Chlorpyrifos (CPF) is an organophosphate pesticide commonly used to control mosquitoes, flies, and other household and agricultural pests (Albasher, Almeer, Al-Otibi, Al-Kubaisi, & Mahmoud, 2019; Spodniewska, Barski, & Gizejewska, 2015). Despite the United States Environmental Protection Agency's (US EPA) ban on domestic and agricultural uses of CPF in 2000, it is still one of the most commonly used OP insecticides worldwide (Bjorling-Poulsen, Andersen, & Grandjean, 2008; Maroni, Colosio, Ferioli, & Fait, 2000). As a result of its widespread use, acute and chronic exposure to CPF has been associated with a variety of abnormalities which include hepatotoxicity (Deng, Zhang, Lu, Zhao, & Ren, 2016; Goel, Dani, & Dhawan, 2006; Owumi & Dim, 2019), nephrotoxicity (Deng et al., 2016; Owumi & Dim, 2019), neurotoxicity (Shou et al., 2019), haematotoxicity (Aroonvilairat et al., 2018), reproductive toxicity (Adedara et al., 2018) and cardiotoxicity (Zafiropoulos et al., 2014). The current study thus assessed the mitigating effect of 3-IPA in CPF induced hepatorenal damage.
In the current study, the body weight gain of CPF-treated rats increased (p < 0.05) compared with the control. Our results are in accordance with other studies, revealing an increase in body weight gain in rats mediated by CPF (Ambali, Ayo, Ojo, & Esievo, 2011; Ezzi et al., 2016; Meggs & Brewer, 2007). The weight increase represents an increase in fatty tissues. OP insecticides such as CPF may induce accelerated differentiation of immature adipocytes into mature fat cells (Meggs & Brewer, 2007). However, other studies recorded a marked reduction in net body weight gain in CPF-exposed rats (Heikal & H. Mossa, 2012; Tanvir et al., 2016).
The increase in the body weight gain in the co-treated group occurred at the higher dose of IPA (50mg/ kg) instead of the lower dose of IPA (25mg/ kg). Our results are in tandem with previous findings, which reported reduced body weight (Ayaso, Ghattas, Abiad, & Obeid, 2014; Konopelski et al., 2019). In the present study, oral administration of CPF resulted in a significant reduction in the relative liver weight. However, there was no substantial change in the relative kidney weight in CPF treated rats compared with the control. The decrease in relative liver weight may be an indication of liver toxicity. Our results are consistent with results from previous studies, which also reported a reduction in organ weight (Ezzi et al., 2016; Joshi, Mathur, & Gulati, 2007). Co-administration of IPA improved relative liver and kidney weights in co-treated groups compared to CPF alone treated rats.
The present investigation assessed the levels of serum marker enzymes. These enzymes include AST, ALT, ALP and GGT and are found in the liver under normal physiological conditions. However, cellular stress and tissue injury induced by various toxins could release these enzymes into the bloodstream, indicating liver injury (Kaplan, 1993). In the present study, CPF administration resulted in a statistically significant increase in ALP and GGT, indicating hepatic toxicity, resulting in liver necrosis and inflammations (Kuzu et al., 2007). There was, however, a non-significant increase in the activities of AST and ALT. Thus, increased activities of hepatic transaminases in serum correlates with hepatocyte injury in CPF treated rats. These findings are consistent with earlier CPF in experimental rodents (S. A. Mansour & Mossa, 2009; Tanvir et al., 2016). Also, in this study, increases in activities of serum hepatic transaminases observed in CPF treated rats were dose-dependent reduced upon co-treatment with IPA, reflecting the protective potential of IPA against CPF hepatoxicity.
The kidneys play an essential role in the excretion of waste products and toxins such as urea and creatinine. Urea is a by-product of protein degradation, whereas muscles produce creatinine as a by-product of creatinine breakdown (Dorgalaleh et al., 2013). Both are excreted from the body by the kidney in urine and serve as indicators of renal function. They are also helpful in detecting nephrotoxicity caused by exogenous compounds in living organisms (Sandilands, Dhaun, Dear, & Webb, 2013). This study found a significant increase in serum urea level and a non-significant increase in serum creatinine level in CPF-treated rats than controls, indicating that CPF caused kidney damage due to various factors, including oxidative stress (S. G. Mansour et al., 2017). The reduced serum urea and creatinine levels in rats co-administered IPA and CPF imply that IPA alleviated CPF-mediated renal dysfunction in the treated rats.
Antioxidants, both endogenous and exogenous, and enzymatic and non-enzymatic, are essential in mitigating oxidative stress. The enzymatic antioxidants (SOD, CAT, GPx, and GST) strongly defend against liver injury and other disorders; thus, they receive a lot of attention (Zhou, Zhang, Yin, Jia, & Shan, 2015). Furthermore, these antioxidant enzymes limit the effects of oxidants in cells and tissues and act in the defence against oxidative cell injury scavenging free radicals (Halliwell & Gutteridge, 2015). Our data indicated a decrease in SOD, CAT, GST and GPx in the liver and kidney of rats treated with CPF alone compared to the control in the current study. The reduction in these antioxidant enzymes may be related to decreases in the synthesis of these enzymes or inactivation/inhibition induced by exposure to CPF (Heikal & H. Mossa, 2012; Owumi & Dim, 2019). This could result in the accumulation of free radicals in the liver and kidney cells of rats treated with CPF alone. Interestingly, our results show an increase in the activities of these antioxidant enzymes in rats co-exposed to CPF and IPA, revealing the antioxidant effects of IPA against CPF mediated oxidative stress in the treated rats.
