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
1
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.
4
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.
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.
Measuring the length and height (cm) ofan oyster.
Location of the digestive gland (e.g. hepatopancreas, HP) in an oyster.
height
length
HP
5
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).
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.
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.
Use rostrum to identify to species. Record length of shrimp.
rostrum
posterior margin of
orbit
1st abdominal segment carapace
hepatopancreas
Cut
intestine
stomach
6
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.
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.
Remove the liver from the body with forceps, and trim away any extraneous tissue.
liver
Dissection of mummichog. Once cut, the liver is easily accessible.
liver
The length (cm) and sex of each fish is taken before each bioassay. Male (top) and female (bottom).
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• A small piece (0.02g) of the liver should be rinsed well by dipping in calcium- and 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 species-
specific 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
Calculate % of cells with destabilized lysosomes.
Record oyster height and length, and gonadal index; dissect out digestive gland, remove excess gonadal tissue.
Shear samples with a glass pipette; Transfer to a microcentrifuge tube/filter apparatus (23 µm screen). Centrifuge at 200-225 g, 5 minutes, (15oC).
Shake samples at 100-120 rpm on a reciprocating shaker for 20 minutes.
Perform 1 – 2 rinses, i.e. centrifuge at 200-225 g, 5 minutes, (15oC). Discard supernatant, resuspend cells in 50-300 µl CMFS.
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.
Remove filter, discard supernatant, resuspend cells in 1 ml CMFS.
Add 400 µl trypsin (in CMFS) to each sample, shake for 20 minutes; keep cool.
Using a 40X lens, score cells (> 50) as either dye present in the lysosome or dye present in the cytosol.
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.
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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]). 3. Separate oysters, clean, and remove all adhering debris. 4. Record length and height in cm and gonadal index (1-4). 5. Dissect out digestive gland tissue. Avoid or trim off excess gonadal and mantle tissue. 6. Rinse digestive gland tissue with clean CMFS, mince into small pieces, rinse again with
CMFS, and place in the 24-well cell culture plate.
12
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. 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).
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).
Cell culture plate with oyster hepatopancreas samples.
Microcentrifuge tube/filter apparatus, consisting of a microcentrifuge tube, square piece of nylon filter, and a cut off pipet tip.
13
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.
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.
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.
14
Record shrimp length (and identify gravid females); dissect out hepatopancreas.
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.
Shake samples at 100-120 rpm on a reciprocating shaker for 20 minutes.
Perform 1 – 2 rinses, i.e. centrifuge at 200-225 g, 5 minutes, (15oC). Discard supernatant, resuspend cells in 25-200 µl CMFS.
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.
Remove filter, discard supernatant, resuspend cells in 1 ml CMFS.
Grass shrimp - Lysosomal Destabilization Assay Flow Chart
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.
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.
15
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. Oxygenate all buffers by bubbling with air and keep cool.
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]).
16
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 100-120 rpm on a reciprocating shaker for 20 minutes.
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).
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).
Cell culture plate with shrimp hepatopancreas samples.
Microcentrifuge tube/filter apparatus, consisting of a microcentrifuge tube, square piece of nylon filter, and a cut off pipet tip.
17
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.
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.
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.
18
Record fish length and sex, dissect out liver.
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.
Shake samples at 100-120 rpm on a reciprocating shaker for 20 minutes.
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.
Remove filter, discard supernatant, resuspend cells in 1 ml CMFS.
Mummichog - Lysosomal Destabilization Assay Flow Chart
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.
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.
Perform 1 – 2 rinses, i.e. centrifuge at 200-225 g, 5 minutes, (15oC). Discard supernatant, resuspend cells in 25-200 µl CMFS.
19
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. Oxygenate all buffers by bubbling with air and keep cool.
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]).
20
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 100-120 rpm on a reciprocating shaker for 20 minutes.
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).
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).
Cell culture plate with fish liver samples.
Microcentrifuge tube/filter apparatus, consisting of a microcentrifuge tube, square piece of nylon filter, and a cut off pipet tip.
21
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.
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.
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.
22
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.
23
GSH Flow Chart
Weigh tissues. Homogenize in 10 volumes 5% SSA.
Transfer 900 µl to 1.5 ml cuvette.
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
Determine rates for standards and generate standard curve. Determine concentrations of tissue samples (see calculations).
Prepare GSH standards (see GSH protocol).
Centrifuge at 13,000 g for 5 minutes, (4oC).
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).
Combine 100 µl sample supernatant with 100 µl 5% SSA.
24
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.
25
(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).
(b) 100 µM GSH 150 µl (a) + 150 µl SSA
(a) 200 µM GSH
2° Stock
(c) 50 µM GSH 150 µl (b) + 150 µl SSA
(d) 25 µM GSH 150 µl (c) + 150 µl SSA
(e) 12.5 µM GSH 150 µl (d) + 150 µl SSA
(f) 6.25 µM GSH 150 µl (e) + 150 µl SSA
a b c d e f
26
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.
27
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.
28
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
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
0.0040
1999
Slop
e of
Lin
e
GSH Control Chart - Yeast Reductase
0.0000
0.0010
0.0020
0.0030
0.0040
0.0050
0.0060
0.0070
2000
Slop
e of
Lin
e
29
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).
30
Weigh tissues; Homogenize in 4 volumes K2PO4
Heat samples in a 100oC water bath for 15 minutes.
Determine MDA concentrations (see calculations).
Add the following solutions to a microcentrifuge tube and vortex: 100 µl sample, MDA standard or blank 1400 µl TBA 14 µl BHT
Transfer supernatant to 1.5 ml cuvette; read absorbance at 532 nm.
