ISOLATION OF CYTOTOXIC COMPOUNDS FROM MARINE ANIMALS

Abstract

During last three decades, a large number of toxins have been purified and characterized from several marine organisms. Among different classes of marine toxins, the jellyfish (coelentrate), shel1fish (mollusks) and sea-snake (Hydrophiidae) toxins are considered dangerous to humans due to their mild to severe clinical complications in victim. The toxins purified from these animals are broadly grouped as cytotoxins and biotoxins. This research project was the aim of to identify venomous and poisonous marine animals suspected to induce cytotoxicological reaction in living system and to isolate and characterize the cytotoxic compounds causing these reactions. To achieve both objectives a regular survey of envenomation and poisoning on coastal areas of Karachi was performed on regular basis folJowed by location and co1lection of species responsible for the clinical manifestations. Later, venoms and poisons from these species and their toxic compounds were isolated and characterized using specific biochemical and biological assay techniques. TI1e survey of envenomation incidences occurred due to jelly-fish, shellfish and sea-snake species has been done with the help of questionnaires. Site selected for the survey includes all recreational beaches such as Clifton, Hawksbay, Sandspit and Paradise point. The intensity of jellyfish envenomation incidences have been recorded both in beach visitors and community residents and a total of 1558 (mild and severe) cases have been recorded which includes 1183 beach visitors and 369 residents of coastal community. The survey record indicates that Physalia species was the most abundant jellyfish species on Karachi coastal areas and implicated in 98 percent of the cases. All victims exhibited typical signs and symptoms such as erythema, whealing, papular eruption, spasms etc. A few cases of shellfish poisoning (33 in number) and non-fatal sea snake envenomation cases (21 cases) were also recorded. The clinical signs and symptoms recorded are nausia, vomiting. diarrehea, watery stool and muscular cramps. The major biochemical constituent of Physalia venoum and shellfish tissue were in a the range of total protein 48.45 to 52.34%, salt soluble protein 11.00 - 18.42% total lipd 9.8 to 11.6% and 11.2 to 13.5% respectively. The lethality of toxic extracts were noted and found variable values for LD50 and LD,oo doses in albino rats and mice. Animals received lethal doses ofjellyfish extract died after exhibiting periodical appearence of convulsions, ataxia, paralysis of hind limb and withdrawal. Physalia venum induced marked respiratory distress, tachycardia, ataxia, and in some cases coughing like symptom with spir of blood from nose were recorded. Death occured within 12 hours. General cytotoxicity data showed a marked increases in lactate dehydrogenase, creatinine phospholcinase, serum glutamate oxaloacetate transaminease, serum glutamate pyruvate transaminase, the protein and lipid content of serum significantly decrease inserum glucose. These finding suggest that Physa/ia venom is the most potent cytotoxic agent. A marked histopathological changes noted in vital organ of guinea pigs after receiving venoms and toxic extracts. Fatty changes, necrosis and hemorrhage ocuurd in all animals. In addition, branchoneumenia and glomerulonephritis with tubular degeneration was also obsereved. Standard chromatographic methods, namely, gel filteration, ion exchange and high performance liquid chromatography were used for the purification of cytotoxic compounds. TI1e recovery range is from 78% to 82%. Different gel matrices were employed for various toxins. Crude toxins were· resolved into a number of peaks, and it depends upon the type of matrix and eluent used. Each peak was tested for lethality and cytotoxicity, and peaks with these activities were further purified with HPLC. Polyacrylamide gel electrophorasis (PAGE) was used to check the purity of compounds and for the determination of their molecular masses. SOS-PAGE pattern of crude Physalia venom exhibited several bands in the molecular weight range of 200.00 Kda to 20. 00 Kda, whereas H. lapemoides venom exhiited heterogenous bands in the molecular weight range of 80.0 k.Da to 5.0 kDa. From the crude venom of Physalia (PrCV) one highly lethal and cytotoxic peak was obtained (Pp-CLTx-VI). Similarly, shellfish venom was purified and the Jethal and cytotoxic activity was confirmed in peak II which was subsequently purified into one compound (PvHTxIIE). TI1e sea snake venom was finally resolved into three purified peaks (HI-CTx-VIIA, HI-CTXVIIB, HI-CTx-VIID). 111e isolated fractions togather with crude venoms and toxins were characterized on the basis of physical and chemical stability, hemolytic, hemorragic, and neurotoxic activities, lethality and cytotoxicity and histopathological changes in vital organs of test animals. Changes in serum hepatic perameters and time dependent comparission of verapamil potentiation were also monitored in test animals. Veraparnil potentiation experiments showed that this drug significantly reduce the elevated hepatic parameters. Similarly, veraparnil (Ca++ ion antagonist) and 3, 4, DAP (K+ ion antagonist) were used to antagonized the changes in ion flux within the tissue of the vital organs due to the cytotoxic activity of venoms and toxins. In all cases, ion disbalance was marked in lung and kidney tissues. TI1e increased Ca++ ion influx was antagonized significantly by verapami1 and K+ ion eflux was also antagonised to the complete restoration of normal levels of these ions. This study reveals that potent cytotoxicity is associated with marine organisms. I t would be interesting to elucidate the structure of isolated compounds in future.