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Leaching of chrysotile asbestos in human lungs
EN\‘IR”NMENTAI.
14, 245-254 (1977)
RESEARCH
Leaching
of Chrysotile
Correlation
in Human
Lungs
with in Vitro Studies Using Rabbit Alveolar Macrophages
M.C. Ltrboratoirr Henri
Asbestos
de Biopathologie Mondor. Arseuue
JAURAND', Pulmonairr. De Lattrr
J. BICNON~
Universite De Tussigny,
Ptrris Val dr Mtrrne. C.H. U 94010 Crrteil. FrtrnccJ
P. SEBASTIEN Lubortrtoire
d‘Etude
des Purticules Inhalees. Direction de /‘Action (Dr. G. Bonnaud). Prefecture de Paris, France
Strnittrire
et Sociulr
AND
J. GOW Bureau
de Recherches
Geologiques Received
et Mini&es December
Orleunu.
La Source,
France
1. 1976
The chemical microanalysis of chrysotile fibers obtained from human lungs and from chrysotile phagocytosed in vitro by rabbit alveolar macrophages (AM) was carried out with the use of an energy dispersive spectrometer mounted on a scanning electron microscope (SEMI. This microanalysis was compared to natural fibers in order to investigate the chemical stability of chrysotile in biologic residence. The Mg versus Si content was estimated by the ratio of the peaks intensity Si:Mg. Lung samples were obtained from workmen with definite asbestos exposure, and chrysotile asbestos was extracted by chemical digestion. Phagocytosed chrysotile fibers were obtained after incubating standard chrysotile (A WCC) with rabbit alveolar macrophages for 24 hours or 5 days. Both chrysotile fibers from human lungs and from in vitro incubation with AM have shown an increase of the Si:Mg ratio in respect to standard fibers. Moreover. the results have shown that the magnesium leakage was not constant along the fiber axis and was different from one fiber to another.
INTRODUCTION
The in V~\YI leaching of chrysotile asbestos has been demonstrated by different investigators (Morgan et nl., 1971; Langer et al., 1972a, b; Jaurand et al., 1976); these authors have observed the release of some chemical elements from the fiber, whether constitutive (Mg) or trace (Co, Cr. Ni) elements. Moreover, in ~~itro studies have shown the chemical instability of chrysotile asbestos in different media (Chowdhury, 1975; Harris and Grimshaw. 1975; Thomassin et 01.. 1976): These results have shown a leakage of magnesium when using distilled water and mineral or organic acids. Elsewhere it was found that the specific area of this mineral increased after an attack made with oxalic acid (Johan et al., 1973). Modifications of the physicochemical properties of chrysotile asbestos or other minerals may have an important effect on the in I+\YI reactivity of the mineral: for ’ Attachee 2 Address
de Recherche INSERM. reprint requests to J. Bignon.
246
JALTRAND
ET AL
example increasing the adsorptive capacity for some toxic substances (gas, metals, hydrocarbons, etc.). Moreover itz \vivo changes in the chemical composition of the fibers may be able to modify the cell reactivity and, on the other hand, bring difficulties in the identification of the fiber. The in \ri~ instability of chrysotile has been demonstrated but its causes have remained unknown. Do products of cell metabolism play a part in the leakage of magnesium? An answer can be provided with the help of in vitro tests using chrysotile of well-known chemical compositions. The aim of the present work was to study the chemical behavior of chrysotile asbestos in biologic residence. The study was carried out on chrysotile fibers obtained from human lungs of workers exposed during their life (in \zi~o) to asbestos fibers. The results were compared to an in vitro study using rabbit alveolar macrophages incubated with standard UICC chrysotile A fibers. The stability of chrysotile was assumed to be related to the silicon/magnesium ratio as measured by means of a scanning electron microscope (SEM) fitted with an energy dispersive spectrometer. MATERIALS
AND METHODS
1. Method for Isolatirzg Coated and Urzcoated Fibers from the Lurzg Intrapulmonary fibers were obtained from lung parenchyma according to a previously described method (Bignon et al., 1970, 1973) and slightly modified for the transmission electron microscope (TEM) analysis. Lung samples were fixed in 10% formaldehyde previously filtered through a Millipore membrane (3-,um pore size); the inorganic material was destroyed by the immersion of the samples in a 50% sodium hypochlorite solution at room temperature. A filtration was made through a Nuclepore membrane (porosity, 0.8 Frn) precoated with a carbon film; the tube was rinsed several times with filtered distilled water, in order to recover all the particles. The membrane received a second carbon layer so that particles were sandwiched between the two layers. The Nuclepore membrane was then laid down on indexed grids and dissolved by chloroform. Grids were examined under a JEOL 100 C TEM to identify and localize fibers. The same fibers were analyzed with a JEOL U-3 SEM. 2. Corztrol Chrysotile Fibers Native chrysotile fibers from Thetford mines (Canada) and chrysotile A supplied by the UICC were processed according to the above description in order to find out if the 10% formaldehyde and sodium hypochlorite treatment altered their chemical composition. The fibers were suspended in a 10% formaldehyde solution. An aliquot was sampled every week and added to the sodium hypochlorite solution for 24 hours. The suspended fibers were collected on a Millipore membrane by filtration, and randomly sampled by means of a Fonbrune micro manipulator. Thereafter, they were placed on a copper grid for X-ray chemical microanalysis. 3. Study of Chrysotile Plzagocytosed in Vitro Alveolar macrophages were obtained from anesthetized rabbits (EC 601 EVIC CEBA) by pulmonary lavage. The animals were intubated through a tracheotomy; thereafter, a bilateral pneumothorax was created to collapse the lungs. Then.
