Zebra balıklarında abamektin üreme toksisitesi: Histolojik yönden incelenmesi
Reproductive toxicity of abamectin in zebrafish: A histological examination
Yazar: SEVDA BAĞDATLI
Danışman: PROF. DR. NAZAN DENİZ YÖN ERTUĞ
Sakarya Üniversitesi / Fen Bilimleri Enstitüsü / Biyoloji Ana Bilim Dalı
Derece: Yüksek Lisans, 2024
Konu: Biyoloji = Biology

Özet:
Günümüzde çevre sorunlarının artmasında kimyasal atık ve pestisitlerin günlük hayatımıza fazlasıyla entegre olması büyük bir etkendir. Özellikle pestisitler, tarımsal arazilerde ürün eldesinde alınan verimin arttırılması amacıyla kullanılmaktadır. Her ne kadar üründe kaliteyi arttırmak amacıyla kullanılsa da; pestisitlerin kullanımı arttıkça çeşitli yollarla toprak ve suya karışımı da artmaktadır. Böceklerin kontrolü ve verdiği zararın azaltılması için kullanılan insektisitler de bu alanda kullanımı artan pestisitlerdir. 
Avermektin ailesinden en sık kullanılan bileşenlerden olan Abamektin de bir insektisit ve antihelmintik ajan olarak tarım alanlarında kullanılmaktadır. Bu bileşik, uygulama bölgelerinden farklı yollarla sucul ortama girip kirletici rolü ile canlılar üzerinde olumsuz etkiler gösterebilmektedir. Abamektin maruziyeti sonrasında zehirlenme, koma, hipotansiyon ve solunum yetmezliğine sebep olduğunu gösteren çalışmalar mevcuttur. Ayrıca sucul ortama karışması sonucunda akuatik organizmalarda da toksik etki gösterdiği bilinmektedir. Abamektinin üreme toksisitesine sebep olduğunu gösteren çalışmalar da mevcuttur. Günümüze model organizma olarak tercih edilen zebra balıklarında kimyasalların oluşturduğu toksisite çalışmalarına dayanarak, insanlar üzerindeki olası etkileri de değerlendirilmektedir.
Bu çalışmada, değişen dozları kullanılan Abamektin'in (0,75 µg L-1, 1,5 µg L-1 ve 3 µg L-1) zebra balıklarına uygulanması sonucunda ovaryum ve testis dokuları disekte edilerek histolojik işlemlerden geçirilmiştir. Hematoksilen & Eozin boyaması sonrasında ışık mikroskobu altında incelemeler yapılmış ve histopatolojik etkiler tespit edilmiştir. 
Abametktin uygulaması sonrasında zebra balığı testis dokularında seminifer tübüller arası bölgede bağ doku artışı (intertisyel fibrosis), seminifer tübüllerde vakualizasyon, seminifer tübül bütünlüğünde bozulma, sperm yoğunluğunda artış, spermatogenik hücre kümelerinde azalış, Sertoli hücrelerinde hipertrofi, piknotik hücre yapıları, apoptotik hücre oluşumu, intertisyel alanda hemoraji ve testis dokusunda ileri seviyede atrofi tespit edilmiştir. 
Ovaryum dokularında ise; zona radiata ile ooplazma ve zona radiata ile foliküler epitel arasında açılma, zona radiatada distorsiyon, morfolojik bozulmalar ve kalınlaşma, atretik oosit oluşumunda artış, intertisyel alanda fibrosis, ooplazmada vakualizasyon ve dejenere olmuş folikül oluşumu, olgun ve vitellojenik oosit sayısında azalış ve hücrelerde nükleer alanda bozulmaların meydana geldiği görülmüştür. Ayrıca tüm bu görülen bulguların doz artışı ile paralel olarak artış gösterdiği belirlenmiştir.

Abstract:
In today's world, the increase in environmental problems is significantly influenced by the extensive integration of chemical waste and pesticides into our daily lives. Pesticides are particularly used in agricultural fields to increase crop yields. The use of pesticides involves chemical substances aimed at eliminating pests and preventing unwanted weed growth; therefore, they can be harmful to other species such as non-target plants, birds, fish, beneficial insects, and also negatively affect the atmosphere, water sources, soil fertility, and agricultural products. Pesticides can reach soils and both surface and groundwater through various means such as wind and rainwater or drift.
Pesticides can be categorized based on numerous parameters such as appearance, physical or chemical structure, toxicity levels, target areas, or how they are applied; however, classification based on the active ingredient and target area is much more commonly used. This classification includes rodenticides, insecticides, acaricides, fungicides, herbicides, molluscicides, virucides, bactericides, and nematicides. The ability to categorize pesticides in various ways allows their use while considering their risks, thereby protecting foods or providing high-quality products. This way, the excessive and uncontrolled use of pesticides can be prevented, minimizing the toxic effects by reducing the exposure of living beings to these substances during their use.
Although pesticides are used to improve product quality, their mixing with soil and water increases as their use increases. Insecticides, used to control insects and reduce the damage they cause, are also among the pesticides with increasing usage in this field. Defined as chemical or biological origin substances that control insects, insecticides act by killing, eliminating, or preventing the harmful behaviors of insects.
