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The effects of copper exposures on cellular responses in oysters
Marine Environmental
PII:
SOl41-1136(97)00084-6
Research, Vol. 46, No. l-5, pp. 591-595, 1998 0 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0141-1136/98 $19.00+0.00
ELSEVIER
The Effects of Copper Exposures on Cellular Responses in Oysters A. H. Ringwood, a~b*D. E. Conner@ and A. DiNovob “Marine Resources Research Institute, 217 Fort Johnson Road, Charleston, South Carolina, SC 29412, USA bMedical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina, SC 29425, USA ABSTRACT Copper is an essential and toxic trace metal that can adversely affect metalrequiring enzymes and proteins, and cause oxidative damage. Oysters, Crassostrea virginica, bioconcentrate Cu from environments contaminated by a variety of point and non-point sources. The purpose of these studies was to evaluate a suite of cellular parameters (lysosomal destabilization, glutathione concentrations, lipid peroxidation, and metallothioneins) in Cu-exposed oysters (5 to 80 pgl-’ for two weeks) in order to distinguish between exposure and stress responses. Copper exposures caused increased lysosomal destabilization, increased lipidperoxidation, and induction of Cu metallothioneins, but no effects on glutathione were observed. Experiments were also conducted in which BSO was used to deplete glutathione to consider how other environmental factors that affect amelioration responses may potentiate Cu toxicity. Lysosomal destabilization rates were significantly higher in glutathione-depleted oysters. Lipid peroxidation was higher initially during the first four days of CM exposures, but then the levels declined to control levels. This amelioration was associated with the increased expression of metallothioneins. During the jirst four days of Cu exposure, no signtjicant increases in MTs were observed, but subsequent induction of MTs was associated with reduced lipid peroxidation. Some cellular responses to Cu exposures represent normal compensatory mechanisms that may eflectively ameliorate the insult. Therefore, it is important to appreciate the signtjicance of the response or use a suite of responses to determine when eflects have progressed from an exposure response to really being a signal of stress. 0 1998 Elsevier Science Ltd. All rights reserved
Copper, a common environmental pollutant that is both essential and very toxic to marine organisms, can adversely affect metal-requiring enzymes and proteins, and can cause generation of hydroxyl radicals leading to a variety of types of oxidative damage *To whom correspondence
should be addressed. 591
592
A. H. Ringwood
et al.
(Viarengo, 1989; Moore, 1994; Mason and Jenkins, 1995). Because Cu is an essential trace element, normal regulatory mechanisms serve to maintain homeostasis during short term fluctuations. However, long-term exposures to elevated Cu concentrations may cause significant adverse effects. Cellular biomarkers that function as sensitive indicators of Cu exposures, as well as those that indicate when homeostatic mechanisms have been overwhelmed are needed. Laboratory studies were conducted to evaluate the effects of environmentally realistic concentrations of Cu on lysosomal destabilization rates, glutathione (GSH) concentrations, lipid peroxidation (LPx), and metallothionein expression (MT). These responses have been identified as potentially valuable biomarkers of anthropogenic stress (Stegeman et al., 1992). Glutathione and MTs are involved in amelioration of adverse effects, while destabilized lysosomal membranes and accumulation of lipid peroxides are indicators of damage. Oysters (3-5 cm in height) were collected from a clean site and exposed to a range of copper concentrations (0, 5, 10, 20, 40, and 80 pg 1-l) at 25% salinity and 25°C (water, Cu, and food renewed every other day) for 14 days. Oyster digestive gland tissues (sampled after 4, 7, and 14 days) were used for all analyses. The potential effects of GSH depletion (experimentally induced using buthionine sulfoximine, BSO) were evaluated during short term exposures. In these experiments, oysters were exposed to Cu (20 pg 1-i) and 20 mg 1-l BSO singly or in combination for 48 h, and the lysosomal destabilization rates as well as GSH concentrations were determined. The effects of starvation on GSH levels were evaluated at 12 h intervals over a 48 h period for fed and unfed oysters. Lysosomal destabilization rates were assessed using modifications of a neutral red (NR) retention assay described by Lowe and Pipe (1994). Digestive gland tissues (10 individual oysters per treatment) were disaggregated using trypsin (0.4mgml-‘) and Ca2+/Mg2+ free saline (CMFS). Equal aliquots of the cellular suspension and neutral red dye (0.04 mg ml-‘) were placed on a microscope slide, and incubated at room temperature in a light protected humidity chamber for one hour. At least 5&100 cells per sample were scored for