DE GRUYTER Journal of Basic and Clinical Physiology and Pharmacology.

2019; 20190041

C. Swathi Priyadarshini1 / Thotakura Balaji2 / Jyothi Ashok Kumar1 / Manickam Subramanian1 /

Indumathi Sundaramurthi1 / M. Meera3

Chlorpyrifos and its metabolite modulates

angiogenesis in the chorioallantoic membrane of
chick embryo
1 Department of Anatomy, Chettinad Hospital and Research Institute, Chettinad Academy of Research and Education, Kelam-

bakkam, Chennai 603103, Tamil Nadu, India

2 Department of Anatomy, Chettinad Hospital and Research Institute, Chettinad Academy of Research and Education, Kelam-

bakkam, Chennai 603103, Tamil Nadu, India, Mobile: +91 7358449857, E-mail: drbalaji123456@gmail.com
3 Department of Medical Biotechnology, Faculty of Allied Health Science, Chettinad Hospital and Research Institute, Chettinad

Academy of Research and Education, Kelambakkam, Chennai 603103, Tamil Nadu, India

Abstract:
Background: Chlorpyrifos (CPF) is an organophosphate insecticide, acaricide, and miticide used primarily to
control foliage and soilborne insect pests on a variety of food and feed crops. Since trace amounts of these
compounds are found in water and food products, they easily enter into the organ system unnoticed. In the
same way, the compound or its metabolite gets transmitted from the parent to the embryo mainly through
blood vessels. Since blood vessels form the major route of transport, it is pertinent to study the effect of these
compounds during angiogenesis. The effect of CPF and 3,5,6-trichloro-2-pyridinol (TCPy) on the angiogenesis
of chick embryo was evaluated in the chorioallantoic membrane (CAM) using an ex vivo model.
Methods: Nine-day-old incubated eggs where inoculated with various doses of CPF and TCPy. After 48 h of
incubation, the CAM layers were retrieved and analyzed using angiogenesis software to obtain the density
of blood vessels. Histomorphometric studies were performed to measure the thickness of vessel walls. The
expression of VEGF, VEGFR2, and N-cadherin genes responsible for angiogenesis were analyzed.
Results: The exposure to the parent compound CPF and its metabolite TCPy promoted angiogenesis in groups
administered with lower concentration of the pesticide and its metabolite, whereas a decline in angiogenesis
was observed at higher concentrations. These observations were made by analyzing the density, histomor-
phometry results, and semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) results. The
density, thickness, and lumen size of blood vessels in the groups with low concentration of CPF and TCPy were
28.34, 9 μm, and 30 μm, respectively, whereas in the groups with higher CPF and TCPy concentrations, they
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were 12, 3 μm, and 9 μm, respectively.

Conclusions: Hence, CPF and its metabolites interfere with angiogenesis in the CAM of chick embryos. Because
of their estrogen-mimicking ability, pesticides are the prime etiological suspects of increasing alteration in blood
vessel formation. These results may be of help in future studies on the effect of CPF in embryonic growth, wound
healing, diabetes, and tumors.
Keywords: 3,5,6 trichloro-2-pyridinol, angiogenesis, chick embryo, chlorpyrifos, chorioallantoic membrane
DOI: 10.1515/jbcpp-2019-0041
Received: February 26, 2019; Accepted: July 2, 2019

Introduction
Chlorpyrifos (CPF) is an organophosphate pesticide that is used on crops, animals, and buildings to kill a
number of pests and has been sold under many brand names [1]. The concentration that is recommended for
the direct-spray pinpoint application of CPF is 0.5%, while for wide-area application a 0.03–0.12% mix of CPF is
suggested. It is commonly used throughout India [2]. The World Health Organization (WHO) reports that even
moderate amounts of CPF are hazardous to humans, causing various autoimmune disorders of various organ
systems during development [3]. It has also been reported that CPF induces malformations in pregnant mice.
Skeletal abnormalities such as anophthalmia, sacral hygroma, microcephaly, hydrocephaly, hind-limb twist,
agnathia, micromelia, drooping twist, and kinky tail have been observed [4]. Low-dose exposure of CPF and its
Thotakura Balaji is the corresponding author.
© 2019 Walter de Gruyter GmbH, Berlin/Boston.

