Redox Properties of 3-Iodothyronamine (T1AM) and 3-Iodothyroacetic Acid (TA1)

3-iodothyronamine (T1AM) and 3-iodothyroacetic acid (TA1) are thyroid-hormone-related compounds endowed with pharmacological activity through mechanisms that remain elusive. Some evidence suggests that they may have redox features. We assessed the chemical activity of T1AM and TA1 at pro-oxidant conditions. Further, in the cell model consisting of brown adipocytes (BAs) differentiated for 6 days in the absence (M cells) or in the presence of 20 nM T1AM (M + T1AM cells), characterized by pro-oxidant metabolism, or TA1 (M + TA1 cells), we investigated the expression/activity levels of pro- and anti-oxidant proteins, including UCP-1, sirtuin-1 (SIRT1), mitochondrial monoamine (MAO-A and MAO-B), semicarbazide-sensitive amine oxidase (SSAO), and reactive oxygen species (ROS)-dependent lipoperoxidation. T1AM and TA1 showed in-vitro antioxidant and superoxide scavenging properties, while only TA1 acted as a hydroxyl radical scavenger. M + T1AM cells showed higher lipoperoxidation levels and reduced SIRT1 expression and activity, similar MAO-A, but higher MAO-B activity in terms of M cells. Instead, the M + TA1 cells exhibited increased levels of SIRT1 protein and activity and significantly lower UCP-1, MAO-A, MAO-B, and SSAO in comparison with the M cells, and did not show signs of lipoperoxidation. Our results suggest that SIRT1 is the mediator of T1AM and TA1 pro-or anti-oxidant effects as a result of ROS intracellular levels, including the hydroxyl radical. Here, we provide evidence indicating that T1AM and TA1 administration impacts on the redox status of a biological system, a feature that indicates the novel mechanism of action of these two thyroid-hormone-related compounds.


Introduction
Both 3-iodothyronamine (T1AM) and its oxidative metabolite, 3-iodothyroacetic acid (TA1) are endogenous compounds circulating in mammals [1]. Due to the fact that their structure is somehow related to thyroid hormones (THs; i.e., T3 and T4), it was hypothesized they represented end metabolites of THs and possible players of the non-genomic effects of T3. However, their biosynthetic pathway and physio-pathological role remain elusive. Despite this, T1AM and TA1 show pharmacological features in rodents, which suggests the merit to be exploited for their potential clinical effectiveness.
T1AM is considered a multi-target compound [2] and, even more than TA1, its mechanism of action remains mostly elusive and undefined [3]. Overall, the effects of T1AM and TA1, such as their rapid onset, high potency, non-linear dose-effect relationship and signaling, indicate that their mechanisms may include additional or alternative pathways to the recognition of a target. In this respect, some clues suggested that T1AM and TA1 modify the redox status of the system studied.

T1AM and TA1 Showed Ferric-Reducing Capacity and Superoxide and Hydroxyl Radical Scavenging Activities
The ferric reducing activity power (FRAP) of T1AM and TA1 was measured as described in the "Methods" section. As positive controls, resveratrol (RESV) and α-tocopherol (α-TOC) were used.
As shown in Figure 1, panel a, the FRAP activity of TA1 and T1AM was evident at concentrations as low as 0.1 µM and it was higher, at 2000 nM ( Figure 2, panel b), than that measured for RESV and α-TOC ( ••• p < 0.001 vs. RESV, *** p < 0.001 vs. α-TOC).
The FRAP activity of T1AM and of TA1 and their ROS scavenging features indicated that both compounds, at high pro-oxidant conditions, behave as antioxidants. T1AM and TA1 also showed free radical scavenging features with some interesting peculiarities. In fact, TA1 showed both superoxide and hydroxyl radical scavenging activities (pEC50 ± SEM of −4.72 ± 0.78 and −6.49 ± 0.10 respectively; Figure 2a,b) with the potency towards the hydroxyl radicals being significantly higher (*** p < 0.001) than that measured for the p-coumaric acid (p-Coum) (pEC50 ± SEM −5.71 ± 0.05; Figure 2b).
Instead, our results clearly showed that T1AM had only the capacity to scavenge superoxide radicals (pEC50 ± SEM of −4.12 ± 0.54; Figure 2a) with a potency similar to that of p-Coum (pEC50 −4.80 ± 0.63; Figure 2a). The FRAP activity of T1AM and of TA1 and their ROS scavenging features indicated that both compounds, at high pro-oxidant conditions, behave as antioxidants.