lysosomal NR retention using oil immersion, light microscopy. Lysosomal membrane destabilization was expressed as the percentage of cells with NR leaking into the cytosol. Glutathione concentrations in oyster tissues (five individual oysters per treatment) were determined by the DTNB-GSSG Reductase Recycling Assay, a sensitive and specific enzymatic procedure that follows the rate of 5-thio-nitrobenzoic acid (TNB) formation (Anderson, 1985). GSH concentrations were estimated from a standard curve and reported as nM GSH gg’ wet wt. The degree of lipid peroxidation was assessed using malondialdehyde (MDA) concentrations as a surrogate measure (Gutteridge and Halliwell, 1990). Tissues (five individual oysters per treatment) were homogenized in 50mM potassium phosphate buffer and centrifuged. A subsample of the supernatant was combined with trichloroacetic acid, thiobarbituric acid, and butylated hydroxytoluene, heated at 100°C for 15 min, and monitored at 532 nm. MDA concentrations were derived from a standard curve and expressed as nM gg’ wet tissue weight. The metallothioneins of pooled oyster digestive gland tissues were isolated as described previously using FPLC (Superdex lo/50 size exclusion column) (Ringwood and Brouwer, 1993). Ammonium bicarbonate buffers with B-mercaptoethanol, bubbled with nitrogen were used for all separations. Metal concentrations of chromatographic fractions were analyzed by furnace AAS after acidification. The results were adjusted for tissue weight and expressed as Cu concentrations associated with the MT pool (nM Cu-MTg-‘).
The effects of copper exposures on cellular responses in oysters
593
Adverse effects on cellular parameters were observed following exposure to low Cu concentrations (the data for the various responses of control oysters and oysters exposed to 80 pgl-’ Cu are listed in Table 1). Significant adverse effects on lysosomal destabilization rates were observed for all concentrations of Cu, increasing linearly with increasing concentration and duration. However no effects on GSH concentrations were observed. No effects on lipid peroxidation were observed with the lower Cu concentrations (5, 10, 20 pgl-I), and although LPx levels of oysters exposed to 4Opgl-’ for 4 days were higher than controls, only the 8Opgl-’ treatment had significantly higher levels (p < 0.06). However LPx levels declined to control levels after 7 and 14 days of continued exposure. There was little evidence of significant MT induction after four days of exposure to 80,~gll’, but by seven days there was evidence of MT induction, and high Cu concentrations were associated with the MT pool after 14 days of exposure. These results suggest that adverse effects, as evidenced by elevated lipid peroxidation after four days, were manifested prior to the induction of MTs, but when Cu-MTs were detected, there was a concomitant decrease in lipid peroxidation (Fig. 1). The amelioration of cellular stress responses with increasing MT production reported here is similar to previous studies conducted with mussels (Viarengo et al., 1987). When oysters held in the laboratory were fed, GSH levels were virtually unchanged for 48 h. When starved, there were no changes in GSH levels after 12 h (982 nM gg’), but then GSH concentrations declined linearly so that at 48 h, GSH levels of starved oysters were less than 40% of fed oysters. When oysters were exposed to BSO alone, GSH levels were 35.5% of controls, but there were no effects on lysosomal destabilization. Those exposed to Cu alone had significantly higher rates of lysosomal destabilization (34% compared to 15% for controls); and the rates of those exposed to Cu and BSO in combination were 43%, significantly different from those exposed only to Cu as well as the controls (ANOVA, p < 0.05). These results suggest that conditions which cause GSH depletion may increase the potential for adverse effects during pollutant exposures. In summary, lysosomal destabilization functioned as a sensitive indicator of Cu exposure (other associated studies have indicated that a response is measurable by as early as 18 h after exposures to Cu). The Cu concentrations and durations used in this study did not affect GSH levels, but GSH depletion has been observed in oysters from contaminated
The Effects of Laboratory
Cu Exposures
Total [Cu] fnMg_‘)
TABLE 1 (8Opgl-I) on Cellular virginica)
% Lysosomal destabilization
Responses
GSH (nMg_‘)
in Oysters LPX fnMg-*)
(Crassostrea CM-MT f nMg-‘J
Control Day 4 Day 7 Day 14
390.6 416.4 441.3
20.9 f 5.6 22.4 f 7.6 26.9 f 5.8
968.7 f 184.3 865.4 f 83.3 903.1 1t61.4
464.0 f 207.1 224.1 f 260.0 229.0 f 40.9
5.5 5.4 6.1
Cu-Exposed Day 4 Day 7 Day 14
501.4 1147.6 2952.5
46.41k 11.5 49.8?c 10.1 60.6 f 9.4
870.9 f 320.5 988.0 f 216.3 973.8 f 228.9
820.0 f 272.3 428.4 f 140.6 331.45265.1
13.6 109.3 281.9
Data for total Cu concentrations samples. For all other parameters,
of digestive gland tissues data are means f standard
and Cu-MTs deviations.
