Short communication: Effect of freezer storage time and thawing method on the recovery of Mycoplasma bovis from bovine colostrum.

Mycoplasma bovis is an important cause of mastitis in dairy cattle, and pneumonia, arthritis, and otitis in calves. Milk and colostrum are considered important sources of infection for calves. knowledge on the effect of on-farm freezing (-18°C) and thawing methods on the recovery of M. bovis from colostrum samples is missing. In this study, 2 separate experiments were performed. The first experiment consisted of a longitudinal study examining the survival [as measured by log(10) reduction] of 2 M. bovis strains in frozen colostrum over 14 wk. The second experiment examined the effect of different thawing temperatures (45 and 20°C), thawing frequencies (once or twice), and initial colostrum titer (104 or 106 cfu/mL) on M. bovis survival. A single freeze-thaw cycle led to an approximate 1 log reduction of M. bovis titer, independent of the thawing temperature. Freezing for 14 wk did not significantly further reduce the titer of bacteria compared with freezing for 2 wk. A second freeze-thaw cycle further reduced the M. bovis count by approximately 0.5 log compared with a single freeze-thaw cycle. Thawing temperature and initial bacterial concentration did not significantly affect M. bovis reduction. In conclusion, storage of colostrum samples in the freezer at -18°C during epidemiological studies, herd monitoring, or test and cull programs will probably have little influence on qualitative bacteriological test results for M. bovis. The epidemiological or clinical relevance of an approximate 1 log reduction of M. bovis in colostrum is currently unclear.

Railway noise annoyance modeling: Accounting for noise sensitivity and different acoustical features.

Noise annoyance due to railway traffic is a growing issue in today's society. This annoyance may be predicted using noise-exposure relationships with mean energy-based index. However, there is room for improvement of models as other acoustical and non-acoustical factors also influence noise annoyance responses. In this paper, it is proposed to highlight annoying auditory sensations evoked by the railway noise and determine acoustical and psychoacoustical indices combined in a predictive model. A laboratory experiment involving railway pass-by noise in urban areas was carried out. Annoyance ratings, noise sensitivity ratings, and free verbalization data were gathered. The analysis underlined annoying auditory sensations caused by railway pass-by noises. Two indices were proposed to account for irregular amplitude fluctuation and noise event duration-related sensations. A multilevel regression analysis was conducted, leading to two annoyance models considering noise indices and noise sensitivity. These models were finally compared to a similarly obtained multilevel regression model related to tramway noise annoyance. The comparison was carried out as a cross-validation considering the models and the respective datasets collected in laboratory conditions for model construction. Results showed that the railway noise annoyance model led to a good prediction of tramway noise annoyance and vice versa.

Genotoxic effects of deoxynivalenol in broiler chickens fed low-protein feeds.

Deoxynivalenol (DON) is one of the most abundant and important trichothecenes in food and feed, and it is a significant contaminant due to its frequent occurrence at toxicologically relevant concentrations worldwide. Deoxynivalenol has negative influences on the health and performance of chicks. However, there is little information available regarding the effect of DON on DNA fragmentation in blood lymphocytes. In addition, the effects of Mycofix select (Biomin GmbH, Herzogenburg, Austria) supplementation to DON-contaminated broiler diets on lymphocyte DNA have not yet been demonstrated. Therefore, the aim of the present study was to establish the effect of DON on lipid peroxidation and lymphocyte DNA fragmentation in broilers and to evaluate the potential of Mycofix select in the prevention of toxin-mediated changes. Thirty-two 1-d-old (Ross 308 male) broiler chicks were randomly divided into 4 groups. The control group was fed a noncontaminated diet, and a second group was fed the same diet but supplemented with Mycofix select (0.25%). A third group of broilers was fed a diet artificially contaminated with 10 mg of feed-grade DON/kg of diet, and a fourth group was fed a DON-contaminated diet supplemented with Mycofix select. At the end of the feeding trial, blood was collected and the degree of lymphocyte DNA damage was measured in the plasma by comet assay. Deoxynivalenol increased (P = 0.016) the amount of DNA damage in chicken lymphocytes by 46.8%. Mycofix select protected lymphocyte DNA from the DON effects. To our knowledge, these are the first data on genotoxic effects of a moderate dose of DON on chicken lymphocytes. However, the thiobarbituric acid reactive substances level in liver and liver enzyme activity did not differ among the groups. In conclusion, the present study demonstrated that the diets contaminated with the mycotoxin DON at moderate levels in combination with low-protein feed are able to induce lymphocyte DNA damage in chickens. Supplementation with Mycofix select protected lymphocyte DNA and it was beneficial for maintaining the lymphocyte DNA integrity.

