Free Radical Enhancer Xenobiotic is an Inducer of Cataract in Rabbit

Free radical enhancers, diquat, paraquat, plumbagin and juglone were used to study the oxy radical-induced damage to the rabbit lens in vitro and in vivo. Each compound caused a 6–8 fold increase in malondialdehyde (MDA) and a 30–55% decrease in reduced glutathione of the lens in vim. These peroxidative and oxidative changes were potentiated in the presence of 100% 0., abolished by N, and prevented by desferal-Mn (III) (DF-Mn) or liposomal superoxide dismutase (LSOD) indicating the involvement of O₂^?.

Diquat injected intravitreally as a single dose (300nmole in 30μl of isotonic saline) in the right eye of a 5-wk-old Dutch belted rabbit, induced early cataract after 24–72h. The lens of the contralateral control eye injected with isotonic saline had no change. In the right eye, O₂,^? and OH -productions were significantly (P < 0.01) higher; O₂-, was about 16 fold higher in the aqueous humor and vitreous humor, and 5 fold in the lens and retina, and OH. was 35 fold higher in the aqueous humor, 2 fold in vitreous humor and 5 fold in the lens and retina as compared to the respective tissues of the control eye. Enhanced lipid peroxidation in the lens was apparent from the higher levels of MDA and formation of aminophospholipid-MDA Schiff-base conjugates.

We propose that cyclic oxidation-reduction of xenobiotics coupled to the endogenous redox systems in the eye, could generate oxy radicals in excessive amounts, triggering cataractogcnesis.     

Regulation of hydrogen peroxide in eye humors effect of 3-amino-1H-1,2,4-triazole on catalase and glutathione peroxidase of rabbit eye

Activities of catalase (H₂O₂ : H₂O₂ oxidoreductase, EC 1.11.1.6) and GSH peroxidase (GSH : H₂O₂ oxidoreductase, EC 1.11.1.9) have been measured in iris, ciliary body, retina, corneal epithelium, corneal endothelium, lens capsule-epithelium and decapsulated lens. 3-Amino-1H-1,2,4-triazole is a specific inhibitor of catalase and a potent cataractogenic agent. We observed marked inhibition of catalase activity in these tissues 1–6 h after the administration of a single intravenous dose of 1 g 3-aminotriazole per kg body weight in rabbit. This was associated with a 2–3-fold increase in the H₂O₂ concentrations of aqueous humor and vitreous humor. The increased peroxide concentrations were restored to the physiological levels as the catalase activity of eye tissues gradually returned to normal with time after injection. Under the conditions, GSH peroxidase activity of the afore-mentioned eye tissues was unaltered, GSH and protein sulfhydryl of lens were not changed, and ascorbic acid of aqueous humor and vitreous humor was not significantly altered. Based on these findings our conclusion is that catalase of eye tissues regulates the endogenous H₂O₂ in eye humors to the physiological level. We speculate that H₂O₂ may be the triggering factor in cataract induced by 3-aminotriazole.     

Antioxidant and anticataractogenic effects of topical captopril in diquat-induced cataract in rabbits.

1-[(2s)-3-Mercapto-2-methylpropionyl]-L-proline (captopril), an antihypertensive and free radical scavenger, protected the rabbit lens from peroxidative and oxidative damage induced by 1 mM diquat in vitro. To evaluate the anticataract efficacy of captopril, an experimental group of five rabbits was treated with topical captopril (1% in 0.15 M NaCl, w/v), and 50 microliters was instilled onto both eyes four times a day for a total of 8 weeks. Following the same procedure, the eyes of five rabbits were treated with topical 0.15 M NaCl as a control for captopril treatment. At the end of the first week of treatment, a single intravitreal dose of 120 nmole diquat in 30 microliters of 0.15 M NaCl was injected into the right eye of each rabbit of both the groups. As a control for intravitreal diquat injection, the left eye of all the rabbits were injected with the diluent, 30 microliters per eye. The intravitreal diquat or its diluent injection was only for one time. From slit-lamp biomicroscopic observation of the diquat-injected right eyes, the anticataract effect of captopril in the treatment group was indicated by the finding that in four of five rabbits the cataract did not advance; whereas in four of five rabbits treated with the diluent the cataract progressed to grade 3. The lenses in the diluent-injected control left eyes of the rabbits treated with the captopril or diluent were normal. However, since the number of animals used for the in vivo studies was few, further confirmation of the anticataract effect of captopril is necessary. In diquat-injected right eyes of animals treated with captopril, the integrated rate of O2- production was about 50% less (p less than .001) in the aqueous humor, vitreous humor, and lens, compared with O2-, 33.49 +/- 2.26 microM (mean +/- SEM) in the aqueous humor, 17.12 +/- 0.75 microM in the vitreous humor, and 31.44 +/- 1.29 nmole/g wet weight in the lens of the diquat-injected right eyes treated with the diluent. Similar significant (p less than .01) differences in the production of .OH and H2O2 in eye tissues were also observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Egg Yolk Phosvitin Inhibits Hydroxyl Radical Formation from the Fenton Reaction

