Introduction

The indiscriminate use of insecticides and the resistance of insects mainly to organochlorines, organophosphates, and pyrethroids lead to the widespread use of other alternatives such as phenylpyrazoles, new second-generation insecticides, which are more efficient and selective for insects control (Simon-Delso et al. 2015).

Fipronil (FPN), a member of the phenylpyrazoles, is a relatively recent and broad spectrum insecticide. Owing to its efficacy even at low concentrations, it gained popularity worldwide for several residential, agricultural, and veterinary applications (Magalhães et al. 2018). The wide dissemination or misuses of fipronil-based products lead to multiple avenues for animal and human exposure (Wang et al. 2016). Additionally, FPN has bioaccumulative effect when used in agriculture; thus, it affects animals and humans via the food chain (Qin et al. 2015). Fipronil acts as a noncompetitive blocker to the GABA-gated chloride channels resulting in neuronal hyperexcitation, paralysis, and death of the insects (Gunasekara et al. 2007). Despite its selective toxicity to insects, there are many evidences donated that the primary metabolites of FPN, fipronil sulfone and fipronil desulfinyl, have higher potency at mammalian GABA-gated chloride channels than those of insects, indicating a potential hazard of these metabolites to insects and non-target organisms than the parent compound (Das et al. 2006). Besides being a neurotoxin (Raquel et al. 2011), FPN induces hepatic, renal, mutagenic, carcinogenic, and endocrine-disruptive toxic effects (Silva et al. 2015; Badgujar et al. 2016; El-Ballal et al. 2019). Earlier studies indicated the implication of FPN in oxidative tissue damages through increasing the production of reactive oxygen species (ROS) and decreasing the endogenous antioxidants (Abdel-Daim and Abdeen 2018; Abouelghar et al. 2020). Disturbance of cellular oxidant/antioxidant balance injuries cellular macromolecules, including nucleic acids, lipids, and proteins, resulting in cellular oxidative damage (Salminen and Paul 2014).

Antioxidants have been used to prevent and ameliorate the hazards of exposure to insecticides. Ginseng is a natural dietary supplement, belonging to panax genus and Araliaceae family, which is widely used as a medicinal plant (Wu et al. 2011). Among various ginseng species, American ginseng (P. quinquefolius), Chinese ginseng (P. notoginseng), and Korean ginseng (P. ginseng) are the most widespread worldwide (Lee and Kim 2014). Ginseng extracts contain various active constituents including, triterpene, saponins, essential oils, alkaloids, aminoglycosides, fatty acids, peptidoglycan, polysaccharides, vitamins, minerals, and phenolic compounds. Among them, ginsenosides are the major bioactive constituents of ginseng berry, leaf-stem, and root that are closely linked to the diverse physiological and pharmacological properties of ginseng (Yang et al. 2017). Ginseng is widely used as an alternative medicine and a natural remedy for the prevention and/or treatment of many diseases. It possesses anti-pyretic, anti-allergic, anti-aging, and immunostimulant properties (Kim et al. 2016). As well as, it has therapeutic potential in treatment of diabetes, hyperlipedemia, hypertension, cancer, and neurological and endocrine disorders (Yang et al. 2017; Ahuja et al. 2018). In addition, numerous studies spotlighted on the potential radioprotective (Mansour 2013), hepatoprotective (Abdel-Fattah et al. 2014), and nephroprotective (Raheem et al. 2017) effects of ginseng may be related to its anti-inflammatory, anti-apoptotic, and antioxidant properties (Youssef 2016; Raheem et al. 2017).

Set against the background of the risk of continuous exposure of animals and humans to FPN and the beneficial role of antioxidants against various toxic insults, this study was performed to evaluate the protective value of ginseng aqueous extract against fipronil-induced hepatorenal toxicity in rats with respect to its anti-inflammatory, anti-apoptotic, and antioxidant activities.

