Betaine Mitigates Cyclophosphamide-Induced Hepatotoxicity via Antioxidant Restoration and Modulation of Wnt3A/Lrp5 Signaling Pathways in Rats

Abstract

Objective: Cyclophosphamide (CP) is an effective alkylating agent used in the treatment of malignancies and autoimmune diseases. However, toxic metabolites generated during hepatic metabolism induce oxidative stress, inflammation, and apoptosis, leading to hepatotoxicity that limits its clinical use. Betaine (BTN), a natural methyl donor found in foods such as beets, spinach, and wheat, has antioxidant, anti-inflammatory, and anti-apoptotic properties. This study investigated the protective effects of BTN against CP-induced liver injury in rats. Methods: Thirty-five Wistar albino rats were randomly assigned to five groups: Control, BTN, CP, CP+BTN125, and CP+BTN250. BTN (125 or 250 mg/kg) was administered orally for seven days, while CP (200 mg/kg) was injected intraperitoneally as a single dose on day 7. Blood and liver samples were collected for biochemical, molecular, and histopathological analyses. Serum AST and ALT activities; tissue MDA, SOD, CAT, GPx, and GSH levels; gene expression by RT-PCR; and histopathological changes were evaluated. Results: CP significantly increased ALT, AST, and MDA levels while decreasing SOD, CAT, GPx, and GSH levels (p<0.05). BTN improved these parameters in a dose-dependent manner, with 250 mg/kg showing greater efficacy (p<0.05). TNF-α and NF-κB expression increased in the CP group but was significantly suppressed by BTN (p<0.05). BTN also reduced apoptosis by decreasing Bax and caspase-3 expression and increasing Bcl-2 expression (p<0.05). Moreover, Wnt3A and Lrp5 expression, reduced by CP, was significantly restored following BTN treatment (p<0.05). Histopathological examination confirmed that BTN markedly attenuated CP-induced liver damage. Conclusion: BTN exerted significant hepatoprotective effects against CP-induced liver injury through antioxidant, anti-inflammatory, and anti-apoptotic mechanisms, suggesting its potential as a supportive therapeutic strategy for reducing CP-associated hepatotoxicity

Keywords

• Cyclophosphamide
• Betaine
• Apoptosis
• Inflammation
• Hepatotoxicity

References

1. 1. Ahmed AR, Hombal SM. Cyclophosphamide (Cytoxan): a review on relevant pharmacology and clinical uses. J Am Acad Dermatol. 1984;11(6):1115–1126. https://doi.org/10.1016/S0190-9622(84)80193-0
2. 2. Delahousse J, Molina L, Paci A. Cyclophosphamide and analogues; a matter of dose and schedule for dual anticancer activities. Cancer Lett. 2024;598:217119. https://doi.org/10.1016/j.canlet.2024.217119
3. 3. Fraiser LH, Kanekal S, Kehrer JP. Cyclophosphamide toxicity: characterising and avoiding the problem. Drugs. 1991;42(5):781–795. https://doi.org/10.2165/00003495-199142050-00005
4. 4. Zarei M, Shivanandappa T. Amelioration of cyclophosphamide-induced hepatotoxicity by the root extract of Decalepis hamiltonii in mice. Food Chem Toxicol. 2013;57:179–184. https://doi.org/10.1016/j.fct.2013.03.028
5. 5. Alrefaei A, Alzahrani M, Alsuhaim S. Cyclophosphamide related toxicity; a systematic review. UMDC. 2022;6(5):740–747. https://doi.org/10.24911/IJMDC.51-1641243189
6. 6. Elrashdy MF, Benomran RA, Gheryani NA, Amer AH. A histopathological study on the effects of cyclophosphamide on the hepatic tissue of female golden hamsters. International Journal of Frontiers in Life Science Research. 2022;3(1):22–29. https://doi.org/10.53294/ijflsr.2022.3.1.0047
7. 7. Zhao G, He F, Wu C, Li P, Li N, Deng J. Betaine in inflammation: mechanistic aspects and applications. Front Immunol. 2018;9:1070. https://doi.org/10.3389/fimmu.2018.01070
8. 8. Day CR, Kempson SA. Betaine chemistry, roles, and potential use in liver disease. Biochim Biophys Acta Gen Subj. 2016;1860(6):1098–1106. https://doi.org/10.1016/j.bbagen.2016.02.001

