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105698 BIO-MEDICAL RESEARCH DOCUMENT DESCRIPTION FORM 63 68 61000000 76 Duplicate in all cards:--> IW year as-1961- / File number [Right justify [Numeric only] Author(s), as Last Name FS (No Punctuation) and coden for journal as JAMA preceeded by one blank space 1 'S'cr/Tf/, pp 20 21 *P^A-W / J/- . 40 41 f!</*-ST>t,'A SP 77 78 U0 ] Sub-Index Code 60 61 62 Title of Report; end with space-hyphen-hyphen-space. Follow with Index Terms, separated from each other with'comma-space. Avoid other punctuation; do not abbreviate. 12 ,,,, , 61 62 21 22 ^ ~ P pp p 23 24 Source (Journal, Vol., Number, Pages, Date ) 1 2 _____________________________________________________________ // , 3 3J- 3f/ r/<?s / 61 62 31 32 Brief Summary 12 10 SUMMARY: 61 62 61 62 63 64 enviko.vmentaj. rkseakcii 16, 333-3-41 (1978) - Some Biochemical and Histopathological Changes Induced by Polyvinyl Chloride Dust in Rat Lung D. K. Agarwal, J. L. Kaw, S, P. Sbivastava, and Praiii,ad K. Skth Industrial Toxicology Research Centre, Mahatma Gandhi Mart;, Post llox No. 80. LttcknotC'226001, India Received September 17, 1976 Enzymatic and pathomorphologic alterations in rat lungs were studied at 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 (ATPasc), and three lysosomal enzymes, acid phosphatase. /J-glucuronidase, and ribonuelease, 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. Histopathological!)', the pulmonary response was in the form of acute inflammatory changes during the early stages of dust burden, followed by the development of granulomatous lesions containing small amounts of stromal elements. INTRODUCTION Polyvinyl chloride (PVC) 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 (Weichardt, 1970), bronchial asthma (Dernchl, 1963; Vanhouten and Cudworth, 1974), acro-ostcolysis 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 (Marstellcr ct al., 1973; Creech and Johnson, 1974; Falk et al., 1974; Makk ct al., 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 al., 1970; Miller ct at., 1975) and dust-exposed animals (Bogdan, 1972; Fronzia cl al.. 1974) point to the need for further studies on the toxicological properties of the polymer. MATERIALS AND METHODS Animals and plan of experiment. Female hooded rats (200 10 g) from the Industrial Toxicology Research Centre colony maintained on an ad libitum pellet diet and under standard husbandry conditions were divided into two groups. The animals of group 1 were injected intratracheally with 25 mg of PVC dust suspended in 1.0 ml of normal saline and iliose of group H received an equal volume of saline to serve as control. The particle size of the dust, prepared as described by Zaidi 333 33 0013*9351/78/1613 - 0333502,00/0 Copyright } Ariiitciiih' IVv". Inc. 9V9> AU right* of ffpriHlnilnut in miy form icwyvcJ. -yr /; 'T.z'i - i-.'iinjJi-V.'Lv_ < -- -*- -ijL.r'ij.' i*. vviix Hi 334 AC-AKWAL ITT AL. (1969). was below 5 /tm. Six treated and four control rats were sacrificed at the desired time 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-- Elvehjem-type homogenizer to yield 10% (w/v) or 4% (w/v) homogenates, respectively, for the enzyme estimations. In some experi ments. a portion of 4% (w/v) homogenate was treated with Triton X-100 (final concentration, 0.1%).to release the latent activity of the lysosomal enzymes (de Duve, 1964). The activities of succinic dehydrogenase (SDH, succinate (acceptor) oxidoreductasc; 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 phosphohydrolase; EC 3.1.3.2) was carried out ac cording to Oser (1965), of ribonucleasc (RNasc, polyribonuclcotidc-2oligonucleotide transferase (cyctizing); EC 2.7.7.16) according to McDonald (1955), and of /3-glucuronidase (/3-n-gIucuronide glucuronohydrolasc; EC 3.2.1.31) according to Fishman (1967). Histopathological studies. The left lung was fixed in 10% 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 were of Analar grade obtained from BDH or E. Merck, except phenolphthalein-/?-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-administcred rats. Biochemical 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, /J-glucuronidasc, and ribonuclcase. The activities of these enzymes were increased in treated ani mals and acid phosphatase and |3-glucuronidasc reached their maxima at 60 days, while ribonuclcase reached a maximum at 30 days. Later, the enzymes exhibited decreases, but their activitcs remained significantly above the control values up to 150 days. The data summarized in Table 3 show that no significant alterations wy.v -yfttrijc >--;TT7. 