Lysosomal Destabilization, Glutathione & Lipid Peroxidation - Marine ...

Cellular Biomarkers (Lysosomal Destabilization, Glutathione & Lipid Peroxidation) in Three Common Estuarine Species: A Methods Handbook 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 Basin’s NERR’s 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.

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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.

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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.

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III. Dissection and Tissue Processing: Use a cold surface for processing tissues (e.g. pack a clean petri dish with ice and turn upside down over a larger petri dish). Specific instructions for individual species follow. Oysters (Crassostrea virginica): Oysters should be rinsed with cool tap water to remove excess mud. Record shell length and height and carefully open oyster. length

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.

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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. pugio’s 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

1st abdominal segment

Cut

carapace

Diagram modified from Bell and Lightner, 1988.

•

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.

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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

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•

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.

Homogenizing tissues on ice ensures that tissues remain cold.

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IV. Lysosomal Destabilization Introduction Lysosomes are intracellular organelles that are involved in many essential functions, including membrane turnover, nutrition, and cellular defense (Adema et al., 1991; Auffret and Oubella, 1994). The internal acidic environment of the lysosome, integral for the optimal activity of acid hydrolases, is maintained by a membrane-bound, ATPase-dependent proton pump (Ohkuma et al., 1982). Lysosomes also act to sequester metals and other contaminants, which may lead to membrane destabilization (Lowe et al., 1981, 1995a; Moore 1982, 1985). Disruption of the proton pump by chemical contaminants can lead to the impairment of vital functions and cell death (Moore, 1994; Lowe, 1996). Lysosomal destabilization has been used as a valuable indicator of cellular damage in a variety of fish and shellfish (Lowe et al., 1992; Moore, 1994; Ringwood et al., 1998b) and has been regarded as a valuable indicator of compromised biotic integrity (Moore, 1994). A relatively simple assay using neutral red retention is used to assess lysosomal stability. This assay has been conducted successfully with hemocytes and hepatic cells. Studies with hemocytes and hepatic cells from the same individual oysters give comparable results (Conners and Ringwood, unpublished data). In general, hepatic preparations provide a larger number of cells and are the only real option for small organisms or those that are difficult to bleed. Furthermore, hepatic tissues are frequently a major site of accumulation of toxins and a likely target for adverse effects. Cells incubated in neutral red accumulate the lipophilic dye in the lysosomes, where it is trapped by protonization. In healthy cells, neutral red is taken up and retained in stable lysosomes, whereas in damaged cells it leaks out of lysosomes and into the cytoplasm. The leaking of neutral red reflects the efflux of lysosomal contents into the cytosol, which ultimately causes cell death (Lowe et al., 1995b).

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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 µm for oysters, 73 µm for grass shrimp, and 41 µm 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 µg/ml, a 90 minute incubation period for grass shrimp and mummichogs; 20 µg/ml, a 60 minute incubation period for oysters).

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Oyster - Lysosomal Destabilization Assay Flow Chart Record oyster height and length, and gonadal index; dissect out digestive gland, remove excess gonadal tissue.

Rinse tissue with CMFS, mince into small pieces, and rinse again. Place tissues into a 24-well plate (each with 600 µl CMFS). Cover plate with lid and keep cool.

Shake samples at 100-120 rpm on a reciprocating shaker for 20 minutes.

Add 400 µl 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 µm screen). Centrifuge at 200-225 g, 5 minutes, (15oC).

Remove filter, discard supernatant, resuspend cells in 1 ml CMFS.

Perform 1 – 2 rinses, i.e. centrifuge at 200-225 g, 5 minutes, (15oC). Discard supernatant, resuspend cells in 50-300 µl CMFS.

Add 2o NR stock (0.04 mg/ml) 1:1 to sample (final NR concentration = 20 µg/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.

