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Histopathological changes induced by zinc hydroxide in rat lungs
Exp Toxic Pathol 1997; 49: 261-266 Gustav Fischer Verlag
Department of Public Health, Yamaguchi University School of Medicine, Ube, Japan of Public Health, Faculty of Medicine, School of Medicine, Kanazawa University, Kanazawa, Japan 3 Tokushima Research Institute Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan I
2 Department
Histopathological changes induced by zinc hydroxide in rat lungs H. ISHIYAMA 1, K. OGIN02 , M. SAT03, M. OGURA\ S. DAN 2 , and T. HOBARA I With 4 figures and 1 table Received: February 27,1996; Revised: May 23,1996; Accepted: June 11, 1996 Address for correspondence: HIRONOBU ISHIY AMA, Tokushima Research Institute Otsuka Pharmaceutical Co., Ltd., Tokushima 771-01, Japan. Key words: Zinc hydroxide; Zinc sulfate; Proliferating cell nuclear antigen; Alveolar macrophages; Nitro blue tetrazolium; Lung, fibrosis; Fibrosis, lung.
Summary Rat lungs were histologically examined at I, 7, 14 and 28 days following a single intratracheal instillation of zinc hydroxide (l mM). After one day of treatment, no confirmatory findings were noted. The zinc hydroxide injections were followed by an increase in proliferating cell nuclear antigen labeling indices in both alveolar macrophages and terminal bronchioles. After 7 days, the zinc hydroxide-treated lungs showed thickening of the interstitium with infiltration by alveolar macrophages, and an increase in the grade of Masson's trichrome staining (collagen fiber) in the alveolar interstitium. Thereafter, these morphological changes disappeared. The vehicle- and zinc sulfate (l mM)-exposed lungs had no abnormalities at any time point. Formazan deposits in alveolar macrophages, formed as a result of nitro blue tetrazolium reduction, were increased in zinc hydroxide-treated lung slices, suggesting that zinc hydroxide stimulated super oxide anion generation from alveolar macrophages. These results show that zinc hydroxide can induce morphological alterations of rat lungs.
Introduction Previous studies have shown that zinc hydroxide stimulated a superoxide anion release from rat alveolar macrophages (OGINO et al. 1992) and neutrophils (OGINO et al. 1994 in vitro. It is well known that superoxide metabolites induce various types of tissue damage. Metal fume fever is a syndrome consisting predominantly of respiratory symptoms following exposure to oxidized metal (mainly zinc and copper) fumes (NOEL and RUTHMAN 1988). The precise mechanism of this syndrome is not known. LAM et al. (1985) reported that zinc oxide caused functional
and morphologic changes in the lungs of guinea pigs. The precise effects of zinc hydroxide on lungs is not yet known. In the present study, we investigated whether a single intratracheal instillation of zinc hydroxide affected the lungs of rat subjects.
Material and methods Animals: Male Sprague-Dawley rats (200-250 g) were obtained from Charles River Japan (SPF) and acclimatized in our own facility for 12 days before use. Animals were allowed free access to water and standard food pellets (CRF1, sterilized by irradiation, Oriental Yeast Co., Ltd.) under conditions of controlled temperature (23 ± 2°C), humidity (60 ± lO %), lighting (lights on; 7:00 A. M. to 7:00 P. M.) and ventilated 13-15 times per hour. Forty-eight animals were randomly divided into three treatment groups (16 rats per group): those receiving zinc hydroxide, zinc sulfate, or only vehicle (distilled water). Animals were housed and treated in barrier room. Preparation of zinc hydroxide: Zinc sulfate (Wako Pure Chemical, grade 99.9 %) was dissolved in pyrogen-free distilled water and was then adjusted with NaOH, which was filtered with a Millipore filter (0.22 /lm). A white opaque liquid was obtained. The concentration of zinc hydroxide was expressed as that of zinc sulfate. Experimental protocol for treatment: Animals received an intratracheal injection of zinc hydroxide (l mM), zinc sulfate (I mM) or vehicle (distilled water), at an injection volume of 0.1 ml under ether anesthesia. At 1, 7, 14 and 28 days post-dosing, four animals per group were euthanized by ether anesthesia. The chest was opened and the lungs were embedded in paraffin wax and cut into 4/lm sections. The sections were stained with hematoxylin and eosin and Masson's trichrome staining for light microscopic evaluation. Exp Toxic Pathol49 (1997) 3-4
