Assessment of Genotoxicity and Inflammation in the Brain Hippocampus of Lead-Induced Mice Treated With Diospyros Mespiliformis

Hippocampus is crucial for memory and cognition, is highly vulnerable to oxidative stress and inflammatory insults from neurotoxicants such as lead (Pb). This study assessed genotoxicity and inflammatory markers in the brain hippocampus of lead – induced mice treated with aqueous extract of Diospyros mespiliformis. Twenty-five mice were divided into five groups and administered the following: Group A (Control, water) Group B (Pb, 50 mg/kg b.wt.), Group C (Pb + D. mespiliformis extract, 200 mg/kg b.wt), Group D ( Pb + D. mespiliformis extract, 400 mg/kg b.wt), and Group E (Pb + vitamin E , 100 mg/kg b.wt). After 28 days of exposure and treatments, hippocampal tissues from mice brain were assayed for oxidative stress markers (reduced glutathione, protein thiol), genotoxic marker (DNA fragmentation), inflammation markers (tumor necrosis factor (TNF-α), interleukin -6 (IL-1β), nitric oxide (NO), myeloperoxidase (MPO), acetylcholinesterase (AChE) and total protein (TP). Results showed that Pb exposure caused significant increases in TNF-α and DNA fragmentation, alongside a decline in IL-1β and AChE activity, confirming neuroinflammation and genotoxicity. Treatment with D. mespiliformis (200 mg/kg) restored GSH and protein levels, reduced MPO activity, and lowered DNA fragmentation. The 400 mg/kg b.wt of plant’s extract, however, elevated TNF-α and NO levels, suggesting a paradoxical pro-oxidant effect. Vitamin E attenuated DNA fragmentation and MPO activity, resembling the protective effects of the plant extract. These findings suggest that D. mespiliformis confers dose-dependent neuroprotection against Pb-induced hippocampal toxicity, with the 200 mg/kg dose being the most effective.

Diospyros mespiliformis, Lead toxicity, Genotoxicity, Neuroinflammation, Hippocampus, Oxidative stress.

1. Abdullahi, A., Muhammad, M. J., Tanko, Y., & Jimoh, A. (2023).Phytochemical profiling, antioxidant, antidiabetic, and ADMET study of the crude ethanol extract of Diospyros mespiliformis. Journal of Medicinal Plants Research, 17(2), 45-56. [Google Scholar] [Crossref]

2. Adikwu, E., & Deo, O. (2013). Effect of vitamin E and C supplementation on oxidative damage and total antioxidant capacity in lead-exposed workers. Environmental Toxicology and Pharmacology, 36(3), 869-876. [Google Scholar] [Crossref]

3. Akinmoladun, A.C., Obutor, E.M. and Farombi, E.O. (2010) Evaluation of Antioxidant and Free Radical Scavenging Capacities of Some Nigerian Indigenous Medicinal Plants. Journal of Medicinal Food, 13, 444-451. [Google Scholar] [Crossref]

4. Augustine, C., Imomon, J. A., Airhihen, B., & Igwe, C. (2024). Lead neurotoxicity in experimental models: A systematic review on hippocampal impairment. Neurotoxicology, 100, 1-15. [Google Scholar] [Crossref]

5. Baranowska-Bosiacka, I., et al. (2020). Hippocampal impairment triggered by long-term lead exposure from adolescence to adulthood in rats: Insights into molecular mechanisms. International Journal of Molecular Sciences, 21(18), 6937. [Google Scholar] [Crossref]

6. Basha, M.R., & Reddy, G.R. (2010). Developmental exposure to lead induces spatial memory deficits in rats. Neuroscience, 171(1), 53-61. [Google Scholar] [Crossref]

7. Bello, I. A., Ojo, G. O. S., & Ogunwande, I. A. (2009). Studies on the chemical constituents of Diospyros mespiliformis Hochst. Ex A. DC. (Ebenaceae). Journal of Applied Sciences, 9(16), 2949–2953. [Google Scholar] [Crossref]

8. Birben E., Sahiner U.M., Sackesen C., Erzurum S., Kalayci O. (2012). Oxidative stress and antioxidant defense. World Allergy Organ. J. 5:9–19. [Google Scholar] [Crossref]

9. Block, M. L., Zecca, L., & Hong, J. S. (2007). Microglia-mediated neurotoxicity: Uncovering the molecular mechanisms. Nature Reviews Neuroscience, 8(1), 57–69. [Google Scholar] [Crossref]

