The prevalent use of insecticides, in domestic and household environments represents a danger not only to target species but also to the environment and humans. In the present study, FIP induced elevation in ALT, AST, LDH, γGT, T. Bil, and AFP, whereas liver glycogen was diminished. Transaminases are the most sensitive biomarkers directly implicated in the extent of cellular damage and toxicity because they are cytoplasmic in location and are released into circulation after cellular damage (McGill, 2016). The impairment in the above-cited parameters may be referred to as FIP-induced overstimulation of ROS, further, the disruption in the liver membrane resulted in the leakage of these enzymes to the bloodstream (Yadav et al., 2020). Guelfi et al. (2015) explained the mechanism of FIP-induced hepatotoxicity through its injurious effect on mitochondrial bioenergetics and calcium homeostasis alteration, which is responsible for ATP depletion giving rise to cellular death by necrosis. Furthermore, the rise in the serum LDH may be due to hepatocellular necrosis, which leads to the discharge of the enzyme into the blood (Al-Harbi, 2017), and/or the lactate production through the anaerobic glucose oxidation stimulated by FIP (Abdel-Daim et al., 2018). In addition, swelling and hypertrophy of hepatocytes by FIP action cause obstruction in the bile duct and an increase in γGT level (De Oliveira et al., 2012). The reduction in glycogen concentration in FIP-treated rats may be due to that FIP not only accelerated glycolysis but also induced glycogenolysis, as it stimulated the liver glycogen catabolism for releasing glucose and lactate (De Medeiros et al., 2015).
Oral supplementation of Vit. D3 with FIP attenuated all liver function markers, as well as AFP. This could be due to the therapeutic effect of cholecalciferol by controlling and remolding the hepatic cellular damage (Tsai et al., 2016). Furthermore, these findings were consistent with those of Hafez et al. (2018) who suggested that the administration of Vit. D3-mediated anti-oxidative effect in hepatic tissues against liver damage also restored hepatic enzyme activities and glucose levels in rat serum. Moreover, cholecalciferol has a key role in decreasing total bilirubin in patients with nonalcoholic fatty liver disease (NAFLD) (Dasarathy et al., 2017) as it is adept at mediating the enhancement of heme metabolism and converting indirect bilirubin into direct bilirubin via the induction of uridine diphosphate glucuronyl transferase (UDGT).
In the current study, oral ingestion of FIP showed disturbances in lipid profile as documented by elevation in serum concentrations of total lipid, total cholesterol, TG, LDL-C and VLDL-C, however, concentration of HDL-C was depleted. FIP increases lipid metabolic disturbance-induced insulin resistance in rats, in consequence, carbohydrates are not able to meet the energy demands of the body (Tohamy et al., 2021). Moreover, Wasef et al. (2021) reported that FIP promoted hepatotoxicity in rats due to antioxidant system imbalance-induced hepatic inflammation and damage, resulting in interruptions of lipid profile, and activation of the apoptotic pathway.
On the other hand, Vit. D3 supplementation could reverse FIP-induced hyperlipidemic action. Vit. D3 improves glucose metabolism to be the source of ATP production instead of free FA, as well as relieves hepatic steatosis by reducing the oxidative state and insulin resistance (Mostafa et al., 2016). Furthermore, lipoprotein lipase activity promoted by Vit. D3 (Wang and Eckel, 2009). This enzyme also decreases cholesterol concentration by the conversion of cholesterol to bile acids (Vaskonen et al., 2002) and/or increases lecithin cholesterol acyltransferase, the enzyme managing ester transfer to HDL-cholesterol (Riedl et al., 2010). Besides, Hoseini et al. (2016) indicated that Vit. D3 has an important role in aerobic exercise training on weight loss and inhibition of cardiovascular risk factors as well as elevation of HDL.
The present disturbances in antioxidant-oxidant status induced by FIP may be due to its cytotoxic metabolites that are rapidly distributed to mitochondrial membranes performing an oxidative phosphorylation uncoupling inhibited supply of cellular energy and increasing the rate of respiration (Abdel-Daim et al., 2018). Moreover, Al-Basher et al. (2020) indicated that the oxidative damage caused by FIP could be attributed to its stimulatory action promoting the generation of ROS and/or RNS. The cellular oxidative products overcome the antioxidant defense system and damage macromolecules of cells, lipids, DNA, and proteins, followed by apoptotic or necrotic mechanisms (Weidinger and Kozlov, 2015; Wang et al., 2016). This may be with respect to FIP-induced oxidative damage, which led to a decline in the defense enzyme capacity. Moreover, the remarkable inhibition in hepatic GSH was coming from the cellular damage promoted by the disability of responding to the oxidative insult of dead cells, indicating that there was inadequate detoxification capacity antagonistic to FIP in these tissues (Mossa et al., 2015).
Co-administration of Vit. D3 in combination with FIP showed a decline in MDA and NO levels in liver tissues, while, GSH and antioxidant enzyme levels were elevated. These results match the study of Elbassuoni et al. (2018) who conveyed that ingestion of cholecalciferol with monosodium glutamate-induced hepatic and renal injury caused a significant inhibition in the liver and kidney MDA following to overcome the increased production of ROS. Moreover, the antioxidant action of the Vit. D3 markedly reduced ROS generation to facilitate the defense systems (Abo El-Magd and Eraky, 2020).
