Document 82m4BdoQd5jnKX2ZnG1aKXkzZ

ENVIRONMENTAL RESEARCH 31, 164-175 (1983) '.'(S' A Comparative Study on the Hemolytic Action of Short Asbestos Fibers on Human, Rat, and Sheep Erythrocytes1 J. P. Pel6 and R. Calvert Department d'anaiomie el de biotogie cellutaire. Programme de recherche sur I'amianle, Faculle de Medecine, University de Sherbrooke, Sherbrooke. Quebec. J1H 5N4 Canada Received March 8, 1982 The hemolytic activity of short asbestos fibers isolated by a sedimentation procedure a as studied using human (HRBC), rat (RRBC). and sheep (SRBC) red blood cells. The init velocity (V,) of hemolysis is proportional to the concentration of fibers with HRBC and RRBC. but not with SRBC. Initial velocity (V,) is 14.30 for HRBC, 5.75 for RRBC, and 3.60 for SRBC at a concentration of 1,000 p.g of fibers/ml. Maximum velocity (Vm) is reached at 400 /agiml with HRBC and its value is 16.7. With RRBC and SRBC. V,,, is reached at 600 /igiml and its value is 10 and 3.6, respectively. The 50% hemolytic concentration (HCs,) at 60 min of incubation is 75 /ug/ml for HRBC, 240 pg/ml for RRBC, and 260 ftgml for SRBC. It appears that the sensitivity against the short asbestos fibers of the three types of RBC used is in the following order: HRBC > RRBC > SRBC. INTRODUCTION The hemolytic activity of chrysotile asbestos was reported by Macnab and Harington (1967). Since then, this in vitro model has been extensively used to study the biological activity of asbestos. Although many hypotheses have been put forward to explain the precise nature of the damages caused by the asbestos fibers to the red blood cell membrane, the mechanism by which these fibers cause the damages is still not completely elucidated. Most studies on the hemolytic activity of asbestos fibers were done with eryth rocytes of sheep (Macnab and Harington, 1967; Schnitzer and Pundsack, 1970; Harington et al., 1971a, b; Schnitzer et al., 1971; Light and Wei, 1977a, b) or of man (Secchi and Rezzonico, 1968: Harington et al., 1971b: Schnitzer et al.. 1971; Morgan et al., 1977; Jaurand et al.. 1979b, 1980). Horse blood cells were used in only a few studies (Schnitzer et al.. 1971: Harington et al.. 1971a). Rat blood cells (Hefner and Gehring, 1975: Harington et al., 1971a) and rabbit erythrocytes (Harington et al.. 1971a; Desai et al., 1975) were also used. Because the chemical composition of the red blood cell membrane from differ ent species is known to be different, it appeared pertinent to do a comparative study on the hemolytic effect of well-characterized asbestos fibers on erythro cytes from different sources. This paper describes the kinetics of the hemolytic action of short chrysotile fibers on human, sheep and rat erythrocytes. ; lhi-> work was supported by Grant G0I79 from the CRSNG of Canada. '%Preparation of er\ ;^eep (SRBC) blood e s collected from hi tm the radial vein: . 'jliong Island rats weigh ilpfer, pH 7.28. \.MAsbestos fibers. Chi "Quebec 4T30 chrysoi: 'farface charge represe 'Mid a value of +52 m .$licoeur et al. (1981) !.y0 and that the m ii'llso determined the s: sorption and water m2/g and the secom | 'if Hemolysis. Weighed : ug/ml) were suspended, prepared as described iiiiie stock solution was suspension of eryr ihe suspension of fibe: jjlood cells was 2r( ar jfig/ml). The Falcon it. -shaking incubator set ;r ijttin of incubation. 0.2 `.fempleto stop hemoh fad the optical densit'. $uid Lomb Spectronie Complete lysis was tilled water with two , "hemolytic activity of th three replicates and u (100%) optical density Electron microscopplaced on a Formvar-e grid was deposited ow Philips EM 300 electro The short asbestos IT than 8 p.m in length < h very infrequently obse1 The curves of hemo species make it possibi. 