RSC Advances

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The assessment of the eco-toxicological effect of

Cite this: RSC Adv., 2018, 8, 22777
gabapentin on early development of zebrafish and
its antioxidant system
Xiuwen Li,a Shuangxi Zhou,a Yuting Qian,a Zhuoran Xu,a Yang Yu, a
Yanhua Xu,a
Yide He*ab and Yongjun Zhang*a

Gabapentin (GAB) is an emerging contaminant that is frequently detected in water bodies across the globe.
The present study used zebrafish as a model organism to investigate the effects of GAB on the early
development of zebrafish and on its antioxidant system. Acute toxicity tests indicated that the 96 h LC50
value of GAB for zebrafish embryos was 59.9 g L 1. Further, it was observed that GAB causes
malformation of embryos such as hemagglutination and pericardial edema. Compared to the control
group, a significant enhancement (p < 0.05) of heartbeat rates was found at GAB concentrations
exceeding 50 mg L 1, while the swimming frequency was clearly increased upon exposure to GAB at
1
a concentration of 100 mg L (p < 0.05). Additionally, the development of the zebrafish embryo was
also negatively impacted after exposure to GAB as demonstrated by significantly decreased body
1
lengths. Exposure to GAB at concentrations exceeding 50 mg L significantly influenced the
development of zebrafish, leading to malformation of organs and abnormal movements. Although no
significant developmental effects of GAB were observed at environmentally relevant concentrations (0.1
and 10 mg L 1), further research about the antioxidant system confirmed that severe oxidant injury
happened inside the organisms. catalase (CAT), lactate dehydrogenase (LDH), glutathione S-transferase
(GST), glutathione (GSH) and the ability of inhibition of hydroxyl radicals (IHR) were used as biomarkers in
the present study to illustrate GAB toxicity at environmentally relevant concentrations. The results
Received 18th May 2018
Accepted 2nd June 2018
showed that activities of CAT, LDH and GST as well as IHR were all elevated after GAB exposure, which
proved that ROS were formed in the body as derived from GAB exposure. Among all of these
DOI: 10.1039/c8ra04250k
biomarkers, CAT was the most sensitive one to evaluate the influence of GAB, and showed a significant
[Link]/rsc-advances increase even at a very low exposure concentration (0.1 mg L 1).

a very low removal efficiency was oen obtained.3 For instance,

1. Introduction R. Gurke et al. detected 56 PPCPs in the inuent and effluent of
The vast use of pharmaceuticals and personal care products German STPs, and found that the removal efficiency of GAB was
(PPCPs) has caused their widespread distribution and frequent only 6.4%.9 In Minnesota, researchers sampled 24 STP effluents,
detection in the global aquatic environment. For example, and many kinds of PPCPs were detected, among which the
researchers detected one or more of these chemicals in 80% of concentration of GAB varied in the ng L 1 range.10 The low
the American streams sampled during 1999–2000. Gabapentin removal efficiency in STPs leads to its frequent detection in
(GAB) is one of the antiepileptic drugs, which are used for the surface waters worldwide. In the UK and America, GAB has been
treatment of partial seizures and neuropathic pain.1 In 2009, detected at the concentration level of ng L 1.11,12 In Vidy Bay,
Germany consumed 58.9 t GAB, which increased to 73.3 t in Switzerland, GAB has been even detected at a concentration of
2012.2,3 GAB was also widely used in many Asian countries, like 400 ng L 1 in raw drinking water.13 The frequent occurrence of
China, India, Singapore, and so on.4–6 GAB is not easily GAB in aquatic systems raises concerns about its ecotoxicity,
metabolized in biota and the human excretion rate is therefore but to the best of our knowledge, hardly any studies on the
very high.7,8 When released to sewage treatment plants (STPs), ecotoxicity of GAB can be found in literature.
In the present study, zebrash (Danio rerio) was used as
a model animal. In comparison with traditional animal models,
a
School of Environmental Sciences and Engineering, Nanjing Tech University, Jiangsu, zebrash offers many advantages, such as small size, short
211816, P. R. China. E-mail: heyd@[Link]; [Link]@[Link]; Tel:
generation time, large numbers of eggs in one spawning, rapid
+86-25-58139656
b
Hunan Provincial Key Laboratory of Renewable Energy Electric-Technology,
development of embryos and so on.14–16 In addition, the
Changsha, Hunan, 410076, P. R. China embryos and larvae of zebrash are transparent,17,18 so it is

