Perfluorooctanesulfonic acid exposure leads to downregulation of 3-hydroxy-3-methylglutaryl-CoA synthase 2 expression and upregulation of markers associated with intestinal carcinogenesis in mouse intestinal tissues

Abstract

Perfluorooctanesulfonic acid (PFOS) is a widely recognized environment pollutant known for its high bioaccumulation potential and a long elimination half-life. Several studies have shown that PFOS can alter multiple biological pathways and negatively affect human health. Considering the direct exposure to the gastrointestinal (GI) tract to environmental pollutants, PFOS can potentially disrupt intestinal homeostasis. However, there is limited knowledge about the effect of PFOS exposure on normal intestinal tissues, and its contribution to GI-associated diseases remains to be determined. In this study, we examined the effect of PFOS exposure on the gene expression profile of intestinal tissues of C57BL/6 mice using RNAseq analysis. We found that PFOS exposure in drinking water significantly downregulates mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting ketogenic enzyme, in intestinal tissues of mice. We found that diets containing the soluble fibers inulin and pectin, which are known to be protective against PFOS exposure, were ineffective in reversing the downregulation of HMGCS2 expression in vivo. Analysis of intestinal tissues also demonstrated that PFOS exposure leads to upregulation of proteins implicated in colorectal carcinogenesis, including β-catenin, c-MYC, mTOR and FASN. Consistent with the in vivo results, PFOS exposure leads to downregulation of HMGCS2 in mouse and human normal intestinal organoids in vitro. Furthermore, we show that shRNA-mediated knockdown of HMGCS2 in a human normal intestinal cell line resulted in increased cell proliferation and upregulation of key proliferation-associated proteins such as cyclin D, survivin, ERK1/2 and AKT, along with an increase in lipid accumulation. In summary, our results suggest that PFOS exposure may contribute to pathological changes in normal intestinal cells via downregulation of HMGCS2 expression and upregulation of pro-carcinogenic signaling pathways that may increase the risk of colorectal cancer development.

Keywords: Colorectal carcinogenesis; Environmental pollutants; Gastrointestinal tract; HMGCS2; Ketogenesis; Perfluorooctanesulfonic acid.

Figure 1.. PFOS exposure downregulates HMGCS2 levels.

(A) Schematic of experimental design: seven-week-old male C57BL/6 mice were placed on an irradiated diet supplemented with cellulose (control), inulin or pectin. PFOS exposure occurred through daily oral ingestion of 3 μg/g body weight via drinking water. The mice were maintained on their respective diets for a 7-week period. (B) Volcano plot of differentially expressed genes (DEGs) between mice exposed to PFOS and the control (cellulose) group. Red and green dots represent upregulated and downregulated genes, respectively. (C) mRNA expression levels of HMGCS2 and VEGFR, normalized to GAPDH in intestinal tissues, were measured by qRT-PCR. Data are presented as mean ± SD (**p<0.01). (D) Venn diagram displaying overlapping genes identified in the intestines of mice exposed to PFOS and fed with different diets. (E) Western blot and (F) densitometric quantification analysis of HMGCS2 protein levels on intestinal tissues of mice exposed to PFOS and treated with cellulose (control), inulin- or pectin-supplemented diets. (G) Western blot ,densitometric quantification analysis of HMGCS2 protein levels, and representative images of wild-type mouse intestinal organoids treated with 1 μg/mL of PFOS for 15 days, (H) human intestinal organoids treated with 1 μg/mL of PFOS for 30 days, and (I) normal human colon primary cells treated with 1 μg/mL of PFOS for 6 months (n=3). The levels of HMGCS2 expression were determined based on normalization of each band to β-actin (Scale bar: 40 μm).

Figure 2.. PFOS exposure upregulates expression of proteins associated with intestinal carcinogenesis.

(A) KEGG pathway classification in mice exposed to PFOS and kept on inulin or pectin diets. (B-C) Western blot and densitometric quantification analysis of mouse intestinal tissues. Control (cellulose) versus PFOS (cellulose) groups.

Figure 3.. HMGCS2 knockdown increases cell proliferation and lipid accumulation.

(A) Cell viability of NTC and HMGCS2 knockdown normal human colon primary cells measured by Presto Blue assay. Data are presented as mean ± SD. The one-way ANOVA followed by Dunnett’s post-test was used to analyze statistical significance (**p≤0.01, ***p≤0.001). (B-C) Western blot and densitometric quantification analysis for HMGCS2 and proliferation / carcinogenic markers. At least 3 replicates were performed for each protein (D) Cells were stained with BODIPY FL (green) and Hoescht (blue) before being processed for confocal analysis (Scale bar: 25 μm).

Figure 4.. HMGCS2 mRNA and protein levels are downregulated in CRC.

(A) HMGCS2 mRNA expression in colorectal cancer patient samples from the TCGA dataset, comprising 22 normal tissues and 215 tumor samples. (B) 5-year survival analysis of patients with colorectal cancer based on HMGCS2 protein levels (source proteinatlas.org). (C) Western blot analysis and (D) densitometric quantification of HMGCS2 protein levels in 15 randomly selected CRC cases. Matched normal mucosa (N) and tumor tissues (T). β-actin was used as an endogenous loading control. (E) Distribuition of HMGCS2 immunoreactivity score was analyzed in tumor tissues from patients diagnosed with CRC stage I-IV. The linear mixed model was used to anlyse statistical signifcance. (F) Representative images of HMGCS2 expression in matched normal and tumor tissues.

Figure 5.. The potential mechanisms of how PFOS exposure by drinking water alters intestinal homeostasis.

PFOS exposure in drinking water leads to downregulation of HMGCS2 and upregulation of FASN, β-catenin, c-MYC, mTOR, cyclin-D1 and AKT protein levels. Furthermore, loss of HMGCS2 results in increased cell proliferation and upregulation of CD36, mTOR, cyclin-D1, AKT, ERK and survivin, along with an increase in lipid accumulation. Further studies are warranted to determine if PFOS exposure can increase the risk of disease-associated GI pathology.