0013-9351 83 53.00 Cop\nc,hi < Academic Pvcss. Inc Ail right*. of reproduction -,nv form reserwd 164 HEMOLYTIC ACTIVITY OF ASBESTOS 165 ; MATERIALS AND METHODS tic Action of Short Rat, and RT gramme tie recherche stir le Sherbrooke. uulti ,i sedimentation procedure v IC) red blood cells. The ini! .,1 lion of fibers with HRBC and 1RBC. 5.75 for RRBC. and 3.60 turn velocity (Vm) is reached at id SRBC. Vm is reached at 600 lytic concentration (HC,,,) at 60 1C. and 260 figtml for SRBC. It f the three types of RBC used is as reported by Macnab and aas been extensively used to many hypotheses have been mages caused by the asbestos m by which these fibers cause s fibers were done with erytnchnitzer and Pundsack, 1970: .ight and Wei, 1977a. b) or of 1971b; Schnitzer et til.. 1971; torse blood cells were used in i ci al.. 1971a). Rat blood cells '"'la) and rabbit erythrocytes -.Iso used. od cell membrane from differpertinent to do a comparative .d asbestos fibers on erythro- ihe kinetics of the hemolytic md rat erythrocytes. i of Canada. Preparation of erythrocyte suspensions. Human (HRBC), rat (RRBC). and '&eep (SRBC) blood cells were prepared at a concentration of 4%. Human blood collected from healthy donors by venipuncture: sheep blood was collected jSom the radial vein; and rat blood was collected from the inferior vena cava of vliiong Island rats weighing 250-300 g. Cells were washed three times with Veronal .Jtiufifer, pH 7.28. ]?%Asbestos fibers. Chrysotile fibers, isolated by a sedimentation procedure, from (Quebec 4T30 chrysotile (Dunnigan et al., 1980) were used in this study. The -'^urface charge represented by the zeta potential (Light and Wei, 1977a), at pH 7.3, .vjiad a value of +52 mV. The length distribution of the fibers as determined by "vjolicoeur et al. (1981) showed that more than 90% of the fibers were shorter than ;,-^0 pm and that the maximum fiber length was 8 yum. These same authors have : 'iiso determined the specific area of the short sedimented fibers from nitrogen .''adsorption and water adsorption: the first method yielded a specific area of '-20m2/g and the second 28 nr/g. " ' Hemolysis. Weighed amounts of asbestos fibers (200. 400, 800, 1200, and 2000 jig/ml) were suspended in Veronal buffer using a Dounce tube. Veronal buffer was prepared as described by Harington et al. (1971a) and just before use, one part of Jhe stock solution was diluted with four parts of distilled water. Exactly 1 ml of the 4% suspension of erythrocytes previously warmed at 37=C was added to l ml of ; the suspension of fibers at the desired concentration (final concentration of red blood cells was 2% and that of asbestos fibers was 100. 200, 400, 600, or 1000 #g/ml). The Falcon tubes were immediately placed into a Dubnoff metabolic Shaking incubator set at 37C and at 45 strokes/min. After 0.5, 1-10, 15, 30. and 60 nun of incubation, 0.2 ml of a 2.,5% solution of glutaraldehyde was added to each temple to stop hemolysis. The samples were then centrifuged at 900g for 5 min and the optical density of the clear supernatant was read at 541 nm in a Bausch and Lomb Spectronic 21 spectrophotometer. Complete lysis was obtained by preparing a 2% erythrocyte suspension in dis tilled water with two drops of Triton X-I00 and 0.2 ml of glutaraldehyde. The hemolytic activity of the fibers was calculated from the average optical density of three replicates and was expressed as a percentage of the totally lysed