Genotoxic damage of benzo[a]pyrene in cultured sea bream (Sparus aurata L.) hepatocytes: Harmful effects of chronic exposure

Pastore, Anna Selene; Santacroce, Maria Pia; Narracci, Marcella; Cavallo, Rosa Anna; Acquaviva, Maria Immacolata; Casalino, Elisabetta; Colamonaco, Michele; Crescenzo, Giuseppe

doi:10.1016/j.marenvres.2014.04.003

Cited by 7 publications

(6 citation statements)

References 36 publications

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“…This reinforces the gene expression results, implying that even after exposure to toxic doses of benzo(a)pyrene, surviving cells remain capable of responding. These findings are supported by previous work with primary sea bream hepatocytes in monolayer, which showed induction of apoptosis after 24 h exposure to benzo(a)pyrene (39.6 pM–39.6 μM), followed by cell death over 72 h and proliferation of surviving cells (Pastore et al, 2014). …”

Section: Discussionsupporting

confidence: 89%

A 3D fish liver model for aquatic toxicology: Morphological changes and Cyp1a induction in PLHC-1 microtissues after repeated benzo(a)pyrene exposures

et al. 2017

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To identify the potential environmental impacts of aquatic pollutants, rapid and sensitive screening tools are needed to assess adaptive and toxic responses. This study characterizes a novel fish liver microtissue model, produced with the cell line PLHC-1, as an in vitro aquatic toxicity testing platform. These 3D micro-tissues remain viable and stable throughout the 8-day testing period and relative to 2D monolayers, show increased basal expression of the xenobiotic metabolizing enzyme cytochrome P450 1A (Cyp1a). To evaluate pulsed, low-dose exposures at environmentally relevant concentrations, microtissue responsiveness to the model toxicant benzo(a)pyrene was assessed after single and repeated exposures for determination of both immediate and persistent effects. Significant induction of Cyp1a gene and protein expression was detected after a single 24 h exposure to as little as 1 nM benzo(a)pyrene, and after a 24 h recovery period, Cyp1a expression declined in a dose-dependent manner. However, cell death continued to increase during the recovery period and alterations in microtissue architecture occurred at higher concentrations. To evaluate a pulsed or repeated exposure scenario, microtissues were exposed to benzo(a)pyrene, allowed to recover, then exposed a second time for 24 h. Following pre-exposure to benzo(a)pyrene, cyp1a expression remained equally inducible and the pattern and level of Cyp1a protein response to a second exposure were comparable. However, pre-exposure to 1 μM or 5 μM of benzo(a)pyrene resulted in increased cell death, greater disruption of microtissue architecture, and alterations in cell morphology. Together, this study demonstrates the capabilities of this PLHC-1 microtissue model for sensitive assessment of liver toxicants over time and following single and repeated exposures.

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Section: Discussionsupporting

confidence: 89%

A 3D fish liver model for aquatic toxicology: Morphological changes and Cyp1a induction in PLHC-1 microtissues after repeated benzo(a)pyrene exposures

et al. 2017

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“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

Section: Fish Acclimatizationmentioning

confidence: 99%

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

Figueiredo

Matos

Diniz

et al. 2021

IJERPH

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Primary cell cultures from wild organisms have been gaining relevance in ecotoxicology as they are considered more sensitive than immortalized cell lines and retain the biochemical pathways found in vivo. In this study, the efficacy of two methods for primary hepatocyte cell isolation was compared using liver from two marine fish (Sparus aurata and Psetta maxima): (i) two-step collagenase perfusion and (ii) pancreatin digestion with modifications. Cell cultures were incubated in L-15 medium at 17 ± 1 °C and monitored for up to six days for cell viability and function using the trypan blue exclusion test, MTT test, lactate dehydrogenase (LDH) activity, and ethoxyresorufin O-deethylase (EROD) activity after Benzo[a]Pyrene exposure. The results showed significant differences between the number of viable cells (p < 0.05), the highest number being obtained for the pancreatin digestion method (average = 4.5 ± 1.9 × 107 cells). Moreover, the hepatocytes showed solid adherence to the culture plate and the rounded shape, changing into a triangular/polygonal shape. The cell viability and function obtained by pancreatin digestion were maintained for five days, and the EROD induction after exposure to the B[a]P showed that cells were metabolically active. This study shows that the optimized pancreatin digestion method is a valid, cost-effective, and simple alternative to the standard perfusion method for the isolation of primary hepatocytes from fish and is suitable for ecotoxicological studies involving marine pollutants, such as PAHs.

