ENVIRONMENTAL
RESEARCH
Wood
20, 455-464 (1979)
Dust Toxicity:
In Vivo and in Vitro Studies
J. WYNNE BHATTACHARJEE,R. S.H. Indltstrial
Toxicology
K. SDOGRA,
M.M.
LAL, AND
ZAIDI
Research Centre. Mahatma Lucknorr~ 226001 (U.P.).
Gandhi Indirr
Marg.
Pmt
Bm
80.
Received December 18. 1978 The relationship between ifr vitro cytotoxicity, hemolytic activity, and in rii~ fibrogenicity of wood dusts. Dalbergia sissoo (Sheesham) and Mangifera indica (Mango), were examined. It was observed that hemolytic activity may be indicative of the degree of acute toxicity and macrophage cytotoxicity of the fibrogenicity of the dusts. It has been suggested that these two parameters may be useful for assessing a wood dust for its acute toxicity and fibrogenicity.
INTRODUCTION
In northern parts of India large areas are covered with forests and the timber industry forms one of the important occupations, employing over 30,000 people. There have been several reports of lung damage attributed to inhaled wood dust in timber workers (Cohen et al., 1967; Michaels, 1967: Sossman et al., 1969). Sheesham (Dalbergia sissoo) and Mango (Mangifera in&a) are the commonest woods used for furniture and building materials but no clinical reports of their harmful effects are available. The present report deals with the study of their toxicity, by using (a) hemolytic activity, (b) macrophage cytotoxicity, and (c) animal experiments. MATERIALS AND METHODS Preparation of dust samples. Indian varieties of coarse Sheesham and Mango
wood dusts were collected from saw mills at Lucknow, India. They were crushed to tine size in an electrically operated agate pestle and mortar and sieved through a 400-mesh (B.S.) sieve. The sieved samples were finally reduced to below 30 wrn by hand grinding and the size was assessed by optical microscopy using eyepiece graticule as described by Zaidi (1969). The particle size distribution of the sample is given in Table 1. The coarse fibers of chrysotile were obtained from Brahmanapalli Mine, Cudapah, India. They were crushed in a pinmill, blended in a Waring blender, and then ground to fine size with an agate pestle and mortar. The fine fibers were prepared as for the wood dusts. The particle size distribution of the sample is given in Table 1. Chemical analysis. The chemical analysis was done by the methods described in the A.O.A.C. (1977) and the results are given in Table 2. Experiment 1 Animal studies.
Twenty-four random-bred male guinea pigs from the Industrial Toxicology Research Centre animal house colony, average weight 300 g, were divided into three groups. 455 0013-9351/79/060455-10$02.00/O Copyright All rights
‘a 1979 by Academic Preys. Inc of reproduction in any form wserved
456
BHATTACHARJEE TABLE PARTICLE
SIZE
E-I-AL.
1
DISTRIBUTION
OF DUST
Percentage distribution
Size range km)
Sheesham
Below 4.5 4.5-6.4 6.4-9.1 9.1-12.8 12.8- 18.1 18.1-25.6 25.6-36.2
Mango
Chrysotile
28.10 21.90 21.22 17.46 7.22 3.18 0.92
27.53 24.96 22.78 16.45 5.99 1.96 0.43
26.77 24.61 24.42 15.82 6.50 I .88 -
100.00
100.00
100.00
The suspension of finely ground and autoclaved wood dust was prepared in sterile normal saline using the Potter-Elvehjem type all glass homogenizer to obtain a homogenous dispersion. One group of animals was injected intratracheally with Mango wood dust and the other with Sheesham dust (Zaidi, 1969). Each animal received 75 mg of dust in 1.5 ml of sterile normal saline. The third group received 1.5 ml sterile normal saline only and served as control. Animals were maintained on Hindustan lever pellets, green leafy vegetables, and water ad libitum. Four animals from each group were sacrificed at 60 and 90 days postinoculation. The lungs were gently inflated in situ to normal size with 10 ml of form01 saline and then removed from the thorax. After preliminary fixation in fresh fixative, blocks were selected along the long axes of both lungs at the levels of the hilum to include the maximum area of the lung. Blocks were embedded in paraffin and sections cut at 5 pm. Multiple sections, at least ten from each block, were stained with hematoxylin and eosin and by the Gordon and Sweets method for reticulin (1936). Pathological grading of fibrosis was assessed according to the method of Belt and King (1945) as described by Zaidi (1969) TABLE CHEMICAL
Constituents (%) Moisture Ash Acids Phenols Holocelluloses Lignin Steam volatiles Tannins Nontannins Total solubles Silica dioxide (Si02)
ANALYSIS
OF SHEESHAM
2 AND
MANGO
WOOD
DUSTS
Sheesham
Mango
7.73 1.71 5.75 10.16 58.26 20.23 0.34 1.67 3.55 5.22
7.47 1.24 0.41 3.60 67.86 29.18 00.092 4.03 2.33 6.36
1.40
1.10
WOOD
DUST
TOXICITY
457
Experiment II Hemolytic activity. Sheep red cells were collected in citrate solution, centrifuged, and plasma was removed. Subsequently the cells were washed three times in normal saline. After the last washing the cells were spun for 10 min at 1500 rpm and a 1% suspension was prepared in veronal-buffered saline (VBS), pH 7.4 (Harington et al., 1971). Varying amounts of Sheesham and Mango dusts (5, 10, 20, and 40 mg) of less than 30-pm particle size were suspended in 6 ml VBS, and to this 4 ml of 1% red cell suspension was added. The following controls were prepared: (a) blank correction containing wood dust in VBS; (b) positive hemolysis control containing quartz; (c) negative red cell control. The tubes were incubated at 37°C for 2 hr with continuous gentle shaking. After incubation the tubes were centrifuged and the supernatant was