The role of interactions between O2, H2O2, .OH,e- and O2- in free radical damage to biological systems

The occurrence of the Haber-Weiss reaction and other interactions between free radicals has been investigated in the effects of mixtures of free radicals on the permeability of resealed erythrocyte ghosts and on the activity of membrane-bound glyceraldehyde-3-phosphate dehydrogenase. The following mixtures were found to induce damage greater than that which could be accounted for by the independent actions of the constituent free radicals: (i) · OH + H₂O₂, and (ii) · OH + H₂O₂ + O₂^?. In contrast, the following mixtures were found to induce less damage than that predicted on the basis of independent actions of constituent free radicals: (i) H₂O₂ + O₂^?, and (ii) oxidizing radicals ( · OH, H₂O₂) + reducing radicals (e^?, H · ). These results suggest a Haber-Weiss-like interaction between H₂O₂ and O₂^?and an interaction between H₂O₂ and · OH to produce a species more potent than either in causing increased permeability. The decrease in damage observed in the simultaneous presence of oxidizing and reducing radicals suggests an antagonistic effect by which each tends to moderate damage by the other. Inactivation of glyceraldehyde-3-phosphate dehydrogenase was found to be more sensitive to radiation than permeability by an order of magnitude, while permeability was more sensitive to the enhancement of damage by oxygen. Comparison of the effectiveness of free radical scavengers in inhibiting the increase in permeability caused by free radicals showed the following order of effectiveness, expressed in terms of percentage protection: formate (90%) > nitrogen (65%) > catalase (60%) > dismutase (32%), and with respect to enzymatic inactivation, nitrogen (100%) > formate (77%) > dismutase (48%) > catalase (44%). The relative rates observed anaerobically and aerobically in the presence and absence of the above scavengers suggest that (at least in the case of radiation damage to the membranes of erythrocyte ghost cells) the “oxygen effect” is due to the interaction of oxygen with e^? and H., producing O₂^? which aggravates damage under conditions which allow consequent Haber-Weiss-like reactions. The further increase in damage when oxygen concentration is raised yet higher is due to the interaction of oxygen with the sites of initial damage.     

Influence of planting geometry on photosynthetically active radiation interception and dry matter production relationships in pearl millet

A field experiment was conducted at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) Center, Patancheru, India to study photosynthetically active radiation (PAR) interception and dry matter production relationships in pearl millet (Pennisetum americanum (L.) Leeke). Two pearl millet genotypes, BJ 104 (G₁) and ICH 226 (G₂) were sown at three planting geometries obtained by using combinations of row and plant spacings (S₁: 37·5 cm × 26·6 cm; S₂: 75·0 cm × 13·3 cm; S₃: 150·0 cm × 6·6 cm) such that plant population was constant at 100 000 ha⁻¹ in all treatments. Cumulative intercepted PAR was maximum (330 MJ m⁻²) in G₂S₂ and minimum (268 MJ m⁻²) in G₁S₃. Conversion efficiency values ranged from 1·87 g MJ⁻¹ in G₁S₂ to 2·32 g MJ⁻¹ in G₂S₃. Final above-ground dry matter followed the pattern of cumulative intercepted PAR and maximum dry matter (7·22 Mg ha⁻¹) was produced by G₂S₂ while G₁S₃ produced minimum dry matter (4·97 Mg ha⁻¹).     

In Vitro Effect of H2O2, Some Transition Metals and Hydroxyl Radical Produced Via Fenton and Fenton-Like Reactions,on the Catalytic Activity of AChE and the Hydrolysis of ACh

