Documentos de Académico
Documentos de Profesional
Documentos de Cultura
A.H. Ringwood, J. Hoguet, C.J. Keppler, M.L. Gielazyn, B.P. Ward, A.R. Rourk
submitted by
Marine Resources Research Institute South Carolina Department of Natural Resources 217 Fort Johnson Road Charleston, SC 29412 January, 2003
Acknowledgments
The work described in this handbook is the result of the efforts and dedication of many individuals. The authors wish to acknowledge the valuable assistance of Emily Howard, Kellee James, Bentley Andrews, and Matthew Jenny. We would also like to thank Dr. Betty Wenner (MRRI), Research Director of the ACE Basins NERRs Program, for her input. We gratefully acknowledge the funding for these studies, from the Cooperative Institute for Coastal and Estuarine Environmental Technology (CICEET), University of New Hampshire and NOAA, CICEET grant # NA870R0512.
Table of Contents
I. Introductory Comments .......................................................................................1
II. Collection of Organisms .......................................................................................3 III. Dissection and Tissue Processing Oysters (Crassostrea virginica) .............................................................................4 Grass shrimp (Palaemonetes pugio) ......................................................................5 Mummichogs (Fundulus heteroclitus) ...................................................................6 Homogenization (all species) .................................................................................7 IV. Lysosomal Destabilization Introduction and General Comments .....................................................................8 Oyster Flow Chart........................................................................................... ..10 Oyster Detailed Instructions..............................................................................11 Grass shrimp Flow Chart...................................................................................14 Grass shrimp Detailed Instructions................................................................ ...15 Mummichog Flow Chart................................................................................. ..18 Mummichog Detailed Instructions....................................................................19 V. Glutathione Introduction and General Comments............................................................. ..22 Flow Chart........................................................................................................23 Detailed Instructions DTNB/GSSG Recycling Assay................................. .24 Data Quality Assurance and Control Charts..................................................... ...27
VI. Lipid Peroxidation Introduction and General Comments.................................................................. 29 Flow Chart............................................................................................................30 Detailed Instructions Malondialdehyde Quantification.....................................31 Data Quality Assurance and Control Charts.........................................................34 VII. Data Management, Statistics, and Interpretation ...................................... 36 VIII. Concluding Comments ....................................................................................41 IX. References.. .......................................................................................................43
I. Introductory Comments Coastal and estuarine ecosystems and their inhabitants are subject to increased stress associated with human population growth, in some cases nearly explosive, in coastal areas of the United States. This requires careful monitoring of biological resources and development of strategies to minimize the impacts. Increased contaminants and bioaccumulation in organisms, more extensive areas experiencing dissolved oxygen stress, and poor water quality associated with increased pathogens and harmful algal species are readily documented. While acute toxicity incidents (e.g. fish kills, depauperate communities) are highly visible occurrences, it is more difficult to appreciate the potential long term effects of sublethal stress. Therefore, the critical issues involve determining if the organisms that should live and thrive in a habitat are adversely affected, and identifying the effects of chronic stress on biotic health. In some cases, compensatory mechanisms may function to sequester, detoxify, or ameliorate the effects of stressors so exposures do not always translate into adverse effects. In other cases, individual stressors or combinations of stressors may cause chronic stress that can compromise basic physiological functions, including reproduction, so that long-term population dynamics and sustainability are endangered. Therefore, sensitive tools are needed that will facilitate our ability to recognize when habitat conditions adversely affect biotic integrity, before the effects are irreversible or expensive to remedy. Cellular biomarker responses provide the greatest potential for identifying when conditions have exceeded compensatory mechanisms and the individuals and populations are experiencing chronic stress, which if unmitigated may progress to severe effects at the ecosystem level. They are routinely used as diagnostic tools in biomedical applications, as early warning signals of early disease conditions, for prognosis, and evaluating the effectiveness of remedies. These kinds of frameworks can be applied to estuarine organisms as a means of characterizing habitat quality. To do this effectively requires a sound basis for interpreting cellular data, including expected values and an appreciation of the potential variation. In the biomedical context, this is analogous to defining the normal range of responses.
This handbook contains detailed descriptions for a suite of commonly used cellular biomarkers (lysosomal integrity, glutathione concentrations, and lipid peroxidation) in three common estuarine species: oysters (Crassostrea virginica), grass shrimp (Palaemonetes pugio), and mummichogs (Fundulus heteroclitus). One of the attributes of cellular response assays is that they should be readily applicable to a wide range of organisms, with fairly minor modifications. This was found to be true for these species from three diverse taxonomic groups (mollusks, crustaceans, and fish). Therefore, detailed descriptions of the various assays, including specific adaptations required for different species due to the nature or size of the tissues are provided. The tissue type used for the assays described in the handbook was hepatopancreas (sometimes referred to as digestive gland) or liver tissues. Some comments about the use of other tissues (blood cells, gill tissues, etc.) are provided, but hepatic tissues can be used most broadly and are also one of the most important sites for contaminant deposition and effects. The lysosomal assay is most readily used for hepatic tissues and blood cells, whereas the glutathione and lipid peroxidation assays are readily used for virtually any tissue type (hepatic, gill, gonadal, muscle, mantle, etc.). The handbook is designed to provide specific technical guidance for conducting the assays. The collection and dissection of the animals are described in the first two sections, then there are separate sections for each assay. In each case, a brief description of the assay and its significance is provided, followed by species-specific flow-charts depicting the various steps of each assay and detailed descriptions of the different steps. The final section provides some guidance and recommendations regarding statistical analyses and for establishing a data base management system. The detailed descriptions are designed to function as independent sections that can be used by teachers and students, as well as research scientists. Hopefully, this handbook can be used in a variety of settings, as a framework for comparative biochemistry and physiology studies, a starting point for adaptation to other species, and for assessment and monitoring programs. The overall intent of this effort is to encourage the development of biomarker techniques that can be used by a variety of researchers and teachers, and facilitate greater exchange of data between investigators in order to advance the routine use of cellular biomarker tools in an ecotoxicological framework.
II.
Collection of Organisms: Oysters (Crassostrea virginica) Grass Shrimp (Palaemonetes pugio) Mummichogs (Fundulus heteroclitus):
Use gloves and oyster knives to collect oysters; break off dead shells and rinse off excess mud. Use baited minnow traps and dipnets to collect mummichogs and shrimp. Place animals in 5 gallon buckets with lids. Cover animals with water collected from the site (i.e. site water). All buckets must be kept cool (i.e. in coolers with ice) and aerated on the boat and during the return trip back to the laboratory. Record site name, water temperature, salinity, pH, date, GPS reading, arrival and departure time, and approximate number of animals collected on a data sheet. All buckets should be aerated in the lab overnight in site water and animals dissected the next day.
height HP Measuring the length and height (cm) of an oyster. Location of the digestive gland (e.g. hepatopancreas, HP) in an oyster.