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RSC Advances Paper

relatively easy to observe adverse effects such as abnormalities by means of a division plate. Next day, when the light period
induced by toxicants. In the recent 20 years, zebrash has been started, the division plate was removed, and aer an hour,
widely used in toxicity tests.16–20 The sh embryo toxicity (FET), embryos were collected and washed several times with the
a standardized OECD test, is the most popular alternative embryonic culture medium water, containing 2 mM CaCl2-
means to live sh in acute test.21 In their whole life circle, $2H2O, 0.5 mM MgSO4$7H2O, 0.75 mM NaHCO3 and 0.08 mM
embryo and larva stages are the most sensitive ones to chem- KCl (ISO 6341-1982).34
icals.22–25 Before hatching, the embryos are surrounded by
a chorion for about 72 h, which may provide a protective barrier 2.3. Acute toxicity test
between the developing embryos and chemicals.26,27 The It was reported that the NOEC value of GAB was 10 g L 1.12
present study extended the exposure period to 192 hpf (hours However, there was no acute toxicity testing to calculate the
post fertilization) covering more of the important develop- LC50 of GAB for zebrash embryo. For general acute toxicity
mental stages and thus providing more information about the testing, embryos were exposed in test solutions, containing 50,
ecotoxicity of GAB. 56, 60, 63, 67, 71 g L 1 GAB for a 96 hours period starting from
Fish exposed to the environmental contaminates exhibit 12 hpf. This exposure concentrations were decided by a series
sensitive antioxidant defences, which were oen used as bio- pre-test. A negative control with water and a positive control
logical indicators of aquatic environmental health.28 The anti- with 4 mg L 1 3,4-dichloroaniline were used in the present
oxidant enzymes, including catalase (CAT) and lactate study. 12-well cell culture clusters (Corning Incorporated, made
dehydrogenase (LDH), represent a rst line of defence against in USA) were chosen as the test chamber. 10 viable eggs were put
external oxidant stress,29 while the glutathione system, in one well with 4 mL medium solution. Three duplicates were
including glutathione S-transferase (GST) and glutathione prepared for each test. The culture clusters were covered with
(GSH) represent the second line.30 Besides, several non- lids to prevent evaporation during exposure, and kept in the
enzymatic mechanisms can also indicate antioxidant incubator at a temperature of 28  0.5  C, pH at 7.11  0.05,
defences, such as the ability of inhibition of hydroxyl radicals conductivity at 293.6  2.4 ms cm 1, and a light and dark cycle of
(IHR).31 The liver is where these antioxidant enzymes are 14/10 hours. In this semi-static exposure test, 3 mL medium per
produced in order to eliminate the reactive oxygen species (ROS) well was renewed daily. Dead embryo were removed and
in organism.32 However, larvae at 96 hpf as used in the present recorded every 24 hours.
