Bioactivity of Epidermal Secretion of Two Marine Catfish from Mumbai

Bioactivity of epidermal secretion of two marine catfish from Mumbai

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Authors Deo, A.D.; Venkateshvaran, K.; Devaraj, M.

Download date 25/09/2021 21:40:42

Link to Item http://hdl.handle.net/1834/33363 97 J. Indian Fish Assoc., 35: 97- 111, 2008

BIOACTIVITY OF EPIDERMAl SECRETION OF TWO MARINE CATFISH FROM MUMBAI

Deo, A.D* ., Venkateshvaran, K., and Devaraj, M**. Centra/Institute of Fisheries Education, Versova, Mumbai 400 061 *College of Fisheries(CAU}, Lembucherra-799210Jripura(W}, ** 68/3,Greams Road,Chennai-600006

ABSTRACT

Crude mucus and its partially purified fractions from two marine catfish from Mumbai, Arius dussumieri and Osteogeneiosus militaris were assayed for their crinotoxicity through assays for hemolysis and haemagglutination of chicken erythrocytes, formation of paw edema in mice, and antibacterial activity against one gram-positive and four gram-negative bacteria. Assays were also done to block the edema using Phineramine maleate, Piroxicam, and Atropine sulfate. Crude toxin as well as their fractions from both the fishes exhibited haemolytic and haemagglutinating activities on chicken blood, besides edematous activity in mice models. The edematous activity was blocked by Phineramine maleate and Piroxicam but enhanced by Atropine sulfate; however, all these activities, either blocking or enhancing, were statistically insignificant. Antibacterial activity was absent in all the extracts tested.

Keywords: marine catfish, crinotoxicity, haemolysis, haemagglutination, edema, antidote.

INTRODUCTION determining in such a way the character of the biotic community (Maretic, 1988). lchthyocrinotoxic group of fishes is intermediate between venomous and Scaleless catfishes are known to poisonous fishes (Halstead and Courville, contain toxin in the skin secretions of the body 1970}; about 50 species of fishes belonging to (Othman et a!., 1993} with haemolytic, 13 families are ichthyocrinotoxic (Cameron dermonecrotic, edematic, vasospastic and and Endean, 1973). Their toxic behavior is lethal components (Mann and Werntz, 1991). comparable to that of amphibians, such as Works on catfish skin toxins and their bioactive toads and frogs that bear venomous glands in potential such as wound healing activity are their skin but lack inoculatory apparatus; the limited and include those on Plotosus lineatus venom serves them for defense purposes, (Shiomi et a/., 1986, 1987, 1988), P/otosus intoxicating predators by contact (Tachibana, canius (Auddy et a/., 1994}, Arius thalassinus 1988}. Besides serving as a repellent to and A. bilineatus (AI-Hassan eta/., 1982, 1983, enemies, epidermal secretions exert, with 1985, 1986, 1987, AI Lahham et al., 1987; their toxic metabolites, a most profound Alnaqeeb et a/., 1989}. There is a paucity of effect upon the sea environment, affecting information on toxicity of Indian marine the number and distribution of associated catfishes and hence the present work was plants and animals, influencing and taken up to establish the crinotoxic nature of 98 Deo, A.D., Venkateshvaran, K., and Devaraj, M

0 the two Indian marine catfishes, Arius were collected and stored at -4 C for further dussumieri and Osteogeneiosus militaris use. commonly available along the north west coast of India. Estimation of Protein

