Since use of insecticides is inevitable to saving crops and fruits from harmful insects, toxic effects on non-target organisms or accidental exposure can only be countered by synthesizing relatively non-toxic insecticides or via adopting remedial measures. Contrary to conventional neurotoxic insecticides, moulting inhibitor insecticides were synthesized in a quest to generating target specific insecticides with relatively non-toxic properties for non-target animal species. Buprofezin (BPFN), a thiadizidine insecticide is one such chemical that kills hemipteran and homopteran insects by blocking cuticle formation during metamorphosis. Toxic effects of BPFN on vertebrates have although not been widely studied but a few reports have indicated some toxic effects (Yang and Yang 2007; Zhang et al. 2010). Vast literature available on the effects of pesticides and insecticides indicate that most of these chemicals generate free radicals as they enter the bodies of animals and humans, thus producing tissue toxicity, various diseases and in certain cases death. Discovering and applying free radical scavenging drugs can therefore be a useful strategy against pesticide induced toxicity. The current study investigated this aspect and explored the protective potential of ascorbic acid (Vitamin C, Vit C) and curcumin (CUR, turmeric herb ingredient) for 28 days of BPFN exposure along with Vit C and CUR supplementation.
Except, typical behavioral signs of toxicity like staggering gait, itching, and rotational movement involving nervous behavior that were noticeable at 250 mg BPFN dose, overall, neither untoward signs of toxicity were noticeable nor there occurred any detectable change in either body weight or organ weight of exposed mice, and no deaths were recorded at above doses. While autopsy also, did not reveal any gross abnormalities. Since low BPFN dose remained relatively non-toxic, medium dose more toxic, and high dose turned to be highly toxic, remedial potential of Vit C and CUR singly or in combination, against BPFN was studied only with long-term exposure to high BPFN dose. Our results demonstrated that both medium and high BPFN doses (100 and 250 mg/kg b.w respectively) were significantly damaging to vertebrate animal; in this case laboratory mice. Toxic tissue damage was revealed by negative alterations in hematological parameters, the HB, HCT, RBC, MCH, MCHC, MCV, increased WBC, elevation of liver enzymes, the ALP, ALT, AST, lowering of antioxidant enzymes, the SOD, POD and CAT, elevation of ROS, TBARS and MDA, cellular damage and histological alterations in liver and kidney tissues and DNA fragmentation. Notably, supplementation with both Vit C and CUR prevented all above alterations. However, the preventing effect was stronger in alleviating above biochemical and cellular malformations when Vit C and CUR were administered in combination.
Although toxicity of BPFN on Balb/c mice has been demonstrated (Bibi and Qureshi 2019), the present is the first study that showed convincing evidence in favor of mitigating or preventive effects of Vit C and CUR on BPFN-induced toxicity. Present study in mice therefore, demonstrated two aspects. First, BPFN is toxic to vertebrate animals at medium and high doses, mainly via generating free radicals and causing lipid peroxidation and secondly, both Vit C and CUR are promising preventive measures that can be adopted in case of BPFN exposure but in combination they more effective.
Buprofezin Toxicity
Although, in general, BPFN is considered relatively safe for humans and vertebrates, databases and information available from past studies have indicated different LD50 for buprofezin (Zhang et al. 2010; Lewis et al. 2016). For instance, for rat, oral LD50 is > 1635 mg kg-1bw, for and mice and hamster, it is >10,000 mg kg-1bw, mammals in general > 2198 mg kg-1bw and so on in several other species of vertebrates. Thus, we redetermined the LD50 first for mice in trial experimentation, and we found that acute 24 h oral doses in the range of 50 - 3000 mg kg-1bw (0.05 - 3 g kg-1bw) were safe, while doses in the range of 5000 - 8000 mg kg-1bw (5 - 7 g kg-1bw) were highly toxic and lethal. The animals could not survive beyond 24 h (data not shown). Since most insecticides, apart from accidental exposure, find their way into the bodies of non-target organisms slowly over weeks, months or years, therefore, in the current study we focused on treating the animals with both safe and unsafe BPFN doses not acutely but over several weeks of exposure. Taking also into consideration that the acceptable daily intake of BPFN is 0.01 mg kg-1bw, and acceptable operator exposure level-systemic is 0.04 mg kg-1bw (Lewis et al. 2016), we currently used 50, 100 and 250 mg kg-1bw daily doses on long-term basis for continuous 28 days.