read at 540 nm in the Klett Summerson photoelectric calorimeter. In order to obtain 100% hemolysis standard, 4 ml of the 1% red cell suspension was centrifuged and the cell deposit lysed with 10 ml of distilled water. The readings of the wood dust suspended in VBS alone were subtracted from the readings of the dust plus red cells in VBS as a blank correction. The percentage of hemolysis was calculated by taking the average of three or more estimations. The effect of polyvinylpyridine N-oxide (P 204) and the trisodium salt of ethylenediaminetetraacetic acid (Na,EDTA) on hemolysis was examined to see if the mechanism of hemolysis resembled that of silica (Stalder and StGber, 1965) or chrysotile (Macnab and Harington, 1%7), respectively. P 204. Four milliliters of 1% red cell suspension was centrifuged for 10 min and the deposit resuspended in 10 ml of VBS containing 0.1 ml of P 204. Forty milligrams of wood dust was added and the test for hemolytic activity carried out as above. Negative controls of red cells and wood dust separately suspended in 10 ml VBS containing 1% P 204 were included. Na,EDTA. A solution of 25 pM/lld Na,EDTA of pH 7.4 was prepared by mixing thoroughly equal volumes of 50 p&i/ml NaOH and 50 pM/lld Na,EDTA. The test was carried out as above. Experiment III Macrophage cytotoxicity. Guinea pigs weighing 300 g were stunned by a blow on the head and then exsanguinated by cutting the jugular veins. The alveolar macrophages were harvested by the method of Myrvik et al (1961) in Hanks’ basal salt solution (HBSS). The harvested cells were pooled and leighton tubes inoculated with lo6 macrophages. After 40 min incubation at 37°C allowing the macrophages to adhere to the cover slips, the supernatant was removed and the monolayers were washed twice with HBSS. The wood dusts (particle size below 30 pm) were suspended in HBSS to give a concentration of 0.1 mg/ml. One milliliter of this finely dispersed suspension was added to each monolayer, five monolayers were inoculated with mango wood dust, and five monolayers with Sheesham wood dust. After 3 hr incubation at 37°C the monolayers were stained with erythrosin B and examined microscopically to determine the percentage of the cells stained (Munch et al., 1971). Five control tubes containing cells only were similarly stained. A positive control consisting of five monolayers inoculated
458
BHATTACHARJEE
ET AL.
with chrysotile dust was included for comparison with the wood dust. Each experiment was repeated three times. The results of Experiments II and III were analyzed by Student’s t test as described by Fisher (1950). RESULTS Experiment I Macroscopic
changes. Animals injected with Mango dust revealed at 60 days dirty pink patches of congestion in the lung but by 90 days only a few persisted. The Sheesham-inoculated animals also showed similar but larger areas of congestion. Microscopic changes. At 60 days the lungs of Mango dust-inoculated animals presented two types of reaction, one confined to the parenchyma and the other to the alveoli of respiratory bronchioles. The parenchymal reaction was comprised of dust-laden macrophages and giant cells which did not show any cytotoxicity and took up normal H and E staining. These lesions with reticulin stain showed fine reticulination of grade I (Fig. 1). The lesions in the walls of the bronchioles and alveolar ducts were comprised of aggregrations of macrophages and fibroblastic proliferation resulting in thickening and grade II fibrosis. Some of these areas also developed adenomatosis and lymphocytic infiltration. Areas of centrilobular emphysema also developed. By 90 days the parenchymal reaction had started to resolve as giant cells began to disintegrate. The fibrosis around the bronchioles and emphysema persisted but the lymphoid aggregations disappeared. Lungs from Sheesham-inoculated animals presented a diffuse reaction confined to alveoli abutting onto the respiratory bronchioles and alveolar ducts. The reaction was comprised of foreign body giant cells and cellular debris. Many giant cells showed degenerative changes in the form of vacuolated nuclei which had progressed in places to ghost forms. Adjoining areas developed centrilobular emphysema. On reticulin staining, grade I fibrosis was observed in these areas (Fig. 2). The reaction had resolved in most of the lung by 90 days but small areas of parenchymal granulomatous reaction and emphysema, particularly in the vicinity of the bronchioles, persisted. These areas developed grade I fibrosis. Numerous conspicuous lymphoid aggregrations were scattered throughout the parenchyma. Experiment
II
The hemolysis produced by different amounts of Mango and Sheesham dust is shown in Fig. 3. Forty milligrams of Sheesham gave 55.2% hemolysis which was consistently reproducible and highly significant (P < 0.0005). However 20 mg of dust also exhibited significant hemolytic activity (P < 0.005) although decrease in the amount of dust was accompanied by a rapid drop in the hemolytic activity. With smaller amounts of dust the degree of hemolysis observed was rather variable. Mango dust was only weakly hemolytic and 40 mg of dust gave 4.5% hemolysis which was statistically significant (p < 0.005) but with 20 and 10 mg the hemolysis was only just detectable. There was no hemolysis in the red cell control. The antisilicotic compound P 204 which inhibits hemolysis of quartz and EDTA which inhibits the hemolysis of chrysotile had no effect on Sheesham hemolysis.