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metabolites affects infants and children because of their lower capacity for detoxification compared to adults
[5].
In recent years, the number of poisoning and mortality cases due to exposure to pesticides is on the rise.
However, because of their broad spectrum of applications, these chemicals neither can be completely with-
drawn from the market nor fully banned for use. CPF causes tens of thousands of deaths per year worldwide
[6].
The major metabolites of CPF are chlorpyrifos-oxon and 3,5,6-trichloro-2-pyridinol (TCPy). CPF and CPF-
methyl, as well as the primary metabolite TCPy, have been evaluated for potential developmental toxicity [7].
TCPy exposure tends to lower testosterone levels in men [8].
Angiogenesis is an important mechanism that fulfills the nutritional requirements of developing cells [9].
Initiation of angiogenesis requires the activation of certain genes via cell signaling, resulting in the production
of certain growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and
angiopoietin. Inhibitors such as thrombospondin, endostatin, and angiostatin are also involved in angiogenesis
[10].
Chorioallantoic membrane (CAM) assay is a widely accepted method to study angiogenesis [11], tumor
cell invasion, and metastasis [12]. There are many advantages of the CAM model: angiogenic nature of CAM
helps to promote the efficiency of cancer cell grafting; it offers more productivity; it is cost effective; and small
peptides tend to stay much longer in comparison to animal models. This facilitates the experimental study of
potential anti-metastatic compounds that are available in small quantities [13]. CAM consists of three layers:
the ectoderm, the mesoderm, and the endoderm at the interface with the allantoic sac [14].
The embryonic vascular system develops in expectation to the demands of the embryo for oxygen and nutri-
ents in the growing stage. Angiogenesis in the adult organism occurs in response to the metabolic requirements
of tissues and is efficiently triggered by hypoxia [15]. Angiogenesis studies have provided useful insights in the
understanding and treatment of diseases [16].

Materials and methods

Test substance
The test substances used were CPF (99.9% purity) and its metabolite TCPy (99.7%) (Sigma-Aldrich, Chennai,
India). All other reagents used were of the highest analytical grade.

Test organisms
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Fertilized country eggs (Gallus gallus) were obtained from the Government Poultry Farm, Potheri, Chennai,
India. The eggs were cleaned with ethanol before inoculating the pesticides.

Doses
Dose range study was performed to assess angiogenesis of these insecticides. Ten groups, each consisting of
six eggs, were dosed on day “0” of incubation with different doses of the insecticide and its metabolite, that is,
0.005, 0.001, 0.01, and 0.05 mg per egg. The dilutions were made in corn oil (Sigma-Aldrich, Chennai, India) for
both pesticide and metabolite. On “0” day of incubation, various doses of pesticides were injected into the air
sac, following Blankenship et al. [17].

Angiogenesis effects by CAM assay

The eggs were divided into 10 groups of six eggs in each group: Group 1 – control; Group 2 – vector control
(0.1% corn oil); Group 3 – 0.001 mg/mL CPF; Group 4 – 0.005 mg/mL CPF; Group 5 – 0.01 mg/mL CPF; Group
6 – 0.05 mg/mL CPF; Group 7 – 0.001 mg/mL TCPy; Group 8 – 0.005 mg/mL TCPy; Group 9 – 0.01 mg/mL
TCPy; Group 10 – 0.05 mg/mL TCPy. Procedures were carried out based on the relevant protocols [18]. CAM
layers were detached from the eggshell membrane using forceps and photographed using a camera (Nikon
D5300). Photographs were analyzed using the angiogenesis software (AngioSys 2.0 image analysis software)
for density, branching pattern of blood vessels, tubule length, number of junctions, frequency, and the mean
number of branches.

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Isolation of CAM tissue

CAMs were isolated from the embryos at developmental 11th day, mRNA was isolated using an RNA isola-
tion kit (Takara Clontech, Chennai, India), and cDNA was synthesized using a Prime script cDNA master mix
(Takara Clonetech) following the manufacturer’s instructions.

Histological and histomorphometric analysis

The membranes collected from the different groups were fixed in 10% neutral buffered formalin and then pro-
cessed for obtaining 4-μm paraffin-embedded sections. The sections were stained with hematoxylin and eosin
and then examined under a microscope (Carl Zeiss Axio Lab. A1 microscope). Images of the CAM sections were
digitally captured and histomorphometry was performed using ImageJ software. Three fields per section were
selected, and all the blood vessels in the selected field were measured for their thickness and lumen diameter
using the software. The results were analyzed statistically and represented as graphs.

