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Baseline activities of four biomarkers in three life-stages of the amphipod,

Leptocheirus plumulosus
Jennifer Hoguet a; Peter B. Key b
a
JHT Incorporated, Contractor for the National Ocean Service, Orlando, Florida, USA b National Ocean
Service, Center for Coastal Environmental Health and Biomolecular Research, Charleston, South
Carolina, USA

To cite this Article Hoguet, Jennifer and Key, Peter B.(2008) 'Baseline activities of four biomarkers in three life-stages of
the amphipod, Leptocheirus plumulosus', Journal of Environmental Science and Health, Part B, 43: 6, 465 — 470
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 Journal of Environmental Science and Health Part B (2008) 43, 465–470
Copyright
C Taylor & Francis Group, LLC

ISSN: 0360-1234 (Print); 1532-4109 (Online)

DOI: 10.1080/03601230802174565

Baseline activities of four biomarkers in three life-stages

of the amphipod, Leptocheirus plumulosus

JENNIFER HOGUET1 and PETER B. KEY2

1
JHT Incorporated, Contractor for the National Ocean Service, Orlando, Florida, USA
2
National Ocean Service, Center for Coastal Environmental Health and Biomolecular Research, Charleston, South Carolina, USA
Downloaded By: [OARE Administrative and Technical Support] At: 21:01 16 February 2010

This study investigated differences in baseline levels of four cellular biomarkers (glutathione (GSH), lipid peroxidation (LPx), acetyl-
cholinesterase (AChE), and cholesterol (CHL)) in the larval, juvenile, and adult stages of the estuarine amphipod, Leptocheirus plumu-
losus. Glutathione, LPx, and AChE exhibited the same pattern of decreasing levels with increasing developmental stage. Cholesterol
showed an inverse relationship of increasing levels with increasing developmental stages. This research provides valuable background
information that may be used in future assessments of amphipod biomarker research.
Keywords: Biomarker; glutathione; lipid peroxidation; acetylcholinesterase; cholesterol; amphipods.