control {-100%) optical density. Electron microscopy. A drop of a diluted suspension of the short fibers was placed on a Formvar-coated grid and allowed to dry. A secotrd Formvar-coated grid was deposited over the dried fibers and the "sandwich" was observed in a Philips EM 300 electron microscope. RESULTS The short asbestos fibers prepared by the sedimentation procedure measure less than 8 /Lrm in length (Fig. 1). They are well dissociated and large aggregates are very infrequently observed. The curves of hemolysis obtained with the erythrocytes of the three different species make it possible to identify different stages during the hemolytic process. j! I z Fig. I. Suspension of short chrysotile fibers dispersed with Dounce ball tube. x5460. TIME (mmutesl Fit.. 3. Human RBC. Percentages of hemohsis during the 0- to 5-min period. Correlation coeffi cients: 1000 fig ml 0 to 5 min (r = 0.9950). 600 fig ml I to 5 min (r = 0.9965). 400 fig ml 2 to 5 min (r = 0.99881. 200 fig ml 2 to 5 min (> = 0.9991). 100 fig ml 2 to 5 min (r = 0.9990). 166 Fig. 3. Hun: With HRBC. be; trations of fibers . between 0 and 1 m were calculated. A linear and V', is iden and 60 min of inci; plateau at 10 min. /j.g/ml a maximum With RRBClFig between 2 and 8 m the highest concern at 30 min for all th When SRBC arc (Fig. 6) and V, is io completed and a p tions of fibers. if! HEMOLYTIC ACTIVITY OF ASBESTOS 167 / ji' W' n Dounce ball tube. *5460, 20 H --------- 1 i5 1 minutes) 5-min period. Correlaiion cu:tTi' 0.99651. 400 {*g. ml 2 lo 5 mm tr = - = 0.9990). 1----J-----1----------------------------1------------------------------------------------------1- C 5 10 15 30 60 TIME, (minutes) Fig. 3. Human RBC. Percentages of hemolysis during the 5- to 60-min period. With HRBC, between 0 and 5 min, the kinetics is not linear when the concen trations of fibers are from 100 to 600 /ag/ml (Fig. 2). The initial velocity (Fj) between 0 and 1 min and the maximum velocity (Vm) between 1 or 2 and 5 min were calculated. At a fiber concentration of 1000 /ag/ml, the kinetics becomes linear and V, is identical to Fm. Figure 3 shows the kinetics of hemolysis between 5 and 60 min of incubation. At higher doses (400- 600 /ag/ml). the curves reach a plateau at 10 min. At 200 /xg/ml. a plateau is reached at 30 min only, but at 100 /ag/ml a maximum is still not reached at 60 min. / With RRBC (Fig. 4). V-, was calculated as occurring between 0 and 2 min and V'm between 2 and 8 min. The kinetics of hemolysis with RRBC is not linear even at the highest concentrations of fibers. Later during incubation, a plateau is reached at 30 min for all the concentrations used (Fig. 5). When SRBC are used, the kinetics of hemolysis is linear between 0 and 15 min (Fig. 6) and V', is identical to Vm. Between 30 and 60 min (Fig. 7), hemolysis is not completed and a positive slope is still observed even for the highest concentra tions of fibers. 100-1 1000 * 600 400 J 200 A 100 s <! Z 60-1 5I uJ Fig. 4. Rat RBC. Percentages of hemolysis during the 0- to 10-min period. Correlation coefficients; 1000 ^xg/ml 2 to 5 min (r = 0.9920), 600/xg/ml 2 to 6 min (r = 0.9951), 400 (xgfml 2 to 8 min (r = 0.9958). 200 jug/ml 2 to 8 min (r = 0.9924), 100 jug/ml 2 to 8 min (r = 0.9729). pg OF FIBERS/ml O z fcsJ Fic. 6. Sheep RBC. Pcdents: 100 p-g/ml (r = 0.9`ti ait; 1000 fig/ml (r = 0.99761. ' If V-, for the three c { seen that velocity is fffRRBC are used, but human blood. 