Comparative Biochemistry and Physiology, Part C 166 (2014) 65–74

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part C

journal homepage: www.elsevier.com/locate/cbpc

Heavy metals induce oxidative stress and trigger oxidative

stress-mediated heat shock protein (hsp) modulation in the
intertidal copepod Tigriopus japonicus
Bo-Mi Kim a, Jae-Sung Rhee b, Chang-Bum Jeong c, Jung Soo Seo d, Gyung Soo Park e,
Young-Mi Lee f,⁎, Jae-Seong Lee a,⁎
a
Department of Biological Sciences, College of Science, Sungkyunkwan University, Suwon 440-746, South Korea
b
Department of Marine Science, College of Natural Sciences, Incheon National University, Incheon 406-772, South Korea
c
Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791, South Korea
d
Pathology Team, National Fisheries Research & Development Institute, Busan 619-902, South Korea
e
Department of Marine Biotechnology, College of Liberal Arts and Sciences, Anyang University, Ganghwa 417-833, South Korea
f
Department of Life Science, College of Natural Sciences, Sangmyung University, Seoul 110-743, South Korea

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

Article history: Heat shock proteins (hsps) are induced by a wide range of environmental stressors including heavy metals in
Received 15 May 2014 aquatic organisms. However, the effect of heavy metals on zooplankton at the molecular level remains still
Received in revised form 25 June 2014 unclear. In this study, we measured the intracellular reactive oxygen species (ROS) level and the antioxidant
Accepted 14 July 2014
enzyme activities for 96 h after exposure to five heavy metals: arsenic (As), cadmium (Cd), copper (Cu), silver
Available online 21 July 2014
(Ag), and zinc (Zn) in the intertidal copepod Tigriopus japonicus. Activities of the antioxidant enzymes were high-
Keywords:
ly elevated in metal-exposed copepods, indicating that heavy metals can induce oxidative stress by generating
Tigriopus japonicus ROS, and stimulate the involvement of antioxidant enzymes as cellular defense mechanisms. Subsequently, tran-
Copepod scriptional changes in hsp gene families were further investigated in the metal-exposed groups for 96 h. The ROS
Heavy metal level and glutathione (GSH) content were significantly increased in Ag-, As-, and Cu-exposed copepods, while
Oxidative stress they were only slightly elevated in Cd- and Zn-exposed groups. Based on the numbers of significantly modulated
Heat shock protein hsp genes and their expression levels for 96 h, we measured the effect of heavy metals to stress genes of
Antioxidant enzyme activities T. japonicus in the following order: Cu N Zn N Ag N As N Cd, implying that Cu acts as a stronger oxidative stress
inducer than other heavy metals. Of them, the expression of hsp20 and hsp70 genes was substantially modulated
by exposure to heavy metals, indicating that these genes would provide a sensitive molecular biomarker for
aquatic monitoring of heavy metal pollution.
© 2014 Elsevier Inc. All rights reserved.

1. Introduction
Heavy metals are released into aquatic ecosystems from anthropo-
genic sources such as mine-washing, metal refining, or agricultural
leaching (Phillips, 1977). Due to their persistence, accumulation, and
magnification towards higher trophic levels through food chains,
heavy metals have been considered important stressors to aquatic or-
ganisms (Roesijadi and Robinson, 1994). Furthermore, essential and
non-essential metals can induce oxidative stress by generating reactive
oxygen species (ROS) in aquatic organisms (Lushchak, 2011). ROS can
attack cellular macromolecules such as proteins, lipids, and DNA. Partic-
ularly, structural and enzymatic proteins are main targets of heavy
⁎ Corresponding authors. Tel.: +82 31 290 7011.
E-mail addresses: ymlee70@smu.ac.kr (Y.-M. Lee), jslee2@skku.edu (J.-S. Lee).
metals (Chang and Suzuki, 1996). Prolonged oxidative stress induces
aging, diseases, and even cell death by necrosis and/or apoptosis
(Pulido and Parrish, 2003). To overcome cellular damage induced by ox-
idative stress, living organisms have developed antioxidant defense
mechanisms. Antioxidant defenses consist of a number of enzymes
such as superoxide dismutase (SOD), catalase (CAT), glutathione reduc-
tase (GR), glutathione peroxidase (GPx), and non-enzymatic antioxi-
dant, glutathione (GSH), which are useful as biomarkers for oxidative
stress (Lushchak, 2011). After exposure to heavy metals in several
aquatic organisms, the induction of antioxidant enzymes has been dem-
onstrated (Chelomin et al., 2005; Mosleh et al., 2006; Farombi et al.,
2007; Zhang et al., 2010b; Kim et al., 2011b). However, despite the
ecological importance of copepods, oxidative damage effects of heavy
metals in copepods remain unclear.
Heat shock proteins (Hsps) play a crucial role in maintaining protein
homeostasis under sub-lethal stress conditions in both prokaryotes and

