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44 CHAPTER 2 EFFECT OF CUTTLEFISH INK ON CHICK EMBRYO DEVELOPMENT 45 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) 46 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. 47 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 48 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. 49 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.