CHEM. RES. CHINESE UNIVERSITIES 2011, 27(3), 413—416
Effects of PAHs on Biotransformation
Enzymatic Activities in Fish
LU Guang-hua, CHEN Wei*, LI Ying and ZHU Zhi
Key Laboratory of Integrated Regulation and Resources Development of Shallow Lakes, Ministry of Education,
College of Environment, Hohai University, Nanjing 210098, P. R. China
Abstract Biotransformation and detoxification responses to the exposure to five polycyclic aromatic hydrocarbons
were investigated in crucian(Carassius auratus). Juvenile crucian were treated with a single intraperitoneal injection
of each compound at dosages of 0.1, 1.0, 2.0, 5.0 and 8.0(or 10.0) mg/kg and sacrificed 15 d later to determine
7-ethoxyresorufin-O-deethylase(EROD) and glutathione S-transferases(GST) activities in gill S9 fractions. EROD
activity is significantly increased by benzo(b)fluoranthene and indeno(1,2,3-cd)pyrene at all the doses. High dosages
of PAHs induced GST activity and the inducing ability of them increased in the following order: fluorene<
fluoranthene<indeno(1,2,3-cd)pyrene<benzo(g,h,i)perylene<benzo(b)fluoranthene. In all the cases, dose dependence
appeared to exist. The gill EROD and GST in Carassius auratus are useful biomarkers to estimate sub-acute toxicity
of both polycyclic aromatic hydrocarbons(PAHs) and PAHs-like compounds.
Keywords Polycyclic aromatic hydrocarbon; Gill; 7-Ethoxyresorufin-O-deethylase activity; Glutathione S-transferases activity
Article ID 1005-9040(2011)-03-413-04
1
Introduction
Polycyclic aromatic hydrocarbons(PAHs) are a group of
chemicals that are formed during the incomplete burning of
coal, oil and gas, garbage or other organic substances. They can
also be found in substances such as crude oil, coal, creosote and
road/roofing tar[1]. Benzo(b)fluoranthene(BbF), indeno(1,2,3-cd)
pyrene(IP), benzo(g,h,i)perylene[B(ghi)P], fluoranthene(Fluo)
and fluorene(Fl) are included in the “priority pollutants” list
developed by the United States Environmental Protection
Agency and considered as possible human or animal carcinogens[2]. China suffers serious PAHs contamination from the
combustion of fossil fuel and biomass. PAHs contamination
issue in China has received much attention of local authorities
and scientists, and several investigations on the distribution and
levels of PAHs in soil, sediment and water have been carried
out, showing that the concentration of PAHs vary markedly
from hundreds to tens of thousands ng/g dried surface soil or
sediment and from hundreds to thousands ng/L in river or lake
waters[3―10]. It was suggested that the levels of PAHs commonly found in many aquatic environments are an important
risk factor for various health aspects of fish[11].
In the aquatic environment, the exposure of living organisms to xenobiotics leads to interactions between the chemical
and the biological system, and may give rise to biochemical
disturbances or/and adaptive responses[12]. The biological responses are termed biomarkers and the fundamental assumption
upon which they are based centers on some compromised bio-
chemical process(biomarker) due to pollutant exposure[13].
Biomarkers can be used to assess the health status of organisms
and to obtain early-warning signals of environmental risks[14].
Various biochemical parameters in fish have been tested for
their responses to toxic substances and their potential use as
biomarkers of exposure or effect[15].
Crucian(Carassius auratus) is distributed widely in
freshwaters throughout China and has been demonstrated to be
a very sensitive species in the studies of biotransformation and
oxidative stress responses[16,17]. This work aims to investigate
the gill biochemical responses to PAHs, measured as 7ethoxyresorufin-O-deethylase(EROD) activity and glutathione
S-transferases(GST) activity, to study their dose-response relationships on Carassius auratus, and to identify effective biomarkers for PAHs exposure. Our study can provide important
information for the early diagnosis of PAHs pollution in aquatic
environment and the ecological risk assessment of PAHs and
PAHs-like compounds.
