ARTICLE IN PRESS

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Comparative Biochemistry and Physiology, Part D xxx (2010) xxx–xxx

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Comparative Biochemistry and Physiology, Part D

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c b p d

Expression characteristics of potential biomarker genes in Tra catfish, Pangasianodon

hypophthalmus, exposed to trichlorfon
Amit Kumar Sinha a,⁎, Caroline Vanparys a, Gudrun De Boeck a, Patrick Kestemont b, Neil Wang b,
Nguyen Thanh Phuong c, Marie-Louise Scippo d, Wim De Coen a, Johan Robbens a,e
a
Laboratory for Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium
b
Unit of Research in Organismic Biology (URBO), University of Namur, 61, rue de Bruxelles, B-5000 Namur, Belgium
c
College of Aquaculture and Fisheries, Can Tho University, 3/2 str. Campus 2, Can Tho City, Vietnam
d
Département de sciences des denrées alimentaires, Analyse des denrées alimentaires, Université de Liège, Boulevard de Colonster, 20, B43b Sart-Tilman, B-4000 Liège, Belgium
e
ILVO Fisheries, Ankerstraat 1, B-8400 Oostende, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: Trichlorfon (TRC) is the most common organophosphorous insecticide used in aquaculture practices in
Received 11 February 2010 Southeast Asian countries. Indiscriminate use of TRC can either damage or alter the enzymatic and hormonal
Received in revised form 2 May 2010 activities in the living organisms. In this present study, therefore, toxicogenomic analyses using real time PCR
Accepted 3 May 2010
was used to characterize expression levels of various genes in Pangasianodon hypophthalmus after exposure
Available online xxxx
to three concentrations, the 96 h 1/100LC50 (0.01 mg/L), the 96 h 1⁄10LC50 (0.1 mg/L) and the 96 h 1⁄2LC50
Keywords:
(0.5 mg/L) of TRC for 6 h, 24 h, 96 h, 7 days, 14 days, 28 days and 56 days respectively. The expression
Acetylcholinesterase kinetics of stress and other cellular toxicity representative genes such as heat shock protein70 (HSP70),
Aquaculture growth hormone, acetylcholinesterase (AChE), trypsinogen, cytochrome P4501B (CYP1B) and cytochrome
Biomarkers oxidase subunit 1 (COI) were investigated in liver and gills. TRC at a level of 0.1 mg/L and 0.5 mg/L induced a
Pangasianodon hypophthalmus time and dose-dependent increase in the expression of the HSP70, COI and CYPIB while the transcript level of
Cytochrome oxidase subunit 1 AChE, growth hormone and trypsinogen were significantly down-regulated. These results could permit to
Gene expression develop a “molecular biomarker system” which can be applied as a first-tier method of identifying
Heat shock protein 70
contaminant exposure before effects at population level occur.
Real time PCR
© 2010 Elsevier Inc. All rights reserved.
Trichlorfon

