Beyond Antioxidant Activity: Redox Properties of Catechins May Affect Changes in the DNA Methylation Profile—The Example of SRXN1 Gene
Abstract
:1. Introduction
2. Materials and Methods
2.1. Selected Redox-Active Compounds
2.2. Cell Culture
2.3. Treatment of Cells with Selected Redox-Active Compounds
2.4. Total RNA Isolation
2.5. Microarray Analysis
2.6. Genomic DNA Isolation
2.7. Bisulfite Conversion
2.8. Prediction of CpG Islands and Primer Design for Methylation Analysis
2.9. Methylation-Specific PCR (MSP)
2.10. Methylation-Sensitive High-Resolution Melting (MS-HRM)
2.11. Quantitative Reverse Transcription PCR (RT-qPCR)
2.12. Statistical Analysis
3. Results
3.1. The Impact of Catechins on SRXN1 Expression
3.2. Methylation of SRXN1 Promoter
3.2.1. DNA Methylation within M1/U1 Region of SRXN1 Promoter Analyzed by MSP
3.2.2. DNA Methylation in Both M1/U1 and M3/U3 Regions of SRXN1 Promoter Analyzed by MS-HRM
3.3. RT-qPCR Analysis of SRXN1 Expression
3.4. The Search for Correlations between Investigated Parameters
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Compound | GSH | C | EC | EGC | ECG | EGCG | Ref. |
---|---|---|---|---|---|---|---|
Structure | Tripeptide | Parent compound | Epimer of C | Epimer of GC | Ester of EC with gallate | Ester of EGC with gallate | [9] |
Catechol moiety | 0 | 1 | 1 | 0 | 1 | 0 | |
Pyrogallol moiety | 0 | 0 | 0 | 1 | 0 | 1 | |
Galloyl moiety | 0 | 0 | 0 | 0 | 1 | 1 | |
Cell-free systems | |||||||
Electrochemical parameters | |||||||
E0 at 37 °C (V) | 0.310 ± 0.003 | 0.281 ± 0.008 | 0.277 ± 0.005 | 0.287 ± 0.003 | 0.098 ± 0.002 | 0.104 ± 0.002 | [9] |
Ep,a + ɛ at 25 °C (V) | 0.984 ± 0.000 | 0.273 ± 0.000 | 0.260 ± 0.007 | 0.140 ± 0.007 | 0.230 ± 0.007 | 0.120 ± 0.009 | [29] |
Ip,a at 25 °C (µA) | 2.60 ± 0.17 | 5.40 ± 0.38 | 8.10 ± 0.28 | 12.60 ± 0.26 | 5.70 ± 1.06 | 6.30 ± 0.63 | |
Qox. at 25 °C (µC·cm−2) | 2.6 ± 0.08 | 5.20 ± 0.05 | 6.70 ± 0.10 | 3.90 ± 0.04 | 3.70 ± 0.65 | 4.70 ± 0.35 | |
AOE (µJ·cm−2) | 233.3 ± 3.50 | 184.40 ± 4.42 | 227.00 ± 1.40 | 65.50 ± 0.32 | 113.80 ± 13.83 | 119.30 ± 7.86 | |
t (s) | 6.6 ± 0.0 | 4.0 ± 0.0 | 4.2 ± 0.0 | 2.9 ± 0.0 | 3.9 ± 0.0 | 4.8 ± 0.0 | |
AOP (µW·cm−2) | 35.4 ± 0.53 | 46.10 ± 1.10 | 54.10 ± 0.34 | 22.60 ± 0.11 | 29.20 ± 3.55 | 24.90 ± 1.64 | |
Antioxidant activity by spectrophotometric tests | |||||||
Fe(III) chelation, pFe pH 7.0 | - | 17.6 * | - | 23.1 | 23.2 | 23.4 | [30] |
FRAP (mol TE/mol), pH 3.6 | 0.01 ± 0.00 | 0.79 ± 0.00 | 0.92 ± 0.00 | 1.03 ± 0.01 | 2.34 ± 0.01 | 2.21 ± 0.01 | [2] |
Spectro. test | Antioxidant activity, n10 value at 37 °C | ||||||
ABTS | 1.52 ± 0.02 | 5.44 ± 0.07 | 5.44 ± 0.11 | 5.98 ± 0.10 | 7.76 ± 0.11 | 8.33 ± 0.09 | [9] |
DPPH | 0.01 | 2.15 ± 0.03 | 2.20 ± 0.03 | 3.02 ± 0.03 | 5.48 ± 0.07 | 6.16 ± 0.08 | |
FC | 0.39 | 1.96 ± 0.02 | 1.83 ± 0.03 | 1.50 ± 0.02 | 2.66 ± 0.02 | 2.31 ± 0.03 | |
Protection against oxidation a/fluorescein bleaching b expressed as IC50 (µM) | |||||||
DHR123 a | 14.07 ± 0.36 | 0.81 ± 0.07 | 1.36 ± 0.04 | 1.21 ± 0.05 | 1.08 ± 0.04 | 1.15 ± 0.00 | [2] |
