Bruce A. Svingen

Pulse radiolysis studies of antitumor quinones: Radical lifetimes, reactivity with oxygen, and one-electron reduction potentials

Abstract The formation of semiquinone free radicals from antitumor drugs has been studied by pulse radiolysis. The semiquinone free radicals are reactive and have short half-lives in aqueous media under anaerobic conditions. The half-lives of the radicals formed from adriamycin, mitomycin C, and 2,5-diaziridinyl-3,6-bis(carboethoxy)amine-1,4-benzoquinone (AZQ) are 50,100, and 200 μs, respectively. The mean diffusion distance of the semiquinone free radical is less than 0.6 μm. In the presence of molecular oxygen the half-life of the semiquinone free radical is shortened. Adriamycin semiquinone reacts rapidly with oxygen, k = 4.4 × 107m−1s−1. In air-saturated buffer the half-life of adriamycin semiquinone radical can be calculated to be 8 μs with a mean diffusion distance of less than 0.1 μm. If the half-lives in buffer are comparable to those within a cell, semiquinone free radicals must be generated close to the site at which they produce a biological effect. One-electron reduction potentials (E71) were determined and were AZQ, −168 mV, adrenochrome, −253 mV, mitomycin C, −271 mV, adriamycin, −292 mV, daunomycin, −305 mV, and anthracenedione, −348 mV. Enzymatic one-electron reduction of these antitumor quinones by NADPH-cytochrome P-450 reductase increased at more positive values of quinone E71.

Effects of a Fish Oil Diet on Pigs' Cardiopulmonary Response to Bacteremia

Since an omega 3 fatty acid (FA) diet may have beneficial effects in inflammatory processes, we tested the hypothesis that the physiologic response to sepsis could be modified by altering the eicosanoid precursor pool via an omega 3 FA diet. Two groups (n = 8) of pigs were prefed for 8 days either an omega 3 FA or an omega 6 FA diet (Weaner Pig Feed with either menhaden or corn oil to produce a eucaloric feed with 15% fat) and then injected with live Escherichia coli. The omega 3 FA diet increased the concentration of eicosapentainoic acid (EPA, 20:5 omega 3) in plasma lipids, and increased the ratio of EPA to arachidonic acid (AA, 20:4 omega 6) in platelets from 1:20 to 1:1 over the 8 days. Following the injection of bacteria, there was a fall in PaO2 and blood pressure that was attenuated (p less than 0.05) by the omega 3 FA diet. The omega 3 FA diet, compared to the omega 6 FA diet, also attenuated the rise in thromboxane B2 (3.0 +/- 1.1 vs 12.9 +/- 5.7 ng/mL) and 6 keto-PGF1 alpha (0.8 +/- 0.5 vs 1.7 +/- 1.1 ng/mL) associated with bacteremia. We conclude that dietary omega 3 FA attenuated the physiologic response to sepsis, possibly by modifying arachidonic acid metabolism.

Enzymatic and non-enzymatic reduction of N-acetyl-p-benzoquinone imine and some properties of the N-acetyl-p-benzosemiquinone imine radical

N-Acetyl-p-benzoquinone imine (NAPQI) is the postulated hepatotoxic intermediate in acetaminophen overdosage. NAPQI was rapidly metabolized by NADPH-cytochrome P-450 reductase, with an apparent Km of 1.8 to 4.0 microM and an apparent Vmax of 29.4 mumoles per min per mg, and exhibited substrate inhibition of metabolism at NAPQI concentrations above 10 microM. NADPH was oxidized by NAPQI at a slower rate in the absence of enzyme. NAPQI did not appear to undergo redox cycling at an appreciable rate to form superoxide, and it did not stimulate oxygen utilization or superoxide release by rat isolated hepatocytes. Electron spin resonance studies failed to show formation of a free radical by chemical or enzymatic reduction of NAPQI under anaerobic conditions in aqueous media.

