INTRODUCTION:

Use of animals for the research purpose is an age long practice. Testing of a new moiety or toxicological approach to a new drug involves use of rats, mice, guinea pigs etc. (Sonali K Doke et al 2015). These animals are exploited as a tool to investigate new treatment options for various diseases. Scientists are in continuous search for new alternative approaches (Christian Liedtke et al 2013) according to the concept of 3R (Reduction, Refinement and Replacement) as introduced by Charles Hume and Willian Russell. One such approach is the use of fishes in order to combat the sufferings, pain and death caused in rodents during animal experimentation. (Schaeck M et al 2013) (Kumbhar Swapnil et al 2013)

Channa Punctatus commonly called as Snakehead Fish, occurs in native range of Ganges-Bharahamputra River basin, stagnant water and muddy areas (Bondre et al 2009).

Also found in open water surfaces to dense vegetation in Keoladeo National Park, Bharatpur, North Central India. (M. Afzal Khan et al 2013) (Froese R et al 2010) (Harit G et al 2007), These are carnivores in nature, attributing to this habit of C. Punctatus is considered as pests in India. (Deepak Rao Khadatkar et al 2011). Hence using these fishes for research is a new and synergistic process. (Nilantika Paul et al 2014) (Sanjay Pandey et al 2005) (Nachiket et al 2009)

Thioacetamide (TAA) is a well-known toxin to cause liver injury in rats. (MC Wallace et al 2015) (Ramachandran P et al 2012) (Mallikarjuna Rao Talluri et al 2016). (S.H.Baie et al 2000). The potency of TAA expressed in two step bio-activation in which thioacetamide sulphoxide is first formed (TASO) leading to thioacetamide S, S-dioxide (TASO₂) formation. (Tasleem Akhtar et al 2012) (Jae-Sang Hong et al 2017) (Sahu et al 2012)

In this study, TAA is used as toxicant to induce hepatocellular carcinoma in Snakehead fishes. And it was explored for its receptor and biological network involved.

MATERIALS AND METHODS:

Experimental Fish: Healthy Channa Punctata fishes were acquired from certified fish vendor. The body weight of fishes were ranging in 20-25g and body length 8-10cm. The fishes were first acclimatized in the lab for 15-20 days. The laboratory conditions were maintained at room temperature (27 ± 3ºC) and dechlorinated water was filled in fish tanks. Foe maintaining the hygiene the water of tank was changed regularly and fishes were regularly fed with supplementary food.

Toxin and Stock Solution: Commercially available Thioacetamide (TAA) extra pure AR from Sisco research Lab was procured. The toxin was stored in the temperature range of 2-8°C.Stock solution of 1mg/ml was prepared by dissolving 10mg TAA in 10ml distilled water.

Grouping of Fishes:

The fishes were weighed and grouped together by maintaining six fishes in each group. Pilot experimentation was conducted to deduce the LC 50 value during which fish groups were exposed to different concentration values 50-60µl for 24, 48 and 72 hours before finalisation of actual dose. Simultaneously control group was administered with distilled water.

Study design:

The C. Punctata fishes were divided in three groups having six fishes in each group including control. Group 1 (Control) group was administered with distilled water while in Group 2 (Diseased) and group 3 (Diseased) 50 µl TAA was administered intraperitoneally. The dosing were followed with the constituent pattern of every third day of the previous dose. The study was carried out for 21 days and each day number of dead fishes (if any) were counted and removed from the tank. The animal was sacrificed at 7^th, 14^th and 21^stday.

Biochemical estimations:

Aspartate aminotransferase (AST), alanine aminotransferase (ALT), Catalase, GSH assays were done with diagnostic kits procured from Span diagnostic and the procedure was followed according to manual provided by manufacturer. It was further analysed by One Way ANOVA.

Histopathological estimation:

The fishes were first given mild Chloroform and then they were sacrificed. The body was clipped properly through pins on the wax Petri plate. The liver of fishes at each stage was removed carefully and kept in 10% Formalin solution. The histopathological estimation was performed on control, Stage I, II and III.

