Document Rpnmbogbr8zzxGmeVQYBjzKBE

< ;7lf Environmental Jlnilth Perspectives Vnl. 17, pp. 211-216, 1076 Biological Effects of Vinyl Chloride: An Experimental Study ~ by M. Winell,* B. Holmberg,* and T. Kronevi* Plasma activities of alkaline phosphata.se, (Al')p transaminases and total lactate dehydrogenase (I,DH) with isoenzymes were determined in mice inhaling 50 and 550 ppm \inyl chloride (\C). The animals were also autopsied and the tissue pathology was studied. llu* total I-DH activity was elevanted in both dose groups along with a shift to cath odic onzvines. Al was increased in animats exposed to 500 ppm and transaminases were not at all changed, knzyme changes occurred after the appearance of tumors. Alveologenic adenomas occurred in all animals at the higher dosage and m about half of the animals inhaling tlie lower dose, Subperitnueal and subcutaneous hemungiosarcomas were frequent in both dose groups; hut especially amung 5ft ppm animals. Only one animal had a hemangiosarcoma of the liver. No liver fibrosis was seen. All primary subjioritoneal and subcutaneous tumors were located in fat tissue. Telangiectasis was ohserved in tuo animals in the *)00 ppn\ series. FI he impoi'tance of htood \essel changes in the toxicology of vinyl chloride is discussed. Introduction Among' other toxicological effects (/), vinyl chloride (VC) has been found to induce liver injury in exposed workers (2) and has also been demonstrated to be an experimental (3, h) and a human carcinogen (.5. 6), Although a few studies on experimentally induced liver injury after chronic exposure to VC have been made, no atlomnt has been made to investigate both biochemical and histopathological param eters in mice. The aim of the present toxicologi cal study was to investigate possible changes in plasma enzymes and at the same time the histopathology of laboratorv mice exposed to VC. Design of Study Groups of mice of an outbred albino com mercial strain (NMRI strain) were exposed by inhalation from 12 weeks of age to 50 and .-><K) ppm VC, G hr day, 5 days/week, Raeh group consisted of 12 females and 12 males. T Hootion of Occupational Toxicology Department of Occupational Medicine, National I'.oarii of Occupational Safety ami Health, S-100 26 .Stockholm, Sweden. t National Veterinary Institute, S-HM Oil Stockholm, Sweden. The 50 ppm group was exposed for 52 weeks and the 500 ppm group for 26 weeks. A con trol group of 21 females and 2 1 males was ex posed to air only. The animals were observed during their life time. 'Fhe plasma activities of the following en zymes were analyzed in an LKB 8600 reaction rate analyzer at 37 C. Alkaline phosphatase (AP) was measured on 20 id plasma according to Bessey et ill. (~), except that l-amino-2methyl-1-propanol was used as buffer as recom mended by Morgenstern cl al. (,S). Glutamic-oxalacelic transaminase (GOT) was measured according to Karmen et al. (!>), and the activity of glutamic-pyruvic transaminase (GPT) according to Vroblewski and ba Due (10) on 25 /d plasma. Lactate dehydrogenase (LDII) was mea sured on 20 pi plasma as described by Wroblewski and La Due (11), except that the pi I was 7.! in our study. The isoenzymes of LDH were separated by disc electrophoresis and measured according to the method of Dietz and Lubrano (12). Plasma samples were diluted with a suitable volume of 40'r sucrose, depending on the total LDH activity, in order to obtain an LDH activity of around 200 mU/ml in the sample. A 10 pi portion of the diluted sample was added to the October 1976 211 yi-'&AV '.A'stft.Wrh'`v` ``A:-.,. V-Lr,., ;-t.s - | 1 : \ j * R&S 026742 h*Irt li\*-^ - 1 - 7r% electrophoresis tube. The stained gels were evaluated at 550 nm in a Philip Unicam spec trophotometer equipped with a gel scanner. In all groups, four animals of each sex were taken for pathological examination after 2G weeks of exposure, and in the control group another group was examined 52 weeks after the start of the experiment. The remaining animals wore aittopsied when moribund or when death occurred. Plasma Enzyme Changes The transaminases were not significantly elevated in exposed animals at any time during the experiment. The AP activity in animals exposed to 50 ppm did not difTer from normal values, but the 500 ppm group had an elevated AP activity dG weeks after the beginning of exposure (Fig. 1). mU/ml AP mU/ml Tot LDH weeks Fua'iu: 2. Total Lilli activity in plasma of exposed and control mice during one year. Exposure time 52 weeus (50 ppm) and 20 week.