LEACHING
OF
CHRYSOTILE
ASBESTOS
247
lungs were infused three times with 50 to 60 ml of NaClO.9% at 37°C through the trachea. The lavage fluid was collected at +4”C and centrifuged (27Og for 20 minutes). The sediment was suspended (5 x 10’ cells/ml) in Hanks’ solution. As judged by the May Griinwald staining, 95% or more of the cells were macrophages and by the Nigrosin viability test, 95% of the macrophages were viable. For the Nigrosin test. cells were incubated 7 minutes at 37°C with Nigrosin at a final concentration of 0.2% in Hanks’ solution. The cells were allowed to settle 1 hour in Falcon flasks (5 ml/flask); then. the supernatant fluid was replaced by Eagle (BME) medium supplemented with 0.3 1% w/v of bovine serum albumin (Fraction V Calbiochem) and with 15% tryptose broth (Gibco), (Hollande 1976). After 24 hours, the supernatant fluid was removed and replaced with 5 ml of fresh medium for 1 hour; chrysotile was then added to the medium (75 pgiml). All incubations were made in an atmosphere of 95% air + 5% CO,. After 24 hours or after 5 days, the cells were washed several times with Hanks’ solution, then fixed in 2.3% glutaraldehyde in 0.045 M cacodylate buffer (pH: 7.2, 380 mOsm) for 1 hour. The cells were rinsed with cacodylate buffer, dehydrated with ethanol, and then air dried. A polyvinyl alcohol solution was spread over the cells and left to solidify at 37°C. The film including macrophages was peeled from the Falcon flask, laid on a Nuclepore membrane, and dissolved by filtration with Hanks’ solution or water at 40°C. The Nucleopore membrane was then put onto grids as described in the first paragraph. The results obtained with this method are illustrated in Fig. 1. It shows the cytoplasmic processes which characterize the adherence of AM in culture. It was noticed that the various steps of the method produced a retraction of the cell. 4. Chemical Analysis of Asbestos Fibers Chrysotile fibers were first identified by TEM according to a method previously described (Sebastien et (il., 1975). The fibers selected for SEM examination were first located on the grids under the TEM and analyzed under the SEM. Semiquantitative analysis was carried out by a Si (Li) energy dispersive spectrometer. After the accumulation of the spectrum of fibers, the “LG” program (least-squares fit to Gaussian) was applied to compute the integral value above the background. The ratio of Si:Mg peaks was determined. RESULTS
I. Control Fibers Analysis Table 1 shows the Si:Mg ratio of Thetford chrysotile samples treated with formaldehyde and sodium hypochlorite compared to untreated fibers. The nearly constant values of the ratio showed that treating chrysotile by these two chemicals did not change the Si and Mg contents in chrysotile. This ratio varied from 0.99 to 1.04 for formaldehyde incubation from 1 to 15 weeks. The slight deviation was due to electronic variations of the analytic device. Similar results were found when UICC A chrysotile fibers were studied. 2. Atdysis of Chrysotile Obtained from Htrrmtl Lungs From three patients, 16 samples of lung tissue were digested and 10 fibers (coated and uncoated) were analyzed in each sample. Table 2 shows for each
248
JAC’RAND
E7 AL
FIG. 1. Rabbit alveolar macrophages incubated in ,zitvo with UICC A chrysotile fibers. The cells were peeled off from the Falcon flask as described under Material and Methods. Note the cytoplasmic expansions at the cell periphery (4). (A) SEM secondary emission x 4760. (B) TEM x 4760.
patient the length of exposure to asbestos, the number of asbestos fibers, and the percentage of chrysotile fibers in lung parenchyma (case Nos. 1 and 2) or in sputum (case No. 3), according to the method previously described (Sebastien et (II., 1975). The Si:Mg ratios of these fibers were larger than those of the control fibers (Table 3). It was also shown that these ratios were not identical at various points along the fiber axis (Figs. 2 and 3), indicating that the leakage of magnesium was not homogeneous. In some coated fibers the magnesium signal was no longer detectable. None of the ratios obtained at the level of coated and uncoated fibers from human lungs were near the Si:Mg value found in control fibers, which was about TABLE Si:Mg R.+.~os TREATMEXT
OWAINED WITH
Incubation time in the formaldehyde solution (weeks) 1 2 5 6 7 15
~~olr-1 THUFORU FORMALDEHYDE
I CHRSSOXU .~ND SODIUM
FIBERS
AFTER
HYPOCHLORITE
Natural fibers
Treated fibers
Treated: natural ratio
1.16 1.19 1.42 1.43 1.25 1.30
1.18 1.18 1.48 1.47 1.29 1.33
1.01 0.99 1.04 1.03 1.03 1.02
LEACHING
OF
CHRYSOTILE
TABLE ASBWI~S
EXPOSURE
CHRYSWILE Case
No.