http://dx.doi.org/10.1016/j.cbpc.2014.07.005
1532-0456/© 2014 Elsevier Inc. All rights reserved.
66 B.-M. Kim et al. / Comparative Biochemistry and Physiology, Part C 166 (2014) 65–74
eukaryotes (Hofmann et al., 2002). Moreover, Hsps are involved as mo-
lecular chaperones in protein folding/unfolding, translocation, and deg-
radation of proteins as well as in the protection against a wide range of
environmental stressors including heat, salinity, heavy metals, and toxic
compounds (Feder and Hofmann, 1999; Qiu et al., 2006). Several forms
of Hsps, including Hsp60, Hsp70, and Hsp90 gene families have been
known and classified by their molecular weight. Of them, Hsp70 and
Hsp90 are the most ubiquitous proteins and play important roles in
the protection against stress-induced cellular damages (Ivanina et al.,
2008). Hsp60 is mainly involved in protein folding and stress protection
in mitochondria and chloroplasts of eukaryotes (Cechetto et al., 2000).
Small Hsps with a molecular mass of 15–43 kDa are constitutively
expressed, and belong to the stress-induced groups (Haslbeck, 2002).
As yet, the potential function of Hsps has been documented in diverse
marine invertebrates such as copepod (Seo et al., 2006a; Rhee et al.,
2009), mussels (Hofmann et al., 2002), rotifers (Rhee et al., 2011), sea
urchins (Bonaventura et al., 2006), snails (Tomanek and Somero,
2002), and sponges (Choresh et al., 2004). Despite their important
role in protein chaperoning, transcriptional modulation of hsp gene
families within a marine species, particularly in zooplankton, by heavy
metals is still unexplored.
Tigriopus japonicus (Copepoda, Harpactidae) is an intertidal benthic
copepod and inhabits rock pools. Due to its small size, sexual dimor-
phism, high fecundity, short reproduction time (≈14 days), and envi-
ronmental resistance (can survive broad ranges of temperature,
salinity, pH), T. japonicus has been used as a laboratory model animal
in several areas of aquatic life science such as in ecotoxicological studies
(Raisuddin et al., 2007). More recently, extensive genomic DNA infor-
mation (10,894 unigenes) and RNASeq (59,983 assembled ESTs; total
length 78.3 Mb; N50 = 2319) from this species were obtained by
using GS-FLX Titanium and Illumina (Lee et al., 2010; unpublished
data). In the present study, we measured ROS levels and antioxidant en-
zyme activities after exposure to five heavy metals, and also examined
the transcriptional modulation of hsp gene families upon heavy metal
stressors. The main objective of this study is to provide a better under-
standing of the molecular mechanisms of cellular protection against ox-
idative stress induced by heavy metals in copepod.
2. Materials and methods
2.1. Culture and maintenance
T. japonicus was reared and maintained in 0.2 μm-filtered artificial
sea water (TetraMarine Salt Pro, Tetra™, Cincinnati, OH, USA) adjusted
to 20 °C under a LD 12:12 h photoperiod with 30 practical salinity
units (psu). The copepods were fed with the algae Tetraselmis suecica
(approximately 5 × 104 cells/ml/day). Identification of the species was
made by morphological characters and the sequence identity of mito-
chondrial DNA COI as a universal barcode marker gene.
2.2. Heavy metal exposure
To study the effects of environmental toxicant exposure on hsp
gene expression, we exposed five metals (Ag, silver; As, arsenic; Cd,
cadmium; Cu, copper; Zn, zinc) to T. japonicus. All the chemicals were
purchased from Sigma (Sigma-Aldrich, Inc., St. Louis, MO, USA;
purity N 99%). Stock solutions of metals were prepared in ultrapure
water. The exposed concentrations of heavy metals were uniformly
set within NOEC value based on our previous studies on the toxicity
value of T. japonicus (Seo et al., 2006b; Lee et al., 2007, 2008a,b; Rhee
et al., 2009). The following concentrations were used in all the heavy
metal exposures: 5, 10, 50, and 100 μg/L. The exposures were given
for 96 h in a static renewal culture. Three replicates were used for
each concentration containing approximately 300 adult copepods
(both sexes) in each container. Fifty percent of culture water was
renewed after 24 h and the desired concentrations of toxicants were
maintained accordingly.
2.3. Total RNA extraction and single-strand cDNA synthesis
After exposure, whole bodies were homogenized in three volumes
of TRIZOL® reagent (Invitrogen, Paisley, Scotland) with a tissue grinder
and stored at −80 °C. Total RNA was isolated from the tissues according
to the manufacturer's instructions. Genomic DNA was removed using
DNase I (Sigma-Aldrich). The quantity of total RNA was measured at
230, 260, and 280 nm using a spectrophotometer (Ultrospec 2100 Pro,
Amersham Bioscience, Freiburg, Germany). To check for genomic DNA
contamination, we loaded the total RNA in a 1% agarose gel that
contained ethidium bromide (EtBr) and visualized on a UV transillumi-
nator (Wealtec Corp., Sparks, NV, USA). Also, to verify the total RNA
quality, we loaded the total RNA in a 1% formaldehyde/agarose gel
with EtBr staining and checked the 18/28S ribosomal RNA integrity
and band ratio. Single-strand cDNA was synthesized from total RNA
using an oligo(dT)20 primer for reverse transcription (SuperScript™ III
RT Kit, Invitrogen, Carlsbad, CA, USA).
2.4. Real-time RT-PCR
Whole T. japonicus hsp genes were registered at GenBank as shown
previously (Rhee et al., 2009). Paralogs of each hsp gene were not ob-
served. To investigate the specific expression patterns of hsp genes
(hsp10, 20, 20.7, 40, 60, 70, 70p, 90α, and 105), real-time RT-PCRs were
performed. Each reaction included 1 μL of cDNA which was reverse-
transcribed from 2 μg of total RNA and a 0.2 μM primer (real-time RT-
F/R for each hsp gene and 18S rRNA RT-F/R). Sequence information
and their specific primer sequences were adopted from our previous
study (Rhee et al., 2009). The primers were designed after comparing
the exon/intron boundaries with the genomic DNA using GENRUNNER
software (Hastings Software, Inc., NY, USA) and confirmed by the Prim-
er 3 program (Whitehead Institute/MIT Center for Genome Research,
Cambridge, MA, USA). To determine the amplicon identity, all the PCR
products were cloned into the pCR2.1 TA vector and sequenced with
an ABI 3700 DNA analyzer (Bionics Co., Seoul, South Korea). The opti-
mized conditions were transferred according to the following CFX96™
real-time PCR system protocol (Bio-Rad, Hercules, CA, USA). The reac-
tion conditions were as follows: 95 °C/3 min; 40 cycles of 95 °C/30 s,
55 °C/30 s, and 72 °C/30 s. To confirm the amplification of specific prod-
ucts, the cycles were continued to check the melting curve under
the following conditions: 95 °C/1 min, 55 °C/1 min, and 80 cycles of
55 °C/10 s with a 0.5 °C increase per cycle. SYBR® Green (Molecular
Probes Inc., Invitrogen, Carlsbad, CA, USA) was used to detect the specif-
ic amplified products. Amplification and detection of the SYBR® Green-
labeled products were performed using a CFX96™ real-time PCR system
(Bio-Rad, Hercules, CA, USA). The data from each experiment were
expressed relative to expression levels of the 18S rRNA gene to normal-
ize the expression levels between samples. All experiments were done
in triplicate. Data were collected as threshold cycle (CT) values (PCR
cycle number where fluorescence was detected above a threshold and
decreased linearly with increasing input target quantity), and used to
calculate the ΔCT values of each sample. The fold change in the relative
gene expression was calculated by the 2− ΔΔCT method (Livak and
Schmittgen, 2001). Each value and their statistical analysis are repre-
sented in the Supplementary materials (Table 1). Hierarchical clustering
analysis was employed in order to make the heat map using MeV v.7.4
(Dana-Farber Cancer Institute, MA, USA) software.
2.5. Measurement of ROS and antioxidant enzyme activities
Overall procedures for measuring the intracellular ROS and antioxi-
dant enzyme activities were prepared according to our previous study
(Kim et al., 2011a). A minimum of three replicates were used for each
 B.-M. Kim et al. / Comparative Biochemistry and Physiology, Part C 166 (2014) 65–74 67

Table 1
Transcript patterns and their statistical analysis of hsp genes in Tigriopus japonicus exposed to different concentrations of heavy metals.

Metals Genes Concentration (μg/L)

