Document NG0q5jyomkrnLpaZ9Jx2877dD

u> ytv.11 Proceedings of an APCA International Specialty Conference ASBESTOS PLAINTIFF'S EXHIBIT CHR-226 ITS HEALTH RISKS, ANALYSIS, REGULATION AND CONTROL ENZYMATIC PROFILE OF THE LUNG LAVAGE FLUID: FURTHER BIOCHEMICAL EVIDENCES OF A TOXICITY TOLERANCE THRESHOLD FOR CHRYSOTILE ASBESTOS Denis Nadeau Raymond Begin Sponsored by APCA and The U.S. EPA Mid-Atlantic Asbestos Training Center at UMDNJ--Robert Wood Johnson Medical School Hosted by APCA's Mid-Atlantic States Section Atlantic City, New Jersey November 1986 ENZYMATIC PROFILE OF THE LUNG LAVAGE FLUID: FURTHER BIOCHEMICAL EVIDENCES OF A TOXICITY TOLERANCE THRESHOLD FOR CHRYSOTILE ASBESTOS Denis Nadeau Laboratoire de Biochimie et de Toxicologie Pulmonaires D6partement de biologie Facult des sciences University de Sherbrooke Sherbrooke (QuSbec) Canada J1K 2R1 and Raymond Bygin Units de recherche pulmonaire Centre Hospitalier Universitaire University de Sherbrooke Sherbrooke (Quybec) Canada J1H 5N4 Although some epidemiological studies on asbestos workers seem to point out an overall linear dose-response relationship between cumulative exposure and the incidence of asbestos-related lung diseases, this linear relationship appears somewhat controversial as far as chrysotile exposure is concerned. To examine this relationship, the conscious sheep model was selected. Canadian chrysotile (UICC B) was repeatedly administered in saline by intra-tracheal instillation. The animals were divided In a low dose (LD) exposure group (<500 mg total dust) and a high dose (HD) exposure group (<5000 mg total dust), while a third group of sheeps received only the saline vehicle (control). Following the 12 months-exposure period, the animals were studied further during four months to assess the evolution of the disease. The following enzymatic activities were measured on bronchoalveolar lavages (BAL) collected regularly during the study: lactate dehydrogenase, acid phosphatase, alkaline phosphatase and B-N-acetylglucosaminidase. The results show that, throughout the exposure period, only the HDexposure group presented steady and significant increases of all four BAL enzymes. During the four months following the last exposure to the chrysotile fibers, again, only the HD-exposure group presented signifi cantly elevated enzyme levels in the BAL. Overall, the LD-exposure group showed only transient elevations of some of the enzyme activities, mainly at the beginning of the exposure period. In conclusion, we believe that these biochemical data are suggestive of a tolerance threshold in regard to the toxicity of chrysotile asbestos fibers. Reprinted from Asbestos: Its Health Risks, Analysis, Regulation and Control, published by APCA, P.O. Box 2861, Pittsburgh, PA 15230 (1987). 236 Introduction Some epidemiological studies on asbestos workers have proposed a linear dose-response relationship between asbestos cumulative exposure and the incidence of asbestos-related lung diseases1-3. This was suggestive to some that there is no safe limit for asbestos exposure4. However, majiy other epidemiological studies do not concur for such linear dosere`sp'onsfi..rPiwnnsiiipT especially far ^ rhrycnff i? exposure~TT concerned. For example, Weill et al.5 report no evidence of a dose-response relationship in chrysotile cement workers. Similarly, McDonald et al.6, found little or no excess risk of cancer or asbestosis in workers