Exposure to BPFN indicated that BPFN causes hypochromic anemia in exposed animals as indicated by significant lowering of Hb, HCT, RBC and other hematological indices. This observation is not new and is similar to that has been reported in an earlier study by Berberian and Enan (1989), who demonstrated that there occurred significant drop in hematological counts in male rats treated with antimoulting compounds leading to the development of significant erythrocytopenia, while significant decrease in the Hb, HCT and MCHC indicated the development of hypochromic type of anemia. Physiologically, MCHC is a sensitive measure for the diagnosis of iron deficiency and indicates a hypochromic situation either due to inhibition of iron utilization or reduced hemoglobin synthesis. Dimilin, an insect growth regulator, has been shown to cause significant lowering of Hb, RBC count and MCH values. This effect was attributed to reduced heme in the bone marrow leading to reduction in the rate of RBC synthesis. It was reasoned that disruptive action of insecticides on the erythropoietic tissue could have resulted in a decrease in RBC and Hb content (Shehata and Shalby 2006). Our study also reaches similar conclusion that BPFN causes erythrocytic disruption leading ultimately to a decreased Hb concentration. Moreover, increase in WBC also indicates that leukocyte number increased to remove cellular debris. It is very well-known that the leucocyte system serves to defend the body against foreign organisms or extraneous materials. Thus, an increase in WBC is suggestive of activation of defense and immune system of the body. As a result of toxic damage, bone marrow might have released more WBC from its storage pool to blood. Leukocytosis therefore likely occurred due to acute hemolysis or hemorrhage (Yousef et al. 2003). Currently, increased levels of MDA indicate lipid peroxidation and ultimate cell membrane disruption, which might have led to red cell hemolysis and lowering of Hb.
Presently, significant increase in ALP, AST, ALT enzymes, reduced glutathione, increased cholesterol, triglycerides, urea, creatinine and protein content upon exposure to BPFN indicates liver and kidney damage. In addition, increase in LDH concentration also indicates excessive cell damage, these biochemical observations are corroborated by significant histological alterations of liver and kidney tissues and also by DNA or genotoxic damage in these tissues. It has been reported that the activities of these enzymes are relatively proportional to the extent of damage (Ekam and Ebong 2007; Nwaehujor et al. 2011; El-Naggar et al. 2015). The liver, being the primary detoxifying organ, is the primary target of pesticides and is most vulnerable to their effects (Mansour and Mossa 2010). In addition, the liver has a high metabolic activity and energy demand, and it is critical to maintaining energy homeostasis. Energy fragment metabolism problems are therefore considered major cause of liver damage (Viollet et al. 2009). In the present case, histological alterations coincided well with morphometric observations in liver and kidney tissues. Histological changes that we observed presently are very similar to the study of Afshar et al. (2008) who indicated tissue congestion, altered radiating canals, hemorrhage in kidney tissues of rats exposed to fenitrothion. In addition, they also reported hypertrophy of hepatocytes, granular cytoplasm, congestion of cytoplasm and inflammatory leuckocytic infiltration. Also, exposure of mice for 10, 20 and 30 days to carbosulfan (a carbamate organic compound) has shown adverse histologic and physiological effects on liver functions (Manna et al. 2004). In contrast, other workers (Collins 2004; Patil and David 2013; de Barros et al. 2016) have highlighted that there were no significant changes in liver and kidney tissues when adult male rats were exposed orally to 0, 2, 4 or 8 mg/kg doses of diflubenzuron for 28 days. BPFN has however already been shown to inhibit the activity of cytochrome C oxidase at the cellular level, which is a major cause of energy metabolism shunting (Ji et al. 2016). Similar elevations in liver enzymes and cholesterol and triglyceride concentrations were reported in male mice administered chlorpyrifos and cypermetherin in combination on alternate days (Khan 2006).
Elevation in serum cholesterol has been suggested to be due to the effect of insecticides on liver membrane permeability (Yousef et al. 2006). Increase in serum urea and creatinine concentrations is similar to the effect of fipronil insecticide, where exposure of rats to 1.0 and 10.0 mg/L of fipronil in drinking water for 45 days caused similar effects (Khan 2006; Mossa et al. 2015). Present increase in serum urea and creatinine and lowering of protein concentration upon exposure to BPFN indicates at one end protein degradation and on the other liver and kidney damage, which is accounted for by histological abnormalities and alterations. Yavasoglu et al. (2006) claimed however, that cypermethrin posed insignificant impact on the total protein contents of liver of rat. Thus, BPFN which is different in its biochemical action might also differ from other insecticides in its physiological impact.