0 5 10 50 100

Ag hsp10 1.00 ± 0.13 0.98 ± 0.08 0.96 ± 0.15 1.88 ± 0.10 1.14 ± 0.11
hsp20 1.00 ± 0.07 1.52 ± 0.06 3.68 ± 0.49⁎ 5.92 ± 0.97⁎⁎ 6.28 ± 1.20⁎⁎
hsp20.7 1.00 ± 0.11 1.05 ± 0.05 2.26 ± 0.61 3.27 ± 0.29⁎ 3.05 ± 0.10⁎
hsp40 1.00 ± 0.08 1.11 ± 0.09 1.89 ± 0.14 0.87 ± 0.02⁎ 0.81 ± 0.03⁎⁎
hsp60 1.00 ± 0.17 1.05 ± 0.11 1.52 ± 0.08 1.69 ± 0.14 0.80 ± 0.06⁎⁎
hsp70 1.00 ± 0.06 1.06 ± 0.15 3.62 ± 0.21⁎ 4.26 ± 0.35⁎⁎ 4.05 ± 0.29⁎⁎
hsp70p 1.00 ± 0.21 1.18 ± 0.30 2.13 ± 0.05⁎ 2.35 ± 0.09⁎ 2.19 ± 0.07⁎
hsp90α 1.00 ± 0.10 0.98 ± 0.08 1.13 ± 0.03 0.97 ± 0.18 1.89 ± 0.09
hsp105 1.00 ± 0.06 1.40 ± 0.07 1.29 ± 0.10 1.53 ± 0.08 1.61 ± 0.06
As hsp10 1.00 ± 0.15 1.38 ± 0.06 1.62 ± 0.05 0.95 ± 0.03 0.88 ± 0.08⁎
hsp20 1.00 ± 0.13 1.26 ± 0.11 5.26 ± 0.46⁎⁎ 6.95 ± 0.92⁎⁎ 2.62 ± 0.06⁎
hsp20.7 1.00 ± 0.14 1.11 ± 0.08 3.26 ± 0.25⁎ 4.35 ± 0.29⁎⁎ 2.15 ± 0.35
hsp40 1.00 ± 0.19 1.62 ± 0.10 1.22 ± 0.08 2.38 ± 0.07⁎ 0.72 ± 0.18⁎
hsp60 1.00 ± 0.20 0.97 ± 0.06 2.39 ± 0.05⁎ 2.95 ± 0.08⁎ 0.88 ± 0.03⁎
hsp70 1.00 ± 0.15 1.28 ± 0.08 13.25 ± 1.89⁎⁎⁎ 8.95 ± 1.24⁎⁎⁎ 5.18 ± 0.71⁎⁎
hsp70p 1.00 ± 0.05 1.70 ± 0.04 3.92 ± 0.54⁎ 3.33 ± 0.21⁎ 3.69 ± 0.30⁎
hsp90α 1.00 ± 0.08 1.66 ± 0.11 1.25 ± 0.06 3.68 ± 0.31⁎ 2.10 ± 0.08⁎
hsp105 1.00 ± 0.09 1.41 ± 0.04 1.71 ± 0.08 3.26 ± 0.25⁎ 2.88 ± 0.22⁎
Cd hsp10 1.00 ± 0.09 1.71 ± 0.11 0.96 ± 0.18 0.88 ± 0.15 0.92 ± 0.06
hsp20 1.00 ± 0.11 0.96 ± 0.12 1.62 ± 0.09 2.66 ± 0.11 3.92 ± 0.10⁎
hsp20.7 1.00 ± 0.14 2.16 ± 0.18 1.70 ± 0.16 3.18 ± 0.07⁎⁎ 4.93 ± 0.89⁎
hsp40 0.99 ± 0.16 1.22 ± 0.22 0.95 ± 0.06 0.95 ± 0.06 0.97 ± 0.02
hsp60 1.00 ± 0.08 0.97 ± 0.07 1.82 ± 0.07 2.16 ± 0.08 3.81 ± 0.06⁎
hsp70 1.00 ± 0.11 1.38 ± 0.09 2.35 ± 0.21 3.26 ± 0.06⁎ 6.82 ± 1.02⁎
hsp70p 1.00 ± 0.08 0.94 ± 0.16 1.52 ± 0.05 2.24 ± 0.27 3.06 ± 0.85
hsp90α 1.00 ± 0.09 1.02 ± 0.11 1.63 ± 0.08 1.11 ± 0.07 3.68 ± 0.13⁎
hsp105 1.00 ± 0.07 1.54 ± 0.15 1.11 ± 0.04 1.23 ± 0.12 2.07 ± 0.23
Cu hsp10 1.00 ± 0.13 1.21 ± 0.13 0.98 ± 0.02 2.14 ± 0.13 1.85 ± 0.05
hsp20 1.00 ± 0.15 6.99 ± 0.59⁎⁎ 8.81 ± 1.12⁎⁎ 12.32 ± 3.11⁎ 9.76 ± 1.13⁎⁎
hsp20.7 0.99 ± 0.17 4.92 ± 0.43⁎⁎ 6.08 ± 0.84⁎ 6.59 ± 0.89⁎⁎ 4.26 ± 0.08⁎⁎⁎
hsp40 1.00 ± 0.13 1.16 ± 0.11 2.29 ± 0.11⁎ 0.96 ± 0.05 0.80 ± 0.11⁎
hsp60 1.00 ± 0.11 1.39 ± 0.08 2.06 ± 0.13 2.34 ± 0.09 5.92 ± 0.62⁎
hsp70 1.00 ± 0.08 6.28 ± 1.32⁎ 12.26 ± 2.37⁎⁎ 16.98 ± 3.22⁎⁎⁎ 15.27 ± 1.23⁎⁎⁎
hsp70p 1.00 ± 0.06 1.22 ± 0.05 2.62 ± 0.11⁎ 7.18 ± 1.34⁎ 5.08 ± 0.76⁎
hsp90α 1.00 ± 0.09 4.97 ± 0.60⁎⁎ 5.62 ± 0.82⁎ 6.92 ± 0.87⁎⁎ 7.08 ± 0.92⁎⁎
hsp105 1.00 ± 0.10 3.28 ± 0.86 2.14 ± 0.02 3.53 ± 0.08⁎ 2.87 ± 0.25⁎
Zn hsp10 1.00 ± 0.11 1.23 ± 0.09 2.10 ± 0.08 0.97 ± 0.06 0.84 ± 0.03⁎
hsp20 1.00 ± 0.09 1.08 ± 0.08 1.44 ± 0.07 2.63 ± 0.15 6.24 ± 0.62⁎
hsp20.7 1.00 ± 0.06 1.11 ± 0.11 1.90 ± 0.04 2.27 ± 0.16 2.92 ± 0.11⁎⁎
hsp40 1.00 ± 0.12 2.30 ± 0.07⁎ 3.28 ± 0.11⁎ 0.97 ± 0.04 0.78 ± 0.06⁎⁎
hsp60 1.00 ± 0.05 1.09 ± 0.09 2.11 ± 0.32 4.29 ± 0.61⁎ 2.36 ± 0.66
hsp70 1.00 ± 0.14 6.28 ± 0.89⁎ 8.91 ± 1.28⁎⁎ 11.52 ± 2.39⁎⁎ 10.50 ± 1.32⁎⁎⁎
hsp70p 1.00 ± 0.07 0.91 ± 0.15 1.33 ± 0.05 3.26 ± 0.87⁎ 4.92 ± 0.69⁎⁎
hsp90α 1.00 ± 0.05 1.19 ± 0.06 2.28 ± 0.14 2.60 ± 0.25 6.25 ± 0.88⁎
hsp105 1.00 ± 0.09 1.90 ± 0.08 2.05 ± 0.26 2.27 ± 0.33 3.44 ± 0.86

All the mRNA expression values were expressed as the fold change.
⁎ Indicates P b 0.05.
⁎⁎ Indicates P b 0.01.
⁎⁎⁎ Indicates P b 0.001.

