## 4 Discussion
These results are in line with those of previous _in vitro_ studies that reported enhanced EROD activity after the addition of cortisol to the liver organ culture or microsomal suspension in _A. anguilla_[35]. Using the same _in vitro_ approach as in the present study, cortisol and E\({}_{2}\) (5.997 ng/mL) were able to decrease the EROD activity inhibition by B[a]P [18], thus highlighting the protective and modulatory effect of these two steroid hormones on EROD activity compromised by exposure to PAHs. To the best of our knowledge, no _in vitro_ study has assessed the impact of the presence of E\({}_{2}\) on EROD activity. The other available studies with this hormone and EROD focused on the _in vivo_ effects of this natural estrogen as an aquatic contaminant [15; 36; 37; 38], reporting, in this context, E\({}_{2}\) as a suppressor of EROD activity [15; 38]. Overall, the present study results showed that cortisol and E\({}_{2}\) have the potential to revert the inhibitory effects of 0.9 and 2.7 \(\upmu\)M FL on EROD activity. Cortisol was able to fully recover hepatic EROD activity inhibited by 0.9 \(\upmu\)M FL, whereas E\({}_{2}\), although not promoting a full reversion of the effects, lead to EROD activity that was 45% higher than in the condition with FL alone. However, neither cortisol nor E\({}_{2}\) were able to fully revert the inhibitory effect of 2.7 \(\upmu\)M FL. The data suggest that cortisol may have a stronger protective role on this enzymatic activity. A modulatory and protective effect of steroid hormones over biotransformation enzymes was demonstrated in this study, emphasizing the potential relevance of the organisms' hormonal levels on this enzymatic activity and thus on its reliability as a biomarker of exposure to organic contamination. Further studies are needed to explore other physiologically relevant conditions, such as distinct mixtures of hormones and different hormone concentrations, able to fully recover hepatic EROD activity inhibited by other contaminants (e.g., pharmaceuticals and metals) in order to provide a better understanding of the relation between endocrine regulation and its effective protective role against the toxic mechanism of action of pollutants towards EROD. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app12063098/s1](https://www.mdpi.com/article/10.3390/app12063098/s1)]([https://www.mdpi.com/article/10.3390/app12063098/s1](https://www.mdpi.com/article/10.3390/app12063098/s1)), Table S1. Buffers, solvents and solutions used during Experimental Setup, Table S2. Liver microsomal EROD activity assay procedures to assess effects of cortisol, 17\(\upbeta\)-estradiol (E2) and fluoranthene (FL), alone or in combination. Conceptualization, supervision, resources, methodology, and funding acquisition, M.A.S., M.P., and M.O. ; investigation, data curation, visualization, and writing--original draft preparation, C.S.S.F. ; formal analysis, C.S.S.F. and M.O. ; validation, C.S.S.F., M.A.S., M.P. and M.O. ; writing--review and editing, M.A.S., M.P., and M.O. All authors have read and agreed to the published version of the manuscript. This research was supported by the Centre for Environmental and Marine Studies (CESAM) and the Foundation for Science and Technology (FCT) through national funds (UIDP/50017/2020+UIDB/50017/2020+LA/P/0094/2020). All experimental procedures followed International Guiding Principles for Biomedical Research Involving Animals (EU 2010/63) and were previously approved by the ethics committee and the responsible national legal authority \"Direcao Geral de Alimentacao e Veteriaria\" (authorization N421/2013). **Informed Consent Statement:** Not applicable. **Data Availability Statement:** The data presented in this study are available on request from the corresponding author. **Conflicts of Interest:** The authors declare no conflict of interest.