are from pooled
tissue
A. H. Ringwood et al.
594
Crassostrea virginica Cu-Exposed (SOug/L)
Cu-MT
0
-
Days
Fig. 1. The effects of Cu exposures (80 pg I-‘) on lipid peroxidation (LPx) and Cu-MTs in oysters
after 4, 7, and 14 days of exposure.
sites. Conditions that cause GSH depletion may potentiate pollutant toxicity. Lipid peroxidation was a sensitive indicator of perturbed homeostasis, and there was evidence that the adverse effects were ameliorated by MTs. Since lipid peroxidation damage was observed prior to the induction of MTs, it might be expected that if exposures continued to the point that the detoxification capacities of MTs were overwhelmed, lipid peroxidation rates would again increase, indicating significant perturbation of homeostasis that could progress to irreversible damage. It is therefore important to distinguish short-term fluctuations that may be ameliorated by compensatory mechanisms from responses that signal more serious levels of stress (Livingstone, 1982). An appreciation of the significance of a response or the use of a suite of responses would facilitate our ability to distinguish exposure from significant levels of damage, which if unmitigated, may lead to reduced fitness.
ACKNOWLEDGEMENTS I gratefully acknowledge J. Hameedi and NOAA (National Oceanic and Atmospheric Administration, Office of Ocean Resources Conservation and Assessment) who provided funding for these studies (grant number NA570A0486).
REFERENCES Viarengo, A. (1989) Aquatic Sciences 1, 295317. Moore, M. N. (1994) In Contaminants in the Environment: A Multidisciplinary Assessment of Risks to
The effects of copper exposures on cellular responses in oysters
595
Man and Other Organisms, eds A. Renzoni, N. Mattei, L. Lorena and M. C. Fossi, pp. 111-123. CRC Press, Lewis Publishers, Boca Ratan, FL. Mason, A. Z. and Jenkins, K. D. (1995) In Metal Speciation and Bioavailability in Aquatic Systems, eds A. Tessier and D. R. Turner, pp. 479-608. John Wiley & Sons, Ltd. Stegeman, J. J., Brouwer, M., Di Giulio, R. T., Forlin, L., Fowler, B. A., Sanders, B. M. and Van Veld, P. A. (1992) In Biomarkers Biochemical, Physiological, and Histologic Markers of Anthropogenic Stress, eds R. J. Huggett, R. A. Kimerle, P. N. Nehrle and H. L. Bergman, pp. 235-335. Lewis Publishers, Chelsea. Lowe, D. M. and Pipe, R. K. (1994) Aquatic Toxicology 30, 357-365. Anderson, M. E. (1985) Meth. Enzymology 113, 548-555. Gutteridge, J. M. C. and Halliwell, B. (1990) TZBS 15, 129-135. Ringwood, A. H. and Brouwer, M. (1993) Comparative Biochemistry Physiology 106B, 523-529. Viarengo, A., Moore, M. N., Mancinelli, G., Mazzucotelli, A., Pipe, R. K. and Farrar, S. V. (1987) Marine Biology 94, 251-257.
Livingstone, D. R. (1982) Marine Pollution Bulletin 13, 261-263.