concentration containing approximately 500 adult copepods (both
sexes) in each container. To confirm as to whether toxicants induce
intracellular ROS in the copepod T. japonicus, we measured ROS
levels using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA;
Molecular Probes, Eugene, OR, USA) which was oxidized to fluores-
cent dichlorofluorescein (DCF) by intracellular ROS. After exposure
to different heavy metals, the copepods were homogenized in a buffer
containing 0.32 M sucrose, 20 mM HEPES, 1 mM MgCl2 and 0.5 mM
PMSF (pH 7.4) using a Teflon homogenizer. The homogenate was cen-
trifuged at 10,000 g for 20 min at 4 °C. The supernatant was collected
for measurement. Following 96-well black plates were filled with
phosphate-buffered saline (PBS) buffer, probe (H2DCFDA at a final
concentration of 40 μM), and supernatant fraction to a final volume of
200 μL. The measurements were obtained with an excitation wave-
length at 485 nm and emission wavelength at 520 nm using fluores-
cence spectroscopy (Thermo™ Varioskan Flash, MA, USA). The ROS
measurements were normalized by total protein and represented as a
percentage of fluorescence of DCF. Total proteins were determined
using the Bradford method (Bradford, 1976).
Glutathione (GSH) concentration was determined by an enzymatic
method using the BIOXYTECH® GSH-420™ Kit (OxisResearch®,
Portland, OR, USA). After exposure to different heavy metals, the cope-
pods were washed in 0.9% NaCl. The rinsed samples were homogenized
in trichloroacetic acid at a ratio of 1 to 20 (w/v) using a Teflon homog-
enizer. The homogenate was centrifuged at 3000 g for 10 min at 4 °C.
The upper aqueous layer was collected for the GSH content assay ac-
cording to the manufacturer's protocol. The GSH content was measured
at an absorbance of 420 nm using a spectrophotometer (Thermo™
Varioskan Flash, MA, USA) and the standard curves were generated
using GSH equivalents (0, 150, and 350 μM).
The glutathione S-transferase (GST) activity was measured as
described by Rhee et al. (2013). After exposure to different heavy
metals, the copepods were homogenized in cold buffer (0.25 M sucrose,
10 mM Tris, 1 mM EDTA, 0.2 mM DTT and 0.1 mM PMSF, pH 7.4) at a
ratio of 1 to 4 (w/v) using a Teflon homogenizer. The homogenate was
centrifuged at 10,000 g for 10 min at 4 °C. The cytosolic fraction
containing the enzyme was collected for an enzymatic assay using
1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. The enzymatic
68 B.-M. Kim et al. / Comparative Biochemistry and Physiology, Part C 166 (2014) 65–74
assay monitored the conjugation of CDNB and GSH at 340 nm using
a spectrophotometer (Thermo™ Varioskan Flash, MA, USA) at 25 °C.
The glutathione peroxidase (GPx) and glutathione reductase
(GR) activities were measured with an enzymatic method using
BIOXYTECH® GPx-340™ and GR-340™ kits (OxisResearch®, Portland,
OR, USA), respectively. After exposure to different heavy metals, the co-
pepods were homogenized in cold buffer (50 mM Tris–Cl, 5 mM EDTA,
and 1 mM 2-mercaptoethanol, pH 7.5) at a ratio of 1 to 4 (w/v) using a
Teflon homogenizer. The homogenate was centrifuged at 10,000 g for
10 min at 4 °C. The upper aqueous layer containing the enzyme was col-
lected for the enzymatic assay according to the manufacturer's protocol.
The GPx and GR activities were then measured at an absorbance of
340 nm using a spectrophotometer (Thermo™ Varioskan Flash, MA,
USA) at 25 °C.
Overall SOD enzyme activities were prepared according to our previ-
ous study (Kim et al., 2011b). The total SOD activities were measured
with an enzymatic method using a SOD assay kit (Sigma-Aldrich
Chemie, Switzerland). After exposures to different heavy metals, the
copepods were homogenized in ice-cold buffer (0.25 M sucrose, 0.5%
triton X-100, pH 7.5) at a ratio of 1 to 4 (w/v) using a Teflon homogeniz-
er. The homogenate was centrifuged at 30,000 g for 30 min at 4 °C. The
upper aqueous layer containing the enzyme was collected for the enzy-
matic assay according to the manufacturer's protocol. The total SOD
activities were then measured at an absorbance of 440 nm using a spec-
trophotometer at 25 °C. Enzyme activities were normalized by total
protein and represented as % of control.
2.6. Statistical analysis
Data were expressed as mean ± S.D. Significant differences were
analyzed using one-way ANOVA followed by Tukey's test. P b 0.05 was
considered significant. The SPSS ver. 17.0 (SPSS Inc., Chicago, IL, USA)
software package was used for statistical analysis.
3. Results
3.1. Analysis of heavy metal-triggered oxidative stress induction in
T. japonicus
3.1.1. Silver (Ag) exposure
The intracellular ROS and GSH levels increased concentration-
dependently in response to Ag exposure (10 and 100 μg/L) for 96 h
(Fig. 1A). Concentration-dependent inductions in response to Ag treat-
ment were also observed at all enzymes' activities in T. japonicus
(Fig. 1A).
3.1.2. Arsenic (As) exposure
Waterborne As treatment induced ROS formation at 10 μg/L treat-
ment (Fig. 1B). Slight elevation was observed in 100 μg/L of As exposure,
although statistical difference was not calculated. GSH contents were
elevated concentration-dependently in response to As treatment
(Fig. 1B). Enzymatic activities of GST, GR, and SOD were significantly in-
duced by exposure to 10 and 100 μg/L of As, while a significant induc-
tion of GPx activity was observed in the 10 μg/L of As treated group.
3.1.3. Cadmium (Cd) exposure
In the case of Cd exposure, any significant change in ROS and GSH
levels was not observed in both concentrations of Cd (10 and 100 μg/L)
(Fig. 1C). The enzymatic activities of GST and SOD were significantly
induced in the 10 and 100 μg/L of Cd-exposed groups, while a significant
induction of GPx was observed in the 100 μg/L of Cd treatment. Although
concentration-dependently increased patterns of the GR activity was
analyzed, a significant difference was not observed (Fig. 1C).
3.1.4. Copper (Cu) exposure
Both concentrations of Cu (10 and 100 μg/L) significantly increased
the formation of intracellular ROS for 96 h, and 100 μg/L of Cu treatment
induced GSH levels in T. japonicus (Fig. 1D). Significant elevations of
enzymatic activities were observed in all antioxidant enzymes, and
their levels were higher than those of other metal exposures (Fig. 1D).
3.1.5. Zinc (Zn) exposure
A significant modulation of intracellular ROS or GSH levels was not
observed in Zn-exposed T. japonicus (Fig. 1E). In the case of GST, GPx,
and SOD, enzymatic activities were significantly increased in 10 and
100 μg/L of the Zn-exposed groups, while significant induction of GR
was observed in 10 μg/L of the Zn-exposed group (Fig. 1E).
3.2. Transcriptional profiles of hsp genes after exposure to heavy metals
The mRNA expression of hsp gene families (hsp10, 20, 20.7, 40, 60, 70,
70p, 90α, and 105) was investigated in heavy metal-exposed T. japonicus
(Fig. 2), and their fold values with statistical results were incorporated
in Table 1. Most of the hsp family genes were highly upregulated after
exposure to Cu, while the hsp40 mRNA level was downregulated at
the highest concentration (100 μg/L). Most hsp transcripts were upreg-
ulated in the Zn-exposed group at relatively high concentrations
(100 μg/L).
To analyze the specific sensitivity of each hsp, hierarchical clustering
was employed for each transcriptional expression of hsps (Fig. 3). As a re-
sult, the hsp20 and hsp70 genes showed strong sensitivities for different
concentrations of five heavy metal exposures (Fig. 3B, F). However, the
transcriptional levels of hsp10 gene were downregulated by 100 μg/L of
As, Cd, and Zn exposures (Fig. 3A). In the case of hsp40 gene, 100 μg/L of
Ag, As, Cu, and Zn inhibited its transcriptional expression (Fig. 3D),
while hsp60 mRNA expression was downregulated in 100 μg/L of the Ag
and As-exposed group (Fig. 3E). Hierarchical clustering also revealed
that Cu showed strong inducible potency for hsp response.
A series of time-course analyses also represented similar patterns of
modulation and sensitivity of hsp genes (Fig. 4). The transcriptional
levels of hsp20 and hsp70 genes were highly increased in all metal-
exposed groups for 96 h, while their sensitivities were slightly different
depending on different time exposures. Overall, the hsp70 mRNA level
was quickly increased in response to the five heavy metals, and its tran-
scriptional level in each metal treatment was prolonged for 96 h com-
pared to those of other hsp expressions.
4. Discussion
The heavy metals examined in this study are essential but may be
toxic if in excess. They can catalyze the generation of intracellular
ROS, are highly toxic to most organisms without nutrient value, and
also induce oxidative stress (Valko et al., 2005). In addition, ROS causes
oxidative stress and attacks macromolecules such as DNA, protein, and
lipids within the cell, leading to aging and programmed cell death
(Valavanidis et al., 2006). In the copepod T. japonicus, the intracellular
ROS level was elevated after exposure to different concentrations of
five heavy metals. In fact, intracellular ROS production can be stimulated
by two different mechanisms via the interference of metal-related pro-
cesses and/or the generation of free radicals by metal ions (Lushchak,
2011). Therefore, we assume that certain heavy metals are potential
ROS inducers in T. japonicus. Generally, ROS can be generated by a
wide range of environmental stressors such as thermal stress, UV radia-
tion, heavy metals, or toxic pollutants (Lesser, 2006). Previously, Cd sig-
nificantly increased ROS-triggered lipid peroxidation and decreased
reduced glutathione in the mussel Crenomytilus grayanus, indicating
that Cd would induce intracellular ROS and lead to cellular damage
(Chelomin et al., 2005). Rico et al. (2009) reported that ciliated aquatic
protozoa exposed to heavy metals such as Cd, Cu, and Zn showed an in-
crease in the ROS level. Based on our results, heavy metal-triggered ROS
 B.-M. Kim et al. / Comparative Biochemistry and Physiology, Part C 166 (2014) 65–74 69

