Document 2JZ97a6dZ40vynwd8aBe0pn5a

environmental research 36, 314-326 (1985) An Assessment of the Fibrogenic Potential of Very Short 4T30| Chrysotile by Intratracheal Instillation in Rats1 Irma Lemaire,* Denis Nadeau,! Jacques Dunnigan/ and Serge Mass$ *Unite de recherche pulmonaire, Faculte de medecirte and tLaboratoire de caracterisation de I'amiante, Faculte des sciences, Universite de Sherbrooke, and #Institut de recherche et de developpement sur I'amiante, Sherbrooke, Quebec, JIH 5N4 Canada Accepted April 23, 1984 Three groups of five rats each received, respectively, a single intratracheal instillation of saline (control), 5 mg of UICC chrysotile B asbestos, and 5 mg of a preparation of very short chrysotile fibers (4T30, 100% < 8 pm) isolated by a sedimentation procedure. At various intervals after the treatment (1 to 60 days), assessment of lung morphology uas performed on each animal. Although the two types of chrysotile fibers have similar chemical composition, structure, and surface charge, the lung tissue reaction differed considerably. Lungs of animals exposed to UICC chrysotile B showed significant pathological alterations as early as 7 days following treatment. The lesions were localized in and around terminal bronchioles and consisted of inflammatory cells, fibroblasts and collagen deposition which distorted and obstructed small airways. Reaction to very short 4T30 chrysotile fibers was quite distinct. Seven days after treatment, lungs of these animals showed alveolar and interstitial accumulation of inflammatory cells. The alveolitis persisted 60 days after treat ment and no fibrosis was apparent. It appears that very short 4T30 chrysotile fibers are much less fibrogenic than UICC chrysotile B and that intratracheal instillations in rats may represent a useful mean of Rapidly assessing the fibrogenic potential of various dusts. These observations support the concept that fiber length is an important factor for fibrogenicity of asbestos. 0 1985 Academic Press, Inc. .II ' INTRODUCTION in vitro models have been extensively used to study the biological activity i asbestos and different pollutant particles. In many instances, however, the ob)| served in vitro effects of asbestos did not closely correlate with its fibrogenic an tumorigenic potential in vivo (Harington et al., 1975) mostly because important/^ mechanisms of physiological regulation cannot be duplicated in culture. Wherea^jp in vitro assays have given a number of clues concerning the mechanism of actiolig of asbestos at the cellular level, in vivo experimentation is more likely to givej$ definite answer about the fibrogenicity of any given material. Inhalation experif| ments have been used to examine the fibrogenic potential of dusts and this method provides a physiological model system allowing for the natural clearance of inJ|:;^ haled minerals. However, chronic exposure of animals as performed in inhaIatiom||j studies often,; requires mpnths of. experimentation and necessitates elaborate anelM expensive facilities. For these reasons, this approach is less suitable for studying^ the molecular processes associated with the onset of fibrosis and for the com) ''$8 1 Supported by grants from the "Institut de recherche et de ddveloppement sur I'amiante" (1RDA), and the Medical Research Council of Canada (MRC). l.L. is a scholar of the "Fonds de la recherche,''V' en santd du Quebec" (FRSQ). 0013-9351/85 $3.00 _ Copyright 1985 by Academic Press/lnc". ` * All rights of reproduction in any form reserved. 314 FIBROGI ikon of large numbers o illation offers an interc satisfactory in reprodi sis in various animal ihner, 1977; Begin et c wious work has suggc by the length of the is regard, various invt Iftbrogenic (Holt and N of the same chemic; :cht, 1970; Davis et < studies were perform 'a way as to modify tf:ntly, very short chrys ,;stos by a sedimentatio -e fibers (Jolicoeur et e, ietermine the fibrogenic (short chrysotile using I trice sample of chrysoi |ijS' investigation was tc for reproducing rap pable dusts and particl M/ '.als. Male Wistar ra plknada Inc. (St.