Klaus Stolze

Ischemia/Reperfusion Impairs Mitochondrial Energy Conservation and Triggers O2− Release as a Byproduct of Respiration

The aim of the present study was to elucidate the role of mitochondria in the development of heart failure following ischemia/reperfusion. Although mitochondria were increasingly assumed to be responsible for the establishment of an oxidative stress situation the lack of suitable methods to prove it required new concepts for an evaluation of the validity of this hypothesis. The principal idea was to expose isolated mitochondria to metabolic conditions which are developed during ischemia/reperfusion in the cell (anoxia, lactogenesis) and study how they respond. Heart mitochondria treated in that way responded with an incomplete collapse of the transmembraneous proton gradient, thereby impairing respiration-linked ATP generation. The membrane effect affected also the proper control of e- transfer through redox-cycling ubisemiquinone. Electrons were found to leak at this site from its normal pathway to O2 suggesting that ubisemiquinone becomes an active O2.- generator. It was concluded from these observations that mitochondria are likely to play a pathogenetic role in the reperfusion injury of the heart both, by an impairment of energy conservation and their transition to a potent O2.(-)-radical generator. Furthermore, there is considerable evidence that the exogenous NADH-dehydrogenase of heart mitochondria is mainly responsible for functional changes of these organelles during ischemia/reperfusion.

Epr studies on the oxidation of hydroxyurea to paramagnetic compounds by oxyhemoglobin

N. Hydroxyurea forms methemoglobin from oxyhemoglobin with concomitant formation of the aminocarbonylaminooxyl radical H2N-CO-NHO., as detected with electron paramagnetic resonance spectroscopy (EPR). This radical could be detected for several hours in a low steady-state concentration. Approximately 1 hr after the reaction had been started, the EPR spectra of two additional paramagnetic intermediates could be detected at low temperature (77 degrees K), a low-spin ferric methemoglobin complex with hydroxyurea (MetHb-NHOH-CO-NH2) and the hemoglobin-nitric oxide adduct (Hb2(+)-NO). The intensities of their EPR spectra increased steadily over the range of more than 64 hr. The low-spin ferric methemoglobin complex was immediately formed when hydroxyurea was dissolved in a methemoglobin whereas the nitric oxide complex was possibly an oxidation product of the MetHb-hydroxyurea adduct. Its oxidative degradation is known to lead to the very toxic compounds nitric oxide and nitrogen dioxide which can therefore contribute to the toxic action of hydroxyurea.

Hydroxylamine and phenol-induced formation of methemoglobin and free radical intermediates in erythrocytes

As previously shown with isolated oxyhemoglobin, methemoglobin formation can also be induced in intact erythrocytes by hydroxylamine compounds and substituted phenols such as butylated hydroxyanisole (BHA). Electron spin resonance investigations revealed that, accordingly, free radical intermediates were formed in erythrocytes from hydroxylamine, N,N-dimethylhydroxylamine, and N-hydroxyurea. Due to the low stability of the dihydronitroxyl radicals, their detection required the use of a continuous flow system and relatively high amounts of the reactants. As has already been demonstrated with the solubilized hemoglobin system, hemoglobin of intact erythrocytes also reacts with the more hydrophilic xenobiotics such as hydroxylamine. However, the reaction rate was slightly reduced, indicating the existence of an incomplete permeability barrier for these compounds. The limited solubility of phenolic compounds in the aqueous buffer of suspended erythrocytes (in combination with the strict requirement of osmolarity in order to prevent hemolysis) impeded the direct detection of the respective phenoxyl radicals previously reported in hemoglobin solutions. However, in accordance with earlier findings in homogeneous reaction systems, chemiluminescence was observed as well, indicating the existence of a further reaction intermediate, which was also obtained in pure hemoglobin solutions when mixed with the respective reactants. As has recently been demonstrated, this light emission is indicative of the existence of highly prooxidative compound I intermediates during methemoglobin formation. Prooxidant formation in erythrocytes is reflected by a significant decrease in thiol levels even with those compounds where free radical formation was not directly detectable by ESR spectroscopy. The use of the spin-labeling technique revealed membrane effects as a result of oxidative stress. Oxidative metabolism of hemoglobin with hydroxylamine caused a release of low molecular weight iron. The marked hemolysis observed in the presence of BHA results from a direct membrane effect of this compound rather than a consequence of free radical-induced oxidative stress. A correlation of the different results is discussed in terms of possible toxicological consequences.

