Download chapter 2 effect of cuttlefish ink on chick embryo

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CHAPTER 2
EFFECT OF CUTTLEFISH INK ON CHICK
EMBRYO DEVELOPMENT
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2.1 INTRODUCTION
Cuttlefish eject ink which is used to deceive large predators when they try to
attack the cuttlefish. The ink acts as decoy and it is believed to have an anesthetic effect
as well. The melanin pigment is manufactured in the mature cells of ink gland (Ortonne
et al., 1981; Palumbo et al., 1997a) a highly specialized organ situated at the bottom of
the ink sac and deputed to continuous production of ink. At the end of the maturation
process, ink gland cells degenerate and shed their contents into the ink sac, acting as a
reservoir of the exhausted material (Russo et al., 2003). The question of the toxicity of
cuttlefish ink is still up in the air, although it is clear that some cephalopod ink is indeed
toxic, but again, the major reason the ink is thought to be toxic is because it coats the gills
of predators causing them to suffocate (Hanlon and Messenger, 1996). The cuttlefish ink
finds wide application in homeopathic medicine (medicinal name – ‘sepia’). Sepia is used
to treat hormonal imbalances especially in women. The source of this medicine is Sepia
officinalis (Boericke, 1999).
Takaya et al., (1994a) investigated the antitumour activity of peptidoglycan from
the squid ink. The melanin free ink of the cephalopod, Sepia officinalis is shown to
contain a heat labile proteinaceous component toxic to a variety of cell lines, including
PC12 cells (Russo et al., 2003). As not much is known about the toxicity of the cuttlefish
ink, investigations were carried out to study the effect of cuttlefish ink on chick embryo
development. The findings are reported in this chapter.
2.2 MATERIALS AND METHODS
2.2.1 REAGENTS AND CHEMICALS
Phosphate Buffered Saline (SRL, Mumbai)
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Proteinase K (SRL, Mumbai)
DNA marker (Genie, Bangalore)
All the other chemicals were purchased from SRL, Mumbai.
2.2.2 EMBRYONATED CHICKEN EGGS
Embryonated chicken eggs were purchased from the poultry farm of the College
of Vetenery and Animal Science, Kerala Agricultural University, Thrissur, Kerala and
brought to the laboratory. The eggs were incubated at 37oC in a humidified incubator till
they were nine days old. The eggs were candled to select the healthy embryos for drug
treatment.
2.2.3 PREPARATION OF CUTTLEFISH INK
Fresh female specimens of the cuttlefish, Sepia pharaonis were collected from the
fishing harbour, Kochi, Kerala and brought to the laboratory in ice cold condition. The
animals were carefully dissected to remove the ink sac aseptically. A known weight of
ink was then dissolved in sterile normal saline to obtain a concentration of 250 g / ml.
2.2.4 EFFECT OF CUTTLEFISH INK ON CHICK EMBRYO DEVELOPMENT
To study the effect of cuttlefish ink on chick embryos, the nine days old
embryonated eggs were inoculated with different concentrations of the ink (25 g, 50 g
and 75 g) through the shell aseptically. Four replicates were kept for each treatment.
The perforation was sealed with paraffin wax and eggs were incubated at 370C in a
humidified incubator. The chicken embryo inoculated with normal saline served as
control. The eggs were candled every day for any dead embryos and the eggs with dead
embryos were transfered to the refrigerator. On the fifth day of the experiment, all the
eggs were opened and the embryos were physically examined.
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2.2.5 EFFECT OF CUTTLEFISH INK ON DNA FRAGMENTATION
One gram of tissue was taken from the control and ink treated chick embryos. The
tissue was sliced into small pieces by using scissors and homogenised by using a mortar
and pestle under ice cold condition. To this 500 l neutral lysis buffer (100 mM NaCl; 10
mM Tris-HCl pH 8.0; 20 mM EDTA and 2% SDS) was added. To this 30 µl protenase k
(100 µg /ml) in reaction buffer was added and incubated overnight at 370C. DNA was
isolated using standard procedure (Sambrook & Russell, 2001). The DNA was
precipitated using 3 M sodium acetate and ethanol and it was dissolved in TE (TrisEDTA Buffer) buffer. Electrophoresis was performed in 0.8% agarose gel in Tris Acetate
EDTA (TAE) buffer at 100 V for 1 h. After electrophoresis DNA was visualized by
ethidium bromide staining.