Synthetic chromanol derivatives and their interaction with complex III in mitochondria from bovine, yeast, and Leishmania.

Synthetic chromanol derivatives (TMC4O, 6-hydroxy-2,2,7,8-tetramethyl-chroman-4-one; TMC2O, 6-hydroxy-4,4,7,8-tetramethyl-chroman-2-one; and Twin, 1,3,4,8,9,11-hexamethyl-6,12-methano-12H-dibenzo[d,g][1,3]dioxocin-2,10-diol) share structural elements with the potent inhibitor of the mitochondrial cytochrome (cyt) bc(1) complex stigmatellin. Studies with isolated bovine cyt bc(1) complex demonstrated that these compounds partially inhibit the mammalian enzyme. The aim of this work was to comparatively investigate these toxicological aspects of synthetic vitamin E derivatives in mitochondria of different species. The chromanols and atovaquone as reference compound were evaluated for their inhibition of the cyt bc(1) activity in mitochondrial fractions from bovine hearts, yeast, and Leishmania. In addition, compounds were evaluated in vitro for their inhibitory activity against whole-cell Leishmania and mouse peritoneal macrophages. In these organisms, the chromanols showed a species-selective inhibition of the cyt bc(1) activity different from that of atovaquone. While in atovaquone the side chain mediates species-selectivity, the marked differences for TMC2O and TMC4O in cyt bc(1) inhibition suggests that direct substitution of the chromanol headgroup will control selectivity in these compounds. Low micromolar concentrations of TMC2O (IC(50) = 9.5 ± 0.5 µM) inhibited the growth of Leishmania, and an esterified TMC2CO derivative inhibited the cyt bc(1) activity with an IC(50) of 4.9 ± 0.9 µM. These findings suggest that certain chromanols also exhibit beyond their antioxidative properties antileishmanial activities and that TMC2O derivatives could be useful toward the development of highly active antiprotozoal compounds.

Influence of cryopreservation on mitochondrial functions in equine spermatozoa.

Cryopreservation of spermatozoa is of essential importance for artificial insemination and breeding programs in horses. Besides other factors, spermatozoal motility depends on mitochondrial energy metabolism. Based on changes of single mitochondrial functions it has been suggested that mitochondrial damage during cryopreservation could be a major reason for diminished post thaw semen quality. However, it is still unclear to which extent this influences the whole bioenergetic performance of mitochondria and whether this plays a role during routine cryopreservation procedures. Therefore, it was the aim of this study to compare changes in mitochondrial bioenergetics in spermatozoa during shock freezing and routine cryopreservation. Mitochondrial integrity in spermatozoa was studied by determination of oxygen consumption, mitochondrial membrane potential, and the oxidation of externally added cytochrome c(2+). Shock freezing of spermatozoa resulted in an irreversible loss of mitochondrial functions. However, respiration difference of uncoupled minus resting state and routine respiration also decreased by 48+/-14 and 58+/-6% (p<0.05), respectively, after routine cryopreservation. This was accompanied by a decline in the mitochondrial membrane potential to 83+/-4% (p<0.05) and spermatozoal motility to 56+/-11% (p<0.05) of pre-freezing values. In contrast, the oxidation rates of externally added cytochrome c(2+) by cytochrome c oxidase slightly increased by 26+/-14% (p<0.1) suggesting a partial rupture of cellular and outer mitochondrial membranes. Our data indicate that also widely used cryopreservation protocols for equine spermatozoa need adjustment to optimize post thaw mitochondrial functions.