It is well known that the principal biomolecules involved in Alzheimer’s disease (AD) are acetylcholinesterase (AChE), acetylcholine (ACh) and the amyloid beta peptide of 42 amino acid residues (Aβ42). ACh plays an important role in human memory and learning, but it is susceptible to hydrolysis by AChE, while the aggregation of Aβ42 forms oligomers and fibrils, which form senile plaques in the brain. The Aβ42 oligomers are able to produce hydrogen peroxide (H₂O₂), which reacts with metals (Fe²⁺, Cu²⁺, Cr³⁺, Zn²⁺, and Cd²⁺) present at high concentrations in the brain of AD patients, generating the hydroxyl radical (^·OH) via Fenton (FR) and Fenton-like (FLR) reactions. This mechanism generates high levels of free radicals and, hence, oxidative stress, which has been correlated with the generation and progression of AD. Therefore, we have studied in vitro how AChE catalytic activity and ACh levels are affected by the presence of metals (Fe³⁺, Cu²⁺, Cr³⁺, Zn²⁺, and Cd²⁺), H₂O₂ (without Aβ42), and ^· OH radicals produced from FR and FLR. The results showed that the H₂O₂ and the metals do not modify the AChE catalytic activity, but the ^·OH radical causes a decrease in it. On the other hand, metals, H₂O₂ and ^·OH radicals, increase the ACh hydrolysis. This finding suggests that when H₂O₂, the metals and the ^·OH radicals are present, both, the AChE catalytic activity and ACh levels diminish. Furthermore, in the future it may be interesting to study whether these effects are observed when H₂O₂ is produced directly from Aβ42.     

Chromate-mediated free radical generation from cysteine,penicillamine, hydrogen peroxide,and lipid hydroperoxides

The Cr(VI)-mediated free radical generation from cystein, penicillamine, hydrogen peroxide, and model lipid hydroperoxides was investigated utilizing the electron spin resonance (ESR) spin trapping technique. Incubation of Cr(VI) with cysteine (Cys) generated cysteinyl radical. Radical yield depended on the relative concentrations of Cr(VI) and Cys. The radical generation became detectable at a cysteine: Cr(VI) ration of about 5, reached its highest level at a ratio of 30, and declined thereafter. Cr(VI) or Cys alone did not generate a detectable amount of free radicals. Similar results were obtained with penicillamine. Incubation of Cr(VI), Cys or penicillamine adn H₂O₂ led to hydroxyl (·OH) radical generation, which was verified by quantitative competition experiments utilizing ethanol. The mechanism for ·OH radical generation is considered to be a Cr(VI)-mediated Fenton-like reaction. When model lipid hydroperoxides such as t-butylhydroperoxide and cumene hydroperoxide were used in place of H₂O₂, hydroperoxide-derived free radicals were produced. Since thiols, such as Cys, exist in cellular systems at relatively high concentrations, Cr(VI)-mediated free radical generation in the presence of thiols may participate in the mechanisms of Cr(VI)-induced toxicity and carcinogenesis.     

The oxidation of ferrocytochrome c by Br−2, (SCN)−2, N3 and OH radicals studied by pulsed-electron and γ-ray radiolysis

The reactions of ferrocytochrome c with Br^?₂, (SCN)^?₂, N₃ and OH radicals were followed by measuring the change in the optical spectra of cytochrome c on γ-irradiation as well as the rate of change of absorbance upon pulse irradiation.Ferrocytochrome c is oxidized to ferricytochrome c by Br^?₂, (SCN)^?₂ or N₃ radical with an efficiency of about 100% through a second-order process in which no intermediates were observed. The rate constants in neutral solutions at I = 0.073 are 9.7 · 10⁸ M^?1 · s^?1, 7.9 · 10⁸ M^?1 · s^?1, 1.3 · 10⁹ M^?1 · s^?1 for the oxidation by Br^?₂, (SCN)^?₂ and N₃ radicals, respectively. The rate constants do not vary appreciably in alkaline solutions (pH 8.9). The ionic strength dependence was observed for the rate constants of the oxidation by Br^?₂ and (SCN)^?₂. Those rate constants estimated on the assumption that the radicals react only with the amino acid residues with the characteristic steric correction factors were less than one-tenth of the observed ones. These results suggest that the partially exposed region of the heme is the probable site of electron transfer from ferrocytochrome c to the radical.Hydroxyl radicals also oxidize ferrocytochrome c with a high rate constant (k > 1 · 10¹⁰ M^?1 · s^?1), but with a very small efficiency (5%).     

