The present study was aimed to know the histopathological and ultrastructural (SEM) alterations of lung due to PQ (Herbicide) in male Albino wistar rats. The experiment was carried out at different time intervals (24, 48, and 72 hrs). Histopathologically group 2 lung sections revealed increased thickness of septa and collapsed alveoli. Tertiary bronchiolar epithelial cells showed mild to moderate hyperplasia and focal areas inflammatory cells infiltration in peribronchiolar area. A dark inclusion like bodies and mild fibrous tissue proliferation in peribrochiolar along with severe congestion was also observed. Few bronchioles were shrunken. Scanning electron microscopy of group 2 lung slices showed irregular surface area with hemorrhages in bronchiolar area. Specimens of 48 and 72 hours showed thickened alveolar septa with mild to moderate fibrosis in peribranchiolar area.
Sustainable agricultural production and quality food security has always believed to be challenge in growing population of the country. Pesticides and other chemicals are of precious inputs in sustaining agricultural practices in Indian agricultural scenario. As a post green revolution which ensured the quantity of production but quality which is remain unclear. Paraquat (PQ) is one of the highly toxic potent herbicides promoted by the United States for use in Mexico to destroy marijuana plants in the year 1955. Frequent PQ poisoning incidents have become a severe public health issue all over world, especially in Asian region. The paraquat (1,1′-dimethyl-4,4′-bipyridilium dichloride – PQ), is one of the most widely used herbicides and holds a largest share of the global herbicide market till today. It is a non-selective quaternary nitrogen herbicide, is commonly used as a desiccant and defoliant in a variety of crops all around the world (Dasta, 1978; Bismuth et al., 1982&1990 and Raghu et al., 2013). For the past 60 years PQ is considered a most toxic compound, which is classified as a moderately hazardous herbicide and placed in poison class II by WHO (2009) due to its acute toxicity.
Materials and Methods
In the present study a total of 36 male albino Wistar rats weighing between 180-240 g were procured from G. Pullareddy College of Pharmacy, Hyderabad. The experiment was conducted after approval of IAEC (GPRCP/IAEC/07/17/01/PCL/AE-3-Rats-M-12). The rats were housed in solid bottom polypropylene cages at Ruska Labs and were maintained under controlled environment (Temperature 20-220C) and sterile rice husk was used as standard bedding material, all rats were provided with standard pellet diet and deionized water ad libitum throughout the experimental period.
The experimental design adopted for the present study is shown in Table 1.
Table 1: The experimental design adopted for the present study
|Group||No. of Animals||Treatment|
|Group-II||18||Paraquat (I/P) single dose @24 mg/kbw|
|Group-II(a)||06||Paraquat (I/P) single dose @ 24 mg/kbw|
|Group-II(b)||06||Paraquat (I/P) single dose @ 24 mg/kbw|
|Group-II(c)||06||Paraquat (I/P) single dose @ 24 mg/kbw|
The tissue samples of lung slices (1×1 cm3) were collected and fixed in 10% neutral buffer formalin (NBF) soon after sacrifice. The samples were processed, sectioned (5μm) and stained with Hematoxylin and Eosin (H&E) for histopathological examination as per the standard procedure (Luna, 1968). Soon after sacrifice thin slices (1x1mm3) of lung were dissected and fixed in 2.5% gluteraldehyde (Sigma Aldrich, USA) in 0.1M phosphate buffer (pH 7.3) and stored at 40C for 12 hours. And washed with buffer, post fixed in 1% aqueous osmium tetroxide (Sigma Aldrich, USA) for 2 hours then dehydrated in ascending grades of alcohol (Qualigens fine chemicals, Mumbai), and the dehydrated specimens were subjected to vacuum desiccation for 45 minutes and mounted over stubs on the double sided carbon conductivity tape and coated with Gold by using sputter coater (JEOL-JFC-1600) for 180 seconds. Later specimens were observed under Scanning Electron Microscope (JEOL; JSM-5600, Japan).