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“…5–7 This study focuses on the 5-ring PAH benzo(a)pyrene, which is on the US Environmental Protection Agency’s priority pollutant list and a known carcinogen. 8, 9 Like all PAHs, benzo(a)pyrene is a hydrophobic molecule with low solubility in water, driving it to adsorb onto hydrophobic materials suspended in sediment or water and to partition into the lipophilic compartments of an organism or cell. 10, 11 Sediment-bound PAHs can desorb and remain bioavailable to aquatic organisms, particularly those that live in close contact with contaminated sediments.…”

Section: Introductionmentioning

confidence: 99%

“…14, 15 While benzo(a)pyrene itself is not a mutagen, metabolic activation generates diol-epoxides and quinones that can cause DNA adducts and oxidative stress, leading to cellular damage and toxicity in exposed tissues. 9, 14…”

Section: Introductionmentioning

confidence: 99%

Impact of emerging, high-production-volume graphene-based materials on the bioavailability of benzo(a)pyrene to brine shrimp and fish liver cells

Rodd

Castilho

Chaparro

et al. 2018

Environ. Sci.: Nano

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With increasing commercialization of high volume, two-dimensional carbon nanomaterials comes a greater likelihood of environmental release. In aquatic environments, black carbon binds contaminants like aromatic hydrocarbons, leading to changes in their uptake, bioavailability, and toxicity. Engineered carbon nanomaterials can also adsorb pollutants onto their carbon surfaces, and nanomaterial physicochemical properties can influence this contaminant interaction. We used 2D graphene nanoplatelets and isometric carbon black nanoparticles to evaluate the influence of particle morphology and surface properties on adsorption and bioavailability of benzo(a)pyrene, a model aromatic hydrocarbon, to brine shrimp (Artemia franciscana) and a fish liver cell line (PLHC-1). Acellular adsorption studies show that while high surface area carbon black (P90) was most effective at a given concentration, 2D graphene nanoplatelets (G550) adsorbed more benzo(a)pyrene than carbon black with comparable surface area (M120). In both biological models, co-exposure to nanomaterials lead to reduced bioavailability, with G550 graphene nanoplatelets cause a greater reduction in bioavailability or response than the M120 carbon black nanoparticles. However, on a mass basis the high surface area P90 carbon black was most effective. The trends in bioavailability and adsorption were consistent across all biological and acellular studies, demonstrating the biological relevance of these results in different models of aquatic organisms. While adsorption is limited by surface area, 2D graphene nanoplatelets adsorb more benzo(a)pyrene than carbon black nanoparticles of similar surface area and charge, demonstrating that both surface area and shape play important roles in the adsorption and bioavailability of benzo(a)pyrene to carbon nanomaterials.

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Genotoxic damage of benzo[a]pyrene in cultured sea bream (Sparus aurata L.) hepatocytes: Harmful effects of chronic exposure

Cited by 7 publications

References 36 publications

A 3D fish liver model for aquatic toxicology: Morphological changes and Cyp1a induction in PLHC-1 microtissues after repeated benzo(a)pyrene exposures

A 3D fish liver model for aquatic toxicology: Morphological changes and Cyp1a induction in PLHC-1 microtissues after repeated benzo(a)pyrene exposures

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

Impact of emerging, high-production-volume graphene-based materials on the bioavailability of benzo(a)pyrene to brine shrimp and fish liver cells

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