Dişi sıçanlarda bisfenol S ile oluşturulmuş ovaryum hasarının ışık ve elektron mikroskobi düzeyinde incelenmesi
Examination of ovarian damage induced by bisphenol S in female rats at light and electron microscopic level
Yazar: ŞEVVAL KOÇAK
Danışman: PROF. DR. NAZAN DENİZ YÖN ERTUĞ ; DOÇ. DR. TUĞBA KOTİL
Sakarya Üniversitesi / Fen Bilimleri Enstitüsü / Biyoloji Ana Bilim Dalı
Derece: Yüksek Lisans, 2024
Konu: Biyoloji = Biology

Özet:
Son yıllarda artan çevre kirliliğinin önemli sebeplerinden biri olan çevresel kirleticilerin kullanımı, gün geçtikçe artmaktadır. Çevresel kirleticiler, ekosistemdeki yetersiz su kaynaklarına ciddi zarar verebilmektedir. Bu kirleticiler, canlılarda endokrin sistemin bozulmasına neden olurken üreme sistemi üzerinde de olumsuz etkiler ortaya çıkarabilmektedir. Bisfenol grubundan olan Bisfenol A (BPA)'nın neden olduğu zararlı etkiler fark edildikçe, dünya genelinde birçok ülkede kullanımı yasaklanmış ve kısıtlanmıştır. Günlük hayatımızda kullandığımız birçok malzemede yer alan bisfenol türevlerinin sebep olduğu olumsuz etkiler farklı kaynaklarda da belirtilmiştir. Son zamanlarda BPA içeren ürünlere alternatif olarak Bisfenol S (BPS) kullanılmaya başlanmıştır. BPS kullanımının artmasıyla beraber toksik etkilerinin üzerinde yapılan çalışmalar da artmıştır.  Model organizma olarak tercih edilen sıçanların farklı dokularında kimyasal ajanların sebep olduğu toksisiteye bağlı olarak insan sağlığı üzerinde meydana gelebilecek potansiyel etkileri değerlendirilmektedir. Wistar albino sıçanlar, üreme süresinin kısa olması ve uyum sağlama potansiyelinin yüksek olmasından dolayı toksisite çalışmalarında sıklıkla kullanılan model organizmadır. 
Bu çalışma kapsamında farklı dozlarda BPS'nin (50 μg/kg, 100 μg/kg ve 150 μg/kg) sıçanlara uygulanması sonucunda ovaryum dokuları alınarak histolojik işlemlere tâbi tutulmuştur. Hematoksilen & Eozin ve Uranil Asetat-Reynolds Çözeltisi (Kurşun Sitrat) boyaması sonucunda ışık ve Transmission Elektron Mikroskobu (TEM) ile oluşan histopatolojik hasarlar tespit edilmiştir. 
BPS uygulamasının sıçan ovaryum dokularında genel olarak folikül gelişim aşamalarını sekteye uğrattığı, germinal epitel yapısında kopma ve bozulma meydana geldiği, atretik ve kistik folikül yapısı oluşturduğu, granüloza tabakasının yapısını bozarak hücrelerini değiştirdiği, bazı granüloza hücrelerinde piknotik çekirdek yapısı meydana geldiği, teka tabakasının inceldiği, teka hücrelerinin yapısını bozduğu, kan damarlarında artış ve dokuda hiperemi meydana getirdiği gözlenmiştir. Teka interna ve eksterna tabakalarında açıklıklara ek olarak intrastromal hemoraji olduğu incelenmiştir. Primordiyal foliküllerin epitel hücrelerinin yassı ve kübik epitel arasında şekillendiği, sekonder ve graaf foliküllerin granüloza tabakasının katmanları arasında ayrılmalar ile açıklıklar olduğu görülmüştür. Doz artışına bağlı olarak teka ve granüloza tabakasının arasındaki sınır seçilememiştir. Bağ dokuda vakuolizasyon ve vasküler konjesyon olduğu incelenmiştir. İntertisyel alanda bağ doku hücrelerinde hücre sınırlarının bozulduğu ve kistik hücre kümelerinin varlığı tespit edilmiştir. Oosit fragmantasyonu, vakuolar dejenerasyon ve angiogenesis meydana geldiği görülmüştür.