Phosvitin, a phosphoprotein known as an iron-carrier in egg yolk, binds almost all the yolk iron. In this study, we investigated the effect of phosvitin on Fe(II)-catalyzed hydroxyl radical (^?OH) formation from H₂O₂ in the Fenton reaction system. Using electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and deoxyribose degradation assays, we observed by both assays that phosvitin more effectively inhibited ^?OH formation than iron-binding proteins such as ferritin and transferrin. The effectiveness of phosvitin was related to the iron concentration, indicating that phosvitin acts as an antioxidant by chelating iron ions. Phosvitin accelerates Fe(II) autoxidation and thus decreases the availability of Fe(II) for participation in the ^?OH-generating Fenton reaction. Furthermore, using the plasmid DNA strand breakage assay, phosvitin protected DNA against oxidative damage induced by Fe(II) and H₂O₂. These results provide insight into the mechanism of protection of the developing embryo against iron-dependent oxidative damage in ovo.     

The relative effectiveness of .OH,H2O2,O2−, and reducing free radicals in causing damage to biomembranes: A study of radiation damage to erythrocyte ghosts using selective free radical scavengers

The relative effectiveness of oxidizing (^.OH, H₂O₂), ambivalent (O₂^?) and reducing free radicals (e^? and CO₂^?) in causing damage to membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase of resealed erythrocyte ghosts has been determined. The rates of damage to membranebound glyceraldehyde-3-phosphate dehydrogenase ($\text{R}$(enz)) were measured and the rates of damage to membranes ($\text{R}$(mb)) were assessed by measuring changes in permeability of the resealed ghosts to the relatively low molecular weight substrates of glyceraldehyde-3-phosphate dehydrogenase. Each radical was selectively isolated from the mixture produced during gamma-irradiation, using appropriate mixtures of scavengers such as catalase, superoxide dismutase and formate. ^.OH, O₂^? and H₂ O₂ were approximately equally effective in inactivating membrane-bound glyceraldehyde-3-phosphate dehydrogenase, while e^? and CO₂^? were the least effective. $\text{R}$(enz) values of O₂^? and H₂O₂ were 10-times and of ^.OH 15-times that of e^?. $\text{R}$(mb) values were quite similar for e^? and H₂O₂ (about twice that of O₂^?), while that of ^.OH was 3-times that of O₂^?. Hence, with respect to $\text{R}$(mb): ${}^{\mathrm{.}}\text{OH}\text{>}\text{e}{}^{\mathrm{?}}\text{=}\text{H}{}_2\text{O}{}_2\text{>}\text{O}{}_2{}^{\mathrm{?}}$ , and with respect to $\text{R}$(enz): ${}^{\mathrm{.}}\text{OH}\text{>}\text{O}{}_2{}^{\mathrm{?}}\text{=}\text{H}{}_2\text{O}{}_2\text{>}\text{e}{}^{\mathrm{?}}$. The difference between the effectiveness of the most damaging and the least damaging free radicals was more than 10-fold greater in damage to the enzyme than to the membranes. Comparison between H₂O₂ added as a chemical reagent and H₂O₂ formed by irradiation showed that membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase were relatively inert to reagent H₂O₂ but markedly susceptible to the latter.     