A) D)
800 800
Control Control ***

Residual activities (% of control)

Ag 10 μg/L Cu 10 μg/L
Residual activities (% of control)

700 700
Ag 100 μg/L Cu 100 μg/L

600
** 600 ** **
** **
500 500 *
**
*
400 * 400
* *
* * *
300 * 300 *
* * *
*
200 200

100 100

0 0
ROS GSH GST GR GPx SOD ROS GSH GST GR GPx SOD

B) E)
800 800
Control Control
As 10 μg/L
Residual activities (% of control)

Zn 50 μg/L

Residual activities (% of control)

700 700
As 100 μg/L Zn 250 μg/L
600
** 600 **

500
**
500
** * * *
400 400 *
* * * **
300 * 300 * *
*
200 * 200

100 100

0 0
ROS GSH GST GR GPx SOD ROS GSH GST GR GPx SOD

C)
800
Control
Cd 100 μ g/L
Residual activities (% of control)

700
Cd 1000 μ g/L

600

500
*
400 *
*
300 *
*
200

100

0
ROS GSH GST GR GPx SOD

Fig. 1. Effects of five heavy metal exposures, A) Ag, B) As, C) Cd, D) Du, and E) Zn, on intracellular ROS formation, GSH contents, and GSH-related enzyme activities in adult
T. japonicus for 96 h. Intracellular generation of reactive oxygen species (ROS) was measured using DCF-fluorescence intensity. Significant differences over control values are
indicated by symbol (* and **) on the data bar (P b 0.05 and P b 0.01) analyzed by multiple-comparison ANOVA. Data are means ± S.D. of three replicates of exposed copepods.

elevation would induce detrimental effects in T. japonicus. In addition to leading to oxidative stress with the induction of an antioxidant defense
ROS elevation observed in metal-exposed copepods, we further exam- system in T. japonicus. GSH levels as nonenzymatic antioxidants were
ined the activities of several oxidative stress-related enzymes to assess slightly increased by heavy metal (Ag, As, and Cu) exposures in this spe-
whether heavy metal exposure may modulate the antioxidant defense cies. In fact, GSH plays a key role in the intracellular protection against
system. heavy metals, and is involved in phase II metabolism in terms of redox
To recover cellular damage induced by overproduced ROS, living or- capacity through GST, GPx, and GR activities (Valko et al., 2005). GSH
ganisms have developed antioxidant defense mechanisms and the non- is also involved in metal detoxification by binding between cysteine res-
enzymatic antioxidant GSH. With increased ROS level, all the oxidative idues and metals (Meister and Anderson, 1983). Thus, differently
stress-related enzyme activities including GSH contents were elevated changed GSH contents upon different heavy metal exposures in
by different heavy metal exposures in T. japonicus. Therefore, we sup- T. japonicus would be explained with the recovering capacity of GSH
pose that heavy metals induced the formation of intracellular ROS, by the involvement of detoxification metabolism or direct depletion as
70 B.-M. Kim et al. / Comparative Biochemistry and Physiology, Part C 166 (2014) 65–74
0.5 1.0 5.0
Concentration 5 10 50 100 (µg/L)
Ag
As
Cd
Cu
Zn
Fig. 2. The mRNA expression profiles of hsp genes after exposure to different heavy metals
in adult T. japonicus. Different colors indicate fold changes of mRNA expression and each
value is designated as fold change. The 18S rRNA gene was used as a reference housekeep-
ing gene to normalize the expression. Values are means of three replicate samples and
data are shown as means ± S.D. Statistical values were deposited in Table 1.
a toxic effect of metals (Sies, 1999). Thus, the modulation of GSH may
contribute to the metal tolerance of an organism (Conners and
Ringwood, 2000; White and Cappai, 2003).
Heavy metals can directly generate ROS via redox activity, and indi-
rectly induce oxidative stress by disrupting or blocking the antioxidant
defense mechanisms in an organism (Gutierrez et al., 2003). A signifi-
cant increase of enzyme activities by heavy metal exposures may arise
due to direct ROS formation induced by these chemicals. In aquatic or-
ganisms, heavy metals are considered as inducers of intracellular oxida-
tive stress causing the transcription of certain genes and/or enzyme
activity. GST activity was increased in the liver of the roach Rutilus
rutilus exposed to Cu (Paris-Palacios et al., 2000). Wang and Wang
(2009) also showed a similar induction pattern of GST with 100 μg/L
of Cd exposure for 96 h in T. japonicus. In T. japonicus, Seo et al.
(2006b) reported that a transcript of GR would be a strong biomarker
for heavy metal monitoring. Lee et al. (2008b) also showed different
biomarker potentials of 10 GST isoenzyme genes upon exposure to sev-
eral heavy metals for 96 h. In the aquatic worm Tubifex tubifex, Cu expo-
sure showed a decrease in GST and an increase in GR after 7 days of
exposure (Mosleh et al., 2006). Mosleh et al. (2006) also indicated
that these antioxidant enzymes were involved in a defense mechanism
against Cu toxicity. In Chironomus riparius, the transcript of GPx was sig-
nificantly increased by cadmium exposure for 72 h (Nair et al., 2012). In
T. japonicus, modulation of antioxidant enzyme activities in heavy
metal-treated groups indicated that GSH may be actively involved in
the defense mechanisms against heavy metal toxicity. Compared to
other groups, the Cd-exposed group showed a relatively slight induc-
tion of activities of antioxidant enzymes in T. japonicus, indicating that
the concentrations of Cd used in this study may be weak to induce det-
rimental effect as T. japonicus was less sensitive to Cd (Lee et al., 2007).
Wang and Wang (2009) reported that 100 μg/L of Cd in T. japonicus ini-
tially inhibited GPx activity for 24 h but its activity level was recovered
at 96 h exposure. However, final Cd exposure in a 7 day exposure signif-
icantly induced GPx activity. Based on our findings, we also observed a
similar GPx activity level in the 100 μg/L of Cd-exposed copepods for
96 h, while 1000 μg/L of Cd exposure strongly induced the residual ac-
tivity of GPx. Taken together, the mode of actions of Cd can vary with
different time courses and concentrations, even within the same test or-
ganism. Overall, heavy metals can induce oxidative stress by generating
ROS and stimulating the involvement of antioxidant enzymes as de-
fense mechanisms against their toxicity.
SOD also catalyzes the dismutation of superoxide radicals to hydro-
gen peroxide and oxygen in oxidative stress conditions. In T. japonicus, a
strong response of SOD activity may be explained as an adaptation in
maintaining the homeostasis against oxidative stress. In fact, Kim et al.
(2011b) showed biomarker potentials of both transcript and enzyme
activities of SODs in response to several heavy metal exposures in
T. japonicus. Wang and Wang (2009) also showed a significant increase
of SOD activity in response to 10–100 μg/L of Cd exposure in a 12 day ex-
posure with T. japonicus. In experiments with heavy metals in the clam
Mactra veneriformis, the SOD activity was enhanced in Cd-exposed mus-
sels for 3 days (Fang et al., 2010). The activity of SOD after exposure to
Cu and Zn was significantly elevated in the African catfish, Clarias
garepinus (Farombi et al., 2007). Zhang et al. (2010b) reported that
the bivalve exposed to Zn showed a 13.3% increase in SOD activity.
Also, Cd exposure significantly increased both mRNAs of Cu/ZnSOD
and MnSOD for 24 h in C. riparius (Park et al., 2012). Thus, SODs play
an important role in cellular protection as phase I enzymes, and also cat-
alyze the conversion of superoxide to oxygen and hydrogen peroxide in
living organisms. Taken together, the involvement of SOD after expo-
sure to heavy metals provides a molecular biomarker for the indication
and monitoring of these environmental stressors. Among aquatic ani-
mals, marine intertidal invertebrates have been unexpectedly exposed
to oxidative stress because of fluctuating environmental conditions in-
cluding metal pollution. We assume, therefore, that T. japonicus has
evolved a recovery system from strong oxidative stress.
To analyze the effects of potential oxidative stress inducer on hsp ex-
pression at the molecular level, all the mRNA expressions of hsp family
were measured in different concentrations of heavy metals and in dif-
ferent time-course experiments with T. japonicus. Overall hsp mRNA ex-
pression revealed that the induced oxidative stress may lead to damage
to the cell and its protein in T. japonicus. In view of in vivo deleterious
effects of As and Cu, several parameters on reproductive and physiolog-
ical traits (nauplius phase, development time, survival, sex ratio, num-
ber of clutch, nauplii per clutch, and fecundity) were also influenced
by such heavy metals as shown in testing of two generations of
T. japonicus (Lee et al., 2008a). Thus, comprehensive modulations of
hsps would reflect the involvement of a strong defense mechanism of
T. japonicus. Hsps play a protective role against a wide range of environ-
mental stressors and act as molecular chaperone which assists in pro-
tein folding and stabilization of either oxidatively modified or partially
 B.-M. Kim et al. / Comparative Biochemistry and Physiology, Part C 166 (2014) 65–74 71