A Deep Nasopharyngeal Swab Versus Nonendoscopic Bronchoalveolar Lavage for Isolation of Bacterial Pathogens from Preweaned Calves With Respiratory Disease.

BACKGROUND: Nonendoscopic bronchoalveolar lavage (BAL) is a practical alternative for a deep nasopharyngeal swab (DNS) to sample the airways of a large number of calves in a short period of time. The extent of commensal overgrowth and agreement of BAL with DNS culture results in preweaned calves are unknown. OBJECTIVES: To compare commensal overgrowth and bacterial culture results between DNS and BAL samples. ANIMALS: A total of 183 preweaned calves (144 with bovine respiratory disease and 39 healthy animals). METHODS: Cross-sectional study. Deep nasopharyngeal swab and BAL samples were taken from each calf and cultured to detect Pasteurellaceae and Mycoplasma bovis. Agreement and associations between culture results of DNS and BAL samples were determined by kappa statistics and logistic regression. RESULTS: Bronchoalveolar lavage samples were less often polymicrobial, more frequently negative and yielded more pure cultures compared to DNS, leading to a clinically interpretable culture result in 79.2% of the cases compared to only in 31.2% of the DNS samples. Isolation rates were lower in healthy animals, but not different between DNS and BAL samples. Only Histophilus somni was more likely to be isolated from BAL samples. In clinical cases, a polymicrobial DNS culture result did not increase the probability of a polymicrobial BAL result by ≥30%, nor did it influence the probability of a negative culture. A significant herd effect was noted for all observed relationships. CONCLUSIONS AND CLINICAL RELEVANCE: Nonendoscopic BAL samples are far less overgrown by bacteria compared to DNS samples under the conditions of this study, facilitating clinical interpretation and resulting in a higher return on investment in bacteriologic culturing.

Quantitative neutron capture radiography for studying the biodistribution of tumor-seeking boron-containing compounds.

Biodistribution of two compounds presently considered for use in neutron capture therapy has been studied in mice carrying a transplantable Harding-Passey melanoma. A method is described by which quantitative assessment can be made of the boron distribution in whole-body sections of such animals. An alpha-particle-sensitive film is placed in close contact with a freeze-dried section of an animal and exposed to neutrons. The tracks visible after etching are analyzed optoelectronically in fields of 0.6 X 0.6 mm2 and compared to standards of boron homogeneously distributed in liver homogenates. The dynamic range of this method is about two orders of magnitude in concentration, with a lower detection limit of 0.1 to 0.01 ppm 10B, depending on the rate of induction of spurious tracks by fast neutrons present in the neutron beam chosen. In a transplantable Harding-Passey melanoma in mice, it was found that the sulfhydryl boron hydride Na2B12H11SH presently used for therapy of glioblastoma clears blood, muscle, and brain very rapidly. Its accumulation in tumors was persistent for more than three days. A higher tumor accumulation was observed with its disulfide, which has been suggested for neutron capture therapy. For both compounds, a marked heterogeneity of boron distribution within one tumor was found.

A new predilection site of Mycoplasma bovis: Postsurgical seromas in beef cattle.

Mycoplasma bovis is a highly contagious bacterium, which predominantly causes chronic pneumonia, otitis and arthritis in calves and mastitis in adult cattle. In humans, Mycoplasma species have been associated with post-surgical infections. The present study aimed to identify the bacteria associated with three outbreaks of infected seromas after caesarian section in Belgian Blue beef cattle. A total of 10 cases occurred in three herds which were in close proximity of each other and shared the same veterinary practice. M. bovis could be cultured from seroma fluid in five of the six referred animals, mostly in pure culture and was isolated from multiple chronic sites of infection (arthritis and mastitis) as well. DNA fingerprinting of the isolates targeting two insertion sequence elements suggested spread of M. bovis from chronic sites of infection (udder and joints) to the postsurgical seromas. Identical genetic profiles were demonstrated in two animals from two separate farms, suggesting spread between farms. Mortality rate in the referred animals positive for M. bovis in a seroma was 80% (4/5), despite intensive treatment. A massive increase in antimicrobial use was observed in every affected farm. These observations demonstrate involvement of mycoplasmas in outbreaks of postsurgical seromas in cattle.

