An in-silico and in-vivo investigation on the effects of acute fumonisin b1 exposure on inflammation and epigenetics in C57BL/6 mice hearts.

Abstract

Fumonisin B1 (FB1), a mycotoxin produced by Fusarium species, is a significant contaminant in cereal grains, posing serious health risks. This pilot study investigated the cardiotoxic effects of acute FB1 exposure on inflammation and epigenetic modifications in C57BL/6 mice. Given the pilot nature of this study, acute exposure was prioritised to establish initial findings. Molecular docking was employed to predict the binding interactions of FB1 with key inflammatory proteins, including tumour necrosis factor-alpha (TNF-α), inducible nitric oxide synthase (iNOS), and nuclear factor kappa B (NF-κB) subunits. In vivo experiments involved treating mice with 5 mg/kg FB1 for 24 hours, followed by heart tissue analysis using quantitative polymerase chain reaction (qPCR), Western blotting, nitric oxide synthase assay (NOS assay), and enzyme linked immunosorbent assay (ELISA) to assess selected gene and protein expression levels of inflammatory markers and DNA methylation. Docking results indicated that FB1 binds to inflammatory proteins TNF-α, iNOS, NF-κB (p65), and NF-κB (p50), potentially altering their function. Gene expression analysis revealed significant downregulation of pro-inflammatory cytokines (TNF-α, interleukin-6 (IL-6), interleukin-1β (IL-1β)) and the anti-inflammatory cytokine interleukin-10 (IL-10), while protein analysis showed an upregulation of these cytokines, suggesting a complex regulatory mechanism. Additionally, FB1 exposure led to increased levels of reactive nitrogen species and significant upregulation of DNA methylation, indicating epigenetic modulation. This study elucidates the cardiotoxic effects of FB1 on mice, emphasizing the intricate interplay between inflammatory pathways and DNA methylation. Molecular docking studies suggest that FB1 may bind to key residues on TNF-α, iNOS, and NF-κB subunits, potentially modulating these proteins' activity and triggering inflammatory responses. In vitro analysis demonstrated significant dysregulation of inflammatory and DNA ethylation-related genes, with a notable upregulation of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and anti-inflammatory mediators (IL-10, Transforming growth factor beta 1 (TGF-β1)) at the protein level. The observed disparity between gene and protein expression could be attributed to several factors, including post-transcriptional and post-translational modifications. These modifications are crucial in biological processes and can cause differences between mRNA and protein levels. Translational regulation, which involves the recruitment of various mRNA species to the ribosome, can lead to a decreased correlation between mRNA and protein amounts. Additionally, the study found contradictory DNA methylation results: global DNA methylation levels were upregulated, indicating hypermethylation, while DNA methylation gene (DNMT) expression was decreased. This suggests a complex interplay between DNA methylation and gene expression, potentially influenced by other regulatory mechanisms like microRNAs. These findings highlight the need for further validation using additional tests, such as Northern blots and microarray assays. Overall, the study underscores FB1's ability to activate inflammatory pathways and cause cardiac distress through cytokine dysregulation and epigenetic changes. Further research is essential to fully understand the mechanisms of FB1- induced cardiotoxicity and the potential therapeutic role of DNA methylation in mitigating these effects.