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Evaluation of developmental toxicity and
genotoxicity of aqueous seed extract of Croton
tiglium L. using zebrafish
Thangal Yumnamcha, Maibam Damayanti Devi, Debasish Roy & Upendra
Nongthomba
To cite this article: Thangal Yumnamcha, Maibam Damayanti Devi, Debasish Roy & Upendra
Nongthomba (2020): Evaluation of developmental toxicity and genotoxicity of aqueous
seed extract of Croton�tiglium L. using zebrafish, Drug and Chemical Toxicology, DOI:
10.1080/01480545.2019.1708094
To link to this article: https://doi.org/10.1080/01480545.2019.1708094
Published online: 06 Jan 2020.
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RESEARCH ARTICLE
Evaluation of developmental toxicity and genotoxicity of aqueous seed extract
of Croton tiglium L. using zebrafish
Thangal Yumnamchaa, Maibam Damayanti Devia, Debasish Royb and Upendra Nongthombab
aDepartment of Life Sciences, Manipur University, Canchipur, India; bDepartment of Molecular Reproduction, Development and Genetics,
Indian Institute of Science, Bangalore, India
ABSTRACT
Croton tiglium L. has been used in Ayurvedic and Chinese herbal medicinal formulations from ancient
times. Although its seeds are widely prescribed as traditional medicine, there is a dearth of information,
regarding its toxic effects, and the mechanisms underlying its toxicity. This study aims to investigate
the developmental toxicity and genotoxicity of the aqueous seed extract of C. tiglium L. (AECT) in
zebrafish. We have examined the effects of AECT on the early embryonic development of zebrafish.
Zebrafish embryos, treated with different concentrations of the AECT, suffered embryonic lethality and
displayed various developmental defects. The 96h-LC50 of AECT was found to be 162.78mg/ml.
Interestingly, the developmental abnormalities observed, such as pericardial edema (PE), yolk sac
edema (YSE), spinal curvature (SC), and delayed hatching, varied in severity, in a dose-dependent man-
ner. Zebrafish embryos, treated with different concentrations of AECT, exhibited exaggerated cell death
in the anatomical regions of brain, heart, and trunk. Our data suggest that the phenomenon of apop-
tosis is probably responsible for both embryonic lethality and developmental toxicity in zebrafish
embryos. Furthermore, the genotoxic potential of the AECT, in vivo, was evaluated using micronucleus
assay and comet assay, on the peripheral blood of zebrafish. The results suggest that AECT has the
potential to cause genotoxicity in the peripheral blood of zebrafish.
ARTICLE HISTORY
Received 22 September 2019
Revised 7 December 2019
Accepted 14 December 2019
KEYWORDS
Genotoxicity; developmental
toxicity; apoptosis; Croton
tiglium; zebrafish;
pericardial edema
1. Introduction
Remedies obtained from medicinal plants are being increas-
ingly adopted by the masses over the past decade, as people
believe that natural medicines are much safer than synthetic
drugs (Khan et al. 2011). However, the notion, that herbal
medicines are totally safe, is not only misleading, but wrong
as well (Ekor 2013). In fact, it is becoming clear that they can
have toxic side-effects in animals, humans included. Previous
studies have reported that different herbs, and herbal prod-
ucts, can cause various types of toxicity, including genotoxic-
ity (Hwang et al. 2013; Yumnamcha et al. 2014; Ortiz et al.
2016), developmental toxicity (Randriamampianina et al.
2013; Yumnamcha et al. 2015), reproductive toxicity (Riet-
Correa et al. 2011; Wu et al. 2016), and hepatotoxicity
(Teschke 2014a, 2014b, 2015). Moreover, there have been
reports of increase in cases of poisoning, following consump-
tion of herbal medicines (Ekor 2013). Therefore, in-depth toxi-
cological evaluation of any medicinal plant, before it enters
widespread usage, is crucial, so as to ensure that it is safe for
consumption, and also mitigate the health risk to the public
(Zhou et al. 2013). Croton tiglium L. is a shrub native to South
East Asia, and belongs to the family Euphorbiaceae. As per
records, this plant has been used to treat various disorders in
humans since ancient times (Morimura 2003; Tsai et al. 2004;
Pal et al. 2014). Phytochemical analysis of the aqueous seed
extract of C. tiglium, performed using chemical methods, has
revealed the presence of saponins, alkaloids, phenolic com-
pounds, tannins, triterpenoids, and carbohydrates
(Yumnamcha et al. 2014). Moreover, the presence of phorbol
esters and crotonic acid in the seeds of C. tiglium has also
been reported (El-Mekkawy et al. 2000; Pal et al. 2014).