Evaluate for gonadal ripeness (gonadal index) using a subjective scale of 1 to 4 as follows: 1 no gametes present 2 gametes present, extends over a small portion of the hepatopancreas 3 extensive gonadal development that covers most of the hepatopancreas 4 extensive gonadal development, hepatopancreas obscured Dissect out the digestive gland (hepatopancreas) and trim away extraneous tissues (e.g. mantle or gonadal material). A small piece (approximately 0.02g) of the hepatopancreas should be minced with a scalpel, rinsed with calcium- and magnesium-free saline (CMFS), and immediately processed for the lysosomal assay. A piece of hepatopancreas (0.02g minimum) should be kept for the glutathione assay (GSH), and a slightly larger piece (0.05g minimum) for the lipid peroxidation assay (LPx). Hepatopancreas samples for GSH and LPx may be placed in numbered and labeled plastic petri dishes (e.g. tissue pieces from 5 individuals in a 50mm X 9mm petri dish) or snap-cap tubes and kept on ice. Store samples in a 80oC freezer until analyzed. It is recommended that samples be weighed prior to freezing.
Grass Shrimp (Palaemonetes pugio): Place shrimp in a 15 cm diameter plastic petri dish and place on ice. When shrimp are immobilized, identify to species level. A related species, Paleamonetes vulgaris, can co-occur with P. pugio. Generally, P. pugio and P. vulgaris can be differentiated by looking at the rostrum. P. pugios rostrum has one tooth behind the posterior margin of orbit, while P. vulgaris has two. In addition, P. pugio has a longer unarmed tip of the rostrum, while P. vulgaris has teeth all the way to the end of the rostrum (Williams, 1984).
rostrum posterior margin of orbit Use rostrum to identify to species. Record length of shrimp.
Record length from tip of rostrum to end of tail (e.g. uropod), and note any gravid females. Cut shrimp in half with a scalpel between the carapace and 1st abdominal segment. Dissect out hepatopancreas and remove extraneous tissue.
intestine hepatopancreas stomach
Cut
carapace
Individual hepatopancreas samples should be processed for lysosomal destabilization immediately. Composite multiple hepatopancreas samples on ice to get a sufficient tissue amount for the GSH (0.02g minimum) and LPx (0.05g minimum) assays. Composite samples for GSH and LPx analyses may be placed in microcentrifuge tubes and preweighed prior to freezing. Store samples in a 80oC freezer until analyzed.
Mummichogs (Fundulus heteroclitus): To anesthetize fish, place in a 15 cm diameter plastic petri dish in the freezer (20oC) until fish are immobilized. *Preliminary experiments were conducted using MS-222 (methanesulfonate salt), a widely used fish anesthetic, but it resulted in elevated LPx values. Remove the fish from the freezer after anesthetized, and sever the spine prior to dissection. Record sex and length.
The length (cm) and sex of each fish is taken before each bioassay. Male (top) and female (bottom). Using dissecting scissors, cut open the abdomen along the anterior-posterior axis, from the anus to the base of the gills. Make a second cut on the left side of the fish, from the base of the gills to the spine.
liver
Dissection of mummichog. Once cut, the liver is easily accessible. Remove the liver from the body with forceps, and trim away any extraneous tissue.
liver
A small piece (0.02g) of the liver should be rinsed well by dipping in calciumand magnesium-free saline (CMFS), minced with a scalpel, rinsed again with CMFS, and immediately processed for the lysosomal assay. One piece of liver (0.02g minimum) should be kept for GSH and a slightly larger piece (0.05g minimum) kept for LPx. Liver samples for GSH and LPx may be placed in numbered and labeled plastic petri dishes (e.g. separate tissue pieces from 5 individuals in a 50mm X 9mm petri dish) or snap-cap tubes and kept on ice. Store samples in a 80oC freezer until analyzed. It is recommended that samples be weighed prior to freezing.
Homogenization of tissues: Depending on the species and tissue, either glass or teflon tissue grinders may be used. Typically, both shrimp and fish tissues are adequately homogenized with teflon tissue grinders. However, oyster tissues are sufficiently disrupted only when homogenized with ground glass homogenizers. A sample of homogenate should be examined with a compound microscope to verify that the methods are effective for cellular disruption.
General Comments: The flow charts schematically detail the major steps of the lysosomal destabilization assay for oysters (Crassostrea virginica), grass shrimp (Palaemonetes pugio), and mummichogs (Fundulus heteroclitus). A few guidelines concerning species-specific differences for the lysosomal destabilization assay are provided below. Refer to the speciesspecific protocols for more detail. Different physiological buffers are used for different species. For all species, calcium- and magnesium-free saline (CMFS) is used for the initial dissociation of the tissues. For oysters, trypsin is then used to complete the dissociation process for generating cellular preparations. For the shrimp and fish, collagenase is used for the second phase of tissue dissociation (note: In trials with the combined use of trypsin and collagenase, there was no improvement in the cell preparations and in some cases cell viability decreased). Magnesium-free saline (MFS; contains calcium) is used for that step because collagenase requires the presence of calcium ions. Furthermore, for collagenase to be effective, the pH must be > 7.5, so the pH of the buffers used for the fish and shrimp assay are higher than those used for oysters. The proper pH range of CMFS and MFS is critical and must be measured in the solutions immediately prior to use (7.35 - 7.40 for oysters, 7.50 7.53 for grass shrimp and mummichogs). Different size screens are used during the cell filtration step of the lysosomal assay (23 m for oysters, 73 m for grass shrimp, and 41 m for mummichogs) due to cell size differences between species. The concentration of neutral red used for the grass shrimp and mummichogs is higher than the concentration used for the oysters, and the incubation period is also longer (e.g. 40 g/ml, a 90 minute incubation period for grass shrimp and mummichogs; 20 g/ml, a 60 minute incubation period for oysters).
Rinse tissue with CMFS, mince into small pieces, and rinse again. Place tissues into a 24-well plate (each with 600 l CMFS). Cover plate with lid and keep cool.
Add 400 l trypsin (in CMFS) to each sample, shake for 20 minutes; keep cool.
Shear samples with a glass pipette; Transfer to a microcentrifuge tube/filter apparatus (23 m screen). Centrifuge at 200-225 g, 5 minutes, (15oC).