study were too tiny to isolated liver tissue, so whole larvae were
used to prepare samples for the enzyme activity tests. 2.4. Developmental toxicity test
This experiment was designed and modied referring to the
2. Materials and methods guideline of OECD TG 236, OECD TG 210 and OECD TG 212.35–37
2.1. Chemicals The concentration gradient of GAB was set as 0.1 mg L 1, 10 mg
L 1, 0.1 mg L 1, 1 mg L 1, 10 mg L 1, 50 mg L 1 and
GAB (CAS 60142-96-3, purity > 98%) was purchased from 100 mg L 1, plus the positive control and the negative control
Aladdin Industrial Corporation. NaCl (CAS 7647-14-5, purity > with no GAB. Three replicates were performed with every group.
99.5%) was purchased from Xilong Scientic Co., Ltd. CaCl2- The cultivation condition were kept the same as in case of the
$2H2O (CAS 10035-04-8, purity > 99.0%) was purchased from acute toxicity test.
Sinopharm Chemical Reagent Co., Ltd. MgSO4$7H2O (CAS Embryos/larvae were observed at the following points in
10034-99-8, purity > 99.0%), NaHCO3 (CAS 144-55-8, purity > time: 6 hpf, 8 hpf, 12 hpf, 24 hpf, 48 hpf, 72 hpf, 96 hpf, 120 hpf,
99.5%) and KCl (CAS 7447-40-7, purity > 99.5%) were purchased 144 hpf, 168 hpf, 192 hpf with a biological inverted microscope
from Shanghai Lingfeng Chemical Reagent Co., Ltd. The CAT
(Nikon 120c, Japan) or a stereo microscope (Jiangnan JSZ6S,
kit (Cat. no: KGT017), the GSH kit (Cat. no: KGT006), the GST kit China) and photos were taken. Hatching rate was calculated
(Cat. no: KGT005), the LDH kit (Cat. no: KGT02448), the$OH kit daily. The apical performance of each tested embryos included:
(Cat. no: KGT010) and the Bradford protein quantitation assay coagulation of embryos, no somite formation, non-detachment
kit (Cat. no: KGT801) were purchased from KeyGEN bio TECH of the tail, and no heartbeat. The deformities included:
Co., Ltd. pigmentations; deformity of yolk, pericardial edema; yolk sac
edema; hemagglutination; tail deformation; swim bladder
2.2. Zebrash husbandry and egg collection defects.38,39
Adult wild-type zebrash (Tübingen line) was purchased from In order to observe the effects of GAB on the development of
Nanjing YSY Biotech Company, and acclimatized in our labo- zebrash, several endpoints were used to identify the variation
ratory for at least one month before use in the experiments. The between test groups and control group, as shown in Table 1.40,41
husbandry methods followed the standard guidance described
in details elsewhere.33 Adult zebrash were maintained in an 2.5. Biomarkers in antioxidant system test
incubator at 28  0.5  C with a 14/10 h light and dark cycle. They From 12 hpf to 96 hpf, embryos in the tested groups were
were fed three times daily with live baby brine shrimp for 5 exposed to 0.1 mg L 1, 10 mg L 1 and 1 mg L 1 GAB, representing
minutes. On the day before spawning, one male and one female a realistic environmental concentration, the worst case envi-
were transferred into the breeding box, and they were separated ronmental level and a pharmaceutically relevant concentration