2
2.1
Experimental
Chemicals
BbF, IP, B(ghi)P, Fluo, Fl, nicotinamide adenine dinucleotide phosphate(NADPH), 3,3′-methylenebis-(4-hydroxycoumarin), 1-chloro-2,4-dinitrobenzene(CDNB), resorufin, ethoxyresorufin and glutathione(GSH) were purchased from Sigma
Chemical Company(St. Louis, MO, USA) and the stated purities were >99.9%. Tris(hydroxymethyl aminomethane) and KCl
———————————
*Corresponding author. E-mail: [email protected]
Received June 2, 2010; accepted September 21, 2010.
Supported by the National Natural Science Foundation of China(No.51079049) and the Crucial Special Project of National
Water Pollution Control and Management Science of China(No.2008ZX07421-002).
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CHEM. RES. CHINESE UNIVERSITIES
were purchased from Nanjing Sunshine Biotechnology Co.,
Ltd.(Nanjing, China) and their purities were >99%. Bovine
serum albumin was purchased from Shanghai Huixing
Biochemistry Reagent Co., Ltd.(Shanghai, China) and the purity was >98%. Coomassie brilliant blue G-250(ultra pure grade)
was purchased from Sinopharm Chemical Reagent Co.,
Ltd.(Shanghai, China). All other chemicals were of analytical
grade and were obtained from Nanjing Chemical Reagent Co.,
Ltd.(Nanjing, China).
2.2
In vivo Exposure
Juvenile crucian weighed approximately 40 g were obtained from Nanjing Institute of Fishery Sciences(China). The
fish were acclimatized for two weeks in dechlorinated municipal water prior to experimentation. Crucian were injected with
0.1, 1.0, 2.0, 5.0 and 8.0(or 10.0) mg/kg PAHs dissolved in
corn oil on the first day. Control fish received corn oil only. The
dose ranges chosen were non-lethal based on the results of a
pilot study(unpublished data) and the lowest two dosages were
based on the observed fish burdens in the field in China[18,19].
Fish were weighed before injection to determine the volume of
dosage per kilogram body mass of each fish. Fish were kept in
groups of three in 30-L glass tanks containing dechlorinated
municipal water under constant aeration and were not fed
throughout the experiment. A change of 50% of water was performed every other day. Water temperatures ranged from 20 °C
to 22 °C.
2.3
Vol.27
triplicate and expressed as units(U) per mg of protein. A U is a
picomol(for EROD) or nanomol(for GST) of substrate hydrolyzed per minute. Protein concentrations were determined with
the Coomassie Protein Assay Kit[24], with bovine serum albumin as standard.
2.4
Statistical Analysis
All the data were checked for normality via normal probability plots. For each biomarker, data were expressed as
mean±standard deviation(SD). Data from different treatments
were compared by a one-way analysis of variance(ANOVA)
and statistical different treatments were identified by Dunnett’s
t test. All differences were considered significant at P<0.05.
Statistical analyses were performed with the SPSS statistical
package(ver. 12.0, SPSS Company, Chicago, IL, USA).
3
Results and Discussion
The in vivo effects of BbF and IP on gill EROD are presented in Figs.1 and 2. EROD activity was significantly induced by all the BbF dosages used(P<0.05)(Fig.1), and the
increased level of EROD activity matched the dosage increase.
EROD activity was also significantly increased by all IP tested
doses(P<0.05)(Fig.2). However, the highest IP dosage(10.0
mg/kg) resulted in a significant decrease of EROD activity as
compared with that of 5.0 mg/kg(P<0.05).
Enzyme Assays
For each treatment and control, three fish were killed by
cervical transection on 15th post-injection. The choice of exposure time was based on a previous finding that maximal
EROD induction by 1 mg/kg or 100 mg/kg benzo(a)pyrene
(BaP) occurs on the 14th day in crucian liver[20]. Gills were
removed individually and used for the analysis of EROD and
GST activities. Gills were washed in 0.15 mol/L KCl, weighed,
homogenized in 5-fold volume of buffer(0.25 mol/L sucrose,
0.1 mol/L Tris-HCl, 1 mmol/L EDTA, pH=7.4) and centrifuged
for 15 min(9560g). The gill S9 fraction, consisting of both microsomal and cytosolic fractions, was obtained and stored at
–80 °C.
