THE EFFECT OF CO-TREATMENT WITH RETINOIC ACID ON … · 2019. 10. 14. · The incubator was located in a well-ventilated place and was accurately adjusted at 37.5 ± 0.1°C before

Assiut Univ. J. of Zoology Printed ISSN 1687-4935

Special Issue 1(1), pp. 43-59 (2019)

The 6th International Conference for Young Researchers for Basic and Applied Science

(ICYS-BAS 19), 27-30th

March 2019, Faculty of Science- Assiut University

THE EFFECT OF CO-TREATMENT WITH RETINOIC ACID ON

RESCUING CITRAL INDUCED MORPHOLOGICAL ANOMALIES DURING

CHICK EMBRYO DEVELOPMENT

Reda A. Ali, Dalia Elzahraa F. Mostafa and Heba E. Aboulqasem

Zoology Department, Faculty of Science, Assiut University, Assiut 71516, Egypt

E-mail address: [email protected]

Received: 10/2/2019 Accepted: 21/7/2019

Introduction: Retinoic Acid (RA) are compounds derived from retinol or vitamin A. RA signaling

has a central role during both embryonic and adult growth, activating gene transcription via interacting

with nuclear RA receptors bound to RA response elements near target genes. RA levels require precise

regulation by controlled synthesis and catabolism. Citral is a natural product of the essential oils of

plants. It has been reported to inhibit the formation of RA. Aim of the work: This research aims to

find out which concentration 0.25, 0.5 or 1 µgm of retinoic acid is most efficient in rescuing the chick

embryo treated with citral. Methods: Fertilized eggs of the chick Gallus domesticus were divided into

six groups, control group, DMSO group (RA solvent), citral group (50 µM) and three groups received

a combination of the citral dose and one of three different doses of RA (0.25, 0.5 or 1 µgm),

respectively. After hatching, hatchability and mortality rates were reported. Embryos were

morphologically examined and weighed. Morphometric measurements were carried out for some

parameters and were statistically analyzed. Results: the present study showed highly deformed

embryos in the citral group, while co-treatment with citral and the lowest dose of RA (0.25 µgm)

showed partial mitigation than the higher doses (0.5 and 1 µgm). Co-treatments of citral and RA (0.25,

0.5 and 1 µgm) showed mortality rates 40%, 74% and 75% respectively compared to 62.5% in the

case of citral treatment alone. Different abnormalities were observed in citral treated embryos such as

high growth retardation, brain deformation. The eye was either invaginated, exophthalmic or

completely absent in some embryos. Long and wry neck, absence of feathers, open body cavity and

limb deformation were also observed. Weight, crown rump, head length, head circumference, head

height, wing and all parts of hind limb lengths in all treated groups were significantly lower than

control. Also, co-treatment with the lowest dose of RA (0.25 µgm) and citral significantly elevated the

all morphometric parameters compared to higher doses of RA (0.5 and 1 µgm), but non- significantly

compared to citral treated group alone. Discussion: This study shows that treatment with citral

decreases the level of endogenous RA than the level needed to maintain the normal embryonic

development and that leads to severe malformation. Treatment with exogenous RA might rescue the

embryo from teratogenic effects of citral and that leads to the partial mitigation in some embryos. It

suggested that the response of embryos to RA is very sensitive. The lowest dose of RA (0.25 µgm)

could partially mitigate the effect of citral while higher doses of RA (0.5 and 1 µgm) exerted

teratogenic properties of RA rather than mitigative effects.

KEY WORDS: Retinoic acid, Citral, Chick embryo, Growth retardation, Mortality.

mailto:[email protected]

44 Reda A. Ali, Dalia Elzahraa F. Mostafa and Heba E. Aboulqasem

INTRODUCTION

Diet supply all vertebrates with the macronutrients needed for energy production and

tissue anabolism, with calcium and phosphorus as minerals, vitamins which serve a structural

role, and with many micronutrients that play an important role as cofactors in metabolism and

as regulators of metabolic functions. All vertebrates need vitamin A for normal growth, cell

and tissue differentiation, vision, development and function of the immune system, and

survival [1]. The concept that vitamins can do only good but never harm has been discounted

for most vitamins [2]. Vitamin A, which readily penetrates into the central nervous system,

can be harmful to both the developing and mature CNS. Thus, precise homeostatic control of

vitamin A is essential to maintain correct levels, and is achieved through the use of the liver

as a reservoir of retinol to be drawn on in times of depletion and as a sink in times of excess

[3, 4& 5].