261
Immunohistochemistry
Results
The immunohistochemical staining of proliferating cell nuclear antigen (PCNA) was performed using the avidin-biotin immunoperoxidase method, with a Vectastain ABC kit (Dako Japan). Paraffin-embedded tissue blocks were sectioned at 4 flm, and mounted on glass slides coated with poly-L-Iysine. The sections were air-dried overnight, deparaffinized and dehydrated, and endogenous peroxidase was blocked by treatment with methyl alcohol containing 0.3 % H 20 2 for 20 min at room tempera~ure following by a brief wash in phosphate-buffered ~ahne. The sections were heated in a microwave on the hIghest power for 10 min, and after cooling the sections were rinsed in phosphate-buffered saline. The sections were reacted with 10 % normal horse serum for 30 min at room temperature, and then incubated with anti-proliferating cell nuclear antigen monoclonal antibody (Dako Japan) at 37°C for 2 hours. After being rinsed with phosphate-buffered saline twice for 5 min each time, the sections were incubated with biotinylated horse anti-mouse IgG at 37°C for 30 min. After being rinsed with phosphate-buffered saline twice for 5 min each time, the sections were incubated with avidin-biotin-peroxidase complex at 37°C for 30 min. The sections were then reacted with diaminobenzidine-hydrogen peroxidase with H 20 2, and lightly counterstained with hematoxylin. The number of PCNA-positive nuclei in 1000 mononuclear cells or 1000 terminal bronchioles were counted in randomly selected high-power fields and expressed as the PCNA labeling index.
Histopathology
Detection of superoxide anion release: Superoxide anion release was detected histologically by a modification of the nitro blue tetrazolium reduction method as previously described (ISHIYAMA et al. 1995). The lungs were removed and cut into approximately 0.5 mm slices. The slic~s were incubated with 1 mM of zinc hydroxide or 1 mM of ZInC sulfate for 20 min in Krebs-Henseleit buffer containing 0.05 % nitro blue tetrazolium (pH 7.4). The slices were fixed with 20 % buffered formalin, embedded in paraffin, cut into 4 11m sections and counterstained with kernechtrot. Stati~tics: Results obtained from the peNA labeling index were analyzed for statistical significance by Student's t-test.
The lungs of all treated animals were grossly normal. Histologically, a focal hemorrhage in one out of four rats was observed one day after treatment with zinc hydroxide (fig. IA). The vehicle- and zinc sulfate-treate~ animals had no abnormal findings (fig. IB, C). In the Immunostaining examination, PCNA-positivity in the mononuclear cells and terminal bronchioles was higher in all the zinc hydroxide-treated animals compared to the vehicle-treated animals at one day post-treatment (fig. 2 A-D), and the PCNA labeling index was significantly increased in both alveolar macrophages and terminal bronchioles (table 1). After 7 days, all the zinc hydroxidetreated animals showed thickening of the interstitium with infiltration by mononuclear cells (fig. 3B), and a few neutrophils and lymphocytes were seen in the alveolar spaces. The vehicle- and zinc sulfate-treated lungs were normal (fig. 3A and C). The grade of Masson's trichrome staining (a marker of collagen fibers) in the alveolar interstitium was higher in the zinc hydroxide-treated animals compared to that of the vehicle- or zinc sulfate-treated animals (fig. 3D, E). The PCN A labeling index of the zinc hydroxide-treated group did not differ significantly from that of the vehicle-treated group (table 1). After 14 and 28 days, all groups showed no abnormalities.
Effect of zinc hydroxide and zinc sulfate on superoxide anion release Zinc hydroxide treatment was followed by an increase in formazan depositions in the alveolar macrophages in the slice following incubation with nitro blue tetrazolium for 20 min (fig. 4B). In vehicle- and zinc sulfate-exposed lung slices, a few formazan deposits were observed (fig. 4A, C).