-, (500 ppm). The 1.1)11 activity is significantly elevated in the 500 ppm group at .')!) weeks (/<- (l.dtll) and in the 50 ppm group at 10 weeks ())< 0.001). Each point represents mean values of one determination on each animal. M lorn LDH R&S 026743 .( |,|.i .weeks Ficrm: 1, Alkaline phosphatase (AP) activity in plasma of exposed and control mice during one year. Kxposure time 52 weeks (50 ppm) and 2G weeks (500 PPm) The AP activity is .significantly elevated (/) < 0.05) in the 500 ppm group at week 10. Huch point represents mean values of one determination of each animal. At 39 weeks the 500 ppm group showed significantly elevated total LDH activity, as later did the animals exposed to 50 ppm VC (Fig. 2). The increase in LDH in both groups Wits generally accompanied by a shift toward cathodic isoenzymes as measured by the per centage of the M form (Fig, 3). Pathology The details of the morphological findings in this study are to bo published elsewhere (13). Fiona: Percentage of M form of I.PII isoenzyme pattern in plasma of control and exposed mice during one year. The M form is significantly elevated in the 500 ppm group from -10 weeks </i < 0.001) and in the 50 ppm group from -10 weeks (;) = 0.001) compared to control. Some morphological results are summarized in Tables 1 and 2. In the control group three animals showed spontaneous tumors, namely, one mammary adenocarcinoma with pulmonary metastases, one riisgerminoma of the ovary, and one reticu lum cell .sarcoma of spleen and mesenteric lymph nodes, all 15 weeks after the start of the experiment. 212 Environmental Health Perspectives .* t%&sk * Table 1. Number of animals with tumors 6 months after start of exposure. Number of animals with tumors VC ex posure, bung Ilomnnjrio- ppm adenoma.'5 sarcomas Other tumors Total number of animals autopsied st 0 50 0 008 2 0 08 500 8 0 1* 8 ` Mammary adenocarcinoma. K - ...-a Table 2.. Number of animals with tumors 12 months after start of exposure. X umber of animals w ith tumors VC ex posure, Tuntf Homantfio- PP'" adenomas sarcomas Other tumors Total number of animals autopsied 0 0 0 3* 24 50 13 1411 21 24 500 24 SJ 5` 24 KHii'id: !. Jlcman^iosarcojiia fmbodded in fat tissue* is s'cn on 1`iioh side of the uterus of animal exposed to 50 ppm VC. a One mammary adenocarcinoma, one distferminoma of the ovary, and one reticulum coll sarcoma of the spleen and mesenteric lymph nodoi 11 Subperitoneal and .subcutaneous and pulmonary he- manariosarcomas. c Mammary adenocarcinoma and one rhabdomyosar coma. iJ Subperitoneal, subcutaneous, hepatic, and renal he* man^io>arcomas. !`if;r KK 5, Pararenal homnn^iosarcoma of an animal ! * Mammary adenocarcinoma.- and one kidney ade exposed to 50 ppm VC. The tumor tissue is well sep noma. arated from kidney tissue. PA-S; 171 X- In the lower dose group about naif the ani mals had alveologenic adenomas; two mice of this group were sacriiied 2G weeks after the start of exposure. In animals autopsied between weeks 29 and 5G, subperitonoal hemangiosar- comas occurred in pararenal (Fig. 4 and 5) and paraintestinal sites, and in the pelvic/caudal part of the abdominal cavity as well as in brown frit or other subcutaneous tissues. The mean latency time for tumor death was 46 weeks. Ituptures of hemangiosareomas causing lemocoelia occurred in about one third of the exposed animals in the lower dose group. All mice in the 500 ppm group had alveoloenic adenomas (Fig. G). Also in this dose group subperitoneal hemangiosareomas were found in the pelvic caudal part of the abdom inal cavity although substantially fewer ani mals were bearing these tumors. Mammary adenocarcinomas were found in four animals. Only one animal had a hemangiosarcoma of the liver (Fig. 7). The mean latency time for tumor death was 35 wer,ks in this dose group. In two mice in the 500 ppm group blood vessel dilata tion (telangiectasis) was found in the liver (Fig. 8) without any other pathological liver changes. All primary subcutaneous and subperitoneal hemangiosareomas were located in fat tissue in both exposed groups. So liver fibrosis was noticed in any mouse. Ictober 1976 213 R&S 026744 fi tt 026745 C, Alvf'.Ju^nic .'trlrnomu of an animal exposed to 50 ]>i)m VC. II & E; 1TL X- 4c1 > ` ' I*'k:i ni: 7. Liver of an animal exposed to 500 ppm VC showing humangiosarcoma, II & E; KM X* 'V , - / V* * m ' #. L-. _ < --7 r ,j < ' X :?