10. Bouayed, J., Rammal, H., & Soulimani, R. (2009). Oxidative stress and anxiety: Relationship and cellular pathways. Oxidative Medicine and Cellular Longevity, 2(2), 63-67. [Google Scholar] [Crossref]

11. Bradley, P.P., Priebat, D.A., Christensen, R.D. and Rothstein, G. (1982). Measurement of cutaneous inflammation: Estimation of neutrophil content with an enzyme marker. J. Invest Dermatol 78: 206 – 209. [Google Scholar] [Crossref]

12. Dangoggo, S. M., Muhammad, M. J., Ali, H., & Tijjani, M. A. (2023). Traditional Uses, Pharmacological Activities, and Phytochemical Analysis of Diospyros mespiliformis Hochst. ex A.DC (Ebenaceae): A Review. Molecules, 28(22), 7759. [Google Scholar] [Crossref]

13. Doss, A., Mubarack, H. M., & Dhanabalan, R. (2011). Antioxidant and free radical scavenging activity of different extracts of Diospyros mespiliformis L. (Ebenaceae). International Journal of Pharmacy and Pharmaceutical Sciences, 3(4), 91–95. [Google Scholar] [Crossref]

14. Doumas, B.T., Bayse, D.D., Carter, R.J., Peters Jr, T. and Schaffer, R. (1981) A candidate reference method for determination of total protein in serum. 1. Development and validation. Clin Chem 27(10):1642 – 1650. [Google Scholar] [Crossref]

15. Eid, A., Moustafa, M., Ahmed, A., & Mohamed, E. (2023). Possible role of selenium in ameliorating lead-induced neurotoxicity in rat hippocampus. Scientific Reports, 13, 14529. [Google Scholar] [Crossref]

16. Ellman, G.L. (1959) Tissue sulfhydryl groups. Arch Biochem. Biophys 82: 70 - 77. [Google Scholar] [Crossref]

17. Ellman, G.L., Courtney, K.D., Andres, V.J. and Featherstone, R.M. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7: 88 - 95 [Google Scholar] [Crossref]

18. Engelmann, H., Ader, R., Schalch, W., & Schwartz, J. (1993). Soluble and Cell Surface Receptors for Tumor Necrosis Factor. In: Faist, E., Meakins, J.L., Schildberg, F.W. (eds) Host Defense Dysfunction in Trauma, Shock and Sepsis. Springer, Berlin, Heidelberg. Engstrom, A., Michaelsson, K., Vahter, M., Julin, B., & Akesson, A. (2010). Low-level lead exposure triggers neuronal apoptosis in the developing mouse brain. Neurotoxicology and Teratology, 32(3), 412-418. [Google Scholar] [Crossref]

19. Flora, G., Gupta, D., & Tiwari, A. (2012). Toxicity of lead: A review with recent updates. Interdisciplinary Toxicology, 5(2), 47–58. [Google Scholar] [Crossref]

20. García-Lestón J., Méndez J., Pasaro E., Laffon B. (2010). Genotoxic effects of lead: An updated review. Environ. Int. 36:623–636. [Google Scholar] [Crossref]

21. Garza, A., Vega, R., & Soto, E. (2006). Cellular mechanisms of lead neurotoxicity. Medical Science Monitor, 12(3), RA57–RA65. [Google Scholar] [Crossref]

22. Gilbert, M.E., Kelly, M.E., Samsam, T.E., & Goodman, J.H. (2005). Chronic developmental lead exposure reduces neurogenesis in adult rat hippocampus but does not impair spatial learning. Toxicological Sciences, 86(2), 365-374. [Google Scholar] [Crossref]

23. . Golumbic, S., Barnea, E., & Ruder, A. (2023). Simultaneously Determined Antioxidant and Pro-Oxidant Activity of Randomly Selected Plant Secondary Metabolites and Plant Extracts. Molecules, 28(19), 6940. [Google Scholar] [Crossref]

24. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J. S., & Tannenbaum, S.R. (1982). Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Analytical Biochemistry, 126(1), 131–138. [Google Scholar] [Crossref]

25. Halliwell, B. (2020). Antioxidants: The basics-what they are and how to evaluate them. Advances in Pharmacology, 88, 3-46. [Google Scholar] [Crossref]

26. Hassoun, E. A., & Stohs, S. J. (1996). Lead-induced oxidative stress in cultured cells. Biochemical and Molecular Toxicology, 10(6), 253–258. [Google Scholar] [Crossref]

