Toxicological effect and histopathological alterations on liver and heart of fish, and rat heart contamina by algal cyanotoxin and dimehypo

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY CO MAI TRANG TOXICOLOGICAL EFFECT AND HISTOPATHOLOGICAL ALTERATIONS ON LIVER AND HEART OF FISH, AND RAT HEART CONTAMINATED BY ALGAL CYANOTOXIN AND DIMEHYPO BACHELOR THESIS Study Mode : Full-time Major : Environmental Science and Management Faculty : Advanced Education Program Office Center Batch : 2014 - 2018 Thai Nguyen, 2018 DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Degree Program: Bachelor of Environmental Science and Management Student name: Co Mai Trang Student ID: DTN1454290032 Toxicological Effect and Histopathological Alterations Thesis Title: on Liver and Heart of Fish, and Rat Heart Contamina by Algal Cyanotoxin Dr Phil Arinafril and Dimehypo Krisna Murti, M.D Ph.D Supervisor (s): Do Xuan Luan Ph.D Marieska Verawaty Ph.D Supervisor’s signature (s) Abstract: To investigate the effects of environmental contaminants, histopathological response of Fish exposed to Algal Cyanotoxin and Rat exposed to Dimehypo pesticide have been used as sensitive biomarkers The present study was conducted to assess the histopathological alterations in the heart and liver of tilapia Oreochromis niloticus which were exposed Planktothrix of two concentrations of 1.48x104 cells/mL and 4.16x104 cells/mL under laboratory conditions The resultant histopathological changes in the heart and liver were recorded by light microscope The observed changes in the treated groups were showed moderate atrophy of hepatocytes leading to early necrosis, necrotic tissue with dilation and congestion of sinusoids and the leukocyte infiltration and sinusoidal dilatation/leukocyte infiltration in central veins in liver Atrophy and splitting of muscle fibers are recognized as common changes recorded in the heart of experimental fish reduction of muscle fiber diameter, edema in cardiac muscles, splitting of muscle fibers and infiltration of blood cells were an interesting observation in heart tissue The damages in histology of heart and liver depend on the exposure concentrations to Planktothrix toxin Besides this study was conducted to assess the histopathological alterations in the heart of Rat wistar were exposed to Dimehypo of concentration 3ml/l, The resultant histopathological changes in the heart and liver were recorded by light microscope Histopathological effects in the rat heart were detected after exposure to Dimehypo showed atrophy, splitting of muscles fibers There was vein artery dilatation and necrosis of cardiac cells of rat treated Large blood vessels involved in cardial tissue are strong dilated In necrotic myocites myofibrillar lysis is complete Therefore, the present investigation gives a brief account of the toxic effects of Algal Cyanotoxin on fish and Dimehypo on Rat The present review illustrates that these histopathological alterations would contribute an important role in assessing the harmful effects of Algal Cyanotoxin and Dimehypo As such, Fish and Rat are used as bio-indictors, providing useful purpose in monitoring Algal Cyanotoxin contamination, and Dimehypo pesticide Hence, implementation of regulations regarding the protection of environments must be taken into consideration Keywords: Algal Cyanotoxin, Dimehypo, Cyanobacteria, esticide, Toxicity, Number of pages: 47 pages Date of Submission: 10th August, 2018 ACKNOWLEDGEMENT From bottom of my heart, I would like to express my deepest appreciation to all those who provided me the opportunity to complete this research First and foremost, I would like to express my sincere gratitude and deep regards to my supervisors: Dr Phil Arinafril of Sriwijaya University, Indralaya, Indonesia, and Krisna MURTI, MD., M Biotech Stud., Ph.D and Marieska Verawaty Ph.D in the Department of Anatomical Pathology, Faculty of Medicine, Sriwijaya University/Dr Mohammad Hoesin Public Hospital who kindly assisted me with the histopathological detection in this dissertation and was very patient with my knowledge gaps and in guiding me wholeheartedly when I implemented this research I also want to express my thanks to Ph.D Do Xuan Luan, the second supervisor, for his supervision, encouragement, advice, and guidance in writing this thesis I would also like to acknowledge with much appreciation to the Dean of Faculty of Medicine in Sriwijaya University, Dr Mohammad Zulkarnain M Med Sc, PKK., who gave the permission to use all required equipment and the necessary materials to conduct my research in Department of Anatomical Pathology, Faculty of Medicine, Sriwijaya University/Dr Mohammad Hoesin Public Hospital I wish to thank the technicians who work in Department of Anatomical Pathology, Faculty of Medicine, Sriwijaya