Materials and methods

Chemicals

Fipronil (Fipromex® 20% EC, MAC-GmbH, Company, Germany) was purchased from a local pesticide market. All kits used for biochemical analysis were obtained from Biodiagnostic Company (Giza, Egypt). Other chemicals were of analytical grade.

Preparation of ginseng aqueous extract

Korean red ginseng, Panax ginseng, root powder was obtained from Shannah Company, Egypt. Ginseng aqueous extract (GAE) was prepared following the method described by (King et al. 2006) with slight modification. Dry powdered ginseng roots were extracted in distilled water (1:9 w/v) at 90 °C for 1 h, cooled at room temperature for 30 min, and then centrifuged at 1200g for 15 min. The supernatant was collected and the pellets were re-suspended in distilled water at half the original volume. The extraction process was repeated twice. The supernatants obtained from all steps were collected and concentrated at 40 °C in hot air oven. The dried extract was kept at 4 °C and re-dissolved in distilled water immediately before administration.

Animals

Thirty-two adult male Wistar albino rats (130–150 g) were obtained from Al-Zyade Experimental Animals Production Center, Giza, Egypt. Rats were maintained in plastic cages on wood-chip bedding in well-ventilated animal house at 20 ± 3 °C temperature, 40–50% relative humidity, and daily natural dark/light cycle and provided with standard pellet feed and tap water ad libitum. Rats were acclimatized for 2 weeks prior to the beginning of the experiment. This study was ethically approved by the International Animal Care and Use Committee IACUC, Faculty of Veterinary Medicine, University of Sadat City, Egypt (Approval No.VUSC-007-1-17), which follows the Guide for the Care and Use of Laboratory Animals 8th edition. Washington (DC): National Academies Press (US); 2011.

Experimental animals and design

Rats were randomly allocated into four equal groups, 8 rats each.

Control group: Rats were given distilled water orally day by day for 6 weeks.

Ginseng aqueous extract group: Rats were administered ginseng aqueous extract (GAE) at a dose of 200 mg/kg b.w. orally day by day for 6 weeks (Al-Hazmi et al. 2015).

Fipronil group: Rats were administered fipronil (FPN) at a dose of 4.85 mg/kg b.w. orally day by day for 6 weeks (Tingle et al. 2003).

Ginseng aqueous extraxt and Fipronil group: Rats were given GAE as ginseng group 2 h before FPN administration as the fipronil group.

The selected dose of FPN was 1/20 of LD50 based on oral LD50 of FPN (97 mg/kg b.w.) in rats (Tingle et al. 2003).

Samples collection

Blood sampling

Twenty-four hours after the last administration, rats were fasted overnight and anesthetized by inhalation of isoflurane. Blood samples were collected without anticoagulant, centrifuged at 3000 rpm for 15 min, and sera samples were separated and stored at − 20 °C for further biochemical analysis.

Tissue sampling

After animals were euthanized by decapitation, the livers and kidneys of rats were immediately excised and divided into two parts. One part was washed with physiological saline then stored at − 80 °C for further tissue biochemical analysis. The other part was immediately kept in 10% neutral buffered formalin for histopathological and immunohistochemical examinations.

Assessment of serum liver and kidney functions biomarkers

Serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured according to the methods described by Reitman and Frankel, (1957). Urea and creatinine levels were measured according to Fawcett and Soctt (1960) and Schirmeister et al. (1964), respectively.

Assessment of oxidant/antioxidant status

Hepatic and renal tissue malondialdehyde (MDA) level, reduced glutathione (GSH) content, and catalase (CAT) activity were estimated by using commercial kits following the manufacturer’s instructions according to Ohkawa et al. (1979), Beutler et al. (1963), and Aebi (1984), respectively.

Histopathological examinations

The preserved liver and kidney specimens were routinely processed and stained by hematoxylin and eosin (H&E) stain for histopathological examination according to Bancroft and Gamble (2008).

