Implications for chronic toxicity of benzo[a]pyrene in sea bream cultured hepatocytes: Cytotoxicity, inflammation, and cancerogenesis

Abstract

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.

Effects of benzo[a]pyrene on the blood and liver of Physalaemus cuvieri and Leptodactylus fuscus (Anura: Leptodactylidae)

“…The AhR expression induced P450 thus generating systemic oxidative stress (Dalton et al, 2002), which eventually impaired hematopoiesis (Latif et al, 2010). Nonetheless, in our study leukocyte remained unchanged, which may be related to hemato and cytotoxicity (Santacroce et al, 2015) that probably impaired the leukocyte response. Also, Matiasovic et al (2008) concluded that unchanged lymphocytes, neutrophils, monocytes, and eosinophils may be due to high variances within individual groups, possibly explaining the unchanged leukocyte percentages in this study.…”

Effect of Polyaryl Hydrocarbons on Cytotoxicity in Monocytic Cells: Potential Role of Cytochromes P450 and Oxidative Stress Pathways

“…There are very few reports that account for the toxicity of chronic BaP exposure in human cell lines. For examples, reports of chronic BaP exposure in animal model suggest its association with neurotoxicity [ 30 ], DNA damage [ 31 ], carcinogenicity, and cytotoxicity [ 32 ]. Most of the studies of BaP toxicity have been conducted at higher concentration for an acute period.…”

Marine Fish Primary Hepatocyte Isolation and Culture: New Insights to Enzymatic Dissociation Pancreatin Digestion

“…Several studies have already demonstrated the toxicological response of S. aurata towards B[a]P exposure [30][31][32][33], including the induction of EROD activity in fish both for in vivo and in vitro models [4,19,30,60,61]. To verify the metabolic activity of primary hepatocytes obtained from S. aurata liver by the pancreatin digestion method, the ERODbased assay was used after cell exposure to B[a]P. In fact, after 24 and 48 h of exposure to three different concentrations of B[a]P (0.1, 0.5, and 1 µM), EROD activity increased significantly relative to control cells, showing that S. aurata primary cells were active and able to metabolize this xenobiotic by liver cytochrome P450 dependent-monooxygenases.…”

“…The turbot (Psetta maxima, Linnaeus, 1758) and gilt-headed seabream (Sparus aurata, Linnaeus, 1758) weighing 46.8-75.1 g and 186.7-271.3 g, respectively, were acquired from a local aquaculture farm (Aquinova-Mira and Setúbal, Portugal, respectively). P. maxima is a bottom-dwelling marine fish, and S. aurata is the most economically important pelagic marine sparid fish species cultured along the Mediterranean coast and has been used in ecotoxicological studies [5,6,[30][31][32][33]. The fish were transported to the MARLab laboratories at the Department of Environmental Sciences and Engineering (FCT NOVA) and maintained in controlled conditions (17 ± 1 • C, 34‰ salinity, with a regular photoperiod set at 12 h light/12 h dark).…”