Development of hydroxybenzoic-based platforms as a solution to deliver dietary antioxidants to mitochondria

Abstract

Oxidative stress and mitochondrial dysfunction have been associated with metabolic and age-related diseases. Thus, the prevention of mitochondrial oxidative damage is nowadays a recognized pharmacological strategy to delay disease progression. Epidemiological studies suggested an association between the consumption of polyphenol-rich diet and the prevention of different pathologies, including diseases with a mitochondrial etiology. The development of mitochondrial-targeted antioxidants based on dietary antioxidants may decrease mitochondrial oxidative damage. Herein, we report the design and synthesis of two new mitochondriotropic antioxidants based on hydroxybenzoic acids (AntiOxBENs). The results obtained showed that the novel antioxidants are accumulated inside rat liver mitochondria driven by the organelle transmembrane electric potential and prevented lipid peroxidation, exhibiting low toxicity. Some of the observed effects on mitochondrial bioenergetics resulted from an increase of proton leakage through the mitochondrial inner membrane. The new derivatives present a higher lipophilicity than the parent compounds (protocatechuic and gallic acids) and similar antioxidant and iron chelating properties. AntiOxBENs are valid mitochondriotropic antioxidant prototypes, which can be optimized and used in a next future as drug candidates to prevent or slow mitochondrial oxidative stress associated to several pathologies.

Figure 1

Design of mitochondriotropic antioxidants (AntiOxBEN₁ and AntiOxBEN₂) based on dietary scaffolds (protocatechuic and gallic acids).

Figure 2

Synthetic strategy used for production of mitochondriotropic antioxidants based on protocatechuic acid (AntiOxBEN₁) and gallic acid (AntiOxBEN₂). Reagents and conditions: (a) triethylamine, ethyl chloroformate, 6-aminohexan-1-ol, dichloromethane, r.t. (12 h); (b) 1,2-dibromotetrachloroethane, 1,2-bis(diphenylphosphine)ethane (diphos), tetrahydrofuran, r.t. (24 h); (c) Triphenylphosphine, 120 °C (48 h); (d) BBr₃, anhydrous dichloromethane, from −70 °C (10 min) to r.t. (12 h).

Figure 3

Antioxidant outline of mitochondria-targeted benzoic antioxidants. Radical scavenging activities of hydroxybenzoic acid derivatives on (a) DPPH and (b) ABTS radicals. •protocatechuic acid; ◾gallic acid; οAntioOxBEN₁; and ◽AntioOxBEN₂. Effect of AntiOxBENs on mitochondrial lipid peroxidation: (c) TBARS levels and (d) oxidation-derived oxygen consumption under different oxidative conditions. Data are means ± SEM from three and six independent experiments and are expressed as % of control (control = 100%) for TBARS and time lag-phase (s) for oxidation-derived oxygen consumption assays, respectively. The comparisons between control preparation vs. AntiOxBENs (5 μM) pre-incubations were performed by using one-way ANOVA. Significance was accepted with *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001.

Figure 4

(a) Representative AntiOxBENs voltammograms: (upper panel) Differential pulse and (lower panel) cyclic voltammograms for a 0.1 mM solution of (▬) AntiOxBEN₁ and (●●●) AntiOxBEN₂ at physiological pH 7.4 supporting electrolyte. Scan rate: 5 mV s⁻¹ (DPV) and 20 mV s⁻¹ (CV). (b) Evaluation of AntiOxBENs lipophilicity in water/DCH. Differential pulse voltammograms representing the transfer of 0.1 mM of AntiOxBEN₁ (▬) and AntiOxBEN₂ (●●●) at the water/DCH micro-interface at physiological pH 7.4. [TMA⁺] = 0.13 mM. Scan rate: 8 mV s⁻¹ (DPV).

Figure 5

Effects of AntiOxBENs on RLM transmembrane electric potential (ΔΨ) supported by (a) 10 mM glutamate + 5 mM malate or (b) 5 mM succinate. The white bars refer to the control, while grey bars refer to the experiments where RLM were pre-incubated with AntiOxBENs (2.5 μM – light grey; 5 μM – grey; and 10 μM – dark grey). The presented results are means ± SEM of five independent experiments. The statistical significance relative to the different bioenergetics parameters was determined using Student’s two tailed t-test (*P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001).

Figure 6

Effect of AntiOxBENs on RLM respiration supported by (a) 10 mM glutamate + 5 mM malate or (b) 5 mM succinate. The white bars refer to the control, while grey bars refer to the experiments where RLM were pre-incubated with AntiOxBENs (2.5 μM – light grey; 5 μM – grey; and 10 μM – dark grey). The presented results are means ± SEM of seven independent experiments. The statistical significance relative to the different respiratory rates/states was determined using Student’s two tailed t-test (*P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001).

Figure 7

Cytotoxicity profile of AntiOxBEN₁ (−) and AntiOxBEN₂ (−) on (a) rat embryonic cardiomyoblasts (H9c2), (b) human neonatal dermal fibroblasts (HNDF) and (c) human hepatocellular carcinoma (HepG2) cells. AntiOxBEN₁ (−) and AntiOxBEN₂ (−) cytotoxicity determined by changes in intracellular ATP levels on (d) rat embryonic cardiomyoblasts (H9c2), (e) human neonatal dermal fibroblasts (HNDF) and (f) human hepatocellular carcinoma (HepG2) cells. Data are means ± SEM of four independent experiments and the results are expressed as percentage of control (control = 100%), which represents the cell density without any treatment in the respective time point. Statistically significant compared with control group using one-way ANOVA. Significance was accepted with *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001.

Figure 8

Antioxidant cytoprotective effects of AntiOxBEN₁ and AntiOxBEN₂ on (a) rat embryonic cardiomyoblasts (H9c2), (b) human neonatal dermal fibroblasts (HNDF) and (c) human hepatocellular carcinoma (HepG2) cells against t-BHP-induced metabolic activity decrease. Each compound has three bars, which corresponds to the different concentrations used (from left to right, 25, 50, 100 µM).The comparisons were performed by using one-way ANOVA between the control (t-BHP) vs. preparation where AntiOxBENs were pre-incubated. Data are means ± SEM of four independent experiments and the results are expressed as percentage of control (control = 100%), which represents the cell density without any treatment in the respective time point. Significance was accepted with *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001.