Co-exposure to PCB126 and PFOS increases biomarkers associated with cardiovascular disease risk and liver injury in mice

Abstract

Polychlorinated biphenyl (PCB)126 and perfluorooctane sulfonic acid (PFOS) are halogenated organic pollutants of high concern. Exposure to these chemicals is ubiquitous, and can lead to potential synergistic adverse effects in individuals exposed to both classes of chemicals. The present study was designed to identify interactions between PCB126 and PFOS that might promote acute changes in inflammatory pathways associated with cardiovascular disease and liver injury. Male C57BL/6 mice were exposed to vehicle, PCB126, PFOS, or a mixture of both pollutants. Plasma and liver samples were collected at 48 h after exposure. Changes in the expression of hepatic genes involved in oxidative stress, inflammation, and atherosclerosis were investigated. Plasma and liver samples was analyzed using untargeted lipidomic method. Hepatic mRNA levels for Nqo1, Icam1, and PAI1 were significantly increased in the mixture-exposed mice. Plasma levels of PAI1, a marker of fibrosis and thrombosis, were also significantly elevated in the mixture-exposed group. Liver injury was observed only in the mixture-exposed mice. Lipidomic analysis revealed that co-exposure to the mixture enhanced hepatic lipid accumulation and elevated oxidized phospholipids levels. In summary, this study shows that acute co-exposure to PCB126 and PFOS in mice results in liver injury and increased cardiovascular disease risk.

Keywords: Cardiovascular disease; Lipid; Liver; PCB126; PFAS; PFOS.

Figure 1.. Hepatic gene expression in mice exposed to PCB126 (0.5 mg/kg), PFOS (250mg/kg), and a mixture of PCB126 (0.5 mg/kg) and PFOS (250mg/kg).

Hepatic expression of inflammation, atherogenic, and redox genes, including Icam1 (A), PAI1 (B), Nrf2 (C), Nqo1 (D), Tnfα (E), and Sele (F). Bars represent mean ± SEM of eight animals in each group. Different subscript letters (a, b, c) indicate statistical significance (p<0.05) by one-way ANOVA and post hoc Tukey’s test.

Figure 2.. Effects of PCB126 (0.5 mg/kg), PFOS (250 mg/kg), and PCB126+PFOS (0.5 mg/kg+250 mg/kg) mixture exposure on liver injury.

(A) H&E staining of hepatic sections established the apparent lipid accumulation (arrow) and inflammatory cell infiltration around the periportal area (arrow head) in the mixture exposed group. (B) Elevated plasma ALT and AST levels were observed in the mixture exposed group. Bars represent mean ± SEM of eight animals in each group. Different subscript letters (a, b, c) indicate statistical significance (p<0.05) by one-way ANOVA and post hoc Tukey’s test.

Figure 3.. Effects of PCB126 (0.5 mg/kg), PFOS (250 mg/kg), and PCB126+PFOS (0.5 mg/kg+250 mg/kg) mixture exposure on markers of cardiovascular disease.

Plasma levels of PAI1, Icam1, and sE-selectin were measured using the MAGPIX system. Bars represent mean ± SEM of eight animals in each group. Different subscript letters (a, b) indicate statistical significance (p<0.05) by one-way ANOVA and post hoc Tukey’s test.

Figure 4.. Effects of PCB126 (0.5 mg/kg), PFOS (250 mg/kg), and PCB126+PFOS (0.5 mg/kg+250 mg/kg) mixture exposure on plasma lipid levels.

Lipids including cholesterol ester (ChE), sphingolipids (Cer, SM), neutral lipids (MG, DG, TG), and phospholipids (PC, PE, PI) were analyzed using UHPLC-Q Exactive mass spectrometer. The normalized peak areas of lipid species in each lipid class were summarized. Bars represent mean ± SEM of eight animals in each group. Different subscript letters (a, b) indicate statistical significance (p<0.05) by one-way ANOVA and post hoc Tukey’s test.

Figure 5.. Effects of PCB126 (0.5 mg/kg), PFOS (250 mg/kg), and PCB126+PFOS (0.5 mg/kg+250 mg/kg) mixture exposure on plasma levels of ceramides and the ratio of ceramide C16/C24.

Ceramides were analyzed using UHPLC-Q Exactive mass spectrometer. Bars represent mean ± SEM of eight animals in each group. * p <0.05 versus control mice, # p <0.05 versus PCB126 exposed mice. Different subscript letters (a, b) indicate statistical significance (p<0.05) by one-way ANOVA and post hoc Tukey’s test.

Figure 6.. Effects of PCB126 (0.5 mg/kg), PFOS (250 mg/kg), and PCB126+PFOS (0.5 mg/kg+250 mg/kg) mixture exposure on liver lipid levels.

Lipids including cholesterol ester (ChE), sphingolipids (Cer, SM), neutral lipids (MG, DG, TG), and phospholipids (PC, PE, PI) were analyzed using UHPLC-Q Exactive mass spectrometer. The normalized peak areas of lipid species in each lipid class were summarized. Bars represent mean ± SEM of eight animals in each group. Different subscript letters (a, b, c) indicate statistical significance (p<0.05) by one-way ANOVA and post hoc Tukey’s test.

Figure 7.. Effects of PCB126 (0.5 mg/kg), PFOS (250 mg/kg), and PCB126+PFOS (0.5 mg/kg+250 mg/kg) mixture exposure on liver OxPLs profiles.

OxPLs were analyzed using UHPLC-Q Exactive mass spectrometer. (A) Heat map showing the top 25 OxPLs that were differentially expressed in different groups identified using ANOVA. Each column corresponds to the OxPLs from an individual mouse, and each row corresponds to a given OxPLs. (B) Variable-importance plot of the top OxPLs identified by random forest analysis. The mean accuracy value decrease is a measure of how much predictive power is lost if a given metabolite is removed or permuted in the random forest algorithm; thus, the more important a metabolite is to classifying samples into time point categories, the further to the right its point is on the graph. *: OxPLs identified by both ANOVA and random forest analysis. ^a: PC(34:2+O) with the retention time at 7.49 min, ^b: PC(34:2+O) with the retention time at 8.33 min, ^c: PC(34:2+2O) with the retention time at 8.77 min, ^d: PC(34:1+2O) with the retention time at 6.47 min, ^e: PC(34:1+2O) with the retention time at 8.59 min. See also Table 1 for detailed information on OxPLs.