Increased toxicity and retention of perflourooctane sulfonate (PFOS) in humanized CYP2B6-Transgenic mice compared to Cyp2b-null mice is relieved by a high-fat diet (HFD)

Abstract

PFOS is a persistent, fluorosurfactant used in multiple products. Murine Cyp2b's are induced by PFOS and high-fat diets (HFD) and therefore we hypothesized that human CYP2B6 may alleviate PFOS-induced steatosis. Cyp2b-null and hCYP2B6-Tg mice were treated with 0, 1, or 10 mg/kg/day PFOS by oral gavage for 21-days while provided a chow diet (ND) or HFD. Similar to murine Cyp2b10, CYP2B6 is inducible by PFOS. Furthermore, three ND-fed hCYP2B6-Tg females treated with 10 mg/kg/day PFOS died during the exposure period; neither Cyp2b-null nor HFD-fed mice died. hCYP2B6-Tg mice retained more PFOS in serum and liver than Cyp2b-null mice presumably causing the observed toxicity. In contrast, serum PFOS retention was reduced in the HFD-fed hCYP2B6-Tg mice; the opposite trend observed in HFD-fed Cyp2b-null mice. Hepatotoxicity biomarkers, ALT and ALP, were higher in PFOS-treated mice and repressed by a HFD. However, PFOS combined with a HFD exacerbated steatosis in all mice, especially in the hCYP2B6-Tg mice with significant disruption of key lipid metabolism genes such as Srebp1, Pparg, and Hmgcr. In conclusion, CYP2B6 is induced by PFOS but does not alleviate PFOS toxicity presumably due to increased retention. CYP2B6 protects from PFOS-mediated steatosis in ND-fed mice, but increases steatosis when co-treated with a HFD.

Keywords: CYP2B6; Cytochrome P450; Hepatotoxicity; Non-alcoholic fatty liver disease (NAFLD); Perflourooctane sulfonate (PFOS); Polyunsaturated fatty acids (PUFAs).

Fig. 1:. Survival curve of female mice treated with different concentrations of PFOS via oral gavage.

This survival curve was generated using GraphPad Prism 8. Statistical differences in survival were determined by Gehan-Breslow-Wilcoxon test where ** indicates p < 0.001 (n = 5-7).

Fig. 2:. Concentration-response curve for the inhibition of CYP2B6 by PFOS in comparison to the positive control, nonylphenol.

IC50 and 95% CI values were determined using a sigmoidal dose response least squares fit test performed on GraphPad Prism 7 (n = 3). Multiple Student t-test’s were performed to determine significance between PFOS and Nonylphenol. * indicates a significant difference of p < 0.05.

Fig. 3:. CYP2B6 (A & B) and Cyp2a5 (C & D) hepatic gene and protein expression in male and female mice.

Mice were treated as described in the Materials and Methods and CYP2B6 and Cyp2a5 expression measured by qPCR (n = 4-5) and Western blotting (n = 2-3). ). The values above the Western blots reflect relative change in protein expression compared to 0-PFOS ND. Statistical significance was determined by one-way ANOVA followed by Fisher’s LSD when comparing 3 or more groups (qPCR) and Student’s t-test when comparing two groups (Western blots). For qPCR, ‘p’ indicates difference between PFOS concentrations, ‘g’ indicates difference between gender, ‘d’ indicates difference between diets, ‘b’ indicated difference between genotypes. A letter indicates a p < 0.05, letter w/ * indicates p < 0.01, letter w/ ** indicates p < 0.001 and a letter w/ *** indicates p < 0.0001. * indicates a significant difference of p < 0.05 in Western blots.

Fig. 4:. LC-MS/MS analysis of PFOS concentrations in the serum (A) and liver (B) of hCYP2B6 and Cyp2b-null mice.

Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Fisher's LSD as the post-hoc test (n=4-5). ‘p’ indicates difference between PFOS concentrations, ‘g’ indicates difference between gender, ‘d’ indicates difference between diets, ‘b’ indicated difference between genotypes. A letter indicates a p < 0.05, letter w/ * indicates p < 0.01, letter w/ ** indicates p < 0.001 and a letter w/ *** indicates p < 0.0001.

Fig. 5:. Serum concentrations of Alkaline Phosphatase (ALP) (A) and Alanine Aminotransferase (ALT) (B) from PFOS-treated mice.

Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Fisher's LSD as the post-hoc test (n = 5). ‘p’ indicates difference between PFOS concentrations, ‘g’ indicates difference between gender, ‘d’ indicates difference between diets ‘b’ indicated difference between genotypes. A letter indicates a p < 0.05, letter w/ * indicates p < 0.01, letter w/ ** indicates p < 0.001 and a letter w/ *** indicates p < 0.0001.

Fig. 6:. Principal component analysis (PCA) biplot composed of 19 different parameters compared across the 8 male groups.

Graphical representation of multivariate data. Treatment groups are color coded and the group descriptions are overlaid on the plot.

Fig. 7:. Principal component analysis (PCA) biplot composed of 19 different parameters compared across the 11 female groups.

Graphical representation of multivariate data. Treatment groups are color coded and the group descriptions are overlaid on the plot.

Fig. 8:. Triglyceride content in livers as measured by Oil Red O.

Male ND Cyp2b-null 0-PFOS (A), Male ND hCYP2B6 0-PFOS (B), Male HFD hCYP2B6 0-PFOS (C), Female ND Cyp2b-null 0-PFOS (D), Female ND hCYP2B6 0-PFOS (E), Female HFD Cyp2b-null 0-PFOS (F), Female HFD hCYP2B6 0-PFOS (G), and quantified using Image J Fiji Particle Analysis (H). Images were taken at 400x (0.05mm) magnification. Data are presented as mean ± SEM (n = 3). Statistical significance was determined by one-way ANOVA followed by Fisher's LSD as the post-hoc test. ‘p’ indicates difference between PFOS concentrations, ‘g’ indicates difference between gender, ‘d’ indicates difference between diets, ‘b’ indicated difference between genotypes. A letter indicates a p < 0.05, letter w/ * indicates p < 0.01, letter w/ ** indicates p < 0.001 and a letter w/ *** indicates p < 0.0001.

Fig. 9:. Changes in hepatic gene expression caused by genotype, PFOS, HFD, or a combination of PFOS and a HFD in male and female mice.

Gene expression of Pparg (A), Cyp4a14 (B), and Cd68 (C) in male and female mice was determined by qPCR as described in Material and Methods. Data are presented as mean ± SEM (n = 4-5). Statistical significance was determined by one-way ANOVA followed by Fisher's LSD as the post-hoc test. ‘p’ indicates difference between PFOS concentrations, ‘g’ indicates difference between gender, ‘d’ indicates difference between diets, ‘b’ indicated difference between genotypes. A letter indicates a p < 0.05, letter w/ * indicates p < 0.01, letter w/ ** indicates p < 0.001 and a letter w/ *** indicates p < 0.0001.

Fig. 10:. Changes in hepatic gene expression caused by genotype, PFOS, HFD, or a combination of PFOS and a HFD in male and female mice.

Gene expression of Hmgcr (A), Cpt1a (B), and Srebp1 (C) in male and female mice was determined by qPCR as described in Material and Methods. Data are presented as mean ± SEM (n = 4-5). Statistical significance was determined by one-way ANOVA followed by Fisher's LSD as the post-hoc test. ‘p’ indicates difference between PFOS concentrations, ‘g’ indicates difference between gender, ‘d’ indicates difference between diets, ‘b’ indicated difference between genotypes. A letter indicates a p < 0.05, letter w/ * indicates p < 0.01, letter w/ ** indicates p < 0.001 and a letter w/ *** indicates p < 0.0001.