Sex Age Years of exposure Years postexposure Number of fibers/cm3 of parenchyma Number of fibers in sputum Percentage of chrysotile in all fibers
CONTENI
2
OF THE INVESTIGATED OF THE
249
ASBESTOS
BIOLOGIC 1
F 39 3 20 3 ‘106 ni” 65
CASES AND SPECIMENS
2 M 68 13 42 2 ,106 ni” 55
3 M 38 10 9 ni” 10” 70
‘I ni: not investigated.
one. Moreover, the value was nearly constant along the control fibers as opposed to that for intrapulmonary fibers. The spatial variations of this ratio may be looked upon as an acquired character by the fiber during its period in biologic residence. 3. Analysis of Itltrncelhrlnr Chrvsotile Phngocytosed itI Virro The Si:Mg ratios for chrysotile fibers also increased after phagocytosis by rabbit alveolar macrophages (AM) either for 24 hours or for 5 days. Data are given in Table 4 and compared first to those of fibers incubated at 37°C for 24 hours in the same medium as that used for AM and next to fibers suspended in physiologic saline (NaCl, 0.9%). It was noted that the chemical components of the culture medium did not modify the Si:Mg ratio. When the phagocytosed fibers were analyzed this ratio increased. Figure 4 shows the picture obtained with the TEM and the SEM. and the chemical analysis carried out on a particular point marked on the fiber. In contrast, the values for extracellular fibers were similar to those for control fibers incubated without macrophages. When counts were made at various points along the fiber axis, different values were found, as was the case for intrapulmonary fibers (Table 5). Thus, in vitro phagocytosed fibers showed the same qualitative characteristics (i.e. heterogeneous leakage of magnesium) as intrapulmonary fibers. DlSCUSSlON The results obtained here may be discussed, keeping in mind the results of an in tlitro leaching experiment carried out on chrysotile when using oxalic acid (Johan et al., 1973). In such an experiment, a leakage of magnesium was found and chrysotile became a structure of hydroxylated silica whose adsorbing properties could be greatly increased. It is well known that cell metabolism involves many biochemical factors having various chemical functions which are able to attack fibers phagocytosed either in \‘itr’o. or in ~i\lo. This fact may account for a decrease in magnesium related to silicon content in fibers staying in a biologic residence, as demonstrated by the increase of the Si:Mg ratio in the irz \Gfro experiments and by the related results in the study of intrapulmonary resident fibers. Our results regarding fibers obtained from human lungs agree with the results found previously by Langer et al. (1972a,
250
JAURAN’D
Si:Mg Case No. 1
RATIOS
OB.I AWED
ON
Elements presents Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Fe Fe
Fe Fe Fe Fe Fe
TABLE 3 SEVERAI. FIBERS
CF
-
Mg Mg Mg Mg Mg
40.2 29.42 28.4 28.29 34.46
1. Si 2. Si 3. Si 4. Si 5. Si 6. Si 1. Si 2. Si
-
Mg Mg Mg Mg Mg Mg Mg Mg
18.5 22.4 26.5 23.5 39.5 40.5 25.8 49.6
- Fe - Fe - Fe
PA.TIESTS”
3.89 3.58 2.04 5.44 2.09 8.26 9.56 7.73 5.3 6.1 5.6 17.9 4.3 47 23.4 19 35 8.44 10.32 10 x x 3c a2 cx x 7.8 x z%
1. Si 2. Si 1. Si 2. Si 3. Si
- Fe
THREE
State of the fibeld
~ -
- Fe - Fe
FROM
Si:Mg ratio
1. Si 2. Si 3. Si 4. Si 5. Si 1. Si 2. Si 3. Si 4. Si 1. Si 2. Si 3. Si 4. Si 1. Si 2. Si 3. Si 4. Si 1. Si 2. Si 3. Si 4. Si 5. Si 1. Si 2. Si 3. Si 4. Si 5. Si 6. Si 7. Si
- Mg
-
ET AL
(I On each fiber, the ratios at several points along the fiber axis are reported. ‘! CF. coated fiber: UF. uncoated fiber.
UF
CF
CF
CF
CF
UF
UF
UF
UF
LEACHING
FIG. carried
OF CHRYSOTILE
ASBESTOS
2. Chrysotile fibers obtained from human lung (case No. 2). The SEM microanalysis out, respectively, at a, b, and c. Si:Mg ratios: a, 28.4: b. 34.5; c. 28.3. TEM x 22.500.