Immunohistochemical investigations

The immunostaining method for localization of caspase-3 and COX-2 was performed following the method described by Liu et al. (2017) and Lin and Prichard (2015), respectively. The formalin-fixed liver and kidney sections were deparaffinized, hydrated in alcohol solutions, incubated in 3% H2O2, and then incubated with anti-caspase-3 (1:1000 dilution, Abcam, Ltd., USA) or anti-COX-2 (1:100 dilution, Abcam, Ltd., USA). The immune reactions were visualized by using diaminobenzidene (DAB; Sigma Chemical Co., USA) and semiquantitatively scored from 0 to 4, according to the percentage of positively immune stained cells (dark brown cytoplasm and nucleus) that were estimated in five random high-power fields (× 40), as previously described by (Rahman et al. 2001). Zero indicates negative staining, 1 indicates ˂ 25% of positive cells per field, 2 indicates 25–50%, 3 indicates 51–75%, and 4 indicates ˃ 75%.

Statistical analysis

Data are presented as mean ± standard error (SE). Statistical analysis was determined by one-way ANOVA followed by Duncan’s multiple range test for post hoc to determine the statistically significant (P < 0.05) differences among the experimental groups. All statistical analyses were performed by using SPSS (Statistical package for Social Sciences) Version 16 released on 2007.

Results

Ginseng aqueous extract ameliorated the toxic effects of FPN on general health condition of rats

Along the experimental period, no mortalities were recorded among different groups. No clinical manifestations were observed in either the control or GAE groups. However, rough hair coat, dullness, depression, and mild tremor were the apparent symptoms of toxicity observed in the FPN-intoxicated group. Rats of the GAE and FPN group showed apparent normal health conditions.

Ginseng aqueous extract normalized FPN-induced alterations in serum liver and kidney functions biomarkers of rats

The effects of FPN and/or GAE on serum hepatic and renal functions biomarkers were presented in Fig. 1. Fipronil intoxication significantly (P ˂ 0.05) increased the activities of serum ALT and AST and serum levels of urea and creatinine compared to the control rats. Conversely, administration of rats with GAE 2 h before FPN intoxication normalized the serum activities of ALT and AST and serum levels of urea and creatinine. On the other hand, GAE had no significant effects on liver and kidney functions biomarkers compared to the control group.

Fig. 1
figure 1

Effect of FPN and/or GAE on serum hepatic and renal functions biomarkers. Different letters indicate significant differences among different groups at P ˂ 0.05. GAE ginseng aqueous extract, FPN fipronil

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Ginseng aqueous extract prevented FPN-induced deterioration of hepatic and renal oxidant/antioxidant status of rats

Intoxication of rats with FPN significantly (P ˂ 0.05) increased hepatic and renal MDA levels, while it decreased GSH contents and CAT activity, compared to the control group. On the contrary, administration of rats with GAE 2 h before their intoxication prevented FPN-induced increase in MDA levels and decrease in GSH contents and CAT activity in hepatic and renal tissues. However, no significant changes in hepatic and renal MDA levels, GSH contents, and CAT activity were detected between GAE and the control groups (Fig. 2).

Fig. 2
figure 2

Effect of FPN and/or GAE on hepatic and renal oxidant/antioxidant status. Different letters indicate significant differences among different groups at P ˂ 0.05. GAE ginseng aqueous extract, FPN fipronil

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Ginseng aqueous extract modulated FPN-induced alterations of hepatic and renal tissue architectures of rats

Liver sections of control (Fig. 3a) and GAE (Fig. 3b) groups appeared normal with no definite histopathological alterations. Conversely, the liver of the FPN-intoxicated group revealed extensive vacuolization with marked ballooning of hepatocytes associated with focal sinusoidal congestion as well as apoptotic hepatocytes (Fig. 3c). However, marked attenuation of hepatocellular vacuolization with very mild cytoplasmic granulation in hepatic parenchyma were observed in GAE- and FPN-treated group (Fig. 3d).