Calculate % of cells with destabilized lysosomes. 10

Lysosomal Destabilization Assay for Oysters Detailed Instructions SOLUTIONS NEEDED: Ca2+ / Mg2+ Free Saline (CMFS) (contains 20mM HEPES, 360mM NaCl, 12.5mM KCl, and 5mM tetrasodium EDTA). Combine 4.766g HEPES, 20.00g NaCl, 0.932g KCl and 1.901g EDTA in 995ml DI H2O. Adjust pH to 7.35 – 7.40 with 6N NaOH. Adjust final volume to 1000ml with DI H2O if necessary and filter through a 0.45µm screen. Check pH and salinity just prior to use (salinity should not be below 25o/oo). Store at 2 to 8oC for up to 1 week. Trypsin (1.0 mg/ml) Add 1.0mg of trypsin to 1.0ml CMFS for a final assay concentration of 1.0mg/ml. Can be frozen and thawed one time. Neutral Red Dye (NR) Make a 1o stock solution by adding 4mg of neutral red powder to 1ml of DMSO. Prior to the assay, make a 2o stock solution (NR concentration of 0.04 mg/ml) by adding 20µl of the 1o stock solution to 1.98 ml CMFS. Wrap in foil to protect from light and keep at room temperature, as crystals can form if kept cold. Make 1o and 2o stocks fresh daily. SAMPLE PREPARATION: 1.

Oxygenate all buffers by bubbling with air and keep cool.

2.

In a 24-well cell culture plate, add 600µL of CMFS to each well (keep plate cool throughout entire process [10-15ºC]). Separate oysters, clean, and remove all adhering debris. Record length and height in cm and gonadal index (1-4). Dissect out digestive gland tissue. Avoid or trim off excess gonadal and mantle tissue. Rinse digestive gland tissue with clean CMFS, mince into small pieces, rinse again with CMFS, and place in the 24-well cell culture plate.

3. 4. 5. 6.

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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.

Cell culture plate with oyster hepatopancreas samples.

8.

Add 400µL trypsin (1mg/mL) to each well and shake for 20 minutes.

9.

Gently shear samples with pipette and transfer to microcentrifuge/filter tubes (23µm 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 1000µL CMFS. 11. Repeat centrifugation, discard supernatant and resuspend cells in CMFS (50-300µL volume depending on size of pellet).

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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.

Mix samples with plastic pipette tip and store in a light protected humidified chamber at room temperature.

3.

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.

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 (25-40µm) and contain lysosomes.

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.

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.

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Grass shrimp - Lysosomal Destabilization Assay Flow Chart Record shrimp length (and identify gravid females); dissect out hepatopancreas.

Rinse tissue with CMFS, mince into small pieces, and rinse again. Place tissues into a 24-well plate (with 500 µl CMFS). Cover plate with lid and keep cool.


Add 500 µl 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 µm screen). Centrifuge at 200-225 g, 5 minutes, (15oC).



Add 2o NR stock (0.08 mg/ml) 1:1 to sample (final NR concentration = 40 µg/ml). Mix and incubate in a dark humidified chamber for 90 minutes.



Lysosomal Destabilization Assay for Grass Shrimp Detailed Instructions SOLUTIONS NEEDED: Ca2+ / Mg2+ Free Saline (CMFS) (contains 20mM HEPES, 450mM NaCl, 12.5mM KCl, and 5mM tetrasodium EDTA). Combine 4.766g HEPES, 25.0g NaCl, 0.932g KCl, and 1.90g EDTA in 995ml DI H2O. Adjust pH to 7.5 – 7.53 with 6N NaOH. Adjust final volume to 1000ml with DI H2O if necessary and filter through a 0.45µm screen. Check pH and salinity just prior to use (salinity should not be below 30o/oo). Store at 2 to 8oC, for up to 1 week. Mg2+ Free Saline (MFS) (contains 20mM HEPES, 480mM NaCl, 12.5mM KCl, and 5.0mM CaCl2). Combine 0.477g HEPES, 2.661g NaCl, 0.093g KCl, and 0.055g CaCl2 in 99.5ml DI H2O. Adjust pH to 7.50 – 7.53 with 6N NaOH. Adjust final volume to 100ml with DI H2O if necessary and filter through a 0.45µm screen. Check pH and salinity just prior to use (salinity should not be below 30 o/oo). Store at 2 to 8oC, for up to 1 week. Collagenase (1.0 mg/ml) Add 1.0mg of collagenase to 1.0ml MFS for a final assay concentration of 1.0 mg/ml. Can be frozen and thawed one time. Neutral Red Dye (NR) Make a 1o stock solution by adding 4mg of neutral red powder to 1ml of DMSO. Prior to the assay, add 40µl of the 1o stock solution to 1.96 ml CMFS for a 2o stock solution concentration of 0.08 mg/ml. Wrap in foil to protect from light and keep at room temperature, as crystals can form if kept cold. Make 1o and 2o stocks fresh daily. SAMPLE PREPARATION: 1.


