Document pp3MVreonnK9YZ8b6J5Kz6vMD

w II v- Uinl - ./ t d->. "an. > ivc ,ivy 40. ' tf im .,hd Ik r ,i . .'1/ isvikovuimw kisiskoi 16, 3 JJ - 341 (197X) r. . , . p's I K, ur.,ei li'C vt . , ... 'ri'i'.II. , ijr 3"jr intri(l;ement -9W. Some Biochemical and Histopathologica! Changes Induced by Polyvinyl Chloride Dust in Rat Lung D. K. Acarwai., J. L. Kaw, S. P. Srivascava. and Pkmii.ad K. Skiit Industrial Tnsieolofty Research Centre. Stiilialnia (iandlii Start:, Post tins No, /*!). I.ai hnon-22611111. India Received Sepicmher 17. 1976 I F.nzymulic .mil piithomorphnlogic iilicrutiorys in rat lungs were sludied ai different time intervals up to ISO days after a single intratracheal administration of 25 mg of polyvinyl chloride dust. The activities of two energy-linked enzymes, succinic dehydrogenase (SDH) and adenosine triphosphatase (ATPase), and three lysosomal enzymes, acid phosphatase. /3-glucuronidase, and rihonuclease. were significantly increased in the early period and then started to decline. The activities of SDH and ATPase reached control values at 150 days, while those of the lysosomal enzymes remained significantly higher up to this period. Histopathologically. the pulmonary response was in the form of acute inflammatory changes I during the early stages of dust burden, followed by the development of granulomatous lesions containing small amounts of stromal elements. INTRODUCTION Polyvinyl chloride (PVC1 is widely used for containers, wrapping films, electri cal insulation, pipes, conduits, biomedical devices, and a variety of other indus trial and consumer products. Due to its inertness to a large variety of chemicals and poor absorption through the gastrointestinal tract and skin, this polymer is gener ally considered to be nontoxic. However, during the last few years, dermatitis and allergic responses (Wcichardt. 1970), bronchial asthma (Dernehl. 1963: Vanhouten and Cudworth, 1974), acro-osteolysis with scleroderma, and Raynaud's phenomenon (Wilson ct al.. 1967) have been reported in workers handling PVC. Recent reports of hepatic disorders, including angiosarcoma of the liver due to the monomer, vinyl chloride (Marsteller ct al.. 1973; Creech and Johnson, 1974; Falk ct al.. 1974: Nlakk ct ul.. 1974) and cases of still births, miscarriages, and malfor mations among the workers engaged in the polymerization of vinyl chloride (Selikoff. 1974: Infante ct al.. 1976), have aroused great interest in the toxicologi cal properties of PVC. Reports of the occurrence of pulmonary dysfunction and a new form of pneumoconiosis in PVC workers (Szendc ct til.. 1970: Miller ct til.. 1975) and dust-exposed animals (Bogdan. 1972; Fronzia ct ul.. 1974) point to the need for further studies on the toxicological properties of the polymer. MATERIALS AND METHODS Animals ami plan of expat intent. Female hooded rats (200 r 10 g) from the Industrial Toxicology Research Centre colony maintained on an ad lihittnn pellet diet and under standard husbandry conditions were divided into two groups. The animals of group I were injected intratracheally with 25 mg of PVC dust suspended I in 1.0 ml of normal saline and those of gioup II received an equal volume of saline to serve as control. The particle size of the dust, prepared as described by Zaidi >33 (WH-V35I 7K -o3.W02.O0U t ip\ ;4<".5 ps W.ulciTtK' I'u'tv Inc Vil iigl.'s .9 is-pf>nl*i.li".i ifl ..in ftum icscImJ BOR 