WOOD
DUST
TOXICITY
459
460
BHATTACHARJEE
ET AL.
WOOD
DUST
/r
461
TOXICITY
QUARTZ
L.2 DALBERGIA
SlSSOO
(Sheesham)
MANGIFERA
INDICA
(Mow)
A
-
5
^-
IO
WEIGHT
FIG.
----
20
- 4
30
40
OF WOOD
3. Percentage
DUST
hemolysis
(m0)
and weight
of wood
dust
Experiment III Only 14.1 % of the cells of the monolayer to which no dust was added took up erythrosin stain after 3 hr incubation. Addition of the wood dusts caused an increase in the uptake of erythrosin; after 3 hr 34.2% (p < 0.0005) of the cells exposed to Sheesham took up the stain as compared with 31% (Z’ < 0.005) of the cells exposed to the Mango dust. The cell monolayers exposed to chrysotile showed a high degree of erythrosin uptake as 5 1.5% (P < 0.0005) of the cells were stained. When the uptake of erythrosin by macrophages exposed to the wood dusts was compared with the uptake of dye by the cells exposed to chrysotile a stastically significant difference was found (for Sheesham dust, P < 0.0005, and for Mango dust, P < 0.005). Therefore both the wood dusts show a significantly lesser degree of toxicity than chrysotile (Table 3). DISCUSSION
A variety of respiratory disorders and pulmonary changes have been attributed to the inhalation of wood dusts (Cohen et al., 1967; Michaels, 1967; Sossman et al., 1969: Cornea et al., 1978). It has been suggested that even nontoxic wood particles inhaled over long periods may cause fibrosis of the lung (Michaels, 1967). In addition to mechanical irritation and the foreign body reaction wood dust can cause toxic or allergic damage to the lung tissue. Based on chemical and biological studies, Hanslian and Kadlec (1964) classified woods into three groups, relative nontoxic, relative toxic, and strongly allergic. Some exotic woods appear to be associated with greater occupational hazards (Kadlec and Hanslian, 1972). Most of the available literature, however, deals with the allergic damage caused TABLE UPTAKE
OF ERYTHROSIN
PIG ALVEOLAR
3 MACROPHAGES
EXPOSED
Percentage cells stained erythrosin in 3 hr
Dust Sheesham Mango Asbestos (control) Control (blank) ” 0.1 mg/l.O
BY GUINEA
X IO” ceils.
34.1 i- 2.6 31.0 IT 7.1 51.5 t 6.0 14.1
i
2.5
TO WOOD
with _-~--.--_
DusV
462
BHATTACHARJEE
ETAL.
by woods and only a few studies have been made on the lung lesions after inhalation. In our experiments pulmonary changes in response to intratracheal injection of Sheesham and Mango wood dusts showed disintegration of giant cells by 90 days. The dust particles enclosed in a basophilic mass lay either free in the alveolar lumen or in the macrophages. These findings and the centrilobular emphysema and fibrosis of simple pneumoconiosis produced by the wood dust are in agreement with the observations in man (Michaels, 1967) and experimental animals (Cornea et al., 1978). The present studies therefore, provide an experimental model for the study of woodworkers pulmonary disease. In view of these findings, therefore, the prevalence of pneumoconiosis among workers employed in occupations where wood or its derivatives are handled needs to be reassessed. Measurement of the hemolytic activity of a variety of mineral dusts like silica and asbestos has provided a very simple means of investigating their potential biological activity (Stalder and Stober, 1965; Macnab and Harington, 1967; Kaw et al., 1973; Kaw and Zaidi, 1975). There is, however, hardly any information on the application of this test to organic dusts. In our experiments Sheesham showed marked and consistent hemolytic activity which may be correlated with the extensive and diffuse tissue reaction and rapid onset of degenerative changes observed in the lungs. In contrast the Mango wood dust which was only poorly hemolytic had limited and delayed onset of the acute reaction. It is suggested therefore that hemolysis may be a possible index for detecting early pulmonary tissue reactions of organic dusts. As both the dusts were able to induce thin reticulin fibrosis (simple pneumoconiosis), hemolysis is not a measure of their tibrogenic activity. In the present studies P 204 did not inhibit the hemolysis caused by Sheesham. The hemolytic activity of Sheesham dust was therefore not dependent on its silica content as reported for quartz by Stalder and Stober (1965). Similarly EDTA did not inhibit the hemolytic activity suggesting that the mechanism did not resemble that of chrysotile. Morgan et al. (1977), investigating the hemolytic activity of some fibrous amphiboles, have suggested a correlation between surface area of the fiber and hemolysis. A similar factor may be operative with fibrogenic wood dusts. Alternately some organic compounds, like acids or phenols leaching out of Sheesham wood, may be important determinants of the hemolysis and acute tissue response in our experiments. These compounds were not present in significant amounts in mango dust which showed minimal hemolysis. Several studies have shown that the tibrogenicity of mineral and polymer dusts can be correlated with macrophage cytotoxicity in vitro (Marks and Mason, 1956; Conning et al., 1970). We have therefore used the uptake of erythrosin as a simple method for measuring macrophage damage caused by the dusts. Both Sheesham and Mango wood dusts gave intermediate levels of cytotoxicity when compared with the marked cytotoxicity produced by chrysotile. This suggests that macrophage cytotoxicity may be indicative of the fibrogenic potential of wood dusts as both Sheesham and Mango were tibrogenic. Certain hard woods and old root woods are known to contain significant levels of silica and have been suspected to induce fibrosis in exposed workers (Hanslian and Kadlec, 1964). The present experiments indicate that the silica content is not
WOOD
DUST
TOXICITY
463
critical in the fibrogenicity of Mango and Sheesham dust, because of its low content in these dusts. From the present work it appears that the hemolytic activity of the organic dusts studied may provide a useful screening test for acute toxicity and macrophage cvtotoxicity for fibrogenicity. ACKNOWLEDGMENTS This investigation was supported by a grant from the United States Government. Department of Health, Education and Welfare, Public Health Services of Public Law 83-480, to which the authors are grateful. The authors are grateful to Professor H. W. Schlipkotter. Director. Medizinisches Institute fur Lufthygiene und Silikoseforschung, Dusseldorf, for the generous supply of the compounds polyvinylpyridine N-oxide, and to Dr. M. Swaley, Incharge. Chemistry of Forest Products. Forest Research Institute Dehradun, who very kindly carried out the wood analysis. and to Mr. K. C. Saxena for silica estimation.
