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in vivo study into the short term effects of exposure to mineral fibres
Exp Toxic Pathol1996; 48: 48~86 Gustav Fischer Verlag Jena
MRC Toxicology Unit, University of Leicester, Leicester, UK
An in vitrolin vivo study into the short term effects of exposure to mineral fibres* A. CLOUTER, C. E. HOUGHTON, C. A. BOWSKILL, 1. A. HOSKINS and R. C. BROWN With 5 figures Received: December 1, 1995; Accepted: December 20,1995 Address for correspondence: Dr. ANNA CLOUTER, MRC Toxicology Unit, Hodgkin Building, University of Leicester, Leicester LEI 9HN, UK. Key words: Mineral fibres, short term effects; Fibres, mineral, short term effects.
Introduction Little is known about the mechanisms by which fibres exert their effects but it is known that different fibres are not equallypathogenic (BROWN et at. 1990)and whilethere is considerable evidence that crocidolite is the most dangerous asbestos fibre (MOSSMAN et al. 1990)calcium sulphate is believed to be without long term effect (SCHEPERS et a. 1955). The inflammatory response caused by both crocidolite and calcium sulphate has been investigated using changesin the levels of glutathione (GSH)and y-glutamyl transpeptidase (y-GT). GSH is found in high concentrations in the extracellular fluid (CANTIN 1987) and may be the first line of defence against oxidative damage, while changes in the level of y-GT activity is a marker of damage to lung epithelium (DINSDALE 1992). In addition the effects of the fibres on isolated lung epithelial type II cells were investigated on see whether these cells respond in the absence of other cell types.
Fig. 1. SEM of calciumsulphate fibres.
Material and methods Animals: Male F344 rats - 36 per group (130-150 g) (Harlan Olac). Fibres: Calciumsulphate (Franklin fibre A-30; US Gypsum) (fig. 1) and VICC crocidolite (fig. 2). In vivo studies: Rats wereexposed in a highcontainment nose-only facility to an aerosol of eithercalciumsulphate or crocidolite (l00 mg rrr': 3 weeksfor 6 hlday, 5 days/week). Eighteen animals from each group were sacrificed immediately and the remaining animals allowed 3 weekswithout
* Paper,presented at the 5th International Inhalation Symposium, Hannover, Germany, 20-24 February 1995. 484
Exp Toxic Pathol 48 (1996) 6
Fig. 2. SEM ofVICC crocidolite.
exposure. After killing the lungs were exsanguinated and removed. Lungs from 5 % of the animals were formalin fixed, sectioned and stained (H & E). Other lungs were lavaged (saline; 8 ml,0.15 M),thelavage centrifuged (300 g, 20 min) and the supernatant usedfor biochemical analyses: protein and non-protein sulphydryl (NPSH; AKERBOOM and SIES (1981)) concentrations and y-GT activity. Macrophages/alveoli werecounted in the H & E sections. In vitro studies: Type II cells from un-exposed and calciumsulphate exposed animals wereplated on ECM coated plates andstained foralkaline phosphatase activity (RICHARDS et al. 1987). Also, type II cells from untreated rats were exposed in vitro to either calcium sulphate or crocidolite (2.5, 5, 7.5, 12.5 or 25 ug/ml) overnight, the medium re-
moved and the wells washed with HEPES phosphate bufferedsaline. Thecells werestained for alkaline phosphatase activity as above.
Results Histology: No significant difference was found between groups in the number of macrophages per alveolus in exposed animals (calcium sulphate or crocidolite) (fig. 3). BAL: Exposure to either fibre did not alter the protein concentrationor y-GT activity (figs. 4 & 5). NPSH levels were increased in animals exposed to calcium sulphate
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Fig. 4. Theeffectof calcium sulphate andcrocidolite on the amounts of protein in rat lung lavage.
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Fig. 3. Theeffectof calcium sulphate andcrocidolite on the number of macrophages in the alveoli.
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Fig. 5. Effect of calcium sulphate and crocidolite on )'-GT (GGT) activity in rat lung lavage.
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Fig. 6. The effects of calcium sulphate and crocidolite on the amounts of NPSH in rat lung lavage. Exp Toxic Pathol48 (1996) 6
485
after 3 weeks of no exposure. Exposure to crocidolitedid not affect NPSH levels at either time point (fig. 4). Type II cells: The viability of typeII pneumocytes from rats pre-exposed to calcium sulphate was unchanged compared to cells from unexposed animals. Cells from control animals incubated with calcium sulphate and crocidolite showedno change in alkalinephosphatase stainingat any concentration.
