Identification of Metabolic Markers as Potential Early Predictors of PFAS- Induced Immunotoxicity

Abstract

Since the 1940s, industries and consumer products have utilized manufactured chemicals known as per- and polyfluoroalkyl substances (PFAS). They can be found in consumer products such as clothing, food packaging, cookware, cosmetics, carpet, and fire-fighting foam. PFAS are often referred to as “forever chemicals” because they break down very slowly and can accumulate in people, animals, and the environment over time. Unfortunately, many studies have shown that PFAS exposure has toxicity and can lead to many health defects and immune dysfunction. Moreover, experimental animal and human studies reveal that legacy long-chain PFAS (perfluorooctanoic acid, PFOA, and perfluorooctanesulfonic acid, PFOS) suppress both the T-cell-dependent (TDAR) and T-cell-independent antibody responses (TIAR). Previous studies have demonstrated that exposure to PFAS is associated not only with suppression of the TDAR but also with changes in overall B cell numbers, suggesting disruptions in B cell differentiation. In this study, we investigated how PFAS exposure alters mitochondrial bioenergetics in activated B cells to understand potential PFAS-mediated B cell immunotoxicity mechanisms. We examined five PFAS compounds: PFOA and four short-chain PFAS, perfluoro-2-methoxyacetic acid (PFMOAA), 3,5,7,9-butaoxadecanoic acid (PFO4DA), perfluoro (3,5-dioxahexanoic acid; PFO2HxA), and perfluoro-3,6,-dioxa-4-methyl-7-octensulfonic acid (Nafion Byproduct 1; NBP1). Adult male and female C57BL/6 mice received daily oral doses of PFMOAA (0, 50 mg/kg), PFOA (0, 7.5 mg/kg), PFO4DANa, PFO2HxA, or NBP1 (0 or 5 mg/kg) for 30 days. Naïve B cells were isolated from spleens by negative bead selection and stimulated ex vivo using anti-CD40 and IL-4. After 24 hours in culture, mitochondrial function was assessed by measuring the oxygen consumption rate (OCR), including basal, maximal, and reserve capacities. Exposure to these PFAS altered OCR under basal and high-energy demand conditions. While exposure didn’t affect the ability of B cells to respond to stimulation, it affected the capacity of this stimulation response. These shifts in mitochondrial activity upon exposure may impact the ability of B cells to differentiate, which may affect functional immune responses, including antibody production. The second part of this study aimed to link in vivo and in vitro immunotoxicity results by exploring use of the CH12.LX B cell line as a basis for screening agents for immunotoxicity. This could provide a simple, rapid, and cost-effective screening assay for immunotoxicants. After optimizing assay conditions, CH12.LX B cells were stimulated with 2.5 µM LPS, 1 µM cyclosporin A (CsA), and/or PFOA (50–200 µM), and mitochondrial function was assessed in glucose versus glucose-free (galactose) medium. Under glucose-rich conditions, the cells relied on both glycolysis and oxidative phosphorylation for ATP production, potentially masking mitochondrial dysfunction. However, under glucose-free conditions, the reliance on mitochondrial ATP production revealed a significant increase. After 3 days of exposure, cells treated with 50 and 100 µM PFOA showed a decrease (p < 0.0001) in maximum and spare respiratory capacity. After 5 days, mitochondrial dysfunction was observed at all PFOA concentrations (50-200 µM). These findings demonstrate that immunosuppressive chemicals, such as PFOA, can directly impair mitochondrial function in B cells, which could lead to suppression of the immune response. It also suggests that detecting shifts in mitochondrial bioenergetic profiles shows promise for an immunotoxicity screen.