3J </> 0 01 -O4 o PULMONARY RESfONSF TO PVC DUST 335 TABLE I Effect of PVC Dust' on Succinic Dfiiyokocenase a.vii Aijenom.ne Tkii'iiosi'iiata.xf. Activity of Rat Lung* Days Succinic dehydrogenase (nmole of K,Fe(CN)s reduccd/min/g of fresh tissue) Percentage increase Adenosine triphosphatase (nmole of Pt libcrated/min/g of fresh tissue) Percentage increase 0 207 = 8 (201 -- 3) 15 253 (131 35) 30 2S5 9** (131 35) 60 240 to*** (131 35) 90 236 19**** (158 22) 120 229 23 (183 5) 150 158 13 (144 IS) ISO 213 * 7 (201 3) 2.9 93.1 117.5 83.2 49.3 25.1 9.7 5.9 450 40 (442 6) 331 15** (230 16) 354 13* (230 16) 411 7**** (331 26) 433 23**** (362 17) 396 25 (350 22) 383 66 (355 8) 444 13 (442 6) 1.8 43.9 53.9 24.1 19.6 13.1 7.8 0.4 * All the values arc 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's / test. P < 0.001; ** P < 0.01; ** P < 0.02; **** P < 0.05. R&S 105701 TABLE 2 Effect of PVC Dust on Lvsosomai. Enzymes of Rat Lung" *' _4 Acid pliosph.it.ise (nmole of P, liberated/ituiVe Days of fresh tissue) Percentage increase /3-Glucuronidase (nmole of phenulphthelein libcrated/min/g of fresh tissue) Percentage increase 0 542 - 30 (323 = 51) 15 937 = 7$** (818 = 21) 30 1284 = 78* (818 =21) 60 1160 = 30* (597 = 35) 90 1140 = 32* (681 = 29) 120 1120 = 30* (681 = 29) 150 538 = 25** (406 = 14) ISO 763 = 52 (597 = 35) 3.6 14.5 56.9 94.3 67.4 64,4 32.5 27.8 525 = 27 (541 = 82) 316 = 13* (21I = 14) 386 * ||* (211 14) 577 = 26* (273 22) 1038 23* (SIS = 28) 1427 = 22* (716 = 22) 447 26* <248 It) 840 34 (634 = 82) -- 49.7 82.9 111.3 100.3 99.3 80.2 32.4 Ribonudcase (AO.D./min/g of fresh tissue) 1.36 0.26 (1.40 = 0.24) 2.54 = 0.05* (I.1J =0.05) 2.61 =0.06* (1.15 = 0.05) 2.35 0.23** (1.26 0.18) ZT2=0.I6*** (1.45 = 0.15) 2.05 = 0.05* (1.37 = 0.07) 2.22 = 0.12* (1.55 = 0.05) 2.00 =0.15 (1.40 = 0.24) Percentage increase _ 120.8 126.9 8S.J 46.2 49.6 43.2 42.8 * All the values are means i Sli for si* observations in experimental and four observations in conlrol groups. Values in parentheses indicate respective controls. Statistical significance was evaluated by Student's I lest. * / < C.00I: " /' < 0.01; ** /' < 0.02, f*. r, :iV V.*Y*.. ,r . ` jC, V ; _' ?* 1 * /' --------- - J-. "5i W-I AJUW I 336 AGAKWAL ETA!.. TABLE 3 Activation ok Lysosomal Enzymes in thk Normal and PVC-Tkkat f.u Rat Lung* Day Acid phosphatase /3-Glucuronidase Ribonuclcase 0 2.36 * 0.43 (2.39 0.39) 1.36 0.09 (1.18 0.07) 15 1.53 0.03 (1.59 * 0.20) 2.60 0.05 (2.51 0.08) 30 1.36 0.09 (1.59 0.20) 2.44 0.09 (2.51 0.08) 60 US 0.02 (1.23 0.07) 2.10 0.09 (1.87 0.09) 90 1.16 0.01 (1.19 0.08) 1.25 0.01 (1.17 0.06) 120 1.70 0.07 (1.57 0.0S) t.08 0.02 (1.04 0.02) 150 1.38 0.10 (1.43 0.09) 1.11 0.02 (1.19 0.09) 180 1.89 0.05 1.16 0.01 (1.59 0.20) (1.18 0.02) 1.26 0.12 (1.25 0.0S) 1.07 0.01 (1.25 0.03) 1.16 0.01 (1.25 0.08) 1.20 0.02 (1.23 0.04) 1.16 0.01 (1.44 0.13) 1.27 0.03 (1.28 0.05) 1.38 0.04 (1.14 0.09) 1.25 0.03 (1.25 0.08) * Values are means SE for six observations in experimental and four observations in control groups, indicating the ratio of enzyme activity before and after Triton X-100 treatment. Figures in parentheses indicate corresponding controls. were observed in the latent activities of the enzymes in experimental and control rats upon treatment of the homogenates with Triton X-100. Histopatlwlogical Studies The microscopic study of the tissue sections of the Jungs 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 the membranes of the alveoli bulging from the latter structures. Congestion of 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. 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 were 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 multinuelcatcd cells was evident at 30 days. Many of the cells were heavily laden with PVC dust in their cytoplasm (Fig. 4). At 60 days, the granulomatous lesions containing the dust-filled mullinucleatcd .v*: V'.' fer; tv* v-?-'rv?