T1AM and TA1 Modify the Expression and Activity Levels of Pro-and Antioxidant Cell Proteins
We then investigated the expression and activity levels of pro-and antioxidant proteins, including UCP-1, MAO (A and B), SSAO, and SIRT1 in M, M + T1AM, and M + TA1 cells.  T1AM and TA1 also showed free radical scavenging features with some interesting peculiarities. In fact, TA1 showed both superoxide and hydroxyl radical scavenging activities (pEC50 ± SEM of −4.72 ± 0.78 and −6.49 ± 0.10 respectively; Figure 2a,b) with the potency towards the hydroxyl radicals being significantly higher (*** p < 0.001) than that measured for the p-coumaric acid (p-Coum) (pEC50 ± SEM −5.71 ± 0.05; Figure 2b).
Instead, our results clearly showed that T1AM had only the capacity to scavenge superoxide radicals (pEC50 ± SEM of −4.12 ± 0.54; Figure 2a) with a potency similar to that of p-Coum (pEC50 −4.80 ± 0.63; Figure 2a). The FRAP activity of T1AM and of TA1 and their ROS scavenging features indicated that both compounds, at high pro-oxidant conditions, behave as antioxidants.

T1AM and TA1 Modify the Expression and Activity Levels of Pro-and Antioxidant Cell Proteins
We then investigated the expression and activity levels of pro-and antioxidant proteins, including UCP-1, MAO (A and B), SSAO, and SIRT1 in M, M + T1AM, and M + TA1 cells.

T1AM and TA1 Modify the Expression and Activity Levels of Pro-and Antioxidant Cell Proteins
We then investigated the expression and activity levels of pro-and antioxidant proteins, including UCP-1, MAO (A and B), SSAO, and SIRT1 in M, M + T1AM, and M + TA1 cells.

Evaluation of UCP-1 Protein and Amine Oxidases mRNA Levels in M + T1AM and M + TA1 Cells
We previously demonstrated that UCP-1 protein levels were similar in M + T1AM and M cells [17]. Instead, in this study, as reported in Figure 3 (panels a and b), the M + TA1 cells showed significantly lower UCP-1 protein expression ( Figure 3, panel a, b; ** p < 0.01) than the M cells. We previously demonstrated that UCP-1 protein levels were similar in M + T1AM and M cells [17]. Instead, in this study, as reported in Figure 3 (panels a and b), the M + TA1 cells showed significantly lower UCP-1 protein expression ( Figure 3, panel a, b; ** p < 0.01) than the M cells.    We previously demonstrated that UCP-1 protein levels were similar in M + T1AM and M cells [17]. Instead, in this study, as reported in Figure 3 (panels a and b), the M + TA1 cells showed significantly lower UCP-1 protein expression ( Figure 3, panel a, b; ** p < 0.01) than the M cells.

MAO-B Activity
As shown above, the M + T1AM and M cells expressed mRNA levels for MAO-B at similar levels. However, it is known that the mRNA levels for MAO-B may be not in line with MAO-B enzyme activity [20]. Since MAO-B is the enzyme involved in T1AM degradation producing hydrogen peroxide and then TA1, we decided to evaluate MAO-B enzyme activity by using [ 14 ]C-benzylamine as substrate at conditions allowing the detection of SSAO or MAO-B respectively. As reported in Figure 5, in M + T1AM cells, SSAO activity (panel a) showed a tendency (not significant) to be lower than in M cells. Conversely, in the same cells, MAO-B activity was found to be significantly higher than in the M cells (panel b, * p < 0.05).

MAO-B Activity
As shown above, the M + T1AM and M cells expressed mRNA levels for MAO-B at similar levels. However, it is known that the mRNA levels for MAO-B may be not in line with MAO-B enzyme activity [20]. Since MAO-B is the enzyme involved in T1AM degradation producing hydrogen peroxide and then TA1, we decided to evaluate MAO-B enzyme activity by using [ 14 ]C-benzylamine as substrate at conditions allowing the detection of SSAO or MAO-B respectively. As reported in Figure 5, in M + T1AM cells, SSAO activity (panel a) showed a tendency (not significant) to be lower than in M cells. Conversely, in the same cells, MAO-B activity was found to be significantly higher than in the M cells (panel b, * p < 0.05).