A) hsp10 B) hsp20

5 10 50 100 (µg/L) 5 10 50 100 (µg/L)

C) hsp20.7 D) hsp40

5 10 50 100 (µg/L) 5 10 50 100 (µg/L)

E) hsp60 F) hsp70

5 10 50 100 (µg/L) 5 10 50 100 (µg/L)

G) hsp70p H) hsp90 α

5 10 50 100 (µg/L) 5 10 50 100 (µg/L)

I) hsp105

5 10 50 100 (µg/L)

Fig. 3. Hierarchical clustering analysis for each hsp gene, A) hsp10, B) hsp20, C) hsp20.7, D) hsp40, E) hsp60, F) hsp70, G) hsp70p, H) hsp90α, and I) hsp105.

misfolded proteins in sub-lethal stress condition (Feder and Hofmann,
1999; Hofmann et al., 2002). In T. japonicus, small hsp family (hsp20
and hsp20.7) and large hsp family (hsp70 and hsp70p) showed signifi-
cant modulations upon heavy metal exposures, while hsp10 and hsp40
were downregulated at a high concentration (100 μg/L) of heavy
metal exposure. Small heat shock proteins (sHsps) are ubiquitous and
play an important role in protein homeostasis under stress conditions
(Seo et al., 2006a; Chen et al., 2010; Rhee et al., 2011). In the bay
scallop Argopecten irradians, the expression of small Hsp22 was up-
regulated 10 days after exposure to heavy metals (Cd, Pb, and Cu)
without dose-dependency (Zhang et al., 2010a). Hsp40 stimulates the
ATPase activity of the Hsp70 which is crucial for protein translation,
folding/unfolding, translocation, and degradation (Qiu et al., 2006).
Down-regulation of hsp40 gene was also observed in the highest concen-
tration (100 μg/L) of all metal exposures, suggesting that Hsp-mediated
cellular damage may occur at this exposure condition. Hsp60 is predom-
inantly found in mitochondria and chloroplasts of eukaryotes, and also
involved in protein folding and stress protection in these organelles
(Cechetto et al., 2000). However, hsp60 transcript did not show a signifi-
cant modulation in T. japonicus. Hsp70 and Hsp90 are cytosolic chaperons
72 B.-M. Kim et al. / Comparative Biochemistry and Physiology, Part C 166 (2014) 65–74

and ubiquitous cellular proteins that are involved in protection against di-
verse stresses (Ivanina et al., 2008). In T. japonicus, the expression of hsp20
and hsp70 genes was actively modulated by heavy metals, particularly Cu
exposure, indicating that both genes may be a good potential molecular
biomarker. In fact, we demonstrated that the T. japonicus hsp70 transcript
is a strong biomarker towards diverse environmental stressors including
heavy metals (Rhee et al., 2009). In aquatic animals, Agell et al. (2004)
reported that Hsp70 protein was significantly induced by Cu exposure
(15 μg/L for 5 days) in the Ascidian Pseudodistoma crucigaster. Planelló
et al. (2010) demonstrated that hsp70 gene was significantly increased
12 h and 24 h after 10 mM Cd exposure in the aquatic midge, C. riparius,
indicating that this gene is useful as a potential biomarker for Cd detec-
tion. In the digestive gland and gill of the Pacific oyster Crassostrea gigas,
Cd treatment (0.01 ppm for 11 days, and 0.05 or 0.1 ppm for 7 days)
increased hsp90 gene expression in a concentration- and time-dependent
manner (Choi et al., 2008). Ivanina et al. (2008) reported that Cd expo-
sure (50, 500, 2000 μM for 4 h) increased expression of hsp60 and hsp70
gene concentration-dependently but did not affect expression of hsp90
gene. Compared to other heavy metals, a relatively low modulation of
hsp gene was observed in the experiment with Cd. This tendency was
similar to the results as shown in antioxidant enzyme activity, implying
that the Cd concentration used in our study may be not enough to
induce cellular damage by oxidative stress in T. japonicus and/or Cd
may be less of an oxidative stress inducer than other heavy metals. Pre-
viously, Kwok et al. (2009) suggested that Cu resistance would be de-
veloped through multigeneration acclimation against elevated Cu
concentrations (0 to 100 μg/L) with a trade-off in fitness cost. Based
on our results, we suggest that metal-triggered energetic budget for

A)
20 hsp10
hsp20
Relative mRNA expression

hsp20.7
15 hsp40
hsp60
hsp70
hsp70p
10 hsp90a
hsp105

0
0 6 12 24 48 72 96

Exposure time (h)

B)
20 hsp10
hsp20
Relative mRNA expression

hsp20.7
15 hsp40
hsp60
hsp70
hsp70p
10 hsp90a
hsp105

0
0 6 12 24 48 72 96

Exposure time (h)

C)
20 hsp10
hsp20
Relative mRNA expression

hsp20.7
15 hsp40
hsp60
hsp70
hsp70p
10 hsp90a
hsp105

0
0 6 12 24 48 72 96

Exposure time (h)

Fig. 4. Time-course analysis of hsp transcript modulations after exposure to 100 μg/L of different heavy metals, A) Ag, B) As, C) Cd, D) Cu, and E) Zn, for 96 h. The 18S rRNA gene was used as a
reference housekeeping gene to normalize the expression. Values are means of three replicate samples and data are shown as means ± S.D.
 B.-M. Kim et al. / Comparative Biochemistry and Physiology, Part C 166 (2014) 65–74 73

