Atrazine Induced Histopathological Alterations in the Liver of Adult Male Mice

Atrazine (2 chloro 4 ethylamino 6 isopropylamino 1, 3, 5- triazine) is the second most commonly used herbicide with an annual consumption of 70,000–90,000 tons (Lizotte et al., 2017; Neequaye, 2019; Singh et al., 2018). It is a major contaminant of soil, water resources, and plants with a half-life of 30-740 days (Gely-Pernot et al., 2017; Michael et al., 2018). Atrazine is very environmentally stable due to low loss by volatilization and degradation. Furthermore, because of its accumulation effect, atrazine is significantly affecting the health of many organisms, including humans (Nodler et al., 2013; Juhel et al., 2017). European Union banned Atrazine in 2003 due to its severe toxicity to cells and tissues. However, the United States Environmental Protection Agency (USEPA) still permits the use of atrazine worldwide (Almberg et al., 2018). Various studies have shown that Atrazine interferes with the normal functioning of body organs, including the liver, disrupting the normal structural and architectural components in a non-infectious hepatic injury (Opute and Oboh, 2021). Histopathological liver sections of adult Xenopus laevis exposed to different concentrations of Atrazine, showed hypertrophied hepatocytes, vascular congestion and dilation, disorganization in the arrangement of hepatic cords, apoptosis and/or necrosis, and infiltration of inflammatory cells, with highest concentrations, showed most severe effects (Sena, 2017). The hepatocyte and hepatocyte nucleus diameterwas significantly decreased in the atrazine-treated groups (Destroy et al., 2021). These damaging effects of atrazine may result from its generation of reactive oxygen species (ROS) that causes oxidative stress of various organs. Increased oxidative stress and lipid peroxidation is implicated in the pathogenesis of herbicide-induced hepatic injury (Toughan et al., 2018). To evaluate various histological and micrometric alterations induced in the liver of albino mice due to ATZ exposure was the major objective of this study. The use of the mammalian model was planned to utilize the findings in humans.

This research was carried out in laboratory-reared albino mice (Mus musculus) with a weight of 28-30 grams and age of 5-6 weeks. These animals were kept in the Department of Zoology’s animal house, University of Sargodha, Sargodha, under thestandard protocol of 12-hours a day and night cycles. The temperature of the animal house was maintained at 25±2°C and humidity at 45%. Bodyweight, survival, and clinical signs were recorded daily.

Twenty animals were divided into two groups (n=10) randomly.

This group was provided regular drinking water with normal feed.

These animals were given 200 mg per kg atrazine dissolved in water through gavagesonce daily for 28 days and regular drinking water and feed ad libitum.

After four weeks of experimentation, the animals were dissected to remove the liver. Organs were weighed and fixed in Conroy’s fixative for further processing.

After fixation, organs were processed for dehydration sequentially in 50%, 70%, 90%, and absolute alcohol for 24 hours each. After xylene clearance, organs were processed for wax embedding to get 5 microns thick sections of male mice’s liver through a rotary microtome (ERMA TOKYO 42). Hematoxylin and Eosin stained sections were observed under the stereoscopic compound microscope.

The micrometric data was calculated from the liver’s digital photomicrographs obtained using a Huawei company’s digital camera (Model no DSC-W35) 13 megapixels affixed on a trinocular microscope (Labomid CXR2) at 400×. Micrometric readings were obtained using Coral DRAW 11. The data obtained werethen analyzed statistically by an unpaired t-test for the comparison of the groups.

Histological analysis of liver slides of Cnt showed normal anatomical structures. In the Cnt group’s liver sections,a continuous array of one-cell thick hepatocytes that form the hepatic cord around compact central veins were seen (Figure 1). Hepatocytes are arranged in trabecularrunning radiantly from the central vein and are separated by sinusoids free of any cellular population (Figure 1A and 2A). Hepatocytes with granular cytoplasm were homogeneously distributed around the nucleus. Large well stained spherical nucleiwerepresent almost in the centre. The nucleus contains distinctly marked nucleolus and chromatin material. A compact organization was observed inperipheral hepatic portal triads or tetrads embedded in connective tissues. Binucleated were lesser in number than mono-nucleated hepatocytes, as shown in Figure 5. Kupffer cells were elongated in shape, lined on both sides of the hepatocytes. Between the hepatic cords, sinusoidal usual size spaces were observed where deposition of oval cells was obvious.

Histological observation of the liver after ATZ treatment showed marked alteration. In the ATZ treated group, loosen hepatic tissues, vacuolar degeneration of hepatocytes, diffuse necrosis, and the hepatic portal vein’s degeneration was visible. The number of hepatocytes with deformed nuclei was greatly reducedcompared to the Cnt group (Figure 1B - 2B). ATZ treatment resulted in massive degeneration of hepatocytes.The mean number of mononucleated and binucleated cells, per unit area, was affected adversely (Figures 1 - 2). ATZ treated group showed increased hepatocyte size and a highly significant increase in hepatocytes’ nuclear diametercompared to theCnt. Highly disordered trabecular arrangement (Figure 1B) and a significant reduction in the mean number of oval cells per unit area were also obvious in ATZ treated sections compared to the Cnt group.

