Fish under the influence of environmental pollution try to adapt through immune system activity or nonspecific defense systems. The immune system in fish consists of organs, cellular structures and humoral (liquid) factors. The organs that make up the immune system are also known as lymphoid organs. Lymphoid organs in fish are divided into two groups as primary and secondary. The primary lymphoid organs are the thymus, kidneys, and spleen, while the secondary lymphoid organs are the intestines, physical barriers (skin, gill), and liver (Zapata et al. 2006; Ocak 2006; Uribe et al. 2011). Recent studies show that histological findings of these organs are biomarker parameters for determining the physiological stress in fish caused by water quality changes (Osman et al. 2010; Liebe et al. 2013).
Pathological changes in fish spleen such as proliferation of white pulp, decrease in lymphocyte count, increase in spleen size, hemosiderosis and increase in melanomacrophage centers are generally considered to be the result of environmental contamination (Garcia Abiado et al. 2004; David and Kartheek 2015). At the 7th and 14th days of our study cloudy swelling and hypertrophy (Fig. 1c and 1d) were seen in the exposed tissue which could be lead to increased size of spleen. The increase in spleen cell size-number and the corresponding increase in spleen weight usually reflects xenobiotic-induced changes in the immune system and a proliferative response to xenobiotics (Guo and White 2010). Acute cell swelling is considered as a response to cell membrane damage caused by lipid peroxidation, direct binding of xenobiotics (such as pesticides) to the cell membrane, damage to ion channels, and the addition of transmembrane pore-forming complexes to the cell membrane (Miller and Zachary 2017). As seen in the results the severity of histopathological alterations increased with the duration of the exposure (Table 3). In this context, on the 21st day of the study, the most prominent histopathological change in the spleen tissue was necrosis spreading throughout the tissue (Fig. 1e). There are studies in the literature that pesticides cause necrotic alterations in the spleen tissues of fish species (Capkin et al. 2010; Karim et al. 2016; Farhan et al. 2021). In accordence with these findings the diffuse necrosis throughout the spleen tissue detected at the 21st day of our study. The number of pyknotic nucleus increased in parallel with the duration of the study (Table 3). Pyknosis is suggested as the irreversible condensation of chromatin and nuclei observed in both apoptotic and necrotic cell death (Hou et al. 2016). In this context, pyknosis could be considered as an early sign of necrotic cell death in the tissues exposed to environmental toxicant such as pesticides.
Macrophages which are one of the cellular factors of the immune system are indicator cells of innate immunity in fish and other vertebrates (Dönmez 2016). Macrophages in poikilotherm organisms form clusters with pigment cells called pigmented macrophages or melanomacrophage centers (MMCs) (Satizabal 2013). Because of their pigmentation, MMa can be distinguished from macrophages in histological examinations, and they can also be observed widely in many organs of poikilotherm vertebrates (Kranz 1989; Ferreira 2011). According to the results of our study the number and size of MMCs were increased with the duration of the experiment (Table 3). The number, size and pigment contents of macrophages change in poor health conditions of fish, under stress and in response to environmental contaminants. The size, number and histopathological appearance of MMCs and the levels of macrophage activities such as chemotaxis, phagocytosis, pinocytosis and chemiluminescence are considered as important parameters among immunological biomarkers, especially in determining the effect of environmental pollutants and in bacteriological infections (Van der Oost et al. 2003; Faccioli et al. 2014; Ledic-Neto et al. 2014). There are studies in the literature that confirms these findings. It was determined that there was an increase in the number of MMCs in Carassius auratus where phenylhydrazine, a substance used in the paint and pharmaceutical industry, was applied (Herraez and Zapata 1986). In the microscopic examination of spleen tissues of 7 different fish species in the Gulf of Mexico, it has been accepted that the presence of more than 40 MMCs per mm2 area can be associated with the presence of contamination in the hypoxic environment or sediment (Fournie et al. 2001). There are also studies showing that the metric properties (number, size and percentage of tissue invasion) of MMCs differ in regions where pollutants are concentrated (Rabitto et al. 2005; Suresh 2009; Ali et al. 2014).