2.

Allow shrimp to cool on ice before dissecting.

3.

In a 24-well cell culture plate, add 500µL of CMFS to each well (keep plate cool throughout entire process [10-15ºC]).

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4.

Record length in cm for all shrimp. Record any gravid females.

5.

Dissect out hepatopancreas and rinse with CMFS.

6.

Mince the tissue into small pieces, rinse again, and place in a 24-well cell culture plate.

7.

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.

Add 500µL collagenase (1mg/mL) to each well and shake for 15 minutes.

9.

Gently shear samples with pipette and shake for another 15 minutes.

10. Shear samples again with pipette and transfer to microcentrifuge/filter tubes (73µm 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 1000µL CMFS. 12. Repeat centrifugation, discard supernatant, and resuspend cells in CMFS (25-200µL volume depending on size of pellet).

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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 (60-75µm) and contain lysosomes.

Shrimp hepatopancreas cells scored as dye present in the lysosome, e.g. stable.

Shrimp 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. 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.

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Mummichog - Lysosomal Destabilization Assay Flow Chart Record fish length and sex, dissect out liver.

Dip liver in CMFS several times, mince into small pieces, and rinse again. Place tissues into a 24-well plate (each with 500 µl CMFS). Cover plate with lid and keep cool.


Add 500 µl 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 µm screen), Centrifuge at 200-225 g, 5 minutes, (15oC).



Add 2o NR stock (0.08 mg/ml) 1:1 with sample (final NR concentration = 40 µg/ml). Mix and incubate in a dark humidified chamber for 2 hours.



Lysosomal Destabilization Assay for Mummichogs Detailed Instructions SOLUTIONS NEEDED: Ca2+ / Mg2+ Free Saline (CMFS) (contains 20mM HEPES, 450mM NaCl, 12.5mM KCl, and 5mM tetrasodium EDTA). Combine 4.766g HEPES, 25.0g NaCl, 0.932g KCl, and 1.901g EDTA in 995ml DI H2O. Adjust pH to 7.5 – 7.53 with 6N NaOH. Adjust final volume to 1000ml with DI H2O if necessary and filter through a 0.45µm screen. Check pH and salinity just prior to use (salinity should not be below 30o/oo). Store at 2 to 8oC, for up to 1 week. Mg2+ Free Saline (MFS) (contains 20mM HEPES, 480mM NaCl, 12.5mM KCl, and 5.0mM CaCl2). Combine 0.477g HEPES, 2.661g NaCl, 0.093g KCl, and 0.055g CaCl2 in 99.5ml DI H2O. Adjust pH to 7.5 – 7.53 with 6N NaOH. Adjust final volume to 100ml with DI H2O if necessary and filter through a 0.45µm screen. Check pH and salinity just prior to use (salinity should not be below 30o/oo). Store at 2 to 8oC, for up to 1 week. Collagenase (1.0 mg/ml) Add 1.0mg of collagenase to 1.0ml MFS for a final assay concentration of 1.0 mg/ml. Can be frozen and thawed one time. Neutral Red Dye (NR) Make a 1o stock solution by adding 4mg of neutral red powder to 1ml of DMSO. Prior to the assay, add 40µl of the 1o stock solution to 1.96 ml CMFS for a 2o stock solution concentration of 0.08 mg/ml. Wrap in foil to protect from light and keep at room temperature as crystals can form if kept cold. Make 1o and 2o stocks fresh daily. SAMPLE PREPARATION: 1.


2.

Anesthetize fish by cooling on ice or in the freezer.

3.

In a 24-well cell culture plate, add 500µL of CMFS to each well (keep plate cool throughout entire process [10-15ºC]).

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4.

Record the sex and length of each fish.

5.

Dissect out liver.

6.

Dip liver in CMFS several times to rinse. This reduces the amount of extraneous blood cells in the preparation.

7.

Rinse liver well with clean CMFS and mince into small pieces.

8.

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 500µL 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 (41µm 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 1000µL CMFS. 13. Repeat centrifugation, discard supernatant, and resuspend cells in CMFS (25-200µL volume depending on size of pellet).