Effect of alpha-lipoic acid and dihydrolipoic acid on ischemia/reperfusion injury of the heart and heart mitochondria.

The aim of the present study was to evaluate a possible interference of alpha-lipoic acid (LA) or its reduced form (dithiol dihydrolipoic acid = DHLA) in the cardiac ischemia/reperfusion injury both at the level of the intact organ and at the subcellular level of mitochondria. In order to follow the effect of LA on the ischemia/reperfusion injury of the heart the isolated perfused organ was subjected to total global ischemia and reperfusion in the presence and absence of different concentrations of LA. Treatment with 0.5 microM LA improved the recovery of hemodynamic parameters; electrophysiological parameters were not influenced. However, application of 10 microM LA to rat hearts further impaired the recovery of hemodynamic functions and prolonged the duration of severe rhythm disturbances in comparison to reperfusion of control hearts. Treatment of isolated mitochondria with any concentration of DHLA could not prevent the impairment of respiratory-linked energy conservation caused by the exposure of mitochondria to 'reperfusion' conditions. However, DHLA was effective in decreasing the formation and the existence of mitochondrial superoxide radicals (O2.-). Apart from its direct O(2.-)-scavenging activities DHLA was also found to control mitochondrial O2.- formation indirectly by regulating redox-cycling ubiquinone. It is suggested that impairment of this mitochondrial O2.- generator mitigates postischemic oxidative stress which in turn reduces damage to hemodynamic heart function.

Endogenous and exogenous regulation of redox-properties of coenzyme Q.

Ubiquinol (QH2, reduced coenzyme Q) is increasingly reported to exert antioxidant functions besides its implication in mitochondrial energy metabolism. On the other hand ubisemiquinones (SQ-.) of the respiratory chain are considered to account for the production of superoxide radicals as a byproduct of cellular respiration. Since the formation of potentially prooxidative ubisemiquinones can be expected to result from the antioxidant activity of ubiquinol, the evaluation whether or not QH2 exerts antioxidant activities depends on the fate of antioxidant-derived metabolites and the existence of a natural recycling system for oxidized QH2. We have recently shown that SQ increasingly undergo autoxidation when approaching the external more polar phase of the membrane. In contrast to mitochondria where the QH2/ SQ-./Q pools are dynamically kept in relatively stable relationships the fate of semi and fully oxidized QH2 is not at all clear in LDL particles where QH2 is suggested to exert important antioxidant functions. Therefore, the antioxidant-derived metabolites of QH2 in liposomes following lipid peroxidation were studied with respect to their localization in the bilayer and the possibility to recycle oxidized QH2 via dihydrolipoic acid (DHLA). The results revealed a considerable fraction of QH2 existing in the outer membrane section where protons from the aqueous phase have access to allow autoxidation. DHLA was found to recycle oxidized QH2 although due to slow partition equilibration the reduction velocity appears to be not sufficient for therapeutic application.

Analyses of the molecular mechanism of adriamycin-induced cardiotoxicity.