Later studies revealed that rat fetuses in mothers fed on vitamin A-deficient diets

demonstrate a lot of abnormalities collectively known as "fetal vitamin A deficiency" (VAD)

syndrome, which comprises hindbrain, eye, ear, heart, lung, diaphragm, kidney, testis, limb,

and skeletal defects, these functions are highly sensitive to abnormal changes in vitamin A

concentration [6].

There are two main sources of vitamin A: animal sources and plant sources. All sources

of vitamin A require some fat in the diet to assist absorption. In animal sources, vitamin A is

found as retinol, the 'active' form of vitamin A. Liver, including fish liver. Plant sources

contain vitamin A in the form of carotenoids which have to be converted during digestion into

retinol before the body can utilize it. Carotenoids are the pigments that make plants their

green color and some fruits and vegetables their red or orange color [7].

Retinol can be relocated to embryonic plasma by means of maternal plasma or egg yolk,

according to the species. In embryonic plasma, the lipophilic retinol fastens to retinol binding

protein 4 (RBP4). Stra6 (activated by RA6) is a membrane receptor for RBP4; thus, retinol

penetrates a cell where it is bound to one of the cellular retinol binding proteins (CRBP-I,

CRBP-II, or CRBP-III). Retinol can be metabolically transformed in different ways, one of

which is to be oxidized to more active retinoid forms. In contrast to the extracellular pathway

of retinol transport, it has the ability to be an anabolic output of intracellular β-carotene

metabolism resulting from either beta, β-carotene 15, 15´-monooxygenase 1(Bcmo1) activity,

which produces two molecules of retinaldehyde that can be either reduced to retinol or

oxidized to RA, or by asymmetric cleavage via β-carotene 9, 10´-dioxygenase 2 (Bcdo2) to

form products that can be bio transformed to RA without formation of retinaldehyde [8].

All-trans retinoic acid (RA), a most active form of vitamin A, is a signaling molecule

necessary for the formation a lot of organs, including eyes, heart, and kidneys. Also, is

important for many physiological processes, including keeping the integrity and function of

all epithelial tissues: for example, the skin, the lining of the digestive tract, the bladder, the

inner ear and the eye. Vitamin A provides the daily replacement of skin cells and ensures that

tissues such as the conjunctiva are able to secrete mucous and provide a barrier to infection.

THE EFFECT OF CO-TREATMENT WITH RETINOIC ACID ON RESCUING… 45

Vitamin A is also important for vision, maintenance of immune system, normal growth and

development and for reproduction. [7].

In embryos, cellular RA is created from retinol, the circulatory form of vitamin A, by two

steps of oxidation, the first by alcohol/retinol dehydrogenases and the second by

retinaldehyde dehydrogenases (RALDHs). Inhibition of RA is catalyzed by CYP26, a

cytochrome P450 enzyme. In target cells, RA act as potentially endogenous ligand for nuclear

retinoic acid receptors (RARs), which form heterodimers with retinoid X receptors (RXRs).

The complex fasten to a regulatory DNA segment, the retinoic acid response element

(RARE), to govern transcription of RA target genes. Local RA availability relies upon

RALDH and CYP26. Within embryos, too little or too much vitamin A/RA causes

malformations [6].

Endogenous RA can function properly only when RA exists at exact concentration. When

RA concentrations deviate from normal, in either direction, causes malformations during

growth and development [9].

