Introduction

Fipronil (FPN) is a phenylpyrazole insecticide, widely used for agricultural and veterinary activities (Tingle et al. 2003). It acts by blocking the GABA-regulated chloride channels, causing central nervous system (CNS) depression and death in pests (Das et al. 2006). Toxicity may occur in humans due to inappropriate use or exceeding the dose, recommended by the manufacturer (Anadon and Gupta 2012). Several studies have reported that FPN induces oxidative stress and cellular DNA damage in rat pheochromocytoma cell culture (Lassiter et al. 2009), female rats (Leghait et al. 2009), Japanese quails (Ali et al. 2016), and tadpoles (Gripp et al. 2017). In addition, FPN disrupts the mitochondrial oxidative phosphorylation, leading to ATP exhaustion, glycolysis activation, and lactate accumulation (Vidau et al. 2011). This may later activate enzymes, involved in the apoptotic process, such as caspases 3 and 7 (Das et al. 2006).

Thymoquinone (TQ) is a safe phytochemical compound (Fig. 1), extracted from the seeds of Nigella sativa L. (Darakhshan et al. 2015). Animal studies have shown the protective efficacy of TQ against xenobiotics-induced toxicity by normalizing the reduced glutathione (GSH) tissue concentrations and the activities of endogenous antioxidant enzymes (Ince et al. 2012; Ince et al. 2013). Other reports have demonstrated the hepatoprotective effects of TQ against carbon tetrachloride (Nagi et al. 1999) and cyclophosphamide (Alenzi et al. 2010). Moreover, TQ has been shown to protect the renal functions against cisplatin, ifosfamide, and mercuric chloride toxicities by minimizing the alterations in renal GSH concentration and lipid peroxidation (Badary 1999; Badary et al. 1997; Fouda et al. 2008).

Fig. 1
figure 1

Chemical structure of fipronil, diallyl sulfide, and thymoquinone

Full size image

Another interesting phytochemical compound is diallyl sulfide (DAS), an essential organosulfur component of garlic oil (Fig. 1) (Tamaki and Sonoki 1999) with established health benefits (Banerjee et al. 2003). Several animal studies have reported the hepatoprotective effects of DAS against aflatoxin B (Sheen et al. 2001) and thallium acetate (Abdel-Daim and Abdou 2015). Moreover, DAS has shown promising results in neuroprotection against ethanol (Huentelman et al. 1999) and ischemia-induced neuronal injuries (Lin et al. 2012). Further, it has well-documented anti-hyperlipidemic, anti-hypertensive, anti-diabetic, and anti-thrombotic activities (Jakubowski 2003; Sato and Miyata 2000).

To our knowledge, no studies have been published on the chemoprotective potency of DAS and TQ against FPN toxicity. Therefore, our objective was to investigate the antioxidant and cytoprotective effects of DAS and TQ against FPN-induced oxidative stress in a rat model.

Materials and methods

Chemicals and reagents

Fipronil (5-amino-1-(2,6-dichloro-α,α,α-triflouro-p-tolyl)-4-[(triflouromethyl)sulfinyl]pyrazole-3-carbonitrile) was purchased as a commercial product (Fipromex 20% SC) from MAC-GmbH (Sigmarszell, Germany). Thymoquinone (CAS Number 490–91-5; molecular weight 164.20 g/mol; purity ≥ 98.5%) and DAS (CAS Number 2179–57-9; molecular weight 146.27 g/mol; purity 80%) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Biochemical kits were purchased from Biodiagnostics Co. (Cairo, Egypt) except for lactate dehydrogenase (LDH) kits, which were provided by Randox Laboratories Ltd., UK. Other chemicals in this experiment were of analytical grade.

Animals

Thirty-two healthy male Wistar rats, weighing between 150 and 180 g, were housed in wire-mesh cages under controlled temperature (25 ± 2 °C) and a 12 h light/dark cycle. Rats had free access to a balanced rat chow and water and were acclimatized for 2 weeks prior to the experiment to restore normal behavior and growth. All animal investigations were performed as per the institutional and national rules for using animals in scientific research and were affirmed by the local ethics committee (Approval No. 201617).

