Cadmium and Cadmium/BDE (47 or 209) Exposure Affect Mitochondrial Function, DNA Damage/Repair Mechanisms and Barrier Integrity in Airway Epithelial Cells

: Heavy metals and Brominated diphenyl ether ﬂame-retardants (BDEs) often coexist in the environment and are capable of inducing injury, cytotoxicity or genotoxicity in human epithelial cells of the lung. We studied the effects of single Cadmium chloride (CdCl 2 ) or CdCl 2 /BDE (47 or 209) mixtures in airway epithelial cells, using A549 cell line cultured at submerged conditions and air–liquid interface (ALI) (an in vitro model described as physiologically relevant in vivo-like). We evaluated cell viability, oxidative stress, apoptosis, DNA damage/repair (Comet assay, γ H2AX phosphorylation ser139), mitochondrial redox balance (NOX-4, Nrf2 and TFAM) and cell barrier integrity (TEER, ZO-1, Claudin-1, E-cadherin-1) in A549 cells exposed to CdCl 2 (1 nM to 10 µ M), or to CdCl 2 (100 nM)/BDEs (47 or 209) (100 nM). CdCl 2 (10 µ M) reduced cell viability and increased apoptosis. CdCl 2 (100 nM) signiﬁcantly affected DNA-damage/repair (Olive Tail length production), γ H2AX phosphorylation and oxidative stress (ROS/JC-1 production) in submerged cell cultures. CdCl 2 (100 nM) decreased viability, TEER, ZO-1, Claudin-1 and E-cadherin-1 mRNA expression, and Nrf2 and TFAM while increased NOX-4, in ALI culture of cells. In both cell culture approaches, the cells stimulated with Cadmium/BDEs mixtures did not show a signiﬁcant increase in the effects observed in the cells treated with CdCl 2 alone. CdCl 2 inhalation might exert cytotoxicity and genotoxicity, playing a pivotal role in the uncontrolled oxidative stress, damaging DNA and gene expression in airway epithelial cells. No additional or synergistic adverse effects of CdCl 2 /BDEs mixture were observed in comparison to CdCl 2 alone in lung epithelium.


Introduction
Heavy metals such as cadmium (Cd) and the flame retardant known as brominated diphenyl ethers (BDEs) are toxic air pollutants ranked on the Priority List of Hazardous Substances [1] (WHO Regional Office for Europe, 2015; 2019 Substance Priority List). Heavy metals are released into the environment after the burning of coal, diesel fuel, gasoline and other fossil fuels or to the incineration of municipal waste and to the polluting metal alloy and electroplating facilities. Further, they are added to various materials such as electronic equipment, plastics, carpet liners and textiles [2,3]. Cd and BDEs coexisted in dust surface [4], sediment from river [5] and even in organisms [6]. The coexistence of Cd and BDE in the environment and biological systems poses a substantial risk to human health, but their potential joint toxicities remain so far elusive [7]. It was observed that Cd and BDEs are toxicants that cause pulmonary dysfunctions, lung cancer and chronic

A549 Cell Cultures and Exposure
The human lung adenocarcinoma cell line (A549) was cultured as an adherent monolayer in DMEM supplemented with 10% FBS, 1% MEM, 2 mM L-glutamine and gentamicin 250 µg/mL. For submerged exposures, A549 cells were seeded into standard six-well culture plates at a density of 1 × 105 cells per well and allowed to proliferate to reach 70-80% of confluence. Before exposure to contaminants, cells were starved for 24 h with culture medium FBS free. A549 cells were stimulated with a range of concentrations (0, 1 nM, 10 nM, 100 nM, 1 microM and 10 microM) as previously described in in vitro models of epithelial cells [24,25]. Furthermore, the cells were stimulated with single CdCl 2 (100 nM) or CdCl 2 (100 nM)/BDEs (47 or 209) (100 nM) mixtures. The single effect of BDEs on the measurements of this study was previously described [10,23]. A549 cells were stimulated for 4 h (to detect ROS and JC1 by cytofluorimetry) or 72 h for viability (MTS), apoptosis (by cytofluorimetry), and DNA damage (Comet assay). For air-liquid interface (ALI) culture, an aliquot of A549 cells (1 × 105) was seeded on 12-well collagen-coated Transwell inserts and grown in DMEM supplemented with 10% FBS, 1% MEM, 2 mM L-glutamine and gentamicin 250 µg/mL, to allow the attachment in the transwell. When A549 reached 90% of confluence (day 0), apical medium was removed and cells were cultured at ALI for 5 days, as previously described [10,26]. A fresh medium was provided every 48 h for 5 days (50 µL in the apical chamber and 1.0 mL in the basal chamber) as previously described [26] to simulate a thin layer of air-liquid in the apical chamber of ALI culture.
Trans-epithelial electrical resistance (TEER) was assessed before and after stimulation to evaluate the integrity of the epithelial layers in ALI culture and to measure the integrity of TJ dynamics, as previously described [10,27] using an EVOM2 Voltohmmeter (World Precision Instruments, Sarasota, USA). ALI were stimulated with CdCl 2 (0, 1, 10, 100 nM, 1 and 10 µM) for dose response or with CdCl 2 (100 nM) and CdCl 2 (100 nM)/BDEs (47 and −209) (100 nM) mixtures for 72 h to detect viability (MTS), TEER, protein expression by Western blot, and mRNA expression. The apical surface of ALI culture was washed with 0.5 mL PBS, then the membranes of transwell inserts (0.4-mm pore size) were cut out and processed for protein extraction and RNA isolation, as described below.

