Research Article NBU J. Anim. Sc.

12: 37-43 (2018)

ISSN 0975-1424

CYP1A SPECIFIC ETHOXYRESORUFIN-O-DEETHYLASE (EROD) ACTIVITY IN

DANIO RERIO EXPERIMENTALLY EXPOSED TO SUBLETHAL DOSE OF
CHLORPYRIFOS

Swati Singh1, Soumen Bhattacharjee2 and Min Bahadur1*

1
Genetics and Molecular Biology Laboratory, Department of Zoology, University of North Bengal,
P.O North Bengal University, District Darjeeling, West Bengal, India-734013
2
Cell and Molecular Biology Laboratory, Department of Zoology, University of North Bengal, P.O
North Bengal University, District Darjeeling, West Bengal, India-734013

Received: August 17, 2018 Revised & Accepted: December 06, 2018

ABSTRACT
Danio rerio is an useful experimental model to evaluate the environmental impact of pollutants like inorganic pesticides
in the aquatic environment. In the present study, the Ethoxyresorufin-O-Deethylase (EROD) activity was studied in
Danio rerio subjected to sublethal dose of chlorpyrifos in the laboratory. The assay monitors the induction of the
cytochrome P450 (CYP) 1A, a widely used biomarker for exposure to pesticides in fishes. A sub lethal dose of 96 µg/L
was selected for the treatment of the fishes and the response was recorded accordingly. The data were recorded at 24hr,
48hr, 72hr and 96 hr and analyzed statistically using one way analysis of variance (ANOVA). EROD activity was
significantly (p<0.001) induced in the treated groups (24 hr, 234.84; 48 hr, 396.42; 72hr, 489.07; 96 hr, 258.63) than control
showing highest induction at 72 hrs of treatment. There was an increase in the activity of the enzyme proportionate to
the duration. The data further supports the fact that measurement of EROD activity is a useful biomarker for the
detection of pesticide contamination in fishes.

Keywords: Xenobiotic, Chlorpyrifos, Pesticide, Cytochrome P450, EROD activity, enzyme, substrate, CYP1A, Danio
rerio

INTRODUCTION mortality (Murthy et al. 2013).

Pesticide usage is a critical concern which
Fish, as a bioindicator species, plays an
causes adverse effects in the environment because
increasingly important role in the biomonitoring of
of their toxicity, persistency and tendency to bio-
water pollution because they are highly sensitive to
concentrate in the organisms. While the pesticides
changes in the aquatic environment (Subramanian
are instrumental in achieving significant increase in
and Amutha, 2006a). Sudden fish mortality is
crop productivity, they also cause serious hazards
indicative of the acute level of toxicity due to
to the environment as well as non-target animals
pollutants. Sublethal levels of pollutants following
especially the fish, which forms an important part
chronic exposure produce adverse physiological
of food chain for various animals including human
effects and tissue damage in vital organs including
beings (Sharma and Ansari, 2011). Chlorpyrifos is a
gill, liver, intestine, and kidneys, which can be
synthetic organophosphate with non-systemic
measured by biochemical, physiological, or
anticholinesterase activity having contact, gastro-
histological studies in fish (Mondon et al., 2001;
intestinal and respiratory effects. Chlorpyrifos is
Subramanian and Amutha, 2006b).
extremely toxic to fish and affects fish health through
impairment of metabolism, sometimes leading to Different biotransformation enzymes have

