Biochemical and morphological changes in lung tissue and isolated lung cells of rats induced by short-term nitrogen dioxide exposure

Abstract

To investigate the effects of repeated exposure to nitrogen dioxide (NO₂) on antioxidant enzymes in lung tissue and isolated lung cells, rats were continuously exposed to 20 mg/m³ NO₂ (10.6 ppm) for 4 days. The activities of glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase (GR), and glutathione peroxidase (GSHPx) were measured in the cytosolic fraction of lung tissue of both control and NO₂-exposed rats as well as in isolated alveolar macrophages (AMs) and type II cells. Qualitative and quantitative changes in AM and type II cells were studied by electron microscopy and by morphometric analyses using enzyme and immunohistochemistry. NO₂ exposure resulted in significantly increased pulmonary activities of G6PDH, GR, and GSHPx, both expressed per lung and per gram of lung weight. Morphometric data show that NO₂ exposure significantly increased the number of type II cells, predominantly in the centriacinar region, indicating proliferation of epithelium following cellular injury. Type II cells in lungs of NO₂-exposed rats had a squamous, less cuboidal appearance with more lamellar bodies compared to type II cells in lungs of control rats. Compared to control lungs, a higher number of macrophages could be isolated from NO₂-exposed lungs, while numbers of type II cells isolated from lungs of control and NO₂-exposed rats were the same. Isolated type II cells from control and NO₂-exposed rats were polymorphic, with a small number of lamellar bodies and without polarity. Isolated macro-phages were rounded and contained many filopodia. NO₂ exposure caused increases in the activities of G6PDH and GSHPx in isolated type II cells and of GSHPx in isolated macrophages, when expressed per number of cells. Macrophages and type II cells isolated from control and NO₂-exposed rats and re-exposed in vitro to NO₂, showed no differences in phagocytosis and viability features. Our results indicate that NO₂-induced increases in pulmonary antioxidant enzymes are also reflected in isolated AM and type II cells. Since these lung cells do not display a decreased sensitivities toward an in vitro NO₂ exposure, overall increase in antioxidant enzyme activities do not seem to play the most pivotal role in controlling cellular NO₂ sensitivity and oxidant defence. Combined data from biochemical, morphological, and morphometric analyses of lungs and lung cells suggest that lung cell and tissue oxidant sensitivity and defence largely depends on the cell and tissue organisation, i.e., cell numbers and morphology as well as the ratio of surface area to cytoplasmic volume.

Keywords

rat lung nitrogen dioxide toxicology in vivo in vitro

References

1 Freeman BA , Crapo JD . Biology of disease. Free radicals and tissue injury . Laboratory Investigation 1982; 47: 412-426 .

2 Samet JM , Utell MJ . The risk of nitrogen dioxide: what have we learned from epidemiological and clinical studies? Toxicology and Industrial Health 1990; 6: 247-262 .

3 Infante-Rivard C . Childhood asthma and indoor environmental factors . American Journal of Epidemiology 1993; 137: 834-844 .

4 Samet JM , et al. Nitrogen dioxide and respiratory illnesses in infants . American Review of Respiratory Disease 1993; 148: 1258-1265 .

5 Morrow PE . Toxicological data on NOx: an overview . Journal of Toxicology and Environmental Health 1984; 13: 205-227 .

6 Evans MJ , Cabral LJ , Stephens RJ , Freeman G . Renewal of alveolar epithelium in the rat following exposure to NO2 . American Journal of Pathology 1973; 70: 175-190 .

7 Evans MJ , Johnson LV , Stephens RJ , Freeman G . Renewal of the terminal bronchiolar epithelium in the rat following exposure to NO2 or 03 . LaboratoryInvestigation 1976; 35: 246-257 .

8 Chang L-Y , et al. Effects of low levels of NO2 on terminal bronchiolar cells and its relative toxicity compared to 03 . Toxicology and Applied Pharmacology 1988; 96: 451-464 .

9 Stephens RJ , Freeman G , Evans MJ . Early response of the lungs to low levels of nitrogen dioxide . Archives Environmental Health 1972; 24: 160-179 .

10.

10 Ichinose T , Suzuki AK , Tsubone H , Sagai M . Biochemical studies on strain differences of mice in the susceptibility to nitrogen dioxide . Life Sciences 1982; 31: 1963-1972 .

11.

11 Leung H-W , Morrow PE . Interaction of glutathione and ascorbic acid in guinea pig lungs exposed to nitrogen dioxide . Research Communication in Chemical Pathology and Pharmacology 1981; 31: 111-118 .

12.

12 Chow CK , Dillard CJ , Tappel AL . Glutathione peroxidase system and lysozyme in rats exposed to ozone or nitrogen dioxide . Environmental Research 1974; 7: 311-319 .

13.

13 Sagai M , Ichinose T , Oda H , Kubota K . Studies on biochemical effects of nitrogen dioxide: I. Lipid peroxidation as measured by ethane exhalation of rats exposed to nitrogen dioxide . Lipids 1981; 16: 64-67 .

14.

