Ginger (Zingiber officinale) attenuates the neurotoxicity in rats induced by organophosphate pesticide

Abstract

Background

We are exposed to different chemicals in various ways in our daily life, and these can be toxic at minute concentrations. The pesticides used for different purposes are also toxic and may pose threat to life by inducing oxidative stress. Dichlorvos (DDVP) is an organophosphate pesticide used for various purposes and is also reported to be toxic. In this study, the neurotoxicity of DDVP exposure was studied. The role of ginger, Zingiber officinale (ZO), was also evaluated against the neurotoxic effects of DDVP. Forty-eight Wistar rats of both the sexes were used in this study. The rats were exposed to DDVP and post-treatment with ZO was given. The oxidative stress in terms of lipid peroxidation (LPO), reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) levels were estimated after exposure and treatment.

Results

DDVP resulted in oxidative stress, evidenced by enhanced LPO level. The simultaneous reductions in other non-enzymatic and enzymatic antioxidants were reported. The post-treatment with ZO led to a reduction in oxidative stress in rat brains. The levels of SOD, CAT, GSH, GR, and GPx were increased whereas declined levels of LPO were reported after treatment.

Conclusion

Hence, ginger can help mitigate the pesticide toxicity through the up-regulation of antioxidant levels.

Background

The chemical substances utilized for the control of pests are classified as pesticides such as organochlorines, organophosphates, pyrethroids, and carbamates. These also enter the food chain and bio-accumulate at different levels and in addition to their target group, these also affect the nontarget groups in aquatic and terrestrial ecosystems (Antwi & Reddy, 2015). Dichlorvos (DDVP) is one of the commonly used pesticides in agriculture and storage. It also works against ectoparasites copepods in fish farms and also kills the larvae of flies. Another property of dichlorvos is to control infection by parasitic worms and therefore used as an antihelminthic (Das, 2013; Le Bris et al., 1995).

Dichlorvos inhibits acetylcholinesterase (AChE) activity and causes oxidative stress in the brain like other organophosphates (Allen, 2010; Franco et al., 2010; Soltaninejad & Abdollahi, 2009). The dichlorvos mediates the excessive production of reactive oxygen species (ROS) that causes oxidative modification of lipids, proteins, and DNA. It is prevented by the non-enzymatic and enzymatic antioxidants machinery of the biological system. But, the cells get deprived of these antioxidants due to continuous exposure to toxicants and it accelerates cell death. The exogenous antioxidants have the capability of ROS scavenging and enhancing cell survival. Different medicinal plants are known to have this property and can be a good option to mitigate the pesticide toxicity (Ahmed et al., 2008; Anilakumar et al., 2009; Umamaheshwari & Chatterjee, 2009; Venkatesan et al., 2003).

The exposure to toxicants is increasing in the modern era and damaging the life forms at various levels. Many plants and their parts are known to protect against various infections and health hazards (Keshav et al., 2021; Goyal et al., 2021). These play a key role in the scavenging ROS and free radicals due to their antioxidants properties. One such important remedy is ginger (Zingiber officinale) which is used by humans in their diet, especially in Asian countries. It is reported to have anti-inflammatory, antioxidants, anticancer, and analgesic properties (Zhang et al., 2022). In this study, we assessed the mitigating potential of aqueous ginger against the neurotoxicity caused by dichlorvos in Wistar rats.

Methods

Animals

Laboratory rats of both the sexes were procured from the animal house of Bundelkhand University. After procurement, Wistar rats (Rattus norvegicus) were caged in the departmental animal house of the university and kept under benchmark laboratory norms (12 h each of dark and light cycles and temperature 25 ± 2 ℃). Animals were allowed to acclimatize before the commencement of experiments for one week. The guidelines of the Institutional Animal Ethics Committee (IAEC) were followed throughout the experiments.

Preparation of aqueous ginger

The ginger was peeled & chopped in pieces & grounded in the mixer grinder to get juice of the ginger. The particulate matter was removed by filtration through Whatman filter paper and centrifuged at 2000 rpm for 15 min. After that, the filtrate was lyophilized to obtain a completely dried powder of ginger. The dried powder was stored in air tight bottles. This dried powder of ginger was reconstituted for the dose preparation as per the requirement (100 and 200 mg/kg body weight). A stock solution of 400 mg/mL was prepared by suspending the dry powder in the water and diluted accordingly.