Perform 1 2 rinses, i.e. centrifuge at 200-225 g, 5 minutes, (15oC). Discard supernatant, resuspend cells in 50-300 l CMFS.
Add 2o NR stock (0.04 mg/ml) 1:1 to sample (final NR concentration = 20 g/ml). Mix and incubate in a dark humidified chamber for 60 minutes.
Using a 40X lens, score cells (> 50) as either dye present in the lysosome or dye present in the cytosol.
11
7.
Place cell culture plate with lid in a plastic container with an ice packet. Shake samples at 100-120 rpm on a reciprocating shaker for 20 minutes.
8. 9.
Add 400L trypsin (1mg/mL) to each well and shake for 20 minutes. Gently shear samples with pipette and transfer to microcentrifuge/filter tubes (23m screen).
Microcentrifuge tube/filter apparatus, consisting of a microcentrifuge tube, square piece of nylon filter, and a cut off pipet tip.
10. Centrifuge samples cool (15oC) at 200-225 g for 5 minutes. Remove filter, discard supernatant and resuspend cells in 1000L CMFS. 11. Repeat centrifugation, discard supernatant and resuspend cells in CMFS (50-300L volume depending on size of pellet).
12
NEUTRAL RED ASSAY: 1. Add 2o NR stock solution to oyster sample microcentrifuge tubes. NR volume must equal that of CMFS used in cell resuspension (see Sample Preparation #11). 2. 3. Mix samples with plastic pipette tip and store in a light protected humidified chamber at room temperature. Samples should be scored 60 minutes after the addition of NR. Cells may be incubated in the microcentrifuge tubes, mixed, and placed on a microscope slide just prior to scoring. Alternatively, cells and NR can be placed on the slides at the start of the incubation period and held in the dark humidified chamber until scored. Score cells (50) using a 40 X lens, as either dye present in the lysosome or dye present in the cytosol. Score only hepatic cells that are large (25-40m) and contain lysosomes.
4.
Oyster hepatopancreas cells scored as dye present in the lysosome, e.g., stable.
Oyster hepatopancreas cells scored as dye present in the cytosol, e.g., destabilized.
Calculations: The percent destabilization of each individual organism is determined by dividing the number of cells with neutral red in the cytosol by the total number of cells counted (both neutral red in the cytosol and lysosomes) and multiplying by 100. QA/QC Procedures: 1. 2. Microscopic photography of representative cells is recommended in order to validate the scoring of the cells as stable or destabilized. In order to validate the scoring of cells, a second reader is recommended, along with documentation of the cells, whether it be through still photography or video.
13
Rinse tissue with CMFS, mince into small pieces, and rinse again. Place tissues into a 24-well plate (with 500 l CMFS). Cover plate with lid and keep cool.
Add 500 l collagenase (in MFS) to each sample, shake for 15 minutes; keep cool. Shear samples with a glass pipette; shake for an additional 15 minutes.
Shear samples again, transfer to microcentrifuge tube/filter apparatus (73 m screen). Centrifuge at 200-225 g, 5 minutes, (15oC).
Perform 1 2 rinses, i.e. centrifuge at 200-225 g, 5 minutes, (15oC). Discard supernatant, resuspend cells in 25-200 l CMFS.
Add 2o NR stock (0.08 mg/ml) 1:1 to sample (final NR concentration = 40 g/ml). Mix and incubate in a dark humidified chamber for 90 minutes.
Using a 40X lens, score cells (> 50) as either dye present in the lysosome or dye present in the cytosol.
15
4. 5. 6. 7.
Record length in cm for all shrimp. Record any gravid females. Dissect out hepatopancreas and rinse with CMFS. Mince the tissue into small pieces, rinse again, and place in a 24-well cell culture plate. Place cell plate with lid in a plastic container with an ice packet. Shake samples at 100120 rpm on a reciprocating shaker for 20 minutes.
Cell culture plate with shrimp hepatopancreas samples. 8. 9. Add 500L collagenase (1mg/mL) to each well and shake for 15 minutes. Gently shear samples with pipette and shake for another 15 minutes.
10. Shear samples again with pipette and transfer to microcentrifuge/filter tubes (73m screen).
Microcentrifuge tube/filter apparatus, consisting of a microcentrifuge tube, square piece of nylon filter, and a cut off pipet tip.
11. Centrifuge samples cool (15oC) at 200-225 g for 5 minutes. Remove filter, discard supernatant, and resuspend cells in 1000L CMFS. 12. Repeat centrifugation, discard supernatant, and resuspend cells in CMFS (25-200L volume depending on size of pellet).
16
NEUTRAL RED ASSAY: 1. Add 2o NR stock solution to shrimp sample microcentrifuge tubes. NR volume must equal that of CMFS used in cell resuspension (see Sample Preparation #12). 2. Mix samples with plastic pipette tip and store in a light protected humidified chamber at room temperature. 3. Samples should be scored 90 minutes after the addition of NR. Cells may be incubated in the microcentrifuge tubes, mixed, and placed on a microscope slide just prior to scoring. Alternatively, cells and NR can be placed on the slides at the start of the incubation period and held in the dark humidified chamber until scored. 4. Score cells (50) using a 40 X lens, as either dye present in the lysosome or dye present in the cytosol. Score only hepatic cells that are large (60-75m) and contain lysosomes.
Shrimp hepatopancreas cells scored as dye present in the lysosome, e.g. stable. Calculations:
Shrimp hepatopancreas cells scored as dye present in the cytosol, e.g., destabilized.
The percent destabilization of each individual organism is determined by dividing the number of cells with neutral red in the cytosol by the total number of cells counted (both neutral red in the cytosol and lysosomes) and multiplying by 100.
QA/QC Procedures: 1. Microscopic photography of representative cells is recommended in order to validate the scoring of the cells as stable or destabilized. 2. In order to validate the scoring of cells, a second reader is recommended, along with documentation of the cells, whether it be through still photography or video.
17
Dip liver in CMFS several times, mince into small pieces, and rinse again. Place tissues into a 24-well plate (each with 500 l CMFS). Cover plate with lid and keep cool.
Add 500 l collagenase (in MFS) to each sample, shake for 15 minutes; keep cool. Shear samples with a glass pipette; shake for an additional 15 minutes.
Shear samples again, transfer to microcentrifuge tube/filter apparatus (41 m screen), Centrifuge at 200-225 g, 5 minutes, (15oC).
