Application of quantitative transcriptomics in evaluating the ex vivo effects of per- and polyfluoroalkyl substances on Atlantic cod (Gadus morhua) ovarian physiology. Reproductive toxicity of PFASs in fish

A 2013 Norwegian environment screening project reported that the levels of preselected per- and polyfluoroalkyl substances (PFASs) were high in marine biota (seal liver, plasma, and eggs of marine birds and polar bear plasma), compared to abiotic (marine water and sediments) samples. This high rate of bioaccumulation have been shown to interfere with normal biological functions. Reduced fertility and fecundity caused by these compounds are among the health concerns reported earlier in humans, zebrafish, copepod, and nematode. However, there are knowledge gaps about the underlying biological mechanisms, processes and pathways responsible for abnormal reproductive outcomes after exposures to these group of compounds. In the present work, we employed ex vivo organ culture technique based on the agarose floating method in exposing cod ovarian tissue to three concentrations of perfluorooctanesulfonic acid (PFOS), perfluorononanoic acid (PFNA), given individually at 1, 5 and 25 µM and perfluorooctanoic acid (PFOA) at 1, 5 and 25 µM. A mixture exposure concentrations, based on the proportional presence of these compounds relative to PFOS (which is the most abundant compound in cod liver) as reported in the 2013 screening project. The concentration of PFOS in low mixture exposure was based on environmental realistic levels, as reported in Bratberg et al. (2013). The other two concentrations, medium and high were 20 and 100 times higher the low concentration mixture. To investigate the underlying processes in detail, transcriptome sequencing (Illumina HiSeq 4000) was performed on exposed ovarian tissue. The number of differentially expressed genes (DEGs), with 0.75 log2-fold differential expression was comparatively larger in high, compared to low and medium concentration, for both individual and mixture exposures. A high exposure concentration of PFNA, PFOA, PFOS, and mixture contained, respective 40, 68, 1295 and 802 DEGs. The PFOS and 100x mixture exposure groups shared a maximum of 438 DEGs. In addition, these two groups shared the majority of functionally enriched pathways, biological processes, and molecular functions. The gene ontology analysis identified biological functions belonging to cell adhesion, cytoskeleton remodelling, cell cycle, apoptosis, lipid metabolism, protein modification, development, immunological responses, signalling pathways and cancer. In addition, steroid hormone biosynthesis and progesterone mediated oocyte maturation pathways were significantly affected by high PFOS and high mixture exposure, respectively. Overall, PFOS, despite its high bioaccumulation properties also altered the biological functioning of female gonads that may lead to serious reproductive consequences in teleost.