Treatment schedule

The experiments were performed on Wistar rats of 150–200 g. A total of 48 rats (24 males and 24 females) were grouped for the experiments keeping 6 animals each of both the sexes in each group as follows: Group 1—control; Group 2—Dichlorvos (8.8 mg/kg for 2 weeks); Group 3—Dichlorvos (8.8 mg/kg for 2 weeks) + aqueous ginger (100 mg/kg for next 2 weeks); and Group 4—Dichlorvos (8.8 mg/kg for 2 weeks) + aqueous ginger (200 mg/kg for next 2 weeks). After completion of the dosing schedule, animals were killed to get the brain samples. These were stored at − 40 °C after washing in 0.9% saline solution till further use (Ajao et al., 2017; Ramudu et al., 2011).

Homogenate of tissues

Ice-cold 0.1 M PBS (pH 7.4) was used to prepare 10% (w/v) crude homogenate of the brain samples. The part of fresh crude homogenate was utilized for the estimation of reduced glutathione and lipid peroxidation. All the rest homogenates were processed to get the S9 fraction by centrifugation for 20 min at 9000 rpm for the analysis of all other parameters. After centrifugation, the supernatant (S9 fraction) was collected and stored for further analysis at − 80 ℃ (Reddy et al., 2013).

Non-enzymatic estimations

Lipid peroxidation (LPO)

The TBARS (thiobarbituric acid reactive substance) was employed for the analysis of lipid peroxidation (LPO) by measuring its end product, malondialdehyde (MDA). Crude homogenate (1 mL) was kept at 37 °C. After 10 min of incubation, chilled trichloroacetic acid (1 mL of 10% w/v) was added to it and spun at room temperature for 15 min at 2500 rpm. 1 mL of this supernatant was mixed with TBA (1 mL of 0.67%) and incubated for 15 min in a boiling water bath. The tubes were allowed to cool under the running tap. The absorbance was read at 530 nm after adding 1 mL distilled water (Ohkawa et al., 1979). The results are represented as nmoles MDA/h/g tissue.

Reduced glutathione (GSH)

1 mL of fresh crude homogenate was mixed with 5% TCA (1 mL), and after 30 min, the mixture was spun for 15 min at 2500 rpm. DTNB (5′5′-dithionitrobenzoic acid) was added to 0.5 mL supernatant. The absorbance was taken at 412 nm after thorough shaking. The results are represented as µmol/g tissue (Ellman, 1959).

Enzymatic estimations

Catalase (CAT)

S9 fraction (0.1 mL) was mixed with phosphate buffer (1 mL) and distilled water (0.4 mL). H₂O₂ (0.5 mL) was added to start the reaction and left for 1 min at 37 °C. Dichromate–acetic acid reagent (2 mL) was added to stop the reaction and incubated for 15 min in a boiling water bath. After cooling, the absorbance was taken at 570 nm (Sinha, 1972). The results are expressed as µmol/min/mg protein.

Superoxide dismutase (SOD)

S9 fraction (0.1 mL) was mixed with 0.25 mL of ice-cold chloroform and ice-cold ethanol (0.15 mL). The mixture was mixed by shaking and spinning at 4 °C for 10 min at 3000 rpm. A mixture of carbonate and bicarbonate buffer (1.3 mL), EDTA (0.5 mL), and distilled water (0.8 mL) were mixed with 0.2 mL of supernatant. 0.2 mL of epinephrine was added to start the reaction. ΔOD/ min was recorded for 3 min at 480 nm (Misra & Fridovich, 1972). The results are represented as nmol/min/mg protein.

Glutathione peroxidase (GPx)

A mixture of Tris–HCl buffer (0.4 mL), GSH (0.2 mL), sodium azide (0.1 mL), H₂O₂ (0.1 mL), and distilled water (0.1 mL) was added to 0.1 mL of S9 fraction. This mixture was kept at 37 °C, and after 15 min, TCA (0.5 mL) was added to it. The mixture was spun and the supernatant was separated. Disodium hydrogen phosphate (2 mL), Ellman’s reagent (0.5 mL), and supernatant (0.5 mL) were mixed and read at 420 nm (Rotruck et al., 1972). The results are represented as µmol/min/mg protein.

Glutathione reductase (GR)

0.1 mL of S9 fraction was mixed with phosphate buffer (2.5 mL), NADPH (0.2 mL), and GSSG (0.2 mL). The mixture was left for 30 s. The reading for absorbance was taken at an interval of 30 s for 3 min at 340 nm (Carlberg & Mannervik, 1985). The results are expressed as nmol/min/mg protein.

Statistical analysis

One-way ANOVA was applied to data with post hoc test. The results were expressed as mean ± SEM (n = 6). The p values less than 0.05 were considered significant.

