useful model for elucidating the molecular of experiments with gametes from different action of the phorbol esters in the cells. animals), the most powerful cocarcinogen from the group of the phorbol esters irreSupported by the Stazione Zoologica, Naversibly influenced development even in ples and the European Communities Envinanomolar concentrations. Thus, the searonmental Research Program. Contract urchin embryo reacted at least as sensi021-74-I ENVD. tively as the mammalian cells tested so far. These cells were unaffected or only reversibly affected by TPA in that concentration Received August. 15, 1978 range. The action of TPA on the embryo cells must be a specific one as MeTPA 1. Weinstein, I.B., Wigler, M. : Nature 270, 659 and PDA showed a remarkably lower ac(1977) tivity, and like many other tested organic 2. Zur Hausen, H., ctat.: ibid. 272, 373 and water-insoluble substances, for exam(1978) ple alkaloids [6, 7], alcohols [8], anesthetics 3. Blumberg, P.M., Driedger, P.E.: ibid. 264, [6], carcinogens [8, 9], pesticides [5], or phe446 (1976) nols [6, 7] acted in the micromolar range 4. Brune, K., et al. : Cancer Lett. (in press) b u t showed no effects in nanomolar 5. Bresch, H., Arendt, U. : Environ. Res, 13, 121 (1977) concentrations. Since the embryotoxic activity of the three phorbol derivatives 6. Harvey, E.B.Y. : The American Arbaeia and Other Sea Urchins. Princeton Univ. Press tested ran parallel to their cocarcinogenic 1956 activity, the hypothesis might be postu7. Druckrey, H., et al.: Arzneim.-Forsch. 3, lated that the wimary mechanisms by 151 (t953) which the cocarcinogen acts on mamma- -. 8. Bresch, H., Spielhoff, R. : Namrwissenschaflian cells and on the embryo cells are (he ten 61, 368 (1974) same or at least very similar. The sea-ur9. de Angelis, E., Giordano, G.G.: Cancer Res. 34, 1275 (I974) chin embryo, therefore, might represent a
bution was more rarely found. The HBsAg and HBcAg were found in focal areas in the tissue which did not overlap in frozen sections of the liver simultaneously incubated with both antibodies.
Elution of HBsAg from liver tissue by dispersion into single cells. Primary hepatocyte monolayer cultures were prepared from liver biopsies obtained during the recovery phase from two chimpanzees experimentally infected with HBV [8]. The suspension medium volume for the disclosure of the tissue represented an approximately 340fold dilution of the original tissue volume. It was tested for HBsAg by radioimmunoassay (RIA) after sedimentation of
The Liver as a Barrier Against Antigens in Viral Hepatitis Infection P.R. Lorenz* Forschungslaboratorien der Behringwerke AG, D-3550 Marburg Present knowledge of the pathogenesis of hepatitis type B indicates that the disease is caused by damage to the hepatocyte following viral infection [1, 2]. Other observations show that the hepatitis type B virus (HBV) may not be damaging the liver cell. Intact hepatocytes overloaded with hepatitis type B surface antigen (HBsAg), the surface component of HBV, have been fomad in patients under immunosuppression [2, 3]. Electron microscopical studies on the intracellular distribution of HBsAg have shown that the antigen was maiNy present in the proliferative degranulated endoplasmic reticulum. Hepatitis type B core antigen (HBcAg), the inner component of HBV, predominated in the nuclei and was occasionally found associated with HBsAg in the cysternae of the endoplasmic reticulum [4, 5]. The degranulated endoplasmic reticulum is the hepatocellular instrument of 'biotransformation' of products of foreign origin or resulting * Present address: Battelle Institute e.V. D-6000 Frankfurt 90 662