Different preparations of this plant extract have been shown
to have anti-tumour, anti-HIV, and anti-inflammatory proper-
ties (Sinsinwar et al. 2016). It is also useful in the manage-
ment of constipation, as a laxative (Pal et al. 2014), and in
the treatment of dermatophytosis (Lin et al. 2016). Despite its
evidently widespread use, the toxicological aspects of this
plant have remained largely unexplored. Therefore, the pre-
sent study aims to investigate the developmental toxicity
and genotoxicity of the aqueous seed extract of C. tiglium
(AECT) using zebrafish as the model.
2. Materials and methods
2.1. Chemicals
Acridine orange (AO), phosphate buffered saline (PBS), nor-
mal melting agarose (NMA), low melting agarose (LMA) ethyl-
enediaminetetraacetic acid disodium salt (Na2EDTA), sodium
chloride (NaCl), sodium dodecyl sulfate (SDS), sodium hydrox-
ide (NaOH), and dimethyl sulfoxide (DMSO) were purchased
from Sigma-Aldrich (St. Louis, MO), whereas Vectashield
CONTACT Thangal Yumnamcha tyumnamcha@gmail.com Department of Life Sciences, Manipur University, Canchipur, 795003, India
� 2020 Informa UK Limited, trading as Taylor & Francis Group
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mounting media was obtained from Vector Laboratories
(Burlingame, CA).
2.2. Plant extract preparation
The seeds of C. tiglium L. were collected from Manipur, India.
The collected seeds were washed with distilled water, kept in
the shade for drying at 28 �C, and then ground into a fine
powder. Forty grams of the seed powder was mixed with
80ml of distilled water, and then kept for 24 h at 28 �C after
proper maceration. The filtrate collected was centrifuged at
6000 rpm for 10min. After removing the uppermost (non-
polar) layer, the clear supernatant was collected and sub-
jected to lyophilization. The lyophilate was stored at �20 �C
until further use.
2.3. Zebrafish maintenance
Wild type adult zebrafish (Danio rerio), of both sexes, were
purchased from Petbonding, Bangalore, India. The adult
zebrafish were housed in the Zebrafish facility centre, Indian
Institute of Science, Bangalore, India, under 14 h Light:10 h
Dark cycle, and at a constant temperature of 28 �C± 2 �C. The
fish were fed twice a day: with commercial flakes (Tetra,Melle, Germany) in the morning, and live Artemia nauplii
(cultured from Artemia cysts, Inve Aquaculture Nutrition,
Thailand) in the evening. The pH, dissolved oxygen content,
total hardness, and other parameters of water were main-
tained following the standard method (APHA 2005).
2.4. Embryo collection
Sexually mature males and females were segregated into
separate pre-breeding chambers one day before setting up
breeding. Zebrafish, in a ratio of 2 males per 2 females were
transferred to each breeding chamber, at night. The following
morning, spawning occurs concurrently with turning on of
the lights. The freshly-laid eggs were collected from the bot-
tom of the breeding chamber using a Pasteur pipette. The
healthy eggs were sorted out from the total number of eggs
collected, and incubated in ‘egg water’ (distilled deionized
water containing 60 mg/ml of instant ocean salt) at 28 �C.
Using an stereomicroscope (Olympus SZ51) for observation,
only the healthy embryos which had reached the blastula
stage of development were selected, while the dead and
malformed embryos were discarded.
2.5. Evaluation of embryo lethality and
developmental toxicity
Five embryos, staged 6 hours post fertilization (hpf), were dis-
tributed randomly, per well into a 24-well plates, and then
exposed to six different concentrations of AECT (125, 150,
175, 200, 225, and 250 mg/mL) along with a control (only egg
water). The treatment was continued for up to 4 days post
fertilization (dpf) under a 12 h Light:12 h Dark cycle, and at a
constant temperature of 28 �C. The embryo lethality and
developmental toxicity parameters were examined under a
stereomicroscope (Olympus SZ51), every 24 h, until 4 dpf.
