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SNV)HOSUENTAL RESEARCH 29, 272-2790982) 2ii PW, p tNV Jfiwis nLO t72 Mitochondrial Changes in Hepatocytes of Rats Chronically Exposed to Vinyl Chloride and Ethanol M. L. Miller, M. J. Radike, A. Andrinoa, and E. Bingham Unfvtrsity of Cincinnati Medical Center, Institute of Environmental Health. 3225 Eden Avenue, Cincinnati, Ohio 45267 Received May 19, 1981 Chronic exposure of male rats to 600 ppm vinyl chloride (VC) or VC and ethanol (EtOH) in drinking water induced ultrestructure) changes in the mitochondria of hepatocytea. After 6 months of VC/ElOH treatment, hepatoeyte mitochondria often contained rigid tubular parallel cristae and dilated cristae with dense inclusions; ethanol ingestion alone did not induce these morphological changes. In animals receiving VC alone, large floccular densities in the mitochondrial matrix were seen occasionally after 6 months of VC inhala tion. The numbers and severity of changes in mitochondria Increased with duration of exposure and age. The greatest responses observed io mitochondria occurred with the combined treatment of VC/EtOH. INTRODUCTION It has been well documented that inhalation of vinyl chloride (VC) by man (Creech and Johnson, 1974) and laboratory animals (Maltoni and Lefemine, 1975) induces angiosarcoma of the liver. The toxicity of VC it attributed to reactive metabolites, chloroethylene oxide and 2-chloroacetaldehyde, which also elicit mutagenic and toxic responses in selected bacteria (Malaveille el al., 1975; McCann et al., 1975) and mammalian cells (Huberman et al., 1979). At equal concentrations in bacterial systems chloroethylene oxide was more mutagenic, although less toxic than 2-chloroacetaldehyde. The hepatoeyte, which is predominantly responsible for the metabolism of VC, has been reported to undergo morphologic changes and significant cellular de struction following acute exposures of rats to VC (Du et al., 1979; Feron et al., 1979; Feron and Kroes, 1979). In an attempt to elucidate some of the intracellular mechanisms involved in VC toxicity and carcinogenicity before destruction of parenchyma] elements, a model of chronic VC inhalation exposure and ethanol ingestion was devised (Radike et al., 1977). This report describes the light and electron microscopic alterations in the mitochondria of the liver parenchymal cells resulting from chronic exposure of rats to VC or VC/EtOH. MATERIALS AND METHODS Male Sprague--Dawley rats were used in two separate long-term studies on the ~ effects of ingested ethanol (EtOH) on VC-induced carcinogenesis. Animals (480) were divided into four groups: group one received filtered air and tap water <Ah7HsO); group two received filtered air and 5% EtOH (v/v) in the drinking water (Air/EtOH); group three inhaled 600 ppm VC. 4 hr/dav. 5 davs/week (VC/HaO); 0013-9351182/060272-08$02.0010 Copyright 19*1 by Academic PrtM, Inc. All right* of reproduction In any form fgMrved. 272 AP00019826 AEROSOL INHALATION SIMULATION 271 Dennis. W. L. (1961). The growth of hygroscopic drop in a humid air stream. In "The Physical Chemistry of Aerosols." pp. 78-85. Aberdeen Univ. Press. Aberdeen. Scotland. Halbert, M. K., Mazumder, M. K.. and Bond, R. L. (1981a). Size distribution analysis of respirable particulates in cosmetic aerosols: A methodological comparison. Food Cosmet. Toxicol. 19, 85-88. Halbert, M. K., Mazumder. M K., and Bond, R. L. (1981b). Respirable particulates in household aerosols. Environ. Res. 26, 105--109. Held. J. L., and Cooper. D. W. (1979). Theoretical investigation of the effects of relative humidity on aerosol respirable fraction. Atmos. Environ. 