Reduced glutathione is an endogenous non-enzymatic antioxidant that operates in association with other endogenous antioxidants in preventing oxidative stress. Our results show decreases in the levels of GSH in the liver and kidney of animals treated with CPF alone compared to the control, indicating over-utilisation in the detoxification process to shield the system from oxidative stress. Consequently, exposure to CPF predisposes the liver and kidney to free radical-mediated cellular damage in rats exposed to CPF. Co-administration with IPA and CPF brought about a significant increase in hepatic and renal GSH levels compared to animals treated with CPF alone. IPA has been proven to effectively scavenge hydroxyl radicals effectively and work in tandem with glutathione to minimize oxidative stress (M. Karbownik, J. J. Garcia, et al., 2001). Besides scavenging the hydroxyl radical, IPA has also been shown to scavenge the superoxide anion radical, which aligns with our result as co-administration with IPA and CPF brought about a significant increase in hepatorenal GSH levels compared to animals treated with CPF alone. The present study revealed a significant decrease in TSH level in the liver and kidney of rats treated with CPF alone compared to the control group, signifying a reduction in antioxidant status and an increased accumulation of free radicals. However, there was a significant increase in TSH level in the liver and kidney of rats co-treated with CPF and IPA in a dose-dependent manner compared to the CPF alone treated group, thus showing the antioxidant ability of IPA.
Both ROS and RNS, when present in a typical physiologically system, play essential roles in normal cellular functions such as fighting against infection, regulating different intercellular signalling pathways (Nunes Silva, 2015). However, when present in high concentrations, they overwhelm the innate antioxidant defence system, resulting in oxidative stress, leading to cellular dysfunction via lipid peroxidation, protein, and DNA damage (Martindale & Holbrook, 2002). In this study, rats treated with CPF alone revealed a marked increase in RONS levels compared to the control, implying that CPF activated the formation of free radicals in liver and kidney tissues. A significant increase in hepatic and renal LPO levels was also increased in rats treated with CPF alone. The increase in LPO levels can be attributed to the rise in RONS levels due to CPF treatment. Our findings are in tandem with previous observations (Heikal & H. Mossa, 2012; S. A. Mansour & Mossa, 2009, 2010). The current study revealed that IPA reversed the elevation of RONS and lipid peroxidation in liver and kidney tissues of co-treated rats. This may be attributed to the ability of IPA to scavenge free radicals capable of inducing lipid peroxidation and further oxidative stress.
The induction of inflammatory biomarkers (NO, MPO, XO) and cytokines (IL-1β, IL-10) could be stimulated by exogenous toxicants such as CYP (Gangemi et al., 2016). These biomarkers are often triggered alongside increased oxidative stress and induce inflammatory responses via activation of redox-sensitive transcription factors, such as NF-κB (Forrester, Kikuchi, Hernandes, Xu, & Griendling, 2018). In the current study, CPF administration in rats resulted in increased NO levels, MPO and XO activities, as well as IL-1β levels. It decreased IL-10 levels in the liver and kidney of rats treated with CPF alone compared to the control rats. The increased levels of these inflammatory biomarkers and cytokines could be attributed to the ROS-mediated activation of NF-κB induced by CPF (Wang et al., 2018). CPF-induced oxidative stress has also been reported previously to trigger the inflammasome and subsequent innate immune response (Jang et al., 2015), thus causing inflammation in cells and tissues. As a result of the inflammation being associated with increased oxidative stress, ROS scavengers can mitigate the inflammatory responses induced by CPF. Co-treatment with IPA significantly increased IL-10 and decreased IL-1β levels, reduced MPO and XO activities, and NO level in the liver and kidney of rats relative to the CPF alone group.
RONS generation resulting from CPF exposure upregulates pro-apoptotic proteins, including caspase 3 and caspase 9 and downregulate anti-apoptotic proteins such as Bcl-2 (Fu, Li, Song, Zhang, & Xie, 2019). The induction of apoptosis by CPF is characterized by NF-κB activation and loss of mitochondrial membrane integrity (Lee, Lim, Park, Park, & Koh, 2014). Our results show that CPF caused a significant increase in 8-OHdG adduct, caspase 3, caspase 9 activity, and a considerable decrease in Trx level and Trx-R activity in the experimental rats liver and kidney. Besides, increases in RONS result in lipid peroxidation, loss of membrane integrity, increased production of pro-apoptotic proteins, DNA fragmentation and damage (Wakf et al., 2018). Furthermore, an increased level of 8-OHdG in rats liver and kidney treated with CPF alone demonstrates the genotoxic effect of CPF by inducing DNA damage (Alavanja, Ross, & Bonner, 2013; Ethikic et al., 2012). In the current study, co-treatment with IPA dose-dependently attenuated the expression of caspase-3 and caspase 9, increased Trx and Trx-R and decreased 8-OHdG level in the liver and kidney of rats compared to CPF alone treated rats. These observations further demonstrate the protective effect of IPA against CPF-mediated oxidative damage and induction of apoptosis.