1. Introduction extensive and intensive aquaculture in Asia constitutes a potential

In intensive farming systems, fish are reared at high density and
the chances for infestations caused by parasites are extremely high
(Grave et al., 1991). Consequently, the application of chemother-
apeutics against parasitic infection is of prime concern for aqua-
culturists to reduce economic losses. Organophosphate (OP)
compounds are the most widely used insecticidal class for agriculture
and aquaculture to control pest (Rodrigues et al., 2001). Trichlorfon
(2,2,2-trichloro-1-hydroxyethyl phosphonate), an organophosphate
compound, is one of the most extensively used products in Southeast
Asian countries for controlling fish parasites. The recommended dose
of trichlorfon (TRC) to eradicate fish parasites varies from 0.1 to
1.0 mg/L (Herwig, 1979; Chang et al., 2006), five applications of
0.25 mg/L weekly indicated as the most effective treatment against
fish parasitic infestation (Benz et al., 1995; MacKinnon, 1997; Tojo
and Santamarina, 1998). However, farmers often apply excessive dose
of TRC in aquaculture system. The repeated use of this insecticide in
⁎ Corresponding author. Tel.: + 32 32 653779; fax: + 32 32 653 497.
E-mail address: sinha_cife@rediffmail.com (A.K. Sinha).
threat to non-targeted species such as fish, crabs, and shrimp
(Tronczynski, 1990). TRC elicits neurobehavioural alterations by
inhibiting acetylcholinesterase (AChE) activity, however, TRC itself
is not a potent AChE inhibiting agent. The toxicity of this chemical is
attributed to its main metabolite dichlorvos (2,2-dichlorovinyl
dimethyl phosphate). In aqueous solution, TRC is hydrolysed very
quickly into dichlorvos which is much more toxic and is a potent AChE
inhibitor (Eto, 1974; Hofer, 1981). AChE is a member of the family of
enzymes known as cholinesterases (ChE), and is responsible for the
degradation of the neurotransmitter acetylcholine in cholinergic
synapses of both targeted and non-targeted animals. The inhibition
of the enzyme causes an accumulation of acetylcholine in the synapse
which blocks the cholinergic activity of the central ganglion (MacK-
innon, 1997). Furthermore, the continuous stimulation of the post-
synaptic membrane may lead to death. Depending upon the severity
and duration, TRC exposure can become a source of stress for fish and
its toxicity can be moderate to high and may produce both sub-lethal
and lethal effects (Galgani and Bocquené, 1990; Silva et al., 1993;
Bocquené et al., 1995; Sievers et al., 1995; Hai et al., 1997; Sturm et al.,
1999; Varó et al., 2003). Moreover, it has already been illustrated that
environmental toxicants may induce the expression of certain genes

