Date of Award
12-2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biological Sciences
Committee Chair/Advisor
Dr. Charles D. Rice
Committee Member
Dr. Peter van den Hurk
Committee Member
Dr. Yanzhang Wei
Committee Member
Dr. Emily E. Rosowski
Committee Member
Dr. Subham Dasgupta
Abstract
The demand for processed foods has led to increased use of synthetic phenolic antioxidants (SPAs), notably tert-butylhydroquinone (TBHQ), to inhibit lipid oxidation and prolong shelf life. While TBHQ is deemed safe at regulated levels, concerns arise from studies indicating potential adverse effects such as oxidative stress, genotoxicity, and impaired immune function. To investigate TBHQ's immunomodulatory effects, RAW 264.7 murine macrophages were pre-treated with TBHQ and subsequently stimulated with lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (poly I:C, PIC) to simulate bacterial or viral challenges. Results demonstrated that TBHQ at high non-cytotoxic doses reduced nitric oxide and IL-10 production while promoting phagocytosis and IL-6 secretion at lower concentrations. Transcriptomic analyses revealed downregulation of pro-inflammatory signaling pathways and upregulation of Nrf2-related antioxidant and metabolic genes (e.g., Cbr3, Gstp1/3, Hmox1). Post-stimulation with LPS or PIC led to decreased expression of key pro-inflammatory cytokines and signaling genes due to pre-treatment, suggesting that TBHQ’s modulation of Nrf2 pathways underlies its inhibitory effect on inflammation in macrophages. Furthermore, the role of aconitate decarboxylase 1 (ACOD1) in macrophage activation and type I interferon (IFN) signaling was investigated using ACOD1 wildtype and knockout RAW 264.7 macrophages. The absence of ACOD1 resulted in reduced nitric oxide production and diminished expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), particularly following PIC stimulation. Cytokine assays indicated increased IL-6 levels, while IL-10 production remained unchanged. RNA sequencing analysis revealed extensive transcriptional changes in ACOD1 knockout macrophages, with significant downregulation of inflammatory and IFN-related gene networks upon PIC treatment, including Rsad2 and Ifit genes. This identifies ACOD1 as a crucial regulator of type I IFN responses in macrophages, linking its metabolic function to antiviral signaling. Lastly, the environmental and immunotoxic implications of TBHQ are examined, particularly in the aquatic environment, where its effects on fish macrophages have been under-researched. TBHQ had little effect on primary rainbow trout (Oncorhynchus mykiss) anterior kidney macrophage activation by PIC. Another in vitro approach utilized the fish hepatocellular carcinoma cell line PLHC-1, where monoclonal antibodies (mAbs) were generated, characterized, and employed to assess specific protein expressions. mAb AW12 successfully identified the expression of ACOD1 in PLHC-1 lysates, while mAb CX2-3 confirmed the expression of inducible COX-2 at the expected molecular weight. However, mAb AW-N2, tested against recombinant human Nrf2, failed to detect fish-derived Nrf2 under the conditions of the study. The reliability of GAPDH detection was established using mAb GPD, which supports the development of a mAb panel for future investigations. TBHQ suppressed the intrinsic expression level of each of these proteins in PIC-stimulated PLHC-1 cells.
Collectively, these findings establish a mechanistic foundation for understanding how oxidative stress modulators such as TBHQ alter macrophage immune and metabolic function through Nrf2, NF-κB, and ACOD1 signaling. TBHQ-mediated activation of Nrf2 suppresses inflammatory signaling, while ACOD1 drives the metabolic and transcriptional reprogramming of underlying macrophage activation, including type I IFN responses. Together, these insights provide a translational framework for evaluating the immunotoxic potential of environmental contaminants.
Recommended Citation
Whisel, Alyssa M., "The Influence of tert-Butylhydroquinone (TBHQ) on ACOD1/IRG1-Driven Inflammatory Pathways" (2025). All Dissertations. 4172.
https://open.clemson.edu/all_dissertations/4172
Author ORCID Identifier
0009-0002-3564-9486