/' '***>-- I'lGVKI : 6, Liver trbingirct.Tsis of on animal e\-j 500 ppm VC. II & E; 171 X Discussion Changes in total plasma I.DII enzyme activ ity can have several causes, such as increased physical stress {!'.), tissue necrosis due to disease or chemically induced organ damage (IS, 1C), or tumor growth (IT). In connection with irreversible injury, cells are releasing in tracellular material including LDH (IS, IIn. which may result in an elevation of total plasma LDH and or an isoenzyme shift sometimes characterizing the organ damaged. Added to that, malignant cells leak small proteins (20) and may thus possibly contribute to a change in the total plasma LDH activity. A shift to wards the M form in the LDII-isoenzyme pat tern has been observed, for instance, in mon keys exposed to carbon tetrachloride (21) and in rats exposed to chlorinated pesticides ('22). The isoenzyme shift in malignant exudates is usually cathodic of nature (22), hut the isoen zyme distribution of plasma LDH of cancer patients seems not to be a good diagnostic tool for detecting malignancy (2-J). In the present experiments total LDH was elevated in both exposed groups of animals. A tendency to a dosc-eiTc-ct relationship in the elevation of total LDH but not in the per centage of M form was seen. It is also interest ing to note that a change in LDH activity oc curred long after the first appearance of tu mors, benign or malign, in both exposed groups. The increase in percentage of M form in the LDII isoenzyme pattern suggests a liver in jury. There was, however, no elevation in trans aminase activities, which could serve as a further indication on liver injury. AP normally decreases with increasing age of the animal (22), An increase in AP activity indicates among other things, lesions in the hepatobiliary tract (2U) and has been observed in VC-exposed workers (2T). Only those ani mals exposed to 500 ppm showed a significant increase in AP activity. The joint biochemical data indicate a tissue damage in miee caused by VC although the data are not convincingly indicative of liver injury. Changes in plasma enzymes in VCexposed mice seem furthermore not to be a good diagnostic criterion of tissue injury or early malignancy, as pathological lesions are mani fest long before a deviation from normality in enzyme activity is noticed (2S). 21-1 Environmental Health Perspectives s i b g si a m in th n~ an di in th ca m ve by vil- 1. C, S. U. 10. c Octo srvv su, rj.L.r'"'Arill;- ' .'-.^V-V' 1 Hemangiosarcomas of the abdominal sites seem often to have ruptured leading to death in hemocoelia- The blood vessels may thus have been fragilized during tumor induction or growth. In two animals inhaling 500 ppm blood sinus dilatation in the liver was seen without any other pathological liver change. In studies made on exposed workmen the blood vessels are involved as a target organ for vinyl chloride in the development of aeroosteolysis and of Ray naud's phenomenon as well as it is reflected in an overrepresentation of deaths in circulatory diseases among VC/PVC workers as reported in this symposium (20). One may thus conclude that blood vessel changes are part of the socalled vinyl chloride syndrome, including tu mor disease--or may even be a step in the de velopment of malignant tumor disease induced by this chemical (JO). This work was supported by the Swedish Work Knvironment Fund, Stockholm, RFFKRFNCF.S Holmbcrg, B., and .Molina, G. The industrial loxiculogy of vinyl chloride. A review. Work-Lnvironni.