Document N2rQ9ERzZEg6B2qxGmGkv7a1p

- v',; Euviwnauiniul Health. Perspectives VoL 9- PP- U9-1S3> Investigation and Analysis of Asbestos Fibers and Accompanying Minerals in Biological Materials by L. Le Bouffant4 A method is describsd for isolating asbestos fibers contained in biological tissues. It consists in Lnomcratinff tho biological material in activated oxygen at ISO'C, and attacking the ash by IN HCI for 18 hr. Th residua is than filtered on a membrans covorad with a carbon Him. Electron microscope examination of the deposit mokes it. possible to determine fiber v concentrations when the weight or volume of primary material U'known, and to make aixa ' analyses. By x-ray diffraction, the mineralogical nature of the asbestos is determined by comparison with an aluminum reference diagram. For x-ray diffraction, s micromsthod Is , used, with an ash sample of about 10 fig. .' . `' The techniques arc used for identifying and couhting.&sbeito* fibers in small fragments of lungs or ocher organs, it was found that asbestos fibers generally go along with other - minerals which may be abundant. Most fibers found in lung an lees than S long. Counts f on lungs of asbestos workers give concentrations often greater than 10r particles per gram of dry tissue. The svalulion of inhalsd duysodla seems to be different from that of smphibolei. .. in the case of pleural mesothelioma, a comparison of fiber* within tho tumor with fibers in . the adjacent parenchyma ihows only slight dlffinrences in the particle since, but marked differences in their nature, with a chrysotlU enrichment in the pleural sane. Pleural plaques . were analyzed in the same way. After docalcUlcation, many small sized asbestos fibers were found. The same technique is now being used for determining ingested particles. A groat , number of observations concerning fiber counts, their nature and sizes, and the presence of various clays minsrals will be necessary to establish the role of the different factors in the formation of ltsiont caused by the inhalation or ths ingestion of asbestos fibers. f When one starts on the problem of the iden tification and quantitative determination of asbestos fibers in biological tissues, it is soon found that the technique of preparation is highly significant for the reliability and even Ue validity of the analytical results. Unlike asbestos fibers suspended in air or in homogeneous liquids, which are free in the fluid and thus can be easily collected, particles in histological material are trapped In a tissue which must be destroyed without alteration of me nature and physical characteristics of the Particles and also without losing part of them. Contro d'ElUdca ei RehKrchs des Charbonnsgcs ,lv `rant.. B. V. 27.60. Crnil, Franc*. After comparing various methods, a techni que based on incineration, which was first described in 1969, was adopted. It consists in ashing the biological material in a plasma fur nace at low temperature (160C) for a period of 2-8 hr, depending on the nature and the weight of the sample. Under such conditions, the organic matter is completely destroyed, while asbestos bodies and naked fibers are well preserved. The influence of the ashing temperature on the characteristics of the chcysoti'.e and amosite fibers was investigated. For that purpose, ground samples of these minerals were sub mitted to the. action of heat under the following conditions: 1S0C in activated oxygen under reduced pressure (5 mm Hg) for 9 hr; 450C in December 1974 RECEIVED TIME jUY. '3. 2:34PM 149 :t f 06/13/02 14:38 FAX 817 735 2283 t'NT HEALTH SCIENCE @004 air for 15 hr, then at 600C for 3 hr. The latter i3 the usual condition of sample preparation for quartz content determinations in silicotic lungs. The samples were then examined by electron microscopy and x-ray diffraction. In addition, a systematic study up to 1200C was made, Chrysotile heated at 150C undergoes no ap parent change, either morphological or crystallographieal. On the other hand, at 450C the aspect and the crystalline structure of the fibers are markedly modified. If the temperature is still further raised, the structure changes rapidly, and at about 800C chrysotile is transformed into forsterite, MgrSiCte. Up to 500C, amosite, which is more resistant, undergoes no change. On raising the tempera ture further, there appear progressively the lines of hematite PezOs, which is an indication of the parcial decomposition of amosite, At lOWC, the lines of amosite have disappeared com pletely. Combustion of organic matter is completeafter a few hours at 150oC in an oxygen plasma, while there remains a carbonaceous residue at 