Experimental design

Rats were divided into four groups (n = 8/group). Group (I) animals were used as a control and received oral physiological saline. Group (II) received oral FPN at a dose of 10 mg/kg bw (Badgujar et al. 2015). Groups (III and IV) received oral FPN at the same previous dose plus DAS (200 mg/kg bw) (Szutowski et al. 2002) or TQ (10 mg/kg bw) (Radad et al. 2014). All treatments were given once daily for 28 days.

Serum collection and tissue preparation

Twenty-four hours after the last FPN dose, individual blood samples were collected from rats under sodium pentobarbital anesthesia and the rats were then sacrificed by decapitation. The resulting serum samples were left to clot and were then centrifuged at 5000 rpm for 10 min and stored at − 20 °C until the biochemical parameters were assessed. Then, the liver, kidney, and brain tissues were homogenized in 10% (w/v) homogenizing buffer (0.1 M phosphate buffer, pH 7.4 + 150 mM KCl) and were later centrifuged at 9000 r/min at 4 °C for 20 min.

Lipid peroxidation and antioxidant assays

The concentration of the lipid peroxidation biomarker malondialdehyde (MDA) was assessed in the hepatic, renal, and brain tissues according to Mihara and Uchiyama (1978), while the nitric oxide (NO) concentration was measured according to Ridnour et al. (2000). We used the methods described by Beutler et al. (1963) to measure the tissue level of GSH, while the enzymatic activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were measured according to Nishikimi et al. (1972), Aebi (1984), and Paglia and Valentine (1967), respectively.

Serum biochemical assay

Serum liver and renal injury biomarkers were measured according to the manufacturer’s protocol. Serum alanine transferase (ALT) and aspartate transferase (AST) levels were evaluated according to Reitman and Frankel (1957), while alkaline phosphatase (ALP) and γ-glutamyl transpeptidase (GGT) serum levels were measured according to Tietz et al. (1983) and Vázquez-Medina et al. (2011), respectively. Moreover, serum cholesterol and triglycerides were measured according to Allain et al. (1974), Richmond (1973), and Winartasaputra et al. (1980), respectively. Further, serum total proteins and albumin were evaluated following the methods of Lowry et al. (1951) and Hinton et al. (1990), respectively. In addition, serum lactate dehydrogenase (LDH) levels were determined according to Babson and Babson (1973), while serum creatinine, urea, and uric acid were measured according to Larsen (1972), Coulombe and Favreau (1963), and Whitehead et al. (1991), respectively.

Data analysis

All statistical analyses were performed using SPSS software (version 17.0 for Windows). All data were expressed as the mean ± standard deviation (SD) of the mean.We used the one-way ANOVA followed by Tukey’s multiple range tests to evaluate the significance of differences between means. When the probability value was lower than 0.05, the difference was considered statistically significant.

Results

Protective effects of DAS and TQ against FPN-induced serum biochemical alterations

Compared to the control rats, FIP-intoxicated rats showed significantly elevated (p < 0.05) serum concentrations of hepatic (ALT, AST, ALP, and GGT) and renal (urea, creatinine, and uric acid) injury biomarkers, as well as elevated serum LDH and cholesterol concentrations in comparison to control rats. Moreover, FPN induced significant reductions (p < 0.05) in serum albumin, total proteins, and triglycerides levels. Cotreatment of FPN-intoxicated rats with DAS or TQ significantly ameliorated all serum biochemical changes with more frequent restoration of normal control ranges in the TQ group (Table 1).

Table 1 The biochemical effects of diallyl sulfide (DAS 200 mg/kg bw once daily for 28 days orally) and thymoquinone (TQ 10 mg/kg bw once daily for 28 days orally) during fipronil treatment (10 mg/kg bw for 28 days orally) on serum hepatorenal function biomarkers
Full size table

Antioxidant activity in the hepatic tissue

Fipronil-treated rats showed significant drops (p < 0.05) in hepatic tissue GSH concentrations (by 59.4%) and activities of GPx (by 56%), SOD (by 60.6%), and CAT (by 58%) enzymes, compared to the control rats. Concomitant treatment by DAS or TQ significantly increased GSH concentration (by 55 and 106%, respectively) and the activities of the aforementioned antioxidant enzymes (GPx by 62 and 117%, SOD by 105 and 145%, and CAT by 75 and 114%, respectively) in the liver, compared to FPN-treated rats. In addition, the hepatic tissue concentrations of MDA and NO were significantly increased (p < 0.05) following FPN treatment (by 163 and 99%, respectively), compared to control rats. These elevations were significantly ameliorated by joint treatment with DAS (MDA by 48% and NO by 39%) or TQ (MDA by 48.4% and NO by 48.3%), restoring the normal control levels (Fig. 2).