-Con ''fibs fibers. UICC st Research Institute / short 4T30 fibers ^''sedimentation proce ived for 45 min and s iphate-buffered saline ( ation into the anim. fperimental protocot. / ({were lightly anestheti The trachea was exi skly injected througl of animals received. |ol), UICC chrysotile jjps in each group were mse of the number of a y, five sets of experii png morphology. A met 1) was fixed by immer araldehyde solution for 1 * iit*|i?*^i*^` f Very Short 4T30/'i n in Rats1 and Serge Mass^I die de curaclerisailan de \lilul de recherche el de 5X4 Canada liitracheal instillation of if a preparation of very lentation procedure. At >f lung morphology v..(s rs have similar chemical >n differed considerably, pathological alterations in and around terminal Hagen deposition which t() chrysotile fibers was is showed alveolar and sted 60 days after treat30 chrysotile fibers are instillations in rats may I of various dusts. These factor for fibrogenicity ie biological activity nces, however, the obt'Ajf e with its fibrogertic andlli istly because important.;;^ .ted in culture. Whereas, he mechanism of actionV; i is more likely to give erial. Inhalation experi)t" dusts and this method natural clearance of inperformed in inhalation, jessitates elaborate and,,-ss suitable for studying>rosis and for the com- .ntent sur l'amiante" (IRDA), if the "Fonds de la reoietche FIBROGENESIS BY 4T30 CHRYSOTILE FIBERS 315 son of large numbers of different particles. For these purposes, intratracheal lation offers an interesting compromise since it is rapid, easily performed, satisfactory in reproducing most of the main histopathological features of >osis in various animal models (Burger and Engelbrecht, 1970; Wright and ichner, 1977; Begin et al. 1983). jrevious work has suggested that the fibrogenic potential of asbestos is influ- by the length of the fiber (Beck et al., 1971, Wright and Kuschner, 1977). ids regard, various investigators have reported that short fibers were either ^fibrogenic (Holt and Mills, 1963; Holt et al., 1964), less fibrogenic than long jjfs of the same chemical composition (Klosterkotter, 1968; Burger and En|Srecht, 1970; Davis et al., 1978), or not fibrogenic (Gross, 1974). Many of |e studies were performed with fibers of diverse origin or fibers prepared in a way as to modify their physicochemical properties (Langer et al., 1978). ntly, very short chrysotile fibers were isolated from bulk 4T30 chrysotile stos by a sedimentation procedure that does not modify the structure of the |/e fibers (Jolicoeur et al., 1981). The present study was undertaken in order etermine the fibrogenic potential of this more homogeneous preparation of short chrysotile using Union International Contre le Cancer (UICC) standard :nce sample of chrysotile B as a control fibrogenic material. The second aim is investigation was to evaluate the usefulness of intratracheal instillations its for reproducing rapidly the histopathological lesions caused by various jjjrable dusts and particles and for assessing their relative fibrogenicity. MATERIALS AND METHODS Mntals. Male Wistar rats weighing 250-300 g were purchased from Charles ffe.Canada Inc. (St.-Constant, Quebec). festos fibers. UICC standard sample of chrysotile B was obtairied from the jojiial Research Institute for Occupational Diseases, Johannesburg, South Af p Very short 4T30 fibers were isolated from Quebec 4T30 chrysotile according Ie sedimentation procedure of Jolicoeur et al. (1981). Each sample was au- ived for 45 min and suspended at a concentration of 10 mg/ml in sterile jjiphate-buffered saline (PBS, pH 7.4) with a Dounce, glass homogenizer prior `"^filiation into the animals. |perimental protocol. All injections were made by the transtracheal route, were lightly anesthetized with a mixture of ketamine/xylazine (85/15, 100 vg). The trachea was exposed surgically and the asbestos suspension or saline jriskly injected through a 20-gauge needle in a final volume of 0.5 ml. Three ips of animals received, respectively, a single intratracheal injection of saline Itrol), UICC chrysotile B (5 mg), and very short 4T30 chrysotile (5 mg), ijials in each group were sacrificed at 1,7, 14, 21, and 60 days after instillation, iijse of the number of animals which