from a friction material plant. In a follow-up study of chrysotile mine and mill workers with the highest exposures, Becklake et al.7 found no relationship between cumulative exposure indices and parameters of pleuropulmonary abnormalities. This study prompted these investigators to point out that a linear relationship had been previously proposed because an excessive number of heavyly exposed workers, in comparison to the number of low exposure workers, were included in the analysis8. Furthemore, a study of Canadian chrysotile miners exposed for 20 years to low levels of dust did not revealed an excessive prevalence of radiographic indices of lung diseases, findings that are In agreement with other studies from Italy and the Soviet Union9-H. More recently, Bgin et al.12-14 showed no relationship between cumulative exposure Indices from chrysotile workers and parameters of early or established asbestosis. These Investigators were lead to believe that a cumulative index may not be the best parameter to use with chrysotile asbestos-related diseases. Thus, substantial epidemiological data are available to support the concept of a tolerance threshold for chrysotile exposure. In this report, we wish to extend further the experimental evidences that, in the sheep model, the Intensity and the rate of exposure are two important parameters to relate with the development of lung damage induced by chrysotile asbestos fibersiS. Material and methods The animals (male sheeps 20-40 kg) used in this study were the same as those described in earlier studies^-1?. A pre-exposure study of the bronchoalveolar lavages (BAL) parameters did not show any significant variations between the animals1^. Briefly, the method used to expose concious sheeps to chrysotile asbestos involved the nasotracheal intubation of UICC Canadian chrysotile fibers (UICC B, described by Timbrell20) resuspended in 50 ml of phosphate-buffered saline (PBS). Control sheeps received only the PBS solution. The chrysotile-exposed sheeps were divided in two categories: a) low doses (ID): this group included sheeps which had received between 412 and 424'mg of UICC B chrysotile asbestos during a 12 months-exposure period; b) high doses (HD): comparatively, this group received between 4244 and 4384 mg of asbestos fibers during the same period. During the first 5 months of the study, all the animals were injected intratracheally an average of 14 times; the single doses administered varied between 2 and 16 mg for the LD group and, between 1 and 128 mg for the HD group. During the 6th month following the first exposure to the asbestos fibers, supplementary doses of chrysotile (105 to 128 mg) were administered to bring the average cumulative doses of the LD group between 240 and 250 mg, and for the HD group between 1220 and 1440 mg. Thereafter, the difference in dosage between the two experimental groups was maintened by injecting the LD and 237 HD groups with 2 and 128 mg of UICC B chrysotile, respectively. The exposure parameters are briefly summarized in Table I. The BAL effluents recovered from the sheeps were immediately spun down to recover the free cells. The cell-free supernatants were analysed on a Beckman Trace III Clinical Chemistry System equiped with a model 35 spectrophotometer. The enzyme activities, lactate dehydrogenase (LDH), alkaline phosphatase (AKP), acid phosphatase (ACP) and s-N-acetylglucosaminidase (B-NAG) were determined accordingly21-23. While for the AKP, ACP and B-NAG, one enzymatic unit express nmoles of p-nitrophenol released/min of incubation, for the LDH, one enzymatic unit express mi11i International Units (mlU)22*23. The experimental data were evaluated for statistical significance according to Tallarida and Murray2*. When the F-values of the one-way analyses of variance were significant, the differences between the mean values of the experimental groups were compared accordingly (for the details on the selection of the appropriate comparison tests, see each figure legend). Results and discussion The present study show that increased levels of cytoplasmic (LDH)21, lysosomal (ACP, 8-NAG)23 and lung surfactant (AKP)23 -associated enzymes are detected in the airways of sheep exposed to chrysotile asbestos (Figures 1 to 4). However, the data clearly demonstrate that: A) only the sheeps exposed to high doses of chrysotile fibers presented significant elevations of all four parameters of the BAL fluid, when compared to the low dose group or the control sheeps; B) the responses of the high dose group show linear (ACP, B-NAG) (Figures 3 and 4) or near linear (LDH, AKP) (Figures 1 and 2) Increases with the number of doses administered; C) that even 4 months after the end of the 12 monthsexposure period, again, only the sheeps exposed to high doses of chrysotile fibers presented significant and sustained elevations of the four enzymatic activities (Table II). The use of BAL fluid as an indicator of toxicity is well documented for lung targeted soluble and particulate materialsi'-^.^^.and high LDH, AKP and lysosomal activities have been associated with early and advanced asbestosis in humans28*29. These data provide substantial experimental evidences that there is a tolerance threshold level to chrysotile exposure, a concept that was recently reviewed by Dunnigan . Moreover, this level cannot be considered only in terms of cumulative exposure but must take into consideration the intensity and the rate of exposures15*31. Although these biochemical data can be primarly associated with inflammatory activities initiating activation and liberation of these enzymes at the site of disease activities, the alveolitis is believed to be the key element to interstitial lung disorders, including asbes tosis32"3*. Therefore, since the induction of alveolitis by chrysotile asbestos seems to be dose-dependant19*29, one must consider that there is indeed an exposure level below which no adverse health effects can be detected. Acknowledgments This work was supported by the following Canadian, provincial and federal, research programs: FCAC, CRSQ, CSST and CRSNG, respectively. We wish to thank Mr. Bruno Gagn6 for the art work and, Ms. Rfijeanne Laplante for typing this manuscript. 238 1. H. Weill, C. Waggenspack, W. Bailey, M. Ziskind, R.E. Rossiter, ((Radiographic and physiologic patterns among workers engaged in manufacture of asbestos cement products*), J. Occup. Med. 15: 248. (1973). ----------- --------------- 2. G. Berry, J.C. Gilson, S. Holmes, H.C. Lewinsohn, S.A. Roach, Asbestosis: a study of dose response relationships in an asbestos textile factory*), Br. J. Indust. Med. 36: 98. (1979). 3. J. Peto, Dose-response relationships for asbestos related disease: implications for hygiene standards. Part II. Mortality*), Ann. N.Y. Acad. Sci. 330: 195. (1979). 