Mohammad G. Mustafa

Ozone interactions with lung tissue. Biochemical approaches.

This paper is a summary of recent research on the biochemical effects of ozone on the lung. Molecular mechanisms of O/sub 3/-induced damage, acute exposure effects, chronic-exposure effects, and factors which decrease lung susceptibility to O/sub 3/ damage are discussed. 58 references.

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.

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.

Fate of inhaled nitrogen dioxide in isolated perfused rat lung

The fate of inhaled NO2 was studied with isolated perfused rat lungs. The isolated lungs were exposed to 5 ppm NO2 for 90 min at a ventilation rate of 34 ml/min. The NO2 exposure had no adverse effects on the lungs as judged from their weights, glucose uptake, or lactate production compared to control lungs. Isolated lungs absorbed 36% of ventilated NO2, which was detected in perfusate and lung tissue as NO2- but not NO3-. The NO2- concentration in perfusate increased linearly with time, and after 90 min of ventilation with NO2 and perfusion with erythrocyte-free medium the NO2- accumulation was 6.36 +/- 0.39 micrograms. If perfusate contained 10% erythrocytes, the ventilated NO2 product was mostly NO3- in perfusate but NO2- in lung tissue. Protein solutions absorbed NO2 more effectively than simple salt solutions, but they all yielded mainly NO2- unless erythrocytes were present, when the product was mostly NO3-. The results indicate that absorbed NO2 in the lung is converted predominantly to NO2-, but after its diffusion into the vascular space it is oxidized to NO3- by interactions with erythrocytes.

Biochemical changes in rat lungs after exposure to nitrogen dioxide.

Sixty-day-old male, specific pathogen-free rats were exposed continuously to 5 or 15 ppm NO2 for 1-7 d. Lung tissue from exposed and control rats was then analyzed for biochemical and enzymatic parameters. The exposure resulted in increased lung enzymatic activities, including elevated protein and DNA contents and nonprotein sulfhydryl levels. Biochemical and enzymatic parameters generally increased maximally after 4 d and remained elevated for up to 7 d of continued exposure. The magnitude of these increases was higher for 15 than for 4 ppm NO2. The increases in biochemical and enzymatic parameters may have occurred in response to NO2-induced lung injury.

Ozone-induced DNA strand breaks In guinea pig tracheobronchial epithelial cells.

Ozone (O3), the major oxidant of photochemical smog, is thought to be genotoxic and a potential respiratory carcinogen or promoter of carcinogenic processes. Because of oxidative reactions with the mucus in the upper airway, O3 reaction products are able to penetrate into the tracheobronchial epithelial (TE) cells. The carcinogenic effects of O3 on the TE cells are especially of interest since most previous studies have focused on the morphology or permeability changes of tracheas only. Therefore, the objective of this study was to examine the potential O3 genotoxicity in TE cells after an in vivo exposure, using DNA strand breaks as an index. Two-month-old male Dunkin-Hartley guinea pigs, specific pathogen free, 4 in each group, were exposed to 1.0 ppm O3 for 0, 12, 24, 48, 72, or 96 h. Animals exposed to filtered air without O3 exposure were used as controls. After O3 exposure, the trachea with two main bronchi was removed from each animal, and TE cells were isolated and employed for determination of DNA strand breaks by fluorometric analysis of DNA unwinding (FADU). The statistical significance level was set at alpha = .05. Compared with controls, ozone exposure did not alter the TE cell yield or viability, but caused an increase in protein content in tracheal lavage and an increase in DNA strand breaks. The amount of DNA left in the alkali lysate of TE cells found at 72 h exposure was significantly decreased from controls for 3 different alkali incubation times. An increase of the double-stranded DNA left in the alkali lysate of TE cells was observed at 96 h of exposure and approached the value of 24 h of exposure. The same pattern was seen with all 3 different alkali incubation times at 15 degrees C. One Qd unit was estimated to correspond to 100 strand breaks per cell. The Qd was also used as an indicator for O3 damage. Compared to controls, the Qd increases significantly after 1 ppm O3 exposure for 72 h, regardless of the alkali incubation time at 15 degrees C.

Effects of short‐term, single and combined exposure to low‐level NO2 and O3 on lung tissue enzyme activities in rats

To examine the pulmonary effects of relatively low levels of NO2 and O3, and test for any possible interaction in their effects, we exposed 3-mo-old male Sprague-Dawley rats, free of specific pathogens, to either filtered room air (control) or 1.20 ppm (2256 micrograms/m3) NO2, 0.30 ppm (588 micrograms/m3) O3, or a combination of the two oxidants continuously for 3 d. We studied a series of parameters in the lung, including lung weight, and enzyme activities related to NADPH generation, sulfhydryl metabolism, and cellular detoxification. The results showed that relative to control, exposure to NO2 caused small but nonsignificant changes in all the parameters; O3 caused significant increases in all the parameters except for superoxide dismutase; and a combination of NO2 and O3 caused increases in all the parameters, and the increases were greater than those caused by NO2 or O3 alone. Statistical analysis of the data showed that the effects of combined exposure were synergistic for 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase, glutathione reductase, and superoxide dismutase activities, and additive for glutathione peroxidase and disulfide reductase activities, but indifferent from those of O3 exposure for other enzyme activities.

Dietary antioxidants and the biochemical response to oxidant inhalation

We fed female strain A/St mice selenium (Se) test diets containing either no Se (−Se) or 1 ppm Se (+Se) for 11 wk. Both diets contained 55 ppm vitamin E. We then exposed three groups of mice from each dietary regimen to either 0.8 ppm (1568 μg/m3) O3 (low-level) continuously for 5 d, 10.0 ppm (19,600 μg/m3) O3 (high-level) for 12 h, or filtered room air, where the latter served as a control for both O3 exposures. After O3 exposures we analyzed the lungs for various physical and biochemical parameters, and compared the results to those obtained from the air controls. The results showed that the difference in dietary Se intake produced an eightfold difference in Se content and a three-fold difference in glutathione peroxidase (GP) activity in the lung, but few changes in other lung parameters. With low-level O3 exposure, NADPH production increased significantly in +Se mice, but did not change in −Se mice. With high-level O3 exposure we observed comparable effects for both dietary regimens, including animal mortality, which was 24% for −Se and 14% for +Se mice. Thus, it seems that diminished GP activity resulting from Se deficiency and the ensuing lack of increase in NADPH production were poorly correlated with mouse tolerance to O3. The lung Se content increased in both dietary regimens after O3 exposure, but the increase was greater after high-level O3 exposure. This suggests a “mobilization” of Se to the lung under O3 stress. It is possible that such a mobilization contributes to the lung reserve of antioxidants, and hence the comparable mortality in both dietary Se regimens.