Pharmaco-networking Studies:

The top interacting genes for Thiaocetamide were retrieved from Comparative Toxicogenomics Database. The receptor list was eventually submitted to STRING database. The master network so formed evaluated for different proteins and signalling pathways on administration of Thioacetamide. The pathways were supported by their probable values.

RESULTS:

Behavioural pattern of fishes:

The behaviour of fishes were checked after introduction to toxin and it was found that initially fishes were striking against the wall of tank in a disturbed manner. Along with this irregular patter of swimming, restlessness were also seen. The abdomen of fishes were swollen after fifteen days. (Fig1)

Fig 1(a) Channa Punctatus in Lab

The mortality was observed at third, sixth, eighteenth and twentieth day of experiment. Hundred percent mortality was examined at dose above 55 µl. During the experimental tenure no behavioural change and mortality is observed in Control group.

Biochemical estimation:

AST, Catalase, ALT, GSH were considerably increased in diseased tissues. Moreover the levels were in correlation with the disease progression. (Fig 2) It was found that AST levels were increased 0.56 AU to 0.90 AU (p<0.01), catalase was on higher side up to 0.95 AU (p<0.01), ALT was found to be amplified to 0.98 AU (p<0.01) and GSH was increased upto 1.0 AU (p<0.01)as compared to control group.

Fig 2: Biochemical estimation of AST, Catalase, ALT, GSH in the liver of Channa fish

Histopathological studies:

In this present study, carcinogenesis was achieved using TAA. Histopathological examination of hepatocytes of control (Fig3a) showed mildly effaced architecture of liver and dilated sinusoidal space but No hepatocellular carcinoma was found. In stage I (Fig 3b) fatty changes in the liver with cholestasis and fibrosis of portal tract and its extension from portal to portal region were observed. In stage II (Fig3c) hepatocellular carcinoma was found with effaced architecture of liver characterised by tumour having polygonal shape, scant cytoplasm, round to oval nucleus with coarse chromatin and high N: C ratio. Later on it showed autolysed tissue. (Fig3d)

Fig 3(b): Stage I HCC- Section examined shows fatty changes in the liver with cholestasis and fibrosis of portal tract and its extension from portal to portal region.

(H and E, 400X)

Fig 3 (a): Control liver - Section examined showsmildly effaced architecture of liver and dilatedsinusoidal space. No hepatocellularcarcinoma Seen.

(H and E, 400X)

Fig 3(d): Stage III HCC – shows autolysed tissue

(H and E, 400X)

Fig 3(c): Stage II HCC- Section examined shows effaced architecture of liver by tumour having polygonal shape, scant cytoplasm, round to oval nucleus with coarse chromatin and high N: C ratio. It is hepatocellular carcinoma.

(H and E, 400X)

Pharmaco networking studies:

Literature mining was done for Thioacetamide chemical and its receptors were retrieved. From Comparative Toxicogenomics Database. Then the entire receptor list was submitted for prediction of protein-protein interaction (Figure 4) and their associated processes and pathways. (Table1). The gene recovered are TGFB1, ACTA2, COL1A1, TNF, TIMP1, GPT, IL6, CASP3, TIMP2 and IL1B. It was hypothesised that major probable processes involved are regulationof cell differentiation, chemokine biosynthetic process, regulation of JNK cascade, Regulation of ERK1 and ERK2 cascade, Regulation of apoptotic process and many more which play important role in Carcinogenesis.

Fig 4: Receptor- Network prediction of Thioacetamide

Table 1: Processes targeted by Thioacetamide along with Probability values

Processes	Probability
Positive regulation of cell differentiation	5.58E-05
Regulation of chemokine biosynthetic process	1.48E-05
Extrinsic apoptotic signalling pathway	1.60E-05
Positive regulation of acute inflammatory response	2.38E-05
Regulation of leukocyte cell adhesion	1.71E-05
Response to lipopolysaccharides	2.06E-05
Regulation of vascular endothelial growth factor production	3.20E-05
Response to oxygen containing compounds	4.44E-05
Positive regulation of MAPK cascade	6.93E-05
Aging	1.40E-03
Positive regulation of JNK cascade	6.50E-03
Generation of neurons	1.24E-02
Embryo development	2.39E-02
Carboxylic acid biosynthetic process	2.75E-02
Regulation of ERK1 and ERK2 cascade	1.60E-03
Positive regulation of histone acetylation	7.40E-04
Regulation of apoptotic process	0.00036
Regulation of proteolysis	0.00025
Regulation of Phagocytosis	0.00011

