TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is a tight binding inhibitor of cytochrome P-450d

Based on data indicating that compounds which induce cytochrome P-450d also bind to the enzyme [R. Voorman and S. D. Aust (1987). Toxicol. Appl. Pharmacol. 90, 69-78], we investigated the inhibition of cytochrome P-450d-dependent estradiol 2-hydroxylase by (2,3,7,8-tetrachlorodibenzo-p-dioxin TCDD), using ligand-free cytochrome P-450d from isosafrole-treated rats. Since maximum inhibition of estradiol 2-hydroxylase occurred at TCDD concentrations comparable to the concentration of enzyme (50 nM), a modified form of steady-state kinetic analysis was used. Using I50 = Et/2 + Ki where Et = total enzyme concentration), we showed that TCDD inhibited cytochrome P-450d-dependent estradiol 2-hydroxylase activity with Ki equal to 8 nM. Association of TCDD with P-450d occurred within 2 min of inhibitor addition. Therefore, TCDD can be considered a tight binding inhibitor of cytochrome P-450d.     

Comparative studies of rat liver and lung NADPH-cytochrome c reductase

NADPH-cytochrome c reductase, the flavoprotein component of the liver microsomal mixed-function oxidases, has been compared to the corresponding rat lung microsomal enzyme. Both enzymes were purified by the same methods and have identical ionic strength optima towards the reduction of cytochrome c. Antibody directed against the liver reductase identically inhibited the reduction of cytochrome c and ferricyanide by both enzymes. Double diffusion immunoprecipitation on Ouchterlony plates of deoxycholate-solubilized liver and lung microsomes resulted in converging precipitin lines indicating similar antigenic sites. The apparent molecular weights of the detergent-solubilized and bromelain-solubilized lung enzymes were determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis to be 79 000 and 71 000, respectively. From the above criteria we conclude that the enzymes in these two tissues are very similar or identical proteins.     

Release of iron from ferritin by cardiotoxic anthracycline antibiotics

The use of the extremely effective anthracycline antitumor drugs adriamycin and daunomycin is limited by a severe, dose-dependent cardiomyopathy. Anthracycline-induced toxicity has been proposed to involve iron-dependent oxidative damage to biological macromolecules yet little is known regarding the availability of physiologic iron. We now report that, in the presence of NADPH-cytochrome P-450 reductase, these drugs undergo redox cycling to generate superoxide which mediates a slow, reductive release of iron from ferritin, the major intracellular iron storage protein. Anaerobically, the semiquinone free radical forms of adriamycin and daunomycin catalyze a very rapid, extensive release of iron from ferritin. In contrast, diaziquone, an aziridinyl quinone antitumorigenic agent which is less cardiotoxic, is unable to release iron from ferritin. Thus, the present studies suggest that the cardiomyopathy observed with the anthracyclines, and perhaps their antineoplastic activity as well, may be related to their ability to delocalize tissue iron, thereby contributing to the formation of strong oxidants capable of damaging critical cellular constituents.     

The molecular weight of NADPH-cytochrome C reductase isolated by immunoprecipitation from detergent-solubilized rat liver microsomes

The molecular weight of detergent-solubilized NADPH-cytochrome c reductase from rat liver microsomes has been estimated to be 79,000. The method used for this determination involves immunoprecipitation of deoxycholate-solubilized enzyme from ¹²⁵I-labeled microsomal proteins. The antibody was prepared against a purified preparation of Bromelain-solubilized enzyme (molecular weight 71,000). The immunoprecipitate was then subjected to SDS-polyacrylamide gel electrophoresis and the enzyme located by ¹²⁵I-gamma counting.     

Referate

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In vitro loading of apoferritin.

This study compared the effect of loading apoferritin either with ferrous ammonium sulfate in various buffers or with ceruloplasmin and chelated ferrous iron. It was shown that loading of apoferritin with ferrous ammonium sulfate was dependent on buffer and pH, and was directly related to the rate of iron autoxidation. The ceruloplasmin-dependent loading of apoferritin, however, was unaffected by these factors. Isoelectric focusing and amino acid analysis of the differently loaded ferritins showed that ferrous ammonium sulfate loading of apoferritin resulted in the depletion of the basic amino acids, lysine and histidine, probably as a result of protein oxidation. No significant differences in amino acid composition was noted for ceruloplasmin-loaded ferritin. Furthermore, ferritin loaded with ferrous ammonium sulfate released more iron than either native or ceruloplasmin-loaded ferritin when either paraquat or EDTA was used as an iron mobilizing agent. We suggest that the loading of apoferritin with ferrous ammonium sulfate occurred as a result of iron autoxidation and may result in oxidation of amino acids and loss of integrity of the protein, and that ceruloplasmin may act as a catalyst for the incorporation of iron into apoferritin in a manner more closely related to that occurring in vivo.     