Reactive oxygen species are involved in regulation of pollen wall cytomechanics

Production and scavenging of reactive oxygen species (ROS) in somatic plant cells is developmentally regulated and plays an important role in the modification of cell wall mechanical properties. Here we show that H₂O₂ and the hydroxyl radical (^?OH) can regulate germination of tobacco pollen by modifying the mechanical properties of the pollen intine (inner layer of the pollen wall). Pollen germination was affected by addition of exogenous H₂O₂, ^?OH, and by antioxidants scavenging endogenous ROS: superoxide dismutase, superoxide dismutase/catalase mimic Mn‐5,10,15,20‐tetrakis(1‐methyl‐4‐pyridyl)21H, 23H‐porphin, or a spin‐trap α‐(4‐pyridyl‐1‐oxide)‐N‐tert‐butylnitrone, which eliminates ^?OH. The inhibiting concentrations of exogenous H₂O₂ and ^?OH did not decrease pollen viability, but influenced the mechanical properties of the wall. The latter were estimated by studying the resistance of pollen to hypo‐osmotic shock. ^?OH caused excess loosening of the intine all over the surface of the pollen grain, disrupting polar growth induction. In contrast, H₂O₂, as well as partial removal of endogenous ^?OH, over‐tightened the wall, impeding pollen tube emergence. Feruloyl esterase (FAE) was used as a tool to examine whether H₂O₂‐inducible inter‐polymer cross‐linking is involved in the intine tightening. FAE treatment caused loosening of the intine and stimulated pollen germination and pollen tube growth, revealing ferulate cross‐links in the intine. Taken together, the data suggest that pollen intine properties can be regulated differentially by ROS. ^?OH is involved in local loosening of the intine in the germination pore region, while H₂O₂ is necessary for intine strengthening in the rest of the wall through oxidative coupling of feruloyl polysaccharides.     

Scavenging of superoxide anion radical and hydroxyl radical by novel thiazolyl‐thiazolidine‐2,4‐dione compounds

The antioxidant behavior of a series of new synthesized substituted thiazolyl‐thiazolidine‐2,4‐dione compounds (TZDs) was examined using chemiluminescence and electron paramagnetic resonance spin trapping techniques. 5,5‐Dimethyl‐1‐pyrroline‐N‐oxide (DMPO) was used as the spin trap. The reactivity of TZDs with superoxide anion radical (O) and hydroxyl radical (HO^?) was evaluated using potassium superoxide/18‐crown‐6 ether dissolved in dimethylsulfoxide, and the Fenton‐like reaction (Fe²⁺ + H₂O₂), respectively. The results showed that TZDs efficiently inhibited light emission from the O generating system at a concentration of 0.05–1 mmol L^?1 (5–94% reductions were found at 1 mmol L^?1 concentration). The TZD compounds showed inhibition of HO^?‐dependent DMPO–OH spin adduct formation from DMPO (the amplitude decrease ranged from 8 to 82% at 1 mmol L^?1 concentration). The findings showed that examined TZDs had effective activities as radical scavengers. Copyright © 2009 John Wiley & Sons, Ltd.     

Copper ions and hydrogen peroxide form hypochlorite from NaCl thereby mimicking myeloperoxidase

Sea urchins have elaborated multiple defenses to assure monospermic fertilization. In this work, we have concentrated on a study of the mechanism(s) by which hydrogen peroxide (H₂O₂) prevents polyspermy in Arbacia punctulata. We found that it is not H₂O₂ but probably hypochlorous acid/hypochlorite (HOCl/OCl^?) derived from H₂O₂ that is toxic to the supernumerary sperm. The spermicidal activity of H₂O₂ is potentiated by at least one order of magnitude by cupric ions (Cu²⁺). This increased toxicity is not due to the formation of hydroxyl radicals (·OH) because ·OH scavengers did not counteract the activity of Cu²⁺. More-over, substitution of Cu²⁺ by ferrous ions (Fe²⁺), which are known to cause formation of ·OH from H₂O₂, had no effect on fertilization even at 10²?10³ times higher concentrations. In contrast, 3-amino-1,2,4-triazole (AT), an HOCl/OCl^? scavenger, totally reversed the toxic effects of Cu²⁺. Furthermore, we found that HOCl/OCl^? is generated in solutions of H₂O₂ and Cu²⁺ in the presence of 0.5 M NaCl and that its accumulation is abolished by AT. Thus it is possible that the antifertility properties of copper are due to its ability to mediate formation of HOCl/OCl^?. HOCl/OCl^? generated by Cu²⁺ from H₂O₂ and Cl^?, a low concentration of exogenously added HOCl/OCl^?, or increased concentrations of H₂O₂ has similar inhibitory effects on the fertilization process in sea urchins. Therefore, we suggest that polyspermy is prevented by the action of a myeloperoxidase that affects the formation of HOCl/OCl^? from the Cl^? present in sea water through reaction with H₂O₂ generated by the newly fertilized egg.     

