Investigation of the Neurotoxic Effects of Dimethyl Phthalate and Diisobutyl Phthalate on SH-SY5Y Neuroblastoma Cells

Abstract

Endocrine disruptors, particularly phthalates like Dimethyl phthalate (DMP) and Diisobutyl phthalate (DiBP), are prevalent environmental contaminants posing significant health risks. This study investigates the combined neurotoxic effects of DMP and DiBP on SH-SY5Y neuroblastoma cells by analyzing cytotoxicity, oxidative stress, and apoptosis. Using MTT and Neutral Red Uptake assays, we determined the IC50 values for DMP and DiBP as 11.35 mM and 1.307 mM, respectively. Flow cytometry revealed increased Reactive Oxygen Species (ROS) levels, indicating oxidative stress, while apoptosis assays showed enhanced cell death with combined phthalate exposure. The results demonstrate a synergistic effect, exacerbating cytotoxic and oxidative damage beyond individual exposures. This study highlights the compounded risk of phthalate mixtures, urging comprehensive risk assessments and regulatory policies to mitigate human health risks from combined chemical exposures.

Keywords

• apoptosis
• endocrine disruptors
• neurotoxicity
• oxidative stress
• phthalates

References

1. Chen, X., Xu, S., Tan, T., Lee, S. T., Cheng, S. H., Lee, F. W. F., Xu, S. J. L., & Ho, K. C. (2014). Toxicity and estrogenic endocrine disrupting activity of phthalates and their mixtures. International Journal of Environmental Research and Public Health, 11(3), 3156–3168. https://doi.org/10.3390/IJERPH110303156
2. Cong, B., Liu, C., Wang, L., & Chai, Y. (2020). The impact on antioxidant enzyme activity and related gene expression following adult zebrafish (Danio rerio) exposure to dimethyl phthalate. Animals, 10(4), 717. https://doi.org/10.3390/ANI10040717
3. Hlisníková, H., Petrovičová, I., Kolena, B., Šidlovská, M., & Sirotkin, A. (2021). Effects and mechanisms of phthalates’ action on neurological processes and neural health: A literature review. Pharmacological Reports, 73(2), 386–404. https://doi.org/10.1007/S43440-021-00215-5
4. Kara, M., Boran, T., Öztaş, E., Jannuzzi, A. T., Özden, S., & Özhan, G. (2022). Zoledronic acid-induced oxidative damage and endoplasmic reticulum stress-mediated apoptosis in human embryonic kidney (HEK-293) cells. Journal of Biochemical and Molecular Toxicology, 36(8), e23083. https://doi.org/10.1002/JBT.23083
5. Kleinsasser, N. H., Wallner, B. C., Kastenbauer, E. R., Weissacher, H., & Harréus, U. A. (2001). Genotoxicity of di-butyl-phthalate and di-iso-butyl-phthalate in human lymphocytes and mucosal cells. Teratogenesis, Carcinogenesis, and Mutagenesis, 21(3), 189–196. https://doi.org/10.1002/tcm.1007
6. Kleinsasser, N. H., Weissacher, H., Kastenbauer, E. R., Dirschedl, P., Wallner, B. C., & Harréus, U. A. (2000). Altered genotoxicity in mucosal cells of head and neck cancer patients due to environmental pollutants. European Archives of Oto-Rhino-Laryngology, 257(6), 337–342. https://doi.org/10.1007/S004059900220
7. Mahmoud, A., Ezgi, Ö., Merve, A., & Özhan, G. (2016). In vitro toxicological assessment of magnesium oxide nanoparticle exposure in several mammalian cell types. International Journal of Toxicology, 35(4), 429–437. https://doi.org/10.1177/1091581816648624
8. Meeker, J. D. (2012). Exposure to environmental endocrine disruptors and child development. Archives of Pediatrics & Adolescent Medicine, 166(10), 952–958. https://doi.org/10.1001/ARCHPEDIATRICS.2012.241

