*2.2. Experimental Design*

In order to assess the ultrastructural abnormalities induced by the Phorate and Dimethoate in the fish renal cells, the fish for each insecticide were placed in four experimental conditions: a control and three different exposure concentrations. Based on the selected doses of each insecticide and the time of exposure. All groups had an equivalent no. of fishes, i.e., 5 fish per group in a glass aquarium. After completion of the exposure period of 72 h, the fish were euthanized, and the kidneys were removed to assess ultrastructural abnormalities using transmission electron microscopy (TEM). It was briefly washed in 0.09% normal saline soon after biopsy of the animal in order to remove the associated mucus or blood from it. The kidney was cut into small pieces of 1 × 1 mm. Then, these tissues were fixed in a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer for 8–12 h at 4 ◦C. Then, the tissues were washed in buffer three times, each for one hour duration at 4 ◦C. The tissues were post fixed in 1% osmium tetroxide for 1 h at 4 ◦C. The tissues were then dehydrated through an acetone series (30%, 50%, 70%, 90% and 100%) for 15 min each. The dehydrated tissues were then cleared off the acetone by propylene oxide for 30 min and then infiltrated and embedded in a liquid resin epoxy. After embedding, the resin blocks were then thin-sectioned and these ultrathin sections (70 nm) were placed on metal grids and stained with electron dense stains, with uranyl acetate and lead citrate. These samples were then examined and photographed for ultrastructural changes by TEM (TECNAI 200 kV, FEI/Phillips, Hillsboro, OR, USA) at AIIMS, New Delhi for observations and photography.

## **3. Results**

Kidney samples from the control fish showed normal structural features. The nucleus was oval in shape with heterochromatin evenly distributed throughout the nucleoplasm and in a binucleate condition. The heterochromatin was uniformly distributed (Figure 1b). The mitochondria were tubular with distinct cristae (Figure 1a); stacks of rough endoplasmic reticulum were easily seen around the nucleus and mitochondria (Figure 1d) lysosomes were also seen. The basement membrane also had normal nuclei. Additionally, the pedicels which arise from the specialised epithelial cells (podocytes) were easily seen forming narrow infiltration slits in the glomerulus (Figure 1c).

**Figure 1.** Transmission electron micrographs of control renal cells with intact normal architecture of cellular organelles: (**a**) normal mitochondria with distinct cristae (white filled arrow) (scale 0.2 μm); (**b**) nucleus, though binucleate (white filled arrow) but with uniformly distributed chromatin (white arrow head) (scale 1 μm); (**c**) pedicel with infiltration slits (white filled arrow) (scale 0.5 μm); (**d**) stacks of endoplasmic reticulum (boxed), mitochondria (arrow) and lysosomes (arrow head) (scale 1 μm).

In both the Phorate- and Dimethoate-treated fish, the ultrastructural changes were evident (Figures 2–4). Marked changes were observed in the shape of nuclei in which the nuclear envelope showed shrinkage and, hence, an irregular outline. The mitochondria displayed degenerative changes with condensed and rounded appearance, having disorganized lamellar cristae. The endoplasmic reticulum was also disorganized and fragmented and less in number. Vacuolization was also very much evident in the cytoplasm of the treated fish.

In the glomerulus of Phorate-treated fish, most of the podocytes exhibited swollen, fused and short pedicel with a significant increase in filtration slit width. These degenerative changes became more and more evident with the increase in the concentration of the Phorate for the exposure time period of 72 h. With the increase in dose, the number of mitochondria increased with distinct degenerative changes. The degenerative changes became more pronounced with the increase in the concentration of the genotoxins, as was revealed by severe deformation and nuclear blebbing of the nucleus and other structures. This increase in the dose led to an increase in the apoptotic stimuli, which was confirmed by the apoptotic body near the nucleus (Figure 4a), and nuclear membrane shrinkage was observed in all the treated fish renal cells.

