expression characteristics of potential biomarker genes in tra catfish, pangasianodon hypophthalmus,...

8/9/2019 Expression characteristics of potential biomarker genes in Tra catfish, Pangasianodon hypophthalmus, exposed to t

1/10


2/10

associated with short term and/or long term toxicological responseswhich have profound effect on overall performance offish (Lee et al.,2006).

Thereby, evaluation of gene expression alterations is advisable to

monitor the potentialadverse effect of TRC onfish healthand allow anearly indication of the ecotoxicological impact. In this context, theexpression kinetics of important stress and growth-related biomark-

er genes measured at molecular level has immense importance as a

sensitive

early warning tool

of chemical stress infi

sh (Carajavilleet al., 2000; Chevre et al., 2003; de la Torre et al., 2005). Althoughresearchers have looked into the toxicants-induced gene expression

alterations in fish, the use of biomarker genes to evaluate generalstress in Pangasianodon hypophthalmus, an important freshwaterculture species in Southeast Asian countries, on TRC exposure is ratherscarce.

It is well understood that no singlebiomarkergene has emerged as a

widely used indicator for toxicity without some limitation (Smolder etal., 2003), therefore, the objective of the present study was to evaluate,in conditions simulating aquaculture treatments, the in vivo effects ofTRC on well known stress and growth-related biomarker genes of P.

hypophthalmus. Since several studies have used AChE activity todiagnose exposure of fish to TRC (Bocquen et al., 1990; Boone andChambers, 1997; Sturm et al., 1999, 2000; Var et al., 2003), theexpression kinetics of the AChE gene is a representative biomarker genein this study. In addition, the expression dynamics of growth-related

and other cellular toxicity representative genes, such as heat shockprotein70 (HSP70),growthhormone, trypsinogen, cytochrome P4501B(CYP1B) and cytochrome oxidase subunit 1 (COI) were investigated.HSP70 are important chaperone molecules for cellular protein folding

and repair and are a general indication of protein homeostasisdisruption (Stegeman et al., 1992; Hartl, 1996), whereas, COI (Villaniand Attardi, 2000) and CYP1B genes are involved in the biotransforma-tion anddetoxification of toxic compounds (Shimadaet al., 1996; Buters

et al., 1999; Willett et al., 2006).

2. Materials and methods

2.1. Experimental system and animals

Pangasianodon hypophthalmus (Siluriformes; Pangasiidae;fishbase.

orgname: striped catfish) juveniles (1520 g) were obtained from anartificial seed production centre located in CanTho city. They werereared at the College of Aquaculture and Fisheries, CanTho University,Vietnam. Fish were acclimated for 2 weeks prior to experimentation

in 1000 L holding tanks equipped with a continuous supply of wellaerated water. During this period, fish were fed ad libitum withcommercial fish pellets (Cargill, 30% crude protein). After acclimation,840 fish were randomly distributed into four groups with three

replicate tanks; each tank contained 70 fish (500 L capacity).All the tanks were supplied with water at 270.5 C from a

recirculating system. The system was subjected to a photoperiod of

12 h light:12 h darkness. Water quality was monitored throughoutthe experiment. All the water parameters were in the optimumrange (temperature 26.227.1 C, pH 7.07.5, dissolved oxygen6.97.4 mg L1, total NH3 0.10.2 mg L

1, nitrite 0.070.1 mg L1

and nitrate 13 mg L1). Water flow was adjusted to keep the oxygen

saturation above 80%. Fish were fed thrice a day at a total of 3% on theirwetbody weight day1. Feeding was adjustedbasedon the weightandthe number offish remaining in the tank after each sampling periods.However, one day before the start of the experiment, they were kept

starved.

2.2. Exposure

The organophosphate insecticide, TRC (Trade name: Dich Bach

Trung 90 SD) was used in the present work. The commercial product is

available with a concentration of 90% (w/v). Different dilutions wereprepared by adding water. Among the fourgroups offish, thefirst groupwas reared in TRC free fresh water and used as a control group. Fish inthe second,third andfourthgroupswere exposed to the 96-h 1/100LC50(0.01 mg/L), 96-h 110LC50 (0.1 mg/L), and 96-h 12LC50 (0.5 mg/L) ofTRC, respectively. Fish were reared for a period of 56 days. To avoidmetabolic and microbial breakdown of test chemical, 40%60% of the

water was discarded every two days and replaced with fresh water

containing the respective amount of test chemical. Fivefi

sh per replicafrom each group were randomly sampled after 0 h, 6 h, 24 h, 96 h,7 days, 14 days, 28 days and 56 days. Extra care was taken while

sampling the fish to make sure that catching an individual fish did notcause stress to remaining ones. The sampled fish were killedimmediately by decapitation.

2.3. Growth measurement

Fish in each container were bulk weighed at the beginning of theexperiment and on the final day (56 days) of experiment. Growthperformance of juveniles was evaluated in terms of weight gain based

on following standard formulae:

Weight gain % = Final weightInitial weight 100= Initial weight:

2.4. Tissue samples

The fish were carefully dissected out to isolate liver and gill. Theextracted organs collected from five individual per replica of thedifferent groups were pooled together in a sterile 50 ml falcon tube

and were immediately frozen in liquid nitrogen and stored at 80 Cuntil RNA isolation.

2.5. RNA extraction and real time RTPCR

Total RNA was isolated from liver and gill samples using the Trizolmethod (Invitrogen, Merelbeke, Belgium) according to the manufac-turer's instructions. The extracted RNA samples were subjected to

DNA-free (DNase) treatment to avoid genomic DNA contamination.The quantity of the RNA was evaluated by using Nano-Dropspectrophotometry (NanoDrop Technologies, Wilmington, DE). The

integrity (quality) was checked by denaturing gel electrophoresis (1%agarose gel) and purity by OD260/OD280 nm absorption ratioN1.95.

A starting amount of 1 g RNA was transcribed to First strandcDNA according to Revert Aid H minus First strand cDNA synthesis

kit (Fermentas, Cambridgeshire). For the real time PCR reaction, the

final volume of 20 L was adjusted to 100 L to achieve a workingamount (5 L) of approximately 50 ng RNA-equivalent for eachreaction.

