Toxic&gy. 73 (1992) 161-167 Elsevier Scientific Publishers Ireland
161 Ltd.
Ranking toxicity of industrial dusts by bronchoalveolar lavage fluid analysis R. Bajpai, M. Waseem, G.S.D. Gupta and J.L. Kaw Industrial Toxicology Research Centre. Post Box 80, M.G. Marg. Lucknow(Received
October
9th, 1991; accepted
March
001 (India)
15th, 1992)
Summary Female wistar rats were inoculated intratracheally with 10 mg/ml suspensions of various dusts, viz: quartz, fly ash, mica and corundum in physiological saline. Biochemical markers of bronchoalveolar lavage fluid (BALF) were analysed 8 days after the instillation of the dusts. Elevated levels of proteins, sialic acid and phospholipid contents and the activity of lactate dehydrogenase correlated well with the degree of the known fibrogenic potential of different dusts in the lungs in the following order, quartz > fly ash > mica > corundum. fl-glucuronidase activity, was however, only elevated in the quartz inoculated group of rats. It is suggested that biochemical constituents of BALF analysed shortly after the exposure to different dusts can be useful to mirror alterations in the tissue response to mineral dusts. Key words: Bronchoalveolar
lavage;
Dusts; Toxicity;
Lung
Introduction Studies aimed at evaluating the cytotoxicity of industrial and environmental dusts have usually employed histological and biochemical parameters to assess the degree of toxicity to cells exposed in vitro and monitor the development of pulmonary fibrosis in vivo, using whole lung tissue [l-4]. During recent years, analysis of the bronchoalveolar lavage fluid (BALF) constituents has proved useful to mirror an inflammatory reaction in pulmonary tissue in diseased states and to evaluate the benefits of therapeutic intervention [5-g]. Particles of quartz, mica, fly ash and corundum are generated in numerous mining operations, in the manufacture and processing of many industrial goods and the burning of fossil fuels for electricity generation. The aim of the present study was, therefore, to investigate whether alterations in the biochemical constituents of BALF can have predictive value in ranking the toxicity of different dusts.
Correspondence to: J.L. Lucknow001, India.
Kaw,
Industrial
0300-483X/92/%05.00 0 1992 Elsevier Scientific Publishers Printed and Published in Ireland
Toxicology
Ireland
Ltd.
Research
Centre,
Post
Box 80, M.G.
Marg,
162
Materials and Methods Dusts Mica, coal fly ash, quartz and corundum (emery) dusts were used. All the dusts had a majority of particles less than 5 pm in diameter. Mica of muscovite variety was obtained from the Central Glass and Ceramic Research Institute, Calcutta, India. Coal fly ash was obtained from a thermal power station, using pulverised coal as the fuel for generation of electricity. Mica and fly ash dust samples of particle size comparable to control dusts were prepared by sieving and repeated grinding of coarse dusts. Quartz and corundum were the reference dust samples and were used as positive and negative control dusts, respectively. Quartz and corundum dusts were obtained from the Institute fur Lufthygiene und Silicoseforschting, Dusseldorf, Germany. Animals Female albino rats (average age lo-12 weeks, average weight 150-180 g) of the Industrial Toxicology Research Centre, Lucknow, India animal house colony were used. The animals were housed in plastic cages with rice husks bedding (6 rats/cage) and were fed ad libitum on pellet diet supplied by M/s Hindustan Levers Ltd., Bombay, India and had free access to fresh drinking water. Preparation
of dust samples
Dusts of corundum, mica, coal fly ash and quartz were suspended in sterilised physiological saline at a concentration of 10 mg/ml. The dust suspensions were sonicated and agitated before inoculation. Inoculation
of animals
The animals were divided into five groups, 12 rats each. The animals of the first four groups were inoculated intratracheally with reference dust suspensions. For intratracheal inoculation, the rats were lightly anaesthetized. The trachea was exposed by blunt dissection and 1 ml of the dust suspension (kept constantly agitated to prevent sedimentation within the syringe) was directly injected into the trachea per OS. Injecting forcibly and using 50% air usually helped to ease dispersion of the dust into the lung alveoli. The wound was closed by a single suture. The rats of group five were similarly inoculated with I ml of physiological saline. All the animals were killed 8 days after dust inoculation. Procedure for obtaining
bronchoalveolar
lavage fluid
(BALF)
The lungs along with the trachea were surgically removed by dissecting the chest cavity after rinsing with 20 ml of sterilized physiological saline, in aliquots of 10 ml, at a hydrostatic pressure of 30 cm. The aliquots of lavage from each animal were
163
collected in precooled tubes, pooled and centrifuged at 200 x g for 10 min under cold conditions. The cell-free supernatant was separated and used for the measurement of biochemical constituents and assay of enzyme activities. Biochemical
estimations
Cell-free BALF from all the groups of animals was assayed for lavage soluble proteins [9], total sialic acid contents [IO], total phospholipid contents by estimation of inorganic phosphorous [l l] and the activities of lactate dehydrogenase 1121 and Pglucuronidase [ 131. Statistical
evaluations
Statistical significance of the data was evaluated less than 0.05 were considered significant.