1744-117X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbd.2010.05.001

Please cite this article as: Sinha, A.K., et al., Expression characteristics of potential biomarker genes in Tra catfish, Pangasianodon
hypophthalmus, exposed to trichlorfon, Comp. Biochem. Physiol. D (2010), doi:10.1016/j.cbd.2010.05.001
 ARTICLE IN PRESS
2 A.K. Sinha et al. / Comparative Biochemistry and Physiology, Part D xxx (2010) xxx–xxx
associated with short term and/or long term toxicological responses
which have profound effect on overall performance of fish (Lee et al.,
2006).
Thereby, evaluation of gene expression alterations is advisable to
monitor the potential adverse effect of TRC on fish health and allow an
early indication of the ecotoxicological impact. In this context, the
expression 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 in fish (Carajaville
et al., 2000; Chevre et al., 2003; de la Torre et al., 2005). Although
researchers have looked into the toxicants-induced gene expression
alterations in fish, the use of “biomarker genes” to evaluate general
stress in Pangasianodon hypophthalmus, an important freshwater
culture species in Southeast Asian countries, on TRC exposure is rather
scarce.
It is well understood that no single biomarker gene has emerged as a
widely used indicator for toxicity without some limitation (Smolder et
al., 2003), therefore, the objective of the present study was to evaluate,
in conditions simulating aquaculture treatments, the in vivo effects of
TRC on well known stress and growth-related biomarker genes of P.
hypophthalmus. Since several studies have used AChE activity to
diagnose exposure of fish to TRC (Bocquené et al., 1990; Boone and
Chambers, 1997; Sturm et al., 1999, 2000; Varó et al., 2003), the
expression kinetics of the AChE gene is a representative biomarker gene
in this study. In addition, the expression dynamics of growth-related
and other cellular toxicity representative genes, such as heat shock
protein 70 (HSP70), growth hormone, 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 homeostasis
disruption (Stegeman et al., 1992; Hartl, 1996), whereas, COI (Villani
and Attardi, 2000) and CYP1B genes are involved in the biotransforma-
tion and detoxification of toxic compounds (Shimada et 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.
org name: striped catfish) juveniles (15–20 g) were obtained from an
artificial seed production centre located in CanTho city. They were
reared 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 well
aerated water. During this period, fish were fed ad libitum with
commercial 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 27 ± 0.5 °C from a
recirculating system. The system was subjected to a photoperiod of
12 h light:12 h darkness. Water quality was monitored throughout
the experiment. All the water parameters were in the optimum
range (temperature 26.2–27.1 °C, pH 7.0–7.5, dissolved oxygen
6.9–7.4 mg L− 1, total NH3 0.1–0.2 mg L− 1, nitrite 0.07–0.1 mg L− 1
and nitrate 1–3 mg L− 1). Water flow was adjusted to keep the oxygen
saturation above 80%. Fish were fed thrice a day at a total of 3% on their
wet body weight day− 1. Feeding was adjusted based on the weight and
the number of fish 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
Please cite this article as: Sinha, A.K., et al., Expression characteristics of potential biomarker genes in Tra catfish, Pangasianodon
hypophthalmus, exposed to trichlorfon, Comp. Biochem. Physiol. D (2010), doi:10.1016/j.cbd.2010.05.001
available with a concentration of 90% (w/v). Different dilutions were
prepared by adding water. Among the four groups of fish, the first group
was reared in TRC free fresh water and used as a control group. Fish in
the second, third and fourth groups were exposed to the 96-h 1/100LC50
(0.01 mg/L), 96-h 1⁄10LC50 (0.1 mg/L), and 96-h 1⁄2LC50 (0.5 mg/L) of
TRC, respectively. Fish were reared for a period of 56 days. To avoid
metabolic 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. Five fish per replica
from 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 not
cause stress to remaining ones. The sampled fish were killed
immediately by decapitation.
2.3. Growth measurement
Fish in each container were bulk weighed at the beginning of the
experiment and on the final day (56 days) of experiment. Growth
performance of juveniles was evaluated in terms of weight gain based
on following standard formulae:
Weight gainð%Þ = ðFinal weight−Initial weightÞ × 100 = Initial weight:
2.4. Tissue samples
The fish were carefully dissected out to isolate liver and gill. The
extracted organs collected from five individual per replica of the
different groups were pooled together in a sterile 50 ml falcon tube
and were immediately frozen in liquid nitrogen and stored at − 80 °C
until RNA isolation.
2.5. RNA extraction and real time RT–PCR
Total RNA was isolated from liver and gill samples using the Trizol
method (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-Drop
spectrophotometry (NanoDrop Technologies, Wilmington, DE). The
integrity (quality) was checked by denaturing gel electrophoresis (1%
agarose gel) and purity by OD260/OD280 nm absorption ratio N 1.95.
A starting amount of 1 µg RNA was transcribed to First strand
cDNA according to “Revert Aid H minus First strand cDNA synthesis
kit” (Fermentas, Cambridgeshire). For the real time PCR reaction, the
final volume of 20 µL was adjusted to 100 µL to achieve a working
amount (5 µL) of approximately 50 ng RNA-equivalent for each
reaction.
Highly purified salt-free “OliGold” primers (Eurogentec, Seraing,
Belgium) for the quantification of the target genes AChE, HSP70, growth
hormone, trypsinogen, CYP1B, COI and reference genes beta-actin
(β-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 nucleotide
sequence of trypsinogen and COI were obtained from GenBank
accession no: AY316360 and EF609427. For rest of the genes we
designed primer pairs based on conserved regions of known respective
gene sequences, compared among other related fish species. The primer
sequences of each gene are listed in Table 1. Real time PCR mastermix
was prepared as follows: 5.5 µL nuclease free water, 1 µL forward and
1 µL reverse primer and 12.5 µL Maxima SYBR Green qPCR Master mix
(Fermentas, Cambridgeshire). Mastermix (20 µL) was mixed with 5 µL
of cDNA template in the glass capillaries. A four-step experimental run
protocol was used in light cycler (Roche version 3.5): denaturation
program (10 min at 95 °C); amplification and quantification program
repeated 40 times (15 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C); melting
 ARTICLE IN PRESS
A.K. Sinha et al. / Comparative Biochemistry and Physiology, Part D xxx (2010) xxx–xxx 3

Table 1
PCR primer sequences, accession numbers, amplicon size, melting temperatures and calculated efficiency.