9. Nahla, E., Arya, P., Maneesha, P., & Chitra, K. C. (2024). Exposure to the plasticizer dibutyl phthalate causes oxidative stress and neurotoxicity in brain tissue. Environmental Science and Pollution Research, 31(14), 21399–21414. https://doi.org/10.1007/S11356-024-32604-7
10. Rodrigues, R. M., Stinckens, M., Ates, G., & Vanhaecke, T. (2023). Neutral red uptake assay to assess cytotoxicity in vitro. Methods in Molecular Biology, 2644, 237–245. https://doi.org/10.1007/978-1-0716-3052-5_15
11. Sellinger, E., Riesgo, V., Brinks, A. S., & Willing, J. (2021). Perinatal phthalate exposure increases developmental apoptosis in the rat medial prefrontal cortex. Neurotoxicology, 87, 167–173. https://doi.org/10.1016/j.neuro.2021.09.007
12. Sevim, C., Taghizadehghalehjoughi, A., Kara, M., Nosyrev, A. E., Nițulescu, G. M., Margină, D., & Tsatsakis, A. (2024). Investigation of the effects of metformin on the miR-21/PTEN/AKT pathway in HT-29 human colorectal adenocarcinoma cell and HUVEC co-culture. Farmacia, 72(1). https://doi.org/10.31925/farmacia.2024.1.5
13. Tran, C. M., Do, T. N., & Kim, K. T. (2021). Comparative analysis of neurotoxicity of six phthalates in zebrafish embryos. Toxics, 9(1), 1–11. https://doi.org/10.3390/TOXICS9010005
14. Wang, G., Shen, J., Lin, Y., Zhai, L., Guan, Q., & Shen, H. (2024). Dimethyl phthalate exposure induces cognitive impairment through COX2-mediated microglial activation. Research Square. https://doi.org/10.21203/RS.3.RS-4081530/V1
15. Wang, Y., & Qian, H. (2021). Phthalates and their impacts on human health. Healthcare, 9(5), 603. https://doi.org/10.3390/HEALTHCARE9050603
16. Yost, E. E., Euling, S. Y., Weaver, J. A., Beverly, B. E. J., Keshava, N., Mudipalli, A., Arzuaga, X., Blessinger, T., Dishaw, L., Hotchkiss, A., & Makris, S. L. (2019). Hazards of diisobutyl phthalate (DIBP) exposure: A systematic review of animal toxicology studies. Environment International, 125, 579–594. https://doi.org/10.1016/J.ENVINT.2018.09.038
17. Chen, X., Xu, S., Tan, T., Lee, S. T., Cheng, S. H., Lee, F. W. F., Xu, S. J. L., & Ho, K. C. (2014). Toxicity and estrogenic endocrine disrupting activity of phthalates and their mixtures. International Journal of Environmental Research and Public Health, 11(3), 3156–3168. https://doi.org/10.3390/IJERPH110303156
18. Chi, Z., Lin, H., Wang, X., Meng, X., Zhou, J., Xiang, L., Cao, G., Wu, P., Cai, Z., & Zhao, X. (2022). Dimethyl phthalate induces blood immunotoxicity through oxidative damage and caspase-dependent apoptosis. Science of The Total Environment, 838, 156047. https://doi.org/10.1016/J.SCITOTENV.2022.156047
19. Cong, B., Liu, C., Wang, L., & Chai, Y. (2020). The impact on antioxidant enzyme activity and related gene expression following adult zebrafish (Danio rerio) exposure to dimethyl phthalate. Animals, 10(4), 717. https://doi.org/10.3390/ANI10040717
20. Hlisníková, H., Petrovičová, I., Kolena, B., Šidlovská, M., & Sirotkin, A. (2021). Effects and mechanisms of phthalates’ action on neurological processes and neural health: A literature review. Pharmacological Reports, 73(2), 386–404. https://doi.org/10.1007/S43440-021-00215-5
21. Kara, M., Boran, T., Öztaş, E., Jannuzzi, A. T., Özden, S., & Özhan, G. (2022). Zoledronic acid-induced oxidative damage and endoplasmic reticulum stress-mediated apoptosis in human embryonic kidney (HEK-293) cells. Journal of Biochemical and Molecular Toxicology, 36(8), e23083. https://doi.org/10.1002/JBT.23083
22. Kara, M., Oztas, E., Ramazanoğulları, R., Kouretas, D., Nepka, C., Tsatsakis, A. M., & Veskoukis, A. S. (2020). Benomyl, a benzimidazole fungicide, induces oxidative stress and apoptosis in neural cells. Toxicology Reports, 7, 501–509. https://doi.org/10.1016/J.TOXREP.2020.04.001