Results and Discussion
The rats were sacrificed at 24, 48 and 72 hrs time intervals of experiment and abnormalities were recorded if any. In lung diffused hemorrhages were seen in all lobes. Small grey necrotic spots were also observed on apical. On cut section blood tinged and oozed out. At 48 hrs of time interval one rat was found dead where the diaphragmatic lobes, middle lobes of lungs showed severe haemorrhages and the other part of lung showed emphysema (Fig.1). These observations were similar to the results of Hampson and Pond, (1988) and Lalrautfela et al. (2014). These changes might be due to the toxic effects of PQ as lung is the primary target organ (Lewis & Nemery, 1995 and Dinis-Oliveira et al., 2006).
|Fig. 1: Showing 48 hrs treated lung with mild to moderate congestion.
|Fig. 2: Photo micrograph of lung showing fibrin deposition (H&E 50µm) (24 hrs).||Fig. 3: Photo micrograph of lung showing increased thickness of septa, showing numerous round cells and severe congestion (H&E 100µm) (24 hrs).
Group two (24 hrs) lung sections revealed an increased thickness of septa, severe congestion along with numerous round cell infiltrations was observed. A prominent dark dense inclusion like bodies with vacuolar degeneration and fibrin deposition was also noticed (Fig. 2&3). The sections of 48 hrs treated lung depicted the perivascular infiltration of inflammatory cells with numerous RBC’s giving red hepatisation texture to the lungs. Majority sections showed proliferation of fibrous tissue which led to shrinkage of bronchiole (Fig. 4&5). At 72 hrs intervals the treated lungs showed altered alveolar septa and severe hemorrhages, congestion and hyperplasia of tertiary bronchiolar epithelial cells. Hyperplasia and fibrosis of peribronchiole was the principle lesion in addition to deposition of exudates (Fig. 6). Theses lesions might be due to the PQ induces mitochondrial damage of the alveolar cells. The similar changes were explained by several authors (Dinis-Oliveira et al. (2006); Chang et al. (2009); Lalrautfela et al. (2014); Zhang et al. (2014); Han et al. (2015) and Li et al.) in both sexes of Wistar rats in their experiments.
|Fig. 4: Photo micrograph of lung showing perivascular infiltration of cells, red hepatisation and fibrous tissue proliferation (H&E 100µm) (48 hrs)||Fig. 5: Photo micrograph of lung showing fibrous tissue proliferation (H&E 50 µm) (48 hrs).||Fig. 6: Photo micrograph of lung showing peribranchiolar fibrosis and hyperplasia of brancheole (H&E 50µm) (72 hrs).|
Scanning Electron Microscopy
On the perusal of literature published data in relation to SEM in rats is not available, but it has been documented in dog (TEM and SEM) by Hampson and S. M. Pond (1988). SEM slices of 24 hours lungs showed hemorrhages in the septa with increased thickness and a note worthy feature of fibrosis in peribroncheolar area during 48 and 72 hrs of experiment (Fig. 5&7). The proliferation of fibrous tissue at the end of 24 hours were also observed, but during 48 and 72 hours it has reached to maximum extent could be due to the activation of ROS and also TGF-β as the PQ is acting on the specific growth factors. Hampson and S. M. Pond (1988) documented irregular alveolar walls, detached type I alveolar cells from the basement membrane and accumulation of large numbers of erythrocytes and inflammatory cells including macrophages which overlay the irregular walls of pneumocytes under SEM in paraquat exposed canine.
|Fig. 7: SEM of lung showing haemorrhages in septa at x1500 (24 hrs).
|Fig. 8: SEM of lung showing proliferated fibrous tissue at x1000 (48 hrs)||Fig. 9: SEM of lung showing proliferated fibrous tissue at x2500 (72 hrs).|
The PQ is a proven compound to induce acute fibrosis in experimental rats. Grossly the lungs have showed typical changes like red hepatization in a short period of experiment. Histopathologically, these observations were supported by showing numerous RBC’s in the interstitial area and infiltration of inflammatory cells and proliferation of fibrous tissue along with increased alveolar septa in peribronchiolar areas. Scanning electron observations have revealed the presence of hemorrhages and proliferating fibrous tissue at various degrees. These observations conclude that the PQ is a potential
compound to induce acute fibrotic changes in experimental rats.