2.3 RESULTS
2.3.1 CUTTLEFISH INK INHIBITS CHICK EMBRYO DEVELOPMENT
The embryos in the untreated or control group were live and healthy whereas the
embryos in the ink treated groups did not survive and they were all dead within five days.
On closely examining the embryos, it was found that embryos in the ink treated group
showed stunted growth and the ink has significantly affected the normal development of
different organs. Size of the drug treated chick embryo was considerably reduced
compared to the control embryo (Figure 2.1). The photographs reveal that the treatment
of the eggs with cuttlefish ink has affected the normal development of head, neck and
web formation of the embryo. The results thus, showed that cuttlefish ink markedly
inhibit the development of chick embryo.
LEGEND FOR FIGURE 2.1
Inhibitory effect of cuttlefish ink on chick embryo development.
Control
- untreated chick embryo
Drug treated - chick embryo treated with 50 g of cuttlefish ink
LEGEND FOR FIGURE 2.2
Effect of cuttlefish ink on DNA fragmentation
M- Marker DNA
A-Control-DNA isolated from untreated embryo.
B-Chick embryo treated with 20 g of cuttlefish ink
C-Chick embryo treated with 50 g of cuttlefish ink
D-Chick embryo treated with 75 g of cuttlefish ink
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2.3.2 CUTTLEFISH INK INDUCES DNA FRAGMENTATION IN CHICK
EMBRYO
DNA was isolated from the tissues of both ink treated and untreated (control)
chick embryos. Agarose gel electrophoresis of the isolated DNA from ink treated embryo
showed fragmentation of DNA, whereas the DNA from untreated embryos did not show
any fragmentation. The control samples show intact DNA (Figure 2.2). The result also
showed that there is a dose dependent increase in the length of fragmented DNA of the
ink treated embryos.
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2.4 DISCUSSION
Squid ink is known to have antibacterial activity (Mochizuki, 1979) and also able
to regulate gastric juice secretion in rats (Mimura et al., 1982a). The results of the present
investigation revealed that the crude ink of cuttlefish, Sepia pharaonis, inhibits the
development of chicken embryo. The cuttlefish ink inhibits the normal development of
head, neck and web formation of the embryo. Agarose electrophoresis of the isolated
DNA from ink treated embryo showed fragmentation of DNA, whereas the DNA from
the untreated embryos did not show any fragmentation. Internucleosomal DNA damage
and fragmentation are the hallmarks of apoptosis. Agarose gel electrophoresis of DNA is
one of the usual methods of demonstrating apoptosis (Kerr et al., 1972). DNA
fragmentation observed in the present study thus indicates the apoptosis inducing activity
of cuttlefish ink on chick embryo. Takaya et al. (1994a) investigated the antitumour
activity of peptidoglycan from the squid ink. They extracted squid ink using Tris-HCl
buffer and fractionated using DEAE Sephacel ion exchange chromatography and
sephacryl S-300 gel filtration to give peptidoglycan fraction which exhibited strong
antitumour activity against Meth A fibro sarcoma in mice. The melanin free ink of the
cephalopod, Sepia officinalis is shown to contain a heat labile proteinaceous component
toxic to a variety of cell lines, including PC12 cells (Russo et al., 2003). The results of
this preliminary study on chicken embryo suggest that the ink has an inhibitory effect on
the growth of the dividing and undifferentiated cells. The ink also induced
internucleosomal DNA damage, indicating apoptosis. Thus, the cuttlefish ink has certain
apoptosis inducing properties which needs to be further investigated for using it as an
anticancer agent.