Gene Accession no. Sequence of primer (5´ → 3´) Amplicon size (bp) Melting temperature (Tm) Calculated efficiency

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 1.81
R: TGGTTGGTCCTGGATGG 59.3
Trypsinogen AY316360 F: CACTGCTACCAGTCTCG 164 59.7 1.92
R: GCGGATTGACTCAGCTTG 59.5

Reference genes
β-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 1.89
R: CGCGTCTCAGAGCCTAA 59.7

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

curve program (55–95 °C with a heating rate of 0.10 °C/s and a
continuous fluorescence measurement) and finally a cooling step
(4 °C). To reduce the pipetting errors, mastermixes were prepared in
duplicate 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 efficiency
LightCycler melting curve analysis of the target genes and reference
genes were performed which resulted in single product specific melting
temperature as follows: AChE, 89.78 °C; HSP70, 80.54 °C; growth
hormone, 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; β-actin, 84.71 °C.
Moreover, no primer–dimers were generated during the applied 40
real time PCR amplification cycles.
The efficiency of amplification of target genes and internal controls
was examined from the slopes obtained from LightCycler Software.
Investigated transcripts showed high efficiency rates (Table 1).
2.6. Statistical analysis
Results for gene quantification are expressed as fold expression
relative to 16S rRNA and represent the mean ratio of 3 biological
replicates (each replicate represents a sample pool of five fish) per
treatment. The expression level at the control (0 h) condition has
been designated value “1” and thereby the expression ratio of
treatments was expressed in relation to the control. Significant
differences in expression between control and treatments were
analyzed by Relative Expression Software tool–Multiple condition
solver (REST–MCS) Version 2 using Pair Wise Fixed Reallocation
Randomisation Test©. A probability level of 0.05 was used for
rejection of the null hypothesis.
The data of weight gain % were subjected to T-test for mean
comparisons of treatments.
3. Results

2.5.2. Relative quantification 3.1. Growth

Relative 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; Pfaffl
et al., 2002). The mathematical algorithm, which needs no calibration
curve, computes an expression ratio, based on real time PCR efficiency
and the crossing point deviation of the sample versus a control, as
illustrated in the following formula:
 
Ratio = Etarget ΔCT = ðEref ÞΔCT
targetðcontrol−sampleÞ ref ðcontrol−sampleÞ
where CT (cycle threshold) value corresponds to the number of cycles
at which the fluorescence emission monitored in real time exceeded
the threshold limit. E is PCR efficiency determined by standard curve
using serial dilution of cDNA. The value of E is calculated according to
equation E = 10(− 1/slope). CT value of dilution series (1×, 2×, 4×, 8×
etc) were used to calculate the slope for target and reference genes.
Each point of dilution was tested in 3 replicates. ΔCT is the crossing
point deviation of the sample versus a control. Comparison of several
reference genes (β-actin, 16S rRNA and 12S rRNA) favoured 16S rRNA
because of its stable expression level in these test conditions.
After 56 days of TRC exposure weight gain was lowered (P b 0.05)
in the groups of fish which were subjected to a concentration of
0.1 mg and 0.5 mg/L TRC (Fig. 1). No significant differences were
observed 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 different
expression patterns in liver and gill in response to increasing exposure
concentrations (0.01, 0.1 and 0.5 mg/L) of TRC. The AChE gene
showed no significant change in the liver in response to TRC (Fig. 2A),
while displayed a dose-dependent change in gills (Fig. 2B). As shown
in Fig. 2B, exposure of P. hypophthalmus juveniles to the highest dose
of TRC (0.5 mg/L) resulted in a dose- and time-dependent decrease in
the level of expression of the AChE in gills which was significantly
down-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/L
reduced (P b 0.05) 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