Iron-EDTA stimulated reduction of indicine N-oxide by the hepatic microsomal fraction, isolated hepatocytes, and the intact rat.

Fe(III) complexes of EDTA and diethylenetriamine pentaacetic acid (DETAPAC) at low concentrations (between 1 and 100 microM) produced up to a 20-fold increase in anaerobic microsomal NADPH- and NADH-dependent reduction of indicine N-oxide. Under aerobic conditions microsomal indicine N-oxide reduction was stimulated to half the levels seen under anaerobic conditions. EDTA alone was much less effective at stimulating indicine N-oxide reduction, while FeCl3 alone had no effect on reduction. Other complexes of Fe(III) had little or no effect in stimulating microsomal indicine N-oxide reduction. Fe(III)-EDTA stimulated indicine N-oxide reduction by purified NADPH-cytochrome P-450 reductase and NADPH. It is probable that iron serves to transfer electrons between microsomal flavoprotein reductases and indicine N-oxide. The redox potential and the presence of an exchangeable ligand, such as water, in the inner ligand sphere of the iron complex are suggested to be important factors in determining which iron complexes will stimulate indicine N-oxide reduction. EDTA complexes of other transition metal ions do not stimulate indicine N-oxide reduction. Hydroxyl radicals, detected as the spin adduct of 5,5-dimethyl-1-pyroline-N-oxide, appear to be formed during Fe(II)-EDTA-dependent reduction of indicine N-oxide under anaerobic conditions. Fe(III)-EDTA at concentrations between 50 and 250 microM stimulated indicine N-oxide reduction by rat isolated hepatocytes up to 5-fold under anaerobic conditions and to half these values under aerobic conditions. By themselves, EDTA and FeCl3 at similar concentrations produced a small stimulation of indicine N-oxide reduction by hepatocytes under anaerobic conditions. Fe(III)-EDTA stimulated indicine N-oxide reduction by murine leukemia P-388 cells under aerobic conditions and by rat caecal flora under anaerobic but not aerobic conditions. Fe(III)-EDTA, EDTA or FeCl3 administered to rats produced a 3-fold increase in the 24-hr urinary excretion of indicine following an i.p. dose of indicine N-oxide.

Essential fatty acid status in isolated closed head injury

Abnormal profiles of polyunsaturated fatty acids in the plasma phospholipid fraction were observed in 14 patients with hypermetabolism due to severe isolated closed head injury. Depressed linoleic acid levels were observed within 24 hours along with elevation of oleic acid levels. By one week postinjury, subnormal levels of arachidonic acid were also observed. These data indicate that the metabolic response to injury encompasses changes in the metabolism of polyunsaturated fatty acids which are rapid in onset and qualitatively similar to those seen in mild essential fatty acid deficiency.

Factors Affecting the Intracellular Generation of Free Radicals from Quinones

Isolated hepatocytes do not liberate appreciable amounts of superoxide into the external medium. Simple quinones stimulate the release of superoxide up to 15 nmol/min/10(6) hepatocytes. Superoxide release stimulated by a variety of simple quinones and more complex antitumor quinones was maximal at a quinone one-electron reduction potential of -70 mV. This was qualitatively similar to the pattern of superoxide formation seen with NADH-cytochrome b5 reductase and NADH: ubiquinone oxidoreductase. Superoxide production by NADPH-cytochrome P-450 reductase was maximal at a quinone single-electron reduction potential at -200 mV. Phenobarbital pretreatment had no effect on superoxide formation by hepatocytes suggesting that NADPH-cytochrome P-450 reductase activity is not rate limiting for quinone stimulated superoxide formation. Sulfonated stilbenes, specific inhibitors of anion exchange, had no effect on the release of superoxide by hepatocytes suggesting that superoxide is not transported through anion channels in the plasma membrane. Pretreatment of hepatocytes with 10(-5) M diethyldithiocarbamate produced over a two fold increase in the release of superoxide.