Cell Proliferation Assay
MTS assay for cell viability was assessed in submerged and ALI experimental conditions. The cells in submerged conditions were plated at a density of 1 × 103 cells per wells in a 96-well plate in 200 µL DMEM 10% FBS and stimulated for 72 h with CdCl 2 and BDEs (−47 and −209) alone for dose response or combined in mixtures. For ALI culture, the viability was evaluated in cells seeded on 12-well collagen-coated Transwell inserts, as previously described with minor modification [10,15] and stimulated with CdCl2 for dose response and with BDEs mixtures. Cell viability was determined in vitro by 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium MTS assay, using the Cell Titer 96 ® Aqueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA), according to the manufacturer's instruction. The MTS is reduced by cells into a formazan product, soluble in tissue culture medium, quantified using Wallac 1420 Victor2 multilabel counter (PerkinElmer Life Sciences, Turku, Finland) at 450 nm. The absorbance is directly proportional to the number of living cells in culture; results are expressed as % of baseline. Untreated controls were incubated in the same plates and under the same conditions [10,15].

Detection of JC-1 Mitochondrial Membrane Potential to Assess Mitochondrial Function
JC-1 was detected in A549 stimulated for 4 h CdCl 2 (0, 1, 10, 100 nM, 1 and 10 µM) for dose response or with CdCl 2 (100 nM) and CdCl 2 (100 nM)/BDEs (47 and −209) (100 nM) mixtures. The mitochondrial function was assessed by membrane potential analysis using MitoProbe JC-1 Assay kit M34152 (Molecular Probes, Eugene, OR, USA), a lipophilic cationic dye as previously described [28]. JC-1 localizes to the mitochondrial inner membrane forming either aggregates (reflecting a high membrane potential as indicated by red fluorescence) or monomers (reflecting a low membrane potential and fluorescing green) depending on membrane potential. The fluorescence of 10,000 cells was recorded upon excitation at fluorescent emission shift of JC-1 from Red (~590 nm) to green (~529 nm), and evaluated by cytofluorimetry (FACScalibur, Becton Dickinson, Mountain View, CA, USA) with 488 excitations lasers as previously described [23].

DNA Damage Analysis
Comet assay was performed as previously described [23,29] in A549 cells exposed for 72 h to CdCl 2 (100 nM) and CdCl 2 (100 nM)/BDEs (47 and −209) (100 nM) mixtures. After incubation, the cell suspension was then layered on one-third frosted slides pre-coated, OxiSelectTM Comet Assay Kit Cat. N. STA-35 (Cell Biolabs, Inc., San Diego, CA, USA). The cells were lysed and subjected to electrophoresis at 0.7 V/cm for 30 min. The Comet images were acquired at 10× magnification by using a fluorescence microscope (Zeiss) and subjected to image analysis using Image J. The DNA damage was quantified by measuring the displacement between the genetic material of the nucleus ('Comet head') and the resulting 'tail". Tail moment length (µm), Tail DNA intensity and Cell DNA intensity were taken by the operator using image analysis, to evaluate Tail DNA% (100 × Tail DNA Intensity/Cell DNA Intensity) and then were used to calculate Olive Tail Moment (OTM) by the formula Tail DNA % × Tail Moment Length.

Measurement of TJ Integrity by TEER
TJ integrity was evaluated in ALI culture, measuring TEER values before (TEER pre) and after 72 h of stimulation (TEER post), as previously described with minor modifications [10,30]. A total of 500 µL of pre-warmed PBS was added in the apical compartment (10 min at 5% CO 2 , 37 • C), and TEER values were measured. After the measurement of TEER pre, the apical solution was removed, and ALI cultures were stimulated. TEER post-stimulation was measured, the apical wash was discharged and the membranes of transwell inserts (0.4 mm pore size) were cut out and processed for protein extraction and RNA isolation, as described below.

