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Relevant differences in pathogenicity of nuisance dusts; model investigations on samples of silicon carbide dusts
ExpToxicPatho11996; 48:477-480 Gustav Fischer Verlag Jena
Institute of Hygiene and Occupational Medicine, Essen Medical School, Essen, Germany
Relevant differences in pathogenicity of nuisance dusts; model investigations on samples of silicon carbide dusts* J. BRUCH and B. REHN With 3 figures and I table Received: December I, 1995; Accepted: December 20, 1995 Address for correspondence: Dr. 1. BRUCH, Institute of Hygiene and Occupational Medicine, Medical School, Hufelandstr. 55, D - 45147 Essen, Germany. Key words: Dusts, nuisance; Nuisance dusts; Silicon carbide dust; Dusts, silicon carbide.
The identification of potential endangerment to health through dusts of less conspicuous toxicity than asbestos and quartz is difficult in humans. In principle, only animal experiments can be used to identify risks and assess risk quantification. Actually, all mineral fine dusts are classified as carcinogenic (6) on the basis of testing results in rats of a series of dusts including diesel soot, silicon carbide (SiC) and others (11); the tumorigenic ranking ofthe samples tested was lowest for SiC (15 %) whereas quartz was the highest. Conceptually, the classification scheme under discussion encompasses all mineral dusts not otherwise classified as specifically toxic and generally denoted as nuisance dusts. It is hypothesized that overloading and hampered lung clearance might "explain" the carcinogenic action of the "particles not otherwise classified" (PNCO TLV definition). In addition, very small particles seem to be particularly associated to the carcinogenic effects of dusts (8). For further testing the presumed non-specific dust nocivity, samples of SiC would seem particularly suitable. One sample tested labelled NF2 represented the lower end of carcinogenicity scale in the comparative investigation (11). The other sample denoted as FI200 has been formerly intensively studied by us (4, 5) and was classified as not specifically toxic in the frame of the methods used. Carcinogenicity tests has not been performed with this substance.
Material and methods
according to FEPA; sample SiC-B was provided by Wacker-Chemie under the label NF2. Grain size distribution is shown in fig I mean diam: FI200 2.26 11m, NF2 1.14Ilm: the probes were free of fibrous SiC varieties. SiC-A and SiC-B were indistinguishable by conventional physico-chemical methods (EDX, x-ray diffractometry) (2). The negative or positive control samples in the in vitro testing were standard samples of quartz (DQ 12) and corundum. For one part of the in vitro testing the samples were separated into fractions of distinct grain size diameter also with the cascade impactor (see figure I). 50
~
D
40
- 0 - SiC.A
- - . - SiC·B
~
E
Cl
'Q) ~ 0
(I)
Cl
30
o 2.26 IJIll
E 1.141J1ll FO.551J1ll GO.261J1ll
20
~ (I)
~
(I)
Q.
0
0
2
4
6
8
10
aerodynamic diameter of particles (11m)
Fig. 1. Grain size distribution of SiC-A and SiC-B subfractions G to D prepared for specific in vitro testing (comp. fig 3).
Dust samples: Sample SiC-A is a purchasable standardized product of the Wacker Chemie, Germany, labelled as F1200
Biological testing procedures
* Paper, presented at the 5th International Inhalation Symposium, Hannover, Germany, 20-24 February 1995.
In vivo testing: The dust samples were instilled into rat lungs, 20 mg per animal, under controlled conditions providing exact doses to each animals. The animal were sacrificed after 2, 14, 21 and 90 days. The animals were evaluated ExpToxic Pathol48 (1996) 6
477
Table 1. Balf of rat lungs after it application of 20 mg silicon carbide samples SiC-A and SiC-B. sample Parameter
Time: days 5
14
21
90
TotLSL cont (mg/ml)
SiC-A SiC-B
74,1 ± 19,4 65,5 ± 11,7
102,0 ± 34,0 44,3 ± 12,2
82,8 ± 20,6 117,0 ± 33,3
49,8 ± 7,8 92,4 ± 24,4
Total cells (l06/ml)
SiC-A SiC-B
3,2± 0,9 4,3 ± 1,5
2,1 ± 0,3 2,2 ± 0,7
2,5 ± 0,3 2,5 ± 0,4
1,5 ± 0,6 1,7 ± 0,4
Granulocytes
SiC-A SiC-B
27,6 ± 11,1 41,8 ± 6,7
25,2 ± 11,2 30,8 ± 12,5
25,0 ± 10,0 39,5 ± 10,0
17,1 ± 11,0 39,9 ± 7,7
(%)
----cr -
~
corundum
200PXI---Q----Q----QI-=::=::hl
S
Q)
"0
'x
e
~ 100
c:
~
-g,
.c
40
80
120
dustconcentration
(~g
160
200
240
/ million cells)
Fig. 2. Hydrogen peroxide release in AM at different doses after testing with quartz, corundum and SiC samples A and B.