Results

LPO

The DDVP exposure increased the MDA level significantly (p < 0.05) in the brain of male and female rats in comparison with control animals. The animals post-treated with both the doses of ZO were reported to have declined MDA levels than untreated and only exposed animals of both the sexes. The decline in LPO level was found to be significant (p < 0.05) after treatment with ZO (200 mg/kg) in contrast to only exposed animals (Fig. 1).

Fig. 1

Lipid peroxidation (LPO). Levels of LPO in male and female rats (n = 6) exposed to dichlorvos (DDVP) and post-treated with Zingiber officianle (ZO). *p value (< 0.05); #p value (< 0.05)

GSH

The levels of non-enzymatic antioxidant (GSH) were scaled and found to be the least (p < 0.05) in the animals of the group exposed to DDVP when compared with the control group. The treatment of DDVP exposed animals with ZO led to increased levels of GSH in animals of both sexes, but the higher dose of ZO resulted in a significant (p < 0.05) increment in GSH levels (Fig. 2).

Fig. 2

Reduced glutathione (GSH). Levels of GSH in male and female rats (n = 6) exposed to dichlorvos (DDVP) and post-treated with Zingiber officianle (ZO). *p value (< 0.05); #p value (< 0.05)

Levels of enzymatic antioxidants

Different enzymatic antioxidants were also assessed for their levels in the brain of male and female animals exposed to DDVP. Moreover, the efficacy of ZO was evaluated by assessing two doses as post-treatment after DDVP exposure.

CAT

The catalase activity was found to be inhibited upon DDVP exposure in rats of both the sexes and was less (p < 0.05) in contrast to the control group of animals. The treatment of animals with ginger (ZO) resulted in enhanced (p < 0.05) levels of catalase in animals at both doses, i.e. 100 and 200 mg/kg, in male and female animals when compared with the untreated and DDVP exposed animals (Fig. 3).

Fig. 3

Catalase (CAT). Levels of CAT in male and female rats (n = 6) exposed to dichlorvos (DDVP) and post-treated with Zingiber officianle (ZO). *p value (< 0.05); #p value (< 0.05)

SOD

A similar pattern of observations was recorded for SOD in male and female animals after exposure to DDVP and treatment with ginger (ZO). The least (p < 0.05) levels of SOD were found in DDVP exposed animals in comparison with control animals which were recovered after treatment with both the doses of ZO in both the sexes of animals (Fig. 4).

Fig. 4

Superoxide dismutase (SOD). Levels of SOD in male and female rats (n = 6) exposed to dichlorvos (DDVP) and post-treated with Zingiber officianle (ZO). *p value (< 0.05); #p value (< 0.05)

GPx

The levels of glutathione peroxidase (GPx) were also decreased in animals after exposure to DDVP and the significance (p < 0.05) was observed. The lower dose (100 mg/kg) of ginger led to an increase (p < 0.05) in the level of GPx which was further enhanced by the higher dose (200 mg/kg) in both the sexes of animals (Fig. 5).

Fig. 5

Glutathione peroxidase (GPx). Levels of GPx in male and female rats (n = 6) exposed to dichlorvos (DDVP) and post-treated with Zingiber officianle (ZO). *p value (< 0.05); #p value (< 0.05)

GR

Glutathione reductase (GR) level was also enhanced by the ginger treatment to DDVP exposed animals at the lower dose but a significant (p < 0.05) difference was observed at the higher dose. The least (p < 0.05) level of GR was reported in only exposed animals in contrast to the control group (Fig. 6).

Fig. 6

Glutathione reductase (GR). Levels of GR in male and female rats (n = 6) exposed to dichlorvos (DDVP) and post-treated with Zingiber officianle (ZO). *p value (< 0.05); #p value (< 0.05)

Discussion

Different toxicants are polluting our environment by their release from various sources, which, in turn, reach the biological systems and posing threat to living beings. The toxic evaluation of DDVP on the brain was done in this study on Wistar rats and was followed by the post-treatment with Zingiber officinale (ZO) at two doses. Furthermore, we assessed the sex-specific susceptibility towards the tested toxicant by performing the experiment on male and female animals. However, we did not observe any significant difference in between both the sexes.