t-test [ 141. P values
by Student’s
Results Lavage soluble proteins, total sialic acid and total phospholipid contents and the activity of lactate dehydrogenase were significantly elevated in quartz, mica and fly
**
1LCI-
r80
I
120 iz 3
60
100
50
& s: o &
** 80
O”
**
7 /0 0l
-20: -30 -LO? .5 02 32 C
l
zl I
0
A
B
E
ABCDE
Fig. 1. Changes in the activities of lactate dehydrogenase and &glucuronidase in the bronchoalveolar lavage fluid (BALF) from lungs of dust exposed rats. A = control: B = corundum; C = mica; D = fly ash: E = quartz. *P < 0.01, **P < 0.001.
164
q Total
Protein
I:
Total Phosphofipids
Ll
Total Sialic Acid 2.8 **
2.L & 200 g ,” 160 -z \
120
z 80
0 ABCDE
AB
CDE
Fig. 2. Changes in the bronchoalveolar lavage fluid (BALF) constituents in the different dusts. A, B, C, D & E same as in Fig. 1. *P < 0.01, **P < 0.001.
of rats exposed
to
ash exposed rats in comparison to animals inoculated with corundum or physiological saline alone (P c 0.001) see Figs. 1 and 2. The degree of increase in these parameters was of the order: quartz > fly ash > mica. There was no significant difference in the protein values in the BALF obtained from corundum and saline inoculated animals. Total sialic acid contents in the corundum treated group, although elevated compared to the control group, were statistically not significant. The total phospholipid content of corundum exposed animals was significantly more than controls (P < 0.01) but less than obtained in quartz, fly ash and mica exposed rats. A significant increase in the fl-glucuronidase activity in BALF of quartz exposed rats was observed (P < 0.001) see Fig. 1. The activity of /3-glucuronidase did not differ in the BALF obtained from fly ash, mica, corundum and physiological saline inoculated animals. Discussion In vitro studies on dust-cell interactions offer quick, economic and useful means of ranking dusts according to their potential toxicity [l-3]. Our earlier studies on the in vitro functional activity of cells isolated from BALF of animals exposed in vivo to mineral dusts revealed changes in the total number of cells, phagocytic poten-
165
tial and adherence which were in the order: quartz > mica > fly ash > corundum [15]. An assessment of the toxic potential of the dusts in terms of alterations in BALF constituents of rats during an observation period of 8 days in the present studies complemented the pulmonary fibrogenic potentials of these dusts in longer studies reported earlier by our group and others [16-201. Sialic acids are one of the major components of mucus glycoproteins [21]. Alterations in sialic acid contents following exposure to toxicant are an indication of hyperreactivity and subsequent damage to airways [22] and could be used as a marker to quantitate airways injury [23,24]. The degree of elevation of sialic acid, in present studies, corresponded closely to the known fibrogenic potential of dusts in the lung of intact animals [4,15] and was in the order: quartz > fly ash > mica > corundum. Such increases in sialic acids also paralleled increases in the protein contents of BALF of dust exposed animals. Simultaneous increases in the protein contents reflect transudation of serum proteins following injury to the alveolar capillary barrier [25]. It is apparent, therefore, that