At the start of the experiment,the mean initial body weight of animals belonging to both experimental groups showed non-significant variations. At the end of the experiment ATZ treatment showed a highly significant (p<0.001) decrease in mean body weight of treated animals compared to the Cnt group. Data analysis by unpaired t-test revealed the toxic effect of atrazine on animals’ body weight at the given dose level.

Unpaired T-test showed a highly significant (p˂0.001) reduction in mean liver weight in the ATZ treated group compared toCnt. This indicated toxicity of herbicide to the general health of animals.

An unpaired t-test revealed that the number of mononucleated cells was highly significantly (p<0.001) reduced by 28 days treatment of atrazine compared to the Cnt group. These parenchymal cells were noticed to be the targeted cellular population of the herbicide.

An unpaired t-test revealed that the mean number of binucleated cells was significantly (p<0.001) reduced by 200 mg/kg atrazine treatment for 28 days. Hepatocytes number either uninucleated or binucleated both were highly decreased by atrazine treatment reflecting toxicity of atrazine tothe liverat the cellular level.

An unpaired t-test revealed that the mean number of oval cells was significantly (p<0.01) reduced by 4 weeks of ATZ compared to the Cnt group.

An unpaired t-test revealed that hepatocytes’ cellular diameter was highly significantly (p<0.001) reduced by atrazine treatment compared to theCntgroup.

An unpaired t-test revealed that hepatocytes’ nuclear diameter was highly significantly (p<0.001) reduced by ATZ treatment compared to the Cnt group.

The liver is a critical organ in the human body that is responsible for an array of functions that help support metabolism, detoxification, digestion, immunity,vitamin storage, among other functions, soit is prone to a lot of environmental toxicants as well (Kalra et al., 2021; Chen et al., 2018). In the present study, atrazine’s effects, a major water pollutant on male mice liver were explored.

The mean body and liver weight in ATZ treated mice showed a highly significant reduction (p<0.001) compared to the Cnt group. Decreased body and liver weight in mice might be attributed to the atrazine-generated oxidative stress that affected animals’ general body health (Zhang et al., 2017).

In the histological examination of the present study, treatment of ATZ (200mg/kg) severely affected liver anatomy. In ATZ treated group, the arrangement of hepatocytes was distorted. The number of mononucleated and binucleated hepatocytes was highly decreased as compared to the Cnt group. ATZ treated sinusoidal group spaces were affected by the ATZ treatment and became disorganized, wider, and irregular (Figures 1 - 2). The number of oval cells also decreased in the treated mice liver (Figure 6). In the ATZ treated group, the hepatocytic nuclei size was highly significantly increased compared to Cnt (Figure 8). Congestion in the bile duct vein was prominent with a 200 mg per kg dose of ATZ (Figure 2). Exposure to ATZ resulted in pronounced histopathological abnormalities such as the expansion of sinusoids, dilation of hepatocytes, nuclear necrosis, emptied hepatic portal vein, reduced cytoplasm, and vacuolation in hepatocytes (Figure 1). In another study, similar findings as vacuolar degeneration of hepatocytes, diffuse necrosis and degeneration of the hepatic portal vein were also reported after administration of the oral dose of 25% (124 mg/kg/body weight) of LD50 (3090 mg/kg/body weight) of the Atrazine dissolved in water for 120 days to male Wistar Albino rats (Deshmukh and Ramteke, 2015).Our findings were in agreement with the report of Senarat,who observed histopathological alterations of the liver in freshwater catfish collected from Tapee River, which is vulnerable to pesticides pollution, as it receives agricultural runoff from paddy fields, Indian rubber plantations, and vegetable and fruit crops (Senarat et al., 2015). Histopathological alterations in the liver of catfish after exposure to pesticides consisted of cellular swelling, eosinophilic cytoplasm of hepatocytes, and constriction of sinusoidal capillaries damage of endothelial cells of blood vessels. Some liver areas showed focal necrosis and contained severe infiltration of macrophage, pyknotic nuclei, and large lipid vacuoles in the cytoplasm of hepatocytes (Figures 1 - 2).

One liver injury mechanism is mitochondrial dysfunction through free radicals generation (Sagarkar et al., 2016).These radicals damage mitochondrial DNA (Rasgele et al., 2015). Consequently, some chemicals may cause hepatocellular necrosis, rapid disorganization of the hepatic architecture, breakdown of sinusoidal structures and pooling of blood in the liver through these mechanisms (Abarikwu et al., 2017). It is reported that ATZ induces oxidative stress in rat tissues and that the oxidative stress was associated with increased lipid peroxidation and changes in the anti-oxidative system. These findings indicated that ATZ exposure caused various anatomical derangements in male mice’s liver possibly due to the generation of excessive reactive oxygen species (Zhang et al., 2017).

In conclusion, results revealed that all liver cell types are highly sensitive to atrazine exposure. So, some measures should be taken to avoid atrazine exposure at the individual level to all possible extents.

We are thankful to the University of Sargodha for providing funding and all the necessary facilities for this research work. We are very obliged to provide all the key research equipment and chemicals for this research project.

The authors have declared no conflict of interest.