Table 1. peNA labeling indices in the alveolar mononuclear cells and terminal bronchioles in zinc hydroxide (lmM)-treated lungs. Treatment
Days after exposure
peNA labeling index Alveolar mononuclear cells
Terminal bronchioles
Vehicle Zinc hydroxide Vehicle Zinc hydroxide
1 1 7 7
10 ± 1 36 ± 8* 1O± 3 11 ± 1
13± 40 ± 1O± 14 ±
3 11 * 2 2
Each value represents the mean ± S. D., N = 4. * P < 0.05 vs. vehicle at the corresponding time. 262
Exp Toxic Pathol49 (1997) 3-4
Discussion In the present study, we showed that a single intratracheal injection of zinc hydroxide (1 mM) caused histopathological alterations in the rat lungs. After one day of zinc hydroxide treatment, the PCNA labeling indices of both mononuclear cells and terminal bronchioles were increased. PCNA is an auxiliary protein of DNA polymerase delta (BRA YO et al. 1987), and is synthesized in " correlation with the proliferative state of cells (KURIKI et al. 1986). PCNA-immunostaining is thought to be a reliable marker of proliferating activity. Thus, PCNA-positive mononuclear cells in the lungs are the proliferative state of alveolar macrophages. In addition, zinc hydroxide treatment was followed by an increase in the proliferation of terminal bronchioles. This phenomenon suggests centriacinal injury (LAM et al. 1985). After 7 days of exposure, zinc hydroxidetreated lungs had an increased infiltration of alveolar macrophages and fibrogenesis in the interstitium. After 14 and 28 days, no abnormalities were observed. Lung injuries such as fibrogenesis are commonly reversible with the cessation of a single exposed to various pulmonary toxins (RICHARDS et al. 1991). OGINO et al. (1992) suggested that zinc hydroxide (1 mM), a coloidal form of zinc under neutral conditions, increased superoxide anion generation from rat isolated alveolar macrophages by a receptor-mediated and Ca2+-dependent process. The release of superoxide anion from alveolar macrophages was confirmed in the present study using the nitro blue tetrazolium reduction method in the lung slice system. SMITH (1986) reported that superoxide metabolites may contribute to the production of lung injuries induced by paraquat and some environmental pollutants. It seems likely from these findings that superoxide anion generation from alveolar macrophages may be involved in the pathogenesis of zinc hydroxide-induced lung injury. However, further studies are necessary to evaluate the precise mechanism of the pathogenesis of this lung injury. A histopathological study revealed that a number of pulmonary toxins such as paraquat cause intraalveolar hemorrhage in the acute phase of the Fig. 1. Light microscopic appearances of hematoxylin and eosin staining injury (SMITH 1986). In the present study, in- (A, Zinc hydroxide; B, Vehicle; C, Zinc sulfate) after one day of treattraalveolar hemorrhage was observed in the ment. X 200.
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Exp Toxic Pathol 49 (1997) 3-4
263
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Fig. 2. PCNA-positive cells (arrows) were observed in the alveolar interstitium (A, Vehicle; B, Zinc hydroxide) and terminal bronchioles (C, Vehicle; D, Zinc hydroxide) after one day of treatment. X 200.
..
•... ;)
"
'
Fi .3. Light mi r copi app aran e fh mat xylin and in laining ( , Vehi Ie; 8, Zin hydr ide; , Zinc ulfal ) and Ma n' lrichr m taining (0, ehicle; rrow Zin hydr xid ) aft r 7 day f lrealmenl. X 2 indicate libr g ne i in lh al e lar int r litium.
zinc hydroxide-treated rats. On the other hand, this finding is well known to be caused by ether anesthesia. Thus, it was not clear whether this hemorrhage was dependent on zinc hydroxide treatment. Zinc ions have been shown to inhibit superoxide anion production by stimulated macrophages (CHV APIL et al. 1977) through its membrane stabilizing effect (BETIGER and O'DELL 1981). Therefore, it is possible that the toxic potential of zinc sulfate is very low. In the present study, the zinc sulfate-treated group showed no abnormalities at any time point. Inhalation of zinc oxide fumes in an industrial setting causes respiratory symptoms (NOEL and RUTHMAN 1988).
Our present morphological changes were similar to those seen in a previous study of zinc oxide in guinea pigs (LAM et al. 1985). Zinc hydroxide chemically resembles zinc oxide. It is not known, however, whether zinc hydroxide is distributed in the alveoli oflungs exposed to zinc fumes. In conclusion, a single intratracheal instillation of zinc hydroxide induced infiltration of alveolar macrophages and fibrogenesis in rat pulmonary interstitium.
