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. Author manuscript; available in PMC: 2011 Aug 11.
Published in final edited form as: In Vivo (Brooklyn). 2010 Winter;31(2):34–42.

Effects of Potential Therapeutic Agents on Copper Accumulations in Gill of Crassostrea virginica

Juan D Luxama 1, Margaret A Carroll 1, Edward J Catapane 1
PMCID: PMC3154757  NIHMSID: NIHMS227898  PMID: 21841975

Abstract

Copper is an essential trace element for organisms, but when in excess, copper’s redox potential enhances oxyradical formation and increases cellular oxidative stress. Copper is a major pollutant in Jamaica Bay and other aquatic areas. Bivalves are filter feeders that accumulate heavy metals and other pollutants from their environment. Previously it was determined that seed from the bivalve Crassostrea virginica, transplanted from an oyster farm to Jamaica Bay readily accumulated copper and other pollutants into their tissues. In the present study we utilized Atomic Absorption Spectrometry to measure the uptake of copper into C. virginica gill in the presence and absence of three potential copper -blocking agents: diltiazem, lanthanum, and p-aminosalicyclic acid. Diltiazem and lanthanum are known calcium-channel blockers and p-aminosalicylic acid is an anti-infammarory agent with possible metal chelating properties. We also used the DMAB-Rhodanine histochemistry staining technique to confirm that copper was entering gill cells. Our result showed that diltiazem and p-aminosalicyclic acid reduced copper accumulations in the gill, while lanthanum did not. DMAB-Rhodanine histochemistry showed enhanced cellular copper staining in copper-treated samples and further demonstrated that diltiazem was able to reduce copper uptake. The accumulation of copper into oyster gill and its potential toxic effects could be of physiological significance to the growth and long term health of oysters and other marine animals living in a copper polluted environment. Identifying agents that block cellular copper uptake will further the understanding of metal transport mechanisms and may be beneficial in the therapeutic treatment of copper toxicity in humans.

Introduction

Jamaica Bay, a 26 square mile estuarial embayment situated between southern Brooklyn and Queens, NY and a major inlet opening to the Atlantic Ocean, lies just east of the entrance to NY Harbor and the mouth of the Hudson River. Copper is a major pollutant in Jamaica Bay and other aquatic areas. Sediment is an important sink and reservoir for metal contaminants and Jamaica Bay sediment is reported to be contaminated with various metal pollutants1–3 including copper at levels higher than 10 ppm4. Bivalves are particularly good accumulators of heavy metals5–7 and being sessile, tend to reflect local contaminant concentrations more accurately than crustaceans and free swimming finfish. Historically the Eastern Oyster, Crassostrea virginica, flourished in Jamaica Bay and the NY/NJ Harbor area as either self-sustaining or farmed populations8,9, but pollution and other problems caused a steady decline in the oyster industry after its peak in the early 1900’s10–12. Today very few wild oysters are found in Jamaica Bay and studies are being done to look at the rehabitation potention of C. virginica to Jamaica Bay. Previously it was determined that C. virginica seed, transplanted from an oyster farm in Oyster Bay, NY to Jamaica Bay, grew well13 despite accumulating significant amounts of copper and other pollutants in their tissues14.

Copper is an essential micronutrient. In addition to its role in activation or repression of transcription of various genes, copper is required as an integral component of at least 12 major proteins involved in such processes as cellular respiration, catecholamine production, connective tissue biosynthesis, superoxide dismutation, iron metabolism and blood coagulation15,16. In humans about one-third of all the copper in the body is contained in the liver and brain, another third is in the muscles, and the rest is dispersed in other tissues17. Adverse health effects are related to copper deficiency as well as excess.