**Figure 2.** Transmission electron micrographs of 0.2 mg/L Phorate exposed renal cells exposed for 72 h revealing degenerative changes: (**a**) deformed nucleus (white filled arrow) and condensed heterochromatin (arrow head) (scale 0.5 μm; (**b**) degenerated endoplasmic reticulum (-) and condensed degenerated mitochondria(white star) (scale 0.2 μm). (**c**) more degenerative and deformed nucleus (white arrow head), dilated and deformed ER (-), increased lysosomes (circled) and deformed mitochondria (white star) (scale 0.5 μm); (**d**) deformed nucleus (white filled arrow) and electron dense condensed chromatin (white arrow head) and vacuole (boxed ♦) (**e**) showing severe electron dense deposits in the deformed nucleus (white filled arrow) with whorl of degenerated ER (-), severe cytoplasmic vacuolation (thick black line) and deformed mitochondria (star) (scale 1 μm); (**f**) deformed nucleus (white filled arrow), degenerated mitochondria (star), severe vacuolation (thick black line) (scale 1 μm).

**Figure 3.** Transmission electron micrographs of 0.6 mg/L Phorate exposed renal cells exposed for 72 h: (**a**) deformed nucleus (white head arrow) and other structures not distinct, mitochondria (star) (scale 1 μm); (**b**) electron dense deposits in deformed nucleus (white filled arrow), deformed fused swollen pedicels (flashed white arrow) and extreme vacuole presence(thick black line) (scale

1 μm). (**c**,**d**) transmission electron micrographs of 0.005 mL/L Dimethoate exposed renal cells exposed for 72 h: (**c**) degenerated nucleus with electron dense deposits throughout nucleoplasm (white filled arrow); condensed degenerated mitochondria (star), vacuole (thick black line) and curved degenerated ER (-) (scale 0.5 μm); (**d**) deformed nucleus (white filled arrow) with condensed electron dense chromatin (arrow head) and peripheral nucleolus (flashed white arrow), presence of vacuoles(thick black line) (scale 0.5 μm) (**e**,**f**) transmission electron micrographs of 0.01 mL/L Dimethoate exposed renal cells exposed for 72 h: (**e**) less distinct cytoplasmic structures-deformed nucleus (white filled arrow), degenerated mitochondria (star), severe cytoplasmic vacuoles (white arrow head) (scale 1 μm); (**f**) extreme vacuolation (white filled arrow) and electron dense degenerated mitochondria (star) (scale 0.5 μm).

**Figure 4.** Transmission electron micrographs of 0.015 mL/L Dimethoate exposed renal cells exposed for 72 h: (**a**) highly deformed nucleus (white filled arrow) with apoptotic body (big star), cytoplasmic vacuole (thick black line), vacuolated mitochondria (small star) (scale 1 μm); (**b**) severe cytoplasmic vacuolization (star head), deformed nucleus(white filled arrow) (scale 1 μm); (**c**) deformed nucleus (white filled arrow) with nuclear vacuoles (O) and condensed chromatin (arrow head) and nucleolus (scale 1 μm); (**d**) highly deformed mitochondria (star), increased intercellular space (thick straight and curved line), and extreme cytoplasmic vacuolation (white filled arrow) (scale 0.5 μm).

## **4. Discussion**

Ultra-morphological analyses through electron microscopy are the best tool to detect various cell intoxication symptoms. Transmission electron microscopy (TEM) produces a two-dimensional image of the cell. It provides the minute details of surface alterations which cannot be observed by the simple light microscope such as an increase or decrease in the number of organelles, nuclear degeneration, loss of cytoplasmic integrity, etc. However, one of the main drawbacks of electron microscopy is that this technique cannot be used to quantify the apoptotic cells [32]. Microscopy is therefore used in the interpretation of the various parameters used to measure the extent of toxicity and contributes to the establishment of measures that aim to prevent environmental contamination, a risk not only to the ecosystem but also to human health [33]. It will support follow-up studies to further elucidate the underlying cellular mechanisms of pesticide toxicity in wildlife. It is pertinent to mention here that the number of fish used in the present study was limited, therefore more work needs to be carried out on true replicates so that there is no room of doubt for the confirmation of the results.

