Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Nayeli K. Sanchez - Eastern Michigan University
Co-Author(s): Apurva Lad, The University of Toledo, Toledo, OH; Fatimah Khalaf, The University of Toledo, Toledo, OH; Andrew Kleinhenz, The University of Toledo, Toledo, OH; Steven T. Haller, The University of Toledo, Toledo, OH; David J. Kennedy, The University of Toledo, Toledo, OH.
Microcystin-LR (MC-LR) is a hepatotoxin produced by cyanobacteria and has known negative effects on human health. It poses a risk of contaminating municipal drinking water throughout the United States due to its presence in freshwater systems. Toxicology studies have described its effects in healthy animal models, but the impact of MC-LR on populations with pre-existing liver disease is largely unknown. MC-LR inhibits protein phosphatases 1 (PP1) and 2A (PP2A) which lead to increased protein phosphorylation and cytoskeletal alterations. It has been demonstrated that oxidative stress contributes to the toxicity of MC-LR and that the formation of excessive free radical species from oxidative lipid alterations increases lipid peroxidation in mice serum. Since PP2A regulates several mitogen-activated protein kinases (MAPKs), its inhibition by MC-LR may affect downstream signaling pathways involved in essential cellular processes and inflammatory responses. In this study, we investigated the mechanisms of liver injury due to MC-LR and tested the hypothesis that long-term oral exposure to MC-LR exacerbates hepatotoxicity in a well-accepted Non-alcoholic Fatty Liver Disease (NAFLD) murine model of pre-existing liver disease. Similar to a study done by Sedan et al., three groups of Lepr db/J mice were administered MC-LR by gavage every 48 hours in concentrations of 0, 50 and 100 µg/Kg over 4 weeks for a total of 15 administrations based on the current No Observed Adverse Effect Level (NOAEL) of 40 µg/Kg. Negative controls were vehicle-treated Lepr db/J mice and untreated wild-type C57 mice. Forty-five phosphoproteins were validated using a phospho-kinase array and the relative mRNA expression of several proteins was determined. We found significant (Unpaired T-test) differences in expression of key signaling proteins in livers with increased expression of STAT5A and p53 in the 100 µg/Kg group. Since MC-LR affects MAPKs and contributes to the phosphorylation of downstream signaling pathways such as that of p53, our results suggest that apoptosis may be a form of MC-LR induced hepatic injury caused by increased phosphorylation of p53 at serine residues. The expression of AMPα2 was significantly (Unpaired T-test) decreased in the 50 ug/Kg group, indicating a response to endoplasmic reticulum stress. In another experiment, total mouse liver RNA was reverse transcribed into cDNA for qPCR analysis of PPARGC-1. The expression of PPARGC-1 was significantly (Unpaired T-test) higher in the 50 µg/Kg group compared to the control or wild type. Since PPARCG-1 is activated by reactive oxygen species and PCG-1 transcriptional coactivators influence inflammation and lipid homeostasis through NF-κB signaling, our results suggest a mechanism for cellular recovery from inflammation caused by MC-LR. To better understand how MC-LR affects vulnerable populations, future studies will evaluate the mechanisms of low-dose MC-LR toxicity in multiple organ systems.Abstract_NayeliSanchez_ERN.docx
Funder Acknowledgement(s): National Science Foundation REU program (DBI-1461124); The Ohio Department of Higher Education; David and Helen Boone Foundation.
Faculty Advisor: Dr. Steven Haller, firstname.lastname@example.org
Role: The prior research for my project was conducted by my mentor Apurva and other members of the laboratory. With support and help from my mentors, I was primarily responsible for the phospho-kinase array and determining the relative mRNA expression of several signaling proteins. Apurva, Andrew and I extracted the total RNA, reverse transcribed it into cDNA, conducted the qPCR analysis and analyzed all data. All laboratory members worked as a team to create the poster and its figures.