Mitochondrial dysfunction and oxidative stress in liver of male albino rats after exposing to sub-chronic intoxication of chlorpyrifos, cypermethrin, and imidacloprid

Highlights

• Mitochondrial bioenergetics and oxidative stress biomarkers were tested in rat liver after exposure to insecticides.

• The insecticides caused a significant decrease of NADH dehydrogenase, ATPase, SOD, and GST.

• Levels of malondialdehyde and protein carbonyl content were significantly increased.

• A significant increase of 8-OH-2DG as a biomarker of the DNA damage was found.

Abstract

The adverse effects of chlorpyrifos, cypermethrin, and imidacloprid on mitochondrial dysfunction and oxidative stress biomarkers were studied in rat liver. The liver deficiency was also confirmed by histological analysis and gel electrophoresis. Each insecticide was administered orally with five doses per week for 28 days to male albino rats at 1/50 of the LD₅₀ per insecticide. The results demonstrated that the mitochondrial dysfunction was confirmed by a significant decrease in NADH dehydrogenase and ATPase activities. Oxidative stress biomarkers include malondialdehyde (MDA), and protein carbonyl content (PCC) were significantly increased. However, superoxide dismutase (SOD) and glutathione S-transferase (GST) as antioxidant enzymes were significantly decreased in the mitochondria of the rat liver. HPLC analysis showed a significant increase of the 8-hydroxy-2′-deoxyguanosine (8-OH-2DG) as a biomarker of the DNA damage in rat liver. In addition, the residue levels of 0.96 and 0.29 μg/mL serum were found for cypermethrin and imidacloprid, respectively. However, chlorpyrifos not detected using the HPLC analysis. Blue native polyacrylamide gel electrophoresis (BN-PAGE) analysis showed a change in the pattern and sequence of complexions of the electron transport chain in liver mitochondria with treatment by such insecticides. The hepatic histological examination also showed symptoms of abnormalities after exposure to these insecticides.

Introduction

The liver is the main detoxifying organ and the primary involved in xenobiotic metabolism in the mammalian body. It is one of the richest organs in terms of the number and density of mitochondria (Chinnery and DiMauro, 2005). In physiological conditions, mitochondria have significant roles in the metabolism and energy regulation as a source of reactive oxygen species (ROS) that generating approximately 85% of the total cellular superoxide radical (O₂•-) (Samarghandian et al., 2016). Mitochondrial impairment can induce oxidative stress and reduce adenosine triphosphate (ATP) content (Samarghandian et al., 2015). The oxidative attack on the mitochondria is also essential in these events. Under conditions of mitochondrial dysfunction, ATP deficiency can lead to failure of the Na⁺/K⁺ ATPase, and in primary sensory neurons, this may contribute to the ectopic activity characteristic of neuropathic pain (Lim et al., 2015).

The oxidative stress of proteins, lipids, DNA, and antioxidant enzymes can be seriously harmful. Protein carbonyl content (PCC) is the most commonly used biomarker of protein oxidation (Sharma et al., 2021). Accumulation of PCC has been observed in several human diseases, including Alzheimer's disease, diabetes, inflammatory bowel disease, and arthritis (Sharma et al., 2020). Indeed, detection of elevated levels of PCC is generally a sign of oxidative stress and protein dysfunction. Lipid peroxidation (LPO) is caused by free radicals that lead to oxidative destruction of polyunsaturated fatty acids, resulting in toxic and reactive aldehyde metabolites such as malondialdehyde (MDA) (Yaman and Ayhanci, 2021). MDA causes mitochondrial dysfunction by enhancing reactive oxygen species (ROS) generation and modulation of mitochondrial proteins (Caldiroli et al., 2020). DNA oxidative stress generates hydroxyl radicals that cause a wide range of DNA lesions, including base alterations, deletions, tuft fractures, and chromosomal rearrangements. A high level of 8-hydroxy-2′-deoxyguanosine (8-OH-2DG) as a biomarker is associated with oxidative DNA damage. 8-OH-2DG is an oxidized derivative of deoxyguanosine. It is one of the major products of DNA oxidation (Hinch et al., 2013). Intracellular concentrations of the 8-OH-2DG are a measure of oxidative stress. Increased levels of 8-OH-2DG in the liver DNA of pesticide-treated rats indicate the generation of ROS capable of causing genetic damage to cells based on the formation of 8-OH-2DG and then causing an abnormality in the DNA that may lead to a mutation or cancer (AbuArrah et al., 2021).

As with the chemical antioxidants, cells are protected from oxidative stress through an interacting network of antioxidant enzymes. Mitochondria produce superoxide radical (O₂•-), hydrogen peroxide (H₂O₂), and hydroxyl radical (OH^•) as byproducts of molecular oxygen consumption in the electron transport chain (Barbosa et al., 2020). The accumulated O₂^• is eliminated by manganese superoxide dismutase (MnSOD), which generates H₂O₂. The mitochondrial glutathione system plays a crucial role in reducing H₂O₂ and protects mitochondria against oxidative stress.

Various pesticides can cause impaired energy regulation and cell dysfunction, and finally, cell death has been observed in many neurological disorders (De Castro et al., 2011). Impairment of mitochondrial function is the primary mechanism involved in the pathogenesis of neurological disorders (Samarghandian et al., 2016). Pesticides are rapidly metabolized in the liver by hydrolytic ester cleavage, and oxidative pathways by cytochrome P450 enzymes produce ROS (He et al., 2020). Chlorpyrifos is a non-systemic chlorinated organophosphate insecticide designed to be effective by direct contact, ingestion, and inhalation. It elicits broad-spectrum insecticidal activity against several important arthropod pests (Foong et al., 2020). Which, in turn, chlorpyrifos is responsible for toxicity to mammals by inhibiting the enzyme cholinesterase. Cypermethrin is a class II synthetic pyrethroid used as an insecticide in agricultural applications and consumer products for household purposes. It causes a prolonged opening of the central nervous system's sodium channels, resulting in hyper-depolarization and over-excitation of the nerve cells (Narahashi et al., 1992). It also induces neurotoxicity by modulating the level of gamma-aminobutyric acid (GABA) (Manna et al., 2005). Imidacloprid is a potent neonicotinoid neurotoxic insecticide that belongs to a nitroguanidine active group, which acts as agonists on the nicotinic acetylcholine receptors. Imidacloprid is characterized by high selective insecticidal activity at a low application rate, low soil persistence, and apparent safety in humans (Tomizawa and Casida, 2005).

Therefore, the present study was conducted to evaluate and compare the mitochondrial dysfunction and oxidative stress after exposure to subchronic intoxication by three insecticides (chlorpyrifos, cypermethrin, and imidacloprid) representing different chemical groups in liver mitochondrial of male albino rats. Bioenergetics' biomarkers include NADH dehydrogenase and ATPase, were determined. Oxidative stress biomarkers (PCC, MDA, and 8-OH-2DG) and antioxidant enzymes (SOD and GST) were measured. 8-OH-2DG levels as a biomarker of the DNA damage were measured by HPLC analysis in rat liver. HPLC also determined the residue levels of the tested insecticides in rat serum. In addition, histological analysis and BN-PAGE electrophoresis analysis were studied in detail.