increased protein and sialic acid contents in BALF of dust exposed animals reflect the toxic potential of respective dusts in causing pulmonary injury. Increases in the LDH activity in the supernatant culture medium have been demonstrated to be an indicator of cell membrane impairment following interaction of mineral dusts with cells cultured in vitro [26-28). Unlike the homogeneity of cells used in culture studies, the increases in LDH activity in the BALF may not be due to impairment in the permeability of any specific cell type. Nevertheless, the magnitude of its increase being in the same order with different dusts both in the in vitro cultured cell supernatant and in the BALF, emphasizes the importance of the alteration of this parameter in the BALF to screen the cytotoxic potential of mineral dusts. Unlike LDH, the activity of /3-glucuronidase was not markedly altered in mica and fly ash exposed animals. Beta-glucuronidase is known to be a membrane bound lysosomal enzyme and an increase in the extracellular level of this enzyme in quartz exposed animals would imply damaged lysosomal membrane permeability [29]. A difference in this biological reactivity of quartz in contrast to mica and fly ash could possibly be found in a difference in the chemical composition/surface structure of the investigated dusts. Quartz is hexagonal crystalline, muscovite mica is monoclinic crystalline and has a lamellar structure, corrundum occurs in the form of small barrel shaped crystals and fly ash particulates are spherical [30,3 11. The degree of accumulation of phospholipids in the lungs exposed to dusts, particularly silica, has been shown to parallel the fibrogenic potential of the dusts [32,33]. An increase in the total phospholipid contents in the BALF of rats inoculated with different dusts reflects the degree of their toxic potential which might be due to an increased turnover of pulmonary surfactant as has been shown in a number of studies [34,35]. The results of the present studies show that BALF analysis carried out shortly after exposure to dusts can be used with a great degree of confidence to mirror alterations in the tissue response of lungs to mineral dusts. It also possesses a predictive value to screen toxic dusts. The value of BALF analysis, made long periods after exposure in evaluating pulmonary response to mineral dusts, has not been assessed in these investigations.
166
Acknowledgements The authors are grateful to Dr. P.K. Ray, Director, Industrial Toxicology Research Centre, Lucknow for his interest in this work. The technical assistance of Mr. K.K. Srivastava and Mr. R.P. Singh and the financial assistance by the Department of Environment, Government of India are thankfully acknowledged. References I
L. Adamis and M. Timar, Effect of various Environ. Health, 37 (1976) 301.
2 3
K.M. Reiser and J.A. Last, Silicosis and librogenesis: facts and artifact. Toxicology. 13 (1979) 51. C.D. Woodworth, B.T. Mossman and J.E. Craighead. Comparative effect of fibrous and nonfibrous minerals on cells and liposomes. Environ. Res., 27 (1982) 190. J.L. Kaw. A.K. Khanna and M. Waseem, In vitro cytotoxic and hemolytic potential of coal fly ash.
4 5 6
I
8 9 IO II 12 13 14 I5 16 17 18
19 20 21
mineral dusts on macrophages
in vitro. Int. Arch. Occup.