9. 9. Rehman A, Mehta KJ. Betaine in ameliorating alcohol-induced hepatic steatosis. Eur J Nutr. 2022;61(3):1167–1176. https://doi.org/10.1007/s00394-021-02738-2
10. 10. Mukherjee S. Role of betaine in liver disease—worth revisiting or has the die been cast? World J Gastroenterol. 2020;26(38):5745–5748. https://doi.org/10.3748/wjg.v26.i38.5745
11. 11. Wang C, Ma C, Gong L, Dai S, Li Y. Preventive and therapeutic role of betaine in liver disease: a review on molecular mechanisms. Eur J Pharmacol. https://doi.org/10.1016/j.ejphar.2021.174604
12. 12. Veskovic M, Labudovic-Borovic M, Mladenovic D, Jadzic J, Jorgacevic B, Vukicevic D, et al. Effect of betaine supplementation on liver tissue and ultrastructural changes in mice on methionine-choline deficient diet. Microsc Microanal. 2019;25(2):386–394. https://doi.org/10.1017/S1431927620024265
13. 13. Sharma S, Kaur T, Sharma AK, Singh B, Pathak D, Yadav HN, et al. Betaine attenuates sodium arsenite-induced renal dysfunction in rats. Drug Chem Toxicol. 2022;45(6):2488–2495. https://doi.org/10.1080/01480545.2021.195969 9
14. 14. Temel Y, Kucukler S, Yildirim S, Caglayan C, Kandemir FM. Protective effect of chrysin on cyclophosphamide-induced hepatotoxicity and nephrotoxicity via the inhibition of oxidative stress, inflammation, and apoptosis. Naunyn Schmiedebergs Arch Pharmacol. 2020;393(3):325–337. https://doi.org/10.1007/s00210-019-01741-z
15. 15. Placer ZA, Cushman LL, Johnson BC. Estimation of product of lipid peroxidation (malonyl dialdehyde) in biochemical systems. Anal Biochem. 1966;16(2):359–364. https://doi.org/10.1016/0003-2697(66)90167-9
16. 16. Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem. 1968;25(1):192–205. https://doi.org/10.1016/0003-2697(68)90092-4
17. 17. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193(1):265–275. https://doi.org/10.1016/S0021-9258(19)52451-6
18. 18. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001;25(4):402–408. https://doi.org/10.1006/meth.2001.1262
19. 19. Dhouibi R, Affes H, Ben Salem M, Charfi S, Marekchi R, Hammami S, et al. Protective effect of Urtica dioica in induced neurobehavioral changes, nephrotoxicity and hepatotoxicity after chronic exposure to potassium bromate in rats. Environ Pollut. 2021;287:117657. https://doi.org/10.1016/j.envpol.2021.117657
20. 20. Karaca O, Akaras N, Simsek H, Gur C, Ileriturk M, Kucukler S, et al. Therapeutic potential of rosmarinic acid in tramadol-induced hepatorenal toxicity: modulation of oxidative stress, inflammation, RAGE/NLRP3, ER stress, apoptosis, and tissue function parameters. Food Chem Toxicol. 2025;197:115275. https://doi.org/10.1016/j.fct.2025.115275
21. 21. Alqahtani S, Mahmoud AM. Gamma-glutamylcysteine ethyl ester protects against cyclophosphamide-induced liver injury and hematologic alterations via upregulation of PPARgamma and attenuation of oxidative stress, inflammation, and apoptosis. Oxid Med Cell
22. 22. Golmohammadi MG, Banaei S, Timar M, Abedi A. Saponin protects against cyclophosphamide-induced kidney and liver damage via antioxidant and anti-inflammatory mechanisms. Physiol Int. 2023;110(2):108–120. https://doi.org/10.1556/2060.2023.00190
23. 23. Bal TT, Akaras N, Demir O, Ugan RA. Protective effect of astaxanthin and metformin in the liver of rats in which the polycystic ovary syndrome model was formed by giving letrozole. Iran J Basic Med Sci. 2023;26(6):688–694. https://doi.org/10.22038/IJBMS.2023.68032.1487 2
24. 24. Kankilic NA, Simsek H, Akaras N, Gur C, Ileriturk M, Kucukler S, et al. Protective effects of naringin on colistin-induced damage in rat testicular tissue: modulating the levels of Nrf-2/HO-1, AKT-2/FOXO1A, Bax/Bcl-2/Caspase-3, and Beclin-1/LC3A/LC3B signaling pathways. J Biochem Mol Toxicol. 2024;38(2):e23643. https://doi.org/10.1002/jbt.23643
25. 25. Gencer S, Akaras N, Simsek H, Gür C, İleritürk M, Küçükler S, et al. The protective effects of chrysin on acrylamide-induced hepatotoxicity: insights into oxidative stress, inflammation, apoptosis and autophagy, and histological evaluation in rats. J Biochem Mol Toxicol. 2025;39(6):e70334. https://doi.org/10.1002/jbt.70334
26. 26. Gencer S, Gur C, Ileriturk M, Kucukler S, Akaras N, Simsek H, et al. The ameliorative effect of carvacrol on sodium arsenite-induced hepatotoxicity in rats: possible role of Nrf2/HO-1, RAGE/NLRP3, Bax/Bcl-2/Caspase-3, and Beclin-1 pathways. J Biochem Mol Toxicol. 2024;38(10):e23863. https://doi.org/10.1002/jbt.23863
27. 27. Gencer S, Simsek H, Akaras N, İleritürk M, Gür C, Küçükler S, et al. Hesperidin ameliorates vancomycin-induced kidney injury via multipathway modulation: Nrf-2/HO-1, Caspase-3/Bax/Bcl-2, ATF-4, KIM-1 and improved renal tissue function. Biochem Pharmacol. 2025;240:117131. https://doi.org/10.1016/j.bcp.2025.117131
28. 28. Gonzale z-Sancho J, Aguilera O, Garcia J, Pendas-Franco N, Pena C, Cal S, et al. The Wnt antagonist DICKKOPF-1 gene is a downstream target of beta-catenin/TCF and is downregulated in human colon cancer. Oncogene. 2005;24(6):1098–1103. https://doi.org/10.1038/sj.onc.1208303
29. 29. Monga SP, Pediaditakis P, Mule K, Stolz DB, Michalopoulos GK. Changes in WNT/beta-catenin pathway during regulated growth in rat liver regeneration. Hepatology. 2001;33(5):1098–1109. https://doi.org/10.1053/jhep.2001.23786
30. 30. Zaghloul R, Elsherbiny N, Kenawy H, El-Karef A, Eissa L, El-Shishtawy M, et al. Hepatoprotective effect of hesperidin in hepatocellular carcinoma: involvement of Wnt signaling pathways. Life Sci. 2017;185:114–125. https://doi.org/10.1016/j.lfs.2017.07.026