450C after a heating period of 18 hr. From these observations we were led to adopt a technique in which the material is heated at 150C in active oxygen. FiotlBE 1. Intensity ol lines in x-ray diffraction pattern: . (O) (002), 7.36 A: (x) (004), 8.68A; (-) MgO content (%! (+! of IN hydrochloric acid shows that the intensity of the002(7,86A) and 004 (3.68A Jlines of thexray diffraction pattern decreases with the time of attack and that there is a.correlation between these intensities and the magnesium content (Pig. 1). The curve shows a flat portion between 5 and 20 hr. Beyond 22 hr a steep decrease in the line intensity is observed. Figure 2 shows the combined effect of heating at 150C for 24 hr and treating with LV hydrochloric acid for 1 hr.(up to the beginning of Ash Treatment Besides asbestos fibers, the ash thus obtained contains various proportions of minerals, such as phosphates, sulfates, carbonates, and iron compounds. This mineral burden makes dif ficulties for several reasons'- during the microscopic examination, it may conceal part of the asbestos fibers, in particular small fibers; during the analysis of the component, it has a dilution effect which in some cases--for exam ple, pleural plaques--may be significant. It is then desirable to eliminate mo3t of the mineral burden without damaging the asbestos fibers. The method adopted consists in attacking the ash by hydrochloric acid under mild con ditions. To determine the optimum conditions of at tack, the action of hydrochloric acid on chrysotile and amosite was studied. The results show that chrysotile attached by liV HC1 at room temperature undergoes no visible change. On the contrary, rapid destruction is observed under the action of 12*V HC1 after 30 min. A more elaborate investigation into the action FICUH52. Micrographs: (a-) untreated chrysotile; (b) chryso lite alter heating et 150'C and etching with IN HC1; (cl chrysotile after heating at 450--600"C and etching with liV HCI. KSU Environmental Health Perspectives OP VL'1J ]3 2 JifV the fla1 respond 450-- 601 same c< Amoi the act Fina' attack re3ponc tensity The first i more c coneen samph the hy miner: mater or pie pleura attack cases ment. also e ccnsic diagr; Furthf and 0 Thi acid carbc Thus cona mem iron, fract filtrz neou susp on ti Tl is tr eiitr. x-ra ed a elirr Elec Diffi Dei 06/13/02 14:38 FAX 817 735 2283________ UNT HEALTH SCIENCE @005 the flattened portion of the curve, the cor responding weight lose is 16%) and of heating at 450- 600C followed by acid attack under the ^ same conditions (weight loss 74%). i Amosite undergoes no visible change under the action of hydrochloric acid. ; Finaliy, the time of 18 hr was chosen for the ; attack by hydrochloric acid. The time cor ' responds to the end of the flat portion of the in , tensity curve of the diffraction lines. The effect of this treatment is twofold. As a . first result, the microscopic images become more clear. A second effect is an increase in the ' concentration of asbestos fibers in the prepared sample. The ash content of the material before the hydrochloric attack varies according to the mineral matter content (exogen dust or endogen ` material): between less than 1% in parenchyma ' or pleura, for example, and more than 50%in " pleural plaques, for example. As a result of the j. attack by acid, the average weight loss in many cases represents about a hundredfold enrich* ment. Part of the iron of the asbestos bodies is also eliminated by the etching, thus improving considerably the quality of the diffraction ; diagram, as can be seen in Figure 8. Further Preparation for Electron Microscopy and Diffraction Analysis The residue from the attack by hydrochloric acid is filtered on a membrane covered' with a carbon film obtained by vacuum evaporation. Thus the fibers lie flat on the membrane. A se* cond carbon layer Is then deposited, and the membrane is cut into two fractions: one for elec tron microscope examination and electron dif fraction, the other for x-ray diffraction. This Iteration method gives a much more homoge neous deposit than collecting a drop of aqueous suspension, and there is no selection depending on the size distribution. The fraction intended for electron microscopy s transferred to a grid, and the membrane is eliminated by dissolution. The fraction used for x-ray diffraction has also its membrane dissolv ed and it is incinerated in a plasma furnace for semination of the carbon films. Elect/on Microscope Examination and Electron Diffraction There is nothing particular to say concerning the electron microscopic examination. Of course, for the determination of particle