Introduction biological systems.[6,7] In addition to serving as a cellular

Increased urbanization of coastal areas has led to increased
contaminant levels in adjacent sediments and waters. Con-
sequently, many studies have been conducted to determine
the potential impacts of contaminants on estuarine organ-
isms. One particular organism, the benthic estuarine am-
phipod, Leptocheirus plumulosus, is of particular impor-
tance, given its wide distribution along the Atlantic coast,
high abundance (sometimes exceeding 2.5 × 104 /m2 ),[1] and
sensitivity to various pollutants.[2,3] As such, it has been
proposed as a potential bioindicator species,[4] and is often
used in sediment toxicity testing.[3,5] Many studies have fo-
cused on the adult stage, resulting in a paucity of knowledge
on the remaining stages (e.g., larval and juvenile). There-
fore, the aim of this study was to investigate differences in
baseline levels of four commonly used cellular biomarkers
in multiple life stages (i.e., larval, juvenile, and adult) of L.
plumulosus. Levels of glutathione (GSH), lipid peroxidation
(LPx), acetylcholinesterase (AChE), and cholesterol (CHL)
were determined. These four biomarkers have been used to
assess a variety of cellular responses that have the potential
to be affected by an array of anthropogenic stressors (e.g.,
heavy metal, pesticide, and pharmaceutical exposures).
Glutathione (GSH) is a ubiquitous tripeptide that is re-
garded as one of the most important non-protein thiols in
Address correspondence to Jennifer Hoguet, JHT Incorporated,
Contractor for the National Ocean Service, Orlando, FL, USA;
E-mail: jen.hoguet@noaa.gov
Received February 5, 2008.
homeostatic modulator, GSH aids in the detoxification of
oxyradicals, metals and xenobiotics.[8,9] Glutathione deple-
tion has been demonstrated in mammalian and marine or-
ganisms exposed to pollutants and other adverse conditions
and may lead to cellular apoptosis and tissue damage.[10−12]
Lipid peroxidation (LPx), an indicator of cellular mem-
brane damage, results from the reaction of excess oxyrad-
icals or reactive oxygen species (ROS) with unsaturated
lipids.[13] An excess of oxyradicals occurs when ROS can-
not be maintained by antioxidant defense systems.[14] Fur-
thermore, ROS and cytotoxic by-products of lipid perox-
idation can cause additional damage to lipids, DNA, and
enzymes,[14] which may ultimately lead to cell injury and
death, and tissue damage. Increased lipid peroxidation has
been demonstrated in response to contaminant exposures,
such as PCBs, polycyclic aromatic hydrocarbons (PAHs)
and metals in a multitude of marine organisms.[11,12,15−17]
Acetylcholinesterase (AChE), an important enzyme of
the nervous system, hydrolyzes the neurotransmitter acetyl-
choline (ACh). The inhibition of AChE leads to an accu-
mulation of ACh which, in turn, over-stimulates sensitive
neurons at the neuromuscular junction resulting in tonic
spasm and tremors. In invertebrates, the accumulation of
ACh can induce a pattern of nerve poisoning with hyper-
activity, tremors, convulsions and paralysis,[18] which may
finally lead to death. Therefore, acetylcholinesterase inhi-
bition is commonly used to assess neurotoxic stress due to
a suite of contaminants including metals,[19] organophos-
phates and carbamates[20] and has been well documented in
crustaceans exposed to various organophosphates.[18,21−24]
 466
Cholesterol (CHL) is an important molecule in main-
taining crustacean health. In various gammarid amphi-
pod species, cholesterol constitutes 70–91% of all sterol
contents.[25] It is a precursor to ecdysteroids, which are es-
sential for molting, and plays an important role in main-
taining the integrity and chemical permeability of cell
walls.[26] Cholesterol may also be indispensable to normal
gonadal development, reproductive performance and off-
spring quality in crustaceans,[27] with a deficiency leading to
reduced growth rates.[28] Although cholesterol has not been
used as a biomarker of contaminant stress in amphipods,
studies have been conducted on the effects of various con-
taminants (i.e., PCBs, and heavy metals) on cholesterol lev-
els in fishes[29,30] and bivalves[31] which indicate the potential
for cholesterol’s use as a biomarker.
Downloaded By: [OARE Administrative and Technical Support] At: 21:01 16 February 2010
Materials and methods
Amphipods, Leptocheirus plumulosus, were maintained in
culture at the NOS laboratory in Charleston, South Car-
olina. Cultures were kept in 11.4 L Rubbermaid/STERITE
storage trays with each containing approximately 1100 ml
of 250 µm-sieved sediment from Leadenwah Creek (N 32◦
38.93; W 80◦ 10.36), a reference site located on Wadmalaw
Island, SC; and 6 liters of 1 µm-filtered 20‰ seawater. Cul-
tures were aerated and kept on a 16 hr light: 8 hr dark cycle