5.75 ft -Mi 1000 jjig of fibers/ml: concentrations betw t and SRBC. Figure 9 shows V. HRBC and its value their values are 10 .i The percentages i centration is 1000 p blood used. Howe\-. of the three types o! of hemolysis reach sents the 50? heme of the curves corres- 5 10 15 30 60 TIME fmmutesi Fig. 5. Rat RBC. Percentages of hemolysis during the 5- to 60-min period. 168 HEMOLYTIC ACTIVITY OF ASBESTOS 169 v\ 4 OO &6 T----- 1----- 1 10 'nmytesl in period. Correlation coefficients: ,, 4()0 ig/ml 2 to 8 min (r = 0.9958!, NL F Ft8ERS/ml .--------------- 1000 ----------- 600 .---------- 400 200 100 c 60 ; to 60-min period. Fig. 6. Sheep RBC. Percentages of hemolysis during the 0- to I5-min period. Correlation coeffi cients: 100 jug/ml (r = 0.9905). 200 fxg/ml (r = 0.9873), 400 /xg/ml (r = 0.9900), 600 Atg/ml (r = 0.9956). 1000 ftgfm! (r 0.9976). If V-t for the three types of blood is plotted on the same graph, (Fig. 8), it is easily seen that velocity is proportional to the concentration of fibers when HRBC and RRBC are used, but it is not when SRBC are used. The value of V, is 14.30 for human blood, 5.75 for rat blood, and 3.60 for sheep blood at a concentration of 1000 ix.g of fibers/ml; V, is expressed as the percentage of hemolysis per minute. At concentrations between 0 and 400 fig/ml, V, of hemolysis is comparable for RRBC ' and SRBC. Figure 9 shows for the three types of blood. Vm is reached at 400 /xg/ml with HRBC and its value if 16.7; with RRBC and.SRBC, Vm is reached at 600 p.g/m! and their values are 10 and 3.6, respectively. The percentages of hemolysis observed at 60 min of incubation (when the con centration is 1000 fig of asbestos/ml) are not very different for the three types of blood used. However, with lower concentrations, one can see that the sensitivity of the three types of blood are different. Between 30 and 60 min. the percentages of hemolysis reach a plateau; at 60 min, we can determine the HC5(I (HCS repre sents the 50% hemolytic concentration; see Schnitzerer /. (1971)). In the portion of the curves corresponding to 50% of hemolysis, the percentage is almost propor- 170 PELfi AND CALVERT TOO-, F OF FIBERS/ml Fig. 8. Initial cel,and HRBC (.1) fr = Fig. 7. Sheep RBC. Percentages of hemolysis during the 5- to 60-min period. tional to the concentration of fibers. Therefore, this HC5,, can be interpolated for the three types of blood: for HRBC it is 75 ig/ml, for RRBC it is 240 /ag/ml, and for SRBC it is 260 /t,g/ml. DISCUSSION , The results on the hemolytic activity of chrysotile asbestos fibers found in the literature are difficult to compare because of (1) the different types of-blood used. (2) the utilization of fibers of diverse origin often poorly characterized, (3) the different protocols used to measure the hemolytic activity. (4) the important dif ferences in the controls used, and (5) the temperature and the duration of the incubation. To illustrate this diversity we have summarized the results reported by different authors on the hemolytic activity of chrysotile fibers (Table 1). In the present work, we have used well-characterized chrysotile fibers isolated by a procedure that does not modify the structure of the native fibers. Further more. these short fibers give stable and homogeneous suspensions. It is veil known that ball-milling modifies considerably the physicochemical properties of Fig. 9. Maximum PIBERS/ml 1000 600 400 Fig. 8. Initial velocity (V,). Percentage of hemolysis per minute. SRBC (O). RRBC () (r = 0.9970), and HRBC (A) (r = 0.9924). 