Fig. 2
figure 2

The protective effects of diallyl sulfide (200 mg/kg bw) and thymoquinone (10 mg/kg bw) against fipronil (10 mg/kg bw) on hepatic tissue malondialdehyde, nitric oxide, and antioxidant biomarkers (n = 8). MDA malondialdehyde, NO nitric oxide, GSH reduced glutathione, GPx glutathione peroxidase, SOD superoxide dismutase, CAT catalase, TQ thymoquinone, DAS diallyl sulfide. Columns (means ± SD) with different superscripts indicate significant differences (p < 0.05) between groups

Full size image

Antioxidant activity in the renal tissue

In comparison to the control rats, FIP intoxication caused significant elevations (p < 0.05) in renal tissue MDA (by 93%) and NO (by 83%) concentrations, as well as significant decreases in renal tissue GSH concentration (by 47%) and activities of GPx (by 55.5%), SOD (by 53.4%), and CAT (by 62.5%) enzymes. However, treatment of FPN-intoxicated rats by DAS or TQ significantly decreased MDA (by 34.4 and 45.4%, respectively) and NO (by 36.6 and 43%, respectively) concentrations, while it significantly increased GSH concentration (by 53 and 77%, respectively) and activities of GPx (by 52 and 99%, respectively), SOD (by 60.7 and 94.6%, respectively), and CAT (by 96 and 144%, respectively) in the renal tissue, restoring the normal ranges of these parameters (Fig. 3).

Fig. 3
figure 3

The protective effects of diallyl sulfide (200 mg/kg bw) and thymoquinone (10 mg/kg bw) against fipronil (10 mg/kg bw) on renal tissue malondialdehyde, nitric oxide, and antioxidant biomarkers (n = 8). MDA malondialdehyde, NO nitric oxide, GSH reduced glutathione, GPx glutathione peroxidase, SOD superoxide dismutase, CAT catalase, TQ thymoquinone, DAS diallyl sulfide. Columns (means ± SD) with different superscripts indicate significant differences (p < 0.05) between groups

Full size image

Antioxidant activity in the brain tissue

Compared to the control rats, we observed significant increases (p < 0.05) in MDA (by 104%) and NO (by 119%) concentrations, as well as significant decreases in GSH concentration (by 45%) and activities of GPx (by 47.4%), SOD (by 57%), and CAT (by 65%) enzymes in the brain tissue following FPN administration. On the other hand, treatment of FPN intoxication by DAS or TQ significantly reduced the brain tissue concentrations of MDA (by 34.7 and 49%, respectively) and NO (by 27 and 46.6%, respectively) and increased its GSH concentration (by 43 and 60%, respectively) and activities of GPx (by 46 and 76%, respectively), SOD (by 98 and 125%, respectively), and CAT (by 109 and 151%, respectively) enzymes in comparison to normal control levels (Fig. 4).

Fig. 4
figure 4

The protective effects of diallyl sulfide (200 mg/kg bw) and thymoquinone (10 mg/kg bw) against fipronil (10 mg/kg bw) on brain tissue malondialdehyde, nitric oxide, and antioxidant biomarkers (n = 8). MDA malondialdehyde, NO nitric oxide, GSH reduced glutathione, GPx glutathione peroxidase, SOD superoxide dismutase, CAT catalase, TQ thymoquinone, DAS diallyl sulfide. Columns (means ± SD) with different superscripts indicate significant differences (p < 0.05) between groups

Full size image

Discussion

Our results showed that FPN treatment induced oxidative injuries in the liver, kidney, and brain tissues of male rats and promoted the intracellular lipid peroxidation (as evidenced by increased tissue MDA concentrations) and thereby disturbing the cellular membrane function in the examined tissues (Badgujar et al. 2015). Antioxidant enzymes, such as SOD, CAT, and GPx, are recognized as the first line of defense for cellular macromolecules against oxidative breakdown. In accordance with previous studies (Gill and Dumka 2016; Ki et al. 2012), FPN significantly reduced GSH concentration and the activities of antioxidant enzymes in animal tissues. The malfunction of the cellular antioxidant machinery increases the cellular sensitivity to the detrimental effects of free radicals, whose production is augmented by FPN (Mohamed et al. 2004).

