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Differences in the biological effects of crocidolite asbestos and two glass fibres on epithelial lung cells
Exp Toxic PathoI1993/94; 45: 467-472 Gustav Fischer Verlag Jena
Institute of Experimental Pathology, Hannover Medical School, Germany
Differences in the biological effects of crocidolite asbestos and two glass fibres on epithelial lung cells M. AUFDERHEIDE, M. RIEBE-IMRE, M . STRAUB and A. PERAUD With 6 figures and 1 table Received: September 1, 1993; Accepted: September 16, 1993 Address for correspondence: Dr. M. AUFDERHEIDE, Institute of Experimental Pathology, Hannover Medical School, Konstanty-Gutschow-Str. 8, D - 30625 Hannover, Germany. Key words: Crocidolite asbestos; Asbestos, crocidolite; Glass fibres; Epithelial lung cells; Lung, epithelial cells; Mineral fibres; Fibrous glass.
Introduction
Material and methods
Asbestos, a naturally occurring mineral, is known to be potentially hazardous to man and experimental animals. Inhaled fibres induce proliferation of connective tissue and increase the risk of acquiring pulmonary carcinoma and mesothelioma in the lung (1, 2). In contrast, new types of fibrous glass reveal only a weak or no carcinogenic potency in animal experiments (3). The mechanisms by which fibres induce these effects are still under discussion. Since the role of fibre dimension, fibre durability and surface properties have been investigated in different studies, it was suggested that multiple mechanisms must be operative to explain the diverse effects of mineral fibres. The cytotoxic and carcinogenic potency have been studied thoroughly for the naturally occurring fibres like crocidolite, chrysotile or amosite, but only few data are available for synthetic fibres. In this study we tested crocido lite asbestos in comparison with two glass fibres JM 104/475 and B-IM for different biological effects in an in vitro system of epithelial hamster cells to elucidate differences in the interaction between cells and fibres.
Cell line: The Syrian hamster epithelial cell line (M3E3/C3) was derived from the lung of a foetus on day 15 of gestation. Cells were grown in a medium consisting mainly of RPMI 1640 (80 %, Flow, Meckenbeim) and fetal bovine serum (BSA; 20 %, Flow) as described previously (4). Cultures were maintained at 37 °C in a humidified atmosphere with 10 % CO 2 and 90 % air. Confluent cultures were harvested by trypsinization (0,25 % Trypsin, 2 % EDTA in PBS) and resuspended in the medium after low speed centrifugation. The epithelial character of the cell line and its application to pulmonary toxicology have been described elsewhere (5 , 6). Mineral fibres: All fibres tested were provided by Prof. Pon from the Medical Institute for Environmental Hygiene (University of Diisseldorf). The characterization of the fibre types was performed by Dr. BELLMANN, Fraunhofer Institute of Inhalation Toxicology and Aerosol Research, and is given in table 1. Cytotoxicity: M3E3/C3 cells (5 x 1()4 cells) were seeded in 25 cm2 Falcon flasks with 5 rol growth medium. 24 h later the cells were treated for 96 h with different doses of mineral
Table 1. Characterization of fibre dusts. fibre length (,..lm) 10 % < 50 % < 90 % <
fibre diameter (/lm) lO % < 50 % < 90 % <
99
0,9
1,8
4,7
0,11
0,19
0,32
158
0,7
2,3
8,2
0,06
0,14
0,38
6,7
10,7
21,1
0,88
1,68
2,49
Fibre
mass of critical fibres (%)
critical fibres/ng mean
Crocidolite
42
JM 104/475
88
B-IM
15
27
Exp Toxic Pathol45 (1993/94) 8
467
fibres (10, 20, 30, 50 Jlg crocidolite asbestos/ml; 50, 100, 150,200 Jlg JM 104/475 and B-IM/ml) which were suspended in medium after being sterilized. At the end of exposure, cells were trypsinized and cell viability was determined by the trypan blue exclusion method in a haematocytometer. Plating efficiency: From each treatment group 200 viable cells were plated in an 8 cm2 Petri dish (Falcon) and cultured under the above-mentioned culture and test conditions for 8 days without medium change. The cells were fixed in methanol, stained with Giemsa and colonies were counted. Plating efficiency was calculated as the percentage of colony formation relative to the control groups. Transformation: The determination of transformation was based on anchorage independent growth in soft agar. The treatment was carried out in the same way as for the cytotoxicity study (doses: a. crocidolite asbestos: 1, 1,5, 2 Jlg/ml; b. JM 104/475: 50, 100, 150 Jlg/ml; B-IM: 300, 400, 500 Jlg/ml). The cells of the different treatment groups were passaged by trypsinization 5 times at intervals depending on the growth speed in reaching about 80 % confluency. The cells were then brought into soft agar cultures to