by broncho-alveolar lavage (BAL), in the BAL-fluid(BALF), protein, cells, and lung surfactant lipids (LSL) were determined. Technical details on the experimental procedures are given in (2) and (I). In vitro testing: The harmfulness was assessed on alveolar macrophages (male guinea pig) by a set of toxicity parameters (LDH, FDA) and through determination of inducible H20 2 release. Procedures are described in detail in (2). Dusts were tested in doses ranging from 20 to 180 mg/l 06cells, tests were performed in triplicate in one term at four independent terms (independent: animals, cell harvest, dust weighing, dust dosing, plate reader assessing).Positivecontrols were quartz (DQI2), the negative ones corundum.
Results In vivo investigation: The animal studies were focused on the inflammatory reaction in the lung parenchyma following bulk exposure of the test samples. The parameters used were taken from the BALF of the animals. 478
Exp Toxic Pathol48 (1996) 6
Basically, the number of cells in the BALF in conjunction with differential cell count reflects disturbances in the alveolar microenvironment. The determination of the lung surfactant lipids shows an epithelial reaction which would seem to respond to alveolar stress and simultaneously contributes to alveolar pathology. These data as shown in table 1 reflect at once the reponse to the massive strain on the lung microenvironment by the exposure as such and the specific reaction following the applied samples. Total cells were increased 5 days (d) post exposure (p. e.) but decreased continuously during the the observation time of 90 d to values close to control animals; no substantial difference could be observed between the samples tested when compared to overall change during the study. The granulocytic reponse was high in both groups immediately following the exposure whereas the following terms show considerable differences. Si-B causes a significant drop at d 14 followed by a new elevation of granulocytic percentage up to the primary level which persisted unchanged until d 90. In contrast to this, the granulocyctic percentage in the group SiC-A decreased continuously and was statistically different to SiC-B at d 21 and 90 (p < 0.001). Changes in LSL in BALF p. e. to SiCA and SiC-B are shown in table 1. Both samples induced distinct and qualitatively different reactions of the epithelial system. SiC-A produces an elevation of LSL in th BALF until d 14 p. e. whereas SiC-B effects a sharp drop in LSL even to subnormal level at d 14 p. e. followed by strong and persistent increase. In vitro testing: Using the original non-sized silicon carbide samples, cell viability measured by FDA showed no significant differences; quartz reduced cell viability even in low doses (not shown). Taking loss of hydrogen peroxidase secretion as a measure for cell damage, macrophages burdened with quartz or SiC-B sample exert a significant and dose dependent reduction in hydrogen peroxide release. In contrast the SiC-A sample did not show this effect (figure 2). Testing the grain size fractioned silicon samples, cell viability showed no marked differences between the two samples and no particular cell toxicity (not shown). In contrast to the results obtained for cell viability, marked differences between the two samples could be measured in the H 20 2 secretion. As
-E (5
-
250 -+-- ~---
c
Q)
"0
'x
e Q)
a. cQ)
200 -r--V.A/ I" r -
e "0
C>
>.c
F1200D F1200 E F1200 F NF2D NF2 E NF2 F NF2G
150+-........~ 180 Jlg/million cells
Fig. 3. Hydrogen peroxide release in AM; tested for grain sizesubfraction G to 0 from SiC-A (F1200)andSiC-B (NF2) compare fig. 1.
seen in fig. 3 the doses and the fractions of the SiC-B sample had significant influence on this parameter which was most pronounced testing the finest fraction of particles (G fraction; 0.26 urn diameter). SiC-A had no effect in any dose grain size combination.