The antioxidant properties of polypore mushroom <Emphasis Type="Italic">Daedaleopsis confragosa</Emphasis>

Daedaleopsis confragosa belong to a large and remarkable group of mushrooms called polypores. This type of mushroom could be easily said to be quite unexplored and unused when it comes to its antioxidant properties. Thus, in order to evaluate its antioxidant activity, the investigation had to include the total phenolics and flavonoide content, the content of Selenium, the content of Zinc, the scavenging capacity on DPPH^· and OH^· radicals, reducing power and capacity to remove lipid peroxidation. The investigated mushroom extract contained 54.17 mg GAE/g of total phenols and 48.46 mg CE/g of total flavonoides. Zinc and Selenium were detected and quantified by using inductively coupled plasma mass spectroscopy. The scavenging activity of the radicals was found to exhibit 50% of the inhibition value (IC₅₀ value) at the extract concentration of 0.015±0.007 mg/ml for the investigated D. confragosa extract. By using electron paramagnetic resonant spectroscopy it was found that the investigated extract does not have a significant role in the prevention of lipid peroxidation. It was effective in scavenging on ^·OH radical, RI was 56±2%.     

Pulse-radiolytic investigations of catechols and catecholamines II. Reactions of Tiron with oxygen radical species

Transient spectra and kinetic data of Tiron (1,2-dihydroxybenzene-3,5-disulphonic acid) are reported, obtained after pulse-radiolytic oxidation by hydroxyl radicals (°OH), superoxide anions (O₂^?) or a combination of both oxygen radicals. The rate constant with °OH radicals was determined at 1.0·10⁹ M^?1·s^?1. Contrary to a previous report (Greenstock, C.L. and Miller, R.W. (1975) Biochim. Biophys. Acta 396, 11–16), the rate constant with O₂^? of 1.0·10⁷ M^?1·s^?1 is lower by one order of magnitude; also the semiquinone absorbs at 300 nm rather than at 400 nm. The ratio of the rate constants with °OH and O₂^? of 100 again demonstrates that any oxidation reaction by the latter radical is unspecific due to the more efficient reaction of °OH radicals, leading to the same products with catechol compounds.     

Formation of Hydroxyl Radicals in Biological Systems. Does Myoglobin Stimulate Hydroxyl Radical Formation from Hydrogen Peroxide?

Incubation of horse-heart oxymyoglobin or metmyoglobin with excess H₂O₂ causes formation of myoglobin(IV), followed by haem degradation. At the time when haem degradation is observed, hydroxyl radicals (.OH) can be detected in the reaction mixture by their ability to degrade the sugar deoxyribose. Detection of hydroxyl radicals can be decreased by transferrin or by OH scavengers (mannitol, arginine, phenylalanine) but not by urea. Neither transferrin nor any of these scavengers inhibit the haem degradation. It is concluded that intact oxymyoglobin or metmyoglobin molecules do not react with H₂O₂ to form OH detectable by deoxyribose, but that H₂O₂ eventually leads to release of iron ions from the proteins. These released iron ions can react to form OH outside the protein or close to its surface. Salicylate and the iron chelator desferrioxamine stabilize myoglobin and prevent haem degradation. The biological importance of OH generated using iron ions released from myoglobin by H₂O₂ is discussed in relation to myocardial reoxygenation injury.     

Partitioning of taxifolin-iron ions complexes in octanol-water system

The composition of taxifolin-iron ions complexes in an octanol-water biphasic system was studied using the method of absorption spectrophotometry. It was found that at pH 5.0 in an aqueous biphasic system the complex of [Tf · Fe₂(OH) _k (H₂O)_{8 ? k} ] is present, but at pH 7.0 and 9.0 the complexes of [Tf₂ · Fe(OH) _k (H₂O)_{2 ? k} ] and [Tf · Fe(OH) _k (H₂O)_{4 ? k} ] are predominantly observed. The formation of a stable [Tf₃ · Fe] complex occurred in octanol phase. The charged iron ion of this complex is surrounded by taxifolin molecules, which shield the iron ion from lipophilic solvent. During transition from water to octanol phase the changes of the composition of complexes are accompanied by reciprocal changes in portion of taxifolin and iron ions in these phases. It was shown that the portion of taxifolin in aqueous solution in the presence of iron ions is increased at high pH values, and the portion of iron ions is minimal at pH 7.0. In addition, the parameters of solubility limits of taxifoliniron ions complexes in an aqueous solution were determined. The data obtained gain a better understanding of the role of complexation of polyphenol with metal of variable valency in passive transport of flavonoids and metal ions across lipid membranes.     