Excess copper can cause both structural and functional impairment due to displacement of ions at metal binding sites or non-specific binding to enzymes, DNA, or other biomolecules18. Alternatively, free copper ions can cause oxidative damage by catalyzing reactions that generate oxyradicals19. The 2 most common oxidation states for copper are Cu (I) and Cu (II) and the easy exchange between these two oxidation states endows copper with redox properties that may be of an essential or deleterious nature in biological systems. Figure 1 shows how soluble copper ions can increase oxidative stress by substituting for iron in the Fenton reaction20 which catalyzes the conversion of hydrogen peroxide and superoxide into the highly cytotoxic hydroxyl radical21, 22. Indeed, the oxidative damage caused by hydroxyl radicals and other reactive oxygen species are thought to be major contributing factors to the development of cancer, diseases of the nervous system and aging23. Mitochondria are particularly sensitive to oxidative damage and depend upon various antioxidants and anti-oxidizing systems to defend against oxidative stress. As the major site of O2 utilization, mitochondria are not only a source of reactive oxygen species24 but are improtant targets for oxidative damage. The presence of excess copper and resulting oxyradicals can overwhelm cellular defensive mechanisms, especially in mitochondrial, compromising respiratory function and further impairing cellular health and survival. Previously, our lab found that oyster gill mitochondrial treated with high doses of copper had impaired O2 utilization25.

Fig. 1.

Fig. 1

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The Fenton Reaction utilizing copper.

In the present study we used atomic absorption spectrometry and histological methods to study copper accumulation in gilll of C. virginica and to compare the therapeutic actions of three potential copper-blocking agents: diltiazem, lanthanum and p-aminosalicylic acid. We also used a p-dimethylaminobenzylidene (DMAB) rhodanine staining technique to histologically localize copper in copper-treated gill sections in the presence and absence of diltiazem.

Lanthanum and diltiazem are well known calcium channel blockers. Lanthanum is an element that forms a trivalent cation that strongly reacts with calcium binding sites and affects most membrane transport processes involving Ca2+ ions26–30. Diltiazem is a benzothiazepine that acts selectively on the voltage-dependent L-type Ca2+ channels31 and is used therapeutically in the treatment of angina pectoris, hypertension and supraventricular arrhythmias32. Previously, our lab found that treatments with diltiazem but not lanthanum were able to protect oyster gill from the deleterious effects of copper additions on mitochondrial respiration33. However, the mechanism of action on how diltiazem blocked the negative effects of copper on mitochondrial respiration is unknown. While these two calcium channel blocker acted differently in our respiration experiments, they both may have the potential of reducing copper accumulations in oyster gill. p-aminosalicyclic acid (PAS), an analogue of aminobenzoic acid, is another potential copper-blocking agent. PAS has antibacterial properties and has been used to inhibit the growth and multiplication of the tubercle bacillus34,35. Recently PAS has been used in the successful treatment of Manganism36, a disease of manganese toxicity, which is clinically similar to Parkinson’s disease. While the mechanism by which PAS alleviates symptoms of Manganism remains unknown, evidence is accumulating that PAS is acting as a manganese chelator37,38. Previous studies of our lab found that PAS reduced manganese accumulations in oysters39 and also protected manganese treated gill mitochondria from a dose dependent decrease in O2 consumption40. The present study examined the potential of PAS as a copper-blocking agent.

Materials and Methods

Instant Ocean Artificial Seawater (ASW) was obtained from Aquarium Systems Inc. (Mentor, OH). Lanthanum chloride, diltiazem, PAS, copper sulfate, DMAB-Rhodanine, nitric acid (trace-metal grade) and all other chemicals were obtained from Fisher Scientific (Pittsburgh, PA). Adult C. virginica of approximately 80 mm shell length were obtained from Frank M. Flower and Sons Oyster Farm in Oyster Bay, NY. They were maintained in the lab for up to two weeks in temperature -regulated aquaria in ASW at 16 – 18°C, specific gravity of 1.024 ± 0.001 and pH of 7.2 ± 0.2. Each animal was tested for health prior to experimentation by the resistance it offered to being opened. Animals that fully closed in response to tactile stimulation and required at least moderate hand pressure to being opened were used for the experiments.