could be handled experimentally in any Pay, five sets of experiments were done, each corresponding to a given time ) iimg morphology. A median longitudinal section of the upper left lobe (I mm :Jk) was fixed by immersion in a phosphate-buffered 4% formaldehyde-1% raldehyde solution for histologic examination. After fixation, lung tissue was 316 LEMAIRE ET AL. embedded in paraffin and representative sections 5 pm thick were cut and ->iainec|!; with hematoxylin-eosin. RESULTS Properties of injected materials. The preparation of 4T30 chrysotile used our study did not contain fibers longer than 8 pm and 98% were smaller than$ pm. The surface charge of these fibers was +40 mV as represented by th^i,' potential at pH 7.4 and their specific surface area equal to 38 m2/g (Table 1). ' chemical and structural properties of very short 4T30 chrysotile fibers (Jolicoe et al., 1981) were not significantly different from those reported for UICC sotile B (Timbrell, 1970). Thus, the major differences between these two typej^ of chrysotile asbestos reside in their fiber length and specific surface area an(f this is best illustrated in Fig. 1. Very short 4T30 asbestos fibers are well dissQ^' ciated and give homogeneous and stable suspensions which are more easi(y( instilled into rats than UICC chrysotile B suspensions. ` Survival and incidence of histopathological lesions. There was no signifies difference in survival between animals treated with the two asbestos samples i all animals survived the exposure period. When the average weights of anim in the different groups were considered, however, some differences were noti able at Days 14 and 21 following exposure. Rats exposed to chrysotile B asbest1 averaged lower body weight (284-288 g) than animals injected with saline (32 329 g) or very short 4T30 chrysotile fibers (308-311 g). This difference was,i)i longer apparent at 60 days after exposure. A summary of the histological findin is given in Table 2. Histological changes which could be related to treatment ' seen at the 7-day interim and thereafter. Descriptions of typical lesions are giyf below and almost ail animals in a particular group were affected. Saline instillation. Rats injected with saline showed normal lung morpholtf TABLE t Properties op Injected Materials Short 4T30 chrysotile Length Specific surface area (m2/g) Surface charge (mV, pH 7.4) Fe MgO X-ray diffraction (d) <0.5 p.m, 50% <3 pm, 98% <8 pm, 100% 38 +40 2.5%" 40.8%" 7.39 (A)" 4.S6 3.65 2.44 1.53 " As reported by Jolicoeur et al. (1981). b As described by Timbrell (1970). UICC chrysotile B >2 p.m, 100% >5 p.m, 42% >10 pm, 21% 26.86 + 36 2.6%b 32.0%'' 7.36 (A)6 4.55 3.66 2.46 1.54 >-s4 k were cut and stained'' DO chrysotile used ini| c were smaller than J - represented by the 38 m2/g (Table 1). The . sotile fibers (Jolicoeurj ported for UICC chry4 tween these two types.' ccific surface area and s fibers are well disso^ i .vhich are more easily, icre was no significantlj o asbestos samples andf age weights of animals! ifferences were notice*;! o chrysotile B asbesto$i|j -acted with saline (324f> This difference was nq'j he histological finding?! lated to treatment were! ypical lesions are givetfj ffected. irmal lung morphologyt; UICC chrysotile B >2 jJim. 100% >5 (Jim, 42% >10 |im, 21% 26.86 + 36 2.6%'' 32.0%* 7.36 (A)* 4.55 3.66 2.46 1.54 FIBROGENESIS BY 4T30 CHRYSOTILE FIBERS 317 I M ^vrrrsssa 318 LEMAIRE ET AL. TABLE 2 Summary of Numbers of Animals with Histological Lesions Treatment Days postinjection Number of animals examined Saline UICC chrysotile B Very short 4T30 1 7 14 21 60 1 7 14 21 60 1 7 14 21 60 5 6 5 6 2 5 5 6 6 5 5 6 5 6 4 Mean body wt (g) 279 * 14 278 14 324 5 329 11 397 12 275 8 250 21 288 5* 284 14* 401 15 281 7 278 3 308 5 311 9 416 13 Number of animals with histopathological lesions , Alveolitis Bronchiolitis ' obliterans i 0 o3 0 0* 0 0! 0o 00 00 04 06 05 0 5 0 o ; 6 0 i) 50 5 0 v-i 4 0f * Significantly different from control (P < 0.05). at each time point studied. Typical lung histopathology of these animals witl normal terminal bronchioles, alveolar ducts, and alveoli is shown in Fig. 2. UICC chrysotile B instillation. The rat lung reacted rapidly to chrysotile fibers! and significant lesions appeared within 7 days in lungs of animals exposed tp.