Chemiluminescence from activated heme compounds detected in the reaction of various xenobiotics with oxyhemoglobin : comparison with several heme/hydrogen peroxide systems

Chemiluminescence was detected in the reaction of oxyhemoglobin with various hydroxylamines and phenols, which have previously been shown to produce free radicals. The emitted light intensity correlated roughly with the methemoglobin formation rate, indicating the involvement of a photoemissive species as a reaction intermediate. In our previous work, we postulated the involvement of a catalase-insensitive, heme-bound hydrogen peroxide species in the methemoglobin formation reaction. In a series of experiments, we showed that intensive chemiluminescence occurred when hydrogen peroxide was mixed with either methemoglobin or metmyoglobin but not with hematin, which lacks the globin moiety. This suggests the involvement of the globin moiety in the light-emitting reaction sequence. The detection of paramagnetic globin species exhibiting similar kinetics as the corresponding light-emitting compound demonstrated that the assumed H2O2-heme compound has strong oxidizing properties. Accordingly, addition of bovine serum albumin to the hematin-hydrogen peroxide system also resulted in a strong chemiluminescence due to the formation of a paramagnetic transient species which could be detected by electron spin resonance (ESR). Several other heme compounds, such as cytochrome c or cytochrome c oxidase which have no vacant ligand site, did not show any light emission under similar conditions. This means that hydrogen peroxide must have access to a free-binding position on the heme. Chemiluminescence most probably stems from the transition of the initially formed heme-H2O2 adduct to the compound II type species. Due to their oxidizing nature, these species might be responsible for deleterious toxic effects such as lipid peroxidation and protein degradation.

Reactive oxygen species are involved in the stimulation of the mitochondrial permeability transition by dihydrolipoate

Dihydrolipoic acid (DHLA) has been found to stimulate the Ca(2+)-induced mitochondrial permeability transition (MPT) in rat liver mitochondria (RLM) [Biochem. Mol. Biol. Int. 44 (1998) 127] which could be due to its prooxidant properties. We therefore investigated whether DHLA stimulated superoxide anion (O(2)(.-)) generation in RLM and in bovine heart submitochondrial particles (SMP). In RLM DHLA caused a concentration-dependent O(2)(.-) generation assayed by lucigenin chemiluminiscence. The stimulation was seen with the lowest concentrations of DHLA (5 microM) with pyruvate as the respiratory substrate, with 2-oxoglutarate or especially succinate the stimulation was less pronounced. Stimulation of O(2)(.-) production by DHLA was also observed in bovine heart SMP using an electron spin-trapping technique. Radical scavengers (butylhydroxytoluene and TEMPO) decreased O(2)(.-) generation induced by DHLA and inhibited MPT. Slight reduction of the mitochondrial membrane potential by a small amount of a protonophorous uncoupling agent also delayed the DHLA-induced MPT. These data indicate that the stimulation of MPT by DHLA is due to DHLA-derived prooxidants, i.e. stimulated production of O(2)(.-) and possibly other free radicals.

FREE RADICAL INTERMEDIATES IN THE OXIDATION OF N-METHYLHYDROXYLAMINE AND N,N-DIMETHYLHYDROXYLAMINE BY OXYHEMOGLOBIN

Nitroxide radicals have been detected in the methemoglobin formation reaction between oxyhemoglobin and the substituted hydroxylamine compounds, N-methylhydroxylamine and N,N-dimethylhydroxylamine, by ESR spectroscopy. The stability of these nitroxide radicals was considerably higher than that of the NH2O. radical derived from unsubstituted hydroxylamine. Only in the case of N-methylhydroxylamine the detection of the nitroxide radical required the use of a flow system, because the radical was found to undergo a rapid degradation with the concomitant formation of a secondary product, the beta-aminonitroxide CH3NO.CH2NH2. The nitroxide radical derived from N,N-dimethylhydroxylamine and oxyhemoglobin was stable for more than 1 hour. In addition, formation of low-spin iron-(III)-complexes from methemoglobin and excess substituted hydroxylamine was observed in both cases. Neither N-methylhydroxylamine nor N,N-dimethyldroxylamine formed the hemoglobin-nitric oxide complex found with unsubstituted hydroxylamine. Parallels and differences in the reaction path of un-, mono- and disubstituted hydroxylamines are discussed.