014022 i 5 - f r * if s F l i 334 .M. \KW \|, t I At . (1969), was below 5 Six treated and four control rats were sacrificed at the desired lime intervals, and the lungs were removed immediately and processed for enzyme estimations and histopathological study. The lungs of the animals that died spontaneously during the course of the experiment were processed only for histopathological studies. Biochemical .studies. Suitable portions of the right lung were minced and homogenized in chilled isotonic sucrose (0.25 m) or sucrose (0.25 m) --EDTA (0.001 m) with a Potter-Hlvehjem-type homogenize!' to yield 10Cf (w/v) or 47? (wvv) homogenates, respectively, for the enzyme estimations. In some experi ments, a portion of 47? (w/v) homogenate was treated with Triton X-100 (final concentration. O.I7f) to release the latent activity of the lysosomal enzymes (de Duve. 1964). The activities of succinic dehydrogenase (SDH. succinate (acceptor) oxidoreductase; EC 1.3.99.1) and adenosine triphosphatase (ATPase. ATP phosphohydrolase; EC 3.6,1.3) were measured by the methods of Slater and Bonner (1952) and Seth and Tangari (1966). respectively. Estimation of acid phosphatase (orthophosphoric monoester phosphohydrolasc: EC 3.1.3.2) w'as carried out ac cording to Oser (1965), of ribonuclease (RNase. polyribonucleotide-2oligonucleotide transferase (cyclizing); EC 2.7.7.16) according to McDonald (1955). and of /3-glucuronidase (/3-i>-glucuronide glucuronohydrolase: EC 3.2,1.31) according to Fishman (1967). Histoinitholo^ical studies. The left lung was fixed in 107? formol-saline by injecting the fixative through a small raminent of the major airway. It was then dehydrated and embedded in paraffin, and 5 gm sections were prepared. The sections were stained with hematoxylin-eosin or impregnated with silver (Gor don and Sweets. 1936). Chemicals. All the chemicals used in this study w'ere of Analar grade obtained from BDH or E. Merck, except' phenolphthalein-/3-glucuronide, which was ob- tained from Schwarz/Mann. Polyvinyl chloride sample grade GEON-121 was gifted by Chemicals and Plastics India Ltd., Madras. RESULTS No significant difference was observed in the rate of mortality of control and PVC-administered rats. Bim hcmical Studies Table I shows the effects of PVC dust on the activities of SDH and ATPase in lung homogenates. The activities of the two enzymes exhibited a sudden increase at 15 days and a further increase at 30 days: they then started to decline and attained normal levels at 150 days. Although the two enzymes followed a similar pattern of change. SDH showed a greater sensitivity to the PVC dust at all time intervals. Table 2 shows the effect of PVC dust on acid phosphatase. /3-glucuronidase. and ribonuclease. The activities of these enzymes were increased in trented animals and acid phosphatase and /3-glucuronidasc reached their maxima at 60 days. while ribonuclease reached a maximum at 30 days. Later, the enzymes exhibited decreases, but their activitex remained significantly above the control values up to 150 days. The data summarized in Table 3 show that no significant alterations I ; I i ; | ! j j ( j j j j Ilf) <WIIIIB|^W.'J I BIUI MW BOR 014023 4* . tine "41 > tr 1 and OTA ; 4C? peri final - ide plor) >hos>r. iier .itiise t aclv-2maid ! 31) i by then The Gor- A m i and in C JSC mod i n of , .