oysters exposed to 0.1 mg/L cadmium (T = 3.1517, P = 0.0161, df = 14; Fig. 4). However, there was no difference in cadmium levels between the 2 tissues in animals exposed to 0.5 mg/L cadmium (T = 0.5326, P = 0.6108, df = 14; Fig. 4).

DISCUSSION

Cadmium is a nonessential element with no known biological importance (Xie & Klerks 2004). A variety of toxic effects of cadmium have been reported in marine bivalves, including mortality (Calabrese et al. 1973), reduced growth (Roseijadi & Klerks 1989), oxidative stress (Valavanidis et al. 2006), and effects on blood cells (Weber et al. 1990, Coles et al. 1995). We found that the level of cadmium in the tissues correlated with the ambient concentration of the metal. This is consistent with other studies in which tissue metal accumulation is dependent on the environmental concentrations (Frazier 1976, Azarbad et al. 2010).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Because the gill and digestive glands are the main sites of metal accumulation in molluscs (Miramand & Bentley 1992, Sokolova et al. 2005), we analyzed the metal accumulation in both the gills and the digestive glands of these animals. We found that the digestive glands accumulated more cadmium than the gill. In cephalopods, more than 80% of the total body burden for cadmium is accumulated in the digestive glands (Miramand & Bentley 1992). This trend could have a serious deleterious effect on feeding activities in oysters, because most digestive activities take place in the digestive glands.

In the cells of the digestive gland, cadmium is accumulated in the mitochondria and lysosomes (Sokolova et al. 2005). The function of mitochondria is compromised by cadmium (Kurochkin et al. 2011). Long-term exposure to cadmium has also been shown to decrease the activity of cellulolytic enzymes in the freshwater clam Corbicula fluminea (Barfield et al. 2001). These results suggest that impairment of the digestive gland and the activity of digestive enzymes play a role in the decrease in growth rate observed in bivalves exposed to cadmium (Naimo et al. 1992). Because metallothioneins have been shown to prevent damage to catabolic enzymes by reactive oxygen species in bivalves (Zapata-Vivenes & Nusetti 2007), and the tissue levels of metallothioneins increase in bivalves exposed to cadmium (Viarengo et al. 1997), metallothioneins may be involved in the protection of digestive enzymes in the digestive gland from oxidative damage induced by the production of reactive oxygen species produced by mitochondria that accumulate cadmium.

It is possible that decreased filtration by the oysters exposed to the high concentration of cadmium may explain in part the decrease in enzyme activities of the digestive gland that we observed. A lower rate of filtration and processing of food might in these animals relative to the controls and animals exposed to a lower concentration of cadmium may have reduced the digestive activity of the hepatopancreas. The filtration rate of bivalves is reduced by exposure to cadmium (Kraak et al. 1992, Naimo et al. 1992). In this study, we reported the disruption of feeding activities by cadmium in the Eastern oyster. Exposure to 0.5 mg/L cadmium resulted in a 71% reduction in amylase activity, an 88% reduction in laminarinase activity, and a 34% reduction in protease activity compared with the control. Oysters that were exposed to 0.1 mg/L cadmium did not show any evidence of impairment of feeding activities by cadmium when compared with the control oysters. These results imply a concentration-dependent response in these organisms. A similar result was obtained in a similar study by van Gestel et al. (1993), who reported that cadmium's inhibition of growth in Eisenia andrei is concentration dependent. However, it is rather surprising that, in this study, exposure to 0.1 mg/L cadmium did not affect enzyme activities in these oysters when cadmium concentrations as low as 20 [micro]g/L decreased oxygen uptake and clearance rate in freshwater clams (Naimo et al. 1992). The lack of disruption of feeding activities at this concentration could be explained by competitive interaction among ions in seawater, which could result in reduction in bioavailability of cadmium ions, such that the concentration of bioavailable cadmium at this concentration is rather low to evoke any significant impairment of feeding activities in these oysters.

[FIGURE 4 OMITTED]

In conclusion, the results of this study showed that cadmium contamination of marine ecosystems can have serious effects on essential activities in marine bivalves--specially on feeding activities--and that these effects are concentration dependent.

ACKNOWLEDGMENT

This study is supported in part through the funds provided by the Graduate Student Organization of the University of Louisiana at Lafayette to J. A.

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JOSEPH A. ADEYEMI (1) * AND LEWIS E. DEATON (2)

(1) Department of Biological Sciences, Osun State University, P.M.B. 4494, Osogbo, Osun State, Nigeria;

(2) Department of Biology University of Louisiana at Lafayette, 300 East St. Mary Blvd, Lafayette, LA 70504

* Corresponding author. E-mail: joseph.adeyemi@gmail.com

DOI: 10.2983/035.031.0306
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