The molecular basis of the adriamycin (AQ)-dependent development of cardiotoxicity is still far from being clear. In contrast to our incomplete understanding of the organ-specific mechanism mitochondria are unequivocally accepted as the locus where the molecular disorder is triggered. A growing number of reports intimate the establishment of unbalanced oxygen activation through heart mitochondria in the presence of anthraquinones. In fact, in contrast to liver mitochondria, isolated heart mitochondria have been unequivocally shown to shuttle single electrons to AQ, giving rise to O2.- formation by autoxidizing AQ. semiquinones. Earlier we have demonstrated the involvement of the exogenous NADH dehydrogenase in this deleterious electron deviation from the respiratory chain. This enzyme that is associated with complex I of the respiratory chain catalyzes the oxidation of cytosolic NADH. AQ activation through isolated heart mitochondria was reported to require the external addition of NADH, suggesting a flux of reducing equivalents from NADH to AQ in the cytosol. Unlike heart mitochondria, intact liver mitochondria, which are lacking this NADH-related pathway of reducing equivalents from the cytosol to the respiratory chain, cannot be made to activate AQ to semiquinones by NADH or any other substrate of respiration. It appears, therefore, that the exogenous NADH dehydrogenase of heart mitochondria exerts a key function in the myocardial toxicogenesis of anthraquinones via oxygen activation through semireduced AQ. Assessing the toxicological significance of the exogenous NADH dehydrogenase in AQ-related heart injury requires analysis of reaction products and their impact on vital bioenergetic functions, such as energy gain from the oxidation of respiratory substrates. We have applied ESR technique to analyze the identity and possible interactions of radical species emerging from NADH-respiring heart mitochondria in the presence of AQ. The following metabolic steps occur causing depression of energy metabolism in the cardiac tissue. After one-electron transfer to the parent hydrophilic anthraquinone molecule destabilization of the radical formed causes cleavage of the sugar residue. Accumulation of the lipophilic aglycone metabolite in the inner mitochondrial membrane diverts electrons from the regular pathway to electron acceptors out of sequence such as H2O2. HO. radicals are formed and affect the functional integrity of energy-linked respiration. The key and possibly initiating role of the exogenous NADH dehydrogenase of cardiac mitochondria in this reaction pathway provides a rationale to explain the selective cardiotoxic potency of the cytostatic anthraquinone glycosides.

Effects of tocopheryl quinone on the heart: model experiments with xanthine oxidase, heart mitochondria, and isolated perfused rat hearts.

It is generally accepted that the protection effect of biological tissues by vitamin E is due to its radical scavenging potency in membranes, thereby being transformed to a vitamin E radical. A deficiency of appropriate reductants, which recycle vitamin E radicals back to its antioxidative active form, causes an irreversible degradation of vitamin E leading to tocopheryl quinone (TQ). TQ-like compounds were shown to result from both vitamin E and corresponding hydrophilic analogues of this antioxidant in vitro. In vivo elevated concentrations of tocopheryl quinones were detected after oxidative stress and TQ supplementation as well. Quinones in general are known to be efficient one-electron donors and acceptors. Therefore the question arises whether TQ-like compounds can undergo redox-cycling in conjunction with redox-active enzymes in the heart, thereby producing harmful oxygen radicals, or whether these compounds exhibit antioxidant properties. In order to elucidate this question we focused our interest on the interaction of TQ and a corresponding short-chain homologue (TQ(0)) with xanthine oxidase and heart mitochondria. Furthermore, we tested the influence of TQ on the recovery of isolated perfused rat hearts after ischemia/reperfusion. Our experiments revealed that hydrophilic TQ(0) was univalently reduced by xanthine oxidase (XOD) yielding semiquinone radicals in the absence of oxygen. However, under aerobic conditions TQ(0) enhanced the O(2)(*)(-) radical output of XOD. In the mitochondrial respiratory chain TQ was shown to interact with high potential cytochrome b in the bc(1) complex specifically. In contrast to the system XOD/TQ(0), lipophilic TQ in submitochondrial particles decreased the O(2)(*)(-) radical release during regular respiration possibly due to its interaction with b-cytochromes in the mitochondrial respiratory chain. In isolated rat hearts perfused with liposomes containing lipophilic TQ, it was efficiently accumulated in the heart tissue. When hearts were subjected to conditions of ischemia/reperfusion, infusion of TQ prior to ischemia significantly improved the recovery of hemodynamic parameters. Our results demonstrate that TQ derivatives may induce pro-oxidative and antioxidative effects depending on the distribution of TQ derivatives in the heart tissue and the interacting redox system.