Copper Treatments of Oyster Gill Tissue in the Presence of Therapeutic Agents

Prior to tissue isolation, all glassware was acid-washed in dilute (5%, wt/wt) nitric acid in deionized water to removed bound metals. Washing was followed by thorough rinses with deionized water. Healthy oysters were shucked by removing their right shells. Gills were dissected using stainless steel instruments and cut into uniform-size pieces (approximately 0.30 wet weights). Gill segments were placed in containers of ASW containing 0.5 mM copper with or without various concentrations (0.5, 1.0 and 2.0 mM) of lanthanum, diltiazem or PAS for 3 days at 15°C. Control animals were similarly prepared without exposure to copper or test agents. After 3 days, gill segments were removed from the containers, weighed, washed 3 times with ASW, and prepared for metal analysis.

Analysis of Copper Accumulation by Atomic Absorption Spectrometry

Gill sections were placed in an oven at 200°C for 2 hours and dry weight of each sample determined. Dried gill sections were digested with concentrated nitric acid on hot plates in a fume hood. Digested samples were adjusted to a final volume of 10 ml in dilute (0.2 %, wt/wt) nitric acid. Aliquots of each sample were analyzed for copper by electrothermal vaporization with deuterium lamp background correction in a Perkin Elmer AAnalyst 800 Atomic Absorption Spectrophotometer with a THGA Graphite Furnace. Copper levels are reported as μg/g dry weight. Statistical significance was determined by two-way ANOVA with Tukey Post test.

The Histological Staining of Copper in Copper-Treated Gill

Gills were dissected from animals and sectioned into uniform smaller pieces. Gill sections were placed in containers of ASW containing 0.5 mM copper, or 0.5 mM copper and 2.0 mM of diltiazem for 3 days at 15°C. Control animals were similarly treated without exposure to copper and test agents. After 3 days the gill sections were removed, washed 3 times with ASW and prepared for fixation. Gill sections were fixed by placing them in containers containing 4% paraformaldehyde in ASW for 20 minutes, and then washed 3 times with ASW. The gills sections were then dehydrated in an alcohol series (70%, 95%, 100% (3Xs) alcohol for 5 minutes each). After dehydration, gill sections were placed in xylene, xylene/paraffin (50/50) and paraffin in a paraffin oven for 4 hours. After 2 days, embedded gill were sectioned with a microtome at 10 microns, placed on warm microscope slides to flatten, deparaffinized in xylene and rehydrated in an alcohol series (absolutely alcohol, 100%, 95%, 70%, distilled water) for 5 minutes.

A stock of 0.1% 5-DMAB-rhodanine was prepared by mixing 0.1g in 100ml absolute ethanol and allowing to stand overnight. A working solution of 4% paraformaldehyde in phosphate buffer was prepared by adding 4.0 g of the DMAB-rhodanine stock to 100 ml of deionized water. DMAB-Rhodanine solution was placed on the slides at 60°C for 1 hour. Gills sections were then rinsed 4 times in distilled water, and then 0.5% of sodium borate solution was briefly added and rinsed off. The sections were washed 3 times in distilled water. The slides were viewed for copper staining with a Zeiss microscope under bright field and phase contrast. Sections were photographed with a ProgRes C3 Peltier cooled camera.

Results

Gill copper levels were determined by Atomic Absortion Spectrometry in gill sections from 20 different specimens of C. virginica, treated under various conditions. Gill samples that were not exposed to copper treatments in the lab had base-line copper levels that ranged from 197–217 ug/g dry weight (dwt).

Effects of Lanthanum, Diltiazem, and PAS on Copper Accumulations in Gill

Gill samples that were incubated for 3 days in ASW containing 0.5 mM CuSO4 readily accumulated significant amounts of copper, exceeding 300%, compared to base-line controls. This increase was not reduced when co-treated with up to 2 mM of lanthanum (Fig. 2). However, both diltiazem and PAS demonstrated copper-blocking activity when gill samples were similarly treated. There was a dose dependent decrease in copper accumulations in gill tissue of up to 64% with the 2 mM dose of diltiazem (Fig. 3). Similar results were obtained when gill samples were co-treated with PAS. The presence of PAS produced a dose dependent decrease in copper accumulations of up to 75% with the 1 mM and 2 mM doses of PAS (Fig. 4).