Essential oils extracted from nominated kinds of plants contain sturdy antimicrobial,

antifungal and antiparasitic activities. One of the most substantial active ingredients of these

essential oils responsible for such activities was proved to be citral [10]. Citral (C10H16O),

also called 3, 7-dimethyl-2,6-octadienal, it is found in the volatile oils of sundry herbal plants.

It is a major content in essential oils extracted from Different Plant Parts of lemongrass

(Cymbopogon citratus), Melissa (Melissa officinalis) and Verbena (Verbena officinalis) [11&

12]. It is a pale yellow mobile liquid. Because of its characteristic strong lemon-like odor and

bitter sweet taste, citral is commonly used as food additive, as fragrance in the cosmetic

industry, preserve flavor or enhance its taste, as an odorant in perfumes and as an insect

repellent [13]. Citral is less dense than water and insoluble in water but soluble in ethanol

(ethyl alcohol), diethyl ether, and mineral oil. Citral is classification is "Generally Recognized

as Safe" substance due to its special odor, antimicrobial, antifungal and insecticide effects, as

well as its low toxicity and low carcinogenicity [14& 15].

Citral has been stated to prevent the oxidation of retinol in mouse epidermis, thereby

interfering with the biological activity of retinol in this tissue. Citral prevents both steps in

retinoic acid synthesis from retinol, since it able to act as a substrate for both the alcohol and

aldehyde dehydrogenases [9]. In Xenopus laevis embryos, citral prevents the formation of RA

and thus treatment with citral can rescue embryos from the exogenous retinol teratogenicity

effects [13].

Why Aves?

Avian embryos are assumed ideal models to study the effects of vitamin A on early

embryonic development. In addition there is numerous evidence that somite differentiation in

birds is comparable to that of mammals [16]. Thus any effects on the survivability or growth

of chicks may be practicable to humans [17].

https://www.britannica.com/science/ethyl-alcohol

https://www.britannica.com/science/ethyl-ether

https://www.britannica.com/technology/mineral-oil


In general, Injection times range from before of incubation (embryonic day zero, E0) to

after 4 days of incubation (E4). Because the plurality of organogenesis in chicken embryos

happens during the first 4 days of development [18].

MATERIAL AND METHODS

1. Chemicals:

Stock solutions of RA were prepared in dark room by dissolving it into DMSO for in ovo

experiments. These solutions were protected from extended exposure to light when being

prepared and used and then kept in aliquots at -20°C.

2. Egg Injections:

Fertilized eggs of the chick Gallus domesticus (Dandrawi strain), obtained from the farm

of Faculty of Agriculture, Assiut University, and were used in the experiments of the present

investigation. All embryological materials needed for the experiments were obtained by

artificial incubation using an electrical thermostatically controlled incubator. The incubator

was located in a well-ventilated place and was accurately adjusted at 37.5 ± 0.1°C before use.

Both the trays of the eggs and inside of the incubator were thoroughly cleaned using dettol

and ethyl alcohol. Labeled fertile eggs were placed vertically in the trays inside the incubator.

Ventilation was allowed in the incubating chamber. Relative humidity was automatically

adjusted at 52%. Incubated eggs were automatically turned approximately bihourly from side

to another until their operation time. The incubator used in the present study belongs

to PTO, Egypt, model C5 [18].

3. Experimental design:

The incubated eggs were randomly divided into 6 groups:

1. The first group: was left untreated as a control one.

2. The second group: received 1 µgm DMSO (RA solvent).

3. The third group: received 50µM citral.

4. The fourth, fifth and sixth groups: received a combination of the citral dose and one of

three different doses of RA (0.25, 0.5 or 1 µgm), respectively.

All the injections were carried out just before incubation. Eggs were thoroughly cleaned

with alcohol. A hole was done at the blunt area of the egg. Injection was carried out by means

of micropipette. The needle was inserted vertically for a suitable distance into the yolk sac.

The hole was then sealed with a sealing tape. The eggs were incubated until they were taken

out at 21 days of incubation to obtain the required embryonic stages.