DISCUSSION:

India’s ranking in world fish production comes at third position. (StevenJ. Cooke et al 2016). The role of fresh water aquaculture in food, economy, generating employment and living hood of people cannot be ignored.( Raghav et al 2009) Channa –an Asian Genus has 26 valid species and it is distributed in Iran and Southeren Asia, South Eastern Asia, China, Taiwan, Korea, and Southern Russia (Mallikarjuna Rao Talluri et al 2016) (Thirumurugan et al 2013). Thioacetamide chemically is a organosulfur compound and earlier it is used in textile and paper industries and laboratories where leather is processed. It effects protein synthesis, (Edokwe Chinelo et al 2010) RNA, DNA, and gamma glutamyl transpeptidase activity. Hence it is a effective hepatotoxicant for the induction of acute and chronic liver injury (MC Wallace et al et al). (Dibyajyoti Saha et al 2011)(Natarajan K et al 2011).It was first discovered as hepatotoxic in rats in 1948. Acute exposure to TAA is depicted by ISHEN (International Society for Hepatic Enchephalopathy and Nitrogen Metabolism) as a well proofed animal model for liver failure. In the present study TAA is used to induce hepatocellular carcinoma in Channa Punctatus and further its various stages were studied through histopathology. Its Pharmaco Networking analysis was carried using STRING software and it was found to be disturbing to various biological processes.

CONCLUSION:

Thioacetamide is a proven toxin for induction of Hepatocellular carcinoma in rats and mice. However the use of Thioacetamide in Fishes especially Channa Punctatus is still unexplored. Usage of fish models as a preclinical screening approach is widely appreciated worldwide. In the present study a new toxicity model for HCC has been developed in C. Punctatus in which clear changes in hepatocytes were found during histopathological analysis. Also it was hypothesised through computational analysis that Thioacetamide disturbs many biological processes linked to liver toxicity.

REFERENCES:

1. AV Bondre, SC Akare, P Mourya, AD Wanjari, PS Tarte, GV Paunikar. In Vitro Cytotoxic Activity of Leaves of Abutilon indicum Linn. Against Ehrlich Ascites Carcinoma and Dalton’s Ascitic Lymphoma Cell Line. Research J. Pharmacognosy and Phytochemistry 2009; 1(1): 72-74.

2. Christian Liedtke et al; Experimental liver fibrosis research: update on animal models, legal issues and translational aspects; Fibrogenesis and Tissue Repair2013; 6-19.

3. Deepak Rao Khadatkar, Yogesh Rathore . An Efficient and Useful Hybrid Approach for Detection of Lung Cancer. Research J. Engineering and Tech. 2(4):Oct.-Dec. 2011 page199-202.

4. Dibyajyoti Saha, Tarashankar Maity, Mayukh Jana, Supradip Mandal. Cancer Treatment Strategy-An Overview. Asian J. Pharm. Tech. 1(2): April-June 2011; Page 28-33.

5. Edokwe Chinelo, Obidoa Onyechi, Joshua Parker Elijah. Biochemical Effects of Persea americana (Avocado) and Dacryodes edulis (African Pear) Extracts on Liver Function Test of Albino Wistar Rats. Research J. Science and Tech. 2010; 2(2): 34-37 .

6. Froese R et al 2010: FishBase. World Wide Web elec-tronic publication. Available at: http://www.fishbase.org (accessed on 10 October 2010).

7. Harit G et al ; Endosulfan Uptake and biochemical profile of the muscle in a freshwater murrel Channa punctatus; Bulletin of Pure and Applied Sceinces , 2007, 26A; 23-27.

