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Toxicology. 13 (1979) 143--154 Elsevier/North-Holland Scientific Publishers Ltd ONE-YEAR TIME-SEQUENCE INHALATION TOXICITY STUDY OF VINYL CHLORIDE IN RATS. III. MORPHOLOGICAL CHANGES IN THE LIVER* V.J. FERON**, B.J. SPIT, H.R. LMMEL and R. KROES Central Institute for Nutrition and Food Research TNO, Zeist (The Netherlands) (Received April 30th, 1979) (Revision received June 19th, 1979) (Accepted June 20th, 1979) SUMMARY Wistar rats were exposed to atmospheres containing 0 (control) or 5000 ppm vinyl chloride monomer (VCM), 7 h/day, 5 days/week, for a period of 52 weeks. After 4, 13, 26 and 52 weeks each time 10 rats/sex/group were killed and subjected to extensive examinations. The present paper describes the morphological changes found in the liver. The major parenchymal changes comprised swelling and malformation of mitochondria, an increased amount of smooth endoplasmic reticulum, necrosis, nuclear and cellular polymorphism of hepatocytes, "foci of cellular alteration", neoplastic nodules and hepatocellular carcinomas. A reduced glucose-6-phosphatase activity in hepatocytes and a strong sinusoidal activity of alkaline phosphatase were found within "foci of cellular alteration". The non-parenchymal alterations included focal dilatation of sinusoids, focal proliferation of atypical sinusoidal cells and multicentric angiosarco mas. The effects of VCM on the hepatic parenchyma seemed to precede those on the hepatic stroma. * The study was sponsored by a group of co-operating European industries, including Verband Kunststofferzeugende Industrie e.V. (F.R.G.), Shell Nederland Chemie, Dutch State Mines, Akzo Zout Chemie Nederland B.V. and Dow Chemical Europe S.A. ** Present address: Dr. V.J. Feron, Department of Toxicology, Central Institute for Nutrition and Food Research TNO, P.O. Box 360, 3700 AJ Zeist, The Netherlands. Abbreviations: MFO, mixed function oxidase; RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum, VCM, vinyl chloride monomer. This is an abridged paper. Copies of the full paper are available from the Editor on request, which should be accompanied by J5.00, or equivalent, to cover reproduction and postage. 143 ucc INTRODUCTION Non-malignant liver disease and hepatic angiosarcoma are among the most important disorders in man associated with exposure to vinyl chloride monomer (VCM) [2,7,10,12,13,17,20--23]. In addition, neoplastic and non-neoplastic liver lesions have been found in a number of animal species after exposure to atmospheres containing VCM at sufficiently high con centrations [9,12,14--16,18,24,25,31--33], In a recent VCM-inhalation experiment haematological, biochemical and histopathological examinations were carried out at intervals over a 1-year period. The main purpose of the study was to determine the earliest VCMrelated changes and to examine the sequence of events resulting in tumour formation. The study is described in 3 papers of which this is the third. It deals with the morphological changes in the liver. The other papers describe the design of the study, clinical, haematological and biochemical findings, organ weights and morphological alterations in organs other than the liver [5,6], MATERIALS AND METHODS 2 groups of Wistar rats, each consisting of 62 males and 62 females, were exposed to atmospheres containing 0 or 5000 ppm VCM. After 4, 13, 26 and 52 weeks, 10 rats/sex/group were killed. Livers of these rats were examined by light and electron microscopy. Enzyme histochemical reactions included alkaline phosphatase, acid phosphatase adenosine monophosphatase and glucose-6-phosphatase. The livers of rats found dead or killed in moribund condition were only examined by light microscopy. RESULTS Cross liver lesions The liver of about 80% of the VCM-exposed animals that died or were killed during the second half of the experimental period showed nodules widely varying in size, appearance and consistency. Some of them were solid and pale, others cystic, haemorrhagic and necrotic. Adhesions between an affected liver and other abdominal organs were not uncommon. The abdominal cavity of several rats found dead contained blood, most likely derived from a ruptured cyst-like structure in the liver. Light microscopy of the liver Treatment-related histopathological liver changes are listed in Table I. Extensive areas of necrosis were found in the liver of many VCM-exposed rats which died or were killed in extremis