University/Dr Mohammad Hoesin Public Hospital for their help in tissue preparation, and Mrs Krisna Murti MD and Marieska Verawaty who provided me fish and rat for free Without them, this research could not be accomplished on time Of course, I would like to thank to my Indonesian friends - Hana, Shesyll, Naufal, Bayumi, Rizka, Hanif and others for their invaluable support and encouragement when I stayed in Palembang Finally, special thanks to my family, my friends for their love and moral support throughout my study Thai Nguy en, 10th August, 2018 Student Co Mai Trang TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES 1994 and Bury et al., 1995) In this concern, Butler et al (2009) reported that, several studies have observed severe liver damage in fish following oral administration of microcystins Dimehypo is pesticide with a degree of toxicity determined belongs to class II, mainly induces acetylcholine receptor blockade of the neuromuscular system, preventing the growth of pests The exposure pattern in this study is injections in Peritoneal Absorption is relatively rapid with intraperitoneal injection because of the rich blood supply to the abdomen It allows chemicals are absorbed into the blood relatively rapidly The rate of absorption by this route is usually one-half to one-fourth as rapid as from the intravenous one (Shimizu, 2004; Woodard, 1965) Limitations are the sensitivity of the tissue to irritating substances, less tolerance to solutions of non-physiological pH According to the result of present study, the exposure of tilapia to Microcystis and the exposure of rat to Dimehypo resulted many pathological changes in different tissues of the tilapiaConsequently, exposure to contaminated water may greatly affect many organs in fish which could enable toxic impacts to the organs of aquatic animals (Parvathi et al., 2011), and exposure to pesticides can have a detrimental effect on mammalian organs as well as on humans Futher more, the living organism is endowed with a number of defensive mechanisms that protect its DNA from genotoxic attack by chemical reactive intermediates and reactive oxygen species The most prominent enzyme system in the defence against reactive intermediates is the glutathione S-transferases, a superfamily of largely cytosolic proteins expressed in most tissues (John Wiley & Son, 2002) Due to this detoxification, we can saw in this study that there are parts of the tissue are damaged and there are parts that are not affected or not affected seriously 4.2.1 Liver Oreochromis niloticus (Nile Tilapia) - Microcystins (Planktothrix) Liver is the organ most associated with the detoxification, it is also one of the organs most influenced by contaminants in the water (Camargo & Martinez, 2007) The liver of the fish exposed to different concentrations of Microcystis showed moderate atrophy of hepatocytes leading to early necrosis, necrosis in large areas occurred in liver as irregular arrangements of hepatocytes, necrotic tissue with dilation and congestion of sinusoids (figure 4.2) The leukocyte infiltration and sinusoidal dilatation/leukocyte infiltration in central veins (figure 4.2A) and in peripheral areas (figure 4.2B) occurred in liver sections These changes may beattributed to direct toxic effects of pollutants on hepatocytes, since the liver is the site of detoxification of all types of toxins and chemicals Besides this, oxygen deficiency is the most common cause of the cellular degeneration in the liver Cellular swelling and irregular arrangements of hepatocytes Rodrigues and Fanta (1998); Olojo et al (2005); Camargo and Martinez, (2007); Atamanalp et al (2008); Mohamed, (2009) Heart Histopathological changes observed, generally were atrophy, reduction of muscle fiber diameter (figure 4.5), edema in cardiac muscles, splitting of muscle fibers and infiltration of blood cells were an interesting observation in heart tissue (Figure 4.6) The histopathological alterations in the cardiac muscle are in agreement with those observations by many investigators who have studied the effect of different pollutants on fish’s muscle (Elnemaki & Abuzinadah, 2003; Patnaik et al., 2011; Jeheshadevi et al, 2014; Reethamma, 2014) Through the digestive tract due to uptake of contaminated water and food or non- dietary routes across permeable membranes including the gill or skin is how pollutants directly get inside the fish body (Burger et al., 2002) Toxin pass through the blood stream in different manners; storage place (i e bone) or to the liver for transformation and/or storage (Nussey et al., 2000) or excreted by the kidney, the gill or stored in an extra hepatic tissues when people consume fish (Dimari et al., 2008) The blood cells after moving through the blood vascular system the toxin could have aimed to affect the