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

Abstract

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.

Keywords: 3-iodothyroacetic acid; 3-iodothyronamine; antioxidant; lipid peroxidation; sirtuin-1; thyroid hormone.

Figure 1

(a) Curves produced during the FRAP assay with T1AM, TA1 (10, 50, 100, 200, 500, 1000, 2000 nM), RESV, α-TOC (10, 50, 100, 1000, 2000 nM). (b) Bar graph showing FeSO₄ equivalent (µM) for T1AM, TA1, RESV, and α-TOC at 2000 nM. All experiments were performed in triplicate. Data are expressed as mean ± SEM; °°° p < 0.001 vs. RESV, *** p < 0.001 vs. α-TOC.

Figure 2

Determination of (a) O2-• and (b) •OH scavenging activities of T1AM, TA1, and p-Coum (3, 10, 30, and 100 μM and 0.1, 1, 10, and 100 μM, respectively). All experiments were performed in triplicate. Data are expressed as mean ± SEM.

Figure 3

The levels of UCP-1 in M and M + TA1 cells. Cells were obtained as described in the “Methods” section, and analyzed for the expression levels of UCP-1 by Western blot analysis. (a) Representative experiment loaded with proteins obtained for two different cell preparations. (b) Densitometric analysis is reported as the mean± standard error of the mean (SEM: n = 5 and 6 cell preparations from M and M + TA1, respectively) of arbitrary units (AU; see Methods) ** p < 0.01 vs. M cells.

Figure 4

Differentiation marker levels in M, M + TA1, and M + T1AM cells. M, M + TA1, and M + T1AM cells obtained as described in the “Methods” section. The expression levels of mRNA for (a) SSAO, (b) MAO-A, and (c) MAO-B were evaluated by RT-PCR. The mRNA expression of (a) SSAO, (b) MAO-A, and (c) MAO-B were reported as the mean± standard error of the mean (SEM) from four different cell preparations each run in triplicate: ** p < 0.01, and *** p < 0.001 vs. M cells.

Figure 5

SSAO and MAO-B activities in M and M + T1AM cells. (a) SSAO and (b) MAO-B activities were measured radiochemically, as described in the “Methods” section. The results are expressed as the means ± SEM of experiments (n = four different cell preparations, each run in triplicate). * p < 0.05 vs. M cells.

Figure 6

SIRT1 expression levels in M, M + TA1, and M + T1AM cells. M, M + TA1, and M + T1AM cells, obtained as described in the “Methods” section, were analyzed for the expression levels of SIRT1 by Western blot analysis. (a,b) Representative experiments are shown. Each gel was loaded with proteins obtained from two different cell preparations. (c,d) Densitometric analysis is reported as the mean± standard error of the mean (SEM: n = four cell preparations) of arbitrary units (AU; see “Methods”. *** p < 0.001 vs. M cells.

Figure 7

H3K9ac and Acetyl-p53 levels in M, M + TA1, and M + T1AM cells. M, M + TA1, and M + T1AM cells, obtained as described in the Section 4, were analyzed for their levels of H3K9ac and Acetyl-p53 by Western blot analysis, as described in the “Materials and Methods”. In Panels (a,b,e,f) representative experiments are shown. Each gel was loaded with proteins obtained for two different cell preparations. (c,d,g,h) Densitometric analysis is reported as the mean ± standard error of the mean (SEM: n = 4 cell preparations) of arbitrary units (AU; see “Methods”. ** p < 0.01, *** p < 0.001 vs. M cells).

Figure 8

TBARS levels in M, M + TA1, and M + T1AM cells. M, M + TA1, and M + T1AM cells were analyzed for TBARS levels, as described in the “Methods” section. The results are expressed as the mean± standard error of the mean (SEM) from four different cell preparations, each run in triplicate. * p < 0.05 vs. M cells.