Oxidative Stress–Mediated Metabolic and Mitochondrial Dysregulation by Tartrazine, Sunset Yellow, Aspartame, and Sodium Benzoate in Zebrafish Embryos: Implications for Diabetes Risk

Jayshree  Nellore; Divya  Christy; Karthikeyan S; Valli  Nachiyar

doi:10.65763/jfhb.2026.e266467

Authors

Jayshree Nellore Department of Biotechnology, School of Bio and Chemical Engineering, Sathyabama Institute of Science and Technology, Tamil Nadu, 600119 India
Divya Christy Department of Biotechnology, School of Bio and Chemical Engineering, Sathyabama Institute of Science and Technology, Tamil Nadu, 600119 India
Karthikeyan S Department of Biotechnology, School of Bio and Chemical Engineering, Sathyabama Institute of Science and Technology, Tamil Nadu, 600119 India
Valli Nachiyar Dean-Publications, Department of Research, Meenakshi Academy of Higher Education and Research, Tamil Nadu, 600078 India

DOI:

https://doi.org/10.65763/jfhb.2026.e266467

Keywords:

Food additives, Zebrafish embryos, Oxidative stress, Diabetes risk

Abstract

The growing global prevalence of diabetes underscores the importance of understanding dietary contributors to metabolic disorders. This study investigates the impact of four commonly used food additives—tartrazine (TTZ), sunset yellow (SY), aspartame (AST), and sodium benzoate (SB)—on diabetes related mechanisms using zebrafish embryos. These compounds were chosen for their frequent dietary consumption, distinct chemical classes, and documented associations with oxidative stress and metabolic disturbances. Exposure to the additives induced dose- and stage-dependent oxidative stress, evidenced by elevated malondialdehyde (MDA) levels and decreased glutathione (GSH) activity, with SB producing the most pronounced effects. Metabolomic profiling via nuclear magnetic resonance (¹H-NMR) revealed additive-specific disruptions in glucose, triglyceride, cholesterol, branched-chain amino acid, and aromatic amino acid metabolism, with SB and AST causing the most extensive metabolic perturbations indicative of mitochondrial dysfunction and impaired energy homeostasis. Gene expression analyses demonstrated upregulation of ptf1a, glrx, and cox, linking additive exposure to pancreatic dysregulation, glutathione metabolism disruption, and variable mitochondrial effects. These findings suggest that early-life exposure to TTZ, SY, AST, and particularly SB may predispose organisms to metabolic dysregulation and diabetes risk through oxidative stress, altered energy metabolism, and mitochondrial impairment. The results underscore the importance of re-evaluating the dietary safety of commonly consumed food additives.

References

Aleström, P., D’Angelo, L., Midtlyng, P.J., Schorderet, D.F., Schulte-Merker, S., Sohm, F., & Warner, S. (2020). Zebrafish: Housing and husbandry recommendations. Laboratory Animals, 54(3), 213–224. https://doi.org/10.1177/0023677219869037

Alsagheir, A.I., AlMutair, A., Bakhamis, S., Aletani, L., Alhumaidi, S., & Bin Abbas, B. (2023). Isolated pancreatic agenesis secondary to PTF1A gene mutation: A case series and literature review. Cureus, 15(10): e47202. https://doi.org/10.7759/cureus.47202

Alsunni, A.A. (2020). Effects of artificial sweetener consumption on glucose homeostasis and its association with Type 2 diabetes and obesity. International Journal of General Medicine, 13, 775–785. https://doi.org/10.2147/IJGM.S274760

Angelin, M., Kumar, J., Vajravelu, L.K., Satheesan, A., Chaithanya, V., & Murugesan, R. (2024). Artificial sweeteners and their implications in diabetes: A review. Frontiers in Nutrition, 11, 1411560. https://doi.org/10.3389/fnut.2024.1411560

Bondue, T., Berlingerio, S.P., van den Heuvel, L., & Levtchenko, E. (2023). The zebrafish embryo as a model organism for testing mRNA-Based therapeutics. International Journal of Molecular Sciences, 24(13), 11224. https://doi.org/10.3390/ijms241311224