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Paper RSC Advances

Table 1 Endpoints used in the present study

Time (hpf) Endpoints Approaches

48 Heartbeat rate Heartbeats during 30 s were counted under a microscope

72 Body length Photos of larvae were taken and measured by Jifei soware
144 Swimming frequency The times of spontaneous swimming behavior in 30 s were recorded under
a microscope
192 Dry weight Weighed by balance aer drying for 24 h at a temperature of 60  C

respectively. Three replicates were used in every group. The 3.2. Heartbeat rates and movements
cultivation conditions were kept the same as those for the acute
The effects of GAB on the heartbeat rate and the swimming
toxicity tests described above. At the end of exposure, 96 hpf
frequency of zebrash embryos and larvae are illustrated in
larvae and embryos were killed on ice, homogenized in 0.6%
Fig. 3. A clear increment of heartbeat rate can be found along
NaCl using a Hand-held cell crusher (MP FastPrep-1, America), the increasing concentrations of GAB (Fig. 3(a)). A signicant (p
and centrifuged at 6000 rpm for 10 min at 4  C. The superna-
< 0.05) increment appeared at the exposure level of 50 mg L 1
tants were collected for enzyme testing following the instruc-
GAB, resulting in 5.1% increase as compared to the control
tions of those kits.
group. The increment of heartbeat rates became more severe at
the highest concentration of 100 mg L 1.
In the developmental process of zebrash, heart is the rst
2.6. Statistical analyses organ to form, which was visible because of the transparent
All data were analyzed with PASW Statistics 18, including the development of zebrash.42 The heartbeat of zebrash develops
focus on one-way ANOVA of 48 hpf heart beats; 72 hpf body to be regular at around 36 hpf43 and this parameter is directly
length; 144 hpf swimming frequency; 192 hpf dry weight as well linked to the temperature.44 The values measured in this study
as all kinds of antioxidant biomarkers. The signicance level were determined at 30  C and at 48 hpf. The heartbeat rate in
was set at p < 0.05. the control group is about 72  1.6 beats per 30 s, and showed
a distinguishable tendency of increasing with the exposure
concentration, which indicated that heartbeat rate can be used
2.7. Live subject statement as a sensitive indicator in this test.
This study was performed in strict accordance with the Labo- Within ecotoxicity assessment of drugs the heartbeat rate at
48 hpf is oen used as an indicator to assess adverse effects of
ratory Animal—Guideline for ethical review of animal welfare
pollutants, and various publications showed signicant
(GB/T 35 892-2018), and was approved by Animal Care and Use
changes in tested groups.43,45 Interestingly, another two
Committee in School of Environmental Science and Technology
psychotropic drugs, carbamazepine and valproic acid
of Nanjing Tech University (Nanjing, China).
commonly used for the treatment of epilepsy, induced a signif-
icant and concentration-dependent decrease of the heartbeat
rate in a laboratory test,46 which was exactly opposite to the
3. Results and discussion result of the present study. It was reported that when rats were
3.1. Mortality and LC50 exposed to gabapentin, the heartbeat rate was elevated too.47
The mortality of zebrash was assessed once every 24 h. The This nding showed that GAB not only increased the heartbeat
dead embryos mostly showed coagulation, which was followed rate of zebrash, but also of mammals.
by a lack of somite formation or heartbeat while no embryo The swimming frequency was slightly increased from
showed non-detachment of the tail (Fig. 1). 10 mg L 1 to 100 mg L 1, and showed a signicant elevation at
In the acute toxicity test, all of embryos exposed to 67 g L 1
GAB died during 96 h exposure, while the negative control and
the positive control own the death rate of 0 and 100%. This
result validated the quality of embryos. Aer 96 h exposure,
LC50 of 59.9 g L 1 was calculated (Fig. 2).

Fig. 1 Appearance of dead embryos: (1) normal, (2) coagulation, (3)

lack of somite formation, (4) lack of heartbeat. Fig. 2 Correlation between zebrafish embryo death rate and log C.

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3.3. Body length and dry weight

The effect of GAB on the body length of zebrash is illustrated
in Fig. 4. There was no signicant decent in body length, but
a reducing tendency was observable. The body length was
reduced by 2.7% at 10 mg L 1, 2.0% at 50 mg L 1, and 2.8% at
100 mg L 1, as compared with the control group. Nevertheless,
the dry weight of larvae of all test groups showed no signicant
changes: 100  6.7 mg per larva.
Body length is associated with risk-related behavior in
zebrash,54 and it is a useful and intuitional parameter in the
experiment. Carbamazepine, an antiepileptic drug, which has
a longer history in human use than GAB, induced an increase of
body length.55 However, in the present study, exposure to GAB
caused a concentration-dependent decrease of body length of
zebrash at 72 hpf. This strongly indicates that GAB has adverse
effects on the development of zebrash. Besides body length,
Fraysse et al. used a similar parameter, tail length, to assess
toxicity of chemicals. The result of their study illustrated tail
length is shorten following exposure to pollutants.56 Dry weight
as another important physiological index was measured at 192
hpf, the end of the exposure period. The dry weight showed no
obvious difference among the control group and all test groups.
This might partly be due to the fact that the animals were not
fed during the whole experiment.