detect anchorage-independent growth indicating transformation. The plating efficiency of the cells for each dose group was determined as described above. Six weeks later, evaluation of cultures was performed by counting only colonies with a diameter greater than 30 Jlm. Transformation frequency was calculated as the percentage of transformed colonies relative to the control cells unexposed to mineral fibres. Fluorescence Microscopy: Cells were grown on 13-mmdiam glass coverslips in 8 cm 2 Petri dishes for 86 h. Mineral fibres sterilized by autoclaving were suspended in growth medium. The resulting suspensions were added to a final volume of 5 ml at a final concentration of 10 Jlg crocidolite asbestos/ml and 50 Jlg JM 104/474/ml and 100 Jlg B-IM/ml. Immunofluorescence microscopy was performed after 14,32,62 and 86 hours of exposure. Cells were fixed in 4 % paraformaldehyde in PBS (20 min) for actin and tubulin detection. The whole preparation procedure was at 37 DC. After washing the cells in the same buffer, they were permeabilized with 1 % NP-40 in PBS for 10 min at room temperature, washed three times with PBS, incubated for 20 min with BSA to avoid non-specific reaction and washed again with PBS. For actin labelling, probes were incubated
100
90 80
~
70
en
60
~
Co>
b.O
= :~ ... ;:::I
en
50 40 30 20 10 0 0
5
10
15
20
25
30
35
40
45
50
crocidolite [1lg/m1] 100
90 80
~
70
.!!.l
60
Q) Co>
b.O
.s==
'E ;:::I
en
50 40 30
~
20 10
•
.....
0 0
20
40
60
80
100
120
nnneralfibres[Ilg/m1] -------- JM 104/475 - - - - B-1M
468
Exp Toxic Pathol45 (1993/94) 8
140
160
180
200
Fig. 1. Cytotoxicity of fibrous dusts in M3E3/C3. a. crocidolite asbestos; b. JM 104/475 (___._) and B-IM (-
for 60 min with the monoclonal antiactin IgM (Amersham) at a dilution of 1:200 in PBS. After intensive washing, cells were incubated with the secondary anti-mouse IgG conjugated with AMCA (Aminomethyl-coumarin acetic acid; Dianova) for 45 min. For tubulin labelling, a primary monoclonal antibody directed against a-tubulin (Sigma) was used after dilution to 1:200 in PBS (60 min) followed by incubation with a second anti-mouse IgG-AMCA for 45 minutes. The procedure was exactly the same as for the actin. For detection of vimentin, cells were fixed in methanol for 10 min at -20 DC. Permeabilization was performed in acetone for 2 min at -20 DC. An anti-vimentin IgG (Boehringer) in a dilution of 1:20 was used as the primary antibody (60 min) followed by incubation with an anti-mouse IgGAMCA for 45 min.
Results The application of increasing doses of crocidolite asbestos and the glass fibre JM 104/475 caused a significant decrease in the cell survival as compared with the untreated cells (figures la and Ib). Both fibre types showed a clear cytotoxicity which turned out to be three-
fold greater than for the concurrently examined glass fibre. No toxic effect has been estimated for the second glass fibre B-IM. These results were confirmed by the measurement of the colony forming efficiency (figures 2a and 2b). M3E3/C3 cells were more sensitive to crocidolite asbestos than to JM 104/475 or even B-IM. Transformation to anchorage independent growth could only be induced by crocidolite asbestos (figure 3) whereas both glass fibres revealed no effects in the transformation assay. As further biological parameters, the components of the cytoskeleton, actin, vimentin and tubulin were investigated for lesions induced by fibrous dusts. There is evidence that the cytoskeleton, a complex structure which is of major importance for the overall integrity of the cell, may be involved in cytotoxic processes after application of chemical and particulate substances. As a matter of fact, all fibres affected the actin filament system in a time-dependent manner. Different degrees of degeneration were observed in the tubulin and the vimentin organization. The untreated cells demonstrated characteristic features of actin stress fibre arrangement which appeared
100
90 ..--, ~
80
>. c.;
~
70
Q)
·8
S
60
Q)
<:Ul ~
·s....