(Is. lose. ' ..ni- ; j> st bned op to nons | ]' !1 ' ' 1 !i j I i Pl'IMONAKV KKM'ONSK TCI l'V(. Ill SI 335 TABLE 1 Kim i iu I* VC lit vi o\ Sri < is ii Dimimni.iMsi imi Am sosist rnii'iuiMMiM am Ai mm m Rti l.rsi." Days Succinic dehydrogenase (nmole of K.FeiCN),, reduced/min, g of fiesh tissue) Percentage increase Adenosine triphosphatase (nmole of P, liheraled/min'g of fresh tissue) Pereemage increase 0 15 30 : AO 90 120 150 180 207 = 8 (201 3) 253 r 23**** (131 -- 35) 285 = 9** (131 i 35) 240 t ID*** (131 - 35) 23A - 19-*** (158 = 22) 229 i 23 (183 5) 158 t 13 (144 r 18) 213 - 7 (201 * 3) 2.9 93.1 117.5 83.2 49.3 25.1 9.7 5.9 450 - 40 (442 * A) 331 * 15** (230 - 16) 354 = 13* (230 16) 411 * 7**" (331 * 26) 413 * 21**** (362 17) 396 * 25 (350 * 22) 383 = 66 (355 8) 444 * 13 (442 e: 6) 1.8 43.9 53.9 24.1 19.6 13.1 7.8 0.4 " All the values are means - SE for six observations in experimental and four observations in control groups. Values in parentheses indicate their respective controls. Statistical significance was evaluated by Student--, / test. * P < 0.001; * P < 0.01; *** P < 0.02; **** P < 0.05. TABLE 2 Ei ik i oi PVC Dtsi o\ SOM >M V| Es/vmis or R,\ i Lest." ' Acid phosph.aase 0-G!ueuromJase (nmole of P, inmole of phcnoiphtheiein liberated mm g Percentage liberated min g Percentage Days of fresh tissue) increase of fresh tissue) increase Rihonuclease (40.D./min/g of fresh tissue) Percentage increase 0 54: * 10 (523 =51) 15 917 * 78** <ik t :i) 30 1284 i 78* (his - :n 60 1 160 10' ('97 * 151 90 i i4n i:* <6xi ;o co l120 In* j'hi t HO (4l>6 i 14) |X0 7m ('97 - H> 3.6 (4 5 56.9 94 1 67 4 64 4 I2_5 27,8 525 = 27 (541 i 82) 116 -- (3(211 i 14) 18* *11* (2:1 * 14) 577 * :*w (273 t 221 1018 i 2'* 151M " :x 1427 * 22* (7lh - 22) 447 * 26* (24X t II) 840 - 14 (614 - 82) _ 1.36 = 0.26 (1.40 0.24) _ 49,7 2.54 * 0 05* (1.15 i 0.U5) 120.8 82.9 2.61 = 0.06* 126.9 (1.15 * 0.05) 111.3 2.15 + 0.2.1** (1,26 = 0,18) 86.5 100 3 2 12 Ml 16*** 46 2 (I.4S = 0.15) 99 1 2.0' 1 0.05* (1.17 * 0 07) 49.6 80.2 2 22 = 0 !' 43 2 (1.45 = 0 0?) 32.4 2.00 i 0 15 (1 40 = 0.24) 42.8 " -\ll V.Jiics the trt values die moils * parentheses indicate si for MS respective ochosnetrrvouls(.winS'sl.OiniMelCx.pilersimigennifticaal n,rcned four w,s ohsci i.iimns m Condo! groups. evaluated by Student's t lest. r 0 4M)|; * /* 0 01 ***/' II n: BOR 014024 336 ACAKWAl. 71 AL. ; TAHI.H 3 \( IIVIIIOV III LlMIMIMII. [WlMI's IS III! NlIKSIM IS11 I'VC-Tm vim K\l i.l St." l);ty Acid phosphatase /i-Glucuronidasc Rihomiele.isc i 0 2.30 * 0 43 (2.39 * 0.39) 1.16 _r U.IW (1.18 = 0 07) 1,26 - 0 12 (1.25 - 0 08) 15 1.53 - 0 04 (1.59 -- 0.20) 2.60 - 0.05 (2.51 * 0.08) 1 07 ; 0,01 (1.25 = 0.081 30 1.3ft 0.09 2.44 0.09 1.16 - 0 01 (1.59 0.20) (2.51 0.08) (1.25 r 0.08) 60 1 15 - 0.02 2.10 = 0.09 1.20 - 0.02 \ (1.23 0.07) (1.87 0 09) (1.23 * 0,04) *Ki t 90 1.16 - 0,01 (1 19 0.08) 1.25 - 0.01 (1.17 r 0.06) 1.16 - 0.01 (1.44 0.13) i:o 1.70 * 0.07 1.08 * 0.02 1 27 = 0.03 (1.57 0 08) (1.04 - 0.02) (1.28 r 0.05) 150 1.38 - 1). 10 (1.43 * 0.09) 1.1! 