The Val16Ala-SOD2 polymorphism affects cyto-genotoxicity of pyridostigmine bromide on human peripheral blood mononuclear cells.

Pyridostigmine bromide (PB), an acetylcholinesterase (AChE) enzyme inhibitor. Experimental evidence showed that when combined with other drugs or exercise, PB caused extensive neural and/or systemic oxidative stress. However, no studies have been conducted on the genetic influence associated with basal oxidative superoxide-hydrogen peroxide (S-HP) imbalance, such as that triggered by Val16Ala-SOD2 single nucleotide polymorphism (SNP, rs4880). This SNP, (homozygous genotypes) has been associated with several chronic degenerative disorders. Therefore, we evaluated whether the SOD-SNP could alter cyto-genotoxic effects triggered by different PB-concentrations in peripheral blood mononuclear cells (PBMCs). PBMCs were obtained from volunteers carrying different SOD2-genotypes and were cultured with various concentrations of PB. PB effects in quantity of enzyme AChE, mortality rate, oxidative stress markers, and DNA damage were assessed. Protein and gene expression of antioxidant enzymes, apoptotic markers and DNA repair enzyme, were evaluated in 24 h cultures. In general, PB up-regulated expression of antioxidant enzymes, and did not trigger apoptotic events. However, AA-PBMCs seemed more sensitive to PB exposure, in a protein decrease of the enzyme AChE by 10%, cell-mortality at concentrations of 20 and 40 ng/mL, protein carbonylation, and DNA damage, as analyzed by the Comet assay. Contrarily, PB demonstrated cyto-genoprotective effects on V-allele cells. These results indicated that genetic factors that increase HP-release may affect PB efficiency and safety.

Effects of Pyridostigmine bromide on SH-SY5Y cells: An in vitro neuroblastoma neurotoxicity model.

Pyridostigmine bromide (PB) is a reversible acetylcholinesterase (AChE) inhibitor and the first-choice for the treatment of symptoms associated with myasthenia gravis and other neuromuscular junction disorders. However, evidence suggested that PB could be associated with the Gulf War Illness characterised by the presence of fatigue, headaches, cognitive dysfunction, and musculoskeletal respiratory and gastrointestinal disturbances. Given that a potential neurotoxic effect of PB has not yet been completely elucidated, the present investigation used neural SH-SY5Y cells to evaluate the effect of PB on the cellular viability, cell apoptosis, modulation of the cell cycle, oxidative stress, and genotoxicity variables, which indicate neurodegeneration. As expected, a PB concentration curve based on the therapeutic dose of the drug showed an inhibition of the AChE activity. However, this effect was transient and did not involve differential AChE gene regulation by PB. These results confirmed that undifferentiated SH-SY5Y cells can be used as a cholinergic in vitro model. In general, PB did not trigger oxidative stress, and at a slightly higher PB concentration (80ng/mL), higher levels of protein carbonylation and DNA damage were detected, as determined by the marker 8-deoxyguanosine. The PB genotoxic effects at 80ng/mL were confirmed by the upregulation of the p53 and DNA methyltransferase 1 (DNMT1) genes, which are associated with cellular DNA repair. PB at 40ng/mL, which is the minimal therapeutic dose, led to higher cell proliferation and mitochondrial activity compared with the control group. The effects of PB were corroborated by the upregulation of the telomerase gene. In summary, despite the methodological constrains related to the in vitro protocols, our results suggested that exposure of neural cells to PB, without other chemical and physical stressors did not cause extensive toxicity or indicate any neurodegeneration patterns.