Ultrastrüktürel inceleme sonucunda ise doz artışına bağlı olarak, ooplazma içerisinde annulat lamel birikiminin arttığı, kortikal granüllerin sayısının azaldığı, bazı hücrelerde lipid birikiminin arttığı, mitokondri hasarı ve mitokondrilerde krista kaybı, granüloza ve teka hücrelerinin yapısında değişiklikler olduğu tespit edilmiştir. Ayrıca bazı yapısı bozulan mitokondrilerde miyelin benzeri yapılar oluştuğu, granüloza hücreleri arasında bulunan intersellüler alanlarda vakuolizasyon ve apoptotik hücreler meydana geldiği görülmüştür. Bazal lamina şekli normal histolojik yapısını kaybederek kıvrımlı bir şekilde (ondüla görüntüsü) karşımıza çıkmıştır.

Abstract:
The use of environmental pollutants, which is one of the major causes of increasing environmental pollution in recent years, is on the rise. These pollutants can cause significant damage to the insufficient water resources in ecosystems. They disrupt the endocrine system in living organisms and can have adverse effects on the reproductive system. Endocrine-disrupting chemicals (EDCs), often produced in industrial settings, are widely used in various fields. EDCs can interfere with the functions of various organs by mimicking or blocking endogenous hormones and adversely affecting the production of naturally produced hormones. Additionally, by mimicking hormones in the body, they can bind to hormone receptors, irreversibly activate the signaling cycle, and accelerate and trigger uncontrolled reactions. These endocrine disruptors, which have negative effects on living organisms, can lead to chronic health problems. By disturbing the internal balance of the body, they can cause hormone diseases, cancer, reproductive system, neurological, and cardiovascular diseases, and teratogenic effects, making them extremely dangerous chemicals. One of the main reasons for the widespread use of endocrine disruptors is their long half-life. However, these substances cannot be directly integrated into the food chain, making them biologically difficult to metabolize, and the resulting metabolites can become more harmful by increasing the endocrine-disrupting effect. The approach of considering all chemicals harmless until their harmful effects are proven, especially EDCs, endangers living beings. One of the most important and widely used EDCs worldwide is Bisphenol A (BPA). As the harmful effects of BPA, which belongs to the bisphenol group, became apparent, its use has been banned and restricted in many countries globally. Burning plastics can release significant amounts of BPA into the atmosphere. BPA, a type of xenoestrogen, can cause genotoxicity, cytotoxicity, mutagenicity, and cancer in living organisms. Additionally, it can lead to obesity, diabetes, and heart diseases in humans. The negative effects of bisphenol derivatives found in many materials we use daily are noted in various sources. Recently, Bisphenol S (BPS) has started to be used as an alternative to BPA in products labeled as 'BPA-free.' With the increased use of BPS, studies on its toxic effects have also increased. BPS, a weak estrogenic substance that alters endocrine system functions, can lead to breast cancer and infertility, and therefore, restrictions have been imposed, and its use in some plastic products like baby bottles has been banned in certain countries. Although BPS has a similar phenolic ring structure to BPA, it is a more stable chemical. BPS is more resistant to sunlight and high temperatures and shows lower estrogenic activity, making it the most studied derivative to replace BPA. Due to its sulfon group and two hydroxyl groups, it has a strong electron-withdrawing property. These properties make it a stronger and more stable substance in terms of acidity compared to other bisphenol analogs.
BPS released into the environment can contaminate air, soil, water, biota, or food, potentially causing pollution and leading to human exposure through these pathways. Although BPS was produced as an alternative to BPA, residues of BPS can be found in thermal paper and beverages. Neonatal exposure to BPS can affect the development of reproductive organs and, as a result, cause serious reproductive problems in adults. In addition, BPS is a weak estrogenic substance that can alter endocrine system functions, lead to breast cancer, and cause infertility. Due to these properties, restrictions have been imposed, and its use in some plastic products, such as baby bottles, has been banned in some countries. BPS has been detected at low concentration levels compared to BPA in various environments, including water, sediment, sludge, indoor dust or air, consumer products, and human urine.
