Oxidative Stress, Induced by Sub-Lethal Doses of BDE 209, Promotes Energy Management and Cell Cycle Modulation in the Marine Fish Cell Line SAF-1

Abstract

The effects of sub-lethal doses of polybrominated diphenyl ether (PBDE)-209 in terms of toxicity, oxidative stress, and biomarkers were evaluated in the Sparus aurata fibroblast cell line (SAF-1). Vitality and oxidative stress status were studied after incubation with PBDE for 72 h. Concomitantly, the quantification of proteins related to cell cycle and DNA repair (p53), cell proliferation (extracellular signal–regulated kinase 1 (ERK1)), energetic restriction (hypoxia-inducible factor 1 (HIF1)), and redox status (Nuclear factor erythroid 2–related factor 2 (NRF2)) was also determined after prolonged exposure (7–15 days) by immunoblotting. Our results demonstrated that rising concentrations of PBDEs exposure-induced oxidative stress, and that this event modulates different cell pathways related to cell cycle, cell signaling, and energetic balance in the long term, indicating the negative impact of sub-lethal dose exposure to cell homeostasis.

Sub-lethal Doses of Polybrominated Diphenyl Ethers, in Vitro, Promote Oxidative Stress and Modulate Molecular Markers Related to Cell Cycle, Antioxidant Balance and Cellular Energy Management

“…Since the toxic mechanism of PBDEs has been suggested to be mediated by the oxidative damage [36,52,53,68,112], we hypothesized that low concentrations of PBDEs might also activate ‘adaptive responses’ through oxidative stress-related signaling such as NRF2 and metabolism adaptation by HIF modulation, that can promote proliferative pathways.…”

“…The cell suspension in completed medium was dispensed in a 96 multiwell plate at a density of 8000 cells/well and incubated, as described above, for 24 h before the exposure to the compounds. The PBDEs standards were provided by SPECTRA (Rome, Italy); stock solutions were prepared by dissolving the powder compounds in dimethyl sulfoxide (DMSO) and then diluting in complete medium (final concentration of DMSO 0.1%), in order to realize the various concentrations: BDE-209 was tested from 0.25 to 2 µmol/L, BDE-47 and 99 from 1 to 100 µmol/L (BDE-209 was tested at lower concentrations respect to the others two compounds, due to its lower solubility [53]). Cells were then incubated for 72 h and the toxicity of the compounds were determined using the (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT, Sigma-Aldrich) assay according to Mosmann [54], as reported in Messina et al [54].…”

“…Among these, we monitored: 1) the protein p53, known as ‘the genome guardian’, that plays a main role in maintaining genetic stability integrity [44]; 2) the retinoblastoma protein pRB, a tumor suppressor that contributes to the cell cycle progression [45]; 3) the poly(ADP-ribose) polymerase (PARP), that is able to detect the DNA damage caused by many factors and permits the DNA repair [46]; 4) the extracellular signal-regulated kinase (ERK), also known as mitogen-activated protein kinase (MAPK), [47,48]; 5) c-Jun and 6) c-Fos, which are involved in oxidative stress-promoted apoptosis [49]; 7) adenosine 5′-monophosphate-activated protein kinase (AMPK), a serine/threonine protein kinase that serves as the most important energy sensor in the regulation of cellular metabolism [50]; 8) the hypoxia inducible factor (HIF), that regulates the central metabolism in relation to the oxygen availability, promoting also angiogenesis [51]; 9) the nuclear factor erythroid-2-related factor-2 (NRF-2), a redox sensitive transcription factor, which plays an important role in defending cells against oxidant stress [52]. In view of all these properties, the evaluation of these keys proteins could indicate the interference of PBDEs on cell homeostasis, energy management, antioxidant balance and cell cycle progression, giving indication on perturbation of standard pathways and functioning as an early warning system, to detect deviations that can compromise the normal life cycle, as we have reported in a recent study carried on a fish cell line [53]. Since most of the in vitro studies evaluated the effects of different PBDEs in acute exposure, in this study we also evaluated, in addition to a preliminary exposure to a wide range of concentrations for a short time, the effects of an exposure to sub-lethal doses, for a longer time, on biochemical changes.…”

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“…Linhartova et al (2015) encontraron daños en membranas celulares, proteínas y ADN, así como en la actividad de SOD en espermatozoides del esturión por efecto del TBBFA (0.5-10 µg/L por 2 horas). Respecto a los EDPB, se encontró que los congéneres BDE-47, -99 y -209 fueron capaces de incrementar las concentraciones de ROS, disminuir el potencial de membrana mitocondrial e incrementar la expresión de la proteína NRF-2 que se encuentra relacionada con la activación de las defensas antioxidantes enzimáticas (Shao et al, 2008;Espinosa-Ruiz et al, 2019a;Espinosa-Ruiz et al, 2019b;Zhou et al, 2019;Espinosa-Ruiz et al, 2020). Estos estudios son sumamente relevantes ya que permiten identificar posibles mecanismos de producción de ROS a nivel celular.…”

“…Adicionalmente otra de las especies de peces más estudiadas fue el pez dorado (C. auratus), particularmente en los peces que fueron dosificados por vía intraperitoneal (Shi et al, 2005;Feng et al, 2013a;2013b;Yang et al, 2013;Feng et al, 2014;Xie et al, 2014;Dong et al, 2018). Con menor frecuencia se estudiaron otras especies de peces con fines comerciales como la trucha arcoíris, el bacalao, y la dorada (Ronisz et al, 2004;Olsvik et al, 2009;Giraudo et al, 2017;Espinosa-Ruiz et al, 2019a;2019b; y otras de especies de peces de origen asiático, con una sola mención como Gobiocypris rarus, Oryzias a algún factor en particular, como ampliamente ha sido descrito en las respuestas de biomarcadores en peces silvestres (Celander, 2011); no obstante, los estudios que consideran mezclas de contaminantes permiten realizar aproximaciones a los fenómenos que ocurren en el ambiente, lo que representa una importante ventaja de este enfoque. En los estudios in vivo que reportaron el estrés oxidativo de peces por efecto de los RFB en mezcla, solamente fueron realizados por vía intraperitoneal y a través del alimento (Feng et al 2013a;Xie et al 2014;Kling et al 2008;Bearr et al 2010;Ghosh et al 2013).…”