3.4. Morphology of the embryos

The morphology of zebrash from the embryos to the juveniles'
development stage was observed at 6 hpf, 8 hpf, 12 hpf, 24 hpf,
48 hpf and 192 hpf. Only up till 48 hpf, morphological malfor-
mations were observed in the tested groups. The physical
appearance before 24 hpf is shown in Fig. 5.
Fig. 3(a) The effect of GAB on the heartbeat rate at 48 hpf of zebrafish At 48 hpf, malformations, including pericardial edema and
embryos, (b) swimming frequency at 144 hpf of zebrafish larvae hemagglutination were visible, whereas pericardial edema was
exposed to GAB of different concentrations. The result of one-way
ANOVA is *p < 0.05, **p < 0.01 and ***p < 0.001. the common malformation in the present study, that was
observed in every test group (Fig. 6). Pericardial edema is the
most popular indicator in zebrash toxicity test, which is for

100 mg L 1 (p < 0.05). Testing of sh behavior is a great tool for

ecotoxicological studies, because it interprets the exerted
complex physiological effects of pollutants at the individual
level.48 Among different kinds of behavior of zebrash, swim-
ming plays an important role including swimming speed,
acceleration, routine turns, swimming bouts, the shape of body
wave and swimming frequency.49–51 In the present study, the
swimming frequency was measured at 144 hpf. At this time
point, larvae became active and the swimming behavior were
easy to be observed. The result of the test showed a whole
tendency upon increasing exposure concentration, which is
consistent with the result of the heartbeat rate test. It seems that
GAB activated zebrash and this might be caused by the
mechanism action of GAB to the nervous system of zebrash.52
In the drug toxicity testing of gabapentin, Wolf et al.53 found
that the children became hyperactive and explosive outbursts
consisting of aggressive and oppositional behavior.
Fig. 4 Body length of 72 hpf zebrafish exposed to different concen-
trations of GAB.

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Paper RSC Advances

Fig. 5 The physical appearance of zebrafish embryos from 0.5 hpf to

24 hpf.

Fig. 7 IHR in zebrafish at 96 hpf. The results of one-way ANOVA are

indicated at the significance levels **p < 0.01 and ***p < 0.001.

oen be used as a biomarker for evaluate toxicity of chemicals.59

The activities of LDH in zebrash exposed to environmentally
relevant concentrations GAB were mainly at the same level as in
the control groups. However, when the concentration was
increased up to 1 mg L 1, activity of LDH was signicant
increased (p < 0.001), as shown in Fig. 8(b).
GSH is an essential reducing chemical, whose concentration
is always regulated at a stable level when the antioxidant system

Fig. 6 The malformations of zebrafish at 48 hpf. HE: hemagglutina-

tion, PE: pericardial edema.

instance also deemed as the most sensitive indicator for ethanol

exposure in zebrash.57

3.5. Antioxidant biomarkers

ROS was produced during the use of oxygen in the metabolism
process, among which hydroxyl radicals ($OH) were deemed as
being the most damaging ROS.29 The activity of IHR in the body
can indirectly reect the content of $OH and explain the
oxidative stress derived from environmental pollutants. In the
present study, aer exposure, the IHR in zebrash was signi-
cantly elevated at 10 mg L 1 (p < 0.01) and 1 mg L 1 (p < 0.001), as
shown in Fig. 7. This results indicated that when the zebrash
exposed to oxidative stress, they tried to protect themselves
from tissue injury by increasing the activity of antioxidant
enzymes as a compensatory mechanism.
CAT can convert H2O2 to H2O and O2 very efficiently, so
accumulation of superoxide chemicals will enhance CAT
activity.58 Compared to the control groups, the activities of CAT
in all test groups were elevated, and the higher the exposure
concentration, the higher CAT activity. Even at the lowest
concentration (0.1 mg L 1) the change was highly signicant (p <
0.001), as shown in Fig. 8(a).
LDH is an essential enzyme in the anaerobic pathway of Fig. 8 Activity of CAT and LDH in zebrafish at 96 hpf. The result of
energy production, and can indicate oxidative stress, so that one-way ANOVA is in all cases: ***p < 0.001.