Fig. 3
figure 3

Representative photomicrographs of liver sections of rats in different groups (H&E stain × 40). a Control group and b GAE group showing normal central vein (CV) and hepatocytes, c FPN group showing extensive vacuolization with marked ballooning of hepatocytes (black arrows) associated with focal sinusoidal congestion (yellow arrow), and d GAE and FPN group showing very mild cytoplasmic granulation (black arrows)

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Renal tissue of control rats showed normal glomerular tuft and tubular epithelium (Fig. 4a). Also, kidneys of GAE-treated group appeared normal and identical to the control group (Fig. 4b). Marked histopathological lesions were demonstrated in the kidney of FPN-intoxicated rats. The main demonstrated lesions were focal necrosis of renal tubular epithelial cells intensely infiltrated by mononuclear infiltrates (Fig. 4c). Interestingly, the kidneys of GAE and FPN group appeared normal in all examined sections with few sporadic intertubular inflammatory cell infiltrates, and necrosis was restricted to sparse cells (Fig. 4d).

Fig. 4
figure 4

Representative photomicrographs of kidney sections of rats in different groups (H&E stain, × 40). a Control group and b GAE group showing normal glomerular and tubular epithelium, c FPN group showing focal necrosis of renal tubular epithelial cells intensely infiltrated by mononuclear infiltrates (black arrows), and d GAE and FPN group showing sporadic intertubular inflammatory cell infiltrates (yellow arrow)

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Ginseng aqueous extract reduce FPN-increased protein expressions of caspase-3 and cyclooxygenase-2 in hepatic and renal tissues of rats

Negative immune reactivity for caspase-3 was demonstrated in the hepatic and renal tissues of the control and GAE groups (Figs. 5a, b and 6a, b, respectively). In contrast, significant increase of caspase-3 immune reactivity was recorded in the hepatic (Fig. 5c) and renal (Fig. 6c) tissues of the FPN-intoxicated group, and the percentages of positive cells were 3.40 ± 0.40 and 2.60 ± 0.50, respectively (Table 1). This immune reactivity was significantly reduced in the GAE and FPN group in both liver (Fig. 5d) and kidneys (Fig. 6d), and the percentages of positive cells were 1.80 ± 0.37 and 0.83 ± 0.37, respectively (Table 1).

Fig. 5
figure 5

Representative photomicrographs of caspase-3 (a–d) and COX-2 (e–h) immunohistochemically stained liver, × 40. a and e control group showing no caspase-3 (a) and no COX-2 immune reactive cells (e). b, f GAE group showing no caspase-3 (b) and no COX-2 immune reactive cells (f). c, g FPN group showing increase of positively immune reactive cells for caspase-3 (c) and COX-2 (g). d, h GAE and FPN group showing marked decrease of positively immune reactive cells for caspase-3 (d) and COX-2 (h)

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Fig. 6
figure 6

Representative photomicrographs of caspase-3 (a–d) and COX-2 (e–h) immunohistochemically stained kidney, × 40. a, e control group showing no caspase-3 (a) and no COX-2 immune reactive cells (e). b, f GAE group showing no caspase-3 (b) and no COX-2 immune reactive cells (f). c, g FPN group showing strong positively stained cells for caspase-3 (c) and COX-2 (g). d, h GAE and FPN group showing weak positively stained cells for caspase-3 (d) and COX-2 (h)

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Table 1 Semiquantitative score of caspase-3 and cyclooxygenase-2 immunostaining in the liver and kidneys of different experimental groups
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Liver of the control (Fig. 5e) and GAE (Fig. 5f) groups showed no COX-2 immune reactive cells. Similarly, no COX-2 immune reactivity was demonstrated in the kidneys of the control (Fig. 6e) and GAE (Fig. 6f) groups. On the contrary, COX-2 immune reactivity was markedly increased in the liver and kidneys of FPN group (Fig. 5g and 6g, respectively) and the percentages of positive cells were 3.00 ± 0.31and 3.60 ± 0.24, respectively (Table 1). Pronounced reduction of COX-2 immune reactivity was recorded in the liver (Fig. 5h) and kidneys (Fig. 6h) of GAE and FPN group and the percentages of positive cells were 1.60 ± 0.40 and 1.60 ± 0.24, respectively (Table 1).