Highly purified salt-free OliGold primers (Eurogentec, Seraing,Belgium) for the quantification of the target genes AChE, HSP70, growthhormone, trypsinogen, CYP1B, COI and reference genes beta-actin

(-actin), 16S ribosome RNA (16S rRNA) and 12S ribosomal RNA(12S rRNA) were designed using the Lightcycler probe design software,version 1.0 (Roche Diagnostics, Vilvoorde, Belgium). The nucleotidesequence of trypsinogen and COI were obtained from GenBankaccession no: AY316360 and EF609427. For rest of the genes we

designed primer pairs based on conserved regions of known respectivegenesequences, compared among other relatedfish species.The primersequences of each gene are listed in Table 1. Real time PCR mastermixwas prepared as follows: 5.5 L nuclease free water, 1 L forward and

1 L reverse primer and 12.5 L Maxima SYBR Green qPCR Master mix(Fermentas, Cambridgeshire). Mastermix (20 L) was mixed with 5 Lof cDNA template in the glass capillaries. A four-step experimental runprotocol was used in light cycler (Roche version 3.5): denaturation

program (10 min at 95 C); amplification and quantification program

repeated40 times(15 s at 95 C, 30 s at60 C,and 30 s at72 C);melting

2 A.K. Sinha et al. / Comparative Biochemistry and Physiology, Part D xxx (2010) xxx xxx

ARTICLE IN PRESS

Please cite this article as: Sinha, A.K., et al., Expression characteristics of potential biomarker genes in Tra catfish, Pangasianodonhypophthalmus, exposed to trichlorfon, Comp. Biochem. Physiol. D (2010), doi:10.1016/j.cbd.2010.05.001
http://dx.doi.org/10.1016/j.cbd.2010.05.001http://dx.doi.org/10.1016/j.cbd.2010.05.001


3/10

curve program (5595 C with a heating rate of 0.10 C/s and acontinuous fluorescence measurement) and finally a cooling step(4 C). To reduce the pipetting errors, mastermixes were prepared induplicate for each sample and for every test sample; a quantitative PCR

for both the target and the reference genes was performed.

2.5.1. Confirmation of primer specificity and efficiencyLightCycler melting curve analysis of the target genes and reference

genes were performed which resulted in single productspecific meltingtemperature as follows: AChE, 89.78 C; HSP70, 80.54 C; growthhormone, 82.75 C; COI, 79.62 C ; trypsinogen, 80.73 C; CYP1B,

86.7 C; 16S rRNA, 81.8 C; 12S rRNA, 78.46 C; -actin, 84.71 C.Moreover, no primerdimers were generated during the applied 40real time PCR amplification cycles.

The efficiency of amplification of target genes and internal controlswas examined from the slopes obtained from LightCycler Software.

Investigated transcripts showed high efficiency rates (Table 1).

2.5.2. Relative quantificationRelative quantification of the target genes transcript with a chosen

reference gene transcript was done following the Pfaffl method with

the Relative Expression Software tool (REST) (Pfaffl, 2001; Pfafflet al., 2002). The mathematical algorithm, which needs no calibrationcurve, computes an expression ratio, based on real time PCR efficiencyand the crossing point deviation of the sample versus a control, as

illustrated in the following formula:

Ratio = Etarget

CT

target controlsample = Eref

CTref controlsample

where CT (cycle threshold) value corresponds to the number of cyclesat which the fluorescence emission monitored in real time exceeded

the threshold limit. Eis PCR efficiency determined by standard curveusing serial dilution of cDNA. The value of Eis calculated according toequation E= 10(1/slope). CT value of dilution series (1, 2, 4, 8etc) were used to calculate the slope for target and reference genes.

Each point of dilution was tested in 3 replicates. CT is the crossingpoint deviation of the sample versus a control. Comparison of severalreference genes (-actin, 16S rRNA and 12S rRNA) favoured 16S rRNA

because of its stable expression level in these test conditions.

2.6. Statistical analysis

Results for gene quantification are expressed as fold expressionrelative to 16S rRNA and represent the mean ratio of 3 biological

replicates (each replicate represents a sample pool of five fish) pertreatment. The expression level at the control (0 h) condition hasbeen designated value 1 and thereby the expression ratio oftreatments was expressed in relation to the control. Significant

differences in expression between control and treatments wereanalyzed by Relative Expression Software toolMultiple conditionsolver (RESTMCS) Version 2 using Pair Wise Fixed Reallocation

Randomisation Test. A probability level of 0.05 was used forrejection of the null hypothesis.

The data of weight gain % were subjected to T-test for meancomparisons of treatments.

3. Results

3.1. Growth

After 56 days of TRC exposure weight gain was lowered ( Pb0.05)

in the groups of fish which were subjected to a concentration of0.1 mg and 0.5 mg/L TRC (Fig. 1). No significant differences wereobserved between control (0 mg/L) and 0.01 mg/L exposed fish.

3.2. Expression pattern of studied genes

The expression levels of the 6 biomarker genes show differentexpression patterns in liver andgill in response to increasing exposure

concentrations (0.01, 0.1 and 0.5 mg/L) of TRC. The AChE geneshowed no significant change in the liver in response to TRC (Fig. 2A),while displayed a dose-dependent change in gills (Fig. 2B). As shownin Fig. 2B, exposure ofP. hypophthalmus juveniles to the highest dose

of TRC (0.5 mg/L) resulted in a dose- and time-dependent decrease inthe level of expression of the AChE in gills which was significantlydown-regulated at 96 h, 7 days, 14 days and 28 days of exposure.Expression level during these periods was 3.22, 4.55, 6.25 and 4.16

times less than their respective control. TRC at a level of 0.1 mg/Lreduced (Pb0.05) the level of AChE expression in gills at an exposure

period of 28 days. It is clear that the effect of 0.5 mg/L of TRC in gills

Table 1

PCR primer sequences, accession numbers, amplicon size, melting temperatures and calculated efficiency.

Gene Accession no. Sequence of primer (53 ) Amplicon size (bp) Me lting temperature (Tm) Calculated ef ficiency

Target gene

HSP 70 DQ885945 F: CATCAGTGATGGTGGACG 186 60.2 1.94

R: TGACGCTGAGAGTCGTTG 60.3

Growth hormone M63713 F: CCAGCCTGGATGAGAACG 162 60.6 1.79

R: GGGATCTCCTGCACTTGG 60.7

AChE FD283846 F: TGAGTGCGTGGGCTGTA 166 59.7 1.91

R: GCCAGTCGAGTCGATGA 59.7

COI EF609427 F: TGGAGCGCCTGATATGG 153 59.9 2.07

R: TGTGCGAGGTTTCCAGC 59.6

CYP1B DQ088663 F: ATCGGAGACATCTTCGGC 159 59.8 1.81R: TGGTTGGTCCTGGATGG 59.3

Trypsinogen AY316360 F: CACTGCTACCAGTCTCG 164 59.7 1.92

R: GCGGATTGACTCAGCTTG 59.5

Reference genes

-actin EU527191 F: TGTATCGCCTCTGGTCGT 176 59.8 1.88

R: AAGCTGTAGCCTCTCTCG 59.9

16S rRNA FJ432682 F: TATCTTCGGTTGGGGCG 223 60.0 1.96

R: CCTGATCCAACATCGAGG 60.0

12S rRNA EU934794 F: CGTCGTCAGCTTACCCT 211 60.0 1.89R: CGCGTCTCAGAGCCTAA 59.7

The accession number refers to the registered sequence used from Genbank. F: forward, R: reverse.