In the current study, a remarkable elevation in the concentrations of liver tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) was noticed in rats treated with FIP. Fipronil can induce the activity of the NF-κB inflammatory-signaling pathway that play a role in the associated tissue inflammation. This pathway catalyzes the elevation of pro-inflammatory cytokine levels as well as prostaglandin concentrations (Ji et al., 2016). The present results are came accordance with El-gawish et al. (2018) who reported that FIP ingestion elevated TNF-α and IL-6 expression via stimulation of the NF-κB pathway. The increase in the levels of these mediators acts to amplify the inflammatory process and contribute to tissue destruction. Whereas, attacking Kupffer cells, which represent the more potential cells susceptible to ROS, FIP causes violent oxidative damage rises to produce a variety of cytokines like TNF-α augmented inflammation and apoptosis (Li et al., 2015).
Co-treatment of Vit. D3 with FIP showed inhibition in the levels of TNF-α and IL-6. The reduction in TNF-α and IL-6 concentrations may be due to the ability of Vit. D3 as an immune modulator via the TGF-β/Smad7 signaling pathway (Chen et al., 2016). Vit. D3 caused a reduction in inflammatory cytokines, including monocyte chemoattractant protein 1 (MCP-1)-stimulated IL-6 expression, and TNF-α via NF-κB downregulation (Sari et al., 2019; Zhang et al., 2021; Khan et al., 2021). Moreover, these results are in the same line with those of Almasmoum et al. (2019) who reported that the inhibition of IL-6 and TNF-α inflammatory cytokines resulted from the endogenous action of Vit. D3, which regulates vital functions in each organ especially inflammation and oxidative stress suppression.
The present study showed that FIP down-regulated 5' adenosine monophosphate-activated protein kinase alpha (AMPK-α) and peroxisome proliferator-activated receptor gamma (PPAR-γ) gene expressions in liver tissue. These results may be attributed to the role of FIP in the elevation of the intracellular Ca2+ developed an inhibition of calcium/calmodulin kinase-β, the upstream regulator of AMPK-α. Therefore, the decline in AMPK-α expression which has a central role in the regulation of energy balance, resulted in alteration in adipogenesis and lipid metabolism (Sun, 2017).
PPARs and their ligands have a principal role in inflammation and immune regulations in the gastrointestinal tract system. Hepatic PPARs play an essential role in fatty acids and glucose metabolism, supplying energy to peripheral tissues and encountering contaminant-induced toxicity (Xi et al., 2020). AMPK and sirtuin-1 (SIRT-1) have been described to directly increase PPAR-γ activity (Cantó and Auwerx, 2009), suggesting that FIP may suppress the expression of PPAR-γ in rat liver tissue. Moreover, FIP may enhance the raising of liver TNF-α-induced inhibition in PPAR-γ hepatic expression. The result agrees with the study of Sun (2017) who revealed that imidacloprid-mediated hepatic inflammation remarkably increased the expression of TNF-α, as well as inhibited PPAR-γ expression during acid oxidation in the liver.
In the current study, treatment with Vit. D3 + FIP showed up-regulation in AMPK-α and PPAR-γ expressions in liver tissue. Vit. D3 decreases the expression of sterol regulatory element-binding protein 1 (SREBP-1) along with its target genes, acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), resulting in inhibition in triglycerides synthesis and glucose output by the induction of hepatic AMPK-α expression (Mostafa et al., 2016). Manna et al. (2017) reported that Vit. D3 supplementation up-regulates SIRT-1 expression and AMPK-α phosphorylation by improving glucose metabolism via insulin receptor substrate-1 (IRS-1)/glucose transporter-4 (GLUT4) cascade.
The present results are also in agreement with the study of Hoseini et al. (2016) who reported that there was an increase in hepatic PPAR-γ expression when rats were supplemented with a high dose of Vit. D3 suggested that Vit. D by itself is capable of increasing FA oxidation and increasing glucose uptake by AMPK phosphorylation
The present histological analysis indicated that ingestion of FIP induced liver cell degeneration, and necrosis, besides the presence of inflammatory cells. Such results go parallel with the previous results of Moussa et al. (2015); and Abdel-Daim et al. (2018) who reported that FIP caused hepatocyte degeneration, congestion of central veins, and sinusoids further focal hemorrhage and necrosis in hepatic tissue which might be due to oxidative stress-induced elevation in lipid peroxidation and depletion in the activities of enzymatic and non-enzymatic antioxidants.
Meanwhile, the changes in liver function marker enzymes as well as inflammatory biomarkers confirmed the histological alteration (Elgawish et al., 2018). However, administration of Vit. D3 with FIP showed a more or less normal appearance of the liver. These results are in agreement with Özerkan et al. (2017) who reported that Vit. D3 showed anti-hepatotoxic effects because it plays an important role as a potent antioxidant by scavenging free radicals and blocking lipid oxidation. Furthermore, Abo El-Magd and Eraky (2020) established that Vit. D3 supplementation prevents the progression of hepatic fibrosis and inhibits necrosis of liver tissue. This may be referred to its hepatoprotective effects by limiting oxidative damage, reducing the pro and inflammatory responses, and preventing fibrogenesis via its antioxidant properties.