Caturano, A., D’Angelo, M., Mormone, A., Russo, V., Mollica, M.P., Salvatore, T., ... Sasso, F.C. (2023). Oxidative stress in type 2 diabetes: impacts from pathogenesis to lifestyle modifications. Current Issues in Molecular Biology, 45(8), 6651–6666. https://doi.org/10.3390/cimb45080420

Christy, L. D., Vignesh, K., Nellore, J., & Tippabathani, J. (2024). Impact of food additives on neurodevelopmental processes in zebrafish (Danio rerio): Exploring circadian clock genes and dopamine system. Developmental Neurobiology, 84(3), 191–202. https://doi.org/10.1002/dneu.22947

Delaroque, C., & Chassaing, B. (2024). Dietary emulsifier consumption accelerates type 1 diabetes development in NOD mice. npj Biofilms and Microbiomes, 10, 1. https://doi.org/10.1038/s41522-023-00475-4

Duque, M., Amorim, J.P., & Bessa, J. (2022). Ptf1a function and transcriptional cis‐regulation, a cornerstone in vertebrate pancreas development. The FEBS Journal, 289(17), 5121–5136. https://doi.org/10.1111/febs.16075

Duy-Thanh, D., Bich-Ngoc, N., Van den Bossche, F., Lai-Thanh, N., & Muller, M. (2022). Discovering novel bioactivities of controversial food additives by means of simple zebrafish embryo toxicity (ZET) assays. Toxics, 11(1), 8. https://doi.org/10.3390/toxics11010008

Fujita, K.K., Xia, Z., Tomy, G., Montina, T., & Wiseman, S. (2021). 1H NMR based metabolomic profiling of early life stage zebrafish (Danio rerio) exposed to a water-soluble fraction of weathered sediment-bound diluted bitumen. Aquatic Toxicology, 232, 105766. https://doi.org/10.1016/j.aquatox.2021.105766

Ganesan, R., Vasantha-Srinivasan, P., Sadhasivam, D.R., Subramanian, R., Vimalraj, S., & Suk, K.T. (2021). Carbon nanotubes induce metabolomic profile disturbances in zebrafish: NMR-based metabolomics platform. Frontiers in Molecular Biosciences, 8, 688827. https://doi.org/10.3389/fmolb.2021.688827

Griebsch, L.V., Theiss, E.L., Janitschke, D., Erhardt, V.K.J., Erhardt, T., Haas, E.C., ... Grimm, H.S. (2023). Aspartame and its metabolites cause oxidative stress and mitochondrial and lipid alterations in SH-SY5Y cells. Nutrients, 15(6), 1467. https://doi.org/10.3390/nu15061467

Halliwell, B., & Gutteridge, J.M.C. (2015). Free radicals in biology and medicine (5th Ed.). Oxford University Press.

Heindel, J.J., Blumberg, B., Cave, M., Machtinger, R., Mantovani, A., Mendez, M.A., ... vom Saal, F. (2017). Metabolism disrupting chemicals and metabolic disorders. Reproductive Toxicology, 68, 3–33. https://doi.org/10.1016/j.reprotox.2016.10.001

Jin, K., & Xiang, M. (2019). Transcription factor Ptf1a in development, diseases and reprogramming. Cellular and Molecular Life Sciences, 76(5), 921. https://doi.org/10.1007/s00018-018-2972-z

Junot, C., Pinu, F.R., van der Hooft, J.J.J., & Moco, S. (2023). Editorial: NMR-based metabolomics. Frontiers in Molecular Biosciences, 10, 1337566. https://doi.org/10.3389/fmolb.2023.1337566

Lee, J.D., Lee, J., Vang, J., & Pan, X. (2024). Sodium benzoate promotes fat accumulation and aging via the SKN-1/Nrf2 signaling pathway: Evidence from the Caenorhabditis elegans model. bioRxiv https://doi.org/10.1101/2024.09.16.613358

Lennerz, B.S., Vafai, S.B., Delaney, N.F., Clish, C.B., Deik, A.A., Pierce, K.A., ... Mootha, V.K. (2015). Effects of sodium benzoate, a widely used food preservative, on glucose homeostasis and metabolic profiles in humans. Molecular Genetics and Metabolism, 114(1), 73–79. https://doi.org/10.1016/j.ymgme.2014.11.010

