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

Jennifer Hoguet a; Peter B. Key ba JHT Incorporated, Contractor for the National Ocean Service, Orlando, Florida, USA b National OceanService, Center for Coastal Environmental Health and Biomolecular Research, Charleston, SouthCarolina, 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 470To link to this Article: DOI: 10.1080/03601230802174565URL:http://dx.doi.org/10.1080/03601230802174565

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466 Hoguet and Key

Cholesterol (CHL) is an important molecule in main-taining crustacean health. In various gammarid amphi-pod species, cholesterol constitutes 7091% of all sterolcontents. [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 normalgonadal development, reproductive performance and off-spring quality in crustaceans, [27] with a deciencyleading toreduced growth rates. [28] Although cholesterol has not beenused 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-elsin shes [29, 30] and bivalves [31] which indicate thepotentialfor cholesterols use as a biomarker.

Materials and methods

Amphipods, Leptocheirus plumulosus , were maintained inculture at the NOS laboratory in Charleston, South Car-olina. Cultureswere kept in 11.4L Rubbermaid/STERITEstorage trays with each containing approximately 1100 mlof 250 m-sieved sediment from Leadenwah Creek (N 32

38.93; W 80 10.36), a reference site located on WadmalawIsland, SC; and 6 liters of 1 m-ltered 20 seawater. Cul-tures were aerated and kept on a 16 hr light: 8 hr dark cycleand amphipods were fed ground Tetramin R ake 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 onthe 1.0 mm sieve were dened as adults, those retainedon the 0.5 mm sieve were dened as juveniles, and thoseretained on the 0.25 mm sieve were dened as larvae. [4] Atotal of 40 samples for each stage (pooled from 8 trays forlarvae and 4 trays for juveniles and adults to obtain enoughtissue) were stored frozen at 70 C for GSH, LPx, AChE,and CHL (n = 10/stage/assay) until analyzed.

Glutathione

Glutathione concentrations (GSH) were assessed usingthe5,5 -dithiobis(2-nitrobenzoic)acid-glutathione(DTNB-GSSG) reductase recycling assay described in Ringwoodet al. [32] Frozen amphipods were homogenized cold ( 4 C)in 5% sulfosalicylicacid (SSA) at100mg tissue/mlandcen-trifuged cold (4 C) for 5 minutes at 13,000 g. Standardswere prepared in SSA by serial dilution using reduced glu-tathione (Sigma-Aldrich). Aliquots (25 L) of standards,blank (SSA), and sample supernatants were mixed with175 L of deionized water, 100 L of 10 mM DTNB (5,5 -dithiobis(2-nitrobenzoic) acid, Sigma-Aldrich) and 700 Lof 0.285 mM NADPH buffer ( -nicotinamide adenine din-ucleotide phosphate) reduced form (Sigma-Aldrich). Themixture was then vortexed and transferred to 1.5 mL cu-vettes. Fifty units/mLGSSGreductase(fromBakers yeast,

Sigma-Aldrich) was then added, the cuvettes placed imme-diately in a spectrophotometer, and the absorbance read at405 nm every 15 seconds for a total of 90 seconds on anUltraspec 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) testwas used to quantify malondialdehyde (MDA), [32, 33] a by-product of lipid peroxidation (LPx). Frozen amphipodswere homogenized cold ( 4 C) in 50 mM potassium phos-phate (K 2PO 4) buffer (pH 7.0) at 250 mg tissue/ml and cen-trifuged cold (4 C) for 5 minutes at 13,000 g. A 10 mMstock solution of MDA was prepared by heating 1,1,3,3-tetraethoxypropane (TEP), 1 N HCl, and ultra-pure H 2Oin a 50 C water bath for 60 minutes. Upon cooling, stan-dards were prepared by serial dilution with K

2PO

4buffer.

Aliquots (75 L) of standards, blank (K 2PO 4), and samplesupernatants were mixed with 1050 L of 0.375% thiobar-bituric acid (TBA)/trichloroacetic acid (TCA) mixture and10.5 L of 2% butylated hydroxytoluene (BHT), heated at100 C for 15 minutes, and centrifuged at 13,000 g for 5minutes to remove the precipitate. The absorbance of theresultant supernatant was then read at 532 nm on an Ultra-spec 4300 pro UV/visible spectrophotometer (AmershamBiosciences) using Swift II software (Biochrom Ltd). Dataare expressed as MDA (nmol/g wet weight).

