Studies on biochemical effects of nitrogen dioxide: III. Changes of the antioxidative protective systems in rat lungs and of lipid peroxidation by chronic exposure

doi:10.1016/0041-008X(82)90056-4

Toxicology and Applied Pharmacology

Volume 66, Issue 1, October 1982, Pages 1-8

https://doi.org/10.1016/0041-008X(82)90056-4 Get rights and content

Abstract

We examined changes and dose-response relationships in lipid peroxidation and antioxidative protective systems in lungs of rats continuously exposed to low levels of nitrogen dioxide for 4 months. JCL:Wistar male rats (13 weeks old) were exposed continuously to 0 (control), 0.4, 1.2, and 4 ppm nitrogen dioxide for 1, 2, 4, 8, 12, and 16 weeks. Lipid peroxidation as measured by ethane exhalation in the breath of rats increased to a maximum during the first week, and then returned to near the initial level at the 4th week. Thereafter, it showed a realised tendency toward increase. Lipid peroxidation in lung homogenates, measured by the thiobarbituric acid reaction, showed a similar pattern to that of ethane exhalation, but the appearance of the maximum was later than that of ethane, and the relative peak height was lower. The activities of some antioxidative protective enzymes, glutathione peroxidase (GPx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), superoxide dismutase (SOD), and disulfide reductase (DSR) increased from the first week, reached their maximum levels at the 4th week, and then decreased gradually until the 16th week. The order of the increased ratios against the control group at the 4th week was G6PD > 6PGD > GR > SOD > DSR > GPx, and the activities were dose dependent against nitrogen dioxide levels of 0.4 to 4 ppm in each period. Glutathione S-transferase activity decreased from the 1st to the 4th week, but then recovered to the initial level from the 8th to the 16th week. The content of nonprotein sulfhydryls reached maximum levels at the 4th week, and remained elevated until the 16th week. The time course of vitamin E was similar to that of lipid peroxidation as measured by ethane exhalation rather than those of the protective enzymes. These results show that the protective ability against oxidative stress decreases with long-term exposure to nitrogen dioxide and that lipid peroxidation increases inversely with the protective systems.