Please cite this article as: Sinha, A.K., et al., Expression characteristics of potential biomarker genes in Tra catfish, Pangasianodon
hypophthalmus, exposed to trichlorfon, Comp. Biochem. Physiol. D (2010), doi:10.1016/j.cbd.2010.05.001
 ARTICLE IN PRESS
4 A.K. Sinha et al. / Comparative Biochemistry and Physiology, Part D xxx (2010) xxx–xxx

treatments slowly reduced till 14 days of exposure and then slowly

Fig. 1. Weight gain (%) of Pangasianodon hypophthalmus juveniles after 56 days under
different trichlorfon treatments. Data are means of 3 replicates ± standard deviation.
Significant differences are indicated with * (P b 0.05; T-test).
was more prominent at 14 days of exposure whereas for 0.1 mg/L the
clearest effect was noticed at 28 days of exposure. The effect in gills
seemed to recover after 28 days of TRC administration.
For HSP70, a significant increase in mRNA levels between control
and 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). The first significant effect in liver
and gills was observed at 96 h of 0.5 mg/L exposure. The highest fold
increase in liver and gill was seen at 7 days of 0.5 mg/L TRC exposure
which was 4.2 and 4.8 times higher (P b 0.01) than their respective
control. At the concentration of 0.1 mg/L, the increase (P b 0.05) in the
HSP70 for liver and gill was seen after 14 days of exposure, which was
numerically 2.8 and 3.07 times higher in liver and gills as compared to
non 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 (P b 0.05) in growth hormone gene
expression 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
recovered. Although the growth hormone gene expression seemed to
change in liver tissue, no significant reduction were verified in any of
the exposure periods after subjected to 0.01 mg/L of TRC. Moreover, in
gills, apparently, no remarkable change was detected in any of the
treatments (Fig. 4B).
The trypsinogen gene expression in liver reduced as a result of TRC
exposure (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 of
exposure till 28 days and afterwards there was a slight increase. The
expression level dropped (P b 0.05) at 14 and 28 days exposure of
0.5 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/L
also reduced the expression level (P b 0.05) in liver when exposed for a
period of 28 days, with a reduction of 2.5 times than the unexposed
group. However, in gills no remarkable change was observed on TRC
exposure during any of the experimental periods (Fig. 5B).
The exposure of fish with 0.5 mg/L of TRC amplified the COI gene
expression level significantly in liver at 14 and 28 days of exposure
which 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 of
2.2 (P b 0.05). It was also noticed that during the longest exposure
period (56 days) there was a gradual recovery in the COI gene
expression 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 noticed from 7 days of exposure onwards (Fig. 6B). After 7
and 14 days of exposure, 0.1 mg/L TRC augmented (P b 0.05) the
expression level by factor of 2.22 and 2.44 respectively, as compared
to control. Similarly, at the dose of 0.5 mg/L, a significant increase in
mRNA transcript was noticed following 7 and 14 days of exposure,
during which it was 2.34 and 3.22 times higher than the control. It was
seen that COI mRNA quantity in gills for all treatment groups after

Fig. 2. Relative expression of AChE 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 of Pfaffl et al. (2002) and are mean ratio of
3 replicates (each replicate represents pooled samples of five fish). Bars indicate standard error. Significant differences between treatments are indicated with superscripts (*P b 0.05;
**P b 0.01).

Please cite this article as: Sinha, A.K., et al., Expression characteristics of potential biomarker genes in Tra catfish, Pangasianodon
hypophthalmus, exposed to trichlorfon, Comp. Biochem. Physiol. D (2010), doi:10.1016/j.cbd.2010.05.001
 ARTICLE IN PRESS
A.K. Sinha et al. / Comparative Biochemistry and Physiology, Part D xxx (2010) xxx–xxx 5

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. Results are expressed as fold expression relative to 16S rRNA, according to the equation of Pfaffl et al. (2002) and are mean
ratio of 3 replicates (each replicate represents pooled samples of five fish). Bars indicate standard error. Significant differences between treatments are indicated with superscripts
(*P b 0.05; **P b 0.01).