Acknowledgement: This work was supported, in part, by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan. Exp Toxic Pathol49 (1997) 3-4
265
References BETIGER WJ, O' DELL BL: A critical physiological role of zinc in the structure and function of biomembranes. Life Sci 1981; 28: 1425-1438. BRA vo R, FRANK R, BLUNDELL PA, et al.: CyciinIPCNA is the auxiliary protein of DNA polymerase-o. Nature 1987; 326: 515-517 . CHVAPILM, STANKOVAL, BERNHARDDS, et al.: Effect of zinc on peritoneal macrophages in vitro. Infect Immun 1977; 16: 367-373. ISHIY AMA H, OGINO K, HOBARA T: Role of Kupffer cells in rat liver injury induced by diethyldithiocarbamate. Eur J Pharmacol 1995; 292: 135-14l. KURIKI P, VANDERLAAN M, DOLBEARE F, et al. : Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the cell cycle. Exp Cell Res 1986; 166: 209-219. LAM HF, CONNER MW, ROGERS AE, et al. : Functional and morphologic changes in the lungs of guinea pigs exposed to freshly generated ultrafine zinc oxide. Toxicol Appl Pharmacol 1985; 78: 29-38. NOEL NE, RUTHMAN JC: Elevated serum zinc levels in metal fume fever. Am J Emerg Med 1988; 6: 609-610. OGINO K, IZUMI Y, ISHIYAMA H, et al.: Zinc hydroxide stimulates superoxide production by rat alveolar macrophages. Biochem Biophys Res Commun 1992; 185: 1 I 15-112l. OGINO K, IZUMI Y, SEGAWA H, et al.: Zinc hydroxide induced respiratory burst in rat neutrophils. Eur J Pharmacol 1994; 270: 73-78. RICHARDS RJ, MASEK LC, BROWN RFR: Biochemical and cellular mechanisms of pulmonary fibrosis. Toxicol Pathol 1991; 19: 526-539. SMITH LL: The response of the lung to foreign compounds that produce free radicals. Ann Rev Physiol 1986; 48: 681-692.
Fig. 4. Light microscopic appearances of lung slice preparations incubated with nitro blue tetrazolium for 20 min. Arrows indicate formazan deposits in alveolar macrophages. (A) Normal control. (B) Addition of zinc hydroxide (1 mM). (C) Addition of zinc sulfate (1 mM). X 200. 266
Exp Toxic Pathol 49 (1997) 3-4
Department of Public Health, Yamaguchi University School of Medicine, Ube, Japan of Public Health, Faculty of Medicine, School of Medicine, Kanazawa University, Kanazawa, Japan 3 Tokushima Research Institute Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan I
2 Department
Histopathological changes induced by zinc hydroxide in rat lungs H. ISHIYAMA 1, K. OGIN02 , M. SAT03, M. OGURA\ S. DAN 2 , and T. HOBARA I With 4 figures and 1 table Received: February 27,1996; Revised: May 23,1996; Accepted: June 11, 1996 Address for correspondence: HIRONOBU ISHIY AMA, Tokushima Research Institute Otsuka Pharmaceutical Co., Ltd., Tokushima 771-01, Japan. Key words: Zinc hydroxide; Zinc sulfate; Proliferating cell nuclear antigen; Alveolar macrophages; Nitro blue tetrazolium; Lung, fibrosis; Fibrosis, lung.
Summary Rat lungs were histologically examined at I, 7, 14 and 28 days following a single intratracheal instillation of zinc hydroxide (l mM). After one day of treatment, no confirmatory findings were noted. The zinc hydroxide injections were followed by an increase in proliferating cell nuclear antigen labeling indices in both alveolar macrophages and terminal bronchioles. After 7 days, the zinc hydroxide-treated lungs showed thickening of the interstitium with infiltration by alveolar macrophages, and an increase in the grade of Masson's trichrome staining (collagen fiber) in the alveolar interstitium. Thereafter, these morphological changes disappeared. The vehicle- and zinc sulfate (l mM)-exposed lungs had no abnormalities at any time point. Formazan deposits in alveolar macrophages, formed as a result of nitro blue tetrazolium reduction, were increased in zinc hydroxide-treated lung slices, suggesting that zinc hydroxide stimulated super oxide anion generation from alveolar macrophages. These results show that zinc hydroxide can induce morphological alterations of rat lungs.