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RSC Advances Paper

is normal.60 When organisms face oxidant pressure, over- was activated. Carbamazepine caused the elevation of CAT
whelming amount of oxidizing substances will be formed. GST activity in rainbow trout aer long term exposure.62 Valproic
can convert these harmful substances with GSH to full acid is commonly used for the treatment of epilepsy, and was
detoxication. Besides, GST can also reduce organic perox- found to inhibit the CAT activity in rat, which contradicts the
ides.61 As shown in Fig. 9, the activity of GST was signicantly results of the present study.63 Opposed to CAT, the change of
increased aer GAB exposure, especially at high exposure LDH activity was not statistically signicant in environmental
concentrations. At the lowest concentration, such elevation was concentration level used in this study. LDH activity is however
not obvious, when the concentration was however increased to still a key indicator of the oxidative stress as for instance shown
the worst case environmental level of 10 mg L 1, this change in a study with the fresh water sh, Cyprinus carpio L. the
became remarkable and kept increasing. toxicity of carbamazepine (5.97 mg L 1) was tested and in which
Environmental pollution may induce the production of ROS, it was found that the LDH activity was signicantly increased.64
which will further cause the elevation of antioxidant enzymes as Elevated LDH activity suggests that the aerobic catabolism of
a defense mechanism. The present study demonstrated that the glycogen and glucose has shied towards the formation of
ability of IHR, and the activity of CAT, LDH and GST were lactate, which may induced toxicity to sh.65 Increased LDH
enhanced by the treatment of GAB. The ability of IHR demon- activity also inuences the muscles, which may indicate
strated that mass $OH formed in the body. To avoid oxidant damage with regard to heartbeat rate.66 The heartbeat rate and
injury, the anti-oxidant system took measures to scavenge so the swimming frequency recoded at 48 hpf and 144 hpf were all
many $OH. CAT was the most sensitive indicator in the present elevated, which may have an underlying connection with LDH.
study, and showed a signicant increase even at a very low To protect the body from the damage induced by pollutants, the
concentration (0.1 mg L 1). The enhanced CAT enzymatic activity of GST is usually increased,67 and the present study
activity reects the oxidative stress that occurred in sh. To conrmed that GST activity is closely related to the exposure
withstand such stress, the anti-oxidative capacity of zebrash concentration. Elevated GST is able to eliminate harmful
substances in the body with GSH.

4. Conclusions
The present study found that the 96 h LC50 value of GAB was
59.9 g L 1, so GAB does not induce acute toxic effects at low
exposure concentrations as neither mortality nor malformation
of zebrash embryos. Parameters like heartbeat rate and body
length illustrated however that GAB is capable of inducing
developmental toxicity to zebrash embryos. GAB can affect the
cardiac function, and the body length of zebrash. Further
research about the antioxidant system of zebrash found that
even at a very low concentration of GAB leads to oxidant injury
in zebrash. CAT, GST and IHR were found to be sensitive
biomarkers in such toxicity tests. LDH not only indicated the
effect of GAB on the antioxidant system, but is also able to
clarify the mechanism of enhancement of heartbeat rate and
swimming frequency. These results draw our attention to
possible adverse effects of GAB in the environment and help
scientists as well as regulators to prepare measures to reduce
the adverse environmental effects of GAB.

Conflicts of interest
There are no conicts to declare.

Acknowledgements
This work was nancially supported by Natural Science Foun-
dation of Jiangsu Province (No. BK20160989), Hunan Provincial
Key Laboratory of Renewable Energy Electric-Technology (No.
2016ZNDL003), Major Research Program of Natural Science of
Fig. 9 Activity of GST and content of GSH in zebrafish at 96 hpf. The University in Jiangsu Province (No. 16KJA610002), and Cultiva-
results of one-way ANOVA are **p < 0.01 and ***p < 0.001. tion Project for International Cooperation from Nanjing Tech

22782 | RSC Adv., 2018, 8, 22777–22784 This journal is © The Royal Society of Chemistry 2018
Paper RSC Advances

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