13, 1419-1425, Hiller, F. C., Mazumder, M. K., Wilson, J, D., and Bone, R. C. (1980). Effect of low and high relative humidity on metered-dosc bronchodilator solution and powdered aerosols, i. Pharm. Sci. 69, 334-337. Mazumder, M. K., Ware, R. E., Wilson, J. D., Renninger. R. G.. Hiller, F. C., McLeod. P. C., Rai- ble, R. W., and Testennah, M. K. (1979). SPART analyzer: Its application to aerodynamic size distribution measurement. J. Aerosol. Sci. 10, 561. Mercer, T. T. (1973). "Aerosol Technology in Hazard Evaluation,'* pp. 5, 34--36, 86--104, 191, 213-256. Academic Press, New York. Mokler, B. V., Wong, B. A., and Snow, J. M. (t979a). Respirable particulates generated by pres* surized consumer products. 1. Experimental method and general characteristics. Amer. Ind. Hyg. Assoc. J. 40, 330. Mokler, B. V., Wong, B. A., and Snow, M. J. (1979b). Respirable particulates generated by pres* surized consumer products. 11. Influence of experimental conditions. Amer. Ind. Hyg. Assoc. J. 40, 339. Morrow, p. E. (1974). Aerosol characterization and deposition. Amer. Rev. Respir. Dfs. 110, 88 -99. Natusch, D. F. S., and Wallace, J. R. (1974). Urban aerosol toxicity: The influence of particle size. Science 186, 695 -699. Savitzky, A., and Golay, MCJ. E. (1964), Smoothing and differentiation of data by least squares procedures, Anal. Chem. 36, 1627-1639. Scherer, P. W., Haselton, F. R., Hanna, L. M-, and Stone, D. R, (1979). Growth of hygroscopic aerosols in a mode) of bronchial airways. J, Appl. Physiol. Respir. Environ. Exercise Physiol. 43, 544 -550. Sciarra, J. J., McGinley, P.,and Izzo, L. (1969). Determination of particle size distribution of selected aerosol cosmetics. I. Hair sprays. J. Soc. Cosmet. Chem. 20,365-394. Silverman, L., Billings, C., and First, W. (1971). "Particle Size Analysis in Industrial Hygiene." pp. 260--268. Academic Press, New York. Task Group on Lung Dynamics (1966). Deposition and retention models for internal dosimetry of the human respiratory tract. Health Phys. 12, 1973. Vos, K. D., and Thomson, D. B. (1974). Particle size measurement of eight commercial pressurized products. Powder Technol. 10, 103. AP00019827 MITOCHONDRIAL CHANGES FROM VC AND EtOH 273 and group four inhaled VC and ingested EtOH (VC/EtOH). All animals were fed Ralston Purina rat chow ad libitum except during exposure to VC, when food and water were withheld. Animals in groups three and four were held in individually compartmented stainless-steel cages for VC exposure by inhalation. Ingestion of EtOH began 4 weeks prior to the initiation of VC inhalation and was continued until death or killing of the animal. In the first experiment, animals were treated with VC for 1 year (80/group). Unless the animals appeared to be moribund, they were killed \Yi years postexpo sure. In the second experiment (40/group), sacrifices were scheduled at 3, 6, 9, and 12 months after the initiation of VC inhalation. The method of killing, proce dures for obtaining paraffin-embedded tissues, special staining procedures for light microscopy, and the evaluation of the types of tumors which developed have been reported (Radike et al., 1981). Preparation of tissues for electron microscopy was as follows: as quickly as possible after pentobarbital anesthesia, pieces ofthe tiver were removed and diced into 1-mrn cubes. Tissues were fixed in Chick fix (Russell, 1972) then post-fixed in phosphate-buffered 1% osmium tetroxide for 2 hr. In addition, duplicate tissues were fixed directly in phosphate-buffered osmium tetroxide. All tissues were de hydrated stepwise through graded ethanols and embedded in Spun* (Spurr, 1969). . Toluidine blue-stained sections (0.5 /xm) were examined with light microscopy and thin sections on naked grids were contrasted with uranyl acetate and lead citrate for transmission electron microscopy. A total of 20 animals each from the Air/ HtO, Air, EtOH, and VC/EtOH groups and 19 from the VC group (79 of 480 animals) were prepared for electron microscopy. RESULTS The ultrastructure