in the second half of the test period. In several of these livers cyst-like spaces occurred which were filled with haemorrhagic and necrotic material and which were occasionally surrounded by a layer of connective tissue. In a number of these rats no 144 UCC 031009 table I TYPE AND INCIDENCE of VCMRELATED H1STOPATHOLOGICAL LIVER CHANGES OBSERVED IN RATS KILLED AFTER 13, 26 OR 52 WEEKS* Type of lesions Incidence of lesions Males Females VCM (ppm) VCM (ppm) 0 5000 0 5000 Animals killed after 13 weeks Number of animals examined Foci of cellular alteration: (a) Clear cell foci 1. A few foci Animals killed after 26 weeks Number of animals examined Foci of cellular alteration: (a) Clear cell foci 1. One or a few 2. Several (b) Basophilic foci 1. One or a few 2. Several foci Animals killed after 52 weeks Number of animals examined Foci of cellular alteration: (a) Clear cell foci 1. One or a few 2. Several 3. Many (b) Basophilic foci I. One or a few Neoplastic nodule Hepatocellular carcinoma Proliferation of atypical sinusoidal cells only Angiosarcoma0 Extensive necrosis Distended sinusoids 10 0 10 0 0 0 0 10 2 0 0 0 0 0 0 0 0 0 10 1 10 5 3 2 1 9 0 2 5 3 0 1 1 3 1 3 10 10 00 10 10 06 00 02 00 10 10 20 04 01 04 01 01 01 06 00 04 * In rats killed after 4 weeks no treatment-related histopathological liver changes were observed. 15 In 3 cases anaplastic carcinoma could not be excluded. indications were found for a neoplastic response of the liver. In others, however, the liver contained large and highly malignant tumours. Apart from neoplastic liver cell nodules 2 major types of tumours were found in the liver: hepatocellular carcinoma and angiosarcoma (Table II). The liver cell lesions were classified according to Squire and Levitt [28], The angio sarcomas were multicentric and often contained fairly large foci of necrosis, 145 TABLE O TYPE AND INCIDENCE OF LIVER TUMOURS FOUND IN 2 GROUPS OF RATS, EACH INITIALLY CONSISTING OF 62 MALES AND 62 FEMALES. WHICH WERE EXPOSED TO 0 AND 5000 PPM VCM RESPECTIVELY* Type of tumours Incidence of tumours Males Females VCM (ppm) VCM (ppm) 0 5000 0 5000 Neoplastic nodule Hepatocellular carcinoma Angiosarcoma*1 0 0 0 0 1 6 03 02 0 16 *10 rats/sex/group were killed after 4, 13, 26 and 52 weeks (only 9 males and 10 females of the test group were still alive in week 52). The remaining controls were all killed in week 53. b In 5 cases anaplastic carcinoma could not be excluded. haemorrhages and thrombi. In most of the tumours the neoplastic cells tended to line spaces, the wall of which was thickened by fibrous tissue. The neoplastic cells were polymorphic and had hyperchromatic, pleo morphic nuclei. The amount of fibrous connective tissue in the neoplasms varied widely. The direct extension of some of the more angiomatous tumours to the adjacent liver tissue was seen to occur in a destructive way, whereas other more solid tumours extended to the adjacent tissue by infil tration between preserved liver cords, thus gradually causing atrophy of the liver parenchyma. In view of their growth pattern, the tumours were con sidered to have developed from sinusoidal cells, most probably endothelial cells, though other sinusoidal cells could not be fully excluded. Many of the rats bearing hepatic angiosarcoma also showed focal disten sion of sinusoids and focal proliferations of either relatively normal or highly atypical sinusodial cells. Enzyme histochemistry of the liver In VCM-exposed rats an increased activity of acid phosphatase was seen m Kupffer-cells (or other sinusoidal cells) located in the periphery of the hepatic lobules. In areas where the sinusoids were distended, sinusoidal cells did not show any activity of acid phosphatase, indicating Kupffer-cells being either absent or inactive with regard to this enzyme. Moreover, "foci of cellular alteration" or neoplastic nodules exhibited much less pericana licular acid phosphatase activity than normal liver tissue. In comparison with normal liver cells the hepatocvtes in these foci of altered liver tissue showed a reduced glucose-6-phosphatase activity. It was interesting to find a strong sinusoidal activity of alkaline phosphatase within the foci of cellular alter- 146 ati are ph, Ele ji cor, rats cell l cho radi area occ. or n A BfeC* Fig. 1. 