Our present experiments with BPFN exposure to mice demonstrated significant decreases in the levels of antioxidative enzymes CAT, SOD, POD, and GSH in kidney and liver tissues. While in contrast, there occurred excessive increase in ROS, TBARS and MDA, all indicating severe oxidative stress and a disbalance in the antioxidant-oxidant defensive system of the tissues. It is therefore quite obvious that elevation in free radicals was the underlying cause to producing severe oxidative stress, lipid peroxidation, release of enzymes, cellular disruption and DNA degradation. SOD and CAT are the most important antioxidant enzymes in their battle with the free radicals. Thus, in our findings, exposure to BPFN caused over production of free radicals which most likely inhibited the SOD and CAT activities in liver by lowering their normal levels. Although ROS are produced in animal cells in small amounts during normal metabolism as result of catalytic reactions by electron transport chain enzymes like NAD(P)H oxidases, and they are also known to regulate intracellular signaling, but they are harmful if produced in excess when triggered by pesticides, insecticides, drugs and chemicals (Nordberg and Arnér 2001). Notably, oxidative stress due to ROS generation is common feature of all pesticides and insecticides (Abdollahi et al. 2004; Sule et al. 2022). It therefore the disbalance or diminished scavenging ability of the antioxidant enzymes caused by BPFN that led to oxidative stress and cell damage. It is known from a number of studies that chief ROS generating radicals are superoxide anion, hydroxyl and hydrogen peroxide (Le Bras et al. 2005) and that ROS are cellular toxins that impede cell survival; they are naturally counteracted by the internal antioxidant defense system primarily POD, CAT, SOD and GSH. Once an imbalance among ROS production and antioxidants occurs, the cells become susceptible to extreme oxidative stress induced damage (Small et al. 2012). This is similar to a study by Gülçin et al. (2012) which demonstrated that the toxic effects of Spinosad (SPD) (macrolide insecticide) at 35 and 350 mg/kg doses on liver tissues were mainly due to overproduction of ROS. In a previous study, mice were intragastrically administered every other day; a total of nine administrations, and three exposure groups were given 0.0463, 0.139 or 0.417 g/kg b.w. of BPFN where, due to accumulation of the toxicant, a severe oxidative stress response was detected in liver (Ji et al. 2016). Thus, BPFN is no different from other functionally non-related insecticides in producing ROS, lipid peroxidation, lowering of antioxidant enzymes thereby causing cellular and biochemical toxicity.
The genotoxic or DNA damage (comet assay analyses), that was observed upon exposure of mice to BPFN was most likely due to ROS. This is similar to previous studies which indicate that pesticides not only interact with the functioning of cells but also interfere with their genetic material (Rajaguru et al. 2003) or as we presume, vice-versa damage the DNA and whole machinery of the cell would stop functioning. Moreover, ROS production leads to production of lipid peroxidation which also causes DNA damage (Patil and David 2013). Previously, organ specific genotoxic effects of fipronil investigated through the comet assay have been shown in the liver, spleen and lungs of mice (Lovinskaya et al. 2016). Ji et al. (2016) have also shown ROS mediated changes in the mitochondrial DNA. Current comet assay carried out on BPFN treated mice revealed DNA damage indicated by an increase in % tail DNA and mean tail length of comet. Significant increase occurred in comet length; head length; tail length; tail moment and olive tail moment. Thus, our present study at one establishes that in long-term exposure, BPFN is toxic to mammalian tissues and that it causes toxicity via increased generation of free radicals.
Role of Vitamin C and curcumin in alleviating buprofezin-induced toxicity
Turning toward the preventive role of Vitamin C and curcumin in BPFN-induced toxicity, our study demonstrates that both supplementations are equivalent in reducing ROS production, decreasing lipid peroxidation, elevation of antioxidant enzymes, liver enzymes, lowering LDH levels, preventing cellular and genotoxic damage and normalizing hematological parameters. However, Vit C appears superior to CUR in its ability to scavenge ROS, but Vit C and CUR administered in combination to BPFN-exposed mice are more protective.
As regards hematological alterations, it is possible that Vit C lowered the BPFN-induced production of harmful oxidants and stimulated iron absorption thereby causing an increase in red blood cell synthesis (Navya et al. 2012). Several studies have indicated that Vit C is an effective remedial measure against toxic tissue damage caused by toxicants, pesticides, insecticides and environmental pollutants to animal bodies (Li et al. 2009, Mirvaghefi et al. 2016). It is very well-known that Vit C acts as a free radical scavenger and protects hepatocytic membranes from oxidative damage and prevent leakage of cellular content (Soudani et al. 2011; Elzoghby et al. 2014 ; El-Shenawy et al. 2015; Ghazanfar et al. 2018). The present study also suggests preventive role of Vit C against BPFN-induced hepato- and nephrotoxicity in mice. Our results are consistent with a previous study conducted on male Wistar rats treated orally with fenitrothion (20 mg/kg b.w) for 30 days, whereby, protective effects of vitamin C (100 mg/kg b.w) and selenium (0.5 mg/kg b.w.) on biochemical, hematological and oxidative stress parameters in the blood were demonstrated (Milošević et al. 2017).
