RNA Yield Quantification as a Marker of First-Contact Toxicity from Food Preservatives in Oral Fibroblasts

Synthetic food preservatives such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) remain widely used, yet their early molecular effects on oral soft-tissue cells, where exposure first occurs, are insufficiently defined. Gingival fibroblasts represent a biologically relevant first-contact model capable of revealing early transcriptional disturbances induced by dietary preservatives. To evaluate the concentration- and time-dependent impact of BHA and BHT on transcriptional capacity in human gingival fibroblasts using total RNA yield as an early biosynthetic biomarker and to integrate area-under-the-curve (AUC) analysis to quantify cumulative transcriptional burden. Human gingival fibroblasts were exposed to BHA and BHT at 1% and 10% for 24, 48, and 72 hours. RNA was extracted and quantified to determine absolute yield and percentage of control. AUC analysis was applied to RNA-yield kinetics to capture cumulative transcriptional suppression across exposure duration. Dose-response curves, toxicity indices, and heatmap visualisation were used to show the data in a descriptive manner. RNA yield was significantly reduced by both preservatives in a dose-dependent and time-progressive manner, with BHT continuously having more potent suppressive effects than BHA. At 72 hours, the most noticeable decrease was under 10% BHT exposure, but transcriptional output was relatively preserved with 1% BHA. Visual summary showed the two antioxidants' increasing disparity over time. 10% BHT imposed the highest cumulative transcriptional burden, followed by 10% BHA, 1% BHT, and 1% BHA, according to AUC analysis. Both BHA and BHT cause detectable early transcriptional stress in gingival fibroblasts, although BHT is more effective in every scenario.The combined use of RNA yield as a sensitive early biosynthetic marker and AUC-based cumulative toxicity profiling introduces a refined evaluative framework for preservative safety assessment at the oral first-contact interface. This transcription-centred, time-integrated approach strengthens the molecular basis for understanding preservative-induced stress in oral tissues. Keywords:BHA, BHT, gingival fibroblasts, RNA yield, transcriptional toxicity, food preservatives, AUC analysis, cumulative toxicity, oral first-contact exposure, molecular toxicology.