4 weeL * The of a tit- ucc 031011 :r. ations. An increased sinusoidal activity of this enzyme was also observed in areas showing distended sinusoids. No difference in adenosine monophosphatase activity was found between livers of test and control animals. Electron microscopy of the hepatic parenchyma* Light microscopy of semi-thin sections showed small foci of hepatocytes, containing a finely "vacuolized" cytoplasm in most of the VCM-exposed rats killed after 4, 26 and 52 weeks (Fig. 1). No such "vacuolized" liver cells were encountered in any of the controls. Ultras true turally, the "vacuoles" appeared to represent swollen mito chondria with a pale augmented matrix and relatively short cristae arranged radially and restricted to the periphery, thus leaving a fairly large central area free from cristae (Fig. 2). Hepatocytes with swollen mitochondria were occasionally found to contain myelin-like figures localized in lipid droplets or mitochondria, After 26 weeks swollen mitochondria were often empty or deprived of 'Oaf; m Fig. 1, Focus of "vacuolized** hepatocytes. Female rat exposed to 5000 ppm VCM for 4 weeks. One-micron Epon section, Paragon, X 640. * The ultrastructure of VCM-induced hepatic angiosarcoma in rats will be the subject of a separate paper. 147 ucc 031012 r-- Fig. 2, Hepatocyte. Swollen mitochondria with pale matrix and short cristae radially arranged along the periphery. Note nucleus with 2 pronounced nucleoli, Male rat exposed to 5000 ppm VCM for 4 weeks. Uranyl acetate and lead citrate, X 10 500. cristae. Irregularly shaped mitochondria with a heterogeneous matrix (lightand dark-grey areas) was a frequent finding in hepatocytes not containing swollen mitochondria. In these hepatocytes the RER consisted of clearly shortened packets of wavy lamellae, and mitochondria were often surround ed by single cistemae of RER. In general, no glycogen particles were present. Occasionally, small areas not showing the normal hepatic architecture were found in the liver. The liver cells in these regions were joined together m an unusual way (Fig. 3). There was a remarkable cell demarcation, invariably seen as a thin layer of condensed, somewhat filamentous cytoplasm adjacent to the plasma membrane. Bile eanaliculi were nearly always present, but sinusoids or sinusoid-like spaces occurred only sporadically in these areas. The amount FS \ V"nu-fci'-j'- . . ... . Fig, 3 Hepatocytes joined together. Sinusoids are almost completely absent. Note aged mitochondria and increased tubular SER. Female rat exposed to 5000 ppm for 26 weeks. Uranyl acetate and lead citrate, X 4500. 149 JCC 031014 Fig. 4. Hepatocytes containing whorl-like bands of tubular SER. Female rat exposed to 5000 ppm VCM for 26 weeks. Uranyl acetate and lead citrate, X 17 000. of tubular smooth endoplasmic reticulum (SER) was increased, whereas the amount of RER, generally occurring as short stacks around the mito chondria, was decreased. In some cases, liver cells were filled with whorllike bands of tubular SER (Fig. 4). The liver changes found after 52 weeks were similar to those seen after 26 weeks, but generally much more pronounced. Remarkable findings were cells tightly packed with swollen mitochondria often filled with a kind of flocky material, RER showing loss of ribosomes, absence of glycogen par ticles and accumulation of free ribosomes (Fig. 5). DISCUSSION Degenerative, hyperplastic and neoplastic alterations of the liver paren chyma have been reported to occur in several animal species and also in man following exposure to VCM. The degenerativa changes varied from fatty 150 UCC Fig. 5. Hepatocytes tightly packed with swollen mitochondria. Male rat exposed to 5000 ppm VCM for 52 weeks. Uranyl acetate and lead citrate, X 2600. infiltration [18,20], hydropic swelling with or without vacuolisation [12,20] isolated-cell necrosis [20,24] and centrolobular vacuolisation [9] to severe chronic hepatitis [32], Hyperplasia was seen as "proliferation of hepato cytes" [2] and enlarged polymorphic parenchymal cells with big hyperchromatic nuclei containing several nucleoli [13,20]. A few hepatomas have been found in rats exposed to VCM for a prolonged period of time [15,16,29,33]. The results of the present study in rats were in full agreement with the observations reported in the literature mentioned, and likewise produced convincing evidence of VCM adversely affecting the hepatic parenchyma. Swelling of mitochondria in foci of parenchymal cells was the earliest change observed and mitochondrial alterations remained characteristic features of the VCM-damage seen at later stages. Slight swelling of hepato- 151 cytic