Donald D. Gibson

Effect of glutathione peroxidase activity on lipid peroxidation in biological membranes

Results are presented indicating that, although glutathione peroxidase activity inhibits lipid peroxidation in membranes, it does not appear to do so by reducing membrane lipid peroxides to lipid alcohols, as has been shown by others to be the case for free fatty acid peroxides in solution. Lipid peroxidation was studied in an enzymic system (microsomal NADPH oxidase) and in a non-enzymic system (mitochondria plus ascorbate). A study of the fatty acids in the phospholipids of microsomes and mitochondria demonstrated that detectable amounts of hydroxy fatty acids were not formed in the membranes when the latter were incubated in the presence of the glutathione peroxidase system even under conditions known to have generated significant levels of lipid peroxides in the membrane. Fatty acid analyses of the microsomal and mitochondrial particles indicated that glutathione peroxidase activity inhibited loss of polyunsaturated fatty acids when these organelles were exposed to peroxidizing conditions. If glutathione peroxidase activity were inhibiting the formation of malondialdehyde (a product of lipid peroxidation) by converting peroxide groups to alcohols, the loss of the constitutive polyunsaturated fatty acids in the membrane should not have been appreciably affected by addition of the peroxidase system. The protective effect cannot be due to quenching of an autocatalytic type of lipid peroxidation (at least in the microsomal system) since it has been established that the microsomal enzyme system (NADPH oxidase) catalyzes a continuous attack on microsomal polyunsaturated fatty acyl groups during the reaction and that the peroxidative process is not autocatalytic in nature. It appears, therefore, that glutathione peroxidase activity must exert its effect on this system by preventing free radical attack on the polyunsaturated membrane lipids in the first place. A possible mechanism for the interruption of a free radical attack on the lipids is proposed.

Glutathione-dependent inhibition of lipid peroxidation by a soluble, heat-labile factor in animal tissues.

A glutathione-dependent, cytosolic factor (previously thought to be glutathione peroxidase), inhibits lipid peroxidation in both microsomal and mitochondrial membranes. Studies in this laboratory had shown that the inhibition was due to prevention of peroxidative attack on the polyunsaturated fatty acids in the membrane lipids even under conditions that would otherwise promote rapid lipid peroxidation. A glutathione-dependent factor is also present in rat liver cytosol which can utilize peroxides of both free fatty acid salts in solution and free fatty acids in micellar suspension as substrates. It does not, however, utilize peroxidized lipids of microsomal and mitochondrial membranes as substrates. Whether or not this is the same factor which inhibits lipid peroxidation is not known with certainty, but current information indicates that they are not the same. Data presented in this report support the conclusion that neither glutathione peroxidase nor glutathione S-transferase activities appear to be responsible for the inhibition of lipid peroxidation in biological membranes. After partial purification of active preparations of both of these peroxidases, it was observed that neither preparation inhibited lipid peroxidation. The results of this study further support the conclusion that the glutathione-dependent cytosolic factor which inhibits lipid peroxidation in biological membranes does so by preventing the peroxidation rather than by reducing lipid peroxides.

GSH-dependent inhibition of lipid peroxidation: Properties of a potent cytosolic system which protects cell membranes

Properties of a heat labile, nondialyzable cytosolic factor which prevents lipid peroxidation in membranous organelles are described. The factor is present in liver and other animal tissues, and its capacity to inhibit lipid peroxidation in membranes subjected to oxidative stress is greatly potentiated by glutathione (GSH), although GSH by itself has no inhibitory effect on lipid peroxidation. The data obtained thus far indicate that one or more sulfhydryl groups associated with the factor is required for the inhibition. The mechanism by which lipid peroxidation is inhibited must involve prevention of initiation of peroxidation in the membranes, presumably by a process requiring one or more sulfhydryl groups associated with the heat labile factor. The latter appears to be protected by GSH while the factor is exerting its inhibitory effect on lipid peroxidation. The factor is not one of the known GSH-dependent enzymes, and appears to be a potent and ubiquitous system for stabilizing cell membranes against oxidative damage.

Magnetic nanoparticles: inner ear targeted molecule delivery and middle ear implant.

