Nabil M. Elsayed

Automated assays for superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activity.

Automated assays for catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase are presented. The assay for catalase is based on the peroxidatic activity of the enzyme. The glutathione peroxidase and reductase assays measure the consumption of NADPH following the reduction of t-butyl hydroperoxide and oxidized glutathione, respectively. The assay for superoxide dismutase is based on the reduction of cytochrome c. All assays utilize the Cobas FARA clinical automated analyzer and provide considerable time savings over the manual assays.

Toxicology of blast overpressure.

Blast overpressure (BOP) or high energy impulse noise, is the sharp instantaneous rise in ambient atmospheric pressure resulting from explosive detonation or firing of weapons. Blasts that were once confined to military and to a lesser extent, occupational settings, are becoming more universal as the civilian population is now increasingly at risk of exposure to BOP from terrorist bombings that are occurring worldwide with greater frequency. Exposure to incident BOP waves can cause auditory and non-auditory damage. The primary targets for BOP damage are the hollow organs, ear, lung and gastrointestinal tract. In addition, solid organs such as heart, spleen and brain can also be injured upon exposure. However, the lung is more sensitive to damage and its injury can lead to death. The pathophysiological responses, and mortality have been extensively studied, but little attention, was given to the biochemical manifestations, and molecular mechanism(s) of injury. The injury from BOP has been, generally, attributed to its external physical impact on the body causing internal mechanical damage. However, a new hypothesis has been proposed based on experiments conducted in the Department of Respiratory Research, Walter Reed Army Institute of Research, and later in the Department of Occupational Health, University of Pittsburgh. This hypothesis suggests that subtle biochemical changes namely, free radical-mediated oxidative stress occur and contribute to BOP-induced injury. Understanding the etiology of these changes may shed new light on the molecular mechanism(s) of injury, and can potentially offer new strategies for treatment. In this symposium. BOP research involving auditory, non-auditory, physiological, pathological, behavioral, and biochemical manifestations as well as predictive modeling and current treatment modalities of BOP-induced injury are discussed.

Free radical-mediated lung response to the monofunctional sulfur mustard butyl 2-chloroethyl sulfide after subcutaneous injection

Vesicant-induced pathogenesis is initiated by rapid alkylation and cross-linking of DNA purine bases causing strand breaks leading subsequently to NAD depletion and cell death. We postulated that vesicants may also be associated with free radical-mediated oxidative stress distal to the site of exposure. To test this postulate in the lung, we injected 3 groups (n = 8) of 5-month-old, male, athymic, nude mice, weighing 30-35 g with a single subcutaneous (s.c.) injection (5 microliters/mouse) of butyl 2-chloroethyl sulfide (BCS), a monofunctional sulfur mustard analog. After 1, 24 and 48 h, we euthanized the treated mice along with 2 untreated control mice at each time point. We then pooled the control mice in one group (n = 6) and analyzed the lungs for biochemical indices of oxidative stress. We found that total lung weight was not altered after treatment, but wet/dry weight ratio decreased 18% (P less than 0.05) and hemoglobin content increased 50% and 36% at 1 and 24 h, respectively. The activity of glucose-6-phosphate dehydrogenase increased significantly, 40% at 1 and 24 h and 84% at 48 h and that of glutathione S-transferases was 60%, P less than 0.05 greater at all time points. Lipid peroxidation (estimated by the thiobarbituric acid test) and total protein content increased 3-fold and 2-fold, at 1 and 24 h, respectively. Total and oxidized glutathione contents were significantly elevated, 38% at 1 h and 64% at 24 h for the former and 45% at 24 h and 56% at 48 h for the latter. Because these changes are consistent with the cellular response to oxidative stress, we conclude that BCS injected subcutaneously, can cause changes in the lung possibly via a free radical-mediated mechanism.

Influence of vitamin E and nitrogen dioxide on lipid peroxidation in rat lung and liver microsomes

Rat lung and liver microsomes were used to examine the effects of dietary vitamin E deficiency on membrane lipid peroxidation. Microsomes from vitamin-E-deficient rats displayed increased lipid peroxidation in comparison to microsomes from vitamin-E-supplemented controls. The extent of lipid peroxidation, as determined by measurement of thiobarbituric acid reacting materials, was enhanced by addition of reduced iron and ascorbate (or NADPH). Rats fed a vitamin-E-supplemented diet and exposed to 3 ppm NO2 for 7 days did not exhibit increases in microsomal lipid peroxidation compared to air-breathing controls. However, increases were found in microsomes prepared from rats fed a vitamin-E-deficient diet and exposed to NO2. Lung microsomes from vitamin-E-fed rats contained almost 10 times as much vitamin E as liver microsomes when expressed in terms of polyunsaturated fatty acid content. The extent of lipid peroxidation was, in turn, considerably less in lung than in liver microsomes. Lipid peroxidation in lung microsomes from vitamin-E-deficient rats was comparable to liver microsomes from vitamin-E-supplemented rats as was the content of vitamin E in these respective microsomal samples. A combination of vitamin E deficiency and NO2 exposure resulted in the greatest increases in lung and liver microsomal lipid peroxidation with the largest relative increases occurring in lung microsomes. An inverse relationship was found between the extent of lipid peroxidation and vitamin E content. Most of the peroxidation in lung microsomes appeared to proceed nonenzymatically whereas peroxidation in liver was largely enzymatic. Vitamin E appears to be assimilated by the lung during oxidant inhalation, but with dietary vitamin E deprivation, the margin for protection in lung may be less than in liver.