REFERENCES A.O.A.C. (1977). “Official Methods of Analysis of the Association of Official Analytical Chemists.” I lth ed. William Horwitz. Washington, D.C. Belt. T. H., and King, E. J. (1945). Chronic pulmonary disease in South Wales coal miners. III. Experimental studies. D. Tissue reactions produced experimentally by selected dusts from South Wales coalminers. In “Spec. Rep. Ser. Med. Res. Coun.” No. 250, pp. 29-68. London. Cohen, H. I., Merigan, T. C., Kosek, J. C.. and Eldridge, F. (1967). Sequosis: A granulomatons pneumonitis associated with red wood saw dust inhalation. Atnrr. J. Med. 43, 785-794. Conning. D. M., Hays, M. J., Styles, .I. A., and Nicholas, J. A. (1970). Comparison between in rirvo toxicity of dusts of certain polymers and minerals and their fibrogenicity. Itr “Inhaled particles. III” (W. H. Walton, ed.), Vol. I. pp. 499-506. Unwin, Surrey. Cornea, G., Elmekki. L., Elgharbi, B.. Benkheder, A.. and Silvia, G. (1978). Une Nouvelle Pneumoconiose Vegetable, Le Poumon De Neefa. “ln 5th International Conference on Pneumoconiosis,” Oct. 29 to Nov. 3, 1978. Caracas, Venezuela. Fisher, R. A. (1950). “Statistical Methods for Research Workers,” 1 lth ed. Oliver & Boyd, London. Gordon, H., and Sweets, H. H., Jr. (1936). A simple method for the silver impregnation of reticulin. Amer. J. Pnthol. 12, 545-552. Hanslian, L.. and Kadlec. K. (1964). Drevo a jeho prach (Timber and timber dust). Prac. Lek. 16, 276-282. Hanshan. L., and Kadlec. K. (1964). Drevo 2 Hlediska Hygienic Rheo (1V)Biol. C’eirtXv Pruch Drr~~r 19, 45 I-452. Harington, J. S., Miller. K., and Macnab, G. (1971). Haemolysis by asbestosE,ri?ro,t. RVS. 4, 95- I 17. Kadlec. K.. and Hanslian, L. (1972). Encyclopaedia of Occupational Health and Safety, Vol. II. L-Z. International Labour Office. Geneva, pp. 1507-1511. Kaw. J. L.. Gupta, G. S. D.. Narang, S.. and Zaidi, S. H. (1973). Haemolytic activity of mica dust and its relation to polyvinylpyridine N-oxide. Exp. Prrthal. 8, 349-353. Kaw, J. L., and Zaidi. S. H. (1975). In i.itro studies on the cytotoxic reaction of different varieties of asbestos dust on macrophages. Acta Plwrmac~ol. To.xicol. 36, X-235. Macnab. G.. and Harington, J. S. (1967). Haemolytic activity of asbestos and other mineral dusts. h’utrrrc (London) 214, 522-523. Marks, J.. and Mason, M. A. (1956). A quantitative technique for studying the effect of dust on phagocytic cells iti \‘itro. Brit. J. Id. Med. 13, 192- 195. Michaels, L. (1967). Lung changes in wood workers. Canad. Med. Asso~. J. 96, 1150-1155. Morgan, A.. Holmes, A.. and Talbot. R. J. (1977). The haemolytic activity of some fibrous amphiboles and its relation to their specific surface areas. Amer. Occup. h’yg. 20. 39-48. Muncn. R.. Beck, E. G., ana Manojlovic, N. ( lY71). Unrerschungen uber die Reaktion iii i,itrrj
464
BHATTACHARJEE
ET Al.
gezuchteter Zellen nach Kurzeitiger. Inkubation Zellen nach Kurzzeitiger. Inkubation mit SiOzstaub. Beitr. Silikose Forsch. 23, 175-205. Myrvik, Q. N., Leake, E. S., and Far&, B. (1961). Studies on pulmonary alveolar macrophages from normal rabbit. A technique to procure them in a high state of purity. J. Immunof. 86, 128 132 Sossman, A. J., Donald, P., Schlueter, J., Fink, N., and Barboriak, J. (1969). Hypersensitivity to wood dust. N. Engl. J. Med. 281, 977-980. Staider, K., and Stober, W. (1965). Haemolytic activity of suspensions of different silica modifications and inert dusts. Nature (London) 207, 874-875. Zaidi, S. H. (1969). “Experimental Pneumoconiosis.” John Hopkins Press, Baltimore.