|| mg of UICC chrysotile B (Fig. 3B). Lesions found at 21 days (Fig. 3C) andljf days (Fig. 3D) following instillation were not significantly different from tho$|j observed earlier (7 and 14 days). No attempt was made to quantify the numbi|p or extent of fibrotic lesions and their distribution at various intervals following! exposure although lung sections of animals sacrificed at 60 days showed, upollp examination, a higher incidence of fibrotic foci (an average of 10 compared to 4a at Day 7 and none at Day 1). Distal lung structures were more affected and thei,|| predominant lesions were located in and around terminal bronchioles. These were)! characterized by focal fibroblastic proliferation in peribronchiolar tissues whicjifl distorted and obstructed small airways (Fig. 4A). These features of peribronchioiff itis and bronchiolitis obliterans were seen at 7 days and thereafter in all animal$|p exposed to UICC chrysotile B (Fig. 3A-D). Some lesions were located at bifur-;^ cations of the distal airways (Fig. 4B) and consisted of granulation tissue withV>| fibroblastic proliferation (Fig. 4C). .Very short 4T30 chrysotile instillation. Animals treated with very short 4T30,i| asbestos fibers had the following pattern of lung histological changes: Within 7/$ days lesions appeared as focal areas of interstitial inflammatory reactions cony| sisting of mononuclear cells (Fig. 5B). Intraalveolar accumulation of mononuclear^ cells was seen (Fig. 6A) and small sheets of packed mononuclear cells began t<fel obliterate alveolar lumena, resulting in multifocal septal thickening and alveolar'ifl &T w: 2. Lung histopii vays and alve sacs (B, x 11 Si? \i. Lesions Number of animals with histopathological lesions Mveolitis 0 0 0 0 0 0 0 0 0 0 0 6 5 5 4 Bronchiolitis obliterans 0 0 0 0 0 0 4 ft 5 5 0 0 0 0 0 ' ' ;y of these animals withij i is shown in Fig. 2. ' tpidly to chrysotiie fibers;| of animals exposed to 5*1 2i days (Fig. 3C) and 6(j| .ntly different from thosef;1 e to quantify the number-''! arious intervals following '' at 60 days showed, upon :rage of 10 compared to 4 ere more affected and the d bronchioles. These were ihronchiolar tissues which features of peribronchioltd thereafter in all animals ions were located at bifurof granulation tissue with ated with very short 4T30 'logical changes: Within 7 '.lammatory reactions con . umulation of mononuclear nononuclear cells began to tal thickening and alveolar FIBROGENESIS BY 4T30 CHRYSOTILE FIBERS 2. Lung histopathology of saline-treated rats showing only perivascular edema with normal Jl airways and alveoli (A, x46) and normal terminal and respiratory bronchioles with alveolar Its and sacs (B, x 117). FIBROGENESIS BY 4T30 CHRYSOTILE FIBERS 321 n%oo m m ti$r 2 0) J2 i I'.4; Fibrotic lesions caused by UICC chrysotile B in rat lungs. (A) Bronchiolitis obliterans by j|s tissue (x91). (B) A small airway bifurcation reveals thickening and fibroblastic proliferation Resulting stenosis (x44). (C) High-power view of Fig. 4B (x 112). FIBROGENESIS BY 4T30 CHRYSOTILE FIBERS 323 324 LEMAIRE ET AL. distortion (Fig. 6B). No evidence of collagen deposition was found. In all animalsi'lj the small airways were normal (Fig. 6C) indicating that the lesions were confinedl>| to the alveolar structures. The alveolitis was still present on Days 21 and 60 (Fig.iJ 5A-D) and the extent of lung injury did not resolve during that period. DISCUSSION In the present study a preparation of very short 4T30 chrysotile fibers wasjl tested in comparison with UICC chrysotile B asbestos for its ability to inducoll lung fibrosis. Using the rat as an animal model and intratracheal instillation a|f the method of exposure, we have demonstrated that the lung reactions to veryjfl short 4T30 chrysotile and UICC chrysotile B differed considerably both in natures and localization. While the predominant pulmonary lesion produced by very shorttlj 4T30 chrysotile was minimal in character consisting of mononuclear cells, few4f giant cells, and no fibrosis, UICC chrysotile B caused severe fibrotic lesions of|l terminal bronchioles. Moreover the lesions found in chrysotile B-treated rats located in and around terminal bronchioles with some present at small airwawF bifurcations. By contrast very short 4T30 chrysotile fibers affected predominant!