Members of the avermectin family are categorized as highly effective but toxic natural substances used as medications in humans and animals and for plant protection. One of the most commonly used components of the avermectin family is Abamectin, used in agricultural fields as an insecticide and antihelminthic agent. Abamectin (80% Avermectin B1a (5-O-demethyl Avermectin A1a) and 20% Avermectin B1b (5-O-dimethyl-25-de-(1-methylpropyl)-25-(1-methylethyl) (ABM)) is a macrocyclic lactone disaccharide compound and results from the natural fermentation of the soil actinomycete S. avermitilis. This compound, by entering aquatic environments through various pathways from application areas, can act as a pollutant and show adverse effects on living organisms. Studies have shown that exposure to Abamectin can lead to poisoning, coma, hypotension, and respiratory failure. Furthermore, it is known to exhibit toxic effects on aquatic organisms when it contaminates aquatic environments. Studies have also shown that Abamectin can cause reproductive toxicity.
Zebrafish are organisms that can tolerate a wide range of temperatures. They can survive without problems at temperatures ranging from 18 to 30°C. Reproduction is quite a simple process for zebrafish. The optimal breeding temperature is 26-28°C. At the end of a 12-hour light and 12-hour dark period, each adult female fish lays approximately 50-80 eggs, which are then fertilized by males.
Based on studies on the toxicity caused by chemicals in zebrafish, which are currently preferred as model organisms, the potential effects on humans are also evaluated. Zebrafish are also used as genetic models in aquaculture, toxicogenomic studies, and drug development for humans. With their easy maintenance, production, and transparent appearance during early development stages, zebrafish stand out as versatile model organisms suitable for many different research fields.
Especially in the last twenty years, zebrafish have been preferred as model organisms to examine the reproductive toxicity caused by drugs and chemicals in both short and long terms. The reproductive organs of bony fish, such as zebrafish, develop bilaterally from germinal ridges originating from the dorsolateral lining of the peritoneal cavity. Germ cells migrate from these ridges to the germinal epithelium and settle in the developing ovary. It is known that oogenesis in zebrafish consists of four main stages based on ultrastructural analysis: primary oocyte, cortical alveolar oocyte, vitellogenic oocyte, and mature oocyte. The testis in fish is an organ located bilaterally between the abdominal wall and the swim bladder. Histological examination of testis tissue reveals seminiferous tubules, testicular ducts, connective tissue, cells in various stages of spermatogenesis, and Leydig cells. The cells involved in spermatogenesis include spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and sperm cells. Spermatogonia, primary and secondary spermatocytes, and spermatids are located at the base of the seminiferous tubules, while sperm cells are found in the lumen. Additionally, Leydig cells are present in the connective tissue between the seminiferous tubules. The reproductive cells in spermatogenesis obtain nutrition, support, and hormonal interactions through Sertoli cells. Sertoli cells are located in the seminiferous tubule walls, settled on the basal membrane, and surround the reproductive cells, filling the space between them.
In this study, adult zebrafish were used as live material. A total of 40 zebrafish, with 5 males and 5 females in each of the 4 aquariums, were used. Heaters were used to maintain the water temperature at 28±1°C in the aquariums. The aquarium water was oxygenated using air pumps, and no filtration system was used in the aquariums. Additionally, an automatic lighting system was installed in the room, maintaining a cycle of 14 hours of light and 10 hours of dark. While no treatment was applied to the control group aquarium, Abamectin was added to the other experimental groups' aquariums at doses of 0.75 µg L-1, 1.5 µg L-1, and 3 µg L-1. After 96 hours of exposure, the fish were euthanized using a high dose of anesthetic (MS-222, 300 mg/L). Following this procedure, the ovary and testis tissues were dissected.
The ovary and testis tissues were fixed in Bouin's solution, dehydrated by passing through ascending ethanol series, and then cleared using xylene before being embedded in paraffin blocks. Sections of 5 µm thickness were taken using a Leica microtome, followed by staining. The tissues were stained using Hematoxylin and Eosin (H&E) and examined with a Leica light microscope.
Histological examination of the zebrafish control group testis tissue showed seminiferous tubules surrounded by seminiferous epithelium. In the interstitial area between the seminiferous tubules, oval nuclei, round-shaped interstitial cells known as Leydig cells, responsible for androgen synthesis, were observed. Following Abamectin application, interstitial fibrosis (connective tissue increase) in the interstitial area of the zebrafish testis tissues, vacuolization in the seminiferous tubules, disruption of seminiferous tubule integrity, increased sperm density, decreased spermatogenic cell clusters, hypertrophy in Sertoli cells, pyknotic cell structures, apoptotic cell formation, hemorrhage in the interstitial area, and severe atrophy in testis tissue were detected.
Hematoxylin and Eosin (H&E) staining results showed that the ovarian tissue of the zebrafish control group exhibited normal histological structure. Different developmental stages of oocytes, such as primary oocytes, cortical alveolar oocytes, vitellogenic oocytes, and mature oocytes, were identified in the control group. In the tissues exposed to Abamectin, separation between the zona radiata and ooplasm, and between the zona radiata and follicular epithelium, distortion in the zona radiata, morphological disruptions and thickening, increased formation of atretic oocytes, fibrosis in the interstitial area, vacuolization in the ooplasm, formation of degenerated follicles, decreased numbers of mature and vitellogenic oocytes, and nuclear area disruptions in the cells were observed. Moreover, all these findings were found to increase in parallel with the dose increase.