J. Environ. Sci. Health.. A23 (1988) 71 I. B.D. Beck, J.D. Brain and D.E. Bohannon, An in viva hamster bioassay to assess the toxicity of particles from the lungs. Toxicol. Appl. Pharmacol.. 66 (1982) 9. L.A. Dethloff. B.C. Gladen, L.B. Gilmore and G.E.R. Hook, Quantitation of cellular and extracellular constituents of pulmonary lining in rats using bronchoalveolar lavage. Effects of silica induced pulmonary inflammation. Annu. Rev. Resp. Dis., 136 (1987) 899. R.F. Henderson, Use of bronchoalveolar lavage to detect lung damage. in D.E. Gardner. J.D. Crapo and E.J. Massaro (Eds.), Toxicology of the lung. Target Organ Toxicology Series, Raven Press. New York, 1988, p. 239. SK. Sharma and J.N. Pande, Bronchoalveolar lavage: application in pulmonary diseases, Indian J. Chest Dis. & Allied Sci., 32 (1990) 157. O.H. Lowry, N. Rosebrough, A.L. Farr and R.J. Randall. Protein measurement with Folin-phenol reagent. J. Biol. Chem.. 193 (1951) 265. D. Aminoff, Methods for the quantitative estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids. Biochem. J., 81 (1961) 384. C.H. Fiske and Y. Subbarow, The calorimetric determination of phosphorus. J. Biol. Chem.. 66 (1925) 375. A. Kornberg, Lactic dehydrogenase of muscle, in S.P. Colowick, N.O. Kaplan (Eds.), Methods in Enzymology, Vol. I. New York, Academic Press, Inc.. 1955. p. 441. W.H. Fishman, B. Springer and R. Brunetti. Application of an improved glucuronidase assay method to the study of human blood. J. Biol. Chem., 173 (1948) 449. G.W. Snedecor and W.G. Cochran, Statistical methods. The Iowa State College Press. Ames. 1976. p. 100. R. Dixit, A.K. Khanna, M. Waseem, S. Dogra and J.L. Kaw, Alterations in in vitro functional activities of alveolar macrophages exposed in vivo to mineral dusts. Vet. Hum. Toxicol.. 32 (1990) 517. J.L. Kaw and S.H. Zaidti, Effect of mica dust on the lungs of rats. Exp. Pathol.. 8 (1973) 224. A.G. Heppleston, Silica and asbestos. Contrasts in tissue response. Ann. N.Y. Acad. Sci.. 330 (1979) 725. S.G. Shami, S.A. Silbaugh, F.F. Hahn, W.C. Griffith and C.H. Hobbs, Cytokinetic and morphological changes in lungs and lung associated lymph nodes of rats after inhalation of fly ash. Environ Res., 35 (1984) 373. J.L. Kaw and M. Waseem, Pulmonary response to agate dust in vivo and cytotoxic and haemolytic effects in vitro. Br. J. Ind. Med., 42 (1985) 700. J.L. Kaw, A.K. Khanna and M. Waseem, Modification of pulmonary silicotic reaction in rats exposed to coal fly ash. Exp. Pathol., 39 (1990) 49. L. Reid and J.R. Clamp, Biochemical and histochemical nomenclature of mucus. Br. Med. Bull.. 34 (1978) 5.
167
22 23 24
R.C. Boucher, Mechanisms of pollutant induced airway toxicity. P. Heulur (Eds.), Clin. Chem. Med., 2 (1982) 377. J.B. Morris, Quantitation of mucus glycoproteins removed from pulmonary lavage. Fundam. Appl. Toxicol.. 7 (1986) 207. G.S.D. Gupta, J.L. Kaw, S.H.N. Naqvi. R. Dixit and P.K. Ray. cyanate: biochemical and cytological profile of bronchoalveolar icol., 11 (1991) 159.
in S.M. Brookes. the respiratory
J.E. Lackey
and
tract of the rat by
Inhalation toxicity of methyl isolavage fluid in rats. J. Appl. Tox-
25 26
A. Hayem. Study of proteins in bronchoalveolar washings, Ann. Biol. Clin. (Paris), 40 (1982) 231. R.C. Lindenschmidt, K.E. Driscoll, M.A. Perkins, J.M. Higgins, J.K. Maurer and K.A. Behiore. The comparison of a librogenic and two non-librogenic dusts by bronchoalveolar lavage. Toxicol. Appl. Pharmacol., 102 (1990) 268.
21
P. Larivee, A. Cantin. A. Dufresne and R. Begin, Enzyme activities of lung lavage in silicosis. Lung, 168 (1990) 151. L.E. Sendelbach and H.P. Witschi, Bronchoalveolar lavage in rats and mice following beryllium sulfate inhalation. Toxicol. Appl. Pharmacol., 90 (1989) 322.