^ the alveolar structures. These findings are consistent with previous suggestio^ (Allison, 1974) that long chrysotile fibers are deposited by interception mainly bifurcations of smaller bronchioles due to their stretched and curved form whijp shorter fibers penetrate more deeply into the lungs and reach the alveolar con! partment. Our results also demonstrate that 2 months after exposure, there was still n|J| evidence of a progressive fibrotic reaction in rats exposed to very short 4T3| chrysotile. The 2-month observation period chosen may appear to be a relativeij short time scale to assess the fibrogenicity of asbestos dusts. Nevertheless! differentiates rapidly the lung reactions to these two chrysotile samples. Whetla very short 4T30 chrysotile fibers will produce lung fibrosis at a later period oifjj a higher dose is unknown but is currently under investigation. Very short 41 asbestos fibers have been reported to have hemolytic (Pele and Calvert. 19^| and cytotoxic activities (Kimmerle et al., in preparation) and to alter lung fibf blast DNA synthesis (Lemaire et al., 1982) although to a lesser extent than UIGjji chrysotile B asbestos. Extrapolation of these in vitro observations to predicttj possible fibrogenic effect of very short 4T30 fibers after a longer period of o|g servation is difficult, especially since there is still controversy concerning th* correlation of hemolytic and cytotoxic potential with fibrogenicity (Robock art Klosterkotter, 1973; Harington et al., 1975). The use in our study of a preparation of chrysotile with well defined fiber lengffigg and unchanged chemical composition brings additional support to the concern that fiber length is an important factor for fibrogenicity (Harington et al., 197SJ Davis et al., 1978). Our observations that small-sized 4T30 chrysotile fibers, | spite of their large surface area, are less fibrogenic than chrysotile B furthts| suggest that fibrogenicity is related to fiber length but not to the specific surfajat, area of the asbestos dusts. Although various animal models (Holt et al., 1964, 1965; Vorwald et al.. I951$ij| Wagner et al., 1974; Davis et al., 1978; Davis, 1963; Goldstein et al., 1978) havijyj |ten used to reproi iibe the best avail; pbioeffects of a lai tea single intratrac Bte similar to that pi, 1974; Davis , ipons (Goldstein Ipsis. A striking &ess with the apj mi. (1964, 1965) al poon as 14 days fenomenon is prol feel but rather to ; m amount of chrys lization at bifurcat Krete areas of the formal connective iljity of asbestos c ]|is to low asbesto ssults of our stu |hg time span in |ly the fundamen ' us to investig; |jt;of fibrosis. Thi illative fibrogeni fpibers, and ma H'(iiis respirable pV'e importance it>|femcity of asbi A. C. (1974). Pall ' -308. J|)3. G., Bruch, J., Fr pcperiments and cell c few chemical influent Rola-Pleszczyn TgSessment of toxicit) m'brmacol. 40, 261-'. H,- B. F., and Engeli reference asbestos sai |pi. M. G. (1963). Ai' pfexp. Pathol. 44, 4,' |;J. M. G. (1979). Th |W. 330, 795-798. j J. M. G., Beckett, a.fibres in the pathog'. |ler, E. A., Carringu V.` n was found. In all animals, t the lesions were confinedi-;l .nt on Days 21 and 60 (Fig.' J luring that period. 4T30 chrysotile fibers was':,! os for its ability to induces! intratracheal instillation as.il the lung reactions to very -Jpi considerably both in nature ' MOn produced by very short ,;! . of mononuclear cells, fewfm cd severe Fibrotic lesions ofifT nrysotile B-treated rats were$ me present at small airway/*! bers affected predominantly/|f it with previous suggestions;-,! ed by interception mainly at(!lf* ched and curved form whilejf and reach the alveolar com-lf l)\ exposure, there was still no/8 exposed to very short 4T3(I1 nay appear to be a relative! estos dusts. Nevertheless lip chrysotile samples. Whether ibrosis at a later period orgi<| vestigation. Very short 4T30)|| /tic (Pele and Calvert. I983)||j ition) and to alter lung fibro--f to a lesser extent than UICC;j| no observations to predict flip after a longer period of objI'S controversy concerning thei;;$| th fibrogenicity (Robock and/-|f t><A with well defined fiber length zonal support to the concept!