cardiac tissue leading to extensive damage The results of this study indicated that the necrosis occurred in heart of tilapia could beattributed to the too much calcium accumulation and/or increased catecholamine release, likewise, as stated in the past findings of Lennard et al (1992) 4.2.2 Wistar Rat Heart - Dimehypo Histopathological effects in the rat heart were detected after exposure to Dimehypo The cardiac tissue sections from control mice showed normal histological architecture, with no identifiable degenerative, necrotic, or apoptotic cells (figure 4.7) Examination of heart sections after exposure to 3ml of dimehypo (figure 4.8) showed atrophy, splitting of muscles fibers were observed The splitting of muscle fibers due to atrophy of cardiac muscle were observed clearly by looking at the distance among cardiac muscles Cells are edematous, lost of the normal striation pattern and posses nuclei with inhomogeneous content There was vein artery dilatation and necrosis of cardiac cells of rat treated Large blood vessels involved in cardial tissue are strong dilated (figure 4.8C) In necrotic myocites myofibrillar lysis is complete (figure 4.8B) Nuclei of these areas are in phases of picnosis or lysis or completely disappearance PART V CONCLUSION AND RECOMMENDATIONS 5.1 Conclusion The present study showed that the exposure of tilapia oreochromis niloticus to cyanobacteria, specific is planktothrix, caused toxicological effects as histopathological alterations in fish organs such as liver, heart and the exposure of wistar rat to dimehypo caused toxicological effects as histopathological alterations in rat's heart Planktothrix posed a potential risk to tilapia Oreochromis niloticus It is obvious that this kind of cyanobacteria has a direct impact on the histological alterations in Oreochromis niloticus As the exposure concentrations increased, fish mortality occurs more rapidly, the more adverse damage occurred in the tissue of tilapia Based on the report, cyanobacteria such as plankthrix could increase the toxicity to aquatic environment and affect fish health and could cause danger to human health The toxic effects of dimehypo in wistar rat have been demonstrated in the present study It is obvious that this kind of insecticide has a direct impact on the histological alterations in wistar rat Furthermore, besides the investigation of the histopathological effects of dimehypo in the heart of wistar rat, the important finding is the mortality appeared in the short time after exposure The observed effects are serious when the concentration of the product used in the test is safely recommendable concentrations according to a perspective of environmental safety and dimehypo also is mori considered to have an acceptable level of safety withincludes regard to humans, domestic animals and crops 5.2 Recommendations Therefore, the present study recommends the importance of attention to toxins produced from cyanobacteria, especially drinking water, some food Knowledge of cyanobacteria toxin effects in aquatic organisms is essential both with respect to environmental management and health of human Also the importance of attention to toxins efect of dimehypo pesticide, it is not only necessary to recommend that the allowed levels for pesticides should be lower, the exposure should be the shortest as possible to make them become more secure and moreover REFERENCES AL-Aarajy, M J and AL-Sultan, E.Y.A (2008) Effect of some toxic microalgae on larval stage of the common carp (Cyprinus carpio L.) and silver carp (Hypophthalmithyes molitrix Val.) J of Basrah journal of Agricul Science 21: 67-87 Walter K Dodds, Matt R Whiles, 2010 in Freshwater Ecology (Second Edition) Kurmayer, R & Gumpenberger, 2006 M Diversity of microcystin genotypes among populations of the filamentous cyanobacteria Planktothrix rubescens and Planktothrix agardhii Impact of pesticides use in agriculture: their benefits and hazards, Md Wasim Aktar, Dwaipayan Sengupta, and Ashim Chowdhury, 2009 Costa M., Costa-Rodrigues J., Fernandes M.H., Barros P., Vasconcelos V., Martins R Marine cyanobacteria compounds with anticancer properties: A review on the implication of apoptosis Mar Drug, 2012 Alan J Hargreaves, John Flaskos, 2014, in Biomarkers in Toxicology Ramesh C Gupta, Dejan Milatovic, 2011 , in Reproductive and Developmental Toxicology Role of toxic and bioactive secondary metabolites in colonization and bloom formation by filamentous cyanobacteria Planktothrix Rainer Kurmayer, Li Deng, and Elisabeth Entfellnera W.F Vincent, in Encyclopedia of Inland Waters, 2009 10 Steven L Percival*, David W Williams**, in Microbiology of Waterborne Diseases (Second Edition), 2014 11 Birgit Puschner DVM, PhD, DABVT, Caroline Moore BS, in Small Animal Toxicology (Third Edition), 2013 12 Peter E