was
b). using the same principles of analysis, i.e., X-ray spectrometry, but with a different device and treatment of the emission signals. The ikz vitro tests carried out in this study with alveolar macrophages have shown the rapid effect of cells on fibers. The kinetic studies of magnesium leakage obtained with ESCA (Emission Spectroscopy for Chemical Analysis) (Thomassin et nl., 1976) after treating asbestos with biologic organic acids have also shown an early action of these acids (from the first minutes). It should be noticed that the measurements made on intrapulmonary fibers as
252
JAURAND
ET AL
I
%
4
*
FIG. 3. Asbestos body obtained from hur nan lung (case No. 1). Si:Mg ratios were a. 6.1; b, 5.6.; c. 17,9; d, 4.2. (A) TEM x 3808, (B) SEM x 2040 (transmitted electrons). TABLE
4
RATIOS OBTAISED FROM UICC A CHR~SOTIII INCUBATED IN TEST MEDIA (EAGLE BME) AND PHACOCV-IOSED BY RABBIT ALVEOLAR MACROPHAGES IN .THE SAME MEDIAN
III Vitro
SmnrEs-si:Mg
Medium
Si : Mg ratios
NaCl 0.9%
1.82 - 2.05 1.93 - 2.51 2.20 - 1.49 2.67 - 2.54 2.65 - 1.89 1.98 - 1.94 24 hours 5.40 9.37 5 days5.11 4.97
Eagle BME
Phagocytosed fibers
1.99 2.00 2.88 1.78 - 8.54 - 6.75 4.14 - 3.91
” The lowest and highest values in each group are in italic type. TABLE Si:Mg RA-1-10s OWAISED Fiber No. 1 2 3 4
FROM irt Vitro
5 PHAW~YT~SED
CHR~SO.I.ILE
FIBERS
Si : Mg ratios 3.31 5.23 5.64 6.65 7.40 4.34
-
4.89 4.25 5.79 5.07 6.49 11.6
4.96 4.84 9.99 8.70 6.38
” The results on four fibers and the values related to different counts along the axis of the fiber are reported
LEACHING
OF
CHRYSOTILE
ASBESTOS
253
FIG. 4. SEM microanalysis on Bchrysotile fibert;hago$tzed in by rabbit alveolar macrophage. (A) secondary emission SEM x 1768, (B) transmitted electrons TEM x 2720, (C) X-ray spectra of the fiber at a point (arrow) which likely was intracellular before processing.
well as those made on phagocytosed fibers gave very different Si:Mg ratios from one fiber to another, and, for a single fiber. values differed along the fiber axis. It is unlikely that all inhaled fibers undergo the same aggression from the biologic environment at the same time. Indeed, the fibers’ localization in the lung tissue, their physical state when inhaled, the time they spend in biologic residence, and their properties may be important factors in modifying the intensity of magnesium leakage. The nonhomogeneous leakage of magnesium may lead to a different reactivity at different points along the fiber axis according to the chemical content: this may be due to the products of the AM metabolism. These cells have an important aerobic metabolism involving various carboxylic and a-cetonic acids which should be able to attack the surface of chrysotile fibers. Then, chrysotile reactivity may be highly modified: Variations of the superficial net charge are able to alter the nature of the cell-mineral interaction; modifications of the sorptive reactions toward exogenous toxic substances (components of cigarette smoke, metals. gas. and so on), or toward some endogenous proteins. So Desai rt crl. (1975) have shown the adsorption of hemoglobin by natural chrysotile. Likewise, using albumin, a protein of equivalent molecular weight, Morgan (1974) also demonstrated this adsorption; in his study he found a decrease in the amount of adsorbed albumin versus magnesium leakage with fibers treated by 0. I N HCl. Therefore, the structural and chemical in rirc~ modifications of chrysotile fibers may have an effect on the mineral reactivity. In a study still in progress, we have shown that the residue of silica remaining after mineral acid attack (HCl) was amorphous silica. thus physically entirely different from the network of cristalline silica remaining after oxalic acid attack (Johan et crl., 1973). The conditions for chrysotile leaching with organic acid seem more like the it? iris) situations than the conditions for leaching with mineral acid.
254
JAURAND
ET AL
ACKNOWLEDGMENTS Chrysotile fibers used in this investigation were supplied by Dr. V. Timbre11 of the Medical Research Council. Pneumoconiosis Research Unit, Penarth. The authors wish to acknowledge the technical assistance of Mrs L. Magne in cell culture and of Mr. X. Janson in electron microscopy. The microanalysis study has been carried out in the laboratories of the Department of BRGM. with the helpful collaboration of P. Jeanrot.
