Document 9JjXa0b32zM6RRb39rxy2YvL6

SEP 13 2002 12:45 FR CIST1 ICIST 613 941 4484 TO 12145201181 environmental research 30, 224-232 (1983) P.03/11 Re Cytotoxicity of a Short-Fiber Chrysotile Asbestos for Human Alveolar Macrophages: Preliminary Observations ph te; ch Henry Yeager, Jr.,* Denise A. Russo,* Miguel YaNez,* Donna Gerardi,* R. P. Nolan,! Elliott Kagan,! and A. M. LangerI U y as ch m- *Department of Medicine, Pulmonary Disease Division, and tDepartment of Pathology, Georgetown University Medical Center, Washington, D.C. 20007, ^Environmental Sciences Laboratory, Mount Sinai School of Medicine. City University of New York, New York, New York 10029 Received June 16, 1982 . | N. | fre V sc : th Studies were performed to compare the cytotoxicity for human alveolar macrophages of a naturally occurring short-fiber chrysotile asbestos (RG 144) to that of a standard reference - ch ar r* mixed-fiber (long and short) chrysotile asbestos (UICC chrysotile A, Rhodesian). Parallel studies were also performed with quartz (Min-U-Sil 15), a known macrophage toxin. On a [ i pc mass basis, and after 24 hr incubation, RG 144 was more cytotoxic than the UICC standard reference fiber and less toxic than quartz (silica). The cytotoxic potential of RG 144 chrysotile was further enhanced after size reduction by milling. These findings may have important biologic implications with respect to the use of short-fiber asbestos in industry. INTRODUCTION } fn | si: f ea | al cc Controversy exists regarding the biologic potential of asbestos fibers of differ- \ ent lengths (recently summarized by several authors, e.g., Harington et al,, 1975; Beck, 1980; Hillerdal, 1980). The central issue of the controversy focuses on the f us ch of cytotoxicity and pathogenicity of short-fiber asbestos, that is, asbestos in which . the longest fibers do not exceed 5 pm in length. In addition to this scientific question, the problem of short-fiber asbestos also has important practical implica Rtie tions. The current protective standard used by the U.S. Department of Labor for workers who may be exposed to asbestos-containing dust in the workplace takes ar into account only those fibers greater than 5 pm in length (OSHA, 1972; Langer et al., 1978). With increasing usage of short-fiber grades in industry and the preponderance of such fibers in the environment, the biologic potential of short fiber | I Pr d Pt should be further studied. I The present study was undertaken to determine whether there is a difference in , :j the cytotoxicity of a naturally occurring short-fiber chrysotile asbestos, before and after milling, and that of a preparation of chrysotile containing both long and short fibers. In addition, these two preparations have been compared to quartz, a known macrophage toxin. Well-characterized asbestos fibers and quartz particles were incubated with human alveolar macrophages, cells considered to be among the most important targets for inhaled asbestos (Hocking and Golde, 1979; Kagan, 1981). Cytotoxicity was determined by the trypan blue exclusion method. ' pi ar of B sr P> ft 0013-9351/83/010224-09S03.OV0 Copyrih1 19*3 by Acadcmfc Piet*. Inc. All rtthu of reprodoetkra ia any form reserved. RECEIVED TIME SEP. 13. 11:48AM 224 PRINT TIME SEP. 13. 11:53AM SEP 13 2002 12:46 FR CISTI ICIST 613 941 4484 TO 12145201181 ffer975; i the hich itific licar for akes er et ponfiber ce in ifore long :d to jartz to be 1979; n lod. CYTOTOXICITY OF SHORT-FIBER CHRYSOTILE ASBESTOS 225 MATERIALS AND METHODS Test materials. UICC chrysotile A fiber was obtained from the Pneumoconiosis Research Unit, Cardiff, Wales, U.K. The mineralogic purity and general physicochemical characteristics of these fibers have been extensively charac terized (Rendall, 1970; Timbrell, 1970). Specimen suitability and purity were checked by polarized light microscopy and continuous scan X-ray diffraction. The UICC preparation consists of98--99% chrysotile fiber, with the remaining mineral assemblage made up of sheet silicates (primarily antigorite, lizardite, and traces of chlorite). Nemalite or fibrous amphibole minerals were not detected. Approxi