The effect of methylene blue on oxidative stress and neuronal injury in rats following acute malathion intoxication was examined. Rats were intraperitoneally injected with malathion at 150 mg/kg body weight along with methylene blue at 5 or 10 mg/kg body weight, and euthanized 4 hr later. The levels of lipid peroxidation [malondialdehyde (MDA)], nitric oxide, and reduced glutathione (GSH), and the activities of paraoxonase 1 (PON1), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) were measured in the brain tissue. Histopathological examination and glial fibrillary acidic protein (GFAP) immunostaining were also carried out in this study. Malathion induced a significant elevation in brain lipid peroxidation (MDA) by 32.8% associated with an increased nitric oxide level by 51.4%. Following malathion exposure, brain GSH level fell by 67.7%, and brain AChE and BChE activities decreased by 25% and 60.4%, respectively. Malathion exposure also inhibited PON1 activity by 39.6% and decreased brain glucose level by 30%. Neuronal degeneration in the cortex and hippocampus and strong GFAP immunostaining in the hippocampus were observed in malathion-exposed animals. Methylene blue co-treatment at 10 mg/kg body weight decreased brain MDA by 17.8%, and at 5 and 10 mg/kg body weight, decreased nitric oxide level by 29.2% and 35.8%, respectively. There was no significant effect on the GSH level, but PON1 activity was increased by 22.9%-30.9% upon methylene blue co-treatment. BChE activity did not change but that of AChE increased after cotreatment with the lower dose of methylene blue. Rats receiving methylene blue co-treatment at 10 mg/kg body weight showed no degenerating neurons in the cortex, and occasional degenerating neurons and weak GFAP immunostaining in the hippocampus. Taken together, our results suggest that methylene blue is neuroprotective in acute malathion intoxication, and the neuroprotective effect is likely due to inhibition of oxidative stress and decreased glial cell activation.

We examined the effect of treatment with neostigmine alone or with atropine on brain oxidative stress and on brain and liver tissue damage following acute malathion toxicity. Rats were intraperitoneally treated with malathion 150 mg/kg along with neostigmine (200 or 400 μg/kg) or neostigmine (200 μg/kg) + atropine (1 mg/kg) and euthanized 4 h later. Results indicated that compared with the saline group, malathion resulted in (i) higher brain malondialdehyde (MDA) and nitric oxide (46.4% and 86.2%); (ii) decreased brain reduced glutathione (GSH) (67.6%); (iii) decreased brain paraoxonase-1 (PON1), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities (31.2%, 21.6% and 60%); (iv) decreased brain glucose (-38.1%); (v) neuronal degeneration in cortex and hippocampus and markedly increased glial fibrillary acidic protein (GFAP) immunostaining in the hippocampus; (v) hydropic and fatty degeneration in liver. Rats treated with malathion along with neostigmine or neostigmine + atropine showed no change in brain MDA but decreased nitric oxide (-34.2%-48%). GSH increased after neostigmine 200 μg/kg or neostigmine + atropine (35.8% and 41%). PON1 activity increased (42%-35.2%) and glucose concentrations increased (91.5%-81.5%) by 400 μg/kg neostigmine or neostigmine + atropine. Brain AChE activity remained unchanged but BChE activity showed 18.3% increment after 400 μg/kg neostigmine. Rats treated with 400 μg/kg neostigmine or neostigmine + atropine had normal neuronal appearance in cortex and hippocampus and weak GFAP expression in hippocampus. Liver damage was prevented by neostigmine + atropine. These results suggest that treatment with neostigmine + atropine afforded protection against the deleterious effects of acute malathion on the brain and liver.