The female reproductive system is a multifunctional system that produces female gametes (oocytes) through oogenesis, prepares the environment for fertilization and fetus development, carries the fertilized oocyte, and nourishes the fetus until birth. For fertility to be observed normally, the oocyte must complete its development properly. The growth and development of the oocyte are at their peak in the stage just before ovulation, where oocyte maturation is completed. Oocyte maturation is the transition from prophase 1 of meiosis to metaphase 2. The surface of the ovary is covered with a single layer of flat or cuboidal epithelium called germinal epithelium. Below the germinal epithelium lies the tunica albuginea, a dense connective tissue layer that gives the ovary its whitish color. Under the tunica albuginea, the ovary is divided into the cortex (outer) and medulla (inner) parts, and a distinct boundary between these histologically different regions cannot be observed. When the ovarian morphology is examined under a light microscope, the width of the cortex region appears larger, and there are follicles at various stages of development in its stroma. The stroma contains fibroblast cells and reticular fibers. These fibroblast cells respond differently to hormonal stimuli than other fibroblast cells. In the cortex, only primordial follicles are observed before puberty, while primary, secondary, and graafian follicles are seen after puberty. In addition to follicles, the corpus luteum and atretic follicles are also present. The medulla region contains interstitial cells, nerves, lymph, and blood vessels.
Rodents are frequently used in human experiments. Wistar Albino rats, part of the rodent group, are used as a model organism in experimental studies due to their relatively short life cycle, short gestation period, adaptability, non-aggressiveness, well-known health sector, and well-documented genetic background. They are often used in biological studies such as drug, toxicology, and organ culture due to their ease of care, high adaptability, short gestation period, and low cost. Rats are crucial for modeling human diseases. They are used to develop therapeutic agents for metabolic diseases such as cardiovascular diseases, obesity, diabetes, neurological diseases, depression, and similar conditions. They are widely known to be used in carcinogenicity, teratogenicity, and toxicity tests in the pharmaceutical industry and government institutions. Because rats are frequently used as model organisms today, this study evaluated the potential effects on human health based on the damage that may occur in the ovaries.
Rats obtained from the Experimental Medicine Application and Research Center (SÜDETAM) of Sakarya University Medical Faculty were kept in an environment with 40-60% humidity, at 21±2 °C room temperature, with a 12-hour light and 12-hour dark cycle. They were fed ad libitum with pellet feed and water (tap water) provided in glass bottle feeders. In our study, female rats weighing 120-210 grams, raised for 8 weeks, were used. Four groups were randomly formed, with 7 animals in each group.
Eight-week-old rats were administered different doses of BPS (50 μg/kg, 100 μg/kg, and 150 μg/kg) for 14 days. Then, ketamine and xylazine were applied to the rats for decapitation. The ovarian tissues were removed and taken into Bouin's fixative for light microscopy examination and into glutaraldehyde fixative for electron microscopy examination, and the necessary histological follow-up steps were carried out. Histopathological damages were detected using Hematoxylin & Eosin and Uranyl Acetate & Reynolds Solution (Lead Citrate) staining, examined with light microscopy and transmission electron microscopy (TEM).
It was observed that BPS administration generally disrupted follicle development stages in rat ovarian tissues, caused detachment and disruption in the germinal epithelium, formed atretic and cystic follicle structures, disrupted the structure of the granulosa layer and altered its cells, caused pycnotic nuclei in some granulosa cells, thinned the theca layer, disrupted the structure of theca cells, increased blood vessels, and caused hyperemia in the tissue. Intrastromal hemorrhage and openings in the theca interna and externa layers were observed. The epithelial cells of primordial follicles were shaped between flat and cuboidal epithelium, and there were separations and openings between the layers of the granulosa layer of secondary and Graafian follicles. The boundary between the theca and granulosa layer was indistinguishable with the increase in dose. Vacuolization and vascular congestion were observed in the connective tissue. It was determined that the cell boundaries of connective tissue cells in the interstitial area were disrupted and cystic cell clusters were present. Oocyte fragmentation, vacuolar degeneration, and angiogenesis were observed.
In ultrastructural examination, with increasing doses, the accumulation of annulate lamellae in the ooplasm increased, the number of cortical granules decreased, lipid accumulation increased in some cells, mitochondrial damage and crista loss in mitochondria were detected, and changes in the structure of granulosa and theca cells were observed. Additionally, myelin-like structures were formed in some damaged mitochondria, vacuolization, and apoptotic cells were observed in the intercellular areas between granulosa cells. The basal lamina lost its normal histological structure and appeared wavy (undulated appearance).