Different stages of embryos have been described as per
Kimmel et al. (1995). Endpoint analysis of embryo lethality
and developmental abnormalities were recorded according
to Nagel (2002). The embryo lethality endpoint includes
coagulation of egg proteins, non-detachment of the tail, and
inability to form somites. The endpoints of developmental
abnormalities include pericardial edema (PE), yolk sac edema
(YSE), spinal curvature (SC), and hatching rate delay, etc. PE,
YSE, and SC were evaluated at 4 dpf. Hatching rate was
defined as the number of treated embryos that hatched by
72 hpf, as compared to the untreated control ones. The
hatching rate calculations were based on a total of 30
embryos subjected to each treatment, and included those
that failed to develop and died even before hatching, and
expressed as the percentage of embryos that had hatched
out at a given time point. All instances of embryo lethality,
and other observable abnormalities, were recorded and pho-
tographed using a digital camera (Leica DFC 300FX) attached
to a fluorescence microscope (Olympus SZX12 Fluorescence
Stereozoom Microscope).
2.6. Assessment of cell death
Apoptotic cells in zebrafish embryos were identified using
AO staining (A6014, Sigma-Aldrich, St. Louis, MO). The zebra-
fish embryos, collected after treating with different concen-
trations of AECT, were rinsed with egg water, anesthetized in
a 0.03% tricaine solution, and then incubated with 5 lg/ml
AO dissolved in PBS (P4417 Sigma) for 20min at room tem-
perature. Embryos treated with egg water only were used as
control to normalize for autofluorescence. Before imaging,
the embryos were washed three times, for 5min each, with
egg water. Then the embryos were mounted in Vectashield
mounting media (Vector Laboratories, Burlingame, CA), and
imaged under a fluorescence microscope (Olympus BX51).
Figures were assembled using Adobe Photoshop CS (ver-
sion 11.0).
2.7. In vivo genotoxicity test of AECT
The experimental doses of AECT were selected based on the
preliminary range-determining experiments performed on
mature zebrafish, aged 6-months-old. Three different concen-
trations of AECT (12mg/ml, 16mg/ml and 20mg/ml) were
chosen for assessing the genotoxicity of AECT. Fish belonging
to the experimental cohort were exposed to the three differ-
ent concentrations of the AECT mentioned above, while the
negative control was exposed to tap water, and the positive
control to 5mg/ml of cyclophosphamide. The treatment regi-
men lasted for 3 days. In order to fully estimate the genotoxic
potential of AECT, in vivo genotoxicity assays, micronucleus
assay, and comet assay were performed.
2.7.1. Micronucleus test
The micronucleus test was performed according to the proto-
col described by Ueda et al. (1992), with slight modification.
Peripheral blood was collected from adult zebrafish by
2 T. YUMNAMCHA ET AL.
making an incision at the region of the dorsal aorta and
inferior vena cava, just posterior to the dorsal fin. Then, the
collected blood was immediately smeared onto pre-cleaned
glass slides. The slides were allowed to dry overnight. After
fixation in methanol for 10min, the slides were air-dried, and
then stained with AO, prepared at a concentration of 0.003%
in PBS. The stained slides were covered with glass cover slips,
and scored at 100� magnification under an epi-fluorescent
microscope. The red blood cells were evaluated for the pres-
ence of micronuclei as described previously (Ueda et al. 1992;
Çavas et al. 2005). The frequency of micronuclei in the eryth-
rocytes of adult zebrafish was assessed after 2 days of expos-
ure to AECT. The micronucleus frequency is represented as a
percentage (MN [%]). For the purpose of analysis, 2000 eryth-
rocytes were examined per slide. Since six zebrafish per con-
centration were used for the present study, so a total of 12
000 erythrocytes per treatment regimen were assessed. The
criterion for the identification of fish blood micronucleus was
based on the report of Al-Sabti and Metcalfe (1995). Small,
non-refractive, circular, or ovoid chromatin bodies, showing
the same staining pattern as the main nucleus, were consid-
ered as micronuclei.