* ticity (Harington et al., 1975;/ed 4T30 chrysotile fibers, in lie than chrysotile B further-,.;^ ->ut not to the specific surface' <4. 1965; Vorwald et al.. 1951; *; Goldstein et al.. 1978) have FIBROGENESIS BY 4T30 CHRYSOTILE FIBERS 325 in used to reproduce the fundamental lesion of asbestosis, the rat was found Lbe the best available model (Davis, 1979) for studies aiming at the assessment Ibioeffects of a large number of particulate materials. In our rat model exposed |;a single intratracheal instillation of UICC chrysotile B, the lung reaction is |te similar to that seen in chronically exposed rats (Holt et al., 1964; Wagner fjal., 1974; Davis et al., 1978), guinea pigs (Davis, 1963; Holt et al., 1965), or boons (Goldstein et al., 1978) and is best characterized by peribronchiolar ifbsis. A striking result of our investigation is the rapid onset of the fibrotic Rjcess with the appearance of well defined fibrotic lesions within 7 days. Holt fiftl. (1964, 1965) also reported the appearance of fibrosis in rats and guinea pigs I'soon as 14 days following inhalation of chrysotile asbestos. Therefore, this |tenomenon is probably not related to the method of exposure or the animal [>del but rather to the type and the concentration of fibers used. It may be that amount of chrysotile B fibers together with their length and preferential lo cation at bifurcation sites result in significant accumulation of long fibers at Crete areas of the lung which in turn leads to an acute irritation process with iormal connective tissue response and fibrosis. Because of the mode and injslty of asbestos exposure, our data do not relate to the very early lung reacIjis to low asbestos inhalation exposure in humans and animals. Furthermore, |;'results of our study do not suggest that short fibers cannot be fibrogenic over jfiihg time span in man. Rather the rat model described here reproduces very Mlly the fundamental lesion of asbestosis, the peribronchiolar fibrosis, and may My us .to investigate some of the changes more directly associated with the ret of fibrosis. This test system clearly differentiates in a short period of time `dative fibrogenicity of two types of asbestos, namely long and short chryjjB'fibers, and may therefore be valuable in assessing the fibrogenic potential us respirable particles. Furthermore it could permit investigation of the |tive importance of surface chemistry versus physical characteristics in the logenicity of asbestos fibers. REFERENCES |on, A. C. (1974). Pathogenic effects of inhaled particles and antigens. Ann. N.Y. Acad. Sci. 221, 399-308. IyE. G., Bruch, J., Friedrichs, K. H., Hilscher, W., and Potl, F. (1971). Fibrous silicates in animal Experiments and cell culture--morphological cell and tissue reactions according to different phys||,6al chemical influences. In "Inhaled Particles" (W. H. Walton, Ed.), Vol. 3, p. 477-487. ^R., Rola-Pleszczynski, M., Masse, S., Berthiaume, Y.. and Drapeau, G. (1983). Rapid in vivo ssessment of toxicity of respirable particles: A concise report. Res. Commnn. Chem. Pathol. Pharmacol. 40, 267-278. |pr, B. F., and Engelbrecht, F. M. (1970). The biological effects of the international standard Reference asbestos samples (UICC) on the lungs of rats. S. Afr. Med. J. 44, 1271-1274. fas, J. M. G. (1963). An electron microscopy study of the effect of asbestos dust on the lung. Brit, f. Exp. Pathol. 44, 454-464. |s, J. M. G. (1979). The use of animal models for studies on asbestos bioeffects. Ann. N. Y. Acad. gSci. 330, 795-798. |s, J. M. G., Beckett, T., Bolton, R. E., Codings, and Middleton, A. P. (1978). Mass and number ||pf fibres in the pathogenesis of asbestos-related lung disease in rats. Brit. J. Cancer 37, 673-688. nsler, E. A., Carrington, C. B., Coutu, R. E., Tomasian, A., Hoffman, L., and Smith, A. A. 326 LEMAIRE ET AL. (1972). Pathological, physiological and radiological correlations in the pneumoconiosis. Ami. Y.y, Acad. Sci. 200, 575-607. ' Goldstein, B., Webster, I., Rendall, R. E. G., and Skikne, M. I. (1978). The effects of asbestos-. cement dust inhalation on baboons. Environ. Res. 16, 216-225. . Gross. P. (1974). Is short-fibered asbestos dust a biological hazard? Arch. Environ. Health 29JJ H5-117. Harington, J. S., Allison, A. C., and Badami, D. V. (1975). Mineral fibers: Chemical, physicochemic^i and biological properties. Adv. Pharmacol. Chemother. 