SIRT1 Expression and Activity in M + T1AM and M + TA1 Cells
SIRT1 is a NAD+-dependent de-acetylase involved in several cellular processes and functions, a cell redox sensor and among the positive controllers of MAO-A-promoter activity [21]. Therefore, we investigated the expression/activity of SIRT1-1 in our cell populations Our results indicated that the M + T1AM and M + TA1 cells expressed SIRT1 protein levels lower and higher, respectively, than the M cells ( Figure 6, Panels a, c; *** p < 0.001; Figure 6, Panels b, d; *** p < 0.001).

SIRT1 Expression and Activity in M + T1AM and M + TA1 Cells
SIRT1 is a NAD+-dependent de-acetylase involved in several cellular processes and functions, a cell redox sensor and among the positive controllers of MAO-A-promoter activity [21]. Therefore, we investigated the expression/activity of SIRT1-1 in our cell populations.
Our results indicated that the M + T1AM and M + TA1 cells expressed SIRT1 protein levels lower and higher, respectively, than the M cells ( Figure 6, Panels a, c; *** p < 0.001; Figure 6, Panels b, d; *** p < 0.001). similar levels. However, it is known that the mRNA levels for MAO-B may be not with MAO-B enzyme activity [20]. Since MAO-B is the enzyme involved in T1AM dation producing hydrogen peroxide and then TA1, we decided to evaluate MAO zyme activity by using [ 14 ]C-benzylamine as substrate at conditions allowing the de of SSAO or MAO-B respectively. As reported in Figure 5, in M + T1AM cells, SSAO a (panel a) showed a tendency (not significant) to be lower than in M cells. Conver the same cells, MAO-B activity was found to be significantly higher than in the M (panel b, * p < 0.05).

SIRT1 Expression and Activity in M + T1AM and M + TA1 Cells
SIRT1 is a NAD+-dependent de-acetylase involved in several cellular process functions, a cell redox sensor and among the positive controllers of MAO-A-promo tivity [21]. Therefore, we investigated the expression/activity of SIRT1-1 in our cell lations Our results indicated that the M + T1AM and M + TA1 cells expressed SIRT1 p levels lower and higher, respectively, than the M cells ( Figure 6, Panels a, c; *** p < Figure 6, Panels b, d; *** p < 0.001).  The SIRT1 activity was then followed by measuring the expression levels of two of its substrates: H3K9ac and Acetyl-p53. According to our results, the M + T1AM and M + TA1 cells showed higher and lower H3K9ac and Acetyl-p53 protein levels, respectively, than the M cells ( Figure 7, panels a, c; ** p < 0.01) (Figure 7, panels e, g; *** p < 0.001) (Figure 7, panels b, d and panels f, h; *** p < 0.001 and ** p < 0.01, respectively).

Discussion
Here we report, for the first time, that T1AM and TA1, two endogenously occurr thyroid-hormone-related compounds, are endowed with antioxidant/ROS scaveng properties. These features are evident at concentrations ranging from 1 to 100 μM. C sistently, we demonstrate that both compounds can reduce ferric ions with an efficien greater than that of two known antioxidants, namely RESV and α-TOC. Furthermo while both molecules are also equipped with scavenging activities in terms of superox ions with a potency higher than that of p-coumaric acid, only TA1 is active against droxyl radicals, with a potency higher than that of p-coumaric acid.
Reactive oxygen species are produced by basal cell metabolism and have essen signalling roles in differentiation/trans-differentiation processes and aging. Furthermo ROS are messengers among cell compartments, of cell-to-cell communication, at phys logical and pathological conditions. In this respect, terminal adipose differentiation strictly dependent on ROS cell levels too. Consistently, a reduction in adipocyte matu tion and then in obesity is one of the therapeutic indications of the pharmacological su plementation of antioxidants/ROS scavengers [22].
We previously reported that BAs, exposed for 6 days to 20 nM T1AM (M + T1A cells), acquired pro-oxidant metabolism, including reduced ATP levels and increased sal lipolysis, in the absence of cell toxicity or of any significant reduction in cell differ tiation extent. Therefore, we retained this model suitable for extending our study on mechanistic of T1AM and TA1. In particular, in this study, we investigated the effect treating BAs with T1AM or TA1 on pro-oxidant cell enzyme expression/activities, i.e., amine oxidases involved in T1AM pharmacokinetic, and on SIRT1. Further, to confirm pro-oxidant effects of the M + T1AM cells, we checked whether these cells showed a ROS-dependent damage, including lipoperoxidation.
Our presented results indicate that M + T1AM cells exhibit lipoperoxidation lev higher than those of M cells. This finding clearly indicates that the long-term presence T1AM induced irreversible ROS-dependent damages to cell lipids, suggesting that dr exposure exhausted the antioxidant defences of cells.
M and M + T1AM cells differ in their MAO-B activity levels. In particular, M + T1A have higher MAO-B activity than M cells. MAO-B is the enzyme degrading T1AM, p ducing hydroxyl radicals that are mostly implicated in lipid peroxidation and not sc enged by T1AM. In addition, the potential higher degradation of T1AM in M + T1A compared with M cells suggests that the long term-treatment of cells with amine exe