Western Blot Analyses
Proteins were separated by SDS and polyacrylamide gel electrophoresis (PAGE) and transferred by electrophoresis onto Immobilon-P membranes (Millipore, Bedford, MA, USA) as previously described [30]. Briefly, after transfer, the membranes were blocked overnight at room temperature in PBS containing 3% BSA and 0.5% Tween 20 before being incubated for 1 h at room temperature with the primary Abs. After washing, the blot was incubated for 45 min with the appropriate horseradish peroxidase-conjugated secondary Ab; bound Ab was detected using the ECL chemiluminescence detection system (Amersham-Pharmacia, Biotech, MI, Italy), according to the manufacturer's instructions. Membranes were stripped and reprobed with housekeeping proteins anti-β-actin Ab to normalize differences in protein loading.

Western Blot Antibodies
The following Abs were used: anti-nicotinamide adenine dinucleotide phosphate

Gel Images Evaluation
Autoradiography films were scanned by means of densitometry and analyzed with Image/Gel Plotting analysis software (National Institutes of Health, Bethesda, MD, USA) to determine band intensities. Data are expressed as arbitrary densitometric units (A.D.U.) corrected against the density of β-actin bands.
2.14. RNA Isolation and Quantitative RT-PCR (ZO-1, Claudin-1 and E-Cadherin-1) Total RNA was extracted from A549 cells in ALI culture using TRIzol Reagent (Invitrogen) following the manufacturer's instructions. Then, it was reverse-transcribed into complementary DNA (cDNA), using M-MLVRT and oligo (dT)12e18 primer (Invitrogen). Quantitative real-time PCR of the ZO-1, Claudin-1 and E-Cadherin transcripts was carried out in a StepOne Plus Real-time PCR System (Applied Biosystems, Foster City, CA, USA) using specific FAM labeled probe and primers (prevalidated TaqMan Gene expression assay for ZO-1 (Hs01551861_m1, Claudin-1 (Hs 00221623_m1) and E-Cadherin (Hs 01023895_m1) and with a dye label VIC for GAPDH (Hs 02786624_g1) were purchased from Thermo Fisher Scientific (Pleasanton, CA, USA). ZO-1, Claudin-1 and E-Cadherin gene expression were normalized to GAPDH endogenous control gene. The relative quantitation of gene expression was performed with the comparative CT method (2DDCt) and plotted as fold-change compared with untreated cells as the reference sample [10].

Morphological Evaluation
Morphological changes of A549 stimulated with cadmium and BDE congeners (100 nM concentration) were recorded by light microscopy (Leica microscope, Wentzler, Germany) at 20× of magnification.

Statistical Analysis
Kolmogorov-Smirnov normality test was performed to assess normal data distribution. Analysis of variance (ANOVA) corrected with Fisher's test and paired t-test analysis were used for comparisons. Data are expressed as mean ± standard deviation (S.D.) All statistical analyses were performed using StatView ® 5 software (SAS institute Inc., Cary, North Carolina, USA). A p-value < 0.05 was considered to be statistically significant.

Cytotoxic Effects of CdCl 2 and CdCl 2 /BDE Mixtures in Submerged Culture of A549 Cells
Submerged culture of A549 cells exposed to different concentrations of CdCl 2 (1, 10, 100 nM,1 µM and 10 µM) for 72 h did not significantly induce alteration of cell metabolism or viability when compared with untreated cells; however, the A549 cells stimulated with a higher concentration of CdCl 2 (10 µM) showed a significant reduction in cell viability by MTS assay ( Figure 1A).
(Invitrogen). Quantitative real-time PCR of the ZO-1, Claudin-1 and E-Cadherin transcripts was carried out in a StepOne Plus Real-time PCR System (Applied Biosystems, Foster City, CA, USA) using specific FAM labeled probe and primers (prevalidated TaqMan Gene expression assay for ZO-1 (Hs01551861_m1, Claudin-1 (Hs 00221623_m1) and E-Cadherin (Hs 01023895_m1) and with a dye label VIC for GAPDH (Hs 02786624_g1) were purchased from Thermo Fisher Scientific (Pleasanton, CA, USA). ZO-1, Claudin-1 and E-Cadherin gene expression were normalized to GAPDH endogenous control gene. The relative quantitation of gene expression was performed with the comparative CT method (2DDCt) and plotted as fold-change compared with untreated cells as the reference sample [10].

Morphological Evaluation
Morphological changes of A549 stimulated with cadmium and BDE congeners (100 nM concentration) were recorded by light microscopy (Leica microscope, Wentzler, Germany) at 20× of magnification.

Statistical Analysis
Kolmogorov-Smirnov normality test was performed to assess normal data distribution. Analysis of variance (ANOVA) corrected with Fisher's test and paired t-test analysis were used for comparisons. Data are expressed as mean ± standard deviation (S.D.) All statistical analyses were performed using StatView ® 5 software (SAS institute Inc., Cary, North Carolina, USA). A p-value < 0.05 was considered to be statistically significant.