Perform 1 2 rinses, i.e. centrifuge at 200-225 g, 5 minutes, (15oC). Discard supernatant, resuspend cells in 25-200 l CMFS.
Add 2o NR stock (0.08 mg/ml) 1:1 with sample (final NR concentration = 40 g/ml). Mix and incubate in a dark humidified chamber for 2 hours.
Using a 40X lens, score cells (> 50) as either dye present in the lysosome or dye present in the cytosol.
19
4. 5. 6. 7. 8.
Record the sex and length of each fish. Dissect out liver. Dip liver in CMFS several times to rinse. This reduces the amount of extraneous blood cells in the preparation. Rinse liver well with clean CMFS and mince into small pieces. Place cell plate with lid in a plastic container with an ice packet. Shake samples at 100120 rpm on a reciprocating shaker for 20 minutes.
Cell culture plate with fish liver samples. 9. Add 500L collagenase (1mg/mL) to each well and shake for 15 minutes.
10. Gently shear samples with a pipette and shake for another 15 minutes. 11. Shear samples again with a pipette and transfer to microcentrifuge/filter tubes (41m screen).
Microcentrifuge tube/filter apparatus, consisting of a microcentrifuge tube, square piece of nylon filter, and a cut off pipet tip. 12. Centrifuge samples cool (15oC) at 200-225 g for 5 minutes. Remove filter, discard supernatant, and resuspend cells in 1000L CMFS. 13. Repeat centrifugation, discard supernatant, and resuspend cells in CMFS (25-200L volume depending on size of pellet).
20
NEUTRAL RED ASSAY: 1. Add 2o NR stock solution to fish sample microcentrifuge tubes. NR volume must equal that of CMFS used in cell resuspension (see Sample Preparation #13). 2. Mix samples with plastic tip pipette and store in a light protected humidified chamber at room temperature. 3. Samples should be scored 2 hours after the addition of NR. Cells may be incubated in the microcentrifuge tubes, mixed, and placed on a microscope slide just prior to scoring. Alternatively, cells and NR can be placed on the slides at the start of the incubation period and held in the dark humidified chamber until scored. 4. Score cells (50) using a 40 X lens, as either dye present in the lysosome or dye present in the cytosol. Score only liver cells that are large (35-45m) and contain lysosomes.
Fish liver cells scored as dye present in the lysosome, e.g., stable.
Fish liver cells scored as dye present in the cytosol, e.g., destabilized.
Calculations: The percent destabilization of each individual organism is determined by dividing the number of cells with neutral red in the cytosol by the total number of cells counted (both neutral red in the cytosol and lysosomes) and multiplying by 100.
QA/QC Procedures: 1. Microscopic photography of representative cells is recommended in order to validate the scoring of the cells as stable or destabilized. 2. In order to validate the scoring of cells, a second reader is recommended, along with documentation of the cells, whether it be through still photography or video.
21
V. Glutathione Assay
Introduction: Glutathione (GSH) is a ubiquitous tripeptide that is regarded as one of the most important non-protein thiols in biological systems (Kosower and Kosower, 1978; Mason and Jenkins, 1996). GSH functions as an important overall modulator of cellular homeostasis, and serves numerous essential functions including detoxification of metals and oxy-radicals (Meister and Anderson, 1983; Christie and Costa, 1984). While exposure to pollutants or stressful conditions can result in elevated GSH levels, there is evidence that adverse effects are associated with GSH depletion in marine bivalves (Viarengo et al., 1990; Ringwood et al., 1999), as well as mammalian systems (Dudley and Klaasen, 1984). Organisms may also be more susceptible to additional stressors when GSH is depleted (Conners and Ringwood, 2000; Ringwood and Conners, 2000), and GSH status has been proposed as a potential risk factor in human-based risk assessments (Jones et al., 1995).
General Comments: The following flow chart schematically details the major steps of the glutathione (GSH) assay for oysters (Crassostrea virginica), grass shrimp (Palaemonetes pugio), and mummichogs (Fundulus heteroclitus). The GSH assay is a basic spectrophotometric assay that is readily applied to different tissue types as well as different species.
22
Add the following solutions to microcentrifuge tubes and vortex: 700 l NADPH Buffer 100 l DTNB 175 l DI H2O 25 l sample, GSH standard or 5% SSA blank
Add 15 l GSSG reductase to cuvette and shake. Immediately read absorbance at 405 nm for a 90-120 second period (continuous read or at 15 second intervals).
Determine rates for standards and generate standard curve. Determine concentrations of tissue samples (see calculations).
23
24
(2) Alternatively, the purified reductase can be left undiluted. The volume of 50 U/ml GSSG reductase required for the number of samples to be analyzed can be calculated, and the appropriate volume of undiluted reductase can be used to make the working solution for the assay. Store undiluted stock at 2 to 8oC; make working stocks fresh daily.
SAMPLE PREPARATION: 1. Weigh tissue samples and homogenize in 10 volumes 5% SSA (e.g. if sample is 0.1g add 1.0ml 5% SSA). 2. Centrifuge samples at 13,000 g, 5 minutes, (4oC). 3. Combine 100l supernatant with 100l 5% SSA. Store at 2 to 8oC until used. Samples can then be stored for up to 24 hours at 4oC prior to running assay.
PREPARATION OF GSH STANDARDS: Prepare GSH standards from the primary 1mM stock of GSH and 5% SSA for the following concentrations: Primary (1) Stock: 1 mM GSH - (3.073 mg GSH in 10 ml 5% SSA)
Secondary (2) Stock: 200 M GSH - (60 l of 1 Stock + 240 l 5% SSA) Prepare serial dilutions as follows (e.g. 200 M GSH standard should be made directly from the 1mmol stock; all other standards should be made by adding 150l of the previous standard mixture to 150l of 5% SSA).
25
GSH ASSAY:
1. Each sample and standard is then mixed with the following solutions so that the relative proportions are: 700l NADPH Buffer 100l DTNB 175l DI H2O 25l sample, standard, or 5% SSA for blanks* Note: A bulk cocktail solution of NADPH Buffer, DTNB, and DI H2O can be made and added as a single volume to each sample or standard. Determine the estimated number of samples to be run (including the standards and blanks), prepare the cocktail mixture, and add 975l of the cocktail to each sample, standard or blank. *Two blanks must be made: (1) cocktail blank used to zero spectrophotometer (2) GSH blank (0M GSH) to be treated as a sample (i.e. add GSSG reductase); the standard and sample rates are then adjusted for the GSH blank. 2. Vortex samples. 3. Transfer 900l of cocktail/sample mixture to 1.5ml cuvettes. 4. Quickly add 15l GSSG reductase to cuvettes, shake, and read absorbance at 405nm every 30 seconds for at least 90 seconds. 5. Zero spectrophotometer between samples with cocktail blank.