Cholecystokinin and tryptic activity in the gut and body of developing Atlantic halibut larvae: evidence for participation in the regulation of protein digestion

At 7 days after first feeding (DAFF), the peptide hormone cholecystokinin (CCK) content (fmol individual^?1) and the tryptic activity [μmol arginine‐methyl‐coumarinyl‐7‐amide (MCA) min^?1 individual^?1] per individual gut of Atlantic halibut Hippoglossus hippoglossus larvae were low: 0·2 ± 0·1 and 0·14 ± 0·10, respectively. Thereafter, both parameters increased with the increase in gut mass and reached 19·67 ± 5·58 and 2·71 ± 0·64 at 26 DAFF, respectively. Due to the small sample size, the dry mass (M_G, mg) of the individual gut could not be determined accurately at 7 DAFF. At 13 DAFF M_G represented 5·5% of whole body dry mass (M_w, mg) while at 26 DAFF it had increased to 23%. The mass specific tryptic activity [μmol MCA min^?1 per mg dry mass (M)] in the gut increased from 2·74 ± 1 ± 98 at 13 DAFF to 5·00 ± 0·78 at 26 DAFF. There was more individual variation in the mass specific CCK content (fmol M^?1) but no significant differences were found, although the data indicated an increase (from 23·38 ± 11·26 at 13 DAFF to 36·27 ± 8·96 fmol M^?1 at 26 DAFF). At 7 DAFF the CCK content of the gut represented c. 2% of the whole body CCK content while it increased to c. 62% of the whole body CCK content at 26 DAFF. This demonstrates that it is necessary to separate neural and gastrointestinal sources of CCK in order to determine its alimentary role in fish larvae. Trypsin activity was only found in the gut compartment. In larvae aged 45 DAFF dietary proteins delivery into the gut by tube‐feeding appeared to stimulate post‐prandial secretion of CCK from the gut as well as stimulate pancreatic trypsin secretion, suggesting that both factors contribute to protein digestion.     

Antioxidant and iron-chelating activities of cyanobacterial exopolymers with potential for wound healing

Progress of wound healing is critically dependent on the balance between oxidants and antioxidants at the wound site, and transition metals such as iron can exacerbate ROS generation. In the present study, cyanobacterial exopolymers from three strains of Anabaena and Tolypothrix tenuis have been characterized for their antiradical and Fe²⁺-chelating activity. All the four exopolymers exhibited antioxidant activities against O₂·, H₂O₂, OH·, and NO·, with the exopolymer from Anabaena oryzae showing strong inhibition of NO· and ·OH radicals followed by that from Anabaena anomala. Correlation analysis of antioxidant activities and sulphate, uronic and phenolic content of the exopolymers showed a strong correlation of sulphate content to superoxide scavenging and activity against nitric oxide radicals. H₂O₂ scavenging was related to the presence of phenolics in the preparation which also contributed to the reducing power. Iron chelation had a strong bearing upon the overall reducing power and superoxide control.     

Evaluation of new thiadiazoles and benzothiazoles as potential radioprotectors: Free radical scavenging activity in vitro and theoretical studies (QSAR,DFT)

Thiol and aminothiol compounds are among the most efficient chemical radioprotectors. To increase their efficiency, we synthesized two new classes of thiol and aminothiol compounds derived from benzothiazole (T1, T2, AM1, AM2) and thiadiazole (T3, T4, AM3) structures. We examined them for their ability to scavenge free radicals (DPPH^·, ABTS^·+, ^·OH). Thiol derivatives with a thiadiazole structure are the most active compounds scavenging DPPH^· and ABTS^·+ free radicals, with an IC₅₀ of 0.053 ± 0.006 and 0.023 ± 0.002 mM, respectively, for the derivative T3. Moreover, compounds T1, T2, and T3 at 60 μM gave 83% protection against 2-deoxyribose degradation by ^·OH. The ability of these compounds to protect DNA against ^·OH produced by a Fenton reaction and γ-irradiation (15 Gy)-induced strand breaks was also evaluated on pBR322 plasmid DNA. In both tests thiol derivatives were the most efficient compounds. Derivatives T2 and T3 totally inhibit DNA strand breaks at the concentration of 50 μM. The protection afforded by these derivatives was comparatively higher than that of the radioprotectors WR-2721 and WR-1065. Our data indicate that these two compounds are free radical scavengers and potential antioxidant agents. Finally, DFT and QSAR studies were performed to support the experimental observations.     