R
and amphipods were fed ground Tetramin flake food ev-
ery other day.
Amphipod cultures were separated through a series of
sieves (1.0, 0.5 and 0.25 mm). The amphipods retained on
the 1.0 mm sieve were defined as adults, those retained
on the 0.5 mm sieve were defined as juveniles, and those
retained on the 0.25 mm sieve were defined as larvae.[4] A
total of 40 samples for each stage (pooled from 8 trays for
larvae and 4 trays for juveniles and adults to obtain enough
tissue) were stored frozen at −70◦ C for GSH, LPx, AChE,
and CHL (n = 10/stage/assay) until analyzed.
Glutathione
Glutathione concentrations (GSH) were assessed using
the 5,5 -dithiobis(2-nitrobenzoic)acid-glutathione (DTNB-
GSSG) reductase recycling assay described in Ringwood
et al.[32] Frozen amphipods were homogenized cold (∼4◦ C)
in 5% sulfosalicylic acid (SSA) at 100 mg tissue/ml and cen-
trifuged cold (4◦ C) for 5 minutes at 13,000 × g. Standards
were prepared in SSA by serial dilution using reduced glu-
tathione (Sigma-Aldrich). Aliquots (25 µL) of standards,
blank (SSA), and sample supernatants were mixed with
175 µL of deionized water, 100 µL of 10 mM DTNB (5,5 -
dithiobis(2-nitrobenzoic) acid, Sigma-Aldrich) and 700 µL
of 0.285 mM NADPH buffer (β-nicotinamide adenine din-
ucleotide phosphate) reduced form (Sigma-Aldrich). The
mixture was then vortexed and transferred to 1.5 mL cu-
vettes. Fifty units/mL GSSG reductase (from Baker’s yeast,
Hoguet and Key
Sigma-Aldrich) was then added, the cuvettes placed imme-
diately in a spectrophotometer, and the absorbance read at
405 nm every 15 seconds for a total of 90 seconds on an
Ultraspec 4300 pro UV/visible spectrophotometer (Amer-
sham Biosciences) using Swift II software (Biochrom Ltd).
Data are expressed as GSH (nmol/g wet weight).
Lipid peroxidation
The spectrophotometric thiobarbituric acid (TBA) test
was used to quantify malondialdehyde (MDA),[32,33] a by-
product of lipid peroxidation (LPx). Frozen amphipods
were homogenized cold (∼ 4◦ C) in 50 mM potassium phos-
phate (K2 PO4 ) buffer (pH 7.0) at 250 mg tissue/ml and cen-
trifuged cold (4◦ C) for 5 minutes at 13,000 × g. A 10 mM
stock solution of MDA was prepared by heating 1,1,3,3-
tetraethoxypropane (TEP), 1 N HCl, and ultra-pure H2 O
in a 50◦ C water bath for 60 minutes. Upon cooling, stan-
dards were prepared by serial dilution with K2 PO4 buffer.
Aliquots (75 µL) of standards, blank (K2 PO4 ), and sample
supernatants were mixed with 1050 µL of 0.375% thiobar-
bituric acid (TBA)/trichloroacetic acid (TCA) mixture and
10.5 µL of 2% butylated hydroxytoluene (BHT), heated at
100◦ C for 15 minutes, and centrifuged at 13,000 × g for 5
minutes to remove the precipitate. The absorbance of the
resultant supernatant was then read at 532 nm on an Ultra-
spec 4300 pro UV/visible spectrophotometer (Amersham
Biosciences) using Swift II software (Biochrom Ltd). Data
are expressed as MDA (nmol/g wet weight).
Acetylcholinesterase
Acetylcholinesterase (AChE) concentrations were as-
sessed as described in Key and Fulton.[34] Frozen
amphipods were homogenized cold (∼4◦ C) in tris-
(hydroxymethyl)aminomethane (TRIS) buffer (pH 8.1) at
20 mg tissue/mL. For each sample, 75 µL aliquots of the
homogenate were added to four separate test tubes, three
of which contained 1.425 ml TRIS and 15 µL of 100%
ethanol and one of which contained 1.425 mL TRIS and
15 µl of 10−3 M eserine sulfate (AChE inhibitor – served
as a blank). Samples were then heated at 30◦ C for 15
minutes and transferred to cuvettes. Thirty-three µL of
20 mM DTNB (5,5 -dithiobis(2-nitrobenzoic) acid, Sigma-
Aldrich) and 10 µL of 75 mM acetylthiocholine iodide
(ACTH) were then added to the cuvettes, which were then
covered with parafilm, inverted to mix, and placed in the
spectrophotometer. The absorbance was read at 412 nm ev-
ery 20 seconds for a total of 100 seconds on an Ultraspec
4300 pro UV/visible spectrophotometer (Amersham Bio-
sciences) using Swift II software (Biochrom Ltd). A 50 µl
aliquot of the initial homogenate was reserved and frozen
at −20◦ C for subsequent total protein analysis with modi-
fications from Lowry et al.[35] Data are expressed as AChE
(nmol/mg protein/min).
 Four biomarkers in Leptocheirus plumulosus 467
Cholesterol p = 0.0021
200
A
Cholesterol concentrations were assessed using the Amplex 180