mitochondria has been reported to occur in a VCM-reactor cleaner suffering from splenomegaly and non-cirrhotic portal hypertension [10]. Swelling of mitochondria has been reported to occur in hepatocytes under various pathological conditions, such as dietary deficiency in ribo flavin [30], hypoxia [19, Kief, H. pers. comm.], and exposure to ethanol [4], Furthermore, the mitochondrial changes described (swelling, electronlucent matrix, shortening and partial loss of cristae) have also been found after exposure to other carcinogens [1]. Despite the extensive mitochondrial changes no increase in autophagic vacuoles containing remnants of mito chondria was found in the present study. This somewhat remarkable find ing, however, seems to be normal for hepatocytic mitochondrial damage caused by carcinogens [1]. It seems, therefore, justified to conclude that the mitochondria] changes observed are far from specific for VCM-damage. In addition to swelling of mitochondria, prolonged exposure to VCM (26 and 52 weeks) appeared to cause a decrease in RER, loss of ribosomes from the RER, while the most important observation was an increase in SER. Hypertrophy of SER has also been found in mice exposed to 2500-- 6000 ppm VCM [26]. These findings suggest an enhancing effect of VCM on the mixed function oxidase system (MFO-system) which has indeed been shown to be involved in the detoxification of VCM [3,25], In this respect one might speculate that the MFO-system is in competition with other cellular functions (e.g. the mitochondria] respiration) in satisfying their need of oxygen. The reduction in glucose-6-phosphatase activity in "foci of cellular alteration" occurring in the livers of VCM-exposed rats is a finding com parable to the decreased activity of this enzyme seen by Scherer and Emmelot [27] in "islands" induced by diethylnitrosamine in the liver of rats. These investigators consider the "islands" to represent an obligatory stage in liver carcinogenesis. The hepatic "foci of cellular alteration" also exhibited an increased alkaline phosphatase activity and a decreased acid phosphatase activity. The physiological or toxicological significance of these observations is not clear, and we are not aware of similar reactions found in "foci of cellular alteration" induced by carcinogens. In addition to the parenchymal alterations, the livers of the test animals exhibited focal dilatation of sinusoids, marked alkaline phosphatase activity of sinusoidal cells, focal proliferation of sinusoidal cells with or without atypia and multicentric angiosarcomas containing widely varying amounts of fibrous tissue. Although these impressive changes of the he patic stroma are in accordance with the observations of other investigat ors in man and experimental animals exposed to VCM [2,7,10,13,17,20, 21,23], it might be of interest to draw attention to certain differences between the previous and the present findings. Maltoni and Lefemine [15] have reported the occurrence of hepatic and extrahepatic blood vessel ectasias and angiomas, and of angiosarcomas outside the liver in rats, mice and hamsters. VCM-induced extrahepatic angiosarcomas in mite have more recently also been reported by Lee et al. [11] and Holmberg et al. [8]. No extrahepatic angiosarcomas were observed in the present study, with the possible exception of a primary angiosarcoma in the lungs [6]. In view of their morphology, the sinusoidal cell tumours were classified as angiosarcomas, and were thus considered to have developed from endo thelial cells. In this respect, an interesting finding was the absence or inactiv ity (as to acid phosphatase) of Kupffer-cells in areas where the sinusoids were distended, possibly indicating Kupffer-cells not being involved in the neoplastic process. A "fibrotic precursor stage" has been suggested to precede angiosarcoma development in humans [21--23,31], In rats marked fibrosis has been seen only within fully developed angiosarcomas or as a reaction to extensive necrosis of liver parenchyma. Therefore, in rats such a conspicuous "fibrotic precursor stage" does not seem to exist, although electron microscopical examinations of livers from rats which were exposed to VCM by the oral route for a prolonged period of time, occasionally revealed the presence of bundles of collagen fibres in contact with angiosarcoma cells extending between liver cords (Spit, B.J., unpublished). 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