3A.K. Sinha et al. / Comparative Biochemistry and Physiology, Part D xxx (2010) xxxxxx

ARTICLE IN PRESS



4/10

was more prominent at 14 days of exposure whereas for 0.1 mg/L theclearest effect was noticed at 28 days of exposure. The effect in gillsseemed to recover after 28 days of TRC administration.

For HSP70, a significant increase in mRNA levels between controland fish exposed for 96 h, 7 days and 14 days to 0.5 mg/L TRC were

observed in liver and gills (Fig.3A,B). Thefirst significant effect in liverand gills was observed at 96 h of 0.5 mg/L exposure. The highest foldincrease in liver and gill was seen at 7 days of 0.5 mg/L TRC exposurewhich was 4.2 and 4.8 times higher (Pb0.01) than their respective

control. At the concentration of 0.1 mg/L, the increase (Pb0.05) in theHSP70 for liver and gill was seen after 14 days of exposure, which wasnumerically 2.8 and 3.07 times higher in liver and gills as compared tonon exposed animals. However, animals exposed longer than 28 days

did not show significant variations compared to controls.The exposure of P. hypophthalmus to TRC at 0.1 and 0.5 mg/L for

14 days indicated an inhibition (Pb0.05) in growth hormone geneexpression in liver samples which was 2.04 and 2.56 fold lower than

the control group (Fig. 4A). It was observed that the value for all

treatments slowly reduced till 14 days of exposure and then slowlyrecovered. Although the growth hormone gene expression seemed tochange in liver tissue, no significant reduction were verified in any ofthe exposure periods after subjected to 0.01 mg/L of TRC. Moreover, in

gills, apparently, no remarkable change was detected in any of thetreatments (Fig. 4B).

The trypsinogen gene expression in liver reduced as a result of TRCexposure (Fig. 5A). In particular, exposure at a level of 0.1 and 0.5 mg/L

TRC induced an apparent decrease in the trypsinogen mRNA at 96 h ofexposure till 28 days and afterwards there was a slight increase. Theexpression level dropped (Pb0.05) at 14 and 28 days exposure of0.5 mg/L TRC which was numerically 2.91 and 2.20 times lower than

their respective control. It was seen that exposure to a level of 0.1 mg/Lalso reduced the expression level (Pb0.05) in liver when exposed for aperiod of 28 days, with a reduction of 2.5 times than the unexposedgroup. However, in gills no remarkable change was observed on TRCexposure during any of the experimental periods (Fig. 5B).

The exposure offish with 0.5 mg/L of TRC amplified the COI geneexpression level significantly in liver at 14 and 28 days of exposurewhich was 2.35 and 2.67 times higher than the control (Fig. 6A).Moreover, TRC at an exposure concentration of 0.1 mg/L and for a

period of 28 days increased the expression level in liver by a factor of2.2 (Pb0.05). It was also noticed that during the longest exposureperiod (56 days) there was a gradual recovery in the COI geneexpression level of the 0.01, 0.1 and 0.5 mg/L exposed fish group. As

compared to liver, a profound impact of TRC (0.1 mg/L and 0.5 mg/L)on gills was noticedfrom7 days of exposure onwards (Fig. 6B). After 7and 14 days of exposure, 0.1 mg/L TRC augmented (Pb0.05) the

expression level by factor of 2.22 and 2.44 respectively, as comparedto control. Similarly, at the dose of 0.5 mg/L, a significant increase inmRNA transcript was noticed following 7 and 14 days of exposure,duringwhich it was 2.34and 3.22times higher thanthe control.It wasseen that COI mRNA quantity in gills for all treatment groups after

Fig. 1. Weight gain (%) of Pangasianodon hypophthalmus juveniles after 56 days under

different trichlorfon treatments. Data are means of 3 replicatesstandard deviation.Significant differences are indicated with * (Pb0.05; T-test).

Fig. 2. Relativeexpression ofAChEgenein (A)liverand(B) gill exposed todifferent concentration (0,0.01, 0.1and 0.5 mg/l) ofTRC duringdifferent exposure periods. Theexpressionlevel in the control (0 h) was regarded as 1.00. Results are expressed as fold expression relative to 16S rRNA, according to the equation ofPfaffl et al. (2002) and are mean ratio of

3 replicates(each replicate representspooled samples offive fish).Bars indicate standarderror. Significantdifferences between treatments are indicatedwith superscripts (*Pb0.05;

**Pb

0.01).


ARTICLE IN PRESS



5/10

reaching a peak at 14 days of exposure gradually lowered in 28 dayand 56 days of exposure period.

Theeffect of TRCexposure on CYP1B geneexpression infish liver was

insignificant (Fig. 7A).However, a notable effectwas observed in gillsathigher exposure levels (0.1 mg/L and 0.5 mg/L) (Fig. 7B). Theremarkable effect (Pb0.05) in gill samples was first observed after96 h of exposure for the highest exposure level (0.5 mg/L). At this pointthe gene expression was increased 2.2 times. The effect was also

noticeable at one week and two weeks of exposure for both 0.1 mg /Land 0.5 mg/L TRC. The highest peak of CYP1B gene expression for0.1 mg/L and 0.5 mg/L was foundafterone week of exposure whichwas3.55 times (Pb0.05) and 4.24 times (Pb0.01) higher thancontrol value.

4. Discussion

In this study we have focused on stress and toxicity representativegenes such as AChE, HSP70, growth hormone, COI, CYP1B and

trypsinogen to be evaluated as potential biomarker genes fortrichlorfon induced stress in P. hypophthalmus. The kinetics of thestudied genes expression revealed overall a dose- and time-dependent response during 56 days of TRC exposure with some

signs of recovery towards the end of exposure period. We found thatresponse of these genes expression differs between different organs(liver and gills). To our knowledge, this is one of the first studies thatassess the effects of TRC at the expression kinetics of stress responding

genes in Tra catfish.AChE is usually located in the membranes of vertebrates and non-

vertebrates. This enzyme controls ionic currents in excitable mem-branes and plays an essential role in nerve conduction processes at the

neuromuscular junction. The exposure of animal to OP insecticidesuch as TRC causes the inhibition of AChE as its main mode of action.

