Synthetic Dye Yellow No.5 (Tartrazine) Short-Term Exposure Lowers Sod1 mRNA Expression in Mouse Neuro2A Neurons in Vitro

Yellow Dye No. 5, also known as tartrazine (TRZ), is widely used (Matsyura et al., 2020) and has an accepted daily intake (ADI) of 0-7.5 mg/kg of body weight per day (JEFCA, 2017). Consuming TRZ dosages greater than the ADI can lead to reduced levels of brain antioxidant enzymes, chromosomal alterations, or decreased neuronal dendritic lengths after 90 days (Mpountoukas et al., 2010, Abd-Elhakim et al., 2019) which can result in oxidative stress, impaired neuronal functioning, and potential mutagenic effects. Within the ADI, there have been observed reductions in the copper-zinc superoxide dismutase-1 (SOD1) enzyme level in conjunction with TRZ treatment (Albasher et al., 2020). TRZ can bind to DNA within the minor groove, which may induce changes in gene expression (Kashanian and Zeidali, 2011). Therefore, we hypothesize that TRZ interacts pre-translationally inside the cell, resulting in a regulatory reduction of Sod1 mRNA levels and decreasing neuronal outgrowth. In this study, differentiated Neuro2A-derived neurons were exposed to TRZ for 3 or 7 days, with a concentration curve from 0 to 11 μg/mL. Treated cells were grown on poly-L-lysine (PLL)- and laminin-coated glass coverslips, immunostained with anti-β-tubulin III, and phalloidin, imaged, and analyzed using NeuronJ/ImageJ (NIH). Sod1 mRNA was quantified using reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Our results indicated that TRZ caused an acute increase in neurite length after 7 days of TRZ exposure and reduced Sod1 mRNA expression within the ADI at 3 and 7 days, but there was a significant reduction in the mRNA expression above the ADI at 7 days exposure. The reduction in Sod1 mRNA expression could indicate possible pre-translational modifications, which could be explained by TRZ’s ability to bind DNA which may cause regulatory alterations. These findings help fill the gap in understanding the mechanism of SOD1 downregulation due to TRZ exposure in short-term conditions.

Abbreviations: TRZ—Tartrazine; ADI—Accepted daily intake; DIV—days in vitro; B.W.—body weight; SOD—Superoxide dismutase; FDA—Food and Drug Administration; ROS—reactive oxygen species; DMEM—Dulbecco’s modified eagle serum; HBSS—hanks balanced salt solution; CCFA—Forty-seventh Session of Codex Committee on Food Additives

Keywords: Tartrazine; Neuron; Immunofluorescence; Reverse Transcription Quantitative Polymerase Chain Reaction; Dose response

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