Dietary antioxidants and the biochemical response to oxidant inhalation: I. Influence of dietary vitamin E on the biochemical effects of nitrogen dioxide exposure in rat lung

Abstract Sprague-Dawley rats derived from a specific pathogen-free colony were raised from birth on a test diet containing either 0 or 50 IU vitamin E/kg diet for 8 weeks. Rats from each dietary group were exposed to 3 ppm (5640 μg/m 3 ) nitrogen dioxide (NO 2 ) continuously for 7 days. They were then killed, and the lungs analyzed for changes in weight, DNA and protein contents, tissue oxygen utilization, sulfhydryl metabolism, and the activities of NADP-reducing enzymes. The difference in dietary vitamin E alone did not cause any significant changes in these parameters. However, after NO 2 exposure the changes in these parameters relative to their corresponding unexposed controls were greater for the deficient rats than for the supplemented rats. The biochemical changes observed may be a response of the lung to injury from NO 2 exposure. The larger changes in the lungs of deficient rats may reflect a greater sensitivity of these animals to inhaled NO 2 . The vitamin E contents of lung tissue in deficient and supplemented rats reflected the dietary levels. After NO 2 exposure, the vitamin E content in the lung increased significantly in supplemented rats but decreased in the deficient rats relative to their corresponding unexposed controls. The elevation of vitamin E levels in the lungs of supplemented rats with NO 2 exposure suggests its mobilization from other body sites, whereas in deficient rats this process may not have been possible.

Visual system degeneration induced by blast overpressure

The effect of blast overpressure on visual system pathology was studied in 14 male Sprague-Dawley rats weighing 360-432 g. Blast overpressure was simulated using a compressed-air driven shock tube, with the aim of studying a range of overpressures causing sublethal injury. Neither control (unexposed) rats nor rats exposed to 83 kiloPascals (kPa) overpressure showed evidence of visual system pathology. Neurological injury to brain visual pathways was observed in male rats surviving blast overpressure exposures of 104-110 kPa and 129-173 kPa. Optic nerve fiber degeneration was ipsilateral to the blast pressure wave. The optic chiasm contained small numbers of degenerated fibers. Optic tract fiber degeneration was present bilaterally, but was predominantly ipsilateral. Optic tract fiber degeneration was followed to nuclear groups at the level of the midbrain, midbrain-diencephalic junction, and the thalamus where degenerated fibers arborized among the neurons of: (i) the superior colliculus, (ii) pretectal region, and (iii) the lateral geniculate body. The superior colliculus contained fiber degeneration localized principally to two superficial layers (i) the stratum opticum (layer III) and (ii) stratum cinereum (layer II). The pretectal area contained degenerated fibers which were widespread in (i) the nucleus of the optic tract, (ii) olivary pretectal nucleus, (iii) anterior pretectal nucleus, and (iv) the posterior pretectal nucleus. Degenerated fibers in the lateral geniculate body were not universally distributed. They appeared to arborize among neurons of the dorsal and ventral nuclei: the ventral lateral geniculate nucleus (parvocellular and magnocellular parts); and the dorsal lateral geniculate nucleus. The axonopathy observed in the central visual pathways and nuclei of the rat brain are consistent with the presence of blast overpressure induced injury to the retina. The orbital cavities of the human skull contain frontally-directed eyeballs for binocular vision. Humans looking directly into an oncoming blast wave place both eyes at risk. With bilateral visual system injury, neurological deficits may include loss or impairments of ocular movements, and of the pupillary and accommodation reflexes, retinal hemorrhages, scotomas, and general blindness. These findings suggest that the retina should be investigated for the presence of traumatic or ischemic cellular injury, hemorrhages, scotomas, and retinal detachment.

