Transcriptional and enzymatic responses to oxidative stress induced by the emerging contaminant Gen-X in the hepatopancreas of Procambarus clarkii

The 2,3,3,3-Tetrafluoro-2-(heptafluoropropoxy)propanoate, better known as Gen-X, a recently introduced per- and polyfluoroalkyl substance (PFAS) developed as a substitute for perfluorooctanoic acid (PFOA), has an environmental impact that is still poorly characterised. To investigate its potential ecotoxicological effects, the red swamp crayfish, Procambarus clarkii, was chosen as an experimental model due to its ecological adaptability and relevance as a bioindicator in freshwater ecosystems. Individuals were placed in four tanks, three of which contained increasing concentrations of Gen-X (0.5 μg/L, 1 μg/L, and 10 μg/L) and one control tank; after one month of exposure, the crayfish were dissected, and hepatopancreas as well as other tissues, including ovaries, muscle, and gills, were collected. This study aimed to evaluate the impact of Gen-X exposure on antioxidant responses in the hepatopancreas of P. clarkii, integrating biochemical analyses of enzymatic activities with gene expression studies. Molecular analyses included RNA extraction from the hepatopancreas, DNase treatment, reverse transcription to cDNA, primer design, and real-time PCR. The results showed significant variations in the expression of mt1, mt2, cat, cSe-gpx and mSe-gpx genes. Gene expression analyses were also performed for mMn-sod and cMn-sod, which did not show statistically significant changes. Similarly, enzymatic assays for catalase, glutathione peroxidase, and superoxide dismutase revealed activity changes depending on the Gen-X dose to which the individuals were exposed. Additional biochemical assays, such as the measurement of advanced oxidation protein products and lipid peroxidation, provided further insights into the relationship between Gen-X concentration and oxidative stress, confirming a dose-dependent effect.