Discussion
Here we report, for the first time, that T1AM and TA1, two endogenously occurring thyroid-hormone-related compounds, are endowed with antioxidant/ROS scavenging properties. These features are evident at concentrations ranging from 1 to 100 µM. Consistently, we demonstrate that both compounds can reduce ferric ions with an efficiency greater than that of two known antioxidants, namely RESV and α-TOC. Furthermore, while both molecules are also equipped with scavenging activities in terms of superoxide ions with a potency higher than that of p-coumaric acid, only TA1 is active against hydroxyl radicals, with a potency higher than that of p-coumaric acid.
Reactive oxygen species are produced by basal cell metabolism and have essential signalling roles in differentiation/trans-differentiation processes and aging. Furthermore, ROS are messengers among cell compartments, of cell-to-cell communication, at physiological and pathological conditions. In this respect, terminal adipose differentiation is strictly dependent on ROS cell levels too. Consistently, a reduction in adipocyte maturation and then in obesity is one of the therapeutic indications of the pharmacological supplementation of antioxidants/ROS scavengers [22].
We previously reported that BAs, exposed for 6 days to 20 nM T1AM (M + T1AM cells), acquired pro-oxidant metabolism, including reduced ATP levels and increased basal lipolysis, in the absence of cell toxicity or of any significant reduction in cell differentiation extent. Therefore, we retained this model suitable for extending our study on the mechanistic of T1AM and TA1. In particular, in this study, we investigated the effect of treating BAs with T1AM or TA1 on pro-oxidant cell enzyme expression/activities, i.e., the amine oxidases involved in T1AM pharmacokinetic, and on SIRT1. Further, to confirm the pro-oxidant effects of the M + T1AM cells, we checked whether these cells showed any ROS-dependent damage, including lipoperoxidation.
Our presented results indicate that M + T1AM cells exhibit lipoperoxidation levels higher than those of M cells. This finding clearly indicates that the long-term presence of T1AM induced irreversible ROS-dependent damages to cell lipids, suggesting that drug exposure exhausted the antioxidant defences of cells.
M and M + T1AM cells differ in their MAO-B activity levels. In particular, M + T1AMs have higher MAO-B activity than M cells. MAO-B is the enzyme degrading T1AM, producing hydroxyl radicals that are mostly implicated in lipid peroxidation and not scavenged by T1AM. In addition, the potential higher degradation of T1AM in M + T1AM compared with M cells suggests that the long term-treatment of cells with amine exerts control over the enzymes involved in its own degradation, likely to avoid the accumulation of the amine in cell compartments, an event potentially causing cell death [23,24]. Moreover, the up-regulation of MAO-B activity following T1AM treatment, if confirmed in brain cells, would shed a novel light on the role of MAO-B and T1AM in neurodegenerative diseases, including Parkinson's disease.
MAO-B activity represents the first step in the enzyme chain, including catalase and aldehyde dehydrogenase (i.e., the MAO cycle) producing TA1 from T1AM. Currently, we do not know how much TA1 is produced in M + T1AM; however, despite this uncertainess, TA1 cannot be excluded as part of the effects described in M + T1AM cells.
The M + TA1 cells presented significantly lower UCP-1 protein levels, mRNA for SSAO, MAO-A and B, early and late markers of adipocyte differentiation [25,26], compared to M cells. These results suggest that TA1 could reduce the extent of adipocyte differentiation, an effect produced by other known antioxidants [27]. Further, consistently with the signalling activated by antioxidants, SIRT1 levels and activity were found to be upregulated in the M + TA1 cells, without any signs of lipid peroxidation. Notably, this is the first evidence indicating that TA1 can also modulate SIRT1 expression/activity and then exert control over genome transcription. Notably, T1AM and TA1 modulate SIRT1 expression/activity in an opposite manner. Since the chemical structure of T1AM and TA1 allows us to exclude the possibility that they can recognize common targets, it is plausible to hypothesize that the modulation of SIRT1 depends on intracellular levels of ROS. In this respect, another important difference between M + T1AM and M + TA1 cells is the expression of MAO-B. In particular, TA1, unlike T1AM, downregulates MAO-B expression in terms of M cells, potentially reducing the intracellular production of hydroxyl radicals, the radical species mostly implicated in BAs differentiation, in the control of SIRT1 and in the lipid oxidation induced by T1AM. In synthesis, at our settings, TA1 behaves as "MAO-B inhibitors" drugs which are known for their protective effects against neurodegeneration and ischemic cell death while working as antidepressants. Consistently, these properties are among the pharmacological effects described so far for TA1 [3,28,29].
Even though our data cannot allow us to conclude on the sequence of the events described, they indicate that the pharmacological activities of T1AM and TA1 include the regulation of the cell redox status, a finding that represents the first mechanism described for TA1. In addition, since the concentrations of both drugs used in this study are close to their endogenous levels, the mechanism described here might also have physio-pathological relevance, including T1AM and TA1 as part of the cell redox balancing systems. In light of these results, the definitive assessment of T1AM and TA1 synthetic pathways assumes increasing importance.