RESEARCH
Wood
20, 455-464 (1979)
Dust Toxicity:
In Vivo and in Vitro Studies
J. WYNNE BHATTACHARJEE,R. S.H. Indltstrial
Toxicology
K. SDOGRA,
M.M.
LAL, AND
ZAIDI
Research Centre. Mahatma Lucknorr~ 226001 (U.P.).
Gandhi Indirr
Marg.
Pmt
Bm
80.
Received December 18. 1978 The relationship between ifr vitro cytotoxicity, hemolytic activity, and in rii~ fibrogenicity of wood dusts. Dalbergia sissoo (Sheesham) and Mangifera indica (Mango), were examined. It was observed that hemolytic activity may be indicative of the degree of acute toxicity and macrophage cytotoxicity of the fibrogenicity of the dusts. It has been suggested that these two parameters may be useful for assessing a wood dust for its acute toxicity and fibrogenicity.
INTRODUCTION
In northern parts of India large areas are covered with forests and the timber industry forms one of the important occupations, employing over 30,000 people. There have been several reports of lung damage attributed to inhaled wood dust in timber workers (Cohen et al., 1967; Michaels, 1967: Sossman et al., 1969). Sheesham (Dalbergia sissoo) and Mango (Mangifera in&a) are the commonest woods used for furniture and building materials but no clinical reports of their harmful effects are available. The present report deals with the study of their toxicity, by using (a) hemolytic activity, (b) macrophage cytotoxicity, and (c) animal experiments. MATERIALS AND METHODS Preparation of dust samples. Indian varieties of coarse Sheesham and Mango
wood dusts were collected from saw mills at Lucknow, India. They were crushed to tine size in an electrically operated agate pestle and mortar and sieved through a 400-mesh (B.S.) sieve. The sieved samples were finally reduced to below 30 wrn by hand grinding and the size was assessed by optical microscopy using eyepiece graticule as described by Zaidi (1969). The particle size distribution of the sample is given in Table 1. The coarse fibers of chrysotile were obtained from Brahmanapalli Mine, Cudapah, India. They were crushed in a pinmill, blended in a Waring blender, and then ground to fine size with an agate pestle and mortar. The fine fibers were prepared as for the wood dusts. The particle size distribution of the sample is given in Table 1. Chemical analysis. The chemical analysis was done by the methods described in the A.O.A.C. (1977) and the results are given in Table 2. Experiment 1 Animal studies.
Twenty-four random-bred male guinea pigs from the Industrial Toxicology Research Centre animal house colony, average weight 300 g, were divided into three groups. 455 0013-9351/79/060455-10$02.00/O Copyright All rights
‘a 1979 by Academic Preys. Inc of reproduction in any form wserved
456
BHATTACHARJEE TABLE PARTICLE
SIZE
E-I-AL.
1
DISTRIBUTION
OF DUST
Percentage distribution
Size range km)
Sheesham
Below 4.5 4.5-6.4 6.4-9.1 9.1-12.8 12.8- 18.1 18.1-25.6 25.6-36.2
Mango
Chrysotile
28.10 21.90 21.22 17.46 7.22 3.18 0.92
27.53 24.96 22.78 16.45 5.99 1.96 0.43
26.77 24.61 24.42 15.82 6.50 I .88 -
100.00
100.00
100.00
The suspension of finely ground and autoclaved wood dust was prepared in sterile normal saline using the Potter-Elvehjem type all glass homogenizer to obtain a homogenous dispersion. One group of animals was injected intratracheally with Mango wood dust and the other with Sheesham dust (Zaidi, 1969). Each animal received 75 mg of dust in 1.5 ml of sterile normal saline. The third group received 1.5 ml sterile normal saline only and served as control. Animals were maintained on Hindustan lever pellets, green leafy vegetables, and water ad libitum. Four animals from each group were sacrificed at 60 and 90 days postinoculation. The lungs were gently inflated in situ to normal size with 10 ml of form01 saline and then removed from the thorax. After preliminary fixation in fresh fixative, blocks were selected along the long axes of both lungs at the levels of the hilum to include the maximum area of the lung. Blocks were embedded in paraffin and sections cut at 5 pm. Multiple sections, at least ten from each block, were stained with hematoxylin and eosin and by the Gordon and Sweets method for reticulin (1936). Pathological grading of fibrosis was assessed according to the method of Belt and King (1945) as described by Zaidi (1969) TABLE CHEMICAL
Constituents (%) Moisture Ash Acids Phenols Holocelluloses Lignin Steam volatiles Tannins Nontannins Total solubles Silica dioxide (Si02)
ANALYSIS
OF SHEESHAM
2 AND
MANGO
WOOD
DUSTS
Sheesham
Mango
7.73 1.71 5.75 10.16 58.26 20.23 0.34 1.67 3.55 5.22
7.47 1.24 0.41 3.60 67.86 29.18 00.092 4.03 2.33 6.36
1.40
1.10
WOOD
DUST
TOXICITY
457
Experiment II Hemolytic activity. Sheep red cells were collected in citrate solution, centrifuged, and plasma was removed. Subsequently the cells were washed three times in normal saline. After the last washing the cells were spun for 10 min at 1500 rpm and a 1% suspension was prepared in veronal-buffered saline (VBS), pH 7.4 (Harington et al., 1971). Varying amounts of Sheesham and Mango dusts (5, 10, 20, and 40 mg) of less than 30-pm particle size were suspended in 6 ml VBS, and to this 4 ml of 1% red cell suspension was added. The following controls were prepared: (a) blank correction containing wood dust in VBS; (b) positive hemolysis control containing quartz; (c) negative red cell control. The tubes were incubated at 37°C for 2 hr with continuous gentle shaking. After incubation the tubes were centrifuged and the supernatant was read at 