Discussion

Introduction of new insecticides into the environment has raised the concern for identification of their potential health hazards. Fipronil is widely used in agro-vet practices; however, it causes serious environmental and public health hazards (Tingle et al. 2003) through inducing oxidative stress in vivo and in vitro (Ki et al. 2012; Badgujar et al. 2015a,b). On the other hand, ginseng has been indicated to have anti-inflammatory, anti-apoptotic, and antioxidant properties (El-Bialy et al. 2020).

The current study indicated that FPN elicited an increment in serum levels of liver and kidney biomarkers, represented by elevated activities of ALT and AST and levels of urea and creatinine. The obtained results were in accordance with those reported by Mossa et al. (2015) and Abdel-Daim and Abdeen (2018). The marked increase in the serum ALT and AST activities observed in FPN-intoxicated rats indicates the injury of hepatocytes and alteration in its membrane permeability, thus, additional ALT and AST are released into the bloodstream (Kasarala and Tillmann 2016).The elevated urea level indicates impairment in renal tubular reabsorption, while the increase in serum creatinine level reflects impairment of glomerular filtration rate (Adedara et al. 2012). These alterations in the liver and kidney functions may be attributed to the ability of FPN to induce hepatic and renal tissue injuries. This assumption was confirmed by the histopathological findings in our study (Figs. 3 and 4), which were in line with previous studies that reported degenerative changes in hepatic and renal architectures following the exposure to FPN (Mossa et al. 2015; El-Ballal et al. 2019). The possible reason behind FPN-induced hepatic and renal tissue injuries may be the disturbance of oxidant/antioxidant status of the liver and kidney as our study demonstrated that oral administration of FPN induced hepatic and renal oxidative stress evidenced by the marked increase of MDA level with suppression of GSH content and CAT activity in hepatic and renal tissues, which were parallel with the previous findings reported by (Badgujar et al. 2015a, b; Mossa et al. 2015; El-Ballal et al. 2019). This disturbance in oxidant/antioxidant balance in the liver and kidneys is due to some xenobiotics induce toxicity via generation of ROS causing alterations in cellular antioxidants, and hence increase susceptibility to oxidative stress (OS) (Lopez et al. 2007). Lipid peroxidation (LPO) is an essential marker of OS and MDA is the end product of LPO that reflects the degree of cellular oxidative damage (Nabavi et al. 2012). Reactive oxygen species attack cellular components to generate peroxyl radicals, which undergo a cyclization reaction to produce endoperoxides, and finally MDA (Marnett 1999). GSH is a free radical scavenger and acts as a substrate for glutathione s-transferase and glutathione peroxidase for detoxification of free radicles (Parke and Piotrowski 1996). Catalase is a cellular antioxidant enzyme, which along with glutathione peroxidase rapidly converts H2O2 to water (Scibior and Czeczot 2006). Fipronil-induced injury of hepatic and renal tissues in our study may be due to oxidative stress-induced caspase-3 and Cox-2 protein expressions in these tissues (Figs. 5 and 6). These findings were in accordance with previous studies that reported an increase of caspase-3 immune reactivity in the hepatic and renal tissue of FPN-intoxicated rats (Abdel-Daim and Abdeen 2018) and increase of COX-2 expression in human neuroblastoma SH-SY5Y cells treated with FPN (Park et al. 2016). Caspase-3 and Cox-2 mediate apoptosis process, important for the maintenance and development of multicellular organisms, and participate in various physiological, pathological, and toxicological conditions (Majno and Joris 1995). Apoptosis is mediated by a proteolytic caspases essential for initiation, regulation, and execution of proteolytic processes (Thornberry 1999); among them, active caspase-3 is used for detection of apoptotic cells because it is the main executioner of apoptosis (Jakob et al. 2008). Cyclooxygenase-2, an immediate response protein, is upregulated in response to various stimuli, including environmental toxicants and proinflammatory cytokines (Narita et al. 2008). Moreover, COX-2 is implicated not only in inflammation but also in carcinogenesis, affecting cell proliferation, differentiation, apoptosis, angiogenesis, and metastasis (Williams et al. 1999). Oxidative stress is the main cause of cellular damage associated with xenobiotics and inflammatory stimuli. Reactive oxygen species produced during OS has been recorded to initiate signaling cascades resulting in apoptosis (Yu et al. 2004). Furthermore, ROS induce inflammatory response and mediate COX-2 expression (Barbieri et al. 2003). Induction of COX-2 in tissues initiates apoptosis by increasing prostaglandin-2 level (Hu et al. 2017). Thus, FPN induced the marked increases in the hepatic and renal MDA level which may indicate the development of OS in the tissues. Moreover, the observed reduction in the hepatic and renal GSH content and CAT activity may be due to the overutilization of the intracellular GSH and CAT to combat the increased ROS production after exposure to FPN, ultimately oxidative hepatic and renal tissue damages via induction of caspase-3 and COX-2 expressions in both hepatic and renal tissues.