Conditions allowing redox-cycling ubisemiquinone in mitochondria to establish a direct redox couple with molecular oxygen.

The present investigation seeks to elucidate the molecular mechanism responsible of the transformation of redox-cycling ubiquinone (UQ) from a save electron carrier to an O2.- generator as observed in toluene-treated mitochondria as well as in mitochondria exposed to conditions of organ ischemia/reperfusion. Starting from the earlier finding that for thermodynamic grounds autoxidation of ubisemiquinone (SQ.-) requires the accessibility of protons, two possibilities were considered: a) protons from the aqueous phase may penetrate into the phospholipid bilayer and react with SQ.- due to a decreased hydrophobicity of the membrane, b) the physical state of the membrane remains unchanged while the binding of redox-cycling UQ is changed such that SQ.- will come into contact with the aqueous phase in the polar head group section. Spin probes were used to follow changes of the physical order of phospholipids of the inner mitochondrial membrane. Binding changes of mitochondrial SQ.- were assessed from power saturation experiments and spin-spin interactions with a Cr3+ salt of the aqueous phase were studied to recognize orientation changes via the polar head group section of the membrane. Our results show that autoxidation of SQ.- occurs in two different ways. In the case of membrane insertion of toluene, the physical property of the membrane was affected such that protons could penetrate and allow SQ.- to undergo autoxidation. In contrast, mitochondrial respiration of cytosolic NADH accumulating during ischemia involves a low saturating SQ.- species that readily autoxidizes due to its spatial orientation close to the aqueous face of the membrane. We conclude from these observations that in line with thermodynamics autoxidation of SQ.- in mitochondria requires protons that normally have no access.

Antioxidant-derived prooxidant formation from ubiquinol.

Ubiquinol (QH2) is increasingly used as antioxidant for the treatment of a variety of diseases and the modulation of biological aging; however, the biological significance of secondary reaction products has been disregarded so far. Our studies on the antioxidant activity of ubiquinol in peroxidizing lipid membranes demonstrate the existence of ubisemiquinone (SQ*) as the first reaction product of ubiquinol. A fraction of SQ* derived from the antioxidative activity of QH2 was detected in the outer section of the membrane bordering the aqueous phase. This localization allows an access of protons and water from the aqueous phase to SQ* a prerequisite earlier found to trigger autoxidation. Superoxide radicals emerging from this fraction of autoxidizing SQ* form H2O2 by spontaneous dismutation. SQ* not involved in autoxidation may react with H2O2. Transfer of the odd electron to H2O2 resulted in HO* and HO- formation by homolytic cleavage. An analogous reaction was also possible with lipid hydroperoxides which accumulate in biological membranes during lipid peroxidation. The reaction products emerging from this reaction were alkoxyl radicals. Both HO* and alkoxyl radicals are strong initiators and promoters of lipid peroxidation. Indirect evidence of the existence and prooxidative activities of these secondary reaction products came from comparative studies with vitamin E. While in the absence of other reactants, QH2 and vitamin E were equally effective in scavenging lipid radicals; the radical protecting activity of QH2 was found to be significantly lower as compared to vitamin E when these antioxidants operate in peroxidizing lipid membranes. This discrepancy reveals that the antioxidative activity of coenzyme Q is compulsorily linked to the formation of split products counteracting the membrane protective effect of this natural antioxidant.

Imbalance of oxygen activation and energy metabolism as a consequence or mediator of aging.