Discussion In vivo: Based on the numberof alveolarmacrophages there was no inflammatory response (fig. 3), although inflammation may have been localised to the acini region. The alveoli of the crocidolite treated animalscontained a large amount of cell debris which appeared to be the remains of damaged macrophages. Protein levels in the BAL remainedunchanged after exposureto either of the fibres suggesting that the lung was not severely damagedby the treatments (CLOUTER 1993). Similarly, no difference in y-GT activity between groups was found which would show lung epithelial cell damage (DINSDALE et al. 1992) The only observed marker to increase in the calcium sulphate treated animals allowed a 3 week period post-exposure was NPSH concentration. We propose that this increase in NPSH concentration in animals treated with the nontoxic fibre is a responseto protect the lungs from oxidant damage and that the lack of this response in the crocidolite treated animals left the lung epithelium of these animals exposed.This could be due to the damagesuggested by the cell debris found. In vitro: Neitherof the fibres used had any effecton the alkaline phosphatase staining of the primary isolated typeII
486
Exp Toxic Pathol 48 (1996) 6
cells at the concentrations used. Other workers have shown toxicity following a much higher dose of crocidolite (250 ug/ml) (1. FINKLESTEIN - personal communication). It is also possiblethat the lack of an effect is due to the absence of other interactingcell types.
References AKERBOOM TPM, SIES H: Assay of glutathione, glutathione disulphide and glutathione mixed disulphide in biological samples. Methods in Enzymology 1981; 48: 373-381. BROWN RC, HOSKINS JA, MILLER K, et al.: Pathogenetic mechanisms of asbestos and other mineral fibres. Mol Aspect Med 1990; 11: 325-349. CANTIN AM, NORTH SL, HUBBARD RC, et. al.: Normal alveolar epithelial lining fluid contains highlevels of glutathione. J Appl Physiol1987; 63: 152-157. CLOUTER A: Clara cells and their reaction to bronchiolar toxins. Ph D Thesis Cardiff 1993. DINSDALE D, GREEN J, MANSON M, et al.: The ultrastructural immunolocalisation of gamma-glutamyl transpeptidase in rat lung: correlation with the histochemical demonstration of enzyme activity. Histochem J 1992; 24: 144-152. MOSSMAN BT, BIGNON J, CORN M,et al.: Asbestos: Scientific Developments and Implications for Public Policy. Science 1990; 247: 294-301. RICHARDS RJ, DAVIES N, ATKINS J, et al.: Isolation, biochemical characterisation and culture of lung type II cells of the rat. Lung 1987; 165: 143-158. SCHEPERS GWH, DURKAN TM: The biological effects of calcined gypsum dust. Arch Indust Health 1955; 12: 329-338.
MRC Toxicology Unit, University of Leicester, Leicester, UK
An in vitrolin vivo study into the short term effects of exposure to mineral fibres* A. CLOUTER, C. E. HOUGHTON, C. A. BOWSKILL, 1. A. HOSKINS and R. C. BROWN With 5 figures Received: December 1, 1995; Accepted: December 20,1995 Address for correspondence: Dr. ANNA CLOUTER, MRC Toxicology Unit, Hodgkin Building, University of Leicester, Leicester LEI 9HN, UK. Key words: Mineral fibres, short term effects; Fibres, mineral, short term effects.
Introduction Little is known about the mechanisms by which fibres exert their effects but it is known that different fibres are not equallypathogenic (BROWN et at. 1990)and whilethere is considerable evidence that crocidolite is the most dangerous asbestos fibre (MOSSMAN et al. 1990)calcium sulphate is believed to be without long term effect (SCHEPERS et a. 1955). The inflammatory response caused by both crocidolite and calcium sulphate has been investigated using changesin the levels of glutathione (GSH)and y-glutamyl transpeptidase (y-GT). GSH is found in high concentrations in the extracellular fluid (CANTIN 1987) and may be the first line of defence against oxidative damage, while changes in the level of y-GT activity is a marker of damage to lung epithelium (DINSDALE 1992). In addition the effects of the fibres on isolated lung epithelial type II cells were investigated on see whether these cells respond in the absence of other cell types.
Fig. 1. SEM of calciumsulphate fibres.
Material and methods Animals: Male F344 rats - 36 per group (130-150 g) (Harlan Olac). Fibres: Calciumsulphate (Franklin fibre A-30; US Gypsum) (fig. 1) and VICC crocidolite (fig. 2). In vivo studies: Rats wereexposed in a highcontainment nose-only facility to an aerosol of eithercalciumsulphate or crocidolite (l00 mg rrr': 3 weeksfor 6 hlday, 5 days/week). Eighteen animals from each group were sacrificed immediately and the remaining animals allowed 3 weekswithout
* Paper,presented at the 5th International Inhalation Symposium, Hannover, Germany, 20-24 February 1995. 484
Exp Toxic Pathol 48 (1996) 6
Fig. 2. SEM ofVICC crocidolite.
exposure. After killing the lungs were exsanguinated and removed. Lungs from 5 % of the animals were formalin fixed, sectioned and stained (H & E). Other lungs were lavaged (saline; 8 ml,0.15 M),thelavage centrifuged (300 g, 20 min) and the supernatant usedfor biochemical analyses: protein and non-protein sulphydryl (NPSH; AKERBOOM and SIES (1981)) concentrations and y-GT activity. Macrophages/alveoli werecounted in the H & E sections. In vitro studies: Type II cells from un-exposed and calciumsulphate exposed animals wereplated on ECM coated plates andstained foralkaline phosphatase activity (RICHARDS et al. 1987). Also, type II cells from untreated rats were exposed in vitro to either calcium sulphate or crocidolite (2.5, 5, 7.5, 12.5 or 25 ug/ml) overnight, the medium re-
moved and the wells washed with HEPES phosphate bufferedsaline. Thecells werestained for alkaline phosphatase activity as above.