5- to 60-min period. can be interpolated for tRBC it is 240 /ag/ml, and DZ s cc bestos fibers found in the erent types of blood used, rly characterized. (3) the ity. (4) the important, dif- and the duration of the 'ed the results reported by . fibers (Table 11. : chrysotile fibers isolaled he native fibers. Furtfern suspensions. It is v ell icochemical properties of 100 200 30 600 1000 pg OF FIBERS ml Fig. 9. Maximum velocitv (f,,j. Percentage of hemolvsis per minute, SRBC (Cl, RRBC (). and HRBC(_). ' 171 172 V -r zc c- o\ PEL AND CALVERT vy p cs p p' vl dc Tf OV~, p o Vi . f >S'. '' 5 y ly* V 0- HI * a c. * 6 3 S, O U uo = ^ c^ m UO o n fi n n n m rj r-* n"i 2 a2 /> E zb cB 3L zi. zL o o vr. E _ E______________ _ Ia5BL "5cSB. eB =L <n cj E aB "53b. <n 'c:b* "Sb Cj <-l Vi o cn ri rr E c3B. c- E *=&1 O r- E ab zL 2 ci m - n \0 di cs ri E Ci 5 E E t* it E ir t-t C. c. V y s ci y y J1 CO S j= CO ci Q. a c. yyy y jz jy= jyC zn ci CO ci CO ci a y a. u. y y J3 ci ci CO ci C. y y CO ri c & Cl C J9 ' *e y s- 'zb zb zj aQ SEE y l/~, V, i* JO -- ci ri r t y -a O. > CO U ffl cd r-i r TABU- EE -t u >. -2 X E o U , - O ,, fc- rt g fc 74 OJ U U R. y y yy C c. VI 3 a 3 c r 40 rE 6-g _to D U Tf U ~~ rt <=-> -n U j i 'r a JS u 2 3 :7- - 3 V < C2 < jh y .C .o y rr y O - fi U y y JS " K CQ 3 i, 2y y *3 O i; g c: / y J ii -j o 2 3x ^5 ^."J i>l 3 SS U O' u O' 5 >u3- 1 J= J= io y -r y __ i= O' -- i= O >. O u JC o r* c v O' _ii V2 *3 C XX *3 *3 CC ZO c c j 'S'. cc C-* 9- CO 7i ' CO m E a. 5b a. e % a. = EE --. :'-t. y g'-i V5vs 2 -J ** --> 5 t: <? S - e <= ; y \cn _y 3 Xyu51! <<c _' 5s a' ato -rz t* Cyog-r if E aw. xJ2 ;, HEMOLYTIC ACTIVITY OF ASBESTOS O O *r. r. X >^o O' sMo - do c C C 3; ri C r*/%. X-- v^ 74.0 100.0 173 50 min 37"C SO min 37"(.' C uU U - mr** o ?n- Ec a c u - c*\ pi r*. -- nt r 5 v~. fU0r-'>, EU 12,5 mg/ml 12,5 mg/ml \> i E aE aE a aE Ea Ea_s E E EE a E EE E E E^ v-i uo io iti v~, a. *a5b. '5ab. *5ab. 5ab. vri ri ri ri ri ri ri o zll -- 7 =b aE ~.22 5 jjCQO. <*3J` '--r 03 OS rl cn ri r* r4 t3i 2a vd, cir, vd, ^ ^j|i r* r=- 2" Cr 7Jz) Rul .Shctp UIC'C chrysolite . C liry.iolilc K0'7 /xm5 ' 03 a 22 < y X -- 3 U V J,,a,, X v Ea o ,y .y < o *" <J U /S> X*/5 rC----/--_ >./}i x c c c J= HO ^c a n a l; c Cj O U X X X XL>-, 5i--s>1_ 5Ss_ y yy y OU UL>LU> OL/ X---s X_ yy LL./ LL// c f. >, __a.. w tTSO" 5us E -S ;j ^ o ?! k 5 o O L/ D 5 5d 5 r, x r- r- M.k ik iI' ami Ila iin g lo n (1007) 174 PEL AND CALVERT the fibers (Langer et al., 1978). The hemolytic action of these short fibers was . . jaurand. M. C.. Bign, measured using human and sheep red blood cells which are reported in the litera les globules roue ture to be the most frequently used on asbestos and also red blood cells of the rat . Jaurand. M. C.. Mag- ' # which is the most frequently utilized animal in laboratories. '!' of asbestos. Bn: i; Our results show that the three types of blood used have a different sensitivity ..." Jaurand. M. C.. Thou " ' cal and photoele, to the same chrysotile fibers. Human erythrocytes are the most vulnerable to the and red blood ee hemolytic action of these fibers. With human blood, hemolysis is the most rapid and the calculated Vi is 2.5 times that of rat erythrocytes and 4 times that of sheep erythrocytes; for human blood, Vm is the highest and its estimated HC50 is 3 times lower than that of the two other types of blood. When the results for rat and sheep blood are compared, it seems that the latter is slightly less sensitive than the 'r Jolicoeur, C.. Roberg asbestos maten. H40-I148. Koshi, K.. Hayaxhi, Ind. Health 6, 6`t Langer. A. M.. Wolf former to the hemolytic action of the fibers used. ' chrysotile asbest. It is difficult to explain the differences in the responsiveness of the three types of blood to the same chrysotile fibers. Nevertheless, one can point to the different physicochemical properties of the red blood cells of the three species used. For example the mean diameters of human and sheep red blood cells are 8.5 and 5.2 fj.m. respectively, with the results that for HRBC and SRBC. the mean surfaces Light, W. C.. and We . 