REFERENCES 1. Bignon, J.. Goni, J., Bonnaud. G., Jaurand. M. C., Dufour, G.. and Pinchon. M. C. (1970). Incidence of pulmonary ferruginous bodies in France. Environ. Res. 3, 430-442. 2. Bignon, J., Depierre. A., Bonnaud, G.. Goni. J.. and Brouet. G. (1973). Mise en evidence des corps ferrugineux par microfiltration de I’expectoration. Correlation avec le risque asbestosique. N. Press. Med. 2, 1697-1700. 3. Chowdhury. S. ( 1975). Kinetics of leaching of asbestos mineral at body temperature. J. Appl. Chem. Bio. Technol. 25, 347-353. 4. Desai. R.. Hext. P.. and Richards, R. (1975). The prevention of asbestos induced hemolysis. L$e Sci. 16, 1931-1938. 5. Harris. A. M.. and Grimshaw. R. W. (1975). The leaching of ground chrysotile. In “Proceeding of the Third International Conference on the Chemistry and Physics of Asbestos, Lava1 University. Quebec. Vol. 4 (17). pp. l-12. 6. Hollande. E. (1976). Personal communication. 7. Jaurand. M. C., Goni, J.. Jeanrot. P., Sebastien, P.. and Bignon. J. (1976). Solubilite du chrysotile in vitro et dans le poumon humain. Rev. Jr. MaI. Resp. Suppl. 4 (2). II I-120. 8. Johan. 2. A.. Goni. J., Sarcia. C., Bonnaud. G.. and Bignon, J. (1973). Influence de certains acides organiques sur la stabilite du reseau du chrysotile 6th congres int. geoch. organique. Technip Ed. Paris. 883-903. 9. Langer. A. M.. Rubin. I. B.. and Selikoff. 1. J. (1972a). Chemical characterization of asbestos body cores by electron microprobe analysis. J. Histochern. Cytochem. 20. 723-734. IO. Langer. A. M.. Rubin. I. B., Selikoff. I. J., and Pooley. F. D. (1972b). Chemical characterization of uncoated asbestos fibers from the lungs of asbestos workers by electron microprobe analysis. J. Histochem. C’ytochrm. 20, 735-740. Il. Morgan. A., Holmes, A.. and Gold, C. (1971). Studies of the solubility of constituents of chrysotile asbestos in virw using radioactive tracer techniques. Environ. ReJ. 4, 558-570. 12. Morgan. A. ( 1974). Adsorption of human serum albumin by asbestiform minerals and its application to the measurement of surface areas of dispersed samples of chrysotile. Environ. Res. 7, 330341. 13. Sebastien, P., Bignon, J.. Fondimare. M. A.. Mineau M. A., and Janson. X. (1975). Application de la microscopic electronique a I’etude quantitative de l’empoussierage pulmonaire asbestosique. J. Microsc. Biol. Cell. A 22, 35. 14. Thomassin. H.. Goni. J., Baillif, P.. and Touray, J. C. (1976). Etude par spectrometrie ESCA des premiers stades de la lixiviation du chrysotile en milieu acide organique. C. R. Acrid. Sci. Paris 283, 131-134.
14, 245-254 (1977)
RESEARCH
Leaching
of Chrysotile
Correlation
in Human
Lungs
with in Vitro Studies Using Rabbit Alveolar Macrophages
M.C. Ltrboratoirr Henri
Asbestos
de Biopathologie Mondor. Arseuue
JAURAND', Pulmonairr. De Lattrr
J. BICNON~
Universite De Tussigny,
Ptrris Val dr Mtrrne. C.H. U 94010 Crrteil. FrtrnccJ
P. SEBASTIEN Lubortrtoire
d‘Etude
des Purticules Inhalees. Direction de /‘Action (Dr. G. Bonnaud). Prefecture de Paris, France
Strnittrire
et Sociulr
AND
J. GOW Bureau
de Recherches
Geologiques Received
et Mini&es December
Orleunu.
La Source,
France
1. 1976
The chemical microanalysis of chrysotile fibers obtained from human lungs and from chrysotile phagocytosed in vitro by rabbit alveolar macrophages (AM) was carried out with the use of an energy dispersive spectrometer mounted on a scanning electron microscope (SEMI. This microanalysis was compared to natural fibers in order to investigate the chemical stability of chrysotile in biologic residence. The Mg versus Si content was estimated by the ratio of the peaks intensity Si:Mg. Lung samples were obtained from workmen with definite asbestos exposure, and chrysotile asbestos was extracted by chemical digestion. Phagocytosed chrysotile fibers were obtained after incubating standard chrysotile (A WCC) with rabbit alveolar macrophages for 24 hours or 5 days. Both chrysotile fibers from human lungs and from in vitro incubation with AM have shown an increase of the Si:Mg ratio in respect to standard fibers. Moreover. the results have shown that the magnesium leakage was not constant along the fiber axis and was different from one fiber to another.
INTRODUCTION
The in V~\YI leaching of chrysotile asbestos has been demonstrated by different investigators (Morgan et nl., 1971; Langer et al., 1972a, b; Jaurand et al., 1976); these authors have observed the release of some chemical elements from the fiber, whether constitutive (Mg) or trace (Co, Cr. Ni) elements. Moreover, in ~~itro studies have shown the chemical instability of chrysotile asbestos in different media (Chowdhury, 1975; Harris and Grimshaw. 1975; Thomassin et 01.. 1976): These results have shown a leakage of magnesium when using distilled water and mineral or organic acids. Elsewhere it was found that the specific area of this mineral increased after an attack made with oxalic acid (Johan et al., 1973). Modifications of the physicochemical properties of chrysotile asbestos or other minerals may have an important effect on the in I+\YI reactivity of the mineral: for ’ Attachee 2 Address
de Recherche INSERM. reprint requests to J. Bignon.