from decay of cellular substances in preparation for their elimination [1, 6]. This may indicate that the hepatitis type B antigen (HBAg) is brought into the liver via the blood stream after multiplying elsewhere. Experimental data were therefore sought on the intrahepatic distribution of HBsAg and HBcAg, and on the infectiousness of liver-cell-associated HBAg by in vitro cultivation of HBAg-positive hepatocytes obtained from experimentally infected chimpanzees. The distribution of IIBAg in the liver. Perimembranous distribution of HBsAg on hepatocytes has been described [5, 71. HBcAg, on the other hand, is typically associated with the nuclei of the cells [4]. This distribution pattern was confirmed by analysis of cryostatic sections of a human HBAg-positive autopsy liver. Figure t shows the distribution of HBsAg and HBcAg as revealed by direct immunofluorescence staining. T w o HBsAg distribution patterns were found. Perimembranous (periceUular) HBsAg deposition wedominated, while intracytoplasmic distri-
Fig. 1. HBsAg (a) and HBcAg (b) distribution in a frozen section of a human autopsy liver of a patient with chronic aggressive hepatitis type B. (a) Incubation ~Jth FITC-conjugated anti-HBs gamma globulin raised in a rabbit and diluted 20fold in phosphate buffer. Specificity of anti-HBs was determined by neutra~ lization with purified HBsAg prior to staining. Arrow indicates perimcmbranous (pericellular) distribution, triangle intracytoplasmic distribution. (b) Incubation with FITC-conju~ gated human anti~HBc gamma globulin diluted 80fold in phosphate buffer. Specificity of anti-HBc was determined by competition against an excess of non-labelled human antiHBc gamma globulin from a different donor. Magnification 250 x
Naturwissenschaften 65 (1978) 9 by Springer-Verlag 1978
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Fig. 2. HBsAg elimination. Liver-biopsy cylinders (0.005 ml) were washed 2 times in an icecold heparin solution [14], rinsed once in icecold glycine-glucose buffer pH 8.3, 300 mOsmol [15], then stirred for 30 min in 1.7 ml of this buffer at 35 ~ The resulting turbid suspension was cleared by centrifugation, and the supernatant was tested by RIA for HBsAg. Results are expressed in terms of RIA indices=counts of the sample/counts of an HBsAg-negative control serum. (a) Elimination by chimpanzee No. 1, (b) by chimpanzee No. 2 the cells. Plasma samples obtained at the same time were tested in parallel. One of the chimpanzees (No. 1) showed no histopathological damage of the liver. The other animal (No. 2) developed a mild persistent hepatitis [8]. Chimpanzee No. 1 eliminated the HBsAg approximately three months earlier from its liver than from the plasma. Chimpanzee No. 2 did not clear the HBsAg earlier from its liver than from the plasma (Fig. 2). These results may suggest that perimembranouslydistributed HBsAg may be eluted from tissue by dispersion into single cells, and that the elimination pattern from the liver may be characteristic for a histopathological disease pattern when compared with the results of parallel plasma-HBsAg tests. The infectivity o f liver-cell-associated H B A g . Apart from a few successes of preliminary nature [9], the serial passage of hepatitis viruses has not been achieved in liver cell and organ cultures [10]. It was therefore attempted to infect various cell lines by long-term co-cultivation ( 1 4 days before the culture transfer) with HBAgpositive primary hepatocyte cultures in order to investigate the possibility that the hepatocyte-associated antigen may be infectious for these cells. Such hepatocyte cultures were obtained from the experimentally infected chimpanzees referred to above. The cells were sedimented from the suspensions, the supernatants of which were used for HBsAg determinations. Similar cultures were also studied for their potential of multiplying the HBAg in vitro. The hepatocytes did not divide in the cultures and remained unchanged for observation periods up to eight months, which