23. Kleinsasser, N. H., Wallner, B. C., Kastenbauer, E. R., Weissacher, H., & Harréus, U. A. (2001). Genotoxicity of di-butyl-phthalate and di-iso-butyl-phthalate in human lymphocytes and mucosal cells. Teratogenesis, Carcinogenesis, and Mutagenesis, 21(3), 189–196. https://doi.org/10.1002/tcm.1007
24. Kleinsasser, N. H., Weissacher, H., Kastenbauer, E. R., Dirschedl, P., Wallner, B. C., & Harréus, U. A. (2000). Altered genotoxicity in mucosal cells of head and neck cancer patients due to environmental pollutants. European Archives of Oto-Rhino-Laryngology, 257(6), 337–342. https://doi.org/10.1007/S004059900220
25. Mahmoud, A., Öztaş, E., Merve, A., & Özhan, G. (2016). In vitro toxicological assessment of magnesium oxide nanoparticle exposure in several mammalian cell types. International Journal of Toxicology, 35(4), 429–437. https://doi.org/10.1177/1091581816648624
26. Meeker, J. D. (2012). Exposure to environmental endocrine disruptors and child development. Archives of Pediatrics & Adolescent Medicine, 166(10), 952–958. https://doi.org/10.1001/ARCHPEDIATRICS.2012.241
27. Nahla, E., Arya, P., Maneesha, P., & Chitra, K. C. (2024). Exposure to the plasticizer dibutyl phthalate causes oxidative stress and neurotoxicity in brain tissue. Environmental Science and Pollution Research, 31(14), 21399–21414. https://doi.org/10.1007/S11356-024-32604-7
28. Rodrigues, R. M., Stinckens, M., Ates, G., & Vanhaecke, T. (2023). Neutral red uptake assay to assess cytotoxicity in vitro. Methods in Molecular Biology (Clifton, N.J.), 2644, 237–245. https://doi.org/10.1007/978-1-0716-3052-5_15
29. Sellinger, E., Riesgo, V., & Brinks, A. (2021). Perinatal phthalate exposure increases developmental apoptosis in the rat medial prefrontal cortex. Neurotoxicology. https://doi.org/10.1016/j.neuro.2021.03.010
30. Sevim, C., Taghizadehghalehjoughi, A., Kara, M., Nosyrev, A. E., Nițulescu, G. M., Margină, D., & Tsatsakis, A. (2024). Investigation of the effects of metformin on the miR-21/PTEN/Akt pathway in HT-29 human colorectal adenocarcinoma cell line. Farmacia, 72(1). https://doi.org/10.31925/farmacia.2024.1.5
31. Tran, C. M., Do, T. N., & Kim, K. T. (2021). Comparative analysis of neurotoxicity of six phthalates in zebrafish embryos. Toxics, 9(1), 1–11. https://doi.org/10.3390/TOXICS9010005
32. Wang, G., Shen, J., Lin, Y., Zhai, L., Guan, Q., & Shen, H. (2024a). Dimethyl phthalate exposure induces cognitive impairment through COX2-mediated microglial activation. Research Square. https://doi.org/10.21203/RS.3.RS-4081530/V1
33. Wang, G., Shen, J., Lin, Y., Zhai, L., Guan, Q., & Shen, H. (2024b). Dimethyl phthalate exposure induces cognitive impairment through COX2-mediated microglial activation. Research Square. https://doi.org/10.21203/RS.3.RS-4081530/V1
34. Wang, Y., & Qian, H. (2021). Phthalates and their impacts on human health. Healthcare, 9(5), 603. https://doi.org/10.3390/HEALTHCARE9050603
35. Yost, E. E., Euling, S. Y., Weaver, J. A., Beverly, B. E. J., Keshava, N., Mudipalli, A., Arzuaga, X., Blessinger, T., Dishaw, L., Hotchkiss, A., & Makris, S. L. (2019). Hazards of diisobutyl phthalate (DIBP) exposure: A systematic review of animal toxicology studies. Environment International, 125, 579–594. https://doi.org/10.1016/J.ENVINT.2018.09.038
36. Zhang, Y., Lyu, L., Tao, Y., Ju, H., & Chen, J. (2022). Health risks of phthalates: A review of immunotoxicity. Environmental Pollution, 313, 120173. https://doi.org/10.1016/J.ENVPOL.2022.120173