246
JALTRAND
ET AL
example increasing the adsorptive capacity for some toxic substances (gas, metals, hydrocarbons, etc.). Moreover itz \vivo changes in the chemical composition of the fibers may be able to modify the cell reactivity and, on the other hand, bring difficulties in the identification of the fiber. The in \ri~ instability of chrysotile has been demonstrated but its causes have remained unknown. Do products of cell metabolism play a part in the leakage of magnesium? An answer can be provided with the help of in vitro tests using chrysotile of well-known chemical compositions. The aim of the present work was to study the chemical behavior of chrysotile asbestos in biologic residence. The study was carried out on chrysotile fibers obtained from human lungs of workers exposed during their life (in \zi~o) to asbestos fibers. The results were compared to an in vitro study using rabbit alveolar macrophages incubated with standard UICC chrysotile A fibers. The stability of chrysotile was assumed to be related to the silicon/magnesium ratio as measured by means of a scanning electron microscope (SEM) fitted with an energy dispersive spectrometer. MATERIALS
AND METHODS
1. Method for Isolatirzg Coated and Urzcoated Fibers from the Lurzg Intrapulmonary fibers were obtained from lung parenchyma according to a previously described method (Bignon et al., 1970, 1973) and slightly modified for the transmission electron microscope (TEM) analysis. Lung samples were fixed in 10% formaldehyde previously filtered through a Millipore membrane (3-,um pore size); the inorganic material was destroyed by the immersion of the samples in a 50% sodium hypochlorite solution at room temperature. A filtration was made through a Nuclepore membrane (porosity, 0.8 Frn) precoated with a carbon film; the tube was rinsed several times with filtered distilled water, in order to recover all the particles. The membrane received a second carbon layer so that particles were sandwiched between the two layers. The Nuclepore membrane was then laid down on indexed grids and dissolved by chloroform. Grids were examined under a JEOL 100 C TEM to identify and localize fibers. The same fibers were analyzed with a JEOL U-3 SEM. 2. Corztrol Chrysotile Fibers Native chrysotile fibers from Thetford mines (Canada) and chrysotile A supplied by the UICC were processed according to the above description in order to find out if the 10% formaldehyde and sodium hypochlorite treatment altered their chemical composition. The fibers were suspended in a 10% formaldehyde solution. An aliquot was sampled every week and added to the sodium hypochlorite solution for 24 hours. The suspended fibers were collected on a Millipore membrane by filtration, and randomly sampled by means of a Fonbrune micro manipulator. Thereafter, they were placed on a copper grid for X-ray chemical microanalysis. 3. Study of Chrysotile Plzagocytosed in Vitro Alveolar macrophages were obtained from anesthetized rabbits (EC 601 EVIC CEBA) by pulmonary lavage. The animals were intubated through a tracheotomy; thereafter, a bilateral pneumothorax was created to collapse the lungs. Then.
LEACHING
OF
CHRYSOTILE
ASBESTOS
247
lungs were infused three times with 50 to 60 ml of NaClO.9% at 37°C through the trachea. The lavage fluid was collected at +4”C and centrifuged (27Og for 20 minutes). The sediment was suspended (5 x 10’ cells/ml) in Hanks’ solution. As judged by the May Griinwald staining, 95% or more of the cells were macrophages and by the Nigrosin viability test, 95% of the macrophages were viable. For the Nigrosin test. cells were incubated 7 minutes at 37°C with Nigrosin at a final concentration of 0.2% in Hanks’ solution. The cells were allowed to settle 1 hour in Falcon flasks (5 ml/flask); then. the supernatant fluid was replaced by Eagle (BME) medium supplemented with 0.3 1% w/v of bovine serum albumin (Fraction V Calbiochem) and with 15% tryptose broth (Gibco), (Hollande 1976). After 24 hours, the supernatant fluid was removed and replaced with 5 ml of fresh medium for 1 hour; chrysotile was then added to the medium (75 pgiml). All incubations were made in an atmosphere of 95% air + 5% CO,. After 24 hours or after 5 days, the cells were washed several times with Hanks’ solution, then fixed in 2.3% glutaraldehyde in 0.045 M cacodylate buffer (pH: 7.2, 380 mOsm) for 1 hour. The cells were rinsed with cacodylate buffer, dehydrated with ethanol, and then air dried. A polyvinyl alcohol solution was spread over the cells and left to solidify at 37°C. The film including macrophages was peeled from the Falcon flask, laid on a Nuclepore membrane, and dissolved by filtration with Hanks’ solution or water at 40°C. The Nucleopore membrane was then put onto grids as described in the first paragraph. The results obtained with this method are illustrated in Fig. 1. It shows the cytoplasmic processes which characterize the adherence of AM in culture. It was noticed that the various steps of the method produced a retraction of the cell. 4. Chemical Analysis of Asbestos Fibers Chrysotile fibers were first identified by TEM according to a method previously described (Sebastien et (il., 1975). The fibers selected for SEM examination were first located on the grids under the TEM and analyzed under the SEM. Semiquantitative analysis was carried out by a Si (Li) energy dispersive spectrometer. After the accumulation of the spectrum of fibers, the “LG” program (least-squares fit to Gaussian) was applied to compute the integral value above the background. The ratio of Si:Mg peaks was determined. RESULTS
I. Control Fibers Analysis Table 1 shows the Si:Mg ratio of Thetford chrysotile samples treated with formaldehyde and sodium hypochlorite compared to untreated fibers. The nearly constant values of the ratio showed that treating chrysotile by these two chemicals did not change the Si and Mg contents in chrysotile. This ratio varied from 0.99 to 1.04 for formaldehyde incubation from 1 to 15 weeks. The slight deviation was due to electronic variations of the analytic device. Similar results were found when UICC A chrysotile fibers were studied. 2. Atdysis of Chrysotile Obtained from Htrrmtl Lungs From three patients, 16 samples of lung tissue were digested and 10 fibers (coated and uncoated) were analyzed in each sample. Table 2 shows for each
248
JAC’RAND
E7 AL
FIG. 1. Rabbit alveolar macrophages incubated in ,zitvo with UICC A chrysotile fibers. The cells were peeled off from the Falcon flask as described under Material and Methods. Note the cytoplasmic expansions at the cell periphery (4). (A) SEM secondary emission x 4760. (B) TEM x 4760.