Discussion and conclusion The toxicological studies came to the following major results: 1. The dust samples SiC-A and SiC-B show different biological effects; SiC-B leads to marked pathological reactions in the animal test and in the in vitro testing whereas the SiC-A sample is inert in the frame of the specificity and sensitivity of the investigational procedures used here. 2. The differences cannot be solely explained on the basis of different grain size distribution. The sample B contains a higher concentration of the finer particles. The fine fraction is more toxic; but on the basis of the side-toside comparison each fraction of sample B is more toxic than sample A. 3. The in vitro assessment on the basis of mere evaluation of cell viability is insufficient to demark relevant differences of dust toxicity. The good agreement of in vitro and in vivo data presented shows a high degree of selectivity of the methods. The results concerning the "inertness" of sample SiC-A cannot positively demonstrate that this substance is noncarcinogenic. On the other hand the conceptual framework of carcinogenicity of all mineral fine dusts probably should be discussed when the underlying database refers to tested samples which, at the lower end of carcinogenicity, are definitively specifically toxic. Moreover this study demonstrates that in the range of present methodological limits practically inert dusts do exist. Regarding
the representativeness of the selected samples for the substance silicon carbide, the accompanying study including test data of over 30 samples of SiC discloses the same degree of inertness as the SiC-A presented here (3). The complete data published separately also include results on TNF-promoting activity of the sample SiC-B as compared to SiC-A or corundum which show no activity (level of untreated cells) (2). In addition, the silica inhibitor PNO could neutralize to some extent the SiC-B dependent depressor effect on H,O, secretion. The results on subfractions of the LSL support the assumption that silica-like effects contribute to the specific biological activity of the sample SiC-B. In conclusion the data show that relevent differences in biopathogenicity do exist for the tested varieties of SiC. Presently, concepts of dust related cancerogenicity are focused on reactive oxygen species (ROS)-caused gene toxicity including formation of 8 oxo-g adducts; ROS formation by dusts is associated to particular endogenic pathways which all include molecular events such as respiratory burst. Activated macrophages and in particular granulocytes secrete high amounts of ROS which are known to damage neighbouring DNA (7). Massive ROS production is also experienced to damage epithelial cells (9). Epithelial stimulation is also associated to asbestos exposure in patients (10). The experiments clearly show that SiC-B elicits a lasting granulocytic response together with an epithelial stimulation. These cellular events fit into the puzzle which might be important for silica carcinogenicity. SiC-A and corundum lack particular biopathogenic effectivity. We are of opinion that further investigation is required in order to substantiate the concept of general mineral dust carcinogenicity.
References 1. BRUCH J, GONO E, MALKUSCH W, et al.: Improved me-
thod for quantitative analysis of lung surfactant phopholipids in bronchoalveolar lavage fluids by high-performance liquidchromatography. Clin ChimActa 1994; 231: 193-204. 2. BRUCH J, REHN B: Selectivity and sensitivity of biological assessment for discriminating harmfulness of dusts in comparison to conventional physico-chemical methods. Submitted 1996. 3. BRUCH J, REHN B, REHN S: Implementation of a biological monitoring system for hygienic surveillance in abrasive industry. accepted 1996. 4. BRUCH J, REHN B, SONG H, et a1.: Toxicological investigations on silicon carbide. 2. In vitro cell tests and long term injection tests. Br J Ind Med 1993; 50: 807-813. 5. BRUCH J, SONG H, GONO E, et al.: Toxicological investigations on silicon carbide. 1. Inhalation studies. Br J Ind Med 1993; 50: 797-806. 6. DFG: Grenzwerte; Verlag Chemie, 1993. 7. FRENKEL K: Oxidation of DNA bases by tumorpromotoractivated processes. Environ Health Perspect 1989; 81 (45): 45-54. Exp Toxic Pathol 48 (I 996) 6
479
8. HEINRICH U: Do "nuisance" dust particles contribute to
the lungcancerrisk Umwelthygiene - Supplement 2 published by GHUIMIU Dusseldorf 1994; 6. 9. KAMP DW, GRACEFFA P, PRYOR WA, et aI.: The role of free radicals in asbestos-induced diseases. Free Radic Bioi Med 1992; 12: 293-315.
480
Exp Toxic Pathol48 ( 1996) 6
10. KLEIN B, BACH-EsSER K, REHN B, et at.: Increased lung surfactant phospholipids inasbestos exposed patients with mesothelioma. Am Rev Respir Dis 1993; 147 (4): A909. 11. POTI F, DUNGWORTH DL, HEINRICH U, et aI.: LungTumours In Rats After Intratracheal Instillation Of Dusts. Edinburgh GB Bohs 1991; pp. 357-363.