PII:
SOl41-1136(97)00084-6
Research, Vol. 46, No. l-5, pp. 591-595, 1998 0 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0141-1136/98 $19.00+0.00
ELSEVIER
The Effects of Copper Exposures on Cellular Responses in Oysters A. H. Ringwood, a~b*D. E. Conner@ and A. DiNovob “Marine Resources Research Institute, 217 Fort Johnson Road, Charleston, South Carolina, SC 29412, USA bMedical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina, SC 29425, USA ABSTRACT Copper is an essential and toxic trace metal that can adversely affect metalrequiring enzymes and proteins, and cause oxidative damage. Oysters, Crassostrea virginica, bioconcentrate Cu from environments contaminated by a variety of point and non-point sources. The purpose of these studies was to evaluate a suite of cellular parameters (lysosomal destabilization, glutathione concentrations, lipid peroxidation, and metallothioneins) in Cu-exposed oysters (5 to 80 pgl-’ for two weeks) in order to distinguish between exposure and stress responses. Copper exposures caused increased lysosomal destabilization, increased lipidperoxidation, and induction of Cu metallothioneins, but no effects on glutathione were observed. Experiments were also conducted in which BSO was used to deplete glutathione to consider how other environmental factors that affect amelioration responses may potentiate Cu toxicity. Lysosomal destabilization rates were significantly higher in glutathione-depleted oysters. Lipid peroxidation was higher initially during the first four days of CM exposures, but then the levels declined to control levels. This amelioration was associated with the increased expression of metallothioneins. During the jirst four days of Cu exposure, no signtjicant increases in MTs were observed, but subsequent induction of MTs was associated with reduced lipid peroxidation. Some cellular responses to Cu exposures represent normal compensatory mechanisms that may eflectively ameliorate the insult. Therefore, it is important to appreciate the signtjicance of the response or use a suite of responses to determine when eflects have progressed from an exposure response to really being a signal of stress. 0 1998 Elsevier Science Ltd. All rights reserved
Copper, a common environmental pollutant that is both essential and very toxic to marine organisms, can adversely affect metal-requiring enzymes and proteins, and can cause generation of hydroxyl radicals leading to a variety of types of oxidative damage *To whom correspondence
should be addressed. 591
592
A. H. Ringwood
et al.
(Viarengo, 1989; Moore, 1994; Mason and Jenkins, 1995). Because Cu is an essential trace element, normal regulatory mechanisms serve to maintain homeostasis during short term fluctuations. However, long-term exposures to elevated Cu concentrations may cause significant adverse effects. Cellular biomarkers that function as sensitive indicators of Cu exposures, as well as those that indicate when homeostatic mechanisms have been overwhelmed are needed. Laboratory studies were conducted to evaluate the effects of environmentally realistic concentrations of Cu on lysosomal destabilization rates, glutathione (GSH) concentrations, lipid peroxidation (LPx), and metallothionein expression (MT). These responses have been identified as potentially valuable biomarkers of anthropogenic stress (Stegeman et al., 1992). Glutathione and MTs are involved in amelioration of adverse effects, while destabilized lysosomal membranes and accumulation of lipid peroxides are indicators of damage. Oysters (3-5 cm in height) were collected from a clean site and exposed to a range of copper concentrations (0, 5, 10, 20, 40, and 80 pg 1-l) at 25% salinity and 25°C (water, Cu, and food renewed every other day) for 14 days. Oyster digestive gland tissues (sampled after 4, 7, and 14 days) were used for all analyses. The potential effects of GSH depletion (experimentally induced using buthionine sulfoximine, BSO) were evaluated during short term exposures. In these experiments, oysters were exposed to Cu (20 pg 1-i) and 20 mg 1-l BSO singly or in combination for 48 h, and the lysosomal destabilization rates as well as GSH concentrations were determined. The effects of starvation on GSH levels were evaluated at 12 h intervals over a 48 h period for fed and unfed oysters. Lysosomal destabilization rates were assessed using modifications of a neutral red (NR) retention assay described by Lowe and Pipe (1994). Digestive gland tissues (10 individual oysters per treatment) were disaggregated using trypsin (0.4mgml-‘) and Ca2+/Mg2+ free saline (CMFS). Equal aliquots of the cellular suspension and neutral red dye (0.04 mg ml-‘) were placed on a microscope slide, and incubated at room temperature in a light protected humidity chamber for one hour. At least 5&100 cells per sample were scored for lysosomal NR retention using oil immersion, light microscopy. Lysosomal