Allen D. Hacker

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.

Effects of vitamin E deficiency and nitrogen dioxide exposure on lung lipid peroxidation: Use of lipid epoxides and malonaldehyde as measures of peroxidation

The effect of vitamin E deficiency in male Sprague-Dawley rats upon lipid peroxidation in lung tissue was examined by measuring malonaldehyde and lipid epoxide production. In addition to controls, some animals were also exposed to 3 +/- 0.1 ppm NO2 continuously for 7 d in order to study the effects of oxidant stress on lung lipid peroxidation and vitamin E content. The observed changes in malonaldehyde and epoxide content could serve as good indices of lipid peroxidation, particularly under conditions of vitamin E deprivation. The responses measured indicated an inverse relation in the lung between tissue vitamin E content and quantity of lipid peroxidation products. Measurement of lipid epoxides served as a reliable indicator of lung tissue lipid peroxidation. Finally, NO2 inhalation appeared to elicit a response characterized by increased assimilation of vitamin E into lung tissue.

Suppression of polyamine biosynthesis prevents monocrotaline-induced pulmonary edema and arterial medial thickening

Previous work in our laboratory has shown that the continuous administration of alpha-difluoromethylornithine (DFMO), a highly specific irreversible inhibitor of ornithine decarboxylase (ODC), which is the rate-limiting enzyme in polyamine biosynthesis, prevented the development of pulmonary hypertension and right ventricular hypertrophy induced in rats 21 days after a single injection of monocrotaline (MCT). We now report that DFMO treatment did not influence the proposed first step of MCT pneumotoxicity, that is, the hepatic metabolism of MCT to toxic pyrrolic metabolites. In contrast, DFMO treatment blunted the development of lung perivascular edema at Day 7, inhibited the respective four- and twofold increases in lung putrescine and spermidine contents at Day 21 without significantly altering spermine content, and prevented the arterial medial thickening at Day 21. It thus appears that increased lung polyamine biosynthesis may be essential for the expression of MCT-induced perivascular edema as well as the development of the medial thickening stage of MCT-induced hypertensive pulmonary vascular disease.

Polyamine metabolism in rat lungs with oxygen toxicity

Ornithine decarboxylase activity increases 2-fold above control after 1 day and 25-fold after 3 days of exposure to 0.85 atm oxygen. Putrescine content nearly doubled by 72 hours which may reflect increased activity or ornithine decarboxylase. Spermidine and spermine content did not increase until after 3 days of exposure which was consistent with the delayed increase of S-adenosylmethionine decarboxylase activity. The results suggest that antimetabolites of polyamine metabolism may be useful to suppress excessive cellular proliferation in the lung after acute lung injury.

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.

Effects of decreased glutathione peroxidase activity on the pentose phosphate cycle in mouse lung

Abstract The effects of reducing glutathione peroxidase activity in the lung by changing dietary selenium intake has been investigated. In animals that were exposed to room air, selenium effects were confined to glutathione peroxidase activity, whereas under conditions of oxidant stress (ozone) the decrease in glutathione peroxidase activity prevented the stimulation of the pentose phosphate cycle (assayed by measuring glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities) which has been reported to increase in response to oxidant stress. The suppression of glutathione peroxidase activity was found to depend on dietary selenium concentration. The physiological significance of this observation may be related to the process of injury and repair in the lung.

Influence of Age On the Biochemical Response of Rat Lung To Ozone Exposure

We have previously examined the influence of animal age on the pulmonary response to ozone (03) in rats between 7 and 90 days of age (Elsayed et al., 1982a). In the present study, we expanded the age groups of rats, and examined in greater detail the relationship between animal age and pulmonary response to inhaled 03. We exposed 7 groups of specific pathogen free, male Sprague-Dawley rats, aged 24, 30, 45, 60, 90, 180, and 365 days, to 0.8 ppm (1568 μg/m3) 03 continuously for 3 days. After 03 exposure, we sacrificed the exposed rats and a matched number of controls from each age group, and analyzed their lungs for a series of physical and biochemical parameters, including glutathione metabolizing and NADPH producing enzyme activities. We observed that in control rats all the parameters increased as a function of age. However, the rate of increase was generally slower after age 60 days. After 03 exposure there was an increase in all the parameters for all age groups relative to their corresponding controls, but the extent of increase was significantly larger in rats 60 days and older than in younger rats. A regression of the difference in mean values between control and exposed animals for each parameter against age showed a linear correlation, indicating that the response was agedependent. Since the magnitude of such increases is thought to reflect the degree of lung injury, the results suggest that 03 exposure causes greater lung injury in older rats than in younger rats. We tested this assumption by exposing rats from four different age groups (24, 45, 60 and 90 days) to a lethal dose of 03 (4 ppm or 7840 ug/m3 for 8 hours). The mortality rates were 50% and 83% for 24 and 45 day old rats, respectively, and 100% for 60 and 90 day old rats. The results of these studies further demonstrate that older rats are more susceptible to lung injury from 03 than younger rats.