Institute of Hygiene and Occupational Medicine, Essen Medical School, Essen, Germany
Relevant differences in pathogenicity of nuisance dusts; model investigations on samples of silicon carbide dusts* J. BRUCH and B. REHN With 3 figures and I table Received: December I, 1995; Accepted: December 20, 1995 Address for correspondence: Dr. 1. BRUCH, Institute of Hygiene and Occupational Medicine, Medical School, Hufelandstr. 55, D - 45147 Essen, Germany. Key words: Dusts, nuisance; Nuisance dusts; Silicon carbide dust; Dusts, silicon carbide.
The identification of potential endangerment to health through dusts of less conspicuous toxicity than asbestos and quartz is difficult in humans. In principle, only animal experiments can be used to identify risks and assess risk quantification. Actually, all mineral fine dusts are classified as carcinogenic (6) on the basis of testing results in rats of a series of dusts including diesel soot, silicon carbide (SiC) and others (11); the tumorigenic ranking ofthe samples tested was lowest for SiC (15 %) whereas quartz was the highest. Conceptually, the classification scheme under discussion encompasses all mineral dusts not otherwise classified as specifically toxic and generally denoted as nuisance dusts. It is hypothesized that overloading and hampered lung clearance might "explain" the carcinogenic action of the "particles not otherwise classified" (PNCO TLV definition). In addition, very small particles seem to be particularly associated to the carcinogenic effects of dusts (8). For further testing the presumed non-specific dust nocivity, samples of SiC would seem particularly suitable. One sample tested labelled NF2 represented the lower end of carcinogenicity scale in the comparative investigation (11). The other sample denoted as FI200 has been formerly intensively studied by us (4, 5) and was classified as not specifically toxic in the frame of the methods used. Carcinogenicity tests has not been performed with this substance.
Material and methods
according to FEPA; sample SiC-B was provided by Wacker-Chemie under the label NF2. Grain size distribution is shown in fig I mean diam: FI200 2.26 11m, NF2 1.14Ilm: the probes were free of fibrous SiC varieties. SiC-A and SiC-B were indistinguishable by conventional physico-chemical methods (EDX, x-ray diffractometry) (2). The negative or positive control samples in the in vitro testing were standard samples of quartz (DQ 12) and corundum. For one part of the in vitro testing the samples were separated into fractions of distinct grain size diameter also with the cascade impactor (see figure I). 50
~
D
40
- 0 - SiC.A
- - . - SiC·B
~
E
Cl
'Q) ~ 0
(I)
Cl
30
o 2.26 IJIll
E 1.141J1ll FO.551J1ll GO.261J1ll
20
~ (I)
~
(I)
Q.
0
0
2
4
6
8
10
aerodynamic diameter of particles (11m)
Fig. 1. Grain size distribution of SiC-A and SiC-B subfractions G to D prepared for specific in vitro testing (comp. fig 3).
Dust samples: Sample SiC-A is a purchasable standardized product of the Wacker Chemie, Germany, labelled as F1200
Biological testing procedures
* Paper, presented at the 5th International Inhalation Symposium, Hannover, Germany, 20-24 February 1995.
In vivo testing: The dust samples were instilled into rat lungs, 20 mg per animal, under controlled conditions providing exact doses to each animals. The animal were sacrificed after 2, 14, 21 and 90 days. The animals were evaluated ExpToxic Pathol48 (1996) 6
477
Table 1. Balf of rat lungs after it application of 20 mg silicon carbide samples SiC-A and SiC-B. sample Parameter
Time: days 5
14
21
90
TotLSL cont (mg/ml)
SiC-A SiC-B
74,1 ± 19,4 65,5 ± 11,7
102,0 ± 34,0 44,3 ± 12,2
82,8 ± 20,6 117,0 ± 33,3
49,8 ± 7,8 92,4 ± 24,4
Total cells (l06/ml)
SiC-A SiC-B
3,2± 0,9 4,3 ± 1,5
2,1 ± 0,3 2,2 ± 0,7
2,5 ± 0,3 2,5 ± 0,4
1,5 ± 0,6 1,7 ± 0,4
Granulocytes
SiC-A SiC-B
27,6 ± 11,1 41,8 ± 6,7
25,2 ± 11,2 30,8 ± 12,5
25,0 ± 10,0 39,5 ± 10,0
17,1 ± 11,0 39,9 ± 7,7
(%)
----cr -
~
corundum
200PXI---Q----Q----QI-=::=::hl
S
Q)
"0
'x
e
~ 100
c:
~
-g,
.c
40
80
120
dustconcentration
(~g
160
200
240
/ million cells)
Fig. 2. Hydrogen peroxide release in AM at different doses after testing with quartz, corundum and SiC samples A and B.