540 nm in the Klett Summerson photoelectric calorimeter. In order to obtain 100% hemolysis standard, 4 ml of the 1% red cell suspension was centrifuged and the cell deposit lysed with 10 ml of distilled water. The readings of the wood dust suspended in VBS alone were subtracted from the readings of the dust plus red cells in VBS as a blank correction. The percentage of hemolysis was calculated by taking the average of three or more estimations. The effect of polyvinylpyridine N-oxide (P 204) and the trisodium salt of ethylenediaminetetraacetic acid (Na,EDTA) on hemolysis was examined to see if the mechanism of hemolysis resembled that of silica (Stalder and StGber, 1965) or chrysotile (Macnab and Harington, 1%7), respectively. P 204. Four milliliters of 1% red cell suspension was centrifuged for 10 min and the deposit resuspended in 10 ml of VBS containing 0.1 ml of P 204. Forty milligrams of wood dust was added and the test for hemolytic activity carried out as above. Negative controls of red cells and wood dust separately suspended in 10 ml VBS containing 1% P 204 were included. Na,EDTA. A solution of 25 pM/lld Na,EDTA of pH 7.4 was prepared by mixing thoroughly equal volumes of 50 p&i/ml NaOH and 50 pM/lld Na,EDTA. The test was carried out as above. Experiment III Macrophage cytotoxicity. Guinea pigs weighing 300 g were stunned by a blow on the head and then exsanguinated by cutting the jugular veins. The alveolar macrophages were harvested by the method of Myrvik et al (1961) in Hanks’ basal salt solution (HBSS). The harvested cells were pooled and leighton tubes inoculated with lo6 macrophages. After 40 min incubation at 37°C allowing the macrophages to adhere to the cover slips, the supernatant was removed and the monolayers were washed twice with HBSS. The wood dusts (particle size below 30 pm) were suspended in HBSS to give a concentration of 0.1 mg/ml. One milliliter of this finely dispersed suspension was added to each monolayer, five monolayers were inoculated with mango wood dust, and five monolayers with Sheesham wood dust. After 3 hr incubation at 37°C the monolayers were stained with erythrosin B and examined microscopically to determine the percentage of the cells stained (Munch et al., 1971). Five control tubes containing cells only were similarly stained. A positive control consisting of five monolayers inoculated
458
BHATTACHARJEE
ET AL.
with chrysotile dust was included for comparison with the wood dust. Each experiment was repeated three times. The results of Experiments II and III were analyzed by Student’s t test as described by Fisher (1950). RESULTS Experiment I Macroscopic
changes. Animals injected with Mango dust revealed at 60 days dirty pink patches of congestion in the lung but by 90 days only a few persisted. The Sheesham-inoculated animals also showed similar but larger areas of congestion. Microscopic changes. At 60 days the lungs of Mango dust-inoculated animals presented two types of reaction, one confined to the parenchyma and the other to the alveoli of respiratory bronchioles. The parenchymal reaction was comprised of dust-laden macrophages and giant cells which did not show any cytotoxicity and took up normal H and E staining. These lesions with reticulin stain showed fine reticulination of grade I (Fig. 1). The lesions in the walls of the bronchioles and alveolar ducts were comprised of aggregrations of macrophages and fibroblastic proliferation resulting in thickening and grade II fibrosis. Some of these areas also developed adenomatosis and lymphocytic infiltration. Areas of centrilobular emphysema also developed. By 90 days the parenchymal reaction had started to resolve as giant cells began to disintegrate. The fibrosis around the bronchioles and emphysema persisted but the lymphoid aggregations disappeared. Lungs from Sheesham-inoculated animals presented a diffuse reaction confined to alveoli abutting onto the respiratory bronchioles and alveolar ducts. The reaction was comprised of foreign body giant cells and cellular debris. Many giant cells showed degenerative changes in the form of vacuolated nuclei which had progressed in places to ghost forms. Adjoining areas developed centrilobular emphysema. On reticulin staining, grade I fibrosis was observed in these areas (Fig. 2). The reaction had resolved in most of the lung by 90 days but small areas of parenchymal granulomatous reaction and emphysema, particularly in the vicinity of the bronchioles, persisted. These areas developed grade I fibrosis. Numerous conspicuous lymphoid aggregrations were scattered throughout the parenchyma. Experiment
II
The hemolysis produced by different amounts of Mango and Sheesham dust is shown in Fig. 3. Forty milligrams of Sheesham gave 55.2% hemolysis which was consistently reproducible and highly significant (P < 0.0005). However 20 mg of dust also exhibited significant hemolytic activity (P < 0.005) although decrease in the amount of dust was accompanied by a rapid drop in the hemolytic activity. With smaller amounts of dust the degree of hemolysis observed was rather variable. Mango dust was only weakly hemolytic and 40 mg of dust gave 4.5% hemolysis which was statistically significant (p < 0.005) but with 20 and 10 mg the hemolysis was only just detectable. There was no hemolysis in the red cell control. The antisilicotic compound P 204 which inhibits hemolysis of quartz and EDTA which inhibits the hemolysis of chrysotile had no effect on Sheesham hemolysis.