4. Specimens' preparation:

The eggs were carefully opened under physiological saline solution. Embryos were

carefully removed from the yolk and membranes and they were transferred to a new saline

solution for washing and then fixed in 10% neutral formalin and 95% ethyl alcohol.

Specimens were morphologically examined. To investigate the skeletal elements,


transparencies of the body were prepared by using Alizarin Red S stain

according to the modified method by Salaramoli et al. [19].

5. Statistical analysis:

The percentages of the weight, crown rump, head length, head circumference, head

height, wing and all parts of hind limb lengths deformities were calculated

and statistically analyzed using column statistics and one-way analysis of

variance with the Newman-Keuls multiple comparison test as a posttest.

These analyses were carried out using prism & excel programs.

RESULTS

Control group:

At the hatching day (21 days of incubation), all characteristic features of complete

development are observed. Eyes, auditory opening, external naris and the beak are well

developed. Limbs reached the adult form except for size. Toes have fine horny scales ending

with claws (Fig. 1). At this stage, alizarin transparency revealed ossification of all skeletal

elements including beak and phalanges of toes (Fig. 2).

DMSO - injected group:

Some specimens showed growth retardation and absence of feathers (Fig. 3). When such

a case was demonstrated with alizarin transparency; it revealed a curvature of the vertebral

column at the beginning of cervical region and extremely closed toes (Figs. 4& 5).

50 μM citral - injected group:

Highly deformation observed in most specimens including unilateral microphthalmia

(Fig. 6) or completely absent eye (anophthalmia) (Fig. 7), absence of limbs (ectromelia cases)

(Figs. 6& 7), laterally compressed embryo (Fig. 8) and invaginated head (Fig. 9). Upon

demonstration with alizarin transparency; it revealed a little ossification of toes, invagination

with some fractures in skull (parietal and frontal bone) (Fig. 10).

Citral and 0.25 μgm RA - injected group:

Many cases showed growth retardation, abdominal hernia, deformed parrot beak,

hypomorphic limbs (Fig. 11), invagination of head and eye (Fig. 12). Upon demonstration of

such a case with alizarin transparency, it revealed curvature, shrinking in cervical vertebrae

and reversed orientation of hind limb (femur and fibula) were observed (Fig. 13). In other

cases, short neck and loss of feather on some parts of body were observed (Fig. 14). Upon

demonstration of such a case with alizarin transparency, it revealed reduction in ossification

of the neck and toes regions (Fig. 15).


Citral and 0.5 μgm RA - injected group:

Some cases exhibited invaginated head (Figs. 16& 18), fallen feathers from some areas

(Fig. 18), abdominal hernia with viscera outside (Fig. 16), wry neck and reversed orientation

of wing (Fig.17). Alizarin transparency revealed abnormal configuration of cervical vertebrae

and invagination in skull (parietal bone) (Fig. 19).

Citral and 1 μgm RA - injected group:

Most specimens were highly deformed and growth retarded (Figs. 20, 21, 22& 23),

dorsoventrally flattened (Fig. 20). Hypomorphic limbs (Figs. 20, 22& 23), or completely

absent limbs (ectromelia) (Fig. 21). Beaks showed a lot of malformations such as parrot beak

(Fig. 22), absent beak (Fig. 21) and not well developed upper and lower jaws of the beak (Fig.

23). Upon demonstration of a case with alizarin transparency, it showed absence of

ossification except for little limb elements (Fig. 24).

Statistical analysis:

Mortality:

Mortality rate (fig. 25) in all treated groups significantly exceeded control. Citral treated

group (62.5%) had greater mortality rate compared to control group, while the group that

treated with a combination of the lowest dose of RA (0.25 µgm) and citral had 40% mortality

rate compared to citral. The two groups that were treated with combination of citral with the

two higher doses of RA (0.5 &1 µgm) had about 74% & 75% mortality rate respectively with

non-significant difference between them.