Figure 2.

Figure 2

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Gills were dissected from adult oysters and treated for 3 days with 0.5 mM copper or varying amounts of lanthanum at 15°C in individual containers of ASW, after which time they were washed 3 times with ASW and prepared for Atomic Absorption Spectrometry. N = 6. There were no statistical difference among the treatments.

Figure 3.

Figure 3

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Gills were dissected from adult oysters and treated for 3 days 0.5 mM copper or varying amounts of diltiazem at 15°C in individual containers of ASW, after which time they were washed 3 times with ASW and prepared for Atomic Absorption Spectrometry. N = 6. ap<0.05 for comparison to 0 mM Dilt, 0.5 mM Cu.

Figure 4.

Figure 4

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Gills were dissected from adult oysters and treated for 3 days with 0.5 mM copper or varying amounts of PAS at 15°C in individual containers of ASW, after which time they were washed 3 times with ASW and prepared for Atomic Absorption Spectrometry. N = 6. ap<0.01, bp<0.05 for comparison to 0 mM PAS, 0.5 mM Cu.

The Histochemical Staining of Copper in Gill

The widely used DMAB-Rhodanine staining for copper is a histochemistry technique that localizes copper in cells as reddish spots. Excess copper accumulates in the cytoplasm of cells and binds to copper- associated protein. DMAB-Rhodanine is a bidentate chelating agent which has a strong affinity for proteinaceous copper deposits in tissues section41–44. Figure 5a,b,c are photomicrographs of control gill sections prepared for DMAB-Rhodanine staining. The sections were viewed with phase contrast microscopy and show slight staining indicating low amounts of endogenous copper within the cells.

Figure 5.

Figure 5

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A: Control gill without additional copper treatments, phase contrast, 200x, Arrows point to endogenous copper deposits. B: Control gill without additional copper treatments, phase contrast, 500x, Arrows point to endogenous copper deposits. C: Control gill without additional copper treatments, phase contrast, 1250x, Arrows point to endogenous copper deposits.

Treating gills for 3 days with 0.5 mM copper resulted in observable copper accumulations in the gills. DMAB-Rhodanine staining revealed massive accumulations of copper within the cytoplasm of the gill cells and copper pigments in the sections (Fig. 6a,b,c). When gills were co-treated with 0.5 mM of copper plus 2 mM of diltiazem for 3 days, DMAB-Rhodanine staining revealed significantly less copper accumulations in the co-treated gill cells, compared to those treated with copper alone (Fig. 7a,b,c).

Figure 6.

Figure 6

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A: Copper (0.5 mM) treated for 3 days, phase contrast 200x. Numerous red copper deposits are present. B: Copper (0.5 mM) treated for 3 days, phase contrast 500x. Numerous red copper deposits are present. C: Copper (0.5 mM) treated for 3 days, phase contrast 1250x. Numerous red copper deposits are present.

Figure 7.

Figure 7

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A: Copper (0.5 mM) treated plus diltiazem (2.0 mM), phase contrast 200x. Arrows point to copper deposits. B: Copper (0.5 mM) treated plus diltiazem (2.0 mM), phase contrast 500x. Arrows point to copper deposits. C: Copper (0.5 mM) treated plus diltiazem (2.0 mM), phase contrast 1250x. Arrows point to copper deposits.

Discussion

Copper is an essential micronutrient needed as an integral component in a large number of enzymatic and structural proteins. However, the potential for toxicity exists and copper homeostasis must be tightly regulated so that the concentration of free copper remains extremely low. When in excess, copper is a potent cytotoxin, binding and impairing the function of various biomolecules, displacing other metals from their normal binding sites, or causing oxidative damage by catalyzing the conversion of hydrogen peroxide and superoxide into hydroxyl radicals. It is noteworthy that the generation of hydroxyl radicals from hydrogen peroxide and superoxide via the Haber-Weiss reaction can only take place when catalytic concentrations of transition metals like iron or copper are present45.