0.02 (1.19 s 0.09) 1.38 - 0.04 (1.14 - 0.09) 3 180 1.89 0.05 1.16 -* 0.01 1.25 - 0 03 A. (1 59 - 0 20) (UK 0.02) (1.25 r 0 08) \ " V.ilucs .ire incans SE for sis observations in experimental and four observations in control l groups, indicating the r.ilio of enzyme activity before and after Triton X-100 treatment. Figures in I i ? parentheses indicate corresponding controls. ] I were observed in the latent activities of the enzymes in experimental and control J rats upon treatment of the homogenates with Triton X-100. t I His tofuitholofiical Studies The microscopic study of the tissue sections of the lungs of dead animals or those sacrificed at various time intervals revealed that 3 days after the inoculation of dust suspension, the dust deposits were situated in various segments of the airways, predominantly in the terminal and respiratory bronchioles (Fig. 1). Be- | sides these locations, the dust was discernible in the alveolar ducts and attached to ! x the membranes of the alveoli bulging from the latter structures. Congestion of I blood vessels in the alveolar septa, leakage of erythrocytes, presence of edemat ous fluid in the alveolar walls or alveolar space, and hemosiderosis were also seen. I At 15 days after the inoculation, vascular and inflammatory changes persisted, | as at the earlier period. However, the dust deposits seen as translucent foamy material were situated predominantly in the alveolar portion of the pulmonary ' tissue drained by the alveolar ducts. The cellular response to the presence of dust in the lungs was in the form of cellular hyperplasia at the periphery of the dust masses. Alveoli contained macrophages laden with dust (Fig. 2). In the neighbor hood of dust loci there w ere focal areas of interstitial fibrosis due to the prolifera tion of histiocytes and reticulin fibers. Transport of the dust by lymphatics was made evident by the presence of dust within tissue spaces, situated on the periphery of bronchioles (Fig. 3). Encroachment of the dust masses by multinueleated cells was evident at 30 days. Many of the cells w ere heavily laden with PVC dust in their cytoplasm (Fig. j 4). At 60 days, the granulomatous lesions containing the dust-filled multinueleated [ BOR 014025 T I'l I.MOVVHV Kt-.SI'ONM 1(1 |'V( HIM 337 t r*: \ 9 i.'l I-K-. 1. Section of the lung of n rut winch died spontaneously 3 days after intratracheal inoculation of PVC dust. The dust deposits are seen predominantly in the proximal alveoli budding from the in respiratory bronchiole. Hematoxylin and cosin. x 135. cells, lymphocytes, and a few macrophages were surrounded by a zone of fibro ol blasts. The lesions were ditfusely distributed and the stroma were composed of reiieulin and collagen fibers. The lumens of the airways still contained erythro cytes and the epithelia of these structures were detached from the underlying to Of itil. d. st r- ,1S F*tu 2. Section of the lung of a rat sactiiiicd 15 <Ja\ n after mtriitr4iche.il inoculation of PVC Just showing compaction of alveolar lumen with dust-lauen macrophages. Hematoxylin and cosin. x*40. j.J 1 iy < 1 11 - yji 1 .1 BOR 014026 ; s t U bf. ' if. . t rv i, [ t l : i r t i t 338 fW.ARWAl. / / Al. W*<M Fn;. 3. DilTcrcncc low-power microscopic field of the same section as show n in Fig. 2. The distal alveoli show some degree of interstitial fibrosis, hyperplasia of microphages, and the presence of dust in some tissue spaces, presumably lymphatics. Hematoxylin and cosin, x 135. lamina propria. The interalveolar septa xvere thickened and contained a network of argyrophilic fibers. The dust was found to have been encased in cellular en velopes , as at 60 days, and without the penetration of the argyrophilic fibers into its substance (Fig. 5). At 90 days the multinucleated cells were less frequently seen to invade the substance of the dust masses. The general cellular reaction at this time interval was similar to that observed at 60 days. The subsequent development of the lesions did not progress beyor.J this stage; instead, there seemed to be a tendency to clear out the entrapped d;.