In the cytopathological results, At the 7th and 21st days of our study the phagocytic activity of MMa was recorded associated with increased number of MMCs (Fig. 4d and 6c). The increase in the number of MMCs is generally thought to occur due to increased phagocytic activity within the cellular defense system to remove cellular debris (Ghosh and Homechaudhuri 2012; Ledic-Neto et al. 2014). In parallel with the phagocytic activity of MMa, it was seen that at the 14th and 21st days of the study there were remarkable hemosiderin and melanin accumulation in the spleen tissue (Fig. 5a, 5b, 5c, 6a and 6b). In our study the main source of hemosiderin accumulation was thought to be because of increased damaged erythrocyte destruction by MMa (Fig. 4d). Hemosiderin pigment is the most important pigment in the intracellular storage of iron during the degradation of hemoglobin and serves as an intermediate step in the recycling of iron. The pathological condition caused by the high level accumulation of hemosiderin is called hemosiderosis which is associated with increased erythrocyte destruction in the spleen (David and Kartheek 2015). Toxicology studies show that the disruptive action of different insecticides on the erythropoietic tissue such as kidney and spleen may decrease erythrocyte number and hemoglobin content as an anemic sign, and even lead to death of fish (Karim et al. 2016; Marteja and Modina 2021). Catabolism of damaged erythrocytes and iron retention in MMCs are two possible metabolisms of increased hemosiderin-iron accumulation in fish spleen (Agius and Roberts 2003). In addition, in cases where erythrocyte phagocytosis is increased, it has been determined that MMCs containing hemosiderin-iron in the spleen increase in parallel (Dönmez 2016).
The second important pigment is melanin which is thought to be derived from different exogenous sources or formed inside the cell. It is thought that melanin has an important role in neutralizing the free radicals, cations and toxic agents that occur during the destruction of phagocytized cell membranes and plays a role in the production of antimicrobial compounds such as hydrogen peroxide (Solano 2014). It has been also reported that an increase in the amount of MMCs containing melanin is a specific indication of chronic inflammation (Haaparanta et al. 1996; Jansson 2002). It is generally thought that the mechanism that causes inflammation in the tissues is necrosis (Yang et al. 2015). These findings could be the explanation of high amount of melanin accumulation in the spleen tissue of O. niloticus exposed to DZN at 14th day of the study where necrosis was detected (Fig. 5a).
The other important cytopathological alteration caused by DZN exposure at the 7th day of the study was mitochondrial deformation (cristolysis) in the spleen endothelial cells of O. niloticus (Fig. 4a). Mitochondria are vital in eukaryotic organisms as they play a central role in biological fuel production, ATP production by phosphorylation, and programmed cell death (apoptosis) (Bras et al. 2005; Voet et al. 2006; Heath-Engel and Shore 2006). Mitochondrial energy production could be impaired in pathological conditions (oxidizing chemicals, ischemia, hypoxia, calcium and other chemical agents) leading structural disorders in mitochondria which cause cellular damage. As a consquence of this impairment, necrotic cell death; because of ATP depletion, ion disorganization, mitochondrial-cellular swelling, activation of degrading enzymes, plasma membrane failure and cell lysis, may occur (Nieminen 2003). According to these findings, mitochondrial deformation (cristolysis) detected in the early days of our study (day 7) could be another cause of necrosis detected in the spleen tissue in the subsequent days of the study (days 14 and 21). Thus, there are studies in the literature reporting that DZN and other pesticides causes mitachondrial deformation in the tissues of some fish species (Samanta et al. 2018; Jindal and Sharma 2019; Díaz-Resendiz et al. 2020). Furthermore, cristolysis could be suggested as an important cytopathological feature for decreased metabolic activity of the cell since the enzymes linked to oxidative phosphorylation are located on inner mitochondrial membrane (Modica-Napolitano and Singh 2002).