Marine bivalves are filter feeders that take up and accumulate metals and other pollutants from the water column. They also can ingest metal contaminants adsorbed to phytoplankton, detritus and sediment particles. Because they are sessile, they reflect local concentrations more accurately than crustaceans and free swimming finfish. Marine bivalves such as oysters and mussels have been extensively used as model organisms in environmental studies of water quality46–48. Trace metals are taken up and accumulated by oysters and many other marine invertebrates to tissue and body concentrations usually much higher on a wet weight basis than concentrations in the surrounding seawater49–51.

Copper is a major aquatic pollutant. Nriagu reported average copper levels in seawater ranging from 0.15 μg/liter in open ocean to 1.0 μg/liter in polluted near-shore waters52 but other studies show a much wider variation especially in polluted waters with reports as high as 40 μg/liter in estuaries in southwest Spain53.

Previous works of our lab showed that C. virginica growing in Jamaica Bay, a copper polluted environment, readily accumulated copper14. Atomic absorption spectrometry revealed that the soft tissues accumulated copper in the ug/g dwt range, which was comparable to other published reports for C. virginica growing in other areas6,54. Copper accumulations were not homogeneously distributed throughout the oyster’s soft tissues with greater amounts accumulating in the gill, heart and palps; shell tissues also accumulated copper, but to a lesser extent14.

The present study confirmed that isolated gill quickly accumulates high levels of copper and demonstrates that this copper analysis and staining technique is useful in testing chemicals and drugs that may be effective in reducing copper accumulations. Subjecting oyster gill to 0.5 mM copper for 3 days increased gill copper by more than 300%. Lanthanum, a calcium channel blocker, was tested for its ability to reduce copper accumulations in the gill. Lanthanum was not effective in reducing copper accumulations. Diltiazem, which is another calcium channel blocker, was effective in reducing copper accumulations in the gill. This was shown by both atomic absorption spectrometry and by histochemical staining. Earlier works also showed that diltiazem was effective in reducing the deleterious effects of copper on mitochondrial respiration while lanthanum was not33. Diltiazem reduced the cellular accumulations of copper. PAS, which is an anti-inflammatory agent with suspected chelating abilities, is being shown as a possible therapeutic drug for Manganism36. PAS reduced copper accumulations into gill as effectively as did diltiazem.

The DMAB-Rhodanine staining for copper histochemistry technique confirmed that copper was entering gill cells and that diltiazem greatly reduced the cellular uptake of copper. In light of the mitochondrial studies, it appears that the protective actions of the known calcium channel blocker, diltiazem, against the deleterious effects of copper on respiration are likely due to blocking copper accumulations. Lanthanum did not protect mitochondria against copper toxicity, and it did not reduce copper accumulations in oyster gill. PAS is not believed to act as a channel blocker and the mechanism by which PAS reduced copper accumulations is not yet known, but may involve copper chelation. The accumulation of copper into gill and the toxic effects of copper on gill mitochondria could be of physiological significance to the growth and long-term health of oysters and other marine animals living in a copper polluted environment. The toxic effects of copper on oysters in particular and animals in general, can be of physiological and medical significance for humans with copper toxicity or exposed to high copper levels. In humans a number of disorders in copper homoeostasis exist such as Wilson Disease55–57, a condition leading to progressive accumulation of copper with resulting cirrhotic and neurological damage. Identifying agents and mechanisms which reduce copper accumulation and protect mitochondria is beneficial for understanding and therapeutic treatment of copper toxicity in humans.

Acknowledgments

This work was supported in part by grants 2R25GM0600305 of the Bridge Program of NIGMS, 0516041071 of the CSTEP Program of the New York State Department of Education and 0622197 of the DUE Program of NSF.

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