The Effect of Ultraviolet and Solar Radiation and Temperature on Survival of Fungal Propagules

Survival of spores, spore aggregates, sclerotia, and pycnidia of fungi was evaluated after exposure to high temperature in the dark, or exposure to ultraviolet (UV) radiation or sunlight. Under most conditions survival decreased from the most resitant Sclerotium rolfsii to Alternaria macrospora, Mycosphaerella pinodes, Aspergillus niger and Botrytis cinerea, in that order. There was no difference in survival among organisms stored in darkness at 45°C as single spores or aggregated into a mass of spores; or between spores of M. pinodes exposed before and after being released from pycnidia. Exposure to UV reduced survival much more than exposure in darkness. Longevity under UV ranged from more than 7 days for S. rolfsii to approximately 50 min for A. macrospora and 3 min for B. cinerea. Much longer survival occurred for spores of all fungi aggregated together; of spores of M. pinodes irradiated when inside rather than outside pycnidia; and in bigger, darker and older rather than in smaller, paler and younger sclerotia of S. rolfsii. Survival of sclerotia cut into slices and exposed to UV increased with thickness, irrespective of exposure to UV with the outer pigmented or innernonpigmented side. As tested with spore aggregates of A. niger, the rate of survival was linearly proportional to the number of conidia aggregated in each body. Irradiation by sunlight affected survival as described for irradiation by UV. The best protection from the detrimental effects of sun and UV radiation was provided by the infected host tissue.     

Transition metals as catalysts of "autoxidation" reactions

Superoxide (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (.OH) produced from the "autoxidation" of biomolecules, such as ascorbate, catecholamines, or thiols, have been implicated in numerous toxicities. However, the direct reaction of dioxygen with the vast majority of biomolecules, including those listed above, is spin forbidden, a condition which imposes a severe kinetic limitation on this reaction pathway. Therefore, an alternate mechanism must be invoked to explain the "autoxidations" reactions frequently reported. Transition metals are efficient catalysts of redox reactions and their reactions with dioxygen are not spin restricted. Therefore it is likely that the "autoxidation" observed for many biomolecules is, in fact, metal catalyzed. In this paper we discuss: 1) the quantum mechanic, thermodynamic, and kinetic aspects of the reactions of dioxygen with biomolecules; 2) the involvement of transition metals in biomolecule oxidation; and 3) the biological implications of metal catalyzed oxidations. We hypothesize that true autoxidation of biomolecules does not occur in biological systems, instead the "autoxidation" of biomolecules is the result of transition metals bound by the biomolecules.     

Reconstituted microsomal lipid peroxidation: ADP-Fe3+-dependent peroxidation of phospholipid vesicles containing NADPH-cytochrome P450 reductase and cytochrome P450

A reconstituted lipid peroxidation system consisting of rat liver microsomal NADPH-cytochrome P450 reductase and cytochrome P450 incorporated into phospholipid vesicles was developed and characterized. Peroxidation of the vesicles required NADPH and ADP-Fe3+, just as in the NADPH-dependent peroxidation of microsomes. The peroxidation of the vesicles was inhibited 30-50% by superoxide dismutase, depending upon their cytochrome P450 content: those with higher cytochrome P450 contents exhibited greater rates of malondialdehyde formation which were less sensitive to inhibition by superoxide dismutase. When cytochrome P450 was incorporated into vesicles, EDTA-Fe3+ was not required for lipid peroxidation, distinguishing this system from the one previously described by Pederson and Aust [Biochem. Biophys. Res. Comm. 48, 789; 1972]. Since at least 50% of the malondialdehyde formation in the vesicular system was not inhibited by superoxide dismutase, alternative means of iron reduction (O2-.-independent) were examined. It was found that rat liver microsomes or a reconstituted mixed function oxidase system consisting of NADPH-cytochrome P450 reductase and cytochrome P450 in dilauroylphosphatidylcholine micelles reduced ADP-Fe3+ under anaerobic conditions.