Introduction Previous studies have shown that zinc hydroxide stimulated a superoxide anion release from rat alveolar macrophages (OGINO et al. 1992) and neutrophils (OGINO et al. 1994 in vitro. It is well known that superoxide metabolites induce various types of tissue damage. Metal fume fever is a syndrome consisting predominantly of respiratory symptoms following exposure to oxidized metal (mainly zinc and copper) fumes (NOEL and RUTHMAN 1988). The precise mechanism of this syndrome is not known. LAM et al. (1985) reported that zinc oxide caused functional
and morphologic changes in the lungs of guinea pigs. The precise effects of zinc hydroxide on lungs is not yet known. In the present study, we investigated whether a single intratracheal instillation of zinc hydroxide affected the lungs of rat subjects.
Material and methods Animals: Male Sprague-Dawley rats (200-250 g) were obtained from Charles River Japan (SPF) and acclimatized in our own facility for 12 days before use. Animals were allowed free access to water and standard food pellets (CRF1, sterilized by irradiation, Oriental Yeast Co., Ltd.) under conditions of controlled temperature (23 ± 2°C), humidity (60 ± lO %), lighting (lights on; 7:00 A. M. to 7:00 P. M.) and ventilated 13-15 times per hour. Forty-eight animals were randomly divided into three treatment groups (16 rats per group): those receiving zinc hydroxide, zinc sulfate, or only vehicle (distilled water). Animals were housed and treated in barrier room. Preparation of zinc hydroxide: Zinc sulfate (Wako Pure Chemical, grade 99.9 %) was dissolved in pyrogen-free distilled water and was then adjusted with NaOH, which was filtered with a Millipore filter (0.22 /lm). A white opaque liquid was obtained. The concentration of zinc hydroxide was expressed as that of zinc sulfate. Experimental protocol for treatment: Animals received an intratracheal injection of zinc hydroxide (l mM), zinc sulfate (I mM) or vehicle (distilled water), at an injection volume of 0.1 ml under ether anesthesia. At 1, 7, 14 and 28 days post-dosing, four animals per group were euthanized by ether anesthesia. The chest was opened and the lungs were embedded in paraffin wax and cut into 4/lm sections. The sections were stained with hematoxylin and eosin and Masson's trichrome staining for light microscopic evaluation. Exp Toxic Pathol49 (1997) 3-4
261
Immunohistochemistry
Results
The immunohistochemical staining of proliferating cell nuclear antigen (PCNA) was performed using the avidin-biotin immunoperoxidase method, with a Vectastain ABC kit (Dako Japan). Paraffin-embedded tissue blocks were sectioned at 4 flm, and mounted on glass slides coated with poly-L-Iysine. The sections were air-dried overnight, deparaffinized and dehydrated, and endogenous peroxidase was blocked by treatment with methyl alcohol containing 0.3 % H 20 2 for 20 min at room tempera~ure following by a brief wash in phosphate-buffered ~ahne. The sections were heated in a microwave on the hIghest power for 10 min, and after cooling the sections were rinsed in phosphate-buffered saline. The sections were reacted with 10 % normal horse serum for 30 min at room temperature, and then incubated with anti-proliferating cell nuclear antigen monoclonal antibody (Dako Japan) at 37°C for 2 hours. After being rinsed with phosphate-buffered saline twice for 5 min each time, the sections were incubated with biotinylated horse anti-mouse IgG at 37°C for 30 min. After being rinsed with phosphate-buffered saline twice for 5 min each time, the sections were incubated with avidin-biotin-peroxidase complex at 37°C for 30 min. The sections were then reacted with diaminobenzidine-hydrogen peroxidase with H 20 2, and lightly counterstained with hematoxylin. The number of PCNA-positive nuclei in 1000 mononuclear cells or 1000 terminal bronchioles were counted in randomly selected high-power fields and expressed as the PCNA labeling index.
Histopathology
Detection of superoxide anion release: Superoxide anion release was detected histologically by a modification of the nitro blue tetrazolium reduction method as previously described (ISHIYAMA et al. 1995). The lungs were removed and cut into approximately 0.5 mm slices. The slic~s were incubated with 1 mM of zinc hydroxide or 1 mM of ZInC sulfate for 20 min in Krebs-Henseleit buffer containing 0.05 % nitro blue tetrazolium (pH 7.4). The slices were fixed with 20 % buffered formalin, embedded in paraffin, cut into 4 11m sections and counterstained with kernechtrot. Stati~tics: Results obtained from the peNA labeling index were analyzed for statistical significance by Student's t-test.