CALCULATIONS: Standard Curve 1. Run standards, including the GSH blank, and record rate of the GSH standards. 2. Generate adjusted standard rates by subtracting the GSH blank rate from the GSH standard rates. 3. Plot known M concentrations of GSH standards (x axis) against their adjusted rates (y axis). 4. Run a linear regression analysis of standards to generate the equation of a line (e.g. the standard curve); check r2 value of line (e.g. goodness of fit should be close to 1); check slope of line and intercept for consistency with control charts. Re-run a new set of standards if these values (r2, slope and y-intercept) are not acceptable or consistent with control chart limits.
26
Samples 1. Run samples and record sample reaction rates. 2. Generate adjusted sample rates by subtracting the GSH blank rate from measured sample rates. 3. Use equation of line (y = mx + b) from standards to calculate GSH M concentrations for each sample, e.g., solve for x (x = y-b / m]. 4. Since 100l of each sample was diluted with 100l of SSA buffer at the beginning of the assay, multiply the GSH M concentration of each sample by 2. 5. Convert M GSH concentrations (mol/L) of samples to nmol/g wet weight. Note: Since the conversion of mol/L to nmol/g involves multiplying and dividing by 1000, these steps essentially cancel out, so that mol/L = nmol/ml. Therefore, the original GSH concentration in mol/L (after step 4) can be converted to the final concentration of nmol/g wet weight by the following calculation: (GSH nmol/ml x total sample volume (ml)) /g wet weight = GSH nmol/g wet weight
Data Quality Assurance and Control Charts A new standard curve must be generated prior to each experiment by measuring the absorbance of five known standard concentrations. It is recommended that two to three sets of standards be analyzed per experiment in order to validate spectrophotometer readings and to identify potential experimenter errors (i.e. making up the standards). The regression parameters should be similar between the multiple analyses and consistent with the control chart limits. Also, an a priori acceptance criteria of r2 > 0.95 was established for standard curves of GSH. Any standard curves with r2 values below 0.95 should be re-run. Control Charts GSH control charts, based on the slope and y-intercept values from the standard curves, should be used to assess the repeatability of standard curve parameters (Millard and Neerchal, 2001). Upper (UCL) and lower control limits (LCL) were calculated as: UCL = running slope or y-intercept mean + 1 standard deviation LCL = running slope or y-intercept mean - 1 standard deviation Any standard curve that results in a slope or y-intercept value deviating beyond the UCL or LCL should be re-run prior to running the samples.
27
GSH control charts for two forms of GSH reductase (bovine and yeast-derived GSH reductase) are shown below. The solid line indicates the running mean and the dashed lines indicate one standard deviation. During 2000, bovine-derived GSH reductase was not available from any supplier and was replaced with yeast-derived GSH reductase. Yeast reductase was just as effective and also less expensive. The reaction rates with yeast reductase are a little faster.
Slope of Line
1999
Slope of Line
2000
28
VI. Lipid Peroxidation Assay: Introduction: Lipid peroxidation (LPx), an indicator of damage to cell membranes, occurs when free radicals react with lipids, and is a source of cytotoxic products that may damage DNA and enzymes (Kehrer, 1993; Yu, 1994). Increased lipid peroxidation has been demonstrated in response to ischemia-reperfusion events in mammalian tissues, paraquat and contaminant exposures in bivalves, cadmium and PCB exposures in mullet, and exposures of catfish to PAH contaminated sediments (Wenning et al., 1988; Wofford and Thomas, 1988; Regoli, 1992; Di Giulio et al., 1995; Livingstone, 2001). Laboratory exposures to copper have shown increased lipid peroxidation levels in digestive gland tissues from Crassostrea virginica (Ringwood et al., 1998a; Conners and Ringwood, 2000).
General Comments: The following flow chart schematically details the major steps of the lipid peroxidation (LPx) assay for oysters (Crassostrea virginica), grass shrimp (Palaemonetes pugio), and mummichogs (Fundulus heteroclitus). The assay described here is based on the detection of malondialdehyde (MDA), a common end-product of oxidatively damaged membrane lipids. The LPx assay is a basic spectrophotometric assay that is readily applied to different tissue types as well as different species. A wider range of standard concentrations is needed for different species (e.g. oyster standards range from 25 800M; for crustaceans, use standards from 25 3200M; and for Fundulus, standards from 6.25 800M MDA are recommended).
29
Add the following solutions to a microcentrifuge tube and vortex: 100 l sample, MDA standard or blank 1400 l TBA 14 l BHT
30
31
PREPARATION OF STANDARDS: Prepare MDA standards from original 10mM stock of MDA and K2PO4 buffer in the following concentrations: Primary (1) Stock: 10 mM MDA - (see SOLUTIONS NEEDED above)
Secondary (2) Stock: 3200 M MDA - (408 l of 1 Stock + 192 l K2PO4) Prepare serial dilutions as follows (e.g. 3200mol/L MDA standard should be made directly from the 10mmol stock; all other standards should be made by adding 300l of the previous standard mixture).
(h) (f) (d) (e) (g) (a) (b) (c) 3200 M MDA 1600 M MDA 800 M MDA 400 M MDA 200 M MDA 100 M MDA 50 M MDA 25 M MDA 300 l (g) + 300 l (e) + 300 l (c) + 300 l (d) + 300 l (f) + 150 l (a) + 2 Stock 300 l (b) + 150 l K2PO4 300 l K2PO4 300 l K2PO4 300 l K2PO4 300 l K2PO4 300 l K2PO4 300 l K2PO4
*Note: These standards are made by serial dilution. The 3200mol/L MDA standard should be made directly from the 10mmol stock. All other standards should be made by adding 300l of the previous standard mixture.
32
LIPID PEROXIDATION ASSAY: 1. Mix the following solutions with 100l of each sample or standard (including a blank of 100l of K2PO4): 1400l (1.4ml) TBA 14l BHT 2. Vortex, then heat samples and standards in a 100oC water bath for 15 minutes.
Make holes at the top of each tube with a hypodermic needle to allow for pressure release while the samples are being heated.
3. Centrifuge samples and standards at 13,000 g for 5 minutes at room temperature. 4. Transfer supernatant to cuvettes and read absorbance at 532nm on a spectrophotometer. 5. Prepare a standard curve by running a linear regression of concentration vs. absorbance of standards. Calculate the MDA concentration in samples based on the standard curve.