numbers, it is necessary to know the quantity of primary material, the surface of the membrane and of the microscope field Uhder examination, Electron diffraction requires the calibration December 1974 ;v .A 151 06/13/02 14:33 FAX 817 735 2283 UNT HEALTH. SCIENCE 006 In general, the quantity of material required for x-ray diffraction is of a few milligrams. Tak ing into account the small sizes of some biological samples, this method was adapted to analytical purposes for analysis of a volume of initial tissue of about 1 mm*. The weightof the final sample used after Incineration is abouflO *g- ' " The diffraction diagram recorded photographically In a flat chamber is in the form of Debye-Scherrer rings which are more or less punctuated, owing to the very small quanti ty of matter and to the size of some of the mineral particles. The minerals are identified by measurement of the ring diameter by a stan dard method. F.igubk 4. Size distribution diagram of Intrspulmonary Fibers. 5 ftm in length are taken into account in at mospheric dust controls. Fiber counts by electron microscopy on asbeatoB-workers generally give very high intrapulmonary concentrations even within the distril very paren tribul chant mixec Applications and Results - tumor, frequently from 10 to 50 X IQ* particles per gram of dry tissue. , signil tumo These methods are used systematically for It is noteworthy that chrysotile becomes rare oarer locating and identifying asbestos particles in in. comparison with amphiboles in the. lung relat: lungs and other organs. Owing to their sensitivi parenchyma, even in the case of workers who thus ty, it is possible to perform analyses on'amall had been exposed a much longer time to ment samples such as surgical biopsies, expec chrysotile than to amphiboles during their for o torations, bronchial biopsies, even in the case of professional life. that samples with small fiber contents. Since the The reason for the disappearance of chrysotile ehryi methods are quantitative, it is possible to deter in the lung is not yet clear. It is thought, Ta mine the concentration of fibers per unit weight or volume of tissue and the size distribution of the particles. Several facts were thus established by ex amining the lungs of workers with bronchia! however, that this is partly accounted for by the rapid clearance of chrysotile which has a high solubility compared to that of amphiboles. To confirm this, a comparative study of lung clearance with different mineral du3ta was with in tl xi carcinoma or pleural mesothelioma and having carried out on rats. The animals inhaled dust for many years of dust exposure in the asbestos in dustry. It was found in particular that asbestos fibers 6 hr in concentrations of 300 mg/m3, and groups of animals were killed at different times. The dust retained in the lung was determined by -/*i I generally go along with other minerals, such as colorimetry (Si determination in the case of talc, kaolinite, chlorite, or quartz. It often chrysotile). Figure 5 (parts of the curves with happens that these attendant minerals are in the gentlest slope) shows that the rate o: fact present in greater concentrations than in alveolar clearance of chrysotile is significantly asbestos itself. different from that of other insoluble or slightly < Size distribution determinations of in- soluble minerals. is.. trapulmonary flhers show that most fibers are Pleural mesotheliomas in asbestos workers less than 5 /im long (Fig. 4). With reBpectto this were examined in the same way. From a limited observation, it is recalled that only fibers above series of examinations it appears that the size 152 \ECEiVED TIME JUU:. 2:34 = Environmental Health Perspectives De 08/13/02 14:39 FAX 817 735 2283 UNT HEALTH SCIENCE 0007 Case \ 2 3 Table 1. Number ot fiber* N. Number of fibers A'/g tisane Lung Tumor jV' in tumor N la lune 17,4 X 10 1600 X 104 18.6 X 10' 13 X 10* 1.07 0.003 34 X 10* 1.2x10' 0.036 FIGURE 5, Plot of pulmonary dust cnntent: (A) titanium oxide; (o) quartz; () chryeatile. distribution of the fibers within the tumor is not very different from that found in the parenchyma (Fig. 6). On the other hand, the dis tribution of the fibers seems to be markedly changed. As a matter of fact, in several cases of mixed dusts (chrysotile--amphiboleB), there is a significant chrysotile enrichment in the pleural tumor, contrary to the observations in the lung . parenchyma in which a3 reported above, a relative amphiboles enrichment was found. It thus appears that the chrysotile Impoverish ment of the parenchyma cannot be accounted for only by the dissolution of this mineral, but that