2.7.2. Comet assay
The alkaline comet assay was performed according to the
method of Olive and Ban�ath (2006). Fresh blood samples
were collected, from the dorsal aorta and the inferior vena
cava, by making an incision with a pair of fine scissors. About
2ml of the blood was diluted in 5ml of PBS. The cell density
was adjusted to 106 cells/ml in PBS, using a hemocytometer.
0.4ml of the cell suspension was mixed rapidly with 1ml of
1% low gelling-temperature agarose, molten at 40 �C. After
proper mixing, 1.4ml of the cell suspension-agarose mixture
was layered onto slides pre-coated with 1% normal melting
point agarose. Following this, the slides were left undisturbed
for about 3min to ensure proper gelling. After the agarose
had solidified, the slides were immersed in a lysis solution
(1.2M NaCl, 100mM Na2EDTA, 0.1% SDS, 0.26M NaOH, pH >
13 with 2% DMSO added fresh). The slides were taken out,
and then immersed in an alkaline rinse and electrophoresis
solution (0.03M NaOH, 2mM Na2EDTA, pH �12.3) at room
temperature (18–25 �C). After rinsing for three times, the
slides were submerged in some fresh alkaline rinse and elec-
trophoresis solution, contained within an electrophoresis
chamber. The chamber was filled with sufficient buffer, such
that it remained about 1–2mm on the top of the agarose.
Electrophoresis was conducted for 25min, at a voltage of
0.6 V/Cm. The slides were removed from the electrophoresis
chamber, and neutralized in 400mL of distilled water. Then
the slides were stained, by pipetting 100 ml of a 10 mg/ml
stock solution of propidium iodide directly onto the slide and
incubating for 20min. The excess stain was removed, by rins-
ing the slides with 400ml distilled water. The prepared slides
were examined under a fluorescence microscope, (Olympus
BX51) at 100� magnification, equipped with a CCD camera.
Two slides, per zebrafish, were prepared, and 50 non-overlap-
ping cells per slide (100 cells per concentration) were scored
randomly, and analyzed using the image analysis software
‘Comet score’. The percentage of tail DNA content (% tail
DNA) was measured as an indicator of DNA damage (singlestrand breaks). Prior to performing the comet assay, the cell
count and cell viability were checked to ensure that there
was an optimum number of living cells to perform the assay.
The cell count and viability were evaluated with a hemocyt-
ometer and trypan blue dye exclusion test respectively.
Whole blood samples, showing greater than 90% viability
and a cell count of 106 cells/ml, were used for the
comet assay.
2.8. Statistical analysis
Graphs were made and analyzed using GraphPad Prism (ver-
sion 5.0). Data were statistically analyzed with one-way ana-
lysis of variance (ANOVA) and the significant difference
between treated and control groups was examined by the
Dunnett post hoc test. The LC50 was calculated using probit
analysis by SPSS software (SPSSv16). A pHu et al. 2000). Spinal deform-
ation is yet another commonly observed developmental tox-
icity parameter in zebrafish embryos. In the present study,
we too observed a significant increase in the occurrences of
SC, compared with the control (Figure 3). The exaggerated
SC may be due to either the disruption of glutamatergic
receptor-associated calcium and phosphorus signaling, or a
decrease in myosin, both of which control major phases of
pattern formation in developing zebrafish embryos, including
body axis determination and spine formation (Webb and
Miller 2000; Oliveira et al. 2009). The larvae which have SC,
are not only unresponsive to touch, and but cannot swim
properly as well. Hatching rate is one of the most important
indices to evaluate the developmental toxicity of a com-
pound. Exposure of zebrafish to AECT delayed hatching in a
dose-dependent manner, compared with the control (Figure
3). The delay in hatching may be attributed to either the
stunted development of the embryo, or the failure of the
embryos to break through the chorion (Osman et al. 2007).