*Author for correspondence: Min Bahadur, E. Mail: bahadurmin@rediffmail.com

37
 Min Bahadur
been used as biomarkers of several xenobiotics,
converting them into more hydrophilic metabolites,
thus facilitating their excretion (van der Oost et al.,
2003). Cytochrome P450s (CYP450) form a diverse
multigene family of heme-containing proteins that
oxidize, hydrolyze, or reduce compounds by inserting
an oxygen atom into the substrate during the reaction
cycle (Nebert et al., 1993; Nelson et al., 1996).
CYP1A isoenzymes catalyse the O-dealkylation of
7-ethoxyresorufin, a reaction that is commonly used
to assess their activity. For this reason, the
ethoxyresorufin-O-deethylase (EROD) is also used
to indicate CYP1A enzymes and/or their activity.
In fish, the sub-family CYP1A in particular, has been
widely used as environmental biomarkers since their
activity is induced by several groups of
environmental contaminants, including pesticides
(Banni et al., 2011). EROD has an important
function in the biotransformation of many xenobiotics
and its specific induction is considered as a molecular
marker in fish for exposure to Polycyclic Aromatic
Hydrocarbons (PAH) and planar halogenated
hydrocarbons (PHH), which are usually aryl
hydrocarbon receptor agonists (Amutha et al., 2009,
Stegeman and Hahn 1994; Bucheli and Fent 1995;
Jewett et al., 2002).
Aquatic ecosystems are increasingly being
used as dumping grounds for a variety of pollutants,
so this study was designed to investigate the
cytochrome P450 1A specific EROD activity on
exposure to chlorpyrifos using Danio rerio as fish
model in the laboratory.
MATERIALS AND METHODS
Insecticide: The chlorpyrifos 20% EC was
purchased from the local agrochemicals supplier
(Dursban, Trade Mark of Dow AgroSciences LLC,
USA).
Test animal and the dose: Danio rerio having
an average body weight of 289 mg and a total body
length of 2.55 cm was used in the present study.
38
The fish specimens were collected from local
hatchery, stocked in laboratory at appropriate density
in 20 litre glass aquaria containing de-chlorinated
tap water and were acclimatized in this condition
for at least 10 days before they were subjected to
investigations. The fish were fed with commercial
artificial feed (pellets) equal to 1/10th of their body
weight. Renewal of water, clearing of unconsumed
feed and fecal wastes were done daily by siphoning
out 80% of water and refilling with fresh purified
water and the aquaria screens were also cleaned
daily (Sreedevi et al., 2014). The test fishes were
starved for 24 hrs before the start of the experiment
(OECD, 1992).
The 96 hr LC50 of Chlorpyrifos for the fish,
Danio rerio was 289 µg/L based on our previous
report (Singh et al., 2017). One third of the LC50
dose was selected (96 µg/L) for the experiment and
the fishes were treated with this dose.
EROD Assay: Fish collected from the site was
sacrificed and tissue (gills) was collected for analysis
of CYP1A specific EROD activity. The tissue was
washed in 0.15 M KCl, weighed and homogenized
in 5-fold volume of buffer (0.25 M sucrose, 0.1 M
Tris, 1mM EDTA, pH 7.4) and centrifuged for 15
min at 9560 x g at 4ºC. The supernatant (S9 fraction)
consisting of both microsomal and cytosolic fractions
was harvested and used immediately to determine
the enzyme activity (Guang-hua et al., 2011).
CYP1A specific EROD activity was quantified
spectrophotometrically following the method of
Koltz et al., (1984). The reaction mixture consisted
of 2 µM 7-ethoxyresorufin, 0.1 M tris buffer (pH
7.4) and 100-200 µg of sample (S9 fraction). After
pre-incubation at 37oC for 5 min the reaction was
initiated by the addition of (0.01 M) NADPH and
the reaction was stopped following 5 minutes of
incubation at the same temperature with 0.5ml of
ice cold methanol. The amount of resorufin formed
was measured at 572 nm. The same was quantified
using a standard curve of resorufin.
 Cyp1A Specific Ethoxyresorufin-o-deethylase Activity in Danio rerio

Protein Estimation: Protein was estimated as duration of exposure to chlorpyrifos. The maximum
described by Lowry et al., (1951) using bovine activity was recorded at 72 hr. At 96hr of the
serum albumin as standard. treatment, the enzyme activity declined to 258.63
pmol resorufin/min/mgPr, however the difference
Statistical Analysis: Statistical analyses were
was statistically significant (p<0.001) with respect
performed using KyPlot version 2.0 beta 15 (32 bit).
to the control. The EROD activity showed an
Data was analyzed using one way analysis of
increasing trend upto 72 hr of treatment after which
variance (ANOVA), where P>0.05 was considered
the activity started to decline (Fig. 1).
non -significant. All data are presented as Mean ±
Standard Error (SE). DISCUSSION
The main advantage of using a biomarker in
RESULTS
real scenario is to find early adverse effects of
In the present study, the control samples had
chemical or other stress exposure allowing the
a basal activity of 112.98 pmol resorufin/min/mgPr.
adoption of preventive or mitigation measures before
The results showed an EROD activity of 234.84
the effects become expressed at high levels of
pmol resorufin/min/mgPr, 396.42 pmol resorufin/min/
biological organization. It also gives information on
mgPr, 489.07 pmol resorufin/min/mgPr, 258.63 pmol
mechanisms of toxicity providing the explanatory
resorufin/min/mgPr at 24hr, 48hr, 72 hr and 96hr,
basis for effects observed at higher levels of
respectively. Analysis using one way ANOVA
biological organization. Fish is an important
between 24 hr, 48hr and 72 hr treated groups
bioindicator of pollution to xenobiotics in the aquatic
showed statistically significant (p<0.001) increase
ecosystem. Studies of Pluta (1993) revealed that
in the activity of the enzyme proportionate to the

Fig 1: EROD activity in Danio rerio exposed to sublethal dose ( 96 µg/L ) of chlorpyrifos in the laboratory (***
significant at p<0.001).