-
50 40
0
>.
30
~
0
'0 c.;
20 10 0 0
5
10
15
20
25
30
35
40
45
50
140
160
180
200
Exp Toxic Pathol45 (1993/94) 8
469
crocidolite [~glmI) 120
~ ~
100
>. c.; ~
Q)
:~
80
:::: Q)
<:Ul ~
·s.... Fig. 2. Colony-forming efficiency of M3E3/C3 cells after exposure to mineral fibres. a. Crocidolite asbestos; b. JM 104/475 (-----) and B-1 M (-0--). Values represent the percentage of colony formation relative to the control group unexposed to the fibres.
.2
60
40
>.
= 0
"0 c.;
20
0 0
20
40
60
80
100
mineral fibres - - - JM 104/475
120
[~glmI]
- - - - - B-1M
800
.......... 700
~ ~600 I:: C1>
&500
/
~
I:: 400 0
:.::;
'"
E 300
-...'" 0
'"
I::
....,
200
100
0 0
1,5
crocidolite asbestos [Ilg/rnl]
as a well-represented network of fibres throughout the cytoplasm (figure 4a). After 38 h of exposure to crocidolite and JM 104/475 glass fibres, the filamentous structures of the actin system were broken down into a granular configuration (figure 4b). An early stage of restoration of the filaments occurred in cultured cells after 86 h of exposure. After treatment with the B-1 M glass fibres, the epithelial cells revealed a delayed reaction in actin depolymerization after 86 h of cultivation. The changes in actin configuration also correspond to a marked reduction in cell adhesion. The microtubule apparatus underwent no alterations in its organization after crocidolite exposure, whereas both glass fibres induced a complete depolymerization of the filamentous tubulin system after 86 h of fibre treatment (figures 5a and 5b). The vimentin network, which normally appeared in a filigree-like pattern throughout the cytoplasm (figure 6a), concentrated in bundles after treatment with croci do lite asbestos (figure 6b). In contrast, no changes could be observed in cells exposed to the glass fibres.
Discussion The results obtained demonstrated that the biological effects of the tested fibres differ to a great extent depending on fibre size and the type of fibrous dust. Crocidolite asbestos and the glass fibre JM 104/475 have comparable dimensions but the result for acute and chronic toxicity as well as transformation showed a stronger effect for the naturally occurring fibre type. Interestingly,
2
Fig. 3. Transformation frequency of M3E3/C3 cells after treatment with crocidolite asbestos. Values represent the transformation frequency relative to the control cells unexposed to fibres.
the number of critical fibres per mass is higher for the synthetic fibre, i.e. different mechanisms for the interaction between fibres and cells must be present. One hint for this hypothesis is the fact that the components of the cytoskeleton are affected to a different degree by different fibres. Crocidolite asbestos as well as JM 104/475 induce changes in the organization of the actin filament system. But with regard to the vimentin and tubulin systems, only the glass fibres show an influence on the tubulin system whereas the vimentin system is affected after application of crocidolite asbestos. These results are supported by studies on micronuclei formation (7). Glass fibres, especially, demonstrated a higher aneugenic potency which might be due to the observed disturbances in the microtubular apparatus. Crocidolite asbestos induced a pronounced clastogenic effect. The effects of the glass fibres with regard to the cytoskeleton and micronuclei formation are comparable, but differ regarding acute and chronic toxicity. This can be related to their varying dimensions which might influence the mode and mechanisms of interaction between the cells and fibres. Cytotoxic activity might be related to damages in the organization of adhesion plaques, specialized areas of cytoplasmic membranes which serve as attachment sites for stress fibres and are in close contact with the underlying substratum (8). Besides this structural function, adhesion plaques may also provide regions where regulatory signals are activated (9) and transmitted across membranes. Transmembrane signalling via specific ligand/receptor interactions induces the immediate polymerization of
Fig. 4. Fluorescence photomicrograph of actin in untreated M3E3/C3 cells showing a normal arrangement of actin fibres (4a) and in cells exposed to crocidolite asbestos (4b). Actin filament rearrangement was detectable in those cells exposed for 86 h. Fig. 5. Fluorescence photomicrographs. M3E3/C3 control cells showing the normal tubulin network (5a). After 86 h the exposure to B-IM glass fibres induced a depolymerization of the microtubular apparatus (5b). 470
Exp Toxic Pathol45 (1993/94) 8
4a\4b Sa Sb Exp Toxic Pathol 45 (1993/94) 8
471
6b
6a
Fig. 6. Fluorescence photomicrographs of vimentin in M3E3/C3 cells (6a). Exposure to crocidolite asbestos induced a concentration of vimentin within the cells at the expense of the normally anastomosing network (6b). actin and formation of microfilament assemblies close to the plasma membrane. A clue to the nature of the regulatory mechanisms involved was recently found in that phosphatidylinositoI4,5-biphosphate interacts with profilin, dissociates the microfilament precursor profilactin complex and thus liberates actin for polymerization (10). This suggests that the phosphatidylinositol (PI) cycle which plays an important role in cell regulation might control microfilament organization. There is also evidence for an opposite effect which has, up to now however, only been demonstrated for chemicals. But it cannot be excluded that there might be a link between the cycle and the microfilament desorganization induced by fibres. In conclusion, our results demonstrate that beyond the determination of classic toxic parameters the evaluation of the cytoskeleton, a morphologic marker system, offers some clues to the mechanisms for the observed differences in fibre action.