Relative quantitative RT-PCR (qRT-PCR)

The primers used for PCR are shown in Table 1. All cDNA samples were diluted to 5 ng/μL. Twenty microliters
of the final volume contained UP (ultrapure) uracil-DNA glycosylase (UDG), dNTPs and optimized buffer
components, 200 nM forward primer, 200 nM reverse primer, and 10 ng of cDNA. PCR was performed using
a Thermocycler PCR apparatus with the following thermal profile: 5 min of Taq DNA polymerase activation
at 94 °C; 40 cycles of denaturation at 95 °C for 30 s; primer annealing at 62 °C for 30 s. Each cDNA sample
was evaluated by agarose gel electrophoresis, in triplicate. Gene expression was further analyzed by ImageJ
software [19].

Table 1: Primer list for qPCR.

Gene Primer sequence
GAPDH F: 5�-GAGAACGGGAAACTTGTCAT
R: 5�-CGCAGGTCAGGTCAACAA
N-cadherin F: 5�-AGATTCTGGAAATCCACATGC-3�
R: 5�-CTTCCTTCATAGTCAAAGACT-3�
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VEGFR2 F: 5�-GACTTCGGTTGGTGATCAGC-3�
R: 5�-ATGGTTGCACGCTTTTCCTT-3�
VEGF F: 5�-GGAACTTTCTGCTCACTTGG-3�
R: 5�-GCCCGTCTCGGTTTTTCACA-3�

Statistics analysis

Data obtained were expressed as mean ± SE. Statistical analysis was done by one-way analysis of variance
(ANOVA) followed by multiple comparisons by two-tailed t-test. If the p-value is less than 0.05, then they are
considered statistically significant.

Results
Morphometry of CAM

Analyses of CAM using the angiogenesis software showed an increase in the number of blood vessels as well as
their branching pattern in Groups 3, 4, 7, and 8 ranging from 28.34 to 21.25 (Figure 1B), i.e. the group exposed
to low concentrations of CPF and TCPy. Significant reduction in the number of number of blood vessels and
branching pattern ranging from 17.842 to 12.701 (Figure 1B) were observed in the CAM layer exposed to higher
concentrations of TCPy and CPF (i.e. Groups 5, 6, 9, and 10).

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Figure 1: Gross Morphology of CAM layer and its Quantification.

(A) Photograph of CAM of 11-day-old chick embryo exposed to various concentrations of CPF and TCPy. (B) Graphical
representation of the density of blood vessel branches in the CAM of various groups of 11-day-old chick embryo.
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Histology and histomorphometry of CAM

CAM sections of control and vector control showed normal histological features such as the presence of blood
vessels in the mesoderm and no changes in the blood vessels. Interestingly, in the sections of Groups 3, 4, 7, and
8, the mean thickness of vessel walls was 9–9.8 μm (Figure 2B), which is much larger than that of the control
(Group 1). In Groups 5, 6, and 9, the thickness of the vessel walls was slightly less than that of the control
(3.442–5.197 μm; Figure 2B). The vessel walls of Group 10 were significantly thinner than those of the control.

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Figure 2: Histological Morphology of CAM layer and its Morphometry.

(A) Photomicrograph of the CAM of chick embryo at day 11 exposed to various doses of CPF and TCPy. (a) Control
(Group 1); (b) vector control (arrow); (c–f) arrow head; (g–J) hash tag. Magnification ×10. (B) Graph showing the histo-
morphometric analysis of blood vessel thickness in various groups of 11-day-old chick embryo. (C) Graph showing the
histomorphometric measurement of blood vessel lumen diameter in control and experimental groups of 11-day-old chick
embryo.

Among Groups 3–10, blood vessels represented by c, d, g, and h looked larger and thick-walled with low-
dose exposure of CPF and TCPy (Figure 2A), whereas those represented by e, f, i, and j were smaller and
thin-walled with higher dose exposure of CPF and TCPy, in the mesoderm (Figure 2A).