Furthermore, our results indicated that administration of GAE 2 h before FPN intoxication protected rats from FPN-induced hepatorenal toxicity. These findings were evidenced by a significant decrease of serum ALT and AST activities and urea and creatinine levels as a result of the antioxidant properties of GAE, represented by marked reduction of hepatic and renal MDA level concomitantly with significant elevation of GSH content and CAT activity. These antioxidant activities of GAE reduced caspase-3 and COX-2 expression in hepatic and renal tissues that ameliorated FPN-induced alterations of hepatic and renal tissue architectures. Our findings were in line with the previous studies that indicate the hepatoprotective effect of ginseng against CCl4 (Bak et al. 2012), cadmium (Park et al. 2013), aflatoxins (Abdel-Fattah et al. 2014), and methotrexate (Youssef 2016). In addition, GAE has protective effect against cisplatin (Abdel-Wahhab and Ahmed 2004), streptozotocin (Hussein et al. 2011), ochratoxin (Morsy et al. 2012), gamma-irradiation (Mansour 2013), and gentamicin-induced (Raheem et al. 2017) nephrotoxicity. These hepatonephroprotective effects of GAE may be related to the antioxidant effect of ginseng that is closely linked to its ginsenoside content (Song et al. 2019) as it stimulates gene expression of antioxidant enzymes and enhances their activities, which play a crucial role for maintaining cell viability, by decreasing the oxygen radical produced by the intracellular metabolites (Zhang et al. 2008). Ginsenosides inhibit LPO by scavenging ROS, restoring GSH level, and inhibiting NO production (Kang et al. 2007; Zhu et al. 2009; Zhang et al. 2010). These antioxidant activities of ginseng may enable it to prevent the apoptosis of kidney tissue after exposure to gentamicin through its ability to reduce caspase-3 reactivity. (Raheem et al. 2017). In addition, Kang et al. (2006) stated that ginseng prevent renal damage in diabetic rats by reducing the overexpression of COX-2 confirming its anti-inflammatory properties that may be due to its ability to inhibit TNF-α-induced NF-κB transcription activity and NF-κB-dependent COX-2 and iNOS gene expressions (Song et al. 2012). Therefore, GAE exerted its protective effects against FPN-induced hepatorenal toxicity which may be via its antioxidant, anti-apoptotic, and anti-inflammatory activities.

Conclusion

Our findings provided evidences for the implication of oxidative stress, inflammation, and apoptosis to fipronil-induced hepatorenal toxicity and highlighted the ameliorative effect of ginseng, possibly via its antioxidant, anti-apoptotic, and anti-inflammatory properties. Therefore, the overall obtained data may be of crucial value in presenting ginseng as an effective therapeutic strategy against toxic effects of FPN and other phenylpyrazoles.