ofthe liver, particularly the parenchymal cells of the Air/HtO group, was similar to that amply described in the literature (Fig. 1). Mitochondria in all experimental groups demonstrated greater heterogeneity in size and shape than did controls. Numbers of mitochondria with changes and the severity of changes increased with the duration of exposure and age. The ratio of surface/volume appeared to increase in the experimental groups with time. Mitochondria of ethanol-treated animals were rounded with an increase in the number of marginated cristae and an increase in heterogeneity in size and shape. Some areas of parallel cristae were seen in animals which received ethanol for 12 months and longer. Not seen were the intracristal inclusions which were so com mon in the animals treated with ethanol and vinyl chloride together. In addition to minimal changes in the mitochondria, the cytoplasm of hepatocytes demonstrated a modest increase in the amount of smooth endoplasmic reticulum. Vinyl chloride inhalation alone did not induce mitochondrial changes which were observed following VC/EtOH treatment. Intracristal inclusions in their group were never demonstrated unequivocally, although dilated cristae with some indication of dense inclusions were encountered. Mitochondria as the main com ponent in autophagocytic bodies in the cytoplasm of VC-treated animals were common. Mitochondria in the VC/EtOH groups were predominantly indented and cup shaped (Fig. 2), whereas those resulting from VC exposure alone were more often APOOOf 9828 6Z861000dV 'XOOO'fC 'uoupuonaoiiuj atp jo uomod patuaput utqt at)t uo aatcua j|nqm jo uo|iztueSjo |S|jEred pus aejsuo jcnsiuej (biujou jo jpuj ajofq *u ui uaos aq u sajnjsmjs a^j[-poj ajiuyaQ sqiuouj 9 jo; jouwpa pus apuojip jAuja 7 'ol 'xOOOd '|*nsn si saimiwS tBUpuoqaojtiUBJiU! ;o mauiaiduioa i]l pu? jbi[uji art supuoipojjuj utipiM an<H3 *iuas%id si uaSooAjo '(le/said uin[no[iaj oiujseidopua qjoouis pro 'sajpoqaioitu `tunjnoijaj otius(dopua qSnai `tupuoqaotmi (uuot>{ aK a joj touBips pus apuofip jAuja ({];* painj) tpuiju* at paqaiBUJ-afs sbm qajifvi ju [ojjuoo b jo Jnn *1 *9iJ ' IV 3 vaiim Kl MITOCHONDRIAL CHANCES FROM VC AND EtOH 275 Fic. 3. Vinyl chloride and ethanol for 6 months. Tubules within the matrix have deformed the mitochondrion. Note the coiled nature. At opportune areas, a dense central line can be observed. Cross sections of the tubules are visible in the lower comer of the mitochondrion. A central core in these particular tubules is not resolved. 59,000x. Fig. 4. Vinyl chloride and ethanol for 6 months. Longitudinal tubular structures in the mitochondria of the hep&tocytes. Cristae not yet transformed are usually tubular, instead of lamellar. 36,000*. elongated. Parallel cristae in the indented areas were common. Pseudoinclusions of cytoplasm or portions of other mitochondria resulted from section artifact. Animals exposed to VC or VC/EtOH had mitochondria which often demonstrated rigid parallel cristae (Figs. 3,4). Cross sections of these structures confirmed their tubular nature. In extreme cases, rigid cristae showed a periodicity and a faint central line. Increases in membrane density of cristae, periodicity, and the central line were not seen in animals receiving EtOH alone or in Air/HjO controls. These changes first appeared at 6 months after initiation of treatment in the VC/EtOH group in a small number of animals and by 12 months they were found routinely. The mitochondria of VC/EtOH-treated animals showed peculiar intracristal densities which, on very opportune sections, had a regular substructure. Some of these were stacked (Fig. 2), and others were coiled (Fig. 5). Dilated cristae with inclusions were seen in VC/EtOH animals after 6 months of exposure and were not seen in animals after VC or ethanol alone or filtered air. In animals which received VC alone, another type of density, not associated with the cristae, was found in the matrix of the mitochondria. These floccular densities were observed at 6 months after initiation of VC and after 1V4 years of recovery (Fig. 6). AP00019830 276 MILLER ET AL. I fig. 5. Vinyl chloride wd ethanol for 6 monthi. lntmristal Inclusions in mitochondria occasionally appeared helical; or coiled. 