AcetylcholinesteraseAcetylcholinesterase (AChE) concentrations were as-sessed as described in Key and Fulton. [34] Frozenamphipods were homogenized cold ( 4 C) in tris-(hydroxymethyl)aminomethane (TRIS) buffer (pH 8.1) at20 mg tissue/mL. For each sample, 75 L aliquots of thehomogenate were added to four separate test tubes, threeof which contained 1.425 ml TRIS and 15 L of 100%ethanol and one of which contained 1.425 mL TRIS and15 l of 10 3 M eserine sulfate (AChE inhibitor servedas a blank). Samples were then heated at 30 C for 15minutes and transferred to cuvettes. Thirty-three L of 20 mM DTNB (5,5 -dithiobis(2-nitrobenzoic) acid, Sigma-Aldrich) and 10 L of 75 mM acetylthiocholine iodide(ACTH) were then added to the cuvettes, which were thencovered with paralm, inverted to mix, and placed in thespectrophotometer. Theabsorbancewas read at 412nm ev-ery 20 seconds for a total of 100 seconds on an Ultraspec4300 pro UV/visible spectrophotometer (Amersham Bio-sciences) using Swift II software (Biochrom Ltd). A 50 laliquot of the initial homogenate was reserved and frozenat 20 C for subsequent total protein analysis with modi-cations from Lowry et al. [35] Data are expressed as AChE(nmol/mg protein/min).


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Four biomarkers in Leptocheirus plumulosus 467

Cholesterol

Cholesterol concentrations were assessed using the AmplexRed Cholesterol Assay Kit (Molecular Probes, A12216).Frozen amphipods were homogenized as described abovefor the acetylcholinesterase assay. Samples were then di-luted (1:10) with the provided 1 X Reaction Buffer. Stan-dards were prepared with a CHL reference standard.Aliquots (50 l) of the standards, blank (1 X ReactionBuffer), and samples were added in triplicate to a black-sided, clear-bottomed, 96-well plate. As per kit instruc-tions, a 300 M Amplex Red reagent/horseradish peroxi-dase/cholesterol oxidase/cholesterol esterase solution wasprepared and a 50 L aliquot was added to each well. Af-ter a 1-hour incubation period at 37 C, the uorescencewas read at an excitation and emission of 560 and 590 nm,respectively, on an FLx800 microplate uorescence 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(R 2 = 0.9999). Data are expressed as cholesterol ( g/mgtissue).

Statistics

Data wereanalyzedwith SASversion9.1.3software. A one-way analysis of variance (ANOVA) with the Shapiro Wilkstest for normality and Levenes test for equal variance wererst run to detect statistical differences between life-stageswithin 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 (Dunns) for ranks with noties. [36] Due to the large number of multiple comparisons, aBonferroni-type adjustment, [p /k(k-1)], was used for ,[36] with an adjusted value of 0.0083 for all comparisons.

Results

Glutathione

There were signicant differences in glutathione (GSH)concentrations between all life history stages, according tothe Kruskall-Wallis test (p = 0.0021), with decreasing GSHconcentrations with increasing developmental stage. Meanlarval concentrations (150.18 nmol/g wet weight) were sig-nicantly higher than mean concentrations of both juve-niles (138.12 nmol/g wet weight) and adults (92.32 nmol/gwet weight) (p = 0.00722 and 0.00138, respectively). Ad-ditionally, mean juvenile concentrations were signicantlyhigher 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-

Larvae Juveniles Adults

p = 0.0021

0

20

4060

80

100

120

140

160

180

200


Stage

G S H ( n m o l

/ g w e t w e i g h

t )

A

B

C

Fig. 1. Baseline activities of glutathione (GSH) in multiple life-stages of L. plumulosus (larvae, juveniles, and adults). Signicantdifferences between life-stages are denoted with differing letters(i.e., A, B, C). Glutathione is expressed as GSH (nmol/g wetweight).

cording to the Kruskall-Wallis test (p = 0.0014), there weresignicant differences in malondialdehyde (MDA) concen-trations with mean larval concentrations (266.63 nmol/gwet weight) being signicantly higher than mean concen-trations of both juveniles (132.00 nmol/g wet weight) andadults (115.46 nmol/g wet weight) (p < 0.0001). And al-though there were no signicant differences in MDA con-centrations between the juvenile andadult stages, there wasstill a trend of decreasing MDA concentrations with in-creasing developmental stage (Fig. 2).

Acetylcholinesterase

Acetylcholinesterase (AChE) concentrations shared theGSH and LPx trend of decreasing levels with increasingdevelopment stage. According to the Kruskall-Wallis test(p < 0.0001), mean larval AChE concentrations (660.34nmol/mgP/min) were signicantly higher than mean con-centrations of both juveniles (404.70 nmol/mgP/min) and

0

50

100

150

200

250

300

350


p = 0.0014

Stage

M D A ( n m o l

/ g w e t w e i g h

t )A

BB

Fig. 2. Baseline activities of lipid peroxidation (LPx) in multiplelife-stages of L. plumulosus (larvae, juveniles, and adults). Signif-icant differences between life-stages are denoted with differingletters (i.e., A, B, C). Lipid peroxidation is expressed as malon-dialdehyde (MDA) (nmol/g wet weight).


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468 Hoguet and Key

B

C

A

0

100

200

300

400

500

600

700

800


p < 0.0001

Stage

A

C h E ( n m o l

/ m g P

/ m i n )

Fig. 3. Baseline activities of acetylcholinesterase (AChE) in mul-tiple life-stages of L. plumulosus (larvae, juveniles, and adults).Signicant differences between life-stages are denoted with dif-fering letters (i.e., A, B, C). Acetylcholinesterase is expressed asAChE (nmol/mgP/min).

adults (192.66 nmol/mgP/min) (p = 0.0019 and < 0.0001,respectively). Additionally, mean juvenile concentrationswere signicantly higher than that of the adults (p < 0.001)(Fig. 3).