GSH (nmol/g wet weight)

160 B
Red Cholesterol Assay Kit (Molecular Probes, A12216). 140
Frozen amphipods were homogenized as described above 120
C
for the acetylcholinesterase assay. Samples were then di- 100
80
luted (1:10) with the provided 1 X Reaction Buffer. Stan- 60
dards were prepared with a CHL reference standard. 40
Aliquots (50 µl) of the standards, blank (1 X Reaction 20

Buffer), and samples were added in triplicate to a black- 0

Larvae Juveniles Adults
sided, clear-bottomed, 96-well plate. As per kit instruc-
tions, a 300 µM Amplex Red reagent/horseradish peroxi- Stage

dase/cholesterol oxidase/cholesterol esterase solution was

prepared and a 50 µL aliquot was added to each well. Af-
ter a 1-hour incubation period at 37◦ C, the fluorescence
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Fig. 1. Baseline activities of glutathione (GSH) in multiple life-
stages of L. plumulosus (larvae, juveniles, and adults). Significant
differences between life-stages are denoted with differing letters

was read at an excitation and emission of 560 and 590 nm,
respectively, on an FLx800 microplate fluorescence reader
(Bio-Tek Instruments, Inc.) with KC4 v.3.0 software (Bio-
Tek). Samples and standards were referenced with a previ-
ously prepared standard curve of CHL reference standards
(R2 = 0.9999). Data are expressed as cholesterol (µg/mg
tissue).
Statistics
Data were analyzed with SAS version 9.1.3 software. A one-
way analysis of variance (ANOVA) with the Shapiro Wilks’
test for normality and Levene’s test for equal variance were
first run to detect statistical differences between life-stages
within each bioassay conducted.[36] Data from each bioas-
say failed to meet the assumptions of the ANOVA. There-
fore, life stages were compared using the Kruskall-Wallis
test.[36] Multiple comparisons were performed using a non-
parametric all pairwise test (Dunn’s) for ranks with no
ties.[36] Due to the large number of multiple comparisons, a
Bonferroni-type adjustment, [p ≤ α/k(k-1)], was used for
α,[36] with an adjusted α value of 0.0083 for all comparisons.
Results
Glutathione
There were significant differences in glutathione (GSH)
(i.e., A, B, C). Glutathione is expressed as GSH (nmol/g wet
weight).
cording to the Kruskall-Wallis test (p = 0.0014), there were
significant differences in malondialdehyde (MDA) concen-
trations with mean larval concentrations (266.63 nmol/g
wet weight) being significantly higher than mean concen-
trations of both juveniles (132.00 nmol/g wet weight) and
adults (115.46 nmol/g wet weight) (p < 0.0001). And al-
though there were no significant differences in MDA con-
centrations between the juvenile and adult stages, there was
still a trend of decreasing MDA concentrations with in-
creasing developmental stage (Fig. 2).
Acetylcholinesterase
Acetylcholinesterase (AChE) concentrations shared the
GSH and LPx trend of decreasing levels with increasing
development stage. According to the Kruskall-Wallis test
(p < 0.0001), mean larval AChE concentrations (660.34
nmol/mgP/min) were significantly higher than mean con-
centrations of both juveniles (404.70 nmol/mgP/min) and
350 p = 0.0014
300
A
MDA (nmol/g wet weight)

250
concentrations between all life history stages, according to
the Kruskall-Wallis test (p = 0.0021), with decreasing GSH
200

150
B
concentrations with increasing developmental stage. Mean B
larval concentrations (150.18 nmol/g wet weight) were sig- 100

nificantly higher than mean concentrations of both juve- 50

niles (138.12 nmol/g wet weight) and adults (92.32 nmol/g 0

wet weight) (p = 0.00722 and 0.00138, respectively). Ad- Larvae Juveniles Adults

ditionally, mean juvenile concentrations were significantly Stage

higher than that of the adults (p < 0.001) (Fig. 1).