Morphometric measurements showed:

In all treated groups, weight, crown rump, head length, head circumference, head height,

wing and all parts of hind limb lengths were significantly lower than control. Also, co-

treatment with the lowest dose of RA (0.25 µgm) and citral significantly elevated the all

morphometric parameters compared to higher doses of RA (0.5 and 1 µgm), but non-

significantly compared to citral treated group alone (Figs. 26, 27, 28, 29, 30, 31, 32, 33&

table 1).





Groups

Lengths

Cont DMSO Citral Citral

+

0.25 µgm RA

Citral

+

0.5 µgm RA

Citral

+

1 µgm RA

Weight 38.21±0.560ᵅ 3.379±1.444ᵇᶜ 2.509±1.170ᵇᶜ 6.589±2.618ᵇ 1.499±0.6639ᶜ 0.1920±0.0272ᵈ

Crown rump 8.096±0.156ᵅ 3.272±0.5142ᵇ 4.000±0.400ᵇᶜ 4.644±0.615ᵇ 2.288±0.5306ᶜ 0.9040±0.148ᵈ

Head length 5.320±0.0424ᵅ 2.156±0.3063

ᵇ 2.088±0.210

ᵇ 2.232±0.336

ᵇ 1.236±0.3200

ᶜ 0.4520±0.1074

ᵈ

Head

circumference 6.596±0.0617

ᵅ 2.784±0.3770

ᵇ 2.928±0.210

ᵇ 3.224±0.391

ᵇ 1.452±0.3763

ᶜ 0.5640±0.1741

ᵈ

Head height 2.456±0.0462ᵅ 1.032±0.1540

ᵇ 0.9400±0.109

ᵇ 1.312±0.130

ᵇ 0.5400±0.1339

ᶜ 0.3360±0.09710

ᶜ

Wing 4.648±0.0996ᵅ 1.528±0.2663

ᵇᶜ 1.340±0.261

ᵇᶜ 1.820±0.268

ᵇ 0.7960±0.2572

ᶜ 0.1320±0.05619

ᵈ

Leg 9.756±0.0787ᵅ 2.232±0.5212

ᵇᶜ 2.732±0.575

ᵇᶜ 3.212±0.688

ᵇ 1.472±0.4880

ᶜᵈ 0.1680±0.07410

ᵈ

15 14

Table 1


Figure 1: A photograph of a control chick embryo after hatching.

Figure 2: A photograph of alizarin preparation of a control chick embryo after hatching,

showing ossification of skeletal elements of all body parts.

Figure 3: A photograph of a chick embryo treated with DMSO after hatching, showing

growth retardation and absence of feathers.

Figure 4: A photograph showing a wry neck of chick embryo treated with DMSO after

hatching.

Figure 5: A photograph of alizarin preparation of a chick embryo treated with DMSO after

hatching, showing wry neck and extremely closed toes.

Figure 6: A photograph of a chick embryo treated with 50 μM citral after hatching, showing

highly deformation, unilateral microphthalmia and absence of limbs (ectromelia).

Figure 7: A photograph of a chick embryo treated with 50 μM citral after hatching showing

highly deformed embryo and right eye is absent completely compared to left.


highly deformed embryo and laterally compressed.


invaginated head and syndactyly toes.

Figure 10: A photograph of alizarin preparation of a chick embryo treated with a combination

of 50 μM citral and 0.25 μgm RA after hatching, showing little ossification of toes,

invagination with some fractures in skull.

Figure 11: A photograph of a chick embryo treated with a combination of 50 μM citral and

0.25 μgm RA after hatching, showing growth retardation, abdominal hernia, hypomorphic

limbs and absence of feathers.

Figure 12: A photograph showing a chick embryo treated with a combination of 50 μM citral

and 0.25 μgm RA after hatching, showing invaginated head, reversed orientation of hind limb

and absence of feathers in some area.


of 50 μM citral and 0.25 μgm RA after hatching, showing shrinking of vertebral columns and

reversed orientation of hind limb.


and 0.25 μgm RA after hatching, showing invaginated eye and curved toes.


of 50 μM citral and 0.25 μgm RA after hatching, showing reduction in ossification of cervical

vertebrae and toes.