39
 Min Bahadur
CYP1A (measured as the induction of hepatic
ethoxyresorufin-O-deethylase (EROD) activity) is
highly sensitive to the presence of pollutants, clearly
responds to concentration, reproducible, and thus, it
is a consistent biomarker. Monitoring programs of
industrial effluents and water bodies in northern
Germany have used hepatic mono-oxygenase
activity in rainbow trout, flounder (Platichthys
felsus), and dab (Limanda limanda) (Pluta, 1993).
Polychlorinated dibenzodionixines (PCBs),
polycyclic aromatic hydrocarbons (PAHs),
polychlorinated dibenzodioxines (PCDDs) and
polychlorinated dibenzodifurans (PCDFs) are well-
established inducers of CYP1A and EROD,
however, induction by pesticides have also been
reported (Haluzova et al., 2011). Enzyme activity
increases with increase in exposure period to the
xenobiotics as it brings about the physiological
differences in the organism (Arellano-Aguilar et al.
2009).
Significantly high EROD activity after 72 hr
of the chlorpyrifos exposure in the present study is
in accordance with those found by Binelli et al.,
(2006) exposed to PCB in Zebra mussel (Dreissena
polymorpha). The EROD activity increased with
time, probably due to EROD’s higher detection
ability. Pacheco and Santos (1997) showed that
Polycyclic Aromatic Hydrocarbons and Resin Acids
are able to induce a significant increase of total
EROD activity after 3 days of exposure in Anguilla
anguilla L. It was also found that the maximum
EROD activity occurred at 3 days of beta-
naphthoflavone (BNF) exposure, while exposure
beyond 3 days, EROD activity does not increase
which is in concurrence to our study. Recently,
Danion et al., (2014) reported a significantly high
liver EROD activity after 21 days of exposure to
water-soluble fraction (WSF) of Arabian crude oil,
in contaminated fish than in control.
In fish, CYP1A activity has been widely used
as a biomarker since it is induced by several common
40
contaminants (Whyte et al., 2000; Rees et al.,
2003; Billiard et al., 2004). Fish CYP1A activity
has been found to be involved in organophosphate
pesticides metabolism (Flammarion et al., 1998;
Kitamura et al., 2000; Schlenk, 2005). The increase
of EROD activity, indicating the involvement of this
enzyme in fenitrothion (organophosphate)
biotransformation has been reported by Almeida et
al., (2010) and showed a significantly increased liver
EROD activity in seabass juveniles exposed to 1
mg/L (2.6 folds of induction), 2 mg/L (3.3 folds of
induction) and 4 mg/L (4.1 folds of induction)
indicating the involvement of this enzyme in the
biotransformation of fenitrothion in this marine fish.
Hepatic EROD activity of Oreochromis
mossambicus was examined in response to
naphthalene. After exposure to lower naphthalene
concentrations up to 6 ppm, no marked change in
activity was noted, whereas at higher concentrations
EROD activity was shown to be considerably high.
EROD activity does not follow a conventional
saturation curve with the increase of inducer
concentration, but reaches a maximum and then
declines which has also been found in the present
study (Rodman et al., 1989; Hahn et al., 1993;
Schmitz et al., 1995; Kennedy et al., 1996;
Verhallen et al., 1997). Bhutia et al., (2015) also
reported a similar situation when Channa punctatus
was treated with sub-lethal concentration of
cypermethrin for a period of 5, 10 and 15 days, the
total CYP450 content and CYP1A mediated EROD
activity was significantly induced (p<0.05) in all the
treated groups.
CONCLUSION
The EROD activity as a biomarker for
pesticide pollution was demonstrated in the
laboratory. EROD is a means to study the levels of
the CYP1A enzyme activity. The results show
proportionate increase in the enzyme activity to the
maximum with respect to the duration of exposure
upto 72hr and thereafter declines. This study further
supports the fact that EROD is a useful biomarker
 Cyp1A Specific Ethoxyresorufin-o-deethylase Activity in Danio rerio

for the detection of the pesticide contamination in Infrastructure programme (FIST), Department of
fishes. Science and Technology, New Delhi, India and the
Special Assistance Programme (SAP), University
ACKNOWLEDGEMENTS
Grants Commission, New Delhi.
The Department of Biotechnology,
Government of West Bengal, EN 24, Sector V, Salt CONFLICT OF INTEREST
Lake, Kolkata 700091 is sincerely acknowledged The authors report no declaration of interest.
for funding the Project (Sanction no. 241/Sanc)-BT
FUNDING
(Estt.) RD-19/13 dated 04.03.2014). Authors are
This work is supported by the funding agency
also thankful to the Head, Department of Zoology
Department of Biotechnology, Government of West
for providing the Departmental Central Instrument
Bengal (Sanction no. 241/Sanc)-BT (Estt.) RD-19/
facility which is supported by the Fund for
13 dated 04.03.2014).
Improvement of Science and Technology

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