The results of the present study on the ultrastructural changes observed in the kidney cells of common carp, *Cyprinus carpio communis,* exposed to two organophosphate insecticides viz. Phorate and Dimethoate have been compared with the previous findings of other researchers, who observed the toxicological effects of other toxicants in different species. Nandita [34] conducted TEM studies on the kidney of *Ctenopharyngodon iddellus* exposed to an organophosphate monocrotophos (36% *w*/*w* for 15, 30 and 45 days of duration). In the treated fish, several ultrastructural abnormalities were observed in the kidney. The prominent changes included degenerative changes in the nucleus and mitochondria, and cytoplasm in the proximal convoluted tubule showed vacuolization. The number of mitochondria were found to be tremendously increased upon exposure. The degenerative changes were found to be directly linked to the dose concentration of the toxicant as the cellular structures were not clearly visible with a high dosage of the toxicant. Shrinkage of glomerulus, enlargement of Bowman's capsule, dissociation of epithelial lining and vacuolization of the cytoplasm were also observed in the kidney of fish, *Cirrhinus mrigala*, exposed to metal, lead acetate (chronic toxicity study of 30 days with sub lethal concentrations of 14.1 and 28.2 ppm) [35]. Pesticides cause physiological and biochemical changes in the fish species and greatly influence their behavioural activities. The tubular degeneration was also observed in the kidney of zebrafish, *Danio rerio,* when exposed to a combination of various pesticides (sublethal concentration of 8.4 and 4.2 μg/L of Chloropyrifos 50% + Cypermethrin 5% EC for 7, 14, 21 and 28th day) [36]. Kidney of Asian stinging catfish, *Heteropneutes. fossilis* treated with chlorpyrifos (1/50 of LC50 and 1/10 of LC50 for a duration of 30 days) also revealed tubular degeneration, vacuolization and loss of glomerular [37]. Similar effects were observed in the kidneys of the brown trout, *Salmo trutta m. fario* when exposed to cadmium and zinc poisoning (4.4 mg/kg and 11 mg/kg, respectively, for a 46-week exposure period [38]. Aluminium also showed its toxic effects on the kidney of Tilapia, *Tilapia zillii*, by causing glomerular shrinkage. This freshwater fish was exposed to three levels of aluminium (25, 50, 100 μg/L) for 96 h [39]. TEM of Almix herbicide at sublethal concentrations of 66.67 mg/L for 30 days on freshwater teleost kidney was found to induce necrosis in the nucleus, severe vacuolization, whorl pattern of ER, and also mitochondrial degeneration [40].

Glomerular podocytes, on the other hand, are highly specialized cells and their most unique features are interdigitated foot processes with filtration slits in between. They are bridged by the slit diaphragm, which plays a major role in establishing the selective permeability of the glomerular filtration barrier [41]. Swollen and fused podocytes and increased infiltration slits observed through TEM in the glomerulus is likely to cause glomerular disease, resulting in failure of the filtration barrier [41]. The ultrastructural examination of the kidney of rainbow trout, *Oncorhynchus mykiss,* exposed to synthetic dyes revealed melanomarcophages increased in number and melanin was more abundant in the cells of treated fish [42].

The kidney thus serves as a major route of excretion of metabolites of xenobiotics [40] and receives the largest proportion of postbranchial blood. It is more likely to undergo histopathological alterations under pesticide stress [14] and, hence, deserves to be examined at cellular level, which is possible only through transmission electron microscopy. The cellular degeneration which occurred in the kidney due to the Phorate and Dimethoate toxicity might have also affected the osmoregulatory function of the fish as the kidney is not only the excretory organ, but also functions as osmoregulatory organ of the fish. The teleost kidney is one of the first organs to be affected by pollutants in water [41]. Thus, the overall ultrastructural changes observed in the kidney of common carp in the present study proved that these organophosphorus insecticides viz. Phorate and Dimethoate are potent genotoxicants which showed their deleterious effects on the osmoregulatory organ of the fish. Previous findings have also proved Phorate to be genotoxic on various other parameters, including the activities of carbohydrate metabolic enzyme, succinate dehydrogenase in common carp [14] and also altered protein content in various tissues of common carp [8]. Phorate also induced pronounced pathological toxicity in the brain of common carp, *Cyprinus carpio* and the extent of damage and degeneration were progressive with the exposure time period and the concentration of Phorate [16]. Phorate also has deleterious effects on humans as it causes cellular damage, as well as changes in DNA structure [7].