-.: This was evident from the presence of dust within the cytoplasm of rr-crophegcs and the lumen of bronchioles. In many cases the absence of dust in me lung sections may have been due to the efficient clearing mechanism of these animals. DISCUSSION The present results indicate that a single intratracheal administration of PVC dust leads to marked biochemical and histopathological alterations. Increases in the activities of oxidative enzymes in the lungs of the experimental animals have been reported in the presence of silica (Kilroe-Smith and Breycr, 19631 and asbestos (Beg cl aL, 1973). It may be possible that some dusts induce metabolic changes in tissues and to cope with the energy demands, the activities of these enzymes tire increased. The increases in the activities of the two energylinked enzymes. SDH and ATPase. suggest a similar effect during the early phase ol the exposure of the lung to PVC dust. The lysosomal enzymes showed a pattern similar to that of the oxidative enzymes in treated rats. The activities of all three '*V BOR 014027 *fpt s tw>" r 1sii v > /-- mw iji* r w L > ]'t'l.MON.\K\ lU.NI'ONM-. IO I'Vf DIM *.fC* ^ ^ V 4A':) V J-fT tf.i :% * Is iju. 339 'i*v wty w .'"ijjt' r?' $ *>>, mrtm f rs 1 i Me distal vtf of dust network Iiihir en ters into i of the ..nJeney t within '.es the clearing of PVC i mental Hi eycr. induce :tivities cnergyy phase pattern iii three *K $ 4 Fk.- 4. Section of the lung of a rat sacrificed 30 days after the intratracheal inoculation of PVC dust. The alveolar lumen contains mullinucleated cells heavily laden with PVC dust. Hematoxylin and eos>in. x540. i i Fn., 5. Section of the lung of a Ml saciiticcd AO days after intratracheal inoculation of PVC Just. Highly granulomatous lesion contain* within n\ substance an aggregate of dost mass. Hcrnaioxvlin Ii and eosin. Ili t BOR 014028 340 \K\\ At. / 1 Ai.. lysosomal enzymes siiulicil in the present experiments increased gradually and then decreased. However, in contrast to SDH and ATPaxe. the levels of the lysosomal enzymes remained significantly higher than the controls up to 150 days of the experiment. Histopathological changes aKo exhibited a more or less similar pattern. There were acute inflammatory changes, phagocytosis, and encasement of the dust without significant stromal proliferation. The sequestration of the dust masses and a relatively lesser degree of phagocytosis may be responsible for the lower levels of lysosomal enzymes during the later periods of the experiment. Certain toxic agents, biochemical stimuli, and inflammation are reported to : cause lysosomal fragility (Allison cl <il.. I%6: Allison. 1967). Alterations in the activity of acid phosphatase, ^-glucuronidase, and ribonuclease in the present study suggest an action of dust on lysosomes. Cytotoxicity of PVC implants (Felling ct til.. 1973). a degenerative effect on peritoneal and alveolar macrophages (Salthouxe ct at.. 1973). and inhibition of cell growth in culture with a clumping of lysosomes (Dehaan. 