WOOD
DUST
TOXICITY
459
460
BHATTACHARJEE
ET AL.
WOOD
DUST
/r
461
TOXICITY
QUARTZ
L.2 DALBERGIA
SlSSOO
(Sheesham)
MANGIFERA
INDICA
(Mow)
A
-
5
^-
IO
WEIGHT
FIG.
----
20
- 4
30
40
OF WOOD
3. Percentage
DUST
hemolysis
(m0)
and weight
of wood
dust
Experiment III Only 14.1 % of the cells of the monolayer to which no dust was added took up erythrosin stain after 3 hr incubation. Addition of the wood dusts caused an increase in the uptake of erythrosin; after 3 hr 34.2% (p < 0.0005) of the cells exposed to Sheesham took up the stain as compared with 31% (Z’ < 0.005) of the cells exposed to the Mango dust. The cell monolayers exposed to chrysotile showed a high degree of erythrosin uptake as 5 1.5% (P < 0.0005) of the cells were stained. When the uptake of erythrosin by macrophages exposed to the wood dusts was compared with the uptake of dye by the cells exposed to chrysotile a stastically significant difference was found (for Sheesham dust, P < 0.0005, and for Mango dust, P < 0.005). Therefore both the wood dusts show a significantly lesser degree of toxicity than chrysotile (Table 3). DISCUSSION
A variety of respiratory disorders and pulmonary changes have been attributed to the inhalation of wood dusts (Cohen et al., 1967; Michaels, 1967; Sossman et al., 1969: Cornea et al., 1978). It has been suggested that even nontoxic wood particles inhaled over long periods may cause fibrosis of the lung (Michaels, 1967). In addition to mechanical irritation and the foreign body reaction wood dust can cause toxic or allergic damage to the lung tissue. Based on chemical and biological studies, Hanslian and Kadlec (1964) classified woods into three groups, relative nontoxic, relative toxic, and strongly allergic. Some exotic woods appear to be associated with greater occupational hazards (Kadlec and Hanslian, 1972). Most of the available literature, however, deals with the allergic damage caused TABLE UPTAKE
OF ERYTHROSIN
PIG ALVEOLAR
3 MACROPHAGES
EXPOSED
Percentage cells stained erythrosin in 3 hr
Dust Sheesham Mango Asbestos (control) Control (blank) ” 0.1 mg/l.O
BY GUINEA
X IO” ceils.
34.1 i- 2.6 31.0 IT 7.1 51.5 t 6.0 14.1
i
2.5
TO WOOD
with _-~--.--_
DusV
462
BHATTACHARJEE
ETAL.
by woods and only a few studies have been made on the lung lesions after inhalation. In our experiments pulmonary changes in response to intratracheal injection of Sheesham and Mango wood dusts showed disintegration of giant cells by 90 days. The dust particles enclosed in a basophilic mass lay either free in the alveolar lumen or in the macrophages. These findings and the centrilobular emphysema and fibrosis of simple pneumoconiosis produced by the wood dust are in agreement with the observations in man (Michaels, 1967) and experimental animals (Cornea et al., 1978). The present studies therefore, provide an experimental model for the study of woodworkers pulmonary disease. In view of these findings, therefore, the prevalence of pneumoconiosis among workers employed in occupations where wood or its derivatives are handled needs to be reassessed. Measurement of the hemolytic activity of a variety of mineral dusts like silica and asbestos has provided a very simple means of investigating their potential biological activity (Stalder and Stober, 1965; Macnab and Harington, 1967; Kaw et al., 1973; Kaw and Zaidi, 1975). There is, however, hardly any information on the application of this test to organic dusts. In our experiments Sheesham showed marked and consistent hemolytic activity which may be correlated with the extensive and diffuse tissue reaction and rapid onset of degenerative changes observed in the lungs. In contrast the Mango wood dust which was only poorly hemolytic had limited and delayed onset of the acute reaction. It is suggested therefore that hemolysis may be a possible index for detecting early pulmonary tissue reactions of organic dusts. As both the dusts were able to induce thin reticulin fibrosis (simple pneumoconiosis), hemolysis is not a measure of their tibrogenic activity. In the present studies P 204 did not inhibit the hemolysis caused by Sheesham. The hemolytic activity of Sheesham dust was therefore not dependent on its silica content as reported for quartz by Stalder and Stober (1965). Similarly EDTA did not inhibit the hemolytic activity suggesting that the mechanism did not resemble that of chrysotile. Morgan et al. (1977), investigating the hemolytic activity of some fibrous amphiboles, have suggested a correlation between surface area of the fiber and hemolysis. A similar factor may be operative with fibrogenic wood dusts. Alternately some organic compounds, like acids or phenols leaching out of Sheesham wood, may be important determinants of the hemolysis and acute tissue response in our experiments. These compounds were not present in significant amounts in mango dust which showed minimal hemolysis. Several studies have shown that the tibrogenicity of mineral and polymer dusts can be correlated with macrophage cytotoxicity in vitro (Marks and Mason, 1956; Conning et al., 1970). We have therefore used the uptake of erythrosin as a simple method for measuring macrophage damage caused by the dusts. Both Sheesham and Mango wood dusts gave intermediate levels of cytotoxicity when compared with the marked cytotoxicity produced by chrysotile. This suggests that macrophage cytotoxicity may be indicative of the fibrogenic potential of wood dusts as both Sheesham and Mango were tibrogenic. Certain hard woods and old root woods are known to contain significant levels of silica and have been suspected to induce fibrosis in exposed workers (Hanslian and Kadlec, 1964). The present experiments indicate that the silica content is not
WOOD
DUST
TOXICITY
463
critical in the fibrogenicity of Mango and Sheesham dust, because of its low content in these dusts. From the present work it appears that the hemolytic activity of the organic dusts studied may provide a useful screening test for acute toxicity and macrophage cvtotoxicity for fibrogenicity. ACKNOWLEDGMENTS This investigation was supported by a grant from the United States Government. Department of Health, Education and Welfare, Public Health Services of Public Law 83-480, to which the authors are grateful. The authors are grateful to Professor H. W. Schlipkotter. Director. Medizinisches Institute fur Lufthygiene und Silikoseforschung, Dusseldorf, for the generous supply of the compounds polyvinylpyridine N-oxide, and to Dr. M. Swaley, Incharge. Chemistry of Forest Products. Forest Research Institute Dehradun, who very kindly carried out the wood analysis. and to Mr. K. C. Saxena for silica estimation.