13, 1135-1145. Light. W. G.. and V. . 537-539. Macnab. G.. and HaSuture (London are 163 and 67 /am-, respectively (Weinstein, 1974). The surface charges expressed Morgan. A.. Holmes. as the electrophoretic mobility (p.m sec-1 V-1 cm) at 25C are 1.08, 1-1.28, and 1.14, respectively for HRBC, RRBC, and SRBC (Seaman, 1975). The permeability of and its relation t, Rahman, Q.. Narang. to its silica soluK the three types of blood toward glycerol or phosphate ions is different (Van Schnilzer. R. J.. and Deenan and De Gier, 1974). Finally the phospholipid composition of the mem fibers. Arch. Eir. ii brane of the three types of RBC as reported by Van Deenan and De Gier (1974) Schnitzer. R. J.. Bunc also underlines these differences with SP/PC ratios (SP: sphingomyelins, PC: phosphatidylcholine) of 1/4 for RRBC, 2/3 for HRBC, and 12/1 for SRBC. vitro. Ann. S.Y, Schnitzer. R. J.. and Seaman, G. V. F. 119" These different physicochemical properties probably play an important role in the . Press, New York sensitivity of the RBC to chrysotile fibers' but it would be imprudent to venture -. Secchi. G. C.. and R. which one is the most important: surface is probably involved but the chemical composition of the RBC membrane could be as determinant. In conclusion, although there are some differences in the results obtained with the different types of blood used, it can be said that all three types are acceptable Van Deenan. L. L. M ' 1. p. 190. Acadc- Weinstein. R. S. (19Press. New Yors to perform these studies on hemolysis. At this time, it appears that the use of well-characterized fibers and the method used to obtain the suspension of fibers are matters of the utmost importance. REFERENCES / Desai. R.. Hext. P.. and Richards. R. 11975). The prevention of asbestos induced hemolvsis. Life Sri. 16. 1931-1938. ' Dunnigan. J.. Nadeau. D.. Paradis. D.. Pele, J.-P., Calvert. R.. Lalancette. J.-.M.. and Cossette. M. (1980). Cytotoxic and haemolytic effect of native and chemically modified chrysotile. In "Fourth International Conference on Asbestos," IV B 1. pp. 747-772. Hartngion, J. S.. Miller. K.. and Macnab. G. (1971a). Hemolvsis b\ ashestos. Environ. Ret. 4, 95-117. '' Harington. J. S.. Macnab. G. M.. Miller. K.. and King. P. C. f 1971 bI. Enhancement of hemolytic activity of asbestos by heat-labile factors in fresh serum. Mcil. Lav. 62, 171 - 176. Hefner. R. E.. and Gehring. P. J. 11975). A comparison of the relative rates of haemoh sis indue- i b; various fibrogenic and non-fibrogenic particles with washed rat erythrocytes in vitro. Aina Hw. Atwe. J. 36. 734-740. HEMOLYTIC ACTIVITY OF ASBESTOS 175 >1'these short fibei ^ way ire reported in the luerared blood cells of the rat ies. ;ve a different sensitivity le most vulnerable to the nolysis is the most rapid and 4 times that of sheep estimated HC3 is 3 times j results for rat and sheep \ less sensitive than the \eness of the three types can point to the different c three species used For lood cells are 8.5 and 5.2 SRBC, the mean surfaces >urface charges expressed are 1.08, 1-1.28. and 1.14, 975). The permeability of te ions is different (Van composition of the memeenan and De Gier (1974) s (SP: sphingomyelins, (BC, and 12/1 for SRBC. iy an important role in the 1 be imprudent to venture nvolved but the chemical linant. > the