Per- and polyfluoroalkyl substances (PFAS) are a group of thousands of heterogeneous synthetic chemicals with a common carbon–fluorine bond in their structure, which determines their hydrophobicity, oleophobicity, resistance to degradation, and thermal stability. (1) These so-called “forever chemicals” have been broadly used in the manufacturing of industrial and household products, and some have been authorized by the Food and Drug Administration (FDA) for limited use in food contact applications. (2) In addition, individuals that have high occupational exposure to PFAS such as firefighters have a higher risk of cancer, including colon cancer. (3) Since the late 1990s, PFAS that contain eight or more carbon atoms in length have raised increasing concerns about their safety on human and animal health. (4,5) Examples of these chemicals include perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA). PFAS have been frequently detected in the environment (e.g., drinking water, indoor dust, cleaning products, coatings) and the human body (6) and have been associated with the development of adverse health effects and diseases including cancer. (7)

Colorectal cancer (CRC) is the third most common cancer and the second cause of cancer death in the world. (8) It is widely known that sporadic CRC frequently arises upon activation of oncogenes such as KRAS (Kirsten rat sarcoma viral oncogene), whose constitutive activation causes aberrant cell growth and promotes cancer metastasis. (9) The most frequent mutations in the KRAS gene are represented by point substitution in codons 12 and 13, which occur in approximately 35–45% of CRC. (10) Beyond its genetic etiology, CRC has been widely associated with environmental risk factors including, but not limited to, diet, alcohol consumption, tobacco smoking, obesity, and interaction with pathogens. (11) The relationship between PFAS exposure and CRC has been investigated only by a few studies. Consequently, there is currently a lack of knowledge about the underlying biological mechanisms of how exposure to PFAS can affect CRC development and progression.

In this regard, metabolomics is gaining interest in PFAS research to identify metabolites of PFAS exposure or biological effect, (12) which could be linked to mechanisms underlying CRC development or progression. Previous studies have shown a differential metabolic profile of CRC based on its tumor localization and across CRC stages I–IV. (13) Levels of specific amino acids (e.g., phenylalanine, valine), carboxylic acids (e.g., lactate), and lipids were shown to change from the early to the later and more aggressive stages of CRC with potential association with tumor metastases. Moreover, the presence of KRAS mutations in CRC was observed to be associated with suppressed expression of genes involved in immune pathways and suppression of iron-dependent cell death (i.e., ferroptosis). (14) To date, no studies have provided detailed profiling of metabolic pathways in CRC after PFOS and PFOA exposure, which is necessary to generate hypotheses regarding their toxicity mechanisms in CRC development and progression.

In this study, we observed that exposure to PFOS and PFOA at a dosage of 10 ?M can induce the migration of CRC spheroids of SW48 KRAS wild-type (WT) and KRAS G12A mutated cells. Initially, we evaluated the cytotoxic concentrations of PFOS and PFOA. Then, the migratory phenotype was analyzed through a wound healing and trans-well migration assay. Untargeted metabolomics was further performed, which revealed changes in fatty acids and amino acid metabolism in both SW48 KRAS WT and G12A cells after exposure to 10 ?M PFOS and PFOA. Finally, the hypothesis that PFOS and PFOA contribute to CRC metastasis was verified through the analysis of the expression of epithelial-mesenchymal transition (EMT)-related markers. In conclusion, this work provides insights into the metabolic response of CRC to PFAS exposure and provides evidence of their metastatic potential (Figure 1)...

Figure 5. Proposed migration related metabolic pathways induced by PFOS and PFOA exposure. Migration typically requires increased energy consumption. Cancer cells respond by enhancing the ?-oxidation of fatty acids and TCA cycle function to produce more ATP. This leads to decreased levels of free fatty acids. The produced ATP was speculated to combine with amino acids through the aminoacyl-tRNA pathway to produce the proteins necessary for cell migration, resulting in reduced levels of free amino acids. As a result, the pathway for synthesizing monoglycerides was downregulated. Elevated levels of intracellular fatty alcohols were also detected, suggesting inhibition of cholesterol synthesis. Furthermore, the pathway for DNA synthesis was also upregulated in response to metastasis.