mately 25% of the fiber population was greater than 5 in length (Table 1). RG 144, a naturally occurring, predominantly short-fiber chrysotile from the New Idria deposit in California, was obtained in the commercially available form from the Union Carbide, New York. This preparation has been previously de scribed in detail (Langer et al., 1978). Its purity was checked in a fashion similar to that described for UICC chrysotile A; it had identical characteristics in terms of chrysotile content and mineral contaminants. In comparison with the UICC prep aration, a very different particle size distribution was found. Only 2% of the fiber population was greater than 5 ftm in length (Table 1; Fig. 1). Fiber size distributions were measured by sizing and counting objects directly from electron micrographs at final magnifications of 10,000x, Counting was as sisted by use of a calibrated hand lens (a Porton graticule). Particle numbers for each of the preparations were computed from the size-distribution data (Langer et al., 1978). The mean particle dimension within each size class was used in^he computation of mass fractions for each of the classes. For each of the calculations used in determining particle number per mass fraction of the specimen, a chrysotile density of 2.5 g/cms was assumed, as well as an average fibril diameter of 333 A, and an average fiber diameter of 9 fibrils in the unmilled specimen and of 3 fibrils for both the 60-sec and the 1200-sec milled preparations. Surface area of RG 144 was estimated from values issued by Union Carbide, and by recomputa tion of particle number for each of the size distributions. The silica used was quartz, Min-U-Sil 15, obtained from the Pennsylvania Glass and Sand Company, Pittsburgh, Pennsylvania. Its physical properties have been previously described (Nolan et al., 1981). Estimates of surface area were based on data provided by the vendor and recomputations as outlined for the asbestos preparations. Harvesting ofhuman alveolar macrophages. Seven healthy volunteers between 21 and 26 years of age were bronchoscoped with a fiberoptic bronchoscope for procurement of human alveolar macrophages. One volunteer had two lavages and another had three; lavages were separated by an interval of at least 4 weeks. Three of the volunteers were nonsmokers; one smoked tobacco and another marijuana. Because there were no significant differences in the response of the cells from smokers and nonsmokers to challenges by particles the results have been ex pressed together. Volunteers gave appropriate informed consent, and the protocol for the study was approved by the Human Research Committee of the Georgetown University Medical Center, where the lavages were peiformed- P. 04/11 RECEIVEO TIME SEP. 13. 11:48AM PRINT TIME SEP. 13. 11:53AM SEP 13 2002 12:46 FR CISTI ICIST 613 941 4484 TO 12145201181 P. 05/11 . M ' u ` 226 YEAGER ET AL. Bronchial lavage was performed as previously described (Yeager et al., 1974). Anesthesia of the nose and upper airway was accomplished with lidocaine spray. No other medication was administered. An Olympus fiberoptic broncho $ scope was passed transnasally and wedged into one of the segments of the right lower lobe. The lung was irrigated with 30- to 50-ml quantities of warm sterile * physiological saline, and the lavage fluid was withdrawn by gentle suction with a 10-ml syringe. Approximately 150 ml of saline was instilled, of which 30--50% was recovered. The aspirated solution was centrifuged at 20Qg for 15 min in an Inter national refrigerated centrifuge, and the supernatant fluid discarded. :I The bronchoalveolar cells obtained were resuspended and washed three times in Gey's saline (Microbiological Associates, Bethesda, Md.). They were finally rc- suspended in RPMI 1640 medium with 25 ism Hepes (N- 2-hydroxyethylpipera- zine-2V'-2-ethanesulfonic acid) and L-glutamine (GIBCO, Grand Island, N.Y.), ' supplemented with 10% heat-inactivated fetal calf serum (Flow Laboratories, ' Rockville, Md.), penicillin at 100 pg/ml, and streptomycin at 100 pg/ml, hereafter referred to as RPMI supplemented medium. The cells obtained