4. A. Englund, ((Based on available data can we project an acceptable standard for industrial use of asbestos? No*>, Ann. N.Y. Acad. Sci. 330: 219. (1979). 5. H. Weill, J. Hughes, C. Waggenspack, ((Influence of dose and fibertype on respiratory malignancy risk in asbestos cement manufacturing*), Am. Rev. Respir. Pis. 120: 345. (1979). 6. A.D. McDonald, J.S. Fry, A.J. Wooley, J.C. McDonald, Dust exposure and mortality in an American chrysotile asbestos friction products plants*), Br. J. Indust. Med. 41: 151. (1984). 7. M.R. Becklake, D. Thomas, F.D.K. Liddell, J.C. McDonald, Follow-up respiratory measurements in Quebec chrysotile asbestos miners and millers*), Scand. J. Work Environ. Health 8 (suppl. 1): 105. (1982). 8. J.C. McDonald, M. Becklake, G.W. Gibbs, A.D. McDonald', R.E. Rossiter, The health of chrysotile asbestos mine and mill workers of Quebec**, Arch. Environ. Health 28: 61. (1974). 9. S. Cordier, G. Theriault, S. Provencher, ((Radiographic changes in a group of chrysotile miners and millers exposed to low asbestos dust concentrations*), Br. J. Indust. Med. 41: 384. (1984). 10. E.C. Vigliani, in ((Pneumoconiosis*), Proceedings of an International Conference, Johannesburg, Capetown, South Africa, Oxford University Press, 1970, pp. 192-196. 11. F.M. Kogan, N.A. Guselnikova, M.R. Guleoskaga, The cancer mortality rate among workers in the asbestos industry of the Urals*), Gig. Senit. 37: 29. (1972). 12. R. Bfegin, A. Cantin, G. Drapeau, G. Lamoureux, M. Boctor, S. Mass, M. Rola-Pleszczynski, ((Pulmonary uptake of Gallium-67 in asbestos exposed humans and sheep*), Am. Rev. Respir. Pis. 127: 623. (1983). 13. R. Bg1n, M. Boctor, D. Bergeron, A. Cantin, Y. Berthiaume, S. Peloquin, G. Bisson, G. Lamoureux, ((Radiographic assessment of pleuro-pulmonary disease in asbestos workers postero-anterior, four view films and computed tomograms of the thorax#, Brit. J. Indust. Med. 41: 373. (1984). 14. R. Bfegin, A. Cantin, Y. Berthiaume, G. Bisson, G. Lamoureux, M. Rola-Pleszczynski, M. Boctor, D. Dalle, J. Breault, S. Mass, ((Detection of early asbestosis*), ILO Vlth International Pneumo coniosis Conference Monograph 2: 846. (1984T. 239 15. R. Bgin, S. Mass, M. Rola-Pleszczynski, M. Boctor, G. Drapeau, Asbestos toxicity - A monograph, TEES Monograph Series, Chap. 7 (to be published). 16. R. Bgin, M. Rola-Pleszczynski, P. Sirois, D. Nadeau, S. Mass, M.A. Bureau, ((Sequential analysis of the bronchoalveolar milieu in the conscious sheeps, J. Appl. Physiol. Respir. Environ. Exper. Physiol. 50: 665. (1981). 17. R. Bfegin, M. Rola-Pleszczynski, P. Sirois, I. Lemaire, D. Nadeau, M.A. Bureau, S. Mass, Early lung events following low-dose asbestos exposures, Environ. Res. 26: 392. (1981). 18. I. Lemaire, D. Nadeau, R. Bgin, ((Significant increases of cyclic AMP and alkaline phosphatase in bronchoalveolar lavage fluids of sheep exposed to asbestos#, Res. Com. Chem. Pathol. Pharmacol. 33: 567. (1981). 19. R. Bgin, M. Rola-Pleszczynski, S. Mass, D. Nadeau, 6. Drapeau, ((Assessment of progression of asbestosis in the sheep model by bronchoalveolar lavage and pulmonary function tests#, Thorax 38: 449. (1983). 20. V. Timbrel!, in ((Pneumoconiosis#, Proceedings of an International Conference, Johannesburg, Capetown, South Africa, Oxford University Press, 1970, pp. 28-36. 21. W.E.C. Wacker, P.J. Snodgrass, Serum LDH activity in pulmonary embolism diagnosis#, J. Am. Med. Assoc. 174: 2142. (1960). 