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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 (35-45µm) and contain lysosomes.

Fish liver cells scored as dye present in the cytosol, e.g., destabilized.

Fish liver cells scored as dye present in the lysosome, e.g., stable.

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

GSH Flow Chart Weigh tissues. Homogenize in 10 volumes 5% SSA.

Centrifuge at 13,000 g for 5 minutes, (4oC).

Combine 100 µl sample supernatant with 100 µl 5% SSA.

Prepare GSH standards (see GSH protocol).

Add the following solutions to microcentrifuge tubes and vortex: 700 µl NADPH Buffer 100 µl DTNB 175 µl DI H2O 25 µl sample, GSH standard or 5% SSA blank

Transfer 900 µl to 1.5 ml cuvette.

Add 15 µl 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

DTNB-GSSG Reductase Recycling Assay for Glutathione Detailed Instructions EQUIPMENT: A spectrophotometer capable of kinetic analyses (i.e. records multiple readings over a defined time period) is preferred for this assay since reaction rates are required for GSH quantification. If this type of instrument is not available, fixed wavelength analysis can be used. However, absorbances for each sample must be recorded every 15-30 seconds for at least 90 seconds. Standards and sample rates must then be determined by linear regression (i.e. slopes represent reaction rates used in GSH calculations). This assay can also be adapted for a microplate reader. SOLUTIONS NEEDED: 5% Sulfosalicylic Acid (SSA) Add 12.5g sulfosalicylic acid to 250ml DI H2O. Store at 2 to 8oC for up to 2 weeks. 143mM Sodium Phosphate Buffer Dissolve 4.29g monobasic sodium phosphate and 0.5988g tetrasodium EDTA in 250ml DI H2O. Dissolve 5.0765g dibasic sodium phosphate and 0.5988g tetrasodium EDTA in 250ml DI H2O. Mix 248ml monobasic solution and 248ml dibasic solution together. Adjust pH to 7.5. Adjust final volume to 500ml with DI H2O if necessary. Store at 2 to 8oC for up to 2 weeks. 10mM 5,5’-Dithiobis(2-Nitrobenzoic acid) (DTNB) Add 0.03963g DTNB to 10ml sodium phosphate buffer. Make fresh daily. 0.238mg/ml NADPH Buffer Dissolve NADPH in sodium phosphate buffer. Make fresh daily. 50 U/ml Glutathione Reductase Note that the specific activity and concentration of the GSSG reductase varies with different batches, so the volumes and dilution factors will vary. The purified standard may be diluted by either of the ways provided in the following examples: (1) GSH reductase shipped as 6.8 mg protein/ml in 0.38 ml , 198 U / mg protein; therefore (6.8 mg protein/ml x 0.38 ml ) x 198 U/mg protein = 511.63 U; so the total required volume = 511.63 U ÷ 500 U/ml = 1.023 ml. Since the vial already contains 0.38 ml, the amount of buffer to be added for 500 U/ml would be 1.023 ml – 0.380 ml = 0.642 ml. Prepare a 1/10 dilution to yield a 50 U/ml for use in the assay.

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 100µl supernatant with 100µl 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 µM GSH - (60 µl of 1° Stock + 240 µl 5% SSA) Prepare serial dilutions as follows (e.g. 200 µM GSH standard should be made directly from the 1mmol stock; all other standards should be made by adding 150µl of the previous standard mixture to 150µl of 5% SSA).

a

(a) 200 µM GSH 2° Stock

b

(b) 100 µM GSH 150 µl (a) + 150 µl SSA

c

(c) 50 µM GSH 150 µl (b) + 150 µl SSA

d

(d) 25 µM GSH 150 µl (c) + 150 µl SSA

25

e

(e) 12.5 µM GSH 150 µl (d) + 150 µl SSA

f

(f) 6.25 µM GSH 150 µl (e) + 150 µl SSA

GSH ASSAY: 1. Each sample and standard is then mixed with the following solutions so that the relative proportions are: 700µl NADPH Buffer 100µl DTNB 175µl DI H2O 25µl 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 975µl of the “cocktail” to each sample, standard or blank. *Two blanks must be made: (1) “cocktail blank” used to zero spectrophotometer (2) “GSH blank” (0µM 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 900µl of cocktail/sample mixture to 1.5ml cuvettes. 4. Quickly add 15µl 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 µM 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 µM concentrations for each sample, e.g., solve for x (x = y-b / m]. 4. Since 100µl of each sample was diluted with 100µl of SSA buffer at the beginning of the assay, multiply the GSH µM concentration of each sample by 2. 5. Convert µM GSH concentrations (µmol/L) of samples to nmol/g wet weight. Note: Since the conversion of µmol/L to nmol/g involves multiplying and dividing by 1000, these steps essentially cancel out, so that µmol/L = nmol/ml. Therefore, the original GSH concentration in µmol/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.