Dietary restriction, polyamines and monocrotaline-induced pulmonary hypertension

Dietary restriction (DR), i.e. reduction of total caloric intake, has been shown to result in protection against monocrotaline (MCT)-induced pulmonary hypertension (PH). Restriction of the diet to 8 g/rat/day instead of the usual intake (18 g/rat/day), inhibits the progression of cardiopulmonary changes and prolongs survival after a single dose of MCT. We have shown previously that the development of MCT-induced pulmonary hypertension is associated with inhibition of polyamine biosynthesis in the lungs of MCT-treated rats. In the present study, we tested the hypothesis that DR provides protection against the development of chronic PH in the rat by limiting increases in polyamine and DNA synthesis. We randomly divided animals into four groups each (MCT, MCT + DR, control, and control + DR). We injected rats with a single dose of MCT (60 mg/kg, s.c.) and a corresponding number of control rats with vehicle. Animals in all groups were given free access to food and water prior to administration of MCT. Immediately following injection of MCT both the MCT and control groups were given free access to food and water, while the other groups (MCT + DR and control + DR) we given the restricted diet (8 g/rat/day). Daily measurements were made of body weight and of water and food intake. Animals were killed in each group at 1, 4, 7, 14, and 21 days post MCT to determine right ventricular hypertrophy (RVH), lung wet weight, ornithine decarboxylase (ODC) activity, and polyamine and DNA contents. We measured DNA synthesis 7 days after MCT by determining [3H]thymidine incorporation into the whole lung DNA. We found that 7 days after MCT treatment DNA synthesis increased compared to control. However, DR (MCT + DR) treatmen prevented the increase in DNA synthesis following MCT. Right ventricular hypertrophy, lung wet weight, ODC activity and lung polyamine levels were increased following MCT. Treatment with DR (MCT + DR) prevented increases in RVH, lung wet weight, ODC activity and lung polyamine levels. We conclude that DR to 8 g/day/rat protects against MCT-induced PH and is associated with an inhibition of increased lung polyamine and DNA synthesis that occur in the lung during the development of MCT-induced PH. These results are consistent with a recent report which suggests that increased lung polyamine biosynthesis is required for the development of MCT-induced PH. The data are also consistent with the hypothesis that inhibition of polyamine biosynthesis influences the development of MCT-induced PH in part by regulating DNA synthesis in key lung cells.

Relationship of age to rat lung collagen synthesis in response to ozone exposure

We investigated the relationship of age to rat lung collagen synthesis in response to ozone (O3) exposure. Specific pathogen-free Sprague-Dawley male rats 24, 30, 45, 60, 94, 182 and 365 days old were exposed to either 0.8 ±.05 ppm O3 for 3 days or to 0.8 ± 0.5 ppm O3 for 3 days followed by 4.0 ±.2 ppm O3 for 8 hours and 60 days recovery in air. A matched number of rats from each age group received filtered air and served as controls. Lung dry weight, protein content, hydroxyproline content, 14C proline incorporation and 14C hydroxyproline formation were determined in control and exposed rat lungs. Rats exposed to 0.8 ppm O3 for 3 days had greater dry weight, protein content, incorporation of 14C proline, and formation of 14C hydroxyproline per lung relative to controls, with the greatest changes occurring in rats older than 60 days. Rats 24 and 94 days old, exposed to 0.8 ppm O3 for 3 days followed by 4.0 ppm O3 for 8 hours and 60 days recovery in air, had greater lung collagen content than controls: 50% and 80%, respectively. These results suggest that collagen synthesis was increased following O3 exposure and that the degree of change was influenced by age in the rat. Age, therefore, may be an important factor in the lung’s response to pulmonary injury.

Responses of natural wildlife populations to air pollution.

Deer mice (Peromyscus californicus) trapped in areas of Los Angeles with high ambient air pollution are significantly more resistant to ozone (6.6 ppm for 12 h) than are mice trapped from areas with low ambient pollution (56 versus 0% survival, respectively). Laboratory-born progeny of these mice show similar response patterns, indicating a genetic basis to this resistance. Young mice (less than 1 yr of age) are more sensitive than older mice (15 versus 44% survival, respectively). Sensitivity is also affected by degree of inbreeding; progeny of full-sib crosses are more sensitive than randomly bred deer mice. The data suggest that deer mice are more resistant to ozone toxicity than are commercially bred laboratory mice and rats.