62,000*. Fig. 6- Vinyl chloride alone for I year and recovery for IH yean. A less common alteration in mitochondria was floccular densities within the matrix. 3000*. Cytoplasmic changes which were observed at the early sacrifices (3 and 6 months) in the VC and VC/EtOH groups included a mild increase in the density of filaments at the plasmalemma of the hepatocytes and a conspicuously close as sociation of the mitochondria with the filaments adjacent to tight junctions (desmo5omes) (Fig. 7). These anchor mitochondria were encountered less frequently in the 3-month Air/HjO controls. Mitochondria associated with the plasmalemma in VC and VC/EtOH groups were noted by light microscopy of semithin sections by an increase in their numbers at the periphery of the hepatocytes. In lesions of parenchymal cells which developed in these rats, hepatocytes also demonstrated long, peripherally located mitochondria of which a large number were associated with desmosomes, sometimes at more than one point of anchoring. Mitochondria also showed an increase in touching (two or more mitochondria in close contact along their outer membranes). These morphologic changes appeared in the early stages of exposure and in lesions; other mitochondrial changes (parallel cristae, tubular transformations, intracristal inclusions, and matrical densities) did not. DISCUSSION Mitochondria with modified cristae have been seen in animals exposed to toxic agents other than VC or VC/EtOH. The administration of polychlorinated biphenyls in the rat resulted m cristae which were dense, noticeably rigid, and APOOOf9831 MITOCHONDRIAL CHANGES FROM VC AND EtOH 277 Fig. 7. Vinyl chloride and ethanol for 12 months. Anchor mitochondria were a regular feature of the animals which received VC/EtOH. Here two mitochondria from adjacent hepatocytet show a peculiar arrangement next to the filaments of a desmosome. Such configuration* were frequently seen in hepatocellular lesions; however, this was visualized in the "normal" portion of the liver. 37,000*. parallel to the long axis of the mitochondrion (Burse ef al., 1974), but dilated cristae with inclusions were not mentioned. Confer and Stenger (1966) reported a central sheaf of cristae in mitochondria of hepatocytes after carbon tetrachloride exposure; they did not observe, however, an increase in density, or tubular or coiled cristae with a central core. Weinstein et at. (1972) observed bizarre-shaped mitochondria resulting from dichloromethane inhalation. Some of the changes bear at least a superficial resemblance to the mitochondrial alterations seen in VC/EtOH-exposed animals. Occasionally, similar mitochondrial structures have been observed in normal tissues. Tubule-like structures with a dense central core seen in spermatids (Fukumoto, 1979) are somewhat similar to inclusions in VC/EtOH-treated mito chondria. Elongation of the organelle accompanied the presence of regularly ar ranged parallel cristae. It has also been reported that in a small number of cases the normal pineal gland and brown adipose tissue (interscapular) of pre- and neonatal rats have unusual mitochondrial inclusions (Barnard and Lindberg, 1969). iris evident that alignment of mitochondrial cristae represents a common response to toxic substances, however, intracristal inclusions appear In suffi ciently few experimental and normal conditions to make their occurrence after VC/EtOH exposure remarkable. APOOO19832 