were 88% viable by trypan blue staining (Paul, 1965). Culture ofalveolar macrophages and cytotoxicity tests. One hundred thousand (1 x IQ5) bronchoalveolar cells were plated into eight-chamber JLab-Tek tissue culture chambers (Miles Laboratories, Naperville, 111.) in 0.4 ml of RPMI supplemented medium, and incubated for 1 hr at 37C in a 5% C02 incubator. More than 95% of the adherent cells were macrophages as judged by light micro scopic appearance, phagocytosis of latex beads, and nonspecific esterase staining. After a 5-sec resuspension with the Vortex Genie Mixer, the appropriate as bestos or silica suspensions were added in fresh medium to the macrophages. The mixtures were allowed to incubate 1 hr at 37C in a humidified 5% C02 incubator, after which nonengulfed particulate material and medium were removed, and fresh medium was added. The cultures were then incubated for 24 hr at 37C in the 5% C02 atmosphere. At the end of this period, the adherent cells were removed by incubating 1 min in 0.25% trypsin/0.2% EDTA (GIBCO) in Hanks' solution at 25C (Paul, 1965). The detached cells were then counted in a hemocytometer, and the number of cells excluding trypan blue dye was determined (Paul, 1965). Statistics. Data were analyzed using analysis of variance and Duncan's multiple range test (Snedecor and Cochran, 1980). RESULTS The physical characteristics of the minerals studied are given in Table 1. The UICC chrysotile A sample contained a larger percentage of long fibers compared to the unmilled RG 144 specimen. However, when the absolute number of fibers per microgram of mass (NoJpg) was computed, the number >5 pm in length was virtually identical in each preparation. This point is stressed, because com parison of the percentage of specific-size fractions in specimens with orders of magnitude difference in particle number per unit mass of dust is misleading. For example, specimen "A*' with 100,000 total fibers per microgram of dust, with a 30% content of fibers >5 pm in length contains 30,000 such fibers per microgram,.. RECEIVED TIME SEP. 13. 11:48AM PRINT TIME SEP. 13. 11:53AM SEP 1-3 2002 12:47 FR CISTI ICIST 613 941 4484 TO 12145201181 t al., >caine nchoi right sterile ivith a % was Inter nes in Uy reipera*Y.), ones,, eafter viable usand tissue RPMI bator, tnicrolining. ite as s. The ibator, 1, and ! in the vedby lion at jr, and 0. .ultiple 1. The ipared 'fibers length i comlers of ig. For with a :>grani. CYTOTOXICITY OF SHORT-FIBER CHRYSOTILE ASBESTOS li 8 up a rsi oV 0>0 $ 8 ?* aa| P O < 2 zUo-JUJ1) U<X -- Do<un> EU 2 "A .zE89 1: 230 i <* 1 12 X <4 X n OpXi rCX>i x om V % 2>Z>i>h M m ** ** X X XXX ^ fn ^ N oo _-a JL E ' N O NO JLN<=? 1e =52 1 00 N0 -- 0wm\ OQ d 0 N(SO ^ n dbj t* n Oci a joz 8 *S ".O4ts1 .5* i li. C -^ C\ c4 v \ ad ao ss M P* N O o oo a u< 33 >x 93 ih 1 Is c 1-5 = E s 4 S O S3 65 5^3 -a 231 as >sa cs o PK s S4 227 P. 06/11 RECEIVED TIME SEP. 13. 11:48AM PRINT TIME SEP. 13. 11:53AM SEP 13 2002 12:47 FR CISTI ICIST 228 613 941 4484 TO 12145201181 YEAGER ET AL. P. 07/11 fi as does specimen "B" with 1,000,000 fibers per microgram of dust but with a 3% content of this same size class of fibers. j As milling time for RG 144 increased, the particle number per unit mass in creased as well. Although the percentage offibers >5 /xm in length decreased after 60 sec of milling, the absolute number per unit mass remained constant. At milling times of 1200 sec, fibers >5 fim in length were not detected in a population defined by almost 1000 measurements (see Table 1 and Fig. 1). The results of treating human alveolar macrophage cultures with various as bestos fiber preparations and silica are shown in Table 2. The unmilled RG 144 chrysotile fiber preparation was more cytotoxic than the UICC preparation. Moreover, after it was milled, the cytotoxic action of the RG 144 chrysotile was further enhanced. This effect was correlated with an increased number of short fibers and the total fiber number, rather than with the absolute number per micro gram of fibers >5 p.m in length. Values were found for the cytotoxicity of silica that were parallel to those found in previous