was determined in a pilot study using human fetal liver cells and cells prepared from liver tissue of Cynomolgous monkeys. Figure 3 shows the HBsAg associated with cells of a culture of primary chimpanzee hepatocytes as traced by the direct immunofluorescence method and by phase contrast. Such cells were co-cultivated with WI-38 cells, with a human fetal lung cell line, and with a permanent cell line of epithelial-like chimpanzee liver cells (chimpanzee liver 60/4, Paul-Ehrlich-Institut, D-6000 Frankfurt, Germany). Culture supernatants were tested for HBsAg by RIA, and Leighton slide subcultures were investigated for HBsAg and HBcAg by the direct immunofluorescence method as described. Only the WI-38 cells did not survive the co-cultivation for longer than three culture transfers. No indication of infection or of multiplication of the antigens was found during observation periods varying between 16 days and 6 weeks, covering between 2 and 9 culture transfers, of all the described cell types. It has not been possible to find experimental evidence proving that HBV multiplies in the liver. All data demonstrating the presence of the HBAg in and around the hepatocytes would agree with the hypothesis that the HBV may be carried to the liver via the portal vein or the hepatic artery after its reproduction in another organ. Furthermore, the hepatocellular damage following the infection of humans and primates with HBV does not represent a typical viral cytopathic effect [6]. Similar cell damage may ensue from influences completely unrelated to hepatitis virus such as anoxia caused by amyloid deposition, the trapping of cofactors as in galac-
Naturwissenschaften 65 (1978) 9 by Springer-Verlag 1978
Fig. 3. (a) Perimembranous association of HBsAg with primary hepatocytes of an experimentally infected chimpanzee, traced as for Fig. 1. Staining was done 1 day after the initiation of the culture. Medium E [16] without serum was used as nutrient solution. The presence of serum tended to interfere with the adhesion of the cells on the culture-flask surface and to stimulate overgrowth of fibroblast-like cells. Similar hepatocyte cultures were used for the co-cultivation experiments described in the text. (b) Phase contrast picture of the same culture. Magnification 1200x tosamine poisoning, the formation of free radicals as in CC14 intoxication, or covalent binding of protein due to chemical poisoning [1, 2]. The common terminal pathway for the cellular damage inflicted by these causes is a cell-membrane defect [21. Rather than by HBV multiplication in the liver, the liver damage in viral hepatitis may be caused by the involvement of the immunological defenses. Insufficient or reduced function of the reticuloendothelial cells of this organ may lead to persistence of the antigens in the liver resulting in increased access of HBAg to the general circulation and particularly the spleen [2, 11]. The increased induction of cellular and humoral immune reactions directed against the antigen-covered or -loaded hepatocytes resulting in hepatocellular damage would be the consequence [2, 12]. Such hepatocytotoxic immune reactions have been described for a number of antigens completely unrelated to HBV [13]. 663
When it is assumed that the HBV does not multiply in the liver, the question where it arises remains to be answered. Standard virological technics have not been helpful in explaining the pathogenesis of viral hepatitis. The isolation of the HBAg-specific hereditary substance (mRNA) during the incubation period of hepatitis type B in experimentally infected laboratory animals would identify the organ of primary synthesis of HBV. Received May 16 and September 15, 1978 1. Altmann, H.-W., Klinge, O., in: Spezielle Pathologic, Bd. 1 (eds. F. B/ichner, E. Grundmann). Miinchen: Urban & Schwarzenberg 1974 2. Popper, H. : Am. J. Pathol. 81, 609 (1975) 3. Kendrey, G., et al.: Kiln. Wschr. 53, 1081 (1975) 4. Huang, S.-N.: Am. J. Med. Sci. 270, 131 (1975) 5. Roos, C.M., Feltkamp-Vroom, T.M., Helder, A.W.: J. Pathol. 118, 1 (1976) 6. Schaffner, F., in: The Liver and Its Diseases (eds. F. Schaffner, S. Sherlock, C.M. Leevy). Stuttgart: Thieme 1974 7. Alberti, A., et al. : Clin. Exp. Immunol. 25, 396 (1976); Arnold, W., etal.: Klin.