Cytotoxic Effects of CdCl2 and CdCl2/BDE Mixtures in Submerged Culture of A549 Cells
Submerged culture of A549 cells exposed to different concentrations of CdCl2 (1, 10, 100 nM,1 μM and 10 μM) for 72 h did not significantly induce alteration of cell metabolism or viability when compared with untreated cells; however, the A549 cells stimulated with a higher concentration of CdCl2 (10 μM) showed a significant reduction in cell viability by MTS assay ( Figure 1A).   Figure 1B). The effect of BDE-47 or BDE-209 (100 nM) alone was previously described [23] and here, the data are not shown.

Effect of CdCl 2 Treatment on ROS Production and Mitochondrial Injury in Submerged Culture of A549 Cells
To establish whether CdCl 2 stimulates intracellular oxidative stress, we examined ROS levels based on DCFH-DA fluorescence using flow cytometry. The incubation of the cells with CdCl 2 100 nM and 1 µM for 4 h significantly increased intracellular ROS formation in A549 cells compared to untreated cells (Figure 2A,B).

Effect of CdCl2 Treatment on ROS Production and Mitochondrial Injury in Submerged Culture of A549 Cells
To establish whether CdCl2 stimulates intracellular oxidative stress, we examined ROS levels based on DCFH-DA fluorescence using flow cytometry. The incubation of the cells with CdCl2 100 nM and 1 μM for 4 h significantly increased intracellular ROS formation in A549 cells compared to untreated cells (Figure 2A,B). Cell stress showed a decrease in the potential membrane mitochondrial and an increase of JC-1 (monomers) green fluorescence (MMP decreases). Accordingly, we found that CdCl2 (100 nM and 1 μM) significantly increased JC-1 in submerged A549 cells Cell stress showed a decrease in the potential membrane mitochondrial and an increase of JC-1 (monomers) green fluorescence (MMP decreases). Accordingly, we found that CdCl 2 (100 nM and 1 µM) significantly increased JC-1 in submerged A549 cells compared to untreated cells ( Figure 2C,D). The effect of BDE-47 or BDE-209 (100 nM) alone was previously described [23] and here, the data are not shown. compared to untreated cells ( Figure 2C,D). The effect of BDE-47 or BDE-209 (100 nM) alone was previously described [23] and here, the data are not shown.

Effects of CdCl2 and CdCl2/BDE Mixtures on ROS Production, Mitochondrial Injury and Apoptosis in A549 Cells
CdCl2 (100 nM) significantly increased intracellular ROS formation and JC-1 green fluorescence in submerged culture of A549 cells compared to untreated cells. The stimulation of the cells with CdCl2/BDE-47 or CdCl2/BDE-209 mixtures did not affect additional differences in ROS and JC-1 formation when compared to the cells stimulated with CdCl2 ( Figure 3). The stimulation of the cells with different concentrations of CdCl2 (0 to 10 μM) show that only CdCl2 10 μM significantly increased cell apoptosis (Anx + PI − + Anx + PI + ) (early + later apoptosis) in submerged culture of A549 cells compared to untreated cells or to the cells stimulated with lower concentrations of CdCl2 ( Figure 4A,B). The stimulation of the cells with different concentrations of CdCl 2 (0 to 10 µM) show that only CdCl 2 10 µM significantly increased cell apoptosis (Anx + PI − + Anx + PI + ) (early + later apoptosis) in submerged culture of A549 cells compared to untreated cells or to the cells stimulated with lower concentrations of CdCl 2 ( Figure 4A,B).
The stimulation of A549 cells with CdCl 2 (100 nM) for 72 h did not increase the percentage of apoptotic cells; however, CdCl 2 (100 nM)/BDE-47 (100 nM) or CdCl 2 (100 nM)/BDE-209 (100 nM) significantly increase the percentage of apoptotic cells compared to the cells treated with CdCl 2 or to untreated cells ( Figure 4C,D). The effect of BDE-47 or BDE-209 (100 nM) alone was previously described [23] and accordingly, here, the data are not shown. No additional increase was observed in the cells stimulated with mixtures and compared to the cells stimulated with single CdCl 2 . The distribution of DNA damage variation is shown in Table 1. The effect of BDE-47 or BDE-209 (100 nM) alone was previously described [23] and accordingly, here, the data are not shown.