Cholesterol

Cholesterol (CHL) showed an inverse trend, as comparedto GSH, LPx, and CHL, of increasing concentrations withincreasing developmental stage. However, according to theKruskall-Wallis test (p = 0.1651),no life-historystagesweresignicantly different from each other (Fig. 4). Neverthe-less, the means reect 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 lifestages of the amphipod, Leptocheirus plumulosus , to (i)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0


p = 0.1651

Stage

C H L ( g / m g t

i s s u e

)

Fig.4. Baselineactivityofcholesterol (CHL) inmultiplelife-stagesof L. plumulosus (larvae, juveniles, and adults). There were nosignicant differencesbetweenlife-stages.Cholesterolis expressedas CHL ( g/mg tissue).

determine if there are differences in developmental stagesand (ii) provide important background information thatcan be used in future contaminant risk assessments. Lipidperoxidation (LPx) and glutathione (GSH) levels are com-monly used in tandem to characterize the pro-oxidant andanti-oxidant status of an organism, respectively. Ideally, a

balance should exist between pro-oxidants (reactive oxy-gen species) and anti-oxidants, such that oxidative stressdoes not occur. Reactive oxygen species (ROS) may resultfrom contaminant exposure but are also produced duringaerobic respiration; therefore, high metabolic activity mayresult in elevated ROS levels. In the present study, signif-icantly higher larval LPx levels may have resulted fromhigher metabolic activity associated with higher growthrates and continued molting as compared to juvenilesand adults. Arun and Subramanian [37] found that duringembryonic 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 themussel, 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 signicantly higher levels of an-tioxidants (glutathione peroxidase (GPx) and SOD) in juvenile amphipods ( Gammarus locusta ) as compared toadults.

Acetylcholinesterase inhibition is commonly used to as-sess neurotoxic stress due to a suite of contaminants. Neu-rological activity, specically AChE levels, has been pos-itively correlated with the hormone 20-hydroxyecdysone(20HE), the primary mechanism controlling molting incrustaceans. [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 increasesin molting frequency corresponds to increases in AChE.Molting typicallypeaks at the juvenile stage in crustaceans,which might explain our ndings of higher levels of AChEin the larval and juvenile stages with a subsequent decreasein adults, corresponding to reduced molting frequencies.

Cholesterol (CHL) is essential for crustaceans to syn-thesize the molting hormone 20-hydroxyecdysone (20HE).In this study, while not signicant, the increase in CHLlevels from larval through adult stages was not surprisingconsidering that CHL is required for molting and molt-ing frequency decreases with age. In addition, crustaceansare incapable of synthesizing CHL. It must be obtainedby diet. The storage and processing of CHL occurs in thehepatopancreas, [40] which may not be fully developed untiladulthood. This might be another reason for an increasein CHL with increased developmental stage. Nevertheless,CHLis also a precursor of sex steroidsandmembrane com-


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Four biomarkers in Leptocheirus plumulosus 469

ponents and is therefore also integral in maintaining ju-venile and adult health. Therefore, measuring cholesterolas a biomarker of contaminant exposure could be use-ful as a measure of hepatopancreatic function in additionto measuring interference with dietary uptake in chronicexposures.

Conclusions

The challenge of biomarker research is determining whichbiomarker is most appropriate for a particular taxon, life-stage, environment, or pollutant. In reality, there are manyconfounding factors that complicate biomarker selection.In crustaceans, factors such as phylogenetic differencesamong taxa, locomotory activity, body size, morphogen-esis and molting cycle have an impact on metabolismalone, 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 itaids in overall risk assessment and can be used as anearly warning of contaminant exposure prior to lethalconsequences.

From recent research, larval crustaceans (grass shrimp)appear to be generally more susceptible to contaminant ex-posure, evidenced by their lower LC50 values than theiradult counterparts. [23, 42, 43] This may be due to increasedmetabolic rates and molting frequencies, which have beensuggested to increase sensitivity to pesticides. [44] Neverthe-less, adults are used more often for toxicology research per-haps because of their availabilityyeararound. [45] In regardsto this study, all four biomarkers were clearly detectable inlarvae; in fact, levels were generally highest in the larvalstage. 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 indicatorsfor environmental stress. However, further studies incorpo-rating contaminants should be conducted to validate thesestatements.

Acknowledgments

The authors thank Dr. Paul Pennington for his assistancewith statistical analyses and John Venturella for maintain-ingtheamphipodcultures. NOAAs 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 publicationfurnished by NOS, in any advertising or sales promotionwhich would indicate or imply that NOS approves, recom-mends, or endorses any proprietary product or proprietarymaterial mentioned herein or which has as its purpose anyintent to cause directly or indirectly the advertised productto be used or purchased because of NOS publication.

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