12, 291-402. ` 41Holt, P. E, and Mills, J. (1963). Experimental asbestosis in guinea pigs. In "Proceedings, XlVtj International Congress of Occupational Health," Vol. 2, pp. 667-670. Holt, P. E, Mills, J., and Young, D. K. (1964). The early effects of chrysotile asbestos dust on iKe'fi rat lung. J. Pathol. Bacterial. 87, 15-23. ''if Holt, P. E, Mills, J., and Young, D. K. (1965). Experimental asbestosis with four types of fibers:-.ff Importance of small particles. Ann. N.Y. Acad. Sci. 132, 87-97. Houribane, D. O., and McCaughey, W. T. E. (1966). Pathological aspects of asbestosis. Postgrad. Med. J. 42, 613-622. Jolicoeur, C., Roberge, P., and Fortier, J. L. (1981). Separation of short fibers from bulk chrysotili(>| asbestos fiber materials: Analysis and physico-chemical characterization. Cattad. J. Chem. Sfy'M I140-H48. ,;r Klosterkdtter, W. (1968). Experimentelle Untersuchungen uber die Bedentung der Faserlange fur difei Asbestofibrose sowei Untersuchungen uber die Beeinflussung der Fibrose durch Polyvinylpyj| idin-(V-Oxid. In "Proceedings, International Conference on the Biological Effects of AsbestoS$?|| Dresden," pp. 47-52. ,;4| Langer, A. M., Wolff, M. S., Rohl, A. N., and Selikoff, I. S. (1978). Variations of properties chrysotile asbestos subjected to milling. J. Toxicol. Environ. Health 4, 173-188. Lemaire, I., Gingras, D., and Lemaire, S. (1982). Thymidine incorporation by lung fibroblasts as a|| sensitive assay for biological activity of asbestos. Environ. Res. 28, 399-409. Pele, J. P., and Calvert, R. (1983). A comparative study on the hemolytic action of short asbestpijf fibers on human, rat and sheep erythrocytes. Environ. Res. 31, 164-175. ;iMf Robock, K., and Klosterkdtter, W. (1973). Investigations on the cytotoxicity of asbestos dusts. Rejiftg haltung Der Luft 33, 279-283. ^JS Timbrell, V. (1970). Characteristics of the international union against cancer standard reference sa pies of asbestos. In "Pneumoconiosis" (Proceedings of an International Conference, Johanttl| burgJCpp. 28-36. Tukiainen, P., Thskinen, E., Korhola, O., and Valid, M. (1978). Tru cut needle biopsy in asbestotfi and silicosis: Correlation of histological changes with radiological changes'and pulmonary futtp tion in 41 patients. Brit. J. Ind. Med. 35, 292-304. Vorwald, A. J., Durkan, T. M., Pratt, P. C. (1951). Experimental studies of asbestosis. Arch. Inim Hyg. Occup. Med. 3, 1-43. -liU Wagner, J. C., Berry, G., Skidmore, J. W., and Timbrell, V. (1974). The effects of the inhalation.! asbestos in rats. Brit. J. Cancer 29, 252-269. Wright, G. W., and Kuschner, M. (1977). The influence of varying lengths of glass and asbestos fibn on tissue response in guinea pigs 455 -472. (ClRONMENTAL Rl I'! ^ Erythro fPoisoning Jff " Department of fe Chronically K' anomalies in tl i|' cadmium chlori p erythrocytes ii K membrane, bav H of nuclei, and <> H though less frei g^were also assot ?|)and mean corpfbfcntration shov (Small lymphoc\ tpadmium-expO'' ^(jplress, inc. Imium is gi ^recognize pl'ihgested o lg food pjD'fbr varioi inic bronchi i |ri6scierosis ischer et al., ifesive cadmii rashi, 197( ifiong fishes, il-effects of ijson, 1975; I'j-Garofano a ||lts are avail prin higher pawai, 1981 ining elicitet rfAnguilla at is) (Larsson. us) (Calabre i J. Beal- <. Effect of \ND SYI.VA : Chlorinated ,'admium on Catfish. Het- iRTHUR M. \sbestos Ma- .RNF.R, D. G. f Nickel. 11. r, K. Loud, try Host De Combustion \RNI, N. G. \ccumulation nan Kidney i>h M. Alryogenically Uicrobe-Mepplication to ontraceptive. he Metabolic r Concentraicbec . . . ced by Di(2- 1 14 26 32 46 56 67 81 89 111 138 144 160 ,-tl an inside bat k cot er This Number Completes Volume 36 Volume 36, Number 2, April 1985