studies (Kagan and Miller, 1981). Macrophage viability decreased at higher particulate concentrations, but a clear cut dose--response relationship was not observed. The results obtained were reproducible, since repeat studies in the same person yielded similar findings on " three occasions in one individual and on two occasions in another volunteer. DISCUSSION The question of the health effects of short-fiber asbestos has been largely ne glected, and attention has been mainly focused on asbestos fibers of 5 jam or more in length. The latter have been shown to be toxic to macrophages in vitro, and fibrogenic and tumorigenic in animals in vivo (Haiington et ah, 1975; Beck, 1980). Certain observations, however, suggest that short-fiber asbestos may also be harmful. A case of diffuse interstitial lung fibrosis was reported in which only submicroscopic particles of chrysotile asbestos were present (Miller et al., 1975). Crapo et al. (1980) found that rats exposed to short-fiber preparations (NIEHS "short range" asbestos size) developed significant lung toxicity, as manifested by alterations in epithelium, interstitial space, and alveolar macrophages after 3 months of exposure. These toxic effects, however, were not as severe as those noted in rats which had inhaled "intermediate range" chrysotile asbestos. In another study, Sebastien et al. (1979) found a preponderance of short-fiber chrysotile (<5 /un) in the pleural tissue of humans with "asbestos-related dis ease." The present study has shown that a naturally occurring short-fiber asbestos (RG 144) is cytotoxic to human alveolar macrophages in vitro. Per unit mass, the RG 144 preparation was more lethal for macrophages than the UICC chrysotile A ' reference sample, even though both samples had comparable numbers of asbestos < fibers >5 jttm in length. Furthermore, the cytotoxic effect was increased after milling for 60 sec, thus creating more short fibers, and was increased even more after 1200 sec of milling, to a point where it was virtually comparable to that of quartz. Since the-milling process produced both an increase in the total number of fibers and an increase in the total surface area of the sample (Fig. 1), each or both Fr ria. fi Mag ::reai -lillc atruc RECEIVED TIME SEP. 13. 11:48AM PRINT TIME SEP. 13. 11:53AM SEP 13 2002 12:47 FR CISTI ICIST 613 941 4484 TO 12145201181 P. 08/11 h a 3% ass in ti aftas milling defined ms asIG 144 ration, le was f short micro* silica 981). a clear i were ngs on :er." ely ner mor<i o, and 1980). dso be h only 1975). 4IEHS ;ted by after 3 ; those tos. In t-fiber sd dis- ds (RG he RG >tile A bestos i after t more ;hat of, iber of r both ' Fig. 1. Montage of transmission electron micrographs of UICC Rhodesian fiber (A). RG 144 Calidria fiber, as received (B), and RG 144 milled for 1200 sec (C). Fiber in A is about 5 fim in length. Magnification for each micrograph is the same. The Rhodesian fiber specimen consists of many fibers greater than S fim in length: -RG' 144, as received, contains fewer long fibers, as does the 1200-sec n:;led preparation. The selected area electron diffraction pattern (inset in Q shows the chrysolite structure was retained even after the 1200-sec milling treatment. RECEIVED TIME SEP. 13. 11:48AM PRINT TIME SEP. 13. 11:53AM SEP 13 2002 12:48 FR CISTI ICIST 613 941 4484 TO 12145201181 230 YEAGER ET AL. TABLE 2 Percentage Viability of Human Alveolar Macrophages (HAM) after J-hr Pulse with Mineral Dust and 24-hr Incubation in Fresh Medium* Culture No. runs Percentage viability 50 fig/culture 100 ptgfculture (1) HAM + no dust* (2) HAM + V1CC chrysotile A* (3) HAM + unmiUed RG 144 chrysotile* (4) HAM + 60 sec. mpled RG 144e (5) HAM + 1200 sec. milled RG I44c (6) HAM + quartz (Min-U-Sil 15)* 10 88.3 88.3 10 67.2 62.7 10 47.4 42.0 6 39.7 27.4 6 23.7 14.4 10 29.3 9.3 * Incubation conditions as described under Materials and Methods. * SEM for all groups from analysis of variance was 3.05. All values in groups 1, 2, 3, and 6 were significantly different from each other at P < 0.05 by Duncan's multiple range test. c SEM for all groups from analysis of variance was 