1971: Grasso ct <il.. 1973) strengthens this assumption. The increases in the activities of lysosomal enzymes may be due to the labilization of lysosomes. as observed with asbestos dust (Viswanathan d a!.. 1973). or to their increased permeability to the substrates. There were no significant alterations in the ratio of the enzyme activities before and after Triton treatment between control and experimental animals, indicating that PVC dust does not increase the enzyme activity by disrupting the lysosomes. Thus, the increased permeability of lysosomal membranes to their substrates or increases in lysosome content, as observed with aflatoxins (Pokrovsky ct al., 1972). or increases in the activity per se may be responsible for the observed change in the activities of acid phos phatase. /3-glucuronidase, and ribonuclease in PVC-treated animals. Further studies are needed at the molecular and ultraslructural levels to delineate the locus operamli of membrane damage by the polymer. ACKNOWLEDGMENTS The author, are thankful to Dr. S. H. Zaidi. Director. Industrial Toxicology Research Centre for his keen interest in and saluable suggestions fur this ssork and to Dr. C. R. Krishna Murti and Dr. P. N. Visw.iit.ithnn for helpful discussions Thanks are also due to Mf. Kanhaiya Lai and Mr. Pratap Singh for technical assistance and to Mr. Musleh Ahmed for photomicrography. REFERENCES Allison. A. (1967). Lysosomes in normal and abnormal cellular reactions. .S/icilnnn tPrctnnn) 5, 1(11-1(12. Allis.in. A. C-. H.irington. J. S.. and Birback, M. (1966). An examination of the cytotoxic effects of silica on macrophages../. /.'ip A/oi/. 124. 141-153. Beg. M U,. Rahman. Q.. Viwsan.ilhan. P. N.. and /nidi. S, H (1973). The effect of asbestos dust on mitochondrial enzimes of rai lung / nurmi, /'/own/, lUmliciii. 3. |X5-|9| Bogdan, C. (1972). Histological and htstochemical changes in liver during experimental polyvinylchloiide pneumoconiosis. Woe.*. AktuL Mill. Ilmluiciokt r. 17, 93-111 [Polish]. Creech. J. t... Jr . and Johnson. M N. (1974). Angiosarcoma of liver in the manufacture of polyvinyl chloitdc -/. (>i ( nr Wi,/ in. I5l>- 151. do Duse. C. ttvMI. The separatum and characterisation of suh-eellular particles. Ihinc\ in turf i 59, 49-77. DeMa.m. R , t,, 11971). I oxicity of tissue culture media exposed to Poly (vinyl chloride) plasties. SunniVeil />'/,./. 231. X5-S6. Dernehl, r. U. i 1963). Hazards to health associated xnth (he use of epoxy resins.On np. Mfti. 5. -t; W iitipiwi.iii wmj BOR 014029 T n T " '! 1' ....... o n* * PCI MON \RY KIsroNM- TO l*Vt. Ill'S I 341 Falk. IF. Creech. J I... Jr,, 1 Io.ith, C, W,. ,li . lohnxon. M. N., and Key, M. M (1974). Hepatic disease among workers at a sinyl chloride polvmci isalion plain J. Amir. Mu/. .tarn . 250, Tiys 59-65. Fishman. W, H, (1467). Determination of /J-g!uciiroidnxex. hi "Methods in Iiioshemie.il Analysis" .til.tr .iv nt dust the 5Tt (David Click. Fd.l. Vol. 15. pp, 77-146. Inteiss'icnee, Ness Ysiik. Fron/ia. N.. Spina/zola. A., and Buearelli. A. (1474). Experimental lung damage from prolonged inhalation of airborne PVC dust. Mid. I.iivnin. (iS. 521 --542. Gordon. H.. and Sweets. H. H,. Jr. (1956) A simple method for the silver unpiegnanon of reticnlin. Amir. ./. I'mii. 12. 545-552. Grasso. P.. Gaydon. J.. and Hendy, R, J. 11475), The safety testing of Medis'al plasties: II. An J to assessment iiflysosom.il changes as an index of tosieily in cell cultures. I'J. Cos, Tmitnl. It. : . the ` 255-265. sent Infante. I'.. Wagoner, J.. Waxwciler. R , McNhchael. A., and Falk. H. (1976), Wives of vinyl chloride i workeis suffer mote miscarriages--NIOSH Study. ./>>/>. .Su/c/s llllli. lii'pn. 6. 