Thereby AChE is widely used as specific biomarker for assessing

exposure to TRC (Magni et al., 2006; Pfeifer et al., 2005; Sarkar et al.,2006). In the present study it was found that AChE gene expression ingills exposed to 96 h 12LC50 (0.5 mg/L) of TRC for 96 h, 7 days,

14 days and 28 days was significantly down-regulated. The maximumreduction was seen following 14 days of exposure. Moreover, for the0.1 mg/L, the transcript level of AChE in gills reduced significantlyonly after 28 days. This is because the chronic administration of TRC atlower dose for 4 weeks would likely increase the concentration of

dichlorvos in the fish and cause a delayed inactivation of the AChE(Guimaraes et al., 2007). The AChE inhibition observed in ourexperiment was probably associated with the presence of dichlorvos,the main metabolite of TRC(Garcia-Repetto et al., 1995). Dichlorvos is

a neurotoxic, and as such, it is logical to expect a reduction in the ChEactivity since this is its main mode of action. In addition, our resultcorroborates the finding of Var et al. (2003) in European sea bass

fingerlings (Dicentrarchus labrax). They reported a significant inhibi-tion in ChE activity after acute exposure to 0.125 mg/L of dichlorvos.

An early inhibition in brain AChE activity was also reported inEuropean eel ( Anguilla anguilla) in response to the OP pesticidefenitrothion (Sancho et al., 1997). Furthermore, Guimaraes et al.(2007) proposed that a treatment of 5 weekly applications of

0.25 ppm TRC in Nile tilapia (Oreochromis niloticus) is expected tocontinuously inhibit AChE. In the present study, the mRNA transcriptlevel of AChE gene for all treatments gradually increased on day 56, itdepicts that fish have the ability to overcome the stress of toxicants.

Similarly, Venkateswara et al. (2003) reported a 90% inhibition ofAChE activity inthe brain and gills ofOreochromis mossambicus in 24 hand a complete recovery within28 days after exposure to a single LC50and multiple exposures to sub-lethal concentrations (0.108 mg/L) of

profenofos pesticide.HSPs are a wide family of conserved proteins, present in all

organisms and classified according to their molecular weight (Basu

Fig. 3. Relative expression of HSP70 gene in (A) liver and (B) gill exposed to different concentration (0, 0.01, 0.1 and 0.5 mg/l) of TRC during different exposure periods. The

expression level in the control (0 h)was regarded as 1.00. Resultsare expressedas fold expression relative to 16S rRNA, according to the equation ofPfaffl et al.(2002) and aremean

ratio of 3 replicates (each replicate represents pooled samples offive fish). Bars indicate standard error. Significant differences between treatments are indicated with superscripts(*Pb0.05; **Pb0.01).


ARTICLE IN PRESS



6/10

Fig. 5. Relative expression of trypsinogen gene in (A) liver and (B) gill exposed to different concentration (0, 0.01, 0.1 and 0.5 mg/l) of TRC during different exposure periods. Theexpression level in thecontrol (0 h)was regarded as 1.00. Resultsare expressedas foldexpression relative to 16S rRNA, according to the equationofPfaffl et al.(2002) andare mean

ratio of 3 replicates (each replicate represents pooled samples offive fish). Bars indicate standard error. Significant differences between treatments are indicated with superscripts

(*Pb

0.05).

Fig.4. Relative expressionof growth hormonegene in (A) liverand (B) gillexposed to differentconcentration (0, 0.01,0.1 and 0.5 mg/l) of TRCduring differentexposureperiods.The

expression level in thecontrol (0 h)was regarded as 1.00. Resultsare expressedas foldexpression relative to 16S rRNA, according to the equationofPfaffl et al.(2002) andare mean


(*Pb0.05).


ARTICLE IN PRESS



7/10

Fig. 6. Relative expression of COI gene in (A) liver and (B) gill exposed to different concentration (0, 0.01, 0.1 and 0.5 mg/l) of TRC during different exposure periods. The expression

level in the control (0 h) was regarded as 1.00. Results are expressed as fold expression relative to 16S rRNA, according to the equation ofPfaffl et al. (2002) and are mean ratio of

3 replicates(each replicaterepresentspooled samples offivefish). Barsindicatestandarderror. Significantdifferences between treatments are indicated withsuperscripts (*Pb0.05;

**Pb0.01).

Fig. 7. Relative expression of CYP1B gene in (A) liver and (B) gill exposed to different concentration (0, 0.01, 0.1 and 0.5 mg/l) of TRC during different exposure periods. Theexpression level in the control (0 h)was regarded as 1.00. Resultsare expressedas fold expression relative to 16S rRNA, according to the equation ofPfaffl et al.(2002) and aremean


(*Pb

0.05; **Pb

0.01).


ARTICLE IN PRESS



8/10

et al., 2002). The current results show that HSP70 mRNA levelsincreased significantly both in liver and gills at 96 h exposure of0.5 mg/L TRC whereas for 0.1 mg/L the remarkable effect was notedafter 14 days. This suggests that the lower concentration (0.1 mg/L)

demands a longer incubation time than the highest concentration(0.5 mg/L) to reach the threshold required to induce HSP70expression and is consistent with the effect observed for AChE.

Moreover, the elevated response of HSP70 found in the present study

suggests that TRC induces proteotoxicity in P. hypophthalmus. Fishcope with proteotoxicity by the induction of HSPs, which are able torepair partly denatured proteins (Hallare et al., 2004). Stressors like

toxicants may induce HSP70, andthis inductionhas already been usedas a general biomarker of toxicant stress (Hassanein et al., 1999; Varet al., 2002; De Boeck et al., 2003). It should be noted that while manyindicators of fish stress are altered by handling and samplingprocedures, Vijayan et al. (1997) demonstrated that handling stress

does not alter levels of hepatic HSP70 in rainbow trout (Oncorhynchusmykiss). Therefore, from the results presented in our work, it can beacknowledged that HSP70 is a promising biomarker gene for stressinduced by TRC exposure in Tra catfish.

In fish, the class of cytochrome P450 isozymes are responsible forthe transformation of a variety of environmental contaminantsincluding polyaromatic hydrocarbons (PAHs), planar polychlorinatedbiphenyls (PCBs) and arylamines (Leaver and George, 2000; van derOost et al., 2003). The existence of CYP1B genes have already been

reported in fish (Godard et al., 2000; Leaver and George, 2000; Willettet al., 2006). In fact, metabolism and carcinogenesis studies haveshown CYP1B to be a critical and necessary enzyme involved in theoxidation of chemical toxicants (Shimada et al., 1996; Buters et al.,

1999; Godard et al., 2000). In an experiment with channel catfish(Ictalurus punctatus), Willett et al. (2006) observed significantinduction in CYP1B gene expression after 4 days of benzopyrene(20 mg/kg) exposure. However, till date, not a single study has been

conducted to elucidate the effect of organophosphate on CYP1Bactivity. In our case, we observed a significant expression of CYP1BmRNA in the gill tissue at an exposure of 0.1 and 0.5 mg/L TRC whileno such remarkable effect was detected in liver. This is in accordance

with northern blot analysis of plaice (Pleuronectes platessa) andcommon carp (Cyprinus carpio) where CYP1Bs have been shown tohave higher abundance in gills as compared to other tissues (Leaver

and George, 2000; El-kady et al., 2004a,b). Thereby, the mRNAexpression profile of this gene in the gill could potentially be a usefulbiomarker in P. hypophthalmus for exposure to OP pesticide.