GSH Control Chart - Bovine Reductase 0.0040 0.0035

Slope of Line

0.0030 0.0025 0.0020 0.0015 0.0010 0.0005 0.0000

1999

GSH Control Chart - Yeast Reductase 0.0070

Slope of Line

0.0060 0.0050 0.0040 0.0030 0.0020 0.0010 0.0000

2000

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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 – 800µM; for crustaceans, use standards from 25 – 3200µM; and for Fundulus, standards from 6.25 – 800µM MDA are recommended).

29

LPx Flow Chart Prepare MDA standards (see LPx protocol).

Weigh tissues; Homogenize in 4 volumes K2PO4

Centrifuge samples at 13,000 g for 5 minutes, (4oC).

Add the following solutions to a microcentrifuge tube and vortex: 100 µl sample, MDA standard or blank 1400 µl TBA 14 µl BHT

Heat samples in a 100oC water bath for 15 minutes.

Centrifuge samples at 13,000 g for 5 minutes.

Transfer supernatant to 1.5 ml cuvette; read absorbance at 532 nm.

Determine MDA concentrations (see calculations).

30

Lipid Peroxidation Assay (Based on Malondialdehyde Concentrations) Detailed Instructions SOLUTIONS NEEDED: 1N Hydrochloric Acid (HCl) Add 8.2ml 12.1N HCl to 91.8ml ultrapure DI H2O. Store at room temperature. 50 mM Potassium Phosphate (K2PO4) Buffer Dissolve 1.7011g monobasic K2PO4 and 2.177g dibasic K2PO4 in 495ml DI H2O. Adjust pH to 7.0 with 1N HCl. Adjust final volume to 500ml with DI H2O. Filter through a 0.22µm screen. Store at 2 to 8oC for up to 2 weeks. Malondialdehyde Tetraethylacetal (10mM stock solution of MDA) Combine 24µl 1,1,3,3-tetraethoxypropane (TEP), 1ml 1N HCl, and 90ml DI H2O in a volumetric flask, bring volume up to 100ml with DI H2O, and mix well. Cap and seal with parafilm. Heat in a 50o C water bath for 60 minutes. Cool to room temperature. Make fresh daily. 0.375% Thiobarbituric Acid (TBA) Instructions for 20 samples plus standards: Dissolve 6.0g of 15% trichloroacetic acid (TCA) and 0.15g thiobarbituric acid (TBA) in 40ml 0.25N HCl (10ml 1N HCl to 30ml DI H2O). Make fresh daily. Keep at 2 to 8oC. 2% Butylated Hydroxytoluene (BHT) Instructions for 20 samples plus standards: Dissolve 0.04g BHT in 2ml absolute alcohol. Seal solution tightly to avoid evaporation before use. Make fresh daily. Keep at room temperature. SAMPLE PREPARATION: 1. Weigh tissue samples and homogenize in 4 volumes of K2PO4 buffer (i.e. if tissue weighs 0.2g, add 0.8ml buffer). Transfer homogenate to microcentrifuge tubes. Keep samples cold. 2. Centrifuge samples at 13,000 g for 5 minutes, 4o C. 3. Transfer 100µl of supernatant to new set of microcentrifuge tubes for assay.