MATERIAL AND METHODS Protein in the crude toxin and fractions were estimated following Peterson (1977}, using Collection of Fishes Bovine Serum Albumin (BSA) as standard, reading absorbance spectrophotometrically at Arius dussumieri and Osteogeneiosus 750nm in the case of crude toxin and at 280 nm militaris caught by trawl, long line, gill net, etc. in the case of fractions. were obtained from the local landing centers (Versova, Ferry Wharf and Sassoon Dock) on a Assay for Haemolytic Activity monthly basis, brought to the laboratory in 0 ice, and immediately preserved at -20 C until Chicken blood was obtained from a use. nearby slaughter house, using EDTA solution (2.7 g in 100 ml of distilled water) as an anti Extraction of Crude Toxin coagulant @ 5% of the volume of blood, and brought to the laboratory. The blood was Crude toxin was extracted following immediately washed thrice with PBS (pH 7.2) at AI-Hassan eta/. (1982, 1986}. Twelve fishes 5000 rpm for7 minutes. From the packed RBC a from each monthly collection were thawed to 1% RBC suspension was prepared by diluting room temperature and mucus was scrapped with PBS. Haemolytic assay was performed in from them with a dull blade in phosphate "V" shaped microtitre plates. Serial two-fold buffer saline (PBS) pH 7.5, and homogenized. dilutions of the mucus (100 J..ll) were made in The homogenate was centrifuged twice, or PBS (pH 7.2} starting from the first well to last more, at 15000 rpm for ten minutes till a clear well with proper mixing. An equal volume of 1% supernatant was obtained. This clear RBC suspension was added to each well. The supernatant was further lyophilized on a plate was gently shaken and allowed to stand 0 Labconco freeze dryer and stored at -4 C for for two hours at room temperature and the further use as crude toxin. result was recorded. Appropriate controls were also included in the test. Reciprocal of the Partial Purification highest dilution of the mucus showing the haemolysis was taken as one haemolytic unit The crude toxin was chromatographed, based (HU). Formation of a compact button on Shiomi et al. (1987), through a DEAE formation at the bottom of the well indicated Cellulose Column. Five unadsorbed fractions negative haemolytic activity. were eluted with Tris HCL Buffer and then 10 adsorbed fractions were eluted step-wise with 0.1-l.OM NaCI in 0.01 M Tris-HCI buffer. Thus a total of 15 fractions, each of 15 ml, BIOACTIVITY OF EPIDERMAl SECRETION OF TWO MARINE CATFISH FROM MUMBAI 99

Assay for Haemagglutination Activity antiprostaglandin analgesic Piroxicam (Dolonex®, Pfizer/Dumex, Mumbai) and the This assay was done following Liang and Pan mydriatic agent Atropine sulphate (Atropine®, (1995). Procedure for the preparation of Patriot Pharma, Mumbai,) were procured erythrocyte was the same as that for the locally and injected i.p. into mice (20 ± 2g) @ 7 hamolytic activity. Haemagglutination assays mg/kg body weight. After one hour, edema were performed in 'U' bottom microtitre formation assay was conducted as described plates using serial dilution of25 Jll of sample above. Applying paired t Test at 1% and 5% (crude/fractionated mucus extract) in level, significance of the results was tested. physiological saline (0.9% NaCI pH 7.5). Two fold dilutions were maintained from first to Assay for Antibacterial Activity the last well, and finally a 2% RBC suspension was distributed in each well. Then the plates The antibacterial activity of the crude as well as were shaken for one minute and incubated fractionated mucus extracts was assayed by for one hour at 4°C,after which the paper disc method following Iguchi et a/. haemagglutinating activity was recorded. (1982). One species of gram-positive bacteria, The last dilution of sample showing complete Staphylococcus aureus and four species of agglutination wasta ken to contain 1 HA unit. gram-negative bacteria, Aeromonas hydrophi/a, Escherichia coli, Pseudomonas Assay for Edema Forming Activity putrifaciens and Vibrio alginolyticus were drawn from the stock cultures maintained in The edema forming activity of the Fish Processing and Fish Pathology mucus extracts and fractions was assayed Laboratories of CIFE. Nutrient broth was used following Smith {2002) as approved by the for growing the bacteria and antibiotic assay Institutional Ethical Committee. Group offive agar was used to prepare bacterial lawn. mice were injected sub plantar with 25 Ill of the mucus toxin in the right foot pad and with Bacterial lawns were prepared by 25 Jll of normal saline in the left foot pad. pouring 5 ml of overnight grown bacterial Thickness of the foot pad was measured using culture in nutrient broth in the petri dish a Vernier caliper before the injection and 2 containing antibiotic assay agar, allowed to hours after the injection. Edema Ratio (ER) cover the surface and finally the paper disc (8 was expressed as the percentage increase in mm dia, Hi Media, Mumbai) soaked in different thickness of the envenomated foot relative to concentrations, viz. 0.25%, 0.5%, 0.75% and the saline injected foot. The minimum dose 1.0% of lyophilized skin extract dissolved in PBS was defined as the dose causing 105% Edema were placed at the centre of each plate. After Ratio (ER). 24 hours incubation at 3l C, petri dishes were studied for growth inhibition by sample. A Blocking Teston Edema Forming Activity control set was also maintained, but the paper discs used were without mucus extract, having The antihistaminic Pheniramine been soaked only in PBS. maleate (Avil®, M/s. Hoechst, Mumbai), the 100 Deo, A.D., Venkateshvaran, K., and Devaraj, M