22. D. Nadeau, M.J. Reasor, G.E.R. Hook, ((Extracellular alkaline phosphatase from alveolar secretions of patients with pulmonary alveolar proteinosis#, Can. J. Biochem. 59: 290. (1981). 23. J. Dunnigan, D. Nadeau, D. Paradis, J.-P. Pels, R. Calvert, J.-M. Lalancette, M, Cossette, Cytotoxic and haemolytic effect of native and chemically modified chrysotlle#, Proceedings of the 4th International Conference on Asbestos, Torino, Italy," Instituto di Arte Mineraria del Polltecnico & Assoclazione Mineraria Subalpina, 1980, Vol. II, pp. 747-772. 24. R.J. Tallarida, R.B. Murray, Manual of pharmacologic calculations with computer programs, Springer-Verlag, New York. 1981. 25. J. Boudreau, A. Beaudoin, 0. Nadeau, ((Sequential isolation of lamellar bodies and surfactant fractions from rat lungs#. Can. 0. Biochem. Cell Biol. 61: 231. (1983). 26. R.F. Henderson, A.H. Rebar, J.A. Pickrell, G.J. Newton, ((Early damage indicators in the lung. III. Bio-chemical and cytological response of the lung to inhaled metal salts#, Toxicol. Appl. Pharmacol. 50: 123. (1979). 27. M. Sj'dstrand, R. Rylander, ((Enzymes in lung lavage fluid after inhalation exposure to silica dust#, Environ. Res. 33: 307. (1984). 28. J. Bignon, K. Atassi, M.C. Oaurand, J. Yamine, H. Kaplan, P. Geslin, R. Solle, M. Bientz, ((Etude cytologique et biochimique du liquide du 240 lavage bronchoalvfiolaire (LBA) dan? la fibrose pulmonaire idiopathique et l'asbestose, Rev. Fr. Mai. Resp. 6: 353. (1978). 29. R. Begin, G. Drapeau, R. Boileau, Y. VSzina, A. Cantin, Y. Desmarais, M. Martel, Enzyme activities of lung lavage in asbestosiss, Clin. Biochero. 19: 240. (1986). 30. J. Dunnigan, Thresho1d exposure level for chrysotiles, Can. J. Public Health 77: 41. (1986). 31. K. Browne, A threshold for asbestos related lung cancers, Br. J. Ind. Med. 43: 556. (1986). 32. B.A. Keogh, R.G. Crystal, ((Alveolitis: the key to interstitial lung disorderss, Thorax 37: 1. (1982). 33. W.J. Martin II, D.E. Williams, D.E. Diver, D.R. Sanderson, ((Interstitial lung disease. Assessment by bronchoalveolar lavages, Mayo Clin. Proc. 58: 751. (1983). 34. R. B6gin, M. Rola-Pleszczynski, S. Masse, I. Lemaire, P. Sirois, M. Boctor, D. Nadeau, G. Drapeau, M.A. Bureau, Asbestos-induced lung injury in the sheep model: the initial alveolitiss, Environ. Res. 30: 195. (1983). 241 Table I Exposure parameters. Months of exposure 4 7 12 Number of exposure periods combined 3 2 1 for analysis Mean number of doses administrated 10 21 40 Mean cumulative amount of chrysotile administered (range) Lof 154 mg (28-240) hd/ 991 mg (580-1440) 383 mg (382-384) 2347 mg (2244-2464) 416 mg (412-424) 4315 mg (4292-4384) Range of cumulative, amount of chrysotile administered at the beginning of the exposure period LD f 10-96 mg hd{ 68-288 mg 376-378 mg 1860-2080 mg 412-424 mg 4292-4384 mg LD: Sheeps exposed to low doses of chrysotile HD: Sheeps exposed to high doses of chrysotile 242 Table I I Enzymatic p ro file of the lung lavage flu id o f sheeps post-exposure to c h ry s o tile asbestos. <OZc1a **4E>Vc3PJ-I. v(a/->) ui* 4^ </) 4>rJ- H Q) O<0 fl) P9*>0" &c N <0 52 - e <aO. *4<9Cr-/*>* rs* a^. 4(-/>i 9Cf xe jd Uw rr-e* f XE3 4- EC(0- rA--S C p u (C/)L 9 uO 0) a> UaKI. 0L1. to 3(/> Xo O *r~ UCXIL U 01 CL 0.-0I.c.m.in3O O I-I H -H +1 O CO Ol 00.p.H 0.0 HN + OCO CpHO*CoM o H -H -H ph oi 10.o.o .o O O CM lNOOCfOOO*fvNs-H H H -H O Irt H cn H IA 0O0.pC.HOC1.*M0 oo H -H +1 ph <r co in.id..i O O pH Ol CO Ol C.O.S.O NIOHH H M +1 f-.f-.sj- s.