28 29 30 31 32 33 34 35
A.C. Allison, Lysosomes and the toxicity of particulate pollutants. Arch. Intern. Med., 128 (1971) 131. S. Krishnaswamy, India’s mineral resources. Oxford & IBH Publishing Co. New Delhi. 1972 p. 8. G.L. Fischer, D.P.Y. Chang and M. Brummer, Fly ash collected from electrostatic precipitators. Microcrystalline structures and the mystery of the spheres. Science. 192 (1976) 553. R.S. Richards and C.G. Curtis, Biochemical and cellular mechanisms of dust induced lung fibrosis. Environ. Health Perspect., 55, (1984) 393. L.A. Dethloff, L.B. Gilmore, B.C. Gladen. G. George, R.S. Chhabra and G.E.R. Hook, Effects of silica on the composition of pulmonary extracellular lining. Toxicol. Appl. Pharmacol., 84 (1986) 66. B.E. Miller and G.E.R. Hook, Stimulation of surfactant phospholipid biosynthesis in the lungs of rats treated with silica. Biochem. J., 253 (1988) 659. M. Schlame, C. Casals, B. Riistow, H. Rabe and D. Kunze, Molecular species of phosphatidylcholine and phosphatidylglycerol in rat pneumocytes type II. Biochem. J., 253 (1988) 209.
lung
surfactant
and
different
pools
of
161 Ltd.
Ranking toxicity of industrial dusts by bronchoalveolar lavage fluid analysis R. Bajpai, M. Waseem, G.S.D. Gupta and J.L. Kaw Industrial Toxicology Research Centre. Post Box 80, M.G. Marg. Lucknow(Received
October
9th, 1991; accepted
March
001 (India)
15th, 1992)
Summary Female wistar rats were inoculated intratracheally with 10 mg/ml suspensions of various dusts, viz: quartz, fly ash, mica and corundum in physiological saline. Biochemical markers of bronchoalveolar lavage fluid (BALF) were analysed 8 days after the instillation of the dusts. Elevated levels of proteins, sialic acid and phospholipid contents and the activity of lactate dehydrogenase correlated well with the degree of the known fibrogenic potential of different dusts in the lungs in the following order, quartz > fly ash > mica > corundum. fl-glucuronidase activity, was however, only elevated in the quartz inoculated group of rats. It is suggested that biochemical constituents of BALF analysed shortly after the exposure to different dusts can be useful to mirror alterations in the tissue response to mineral dusts. Key words: Bronchoalveolar
lavage;
Dusts; Toxicity;
Lung
Introduction Studies aimed at evaluating the cytotoxicity of industrial and environmental dusts have usually employed histological and biochemical parameters to assess the degree of toxicity to cells exposed in vitro and monitor the development of pulmonary fibrosis in vivo, using whole lung tissue [l-4]. During recent years, analysis of the bronchoalveolar lavage fluid (BALF) constituents has proved useful to mirror an inflammatory reaction in pulmonary tissue in diseased states and to evaluate the benefits of therapeutic intervention [5-g]. Particles of quartz, mica, fly ash and corundum are generated in numerous mining operations, in the manufacture and processing of many industrial goods and the burning of fossil fuels for electricity generation. The aim of the present study was, therefore, to investigate whether alterations in the biochemical constituents of BALF can have predictive value in ranking the toxicity of different dusts.
Correspondence to: J.L. Lucknow001, India.
Kaw,
Industrial
0300-483X/92/%05.00 0 1992 Elsevier Scientific Publishers Printed and Published in Ireland
Toxicology
Ireland
Ltd.
Research
Centre,
Post
Box 80, M.G.