CALCULATIONS: 1. Plot known mol/L concentrations of MDA standards (x axis) against their absorbance reading from the spectrophotometer (y axis). 2. Run a linear regression analysis of standards to generate the equation of a line (e.g. the standard curve); check r2 value of line (e.g. goodness of fit should be close to 1); check slope of line and intercept for consistency with control charts. Re-run a new set of standards if these values (r2, slope and y-intercept) are not acceptable or consistent with control chart limits.
33
3. Use equation of line (y = mx + b) from standards to calculate MDA mol/L concentrations of samples from absorbance readings (y value is absorbance use equation of line to solve for x, which will yield a concentration in mol/L for each sample). 4. Convert MDA mol/L concentrations of samples to nmol/g wet weight.
Convert mol/L to mol/ml by dividing by 1000. Convert to MDA mol/ml by multiplying by the total sample volume (i.e. volume of potassium phosphate buffer (in ml) added to each sample). Convert MDA mol concentrations to nmol by multiplying by 1000. Divide the MDA nmol concentration by the wet weight (in grams) of each sample to give a final MDA concentration in nmol/g. Note: Since the conversion of mol/L to nmol/g involves dividing and multiplying by 1000, these steps essentially cancel out, so that mol/L = nmol/ml. Therefore, the original MDA concentration in mol/L can be simply converted to the final concentration of nmol/g wet weight by the following calculation: (MDA mol/L x volume K2PO4 ml) / g wet weight = MDA nmol/g wet weight
Data Quality Assurance and Control Charts Equipment and Procedural Validation A new standard curve must be generated prior to each experiment by measuring the absorbance of five known standard concentrations. It is recommended that two to three sets of standards be analyzed per experiment in order to validate spectrophotometer readings and to identify potential experimenter errors (i.e. preparing the standards). The regression parameters should be similar between the multiple analyses and consistent with the control chart mean. Also, an a priori acceptance criteria of r2 > 0.95 was established for standard curves of LPx; any r2 values below 0.95 should be rerun.
34
Control Charts Control charts should be used to assess the repeatability of standard curve parameters (e.g. slopes and intercepts) (Millard and Neerchal, 2001). A LPx control chart, based on the slope values of standard curves conducted from 1998 2000, is shown below. Upper (UCL) and lower control limits (LCL) were calculated as: UCL = running slope (or y-intercept) mean + 1 standard deviation LCL = running slope (or y-intercept) mean - 1 standard deviation
Any standard curve that resulted in a slope or y-intercept value deviating beyond the UCL or LCL was re-run prior to running the samples. Values for the standard curve slope for LPx ranged from 0.0010 to 0.0014. A y-intercept below 0.005 was considered acceptable.
Slope of Line
1998
1999
2000
Control chart for LPx standard curve slopes. The solid line indicates the running mean and the dashed lines indicate one standard deviation.
35
VII. Data Management, Statistics, and Interpretation The data should be organized into spreadsheet formats (e.g. Excel) that can be readily exported for statistical analyses software (e.g. Sigma Stat or SAS) and for relational database software (e.g. Access). These approaches provide important means of extracting subsets of the data and performing various queries as well as archiving the data in a form that can be made available to other scientists. Data should be entered into spreadsheets as soon as possible and organized into raw data and summary tables. The raw data tables should include data such as the site name or code, species code, collection and processing dates, each individual organisms height, length, sex or gonadal index, as well as the biomarker responses for each individual or composite sample. The summary data tables should include enough redundant information that they can be clearly linked to the raw data tables (such as the site name or code, species name, dates) and also provide overall summaries (e.g. statistical values such as the mean, standard deviation, median, 25th and 75th percentiles). A QACODE column should be included on all tables and used as a flag for any data points that need some explanation (single letter codes can be used to identify outlying data not used in the final analysis, missing data points, etc). Site names or codes can be designed any number of ways. It is recommended that they should include information regarding the project name, site name, season (if applicable) and year of study. This will allow for easy identification of each data point if the results of several studies over the course of several years are combined. An example of a site code and the explanations for the different fields are illustrated as follows: Site Code CIMOSW00 Explanation of fields CI refers to the CICEET Project; MOS refers to the site, Mosquito Creek; W indicates winter season; 00 indicates year 2000.
For the species code, the first 3 or 4 letters of the genus followed by the first 3 or 4 letters of the species are combined as follows: Species Code crasvirg Explanation Crassostrea virginica
36
Examples of raw data and summary tables for the lysosomal destabilization assay are shown below for two sites, AAA and MOS:
A. Example of raw data table used for the lysosomal destabilization assay.
Sampling Date 2/9/2000 2/9/2000 2/9/2000 2/9/2000 2/9/2000 4/12/2000 4/12/2000 4/12/2000 4/12/2000 4/12/2000 Height (cm) 9.3 9.0 9.1 8.6 9.4 6.7 2.9 2.9 5.2 6.9 Length (cm) 4.1 4.4 3.5 3.6 3.8 2.6 2.2 2.3 2.2 2.5 Gonadal Index 4 3 3 4 3 4 2 4 3 4 % Lysosomal Destablization 24.53 26.56 29.63 26.23 25.97 47.17 39.22 49.06 63.64 36.67 Lysosomal Analysis Date 2/10/2000 2/10/2000 2/10/2000 2/10/2000 2/10/2000 4/13/2000 4/13/2000 4/13/2000 4/13/2000 4/13/2000
Site CIAAAW00 CIAAAW00 CIAAAW00 CIAAAW00 CIAAAW00 CIMOSW00 CIMOSW00 CIMOSW00 CIMOSW00 CIMOSW00
Species crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg
Animal # 1 2 3 4 5 1 2 3 4 5
QACODE
# Animals 5 5
26.58 47.15
26.23 47.17
Another set of examples is provided below for the GSH data. In this case, there are 3 sets of data tables: a raw data table, a summary table, and a QA table that contains the standard curve parameters used to generate the control charts. Important components that link all 3 of these tables include the sampling and processing dates, and site name. The linking elements enable tracking between the raw and summary tables; they also enable verification of the GSH standard curve data found in the QA table so that the validity of the data can be verified. Verification that the control chart parameters between different analysis
37
sets (or even different investigators) are within acceptable ranges increases confidence in the data.