Measurement of OH radical CT for inactivating Cryptosporidium parvum using photo/ferrioxalate and photo/TiO2 systems

Aim: This study investigates the inactivation of Cryptosporidium parvum using the OH radical and reports the OH radical CT (OH radical concentration × contact time) values for C. parvum inactivation. Methods and Results: Although a wealth of information has demonstrated the efficacy of the microbial inactivation activity of the OH radical, no studies have performed a quantitative estimation of the OH radical for C. parvum inactivation. The CT value of the OH radical required for 2 log C. parvum inactivation was measured with two OH radical‐generating systems, photo/ferrioxalate and photo/TiO₂. The OH radical was approx. 10⁴–10⁷‐fold more effective for microbial inactivation than other popular chemical disinfectants such as ozone, chlorine dioxide and free chlorine. Conclusions: The OH radical appears to be suitable for microbial inactivation with a calculated CT value required for 2 log C. parvum inactivation of 9·3 × 10^?5 mg min l^?1. Significance and Impact of the Study: This study is the first report of an investigation on the role of the OH radical in the photo/ferrioxalate and photo/TiO₂ systems and on the OH radical CT required for C. parvum inactivation.     

Non-invasive Analysis of Reactive Oxygen Species Generated in NH4OH-induced Gastric Lesions of Rats using a 300 MHz In Vivo ESR Technique

Free radicals are reportedly involved in mucosal injury, including NH⁴OH-induced gastric lesions, but the kind, location and origin of radical generation have yet to be clarified. We developed the non-invasive measurement of reactive oxygen species (ROS) in stomach, and applied to mucosal injury. NH⁴OH-induced gastric lesions were prepared in rats, which were then given a nitroxyl probe intragastrically or intravenously, and the spectra of the gastric region were obtained by in vivo 300?MHz electron spin resonance (ESR) spectroscopy. The spectral change of the nitroxyl probe administered intragastrically was significantly enhanced 30?min after NH⁴OH administration, but no change occurred when the probe was given by intravenous injection. The enhanced change was confirmed to be due to ?OH generation, because it was completely suppressed by mannitol, catalase and desferrioxamine (DFO), and was not observed in neutropenic rats. NH⁴OH-induced neutrophil infiltration of the gastric mucosa was suppressed by intravenous injection of superoxide dismutase (SOD) or catalase, or by administration of allopurinol. The present study provided the direct evidence in NH⁴OH-treated living rats that ?OH produced from O₂^?- derived from neutrophils caused gastric lesion formation, while O₂^?- or H²O² derived from the xanthine oxidase system in endothelial cells was involved in neutrophil infiltration.     

Detection and identification of oxidants formed during •NO/O2•– reaction: A multi-well plate CW-EPR spectroscopy combined with HPLC analyses

Abstract

New techniques and probes are routinely emerging for detecting short-lived free radicals such as superoxide radical anion (O₂^?–), nitric oxide (^?NO), and transient oxidants derived from peroxynitrite (ONOO^–/ONOOH). Recently, we reported the profiles of oxidation products (2-hydroxyethidium, ethidium, and various dimeric products) of the fluorogenic probe hydroethidine (HE) in the ^?NO/O₂^?– system (Zielonka et al. 2012). In this study, we used HPLC analyses of HE oxidation products in combination with continuous wave electron paramagnetic resonance (CW-EPR) spin trapping with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) to define the identity of the oxidizing species formed in the ^?NO/O₂^?– system. EPR spin-trapping technique is still considered as the gold standard for characterization of free radicals and their intermediates. We monitored formation of BMPO-superoxide (BMPO-^?OOH) and BMPO-hydroxyl (BMPO-^?OH) radical adducts. Simultaneous analyses of results from EPR spin-trapping and HPLC measurements are helpful in the interpretation of the mechanism of formation of products of HE oxidation.     