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 µM MDA - (408 µl of 1° Stock + 192 µl K2PO4) Prepare serial dilutions as follows (e.g. 3200µmol/L MDA standard should be made directly from the 10mmol stock; all other standards should be made by adding 300µl of the previous standard mixture).

a

b

c

d

e

f

g

h

(h) (f) (d) (e) (g) (a) (b) (c) 3200 µM MDA 1600 µM MDA 800 µM MDA 400 µM MDA 200 µM MDA 100 µM MDA 50 µM MDA 25 µM MDA 300 µl (g) + 300 µl (e) + 300 µl (c) + 300 µl (d) + 300 µl (f) + 150 µl (a) + 2° Stock 300 µl (b) + 150 µl K2PO4 300 µl K2PO4 300 µl K2PO4 300 µl K2PO4 300 µl K2PO4 300 µl K2PO4 300 µl K2PO4

*Note: These standards are made by serial dilution. The 3200µmol/L MDA standard should be made directly from the 10mmol stock. All other standards should be made by adding 300µl of the previous standard mixture.

32

LIPID PEROXIDATION ASSAY: 1. Mix the following solutions with 100µl of each sample or standard (including a blank of 100µl of K2PO4): 1400µl (1.4ml) TBA 14µl 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.

LPx samples heating in water bath.

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 µmol/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 µmol/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 µmol/L for each sample). 4. Convert MDA µmol/L concentrations of samples to nmol/g wet weight. •

Convert µmol/L to µmol/ml by dividing by 1000.

•

Convert to MDA µmol/ml by multiplying by the total sample volume (i.e. volume of potassium phosphate buffer (in ml) added to each sample).

•

Convert MDA µmol 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 µmol/L to nmol/g involves dividing and multiplying by 1000, these steps essentially cancel out, so that µmol/L = nmol/ml. Therefore, the original MDA concentration in µmol/L can be simply converted to the final concentration of nmol/g wet weight by the following calculation: (MDA µmol/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.

LPX Control Chart 0.0020 0.0018

Slope of Line

0.0016 0.0014 0.0012 0.0010 0.0008 0.0006 0.0004

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 organism’s 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

Explanation of fields

CIMOSW00

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

Explanation

crasvirg

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.

Site CIAAAW00 CIAAAW00 CIAAAW00 CIAAAW00 CIAAAW00 CIMOSW00 CIMOSW00 CIMOSW00 CIMOSW00 CIMOSW00

Species crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg

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

Animal # 1 2 3 4 5 1 2 3 4 5

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

QACODE

Species crasvirg crasvirg

Assay Lyso Lyso

# Animals 5 5

26.58 47.15

STD 1.87 10.59

Median % Lysosomal Destabilization

Site CIAAAW00 CIMOSW00

Mean % Lysosomal Destabilization

B. Example of summary data table for the lysosomal destabilization assay.

26.23 47.17

25% 25.61 38.58

75% QACODE 27.33 52.71

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.

A. Example of raw data table for GSH

Site CIAAAW00 CIAAAW00 CIAAAW00 CIAAAW00 CIAAAW00 CIMOSW00 CIMOSW00 CIMOSW00 CIMOSW00 CIMOSW00

Species crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg crasvirg

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

Animal # 1 2 3 4 5 1 2 3 4 5

GSH (nmol/g) 1558.7 1640.0 1776.0 1449.3 158.2 1355.0 1270.0 887.8 1079.4 2121.7

GSH Analysis Date 6/13/2000 6/13/2000 6/13/2000 6/13/2000 6/13/2000 6/14/2000 6/14/2000 6/14/2000 6/14/2000 6/14/2000

QACODE

A

QACODE Definitions A Outlier not used in analysis

Assay GSH GSH

6/13/2000 6/14/2000

# Animals 4 1606 5 1343

STD 137.6 471.3

Median GSH (nmol/g)

Species crasvirg crasvirg

Mean GSH (nmol/g)

Site CIAAAW00 CIMOSW00

GSH Analysis Date

B. Example of summary data table for GSH

1599.3 1270.0

25% 1504.0 1031.5

75% QACODE 1708.0 1546.7

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

38

Slope 0.0045 0.0036

y Intercept -0.035 -0.038

2

r 0.994 0.999

QACODE

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 α 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 Dunn’s 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:

Lysosomal Destablization Glutathione (nM/g)

Normal Range

Concern

Stress

< 15 – 30%

30 – 40%

> 40%

< 800

< 500

800- 1600

> 1600 Lipid Peroxidation (nM/g)

< 150

150 – 250

> 250

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).

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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.

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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.

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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.

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Yu, B.P. 1994. Cellular defenses against damage from reactive oxygen species. Physiol. Reveiws 74: 139-162.

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