Agricultural Biotechnology Journal

Agricultural Biotechnology Journal

Dose-dependent effects of neotame on hepatic Th and Cyp1a2 mRNA expression, dopamine level, and hepatic histopathology in male rats

Document Type : Research Paper

Authors
Randah F. Hassan 1
Hassan K. Al-Aawadi 1
Rawaa S. A. Al-Azawi 2
1 College of Science, Al-Qasim Green University, 51013, Babylon, Iraq.
2 College of Science, Al-Qasim Green University, 51013, Babylon, Iraq
10.22103/jab.2026.27817.1996
Abstract
Objective
Neotame is a high-intensity, non-caloric dipeptide sweetener structurally related to aspartame that is approved for use in foods and beverages. Because it is metabolized in the liver and shares structural features with compounds shown to disturb hepatic drug-metabolizing enzymes and catecholaminergic signaling, concerns persist regarding its dose-dependent effects on hepatic gene expression and dopaminergic-related systemic function. This study was designed to evaluate the dose-dependent effects of orally administered neotame on the hepatic expression of tyrosine hydroxylase (TH) and cytochrome P450 1A2 (CYP1A2) genes, serum dopamine concentration, liver function biomarkers, and hepatic histoarchitecture in adult male albino rats.
Material and methods
Four groups of ten adult male Wistar rats (forty rats in total) were randomly selected. One group of animals was a control group treated with solvent and the other three groups were treated with neotame for 60 consecutive days. The doses used were 250, 500, or 750 mg/kg body weight per day, administered by oral gavage. Hepatic TH and CYP1A2 mRNA expression was quantified by quantitative real-time reverse-transcription PCR (qRT-PCR) with normalization to validated reference gene. Serum dopamine was measured by ELISA; liver function was assessed via ALT, AST, ALP, total protein, and total bilirubin; and liver sections were examined histopathologically. Data were analyzed by one-way ANOVA with Duncan's and Tukey's post-hoc test, and Pearson correlation coefficients were calculated between gene expression and biochemical/histological markers.