Effect of CdCl 2 and CdCl 2 /BDE Mixtures on DNA Damage/Repair Mechanisms in A549 Cells
We first investigated the impact of persistent oxidative stress on protein by Western blot analysis of whole-cell extracts from A549 submerged cells stimulated for 72 h. CdCl 2 (100 nM) or with CdCl 2 (100 nM)/BDE-47 (100 nM) and CdCl 2 (100 nM)/BDE-209 (100 nM) mixtures induced a significant increase of γH2AX phosphorylation in the stimulated cells rather than in untreated cells ( Figure 6A). No additional increase was observed in the cells stimulated with mixtures a compared to the cells stimulated with single CdCl2. The distribution of DNA dama variation is shown in Table 1. The effect of BDE-47 or BDE-209 (100 nM) alone was previously described [23] a accordingly, here, the data are not shown.

Cytotoxic Effects and NOX-4, Nrf2 and TFAM Protein Expression in ALI Culture of A549 Cells Exposed to CdCl 2 and CdCl 2 /BDE Mixtures
A549 cells cultured in ALI system and stimulated with different concentrations of CdCl 2 (from 1 nM to 10 µM) for 72 h showed a significant reduction in cell viability only with CdCl 2 10 µM using MTS assay ( Figure 7A). Furthermore, CdCl 2 and CdCl 2 (100 nM)/BDE-47 (100 nM) or CdCl 2 (100 nM)/BDE-209 mixtures did not decrease cell viability in A549 cells compared with untreated cells ( Figure 7B).
We explored NOX-4 protein expression in ALI culture of A549 cells treated with CdCl 2 (100 nM) and CdCl 2 /BDE-47 or CdCl 2 /BDE-209 mixtures for 72 h. As shown in Figure 8A, the treatment of the cells with CdCl 2 and CdCl 2 /BDE-47 or CdCl 2 /BDE-209 mixtures significantly enhanced the expression of NOX-4 in comparison with untreated cells ( Figure 8A).  Figure 6B). The effect of BDE-47 or BDE-209 (100 nM) alone on phosphorylated γH2AX was previously described [23] and accordingly, here, the data are not shown.

Cytotoxic Effects and NOX-4, Nrf2 and TFAM Protein Expression in ALI Culture of A549 Cells Exposed to CdCl2 and CdCl2/BDE Mixtures
A549 cells cultured in ALI system and stimulated with different concentrations of CdCl2 (from 1 nM to 10 μM) for 72 h showed a significant reduction in cell viability only with CdCl2 10 μM using MTS assay ( Figure 7A).  Figure 7B).
We explored NOX-4 protein expression in ALI culture of A549 cells treated with  (Figure 8B,C). The effect of BDE-47 or BDE-209 (100 nM) alone on NOX-4 was previously described [9], and accordingly, here, the data are not shown.

TEER and ZO-1, Claudin-1 and E-Cadherin mRNA and Protein Expression in ALI Culture of A549 Cells Exposed to CdCl2 and CdCl2/BDE Mixtures
TEER post-stimulation was significantly lower in the ALI culture of A549 cells exposed to CdCl2 (100 nM, 1 and 10 μM) for 72 h in comparison with TEER pre-stimulation ( Figure 9A).
3.6. TEER and ZO-1, Claudin-1 and E-Cadherin mRNA and Protein Expression in ALI Culture of A549 Cells Exposed to CdCl 2 and CdCl 2 /BDE Mixtures TEER post-stimulation was significantly lower in the ALI culture of A549 cells exposed to CdCl 2 (100 nM, 1 and 10 µM) for 72 h in comparison with TEER pre-stimulation ( Figure 9A).
No differences were shown between TEER pre and post in baseline conditions. The levels of Delta TEER were not significantly different in ALI culture of A549 exposed to CdCl 2 and to CdCl 2 /BDE-47 or CdCl 2 /BDE-209 mixtures than in untreated cells ( Figure 9B). The effect of BDE-47 or BDE-209 (100 nM) alone on TEER measurements was previously described [10], and accordingly, here, the data are not shown.
ZO-1, Claudin-1 and E-Cadherin proteins are proteins that well-characterized the role of tight junctions in the integrity of the epithelial layer. ZO-1, Claudin-1 and E-Cadherin mRNA expression was detected in ALI culture of A549 cells stimulated with CdCl 2 (100 nM) and CdCl 2 (100 nM)/BDE-47 (100 nM) or CdCl 2 (100 nM)/BDE-209 (100 nM) for 72 h by RT-qPCR (in total RNA). ZO-1, Claudin-1 and E-Cadherin significantly decreased in the cells stimulated with CdCl 2 and with CdCl 2 /BDE-47 or with CdCl 2 /BDE-209 mixtures compared to untreated cells. The effect of BDE-47 or BDE-209 (100 nM) alone was previously described on barrier integrity [9] and accordingly, here, the data are not shown.
The analyses of ZO-1, Claudin-1 and E-Cadherin proteins by immunofluorescence assay support the data on the levels of transcripts. Interestingly, immunofluorescence staining pointed out that exposure of A549 under ALI culture to CdCl 2 (100 nM) and CdCl 2 (100 nM)/BDE-47 (100 nM) and CdCl 2 (100 nM)/BDE-209 (100 nM) affected the expression of ZO-1, Claudin-1 and E-cadherin ( Figure 10). The same % of DMSO was added to the baseline as a vehicle. Statistical analysis was performed by Student's t-test. Significance was at p < 0.05. Bars represent mean ± S.D. of Delta TEER. Statistical analysis was performed by ANOVA followed by Fisher's PLSD correction. Significance was at p < 0.05.
No differences were shown between TEER pre and post in baseline conditions. The levels of Delta TEER were not significantly different in ALI culture of A549 exposed to CdCl2 and to CdCl2 /BDE-47 or CdCl2 /BDE-209 mixtures than in untreated cells ( Figure  9B). The effect of BDE-47 or BDE-209 (100 nM) alone on TEER measurements was previously described [10], and accordingly, here, the data are not shown.
ZO-1, Claudin-1 and E-Cadherin proteins are proteins that well-characterized the role of tight junctions in the integrity of the epithelial layer. ZO-1, Claudin-1 and E-Cadherin mRNA expression was detected in ALI culture of A549 cells stimulated with CdCl2 (100 nM) and CdCl2 (100 nM)/BDE-47 (100 nM) or CdCl2 (100 nM)/BDE-209 (100 nM) for 72 h by RT-qPCR (in total RNA). ZO-1, Claudin-1 and E-Cadherin significantly decreased in the cells stimulated with CdCl2 and with CdCl2 /BDE-47 or with CdCl2 /BDE-209 mixtures compared to untreated cells. The effect of BDE-47 or BDE-209 (100 nM) alone was previously described on barrier integrity [9] and accordingly, here, the data are not shown.

