## 4 Discussion  
Liver EROD activity is considered a valuable biomarker of exposure to organic contamination in fish [9] and is frequently used to assess phase I biotransformation. However, few studies have considered the effects of individual-related factors such as endocrine or hormonal individual status on biotransformation. The interaction between steroid hormones (cortisol and E\({}_{2}\)) and the PAH benzo[a]pyrene (B[a]P) on EROD activity was studied by [PERSON] _et al._[18], who found that these hormones are, to some extent, able to protect EROD activity from inhibition caused by B[a]P. We aimed to investigate whether the protective effect of cortisol and E\({}_{2}\), observed for B[a]P, was also observed for other PAHs. FL was selected based on its reported ability to inhibit EROD activity _in vitro_[7,19]. Environmentally relevant concentrations of FL (0.1, 0.3, 0.9, and 2.7 uM) [18,30] and a physiologically relevant concentration of cortisol and E\({}_{2}\) (5.997 ng/mL) [18] were selected to assess the potential protective effects of these hormones on EROD activity challenged with FL. The data revealed that liver microsomal EROD activity can be increased _in vitro_ in the presence of low concentrations of FL, up to 0.3 uM. However, higher concentrations elicit an opposite response, leading to significant activity inhibition as observed with 0.9 and 2.7 uM concentrations. Other studies have also demonstrated _in vitro_[7] and _in vivo_[19] inhibition of liver EROD activity by FL in _Solea solea_ hepatocytes and in _Fundulus heteroclitus_ isolated microsomes, respectively, although no significant effects on this enzymatic activity after _in vitro_ exposure to FL have been reported [30]. The present study's results support the idea that FL, though able to induce EROD activity in low concentrations, can have a strong inhibitive power in high concentrations. This is most likely associated with an occurrence of non-competitive enzyme inhibition, as suggested by [PERSON] and co-workers [19]. This possible mechanism for EROD inhibition is supported by FL IC50 and respective kinetic analysis [19] along with the lower affinity of FL for the AhR [7] function. This inhibitory potential of FL agrees with other reports showing that PAHs such as B[a]P, which are typically CYP1A inducers, can also act as inhibitors [18,31], especially when occurring in high concentrations [7,18]. The ability of PAHs with a structure and weight similar to FL, such as phenanthrene, to induce EROD activity _in vivo_ was previously reported by [PERSON] _et al._[32] in _Liza aurata_ after short-term exposure to 0.3, 0.9, and  
Figure 2: _In vitro_ effects of fluoranthene (FL) on _A. anguilla_ liver EROD activity in the presence and absence of E\({}_{2}\) (5.997 ng/mL). Bars represent mean \(\pm\) SE (\(n=4\)). Asterisks (*), significant differences between the presence and absence of E\({}_{2}\) (*** \(p<0.001\)); a, differences from control without E\({}_{2}\); b, differences from to control with E2. One-way ANOVA followed by [PERSON]’s post hoc test. 2.7 \(\upmu\)M. However, the available data concerning CYP1A induction in fish by small PAHs (e.g., naphthalene, phenanthrene, pyrene) provided inconclusive and species-dependent results [7; 19; 33; 34]. The present results showed that exposure to environmental levels of FL (0.9 and 2.7 \(\upmu\)M) can affect EROD activity, which may lead to impaired biotransformation and detoxification. This may limit the relevance of its use as a biomarker in environments with high PAH levels. Steroid hormones are endogenous factors with reported functions in regulating the expression of CYP1A [12]. However, there is scarce knowledge on the potential modulatory effect of steroid hormones such as cortisol and E\({}_{2}\) on EROD activity. In this _in vitro_ study, cortisol and E\({}_{2}\) proved to be able to increase EROD activity and decrease the inhibition induced by 0.9 and 2.7 \(\upmu\)M FL.