Results
Neotame produced a dose-dependent downregulation of hepatic TH mRNA and a concomitant reduction in serum dopamine, together with dose-dependent upregulation of hepatic CYP1A2 mRNA and elevation of ALT and AST activities, accompanied by progressively more severe periportal inflammation, hepatocellular vacuolation, and mild fibrosis at the highest dose. TH expression correlated positively with serum dopamine and negatively with CYP1A2 expression and liver enzyme activities.
Conclusion
This study suggests that dose-dependent changes in hepatic Th and Cyp1a2 mRNA expression, serum dopamine concentration, liver biochemical markers, and liver histopathology in male rats may be due to the fact that these animals were chronically exposed to high doses of neotame. These results confirm the need for further studies to use more reference genes, perform protein level measurements, and evaluate the mechanism of the relevant signaling pathways.
Keywords
dopamine
hepatotoxicity
male rats
neotame
qRT-PCR
Abhilash, M., Paul, M. V., Varghese, M. V., & Nair, R. H. (2011). Effect of long term intake of aspartame on antioxidant defense status in liver. Food and Chemical Toxicology, 49(6), 1203-1207. https://doi.org/10.1016/j.fct.2011.02.019
Alkafafy, M. el-S., Ibrahim, Z. S., Ahmed, M. M., & El-Shazly, S. A. (2015). Impact of aspartame and saccharin on the rat liver: Biochemical, molecular, and histological approach. International Journal of Immunopathology and Pharmacology, 28(2), 247-255. https://doi.org/10.1177/0394632015586134
Coccurello, R. (2025). Disrupting the gut-brain axis: How artificial sweeteners rewire microbiota and reward pathways. International Journal of Molecular Sciences, 26(20), Article 10220. https://doi.org/10.3390/ijms262010220
Demir, M., Bornstein, S. R., Mantzoros, C. S., & Perakakis, N. (2023). Liver fat as risk factor of hepatic and cardiometabolic diseases. Obesity Reviews, 24(10), Article e13612. https://doi.org/10.1111/obr.13612
EFSA Panel on Food Additives and Flavourings (FAF), Castle, L., Andreassen, M., Aquilina, G., Bastos, M. L., Boon, P., Fallico, B., FitzGerald, R., Frutos Fernandez, M. J., Grasl-Kraupp, B., Gundert-Remy, U., Gürtler, R., Houdeau, E., Kurek, M., Louro, H., Morales, P., Passamonti, S., Batke, M., Bruzell, E., Chipman, J., Crebelli, R., Fortes, C., Fürst, P., Gaffet, E., Karlien, C., Halldorsson, T., Leblanc, J. C., Lindtner, O., Loeschner, K., Mast, J., Mirat, M., Mortensen, A., Undas, A., Wright, M., Barmaz, S., Civitella, C., Abrahantes, J. C., Le Gall, P., Mazzoli, E., Mech, A., Rasinger, J. D., Rincon, A., Riolo, F., Smeraldi, C., Tard, A., Zakidou, P., & Lodi, F. (2025). Re-evaluation of neotame (E 961) as food additive. EFSA Journal, 23(7), Article e9480. https://doi.org/10.2903/j.efsa.2025.9480
Farid, A., Hesham, M., El-Dewak, M., & Amin, A. (2020). The hidden hazardous effects of stevia and sucralose consumption in male and female albino mice in comparison to sucrose. Saudi Pharmaceutical Journal, 28(10), 1290-1300. https://doi.org/10.1016/j.jsps.2020.08.019
Golzan, S. A., Movahedian, M., Haghighat, N., Asbaghi, O., & Hekmatdoost, A. (2023). Association between non-nutritive sweetener consumption and liver enzyme levels in adults: A systematic review and meta-analysis of randomized clinical trials. Nutrition Reviews, 81(9), 1105-1117. https://doi.org/10.1093/nutrit/nuac107
Haga, C., Tolaymat, L., Walker, A., Hedges, M., Yin, M., McManus, M., & Dawson, N. (2023). Acute adverse effects related to consumption of nonnutritive and low-calorie sweeteners. Southern Medical Journal, 116(6), 450-454. https://doi.org/10.14423/SMJ.0000000000001560
Haq, N., Tafweez, R., Saqib, S., Bokhari, Z. H., Ali, I., & Syami, A. F. (2019). Aspartame and sucralose-induced fatty changes in rat liver. Journal of the College of Physicians and Surgeons Pakistan, 29(9), 848-851. https://doi.org/10.29271/jcpsp.2019.09.848
Iman, M. M. (2011). Effect of aspartame on some oxidative stress parameters in liver and kidney of rats. African Journal of Pharmacy and Pharmacology, 5(6), 678-682. https://doi.org/10.5897/AJPP10.302
Jia, M., Peng, Z., Yang, K., Su, C., Wang, Y., & Yan, C. (2020). A high-throughput targeted metabolomics method for the quantification of 104 non-polar metabolites in cholesterol, eicosanoid, and phospholipid metabolism: Application in the study of a CCl₄-induced liver injury mouse model. Analyst, 145(10), 3575-3591. https://doi.org/10.1039/d0an00385a
Kanehisa, M., Furumichi, M., Sato, Y., Kawashima, M., & Ishiguro-Watanabe, M. (2023). KEGG for taxonomy-based analysis of pathways and genomes. Nucleic Acids Research, 51(D1), D587-D592. https://doi.org/10.1093/nar/gkac963
Khan, A. H. A. A., Al-Alwany, E. A. H., Al-Azawi, R. S. A., Al-Jebori, J. G. A., Alkafagy, S. M., Al-Khakani, S. S. A., … & Abdulhasan, A. S. (2025). Serum level of IL-37 as a modulator in atherosclerosis, correlating its levels with oxidative stress and disease progression. Regulatory Mechanisms in Biosystems, 16(2), Article e25076. https://doi.org/10.15421/0225076