Chemical composition of the leaves of Reynoutria japonica Houtt. and soil features in polluted areas

The study was conducted on six sites that are dominated by Japanese knotweed (Reynoutria japonica) and that vary in the level of industrialization and habitat transformation by humans. The aim of the research was to investigate the chemical-physical features of soil under a closed and dense canopy of R. japonica, the chemical composition of the R. japonica leaves, and to compare the content of certain elements in the soil-plant-soil system. The soil organic carbon (C_org) content varied from 1.38±0.004% to 8.2±0.047% and the maximum in leaves was 49.11±0.090%. The lowest levels of total nitrogen (N_tot) in soil were recorded on the heavily disturbed sites (till 0.227±0.021%). Soil pH varied greatly, ranging from acidic (pH=4.0) to neutral (pH=7.7). Heavy metal content differed significantly among the study sites. At all of the sites, both in the case of soil and plant leaves, Zn was a dominant element and its concentration ranged from 41.5 to 501.2 mg·kg^?1 in soils and from 38.6 to 541.7 mg·kg^?1 in leaves. Maximum accumulations of P (2103.3±15.3 mg·kg^?1) and S (2571.7±17.6 mg·kg^?1) were observed on the site that had been influenced by agricultural practices. The results obtained showed that R. japonica is able to accumulate high levels of heavy metals.     

Hydroxyl radical scavenging activity of flavonoids

The flavonoids scavenge hydroxyl (^.OH) radicals generated by UV photolysis of hydrogen peroxide. Free ^.OH radicals were spin-trapped by 5,5-dimethyl-1-pyrroline N-oxide and the adduct was detected by high pressure liquid chromatography coupled with an electrochemical detector. The scavenging activity of flavonoids decreases in the order: myricetin > quercetin > rhamnetin > morin > diosmetin > naringenin > apigenin > catechin >5,7- dihydroxy -3′,4′,5′-trimethoxyflavone > robinin > kaempferol > flavone. The activity increases with the number of hydroxyl groups substituted in the aromatic B-ring. The presence of a hydroxyl at C-3 and its glycosylation does not further increase scavenging efficiency. It is suggested that the overall antioxidant effect of flavonoids on lipid peroxidation may be due to their ^.OH and O·⁻₂ scavenging properties and the reaction with peroxy radicals.     

Laser-light scattering investigation of the micellar properties of gangliosides

The micellar properties of gangliosides in water solutions were investigated by quasielastic light scattering measurements. G_M1 and G_D1a gangliosides were isolated from calf brain, purified to more than 99% and dissolved in 0.025 M Tris—HCI buffer (pH 6.8) at 37°C. The average intensity of scattered light and the intensity correlation function were measured by an apparatus including a 5145 Å argon laser and a real-time digital correlator. The scattered intensity data allowed the derivation of an upper limit to the critical micelle concentration (c₀) and the evaluation of the molecular weight (M) of the micelle. The intensity correlation function gave the diffusion coefficient D, and hence the hydrodynamic radius R_H, and also contained information on the polydispersity of the sample. We find c_o < 1 × 10^?6 M for both G_M1 and G_D1a, M = 532 000 ± 50 000 and $\text{R}{}_{\mathrm{<mtext>H</mtext>}}\text{= 63.9 ± 2}\text{A}\text{?}$ for G_M1, and M = 417 000 ± 40 000 and $\text{R}{}_{\mathrm{<mtext>H</mtext>}}\text{= 59.5 ± 2}\text{A}\text{?}$ for G_D1a. The mixture 3:1 of the two gangliosides gave intermediate values for all examined parameters. The presence of cations, within the physiological concentration range. and, in particular of Ca²⁺, did not influence significantly the values of c_o and the main features of the micelle.     

Low temperature photodissociation studies of ferrous hemoglobin and myoglobin complexes by Mössbauer spectroscopy

⁵⁷Fe-enriched complexes of hemoglobin and myoglobin with CO and O₂ were photodissociated at 4.2°K, and the resulting spectra were compared with those of the deoxy forms. Differences in both quadrupole splitting and isomer shift were noted for each protein, the photoproducts having smaller isomer shift and larger quadrupole splitting than the deoxy forms. The photoproducts of HbCO and HbCO₂ had narrow absorption lines, indicating a well-defined iron environment. The corresponding myoglobin species had broader absorption lines, as did both deoxy forms. The weak absorption lines of photodissociated NO complexes appeared to be wide, possibly indicating magnetic interaction with the unpaired electron of the nearby NO.     

