Reflections of an aging free radical

Abstract

In this mini-reflection, I explain how during my doctoral work in a Botany Department I first became interested in H₂O₂ and later in my career in other reactive oxygen species, especially the role of "catalytic" iron and haem compounds (including leghaemoglobin) in promoting oxidative damage. The important roles that H₂O₂, other ROS and dietary plants play in respect to humans are discussed. I also review the roles of diet-derived antioxidants in relation to human disease, presenting reasons why clinical trials using high doses of natural antioxidants have generally given disappointing results. Iron chelators and ergothioneine are reviewed as potential cytoprotective agents with antioxidant properties that may be useful therapeutically. The discovery of ferroptosis may also lead to novel agents that can be used to treat certain diseases.

Keywords: Antioxidant; Ascorbate-glutathione cycle; Catalytic iron; Ergothioneine; Ferroptosis; Free radical; Hydrogen peroxide; Plants.

Fig. 1

The front page of my D. Phil. thesis and a photograph of the graduating class. Can you spot the younger me in the photo? (Note the thesis had to be hand-typed with carbon paper; the original went to the examiners and mine is a carbon copy). Photograph courtesy of Professor Chris Leaver. Abstract Leaves of spinach (Spinacia oleracea L.) and other plants readily catalyse the oxidation of formate to CO₂. The enzymic mechanisms by which leaves convert formate to CO₂ have not been fully established. In the first part of this thesis, a detailed study of the properties and mechanism of formate oxidation in leaves of spinach and spinach-beet is described. Studies of the effects of catalase, catalase inhibitors and H₂O₂ led to the conclusion that the peroxidatic action of catalase was responsible for formate oxidation at pH 5 in all fractions. In vivo, this activity takes place in peroxisomes. Its apparent location in chloroplasts and mitochondria is shown to be an artefact due to the generation of H₂O₂by these organelles and the presence of contaminating catalase. The pathways by which formate is produced in plant tissues have been the subject of much previous investigation. Glyoxylate is a good precursor of formate when supplied to plant tissues (Cossins & Sinha, 1965), and so formate could be produced from this compound during photorespiration. It was found that peroxisomes could catalyse the decarboxylation of glycollate and glyoxylate. Detailed studies of these reactions, led to the conclusion that these activities were due to the generation of H₂O₂in peroxisomes by the action of glycollate oxidase, followed by non-enzymic reaction of H₂O₂with glyoxylate, producing CO₂(from the carboxyl group) and formate. This occurs because the peroxisomal catalase is unable to completely destroy H₂O₂, consistent with the observations of other workers on different systems.

Fig. 2

A: Hydrogen Peroxide Generation by Beverages. B: The Effect of Milk. H₂O₂ is present at high levels in freshly-brewed beverages and levels increase with time. Adding 10% (v/v) milk decreases H₂O₂ levels. Data reproduced from Ref. [18] with publisher's permission.

Fig. 3

Hydrogen peroxide is present in human urine. A sample of freshly-voided human urine was placed in the chamber of an oxygen electrode. At point A, buffer (phosphate-buffered saline PBS] pH 7.4) was added and at point B, urine was added. Freshly-voided urine has low O₂ levels, hence the decrease. At point C, catalase dissolved in PBS (103 units of catalase) was injected through the cap. Note the immediate burst of O₂ evolution. Adapted from Ref. [24]. The O₂ electrode method to measure H₂O₂ is not very sensitive, but it is fast and simple; other methods (especially peroxidase-based ones) are susceptible to interference by compounds present in urine [8].

Fig. 4

A ROS view of the human life cycle. ROS are essential for all aspects of the human life cycle, from conception and development until ageing and death [8,[11], [12], [13],[32], [33], [34], [35], [36], [37], [38], [39]]. There is a fine balance between their essentiality and the ability of too many of them to cause harm.

Fig. 5

Foyer-Halliwell-Asada Cycle. The ascorbate–glutathione cycle in chloroplasts. Enzymes involved: 1, ascorbate peroxidase; 2, dehydroascorbate reductase (reaction 2 can also occur non-enzymically at high pH–the pH of the stroma during photosynthesis may rise to as high as 8, due to formation of the proton gradient); 3, glutathione reductase. The first product of oxidation of ascorbate by ascorbate peroxidase is semidehydroascorbate (SDA); two SDA radicals can undergo disproportionation to form ascorbate and dehydroascorbate. NAD(P)H-dependent and ferredoxin-dependent mechanisms for reducing SDA also exist in chloroplasts. Thylakoid-bound ascorbate peroxidase can scavenge H₂O₂ as it is produced, and the resulting SDA can be reduced by electrons from photosystem I. The cycle shown above has been called the Foyer–Halliwell–Asada cycle after the names of the two scientists who first proposed it [42] and the third who did much to establish evidence for its occurrence [44]. However, we should acknowledge the contribution of Groden and Beck, who discovered ascorbate peroxidase in chloroplasts [45]. Adapted from Ref. [8] with permission of Oxford University Press.

Fig. 6

Oxidative damage in human ischemic stroke: a biomarker study. Data taken from Ref. [92]. Note that levels of oxidative damage (as measured by plasma biomarkers) were already high when the first samples were taken and remained high even after clinical improvement. Control refers to age-matched healthy subjects. F₂- and F₄- isoprostanes are well established biomarkers of oxidative lipid damage and allantoin is a biomarker of oxidative degradation of urate [8,[92], [93], [94], [95]].

Fig. 7

Are Haem proteins Fenton reagents?.

Fig. 8

What is the significance of oxidative stress in human disease? Adapted from Ref. [8] with permission of Oxford University Press.