Marg,
162
Materials and Methods Dusts Mica, coal fly ash, quartz and corundum (emery) dusts were used. All the dusts had a majority of particles less than 5 pm in diameter. Mica of muscovite variety was obtained from the Central Glass and Ceramic Research Institute, Calcutta, India. Coal fly ash was obtained from a thermal power station, using pulverised coal as the fuel for generation of electricity. Mica and fly ash dust samples of particle size comparable to control dusts were prepared by sieving and repeated grinding of coarse dusts. Quartz and corundum were the reference dust samples and were used as positive and negative control dusts, respectively. Quartz and corundum dusts were obtained from the Institute fur Lufthygiene und Silicoseforschting, Dusseldorf, Germany. Animals Female albino rats (average age lo-12 weeks, average weight 150-180 g) of the Industrial Toxicology Research Centre, Lucknow, India animal house colony were used. The animals were housed in plastic cages with rice husks bedding (6 rats/cage) and were fed ad libitum on pellet diet supplied by M/s Hindustan Levers Ltd., Bombay, India and had free access to fresh drinking water. Preparation
of dust samples
Dusts of corundum, mica, coal fly ash and quartz were suspended in sterilised physiological saline at a concentration of 10 mg/ml. The dust suspensions were sonicated and agitated before inoculation. Inoculation
of animals
The animals were divided into five groups, 12 rats each. The animals of the first four groups were inoculated intratracheally with reference dust suspensions. For intratracheal inoculation, the rats were lightly anaesthetized. The trachea was exposed by blunt dissection and 1 ml of the dust suspension (kept constantly agitated to prevent sedimentation within the syringe) was directly injected into the trachea per OS. Injecting forcibly and using 50% air usually helped to ease dispersion of the dust into the lung alveoli. The wound was closed by a single suture. The rats of group five were similarly inoculated with I ml of physiological saline. All the animals were killed 8 days after dust inoculation. Procedure for obtaining
bronchoalveolar
lavage fluid
(BALF)
The lungs along with the trachea were surgically removed by dissecting the chest cavity after rinsing with 20 ml of sterilized physiological saline, in aliquots of 10 ml, at a hydrostatic pressure of 30 cm. The aliquots of lavage from each animal were
163
collected in precooled tubes, pooled and centrifuged at 200 x g for 10 min under cold conditions. The cell-free supernatant was separated and used for the measurement of biochemical constituents and assay of enzyme activities. Biochemical
estimations
Cell-free BALF from all the groups of animals was assayed for lavage soluble proteins [9], total sialic acid contents [IO], total phospholipid contents by estimation of inorganic phosphorous [l l] and the activities of lactate dehydrogenase 1121 and Pglucuronidase [ 131. Statistical
evaluations
Statistical significance of the data was evaluated less than 0.05 were considered significant.
t-test [ 141. P values
by Student’s
Results Lavage soluble proteins, total sialic acid and total phospholipid contents and the activity of lactate dehydrogenase were significantly elevated in quartz, mica and fly
**
1LCI-
r80
I
120 iz 3
60
100
50
& s: o &
** 80
O”
**
7 /0 0l
-20: -30 -LO? .5 02 32 C
l
zl I
0
A
B
E
ABCDE
Fig. 1. Changes in the activities of lactate dehydrogenase and &glucuronidase in the bronchoalveolar lavage fluid (BALF) from lungs of dust exposed rats. A = control: B = corundum; C = mica; D = fly ash: E = quartz. *P < 0.01, **P < 0.001.
164
q Total
Protein
I:
Total Phosphofipids
Ll
Total Sialic Acid 2.8 **
2.L & 200 g ,” 160 -z \
120
z 80
0 ABCDE
AB
CDE
Fig. 2. Changes in the bronchoalveolar lavage fluid (BALF) constituents in the different dusts. A, B, C, D & E same as in Fig. 1. *P < 0.01, **P < 0.001.