Liberti, M.V., & Locasale, J.W. (2016). The Warburg effect: How does it benefit cancer cells? Trends in Biochemical Sciences, 41(3), 211–218. https://doi.org/10.1016/j.tibs.2015.12.001

Li, Y., Si, D., Sabier, M., Liu, J., Si, J., & Zhang, X. (2023). Guideline for screening antioxidant against lipid‐peroxidation by spectrophotometer. EFood, 4(2), e80. https://doi.org/10.1002/efd2.80

Livak, K.J., & Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods, 25(4), 402–408. https://doi.org/10.1006/meth.2001.1262

Lyu, C., & Liu, Y. (2024). IDDF2024-ABS-0302 The impact of food additives on gestational diabetes and birth weight. Gut, 73(Suppl 2), A340–A341. https://doi.org/10.1136/gutjnl-2024-IDDF.297

Massarsky, A., Kozal, J.S., & Di Giulio, R.T. (2017). Glutathione and zebrafish: Old assays to address a current issue. Chemosphere, 168, 707–715. https://doi.org/10.1016/j.chemosphere.2016.11.004

Magnuson, B.A., Burdock, G.A., Doull, J., Kroes, R.M., Marsh, G.M., Pariza, M.W., ... Williams, G.M. (2007). Aspartame: A safety evaluation based on current use levels, regulations, and toxicological and epidemiological studies. Critical Reviews in Toxicology, 37(8), 629–727. https://doi.org/10.1080/10408440701516184

Miao, W., He, L., Zhang, T., & Li, C. (2022). Lentinan Impairs the early development of zebrafish embryos, possibly by disrupting glucose and lipid metabolism. Processes, 10(1), 120. https://doi.org/10.3390/pr10010120

Ong, K.L., Stafford, L.K., McLaughlin, S.A., Boyko, E.J., Vollset, S.E., Smith, A.E., ... Brauer, M. (2023). Global, regional, and national burden of diabetes from 1990 to 2021, with projections of prevalence to 2050: A systematic analysis for the global burden of disease study 2021. The Lancet, 402(10397), 203–234. https://doi.org/10.1016/S0140-6736(23)01301-6

Panyod, S., Wu, W.K., Chang, C.T., Wada, N., Ho, H.C., Lo, Y.L., ... Sheen, L.Y. (2024). Common dietary emulsifiers promote metabolic disorders and intestinal microbiota dysbiosis in mice. Communications Biology, 7(1), 749. https://doi.org/10.1038/s42003-024-06224-3

Paula Neto, H.A., Ausina, P., Gomez, L.S., Leandro, J.G.B., Zancan, P., & Sola-Penna, M. (2017). Effects of food additives on immune cells as contributors to body weight gain and immune-mediated metabolic dysregulation. Frontiers in Immunology, 8, 1478. https://doi.org/10.3389/fimmu.2017.01478

Peterson, S.M., & Freeman, J.L. (2009). RNA Isolation from embryonic zebrafish and cDNA synthesis for gene expression analysis. Journal of Visualized Experiments, 30, 1470. https://doi.org/10.3791/1470

Pitocco, D., Tesauro, M., Alessandro, R., Ghirlanda, G., & Cardillo, C. (2013). Oxidative stress in diabetes: implications for vascular and other complications. International Journal of Molecular Sciences, 14(11), 21525–21550. https://doi.org/10.3390/ijms141121525

Puttegowda, S.K.B., Shivaramu, M., Manjunath, B.S., Venkataraman, R., & Nagarajappa, R.B. (2024). Understanding artificial sweeteners and food colorants-their impact on human health: A review. Indian Journal of Pharmacy Practice, 18(1), 8–15. https://doi.org/10.5530/ijopp.20250089

Rivera Nieves, A.M., Wauford, B.M., & Fu, A. (2024). Mitochondrial bioenergetics, metabolism, and beyond in pancreatic β-cells and diabetes. Frontiers in Molecular Biosciences, 11, 1354199. https://doi.org/10.3389/fmolb.2024.1354199