patient the length of exposure to asbestos, the number of asbestos fibers, and the percentage of chrysotile fibers in lung parenchyma (case Nos. 1 and 2) or in sputum (case No. 3), according to the method previously described (Sebastien et (II., 1975). The Si:Mg ratios of these fibers were larger than those of the control fibers (Table 3). It was also shown that these ratios were not identical at various points along the fiber axis (Figs. 2 and 3), indicating that the leakage of magnesium was not homogeneous. In some coated fibers the magnesium signal was no longer detectable. None of the ratios obtained at the level of coated and uncoated fibers from human lungs were near the Si:Mg value found in control fibers, which was about TABLE Si:Mg R.+.~os TREATMEXT
OWAINED WITH
Incubation time in the formaldehyde solution (weeks) 1 2 5 6 7 15
~~olr-1 THUFORU FORMALDEHYDE
I CHRSSOXU .~ND SODIUM
FIBERS
AFTER
HYPOCHLORITE
Natural fibers
Treated fibers
Treated: natural ratio
1.16 1.19 1.42 1.43 1.25 1.30
1.18 1.18 1.48 1.47 1.29 1.33
1.01 0.99 1.04 1.03 1.03 1.02
LEACHING
OF
CHRYSOTILE
TABLE ASBWI~S
EXPOSURE
CHRYSWILE Case
No.
Sex Age Years of exposure Years postexposure Number of fibers/cm3 of parenchyma Number of fibers in sputum Percentage of chrysotile in all fibers
CONTENI
2
OF THE INVESTIGATED OF THE
249
ASBESTOS
BIOLOGIC 1
F 39 3 20 3 ‘106 ni” 65
CASES AND SPECIMENS
2 M 68 13 42 2 ,106 ni” 55
3 M 38 10 9 ni” 10” 70
‘I ni: not investigated.
one. Moreover, the value was nearly constant along the control fibers as opposed to that for intrapulmonary fibers. The spatial variations of this ratio may be looked upon as an acquired character by the fiber during its period in biologic residence. 3. Analysis of Itltrncelhrlnr Chrvsotile Phngocytosed itI Virro The Si:Mg ratios for chrysotile fibers also increased after phagocytosis by rabbit alveolar macrophages (AM) either for 24 hours or for 5 days. Data are given in Table 4 and compared first to those of fibers incubated at 37°C for 24 hours in the same medium as that used for AM and next to fibers suspended in physiologic saline (NaCl, 0.9%). It was noted that the chemical components of the culture medium did not modify the Si:Mg ratio. When the phagocytosed fibers were analyzed this ratio increased. Figure 4 shows the picture obtained with the TEM and the SEM. and the chemical analysis carried out on a particular point marked on the fiber. In contrast, the values for extracellular fibers were similar to those for control fibers incubated without macrophages. When counts were made at various points along the fiber axis, different values were found, as was the case for intrapulmonary fibers (Table 5). Thus, in vitro phagocytosed fibers showed the same qualitative characteristics (i.e. heterogeneous leakage of magnesium) as intrapulmonary fibers. DlSCUSSlON The results obtained here may be discussed, keeping in mind the results of an in tlitro leaching experiment carried out on chrysotile when using oxalic acid (Johan et al., 1973). In such an experiment, a leakage of magnesium was found and chrysotile became a structure of hydroxylated silica whose adsorbing properties could be greatly increased. It is well known that cell metabolism involves many biochemical factors having various chemical functions which are able to attack fibers phagocytosed either in \‘itr’o. or in ~i\lo. This fact may account for a decrease in magnesium related to silicon content in fibers staying in a biologic residence, as demonstrated by the increase of the Si:Mg ratio in the irz \Gfro experiments and by the related results in the study of intrapulmonary resident fibers. Our results regarding fibers obtained from human lungs agree with the results found previously by Langer et al. (1972a,
250
JAURAN’D
Si:Mg Case No. 1
RATIOS
OB.I AWED
ON
Elements presents Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Fe Fe
Fe Fe Fe Fe Fe
TABLE 3 SEVERAI. FIBERS
CF
-
Mg Mg Mg Mg Mg
40.2 29.42 28.4 28.29 34.46
1. Si 2. Si 3. Si 4. Si 5. Si 6. Si 1. Si 2. Si
-
Mg Mg Mg Mg Mg Mg Mg Mg
18.5 22.4 26.5 23.5 39.5 40.5 25.8 49.6
- Fe - Fe - Fe
PA.TIESTS”
3.89 3.58 2.04 5.44 2.09 8.26 9.56 7.73 5.3 6.1 5.6 17.9 4.3 47 23.4 19 35 8.44 10.32 10 x x 3c a2 cx x 7.8 x z%
1. Si 2. Si 1. Si 2. Si 3. Si
- Fe
THREE
State of the fibeld
~ -
- Fe - Fe
FROM
Si:Mg ratio
1. Si 2. Si 3. Si 4. Si 5. Si 1. Si 2. Si 3. Si 4. Si 1. Si 2. Si 3. Si 4. Si 1. Si 2. Si 3. Si 4. Si 1. Si 2. Si 3. Si 4. Si 5. Si 1. Si 2. Si 3. Si 4. Si 5. Si 6. Si 7. Si
- Mg
-
ET AL
(I On each fiber, the ratios at several points along the fiber axis are reported. ‘! CF. coated fiber: UF. uncoated fiber.