Size of the lumen

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Histological sections of Groups 3, 4, 7, and 8 showed larger lumens compared to those of control (Group 1;
Figure 2C). In vessels of the Groups 5, 6, 9, and 10, which were exposed to high doses of TCPy and CPF, the
lumen was found to be of variable size and disrupted with infiltration.

Gene expression

In the gene expression study, all the groups showed upregulation of VEGF, VEGFR2, and N-cadherin genes.
With higher doses of CPF and TCPy, Groups 5, 6, 9, and 10 showed the downregulation of N-cadherin alone
(Figure 3A). From this data, it can be concluded that VEGF and VEGFR2 induced the formation of blood vessels
in both CPF and TCPy groups, whereas N-cadherin was reduced in groups treated with higher doses (Figure
3A,B).

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Figure 3: Semiquantitative PCR gel  and its Quantification.

(A) Electrophoresis results of the PCR products on 1% agarose gel. The result shown in Lane 1 is for the housekeep-
ing gene for all groups. Lanes 2, 3, and 4 show the expressions of VEGF, VEGFR2, and N-cadherin, respectively. (B) Bar
graph of semiquantitative RT-PCR showing the expression of VEGF, VEFR2, and N-cadherin in the CAM of chick embryo
following exposure to various doses of CPF and TCPy.
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Discussion

CPF affects the nervous system of insects by hampering the breakdown of acetylcholine (ACh) [20]. CPF binds
to the active site of the cholinesterase (ChE) enzyme, thereby preventing the breakdown of ACh in the synaptic
cleft [21]. This leads to an increase of ACh in the synaptic cleft, causing overstimulation of the neuronal cells,
which in turn leads to neurotoxicity and ultimately death of the neurons [21]. Agricultural Health Study (AHS)
investigators have established the association of diazinon, chlorpyrifos, and terbufos usage to lung cancer [22].
CPF use has been shown to be associated with an increased risk of breast cancer [23]. In the pathological state,
angiogenesis is observed during solid tumor growth and metastasis, diabetic retinopathy, and chronic inflam-
matory disorders. [24].
The CAM assay serves as an appropriate model to evaluate the effects of angiogenic or anti-angiogenic
agents [25]. In CAM, normal vascular development occurs between day-4 and day-11 embryo [26]. In the CAM
layer, the primary blood vessels are formed as a result of the proliferation and differentiation of angioblasts
into endothelial cells (ECs). The remaining blood vessels for development sprout from existing blood vessels.
In CAM, it is possible to identify the primary vessel formation followed by the appearance of secondary and
tertiary vessels, which are tortuous in nature. Intussusceptive microvascular growth with associated lower EC
proliferation occurs between days 7 and 11 [27]. The surface of the entire inner shell membrane surrounds the
complete embryo formed by CAM [28].
The present study shows the impact of CPF and its metabolite TCPy on the angiogenesis of the CAM mem-
brane. In albino rats fed with food containing CPF-methyl for a short period, increased blood vessel formation
and inflammatory changes were observed [29]. Oxidative stress in tissues can activate an angiogenic switch
linked with experimental plague progression and angiogenesis [30]. In the present study, Groups 3, 4, 7, and