Discussion
It is established that Cd or BDEs inhalation leads to several lung pathologies; however, the effect of the exposure to Cd together with BDE 47 or BDE 209 on the alterations of human health is partially studied and understood in airway diseases. Here, we reported for the first time the joint toxicity of Cd/BDE 47 or Cd/BDE 209 exposure on cell death, mitochondrial function, mechanisms of DNA damage/repair and barrier integrity in an in vitro model of airway epithelial cells obtained using submerged and ALI culture of A549 cell line. We observed that CdCl 2 might have adverse effects on human lung function damaging airway epithelial cell activities, without an additional or synergistic effect in the presence of BDE 47 or 209.
Cd is a typical toxic heavy metal and a persistent organic compound present, together with BDEs, in some areas of the world. The effects of contaminants are often manifested as imbalances in antioxidants [34], resulting in an increase in ROS production that could destroy the redox/balance and promote protein, DNA and lipid degradation [35]. Many studies have shown that environmental pollution due to Cd or BDEs plays a critical role in the pathophysiology of diseases, and the main cell mechanism responsible for their toxicity is oxidative stress. Detection of the environmental coexistence of Cd and BDEs increases public concern about their combined toxicity, which might be considered a higher health risk for humans and wildlife [36].
A549 cells were widely used in studies on human lung damage caused by toxic substances [36,37]. The use of A549 cell line culture is a widely used in vitro model to study the pathophysiological roles of BDEs in human lungs and to reproduce the effect of toxicants observed in cell cultures of primary bronchial epithelial cells [10,38]. First, we evaluated the potential effect of CdCl 2 and of CdCl 2 /BDE mixtures on cytotoxic and genotoxic mechanisms using an in vitro model of A549 cells cultured in submerged conditions readily accessible, straightforward and practical. Then, we studied the effect of oxidative stress generated by CdCl 2 and byCdCl 2 /BDEs mixtures on epithelial barrier integrity using the ALI culture system of A549, a 3D in vitro model of epithelium commonly useful to assess the effect of toxicant on the mechanism of cell-cell interactions [39].
Exposure to air pollutants injured the lung and epithelial cells are the first line of defense against exogenous molecules, playing a central role in pathophysiological mechanisms [2]. Single toxicological effects of Cd and BDE (47 or 209) have been recently studied in airway epithelium [2,10,23], but little is known about the effect of Cd and BDE (47 or 209) mixtures either in vivo or in vitro studies. Here, we showed that lower concentrations of single CdCl 2 (1 nM to 1µM), as well as the single CdCl2 (100 nM) in combination with BDE 47 or 209 (100 nM), did not cause a reduction in cell viability in submerged culture of A549 cells.
As previously described for BDE 47 and BDE 209 in human bronchial epithelial cell line [23], we showed here that CdCl 2 affect, in a dose-dependent manner, ROS production and mitochondrial membrane potential (JC-1 monomer) dysfunction in submerged culture of A549 cells. At the same time, in this study, we observed that single CdCl 2 in combination with BDE 47 or BDE 209 (100 nM) did not show an additional or synergistic increase in ROS production and dysfunction of mitochondrial membrane potential (JC-1). These findings show that there is no combined action of CdCl 2 and BDEs in the mechanism of oxidative stress of airway epithelium, supporting the concept that CdCl 2 or BDEs have high and equal levels of toxicity on human health of the lung.
CdCl 2 -induced hepatotoxicity [40,41] and cytotoxicity of renal tubular epithelial cells, in duck [42] by ROS mediated dependent apoptosis pathway. The flame retardants (BFRs) such as BDEs play a primary role in the regulatory process of A549 cell line apoptosis through the mitochondrial pathway [43]. Many studies show that the mechanisms of cell transformation autophagy-associated are impaired by Cd [44,45] and trigger a wide range of responses related to immunity and oxidation (e.g., ROS, DNA damage, lipid peroxidation, cytokine overproduction, inflammation and proteostasis), damaging the epithelium lining the airways and alveoli [46,47]. Oxidative stress following BDE exposure has also been related to autophagy induction [48] to defend the cells against further ROS and pro-apoptotic proteins production [49][50][51]. We showed here that different CdCl2 concentrations did not affect apoptosis in A549 cells; however, the combined stimulation of the cells with CdCl 2 (100 nM) and BDE-47 (100 nM) or BDE-209 (100 nM) had an additional effect on the A549 cell line apoptosis. Our findings suggest that oxidative stress dysfunction due to CdCl 2 might be associated with autophagy signaling suppressing apoptosis and BDEs alone (as previously described in Montalbano et al., 2020) or, in combination with CdCl 2, reverted this cell activity. These data open new perspectives toward further studies to clarify the mechanism of apoptosis in airway epithelial cells exposed to environmental chemicals as Cd and BDE-47 or BDE-209 mixtures.