Ultimately, therefore, the present study clearly demonstrates
that zebrafish embryos exposed to AECT suffer from develop-
mental toxicity. Identifying the exact mode of cell death is
important to characterize the lethality due to toxicity. The
present study used AO staining as a method to detect
enhanced apoptotic cell death in developing zebrafish
embryos following exposure to AECT. Figure 4 demonstrates
that exposure to AECT results in increased cell death in the
regions of brain, heart and trunk, as shown by distinct bright
green punctae in the fluorescence microscopy images, com-
pared with the control, thereby suggesting cardiotoxic and
neurotoxic properties of AECT. It is well known that toxicants
generate reactive oxygen species (ROS) in vivo, which, in
turn, trigger the apoptotic cascade. This can ultimately con-
tribute to the developmental toxicity in zebrafish embryos
(Yamashita 2003; Yumnamcha et al. 2015).
Proper study of the genotoxic properties of medicinal
plants is very important to prevent potential health risks to
the society, particularly when medicinal plant-based drugs
Figure 4. Fluorescence microscopic images of apoptotic cells of control (A, C) and ACET treated (B, D) 4 dpf zebrafish embryos. B¼ 200mg/ml AECT; D¼ 175mg/ml
AECT. The apoptotic cells stained with AO appeared as bright spot, mainly confined at brain, heart and eyes, as shown by arrows (B) and in trunk, as shown by
arrow (D) compared to the control groups (A, C).
6 T. YUMNAMCHA ET AL.
are taken for a long time. Results obtained from the in vivo
genotoxicity tests, i.e., micronucleus assay (Figure 5) and
comet assay (Figure 6), clearly demonstrated the genotoxic
effect of AECT. Although we have previously reported
(Yumnamcha et al. 2014) the genotoxic potential of the AECT
in in vitro conditions, literature suggests that an in vivo test
can provide data more relevant to the assess the DNA dam-
age potential in humans (Recio et al. 2010). Previous studies
have also shed light on the genotoxic properties of certain
other Croton species (Lopes et al. 2004; Maistro et al. 2013).
In conclusion, the present study clearly demonstrates that
AECT has the potential to generate both developmental tox-
icity and genotoxicity in zebrafish. However, further study is
required to determine mechanism behind upregulation of
the major apoptotic genes, and the different pathways whose
perturbation is responsible for causing developmental toxicity
and genotoxicity of AECT. Moreover, identification of the
exact active compounds responsible for causing developmen-
tal toxicity and genotoxicity of AECT is also required.
However, the findings from the present study may prove
helpful in providing valuable insights into the potential
health hazards of using C. tiglium as a traditional medicine
for treating various diseases.
Acknowledgements
The authors are grateful to Department of Life Sciences, Manipur
University and UGC for providing financial assistance during the research
period. The authors also acknowledged the Indian Institute of Science
(IISc) for financial assistance. Authors are thankful to Ms. Meenakshi Sen
and Ms. Deepti Bapat at IISc Confocal Facility for their help in taking con-
focal images and image processing. Amartya Mukherjee helps in editing
the manuscript.
Disclosure statement
No potential conflict of interest was reported by the authors.
Author contributions
TY, MDD and UN conceived the idea and planed the experiment, TY did
all the experiments, TY and DR did microscopic imaging and image proc-
essing. TY and UN analyzed the data and manuscript writing.
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Figure 5. Fluorescent microscopic images showing AO stained micronuclei in peripheral blood erythrocytes of Danio rerio after 2 days of exposure with AECT. (A)
Negative control (RO water) without micronucleus; (B) Positive control (Cyclophosphamide) 5mg/mL having micronuclei shown by arrowhead; (C) 12mg/mL of
AECT having micronuclei shown by arrowhead. (D) Graph showing frequency of micronucleated cells (MN) scored from erythrocytes from fish exposed with different
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Figure 6. Fluorescent microscopic images showing DNA strand breakage analyzed by comet assay in peripheral erythrocytes of Danio rerio after 2 days of exposure
with AECT by staining with propidium iodide. (A) Negative control showing no DNA strand breakage; (B) Positive control (Cyclophosphamide) 5mg/mL showing
DNA strand breakage as indicated by comet tail; (C) 12mg/mL of AECT showing DNA strand breakage as indicated by comet tail. (D) Graph showing DNA strand
breakage as indicated by comet tail DNA (%) scored from erythrocytes of zebrafish exposed with different concentrations of AECT along with negative control (NC)
and positive control (PC). ��� Represents significantly different p values compared to negative control (one-way ANOVA, followed by post-test: Dunnett test,���p