278 MILLER ET AL. Feron et al. (1979) examined mitochondria after high doses of VC (5000 ppm) and they stated that the swelling and irregularity in mitochondrial shape were not specific for vinyl chloride. Their exposures were very high in comparison to the 600 ppm used here. After high doses of VC, greatly enlarged pale mitochondria and spotty degeneration of the hepatocytes were reported (Du et al., 1979). We found that at low doses necrosis was not a prominent feature of hepatocyte mor phology, though hepatocellular carcinomas, angiosarcomas, and other tumors de veloped in rats after prolonged exposure to low doses of VC and VC/EtOH (Radike et al., 1981). In hepatocytes of animals treated with ethanol alone, the low level (5%) EtOH used in this study did not produce the megamitochondria commonly observed in animals receiving high concentrations of ethanol nor the filaments of Mallory bodies characteristic of the response of human hepatocytes. These effects of ethanol on mitochondria, in vivo and in vitro, have long been recognized (Rubin, 1973). At this time, no correlation can be drawn between the structural changes in mitochondria reported here in rats concomitantly exposed to VC and ethanol and the increased incidence of hepatic angiosarcomas and carcinomas which were observed (Radike et al,, 1981). ACKNOWLEDGMENTS The authors-gratefully acknowledge the assistance of Frank Grande and David McVey. This-re search was supported in part by EPA Contract 68-03-2402, USPHS Grant ESQ01J9, and American Cancer Society Grant CH116. REFERENCES Barnard, T., and Lindberg, O. (1969). Ultrastructural changes in the chondriome during perinatal development in brown adipose (issue of rats. J. Ultrastruct. Res. 29, 293-310. Burse, V. W., Kimbrough. R. D., Villanueva, E. C.,et al. (1974). Polychlorinated biphenyls: Storage, distribution, excretion, and recovery: Liver morphology after prolonged dietary ingestion. Arch. Environ. Health 29, 301. Confer. D. B., and Stenger, R. J. (1966). Nodules in the livers of C3H mice after long-term carbon tetrachloride administration: A light and electron microscopic study. Cancer Res. 26, 834 -843. Creech, J. L., Jr., and Johnson, M. N. (1974). Angiosarcoma of liver in the manufacture of polyvinyl chloride. J. Occup. Med. 16, 150-151. Du. J. T., Sandoz, J. P., Tseng, M. T., and Tamburro, C. H- (1979). Biochemical alterations in liven of rats exposed to vinyl chloride. J. Toxicol. Environ. Health 5, 1119-1132. Feron, V. J., and Kroei, R. (1979). One-year time-sequence inhalation toxicity study of vinyl chloride in rats. II. Morphological changes in the respiratory tract, ceruminous glands, brain, kidneys, heart and spteen. Toxicology 13, 131--141. Feron, V. J., Spit, B. J., Imreel, H. R., and Kroes, R. (1979). One-year time-sequence inhalation toxicity study of vinyl chloride in rats. III. Morphological changes in the liver. Toxicology 13, 143-154. Fukumoto, M. (1979). Tube-like structures in mitochondria of tunicate iPyura viitata) spermatids. J. Ultrastruct. Res. 68, 1-5. Huberman. E., Bartseh, H., and Sachs, L. (1979). Mutation induction in Chinese hamster V79 cells by two vinyl chloride metabolites, chloroethytene oxide and 2-chloroacetaldehyde. J. Toxicol Envi ron. Health 5, 1119-1132. Malaveille, C., Bartseh, H., Montesano, H., Barbin. A.. Camos, A. M., Croizy, A., and Jacquignon. AP000I9833 `- MITOCHONDRIAL CHANGES FROM VC AND EtOH 279 P. (1975). Mutagenicity of vinyl chloride, chloroethylene oxide, chloroaceiaidehyde and chloroethanol. Biochem. Biophys. Res. Commun. 