Genotoxic and cytotoxic effects of toxicants can generate lung cancer. Detection of phosphorylated γH2AX and Comet assay is frequently used to evaluate the genotoxic effect of the substances on DNA damage/repair mechanisms. CdCl 2 , through enhancement of ROS formation, induces damage to DNA macromolecules or cell cycle phase [52,53]. To resist DNA alteration, the cell initiates "DNA Damage Response", including a cohort of events such as arrest the cell cycle, apoptosis and γH2AX phosphorylation [54]. γH2AX (serine 139) phosphorylation occurs immediately after the formation of DNA double-strand breaks (DNA DSBs), signaling injury to the proteins responsible for DNA repair [55]. The phosphorylation of γH2AX is a fundamental step in the mechanisms of DNA damage [56]. The result is the production of detectable nuclear foci in euchromatin regions considered transcriptionally active in the mechanism of DNA repair [57][58][59][60]. The persistence of DNA lesions generates strand breaks corresponding to DNA migration by the Comet test. We show here that CdCl 2 induces γH2AX phosphorylation and immunofluorescent Comets in A549 cells. At the same time, the analysis of CdCl2 and BDEs (100 nM) mixtures effect show a slight increase in γH2AX phosphorylation and immunofluorescent Comet. Moreover, the immunofluorescence assay described the presence of nuclear foci in the cells showing γH2AX phosphorylation and Comets. Our findings might suggest that CdCl 2 generates the outbreaks of γH2AX phosphorylated and DSB signaling, promoting DNA lesions which in turn might be recognized as highly mutagenic lesions in the epithelium of the lung; however, we did not identify clear evidence to support that the combined exposure to CdCl2 and BDEs might have an additional effect on the mechanisms of DNA damage/repair of epithelial cells.
In cells, increased ROS production induces activation of autophagy to compensate for the excessive cell damage and death that result from oxidative stress [61]. NOX-4 serves as a gatekeeper in the regulation of autophagy activation through ROS production based on cell types and conditions [62,63]. Aberrant and excessive mechanisms of oxidative stress are involved in cellular/tissue injury and lead to pathological changes [49]. BDEs exert cytotoxic and genotoxic effects damaging the integrity of the airway epithelial barrier [10,23]. An increment in NOX-4 activity represses Nrf2 and TFAM proteins. Nrf2 is an oxidative stress-sensitive transcription factor and controls the expressions of antioxidant defense and detoxification genes [20]. Nrf2 plays a fundamental role in regulating mitochondrial function [64], and TFAM is important in the functional integrity of the mitochondrial respiratory chain and in the maintenance of balance between anti-oxidation and oxidation [65]. After the analysis of CdCl 2 or CdCl 2 /BDE mixtures on cell viability in 3D ALI cultures of the A549 cell line, we studied oxidative stress and redox balance. We observed that CdCl 2 or CdCl 2 /BDE mixtures affected the increase in NOX-4 and downregulated Nrf2 and TFAM expression, without any additional effect. These findings suggest that CdCl 2 might be involved in mitochondrial bioenergetics and biogenesis of NOX-4 ROS-dependent activities, repressing Nrf2 and TFAM endogenous antioxidant responses to cell stress of the stratified epithelium of the airways exposed to environmental pollution. Accordingly, with data on ROS or JC-1 activity observed in submerged culture of A549 cells, the expression of NOX-4, Nrf2 and TFAM did not show an additional or synergistic effect when the cells were stimulated with CdCl 2 and CdCl 2 /BDEs mixtures. Once again, these findings show that there is no combined action of CdCl 2 and BDEs on the epithelial cells supporting the concept that CdCl 2 or BDEs have high and equal levels of toxicity on human health of the lung; however, we suggest further study to describe mitochondrial oxygen consumption and ATP formation to better describe the action of CdCl 2 and CdCl 2 /BDE mixtures on mitochondrial functions.
The effective function of the respiratory epithelium is a fundamental requirement to protect the airways from a wide range of exogenous inhaled substances. The mitochondrial function is important for the activities of the lung epithelium, such as barrier integrity, ciliary function and the regeneration of epithelial cells following injury [66]. The apical junctional complexes between neighboring cells are important to keep the epithelial barrier functioning in the lung. They are made up of tight apical junctions (TJs) proteins (Claudin family, Occludin or ZO-1 proteins) and adherent junctions (AJs) (E-cadherin) [67][68][69]. Interestingly to note that CdCl 2 or CdCl 2 /BDE mixtures caused airway epithelial barrier dysfunction as demonstrated by TEER values reduction and simultaneously affecting ZO-1, Claudin-1 and E-cadherin mRNA and proteins expression. These findings suggested that CdCl 2 or CdCl 2 /BDE mixtures caused airway epithelial barrier dysfunction by multiple TJ and AJ proteins downregulation; however, no additional or synergistic effect of CdCl 2 and CdCl 2 /BDEs mixtures were found. These findings showed that CdCl 2 has a significant negative impact on the maintenance of the integrity barrier imparted by the airway epithelium. We believe that the central issue might be associated with autophagic dysfunction. Hence, a careful prospective analysis of autophagic activity can prevent or resolve the potentially toxic effects due to exposure to environmental contaminants. Furthermore, since cadherin adhesive function depends on the actin cytoskeleton, and this contribution to cell-cell adhesion is not fully understood [70], further study might be necessary to clarify and to elaborate the role of toxicants on actin cytoskeletal reorganization in epithelial cells of the airway; however, our observations might be considered an initial issue for a possible understanding of the real adverse effects associated with Cd and BDEs co-exposure of airway epithelial cells. In fact, although for some parameters there is no statistical significance, we observed a trend in γH2AX phosphorylation, NOX-4/TFAM protein expression and genomic DNA damage (Comet assay) changes in the epithelial cells stimulated with CdCl 2 /BDE209 and compared to CdCl 2 . These differences might suggest further investigation of the specific mechanism involved in γH2AX activity, NOX-4 and TFAM protein expression and DNA damage in the presence of environmental contaminants mixtures.