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The cytoprotective roles of ascorbate and glutathione against nitrogen dioxide toxicity in human endothelial cells
1995, Toxicology
The depletion of human umbilical vein endothelial (HUVE) cell glutathione with buthionine sulfoximine or with sulfur amino acid-free medium potentiated the sub-lethal (³H-deoxyglucose release) and lethal (lactate dehydrogenase release) cytotoxicity responses of the cells to direct exposure to NO₂ over the range 2–20 ppm. When control cells, or glutathione-depleted cells, were either pre-loaded with ascorbate (intracellular ascorbate), or washed with ascorbate-containing medium just before exposure (extracellular ascorbate), the cells were fully protected from NO₂-dependent toxicity. Concomitant with these exposures, NO₂ caused dose-dependent depletions of both glutathione and ascorbate. Further, it was noted that the depletion of the intracellular ascorbate pool was accelerated in these glutathione depleted cells. Conversely, loading ascorbate into the cells significantly diminished NO₂-dependent depletion of intracellular GSH. In contrast to affecting the acute cytotoxicity response of the HUVE cells to NO₂, ascorbate supplementation of the medium of cells exposed to NO₂ at clonal density facilitated considerable protection to the colony-forming efficiency of the cells. We conclude that both ascorbate and glutathione play important protective roles in defending HUVE cells from the toxicity of NO₂ under direct exposure conditions. The results also strengthen the premise that ascorbate and glutathione co-operate in the antioxidative protection of cellular viability.
Experimental studies on tumor promotion by nitrogen dioxide
1991, Toxicology
The effects of nitrogen dioxide (NO₂) on promotion of lung tumorigenesis induced by N-bis(2-hydroxypropyl) nitrosamine (BHPN) were investigated in male Wistar rats. In a preliminary study, the highest non-effective dose of BHPN was found to be 0.5 g per kg body weight. Rats were given a single intraperitoneal injection of BHPN at a dose of 0.5 g per kg body weight or saline at 6 weeks of age, and then exposed to clean air, 0.04 ppm, 0.4 ppm or 4 ppm of NO₂ for 17 months, respectively. The incidence of pulmonary tumors in rats exposed to BHPN plus 4 ppm of NO₂ was 12.5%; the tumors were adenomas and adenocarcinomas. Adenomas were found in 4 out of 40 rats (10%) and adenocarcinomas were found in 1 out of 40 rats (2.5%). The tumor incidence in the lungs of rats kept in BHPN plus clean air and BHPN plus 0.04 ppm of NO₂ was 2.5% (1/40). In both groups adenomas were found. There was no significant difference in tumor incidence between animals exposed to BHPN plus clean air and to BHPN plus 4 ppm of NO₂. No lung tumors were found in the group of BHPN plus 0.4 ppm NO₂ and in animals exposed to NO₂ without BHPN treatment. A high incidence of alveolar cell hyperplasia was observed in the lungs of rats injected with BHPN, and the effect of NO₂ on development of alveolar cell hyperplasia was slight. On the other hand, marked bronchiolar mucosal hyperplasia was found in 17 out of 40 rats (42.5%) in the group of BHPN plus 4 ppm of NO₂, and in 1 out of 40 rats (2.5%) in each of the group exposed to clean air, 0.04 ppm or 0.4 ppm of NO₂ with BHPN treatment, respectively. The hyperplasia in lungs of rats exposed to 4 ppm of NO₂ without BHPN treatment was slighter than that in lung of rat exposed to 4 ppm of NO₂ with BHPN treatment. On the other hand, tumor incidence in the nasal cavity of rats in each of group exposed to clean air and NO₂ with BHPN treatment was 97–100%. Incidence of tumors in other organs in the groups exposed to clean air and NO₂ with and without BHPN treatment was very low, and NO₂ had no effect on tumor development in the nasal cavity and other organs whether animals were treated with BHPN or not. Tumors found in the lungs were not metastases of tumors that had developed in the nasal cavity or in the other organs. These results suggest that NO₂ can promote the development of lung tumors initiated by BHPN, the NO₂ concentration which has promoting activity is 4 ppm or higher.
Biochemical effects of combined gases of nitrogen dioxide and ozone. IV. Changes of lipid peroxidation and antioxidative protective systems in rat lungs upon life span exposure
1991, Toxicology
Lipid peroxide production, antioxidant contents and activities of antioxidative protective enzymes were examined in lungs of rats exposed to clean air (control group), 0.05 ppm O₃, 0.05 ppm O₃ + 0.04 ppm NO₂ and 0.05 ppm O₃ + 0.4 ppm NO₂ for 22 months. The results were compared with our previous data in rats exposed to 0.04 ppm NO₂, 0.4 ppm NO₂ and 4 ppm NO₂ for their life span (Sagai et al., Toxicol. Appl. Pharmacol., 73, (1984) 444–456). TBA values used as an index of lipid peroxidation in the lungs were increased maximally at 9 months, but were decreased below control values in animals exposed for 18 and 22 months. Nonprotein sulfhydryl (NPSH) contents were increased maximally at 9 months, and after 18 and 22 months were decreased significantly below values. Vitamin E (VE) contents showed a similar trend. On the other hand, enzyme activities of glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), glutathione reductase (GR), glutathione peroxidase measured by using cumene hydroperoxide (cum · OOH) substrate (GPx-cum · OOH), glutathione peroxidase measured by using H₂O₂ as a substrate (GPx-H₂O₂), glutathione S-transferase (GSH-Tase) and superoxide dismutase (SOD) did not show any significant changes during this experiment. The results show that lipid peroxidation in lungs was increased synergistically by a combination of NO₂ and O₃ at ambient levels, and that the time of maximum lipid peroxide production was shorter than with NO₂ alone. The protective ability against lipid peroxides was higher with increased lipid peroxide levels, but the inducibility was not maintained through a life span exposure to the combined gases. Additionally, two small adenomas were observed in 2 out of 18 rats in the 0.05 ppm O₃ + 0.04 ppm NO₂ group and a large adenoma was observed in 1 out of 18 animals in the 0.05 ppm + 0.4 ppm NO₂ group exposed for 22 months.
Early biochemical changes in kerosene exposed rat lungs
1991, Chemosphere
The activities of enzymes, which detoxify oxygen free radical species directly or indirectly were studied in lungs of kerosene-treated rats after 1,4,8 and 16 days of exposure. In the kerosene-exposed animals, activities of glutathione peroxidase (GP), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD) and glutathione-s-transferase (GST) were significantly increased; thiobarbituric acid (TBA) - reacting products of lipid peroxidation and reduced glutathione (GSH) content were also significantly increased in the lungs. The increase in reduced glutathione content and elevations in the activities of enzymes and products of lipid peroxidation are suggestive of oxidative stress in the lungs of kerosene exposed animals.
Biochemical effects of combined gases of nitrogen dioxide and ozone III. Synergistic effects on lipid peroxidation and antioxidative protective systems in the lungs of rats and guinea pigs
1989, Toxicology
Rats and guinea pigs were exposed continuously to 0.4 ppm NO₂, 0.4 ppm O₃ or a combination of the two gases for 2 weeks. The concentrations of lipid peroxides in lungs of rats and guinea pigs exposed to NO₂ alone or O₃ alone did not change. The lipid peroxide level of rats inhaling the combined gases also did not change. However, the level of lipid peroxides in guinea pigs exposed to a combination of the two gases was increased to 2.2 times of the control level, showing a synergistic interaction.
No increases of antioxidative protective enzyme activities and of antioxidants (such as NPSH, VE, VC) in guinea pigs exposed to NO₂, O₃ or the combined gases were found. In rats, no changes in enzyme activities and of the antioxidant contents were observed after NO₂ alone, but O₃ exposure produced slight increases of NPSH, VC, and GPx-H₂O₂. On the other hand, in rats exposed to the combined gases, marked synergistic increases of many antioxidative factors such as NPSH, VC, G6PD, GPx-cum · OOH and GPx-H₂O₂ were found.
The results show that those animals which are able to increase antioxidative protective factors in the lung following exposure to the combined gases do not respond with a significant increase in lipid peroxides. On the other hand, in animals with poor induction-ability of these factors lipid peroxides are formed. This might explain why guinea pigs were the most sentitive to the effects of the combined
Superoxide dismutase activity in lung from copper- and manganese-deficient mice exposed to ozone
1988, Toxicology Letters
Nutritional manganese (Mn) or copper (Cu) deficiency was investigated in Swiss-Webster mice exposed to ozone (O₃). Mice rendered Mn-deficient were first reared from Mn-deficient dams and then fed a Mn-deficient (1 μg/g) diet. Mice rendered Cu-deficient were fed a diet containing 0.2 μg Cu/g diet. Control mice were fed a diet containing Mn at 45 μg/g and Cu at 8 μg/g. During the last week of the experiment (week 7, post-weanling), mice in each group were exposed continuously to 1.2 ppm O₃ or filtered air for 7 days. Superoxide dismutase (SOD) activity in lung was then estimated. In mice breathing filtered air, neither lung Cu,Zn- nor Mn-SOD activity (U/g) was affected by diet. In O₃-exposed mice, howver, Mn-SOD activity was lower in the Mn-deficient group and Cu,Zn-SOD activity was lower in the Cu-deficient group. Moreover, total lung Cu,Zn-SOD activity was elevated in the Mn-deficient mice, whereas total Mn-SOD activity was elevated in the Cu-deficient mice in response to O₃. These data indicate that under normal circumstances lung Cu,Zn-SOD and Mn-SOD are not affected by Cu or Mn deficiency. However, when an oxidant stress is superimposed on the Cu- or Mn-deficient condition, Cu,Zn- and Mn-SOD activities are impaired.