Antagonistic effects of selenium and lipid peroxides on growth control in early hepatocellular carcinoma.

Activation of the activator protein 1 (AP‐1) transcription factor as well as increased serum levels of vascular endothelial growth factor (VEGF) and interleukin (IL)‐8 predict poor prognosis of patients with hepatocellular carcinomas (HCCs). Moreover, HCC patients display reduced selenium levels, which may cause lipid peroxidation and oxidative stress because selenium is an essential component of antioxidative glutathione peroxidases (GPx). We hypothesized that selenium‐lipid peroxide antagonism controls the above prognostic markers and tumor growth. (1) In human HCC cell lines (HCC‐1.2, HCC‐3, and SNU398) linoleic acid peroxide (LOOH) and other prooxidants enhanced the expression of VEGF and IL‐8. LOOH up‐regulated AP‐1 activation. Selenium inhibited these effects. This inhibition was mediated by glutathione peroxidase 4 (GPx4), which preferentially degrades lipid peroxides. Selenium enhanced GPx4 expression and total GPx activity, while knock‐down of GPx4 by small interfering RNA (siRNA) increased VEGF, and IL‐8 expression. (2) These results were confirmed in a rat hepatocarcinogenesis model. Selenium treatment during tumor promotion increased hepatic GPx4 expression and reduced the expression of VEGF and of the AP‐1 component c‐fos as well as nodule growth. (3) In HCC patients, increased levels of LOOH‐related antibodies (LOOH‐Ab) were found, suggesting enhanced LOOH formation. LOOH‐Ab correlated with serum VEGF and IL‐8 and with AP‐1 activation in HCC tissue. In contrast, selenium inversely correlated with VEGF, IL‐8, and HCC size (the latter only for tumors smaller than 3 cm). Conclusion: Reduced selenium levels result in accumulation of lipid peroxides. This leads to enhanced AP‐1 activation and consequently to elevated expression of VEGF and IL‐8, which accelerate the growth of HCC. Selenium supplementation could be considered for investigation as a strategy for chemoprevention or additional therapy of early HCC in patients with low selenium levels. (HEPATOLOGY 2012)

The Effects of Xenobiotics on Erythrocytes

1. Methemoglobin formation was observed when erythrocytes were incubated with xenobiotics such as hydroxylamines or phenols, other metabolites resulting from the interaction of these compounds with erythrocytes being reactive free radicals derived from the respective xenobiotic, and a ferryl-heme oxo-complex. 2. Steady-state levels of these reaction products depended on the permeability of the erythrocyte membrane for the various methemoglobin (MetHb) generators and the presence of antioxidants that downregulate the radicals formed. 3. Electron spin resonance (ESR) spectra of xenobiotic-derived free radicals could be obtained only from the readily water soluble hydroxylamines, whereas the poorly water soluble phenolic compounds did not allow the use of concentrations required for the generation of detectable amounts of ESR-sensitive metabolites in erythrocytes. 4. Previous investigations with oxyhemoglobin solutions and with the MetHb/H2O2 model systems have shown that, apart from ESR-sensitive radical species, excited reaction intermediates such as compound 1 ferryl hemoglobin can be detected as well by using chemiluminescence measurements. 5. A strong correlation was found between the intensity of the emitted light and the MetHb formation rate, indicating that the production of compound 1 ferryl hemoglobin is closely related to the MetHb formation step. 6. The sensitivity of the photon-counting method allowed measurements of excited species in intact erythrocytes not only with the readily soluble hydroxylamines, but also with the less soluble phenolic compounds. 7. In addition, parameters indicative of xenobiotic-induced oxidative alterations were found: a significant decrease in intraerythrocytic thiol levels was a result of all compounds that initiate MetHb formation, as also described for slowly reacting xenobiotics. 8. With the most reactive compound investigated, unsubstituted hydroxylamine, a significant release of iron from the oxidatively modified hemoglobin was detected, facilitated by binding of this transition metal to hydroxylamine and its final oxidation product, nitric oxide. 9. The use of the ESR spin-labeling technique revealed membrane alterations of erythrocytes exposed to the reducing MetHb generators presented in this study. 10. A direct action of BHA and BHT on the integrity of the erythrocyte membrane was observed, leading to hemolysis independent of the formation of prooxidant species. 11. The presence of strong prooxidants (radicals) was indicated both by fluidity changes in the membrane and by an oxidative decrease in cytosolic thiol levels.

