Effects of Benzo[a]pyrene on Gilthead Sea Bream (Sparus AurataL.) Hepatocytes Exposedin Vitroto Short and Long Term Trials

Abstract

In the present study, cytotoxic effects of the polycyclic aromatic hydrocarbons benzo [a] pyrene (B[a]P) were investigated in Sparus aurata hepatocytes primary cultures after acute and chronic exposure. Cells were treated with a wide range of B[a]P doses (1 pg/mL to 100 µg/mL) for 24, 48 and 72 h. B[a]P toxicity was quantified in sea bream hepatocytes by MTT assay and immunofluorescence analysis of apoptosis after the various exposure periods, in order to evaluate the hepatic damage and toxicity range. Results showed three cytotoxic responses: B[a]P cell death for primary necrosis after exposure to high concentrations for short times, apoptosis induction with the use of sublethal doses and cell proliferation allied with neoplastic foci formation after exposure to low concentrations for long times. This responses provided an interesting correlation between the damage caused on hepatocytes and the metabolism of this toxic compound, to date mainly studied in vivo. Additionally, the statistical analysis revealed that the effects of time and dose were significant for both parameters and especially the time was extremely significant (P<0.0001), in fact B[a]P induced damage that increased over time. Our findings demonstrated and confirmed that S. aurata is a very sensitive species to B[a]P exposure since adverse effects were found at all tested doses. Furthermore, the new in vitro animal model can be considered a useful tool for studying the cellular effects induced by any contaminant harmful for farmed fish. This work is licensed under a Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). ©Copyright V.

Primary hepatocytes from Arctic char (Salvelinus alpinus) as a relevant Arctic in vitro model for screening contaminants and environmental extracts

“…Interestingly the EC50 for metabolic activity after exposure to BAP was 6 times lower than the EC50 for membrane integrity, which could indicate that BAP specifically target mitochondrial functions. This is supported by findings that exposure to BAP induced formation of reactive oxygen species (ROS) via CYP1A metabolism, resulting in harmful BAP diones which may cause cytotoxicity (full review see Verma et al, 2012) and potentially result in apoptosis (programmed cell death) at low concentrations and necrosis at high concentrations (Zacchino et al, 2013). No difference in the EC50 for metabolic activity and membrane integrity was observed after exposure to BPA in this study.…”

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

“…We have previously shown by MTT assay that at lower doses of B[a]P, identified by increased cell viability, signs of apoptosis and cell damage in a high percentage of viable cells were reported (Zacchino et al, ). Additionally, we found that the cleaved caspase‐3 immunoreactivity was more frequently identified in proliferating oval cells than in non‐proliferating hepatocytes, suggesting an involvement in promoting transformation of oval cells, conceivably by allowing cells to evade the apoptotic process.…”

“…Afterward, we chose to restrict the genotoxicity investigation to the lowest doses of B[a]P, from 4 to 0.004 nM, which is the hypothetic genotoxic range as previously documented (Zacchino et al, ), to investigate the relation between apoptosis, inflammation, and cancer, by evaluating the genotoxic damage in combination with the cytotoxic assessment for a better understanding of the kind of response triggered by chronic exposure to the lowest B[a]P concentrations.…”

“…At present, most of the toxicological data come from wild fish, and very little is known on B[a]P toxicity in sea bream ( Sparus aurata L.), the most economically important sparid species cultured along the Mediterranean costs. According to the only two studies published on B[a]P toxicity in sea bream, one evaluating the in vivo genotoxic potential and one providing the first in vitro preliminary toxicity range, S. aurata was found to be a very sensitive species to acute B[a]P exposure (Banni et al, ; Zacchino et al, ). In S. aurata intraperitoneally injected with 20 mg/kg B[a]P, B[a]P genotoxic potential was demonstrated soon after 24 h exposure to B[a]P, with a significant increase in the DNA damage evaluated as a strong strand fragmentation in the nucleus of liver cells (Banni et al, ).…”

Using ovarian and brain cell culture from the Mullet, Liza klunzingeri, to assess the inhibitory effects of benzo[a]pyrene on aromatase activity

“…B a P at the lowest concentration caused toxic effects on the treated cells and its toxicity increased with increasing B a P concentration. Zacchino, Centoducati, Narracci, Selvaggi, and Santacroce () reported significant changes in the cultivated brain, liver, ovary, and kidney cells from Sparus aurata after 24‐h exposure to B a P. Several researchers have stated that prolonged exposure to higher concentration of toxicants leads to a reduction in the number of live cells and a decrease in cellular attachment to the culture plate, resulting in necrosis and cell death (Abdul Majeed et al, ; Rao, ; Weber & Janz, ). The prolonged exposure of the cell to contaminants leads to disturbance in the inhibition of free radicals produced by the contaminants within the cells.…”