reaching a peak at 14 days of exposure gradually lowered in 28 day
and 56 days of exposure period.
The effect of TRC exposure on CYP1B gene expression in fish liver was
insignificant (Fig. 7A). However, a notable effect was observed in gills at
higher exposure levels (0.1 mg/L and 0.5 mg/L) (Fig. 7B). The
remarkable effect (P b 0.05) in gill samples was first observed after
96 h of exposure for the highest exposure level (0.5 mg/L). At this point
the 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 /L
and 0.5 mg/L TRC. The highest peak of CYP1B gene expression for
0.1 mg/L and 0.5 mg/L was found after one week of exposure which was
3.55 times (P b 0.05) and 4.24 times (P b 0.01) higher than control value.
4. Discussion
In this study we have focused on stress and toxicity representative
genes such as AChE, HSP70, growth hormone, COI, CYP1B and
trypsinogen to be evaluated as potential “biomarker genes” for
trichlorfon induced stress in P. hypophthalmus. The kinetics of the
studied 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 that
response of these genes expression differs between different organs
(liver and gills). To our knowledge, this is one of the first studies that
assess 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 insecticide
such 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 in
gills exposed to 96 h 1⁄2LC50 (0.5 mg/L) of TRC for 96 h, 7 days,
14 days and 28 days was significantly down-regulated. The maximum
reduction was seen following 14 days of exposure. Moreover, for the
0.1 mg/L, the transcript level of AChE in gills reduced significantly
only after 28 days. This is because the chronic administration of TRC at
lower 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 our
experiment 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 ChE
activity since this is its main mode of action. In addition, our result
corroborates the finding of Varó 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 in
European eel (Anguilla anguilla) in response to the OP pesticide
fenitrothion (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 to
continuously inhibit AChE. In the present study, the mRNA transcript
level of AChE gene for all treatments gradually increased on day 56, it
depicts that fish have the ability to overcome the stress of toxicants.
Similarly, Venkateswara et al. (2003) reported a 90% inhibition of
AChE activity in the brain and gills of Oreochromis mossambicus in 24 h
and a complete recovery within 28 days after exposure to a single LC50
and 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

Please cite this article as: Sinha, A.K., et al., Expression characteristics of potential biomarker genes in Tra catfish, Pangasianodon
hypophthalmus, exposed to trichlorfon, Comp. Biochem. Physiol. D (2010), doi:10.1016/j.cbd.2010.05.001
 ARTICLE IN PRESS
6 A.K. Sinha et al. / Comparative Biochemistry and Physiology, Part D xxx (2010) xxx–xxx

Fig. 4. Relative expression of growth hormone 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 of Pfaffl et al. (2002) and are mean
ratio of 3 replicates (each replicate represents pooled samples of five fish). Bars indicate standard error. Significant differences between treatments are indicated with superscripts
(*P b 0.05).

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. 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 of Pfaffl et al. (2002) and are mean
ratio of 3 replicates (each replicate represents pooled samples of five fish). Bars indicate standard error. Significant differences between treatments are indicated with superscripts
(*P b 0.05).

Please cite this article as: Sinha, A.K., et al., Expression characteristics of potential biomarker genes in Tra catfish, Pangasianodon
hypophthalmus, exposed to trichlorfon, Comp. Biochem. Physiol. D (2010), doi:10.1016/j.cbd.2010.05.001
 ARTICLE IN PRESS
A.K. Sinha et al. / Comparative Biochemistry and Physiology, Part D xxx (2010) xxx–xxx 7

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 of Pfaffl et al. (2002) and are mean ratio of
3 replicates (each replicate represents pooled samples of five fish). Bars indicate standard error. Significant differences between treatments are indicated with superscripts (*P b 0.05;
**P b 0.01).

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. 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 of Pfaffl et al. (2002) and are mean
ratio of 3 replicates (each replicate represents pooled samples of five fish). Bars indicate standard error. Significant differences between treatments are indicated with superscripts
(*P b 0.05; **P b 0.01).