there seems to be a preferential drainage of chrysotile towards the pleura- Table 1 gives the number of fibers found within the tumor and in the adjacent lung tissue in three cases of pleural mesothelioma. /*\ * :c\ m tI u *' $ l! ztsTvevnoK _- ,u>e ---- Cvj 'L FlOl'KCfi. SizediniriuuLifm'. (-1 lung: (--I mesothelinma. Many fibers are found in each case in the tumor region. In one case there are as many fibers in the tumor as in the lung. Finally, fibrohyaline and calcified pleural plaques were analyzed in the same way. After concentrating as described above, many small, fibers were found in several cases (Table 2). Table 2. Number of fiber: A'/g dry tissue Caa I Cuaa 2 Fibrohyaline tissue 6 x 10' 3.6 X 10> . Calcified tissue (length of fibers 0.1-2 pm) 30 X 10' 40 X 10' Corrcspondinj lune 10XKF 1.6X10' By comparing the central calcified zone with the external fibrohyaline zone of a pleural pla que, it is found that the fiber concentration is greater in the calcified zone than in the fibrohyaline zone. Conclusion These few findings illustrate the possibilities of the method described for the qualitative and quantitative determinations of asbestos fibers in lung tissues. Although the results stated refer to lung analyses, the method is, of course, applicable to the determination of asbestos fibers in any other organ or tissue, without ex ception, and in particular to the study of in gested particles. It appears necessary now to total up count results and analytical data to es tablish the statistical significance of the different factors, in particular concerning the number, mineral composition, fiber size, and the role of the attendant minerals in the formation of lesions caused by the inhalation or ingestion of asbestos fibers. December 1974 -ic-C/\ rt, VE3 t:v,e '3. 2:34 PM - . .-star' .-2MK .12 14'," FA.\ Sir 7:)S U^S3 I'NT HEALTH SCIENCE igao: 0 h P :NV1RONM ENTAL HEALTH PERSPECTIVES Volume 9, December 1974 294 BIOLOGICAL EFFECTS OF ASBESTOS some way related to the presence of durable particles of fibrous configuration that are particularly long at or perhaps below the smallest diameter which can be recognised optically; and that the carcinogenicity of these fibres has little relation to their chemical com position or to their potential contaminants. SUMMARY Various structural forms of asbestos, fibrous glass and aluminium oxide have been tested for carcinogenicity on the pleura of rats. Results from all three materials indicate that carcinogenicity is related primarily to fibrous structure rather than to physicochemical properties. A comparison of the dimensional distribution of fibres in those samples of asbestos and glass producing high and low tumour incidence indicate that carcinogenicity may be related to fibres below 2.5 pm in diameter and between 10 to 80 um in length. ACKNOWLEDGMENTS We wish to thank Dr Vernon Timbrel! for advice in the analysis of the data; Mrs Constance Wrench and Miss Eliza Miller for their diligence in monitoring the experiments; and the staffs.of the Owens-Coming Fiberglas Corporation, the Johns-Manville Research and Engineering Center, and the MRC Pneumoconiosis Unit, Penarth, UK. for generously providing many of the materials used. REFERENCES Hanngton, J. S. (1965) Chemical studies of asbestos. Annals ofthe New York Academy of Sciences, 132, 31 47 - Stanton. M. F,, Blackwell R. & Miller, E.U969) Experi mental pulmonary carcinogenesis with asbestos. American industrial Hygiene Association Journal, 30, 236-244 Stanton. M. F. & Wrench. C. (1972) Mechanisms of me sothelioma induction with asbestos and fibrous glass. Journal of the National Cancer Institute, 48. 797-821 Timbrel!, V. (1970) Characteristics of the International Union Against Cancer standard reference samples of asbestos. In; Shapiro, H. A., ed.. Pneumoconiosis. Proceedings of the International Conference, Johannes burg, 1969, Cape Town. Oxford University Press, pp. 28-36 Wagner. J. C. (1970) The pathogenesis of tumors follow ing the intrapleural injection of asbestos and silica. Morphology of Experimental Respiratory Carcino genesis. AEC Symposium Monograph Series, 21, Oak Ridge, Tennessee, Oak Ridge National Laboratories, pp. 347-358 Wagner, J.C., Berry. G. &Timbrell, V. (1970) Mesothe liomas in rats following the intrapleural inoculation of asbestos. In: Shapiro. H. A.. ed,, Pneumoconiosis. Proceedings of the International Conference. Johannes burg, 1969, Cape Town. Oxford University Press, pp. 216-219 ' P tior. phy ciat peri asfcx lens aa occi desc aerc the seqt brec fibre intn 1971 tion mes surp offi otfe cal < S( two ated crys pro| Figi the effee part that appt the Pena 2(