References 1. Environmental Health Criteria 53: Asbestos and other Natural Mineral Fibres. World Health Organization, Geneva, 1986. 2. NEUBERGER M, KUNDl M, FRIEDL HP: Environmental asbestos exposure and cancer mortality. Arch Environ Health 1984; 39: 261-265. 472
Exp Toxic Pathol45 (1993/94) 8
3. POTI' F, SCHLIPKOTER H-W, ROLLER M, et al.: Carcinogenicity of glass fibres with different durability. Zbl Hyg 1990; 189: 563-566. 4. EMURA M, RICHTER-REICHHELM H-B, BONING W, et al.: A fetal respiratory epithelial cell line for studying some problems of transplacental carcinogenesis in Syrian golden hamsters. J Cancer Res Clin Oncol 1982; 104: 133-144. 5. EMURA M, RIEBE M, GERMANN P, et al.: Functional culture of hamster and human airway epithelial cells and its application to pulmonary toxicology. Exp Pathol 1989; 37: 224-227. 6. EMURA M, RIEBE M, OCHIAI A, et al.: New functional cell-culture approach to pulmonary carcinogenesis and toxicology. J Cancer Res Clin Onco11990; 116: 557-562. 7. RIEBE-IMRE M, AUFDERHEIDE M, GARTNER-HuBSCH S, et al.: Cyto- and genotoxic effects of insoluble particles in vitro. 4th International Inhalation Symposium, 1st-5th March 1993, Hannover, Germany. 8. BURRIDGE K, MOLONY L, KELLY T: Adhesion plaques sites of membrane interaction between the extracellular matrix and the actin cytoskeleton. J Cell Sci Suppl 1987; 8: 211-229. 9. GENTRY LE, ROHRSCHNEIDER LR: Common features of the yes and scr gene products defined by peptide-specific antibodies. J Viro11984; 51: 539-546. 10. LASSING I, LINDBERG U: Evidence that the phosphatidylinositol cycle is linked to cell motility. Exp Cell Res 1988; 174: 1-15.
Institute of Experimental Pathology, Hannover Medical School, Germany
Differences in the biological effects of crocidolite asbestos and two glass fibres on epithelial lung cells M. AUFDERHEIDE, M. RIEBE-IMRE, M . STRAUB and A. PERAUD With 6 figures and 1 table Received: September 1, 1993; Accepted: September 16, 1993 Address for correspondence: Dr. M. AUFDERHEIDE, Institute of Experimental Pathology, Hannover Medical School, Konstanty-Gutschow-Str. 8, D - 30625 Hannover, Germany. Key words: Crocidolite asbestos; Asbestos, crocidolite; Glass fibres; Epithelial lung cells; Lung, epithelial cells; Mineral fibres; Fibrous glass.
Introduction
Material and methods
Asbestos, a naturally occurring mineral, is known to be potentially hazardous to man and experimental animals. Inhaled fibres induce proliferation of connective tissue and increase the risk of acquiring pulmonary carcinoma and mesothelioma in the lung (1, 2). In contrast, new types of fibrous glass reveal only a weak or no carcinogenic potency in animal experiments (3). The mechanisms by which fibres induce these effects are still under discussion. Since the role of fibre dimension, fibre durability and surface properties have been investigated in different studies, it was suggested that multiple mechanisms must be operative to explain the diverse effects of mineral fibres. The cytotoxic and carcinogenic potency have been studied thoroughly for the naturally occurring fibres like crocidolite, chrysotile or amosite, but only few data are available for synthetic fibres. In this study we tested crocido lite asbestos in comparison with two glass fibres JM 104/475 and B-IM for different biological effects in an in vitro system of epithelial hamster cells to elucidate differences in the interaction between cells and fibres.