- c.o m. r-- m pH CoOM 1pH0 C1O0 COMl CM CJ 1 H -H +1 pH.i *po.H- oO.l 10 ppHH 0C0O O Ol^l <f.c.o.w pH pH CO H -H -H PpHM.Cp.HM CC.MM Ol *PJH- lsOo $ in cm cm cmo 00pH pH H +1 +1 0O0.01.00 CC.OM, f-- OpH CO CO lo in io i0 in m aa UJX oo UJX Xop f 4-> fV1I iV- O. 4- *C> O xC O CM vt3O.i ujh aX u 3VI VI OXa.X*of- f+-> c l 1-0E *V->I Q.CM (OX '-- rroo as*c-shj O O CM H -H -H Oio.Nr.- S.ph ph ph in + 1o0.Op.hl pi.nH ooo H -H -H tf1- 1000* 0C0O O O pH HO.i Hmin.<trpp.-HHf H -H -H rm~.Cc.mM Cr.-M~ CO CM pH 0CM0 %10 CM ID i0 i O CM OpH H -H -H 0CM0.01.00 0C.M0 X- OpH ICDO stf- X- S- ao UJX u 3VI PV->I OXf XQ.*Or* 4CJ 9l 5r 8E VI O Cl*-t a. o X XJ c<0 VI > 01 Ol r- + O O VI vs > >> >- Uf fu o 4- O VI VI 0) 0) oVI VoI x X) OI" r- f oo VQI.X X 0o>) VoI VoI f a. CL VI X01 X0) r-- O VI VI Olt- a. q. uCOv0u)fnf01 >1 ?01 oc~*ox 4J VI +V0->)I > a c o Vp.I .p^ u > . s u01Woc p- CL+-> 4i--- V0)I r-- +> E +-> vi - - VI *-> +-> +J c c XI C3 3 4J O I/O in in oo o o V SJf o. a. o o UJX ** 243 50i 4 0" > l~ H 30% -i '2 O^ H* < E 20- 2 >N Z Ui 10- LDH * + T 1*1 HD NUMBERS OF i T" "I 10 20 30 40 DOSES ii 47 MONTHS OF EXPOSURE Figure 1 Enzymatic profile of the lung lavage fluid of sheeps exposed to chrysotile asbestos: Lactate dehydrogenase (LDH) C: Control sheeps LD: Sheeps exposed to low doses of chrysotile HD: Sheeps exposed to high doses of chrysotile Vertical bars: Standard error (S.E.) of the mean for the enzyme activities (n * 5 or 6) Horizontal bars: S.E. of the mean for each number of exposure p<0.05 Dunnett's multiple comparison test (one-way) C vs LD and HD p<0.05 Students' t-t.est (one-way) LD vs HD** ** p<0.05 Analysis of variance (one-way) HD (12 months) vs HD (4 and 7 months) 244 AKP HD LD C NUMBERS OF DOSES _ MONTHS V2 OF EXPOSURE Figure 2 Enzymatic profile of the lung lavage fluid of sheeps exposed to chrysotile asbestos: Alkaline phosphatase (AKP) C: Control sheeps LD: Sheeps exposed to low doses of chrysotile HD: Sheeps exposed to high doses of chrysotile Vertical bars: Standard error (S.E.) of the mean for the enzyme activities (n * 5 or 6) Horizontal bars: S.E. of the mean for each number of exposure p<0.05 Dunnett's multiple comparison test (one-way) C vs LD and HD p< 0.05 Students' t-test (one-way) LD vs HD 245 ACP Figure 3 Enzymatic profile of the lung lavage fluid of sheeps exposed to chrysotile asbestos: Acid phosphatase (ACP) C: Control sheeps LD: Sheeps exposed to low doses of chrysotile HD: Sheeps exposed to high doses of chrysotile Vertical bars: Standard error (S.E.) of the mean for the enzyme activities (n * 5 or 6) Horizontal bars: S.E. of the mean for each number of exposure p<0.05 Dunnett's multiple comparison test (one-way) C vs LD and HD p<0.05 Students' t-test (one-way) LD vs HD * p<0.05 Analysis of variance (one-way) HD (12 months) vs HD (4 months) 246 B-NAG Figure 4 Enzymatic profile of the lung lavage fluid of sheeps exposed to chrysotlle asbestos: &-N-acetylglucosaminidase (B-NAG) C: Control sheeps LD: Sheeps exposed to low doses of chrysotile HD: Sheeps exposed to high doses of chrysotile Vertical bars: Standard error (S.E.) of the mean for the enzyme activities (n * 5 or 6) Horizontal bars: S.E. of the mean for each number of exposure p<0.05 Dunnett's multiple comparison test (one-way) C vs LD and HD p<0.05 Students' t-test (one-way) LD vs HD * p<0.05 Analysis of variance (one-way) HD (12 months) vs HD (4 months) 247