Furthermore, TRC also induces oxidative stress in fish by

generating reactive oxygen species (ROS) such as superoxide anionradicals (O2

), hydrogen peroxide (H2O2) andhighly reactive hydroxylradical (OH) (Hai et al., 1997; Yarsan et al., 1999; Pena-Llopis et al.,2003; Mohammad et al., 2004; Monteiro et al., 2006; Var et al., 2007;

Thomaz et al., 2009). These radicals can react with susceptiblebiological macromolecules and produce lipid peroxidation, DNAdamage and protein oxidation.

The main function of COI is to transfer electrons from cytochrome cto oxygen in mitochondrial based electron transport chain and togenerate ATP. Besides,COI may alsofunctionindirectlyas an antioxidantby either preventing thedawdlingof electronflow (Bolter and Chefurka,1990; Benzi et al., 1992) or by uncoupling electron transport from

proton transfer (Richter, 1997). In the present study, COI geneexpression was up-regulated at higher doses (0.1 and 0.5 mg/L) ofTRC. Elevated expression of COI has also been associated with apyrethroid insecticide resistant strain of Blatella germanica (German

cockroach) (Pridgeon and Liu, 2003). Danio rerio (zebrafish) fed withdiets contaminated with methyl mercury resulted in increasedexpression of COI gene (Gonzalez et al., 2005). Similarly, Achard-Joriset al. (2006) reported that COI gene expression was up-regulated by

exposure to an oxidative stressor such as cadmium in two freshwater

bivalves (Corbiculafluminea and Dreissena polymorpha) and onemarine

bivalve (Crassostrea gigas). To our knowledge, the direct relationshipbetween COI expression and TRC contamination has never beenreported before. Since COI is considered as the rate-limiting step formitochondrial respiration (Villani and Attardi, 2000), elevated expres-

sion COI gene could be a compensating mechanism to restore thedecrease in mitochondrial activity and to efficiently consume oxygen(Achard-Joris et al., 2006), thus limiting dichlorvos induced damage inthe cell. The onset of up-regulation of COI expression was found to be

early (7 days) and more profound in gills than liver, because gills are theorgan at the interface with the contaminated environment and are thusthe main tissues from which the toxicants loading in the organism willproceed (Marigomez et al., 2002). Our findings suggests thatin additionto AChE, HSP 70 and CYP1B genes, COI gene expression level might

constitute a key biomarker gene for detecting TRC induced stressresponses in Tra catfish. Moreover, an advantage of using COI to studythe impact of TRC on mitochondrial metabolism is that COI genesequence is highly conserved between lower and higher eukaryotes

(Capaldi, 1990). Thus a similar COI gene regulation between differentspecies of catfish can be expected.

Results from the weight gain demonstrated that prolongedexposure to TRC at a level of 0.1 or 0.5 mg/L in rearing water

hampered the growth performance of P. hypophthalmus juveniles. Asignificant reduction in growth was commensurate with an increasein the TRC concentration. It indicates that the applied pesticide at ahigher dose (0.1 and 0.5 mg/L) may hinder the growth hormone

regulating gene. To verify this, we checked the transcript level ofgrowthhormone and our results confirm that TRC at higher doses(0.1and 0.5 mg/L) significantly down-regulates the gene expression level

of growth hormone. Therefore, a positive correlation was foundbetween genotypic and phenotypic expression for growth hormoneon TRC exposure. Consequently, the mRNA expression of growthhormone in the present study can be speculated as a specificbiomarker to detect fish performance under TRC treatment; however

more in-depth research on the interaction of growth hormone ongrowth in P. hypophthalmus is needed. The negative effect of TRC onthe growth performance of juveniles in the presentstudy is consistentwith the result obtained by Guimaraes and Calil (2008). They reported

that five weekly doses of 0.25 ppm of TRC reduced the growth of O.niloticus by 54.7% under laboratory conditions. Similar adverse effectof OP pesticide in fish has also been reported (Pal and Konar, 1985;

Silva et al., 1993; Sturm et al., 1999). However, these results were incontrast with the results obtained by Ludwig (1993) who documen-ted that the use of the pesticides TRC did not cause any significantdifference in growth in hybrids of Morone saxatilis in culture

environment. Similarly, Ruddle and Zhong (1988) suggested that noadverse effects existed in the use of TRC in aquaculture. Thediscrepancy betweenthese results may be associated with differencesin the rearing conditions because the mode of action of organophos-

phate pesticides depends on pH and temperature, being more activein temperatures above 16 C (Messenger and Esnault, 1992; Howeet al., 1994).When pH is above 8.0, hydrolysis of TRC is extremely fast,

increasing then the toxicity, and requiring, therefore, lower concen-trations for the treatments (Messenger and Esnault, 1992).

Moreover, the down-regulation of trypsinogen gene expressionunder the exposure level of 0.1 and 0.5 mg/L TRC supports the findingof growth performance. However, it is not clear from our study that if

it was direct or indirect overriding effect due to other genes orhormones. Like other vertebrates, trypsinogen (zymogen) is theinactive precursor for the production of proteolytic enzyme, trypsin.Tryptic enzyme activity has been demonstrated as a useful indicator

for the evaluation of digestive capacity and can be used to measurefish juvenile condition in response to changing environmentalconditions (Nolting et al., 1999). Moreover, the direct correlation ofdigestive enzyme activities with growth have been examined in sea

bream (Sparus aurata) (Sarasquete et al., 1993), walleye Pollock

(Theragra chalcogramma) (Oozeki and Bailey, 1995) and Senegal sole


ARTICLE IN PRESS



9/10

(Solea senegalensis) (Ribeiro et al., 1999). Thereby, it can be speculatedthat trypsinogen regulating gene could be a promising biomarker toaccess the stress induced changes in fish growth.

5. Conclusion

We have determined the expression kinetics of mRNAs coding for

stress and growth-related genes in P. hypophthalmus, one of the main

candidate species for aquaculture in Southeast Asian countries. Ourstudy clearly demonstrates that the application of high dose (0.1 and0.5 mg/L) of organophosphate pesticide, trichlorfon in culture water

of P. hypophthalmus significantly influences the expression levels ofstudied genes (AChE, HSP70, growth hormone, trypsinogen, CYP1B,COI). It was seenthat each gene wasdifferentially expressed in a dose-and time- dependent way to TRC exposure. In short, it can be said thatstress-inducible proteins appear to be excellent candidates for

molecular biomarkers. Nevertheless, the importance of these studiedgenes in terms of both functionality and isoform variation might becrucial in further examinations.