Assessment of DNA damage in sperm after repeated non‐invasive sampling in zebrafish Danio rerio

Repeated non‐invasive sampling of zebrafish Danio rerio sperm was conducted, sperm counts were obtained and a method for measurement of DNA damage in sperm was developed and validated (single‐cell gel electrophoresis, comet, assay). DNA damage in sperm increased with concentration of hydrogen peroxide (H₂O₂, 0–200 µM), and in vitro exposure of sperm to 200 µM H₂O₂ produced 88·7 ± 3·9% tail DNA compared to unexposed controls [12 ± 0·7% tail DNA (mean ± s.e ., n = 3)]. Frequency of sperm sampling (sampled every 2, 4 or 7 days) did not affect DNA damage in sperm, but sperm counts decreased 57 and 22% for fish sampled every 2 or 4 days, respectively.     

Ventilation and oxygen consumption in the hagfish, Myxine glutinosa L.

Ventilation was measured directly in the hagfish, Myxine glutinosa L., by means of an electro-magnetic blood flowmeter. Ventilatory flow and frequency increased from 0.86 ± 0.27 ml·min^?, and 18.2 ± 5.1·min^?, respectively, at 7°C to 1.70 ± 0.20 ml·min^?, and 70.1 ± 9.5·min^? at 15 ·C.Standard oxygen consumption,V?O₂, was measured in non-buried hagfish. V?O₂ was 0.57 ± 0.17μl O₂·g^?1·min^?1 at 7°C, and 0.85 ± 0.12μl O₂·g^?1·min^?1 at 15°C.     

Protective effect of histidine on para-nonylphenol-enhanced hydroxyl free radical generation induced by 1-methyl-4-phenylpyridinium ion (MPP+) in rat striatum

The present study examined the antioxidant effect of histidine, a singlet oxygen (¹O₂) scavenger, on para-nonylphenol (an environmental estrogen-like chemical)-enhanced hydroxyl radical (·OH) generation induced by 1-methyl-4-phenylpyridinium ion (MPP⁺) in extracellular fluid of rat striatum. Rats were anesthetized, and sodium salicylate in Ringer’s solution (0.5 nmol/μl/min) was infused through a microdialysis probe to detect the generation of ·OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. Introduction of para-nonylphenol (10 μM) significantly enhanced MPP⁺-induced ·OH generation. Histidine (25 mM) decreased the para-nonylphenol-enhanced ·OH formation. Although the level of MPP⁺-induced ·OH formation trapped as DHBA after para-nonylphenol treatment increased, para-nonylphenol failed to increase either the level of dopamine and DHBA formation in the reserpinized animals. These results indicate that para-nonylphenol and MPP⁺-enhanced ·OH generation was based on ¹O₂ production, and histidine may have a preventive effect on para-nonylphenol and MPP⁺-induced ·OH generation in rat striatum.     

Copper-Ligand Interactions and Physiological free Radical Processes. Part 2. Influence of Cu2+ Ions on Cu+-Driven Oh Generation and Comparison with Their Effects on Fe2+-Driven Oh Production

In our search to establish a reference ·OH production system with respect to which the reactivity of copper(II) complexes could then be tested, the influence of free Cu²⁺ ions on the Cu⁺/H₂O₂ reaction has been investigated.

This influence depends on the C_Cu²⁺/C_Cu⁺ ratio. At low Cu²⁺ concentrations, ·OH damage to various detector molecules decreases with increasing Cu²⁺ concentrations until C_Cu²⁺/C_Cu⁺ reaches unity. Above this value, ·OH damage increases sharply until C_Cu²⁺/C_Cu⁺ becomes equal to 5 with salicylate and 2 with deoxyribose, ratios for which the protective effect of Cu²⁺ cancels. Finally, at higher concentrations, Cu²⁺ ions logically add their own ·OH production to that normally expected from Cu⁺ ions. The possible origin of this unprecedented alternate effect has been discussed. The possible influence of Cu⁺ ions on the generation of ·OH radicals by water gamma radiolysis has also been tested and, as already established for Cu²⁺ in a previous work, shown to be nonexistent. This definitely confirms that either form of ionised copper cannot scavenge ·OH radicals in the absence of a Iigand.