*#y-n R&S 105702 PULMONARY RESPONSE TO PVC DUST 337 Fig. 1. Section of the lung of a rat which died spontaneously 3 days after intratracheal inoculation of PVC dust. The dust deposits are seen predominantly in the proximal alveoli budding from the respiratory bronchiole. Hematoxylin and eosin. X135. cells, lymphocytes, and a few macrophages were surrounded by a zone of fibro blasts. The lesions were diffusely distributed and the stroma were composed of retictilin and collagen fibers. The lumens of the airways still contained erythro cytes and the epithelia of these structures were detached from the underlying 33 So CO O? 3 Co j>. /"fr? . < ; '`V \ - t* ... ^ Tc-.V-v'-'v'- "tiii-JsSELSii Fig. 2. Section of the lung of a rat sacrificed 15 days after imiatrachcal inoculation of PVC dust showing compaction of alveolar lumen with dust-laden macrophages. Hematoxylin and eosin. *540. vt-v Pi: t v.. K }?V --~xr. V V a-h" . 338 AGARWAL TA/.. ` V-.V < > CvAi\Z'F.' 3 30 fi 0) O <ji o-si u re'4^.->. ) Y -'. ' ^ ^ Fie. 3, Difference low-power microscopic field of the same section as shown 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 eosin. *135. lamina propria. The interalveolar septa were 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 multinucleatcd 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 beyond this stage; instead, there seemed to be a tendency to clear out the entrapped dust. This was evident from the presence of dust within the cytoplasm of macrophages and the lumen of bronchioles. In many cases the absence of dust in the 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 histopalhological alterations. Increases in the activities of oxidative enzymes in the lungs of the experimental animals have been reported in the presence of silica (Kilroc-Smith and Breyer, 1963) and asbestos (Beg ct 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 arc increased. The increases in the activities of the two energylinked enzymes, SDH and ATPasc, suggest a similar effect duting the early phase of 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 ;'g'fv.'- rrv^rx PULMONARY RESPONSE TO PVC OUST 339 '-sr.'i''' V*'-' V*' ;; fsif* vOsV . VA- ( V.-*? **?* viv .. I V`*>>C V # Vi- 4 { 6 -,. ' _, * 4.t - -* *W -V.\ NV fLi&V ^ Fio. 4. Section of the lung of a rut sacrificed 30 days after the intratracheal inoculation of PVC dust. The alveolar lumen contains multinucleated cells heavily laden with PVC dust. Hematoxylin and eosin. X540. R&S 105705 I-ir.. 5. Section of the lung of a rat sacrificed 60 days after inliairacheal inoculation of PVC dust. Highly granulomatous lesion contains within its substance an aggregate of dust mass. Hematoxylin and cosin. x 135. *n*t 340 AC.AKWAl. ET At- lysosomal enzymes studied in the present experiments increased gradually and then decreased. However, in contrast to SDH and ATPasc. the levels of the lysosomal enzymes remained significantly higher than the controls up to 150 days of the experiment. Histopathological changes also 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 arc reported to cause lysosomal fragility (Allison et al.. 1966; Allison, 1967). Alterations in the activity of acid phosphatase, //-glucuronidase, and ribonuclcasc in the present study suggest an action of dust on lysosomes. Cytotoxicity of PVC implants (Polling et al., 1973), a degenerative effect on peritoneal and alveolar macrophages (Salthouse et al., 1973), and inhibition of cell growth in culture with a clumping of lysosomes (Dehaan, 1971; Grasso et al.. 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 et al.. 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 docs 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 afiatoxins (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 ribonuclcasc in PVC-trcatcd animals. Further studies are needed at the molecular and ultrastructural levels to delineate the locus operandi of membrane damage by the polymer. ACKNOWLEDGMENTS The authors are thankful to Dr. S. H. Zaidi, Director, Industrial Toxicology Research Centre for his keen interest in and valuable suggestions fur this work and to Dr. C. R. Krishna Muni and Dr. P. N. Viswanathan for helpful discussions. Thanks are also due to Mr. Kanhaiya Lai and Mr, Pratnp Singh for technical assistance and to Mr. Musleh Ahmed for photomicrography. REFERENCES Allison, A. (1967). Lysosomes in normal and abnormal cclhd.tr reactions. Spectrum (Pretoria) S, 101-102. Allison, A. 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