Acknowledgments

The study is a part of a joint BelgianVietnamese collaborativeproject entitled Analytical and biological methods in support ofsustainable aquaculture practices in Vietnam (contracts BELSPO BL/

13/V17). We are grateful to the Belgian Science Policy Office (MrsDesmeth M. and Decadt B.) and to the Vietnamese Ministry of Scienceand Technology.

References

Achard-Joris, M., Gonzalez, P., Marie, V., Baudrimont, M., Bourdineaud, J.P., 2006.Cytochrome c oxydase subunit I gene is up-regulated by cadmium in freshwaterand marine bivalves. Biometals 19, 237244.

Basu, N., Todgham, A.E., Ackerman, P.A., Bibeau, M.R., Nakano, K., Schulte, P.M., Iwama,G.K., 2002. Heat Shock Protein genes and their functional significance in fish. Gene295, 173183.

Benz, G.W., Otting, R.L., Case, A., 1995. Redescription of Argulus melanosticus

(Branchiura : Argulidae), a parasite of California grunion (Leuresthes tenuis:Atherinidae), with notes regarding chemical control ofA. melanosticus in a captivehost population. J. Parasitol. 81, 754761.

Benzi, G., Pastoris, O., Marzatico, R.F., Villa, R.F., Curti, D., 1992. The mitochondrialelectron transfer alteration as a factor involved in the aging brain. Neurobiol. Aging13, 361368.

Bocquen, G., Galgani, F., Truquet, P., 1990. Characterization and assay conditions foruse of AChE activity from several marine species in pollution monitoring. Mar.Environ. Res. 30, 7589.

Bocquen, G., Bellanger, C., Cadiou, Y., Galgani, F., 1995. Joint action of combinations ofpollutantson the acetylcholinesterase activity of several marine species. Ecotoxicology4, 226279.

Bolter, C.J., Chefurka, W., 1990. Extramitochondrial release of hydrogen peroxide frominsect and mouse liver mitochondria using the respiratory inhibitors phosphine,myxothiazol, and antimycin and spectral analysis of inhibited cytochromes. Arch.Biochem. Biophys. 278, 6572.

Boone, J.S., Chambers, J.E., 1997. Biochemical factors contributing to toxicity differencesamong chlorpyrifos, parathion, and methyl parathion in mosquitofish (Gambusiaaffinis). Aquat. Toxicol. 39, 333343.

Buters, J.T.M., Sakai, S., Richter, T., Pineau, T., Alexander, D.L., Savas, U., Doehmer, J.,Ward, J.M.,Jefcoate, C.R., Gonzalez,F.J., 1999. Cytochrome P450 CYP1B1 determinessusceptibility to 7, 12-dimethylbenz[]anthracene-induced lymphomas.Proc. Natl.Acad. Sci. USA 96, 19771982.

Capaldi, R.A., 1990. Structure and assembly of Cytochrome c oxidase. Arch. Biochem.Biophys. 280, 252262.

Carajaville, M.P., Bebianno, M.J., Blasco, J., Porte, C., Sarasquete, C., Viarengo, A., 2000.The use of biomarkers to assess the impact of pollution in coastal environment ofthe Iberian Peninsula: a practical approach. Sci. Total Environ. 247, 295311.

Chang, C., Lee, P., Liu, C.H., Cheng, W., 2006. Trichlorfon, an organophosphorusinsecticide, depresses the immuneresponsesand resistance toLactococcus garvieaeof the giant freshwater prawn Macrobrachium rosenbergii. Fish Shellfish Immunol.20, 574585.

Chevre, N., Gagne, F., Gagnon, P., Blaise, C., 2003. Application of rough sites analysis toidentify polluted aquatic sites based on a battery of biomarkers: a comparison withclassical methods. Chemosphere 51, 1323.

De Boeck, G., De Wachter, B., Vlaeminck, A., Blust, R., 2003. Effect of cortisol treatmentand/or sublethal copper exposure on copper uptake and heat shock protein levels

in common carp, Cyprinus carpio. Environ. Toxicol. Chem. 22, 11221126.

de la Torre, F.R., Ferrari, L., Salibin, A., 2005. Biomarkers of a native fish species(Cnesterodon decemmaculatus) application to the water toxicity assessment of aperi-urban polluted river of Argentina. Chemosphere 59, 577583.

El-Kady, M.A.H., Mitsuo, R., Kaminishi, Y., Itakura, T., 2004a. cDNA cloning, sequencesanalysis and expression of 3-methylcholanthrene- inducible cytochrome P450 1B1in carp (Cyprinus carpio). Environ. Sci. 11, 231240.

El-Kady, M.A.H., Mitsuo, R., Kaminishi, Y., Itakura, T., 2004b. Isolation of cDNA of novelcytochrome P450 1B2 gene from carp (Cyprinus carpio) and induced expression ingills. Environ. Sci. 11, 345354.

Eto, M., 1974. Organophosphorous Pesticides: Organic and Biological Chemistry. CRCPress, Boca Raton, FL.

Galgani, F., Bocquen, G., 1990. In vitro inhibition of acetylcholinesterase from fourmarine species by organophosphates and carbamates. Bull. Environ. Contam.Toxicol. 45, 243249.

Garcia-Repetto, R., Martinez, D., Repetto, M., 1995. Malathion and dichlorvostoxicokinetics after the oral administration of malathion and trichlorfon. Vet.Hum. Toxicol. 37, 306309.

Godard, C.A.J., Leaver, M.J., Said, M.R., Dickerson, R.L., George, S., Stegeman, J.J., 2000.Identification of cytochrome P450 1B-like sequences in two teleost fish species(scup, Stenotomus chrysops and plaice, Pleuronectes platessa) and in a Cetacean(striped dolphin, Stenella coeruleoalba). Mar. Environ. Res. 50, 710.

Gonzalez, P., Dominique, Y., Massabuau, J.-C., Boudou, A., Bourdineaud, J.-P., 2005.Comparative effects of dietary methylmercury on gene expression in liver, skeletalmuscle,and brain of thezebrafish (Daniorerio). Environ. Sci.Technol.39, 39723980.

Grave, K., Engelstad, M., Soli, N.E., 1991. Utilization of dichlorvos and trichlorfon insalmonid farming in Norway during 19811988. Acta Vet. Scand. 32, 17.

Guimaraes, A.T.B., Calil, P., 2008. Growth evaluation ofOreochromis niloticus (Cichlidae,Neopterygii) exposed to trichlorfon. Braz. Arch. Biol. Technol. 51, 323332.