Results Histology: No significant difference was found between groups in the number of macrophages per alveolus in exposed animals (calcium sulphate or crocidolite) (fig. 3). BAL: Exposure to either fibre did not alter the protein concentrationor y-GT activity (figs. 4 & 5). NPSH levels were increased in animals exposed to calcium sulphate
2.5 . . . . - - - - - - - - - - - - - - ,
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No recovery 3 weeks recovery
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Fig. 4. Theeffectof calcium sulphate andcrocidolite on the amounts of protein in rat lung lavage.
2.5 . . . - - - - - -- - - - - - - - - ,
D
...L---L....,.......L..
Calcium sulphate
Croc ldo llte
Fig. 3. Theeffectof calcium sulphate andcrocidolite on the number of macrophages in the alveoli.
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Fig. 5. Effect of calcium sulphate and crocidolite on )'-GT (GGT) activity in rat lung lavage.
Calcium lulphate
Cr ocl doli te
Fig. 6. The effects of calcium sulphate and crocidolite on the amounts of NPSH in rat lung lavage. Exp Toxic Pathol48 (1996) 6
485
after 3 weeks of no exposure. Exposure to crocidolitedid not affect NPSH levels at either time point (fig. 4). Type II cells: The viability of typeII pneumocytes from rats pre-exposed to calcium sulphate was unchanged compared to cells from unexposed animals. Cells from control animals incubated with calcium sulphate and crocidolite showedno change in alkalinephosphatase stainingat any concentration.
Discussion In vivo: Based on the numberof alveolarmacrophages there was no inflammatory response (fig. 3), although inflammation may have been localised to the acini region. The alveoli of the crocidolite treated animalscontained a large amount of cell debris which appeared to be the remains of damaged macrophages. Protein levels in the BAL remainedunchanged after exposureto either of the fibres suggesting that the lung was not severely damagedby the treatments (CLOUTER 1993). Similarly, no difference in y-GT activity between groups was found which would show lung epithelial cell damage (DINSDALE et al. 1992) The only observed marker to increase in the calcium sulphate treated animals allowed a 3 week period post-exposure was NPSH concentration. We propose that this increase in NPSH concentration in animals treated with the nontoxic fibre is a responseto protect the lungs from oxidant damage and that the lack of this response in the crocidolite treated animals left the lung epithelium of these animals exposed.This could be due to the damagesuggested by the cell debris found. In vitro: Neitherof the fibres used had any effecton the alkaline phosphatase staining of the primary isolated typeII
486
Exp Toxic Pathol 48 (1996) 6
cells at the concentrations used. Other workers have shown toxicity following a much higher dose of crocidolite (250 ug/ml) (1. FINKLESTEIN - personal communication). It is also possiblethat the lack of an effect is due to the absence of other interactingcell types.
References AKERBOOM TPM, SIES H: Assay of glutathione, glutathione disulphide and glutathione mixed disulphide in biological samples. Methods in Enzymology 1981; 48: 373-381. BROWN RC, HOSKINS JA, MILLER K, et al.: Pathogenetic mechanisms of asbestos and other mineral fibres. Mol Aspect Med 1990; 11: 325-349. CANTIN AM, NORTH SL, HUBBARD RC, et. al.: Normal alveolar epithelial lining fluid contains highlevels of glutathione. J Appl Physiol1987; 63: 152-157. CLOUTER A: Clara cells and their reaction to bronchiolar toxins. Ph D Thesis Cardiff 1993. DINSDALE D, GREEN J, MANSON M, et al.: The ultrastructural immunolocalisation of gamma-glutamyl transpeptidase in rat lung: correlation with the histochemical demonstration of enzyme activity. Histochem J 1992; 24: 144-152. MOSSMAN BT, BIGNON J, CORN M,et al.: Asbestos: Scientific Developments and Implications for Public Policy. Science 1990; 247: 294-301. RICHARDS RJ, DAVIES N, ATKINS J, et al.: Isolation, biochemical characterisation and culture of lung type II cells of the rat. Lung 1987; 165: 143-158. SCHEPERS GWH, DURKAN TM: The biological effects of calcined gypsum dust. Arch Indust Health 1955; 12: 329-338.