Ever increasing numbers of aging theories suggest that free radicals are only one factor among others that may initiate stochastic disorders finally terminating life. It is therefore compelling not only to demonstrate the existence of increasing steady-state concentrations of free oxygen radicals during senescence, but it is essential to show that they act in concert with other postulated triggering factors of aging. We have recently shown that various factors may have a life-long influence and challenge oxygen homeostasis of cell respiration. Among these factors are environmental pollutants, therapeutics, and transient hypoxia. Although the nature of these "hits" is different, mitochondrial respiration was found to respond in a similar manner to each of them. The major derangement was an univalent electron leak to oxygen giving rise to the establishment of oxidative stress. Associated with this transformation, oxidative phosphorylation was impaired with the resultant reduction of cellular ATP. Mitochondria from senescent rats exhibited similar alterations of all cell parameters found when adult animals were exposed to "environmental stress" or transient ischemia. Age-related stimulation of mitochondrial oxygen radical generation is therefore suggested to result from accumulation of minihits during life. Based on our data, together with those from other laboratories, it is possible to assess the ranking order of oxygen radicals in the development of stochastic events associated with (or causing) aging.

The biochemical, pathophysiological, and medical aspects of ubiquinone function.

Ubiquinone (Q) shares its biological implication in membrane-associated redox reactions with a variety of other redox carriers, such as dehydrogenases, non-heme-iron proteins, and cytochromes. Peculiarities arise from the lack of transition metals, which in contrast to the other electron carriers do not participate in redox-shuttle activities of Q. Another peculiarity is the lipophilicity of Q, which allows free movement between reductants and oxidants of a membrane. The chemistry of Q reduction and ubiquinol oxidation requires the stepwise acceptance and transfer of two single electrons associated with the addition or release of two single H+. These special qualities are widely used in biological membranes for linear electron transfer and transmembranous H+ translocation. In mitochondria it was long reported that under certain conditions linear e- transfer from the semireduced form (SQ.) to native oxidants of the respiratory chain may run out of control, thereby establishing a permanent source of oxygen radical release. It should be mentioned that in mitochondria e- transfer to dioxygen out of sequence requires a particular treatment with inhibitors and uncouplers of the respiratory chain. Nevertheless, it is generally assumed that Q is mainly involved in mitochondrial O2.- generation and that mitochondria represent the major source of O2.- radicals under physiological and various pathophysiological conditions. The ever-increasing application of coenzyme Q as an antioxidant for the prophylaxis and treatment of a great variety of functional disorders, including senescence, has considerably stimulated our interest in the potential prooxidative potency of this natural electron carrier. Experimental evidence will be presented that under physiological conditions Q implicated in mitochondrial e- transfer of the respiratory chain is not involved in cellular oxygen activation. It will also be shown that alterations of Q from an e- carrier to an active radical promotor is possible under various conditions. In addition, reaction products emerging from the antioxidant activity of ubiquinol were found to stimulate the formation of inorganic as well as organic oxygen radicals.

mRNA levels of the hypoxia inducible factor (HIF-1) and DNA repair genes in perinatal asphyxia of the rat.

Hypoxia inducible factor 1 (HIF-1) is a transcription factor which is expressed, when mammalian cells are subjected to hypoxia, activating the transcription of genes encoding proteins thought important for maintaining oxygen hemostasis. The aim of the study was to evaluate HIF-1 mRNA levels in a non-invasive model of perinatal asphyxia (PA). Brain was taken for studies on HIF-1 alpha and beta 10 min following the asphyctic period. To rule out influences by the redox status we also determined antioxidant enzyme mRNA levels for superoxide dismutase, catalase, glutathion peroxidase and performed electron spin resonance studies. To study the link to protein phosphorylation as previously proposed, we evaluated mRNA levels for protein kinase C. As DNA breaks were reported to occur in PA, we determined mRNA levels of two genes representing DNA nucleotide excision repair, ERCC2 and ERCC3, and a DNA repair gene involved in the repair of oxidation mediated DNA damage, XRCC1. mRNAs for HIF-1 were not detectable following 5-20 minutes of asphyxia. The antioxidant enzymes did not show any changes during the asphyctic periods either and electron spin resonance failed to detect the presence of the hydroxyl radical. PKC significantly decreased with the length of the asphyctic period. ERCC2 and XRCC1 mRNAs were inducible during the acute phase of asphyxia indicating early repair phenomena. HIF-1 may not be relevant for periods of PA up to 20 minutes, the maximal survival time in our model. Neonatal factors may be responsible for that phenomenon although we cannot rule out that HIF-1 changes may occur at the protein level.

