Methotrexate-Related Response on Human Peripheral Blood Mononuclear Cells May Be Modulated by the Ala16Val-SOD2 Gene Polymorphism

Abstract

Methotrexate (MTX) is a folic acid antagonist used in high doses as an anti-cancer treatment and in low doses for the treatment of some autoimmune diseases. MTX use has been linked to oxidative imbalance, which may cause multi-organ toxicities that can be attenuated by antioxidant supplementation. Despite the oxidative effect of MTX, the influence of antioxidant gene polymorphisms on MTX toxicity is not well studied. Therefore, we analyzed here whether a genetic imbalance of the manganese-dependent superoxide dismutase (SOD2) gene could have some impact on the MTX cytotoxic response. An in vitro study using human peripheral blood mononuclear cells (PBMCs) obtained from carriers with different Ala16Val-SOD2 genotypes (AA, VV and AV) was carried out, and the effect on cell viability and proliferation was analyzed, as well as the effect on oxidative, inflammatory and apoptotic markers. AA-PBMCs that present higher SOD2 efficiencies were more resistance to high MTX doses (10 and 100 µM) than were the VV and AV genotypes. Both lipoperoxidation and ROS levels increased significantly in PBMCs exposed to MTX independent of Ala16Val-SOD2 genotypes, whereas increased protein carbonylation was observed only in PBMCs from V allele carriers. The AA-PBMCs exposed to MTX showed decreasing SOD2 activity, but a concomitant up regulation of the SOD2 gene was observed. A significant increase in glutathione peroxidase (GPX) levels was observed in all PBMCs exposed to MTX. However, this effect was more intense in AA-PBMCs. Caspase-8 and -3 levels were increased in cells exposed to MTX, but the modulation of these genes, as well as that of the Bax and Bcl-2 genes involved in the apoptosis pathway, presented a modulation that was dependent on the SOD2 genotype. MTX at a concentration of 10 µM also increased inflammatory cytokines (IL-1β, IL-6, TNFα and Igγ) and decreased the level of IL-10 anti-inflammatory cytokine, independent of SOD2 genetic background. The results suggest that potential pharmacogenetic effect on the cytotoxic response to MTX due differential redox status of cells carriers different SOD2 genotypes.

Haloperidol and Risperidone at high concentrations activate an in vitro inflammatory response of RAW 264.7 macrophage cells by induction of apoptosis and modification of cytokine levels

“…The haloperidol and risperidone effect on some Bcl-2 and BAX genes that are related to intrinsic route of apoptosis triggering was measured by QT-PCR, as previously described (Barbisan et al 2014). Total RNA was isolated using TRIzol reagent and RNA yields were measured using a Nanodrop 2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).…”

The antioxidant effect of Brazil nut (Bertholletia excelsa) is influenced by a genetic superoxide-hydrogen peroxide imbalance in healthy humans

“…It is important to point out that some volunteers participated in prior studies by donating blood samples for in vitro protocols. [16][17][18] These subjects were previously SOD2genotyped by the protocol described elsewhere. 18 Briefly, genomic DNA was isolated from peripheral-blood leukocytes using the DNA Mini Kit Purification (Mo Bio).…”

“…[16][17][18] These subjects were previously SOD2genotyped by the protocol described elsewhere. 18 Briefly, genomic DNA was isolated from peripheral-blood leukocytes using the DNA Mini Kit Purification (Mo Bio). SOD polymorphism was detected by PCR with restriction fragment length polymorphism (RFLP) analysis.…”

“…ROS production was determined using a nonfluorescent cellpermeating compound called 2-7-dichlorofluoresceindiacetate (DCFDA). 18 In this assay, DCFDA is hydrolyzed by intracellular esterases to DCFH, which is trapped within the cell. This nonfluorescent molecule is then oxidized to fluorescent dichlorofluorescein (DCF) by cellular oxidative factors.…”

Association between Polymorphisms in Antioxidant Genes and Inflammatory Bowel Disease

“…Diseases such as diabetic nephropathy, cardiomyopathy, Behcet’s disease, and various cancers have already been associated with polymorphisms in antioxidant genes [16]. Genetic polymorphisms in SOD2 have also been referred [17] as a potential mechanism interfering in the pharmaco-response of cells exposed to methotrexate (MTX), an immunomodulator commonly prescribed to IBD patients, and an interdependent mechanism of action between SOD2 and GPX1 has been suggested, indicating that these genes could also have an important role for therapeutic management.…”