Presently, significant decrease that was found in the total protein content of kidney and liver tissue of male mice, exposed to BPFN as compared to control, and increased levels of creatinine and urea were most likely due to protein degradation. Protein plays an extensive role in cellular biological functions in fish and other vertebrates (Desboeufs et al. 2005). Previous data showed considerable effects of Vit C in replenishment of such biochemical parameters interrupted by organophosphates (Halliwell and Gutteridge 2015). Since Vit C scavenges free radicals retaining the integrity and cell membrane functioning, presently, increased lipid peroxidation (MDA assay) provided clear indication of oxidative stress caused by BPFN. Mammalian cells possess antioxidative properties to get rid of free radicals and their harmful effects (Sefi et al. 2011). Al-Harbi et al. (2014) demonstrated insecticide induced physiological stress, stimulated oxidative degradation of lipids that carried antioxidant enzymes. Our study is in also accordance with Djeffal et al. (2015) who demonstrated that selenium (Se) and vitamin C decreased ROS generation while increasing the GSH content in methomyl-induced oxidative stress in rats, indicating the antioxidative potential of Se and Vitamin C. Our results also show that Vit C doses restored enzymic activities by elevating their levels and suppressed oxidative stress caused by BPFN through scavenging ROS. Thus a restoration or increase in the levels of endogenous antioxidant enzymes, and decrease in LDH observed presently, was quite possibly due to the free radical scavenging properties of vitamin C (Djeffal et al. 2015; Hamza et al. 2017).
Present tissue histology demonstrated hepatocytic and nephrotoxic damage in mice treated with BPFN (toxicity groups). In contrast, both Vit C and CUR supplementation remained effective in preventing histological tissue damage. Our study clearly demonstrated hepato- and nephroprotecive effect of Vit C and CUR due to their antioxidant or free radical scavenging potential.
Severe cellular damage and tissue degeneration caused by BPFN further revealed the genotoxic damage. Co- treatment with Vit C and CUR upon BPFN exposure reduced the genotoxic damage as revealed by normalization in the mean tail DNA, mean tail moment, mean tail length and Olive tail moment when compared with the control. It has been reported that Vit C protects biomolecules like DNA from oxidative damage caused by free radicals (Konopacka 2004). Hence, these facts support our present findings in mice that generation of ROS and free radicals in BPFN treated mice led to marked DNA fragmentation which was attenuated by the antioxidant action of Vit C and CUR. These results are also in agreement with Poljšak and Fink (2014) who showed that Vit C can protect proteins and DNA from pesticide-induced oxidative stress. The present findings clearly suggest that Vit C and CUR can reverse the DNA damage and may act as good ameliorating agents against BPFN-induced bicochemical, cellular and histologic toxicity in laboratory mice.
Not much data is available as regards the protective effects of CUR against pesticides/insecticide-induced toxicity. However, a few studies have reported its ameliorating effects in case of insecticidal exposure. For instance, CUR has been shown to protect rat liver tissue from oxidative stress and recovers histological damage caused by pyrethroid insecticides (Abdel Rheim et al. 2015). Similarly, combinatorial administration of resveratrol and CUR was shown to prevent male albino rats from fipronil-induced oxidative damage and restored elevated ALT, AST, ALP, cholesterol, triglycerides, urea, creatinine and total protein concentrations to values near control animals (AlBasher et al. 2020). Recently, a study in rainbow trout has also demonstrated that genotoxic or DNA damage (comet assay) due to chlorpyrifos-induced oxidative stress, is modified to a moderate or high degree with CUR, but it is dose-dependent (Sahinoz et al. 2019).
Thus, our present study against BPFN toxicity is a step further in the on-going research to find out remedial measures upon insecticidal exposure and demonstrates that Vit C and CUR are protective and preventive agents against long-term BPFN-induced toxicity in mice. As BPFN is an effective insecticide, and is being widely used worldwide, future studies are definitively required to explore BPFN toxicity at cell, molecular and biochemical level in other animals and human users, safe concentration in the environment and remedial measures that can be adopted upon exposure to this insecticide. This is so because until to date has been reported one case of unusual case of insecticide poisoning where a 44-year-old person self-harmed with burpofezin. Symptoms that were observed included high cough, tongue fasciculations, myalgia, giddiness and burning sensation in epigastric region. He reverted to normal after 5 days and was discharged. It was reported that buprofezin could be toxic to humans and can tongue fasciculations and burning sensation in epigastric region (Shagufta et. 2019). Besides, BPFN studies on animal models indicate its genotoxic and carcinogenic potential (Culleres et al., 2007).