Pesticides have gained importance in contemporary agricultural practices by effectively preventing pre-harvest and post-harvest losses. Many new chemical insecticides are emerging in the market today like phenylpyrazoles, 4th generation pyrethroids, avermectins, diamides, spinosyns etc. Fipronil, classified as a phenylpyrazole insecticide, operates on γ-aminobutyric acid (GABA) receptors (Law and Lightstone 2008). It poses significant toxic effects in rats (LD50 97 mg/kg body weight) and mice (LD50 95 mg/kg body weight). At elevated doses, fipronil induces heightened neural excitation, paralysis, and eventual fatality in non targeted species. (Pisa et al., 2015; Simon-Delso et al., 2015). It is a matter of great concern regarding the effects of neurotoxic systemic pesticides, particularly for neonicotinoids and fipronil (Simon-Delso et al., 2015; Pisa et al., 2021). Medicinal plants play a crucial role in maintaining the health of a significant portion of the global population. Anthocephalus cadamba (kadamba) possesses analgesic, anti-inflammatory, antimicrobial, antioxidant, antimalarial, antihepatotoxic, antidiarrheal, and wound-healing properties (Umachigi et al., 2007; Alam et al., 2008a; Alam et al., 2008b). Brassica juncea, commonly known as Indian mustard, stands as a prominent oilseed crop cultivated worldwide in tropical, subtropical regions, also in the Indian subcontinent (Tiwari et al., 2022). It is an economical and nourishing food source enriched with bioactive components like glucosinolates and their breakdown products, polyphenols (flavonoids and anthocyanins), substantial dietary fiber, chlorophylls, β-carotene, ascorbic acid, minerals, and volatile compounds (Yokozawa et al., 2003). The fruit of Pithecellobium dulce is endowed with both nutritional and medicinal values and is widely consumed as a food in various regions of India. It contains therapeutically potential bioactive phytocomponents, including naringenin, quercetin, rutin, gallic acid, stigmasterol, clonazepam, quinolinone, nootkatone, junipene, calarene, eremophiline, valencene, and baicalin (Pradeepa et al., 2013). This study was conducted to examine the histopathological changes induced by sub-acute exposure to fipronil in rats and to assess the effectiveness of extracts from Anthocephalus cadamba, Brassica juncea, and Pithecellobium dulce in mitigating the histopathological alterations caused by sub-acute exposure to fipronil in rats.

Experimental animals. The study was conducted on adult male and female Wistar rats weighing 100-250 g procured from Birds Park Meerut Cantt. (U.P.), India. The animals were maintained under standard management conditions and provided feed and water ad libitum. Animals were kept in laboratory conditions for 7 days for acclimatization before the start of the experiment.  The bedding material (wheat straw) was changed every alternate day. Throughout the entire study, the experimental animals were continuously observed and handled according to institutional animal ethics guidelines. The use of animals in this study received prior approval from the institutional animal ethics committee.

Preparation of extracts. Anthocephalus cadamba leaves and Pithecellobium dulce pods were collected from in and around the campus of the College of Veterinary and Animal Science, Navania, Udaipur. Dried seeds of Brassica juncea were procured from the local market. All plant materials (Leaves, pods, and seeds) were authenticated by the Department of Horticulture, Maharana Pratap University of Agriculture and Technology in Udaipur, Rajasthan. Plant materials were cleaned, shade-dried, and ground to make a coarse powder. The extracts were prepared through maceration using distilled water for Anthocephalus cadamba leaves, and 70 percent ethanol for Pithecellobium dulce pods and Brassica juncea seeds. After the seven-day maceration, the aqueous extract of the Anthocephalus cadamba and hydroethanolic extracts of Pithecellobium dulce pods and Brassica juncea seeds were filtered through Whatman filter paper no. 1, and the filtrate was subjected to evaporation using a rotary vacuum evaporator (Macro Scientific Works Pvt. Ltd.).

Experimental design:

Table 1: Experimental design for the study of ameliorating potential of Anthocephalus cadamba, Brassica juncea, and Pithecellobium dulce against histopathological alterations induced by sub-acute exposure of fipronil in rats.

Histopathological assessments are commonly employed to identify organ-specific effects associated with chemical exposure (Crissman et al., 2004). Fipronil seems to produce oxidative stress in the liver, kidney, brain, and spleen of rats. These findings also seem to be correlated with the histopathological changes observed in various organs viz. liver, kidney, brain, and spleen of rats as compared to control animals. Examination of all organs revealed significant alterations in the normal histological architecture.