The pyknotic nucleus recorded in spleen endothelial cells of fish at the 7th day of the study could be considered as an early sign of wide-spread necrosis at the 14th and 21st days of the study. Unlike apoptosis, in necrosis the cell nuclei condense into smaller chromatin clusters with irregular and scattered morphologies that can later be dissolved (Fujikawa et al. 2000; Bortul et al. 2001; Niquet et al. 2003). It was also suggested that necrotic pyknosis was likely to be initiated by the detachment of chromatin from the nuclear envelope (anucleolytic pyknosis) (Fujikawa et al. 2010; Hou et al. 2016). In this case, the nuclear envelope shrinks slightly, causing the chromatin to detach from the nuclear envelope. Subsequently, the nuclear envelope and chromatin are further condensed together, causing both structures to collapse (Sohn et al. 1998; Fujikawa et al. 2010; Hou et al. 2016). At the 7th day of our study the detachment of nuclear envelope and condensed nucleus are clearly seen in parallel with this finding (Fig. 4a). In this context, according the results gained from our study, it could be assumed that DZN caused cell death in the spleen tissue of O. niloticus because of necrosis rather than apoptosis.
It was previously reported in many studies that DZN caused vacuolar changes in the fish tissues (Banaee et al. 2013; Banik et al. 2016; Omar-Ali and Petrie-Hanson 2019). In accordence with these findings vacuolation was detected in the spleen tissue of O. niloticus at 7th, 14th and 21st days of our study (Fig. 4a, 4c, 5d and 6d). Vacuolization in eukaryotic cells can be temporary or irreversible. Irreversible vacuolization can cause cell death in the presence of a cytotoxic substance. The death of the cell could be because of the endoplasmic reticulum (ER), endosomal-lysosomal system and Golgi apparatus affected by irreversible vacuolization (Shubin et al. 2016). At the 21st day of the study the vacuolation was detected in the lymphocyte of O. niloticus (Fig. 6d). This result is especially important because it was known that OPs, including DZN, have immunotoxic effects on some fish species (Al-Ghanim 2012; Ahmadi et al. 2014; El-Bouhy et al. 2016; Díaz-Resendiz et al. 2019). In some studies it was suggested that the immunotoxicity of OPs was because of their disruptive effect on leukocyte cholinergic system (Toledo-Ibarra et al. 2016; Díaz-Resendiz et al. 2019). However, another study reported that some OPs deregulated lysozyme activity in immune cells of fish species (Li et al. 2013). The later finding is more relevant with our results in the manner of the cause of vacuolation in the spleen endothelial cells and lymphocytes of O. niloticus exposed to DZN.
Nuclear deformation was another cytopathological alteration detected at the 7th day of the study in the spleen tissue of O. niloticus exposed to DZN. Micronucleus, deformed nucleus and nuclear shift were some of the chemical induced nuclear deformations reported previous studies (Ali et al. 2008; Anbumani and Mary 2011). Nuclear abnormalities such as lobbed, blebbed, notched nuclei and binucleated cells have been shown in some studies as an indicator of genotoxicity (Da Silva Souz and Fontanetti 2006). Nuclear deformation and abnomalities were reported in some previous studies in fish species exposed to OPs and other pesticide groups (Muranli and Guner 2011; Kumar 2012; Khatun et al. 2021). In contrast to our study, in previous studies, it was detected that with the duration of the exposure such abnormalities were increased in parallel (Khatun et al. 2021). It was also reported positive correlation between dosage level and number of nuclear abnormalities (Ruiz de Arcaute et al. 2016; Khan et al. 2021). According to the results gained from our study, it could be suggested that DZN had genotoxic effect on the spleen tissue f O. niloticus.