The degree of damage to the gill morphology of the Dimethoate-treated fish showed direct correlation to the exposure period, (24, 48 and 96 h when exposed to sub lethal concentration of 0.96 mg/L), while the oxygen consumption and ventilation rate showed a significant decrease during the exposure period to *Cyprinus carpio* [24]. Studies have also shown that Dimethoate can adversely affect the reproductive fitness of male common carps, even at sublethal concentration, both in short-term and long-term exposure, 96 h and 36 days, respectively, when exposed to 0.96 mg/L and 0.48 mg/L [25]. The carp fingerlings exhibit a number of abnormalities in their behaviour when exposed to Dimethoate such as gulping of surface water, uncoordinated movements, tremors and convulsions, excessive mucus secretion, and imbalanced swimming, ending in a collapse to the bottom of the aquarium with wide mouth open [10–12]. Histopathological examination of liver of carp exposed to a sublethal concentration of Dimethoate revealed disorganization of hepatocytes, rupture of blood vessels, vacuolization of cytoplasm, pyknotic nuclei and necrosis of the hepatic tissue [23]. *Oncorhynchus mykiss* fish showed remarkable behavioural abnormalities such as loss of balance, erratic swimming, and convulsion and a decrease in protein concentration with the increase in exposure to Dimethoate [19]. Shrinkage of glomerulus, dilation of lumen, vacuolization and tubular degeneration were histopathological changes observed in the kidney of common carp exposed to Dimethoate [21]. Dimethoate-exposed Labeo showed a decrease in protein content [22]. Nortan [27] studied ultrastructural effects of diethylnitrosamine (DENA) and trichloro ethylene (TCE) on the kidney of Japanese medaka, *Oryzias latipes.* Tissues of control and DENA and TCE exposed medaka for 32 weeks were observed by TEM. The kidney of the treated group showed cytotoxic effects along the length of proximal tubule, which were more pronounced in region II, distented proximal tubule, cellular necrosis, membrane fragments, pyknotic nuclei, nuclear membrane disintegration. Marlasca et al., [42] reported degranulation and dilation of endoplasmic reticulum of hepatocytes and renal cells with packed melanosomes in all the developmental stages of rainbow trout exposed to two synthetic dyes (for 30 days) by TEM. Spazier et al., [43] reported severe cellular disintegration of spleen of Anguilla fish exposed to contaminated waters. The cells revealed necrotic changes marked by swelling of the mitochondria. Godar et al., [44] observed apoptosis by dissociation of ribosomes, ER dilation, degeneration of nucleus and chromatin condensation through TEM on inducing the photo toxicity of ultraviolet radiations (UV) of different wave band regions on L5178Y-R murine lymphoma (LYR) cells. Anglade et al., [45] observed apoptotic changes such as loss of contact with surrounding tissues, shrinkage and chromatin condensation in myelinated neurons of substantia nigra in four normal aged humans. Hagar et al., [46] reported ultrastructural anomalies in β-cells of rat pancreas on exposure to dimethoate, as there was a tremendous increase in the number of vacuoles and dilated ERs. The treated group received dimethoate orally via gavage (21 mg/kg) daily for two months. Brajuskovic et al., [47] reported ultrastructural changes such as a reduction in the size of mitochondria of B-chronic lymphocytic leukaemia (B-CLL). The proliferation of smooth

endoplasmic reticulum and the presence of several cytosolic electron dense granules were reported in the liver hepatocytes of two the fish species exposed to contaminated waters [48]. The ultrathin sections of the kidney of a malathion-exposed (dose of 27 mg/kg b.wt/day for one month) rat revealed positive signs of degeneration including narrowing of capillary space and deposition of electron dense material in the podocytes and vacuolation of podocyte cytoplasm with fusion of its foot processes [49]. In a nutshell, these examples clearly reveal that our findings are in line with previous studies that suggest that, like other toxicants, these two insecticides, i.e., Phorate and dimethoate, also induce abnormal degenerative changes in cells.