Critical aspects of the antioxidant function of coenzyme Q in biomembranes.

Coenzyme Q (ubiquinone, UQ) is increasingly considered as a significant natural antioxidant, which protects biomembranes in concert with alpha-tocopherol. In vitro experiments demonstrated that reduced UQ (ubiquinol) can improve the chain-breaking activities of alpha-tocopherol by recycling the antioxidant-derived reaction product, the chromanoxyl radical, to the native antioxidant. Less attention, however, was devoted to the antioxidant-derived reaction products of reduced UQ. Although both alpha-tocopherol and ubiquinol were found to be equally effective in scavenging chain-propagating lipid radicals. alpha-tocopherol protected lipid membranes from lipid peroxidation more efficiently than ubiquinol. The present study not only provides data which document this discrepancy but also contributes experimental data on the existence of ubiquinol derived pro-oxidants, which give an explanation of this phenomenon.

Evaluation of the antioxidant capacity of ubiquinol and dihydrolipoic acid.

Ubiquinone and alpha-lipoic acid are natural constituents which are involved in mitochondrial energy metabolism. Their bioenergetic activities require redox-cycling. In the case of alpha-lipoic acid redox-cycling leads to dihydrolipoic acid which occurs in multienzyme complexes involved in the citric acid cycle while UQ recycles through semi- and divalently reduced ubiquinones in the respiratory chain. We have proved the validity of the concept about the antioxidant function of these natural compounds in their reduced form. Ubiquinol was found to interfere with lipid peroxidation of liposomal membranes being itself degradated by two consecutive oxidation steps. Dihydrolipoic acid was found to totally recycle ubiquinone to the antioxidant active divalently reduced form. In contrast to the antioxidative derived reaction products of ubiquinols which in turn promoted lipid peroxidation, the antioxidant derived reaction product of dihydrolipoic acid was the unreactive two electron oxidation product alpha-lipoic acid. Our experiments demonstrate the existence of an dihydrolipoic acid driven recycling of UQ to the antioxidative-active UQH2. The efficiency of the antioxidative capacity of the latter was found to be diminished through prooxidant activities of the antioxidant-derived metabolites.

The exogenous NADH dehydrogenase of heart mitochondria is the key enzyme responsible for selective cardiotoxicity of anthracyclines.

The molecular mechanism of the anthracycline-dependent development of cardiotoxicity is still far from being clear. However, it is generally accepted, that mitochondria play a significant role in triggering this organ specific injury. The results presented in this study demonstrate that, in contrast to liver mitochondria, isolated heart mitochondria shuttle single electrons to adriamycin, giving rise to oxygen radical formation via autoxidation of adriamycin semiquinones. This one electron reduction of anthracyclines is catalyzed by the exogenous NADH dehydrogenase associated with complex I of heart mitochondria, an enzyme which is lacking in liver mitochondria. Upon addition of NADH heart mitochondria generate significant amounts of adriamycin semiquinones while liver mitochondria were ineffective. Adriamycin semiquinones undergo both autoxidation leading to superoxide radical release and complex reactions under formation of adriamycin aglycone. Due to the high lipophilicity adriamycin aglycones accumulate in the inner mitochondrial membrane where they interfere with electron carriers of the respiratory chain. Adriamycin aglycone semiquinones emerging from an interaction with complex I were found to trigger homolytic cleavage of H2O2 which results in the formation of hydroxyl radicals. As demonstrated in this study the activation of adriamycin by the exogenous NADH dehydrogenase of cardiac mitochondria initiates a cascade of reaction steps leading to the establishment of oxidative stress. Our experiments suggest the exogenous NADH dehydrogenase of heart mitochondria to play a key role in the cardiotoxicity of adriamycin. This organ-specific enzyme initiates a sequence of one electron transfer reactions ending up in the establishment of oxidative stress.