Cell line: The Syrian hamster epithelial cell line (M3E3/C3) was derived from the lung of a foetus on day 15 of gestation. Cells were grown in a medium consisting mainly of RPMI 1640 (80 %, Flow, Meckenbeim) and fetal bovine serum (BSA; 20 %, Flow) as described previously (4). Cultures were maintained at 37 °C in a humidified atmosphere with 10 % CO 2 and 90 % air. Confluent cultures were harvested by trypsinization (0,25 % Trypsin, 2 % EDTA in PBS) and resuspended in the medium after low speed centrifugation. The epithelial character of the cell line and its application to pulmonary toxicology have been described elsewhere (5 , 6). Mineral fibres: All fibres tested were provided by Prof. Pon from the Medical Institute for Environmental Hygiene (University of Diisseldorf). The characterization of the fibre types was performed by Dr. BELLMANN, Fraunhofer Institute of Inhalation Toxicology and Aerosol Research, and is given in table 1. Cytotoxicity: M3E3/C3 cells (5 x 1()4 cells) were seeded in 25 cm2 Falcon flasks with 5 rol growth medium. 24 h later the cells were treated for 96 h with different doses of mineral
Table 1. Characterization of fibre dusts. fibre length (,..lm) 10 % < 50 % < 90 % <
fibre diameter (/lm) lO % < 50 % < 90 % <
99
0,9
1,8
4,7
0,11
0,19
0,32
158
0,7
2,3
8,2
0,06
0,14
0,38
6,7
10,7
21,1
0,88
1,68
2,49
Fibre
mass of critical fibres (%)
critical fibres/ng mean
Crocidolite
42
JM 104/475
88
B-IM
15
27
Exp Toxic Pathol45 (1993/94) 8
467
fibres (10, 20, 30, 50 Jlg crocidolite asbestos/ml; 50, 100, 150,200 Jlg JM 104/475 and B-IM/ml) which were suspended in medium after being sterilized. At the end of exposure, cells were trypsinized and cell viability was determined by the trypan blue exclusion method in a haematocytometer. Plating efficiency: From each treatment group 200 viable cells were plated in an 8 cm2 Petri dish (Falcon) and cultured under the above-mentioned culture and test conditions for 8 days without medium change. The cells were fixed in methanol, stained with Giemsa and colonies were counted. Plating efficiency was calculated as the percentage of colony formation relative to the control groups. Transformation: The determination of transformation was based on anchorage independent growth in soft agar. The treatment was carried out in the same way as for the cytotoxicity study (doses: a. crocidolite asbestos: 1, 1,5, 2 Jlg/ml; b. JM 104/475: 50, 100, 150 Jlg/ml; B-IM: 300, 400, 500 Jlg/ml). The cells of the different treatment groups were passaged by trypsinization 5 times at intervals depending on the growth speed in reaching about 80 % confluency. The cells were then brought into soft agar cultures to
detect anchorage-independent growth indicating transformation. The plating efficiency of the cells for each dose group was determined as described above. Six weeks later, evaluation of cultures was performed by counting only colonies with a diameter greater than 30 Jlm. Transformation frequency was calculated as the percentage of transformed colonies relative to the control cells unexposed to mineral fibres. Fluorescence Microscopy: Cells were grown on 13-mmdiam glass coverslips in 8 cm 2 Petri dishes for 86 h. Mineral fibres sterilized by autoclaving were suspended in growth medium. The resulting suspensions were added to a final volume of 5 ml at a final concentration of 10 Jlg crocidolite asbestos/ml and 50 Jlg JM 104/474/ml and 100 Jlg B-IM/ml. Immunofluorescence microscopy was performed after 14,32,62 and 86 hours of exposure. Cells were fixed in 4 % paraformaldehyde in PBS (20 min) for actin and tubulin detection. The whole preparation procedure was at 37 DC. After washing the cells in the same buffer, they were permeabilized with 1 % NP-40 in PBS for 10 min at room temperature, washed three times with PBS, incubated for 20 min with BSA to avoid non-specific reaction and washed again with PBS. For actin labelling, probes were incubated
100
90 80
~
70
en
60
~
Co>
b.O
= :~ ... ;:::I
en
50 40 30 20 10 0 0
5
10
15
20
25
30
35
40
45
50
crocidolite [1lg/m1] 100
90 80
~
70
.!!.l
60
Q) Co>
b.O
.s==
'E ;:::I
en
50 40 30
~
20 10
•
.....