The lungs of all treated animals were grossly normal. Histologically, a focal hemorrhage in one out of four rats was observed one day after treatment with zinc hydroxide (fig. IA). The vehicle- and zinc sulfate-treate~ animals had no abnormal findings (fig. IB, C). In the Immunostaining examination, PCNA-positivity in the mononuclear cells and terminal bronchioles was higher in all the zinc hydroxide-treated animals compared to the vehicle-treated animals at one day post-treatment (fig. 2 A-D), and the PCNA labeling index was significantly increased in both alveolar macrophages and terminal bronchioles (table 1). After 7 days, all the zinc hydroxidetreated animals showed thickening of the interstitium with infiltration by mononuclear cells (fig. 3B), and a few neutrophils and lymphocytes were seen in the alveolar spaces. The vehicle- and zinc sulfate-treated lungs were normal (fig. 3A and C). The grade of Masson's trichrome staining (a marker of collagen fibers) in the alveolar interstitium was higher in the zinc hydroxide-treated animals compared to that of the vehicle- or zinc sulfate-treated animals (fig. 3D, E). The PCN A labeling index of the zinc hydroxide-treated group did not differ significantly from that of the vehicle-treated group (table 1). After 14 and 28 days, all groups showed no abnormalities.
Effect of zinc hydroxide and zinc sulfate on superoxide anion release Zinc hydroxide treatment was followed by an increase in formazan depositions in the alveolar macrophages in the slice following incubation with nitro blue tetrazolium for 20 min (fig. 4B). In vehicle- and zinc sulfate-exposed lung slices, a few formazan deposits were observed (fig. 4A, C).
Table 1. peNA labeling indices in the alveolar mononuclear cells and terminal bronchioles in zinc hydroxide (lmM)-treated lungs. Treatment
Days after exposure
peNA labeling index Alveolar mononuclear cells
Terminal bronchioles
Vehicle Zinc hydroxide Vehicle Zinc hydroxide
1 1 7 7
10 ± 1 36 ± 8* 1O± 3 11 ± 1
13± 40 ± 1O± 14 ±
3 11 * 2 2
Each value represents the mean ± S. D., N = 4. * P < 0.05 vs. vehicle at the corresponding time. 262
Exp Toxic Pathol49 (1997) 3-4
Discussion In the present study, we showed that a single intratracheal injection of zinc hydroxide (1 mM) caused histopathological alterations in the rat lungs. After one day of zinc hydroxide treatment, the PCNA labeling indices of both mononuclear cells and terminal bronchioles were increased. PCNA is an auxiliary protein of DNA polymerase delta (BRA YO et al. 1987), and is synthesized in " correlation with the proliferative state of cells (KURIKI et al. 1986). PCNA-immunostaining is thought to be a reliable marker of proliferating activity. Thus, PCNA-positive mononuclear cells in the lungs are the proliferative state of alveolar macrophages. In addition, zinc hydroxide treatment was followed by an increase in the proliferation of terminal bronchioles. This phenomenon suggests centriacinal injury (LAM et al. 1985). After 7 days of exposure, zinc hydroxidetreated lungs had an increased infiltration of alveolar macrophages and fibrogenesis in the interstitium. After 14 and 28 days, no abnormalities were observed. Lung injuries such as fibrogenesis are commonly reversible with the cessation of a single exposed to various pulmonary toxins (RICHARDS et al. 1991). OGINO et al. (1992) suggested that zinc hydroxide (1 mM), a coloidal form of zinc under neutral conditions, increased superoxide anion generation from rat isolated alveolar macrophages by a receptor-mediated and Ca2+-dependent process. The release of superoxide anion from alveolar macrophages was confirmed in the present study using the nitro blue tetrazolium reduction method in the lung slice system. SMITH (1986) reported that superoxide metabolites may contribute to the production of lung injuries induced by paraquat and some environmental pollutants. It seems likely from these findings that superoxide anion generation from alveolar macrophages may be involved in the pathogenesis of zinc hydroxide-induced lung injury. However, further studies are necessary to evaluate the precise mechanism of the pathogenesis of this lung injury. A histopathological study revealed that a number of pulmonary toxins such as paraquat cause intraalveolar hemorrhage in the acute phase of the Fig. 1. Light microscopic appearances of hematoxylin and eosin staining injury (SMITH 1986). In the present study, in- (A, Zinc hydroxide; B, Vehicle; C, Zinc sulfate) after one day of treattraalveolar hemorrhage was observed in the ment. X 200.