membrane destabilization was expressed as the percentage of cells with NR leaking into the cytosol. Glutathione concentrations in oyster tissues (five individual oysters per treatment) were determined by the DTNB-GSSG Reductase Recycling Assay, a sensitive and specific enzymatic procedure that follows the rate of 5-thio-nitrobenzoic acid (TNB) formation (Anderson, 1985). GSH concentrations were estimated from a standard curve and reported as nM GSH gg’ wet wt. The degree of lipid peroxidation was assessed using malondialdehyde (MDA) concentrations as a surrogate measure (Gutteridge and Halliwell, 1990). Tissues (five individual oysters per treatment) were homogenized in 50mM potassium phosphate buffer and centrifuged. A subsample of the supernatant was combined with trichloroacetic acid, thiobarbituric acid, and butylated hydroxytoluene, heated at 100°C for 15 min, and monitored at 532 nm. MDA concentrations were derived from a standard curve and expressed as nM gg’ wet tissue weight. The metallothioneins of pooled oyster digestive gland tissues were isolated as described previously using FPLC (Superdex lo/50 size exclusion column) (Ringwood and Brouwer, 1993). Ammonium bicarbonate buffers with B-mercaptoethanol, bubbled with nitrogen were used for all separations. Metal concentrations of chromatographic fractions were analyzed by furnace AAS after acidification. The results were adjusted for tissue weight and expressed as Cu concentrations associated with the MT pool (nM Cu-MTg-‘).
The effects of copper exposures on cellular responses in oysters
593
Adverse effects on cellular parameters were observed following exposure to low Cu concentrations (the data for the various responses of control oysters and oysters exposed to 80 pgl-’ Cu are listed in Table 1). Significant adverse effects on lysosomal destabilization rates were observed for all concentrations of Cu, increasing linearly with increasing concentration and duration. However no effects on GSH concentrations were observed. No effects on lipid peroxidation were observed with the lower Cu concentrations (5, 10, 20 pgl-I), and although LPx levels of oysters exposed to 4Opgl-’ for 4 days were higher than controls, only the 8Opgl-’ treatment had significantly higher levels (p < 0.06). However LPx levels declined to control levels after 7 and 14 days of continued exposure. There was little evidence of significant MT induction after four days of exposure to 80,~gll’, but by seven days there was evidence of MT induction, and high Cu concentrations were associated with the MT pool after 14 days of exposure. These results suggest that adverse effects, as evidenced by elevated lipid peroxidation after four days, were manifested prior to the induction of MTs, but when Cu-MTs were detected, there was a concomitant decrease in lipid peroxidation (Fig. 1). The amelioration of cellular stress responses with increasing MT production reported here is similar to previous studies conducted with mussels (Viarengo et al., 1987). When oysters held in the laboratory were fed, GSH levels were virtually unchanged for 48 h. When starved, there were no changes in GSH levels after 12 h (982 nM gg’), but then GSH concentrations declined linearly so that at 48 h, GSH levels of starved oysters were less than 40% of fed oysters. When oysters were exposed to BSO alone, GSH levels were 35.5% of controls, but there were no effects on lysosomal destabilization. Those exposed to Cu alone had significantly higher rates of lysosomal destabilization (34% compared to 15% for controls); and the rates of those exposed to Cu and BSO in combination were 43%, significantly different from those exposed only to Cu as well as the controls (ANOVA, p < 0.05). These results suggest that conditions which cause GSH depletion may increase the potential for adverse effects during pollutant exposures. In summary, lysosomal destabilization functioned as a sensitive indicator of Cu exposure (other associated studies have indicated that a response is measurable by as early as 18 h after exposures to Cu). The Cu concentrations and durations used in this study did not affect GSH levels, but GSH depletion has been observed in oysters from contaminated
The Effects of Laboratory
Cu Exposures
Total [Cu] fnMg_‘)
TABLE 1 (8Opgl-I) on Cellular virginica)
% Lysosomal destabilization
Responses
GSH (nMg_‘)
in Oysters LPX fnMg-*)
(Crassostrea CM-MT f nMg-‘J
Control Day 4 Day 7 Day 14
390.6 416.4 441.3
20.9 f 5.6 22.4 f 7.6 26.9 f 5.8
968.7 f 184.3 865.4 f 83.3 903.1 1t61.4
464.0 f 207.1 224.1 f 260.0 229.0 f 40.9
5.5 5.4 6.1
Cu-Exposed Day 4 Day 7 Day 14
501.4 1147.6 2952.5
46.41k 11.5 49.8?c 10.1 60.6 f 9.4
870.9 f 320.5 988.0 f 216.3 973.8 f 228.9
820.0 f 272.3 428.4 f 140.6 331.45265.1
13.6 109.3 281.9
Data for total Cu concentrations samples. For all other parameters,
of digestive gland tissues data are means f standard
and Cu-MTs deviations.