Prepare MDA standards (see LPx protocol).
Centrifuge samples at 13,000 g for 5 minutes, (4oC).
Centrifuge samples at 13,000 g for 5 minutes.
LPx Flow Chart
31
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.
32
PREPARATION OF STANDARDS: Prepare MDA standards from original 10mM stock of MDA and K2PO4 buffer in the following concentrations:
*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.
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).
f g ha b c d e
(b) 1600 µM MDA 150 µl (a) + 150 µl K2PO4
(a) 3200 µM MDA
2° Stock
(c) 800 µM MDA
300 µl (b) + 300 µl K2PO4
(d) 400 µM MDA
300 µl (c) + 300 µl K2PO4
(e) 200 µM MDA
300 µl (d) + 300 µl K2PO4
(f) 100 µM MDA
300 µl (e) + 300 µl K2PO4
(g) 50 µM MDA 300 µl (f) + 300 µl K2PO4
(h) 25 µM MDA 300 µl (g) + 300 µl K2PO4
33
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. 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.
LPx samples heating in water bath. Make holes at the top of each tube with a hypodermic needle to allow for pressure release while the samples are being heated.
34
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.
35
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
0.0004
0.0006
0.0008
0.0010
0.0012
0.0014
0.0016
0.0018
0.0020
Slop
e of
Lin
e
Control chart for LPx standard curve slopes. The solid line indicates the running mean and the dashed lines indicate one standard deviation.
1998 1999 2000
36
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
37
Examples of raw data and summary tables for the lysosomal destabilization assay are
shown below for two sites, AAA and MOS:
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
Site SpeciesSampling
Date Animal #Height (cm)
Length (cm)
Gonadal Index
% Lysosomal Destablization
Lysosomal Analysis Date QACODE
CIAAAW00 crasvirg 2/9/2000 1 9.3 4.1 4 24.53 2/10/2000CIAAAW00 crasvirg 2/9/2000 2 9.0 4.4 3 26.56 2/10/2000CIAAAW00 crasvirg 2/9/2000 3 9.1 3.5 3 29.63 2/10/2000CIAAAW00 crasvirg 2/9/2000 4 8.6 3.6 4 26.23 2/10/2000CIAAAW00 crasvirg 2/9/2000 5 9.4 3.8 3 25.97 2/10/2000CIMOSW00 crasvirg 4/12/2000 1 6.7 2.6 4 47.17 4/13/2000CIMOSW00 crasvirg 4/12/2000 2 2.9 2.2 2 39.22 4/13/2000CIMOSW00 crasvirg 4/12/2000 3 2.9 2.3 4 49.06 4/13/2000CIMOSW00 crasvirg 4/12/2000 4 5.2 2.2 3 63.64 4/13/2000CIMOSW00 crasvirg 4/12/2000 5 6.9 2.5 4 36.67 4/13/2000
A. Example of raw data table used for the lysosomal destabilization assay.
Site Species Assay # Animals Mea
n %
L
ysos
omal
D
esta
biliz
atio
n
STD Med
ian
%
Lys
osom
al
Des
tabi
lizat
ion
25% 75% QACODECIAAAW00 crasvirg Lyso 5 26.58 1.87 26.23 25.61 27.33CIMOSW00 crasvirg Lyso 5 47.15 10.59 47.17 38.58 52.71
B. Example of summary data table for the lysosomal destabilization assay.
38
sets (or even different investigators) are within acceptable ranges increases confidence in the
data.
B. Example of summary data table for GSH
Site Species Assay GSH
Ana
lysi
s D
ate
# Animals Mea
n G
SH
(nm
ol/g
)
STD Med
ian
GSH
(n
mol
/g)
25% 75% QACODECIAAAW00 crasvirg GSH 6/13/2000 4 1606 137.6 1599.3 1504.0 1708.0CIMOSW00 crasvirg GSH 6/14/2000 5 1343 471.3 1270.0 1031.5 1546.7
A. Example of raw data table for GSH
GSHSampling Animal GSH Analysis
Site Species Date # (nmol/g) Date QACODECIAAAW00 crasvirg 2/9/2000 1 1558.7 6/13/2000CIAAAW00 crasvirg 2/9/2000 2 1640.0 6/13/2000CIAAAW00 crasvirg 2/9/2000 3 1776.0 6/13/2000CIAAAW00 crasvirg 2/9/2000 4 1449.3 6/13/2000CIAAAW00 crasvirg 2/9/2000 5 158.2 6/13/2000 ACIMOSW00 crasvirg 4/12/2000 1 1355.0 6/14/2000CIMOSW00 crasvirg 4/12/2000 2 1270.0 6/14/2000CIMOSW00 crasvirg 4/12/2000 3 887.8 6/14/2000CIMOSW00 crasvirg 4/12/2000 4 1079.4 6/14/2000CIMOSW00 crasvirg 4/12/2000 5 2121.7 6/14/2000
QACODE DefinitionsA Outlier not used in analysis
C. Example of QA table with GSH standard curve parameters used for the control charts.
Date AssayLow Standard
(uM)High Standard
(uM) Slope y Intercept r2 QACODE6/13/00 GSH 6.25 100 0.0045 -0.035 0.9946/14/00 GSH 6.25 100 0.0036 -0.038 0.999
39
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.
40
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 Concern Stress
Lysosomal Destablization < 15 – 30% 30 – 40% > 40%
Glutathione (nM/g) 800- 1600 < 800 < 500
> 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
41
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.
43
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