by broncho-alveolar lavage (BAL), in the BAL-fluid(BALF), protein, cells, and lung surfactant lipids (LSL) were determined. Technical details on the experimental procedures are given in (2) and (I). In vitro testing: The harmfulness was assessed on alveolar macrophages (male guinea pig) by a set of toxicity parameters (LDH, FDA) and through determination of inducible H20 2 release. Procedures are described in detail in (2). Dusts were tested in doses ranging from 20 to 180 mg/l 06cells, tests were performed in triplicate in one term at four independent terms (independent: animals, cell harvest, dust weighing, dust dosing, plate reader assessing).Positivecontrols were quartz (DQI2), the negative ones corundum.
Results In vivo investigation: The animal studies were focused on the inflammatory reaction in the lung parenchyma following bulk exposure of the test samples. The parameters used were taken from the BALF of the animals. 478
Exp Toxic Pathol48 (1996) 6
Basically, the number of cells in the BALF in conjunction with differential cell count reflects disturbances in the alveolar microenvironment. The determination of the lung surfactant lipids shows an epithelial reaction which would seem to respond to alveolar stress and simultaneously contributes to alveolar pathology. These data as shown in table 1 reflect at once the reponse to the massive strain on the lung microenvironment by the exposure as such and the specific reaction following the applied samples. Total cells were increased 5 days (d) post exposure (p. e.) but decreased continuously during the the observation time of 90 d to values close to control animals; no substantial difference could be observed between the samples tested when compared to overall change during the study. The granulocytic reponse was high in both groups immediately following the exposure whereas the following terms show considerable differences. Si-B causes a significant drop at d 14 followed by a new elevation of granulocytic percentage up to the primary level which persisted unchanged until d 90. In contrast to this, the granulocyctic percentage in the group SiC-A decreased continuously and was statistically different to SiC-B at d 21 and 90 (p < 0.001). Changes in LSL in BALF p. e. to SiCA and SiC-B are shown in table 1. Both samples induced distinct and qualitatively different reactions of the epithelial system. SiC-A produces an elevation of LSL in th BALF until d 14 p. e. whereas SiC-B effects a sharp drop in LSL even to subnormal level at d 14 p. e. followed by strong and persistent increase. In vitro testing: Using the original non-sized silicon carbide samples, cell viability measured by FDA showed no significant differences; quartz reduced cell viability even in low doses (not shown). Taking loss of hydrogen peroxidase secretion as a measure for cell damage, macrophages burdened with quartz or SiC-B sample exert a significant and dose dependent reduction in hydrogen peroxide release. In contrast the SiC-A sample did not show this effect (figure 2). Testing the grain size fractioned silicon samples, cell viability showed no marked differences between the two samples and no particular cell toxicity (not shown). In contrast to the results obtained for cell viability, marked differences between the two samples could be measured in the H 20 2 secretion. As
-E (5
-
250 -+-- ~---
c
Q)
"0
'x
e Q)
a. cQ)
200 -r--V.A/ I" r -
e "0
C>
>.c
F1200D F1200 E F1200 F NF2D NF2 E NF2 F NF2G
150+-........~ 180 Jlg/million cells
Fig. 3. Hydrogen peroxide release in AM; tested for grain sizesubfraction G to 0 from SiC-A (F1200)andSiC-B (NF2) compare fig. 1.
seen in fig. 3 the doses and the fractions of the SiC-B sample had significant influence on this parameter which was most pronounced testing the finest fraction of particles (G fraction; 0.26 urn diameter). SiC-A had no effect in any dose grain size combination.