Sachett, A., Gallas-Lopes, M., M M Conterato, G., Benvenutti, R., P Herrmann, A., & Piato, A. (2020). Quantification of thiobarbituric acid reactive species (TBARS) optimized for zebrafish brain tissue v.1. https://doi.org/10.17504/protocols.io.bjp8kmrw

Salame, C., Javaux, G., Sellem, L., Viennois, E., de Edelenyi, F.S., Agaësse, C., ... Touvier, M. (2024). Food additive emulsifiers and the risk of type 2 diabetes: Analysis of data from the NutriNet-Santé prospective cohort study. The Lancet Diabetes & Endocrinology, 12(5), 339–349. https://doi.org/10.1016/S2213-8587(24)00086-X

Sangwung, P., Petersen, K.F., Shulman, G.I., & Knowles, J.W. (2020). Mitochondrial dysfunction, insulin resistance, and potential genetic implications: Potential role of alterations in mitochondrial function in the pathogenesis of insulin resistance and type 2 diabetes. Endocrinology, 161(4), bqaa017. https://doi.org/10.1210/endocr/bqaa017

Simmons, A.L., Schlezinger, J.J., & Corkey, B.E. (2014). What are we putting in our food that is making us fat? Food additives, contaminants, and other putative contributors to obesity. Current Obesity Reports, 3(2), 273–285. https://doi.org/10.1007/s13679-014-0094-y

Souza, J.M., Peluffo, G., & Radi, R. (2008). Protein tyrosine nitration—functional alteration or just a biomarker? Free Radical Biology and Medicine, 45(4), 357–366. https://doi.org/10.1016/j.freeradbiomed.2008.04.010

Vander Heiden, M.G., Cantley, L.C., & Thompson, C.B. (2009). Understanding the Warburg effect: The metabolic requirements of cell proliferation. Science, 324(5930), 1029–1033. https://doi.org/10.1126/science.1160809

Warner, J.O. (2024). Artificial food additives: Hazardous to long-term health? Archives of disease in childhood, 109(11), 882–885. https://doi.org/10.1136/archdischild-2023-326565

Walker, V. (2009). Ammonia toxicity and its prevention in inherited defects of the urea cycle. Diabetes, Obesity and Metabolism, 11(9), 823–835. https://doi.org/10.1111/j.1463-1326.2009.01054.x

Wu, L. (2021). Analysis of food additives. In Galanakis, C.M. (Ed.), Innovative Food Analysis (pp. 157–180). Elsevier. https://doi.org/10.1016/B978-0-12-819493-5.00007-8

Yaribeygi, H., Sathyapalan, T., Atkin, S.L., & Sahebkar, A. (2020). Molecular mechanisms linking oxidative stress and diabetes mellitus. Oxidative Medicine and Cellular Longevity, 2020(1), 8609213. https://doi.org/10.1155/2020/8609213

Zhou, M., Hanschmann, E.M., Römer, A., Linn, T., & Petry, S.F. (2024). The significance of glutaredoxins for diabetes mellitus and its complications. Redox Biology, 71, 103043. https://doi.org/10.1016/j.redox.2024.103043

Zong, Y., Li, H., Liao, P., Chen, L., Pan, Y., Zheng, Y., ... Gao, J. (2024). Mitochondrial dysfunction: mechanisms and advances in therapy. Signal Transduction and Targeted Therapy, 9, 124. https://doi.org/10.1038/s41392-024-01839-8

Zuberi, Z., Eeza, M.N.H., Matysik, J., Berry, J.P., & Alia, A. (2019). NMR-based metabolic profiles of intact zebrafish embryos exposed to aflatoxin B1 recapitulates hepatotoxicity and supports possible neurotoxicity. Toxins, 11(5), 258. https://doi.org/10.3390/toxins11050258

Zuo, M., Ye, M., Lin, H., Liao, S., Xing, X., Liu, J., ... Ren, X. (2024). Mitochondrial dysfunction in environmental toxicology: Mechanisms, impacts, and health implications. Chemical Research in Toxicology, 37(11), 1794–1806. https://doi.org/10.1021/acs.chemrestox.4c00328