0 0
20
40
60
80
100
120
nnneralfibres[Ilg/m1] -------- JM 104/475 - - - - B-1M
468
Exp Toxic Pathol45 (1993/94) 8
140
160
180
200
Fig. 1. Cytotoxicity of fibrous dusts in M3E3/C3. a. crocidolite asbestos; b. JM 104/475 (___._) and B-IM (-
for 60 min with the monoclonal antiactin IgM (Amersham) at a dilution of 1:200 in PBS. After intensive washing, cells were incubated with the secondary anti-mouse IgG conjugated with AMCA (Aminomethyl-coumarin acetic acid; Dianova) for 45 min. For tubulin labelling, a primary monoclonal antibody directed against a-tubulin (Sigma) was used after dilution to 1:200 in PBS (60 min) followed by incubation with a second anti-mouse IgG-AMCA for 45 minutes. The procedure was exactly the same as for the actin. For detection of vimentin, cells were fixed in methanol for 10 min at -20 DC. Permeabilization was performed in acetone for 2 min at -20 DC. An anti-vimentin IgG (Boehringer) in a dilution of 1:20 was used as the primary antibody (60 min) followed by incubation with an anti-mouse IgGAMCA for 45 min.
Results The application of increasing doses of crocidolite asbestos and the glass fibre JM 104/475 caused a significant decrease in the cell survival as compared with the untreated cells (figures la and Ib). Both fibre types showed a clear cytotoxicity which turned out to be three-
fold greater than for the concurrently examined glass fibre. No toxic effect has been estimated for the second glass fibre B-IM. These results were confirmed by the measurement of the colony forming efficiency (figures 2a and 2b). M3E3/C3 cells were more sensitive to crocidolite asbestos than to JM 104/475 or even B-IM. Transformation to anchorage independent growth could only be induced by crocidolite asbestos (figure 3) whereas both glass fibres revealed no effects in the transformation assay. As further biological parameters, the components of the cytoskeleton, actin, vimentin and tubulin were investigated for lesions induced by fibrous dusts. There is evidence that the cytoskeleton, a complex structure which is of major importance for the overall integrity of the cell, may be involved in cytotoxic processes after application of chemical and particulate substances. As a matter of fact, all fibres affected the actin filament system in a time-dependent manner. Different degrees of degeneration were observed in the tubulin and the vimentin organization. The untreated cells demonstrated characteristic features of actin stress fibre arrangement which appeared
100
90 ..--, ~
80
>. c.;
~
70
Q)
·8
S
60
Q)
<:Ul ~
·s....
-
50 40
0
>.
30
~
0
'0 c.;
20 10 0 0
5
10
15
20
25
30
35
40
45
50
140
160
180
200
Exp Toxic Pathol45 (1993/94) 8
469
crocidolite [~glmI) 120
~ ~
100
>. c.; ~
Q)
:~
80
:::: Q)
<:Ul ~
·s.... Fig. 2. Colony-forming efficiency of M3E3/C3 cells after exposure to mineral fibres. a. Crocidolite asbestos; b. JM 104/475 (-----) and B-1 M (-0--). Values represent the percentage of colony formation relative to the control group unexposed to the fibres.
.2
60
40
>.
= 0
"0 c.;
20
0 0
20
40
60
80
100
mineral fibres - - - JM 104/475
120
[~glmI]
- - - - - B-1M
800
.......... 700
~ ~600 I:: C1>
&500
/
~
I:: 400 0
:.::;
'"
E 300
-...'" 0
'"
I::
....,
200
100
0 0
1,5
crocidolite asbestos [Ilg/rnl]
as a well-represented network of fibres throughout the cytoplasm (figure 4a). After 38 h of exposure to crocidolite and JM 104/475 glass fibres, the filamentous structures of the actin system were broken down into a granular configuration (figure 4b). An early stage of restoration of the filaments occurred in cultured cells after 86 h of exposure. After treatment with the B-1 M glass fibres, the epithelial cells revealed a delayed reaction in actin depolymerization after 86 h of cultivation. The changes in actin configuration also correspond to a marked reduction in cell adhesion. The microtubule apparatus underwent no alterations in its organization after crocidolite exposure, whereas both glass fibres induced a complete depolymerization of the filamentous tubulin system after 86 h of fibre treatment (figures 5a and 5b). The vimentin network, which normally appeared in a filigree-like pattern throughout the cytoplasm (figure 6a), concentrated in bundles after treatment with croci do lite asbestos (figure 6b). In contrast, no changes could be observed in cells exposed to the glass fibres.
