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. 2019 Feb 18;16(4):588.
doi: 10.3390/ijerph16040588.

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

Simona Manuguerra  1 , Cristóbal Espinosa Ruiz  2 , Andrea Santulli  3   4 , Concetta Maria Messina  5
Affiliations

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

Simona Manuguerra et al. Int J Environ Res Public Health. .

Abstract

In the present study, we evaluated the effects of different concentrations of the polybrominated diphenyl ethers (PBDEs) BDE-209, BDE-47 and BDE-99, on the vitality and oxidative stress of a HS-68 human cell culture exposed to the compounds for three days. The results showed that for this exposure time, only the highest concentrations produced a significant vitality reduction and oxidative stress induction (p < 0.05), measured as reactive oxygen species (ROS). Subsequently, in order to verify the effects of sub-lethal doses, cells were exposed for a longer time and data collected, after 12 and 20 days, to study ROS production and some molecular markers related to cell cycle and stress (p53, pRB, PARP, c-Jun and c-Fos), antioxidant status and proliferation (ERK, c-Jun and c-Fos), energy balance (NRF2, AMPK, HIF). Most of the biomarkers were influenced by the treatments, indicating that sub-lethal doses of PBDEs, for longer time, can enhance the production of ROS, altering the energetic metabolism, cell cycle and antioxidant balance, determining possible negative effects on the cell proliferation equilibrium.

Keywords: PBDEs; biomarkers; metabolism; oxidative stress; proliferation.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Cytotoxicity and oxidative stress on HS-68 cells exposed for 48 h to different concentrations of PBDEs: (A) vitality percentage (vs. control) of cells exposed to BDE 209 (0.25–2 µmol/L); (B) vitality percentage (vs. control) of cells exposed to BDE 47 and 99 (1–100 µmol/L).; (C) intracellular ROS production (expressed as relative fluorescence) on cells exposed to BDE 209 (0.25–2µmol/L) and (D) to BDE 47 and 99 (1–100 µmol/L). Bars represent the mean ± SEM (n = 6). Different superscript letters represent statistically significant differences (ANOVA; p ≤ 0.05) between groups.
Figure 2
Figure 2
Cytotoxicity and oxidative stress on HS-68 cells exposed for 20 days to 1 µmol/L BDE 209, 99, 47 and MIX: (A) vitality percentage (vs. control); (B) intracellular ROS production (expressed as relative fluorescence). Bars represent the mean ± SEM (n = 6). Different superscript letters represent statistically significant differences (ANOVA; p ≤ 0.05) vs. control. (C) HS-68 cells after 20 days of treatment (phase contrast microscopy at 20× magnification).
Figure 3
Figure 3
Immunoblotting of p53, pRB, PARP, evaluated on HS-68 cells exposed to 1 µmo/L BDE 209, 99, 47 and MIX for 12 and 20 days. Actin was used as internal control. The images are representative of at least three separate experiments. The relative protein quantification is represented in the graphic (* p < 0.05).
Figure 4
Figure 4
Immunoblotting of ERK, c-Jun and c-Fos, evaluated on HS-68 cells exposed to 1 µmol/L BDE 209, 99, 47 and MIX for 12 and 20 days. Actin was used as internal control. The images are representative of at least three separate experiments. The relative protein quantification is represented in the graphic (* p < 0.05).
Figure 5
Figure 5
Immunoblotting of NRF2, AMPK, HIF, evaluated on HS-68 cells exposed to 1 μmol/L BDE 209, 99, 47 and MIX for 12 and 20 days. Actin was used as internal control. The images are representative of at least three separate experiments. The relative protein quantification is represented in the graphic (* p < 0.05).
Figure 6
Figure 6
Proposed pathway explaining some biochemical effects, induced in vitro by PBDEs, on cell cycle, antioxidant status, metabolism and proliferation.
See this image and copyright information in PMC

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