63, 303-370. Maitoni, C., and Lefemine, G. (1975). Carcinogenicity bioassays of vinyl chloride: Current results. Ann. N.Y.Acad. Set. 246, 195-218. McCann, J., Simmon, V., Streiiweiser, D., and Ames, B. N. (1975). Mutagenicity of chloroacetaldehyde. a possible metabolic product of 1,2-dichloroethane (ethylene dichloride), chloroethanol (ethylene chlorohydrin). vinyl chloride, and cyclophosphamide. Proc. Natl. Acad. Set. USA 72, 3190-3193. Radike, M. 3., Stemmer, K. L.. Brown, P. G., Larson, E., and Bingham, E. (19771. Effect of ethanol and vinyl chloride on the induction of liver tumors: Preliminary report .Environ. Health Persped. 21, 153-155. Radike, M. J., Stemmer, K. L., and Bingham, E. (1981). Effect of ethanol on vinyl chloride carcino genesis. Environ. Health Perspect. 41, 59--62. Rubin, E. (1973). The spectrum of alcoholic liver injury. In `'The Liver" (E. A. Gall and F- K. Mostofi, Eds.). Williams & Wilkins, Baltimore. Russell, N. J. (1972). "Epidermal Ultrastructure during Wound Healing in Chick Embryos." Doctoral thesis, University of Minnesota. Spurr, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31 -43. Weinstein, R. S., Boyd, D. C, and Back, K.C. (1972). Effects of continuous inhalation of dichloromethane in the mouse: Morphological and functional observations. Toxicol. Appl. Phar macol. 23, 660-679. AP000198J4 ENVIRONMENTAL RESEARCH 29, 280-286(1982} 1 Effect of Subacute Exposure to NO* on Lymphocytes Required for Antibody Responses Hidekazu Fujimaki, Fujjo Shimizu, and Kentaro Kubota Basic Medical Sciences Division, The National Institute for Environmental Studies, Yaiabe-mochf Tstiknba Ibaraki, 305 Japan Received May 23, 1981 BALB/c mice were continuously exposed to 0,4 and 1.6 ppm NO* for 4 weeks and the effects on lymphocytes which are required for primary and secondary antibody responses to sheep red blood cells were examined in vitro. The primary antibody response was signifi cantly suppressed by both concentrations of NO,, whereas the secondary antibody response was slightly stimulated by 1.6 ppm NO, exposure. In reconstitution experiments no signifi cant differences were observed in the activities of T and B lymphocytes from mice exposed to 1.6 ppm NO,, INTRODUCTION Many investigators have reported the effect of short- and long-term exposure to low levels of NO, on immune defense mechanism's (Fenters el al., 1971, 1973; Ehrlich et al., 1975). Maigetter et al. (1978) have already shown the depression of mitogenic responses in both T and B cells by a long-term exposure to 0.5 ppm NOt. Recently, the effect on the immunological fraction of exposure to 10 ppm NO or NO, for 2 hr daily on weekdays, for varying periods up to 30 weeks, was examined by Holt et al. (1979). They indicated that the immune functions, e.g., serum antibody responses, T cell mitogen, and graft-versus-host responses, were suppressed by chronic exposure but were enhanced by shorter exposure. In vitro analysis ofeffects of acute exposure to NO, on antibody responses was performed in our laboratory and indicated that the acute exposure to 20 ppm NO, for 12 hr markedly suppressed the primary antibody response, which was mainly caused by the inactivation of B cells (Fujimaki et a!., 1981). In this paper, to extend these studies the effect of subacute exposure to rela tively low levels of NO, on lymphocytes required for antibody responses to SRBC was investigated in vitro. The primary antibody response was significantly sup pressed by exposure to 0.4 and 1.6 ppm NO, for 4 weeks, but the secondary antibody response was stimulated by exposure to 1.6 ppm NO,. MATERIALS AND METHODS Mice. BALB/c male mice, purchased from Charles River Japan, Inc., were used mid they were 7 weeks old at the commencement of the exposure. NOt exposure. Mice were continuously exposed to 0.4 and 1.6 ppm NO, for 4 weeks In a stainless-steel and glass chamber (1800 (iters) made by Koito Kogyo Co., Ltd., which was provided with 60 air changes per hour, and the concentra- 0013-9331/82/060280-07502.00/0 Copyright 19K by Academic Prtu. lee. AB h|hti of reproduction la any form reserved. 280 APOOOt 9835