Discussion and conclusion The toxicological studies came to the following major results: 1. The dust samples SiC-A and SiC-B show different biological effects; SiC-B leads to marked pathological reactions in the animal test and in the in vitro testing whereas the SiC-A sample is inert in the frame of the specificity and sensitivity of the investigational procedures used here. 2. The differences cannot be solely explained on the basis of different grain size distribution. The sample B contains a higher concentration of the finer particles. The fine fraction is more toxic; but on the basis of the side-toside comparison each fraction of sample B is more toxic than sample A. 3. The in vitro assessment on the basis of mere evaluation of cell viability is insufficient to demark relevant differences of dust toxicity. The good agreement of in vitro and in vivo data presented shows a high degree of selectivity of the methods. The results concerning the "inertness" of sample SiC-A cannot positively demonstrate that this substance is noncarcinogenic. On the other hand the conceptual framework of carcinogenicity of all mineral fine dusts probably should be discussed when the underlying database refers to tested samples which, at the lower end of carcinogenicity, are definitively specifically toxic. Moreover this study demonstrates that in the range of present methodological limits practically inert dusts do exist. Regarding
the representativeness of the selected samples for the substance silicon carbide, the accompanying study including test data of over 30 samples of SiC discloses the same degree of inertness as the SiC-A presented here (3). The complete data published separately also include results on TNF-promoting activity of the sample SiC-B as compared to SiC-A or corundum which show no activity (level of untreated cells) (2). In addition, the silica inhibitor PNO could neutralize to some extent the SiC-B dependent depressor effect on H,O, secretion. The results on subfractions of the LSL support the assumption that silica-like effects contribute to the specific biological activity of the sample SiC-B. In conclusion the data show that relevent differences in biopathogenicity do exist for the tested varieties of SiC. Presently, concepts of dust related cancerogenicity are focused on reactive oxygen species (ROS)-caused gene toxicity including formation of 8 oxo-g adducts; ROS formation by dusts is associated to particular endogenic pathways which all include molecular events such as respiratory burst. Activated macrophages and in particular granulocytes secrete high amounts of ROS which are known to damage neighbouring DNA (7). Massive ROS production is also experienced to damage epithelial cells (9). Epithelial stimulation is also associated to asbestos exposure in patients (10). The experiments clearly show that SiC-B elicits a lasting granulocytic response together with an epithelial stimulation. These cellular events fit into the puzzle which might be important for silica carcinogenicity. SiC-A and corundum lack particular biopathogenic effectivity. We are of opinion that further investigation is required in order to substantiate the concept of general mineral dust carcinogenicity.
References 1. BRUCH J, GONO E, MALKUSCH W, et al.: Improved me-
thod for quantitative analysis of lung surfactant phopholipids in bronchoalveolar lavage fluids by high-performance liquidchromatography. Clin ChimActa 1994; 231: 193-204. 2. BRUCH J, REHN B: Selectivity and sensitivity of biological assessment for discriminating harmfulness of dusts in comparison to conventional physico-chemical methods. Submitted 1996. 3. BRUCH J, REHN B, REHN S: Implementation of a biological monitoring system for hygienic surveillance in abrasive industry. accepted 1996. 4. BRUCH J, REHN B, SONG H, et a1.: Toxicological investigations on silicon carbide. 2. In vitro cell tests and long term injection tests. Br J Ind Med 1993; 50: 807-813. 5. BRUCH J, SONG H, GONO E, et al.: Toxicological investigations on silicon carbide. 1. Inhalation studies. Br J Ind Med 1993; 50: 797-806. 6. DFG: Grenzwerte; Verlag Chemie, 1993. 7. FRENKEL K: Oxidation of DNA bases by tumorpromotoractivated processes. Environ Health Perspect 1989; 81 (45): 45-54. Exp Toxic Pathol 48 (I 996) 6
479
8. HEINRICH U: Do "nuisance" dust particles contribute to
the lungcancerrisk Umwelthygiene - Supplement 2 published by GHUIMIU Dusseldorf 1994; 6. 9. KAMP DW, GRACEFFA P, PRYOR WA, et aI.: The role of free radicals in asbestos-induced diseases. Free Radic Bioi Med 1992; 12: 293-315.
480
Exp Toxic Pathol48 ( 1996) 6
10. KLEIN B, BACH-EsSER K, REHN B, et at.: Increased lung surfactant phospholipids inasbestos exposed patients with mesothelioma. Am Rev Respir Dis 1993; 147 (4): A909. 11. POTI F, DUNGWORTH DL, HEINRICH U, et aI.: LungTumours In Rats After Intratracheal Instillation Of Dusts. Edinburgh GB Bohs 1991; pp. 357-363.