Antioxidant Therapy Reverses Hepatotoxicity Induced by Microcystin-LR in a Cellular Model of Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD)

Authors

• Apurva Lad, University of Toledo College of Medicine and Life SciencesFollow
• Jason Kindle, University of Toledo College of Medicine and Life Sciences
• Prajwal Hegde, University of Toledo College of Medicine and Life SciencesFollow
• Gabriel G. Kleer, University of Toledo College of Medicine and Life SciencesFollow
• Andrew L. Kleinhenz, University of Toledo College of Medicine and Life SciencesFollow
• Johnna A. Birbeck, Wayne State University
• Judy Westrick, Wayne State UniversityFollow
• Nicholas J. Peraino, Wayne State UniversityFollow
• Terry D. Hinds, University of KentuckyFollow
• Neeraja Purandare, University of Toledo College of Medicine and Life SciencesFollow
• Andrew M. Fribley, University of Toledo College of Medicine and Life SciencesFollow
• Steven T. Haller, University of Toledo College of Medicine and Life SciencesFollow
• David J. Kennedy, University of Toledo College of Medicine and Life SciencesFollow

Abstract

Microcystin-LR (MC-LR) is a potent hepatotoxin that has been shown to cause liver damage even at doses lower than the established Low Observable Adverse Effect Level (LOAEL) of 200 μg/kg in animal models. We have previously observed that low-dose exposure to MC-LR in animals with diet-induced Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) and subsequent treatment with antioxidants like N-acetylcysteine (NAC) and the Na⁺/K⁺ ATPase-Src kinase inhibitor pNaKtide significantly alleviated hepatic infiltration of immune cells, downregulated markers of inflammation and hepatotoxicity, increased the breakdown of the toxin molecule, and restored phase I and II drug metabolism pathways, including the glutathione pathway. Because the liver is composed of heterogeneous cell types, this study aimed to determine the specific role of hepatocytes in the uptake and metabolism of MC-LR, especially in the setting of MASLD. To address this, we used two well-established hepatocyte cell lines—AML-12 murine hepatocytes and human Hep3B hepatocytes. Preliminary dose comparison studies with AML-12 cells showed that MC-LR at 10 μM concentration showed a significant upregulation in the genetic expression of the markers of hepatotoxicity—OSMR (p ≤ 0.01) and SerpinE (p ≤ 0.0001)—in comparison to Vehicle. Treatment with pNaKtide (1 µM) and/or NAC (10 mM) in the presence of MC-LR significantly reduced the expression of both OSMR (p ≤ 0.0001) and SerpinE (p ≤ 0.01 and p ≤ 0.0001, respectively). To model steatotic hepatocytes characteristic of the MASLD phenotype, Hep3B hepatocytes were first treated with 500 µM of oleic acid (OA) before exposing them to the toxin in the presence and absence of antioxidants. MC-LR exposure, induced markers of inflammation and hepatotoxicity to be elevated significantly in the presence of OA as compared to MC-LR exposure alone. This elevation of the genetic markers of inflammation and hepatotoxicity was significantly attenuated on treatment with pNaKtide (1 µM) and NAC (10 mM). Quantification of human SERPINE1 (PAI1) and 8-OHdG, a stable marker of oxidative stress, in the spent media of Hep3B cells corroborated the trends observed in the genetic markers of hepatotoxicity. These observations support the central role that hepatocytes play in the uptake and metabolism of MC-LR, which is complicated by the presence of MASLD-like conditions and can help in the development of future therapeutic strategies.

Document Type

Article

Publication Date

6-1-2026

Notes/Citation Information

Publisher Copyright: © 2026 by the authors.

Digital Object Identifier (DOI)

10.3390/jox16030076

Archival?

Archival

Repository Citation

Lad, Apurva; Kindle, Jason; Hegde, Prajwal; Kleer, Gabriel G.; Kleinhenz, Andrew L.; Birbeck, Johnna A.; Westrick, Judy; Peraino, Nicholas J.; Hinds, Terry D.; Purandare, Neeraja; Fribley, Andrew M.; Haller, Steven T.; and Kennedy, David J., "Antioxidant Therapy Reverses Hepatotoxicity Induced by Microcystin-LR in a Cellular Model of Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD)" (2026). Markey Cancer Center Faculty Publications. 508.
https://uknowledge.uky.edu/markey_facpub/508