Global DNA Methylation Of Brain Neurons In Acute Poisoning With Clozapine And Its Combination With Alcohol: An Experimental Study

Abstract

Background — Acute poisoning with atypical neuroleptic clozapine is characterized by rapid progression, high risk of death and severe neurological manifestations. Neurotoxic effects of this pharmaceutical drug have also been reported at therapeutic doses. The pathogenesis of brain damage in acute clozapine poisoning is not fully understood. Changes in DNA methylation level may play an important role in the mechanisms of drug neurotoxicity. The available data on the effect of clozapine on brain cell DNA provide a rationale for studying the epigenetic aspects of the pathogenesis of acute poisoning with this neuroleptic agent. The objective of our study was to evaluate the global DNA methylation level in rat brain neurons in acute poisoning with clozapine and its combination with ethanol. Material and methods — Clozapine – 150 mg/kg in 2.0 ml of normal saline solution, or clozapine – 150 mg/kg in 2.0 ml of 40% ethanol were administered via a gastric tube to adult male Wistar rats (n=21) under anesthesia with sevoflurane. In the control group, saline was administered via a gastric tube. Animals were euthanized four hours after drug administration. Autopsy was performed with the collection of brain samples for histochemical examination and determination of the DNA methylation level using the fluorometric method. To detect DNA in sections of paraffin-embedded tissue, we used the Feulgen staining. The TUNEL method was employed to detect DNA fragmentation. Results — An increase in the level of global DNA methylation in brain neurons was found in the clozapine and clozapine+ethanol groups. The average level of methylated DNA in the clozapine+ethanol group was higher than in the control group or clozapine group (2.56±0.31 vs. 1.35±0.1, p=0.007 and 1.70±0.33, p=0.044, respectively). An increase in the mean optical density of the cortical neuron nuclei was observed in the clozapine+ethanol group compared with the control group and clozapine group. DNA fragmentation was not detected in any experimental group. Conclusion — Acute poisoning with clozapine in combination with alcohol caused an increase in the global DNA methylation level in brain neurons, which may have played a significant role in the pathogenesis of acute clozapine poisoning and could be an important factor in the neurotoxicity of this medication.