Liver. Histopathological lesions observed in the liver from groups I to V are depicted in Plate 1. The control group exhibited normal cellular architecture with identified hepatocyte structure arranged in cord pattern around the central vein. Congestion, dilation of blood vessels and sinusoids along with infiltration around portal triad in the liver were observed in fipronil treated group. Significant improvement was noticed in groups III, IV, and V with only mild congestion and infiltration of mononuclear cells. The results were in corroboration with another study (Mossa et al., 2015) which reported that rats exposed to 10mg/L of fipronil showed degeneration of hepatocytes and portal infiltration with inflammatory cells. Similar histopathological changes were observed after 15 days of oral exposure of fipronil in Japanese quail (Ali et al., 2016) and after 4 weeks of oral administration of fipronil in mice (Badgujar et al., 2016). Similar findings were also observed in sub chronic acetamiprid toxicity in Wistar rats Chakroun et al. (2016). In the present study, Kadamba, Mustard, and Jungle jalebi co-treatment ameliorated the histopathological changes in the liver of fipronil-treated animals. These results were in agreement with the study where the Anthocephalus cadamba treated group showed relatively normal architecture of hepatocytes in alloxan-induced diabetic rats (Swarnkar et al., 2016). Similar findings were also reported by others where treatment with aqueous extract of Pithecellobium dulce before and after the toxin exposure showed a considerable improvement in liver morphology (Manna et al., 2011).

Kidney. Histopathological lesions in the kidney of control and other treatment groups are presented in Plate 2. The control group exhibited intact glomeruli and renal tubules in the cortical area of kidneys whereas the fipronil-treated group showed tubular dilation, necrosis of tubular cell lining along with degeneration, increased Bowman's space, and formation of hyaline cast in the lumen of tubules in the cortical area of the kidney. Fipronil co-treatment with extracts of A. cadamba, B. juncea, and P. dulce groups exhibited improvement in histopathological changes of kidney by diminishing the necrosis and degeneration. Previous studies also reported dilation of collecting tubules, congestion, and severe degenerative changes along with necrosis of tubular lining cells in mice (Badgujar et al., 2015) and alterations in the histopathological architecture of the kidney which included necrosis, inflammatory cell infiltration and vacuolation in rats exposed to fipronil (Mossa et al., 2015). In the present study, Kadamba, Mustard, and Jungle jalebi co-treatment showed nephroprotective effect. Treatment with these plant extracts improved the kidney tissue histology with extremely mild necrosis of tubular lining cells. It may be attributed to the protective effect of these plants due to the presence of antioxidants in their extracts. 

Brain. Histopathological lesions in the brain of control and other treatment groups are presented in Plate 3. The control group displayed intact neuron and glial cells in the cerebral cortex of the brain while fipronil-treated group exhibited marked neuronal degeneration, vacuolation, and congestion of blood vessels in the cerebral cortex. Fipronil co-treatment with A. cadamba, B. juncea and P. dulce extracts provide improvement in histopathological changes with mild neuronal degeneration, vacuolation, and congestion of blood vessels. Previous studies also reported similar findings where high dose of fipronil caused severe vacuolation in the molecular layer, congestion of blood vessels in the cerebral cortex along with degeneration (Badgujar et al., 2015). Cerebral hemisphere revealed changes comprising of mild neuronal degeneration with surrounding glial cells in Gallus domesticus due to thiacloprid toxicity (Goyal et al., 2010). In the present study, co-treatment with Kadamba, Mustard, and Jungle jalebi in fipronil-treated rats restored the normal histopathological structure of the brain. From these results, it could be suggested that these plants had neuroprotective action.

Our findings suggest a promising avenue for the development of natural remedies to counteract the degenerative changes induced by Fipronil. Further research and exploration of the suggested areas can contribute to the development of novel therapeutic strategies with potential benefits for human health.

Devendra Singh, Pratishtha Sharma, Shweta Anand, Rahul Swarnkar and Naresh Singh Kuntal (2024). Efficacy of Extracts of Anthocephalus cadamba, Brassica juncea and Pithecellobium dulce Against Fipronil-induced Histopathological Changes in Wistar Rats. Biological Forum – An International Journal, 16(1): 120-124.