REFERENCES A.O.A.C. (1977). “Official Methods of Analysis of the Association of Official Analytical Chemists.” I lth ed. William Horwitz. Washington, D.C. Belt. T. H., and King, E. J. (1945). Chronic pulmonary disease in South Wales coal miners. III. Experimental studies. D. Tissue reactions produced experimentally by selected dusts from South Wales coalminers. In “Spec. Rep. Ser. Med. Res. Coun.” No. 250, pp. 29-68. London. Cohen, H. I., Merigan, T. C., Kosek, J. C.. and Eldridge, F. (1967). Sequosis: A granulomatons pneumonitis associated with red wood saw dust inhalation. Atnrr. J. Med. 43, 785-794. Conning. D. M., Hays, M. J., Styles, .I. A., and Nicholas, J. A. (1970). Comparison between in rirvo toxicity of dusts of certain polymers and minerals and their fibrogenicity. Itr “Inhaled particles. III” (W. H. Walton, ed.), Vol. I. pp. 499-506. Unwin, Surrey. Cornea, G., Elmekki. L., Elgharbi, B.. Benkheder, A.. and Silvia, G. (1978). Une Nouvelle Pneumoconiose Vegetable, Le Poumon De Neefa. “ln 5th International Conference on Pneumoconiosis,” Oct. 29 to Nov. 3, 1978. Caracas, Venezuela. Fisher, R. A. (1950). “Statistical Methods for Research Workers,” 1 lth ed. Oliver & Boyd, London. Gordon, H., and Sweets, H. H., Jr. (1936). A simple method for the silver impregnation of reticulin. Amer. J. Pnthol. 12, 545-552. Hanslian, L.. and Kadlec. K. (1964). Drevo a jeho prach (Timber and timber dust). Prac. Lek. 16, 276-282. Hanshan. L., and Kadlec. K. (1964). Drevo 2 Hlediska Hygienic Rheo (1V)Biol. C’eirtXv Pruch Drr~~r 19, 45 I-452. Harington, J. S., Miller. K., and Macnab, G. (1971). Haemolysis by asbestosE,ri?ro,t. RVS. 4, 95- I 17. Kadlec. K.. and Hanslian, L. (1972). Encyclopaedia of Occupational Health and Safety, Vol. II. L-Z. International Labour Office. Geneva, pp. 1507-1511. Kaw. J. L.. Gupta, G. S. D.. Narang, S.. and Zaidi, S. H. (1973). Haemolytic activity of mica dust and its relation to polyvinylpyridine N-oxide. Exp. Prrthal. 8, 349-353. Kaw, J. L., and Zaidi. S. H. (1975). In i.itro studies on the cytotoxic reaction of different varieties of asbestos dust on macrophages. Acta Plwrmac~ol. To.xicol. 36, X-235. Macnab. G.. and Harington, J. S. (1967). Haemolytic activity of asbestos and other mineral dusts. h’utrrrc (London) 214, 522-523. Marks, J.. and Mason, M. A. (1956). A quantitative technique for studying the effect of dust on phagocytic cells iti \‘itro. Brit. J. Id. Med. 13, 192- 195. Michaels, L. (1967). Lung changes in wood workers. Canad. Med. Asso~. J. 96, 1150-1155. Morgan, A.. Holmes, A.. and Talbot. R. J. (1977). The haemolytic activity of some fibrous amphiboles and its relation to their specific surface areas. Amer. Occup. h’yg. 20. 39-48. Muncn. R.. Beck, E. G., ana Manojlovic, N. ( lY71). Unrerschungen uber die Reaktion iii i,itrrj
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gezuchteter Zellen nach Kurzeitiger. Inkubation Zellen nach Kurzzeitiger. Inkubation mit SiOzstaub. Beitr. Silikose Forsch. 23, 175-205. Myrvik, Q. N., Leake, E. S., and Far&, B. (1961). Studies on pulmonary alveolar macrophages from normal rabbit. A technique to procure them in a high state of purity. J. Immunof. 86, 128 132 Sossman, A. J., Donald, P., Schlueter, J., Fink, N., and Barboriak, J. (1969). Hypersensitivity to wood dust. N. Engl. J. Med. 281, 977-980. Staider, K., and Stober, W. (1965). Haemolytic activity of suspensions of different silica modifications and inert dusts. Nature (London) 207, 874-875. Zaidi, S. H. (1969). “Experimental Pneumoconiosis.” John Hopkins Press, Baltimore.