are from pooled
tissue
A. H. Ringwood et al.
594
Crassostrea virginica Cu-Exposed (SOug/L)
Cu-MT
0
-
Days
Fig. 1. The effects of Cu exposures (80 pg I-‘) on lipid peroxidation (LPx) and Cu-MTs in oysters
after 4, 7, and 14 days of exposure.
sites. Conditions that cause GSH depletion may potentiate pollutant toxicity. Lipid peroxidation was a sensitive indicator of perturbed homeostasis, and there was evidence that the adverse effects were ameliorated by MTs. Since lipid peroxidation damage was observed prior to the induction of MTs, it might be expected that if exposures continued to the point that the detoxification capacities of MTs were overwhelmed, lipid peroxidation rates would again increase, indicating significant perturbation of homeostasis that could progress to irreversible damage. It is therefore important to distinguish short-term fluctuations that may be ameliorated by compensatory mechanisms from responses that signal more serious levels of stress (Livingstone, 1982). An appreciation of the significance of a response or the use of a suite of responses would facilitate our ability to distinguish exposure from significant levels of damage, which if unmitigated, may lead to reduced fitness.
ACKNOWLEDGEMENTS I gratefully acknowledge J. Hameedi and NOAA (National Oceanic and Atmospheric Administration, Office of Ocean Resources Conservation and Assessment) who provided funding for these studies (grant number NA570A0486).
REFERENCES Viarengo, A. (1989) Aquatic Sciences 1, 295317. Moore, M. N. (1994) In Contaminants in the Environment: A Multidisciplinary Assessment of Risks to
The effects of copper exposures on cellular responses in oysters
595
Man and Other Organisms, eds A. Renzoni, N. Mattei, L. Lorena and M. C. Fossi, pp. 111-123. CRC Press, Lewis Publishers, Boca Ratan, FL. Mason, A. Z. and Jenkins, K. D. (1995) In Metal Speciation and Bioavailability in Aquatic Systems, eds A. Tessier and D. R. Turner, pp. 479-608. John Wiley & Sons, Ltd. Stegeman, J. J., Brouwer, M., Di Giulio, R. T., Forlin, L., Fowler, B. A., Sanders, B. M. and Van Veld, P. A. (1992) In Biomarkers Biochemical, Physiological, and Histologic Markers of Anthropogenic Stress, eds R. J. Huggett, R. A. Kimerle, P. N. Nehrle and H. L. Bergman, pp. 235-335. Lewis Publishers, Chelsea. Lowe, D. M. and Pipe, R. K. (1994) Aquatic Toxicology 30, 357-365. Anderson, M. E. (1985) Meth. Enzymology 113, 548-555. Gutteridge, J. M. C. and Halliwell, B. (1990) TZBS 15, 129-135. Ringwood, A. H. and Brouwer, M. (1993) Comparative Biochemistry Physiology 106B, 523-529. Viarengo, A., Moore, M. N., Mancinelli, G., Mazzucotelli, A., Pipe, R. K. and Farrar, S. V. (1987) Marine Biology 94, 251-257.
Livingstone, D. R. (1982) Marine Pollution Bulletin 13, 261-263.