of rats exposed
to
ash exposed rats in comparison to animals inoculated with corundum or physiological saline alone (P c 0.001) see Figs. 1 and 2. The degree of increase in these parameters was of the order: quartz > fly ash > mica. There was no significant difference in the protein values in the BALF obtained from corundum and saline inoculated animals. Total sialic acid contents in the corundum treated group, although elevated compared to the control group, were statistically not significant. The total phospholipid content of corundum exposed animals was significantly more than controls (P < 0.01) but less than obtained in quartz, fly ash and mica exposed rats. A significant increase in the fl-glucuronidase activity in BALF of quartz exposed rats was observed (P < 0.001) see Fig. 1. The activity of /3-glucuronidase did not differ in the BALF obtained from fly ash, mica, corundum and physiological saline inoculated animals. Discussion In vitro studies on dust-cell interactions offer quick, economic and useful means of ranking dusts according to their potential toxicity [l-3]. Our earlier studies on the in vitro functional activity of cells isolated from BALF of animals exposed in vivo to mineral dusts revealed changes in the total number of cells, phagocytic poten-
165
tial and adherence which were in the order: quartz > mica > fly ash > corundum [15]. An assessment of the toxic potential of the dusts in terms of alterations in BALF constituents of rats during an observation period of 8 days in the present studies complemented the pulmonary fibrogenic potentials of these dusts in longer studies reported earlier by our group and others [16-201. Sialic acids are one of the major components of mucus glycoproteins [21]. Alterations in sialic acid contents following exposure to toxicant are an indication of hyperreactivity and subsequent damage to airways [22] and could be used as a marker to quantitate airways injury [23,24]. The degree of elevation of sialic acid, in present studies, corresponded closely to the known fibrogenic potential of dusts in the lung of intact animals [4,15] and was in the order: quartz > fly ash > mica > corundum. Such increases in sialic acids also paralleled increases in the protein contents of BALF of dust exposed animals. Simultaneous increases in the protein contents reflect transudation of serum proteins following injury to the alveolar capillary barrier [25]. It is apparent, therefore, that increased protein and sialic acid contents in BALF of dust exposed animals reflect the toxic potential of respective dusts in causing pulmonary injury. Increases in the LDH activity in the supernatant culture medium have been demonstrated to be an indicator of cell membrane impairment following interaction of mineral dusts with cells cultured in vitro [26-28). Unlike the homogeneity of cells used in culture studies, the increases in LDH activity in the BALF may not be due to impairment in the permeability of any specific cell type. Nevertheless, the magnitude of its increase being in the same order with different dusts both in the in vitro cultured cell supernatant and in the BALF, emphasizes the importance of the alteration of this parameter in the BALF to screen the cytotoxic potential of mineral dusts. Unlike LDH, the activity of /3-glucuronidase was not markedly altered in mica and fly ash exposed animals. Beta-glucuronidase is known to be a membrane bound lysosomal enzyme and an increase in the extracellular level of this enzyme in quartz exposed animals would imply damaged lysosomal membrane permeability [29]. A difference in this biological reactivity of quartz in contrast to mica and fly ash could possibly be found in a difference in the chemical composition/surface structure of the investigated dusts. Quartz is hexagonal crystalline, muscovite mica is monoclinic crystalline and has a lamellar structure, corrundum occurs in the form of small barrel shaped crystals and fly ash particulates are spherical [30,3 11. The degree of accumulation of phospholipids in the lungs exposed to dusts, particularly silica, has been shown to parallel the fibrogenic potential of the dusts [32,33]. An increase in the total phospholipid contents in the BALF of rats inoculated with different dusts reflects the degree of their toxic potential which might be due to an increased turnover of pulmonary surfactant as has been shown in a number of studies [34,35]. The results of the present studies show that BALF analysis carried out shortly after exposure to dusts can be used with a great degree of confidence to mirror alterations in the tissue response of lungs to mineral dusts. It also possesses a predictive value to screen toxic dusts. The value of BALF analysis, made long periods after exposure in evaluating pulmonary response to mineral dusts, has not been assessed in these investigations.
166
Acknowledgements The authors are grateful to Dr. P.K. Ray, Director, Industrial Toxicology Research Centre, Lucknow for his interest in this work. The technical assistance of Mr. K.K. Srivastava and Mr. R.P. Singh and the financial assistance by the Department of Environment, Government of India are thankfully acknowledged. References I
L. Adamis and M. Timar, Effect of various Environ. Health, 37 (1976) 301.
2 3
K.M. Reiser and J.A. Last, Silicosis and librogenesis: facts and artifact. Toxicology. 13 (1979) 51. C.D. Woodworth, B.T. Mossman and J.E. Craighead. Comparative effect of fibrous and nonfibrous minerals on cells and liposomes. Environ. Res., 27 (1982) 190. J.L. Kaw. A.K. Khanna and M. Waseem, In vitro cytotoxic and hemolytic potential of coal fly ash.
4 5 6
I
8 9 IO II 12 13 14 I5 16 17 18
19 20 21
mineral dusts on macrophages
in vitro. Int. Arch. Occup.
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