## 4 Discussion  
The presented study aimed to assess if environmentally relevant concentrations of B[a]P (0.1, 0.3, 0.9, and 2.7 uM), as well as a physiologically relevant concentration of cortisol and E\({}_{2}\) (5.997 ng/mL), alone or in combination, had a significant impact on EROD activity. This issue is of considerable relevance based on the importance given to this biomarker to assess the presence of environmental contaminants that induce Phase I biotransformation. Since BNF is recognized as a strong inducer of hepatic EROD [8; 29; 30], it was used in this study as a reliable in vivo positive control for EROD activity. Although EROD activity is known to be induced in the presence of PAHs, including B[a]P [31], in vivo inhibition of hepatic EROD activity by B[a]P has previously been reported [32], which suggests inducers may become inhibitors, since B[a]P, as well as other inducers of CYP1A, may also act either by competitive inhibition or by a mechanism-based inactivation, in which the inhibitor is metabolized by the P450 into a product that covalently modifies the active site and thereby inactivates the enzyme [33]. In vitro inhibition of this enzymatic activity has also been reported in _A. anguilla_, after exposure to retene, abiotic acid [30], and heavy metals (e.g., copper, zinc, chromium) [31]. The EROD activity inhibition observed in the present study, with concentrations of B[a]P of 0.1, 0.3, 0.9, and 2.7 uM, can be considered of high environmental relevance, since it falls within the levels detected in natural waters (up to 8.2 uM) [34]. In this perspective, this study proves that EROD activity can be compromised by environmentally relevant levels of known inducers. It is known that fish EROD activity can also be affected by individual-related factors (e.g., reproductive status, hormonal levels) [8] that in turn influence the physiological condition of the fish itself. Limited information is available concerning the effects of cortisol on the activity of CYP1A enzymes. Some of the performed studies have shown an increase of the plasmonic cortisol level as well as of the EROD activity after exposure to contaminants [16; 35], namely PAHs [15]. However, information about the potential modulatory effect of EROD activity by this steroid hormone remains unclear. In this context, the in vitro assays can provide highly relevant information in order to clarify this linkage. In this study, the microsomal in vitro exposure to cortisol demonstrated the ability of this hormone to increase EROD activity and decrease the inhibition induced by B[a]P. Previous in vitro studies had also reported the increment of EROD activity in _A. anguilla_ liver organ culture, when cortisol was added to culture medium [36]. Unlike the in vivo studies that have been performed concerning the effects of E\({}_{2}\) on EROD activity [17; 18; 35; 37; 38], there is a considerable lack of information concerning in vitro studies. On the other hand, most of the performed studies have focused on the toxicity assessment of this natural estrogen itself as a potential aquatic contaminant, reporting, in this context, E\({}_{2}\) as a suppressor of the constitutive CYP1A-associated EROD activity [17; 38]. The results of the present study showed that in vitro liver microsomalexposure to E\({}_{2}\) did not significantly affect EROD activity, which has been previously observed by [PERSON] et al. (2006) [18] with _D. labrax_, in an in vivo study testing similar concentrations of E\({}_{2}\). Our results revealed that E\({}_{2}\) had a protective effect on hepatic EROD activity inhibited by B[a]P.  
The protective effects of steroid hormones (cortisol and E\({}_{2}\)) over EROD activity inhibited by contaminants has been demonstrated for the first time in this work. [PERSON] and [PERSON] (2002) [32] also reported that EROD activity inhibition by B[a]P could be partially reverted, in this case, by another PAH, naphthalene.