The relationship between standard reduction potentials of catechins and biological activities involved in redox control

Abstract

Redox homeostasis involves factors that ensure proper function of cells. The excess reactive oxygen species (ROS) leads to oxidative stress and increased risk of oxidative damage to cellular components. In contrast, upon reductive stress, insufficient ROS abundance may result in faulty cell signalling. It may be expected that dietary antioxidants, depending on their standard reduction potentials (E°), will affect both scenarios. In our study, for the first time, we systematically tested the relationship among E°, chemical properties, and biological effects in HT29 cells for a series of structurally different catechins and a major endogenous antioxidant - glutathione (GSH), at both physiological and dietary concentrations. Among chemical antioxidant activity tests, the strongest correlation with E° was seen using a DPPH assay. The values of E° were also highly correlated with cellular antioxidant activity (CAA) values determined in HT29 cells. Our results indicated that physiological concentrations (1-10 µM) of tested catechins stabilized the redox status of cells, which was not exhibited at higher concentrations. This stabilization of redox homeostasis was mirrored by constant, dose and E° independent CAA values, uninhibited growth of HT29 cells, modulation of hydrogen peroxide-induced DNA damage, as well as effects at the genomic level, where either up-regulation of three redox-related genes (ALB, CCL5, and HSPA1A) out of 84 in the array (1 µM) or no effect (10 µM) was observed for catechins. Higher catechin concentrations (over 10 µM) increased CAA values in a dose- and E°-dependent manner, caused cell growth inhibition, but surprisingly did not protect HT29 cells against reactive oxygen species (ROS)-induced DNA fragmentation. In conclusion, dose-dependent effects of dietary antioxidants and biological functions potentially modulated by them may become deregulated upon exposure to excessive doses.

Keywords: Catechins; Oxidative stress; Redox homeostasis; Standard reduction potential.

Graphical abstract

Fig. 1

Chemical structures of tested compounds. Characteristic substituents linked to parent flavan-3-ol structure present in catechin derivatives are highlighted in red. The acronyms refer to: C – (+)-catechin, EC – (-)-epicatechin, EGC – (-)-epigallocatechin, ECG – (-)-epicatechin gallate, EGCG – (-)-epigallocatechin gallate.

Fig. 2

The linear relationship between concentration of reduced radicals or gallic acid equivalents and concentration of flavan-3-ols and GSH – Panel A, comparison of stoichiometric values n₁₀ calculated based on ABTS and DPPH methods as well as n₆₀ in the case of the FC method – Panel B. The results are means ± SD of three independent determinations.

Fig. 3

Inhibition of growth of HT29 cells determined by MTT test after 6 (circles), 24 (squares) and 72 h (triangles) exposure to exogenous GSH and catechins. Results represent means of three independent experiments carried out in triplicate (SD are lower than 15%).

Fig. 4

Cellular antioxidant activity of exogenous GSH and catechins in HT29 cells – Panel A, Panel B – grouping antioxidant potential of tested compounds as regards physiological concentrations potentially occurring in blood (1–10 µM) and concentrations reachable in alimentary tract (100–1000 µM); Panel C – the impact of individual compounds on cellular antioxidant activity in HT29 cells. Results are means ± SD of three independent experiments. Different letters for the same concentration indicate a significant difference in the one way ANOVA with Tukey test (p < 0.05).

Fig. 5

Genotoxicity of tested antioxidants expressed as %DNA in comet tail evaluated in HT29 cells. Results represent means ± SD of three independent experiments. Negative control (C−) – non-treated cells. No significant differences between treated and non-treated cells were observed according to one-way ANOVA with Dunnett's test.

Fig. 6

The ability of tested antioxidants to protect DNA of HT29 cells from H₂O₂-induced oxidative damage expressed as %DNA in comet tail. Results represent means ± SD of three independent experiments. Negative control (C−) – non-treated cells, positive control (C+) – cells treated with 150 μM H₂O₂. Significantly different values determined by one-way ANOVA with Dunnett's test are marked as (*) p < 0.05.