8. Jae-Sang Hong et al ; MicroRNA signatures associated with thioacetamide-induced liver ﬁbrosis in mice ; Bioscience, Biotechnology, and Biochemistry, 2017 ; Vol. 81, No. 7, 1348–1355.

9. Kumbhar Swapnil, Salunkhe Vijay , Magdum Chandrakant. Targeted Drug Delivery: A Backbone for Cancer Therapy. Asian J. Pharm. Res. 3(1): Jan.-Mar. 2013; Page 40-46.

10. M. Afzal Khan et al ; Morphometric variation of snakehead fish, Channa punctatus, populations from three Indian rivers; Journal of Applied Ichthyology 29(3) · June 2013.

11. Mallikarjuna RaoTalluri et al ; Thioacetamide induced acute liver toxicity in rats treated with Balanites Roxburghii extracts; Journal of Acute Disease, Vol5(5), 2016; 413-418.

12. MC Wallace et al ; Standard operating Procedures in Experimemtal Liver Research ; Thioacetamide model in mice and rats, Laboratory Animals; 2015, Vol. 49(S1) 21–29.

13. Nachiket S Dighe, Shashikant R Pattan, Deepak S Musmade, Abhijeet N Merekar, Manisha S Kedar, Deepak K Thakur, Pratik V Patel. Recent Synthesis of Marine Natural Products with Anticancer Activity: An Overview. Research J. Science and Tech. 2009; 1(2): 63-70 .

14. Natarajan K., Ramanathan S., Sathish R. , C. Amudhadevi. In Vitro Cytotoxic Study on Crude Extract of Marine Invertebrate Animal Polyclinum madrasensis Sebastian. Asian J. Pharm. Tech. 1(4): Oct. - Dec. 2011; Page 152-154.

15. Nilantika Paul et al ; Lead toxicity on non-specific immune mechanisms of freshwater fish Channa Punctatus; Aquatic Toxicology ; Volume 152, July 2014, Pages 105-112.

16. Raghav M. , Dixit S. Diversity of Fishes in a Freshwater Reservoir of Chhattisgarh, India. Research J. Science and Tech. 2009; 1(2): 103-107.

17. Ramachandran P; Liver fibrosis: a bidirectional model of fibrogenesis and resolution. QJM 2012, 105:813–817.

18. S. K. Sahu, D. Das, N. K. Tripathy. Hepatoprotective activity of aerial part of Glinus oppositifolius L. against Paracetamol-induced Hepatic Injury in Rats. Asian J. Pharm. Tech. 2(4): Oct. - Dec. 2012; Page 154-156.

19. S. H. Baie et al ; The wound healing properties of Channa striatus-cetrimide cream -wound contraction and glycosaminoglycan measurement,” Journal of Ethno pharmacology, vol.73,no.1-2,pp.15–30,2000.

20. Sanjay Pandey et al ; Acute toxicity bioassays of mercuric chlorideand malathion on air breathing fish Channa punctatus(Bloch); Ecotoxicology and Environmental safety; Volume 61, Issue 1, May 2005, Pages 114-120.

21. Schaeck M et al; Fish as research tools: alternatives to in vivo experiments; Alternative Lab Animals ; 2013 Jul;41(3):219-29.

22. Sonali K Doke et al; Alternatives to animal testing: A review; Saudi Pharmaceutical Journal; 2015 Jul; 23(3): 223–229.

23. Steven J. Cooke et al; On the sustainability of inland fisheries: Finding a future for the forgotten; Ambio. 2016 Nov; 45(7): 753–764.

24. Tasleem Akhtar et al ; An overview of thioacetamide - induced hepatotoxicity; Journal Toxin Reviews , Volume 32, 2013, Issue3, 43-46.

25. Thirumurugan D, Vijayakumar R.. Exploitation of Antibacterial Compound Producing Marine Actinobacteria against Fish Pathogens Isolated from Less Explored Environments. Asian J. Pharm. Res. 3(2): April- June 2013; Page 75-78.

Received on 10.06.2019 Modified on 22.06.2019

Research J. Pharm. and Tech. 2019; 12(7): 3559-3563.

DOI: 10.5958/0974-360X.2019.00607.3