4.02. There was a significant difference between the results obtained with the 60-sec milled and 1200-sec milled preparations (F(l,15) -- 12.99, P < 0.005). of these parameters may in part explain the enhanced cytotoxic action of milled asbestos on human alveolar macrophages. No clearly defined dose--response relationship was observed in the dose range used. There are a number of possible explanations. It is conceivable that clumping of particles, or their removal after 1 hr of exposure, or adsorption of protein onto the particle surface may have modified the cytotoxic properties of the dusts at higher mass concentrations. It is of interest that a'similar rank order of toxicity of these particulates was seen in preliminary experiments with rabbit alveolar mac rophages (H. Yeager, unpublished data). Differential phagocytosis of different fiber sizes is a possible bias in these ex periments. Nevertheless, on light microscopy, fibers >5 fim in length were seen both in cultures treated with RG 144 and in those treated with the UICC preparation. The findings in the present study are at variance with those reported elsewhere by other investigators. Thus Chamberlain et al. (1980) reported that ball-milled UICC chrysotile B (Canadian) was less cytotoxic to Chinese hamster lung cells than unmilled UICC chrysotile B. Such lack of biologic potential may have been produced as the result of a vigorous comminution process, since such has been shown to alter fiber structurally and blunt at least some of its activity (Langer et aL, 1978). Tilkes and Beck (1980), using short-fiber chrysotile prepared by physi cal separation (Spumy et al., 1979), observed that "extra-fine" fibers were less toxic for phagocytic tumor cells than the "parent" fiber preparation. The explanation for the differences among the various studies is not apparent. It is possible that the naturally occurring short fiber used in the present study is somehow unique and not representative of all short-fiber chrysotile asbestos. Other factors, such as differences in culture systems, doses used, etc., may have accounted for the discrepancies between our results and those of the aforemen- RECEIVED TIME SEP. 13. 11:48AM PRINT TIME SEP. 13. II:52AM SEP 13 2002 12:48 FR CISTI ICIST 613 941 4484 TO 12145201181 SE -y :ulturc 3 7 0 4 4 .3 6 were I '* I etween 1 12.99, , I milled range mping i onto ists at :ity of mac- se exwere UICC where milled ' cells rbeen been ger et physie less . - ;nt. It tdy is jstos. have :men- CYTOTOXICITV OF SHORT-FIBER CHRYSOTILE ASBESTOS 231 tioned authors. Nevertheless, since the type of short fiber used by us is currently employed in the United States and has been shown to be cytotoxic to human alveolar macrophages in vitro, it would seem of importance that further studies of the possible harmful effects of this material should be pursued. ACKNOWLEDGMENTS The authors thank the Pulmonary Disease Division Fellows, Georgetown Medical Center, and Mrs. Roberta Bjornerud for assistance with the bronchoscopies, and Mrs. Elizabeth Phillips for help with the statistical analyses. We are grateful to Dr. Vernon Timbrell of the MRC Pneumoconiosis Unit, LIandough Hospital, Penorth, S. Glamorgan, Wales, U.K., for supplying the UICC Rhodesian chrysotile A, and to Dr. J. S. Harington for the valuable and incisive review. This work was supported in part by a grant from the NIEHS, ES 00928, and by the assistance from the Mobil Foundation (A.M.L. and R.P.N.). REFERENCES Beck, E. G. (1980). Experimental pathology--in vitro studies--related to asbestos and other mineral fibers. Jn "Biologic Effects of Mineral Fibers" (J. C. Wagner, Ed.), Vol. 1, pp. 385-400- 1ARC, Lyon. Chamberlain, M., Brown, R. C,, and Griffiths, D. M. (1980). The correlation between the carcinogenic activities * in vivo' and the cytopathic effects `in vitro'' of mineral dusts. In * `The In Vitro Effects of Mineral Dusts" (R. C, Brown, I. P. Gormley, M. Chamberlain, and R. Davies, Eds.), pp. 345--349, Academic Press, New York. Crapo, J. D., Barry, B. E., Brody, A. R., and O'Neil, J. J. (1980). Morphological, morphometric, and x-ray microanalytica) studies on lung tissue of rats exposed to chrysotile asbestos in inhalation chambers. In "Biologic Effects of Mineral Fibers" (J. C. Wagner, Ed.), Vol. 1, pp. 273-283. IARC, Lyon. Harington, J. S., Allison, A. C., and Badami, D. V. B. (1975). Mineral fibers. Chemical, physicochemical and biologic properties. Adv, Pharmacol. Chemother. 