Site CIAAAW00 CIAAAW00 CIAAAW00 CIAAAW00 CIAAAW00 CIMOSW00 CIMOSW00 CIMOSW00 CIMOSW00 CIMOSW00
Species crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg
QACODE
6/13/2000 6/14/2000
1599.3 1270.0
C. Example of QA table with GSH standard curve parameters used for the control charts.
Date 6/13/00 6/14/00 Assay GSH GSH Low Standard (uM) 6.25 6.25 High Standard (uM) 100 100 Slope 0.0045 0.0036 y Intercept -0.035 -0.038 r 0.994 0.999
2
QACODE
38
Once the data have been organized into the data management framework, statistical analyses can then be conducted. For our studies, the data were analyzed using Sigma Stat (Jandel Scientific), with set at 0.05 in all tests; this program also automatically performed normality and equal variance tests as an initial step. Generally, a sample size (n) of 15 20 individuals or composites was recommended, although an n of 10 also yielded data sets that were normally distributed. Since the lysosomal data were based on percentages, arcsin transformations were conducted, although these data were normally distributed even as percentage values. The variation observed with the biochemical parameters was sufficiently narrow (e.g. coefficients of variation were generally < 30%); and variances were also generally equal. However, data outliers, particularly those that are likely to be associated with experimental errors (e.g. errors in weight measurements or reagent additions, data entry errors, etc.) should be removed so that important patterns are not obscured (Snedecor and Cochran, 1967). In most cases, experience with the assays and an appreciation of when values are abnormally high or low would be a basis for flagging individual values; negative values were automatically removed, especially when there seemed to be a sufficient amount of tissue, because they were assumed to represent experimental errors. We also used more objective approaches to evaluate extreme values. Individual responses were flagged as potential outliers if individual samples were more than 2.5 standard deviations above or below the mean value for a site; tests based on residuals can also be applied and can be used to verify simpler variance rules (Barnett and Lewis, 1978). As a general rule, we do not recommend removing more than 10 - 20% of the values to avoid biasing the data (e.g. if n= 10 or 20, then no more than 2 samples should be removed). In general removal of the outliers may have little effect on the site-specific mean value, but due to the effect on the variances, may facilitate meeting the assumptions of normality and equal variances. Therefore, t tests or analysis of variance (ANOVA) tests are preferred for comparisons between sites, with the Student-Neuman-Keuls (SNK) or Tukey tests used for a posteriori pairwise multiple comparison analyses. However, when data do not meet the assumptions of normality and equal variances, the non-parametric Kruskal-Wallis ANOVA on ranks should be used for site comparisons, and the non-parametric Dunns test for a posteriori pairwise multiple comparison analyses.
39
Normal Ranges Normal ranges (expected GSH, LPx, and lysosomal destabilization values for a species) can be determined based on data from unpolluted sites. This is analogous to the normal range approaches used in medicine to determine if various test indicators (e.g. blood parameters such as white cell counts) are perturbed. For example, we were interested in determining if the normal ranges were different for winter and summer seasons. To calculate these normal ranges, seasonal data from multiple years were combined, all sites designated a priori as degraded or polluted were removed, and means and standard deviations were calculated. The robustness of the normal range values will be dependent on the size of the data set, but at some point, the values should not change very much with additional data. An important value to the use of normal ranges is that investigators are not limited to evaluating the effects at one unknown site to those of only one or a few reference sites, but can compare any unknown site to a broader array / database of reference sites, thereby reducing uncertainty. In this way, decisions about whether or not the organisms from a site are stressed can be made with greater confidence. For example, using this process, we would currently recomment the following criteria for oysters, based on responses of hepatopancreas tissues: Normal Range Lysosomal Destablization Glutathione (nM/g) < 15 30% 800- 1600 Concern 30 40% < 800 > 1600 Lipid Peroxidation (nM/g) < 150 150 250 > 250 Stress > 40% < 500
The Normal Range is regarded as the optimum conditions and are considered to indicate that there is no evidence of stress. The Concern values represent levels that are somewhat outside the Normal Range limits, and should be regarded as indicating that the animals are experiencing some stressful conditions. Notice that for GSH, Concern levels are defined both above and below the normal range. This reflects the fact that perturbed GSH responses may be elevated (indicating activation of a detoxification response) or showing signs of
40
depletion. Then Stress levels for GSH and the other indicators are believed to indicate that homeostatic and detoxification mechanisms have been overwhelmed and that the oysters are significantly stressed. Our current working model is that Concern levels may be reversible, if the source of the stress is reduced or removed. However, levels associated with significant Stress may or may not be reversible, but would certainly be expected to cause significant impairment of normal physiological functions (e.g. growth and reproduction), and could result in mortality.
VIII. Concluding Comments The biotechnologies and routine use of cellular biomarker responses have advanced to the point that they should be incorporated into environmental assessments. Some of the kinds of objections that were historically raised were factors such as lack of standardized protocols, lack of QA/QC capabilities, and unclear linkages to higher level effects (e.g. population and community changes). These are important issues that should be used as a basic framework for defining the application of biomarker tools. This handbook provides detail protocols for three frequently used cellular biomarkers for three common estuarine species. This kind of document is a critical component for routine use of biomarkers in order to assure that different scientists and laboratories are using the same methods. This document also describes data management strategies and some of the kinds of QA/QC components that can be included to assure comparable data from different analysis sets (e.g. control charts, etc.) and potentially between different laboratories. We have more than five years of data and experience with the oyster responses (e.g., extensive laboratory and field data in addition to that developed for this CICEET-funded program), so at present we have the most confidence in our recommendations for this species. Moreover, we have recently been conducting studies that reinforce the linkages between the cellular biomarker responses in oysters and physiological responses related to population processes (e.g. reproductive success).
41
Finally, we welcome comments and feedback from anyone who uses these protocols, and encourage any recommendations for improving the techniques, developing QA/QC protocols, sharing and evaluating data, etc. Furthermore, as more of these approaches are developed and incorporated for routine screening, the diagnostic potential will increase, especially as the database from stressful and reference conditions increases. Using this framework to add additional biomarker responses, including protein and gene responses, other cellular damage indicators such as DNA damage, etc., will facilitate our ability to characterize the effects of environmental conditions on organismal health and to develop a sound basis for interpretation based on expected normal ranges. The benefits of sensitive indicators, early diagnosis, and early intervention are well established in the human medical arena. These same kinds of approaches applied to marine organisms should provide important benefits for assessing the impacts of increasing anthropogenic activities in estuarine and coastal regions. With improved diagnostic capabilities, valuable strategies for mitigating pollution and other environmental problems can be implemented.