Ferric-Reducing Activity Power (FRAP)
The FRAP activity was measured according to Lodovici et al. [30]. A FRAP reagent solution was freshly prepared by mixing 300 mM acetate buffer, pH 3.

Superoxide Scavenging Activity
The high-throughput superoxide (O 2 − •) scavenging assay was carried as reported by Tao et al. [31]. Four different concentrations were used for each antioxidant compound in the study. We used p-coumaric acid (p-Coum) as reference compound. The scavenger sample solution (TA1, T1AM, p-Coum, (final concentration range 3-100 µM; 6.67 µL) and 193.33 µL of substrate solution (including 6.43 µL of 4 mM xanthine, 12.86 µL of 4 mM NBT, and 174.04 µL of 0.05 M PBS) were added to each well. Next, 20 µL of 0.04 U/mL XO solution was added to start the reaction. After gentle shaking for 10 s, the reaction mixture was incubated at 37 • C for 20 min. Finally, 20 µL of 0.6 M HCl was added to stop the reaction, followed by shaking for another 10 s. The absorbance at 560 nm was measured. The total volume for each reaction mixture in each well was 240 µL. A blank test without XO and a control test without antioxidants were also conducted. The superoxide scavenging activity was expressed as percentage (%) of superoxide quenching, which was calculated as (1 − B/A) × 100, where B and A are the activities of xanthine oxidase with and without the addition of sample solution, respectively.

Hydroxyl Radicals' Scavenging Activity
The hydroxyl radicals' (•OH) scavenging activity was studied using a cell-and-proteinfree in vitro system, with the Fenton reaction, as described by Gonzales et al. [32]. The sample solution (T1AM, TA1, p-Coum (0.1-100 µM)) was added to the physiological Ringer's saline solution at 37 • C, supplemented with the ROS-sensitive fluorescent probe 2 ,7 -dichlorodihydrofluorescein (DCF; 5 µM), ferrous iron (Fe 2 SO 4 ; 10 µM), and H 2 O 2 (100 µM) for 30 min. In these conditions, DCF detects hydroxyl radicals but not H 2 O 2 . A blank test without H 2 O 2 and a control test without antioxidants were also conducted. Fluorescence intensities were measured using a fluorescence microplate reader and excitation/emission filters 485 nm/520 nm. The hydroxyl scavenging activity was expressed as percentage (%) of hydroxyl quenching, which was calculated as (1 − B/A) × 100, where B and A are the activities of Fe 2+ and H 2 O 2 with and without the addition of sample solution, respectively.