Guimaraes, A.T.B., Silva de Assis, H.C., Boeger, W., 2007. The effect of trichlorfon onacetylcholinesterase activity and histopathology of cultivated fish Oreochromisniloticus. Ecotoxicol. Environ. Saf. 68, 5762.

Hai, D.Q., Varga, S.I., Matkovics, B., 1997. Organophosphate effects on antioxidantsystem of carp (Cyprinus carpio) and catfish (Ictalurus nebulosus). Comp. Biochem.Physiol. C 117, 8388.

Hallare, A.V., Kohler, H.R., Triebskorn, R., 2004. Development toxicity and stress proteinresponses in zebrafish embryos after exposure to diclofenac and its solvents,DMSO. Chemosphere 56, 659666.

Hartl, F.U., 1996.Molecular chaperones in cellular protein folding.Nature 381,571580.Hassanein, H.M.A., Banhawy, M.A., Soliman, F.M., Abdel-Rehim, S.A., Muller, W.E.G.,

Schroder, H.C., 1999. Induction of Hsp70 by the herbicide oxyfluorfen (Goal) in theEgyptianNile Fish, Oreochromis niloticus. Arch.Environ.Contam.Toxicol. 37,7884.

Herwig, N.,1979. Handbookof Drugsand ChemicalsUsed inTreatment of Fish Diseases,A Manual of Fish Pharmacology and Material Medica. Thomas Publishers,Springfield, IL, USA.

Hofer, W., 1981. Chemistry of metrifonate and dichlorvos. ActaPharmacol. Toxicol. 49, 714.Howe, G.E., Marking, L.L., Bills, T.D., Rach, J.J., Mayer, F.L., 1994. Effects of water

temperature and pH on toxicity of terbufos, Trichlorfon, 4- nitrophenol and 2,4-dinitrophenol to the amphipod Gammarus pseudolimnaeus and rainbow trout

(Oncorhynchus mykiss). Environ. Toxicol. Chem. 13, 5166.Leaver, M.J., George, S.G., 2000. A cytochrome P4501B gene from a fish, Pleuronectes

platessa. Gene 256, 8391.Lee, S.M., Lee, S.B., Park, C.H., Choi, J., 2006. Expression of heat shock protein and

hemoglobin genes in Chironomus tentans (Diptera, chironomidae) larvae exposedto various environmental pollutants: a potential biomarker of freshwatermonitoring. Chemosphere 65, 10741081.

Ludwig, G.M., 1993. Effects of Trichlorfon, fenthion, and diflubenzuron on thezooplankton community and on production of reciprocal-cross hybrid stripedbass fry in culture ponds. Aquaculture 110, 301319.

MacKinnon, B.M., 1997. Sea lice: a review. Aquaculture 28, 510.Magni, P., De Falco, G., Falugi, C., Franzoni, M., Monteverde, M., Perrone, E., Sgro, M.,

Bolognesi, C., 2006. Genotoxicity biomarkers and acetylcholinesterase activity innatural populations of Mytilus galloprovincialis along a pollution gradient in theGulf of Oristano (Sardinia, western Mediterranean). Environ. Pollut. 142, 6572.

Marigomez, I., Soto, M., Cajaraville, M.P., Angulo, E., Giamberini, L., 2002. Cellular andsubcellular distribution of metals in molluscs. Microsc. Res. Tech. 56, 358392.

Messenger,J.L.,Esnault,F., 1992. Traitementpar le dichlorvosdes copepodosesde latruitearc-en-ciel eleve en mer: modalities de traitement adaptes aux conditions environmen-

tales franaises. In: Michel, C., Alderman, D.J. (Eds.), Chemotherapy in Aquaculture:From Theory to Reality. Office International des Epizootics, Paris, pp. 195205.

Mohammad, A., Ranjbar, A., Shahin, S., Nikfar, S., Rezaie, A., 2004. Pesticides andoxidative stress: a review. Med. Sci. Monit. 10, 141147.

Monteiro,D.A.,Almeida, J.A., Rantin,F.T.,Kalinin,A.L.,2006. Oxidativestressbiomarkers inthe freshwater characid fish, Brycon cephalus, exposed to organophosphorusinsecticide Folisuper600(methylparathion). Comp.Biochem. Physiol. C 143, 141149.

Nolting, M., Ueberscha, B., Rosenthal, H., 1999. Trypsin activity and physiologicalaspects in larval rearing of European sea bass (Dicentrarchus labrax) using live preyand compound diets. J. Appl. Ichthyol. 15, 138142.

Oozeki, Y., Bailey, K.M., 1995. Ontogenetic development of digestive enzyme activitiesin larval walleye pollock, Theragra chalcogramma. Mar. Biol. 122, 177186.

Pal, A.K., Konar, S.K., 1985. Chronic effects of the organophosphorus insecticide DDVPon feeding, survival, growth and reproduction offish. Environ. Ecol. 3, 398402.

Pena-Llopis, S., Ferrando, M.D., Pena, J.B., 2003. Fish tolerance to organophosphate-induced oxidative stress is dependent on the glutathione metabolism andenhanced by N-acetylcysteine. Aquat. Toxicol. 65, 337360.

Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time

RTPCR. Nucleic Acids Res. 29, 20022007.


ARTICLE IN PRESS



10/10

Pfaffl, M.W., Horgan, G.W., Dempfle, L., 2002. Relative expressionsoftware tool(REST)forgroup-wise comparison and statistical analysis of the relative expression resultsin real-time PCR. Nucleic Acids Res. 30, 110.

Pfeifer, S., Schiedek, D.S., Dippner, J.W., 2005. Effect of temperature and salinity onacetylcholinesterase activity, a common pollution biomarker, in Mytilus sp. fromthe south-western Baltic Sea. J. Exp. Mar. Biol. Ecol. 320, 93103.

Pridgeon,J.W., Liu, N., 2003. Overexpressionof the Cytochrome c oxidase subunit I geneassociated with a pyrethroid resistant strain of German cockroaches, Blattella

germanica (L.). Insect Biochem. Mol. Biol. 33, 10431048.Ribeiro, L., Zambonino-Infante, J.L., Cahu, C., Dinis, M.T.,1999. Development of digestive

enzymes in larvae ofSolea senegalensis, Kaup 1858. Aquaculture 179, 465473.

Richter, C., 1997. Reactive oxygen species and nitrogen species regulate mitochondrialCa2+ homeostasis and respiration. Biosci. Rep. 17, 5366.Rodrigues,E.L., Ranzani-Paiva,M.J.T., Pacheco,F.J., Veiga, M.L.,2001. Histopathologic lesions

in the liver ofProchilodus lineatus (Pisces, Prochilodontidae) exposed to a sublethalconcentrationof the organophosphate insecticideDipterex500s(Trichlorfon).Acta Sci.23, 503505.