12, 1--402. Hillerdal, G. (1980). The pathogenesis of pleural plaques and pulmonary asbestos. Possibilities and impossibilities. Eur. J. Sespir. Dls. 61, 129--138. Hocking, W. G., and Goide, D. W. (1979). The pulmonary alveolar macrophage. AT. Engl. J. Med. 301, 580-587, 638-645. Kagan, E. (1981). The alveolar macrophage: Immune derangement and asbestos-related malignancy. Semin. Oncol. 8, 258--267. Kagan, E., and Miller, K. (1981). In vino use of silica. In "Manual of Macrophage Methodology. Collection, Characterization and Function" (H. B. Herscowitz, H. T. Holden, J. A. Bellanti, and A. Ghaffar, Eds.), pp. 137--143. Dekker, New York. Langer, A. M., Wolff, M. S., Rohl, A. N., and Selikoff, I, J. (1978). Variation of properties of chrysotile asbestos subjected to milling. J. Toxicol. Environ. Health 4, 173-188. Miller, A., Langer, A. M., Teirstein, A. S., and Selikoff. I. J. (1975). ''Non-specific" interstitial pul monary fibrosis. Association with asbestos fibers detected by electron microscopy. N. Engl. J. Med. 292, 91-93. Nolan, R. P., Langer, A. M.. Harington, J. S., Oster, G., and Selikoff, I. J. (1981). Quartz hemolysis as related to its surface fractionalities. Environ. Res. 26, 503 -520. OSHA (1972). Occupational safety and health standards. Standard for exposure to asbestos dust. Fed. Reg. 37, 11318-11322.- Paul, J. (1965). "`Cell and Tissue Culture," 5th ed., p. 368. Churchill Livingstone, Edinburgh. Rendall, R. E. G. (1970). The data sheets on the chemical and physical properties of the U.I.C.C. standard reference samples. In "Pneumoconiosis. Proceedings of the Internationa] Conference" (H. A. Shapiro, Ed.),pp.-23-*-27. Oxford Univ, Press, Capetown. P. 10/11 RECEIVED TIME SEP.13. 11:48AM PRINT TIME SEP. 13. 11:52AM SEP 13 2002 12:48 FR CISTI ICIST 613 941 4484 TO 12145201181 P. 1 232 YEAGER ET AL. Sebutien, P., Janaon, X., Bonnaud, G., Riba, G., Masse, R., and Bignon, J. (1979). Translocation of asbestos libers through respiratory tract and gastrointestinal tract according to fiber size and type. In "Dusts and Disease" (R. Lenten and J. M. Dement, Eds.), pp. 65--85. Pathotox, Park Forest South, in. Snedecor, G. W.. and Cochran, W. G. (1980). "Statistical Methods," 7thed., pp. 215-237. IowaStatc Univ. Press, Ames. Spumy, K., Stober, W., Orkla, H. L., and Weiss, G. 0979). Size selective preparation of inorganic fibers for biological experiments. Amer. Ind. Hyg. Assoc. J. 40, 20--38. Tilkes, F., and Beck, E. G. (1980). Comparison oflength-dependent cytotoxicity ofinhalable asbestos and man-made mineral fibers./n "Biologic Effects of Mineral Fibers" (J. C. Wagner, Ed.), Vol. 1, pp. 475--483. IARC, Lyon. Timbrel], V. (1970). Characteristics of the International Union Against Cancer standard reference samples of asbestos. In "Pneumoconiosis Proceedings of the International Conference, Johan nesburg, 1969" (H. A. Shapiro, Ed.), pp. 23-36. Oxford Univ. Press, Capetown. Yeager, H., Jr., 2immet, S. M., and Schwartz, S. L. (1974). Pinocytosis by human alveolar mac rophages. Comparison of smokers and nonsmokers. J. Clin. Invest. 54, 247--251. 1 < Y t `k RECEIVED TIME SEP, 13. 11:48AM PRINT TIME SEP. 13. 11:52A^al page, n M.til! 1:1 nUtKVl'. 'll 1 f-1 .1' `i-Nt 1 < h ' V | * t ,i i I. in nx-ilnl 'iii.-'t . "w \' i I i '. !i'iu >' I i i-i h-'C -ii<.1 ri-.-- i-. lu,' I Vl.-t. :)ii;il [ 'it! 'i i-1 k*! n i VluUi.ji'm . ity h n ) M'.'niMi'-iiiil , ! > 4l l" 'll \K' 11 > is \ i; 'i' (ill- n \\i>. -fI'm.lift i! K-nili.'.i'uni SA v 1 I'll:'K * * *1 il'U.lIJil'Ti Nphji . I'lsll- i n i hi i. 1 IlJ'.-OI ' Kofi-, r-te's t HR 1 i'ei.`uii'.ni! . ! H. \N|:: 1: M i f`-- . T IS!' "H >'llj 443 45a 4kl 48(1 492 499 it m: ! -vml 985 3 oo ACADEMIC PRESS . ' .it . , I { - I. i ji i \<i*)uVkIi I* -' ; 'i <.{! i<<(>']<>>' %' I >*-. Li ^ .t i - 'iinv,.i I1 juln .k\i* l - ' * (