Chemical and molecular mechanisms of antioxidants: experimental approaches and model systems

Abstract

Free radicals derived from oxygen, nitrogen and sulphur molecules in the biological system are highly active to react with other molecules due to their unpaired electrons. These radicals are important part of groups of molecules called reactive oxygen/nitrogen species (ROS/RNS), which are produced during cellular metabolism and functional activities and have important roles in cell signalling, apoptosis, gene expression and ion transportation. However, excessive ROS attack bases in nucleic acids, amino acid side chains in proteins and double bonds in unsaturated fatty acids, and cause oxidative stress, which can damage DNA, RNA, proteins and lipids resulting in an increased risk for cardiovascular disease, cancer, autism and other diseases. Intracellular antioxidant enzymes and intake of dietary antioxidants may help to maintain an adequate antioxidant status in the body. In the past decades, new molecular techniques, cell cultures and animal models have been established to study the effects and mechanisms of antioxidants on ROS. The chemical and molecular approaches have been used to study the mechanism and kinetics of antioxidants and to identify new potent antioxidants. Antioxidants can decrease the oxidative damage directly via reacting with free radicals or indirectly by inhibiting the activity or expression of free radical generating enzymes or enhancing the activity or expression of intracellular antioxidant enzymes. The new chemical and cell-free biological system has been applied in dissecting the molecular action of antioxidants. This review focuses on the research approaches that have been used to study oxidative stress and antioxidants in lipid peroxidation, DNA damage, protein modification as well as enzyme activity, with emphasis on the chemical and cell-free biological system.

Fig 1

Summary of ROS types and sources, and action point of antioxidants. O₂^−•, superoxide anion; HO₂^▪, perhydroxyl radical; ▪OH, hydroxyl radical; H₂O₂, hydrogen peroxide; HOCl, hypochlorous acid; ONOO⁻, peroxynitrite; R▪, lipid alkyl radical; RH, lipid; ROO▪, lipid peroxyl radical; ROOH, lipid hydroperoxide; SOD, superoxide dismutase; CAT, catalase and GPX, glutathione peroxidase.

Fig 2

Direct reactions of vitamin E (TOH) with ▪OH (A) and vitamin C (AscH⁻) with ROO▪ (B) and regeneration of vitamin E from vitamin C (C).

Fig 3

Structure of quinones (Q): LY83583 (A), riboflavin (B) and the formation of superoxide and its reaction with NBT (C), where R, R′ and R″ represent p-nitrophenyl, o-methoxyphenyl and phenyl groups, respectively.

Fig 4

Process of Luc luminescence.

Fig 5

Structures of DHE, EOH and ethidium and hypothesized reaction pathways to account for the formation of DHE-derived red fluorescence. EOH is basically formed from superoxide and possibly involving ONOO⁻/CO₂ while ethidium is mainly formed from H₂O₂ pathways involving metal proteins. Dotted arrows indicate possible intermediate pathways involved in the formation of these products.

Fig 6

The structures of H₂DCFDA, DCFH and DCF; and the reaction pathway of superoxide detection. DCF has an absorption maximum at 494 nm and emission maximum of 521 nm.

Fig 7

Detection of ONOO⁻ and superoxide by DHR123.

Fig 8

Reaction pathway of TBARS assay of ▪OH.

Fig 9

Structures of DPPH, galvinoxyl, ABTS and DMPD.

Fig 10

Superoxide and hydroperoxide generation from NAD(P)H, oxidase (NOX) and XO.

Fig 11

10-acetyl-3,7-dihydroxyphenoxazine (ADHP) fluorescent assay of XO activity.

Fig 12

Mechanism of linoleic acid peroxidation initiated by ▪OH radical.

Fig 13

Potential pathways of nitrotyrosine and chlortyrosine formation. ONOO⁻ , peroxynitrite; HOCl, hypochlorous acid; NO₂Cl, nitryl chloride; Cl⁻, chloride; H₂O₂, hydrogen peroxide; CO₃^−▪, carbonate radical; ▪NO₂, nitrogen dioxide radical; ONOOH) NO₂⁻, peroxynitrous acid; NO₂Cl, nitryl chloride; MPO, myeloperoxidase; Tyr, tyrosine.

Fig 14

Detection of HOCl by DTNB.