RESULTS from 4 HU to 32 HU during the study period; monthwise, F6 fraction of April, U4 fraction of Extraction of Crude Toxin May, and F8 fraction of November showed the highest activity of 32 HU. The lowest The quantity of mucus that was haemolytic activity viz. 4HU was observed in scrapped from 12 fishes in a given month September (F7L December (F3), January {F3 varied in quantity between 7 and 12 ml and F8), February (U2, F6) and March 1998 depending on the size of the fish. Smaller {U2). The results are presented in Table 2. fishes were observed to yield more quantity of mucus. Upon lyophilization, this quantity of Partially purified fractions of 0. mucus yielded 300mg to 500mg of crude toxin militaris showed the highest haemolytic in powderform. activity of 16 HU during May (F6) and September (F6 and FlO). The results are Estimation of Protein presented in Table 3.

The highest protein content in the Assay for Haemagglutination Activity crude toxin was 3631J.g/mg (November) in the case of A. dussumieri and 225 IJ.g/mg The crude mucus extracts of both fishes {September) in the case of 0. militaris exhibited partial haemagglutinating activity whereas the least quantity of protein was 275 that became more prominent upon partial IJ.g/mg (A. dussumieri) and 190 IJ.g/mg (0. purification. In the case of A. dussumieri, the militarisL both in February. The month-wise highest haemagglutinating titre of 32 HAU was data on protein content of the crude toxin are observed in November (F9). However, partially presented in fig. 1 and while that of the purified fractions of 0. militaris were found to fractions in fig. 2. have the highest haemagglutinating titre of 32 HAU in September (F8 and F9). Results of the Assay for Haemolytic Activity assays for haemagglutinating activity are described in Table 4 and 5. Crude mucus extracts as well as partially purified fractions of both fishes to be Assay of Edematous Activity haemolytic on chicken erythrocytes. Crude toxin of A. dussumieri exhibited haemolytic Crude as well as fractions of both the activity of 16 HU in August, September, fish caused paw edema in mice. Crude mucus October, November, and December while that extracts of" A. dussumieri showed a higher of 0. militaris exhibited the highest level of 8 amount of activity {154.29%), compared to HU in May, September and October. Results of those of 0. militaris {137.9%). Only two haemolytic activity of the crude mucus partially purified fractions (F5 and F7) of A. extracts are presented in Table 1. dussumieri showed edema forming activity. Fractions of 0. militaris also had two edema Partially purified fractions of A. factors, one in Unadsorbed (Ul) and another in dussumieri had varying haemolytic activity BIOACTIVITY OF EPIDERMAL SECRETION OF TWO MARINE CATFISH FROM MUMBAI 101