Discussion The results obtained demonstrated that the biological effects of the tested fibres differ to a great extent depending on fibre size and the type of fibrous dust. Crocidolite asbestos and the glass fibre JM 104/475 have comparable dimensions but the result for acute and chronic toxicity as well as transformation showed a stronger effect for the naturally occurring fibre type. Interestingly,
2
Fig. 3. Transformation frequency of M3E3/C3 cells after treatment with crocidolite asbestos. Values represent the transformation frequency relative to the control cells unexposed to fibres.
the number of critical fibres per mass is higher for the synthetic fibre, i.e. different mechanisms for the interaction between fibres and cells must be present. One hint for this hypothesis is the fact that the components of the cytoskeleton are affected to a different degree by different fibres. Crocidolite asbestos as well as JM 104/475 induce changes in the organization of the actin filament system. But with regard to the vimentin and tubulin systems, only the glass fibres show an influence on the tubulin system whereas the vimentin system is affected after application of crocidolite asbestos. These results are supported by studies on micronuclei formation (7). Glass fibres, especially, demonstrated a higher aneugenic potency which might be due to the observed disturbances in the microtubular apparatus. Crocidolite asbestos induced a pronounced clastogenic effect. The effects of the glass fibres with regard to the cytoskeleton and micronuclei formation are comparable, but differ regarding acute and chronic toxicity. This can be related to their varying dimensions which might influence the mode and mechanisms of interaction between the cells and fibres. Cytotoxic activity might be related to damages in the organization of adhesion plaques, specialized areas of cytoplasmic membranes which serve as attachment sites for stress fibres and are in close contact with the underlying substratum (8). Besides this structural function, adhesion plaques may also provide regions where regulatory signals are activated (9) and transmitted across membranes. Transmembrane signalling via specific ligand/receptor interactions induces the immediate polymerization of
Fig. 4. Fluorescence photomicrograph of actin in untreated M3E3/C3 cells showing a normal arrangement of actin fibres (4a) and in cells exposed to crocidolite asbestos (4b). Actin filament rearrangement was detectable in those cells exposed for 86 h. Fig. 5. Fluorescence photomicrographs. M3E3/C3 control cells showing the normal tubulin network (5a). After 86 h the exposure to B-IM glass fibres induced a depolymerization of the microtubular apparatus (5b). 470
Exp Toxic Pathol45 (1993/94) 8
4a\4b Sa Sb Exp Toxic Pathol 45 (1993/94) 8
471
6b
6a
Fig. 6. Fluorescence photomicrographs of vimentin in M3E3/C3 cells (6a). Exposure to crocidolite asbestos induced a concentration of vimentin within the cells at the expense of the normally anastomosing network (6b). actin and formation of microfilament assemblies close to the plasma membrane. A clue to the nature of the regulatory mechanisms involved was recently found in that phosphatidylinositoI4,5-biphosphate interacts with profilin, dissociates the microfilament precursor profilactin complex and thus liberates actin for polymerization (10). This suggests that the phosphatidylinositol (PI) cycle which plays an important role in cell regulation might control microfilament organization. There is also evidence for an opposite effect which has, up to now however, only been demonstrated for chemicals. But it cannot be excluded that there might be a link between the cycle and the microfilament desorganization induced by fibres. In conclusion, our results demonstrate that beyond the determination of classic toxic parameters the evaluation of the cytoskeleton, a morphologic marker system, offers some clues to the mechanisms for the observed differences in fibre action.
References 1. Environmental Health Criteria 53: Asbestos and other Natural Mineral Fibres. World Health Organization, Geneva, 1986. 2. NEUBERGER M, KUNDl M, FRIEDL HP: Environmental asbestos exposure and cancer mortality. Arch Environ Health 1984; 39: 261-265. 472
Exp Toxic Pathol45 (1993/94) 8
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