27. Keller, J. N., Kindy, M. S., Holtsberg, F. W., St Clair, D. K., Yen, H. C., Germeyer, A., & Markesbery, W. R. (2000). Mitochondrial manganese superoxide dismutase prevents neural apoptosis and reduces ischemic brain injury: Suppression of peroxynitrite production, lipid peroxidation, and mitochondrial dysfunction. Journal of Neuroscience, 20(15), 6082–6090. [Google Scholar] [Crossref]

28. Liu, C. M., Ma, J. Q., & Sun, Y. Z. (2013). Protective role of puerarin against lead-induced oxidative stress and apoptosis in rat kidneys. Biological Trace Element Research, 152(2), 270–276. [Google Scholar] [Crossref]

29. Lorke, D. (1983). A new approach to practical acute toxicity testing. Archives of Toxicology, 54(4), 275-287. [Google Scholar] [Crossref]

30. March, C. J., Mosley, B., Larsen, A., Cerretti, D. P., Braedt, G., Price, V., Gillis, C. S., Henney, C. S., Kronheim, S. R., Grabstein, P. J., Conlon, P. J., Hopp, T. P., & Cosman, D. (1985). Cloning, sequence and expression of two distinct human interleukin-1 complementary DNAs. Nature, 315(6021), 641–647. https://doi.org/10.1038/315641a0 [Google Scholar] [Crossref]

31. Muhammad, M.J., Magaji, M.G., & Gyang, M.D. (2017). Methanol Leaf Extract of Diospyros mespiliformis Hochst. Offers protection against some chemoconvulsants. Journal of Pharmacy & Bioresources, 14(2), 123-132. [Google Scholar] [Crossref]

32. Muhammad, M.J., Tanko, Y., Tijjani, M.A., & Dangoggo, S.M. (2025). Diospyros mespiliformis hochst modulates the Hippocampus and Prefrontal Cortex of Wistar Rat following Lithium chloride-pilocarpine-induced Epilepsy. Journal of Neuroscience Research, 103(1), 45-56. [Google Scholar] [Crossref]

33. Nguelefack-Mbuyo, E. P., Ndoye, O., Talla, E., & Mbafor, J. T. (2023). Traditional Uses, Pharmacological Activities, and Phytochemical Analysis of Diospyros mespiliformis Hochst. ex A.DC (Ebenaceae): A Review. Molecules, 28(22), 7759. [Google Scholar] [Crossref]

34. OECD. (2001). Test No. 423: Acute Oral toxicity - Acute Toxic Class Method. OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing, Paris. https://doi.org/10.1787/9789264071001-en [Google Scholar] [Crossref]

35. Patra, R.C., Swarup, D., & Dwivedi, S.K. (2001). Antioxidant effects of alpha tocopherol, ascorbic acid and L-methionine on lead induced oxidative stress to the liver, kidney and brain in rats. Toxicology, 162(2), 81-88. [Google Scholar] [Crossref]

36. Sadiq, M.B., et al. (2019). Chemical ingredients and antioxidant activities of underutilized wild fruits. Heliyon, 5(6), e01874. [Google Scholar] [Crossref]

37. Sajitha, G. R., Jose, R., Andrews, A., Ajantha, K. G., Augustine, P., & Jose, T. (2010). Effect of vitamin E and selenium on oxidative stress and tissue lead concentration in lead exposed rats. Toxicology and Industrial Health, 26(8), 517-524. [Google Scholar] [Crossref]

38. Sanders T, Liu Y, Buchner V, Tchounwou PB. (2009). Neurotoxic effects and biomarkers of lead exposure: a review. Rev Environ Health. 24(1):15-45. [Google Scholar] [Crossref]

39. Sedlak, J., & Lindsay, R. H. (1968). Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Analytical Biochemistry, 25(1), 192-205. [Google Scholar] [Crossref]

40. Wang, B., Du, Y., Zhang, J., & Chen, L. (2020). Early-Life Exposure to Lead Induces Cognitive Impairment in Elder Mice Targeting SIRT1 Phosphorylation and Oxidative Alterations. Frontiers in Physiology, 11, 446. [Google Scholar] [Crossref]

41. WHO. (2013). WHO guidelines on good manufacturing practices (GMP) for herbal medicines. World Health Organization, Geneva. [Google Scholar] [Crossref]

42. Wu, B., Ootani, A., Iwakiri, R., Sakata, Y., Fujise, T., Amemori, S., Yokoyama, F.,Tsoumada, S. and Fujimoto, K. (2005). T cell deficiency leads to liver carcinogenesis inAzoxymethane-treated rats. Experimental Biol Med. 231:91–98. [Google Scholar] [Crossref]

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