EROD activity was quantified using 96-well plates, as described by Chen et al.[21]. The reaction mixture consisted of 140
μL of buffer(0.1 mol/L Tris, 0.15 mol/L KCl, pH=8.0), 10 μL of
2 μmol/L 7-ethoxyresorufin and 10 μL of S9 fraction. The reaction was initiated at 25 °C by the addition of 40 μL of 2.1
mg/mL NADPH. The optical density values were determined at
572 nm. GST activity was measured via the general methodology described by Habig and Jakoby[22] adapted to a microplate
reader[23]. The reaction mixture consisted of 100 μL of 0.1
mmol/L potassium phosphate, 10 μL of 1.0 mmol/L CDNB, 10
μL of 1.0 mmol/L GSH and 880 μL of H2O. Reaction mixture
of 150 μL and S9 fraction of 10 μL were incubated at 30 °C for
30 s. H2O instead of S9 fraction was used in the blank wells.
The increase in absorbance was recorded at 340 nm for 2 min
at 12 s intervals. Both enzymatic activities were determined in
Fig.1
EROD and GST activities after exposure to
BbF for 15 d
Bars not sharing a common letter(a, b, c, d for EROD and a′, b′, c′,
d′ for GST) are significantly different from one another(P<0.05).
Fig.2 EROD and GST activities after exposure to
IP for 15 d
Bars not sharing a common letter(a, b, c, d for EROD and a′, b′, c′,
d′ for GST) are significantly different from one another(P<0.05).
P450 induction is primarily due to the transcriptional
activation of the gene. But it can also be caused by posttranscriptional regulation or post-translational regulation[25,26].
No.3
LU Guang-hua et al.
The mechanism by which cells recognize inducers and transmit
information to genes is well understood in the case of the
members of subfamily CYP1A, which are induced by PAHs
and their halogenated forms[27]. Usually, EROD activity is analyzed in liver microsomes[28―30]. As in liver organ, CYP1A is
also induced in fish gills following exposure to PAH-type contaminants. Mdegela et al.[31] compared EROD activities in gill
filaments and liver tissue of African sharptooth catfish(Clarias
gariepinus) and found the induction of EROD activities was
stronger in the gills than in the liver following exposure to waterborne BaP. Similar results were obtained in terms of CYP1A
induction in rainbow trout(Oncorhynchus mykiss) by exposure
to waterborne BaP and indigo[32]. Ortiz-Delgad et al.[33] compared xenobiotic CYP1A induction in liver, gills, and excretory
kidney of sea bream, Sparus aurata exposed to different concentrations of BaP or 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) via water for 20 d. Although their results in principal
confirm the notion of the liver as the major metabolic organ in
fish, they also provide evidence for substantial metabolic potential in gills. Gill EROD activity in juvenile crucian was significantly induced by 5 mg/kg of IP(4.2-fold) and BbF(2.4-fold)
in this study. This suggests that the gill is important for the
biotransformation of lipid-soluble xenobiotics, not only those
reaching the gill via the water, but also those via the blood[34].
The gill therefore seems to be an appropriate tissue to provide
biomarkers for xenobiotic exposure.
The responses of gill GST exposed to five PAHs are presented in Figs.1―3, respectively. Both BbF and IP at 0.1 mg/kg
did not induce GST activity compared to the controls, but they
did significantly increase GST activity at higher doses(P<0.05)
(Figs.1 and 2). However, GST activity at the highest exposure
dosages was not significantly different from that at 5 mg/kg for
both BbF and IP. From Fig.3, low dosages of Fluo and
B(ghi)P(0.1 and 1 mg/kg) did not induce obvious effects on
GST, while GST activity was significantly induced by exposure
to higher dosages of the two chemicals. However, the highest
dosage of Fluo resulted in a significant reduction of GST
activity. GST activity was only significantly altered by exposure to high dosages of Fl(5.0 and 10.0 mg/kg).
Fig.3 GST activity after respectively exposure to
Fl, Fluo and B(ghi)P for 15 d
* Indicate the values that are significantly higher than control
values(P<0.05).
The metabolites formed by phase I biotransformation are
conjugated via phase II enzymes(e.g., GST) before excretion.