Ruddle, K., Zhong, G., 1988. Integrated AgricultureAquaculture in South China: TheDikePond System of the Zhujiang Delta. Cambridge University Press, Cambridge.

Sancho, E., Ferrando, M.D., Andreu-Moliner, E., 1997. Response and recovery of brainacetylcholinesterase activity in the European eel, Anguilla anguilla, exposed tofenitrothion. Ecotoxicol. Environ. Saf. 38, 205209.

Sarasquete, M.C., Polo, A., Gonzalez de Canales, M.L., 1993. A histochemical andimmunohistochemical study of digestive enzymes and hormones during the larvaldevelopment of the sea bream, Sparus aurata L. Histochem. J. 25, 430437.

Sarkar, A., Ray, D., Shrivastava, A.N., Sarker, S., 2006. Molecular biomarkers: theirsignificance and application in marine pollution monitoring. Ecotoxicology 15,333340.

Shimada, T., Hayes, C.L., Yamazaki, H., Amin, S., Hecht, S.S., Guengerich, F.P., 1996.Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1.

Cancer Res. 56, 29792984.Sievers, G., Palacios, P., Inostroza, R., D'olz, H., 1995. Evaluation of the toxicity of

8 insecticides in Salmo salar and the in vitro effects against the isopode parasite,Ceratothoa gaudichaudii. Aquaculture 134, 916.

Silva, H.C., Medina, H.S.G., Fanta, E., Bacila, M., 1993. Sub-lethal effects of theorganophosphate Folidol 600 (Methyl Parathion) on Callichthys callichthys (Pisces,Teleostei). Comp. Biochem. Physiol. C 105, 197201.

Smolder, R., Bervoets, L., Wepener, V., Blust, R., 2003. A conceptual framework for usingmusselsas biomonitors in whole effluenttoxicity.Hum. Ecol. RiskAssess.9, 741760.

Stegeman, J.J., Brouwer, M., Di Giulio, R.T., Forlin, L., Fowler, B.A., Sanders, B.M., VanVeld, P.A., 1992. Molecular responses to environmental contamination: enzymeandproteinsystems as indicators of chemicalexposureand effect. In: Di Giulio, R.T.,Forlin, L., Fowler, B.A., Sanders, B.M., Van Veld, P.A., Huggett, R.J., Kimerle, R.A.,Merhle, P.M., Bergman, H.L. (Eds.), Biomarkers: Biochemical, Physiological and

Histological Markers of Anthropogenic Stress. A Special Publication of SETAC. LewisPublishers, Chelsea, MI, pp. 235335.

Sturm, A., Silva de Assis, H.C., Hansen, P.D., 1999. Cholinesterases of marine teleostfish:enzymological characterization and potential use in the monitoring of neurotoxiccontamination. Mar. Environ. Res. 47, 389398.

Sturm, A., Wogram, J., Segner, H., Liess, M., 2000. Different sensitivity to organopho-sphates of acetylcholinesterase and butyrylcholinesterase from three-spinedstickleback (Gasterosteus aculeatus): application in biomonitoring. Environ. Toxicol.Chem. 19, 16071617.

Thomaz, J.M., Martins, N.D., Monteiro, D.A., Rantin, F.T., Kalinin, A.L., 2009. Cardio-respiratory function and oxidative stress biomarkers in Nile tilapia exposed to the

organophosphate insecticide trichlorfon (NEGUVON). Ecotoxicol. Environ. Saf.72, 14131424.Tojo, J.L., Santamarina, M.T., 1998. Oral pharmacological treatments for parasitic

diseases of rainbow trout Oncorhynchus mykiss. III Ichthyobodo necator. Dis. Aquat.Org. 33, 195199.

Tronczynski, J., 1990. Programme de recherche sur les produits phytosanitaires enzones littorals et estuariennes. Institut Francais de Recherche pour I'Exploitation dela Mer, Brest (IFREMER DR0e90e05-MR).

vander Oost,R., Beyer, J.,Vermeulen,N.P.E., 2003. Fishbioaccumulation andbiomarkers inenvironmental risk assessment: a review. Environ. Toxicol. Pharmacol. 13, 57149.

Var, I., Serrano, R., Pitarch, E., Amat, F., Lpez, F.J., Navarro, J.C., 2002. Bioaccumulationof chlorpyrifos through an experimental food Chain: study of protein HSP70 asbiomarker of sublethal stress in fish. Arch. Environ. Contam. Toxicol. 42, 229235.

Var, I., Navarro, J.C., Amat, F., Guilhermino, L., 2003. Effect of dichlorvos oncholinesterase activity of the European sea bass (Dicentrarchus labrax). PesticideBiochem. Physiol. 75, 6172.

Var, I., Navarro, J.C., Nunes, B., Guilhermino, L., 2007. Effects of dichlorvos aquaculturetreatments on selected biomarkers of gilthead sea bream (Sparus aurata L.)fingerlings. Aquaculture 266, 8796.

Venkateswara, R.L., Shilpanjali, D., Kavitha, P., Madhavendra, S.S., 2003. Toxic effects ofprofenofos on tissue acetylcholinesterase and gill morphology in a euryhalinefish,Oreochromis mossambicus. Arch. Toxicol. 77, 227232.

Vijayan, M.M., Pereira, C., Forsyth,R.B., Kennedy, C.J.,Iwama, G.K.,1997. Handling stressdoes not affect the expression of hepatic heat shock protein 70 and conjugationenzymes in rainbow trout treated with -naphthoflavone. Life Sci. 61, 117127.

Villani, G., Attardi, G., 2000. In vivo control of respiration by cytochrome c oxidase inhuman cells. Free Radic. Biol. Med. 29, 202210.

Willett, K.L., Ganesan, S., Patel, M., Metzger, C., Quiniou, S., Waldbieser, G., Scheffler, B.,2006. In vivo and in vitro CYP1B mRNA expression in channel catfish. Mar. Environ.Res. 62, S332S336.

Yarsan, E., Tanyuksel, M., Celik, S., Aydin, A., 1999. Effects of aldicarb and malathion onlipid peroxidation. Bull. Environ. Contam. Toxicol. 63, 575581.


ARTICLE IN PRESS

Please cite this article as: Sinha, A.K., et al., Expression characteristics of potential biomarker genes in Tra catfish, Pangasianodonhypophthalmus exposed to trichlorfon Comp Biochem Physiol D (2010) doi:10 1016/j cbd 2010 05 001

expression characteristics of potential biomarker genes in tra catfish, pangasianodon hypophthalmus,...

Documents