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8 evidently showed a continuous increase in the branching of blood vessels compared with the control (Group
1; Figure 1B). This is primarily due to the estrogenic effect of the organophosphate pesticide. The effect of pes-
ticides and their metabolites mimicking estrogen has been widely documented [31].
Decrease in antioxidant, increases the oxidative stress ability. A linkage between malignant growth and
oxidative stress is indicated by the negative relationship between glutathione content and DNA adduct con-
figuration.Simultaneously, this transformation promotes angiogenesis and abnormal angioarchitecture [32]. In
addition, many studies have reported that CPF has a variety of effects, including biochemical and histopatho-
logical alterations, genotoxicity, oxidative stress, reproductive toxicity, and endocrine-disrupting effects [33]. In
the present study, an increase in the concentration of the pesticides led to a reduction of blood vessel formation
and branching pattern (Figure 2A – e, f, i, j).
Very little information is available on the histopathology of CAM exposed to CPF and TCPy. The histologi-
cal sections of the CAM layer in our study showed blood vessels in the mesoderm. In all groups, the mesoderm
appeared normal. At lower concentrations of CPF (Groups 3 and 4) and TCPy (Groups 7 and 8), hypertrophied
blood vessels with intact lumen and thick wall were observed (c, d, g, h – Figure 2A). Estrogen increases the
generation of nitric oxide and prostacyclin in ECs [34], [35] as well as the proliferation of cultured rat smooth
muscle cells [35]. Estrogen slows the senescence of these cells through increased telomerase activity and in-
creases the proliferation of smooth muscle cells during the activation of ERα [36], [37].
Multicellular organisms undergo apoptosis from early development phase to the adult phase to retain home-
ostasis [38]. Changes characteristic of apoptosis have been observed in the heart tissues of zebrafish embryos
treated with high dose of CPF [39]. Our study shows reduced lumen size with thin-walled blood vessels in
Group 5, 6, 9, and 10, which was in contrast to that of low-dose CPF- and TCPy-treated groups (e, f, i, j – Figure
2B,C). The reduced lumen size is probably due to the apoptotic effect of the high dose of CPF and TCPy. In
blood vessels of certain high-dose groups, the lumen was dilated with infiltration and endothelial disruption
(Group 9; Figure 2A). Histological examination of kidney exposed to CPF has shown lymphocytes infiltration
in the interstitium, increased vascularity, mild hyperemia, a few dilated renal vessels, and interstitial edema
[40].
Embryogenesis and angiogenesis are regulated by VEGFs and their receptors (VEGFRs). VEGFR2 is a major
signal transducer for angiogenesis and vascular permeability and is activated by VEGF-A. In anti-angiogenic
treatment of cancer and pro-angiogenic treatment for neuronal degeneration and ischemic diseases, the prime
target is the VEGF-VEGFR system [41].
Upregulation of VEGF and VEGFR leads to an increased number of newly formed vessels and higher vessel
sprouting [42]. In Groups 3, 4, 7, and 8, overexpression of VEGF and VEGFR2 was detected compared to Group
1 (Figure 3B). Overexpression of VEGF is found in various types of tumor, which helps the solid tumor to grow
and metastases to other regions of the body. Vascular abnormalities in the retina and other parts of the body
during development are due to the overexpression of VEGF during development.
Cell-to-cell adhesion is a critical part of the development, maintenance, and functioning of vascular struc-
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tures [43]. Establishment of the vessel wall is mainly facilitated by N-cadherin [44]. Cadherins are transmem-
brane glycoproteins that intervene in homophilic calcium-dependent cell-cell-adhesion [44]. N-cadherin is con-
centrated at cell-to-cell contact sites, but in most other cell sites it is downregulated [9]. In the present study,
downregulation of N-cadherine was observed in Groups 5, 6, 9, and 10 exposed to TCPy and CPF. Administra-
tion of antibodies blocking N-cadherin resulted in vascular abnormalities and hemorrhage in developing chick
embryo because of disrupted pericyte interaction in ECs [45].
N-cadherin downregulation does not affect the initial angiogenesis and endothelial sprouting but results in
impairment of pericyte in the embryonic endothelium. Stability of vessels is mainly promoted by N-cadherin
[46]. In embryoid bodies, cadiomyocytes, muscle cells, and neurons, N-cadherin is potentially expressed. Defi-
ciency of N-cadherin causes malformed somites and yolksac, undulated neural tube, and severe cardiovascular
defects [47].
N-cadherin is expressed in a variety of cell types such as neuronal, lens, skeletal, and heart muscle cells,
osteoblasts, pericytes, and fibroblasts [48]. An anti-angiogenesis effect was observed in groups exposed to high
doses of CPF and TCPy, which might be due to the downregulation of N-cadherin.

Conclusion
Low concentrations of CPF and TCPy induced angiogenesis in the CAM of chick embryo, but this effect was
reversed with increased concentration of CPF and TCPy.

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Acknowledgment

The authors acknowledge the Chettinad Academy Research and Education for funding this project and support.

Author contributions: All authors have accepted responsibility for the entire content of this manuscript and
approved its submission.
Research funding: None declared.
Competing interests: Authors state no conflict of interest.
Ethical approval: According to Indian Animal Welfare Regulations, chicken embryos until hatching are not
assigned the legal status of ”animals”; therefore approval of an Ethics Committee was not required for this
study.

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