Please cite this article as: Sinha, A.K., et al., Expression characteristics of potential biomarker genes in Tra catfish, Pangasianodon
hypophthalmus, exposed to trichlorfon, Comp. Biochem. Physiol. D (2010), doi:10.1016/j.cbd.2010.05.001
 ARTICLE IN PRESS
8 A.K. Sinha et al. / Comparative Biochemistry and Physiology, Part D xxx (2010) xxx–xxx
et al., 2002). The current results show that HSP70 mRNA levels
increased significantly both in liver and gills at 96 h exposure of
0.5 mg/L TRC whereas for 0.1 mg/L the remarkable effect was noted
after 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 HSP70
expression 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. Fish
cope with proteotoxicity by the induction of HSPs, which are able to
repair partly denatured proteins (Hallare et al., 2004). Stressors like
toxicants may induce HSP70, and this induction has already been used
as a general biomarker of toxicant stress (Hassanein et al., 1999; Varó
et al., 2002; De Boeck et al., 2003). It should be noted that while many
indicators of fish stress are altered by handling and sampling
procedures, Vijayan et al. (1997) demonstrated that handling stress
does not alter levels of hepatic HSP70 in rainbow trout (Oncorhynchus
mykiss). Therefore, from the results presented in our work, it can be
acknowledged that HSP70 is a promising biomarker gene for stress
induced by TRC exposure in Tra catfish.
In fish, the class of cytochrome P450 isozymes are responsible for
the transformation of a variety of environmental contaminants
including polyaromatic hydrocarbons (PAHs), planar polychlorinated
biphenyls (PCBs) and arylamines (Leaver and George, 2000; van der
Oost et al., 2003). The existence of CYP1B genes have already been
reported in fish (Godard et al., 2000; Leaver and George, 2000; Willett
et al., 2006). In fact, metabolism and carcinogenesis studies have
shown CYP1B to be a critical and necessary enzyme involved in the
oxidation 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 significant
induction 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 CYP1B
activity. In our case, we observed a significant expression of CYP1B
mRNA in the gill tissue at an exposure of 0.1 and 0.5 mg/L TRC while
no such remarkable effect was detected in liver. This is in accordance
with northern blot analysis of plaice (Pleuronectes platessa) and
common carp (Cyprinus carpio) where CYP1Bs have been shown to
have higher abundance in gills as compared to other tissues (Leaver
and George, 2000; El-kady et al., 2004a,b). Thereby, the mRNA
expression profile of this gene in the gill could potentially be a useful
biomarker 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 anion
radicals (O−2 ), hydrogen peroxide (H2O2) and highly reactive hydroxyl
radical (− OH) (Hai et al., 1997; Yarsan et al., 1999; Pena-Llopis et al.,
2003; Mohammad et al., 2004; Monteiro et al., 2006; Varó et al., 2007;
Thomaz et al., 2009). These radicals can react with susceptible
biological macromolecules and produce lipid peroxidation, DNA
damage and protein oxidation.
The main function of COI is to transfer electrons from cytochrome c
to oxygen in mitochondrial based electron transport chain and to
generate ATP. Besides, COI may also function indirectly as an antioxidant
by either preventing the dawdling of electron flow (Bolter and Chefurka,
1990; Benzi et al., 1992) or by uncoupling electron transport from
proton transfer (Richter, 1997). In the present study, COI gene
expression was up-regulated at higher doses (0.1 and 0.5 mg/L) of
TRC. Elevated expression of COI has also been associated with a
pyrethroid insecticide resistant strain of Blatella germanica (German
cockroach) (Pridgeon and Liu, 2003). Danio rerio (zebrafish) fed with
diets contaminated with methyl mercury resulted in increased
expression of COI gene (Gonzalez et al., 2005). Similarly, Achard-Joris
et al. (2006) reported that COI gene expression was up-regulated by
exposure to an oxidative stressor such as cadmium in two freshwater
bivalves (Corbicula fluminea and Dreissena polymorpha) and one marine
Please cite this article as: Sinha, A.K., et al., Expression characteristics of potential biomarker genes in Tra catfish, Pangasianodon
hypophthalmus, exposed to trichlorfon, Comp. Biochem. Physiol. D (2010), doi:10.1016/j.cbd.2010.05.001
bivalve (Crassostrea gigas). To our knowledge, the direct relationship
between COI expression and TRC contamination has never been
reported before. Since COI is considered as the rate-limiting step for
mitochondrial respiration (Villani and Attardi, 2000), elevated expres-
sion COI gene could be a compensating mechanism to restore the