Lauretani, F., Giallauria, F., Testa, C., Zinni, C., Lorenzi, B., Zucchini, I., Salvi, M., Napoli, R., & Maggio, M. G. (2024). Dopamine pharmacodynamics: New insights. International Journal of Molecular Sciences, 25(10), Article 5293. https://doi.org/10.3390/ijms25105293
Luo, J., Lu, D., Zhang, R., Long, B., Chen, L., Wang, W., & Tian, X. (2025). What exactly happens to rats that drink different types of sweetness water over a long time: A comparison with sucrose, artificial sweeteners and natural sweeteners. Journal of Functional Foods, 127, Article 106715. https://doi.org/10.1016/j.jff.2025.106715
Mathur, P., & Bakshi, A. (2024). Effect of non-nutritive sweeteners on insulin regulation, glycemic response, appetite and weight management: A systematic review. Nutrition & Food Science, 54(1), 100-119. https://doi.org/10.1108/NFS-03-2023-0060
McPherson, S., Hardy, T., Henderson, E., Burt, A. D., Day, C. P., & Anstee, Q. M. (2015). Evidence of NAFLD progression from steatosis to fibrosing-steatohepatitis using paired biopsies: Implications for prognosis and clinical management. Journal of Hepatology, 62(5), 1148-1155. https://doi.org/10.1016/j.jhep.2014.11.034
Miller, M. A., & Zachary, J. F. (2017). Mechanisms and morphology of cellular injury, adaptation, and death. In Pathologic basis of veterinary disease (pp. 2-43.e19). https://doi.org/10.1016/B978-0-323-35775-3.00001-1
Mohammed, A. J., Al-Awadi, H. K., & Al-Shukri, H. H. K. (2025). Comparative study between artificial sweeteners such as aspartame and neotame on neurobehavioral and some haematological parameters in male rats. Iraqi Journal of Industrial Research, 12(1), 109-117. https://doi.org/10.53523/ijoirVol12I1ID566
Mohammed-Ali, Z., Brinc, D., Kulasingam, V., & Selvaratnam, R. (2020). Defining appropriate utilization of AST testing. Clinical Biochemistry, 79, 75-77. https://doi.org/10.1016/j.clinbiochem.2020.02.006
Namikawa-Kanai, H., Miyazaki, T., Matsubara, T., Shigefuku, S., Ono, S., Nakajima, E., Morishita, Y., Honda, A., Furukawa, K., & Ikeda, N. (2020). Comparison of the amino acid profile between the nontumor and tumor regions in patients with lung cancer. American Journal of Cancer Research, 10(7), 2145-2159. https://pubmed.ncbi.nlm.nih.gov/32775007/
Nichol, A. D., Holle, M. J., & An, R. (2018). Glycemic impact of non-nutritive sweeteners: A systematic review and meta-analysis of randomized controlled trials. European Journal of Clinical Nutrition, 72(6), 796-804. https://doi.org/10.1038/s41430-018-0170-6
O’Donnell, K. (2012). Aspartame, neotame and advantame. In K. O’Donnell & M. W. Kearsley (Eds.), Sweeteners and sugar alternatives in food technology. https://doi.org/10.1002/9781118373941.ch6
Özatik, F. Y., Özatik, O., Tekşen, Y., Koçak, H., Arı, N. S., & Çengelli Ünel, Ç. (2023). Dose-dependent effect of hydrogen sulfide in cyclophosphamide-induced hepatotoxicity in rats. Turkish Journal of Gastroenterology, 34(6), 626-634. https://doi.org/10.5152/tjg.2023.22040
Radenkovic, S. (2023). Investigating the effects of artificial sweeteners. Nature Reviews Endocrinology, 19(8), 442. https://doi.org/10.1038/s41574-023-00860-1
Romaniuk-Drapała, A., Kosicka-Noworzyń, K., Sheng, Y.-H., Yohn, C., Brunetti, L., & Kagan, L. (2025). Evaluation of reference genes for qPCR in human liver and kidney tissue from individuals with obesity. Scientific Reports, 15, Article 5347. https://doi.org/10.1038/s41598-025-87911-x
Song, I.-S., Nam, S.-J., Jeon, J.-H., Park, S.-J., & Choi, M.-K. (2021). Enhanced bioavailability and efficacy of silymarin solid dispersion in rats with acetaminophen-induced hepatotoxicity. Pharmaceutics, 13(5), Article 628. https://doi.org/10.3390/pharmaceutics13050628
Souza Cruz, E. M., Bitencourt de Morais, J. M., Dalto da Rosa, C. V., da Silva Simões, M., Comar, J. F., de Almeida Chuffa, L. G., & Seiva, F. R. F. (2020). Long-term sucrose solution consumption causes metabolic alterations and affects hepatic oxidative stress in Wistar rats. Biology Open, 9(3), Article bio047282. https://doi.org/10.1242/bio.047282
Toews, I., Lohner, S., Küllenberg de Gaudry, D., Sommer, H., & Meerpohl, J. J. (2019). Association between intake of non-sugar sweeteners and health outcomes: Systematic review and meta-analyses of randomised and non-randomised controlled trials and observational studies. BMJ, 364, Article k4718. https://doi.org/10.1136/bmj.k4718
Zeng, J., Li, N., Ge, J., Ma, H., Sun, S., Jing, Y., Qian, C., Cui, R., Qu, S., & Sheng, H. (2025). Serum ALT activity and its isoenzymes as potential biomarkers for diagnosis of sarcopenia in older adults: A retrospective, cross-sectional study. BMC Geriatrics, 25(1), Article 53. https://doi.org/10.1186/s12877-025-05697-z                  
Agricultural Biotechnology Journal
Volume 18, Issue 4
Summer 2026
Pages 629-648