The liver and kidneys play a vital role in the biotransformation of insecticides, resulting in chemically-induced hepatorenal injuries and disturbances in the oxidant-antioxidant system (Mansour and Mossa 2010). The elevations in AST, ALT, ALP, and GGT following FPN treatment in our study can be attributed to hepatocellular membrane damage and outflow of these enzymes into the blood (Goel et al. 2005). Moreover, De Oliveira and colleagues showed that FPN induces swelling and hypertrophy of hepatocytes, causing bile duct obstruction and elevation of serum ALP and GGT levels (De Oliveira et al. 2012).

The increases in serum creatinine, uric acid, and urea may be due to degradation of purines and pyrimidines (DNA breakdown) and the deterioration of renal function in excreting catabolic by-products (Hovind et al. 2009; Sun et al. 2013). In addition, we observed significant reductions in serum total proteins, albumin, and triglycerides, as well as a significant elevation in serum cholesterol levels. This may be attributed to the FPN-induced hepatic injury.

Our results revealed that DAS and TQ ameliorated the oxidative and biochemical alterations, induced by FPN. Our proposed mechanism for such effects in this study is the ability of both compounds to ameliorate lipid peroxidation and enhance the cellular antioxidant defenses. Our data are in agreement with the findings of former studies on the antioxidant/anti-inflammatory effects of DAS (Lawal and Ellis 2011; Nasr and Saleh 2014) and TQ (Fouda et al. 2008; Oguz et al. 2012; Woo et al. 2012).

The literature suggests the following antioxidant and cytoprotective mechanisms for DAS: (1) suppressing the enzymatic activity of cytochrome P450-2E1 and thereby reducing the generation of reactive oxygen species (Khanum et al. 2004) and (2) inducing the mRNA expression of the nuclear factor (erythroid-derived 2)-like 2 [Nrf2] and heme-oxygenase 1 enzyme (Gong et al. 2004). Similarly, former studies have concluded that TQ can (1) directly scavenge for superoxide (O2−) and hydroxyl radicals (OH·) (Badary et al. 2003; Kruk et al. 2000), (2) suppress mRNA expression of the inducible nitric oxide synthase (iNOS) enzyme (El-Mahmoudy et al. 2002), and (3) enhance the expression of antioxidant enzymes’ genes, such as CAT and glutathione-S-transferase in the liver (Ismail et al. 2010).

Considering the antioxidant effects of both agents in the brain tissue (shown in our study), several studies have reported the neuroprotective effects of TQ against irradiation (Ahlatci et al. 2014), toluene exposure (Kanter 2008), and allergic encephalitis (Mohamed et al. 2003). Similarly, other studies have reported that DAS can protect the neuronal cells against xenobiotics (Huentelman et al. 1999) and transient focal ischemia (Lin et al. 2012). Along with the established connection between pesticides exposure and neurodegeneration (Ahmed et al. 2017), these findings make DAS and TQ promising candidates for experimental research on neuroprotection (Al-Majed et al. 2006).

Of note, the antioxidant and cytoprotective effects against FPN were consistently higher in the TQ group than those in the DAS group (as evidenced by the more frequent restoration of normal control levels after FPN intoxication in the TQ group than in the DAS group). This may be related to the potent direct scavenging activity of TQ. Moreover, the selection of the DAS dose in this study was based on the work by Szutowski et al. (Szutowski et al. 2002); therefore, increasing the dose of DAS could achieve better results.

In conclusion, treatment with TQ or DAS ameliorated the FPN-induced cerebral and hepatorenal injuries in rats, probably through enhancing tissue antioxidant defenses. Further experimental and clinical research is needed to translate these findings into therapeutic applications.