Evaluating toxicity and level of DNA damage in human fetal lung cells upon exposure to 5-methyltetrahydrofolate (bioactive folate)

<b>Background:</b> Folic acid (FA) supplementation is widely regarded as a key nutritional intervention during pregnancy due to its protective effect against neural tube defects. Recent research has reported FA supplementation outcomes on offspring's health, with increased incidences of allergy/respiratory problems. <b>Aim:</b> This study evaluates if increased levels of 5-methyltetrahydrofolate (5-MTHF) are associated with DNA modification, leading to disruption of cell proliferation in fetal lung cells and increasing susceptibility to asthma. <b>Methods:</b> Two fetal lung cells, MRC5 and IMR90, were treated with nine concentrations of 5-MTHF for six time points. Cell viability was evaluated using Trypan Blue staining. Flow cytometry analysis to quantify DNA content in cells was done with a propidium iodide stain. Followed by 1.6 mM glutathione treatment to alleviate the oxidative stress caused by 5-MTHF. A quantitative test for DNA damage was executed using neutral and alkaline comet assay. Gene expression study for five genes namely MTR, MTHFD1, XRCC1, Pol β, and epidermal growth factor receptor (EGFR) was evaluated using a 2-step quantitative reverse transcription polymerase chain reaction. <b>Results:</b> Fetal lung cell survival rate remained unaffected with 5-MTHF concentration below 1.25 µM. Beyond this concentration, cell viability is reduced with an increase in concentration. Cell cycle analysis revealed cell arrest in the G₁ phase. The antioxidant activity of glutathione led the cells to bypass this arrest. Precisely, 10 and 50 µM 5-MTHF concentrations led to double-strand DNA breaks and single-strand DNA breaks. Gene expression study revealed lower expression of the MTR gene and higher expression of MTHFD1, EGFR, XRCC1, and DNA Pol β gene with an increase in 5-MTHF concentration. <b>Conclusion:</b> 5-MTHF concentration higher than 1.25 µM led to DNA damage in MRC5 and IMR90 human fetal lung cells.