Hydroxyl Radical Generation Depending on O2 or H2O by a Photocatalyzed Reaction in an Aqueous Suspension of Titanium Dioxide

Photocatalytic production of the electron (e^-) and positive hole (h⁺) in an aqueous suspension of TiO₂ (anatase form) under illumination by near-UV light (295-390 nm) generated the superoxide (O₂ ^-) and hydroxyl radical (?OH), which both proceeded linearly with reaction time, while H₂O₂ accumulated non-linearly. Under anaerobic conditions (introduced Ar gas), the yields of three active species of oxygen were decreased to 10-20% of those detected in the air-saturated reaction. The electron spin resonance (ESR) signal characteristics of ?OH were obtained when a spin trap of 5,5-dimthyl-1-pyrroline-N-oxide (DMPO) was included in the illuminating mixture. The intensity of the ESR signal was increased by Cu/Zn superoxide dismutase, and decreased under anaerobic conditions, amounting to only 20% of the intensity detected in the aerobic reaction. The addition of H₂O₂ to the reaction mixture resulted in about an 8-fold increase of ?OH production in the anaerobic reaction, but only about 1.5-fold in the aerobic reaction, indicating that e^- generated by the photocatalytic reaction reduced H₂O₂ to produce ?OH plus OH^-. On the other hand, D₂O lowered the yield of ?OH generation to 18% under air and 40% under Ar conditions, indicating the oxidation of H₂O by h⁺. The addition of Fe(III)-EDTA as an electron acceptor effectively increased ?OH generation, 2.3-fold in the aerobic reaction and 8.4-fold in the anaerobic reaction, the yield in the latter exceeding that in the air-saturated reaction.     

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.     

A blue chemiluminescence in the reaction of umbelliferone with hypochlorite

A strongly fluorescing 7-hydroxycoumarin (umbelliferone, U) oxidized in dilute (10 μmol/L-0, 1 mol/L) aqueous solution with CIO^? or CIO^? + H₂O₂ (but not with H₂O₂ alone) produces a strong chemiluminescence (CL). Light emission kinetics depends on the pH of solution (4.0–10.5) and the reaction has a low activation energy E_a = 31 ± 2 kJ/mol (285–310 K). The spectrum covers the fluorescence of umbelliferone (400–550 nm, λ_max 460nm). No red emission typical of ¹Δg, ¹Σ⁺g (O₂)₂ is observed either in the umbelliferone +CIO^? or the umbelliferone +CIO^? + H₂O₂ solution. The possible mechanism of CL and concomitant degradative oxidation of umbelliferone is discussed.     

On the repair of oxidative damage to apoferritin: a model study with the flavonoids quercetin and rutin in aerated and deaerated solutions

Abstract

Ferritin (Ft) impairment through ^?O₂^–, H₂O₂, and ^?OH production occurs in the cases of ketoses, diabetes mellitus, acute intermittent porphyria and tyrosinemia. In addition to ^?Trp and TyrO^? radical production, ferrous iron liberation and Ft synthesis stimulation, site-specific oxidation reactions are induced leading to toxic iron accumulation in organs with high Ft content, for example, liver and brain. To elucidate the potential pathways to Ft recovery, repair of oxidative damage to horse spleen apoferritin (apoFt) and Ft by quercetin (QH) or rutin (RH) was studied in the presence and absence of oxygen. ^?Trp and TyrO^? radicals were produced in pulse radiolysis through apoFt oxidation by ^?Br₂^– radicals. QH and RH bind to apoFt on eight sites with binding constants of ?80,000 and ?32,000 M^?1, respectively. In deaerated solutions, a repair of apoFt radicals is observed involving both bound and free flavonoids. This repair occurs by a fast intra- and a slow inter-molecular electron transfer from bound and free flavonoids, respectively. With QH, the rate constants are 10⁴ s^–1 and 3.5 × 10⁷ M^–1 s^–1 for the intra- and intermolecular repair reactions, respectively. Oxygen does not interfere with repair of apoFt or Ft by bound QH but inhibits 90% of Ft repair by RH. These results taken together indicate that flavonoid antioxidants may help alleviate Ft impairment in diseases involving an oxidative stress.     