results obtained with three types are acceptable it appears that the use of n the suspension of fibers jaurand. M. C., Bignon, J.. Magne, L.. Renier, A., and Lafuma, J. (1979a). Interaction des fibres avec Ies globules rouges et les macrophages alvdolaires in vitro. Rev. Fr. Mai. Resp. 7, 717-722. Jaurand. M. C.. Magne, L.. and Bignon. J. (1979b). Inhibition by phospholipids of haemolytic action ;. ; of asbestos. Brit. J. hut. Med. 36, 113- 116. ' Jaurand. M. C.. Thomassin, J. H.. Baillif, P., Magne, L., Touray. J. C.. and Bignon, J. (1980). Chemi,, cal and photoelectron spectrometry analysis of the adsorption of phospholipid model membranes IS ' and red blood cell membranes on the chrysotile fibres. Brit. J. hid. Med. 37, 169-174. !' .; 1 Jolicoeur, C-, Roberge. P., and Fortier. J.-L. (1981). Separation of short fibers from bulk chrysotile ' Sv1' asbestos materials: Analysis and physicochemical characterization. Cttnad. J. Client. 59, 1140-1148. , Koshi, K., Hayashi, H., and Sakabe, H. (1968). Cell toxicity and hemolytic action of asbestos dust. ' ] hid. Health 6, 69-79. Langer, A. M.. Wolff. M. S.. Rohl, A. N., and Selikoff, I. J. (1978). Variations of properties of .. chrysotile asbestos subjected to milling. J. Toxicol. Environ. Health 4, 173-188. ` Light. W. C.. and Wei. E. T. (1977a) Surface charge and hemolytic activity of asbestos. Environ. Rex. 13,1135-1145. 'fV Light. W. G., and Wei, E. T. (1977b). Surface charge and asbestos toxicity. Nature (London) 265, 537-539. . Macnab. G-. and Harington. J. S. (1967). Haemolytic activity of asbestos and other mineral dusts. Nature (London) 214, 522-523. Morgan, A., Holmes, A., and Talbot, R. J. (1977). The haemolytic activity of some fibrous amphiboles and its relation to their specific surface areas. Ann. Occup. Hyg. 20, 39-48. 'Rahman, Q., Narang, S., Kaw. J. L., and Zaidi, S. H. (1974). Asbestos induced hemolysis in relation . to its silica solubility. Environ. Physiol. Biochem. 4, 284-288. f'C-',Schnitzer. R. J., and Bunescu. G. (1970). Polymers as selective antagonists of haemolytic asbestos *!'/.' fibers. Arch. Environ. Health 20, 481-482. V`\ V V'Schnitzer, R. J., Bunescu, G., and Baden, V. (1971). Interactions of mineral fiber surfaces with cells in vitro. Ann. N.Y. Acad. Sci. 172,759-771. 'v'y)Schnitzer, R. J.. and Pundsack, F. L. (1970). Asbestos hemolysis. Environ. Res. 3, 1-13. yCij'Seaman, G. V. F. (1975). In "The Red Blood Cell" (D. M. Surgenor. Ed.). Vol. 2. pp. 1186. Academic ' Press, New York. (Secchi, G. C., and Rezzonico, A. (1968). Hemolytic activity of asbestos dusts. Med. Lav. 59, 1-5. (.iyan Deenan, L. L. M., and De Gier, J. (1974). In "The Red Blood Cell" (D. M. Surgenor, Ed.), Vol. 1, p. 190. Academic Press, New York. Weinstein, R. S. (1974). In "The Red Blood Cell" (D. M. Surgenor, Ed.). Vol. I, p. 232. Academic Press, New York. sios induced hemolysis. Idle Su .incette. J.-M.. and Cossette. M modified chrysotile. In "Fourth - by asbestos. Environ. Re-. 4. "Ibl. Enhancement of hemolytic Lav. 62, 171-176. e rates of haemolysis indu. d bt erythrocytes in vitro. Ann InJ I- er Lind, Barry J. vposure Alters Spon- )UTCHER, AND W. E. >. and Mutagenicity Kumai, and M. itants in Nonpolluted ^ and Smoking. . . Dutcher. In Vitro om a Low-Btu Coal 448 460 472 484 uses: Research Apand M. Turner. . . 493 ce. Edited by R. S. F. 493 e--Causes and Prac494 xicology. By Keith 495 498 . 499 r in the December 1983 1983. ' :aDemic prls.s ' `v ^ (irk I (}(.id i* :ns s.m | )u N vn } .iu is, <> s `i i 11 '<r ".* FRO; 4 ' : v - . i CP) iii' \ 1 ik \ 1 t >n ju(i Volume 31. Number 1. June 1983