Reactions of reducing xenobiotics with oxymyoglobin. Formation of metmyoglobin, ferryl myoglobin and free radicals: An electron spin resonance and chemiluminescence study

The oxygen-haem centre of oxymyoglobin reacts with reducing xenobiotics such as hydroxylamines and phenols with the concomitant formation of metmyoglobin and oxidation of the respective xenobiotic. Metmyoglobin formation rates were measured by visible spectroscopy with xenobiotic concentrations ranging from 100 microM to 30 mM. Analogous to previous results obtained with oxyhaemoglobin, the first step in the reaction of hydroxylamines with oxymyoglobin leads to the formation of the one-electron oxidation product of hydroxylamine, a nitroxyl radical detectable by electron spin resonance. A variety of paramagnetic secondary products were also found. The terminal oxidation product of hydroxylamine and hydroxyurea was the myoglobin-nitric oxide complex, one showing similar spectral characteristics to the analogous haemoglobin-nitric oxide adduct found in our previous experiments. On the other hand, the amount of low-spin ferric complexes obtained from metmyoglobin and an excess of the respective hydroxylamine was considerably lower than the corresponding results with methaemoglobin. A second important reaction intermediate was the compound I-type ferryl haem-species detected by a recently-published chemiluminescence assay. Partial spectral resolution of the emitted light using a set of cut-off filters indicated that maximum light emission occurred above 600 nm, most probably involving excited porphyrin states. The intensity of oxymyoglobin-related light emission was considerably higher than that reported earlier with oxyhaemoglobin. This indicates a difference in the excitation mechanism which leads to the formation of the compound I-type ferry haem species.

Methemoglobin Formation from Butylated Hydroxyanisole and Oxyhemoglobin. Comparison with Butylated Hydroxytoluene and P-Hydroxyanisole

The widely used food additives butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) react with oxyhemoglobin, thereby forming methemoglobin. The reaction rates were measured using visible spectroscopy, and second order rate constants were established for BHA and compared with p-hydroxyanisole. Using ESR we investigated the involvement of free radical reaction intermediates. The expected one-electron oxidation product of BHA and BHT, the phenoxyl radical, could only be detected with pure 3-t-butyl-4-hydroxyanisole and oxyhemoglobin. With the commercial mixture of 2- and 3-t-butyl-4-hydroxyanisole a very strong ESR signal of a secondary free radical species was observed, similar to the one observed earlier with p-hydroxyanisole and dependent on the presence of free thiol groups, so that we assumed the intermediate existence of a perferryl species, the MetHb-H2O2 adduct. In a second series of experiments we investigated the reactivity of this postulated intermediate with BHA and BHT, starting with a pure MetHb/H2O2-phenol mixture in a stopped-flow apparatus linked to the ESR spectrometer, detecting the expected phenoxyl radicals from BHA and p-hydroxyanisole. Due to the low solubility and decreased reactivity of BHT only traces of phenoxyl type radical were found together with a high concentration of unreacted perferryl species. The reactivity of BHA, BHT and p-hydroxyanisole with free thiol groups is demonstrated by an increased reaction rate in the presence of the thiol group blocking substance NEM.