Superparamagnetic iron oxide nanoparticles (SNP) composed of magnetite (Fe3O4) were studied preliminarily as vehicles for therapeutic molecule delivery to the inner ear and as a middle ear implant capable of producing biomechanically relevant forces for auditory function. Magnetite SNP were synthesized, then encapsulated in either silica or poly (D,L,-Lactide-co-glycolide) or obtained commercially with coatings of oleic acid or dextran. Permanent magnetic fields generated forces sufficient to pull them across tissue in several round window membrane models (in vitrocell culture, in vivo rat and guinea pig, and human temporal bone) or to embed them in middle ear epithelia. Biocompatibility was investigated by light and electron microscopy, cell culture kinetics, and hair cell survival in organotypic cell culture and no measurable toxicity was found. A sinusoidal magnetic field applied to guinea pigs with SNP implanted in the middle ear resulted in displacements of the middle ear comparable to 90 dB SPL.

Evidence that the large loss of glutathione observed in ischemia/reperfusion of the small intestine is not due to oxidation to glutathione disulfide

Reperfusion injury following ischemia is thought to be the consequence of reactive oxygen species possibly generated either by xanthine oxidase activity or by processes associated with neutrophil activation in the affected organ or tissue. The conversion of xanthine dehydrogenase to the oxidase as well as the interactions between endothelium and neutrophils in the margination and activation of the latter are all considered to be results of conditions resulting from the ischemic episode. Determination of the redox status of glutathione in an ischemic/reperfused organ is frequently employed as an indicator of oxidative stress created by the production of oxygen free radicals during the reperfusion period. In this procedure, the ratio of oxidized glutathione (GSSG) to total glutathione (GSH + GSSG) is utilized to demonstrate the proportion of glutathione oxidized during reperfusion. We determined this ratio in the rat small intestine during ischemia and reperfusion and found that while the ratio of GSSG/(GSH + GSSG) does increase, this increase was the result of GSH disappearance rather than an increase in GSSG, and that essentially all of this loss occurred during the ischemic episode. We demonstrated that no oxidation of GSH occurred that was attributable to reperfusion per se; nor was there an increase of GSSG during this reoxygenation period.

Effects of butylated hydroxytoluene and acetylaminofluorene on NADPH-cytochrome P-450 reductase activity in rat liver microsomes

Cytochrome P-450 content and NADPH-cytochrome P-450 reductase activity were measured in liver microsomes prepared from male weanling rats fed low-fat, high-saturated fat or high-polyunsaturated fat diets with or without butylated hydroxytoluene (BHT) and 2-acetylaminofluorene (AAF). The inclusion of BHT and/or AAF in the diets consistently produced marked decreases in cytochrome P-450 reductase activity, regardless of the amount and type of dietary fat. In contrast, there was no inhibition of reductase activity when the compounds were added in vitro to liver microsomes.

Magnetically-responsive nanoparticles for vectored delivery of cancer therapeutics

We propose that physical targeting of therapeutics to tumors using magnetically‐responsive nanoparticles (MNPs) will enhance intratumoral drug levels compared to free drugs in an effort to overcome tumor resistance. We evaluated the feasibility of magnetic enhancement of tumor extravasation of systemically‐administered MNPs in human xenografts implanted in the mammary fatpads of nude mice. Mice with orthotopic tumors were injected systemically with MNPs, with a focused magnetic field juxtaposed over the tumor. Magnetic resonance imaging and scanning electron microscopy both indicated successful tumor localization of MNPs. Next, MNPs were modified with poly‐ethylene‐glycol (PEG) and their clearance compared by estimating signal attenuation in liver due to iron accumulation. The results suggested that PEG substitution could retard the rate of MNP plasma clearance, which may allow greater magnetically‐enhanced tumor localization. We propose that this technology is clinically scalable to many types of both superficial as well as some viscerable tumors with existing magnetic technology.

Biological Systems Which Suppress Lipid Peroxidation

Cell metabolism and the environmental factors in general place animal tissues at chronic risk of oxidative alteration of membrane lipids and other components. Several oxidation-reduction enzymes in subcellular organelles are capable of initiating lipid peroxidation in those organelles in vitro. For example, the synthesis of ascorbic acid from gulonolactone by gulonolactone oxidase causes a peroxidative degradation of membrane phospholipids in liver microsomes (1). Oxidation of NADPH by both liver microsomes (2) and liver mitochondria (3) results in lipid peroxidation also. The metabolism of some xenobiotic compounds by the drug metabolizing system is also capable of promoting oxidative degradation of both membrane lipids and proteins (4–6). In addition, radiation, airborne chemicals, ozone, and water pollutants may also produce oxidative damage to tissue.