It has been noted that neurological functions are altered in response to pesticide-induced toxicity. Exposure to organophosphates (chronic and subchronic) results into oxidative stress that has been reported to be the key mechanism behind their toxicity. Acute and chronic exposure to organophosphates led to the oxidative stress in different organs disturbance in the levels of non-enzymatic and enzymatic antioxidants. Organophosphate poisoning is also associated with the pathophysiology of neurodegenerative diseases like Parkinson’s disease. Pesticide mediates the declined level of glutathione which is marker of progression of Parkinson’s disease. Brain has high oxygen demand and uses around 20% while constitute only 2% of the entire body weight. Higher levels of ROS are produced in brain and detoxification mediated by glutathione is essential (Abdollahi et al., 2004; Franco et al., 2010; Lukaszewicz-Hussain, 2010; Matés et al., 1999; Petrovitch et al., 2002; Ralf, 2000; Ranjbar et al., 2005). Organophosphate intoxication increases the burden of oxidative stress and creates an imbalance in antioxidant defence system that leads to the toxicity in the brain. Mechanism of organophosphate intoxication has been suggested through the generation of oxidative stress and thereby linked with the neurodisorders.

Other mechanism suggested for the poisoning of organophosphates is through the imbalance in the level of acetylcholinesterase (AChE). The intoxication to these pesticides causes the accumulation of acetylcholine due to the inhibition of AChE that result in the calcium overload and enhanced ROS. This in turn dysregulates the mitochondrial functioning and mediates the ROS production and impaired antioxidant system, and bioenergetics, and ultimately results in the neuronal injury and gliosis (Pearson and Patel, 2016).

Comparatively lower levels of antioxidants and higher levels of polyunsaturated fatty acids (PUFA) make the brain a vulnerable target for oxidative stress, especially for DDVP which is lipophilic in nature. The results of this study depicted the highest and lowest levels of LPO and GSH, respectively, after DDVP exposure to experimental animals. Our findings are in line with the study by Sahoo et al. (2000) where they have observed neurotoxicity in rats after lindane exposure. The reduced GSH level was observed in experimental animals in this study and the results are in corroboration with the study by Dwivedi et al. (2010) that depicted a decrease in GSH level in the brain after exposure to monocrotophos and dichlorvos.

Enzymatic antioxidants (SOD and CAT) are found to be declined in rat brains in this study after DDVP exposure. The levels of GPx and GR were found to decline in this study and this may be due to decreased level of GSH due to DDVP exposure. The pesticides (methoxychlor, lindane and chlorpyrifos) have been reported to decline such enzymatic antioxidants in rats upon exposure (Latchoumycandane & Mathur, 2002; Sahoo et al., 2000; Verma & Srivastava, 2003).

The accumulation of pesticides in tissues abundant in lipid content may lead to damage to the cell membrane, which in turn leads to the death of the cells due to the enhanced ROS generation. Different herbal compounds and plants have been assessed for their potential as antioxidants against pesticide and metal toxicity in different animal models. Plants like Ipomoea batatas, Pennisetum glaucam, Sorghum bicolour, Alchomea cordifolia, and Adansonia digitata have been shown to exert protection against oxidative stress in murine model (Gabriel & Idu, 2021; Idu et al., 2022; Makena et al., 2021). Zingiber officinale (ZO) is culinary and used in most societies in cooking. In this study, ZO was evaluated for its antioxidative property against DDVP toxicity in the rat model. The results of this study showed the protective role of ZO against DDVP-induced oxidative stress in rat brains. The exposure of DDVP to rats and post-treatment with ZO at two doses showed its protective nature by mitigating the pesticide-induced toxicity and the higher dose (200 mg/kg) was more effective in comparison with the lower dose (100 mg/kg). The increased levels of GSH with a decline in LPO were observed after treatment with ZO. The other parameters like SOD, CAT, GPx, and GR were also up-regulated in the treated rats. The antioxidative role of ginger has also been evaluated in rats exposed to cadmium (Gabr et al., 2019). The high phenol content in ginger can be helpful for its antioxidant properties (Ezez & Tefera, 2021). The antioxidant potential of ginger has also been studied against cancer chemotherapy (Danwilai et al., 2017). Ginger has also been used as radioprotective against neutron-induced oxidative stress in rats (Nabil et al., 2009). The similar results were obtained in a study on bisphenol A toxicity on testis of rats. The protection was provided by the root extract of Costus through the reduction in oxidative stress induced by bisphenol A (Osman et al., 2022).

Conclusions

Results of this study showed that Zingiber officinale (post-treatment) mitigates the toxicity in rat brains after exposure to DDVP. The protection was provided by ZO as it improves the antioxidant defence mechanism of the rats exposed to DDVP. Humans and other living beings are unintentionally exposed to different organophosphate pesticides in many ways due to the presence of these persistent compounds in environment. Therefore, daily dietary ginger can help mitigate the pesticide-induced toxicity. This study emphasized the role of ginger in amelioration of organophosphate poising in the brain of the rats. However, more studies are required to perform in this direction to get the exact mechanism of action and other parameters for better understanding.