..
~
Exp Toxic Pathol 49 (1997) 3-4
263
.1.
w
,:;!
'-0 '-0
~
......
~
S.S2..
'"tl
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~
tTl
2:
N
Fig. 2. PCNA-positive cells (arrows) were observed in the alveolar interstitium (A, Vehicle; B, Zinc hydroxide) and terminal bronchioles (C, Vehicle; D, Zinc hydroxide) after one day of treatment. X 200.
..
•... ;)
"
'
Fi .3. Light mi r copi app aran e fh mat xylin and in laining ( , Vehi Ie; 8, Zin hydr ide; , Zinc ulfal ) and Ma n' lrichr m taining (0, ehicle; rrow Zin hydr xid ) aft r 7 day f lrealmenl. X 2 indicate libr g ne i in lh al e lar int r litium.
zinc hydroxide-treated rats. On the other hand, this finding is well known to be caused by ether anesthesia. Thus, it was not clear whether this hemorrhage was dependent on zinc hydroxide treatment. Zinc ions have been shown to inhibit superoxide anion production by stimulated macrophages (CHV APIL et al. 1977) through its membrane stabilizing effect (BETIGER and O'DELL 1981). Therefore, it is possible that the toxic potential of zinc sulfate is very low. In the present study, the zinc sulfate-treated group showed no abnormalities at any time point. Inhalation of zinc oxide fumes in an industrial setting causes respiratory symptoms (NOEL and RUTHMAN 1988).
Our present morphological changes were similar to those seen in a previous study of zinc oxide in guinea pigs (LAM et al. 1985). Zinc hydroxide chemically resembles zinc oxide. It is not known, however, whether zinc hydroxide is distributed in the alveoli oflungs exposed to zinc fumes. In conclusion, a single intratracheal instillation of zinc hydroxide induced infiltration of alveolar macrophages and fibrogenesis in rat pulmonary interstitium.
Acknowledgement: This work was supported, in part, by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan. Exp Toxic Pathol49 (1997) 3-4
265
References BETIGER WJ, O' DELL BL: A critical physiological role of zinc in the structure and function of biomembranes. Life Sci 1981; 28: 1425-1438. BRA vo R, FRANK R, BLUNDELL PA, et al.: CyciinIPCNA is the auxiliary protein of DNA polymerase-o. Nature 1987; 326: 515-517 . CHVAPILM, STANKOVAL, BERNHARDDS, et al.: Effect of zinc on peritoneal macrophages in vitro. Infect Immun 1977; 16: 367-373. ISHIY AMA H, OGINO K, HOBARA T: Role of Kupffer cells in rat liver injury induced by diethyldithiocarbamate. Eur J Pharmacol 1995; 292: 135-14l. KURIKI P, VANDERLAAN M, DOLBEARE F, et al. : Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the cell cycle. Exp Cell Res 1986; 166: 209-219. LAM HF, CONNER MW, ROGERS AE, et al. : Functional and morphologic changes in the lungs of guinea pigs exposed to freshly generated ultrafine zinc oxide. Toxicol Appl Pharmacol 1985; 78: 29-38. NOEL NE, RUTHMAN JC: Elevated serum zinc levels in metal fume fever. Am J Emerg Med 1988; 6: 609-610. OGINO K, IZUMI Y, ISHIYAMA H, et al.: Zinc hydroxide stimulates superoxide production by rat alveolar macrophages. Biochem Biophys Res Commun 1992; 185: 1 I 15-112l. OGINO K, IZUMI Y, SEGAWA H, et al.: Zinc hydroxide induced respiratory burst in rat neutrophils. Eur J Pharmacol 1994; 270: 73-78. RICHARDS RJ, MASEK LC, BROWN RFR: Biochemical and cellular mechanisms of pulmonary fibrosis. Toxicol Pathol 1991; 19: 526-539. SMITH LL: The response of the lung to foreign compounds that produce free radicals. Ann Rev Physiol 1986; 48: 681-692.
Fig. 4. Light microscopic appearances of lung slice preparations incubated with nitro blue tetrazolium for 20 min. Arrows indicate formazan deposits in alveolar macrophages. (A) Normal control. (B) Addition of zinc hydroxide (1 mM). (C) Addition of zinc sulfate (1 mM). X 200. 266
Exp Toxic Pathol 49 (1997) 3-4