14 Sagai M , Ichinose T , Oda H , Kubota K . Studies on biochemical effects of nitrogen dioxide: II. Changes of the protective systems in rat lungs and of lipid peroxidation by acute exposure . Journal of Toxicology and Environmental Health 1982; 9: 153-164 .

15.

15 Kelly FJ , et al. Antioxidant kinetics in lung lavage fluid following exposure to humans to nitrogen dioxide . American Journal of Respiratory and Critical Care Medecine 1996; 154: 1700-1705 .

16.

16 Pagani P , et al. Biochemical effects of acute and subacute nitrogen dioxide exposure in rat lung and bronchoalvelar lavage fluid . Archives of Environmental Contamination and Toxicology 1994; 27: 426-430 .

17.

17 Marra M , Rombout PJA . Design and performance of an inhalation chamber for exposing laboratory animals to oxidant air pollutants . Inhalation Toxicology 1990; 2: 187-204 .

18.

18 Rombout PJA , Dormans JAMA , Marra M , Van Esch GJ . Influence of exposure regimen on nitrogen dioxide induced morphological changes in the rat lung . Environmental Research 1986; 41: 466-480 .

19.

19 Rietjens IMCM , et al. Glutathione pathway enzyme activities and the ozone sensitivity of lung cell populations derived from ozone exposed rats . Toxicology 1985; 37: 205-214 .

20.

20 Mason RJ , Williams MC , Greenleaf RD , Clements JA . Isolation and properties of type II alveolar cells from rat lung . American Review of Respiratory Disease 1977; 115: 1015-1026 .

21.

21 Alink GM , et al. A new exposure model for in vitro testing of effects of gaseous pollutants on mammalian cells by means of gas diffusion through plastic films . Chemosphere 1979; 2: 63-73 .

22.

22 Huygen C . Reaction of nitrogen dioxide with Griess-type reagents . Analytical Chemistry 1970; 42: 407-409 .

23.

23 Miller BE , et al. Quantification of silica induced type II cell hyperplasia by using alkaline phosphatase histochemistry in glycol methacrylate embedded lung . Experimental Lung Research 1987; 12: 135-148 .

24.

24 Mochitate K , Ishida K , Ohsumi T , Miura T . Longterm effects of ozone and nitrogen dioxide on the metabolism and population of alveolar macrophages . Journal of Toxicology and Environmental Health 1992; 35: 247-260 .

25.

25 Mochitate K , Takabashi Y , Ohsumi T , Miura T . Activation and increment of AM induced by nitrogen dioxide . Journal of Toxicology and Environmental Health 1986; 17: 229-239 .

26.

26 Freeman BA , Mason RJ , Williams MC , Crapo JD . Antioxidant enzyme activity in alveolar type II cells after exposure of rats to hyperoxia . Experimental Lung Research 1986; 10: 203-222 .

27.

27 Miller BE , Hook GER . Isolation and characterization of hypertrophic type II cells from the lungs of silica-treated rats . Laboratory Investigation 1988; 58: 565-575 .

28.

28 Van Bree L , Haagsman HP , Van Golde LMG , Rombout PJA . Phosphatidyl choline synthesis in isolated type II pneumocytes from ozone-exposed rats . Archives of Toxicology 1988; 61: 224-228 .

29.

29 Tu B , Wallin A , Moldeus P , Cotgreave I . The cytoprotective roles of ascorbate and glutathione against nitrogen dioxide toxicity in human endothelial cells . Toxicology 1995; 12: 125-136 .

30.

30 Barry BE , Miller FJ , Crapo JD . Effects of inhalation of 0.12 and 0.25 parts per million ozone on the proximal alveolar region of juvenile and adult rats . Laboratory Investigation 1985; 53: 692-704 .

31.

31 Chang L-Y , et al. Effects of subchronic inhalation of low concentrations of nitrogen dioxide. I. The proximal alveolar region of juvenile and adult rats . Toxicology and Applied Pharmacology 1986; 83: 46-61 .

32.

32 Bhalla DK , et al. Tracheal and bronchoalveolar permeability changes in rats inhaling oxidant atmospheres during rest or exercise . Journal of Toxicology Environmental Health 1987; 22: 417-437 .

33.

33 Slade R , Highfill JW , Hatch GE . Effects of depletion of ascorbic acid or nonprotein sulfhydryls on the acute inhalation toxicity of nitrogen dioxide, ozone, and phosgene . Inhalation Toxicology 1989; 1: 261-271 .

34.

34 Rietjens IMCM , et al. Toxicity of ozone and nitrogen dioxide to alveolar macrophages: a comparative study revealing differences in their mechanism of toxic action . Journal of Toxicology and Environmental Health 1986; 19: 555-568 .

35.

35 Prior WA , Mike M , Das B , Church DF . The mixture of aldehydes and hydrogen peroxide produced in the ozonation of dioleolyl phosphatidylcholine causes hemolysis of human red blood cells . Chemical and Biological Interactions 1991; 79: 41-42 .

36.

36 Cheek JM , et al. Ozone injury to alveolar epithelium in vitro does not reflect loss of antioxidant defenses . Toxicology and Applied Pharmacology 1994; 125: 59-69 .