Free radical scavenging activity of novel thiazolidine‐2,4‐dione derivatives

Free radical activity towards superoxide anion radical (), hydroxyl radical (HO^?) and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^?) of a series of novel thiazolidine‐2,4‐dione derivatives (TSs) was examined using chemiluminescence, electron paramagnetic resonance (EPR) and EPR spin trapping techniques. 5,5‐Dimethyl‐1‐pyrroline‐N‐oxide (DMPO) was applied as the spin trap. Superoxide radical was produced in the potassium superoxide/18‐crown‐6 ether dissolved in dimethyl sulfoxide. Hydroxyl radical was generated in the Fenton reaction (Fe(II) + H₂O₂. It was found that TSs showed a slight scavenging effect (15–38% reduction at 2.5 mmol/L concentration) of the DPPH radical and a high scavenging effect of (41–88%). The tested compounds showed inhibition of HO^? ‐dependent DMPO‐OH spin adduct formation (the amplitude of EPR signal decrease ranged from 20 to 76% at 2.5 mmol/L concentration. Our findings present new group compounds of relatively high reactivity towards free radicals. Copyright © 2012 John Wiley & Sons, Ltd.     

Electrochemical detection of extracellular hydrogen peroxide in Arabidopsis thaliana: a real‐time marker of oxidative stress

An electrochemical approach to directly measure the dynamic process of H₂O₂ release from cultures of Arabidopsis thaliana cells is reported. This approach is based on H₂O₂ oxidation on a Pt electrode in conjunction with continuous measurement of sample pH. For [H₂O₂] <1 mm , calibration plots were linear and the amperometric response of the electrode was maximum at pH 6. At higher concentrations ([H₂O₂] >1 mm ), the amperometric response can be described by Michaelian‐type kinetics and a mathematical expression relating current intensity and pH was obtained to quantitatively determine H₂O₂ concentration. At pH 5.5, the detection limit of the sensor was 3.1 µm (S/N = 3), with a response sensitivity of 0.16 Am ^?1 cm^?2 and reproducibility was within 6.1% in the range 1–5 × 10^?3 m (n = 5). Cell suspensions under normal physiological conditions had a pH between 5.5–5.7 and H₂O₂ concentrations in the range 7.0–20.5 µm (n = 5). The addition of exogenous H₂O₂, as well as other potential stress stimuli, was made to the cells and the change in H₂O₂ concentration was monitored. This real‐time quantitative H₂O₂ analysis is a potential marker for the evaluation of oxidative stress in plant cell cultures.     

Drugs with susceptible sites for free radical induced oxidative transformations: the case of a penicillin

Penicillins, as bactericidal antibiotics, have been widely used to treat infections for several decades. Their structure contains both aromatic and thioether moieties susceptible to free radical oxidation. The ^?OH induced oxidation mechanism of amoxicillin was investigated by pulse radiolysis techniques and by final product analysis performed after steady-state γ-irradiation. The predominant sites of the ^?OH attack are suggested to be the thioether group, initially yielding an ^?OH adduct to the sulfur, and the aromatic ring. This adduct to the sulfur converts to sulfur radical cation, which has three competitive reaction paths: (1) by deprotonation at the adjacent carbon α-(alkylthio)alkyl radicals form, which undergo disproportionation leading presumably to sulfoxide as main product; (2) via the pseudo-Kolbe mechanism it may transform to α-aminoalkyl radicals; (3) the radical cation can be stabilized through intramolecular S.˙.O bond formation. The reaction mechanism suggests the presence of a short-living and a stabilized (via hydrogen bonding) long-living ^?OH adduct to the sulfur. The three-electron bonded dimers of amoxicillin were not formed owing to steric hindrance. Thiyl radicals were also present in equilibrium with α-aminoalkyl radicals. In the presence of dissolved oxygen, aromatic ring hydroxylation occurred along with complex reactions resulting in e.g. oxidation of the methyl groups. The formation of the sulfoxide is especially effective in the presence of dissolved oxygen, under anaerobic condition, however, it is also generated owing to H₂O₂ and α-(alkylthio)alkyl radicals. The thioether moiety appears to be more sensitive to oxidation compared to the aromatic ring in case of amoxicillin.