Conclusions
We concluded that CdCl 2 is a heavy metal involved in serious toxicities processes of the lung tissue and likewise BDEs as previously described [10,23]. Our investigation with respect to the toxicity of single CdCl 2 or CdCl 2 /BDE mixtures showed an increase in ROS and JC-1 production, cell apoptosis and necrosis, DNA damage in terms of alkaline Comet assay and the presence of nuclear γH2AX foci in comparison to unexposed airway epithelial cells; however, the CdCl 2 and CdCl 2 /BDE co-exposure might cause mitochondrial dysfunction and barrier integrity damage in the airway epithelium. Although our first idea was to demonstrate a combined effect of toxicant mixtures on the dysfunctional activities of epithelial cells, we showed here that CdCl 2 /BDE47 or CdCl 2 /BDE209 mixtures did not have a significative synergistic or additional effect compared to CdCl 2 alone on cytotoxic and genotoxic activities of airway epithelial cells; however, since we highlighted a changing trend induced by Cd and BDE-47 or BDE-209 mixtures, further investigation is needed in order to better understand these effects.
In this scenario, we concluded that the levels of toxicity generated individually by CdCl 2 or BDEs in airway epithelial cells are so high that the co-exposure to these substances does not generate further effects; however, due to the limits of the in vitro studies, to reinforce the message of our study, we suggest further in vivo epidemiological study to better clarify their combined negative action on human health of the lung.