0.5 μgm RA after hatching, showing invagination of head, abdominal hernia, reversed

orientation of hind limb with overlapped toes and absence of feathers.


and 0.5 μgm RA after hatching, with reversed orientation of wing and parrot beak.


0.5 μgm RA after hatching, showing invaginated head and fallen feathers from some areas.


of 50 μM citral and 0.5 μgm RA after hatching, revealed abnormal configuration of cervical

vertebrae and invaginated skull.


1 μgm RA after hatching, showing highly deformation and dorsoventrally flattened embryo.


1 μgm RA after hatching, showing highly deformation and absence of most parts of embryo.


1 μgm RA after hatching, showing highly deformation and Parrot shaped beak.


1 μgm RA after hatching, showing deformation embryo and not well deformed beak.


of 50 μM citral and 1 μgm RA after hatching, showing absence of ossification except for little

limb elements.

Figure 25: Graphic representation the difference in mortality rates of exposed experimental

groups: citral (50 µM) and the combination of the citral dose with three doses of RA (0.25,

0.5 &1 µgm) as compared to the control group.

Figure 26: A photograph showing a comparison between all treated groups and control

revealed the differences in crown rump and weight.

Figures (27, 28, 29, 30, 31, 32, & 33): The percentages of weight, crown rump, head length,

head circumference, head height, wing and all parts of hind limb lengths deformities. A

comparison between control, DMSO, citral (50 µM) and the combination of the citral dose

with one of the three different concentrations of RA (0.25, 0.5& 1 µgm).

Table 1: Effect of citral and combination of the different doses of RA with citral on different

lengths of body parts and different weights of chick embryo after hatching. a, b, c, d

significant difference between groups. Data are presented as means ±SE.


DISCUSSION

Heart is the first organ to work and is necessary for the transportation of nutrients and

oxygen in the growing in all vertebrate embryos. Normal morphogenesis of cardiac is thus

necessary for embryonic survival. Heart development is a complicated process that needs the

delicate and coordinate interactions between many cardiac and extra-cardiac cell types. Any

disturbance in the cells that contribute to heart formation leads to cardiac disorder. Many

studies have reported that the formation of the heart rely on the vitamin A metabolite RA,

which serves as a ligand for nuclear receptors. The metabolic pathways of RA have been the

subject of several recent reviews. Excess exposure in humans to vitamin A or retinoids,

leading to embryonic abnormalities and congenital heart disease (CHDs), including

conotruncal and aortic arch artery anomalies such as translocation of the great vessels, double

outlet right ventricle, and tetralogy of Fallot [20]. Moreover, RA reduces the survivability of

embryos in embryonic development. Implying mechanisms include opposite effects on

implantation, inner cell mass population and raised permeability of the fetal membranes [17].

Indeed, early disturbances of RA signaling may lead to severe CHDs associated with

embryonic death and thus this explain our observation that the citral elevate the mortality rate

compared to control, while when treated the embryo with citral and the lowest dose of RA

(0.25 µgm) together cause partial mitigation . In contrast, the embryo treated with citral and

higher doses of RA (0.5 µgm& 1 µgm) led to significant increase in mortality rate, which RA

might exert teratogenic effect rather than mitigation.

Early studies recommended that RA controls retina patterning. However, deletion of

Raldh1 and Raldh3 in mice and pharmacological and genetic knockdown of RALDH function

in Zebrafish showed that loss of optic-cup RA efficiency leads to excessive perioptic

mesenchyme growth, which is related with dysgenesis of the cornea and eyelid, and

mechanical stresses that lead to abnormal optic-cup formation. Cunningham and Duster [21]

suggested that RA directly activates Pitx2 expression through a nearby RARE, which in turn

induces dickkopf homologue 2 (Dkk2) to locally suppress WNT signaling. Thus, RA regulates

eye morphogenesis by inhibiting excessive WNT signaling in the perioptic mesenchyme. This

may explain the seemingly perplexing observation that the effects of retinoic excess and

retinoic deficiency are often similar - both are exerting teratogenic effects.