D)
20 hsp10
hsp20

Relative mRNA expression

hsp20.7
15 hsp40
hsp60
hsp70
hsp70p
10 hsp90a
hsp105

0
0 6 12 24 48 72 96
Exposure time (h)
E)
20 hsp10
hsp20
Relative mRNA expression

hsp20.7
15 hsp40
hsp60
hsp70
hsp70p
10 hsp90a
hsp105

0
0 6 12 24 48 72 96
Exposure time (h)

Fig. 4 (continued).

the trade-off in fitness costs would be strongly associated with molecu-
lar defense and cellular repair metabolism in T. japonicus.
In conclusion, heavy metals induced oxidative stress by generating
ROS and led to the synthesis of antioxidant enzymes for cellular protec-
tion in T. japonicus. Hsp genes such as Hsp20 and hsp70 are useful as
potential biomarkers in the monitoring of heavy metals. Our finding
demonstrates that Cu acts as a stronger oxidative stress inducer than
other heavy metals in the copepod T. japonicus.
Acknowledgments
We thank Prof. Hans-U. Dahms for his comments on the manuscript,
and also thank two anonymous reviewers for their valuable comments
on the manuscript. This work was supported by a grant of the
National Research Foundation (NRF) funded to Young-Mi Lee (No.
2011-0071218). This work was supported by a grant of the R&D program
of the Ministry of Land, Transportation and Marine Affairs of Korea
(2013) and was also supported by a grant (2012R1A2A2A02012617)
of the National Research Foundation funded to Jae-Seong Lee.
References
Agell, G., Turon, X., Caralt, S.D., Lopez-Legentil, S., Uriz, M.J., 2004. Molecular and organism
biomarkers of copper pollution in the ascidian Pseudodistoma crucigaster. Mar. Pollut.
Bull. 48, 759–767.
Bonaventura, R.,Poma, V.,Russo, R.,Zito, F.,Matranga, V., 2006. Effects of UV-B radiation on
development and hsp70 expression in sea urchin cleavage embryos. Mar. Biol. 149,
79–86.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.
72, 248–254.
Cechetto, J.D.,Soltys, B.J.,Gupta, R.S., 2000. Localization of mitochondrial 60 kD heat shock
chaperonin protein (Hsp60) in pituitary growth hormone secretory granules and
pancreatic zymogen granules. J. Histochem. Cytochem. 48, 45–56.
Chang, L.W., Suzuki, T., 1996. Toxicology of Metals. Lewis Publishers, p. 1198.
Chelomin, V.P.,Zakhartsev, M.V., Kurilenko, A.V., Belcheva, N.N., 2005. An in vitro study of
the effect of reactive oxygen species on subcellular distribution of deposited cadmium in
digestive gland of mussel Crenomytilus grayanus. Aquat. Toxicol. 73, 181–189.
Chen, J., Feige, M.J., Franzmann, T.M., Bepperling, A., Buchner, J., 2010. Regions outside the
α-crystallin domain of the small heat shock protein Hsp26 are required for its dimer-
ization. J. Mol. Biol. 398, 122–131.
Choi, Y.K., Jo, P.G., Choi, C.Y., 2008. Cadmium affects the expression of heat shock protein
90 and metallothionein mRNA in the Pacific oyster, Crassostrea gigas. Comp. Biochem.
Physiol. C 147, 286–292.
Choresh, O., Loya, Y., Müller, W.E.G., Wiedenmann, J., Azem, A., 2004. The mitochondrial
60 kDa heat shock protein in marine invertebrates: biochemical purification and mo-
lecular characterization. Cell Stress Chaperones 9, 38–47.
Conners, D.E., Ringwood, A.H., 2000. Effects of glutathione depletion on copper cytotoxicity
in oyster (Crassostrea virginica). Aquat. Toxicol. 50, 341–349.
Fang, Y.,Yang, H.,Wang, T.,Liu, B.,Zhao, H.,Chen, M., 2010. Metallothionein and superoxide
dismutase responses to sublethal cadmium exposure in the clam Mactra veneriformis.
Comp. Biochem. Physiol. C 151, 325–333.
Farombi, E.O.,Adelowo, O.A.,Ajimoko, Y.R., 2007. Biomarkers of oxidative stress and heavy
metal levels as indicators of environmental pollution in African cat fish (Clarias
gariepinus) from Nigeria Ogun River. Int. J. Environ. Res. Public Health 4, 158–165.
Feder, M., Hofmann, G., 1999. Heat-shock proteins, molecular chaperones, and the stress
response: evolutionary and ecological physiology. Annu. Rev. Physiol. 61, 243–282.
Gutierrez, J.C., Martin-Gonzalez, A., Diaz, S., Ortega, R., 2003. Ciliates as a potential source
of cellular and molecular biomarkers/biosensors for heavy metal pollution. Eur. J.
Protistol. 39, 461–467.
Haslbeck, M., 2002. sHsps and their role in the chaperone network. Cell. Mol. Life Sci. 59,
1649–1657.
Hofmann, G.E., Buckley, B.A., Place, S.P., Zippay, M.L., 2002. Molecular chaperones in
ectothermic marine animals: biochemical function and gene expression. Integr.
Comp. Biol. 42, 808–814.
Ivanina, A.V., Cherkasov, A.S., Sokolova, I.M., 2008. Effects of cadmium on cellular protein
and glutathione synthesis and expression of stress proteins in eastern oyster,
Crassostrea virginica Gmelin. J. Exp. Biol. 211, 577–586.
Kim, R.-O.,Rhee, J.-S.,Won, E.-J.,Lee, K.-W.,Kang, C.-M.,Lee, Y.-M.,Lee, J.-S., 2011a. Ultravi-
olet B retards growth, induces oxidative stress, and modulates DNA repair-related
74 B.-M. Kim et al. / Comparative Biochemistry and Physiology, Part C 166 (2014) 65–74
gene and heat shock protein gene expression in the monogonont rotifer, Brachionus
sp. Aquat. Toxicol. 101, 529–539.
Kim, B.-M., Rhee, J.-S., Lee, J., Lee, Y.-M., Lee, J.-S., 2011b. Cu/Zn- and Mn-superoxide
dismutase (SOD) from the copepod Tigriopus japonicus: molecular cloning and
expression in response to environmental pollutants. Chemosphere 84, 1467–1475.
Kwok, K.W.,Grist, E.P., Leung, K.M., 2009. Acclimation effect and fitness cost of copper re-
sistance in the marine copepod Tigriopus japonicus. Ecotoxicol. Environ. Saf. 72,
358–364.
Lee, K.-W., Raisuddin, S., Hwang, D.-S., Park, H.G., Lee, J.-S., 2007. Acute toxicities of trace
metals and common xenobiotics to the marine copepod Tigriopus japonicus: evalua-
tion of its use as a benchmark species for routine ecotoxicity tests in Western Pacific
coastal regions. Environ. Toxicol. 22, 532–538.
Lee, K.-W., Raisuddin, S., Hwang, D.-S., Park, H.G., Dahms, H.U., Ahn, I.-Y., Lee, J.-S., 2008a.
Two-generation toxicity study on the copepod model species Tigriopus japonicus.
Chemosphere 72, 1359–1365.
Lee, K.-W., Raisuddin, S., Rhee, J.-S., Hwang, D.-S., Yu, I.-T., Lee, Y.-M., Park, H.G., Lee, J.-S.,