decrease in mitochondrial activity and to efficiently consume oxygen
(Achard-Joris et al., 2006), thus limiting dichlorvos induced damage in
the 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 the
organ at the interface with the contaminated environment and are thus
the main tissues from which the toxicants loading in the organism will
proceed (Marigomez et al., 2002). Our findings suggests that in addition
to AChE, HSP 70 and CYP1B genes, COI gene expression level might
constitute a key biomarker gene for detecting TRC induced stress
responses in Tra catfish. Moreover, an advantage of using COI to study
the impact of TRC on mitochondrial metabolism is that COI gene
sequence is highly conserved between lower and higher eukaryotes
(Capaldi, 1990). Thus a similar COI gene regulation between different
species of catfish can be expected.
Results from the weight gain demonstrated that prolonged
exposure to TRC at a level of 0.1 or 0.5 mg/L in rearing water
hampered the growth performance of P. hypophthalmus juveniles. A
significant reduction in growth was commensurate with an increase
in the TRC concentration. It indicates that the applied pesticide at a
higher dose (0.1 and 0.5 mg/L) may hinder the growth hormone
regulating gene. To verify this, we checked the transcript level of
growth hormone and our results confirm that TRC at higher doses (0.1
and 0.5 mg/L) significantly down-regulates the gene expression level
of growth hormone. Therefore, a positive correlation was found
between genotypic and phenotypic expression for growth hormone
on TRC exposure. Consequently, the mRNA expression of growth
hormone in the present study can be speculated as a specific
biomarker to detect fish performance under TRC treatment; however
more in-depth research on the interaction of growth hormone on
growth in P. hypophthalmus is needed. The negative effect of TRC on
the growth performance of juveniles in the present study is consistent
with 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 effect
of OP pesticide in fish has also been reported (Pal and Konar, 1985;
Silva et al., 1993; Sturm et al., 1999). However, these results were in
contrast with the results obtained by Ludwig (1993) who documen-
ted that the use of the pesticides TRC did not cause any significant
difference in growth in hybrids of Morone saxatilis in culture
environment. Similarly, Ruddle and Zhong (1988) suggested that no
adverse effects existed in the use of TRC in aquaculture. The
discrepancy between these results may be associated with differences
in the rearing conditions because the mode of action of organophos-
phate pesticides depends on pH and temperature, being more active
in temperatures above 16 °C (Messenger and Esnault, 1992; Howe
et 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 expression
under the exposure level of 0.1 and 0.5 mg/L TRC supports the finding
of growth performance. However, it is not clear from our study that if
it was direct or indirect overriding effect due to other genes or
hormones. Like other vertebrates, trypsinogen (zymogen) is the
inactive 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 measure
fish juvenile condition in response to changing environmental
conditions (Nolting et al., 1999). Moreover, the direct correlation of
digestive 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
A.K. Sinha et al. / Comparative Biochemistry and Physiology, Part D xxx (2010) xxx–xxx
(Solea senegalensis) (Ribeiro et al., 1999). Thereby, it can be speculated
that trypsinogen regulating gene could be a promising biomarker to
access 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. Our
study clearly demonstrates that the application of high dose (0.1 and
0.5 mg/L) of organophosphate pesticide, trichlorfon in culture water
of P. hypophthalmus significantly influences the expression levels of
studied genes (AChE, HSP70, growth hormone, trypsinogen, CYP1B,
COI). It was seen that each gene was differentially expressed in a dose-
and time- dependent way to TRC exposure. In short, it can be said that
stress-inducible proteins appear to be excellent candidates for
molecular biomarkers. Nevertheless, the importance of these studied
genes in terms of both functionality and isoform variation might be
crucial in further examinations.
Acknowledgments
The study is a part of a joint Belgian–Vietnamese collaborative
project entitled “Analytical and biological methods in support of
sustainable aquaculture practices in Vietnam” (contracts BELSPO BL/
13/V17). We are grateful to the Belgian Science Policy Office (Mrs
Desmeth M. and Decadt B.) and to the Vietnamese Ministry of Science
and Technology.
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