The teratogenic effect of RA on facial morphogenesis of mammalian embryos is well

known. Embryos of pregnant rats given excess vitamin A during day 8 of gestation show

abnormal orofacial morphogenesis including upper jaw defects such as cleft palate [22].

Previously recognized genetic factors related with beak anomaly include the reported

candidate genes such as fibroblast growth factor 8 (FGF8) and bone morphogenetic protein 4

(BMP4). The over-expression of homeobox A1 (HOXA1) and homeobox D3 (HOXD3) may

result in beak anomaly in chicks [23]. RA is well known [21] to affect these genes which may

explain the beak malformations that were observed in this study.

Later study showed that RA controls gene expression directly at the transcriptional level

through nuclear RA receptors (RARs) that fasten to RA response elements (RAREs).

Cunningham and Duster [21] checked whether the RA regulates development by acting as a


diffusible signaling molecule that controls the activity of a family of RARs. Moreover, RA is

important in adults for tissue maintenance, and for spermatogenesis, immune function and

brain function. Also, chick embryonic limb proximodistal axis is also susceptible to high

levels of exogenous RA when it is applied to distal limb regions (which are normally devoid

of RA activity); leading to distal expression of the proximal-specific Meis1 and Meis2 genes

(Meis1/2), thus the distal-limb development is prevented. Also, RA is necessary for digit

specification late in limb development. Additionally, in vivo studies have identified RAREs

that regulate inhibition of Fgf8 during body axis extension or stimulation of homeobox (Hox)

genes during neuronal differentiation and organogenesis [21]. We suggest that the limb

malformations that were observed in this study is may due to disturbance in Meis1, Meis2,

Fgf8 and Hox genes that control limbs specification.

In the current study alizarin transparencies disclosed some of fracture with invagination

in skull bone, curvatures of the vertebral column at the cervical region, shrinking in cervical

vertebrae and reduction in ossification of limbs. Ali et al. [14] stated that retinoid signaling is

essential for controlling proliferation and differentiation of chondrocytes during endochondral

bone formation and particularly in the controlling of Bmps signaling during chondrogenesis

and osteogenesis.

According to our observations, most the morphological abnormalities were in the head,

eye, beak and limbs. These structures are very sensitive to abnormal changes in RA

concentration. For RA to function correctly it needs to be localized to the correct region at the

correct time and concentration. This is why the pattern of RA synthetic and catabolic enzymes

is so essential for normal development. Interference with this pattern by high or low levels of

either vitamin A or RA will disrupt the developmental events normally regulated by RA. This

explanation would predict that those areas in which RA signaling normally occurs are the

regions that are most sensitive to RA teratogenicity. This indeed is the case, and areas of the

CNS sensitive to RA teratogenicity, such as the eye, ear and spinal cord, are also regions that

require RA for normal development [5]. Our results are in assent with the findings of the

earlier studies aforesaid above.

Conclusion:

This study shows that treatment with citral decreases the level of endogenous RA than the

level needed to maintain the normal embryonic development and that leads to severe

malformation. Treatment with exogenous RA might rescue the embryo from teratogenic

effects of citral and that leads to the partial mitigation in some embryos. It suggested that the

response of embryos to RA is very sensitive. The lowest dose of RA (0.25 µgm) could

partially mitigate the effect of citral while higher doses of RA (0.5 and 1 µgm) exerted

teratogenic properties of RA rather than mitigative effects.


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______________________________


تأثير المعالجة المصاحبة بحامض الرتينويك على تحسين التشوهات المورفولوجية المستحثة بالسيترال

للكتكوت الجنينيأثناء النمو

أبو القاسم لسيدهبة ا , داليا الزهراء فاروق مصطفى ورضا عبدالرحمن علي , يصز61517لظى ػهى انذا, كهح انؼهو, جايؼح أطط, أطط

أدذ يشرماخ فراي أ درا ايا ف يزادم ان انجح أضا انثانغ. دس أ انزذك دايضهؼة

ذرطهة انزذك داخم انجظى دايضهؼة درا ايا ف ظخ انؼذذ ي انجاخ انخرهفح انح ن انج, يظراخ

ي انظرخزجح انؼطزح انشخ ي رئظ يزكة انظرزالذظا دلما ػ طزك انرذكى تؼهاخ انذو انثاء.