21. 1 uiUtS i Kilroe-Smnh. T. A , and lireyer. M, G. (1465). Changes in activities of respiratory enzymes in lungs of ages I guinea pigs exposed to silica dust, /hit ./. hut. Mnl. 20, 245 -- 247. ig of j Makk. L., Creech. J I... Whelan. J. G . Jr., and Johnson. M. N. 11974). Liver damage and angiosar- The ; coma in vinyl chlonde uoikers. ./. Ami'r. Mtil. Avon . 250, 64 - 6N. , m of Marsicller. H J I.elhack. W. K.. Muller. R., Juhe. S.. l.unge. C. F... Rtihmcr, H. G.. and Veltman. | G (19751. Chronic t.yxic liver damage of workers in 1'VC production. Ihuii. \/ct/. Woc/icnst hr. iheir 98.2511-2514. ' .s in ; McDonald, M. R. (1955). Rihonucleases. hi "Methods in Enzymology" (S. P. Colowick and N. O. 5 iirol 1 Kaplan. F!ds,t, Vol. 2, pp. 427-456. Academic Press. New York. ; \ me Miller, A., Tcirstcm. A. S.. Chuang. M.. Selikoff, I. J., and Warshaw, R. (1975). Changes in pulmo . of nary function in workers exposed to vinyl chloride and PVC. Ami. Y. 1". Ai ml. Si i. 246, 42 -- 52. Oser, H. L. (1965) Determination of seium phosphatase tictivity. hi "Howk'x Physiological Chemis ! ;. ns try" (U. L. Oser. Fd.). I4ihed..pp III8-1I2I. McGraw-Hill. New York. per I Pelting. D., Sharr.iti. M.. and Hardy. J. (1475). The safety lesung of medical plastics: I. An assessment hos- I of methods. Id Cm. h-ucul. 11,64-85. thcr I Pokrovsky. A. A.. Kravchenko. L. V.. and Tuielyan. V. A. (14721. Comparative study of the effect of me 1 I afl.uoxiii and some antitumour antibiotics on rat liver lysosomcs in rno and in i itro. llnn hciii. I'hiirmninl. 21. 24S9-2446. I Sallhousc. T N\. Matlaga, H. F.. and O'Leary. R. K. (197.5). Microspccirophotomeiry of mucrophage lysosomal en/yme activity: A measure of polymer implant tissue toxicity. 7ii.\iio/. Appl. i'hhr- -i his V mm nt 25. 201 - 211. Selikofl. 1. .1 (14741 Still birihs and miscarnages in wives of vinyl chloride xvoikers studied, (h i up. li'ih .Vu.vn l,i il 4, I. j Seth. P K.. and Tang,in. K. K. (1466). Biochemical effects of newer salicylic acid congeners. J. 1 rihtrm, /`inn mm nt. 18. 851 -855. j Staler. F C\. and Bonner, W. D (1452). The effeet of fluoiide on the succinic oxidase system. 1 Him him f 52, 185- 145. ' S/ende. R.. I apis. K.. Nemos. A., and Piter. A. (1970) Pneumoconiosis caused by the inhalation of IS of poly vinyl ehloi ide dust. \lnl. 1. iooio 61.455-456 Vanhtniten. Is W.. and Cudworth. A. L. (1974), Evaluation and reduction of air contaminants pro duced by theimal cutting and sealing of PVC packaging film. Amir. /ml. Hyp. Assoc. I. 35. i 218- 222 i nl- Viswar.uthan. P N Rahman, O . Beg. M, U .. and Zaidi. S H. (1975) Pulmonary ly sosomal enzy mes *i in experimental axhestoxix in guinea pig'. Ihniimi. I'hwinl. Iiii>,lhm 5. 120-126. - Hit 1 > Welsh.irdt. H. (19701. Dangers the skin is exposed to in the plastic industry. Iliritlulirinnlmrn. IS, i ! 25 - 54 \ * 5*). Wilson, R, H.. McCormick. W F.,, Tatum. C. F.. and Creech. J [.. I 1467). Occupational acro- Ostcolysis Heps'll of 51 cases) / Ami r, Mi ll. Assoi 201. 577-581. Zaidi. S H. (|4i,4) Fxpenmenial mclhods. In "Expcnmcnial phneumoeonioxis." PP- 55 -49 Johns Hopkins Press. Baltimore. 1 ' si 4 $ I I 1 li ^ ; nun jmn + n '.i 'SW - 'r it VX ' .ia iin jw w .p s y is i c ||jj i. c jj i^ in f p, jctM i im i .-J -- p ^ l*i "f BOR 014030