Blocking of Edematous Activity Pogonoperca punctata (Hashimoto and Oshima,1972, Oshima eta/.,1974), Moses sole Blocking of edematous activity with Pardachirus marmoratus {Primer and Zoltkin, three different drugs, Avit Dolonex and 1975, Lazarovici eta/.,1986) etc. Atropine had different effects. Dolonex was found to be more effective in bringing down Haemaggluting activity was also edema level than Avil. However, both of present, in addition to haemolytic activity, to them were found to be statistically some extent in the crude toxin. However, after insignificant at 1% and 5% level. Atropine in all partial purification, the haemagglutinating cases was found to increase edema level, but activity was more pronounced. AI-Hassan et this increase was also statistically a/. (1986) isolated and purified a lectin from the insignificant. The results on edema formation epidermal secretion of A. tha/assinus which and blocking of edematous activity are comprised about 2% of the total gel protein, presented in Table 6 and 7. lacked carbohydrates and contained no unusual types or amounts of aminoacids; the Assay for Antibacterial Activity lectin was reported to agglutinate a wide range or red blood cell types. Haemagglutinating Crude toxin as well as partially activity was also reported from the skin mucus purified fractions from both the fishes, in all of the Moray eel, Lycodontis nudivomer by the concentrations used, had no antibacterial Randall eta/. (1981L and that of the Japanese activity with reference to the bacteria tested. eel, Anguillajaponica by Suzuki {1985).

DISCUSSION A total of 2 edematous factors were discernible in each ofthe species studied upon Crude as well as partially purified partial purification of the crude toxin. Edema toxins of both the species exhibited a potent forming activity, in conformity with the present haemolytic activity against chick data, has also been reported in case of mucus erythrocytes. Upon partial purification, 3 extracts from P. /ineatus (Shiomi et a!. 1986, haemolytic factors each in the case of A. 1987, 1988) as also from seasnake venom (Tu, dussumieri and 0. militaris were obtained. 1988). Haemolytic activity has been shown to be present in the mucus of various catfishes such It is noteworthy that, in the present as A. thalassinus (AI-Hassan et a!., 1982, investigation all lethal factors (results of which 1986) A. bilineatus (Thomson eta/., 1998) P. are being published elsewhere) also had either lineatus (AI-Lahham eta/., 1987, Shiomi et al., haemolytic, or edematous activities; also, 1986) P. canius {Othman et a/., 1993L eels there were fractions with only haemolytic or such as Anguilla japonica (Suzuki, 1985) only edematous activities without concomitant Lycodontis nudivomer (Randall et a/., 1981) lethal activity. Such combined lethal and soapfishes, edematous activities of the same fraction have been reported in the case of P.lineatus by 102 Deo, A.D., Venkateshvaran, K., and Devaraj, M

400 mm A. dussumieri ~ 0. militaris en 3so E tD 300 ::1. -~ 250 a> c 200 'a') 0... 150 A. 100 50

0 l\1ar April May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar

Month

Fig. 1. Showing month-wise variation in protein content of the crude mucus toxin from Arius dussumieri and Osteogeneiosus militaris

~A. dussumieri ~ 0. militaris

0.6

o; 0.5 E --Ol 0.4 :::!. c 0.3 -a> c -0 0.2 0 !: ·; 0.1 0 -1.. D.. 0 Ul U2 U3 U4 US Fl F2 F3 F4 FS F6 F7 F8 F9 FlO Fraction

Fig. 2. Showing protein content in various fractions of the crude mucus toxin from Arius dussumieri and Osteogeneiosus militaris; all values are average for 13 months 103 BIOACTIVITY OF EPIDERMAL SECRETION OF TWO MARINE CATFISH FROM MUMBAI Table 1. Showing haemolytic of crude mucus extracts of A. dussumieri and 0. militaris on

chicken blo~o~d ______Month A. dussumieri 0. militaris

Haemol·y:tic Unit (HU) Haemolytic Unit (HU) March 4 2 April 4 4 May 8 8 June 8 4 July 8 4 August 16 2 September 16 8 October 16 8 November 16 4 December 16 2 January 8 4 February 4 2 March 4 2

Table 2. Showing haemolytic activities in the fractions of A. dussumieri on chicken blood

Month Fraction

Ul U2 U3 U4 us 0.1M 0.2M 0.3M 0.4M O.SM O.GM 0.7M 0.8M 0.9M l.OM

Mar. 8 16 16 Apr. 16 8 32 May 32 16 8 8 June 8 4 16 July 16 8 16 Aug. 16 8 16 Sept. 8 8 4 Oct. 8 16 8 Nov. 16 8 32 Dec. 16 4 16 Jan. 8 4 4 Feb. 4 8 4 Mar. 4 8 8