UF
CF
CF
CF
CF
UF
UF
UF
UF
LEACHING
FIG. carried
OF CHRYSOTILE
ASBESTOS
2. Chrysotile fibers obtained from human lung (case No. 2). The SEM microanalysis out, respectively, at a, b, and c. Si:Mg ratios: a, 28.4: b. 34.5; c. 28.3. TEM x 22.500.
was
b). using the same principles of analysis, i.e., X-ray spectrometry, but with a different device and treatment of the emission signals. The ikz vitro tests carried out in this study with alveolar macrophages have shown the rapid effect of cells on fibers. The kinetic studies of magnesium leakage obtained with ESCA (Emission Spectroscopy for Chemical Analysis) (Thomassin et nl., 1976) after treating asbestos with biologic organic acids have also shown an early action of these acids (from the first minutes). It should be noticed that the measurements made on intrapulmonary fibers as
252
JAURAND
ET AL
I
%
4
*
FIG. 3. Asbestos body obtained from hur nan lung (case No. 1). Si:Mg ratios were a. 6.1; b, 5.6.; c. 17,9; d, 4.2. (A) TEM x 3808, (B) SEM x 2040 (transmitted electrons). TABLE
4
RATIOS OBTAISED FROM UICC A CHR~SOTIII INCUBATED IN TEST MEDIA (EAGLE BME) AND PHACOCV-IOSED BY RABBIT ALVEOLAR MACROPHAGES IN .THE SAME MEDIAN
III Vitro
SmnrEs-si:Mg
Medium
Si : Mg ratios
NaCl 0.9%
1.82 - 2.05 1.93 - 2.51 2.20 - 1.49 2.67 - 2.54 2.65 - 1.89 1.98 - 1.94 24 hours 5.40 9.37 5 days5.11 4.97
Eagle BME
Phagocytosed fibers
1.99 2.00 2.88 1.78 - 8.54 - 6.75 4.14 - 3.91
” The lowest and highest values in each group are in italic type. TABLE Si:Mg RA-1-10s OWAISED Fiber No. 1 2 3 4
FROM irt Vitro
5 PHAW~YT~SED
CHR~SO.I.ILE
FIBERS
Si : Mg ratios 3.31 5.23 5.64 6.65 7.40 4.34
-
4.89 4.25 5.79 5.07 6.49 11.6
4.96 4.84 9.99 8.70 6.38
” The results on four fibers and the values related to different counts along the axis of the fiber are reported
LEACHING
OF
CHRYSOTILE
ASBESTOS
253
FIG. 4. SEM microanalysis on Bchrysotile fibert;hago$tzed in by rabbit alveolar macrophage. (A) secondary emission SEM x 1768, (B) transmitted electrons TEM x 2720, (C) X-ray spectra of the fiber at a point (arrow) which likely was intracellular before processing.
well as those made on phagocytosed fibers gave very different Si:Mg ratios from one fiber to another, and, for a single fiber. values differed along the fiber axis. It is unlikely that all inhaled fibers undergo the same aggression from the biologic environment at the same time. Indeed, the fibers’ localization in the lung tissue, their physical state when inhaled, the time they spend in biologic residence, and their properties may be important factors in modifying the intensity of magnesium leakage. The nonhomogeneous leakage of magnesium may lead to a different reactivity at different points along the fiber axis according to the chemical content: this may be due to the products of the AM metabolism. These cells have an important aerobic metabolism involving various carboxylic and a-cetonic acids which should be able to attack the surface of chrysotile fibers. Then, chrysotile reactivity may be highly modified: Variations of the superficial net charge are able to alter the nature of the cell-mineral interaction; modifications of the sorptive reactions toward exogenous toxic substances (components of cigarette smoke, metals. gas. and so on), or toward some endogenous proteins. So Desai rt crl. (1975) have shown the adsorption of hemoglobin by natural chrysotile. Likewise, using albumin, a protein of equivalent molecular weight, Morgan (1974) also demonstrated this adsorption; in his study he found a decrease in the amount of adsorbed albumin versus magnesium leakage with fibers treated by 0. I N HCl. Therefore, the structural and chemical in rirc~ modifications of chrysotile fibers may have an effect on the mineral reactivity. In a study still in progress, we have shown that the residue of silica remaining after mineral acid attack (HCl) was amorphous silica. thus physically entirely different from the network of cristalline silica remaining after oxalic acid attack (Johan et crl., 1973). The conditions for chrysotile leaching with organic acid seem more like the it? iris) situations than the conditions for leaching with mineral acid.
254
JAURAND
ET AL
ACKNOWLEDGMENTS Chrysotile fibers used in this investigation were supplied by Dr. V. Timbre11 of the Medical Research Council. Pneumoconiosis Research Unit, Penarth. The authors wish to acknowledge the technical assistance of Mrs L. Magne in cell culture and of Mr. X. Janson in electron microscopy. The microanalysis study has been carried out in the laboratories of the Department of BRGM. with the helpful collaboration of P. Jeanrot.
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