Benzo[a]pyrene (B[a]P) is the most studied dangerous polycyclic aromatic hydrocarbon for its hepatotoxic, carcinogenic, mutagenic, teratogenic, and immunosuppressant effects, which can affect both wild and farmed marine fish through the trophic chain. This study investigated, for the first time, the chronic effects induced in vitro by B[a]P prolonged exposure on gilthead sea bream (Sparus aurata L.) hepatocytes, evaluating the cellular and nuclear latent damage. The purpose was to characterize the kind of B[a]P cyto- and genotoxic damage by morphological and immunocytochemical parameters applied in combination with the use of multiple assay endpoints. In light of our results, the short-term effects at higher B[a]P doses were linked to higher cytotoxicities and necrotic lysis, whereas a sustained inflammatory response at medium-low doses was perceived as a mitochondria-mediated apoptosis, both by surface and nuclear morphological changes. The strong immunoreactivity for the cleaved caspase-3 showed that the labeled cells committed suicide by apoptosis. B[a]P involvement on carcinogenesis comes from prolonged exposure at lower doses, establishing the connection between the escape from apoptosis and the selection of a tumoral phenotype. Cells colabeled with proliferating cell nuclear antigen/caspase-3 within the proliferative foci, were proliferating transformed oval stem cells, which escaped the suicide by apoptosis allowing cancer development. Finally, it was established that sea bream cultured hepatocytes are highly sensitive to chronic B[a]P exposure, as serious genotoxic effects were found even at the lowest doses.

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