Ul- US: Unadsorbed fractions 0.1-1.0 M: Adsorbed fractions (Fl- FlO) 104 Deo, A.D., Venkateshvaran, K., and Devaraj, M

Table 3. Showing haemolytic activities in the fractions of 0. militaris on chicken blood

Month Fraction

Ul U2 U3 U4 us O.lM 0.2M 0.3M 0.4M O.SM 0.6M 0.7M 0.8M 0.9M l.OM

Mar 4 8

Apr 4 8

May 8 16 8

June 8 4 4

July 4 8 2

Aug 4 8 4

Sept 4 16 16

Oct 8 8

Nov 4 8 4

Dec 4 4 4

Jan 8 8 8

Feb 4 8 4

Mar 4 4

Ul- US: Unadsorbed fractions 0.1-1.0 M: Adsorbed fractions (Fl- FlO) BIOACTIVITY OF EPIDERMAL SECRETION OF TWO MARINE CATFISH FROM MUMBAI 105

Table 4. Showing haemagglutinating activities in the fractions of A. dussumieri on chicken blood

Month Fraction

Ul U2 U3 U4 US O.lM 0.2M 0.3M 0.4M O.SM 0.6M 0.7M 0.8M 0.9M l.OM

Mar 16

Apr 8

May 8

June 16

July 16

Aug 4

Sept 8

Oct 4 8

Nov 32 8

Dec 16 8

Jan 4

Feb 4

Mar 4

Ul- US: Unadsorbed fractions 0.1-1.0 M: Adsorbed fractions (Fl- FlO) 106 Deo, A.D., Venkateshvaran, K., and Devaraj, M

Table 5. Showing haemagglutinating activities in the fractions of 0. militaris on chicken blood

Month Fraction

Ul U2 U3 U4 US O.lM 0.2M 0.3M 0.4M O.SM 0.6M 0.7M 0.8M 0.9M l.OM

Mar 8

Apr 4 4

May 4 8

June 8

July 8

Aug 4 8

Sept 32 32

Oct 16 8

Nov 8 4

Dec 8

Jan 4 8

Feb 4 4

Mar 4

Ul- US: Unadsorbed fractions 0.1-1.0 M: Adsorbed fractions (Fl- FlO) BIOACTIVITY OF EPIDERMAL SECRETION OF TWO MARINE CATFISH FROM MUMBAI 107

Table 6. Showing edematic activity of the crude mucus extract and fractions of A. dussumieri and its blocking A: Mice i.p. injected with only the toxin extrac B: Mice i.p. injected previously with Avil@7mg/ kg. C: Mice i.p. injected previously with Dolonex @7mg/ kg. D: Mice i.p. injected previously with Atropine@7mg/ kg.(Results are presented as Mean ± SE)

Edema Ratio(%} Toxin solution A B c D

Crude extra ct 154.29 ± 0.144 149.63 ± 0.227 140.438 ± 0.286 160.726 ± 0.167

F5 145.90 ± 0.523 134.29 ± 0.376 131.52 ± 0.366 145.97 ± 0.400

F7 135.13 ± 0.276 130.56 ± 0.376 125.116 ±0.231 141.10 ± 1.514

FlO

UA 108 Deo, A.D., Venkateshvaran, K., and Devaraj, M

Table 7. Showing edematic activity of the crude mucus extract and fractions of 0. militaris and its blocking A: Mice i.p. injected with only the toxin extrac B: Mice i.p. injected previously with Avil@7mg/ kg. C: Mice i.p. injected previously with Dolonex @7mg/ kg. D: Mice i.p. injected previously with Atropine@7mg/ kg.(Results are presented as Mean± SE)

Toxin Edema Ratio(%} solution A B c D Crude 137.9± 0.279 131.68 ± 0.460 125.49 ± 0.222 146.79 ± 0.545 extract Fl

FlO

UA 135.112 ± 132.25 ± 0.866 126.90 ± 0.433 139.934± 2.365 0.178 BIOACTIVITY OF EPIDERMAL SECRETION OF TWO MARINE CATFISH FROM MUMBAI 109

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