Buss, Roy G Bengis, in Fowler's Zoo and Wild Animal Medicine, 2010 13 Walter K Dodds, Matt R Whiles, in Freshwater Ecology (Second Edition), 2010 Microbes and Plants (Falconer, 1999; Codd et al., 1999a, 1999b) 14 Kirsten Heimann123, Samuel Cirés123, in Handbook of Marine Microalgae, 2015 15 Bancroft, J D., & Gamble, M., (Eds) (2002) Theory and practice of histological techniques(5th ed.) Edinburgh: Churchill Livingstone 16 Barton, B.A., Morgan, J.D., Vijayan, M.M., 2002 Physiological and conditionrelated indicators of environmental stress in fish In: Adams, S.M (Ed.), Biological indicators of aquatic ecosystem stress American Society of Fisheries, Bethesda, MD 17 Berg, K., Skulberg, O.M., Skulberg, R., Underdal, B., Willen, T., 1986 Observations on toxic blue-green algae (cyanobacteria) in some Scandinavian lakes Acta Vet 18 Blom, J.F., Bister, B., Bischoff, D., Nicholson, G., Jung, G., Sussmuth, "R.D., Juttner, F., 2003 Oscillapeptin J, a grazer toxin of the freshwa- "ter cyanobacterium Planktothrix rubescens J Nat 19 Blom, J.F., Robinson, J.A., Juttner, F., 2001 High grazer toxicity of [D"Asp3 (E)-Dhb7] microcystin-RR of Planktothrix rubescens as compared to different microcystins 20 Fastner, J., Erhard, M., Carmichael, W.W., Sun, F., Rinehart, K.L., Ronicke, "H., Chorus, I., 1999a Characterization and diversity of microcystins in natural blooms and strains of the genera Microcystis and Planktothrix from German freshwater 21 Fastner, J., Neumann, U., Wirsing, B., Weckesser, J., Wiedner, C., Nixdorf, B., Chorus, I., 1999b Microcystins (hepatotoxic heptapeptides) in German fresh water bodies Environ 22 Fischer, W., Hitzfeld, B.C., Tencalla, F., Eriksson, J.E., Mikhailov, A., Dietrich, D.R., 2000 Microcystin-LR toxicodynamics, induced pathology, and immunhistochemical localisation in livers of blue-green algae exposed rainbow trout (Oncorhynchus mykiss) Toxicol B Ernst et al / Aquatic Toxicology 79 (2006) 23 Fournie, J.W., Courtney, L.A., 2002 Histopathological evidence of regeneration following hepatotoxic effects of the cyanotoxin microcystin-LR in the Hardhead Catfish and Gulf Killfish J Aquat Anim 24 Landsberg J.H Toxins and harmful mechanisms The effect of harmful algal blooms on aquatic organisms Rev Fisheries 2002 25 Falconer I.R., Humpage A.R., 2005 Health risk assessment of cyanobacterial (Blue-green Algal) toxins in drinking water Int.J Environ Res Public Health 26 JIANG Jin-Lin, SHI Yue, YANG Liu-Yan, SHAN Zheng-Jun, WANG XiaoRong, BU Yuan-Qing, 2013 Histopathological Effects of Combined Pollution of Microcystin and Atrazine on Cyprinus carpio Journal of Ecology and Rural Environment 27 Lycopene Attenuates Tulathromycin and Diclofenac Sodium-Induced Cardiotoxicity in Mice Mohamed M Abdel-Daim 1,*Orcid, Rasha Eltaysh 2, Azza Hassan and Shaker A Mousa 4Orcid 28 Abushouk, A.I.; Ismail, A.; Salem, A.M.A.; Afifi, A.M.; Abdel-Daim, M.M , 2017 Cardioprotective mechanisms of phytochemicals against doxorubicininduced cardiotoxicity Biomed Pharmacother 29 WHO Pesticides Evaluation Scheme: resources Joint FAO/WHO Meeting on Pesticide Residues (JMPR) publications, 2010 30 Abushouk, A.I.; Ismail, A.; Salem, A.M.A.; Afifi, A.M.; Abdel-Daim, M.M Cardioprotective mechanisms of phytochemicals against doxorubicininduced cardiotoxicity Biomed Pharmacother 2017 31 Abushouk, A.I.; Ismail, A.; Salem, A.M.A.; Afifi, A.M.; Abdel-Daim, M.M Cardioprotective mechanisms of phytochemicals against doxorubicininduced cardiotoxicity Biomed Pharmacother 2017, 90, 935-946 32 Office of Pesticide Programs US Environmental Protection Agency Fenech, M., 2000 The in vitro micronucleus technique 33 Garaj-Vrhovac, V., Zeljezic, D., 2001 Cytogenetic monitoring of Croatian population occupationally exposed to a complex mixture of pesticides Toxicology 34 Ware, G.W., Whitacre, D.M., 2004 The Pesticide Book Meister Media Worldwide Willoughby, Ohio, p 496 Weiss, B., Amler, S., Amler, R.W.M., 2004 35 WHO, 2005 The WHO Recommended Classification of Pesticides by Hazard and Guidelines to Classification 2004 36 WHO - World Health Organization (2007) WHO Specifications And Evaluations For Public Health Pesticides International Programme on Chemical Safety, World Health Organization: Geneva, Switzerland ... DTN1454290032 Toxicological Effect and Histopathological Alterations Thesis Title: on Liver and Heart of Fish, and Rat Heart Contamina by Algal Cyanotoxin Dr Phil Arinafril and Dimehypo Krisna... the histopathological alterations in the heart Histopathological examination Dead fish and rat were taked out and the portions of heart and liver are excise out The heart tissure of rat and. .. determine toxicological effects of cyanotoxin from algae on fish liver and heart, and rat heart by dimehypo as well as to reveal the histopathological alterations 1.3 Research questions How can