-Health 11: 138 111)74). Marstollcr, 11, ct al. Chronisch-toxische Lcher- seldiden hei Arbeitern in dor PVC-Produktion. Dcutsch. Med. Wochcnschrift OS: 2311 (10711). Viola, l1, I,,, Bigotti, A., and Caputo, A. Oncogenic response of rat skin, lungs and bones to vinyl chloride. Cancer Res. St: SIC (1971). Maltoni, C. The value of predictive experimental hioassays in occupational and environmental car cinogenesis, An example: vinyl chloride. Ambio 4: 18 (11)75). Lloyd, J. W. Angiosarcoma of the liver in vinyl chloride/polyvinyi chloride workers. J. Occup. .Med. 17: .Tty (11)75).' Byron, D,, and Holm berg, B. Two possible cases of angiosarcoma of the liver in a group of Swedish vinyl chloride-polyvinyl chloride workers. Ann. N.V, Acad. Sci, 240: 'ill) (1975). Bessey, 0. A., Lowry. 0. 1L, Brock, M. J. A method for the rapid determination of alkaline phosphatase with live cubic millimeters of serum. J. Biol. Cliem. ioi: am (1940). Morgenslern, et al. An automated p-nitrophenylphosphatc serum alkaline phosphatase procedure for the autoanalyzer. Clin. Client. 11: S7G (19G5). Karmen, A., A note on the spectrophotometric as say of glutamic-oxalacctic transaminase in human blood serum. .1. Clin. Invest. 34: Lit (1935). Wruldewski, F.t and La Due, J. S. Serum glutamic pyruvic transaminase in cardiac and hepatic dis ease. Bloc. Kxp. Biol. Med. 91: 509 (195G). Wrnblcwski, F., and La Due. J. S. Lactic dehydrog enase in blond. Broc. Soc. Kxp. Biol. Med. 90: 210 (1955). Iliei-., A. A,, and Luhrano, T. Separation and quan titation of laetie dehydrogenase isoenzymes by disc electrophoresis. Anal. Biochem. 20: 24G (1907). Kroncvi, T., Holmbcrg, B., and Winell, M. The pathology of vinyl chloride exposed mice. Acta. Vet. Scand. 17: 328 (197G). Porter, IL, Doty, D. IL, and Bloor, C. M. Interac tion of ago and exerei.se on tissue lactic dehydrog enase activity in rats. Lab. Invest. 25; 572 (1971). Muggli. A. I.Dll-Isoenzyme in tier Diagnose ties Hciv.infarktes. Vcrglciehendo L'ntersuchungen mit Cellegelelekt rophorese, D K A F-Sephadcx and ilBDIl-Bestimmung. Fnzymol. Biol. Clin. 9: 401 (1908). Kingoir, S. Lilli isoenzyme jiattern of rat kidney in mercurial intoxication. Nephron 7: 53S (1970). Burgess, F. A., anti Kylven, B. Laetie dehydrog enase activity in plasma ami interstitial flu it l dur ing growth of mouse tumors. Cancer Res. 23: 714 (19113). 18. Htilmlieig. B. On tile permeability to lissamine gifea ami other dyes in the course of cell injury and cell death. Fxji, Cel Res. 22: 40 (1901). 19. Vacca, C. Possibility that tlilferent "sticssos", anti especially hxpo.xia, allow tile outspread of intra cellular enzymes, due tt> an increase of cell mem brane permeability, without tissue necrosis. Kiv. .Med, Aero 20: 4 Li'(1903). Holmbcrg, B. On the in film release of cytoplasmic enzymes from ascites tumor cells as compared with strain L cells. Cancer Res. 21: 13SG (19G1). Wiemc, It. J., ami Maercke, Y, 'Lite fifth (elcctrophoretically slowest) serum lactic dehydrogenase as an index of liver injury. Ann. N.Y, Acatl. Sci. 91; 898 (1901). Khaikina. B. I., Kuzminskaya, U. A., and Alekhina, S. M. Fifed of some pesticides on activity of isocn-/.vmes uf serum lactate tlehydrogenase. Bull. Exptl. Biol. Med. (USSR), 70: 1140 (1970). Richtcnch, R., and Burger, A. Lactic dehydrogen ase isoenzymes in human cancer cells and malignant clfusions. Fnzymol. Biol. Clin. 3: G3 (19G3). 24. Kowaiewski, S, L. Die Isoenzyme der Lactatdehydrogenase. Thieme Verlag, Stuttgart, 1972. Schmidt, F. H. Fxpciienccs with enzyme determinations in toxicity stutlies. In Proceedings of the Kuropcan Society for the Study of Drug Toxicity. Vol. V. Bad llomhcrg, 1905. (Adv, Toxicological Methodology), Exccpta Mediea Foundation, 19G5, p. 97, 2G. Popper, IL, Koch-Weser, I)., and Huergn, J. Serum and hepatic enzymes in experimental liver damage. J. Mount Sinai llosp. 19: 23G (1952). Lilis, R., et al. Liver function among 1177 vinyl chloride polymerization workers. Abstracts of the XVIII International Congress on Occupational Health, Brighton, Knglnnd, Steptemher 1975: p. 398. 2S. Grice, H. C., et al. Correlation between scrum en zymes, isozyme patterns and histologically detec- -clober 1976 215 i ;1 K-sa f 7} j insigh genics ;i stance ] in oc( knowI( j furnisl * the dir i carcinc