Lipid peroxidation
Lipid peroxidation (LPx) levels shared the GSH trend of
decreasing levels with increasing development stage. Ac-
Fig. 2. Baseline activities of lipid peroxidation (LPx) in multiple
life-stages of L. plumulosus (larvae, juveniles, and adults). Signif-
icant differences between life-stages are denoted with differing
letters (i.e., A, B, C). Lipid peroxidation is expressed as malon-
dialdehyde (MDA) (nmol/g wet weight).
 468 Hoguet and Key
800
A p < 0.0001 determine if there are differences in developmental stages
700 and (ii) provide important background information that
AChE (nmol/mgP/min)

600 can be used in future contaminant risk assessments. Lipid

500
B peroxidation (LPx) and glutathione (GSH) levels are com-
400
monly used in tandem to characterize the pro-oxidant and
300
C anti-oxidant status of an organism, respectively. Ideally, a
200
balance should exist between pro-oxidants (reactive oxy-
100

0
Larvae Juveniles
Stage
Fig. 3. Baseline activities of acetylcholinesterase (AChE) in mul-
tiple life-stages of L. plumulosus (larvae, juveniles, and adults).
Significant differences between life-stages are denoted with dif-
fering letters (i.e., A, B, C). Acetylcholinesterase is expressed as
Downloaded By: [OARE Administrative and Technical Support] At: 21:01 16 February 2010
gen species) and anti-oxidants, such that oxidative stress
Adults does not occur. Reactive oxygen species (ROS) may result
from contaminant exposure but are also produced during
aerobic respiration; therefore, high metabolic activity may
result in elevated ROS levels. In the present study, signif-
icantly higher larval LPx levels may have resulted from
higher metabolic activity associated with higher growth
rates and continued molting as compared to juveniles

AChE (nmol/mgP/min).
adults (192.66 nmol/mgP/min) (p = 0.0019 and <0.0001,
respectively). Additionally, mean juvenile concentrations
were significantly higher than that of the adults (p < 0.001)
(Fig. 3).
Cholesterol
Cholesterol (CHL) showed an inverse trend, as compared
to GSH, LPx, and CHL, of increasing concentrations with
increasing developmental stage. However, according to the
Kruskall-Wallis test (p = 0.1651), no life-history stages were
significantly different from each other (Fig. 4). Neverthe-
less, the means reflect a trend of increasing CHL concen-
trations with increasing developmental stage.
Discussion
The aim of this study was to investigate baseline activ-
ities of four commonly used biomarkers in multiple life
stages of the amphipod, Leptocheirus plumulosus, to (i)
p = 0.1651
8.0
7.0
6.0
CHL (µg/mg tissue )
and adults. Arun and Subramanian[37] found that during
embryonic development, the freshwater prawn, Macro-
brachium malcomsonii, increases oxygen uptake with a sub-
sequent increase in antioxidant activity (superoxide dismu-
tase (SOD), catalase (CAT), and glutathione transferase).
Similarly, Peters and Livingstone[38] found that SOD activ-
ity was highest in embryonic turbot, Scophthalmus max-
imus, with a subsequent decrease 11 days post-hatching.
Viarengo et al.[11] demonstrated that increased age in the
mussel, Mytilus edulis, resulted in lower oxygen consump-
tion rates with subsequent decreased CAT activity. Con-
sistent with the decrease in activity with age shown here,
Correia et al.[39] showed significantly higher levels of an-
tioxidants (glutathione peroxidase (GPx) and SOD) in
juvenile amphipods (Gammarus locusta) as compared to
adults.
Acetylcholinesterase inhibition is commonly used to as-
sess neurotoxic stress due to a suite of contaminants. Neu-
rological activity, specifically AChE levels, has been pos-
itively correlated with the hormone 20-hydroxyecdysone
(20HE), the primary mechanism controlling molting in
crustaceans.[40] Gagne and Blaise[41] found that 20HE lev-
els increased as AChE levels increased in the brine shrimp,
Artemia franciscana. It may then be inferred that increases
in molting frequency corresponds to increases in AChE.
Molting typically peaks at the juvenile stage in crustaceans,
which might explain our findings of higher levels of AChE
in the larval and juvenile stages with a subsequent decrease
in adults, corresponding to reduced molting frequencies.