42
IX. References Adema, C.M., Van der Knaap, W.P., Sminia, T. 1991. Molluscan hemocyte-mediated cytotoxicity: the role of reactive oxygen intermediates. Rev. Aquat. Sci. 4: 201-233. Auffret, M., Oubella, R. 1994. Cytometric parameters of bivalve molluscs: effect of environmental factors. In J.S. Stolon, T. C. Fletcher (eds) Modulators of fish immune responses. SOS Publications, Fairhaven, NJ, p 23-32. Barnett, V., Lewis. T. 1978. Outliers in Statistical Data. John Wiley & Sons, Chicester Bell, T.A., Lightner, D.V. 1988. A handbook of normal penaeid shrimp histology. Baton Rouge, LA. World Aquaculture Society. Christie, N. T., Costa, M. 1984. In vitro assessment of the toxicity of metal compounds. IV. Disposition of metals in cells: interactions with membranes, glutathione, metallothionein, and DNA. Biol. Trace Elements Res. 6: 139-158. Conners, D.E., Ringwood, A.H. 2000. Effects of glutathione depletion on copper cytotoxicity in oysters (Crassostrea virginica). Aquat. Toxicol. 50: 341-349 Di Giulio, R. T., Behar, J. V., Carlson, D. B., Hasspeiler, B. M., Pollard, B. 1995. Determinations of species susceptibility to oxidative stress: a comparison of channel catfish and brown bullhead. Mar. Environ. Res. 39: 321-324. Dudley, R.E., Klaassen, C.D. 1984. Changes in the hepatic glutathione concentration modify cadmium-induced hepatotoxicity. Toxicol. Appl. Pharmacol. 72: 530-538. Jones, D.P., Brown, L.S., Sternberg, P. 1995. Variability in glutathione-dependent detoxication in vivo and its relevance to detoxication of chemical mixtures. Toxicol. 105: 267-274. Kehrer, J.P. 1993. Free radicals as mediators of tissue injury and disease. Crit. Rev. Toxicol. 23: 21-48. Kosower, N.S., Kosower, E.M. 1978. The glutathione status of cells. Int. Rev. in Cytol. 54: 109-160. Livingstone, D.R. 2001. Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. Mar. Poll. Bull. 42: 656-666. Lowe, D.M. 1996. Mechanisms of toxicity in molluscan lysosomes. Mar. Environ. Res. 42: 109. Lowe, D.M., Moore, M.N., Clarke, H.E. 1981. Effects of oil on digestive cells in mussels: Quantitative alteration in cellular and lysosomal structure. Aquat. Toxicol. 1: 213226. Lowe, D.M., Moore, M.N, Evans, B. 1992. Contaminant impact on interactions of molecular probes with lysosomes in living hepatocytes from dab (Limanda limanda). Mar. Ecol. Progr. Ser. 91: 135-140. Lowe, D.M., Soverchia, C., Moore, M.N. 1995a. Lysosomal membrane responses in the blood and digestive cells of mussels experimentally exposed to fluoranthene. Aquat. Toxicol. 33: 105-112.
43
Lowe, D.M., Fossato, V.U., Depledge, M.H. 1995b. Contaminant-induced lysosomal membrane damage in blood cells of mussels Mytilus galloprovincialis from the Venice Lagoon: an in vitro study. Mar. Ecol. Prog. Ser. 129: 189-196. Mason, A.Z., Jenkins, K.D. 1996. Metal detoxification in aquatic systems. In: Metal Speciation and Bioavailability in Aquatic Systems, A. Tessier and D.R. Turner, eds. John Wiley & Sons, Chicester. p. 479-608. Meister, A., Anderson, M.E. 1983. Glutathione. Ann. Rev. Biochem. 52: 711-760. Millard, S.P. and Neerchal, N.K. 2001. Environmental Statistics with S-Plus. CRC Press, New York, New York. Moore, M.N. 1982. Lysosomes and environmental stress. Mar. Poll. Bull. 13: 42-43. Moore, M.N. 1985. Cellular responses to pollutants. Mar. Poll. Bull. 16: 134-139. Moore, M.N. 1994. Contaminants in the Environment: A Multidisciplinary Assessment of Risks to Man and Other Organisms. p. 111-123. Lewis Publishers. Ohkuma, S., Moriyama, Y., Takano, T. 1982. Identification and characterization of a proton pump on lysosomes by fluoroscein isothiocyanate-dextran fluorescence. Proc. Nat. Acad. Sci. USA 79: 2758-2762. Regoli, F. 1992. Lysosomal responses as a sensitive stress index in biomonitoring heavy metal pollution. Mar. Ecol. Prog. Ser. 84: 63-69. Ringwood, A.H., Conners, D.E., DiNovo, A.A. 1998a. The effects of copper exposures on cellular responses in oysters. Mar. Environ. Res. 46: 591-595. Ringwood, A.H., Conners, D.E., Hoguet, J. 1998b. Effects of natural and anthropogenic stressors on lysosomal destabilization in oyster Crassostrea virginica. Mar. Ecol. Progr. Ser. 166: 163-171. Ringwood, A.H., Conners, D.E., Keppler, C.J., DiNovo, A.A. 1999. Biomarker studies with juvenile oysters (Crassostrea virginica) deployed in situ. Biomarkers 4: 400-415. Ringwood, A.H., Conners, D.E. 2000. The effects of glutathione depletion on reproductive success in oysters, Crassostrea virginica. Mar. Environ. Res. 50: 207-211. Snedecor, G.W., Cochran, W.G. 1967. Statistical Methods (sixth edition). The Iowa State University Press. Viarengo, A.L., Canesi, L., Pertica, M., Poli, G., Moore, M.N., Orunesu, M. 1990. Heavy metal effects on lipid peroxidation in the tissues of Mytilus galloprovincialis Lam. Comp. Biochem. Physiol. 97C: 32-42. Wenning, R.J., Di Giulio, R.T., Page, D.S. 1988. Oxidant-mediated biochemical effects of paraquat in the ribbed mussel, Geukensia demissa. Aquat. Toxicol. 12: 157-170. Williams, A.B. 1984. Shrimps, Lobsters, and Crabs of the Atlantic Coast of the Eastern United States, Maine to Florida. p. 71-74. Smithsonian Institution Press. Wofford, H.W., Thomas, P. 1988. Peroxidation of mullet and rat liver lipids in vitro: Effects of pyridine nucleotides, iron, incubation buffer, and xenobiotics. Comp. Biochem. Physiol. 89C: 201-206.
44
Yu, B.P. 1994. Cellular defenses against damage from reactive oxygen species. Physiol. Reveiws 74: 139-162.
45
46