Age-dependent pulmonary response of rats to ozone exposure

The influence of age on O3 effects in the lung was studied in 8 groups of Sprague-Dawley rats: 7, 12, and 18 d of age (neonatal); 24, 30, and 45 d of age (infant); and 60 and 90 d of age (adult). Lung weight, total lung protein and DNA contents, and a series of marker enzyme activities in lung tissue were determined. After exposure of rats from each group to 0.8 ppm (1568 microgram/m3) O3 continuously for 3 d, a biphasic effect was noted. The biochemical parameters, expressed per lung, in O3-exposed rats relative to their corresponding controls decreased in the 7- and 12-d-old groups, increased or remained unchanged in the 18-d-old group, and increased in the 24- to 90-d-old groups. However, the increases were much greater for 60- to 90-d-old rats than for 24- to 30-d-old rats. The increase in lung biochemical parameters is thought to occur in response to lung injury and subsequent repair processes, and greater increases in the lungs of older rats suggest that they are more responsive to O3 exposure than younger rats. The decrease in lung biochemical parameters and increased mortality in 7- and 24-d-old neonatal rats suggest that they are more susceptible to O3 stress than infant and adult rats.

Increased vitamin E content in the lung after ozone exposure: A possible mobilization in response to oxidative stress

Vitamin E (vE) is a biological free radical scavenger capable of providing antioxidant protection depending upon its tissue content. In previous studies, we observed that vE increased significantly in rat lungs after oxidant exposure, and we postulated that vE may be mobilized to the lung from other body sites under oxidative stress. To test this hypothesis, we fed Long-Evans rats either a vE-supplemented or a vE-deficient diet, injected them intraperitoneally with 14C-labeled vE, and then exposed half of each group to 0.5 ppm ozone (O3) for 5 days. After exposure, we determined vE content and label retention in lungs, liver, kidney, heart, brain, plasma, and white adipose tissue. Tissue vE content of all tissues generally reflected the dietary level, but labeled vE retention in all tissues was inversely related to tissue content, possibly reflecting a saturation of existing vE receptor sites in supplemented rats. Following O3 exposure, lung vE content increased significantly in supplemented rats and decreased in deficient rats, but the decrease was not statistically significant, and vE content remained unchanged in all other tissues of both dietary groups. Retention of 14C-labeled vE increased in all tissues of O3-exposed rats of both dietary groups, except in vE-deficient adipose tissue and vE-supplemented brain, where it decreased, and plasma, where it did not change. The marked increases in lung vE content and labeled vE retention of O3-exposed vE-supplemented rats support our hypothesis that vE may be mobilized to the lung in response to oxidative stress, providing that the vitamin is sufficiently available in other body sites.

A comparison of biochemical effects of nitrogen dioxide, ozone, and their combination in mouse lung.

Swiss Webster mice were exposed to either 4.8 ppm (9024 microgram/m3) nitrogen dioxide (NO2), 0.45 ppm (882 microgram/m3) ozone (O3), or their combination intermittently (8 hr daily) for 7 days, and the effects were studied in the lung by a series of physical and biochemical parameters, including lung weight, DNA and protein contents, oxygen consumption, sulfhydryl metabolism, and activities of NADPH generating enzymes. The results show that exposure to NO2 caused relatively smaller changes than O3, and that the effect of each gas alone under the conditions of exposure was not significant for most of the parameters tested. However, when the two gases were combined, the exposure caused changes that were greater and significant. Statistical analysis of the data shows that the effects of combined exposure were more than additive, i.e., they might be synergistic. The observations suggest that intermittent exposure to NO2 or O3 alone at the concentration used may not cause significant alterations in lung metabolism, but when the two gases are combined the alterations may become significant.

Response of mouse brain to a single subcutaneous injection of the monofunctional sulfur mustard, butyl 2-chloroethyl sulfide (BCS)☆

Exposure to mustard-type vesicants results in alkylation of DNA and vesication. However, the biochemical mechanism for vesicant injury and whether it is localized or diffuse are not clear. We postulated that vesicant damage is mediated by free radicals, resulting in oxidative stress. These free radicals-mediated reactions may propagate systemically distal to the site of exposure. To test this hypothesis, we examined the effects of a single subcutaneous injection of the monofunctional sulfur mustard, butyl 2-chloroethyl sulfide (BCS), on the brain. We injected 3 groups (6 mice/group) of 5-month-old male, athymic, nude mice, weighing 30-35 g, subcutaneously with neat (undiluted) BCS (5 microliters/mouse). After 1, 24, and 48 h, we sacrificed the treated mice along with an untreated control group and analyzed the brains for biochemical markers of oxidative stress. Compared to untreated controls, the activity of glutathione peroxidase increased by 76%, P less than 0.005 at 24 h, and that of glutathione S-transferases by 25-37%, P less than 0.05 over the entire period. Total glutathione content in the brain was significantly lower, 17%, after 1 h and 23% after 24 h. We found also, concomitant with decreased glutathione, almost a 3-fold increase in susceptibility to lipid peroxidation. Because these changes are consistent with oxidative stress, we conclude that the effect of BCS administered subcutaneously may be translocated, reaching mouse brain, and causing oxidative stress.