Cell Culture
The interscapular brown adipose tissue was isolated from one-month-old male Wistar rats (Envigo, San Pietro al Natisone, Italy) according to [17]. For this study, we received

Western Blot
M, M + T1AM, and M + TA1 cells on were lysed on Day 6 in a lysis buffer containing 50 mM Tris HCl (pH = 8), 150 mM NaCl, 1 mM EDTA, 0.1% w/v SDS, protease, and phosphatase inhibitor cocktail (Thermo Scientific, Monza, Italy). Total protein levels were quantified using the Pierce Protein Assay (Rockford, IL, USA)/BCA (bicinchoninic acid). Twenty micrograms of proteins were separated on 4-20% SDS-PAGE (Thermo Fisher scientific, Waltham, MA, USA) and transferred into PVDF membranes (60 min at 398 mA) using standard procedures. Blots were incubated overnight at 4 • C with specific primary antibody (Table 1) diluted in PBS containing 5% BSA or 5% non-fat dry milk and 0.05% Tween 20. The antigen-antibody complexes were visualized using appropriate secondary antibodies (1:10,000, diluted in PBS containing 5% albumin or 5% non-fat dry milk and 0.05% Tween 20), and left for 1 h at room temperature. Blots were then extensively washed with PBS containing 0.1% Tween 20 and developed using an enhanced chemiluminescence detection system (Pierce, Rodano, Italy). Exposition and developing time were standardized for all blots. Densitometric analysis was performed using the public domain NIH Image program (Image J software Version 1.50i, National Institute of Health, Bethesda, MD, USA). Each gel was loaded with proteins from two different cell preparations. The densitometric analysis presented in the histograms resumed the mean ± SEM of four different cell preparations and was reported as arbitrary units (AU), consisting of the ratio between the level of the target protein expression and that of the GAPDH.

RT-PCR
Total RNA was extracted from M, M + T1AM, and M + TA1 cells on Day 6 using the Macherey-Nagel Nucleo spin RNA (Macherey-Nagel, Dueren, Germany), according to the manufacturer's protocol. Reverse transcription was performed using the iScript™ Select cDNA Synthesis Kit, Bio-Rad, with 1 µg of total RNA. RT-PCR analyses were performed as described by Laurino et al. [33]. The primers were designed on the basis of the GenBank sequences for Rattus norvegicus (Table 2). M and M + T1AM cells were lysed in saline phosphate buffer (PBS pH = 7.4), and centrifuged at 1000× g × 10 min at 4 • C in order to remove cell debris. Enzyme determination was carried on in the resulting supernatant.
SSAO and MAO-B activity of M and M + T1AM cells were assayed radiochemically using 100 µM ( 14 C)-benzylamine (1 µCi/mL; Amersham Biosciences, Amersham, UK) in the absence or in the presence of 1 mM pargyline or 1mM semicarbazide, to inhibit MAO or SSAO activity, respectively. Both inhibitors were pre-incubated for 30 min at 37 • C before the addition of 100 µM ( 14 C)-benzylamine.
The labeled substrate was left in contact with enzyme preparations for 30 min at 37 • C. Reactions were stopped by the addition of 20 µL 3 N HCl. The aldehyde produced by enzyme reaction was extracted in ethylacetate (300 µL). The organic phase was separated by brief spinning (1000× g × 5 min) and a portion, 150 µL, counted for radioactivity in a β-counter. MAO-B and SSAO activity were then referred to the radioactivity recovered in the organic phase corrected for the radioactivity extracted in the organic phase from a sample where proteins were denatured before the addition of the labeled substrate. Results are expressed as nmol/mg of proteins/30 min.

Thiobarbituric Acid Reactive Substances (TBARS)
Thiobarbituric acid reactive substances (TBARS), markers of lipid peroxidation, were evaluated as previously described by Luceri et al. [34]. M, M + T1AM, and M + TA1 cells on were lysed on Day 6 in 50 µL of PBS. After the addition of 100 µL trichloroacetic acid (TCA), the resulting supernatant (160 µL) was added to 32 µL thiobarbituric acid, 0.12 M (Sigma-Aldrich, Milan, Italy), and heated at 100 • C, for 15 min. The samples were then placed in ice for 10 min and centrifuged at 1600× g at 4 • C, for 10 min. The absorbance of the supernatants was measured at 532 nm by using a Wallac 1420 Victor3 Multilabel Counter (Perkin Elmer, Waltham, MA, USA). The amount of TBARS, expressed as fmol/mg of proteins, was calculated using a molar absorption coefficient of 1.56 × 10 −5 M −1 cm −1 .

Statistical Analysis
Data are expressed as the mean ± SEM of 3-4 independent experiments. Statistical analysis was performed by Student's t-test for paired data or by the one-way ANOVA test followed by the Dunnett multiple comparison test. Before the ANOVA test, a D'Agostino-Pearson omnibus normality test was performed. The threshold of statistical significance was set at p < 0.05. Data analysis was performed using the GraphPad Prism 7.0 statistical program (GraphPad software, San Diego, CA, USA).

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.