NaOCl b | 3.52 ± 0.05 | 0.34 ± 0.00 | 0.51 ± 0.03 | 0.25 ± 0.03 | 0.31 ± 0.01 | 0.36 ± 0.01 | |
AAPH b | 15.44 ± 0.01 | 0.67 ± 0.04 | 1.08 ± 0.06 | 0.66 ± 0.03 | 1.87 ± 0.07 | 2.16 ± 0.17 | |
Cell culture | |||||||
Conc. | Cell growth (% of control) at 37 °C, 24 h | ||||||
1 µM | 94.3 ± 5.8 | 109.7 ± 10.8 | 127.8 ± 10.2 | 97.7 ± 9.3 | 110.5 ± 11.3 | 94.3 ± 5.8 | [9] |
10 µM | 90.8 ± 4.3 | 96.1 ± 9.9 | 106.5 ± 7.8 | 92.9 ± 10.4 | 101.2 ± 10.6 | 90.8 ± 4.3 | |
Conc. | Cellular antioxidant activity, CAA value at 37 °C, 1 h | ||||||
1 µM | 7 ± 7 | 11 ± 8 | 23 ± 3 | 17 ± 5 | 35 ± 9 | 22 ± 10 | [9] |
10 µM | 22 ± 6 | 21 ± 10 | 20 ± 7 | 23 ± 9 | 35 ± 9 | 25 ± 10 | |
Conc. | Genotoxicity % at 37 °C, 24 h | ||||||
1 µM | 6.75 ± 0.88 | 3.58 ± 0.29 | 3.07 ± 0.23 | 3.54 ± 0.05 | 2.92 ± 0.69 | 2.59 ± 0.81 | [9] |
10 µM | 6.35 ± 2.01 | 3.77 ± 0.62 | 3.04 ± 1.00 | 3.02 ± 0.37 | 2.55 ± 0.35 | 2.66 ± 0.63 | |
Conc. | Fold regulation of gene expression at 37 °C, 24 h | ||||||
1 µM | 1.8 | 1.7 | −1.1 | −2.0 | 1.2 | 1.0 | [9] |
10 µM | 1.4 | 1.0 | −1.4 | −2.5 | −1.6 | −1.8 | |
Conc. | Fold change of % DNA methylation at 37 °C, 24 h | ||||||
MSP M1/U1 | Current study | ||||||
1 µM | 0.802 ± 0.136 | 1.005 ± 0.047 | 0.687 ± 0.053 | 1.139 ± 0.202 | 1.109 ± 0.317 | 0.671 ± 0.162 | |
10 µM | 1.159 ± 0.131 | 1.084 ± 0.069 | 0.900 ± 0.024 | 1.074 ± 0.213 | 1.204 ± 0.052 | 0.915 ± 0.269 | |
Conc. | MS-HRM M1/U1 | ||||||
1 µM | 1.199 ± 0.471 | 0.498 ± 0.054 | 0.419 ± 0.051 | 0.547 ± 0.167 | 0.478 ± 0.032 | 0.569 ± 0.106 | |
10 µM | 1.009 ± 0.208 | 0.330 ± 0.088 | 0.236 ± 0.217 | 1.768 ± 0.171 | 1.889 ± 0.029 | 0.269 ± 0.126 | |
Conc. | MS-HRM M3/U3 | ||||||
1 µM | 1.163 ± 0.080 | 0.811 ± 0.219 | 0.809 ± 0.034 | 0.918 ± 0.073 | 0.816 ± 0.084 | 0.882 ± 0.082 | |
10 µM | 1.191 ± 0.084 | 0.797 ± 0.070 | 0.685 ± 0.037 | 1.331 ± 0.130 | 1.410 ± 0.227 | 0.920 ± 0.236 |
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Jakubek, P.; Rajić, J.; Kuczyńska, M.; Suliborska, K.; Heldt, M.; Dziedziul, K.; Vidaković, M.; Namieśnik, J.; Bartoszek, A. Beyond Antioxidant Activity: Redox Properties of Catechins May Affect Changes in the DNA Methylation Profile—The Example of SRXN1 Gene. Antioxidants 2023, 12, 754. https://doi.org/10.3390/antiox12030754
Jakubek P, Rajić J, Kuczyńska M, Suliborska K, Heldt M, Dziedziul K, Vidaković M, Namieśnik J, Bartoszek A. Beyond Antioxidant Activity: Redox Properties of Catechins May Affect Changes in the DNA Methylation Profile—The Example of SRXN1 Gene. Antioxidants. 2023; 12(3):754. https://doi.org/10.3390/antiox12030754
Chicago/Turabian StyleJakubek, Patrycja, Jovana Rajić, Monika Kuczyńska, Klaudia Suliborska, Mateusz Heldt, Karol Dziedziul, Melita Vidaković, Jacek Namieśnik, and Agnieszka Bartoszek. 2023. "Beyond Antioxidant Activity: Redox Properties of Catechins May Affect Changes in the DNA Methylation Profile—The Example of SRXN1 Gene" Antioxidants 12, no. 3: 754. https://doi.org/10.3390/antiox12030754