2008b. Expression of glutathione S-transferase (GST) genes in the marine copepod
Tigriopus japonicus exposed to trace metals. Aquat. Toxicol. 89, 158–166.
Lee, J.-S.,Rhee, J.-S.,Kim, R.-O.,Hwang, D.-S.,Han, J.,Choi, B.-S.,Park, G.S.,Kim, I.-C.,Park, H.G.,
Lee, Y.-M., 2010. The copepod Tigriopus japonicus genomic DNA information
(574 Mb) and molecular anatomy. Mar. Environ. Res. 69, S21–S23.
Lesser, M.P., 2006. Oxidative stress in marine environments: biochemistry and physiolog-
ical ecology. Annu. Rev. Physiol. 68, 253–278.
Livak, K.J.,Schmittgen, T.D., 2001. Analysis of relative gene expression data using real time
quantitative PCR and the 2−ΔΔCt method. Methods 25, 402–408.
Lushchak, V.I., 2011. Environmentally induced oxidative stress in aquatic animals. Aquat.
Toxicol. 101, 13–30.
Meister, A., Anderson, M.E., 1983. Glutathione. Annu. Rev. Biochem. 52, 711–760.
Mosleh, Y.Y.,Paris-Palacios, S.,Biagianti-Risbourg, S., 2006. Metallothioneins induction and
antioxidative response in aquatic worm Tubifex tubifex (Oligochaeta, Tubificidae) ex-
posed to copper. Chemosphere 64, 121–128.
Nair, P.M., Park, S.Y., Choi, J., 2012. Characterization and expression analysis of phospholipid
hydroperoxide glutathione peroxidase cDNA from Chironomus riparius on exposure to
cadmium. Comp. Biochem. Physiol. B 163, 37–42.
Paris-Palacios, S.,Biagianti-Risbourg, S.,Vernet, G., 2000. Metallothionein analyzed in liver
of Rutilus rutilus exposed to Cu with three methods: metal summation, SH determi-
nation and original spectrofluorimetric method. Comp. Biochem. Physiol. C 126,
122–133.
Park, S.-Y.,Nair, P.M.,Choi, J., 2012. Characterization and expression of superoxide dismut-
ase genes in Chironomus riparius (Diptera, Chironomidae) larvae as a potential bio-
marker of ecotoxicity. Comp. Biochem. Physiol. C 156, 187–194.
Phillips, D.J.H., 1977. The use of biological indicator organisms to monitor trace metal
pollution in marine and estuarine environments — a review. Environ. Pollut. 13,
281–317.
Planelló, R.,Martínez-Guitarte, J.L.,Morcillo, G., 2010. Effect of acute exposure to cadmium
on the expression of heat-shock and hormone-nuclear receptor genes in the aquatic
midge Chironomus riparius. Sci. Total Environ. 408, 1598–1603.
Pulido, M.D., Parrish, A.R., 2003. Metal-induced apoptosis: mechanisms. Mutat. Res. 533,
227–241.
Qiu, X.B., Shao, Y.M., Miao, S., Wang, L., 2006. The diversity of the DnaJ/Hsp40 family, the
crucial partners for Hsp70 chaperones. Cell. Mol. Life Sci. 63, 2560–2570.
Raisuddin, S., Kwok, K.W.H., Leung, K.M.Y., Schlenk, D., Lee, J.-S., 2007. The copepod
Tigriopus: a promising marine model organism for ecotoxicology and environmental
genomics. Aquat. Toxicol. 83, 161–173.
Rhee, J.-S.,Raisuddin, S.,Lee, K.-W.,Seo, J.S.,Ki, J.-S.,Kim, I.-C.,Park, H.G.,Lee, J.-S., 2009. Heat
shock protein (Hsp) gene responses of the intertidal copepod Tigriopus japonicus to
environmental toxicants. Comp. Biochem. Physiol. C 149, 104–112.
Rhee, J.-S.,Kim, R.-O.,Choi, H.-G.,Lee, J., Lee, Y.-M.,Lee, J.-S., 2011. Molecular and biochem-
ical modulation of heat shock protein 20 (Hsp20) gene by temperature stress and hy-
drogen peroxide (H2O2) in the monogonont rotifer, Brachionus sp. Comp. Biochem.
Physiol. C 154, 19–27.
Rhee, J.-S.,Yu, I.T.,Kim, B.-M.,Jeong, C.-B.,Lee, K.-W.,Kim, M.-J.,Lee, S.-J.,Park, G.S.,Lee, J.-S.,
2013. Copper induces apoptotic cell death through reactive oxygen species-triggered
oxidative stress in the intertidal copepod Tigriopus japonicus. Aquat. Toxicol. 132/133,
182–189.
Rico, D., Martin-Gonzalez, A., Diaz, S., de Lucas, P., Gutierrez, J.C., 2009. Heavy metals gen-
erate reactive oxygen species in terrestrial and aquatic ciliated protozoa. Comp.
Biochem. Physiol. C 149, 90–96.
Roesijadi, G., Robinson, W.E., 1994. Metal regulation in aquatic animals: mechanism of
uptake, accumulation and release. In: Malins, D.C., Ostrander, G.K. (Eds.), Aquatic
Toxicology, Molecular, Biochemical and Cellular Perspectives. Lewis Publishers,
London.
Seo, J.S., Lee, Y.-M., Park, H.G., Lee, J.-S., 2006a. The intertidal copepod Tigriopus japonicus
small heat shock protein 20 gene (Hsp20) enhances thermotolerance of transformed
Escherichia coli. Biochem. Biophys. Res. Commun. 340, 901–908.
Seo, J.S.,Lee, K.-W.,Rhee, J.-S.,Hwang, D.-S.,Lee, Y.-M.,Park, H.G.,Ahn, I.-Y.,Lee, J.-S., 2006b.
Environmental stressors (salinity, heavy metals, H2O2) modulate expression of gluta-
thione reductase (GR) gene from the intertidal copepod Tigriopus japonicus. Aquat.
Toxicol. 80, 281–289.
Sies, H., 1999. Glutathione and its role in cellular functions. Free Radic. Biol. Med. 27,
916–921.
Tomanek, L.,Somero, G.N., 2002. Interspecific- and acclimation-induced variation in levels
of heat-shock proteins 70 (hsp70) and 90 (hsp90) and heat-shock transcription
factor-1 (HSF1) in congeneric marine snails (genus Tegula): implications for regula-
tion of hsp gene expression. J. Exp. Biol. 205, 677–685.
Valavanidis, A.,Vlahogianni, T.,Dassenakis, M.,Scoullos, M., 2006. Molecular biomarkers of
oxidative stress in aquatic organisms in relation to toxic environmental pollutants.
Ecotoxicol. Environ. Saf. 64, 178–189.
Valko, M., Morris, H., Cronin, M.T., 2005. Metals, toxicity and oxidative stress. Curr. Med.
Chem. 12, 1161–1208.
Wang, M.H., Wang, G.Z., 2009. Biochemical response of the copepod Tigriopus japonicus
Mori experimentally exposed to cadmium. Arch. Environ. Contam. Toxicol. 57,
707–717.
White, A.R., Cappai, R., 2003. Neurotoxicity from glutathione depletion is dependent on
extracellular trace copper. J. Neurosci. Res. 71, 889–897.
Zhang, L., Wang, L., Song, L., Zhao, J., Qiu, L., Dong, C., Li, F., Zhang, H., Yang, G., 2010a. The
involvement of HSP22 from bay scallop Argopecten irradians in response to heavy
metal stress. Mol. Biol. Rep. 37, 1763–1771.
Zhang, Y., Song, J., Yuan, H., Xu, Y., He, Z., 2010b. Concentrations of cadmium and zinc in
seawater of Bohai bay and their effects on biomarker responses in the bivalve Chlamys
farreri. Arch. Environ. Contam. Toxicol. 59, 120–128.