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Toxicology and Applied Pharmacology

Abstract

References (31)

Isocitrate dehydrogenase-UV assay

Glutathione S-transferase: The first enzymic step in mercapturic acid formation

J. Biol. Chem.

Protein measurement with the folin phenol reagent

J. Biol. Chem.

Superoxide dismutase: An enzymic function for erythrocuprein (Hemocuprein)

J. Biol. Chem.

Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction

Anal. Biochem.

The glutathione peroxidase activity of glutathione S-transferases

Biochim. Biophys. Acta

Age-related changes in lipid peroxidation as measured by ethane, ethylene, butane and pentane in respired gases of rats

Life Sci.

Measurement of and protection from in vivo lipid peroxidation

Disulfide reduction in rats liver: Evidence for the presence of non specific nucleotide dependent disulfide reductase and glutathione-disulfide transhydrogenase activities in the high-speed supernatant fraction

Arch. Biochem. Biophys.

Vitamin E and lipid antioxidants in free-radical-initiated reactions

Rapid and micro-method for the determination of tocopherols in liver

Vitamins

Reagents for enzymatic analysis: Glutathione reductase

An enzyme protective mechanism against lipid peroxidation damage to lungs of ozone-exposed rat

Lipids

Glutathione peroxidase system and lysozome in rats exposed to ozone or nitrogen dioxide

Environ. Res.

Superoxide dismutase and pulmonary oxygen toxicity

Amer. J. Physiol.

Cited by (34)

The cytoprotective roles of ascorbate and glutathione against nitrogen dioxide toxicity in human endothelial cells

Experimental studies on tumor promotion by nitrogen dioxide

Biochemical effects of combined gases of nitrogen dioxide and ozone. IV. Changes of lipid peroxidation and antioxidative protective systems in rat lungs upon life span exposure

Early biochemical changes in kerosene exposed rat lungs

Biochemical effects of combined gases of nitrogen dioxide and ozone III. Synergistic effects on lipid peroxidation and antioxidative protective systems in the lungs of rats and guinea pigs

Superoxide dismutase activity in lung from copper- and manganese-deficient mice exposed to ozone