GST may play an important role in detoxifying strong
415
electrophiles having toxic, mutagenic and carcinogenic properties by catalysing the conjugation of the tripeptide glutathione
with the xenobiotic in the phase II of the biotransformation
process promoting its elimination from the organism[35]. The
present results suggest that EROD induced by IP and GST induced by Fluo exhibited bell-shaped dose-response curves.
However, the reduction of GST activity by BbF and IP at 10
mg/kg is not significant. Therefore, higher concentrations need
to be tested to check if the dose-response curve is bell-shaped.
Bell-shaped curves have been reported on EROD activity or
CYP1A induction for various non-fish in vitro systems after
exposure to PAHs[36―38]. Although the mechanism that results
in decreased EROD or GST induction has not been completely
defined, it is likely that high concentrations of the inducer inhibit or inactivate the induced enzyme[39].
In order to compare the biochemical disturbances of different PAHs, the observed highest-fold increase of EROD and
GST activities were calculated(Table 1). The results show that
the order of inducing EROD activity was BbF<IP, and the order
of the ability of inducing GST activity is: Fl<Fluo<IP<
B(ghi)P<BbF.
Table 1
Data of fold increases of EROD and GST
EROD
Compound
GST
Fold
induction
Dose/(mg·kg–1)
Fold
induction
Dose/(mg·kg–1)
4.20
2.43
5
8
1.43
2.11
5
5
B(ghi)P
1.86
10
Fluo
1.42
5
Fl
0.61
10
IP
BbF
Although it is known that fish rapidly metabolize PAHs to
intermediates, recent studies have reported PAHs accumulation
in fish organs. The total contents of PAHs in fish muscles
ranged from 12.85 ng/g to 34.89 ng/g wet weight in the Gomti
river, India[40], from 1.91 ng/g to 224.03 ng/g wet weight in the
Pearl river delta[18], and from 184 ng/g to 194 ng/g dry weight
in Hong Kong[19]. Banni et al.[41] treated immature sea bream
by the intraperitoneal injection of BaP(20 mg/kg) and quantified the bioaccumulation of BaP. The highest accumulation in
the liver was 5.55 μg/g dry weight after exposure for 6 h. On
the basis of this result, the internal exposure levels to induce
enzymatic activities in crucian might be converted from the
treated dosages in this study. The PAHs levels in fish producing
maximal induction were approximately from 1.39 to 2.78 μg/g
dry weight, while the lowest BbF and IP concentrations resulting in significant EROD alternation were equivalently 28 ng/g
dry weight. It was suggested that the levels of PAHs commonly
found in fish could pose an environmental health risk.
The results in this study demonstrate that exposure to
PAHs each results in significant increases in gill EROD and
GST activities in Carassius auratus. This is consistent with
previous findings in the same and different species of fish
treated intraperitoneally by BaP, β-naphthoflavone(βNF) and
phenanthrene. BaP displayed strong induction potency of liver
microsomal EROD and cytosolic GST and a significant increase of the DNA damage on liver cell nuclei in sea bream[41].
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CHEM. RES. CHINESE UNIVERSITIES
The EROD response determined in gill filaments is dosedependent in rainbow trout exposed to βNF[42]. In addition,
phenanthrene was found to cause a dose-dependant increase of
EROD activity in liver S9 fractions in tilapia(Oreochromis
mossambicus)[43]. Microsomes contain phase I oxidative enzymes only, whereas S9 fractions contain the full complement
of phase I and II enzymes. Thus, S9 fractions are generally
used to provide more complete information regarding the metabolism of the tested compound. In addition, the use of S9
fractions is convenient and relatively cheap compared to using
microsomal fractions.
12, 331
[14]
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Conclusions
Clear relationships between the activity of gill EROD and
GST and exposure dosage of selected PAHs were found in this
study. The EROD and GST activities in Carassius auratus were
confirmed as useful biomarkers of exposure to both PAHs and
PAHs-like compounds. In addition, biotransformation enzymes
in gill S9 fractions were as sensitive to PAHs exposure as those
in liver. However, this study investigated only fish biochemical
responses to the exposure to individual PAHs. In actual water
bodies, aquatic organisms are exposed to the mixtures of multiple pollutants simultaneously. Further researches should be
carried out in the field to establish Carassius auratus as a
suitable biomonitoring species in fresh waters.
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