5.0
Cholesterol (CHL) is essential for crustaceans to syn-
4.0
thesize the molting hormone 20-hydroxyecdysone (20HE).
3.0
In this study, while not significant, the increase in CHL
2.0
levels from larval through adult stages was not surprising
1.0
0.0
considering that CHL is required for molting and molt-
Larvae Juveniles Adults ing frequency decreases with age. In addition, crustaceans
Stage
are incapable of synthesizing CHL. It must be obtained
by diet. The storage and processing of CHL occurs in the
Fig. 4. Baseline activity of cholesterol (CHL) in multiple life-stages hepatopancreas,[40] which may not be fully developed until
of L. plumulosus (larvae, juveniles, and adults). There were no adulthood. This might be another reason for an increase
significant differences between life-stages. Cholesterol is expressed in CHL with increased developmental stage. Nevertheless,
as CHL (µg/mg tissue). CHL is also a precursor of sex steroids and membrane com-
 Four biomarkers in Leptocheirus plumulosus
ponents and is therefore also integral in maintaining ju-
venile and adult health. Therefore, measuring cholesterol
as a biomarker of contaminant exposure could be use-
ful as a measure of hepatopancreatic function in addition
to measuring interference with dietary uptake in chronic
exposures.
Conclusions
The challenge of biomarker research is determining which
biomarker is most appropriate for a particular taxon, life-
stage, environment, or pollutant. In reality, there are many
confounding factors that complicate biomarker selection.
In crustaceans, factors such as phylogenetic differences
Downloaded By: [OARE Administrative and Technical Support] At: 21:01 16 February 2010
among taxa, locomotory activity, body size, morphogen-
esis and molting cycle have an impact on metabolism
alone, not to mention the impacts of natural environmen-
tal variables, such as nutrition, pH, temperature, salin-
ity, and osmotic pressure.[40] Nevertheless, biomarker re-
search is important in toxicological research because it
aids in overall risk assessment and can be used as an
early warning of contaminant exposure prior to lethal
consequences.
From recent research, larval crustaceans (grass shrimp)
appear to be generally more susceptible to contaminant ex-
posure, evidenced by their lower LC50 values than their
adult counterparts.[23,42,43] This may be due to increased
metabolic rates and molting frequencies, which have been
suggested to increase sensitivity to pesticides.[44] Neverthe-
less, adults are used more often for toxicology research per-
haps because of their availability year around.[45] In regards
to this study, all four biomarkers were clearly detectable in
larvae; in fact, levels were generally highest in the larval
stage. Because of the high sensitivity of larvae to contam-
inants and the applicability of these four biomarkers, am-
phipod larvae may prove useful as early sublethal indicators
for environmental stress. However, further studies incorpo-
rating contaminants should be conducted to validate these
statements.
Acknowledgments
The authors thank Dr. Paul Pennington for his assistance
with statistical analyses and John Venturella for maintain-
ing the amphipod cultures. NOAA’s National Ocean Service
(NOS) does not approve, recommend, or endorse any pro-
prietary product or material mentioned in this publication.
No reference shall be made to NOS, or to this publication
furnished by NOS, in any advertising or sales promotion
which would indicate or imply that NOS approves, recom-
mends, or endorses any proprietary product or proprietary
material mentioned herein or which has as its purpose any
intent to cause directly or indirectly the advertised product
to be used or purchased because of NOS publication.
469
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