جذ أ ن انؼشثح انثاذاخ ي انؼذذ كا شى ي داخم انجظى. انزذك نرخهك دايض يصثطا يثاشزا ذأشزاانطثؼح

0,25اطح )ي انرزكشاخ انر ذى اطرخذايا ف ذ انذر انزذك دايضي ذا انثذس يؼزفح أ ذزكش ي انذف

ياكزغزاو( الأكصز فؼانح ف ذذظ انرشاخ انظرذصح تانظرزال ف ج انكركخ. 1أ 0,5أ

طد يجػاخ إن انثض ذمظى ذى". ديظركض جالاص" ع ي نهكركخ يخصثا تضا انثذس ذا ف اطرخذو

50)يذة دايض انزذك (, يجػح انظرزال ) انصم شائ طهفكظذ: انجػح انضاتطح, يجػح

انخرهفح جزػاخ انصلاز ي ادذج جزػح يغجزػح انظرزال ي تشج يا كلا دمدياكزيلار(, شلاز يجػاخ

سا, كا ذى يرفنجا انؼاخ دراطحياكزغزاو( ػه انران. ذى 1أ 0,5أ 0,25ي دايض انزذك )

, ذذهها إدصائا. (انفمض) و 21 تؼذ ذظجم يؼذلاخ انفاخ انفمض ف جغ انجػاخ

أظزخ انذراطح أ انجػح انؼانجح تانظرزال كاد يشح نهغاح, ف د أ انجػح انؼانجح تانظرزال

جذ أ يؼذل ياكزغزاو( أظزخ ذذظ ظ 0,25ألم جزػح ي انزذك ) ث يمارح تانجزػر الأػه. كا أ

% ( ػه انران يمارح 65% 60%, 00انفاخ ف انجػاخ انؼانج تانظرزال دايض انزذك يؼا كاد )

%(. لذ ندظد ذشاخ يخرهفح ف الأجح يصم: ذأخز ان, ذش انذياؽ, ضر أ جذظ ف 72,5تانظرزال فمظ )

نؼ, انراء انزلثح, اخرفاء انزش ف تؼض الاياك ا اخرفاؤ كها غاب الأطزاف. ػذ ذذهم انماطاخ انرفنجحا

) انس, طل انجظى, طل انزأص, يذظ انزأص, ارذفاع انزأص طل كم ي انجاح انزجم ( ندع جد ادصائا

زخ ألم جزػح ي جزػاخ انزذك انجػح انضاتطح. تا أظفزق يؼح يهذظح ت انجػاخ انؼانجح

ياكزغزاو(, 1أ 0,5)نماطاخ يمارح تانجزػر الأػه ياكزغزاو( يغ انظرزال ارذفاع يؼ يهذظ ف ا 0,25)

نكا نى ذك يؼح يمارح تجػح انظرزال فمظ .

ذضخ ذ انذراطح أ انؼانجح تانظرزل أدخ إن ذمهم يؼذل دايض انزذك انذاخه انلاسو نه انطثؼ

لذ ذك أ انذرم ينهج يا أد إن ظر ذشاخ خهم خطزج, كا أ انؼانجح تذايض انزذك انخارج

ػانح انزذكذا شز إن أ دظاطح اطرجاتح انج نذايض ػضد انمص انذ أدذش انظرزال داخم انخلاا.

ياكزغزاو( أدخ إن ذذظ ظث, ػه انمض انؼانجح تانجزػر الأػه 0,25جذا , دس أ انؼانجح تألم جزػح )

(ياكزغزاو 1 أ 0,5) ا أكصزي ك محسنا. لذ أظزذا ذأشزا يش

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