Wschr. 53, 231 (1975); Murphy, B.L., et al. : Intervirology 6, 207 (1976) 8. Thomssen, R., et al.: Zbl. Bakt. Hyg. I. Abt. Orig. A 235, 242 (1976) 9. Panouse-Perrin, J., Courouc6-Pauty, A.M., Rachman, F.: Develop. Biol. Stand. 30, 211 (1975); Watanabe, M., et al. : Brit. J. Exp. Pathol. 57, 211 (1976) 10. Buynak, E.B., etal.: JAMA 235, 2832 (1976); Zuckerman, A.J., in: Hepatitis Associated Antigens and Viruses (ed. A.J. Zuckerman). Amsterdam: North-Holland 1972; Zuckerman, A.J.: Am. J. Med. Sci. 270, 197 (1975) 11. Sherlock, S. : Proc. Roy. Soc. Med. 70, 851 (1977); Thomas, H.C. : ibid. 70, 521 (1977) 12. Newble, D.I., et al. : Clin. Exp. Immunol. 20, 17 (1975); Paronetto., F., Vernace, S. : ibid. 19, 99 (1975) 13. Aubertin, A.-M., et al. : Nature 265, 456 (1977); Eckhardt, R., Heinisch, M., Meyer zum Btischenfelde, K.H.: Scand. J. Gastroent. 11, 49 (1976); News. Hepatitis and herpes viruses: Brit. Med. J. 3, 484 (1974); Rabin, B.S., Rogers, S.: Am. J. Pathol. 84, 201 (1976) 14. Bonney, R.J., Walker, P.R., Potter, V.R. : Biochem. J. 136, 947 (1973) 15. McLimans, W.F., in: Axenic Mammalian Cell Reactions, p. 307 (ed. G.L. Tritsch). New York: Dekker 1969 16. Williams, G.M., Gunn, J.M.: Exp. Cell Res. 89, 139 (1974)
Protection of Rats Against PhaHoidin by Ligation of Bile Duct B. Agostini Abteilung Physiologic, Max-Planck-Institut ftir medizinische Forschung, D-6900 Heidelberg A.K. Walli and H. Wieland Klinisch-Chemisches Labor, Medizinische Universit/itsklinik, Heidelberg Parenteral administration of a lethal dose of phalloidiu, one of the main toxic components of the fungus Amanita phalloides [1], causes death of adult rats within a few hours. A rapid increase of actin filaments and endocytotic vacuolization within the liver cells, and a massive accumulation of blood in the liver are observed [2]. Furthermore, phalloidin diminishes bile flow in isolated perfused liver [3]. Daily administration of a sublethal dose of phalloidin leads to an increase in the microfilaments, dilation of the bile eanaliculi with a loss of microvilli and a decrease in bile flow [4, 5]. These changes resemble the morphological alterations seen after extrahepatic cholestasis induced by bile-duct ligation [6]. Since rats survive this daily ad664
ministration of phalloidin, despite the alteration to the bile secretory system, we investigated whether cholestasis brought about by bile-duct ligation could protect against the toxic effect of phalloidin. Male Wistar strain rats weighing 180-200 g were used. Under Evipan natrium (Bayer, Leverkusen, BRD) anaesthesia a midline abdominal incision was made and the common bile duct ligated. The animals were divided into six groups of five rats and 1, 2, 7, 14, 21 and 28 days after bile-duct ligation three rats in each group were injected intraperitoneally with 3 mg phalloidin per kg body weight, while two rats in each group served as a control of cholestasis. This amount of phalloidin is about twice the lethal dosage for adult rats.
Blood and liver were removed under Evipan natrium anaesthesia 24 h after phalloidin administration. The activity of alkaline phosphatase and 7-glutamyl transpeptidase (7-GT), the bilirubin content in serum and examination of liver by light and electron microscopy were taken as indices of cholestasis. As shown in Table 1, up to 21 days after bile-duct ligation the mortality rate was zero. However, at 28 days after bile-duct ligation rats became sensitive to phalloidin toxicity and the mortality rate rose to 66%. As was expected, bile-duct ligation led to a severalfold increase in bilirubin levels and in the activity of alkaline phosphatase and y-GT in serum [7, 8]. During the first 2 weeks of cholestasis, phalloidin administration caused a further slight rise in the activity of alkaline phosphatase with a more pronounced rise in that of 7-GT. Light and electron microscopy of cholestatic nonpoisoned livers revealed characteristic alterations associated with bileduct ligation [6]. No definite morphological alterations that could be attributed to phalloidin poisoning were observed in rats which received phalloidin in the first week after bile-duct ligation. In contrast, typical phalloidin-indnced proliferation of actin filaments, vacuolization of hepatocytes, and accumulation of blood in the liver were found in rats which died from phalloidin 28 days after cholestasis. The increased amount of bile acids [7] in the liver following extrahepatic cholestasis may play a protective role against phalloidin poisoning. It has recently been shown that bile acids prevent the formation of phalloidin-induced protrusions in isolated hepatocyte [9]. However, in these in vitro experiments the unspecific effect on the cell membrane of the bile acids associated with their action as detergent cannot be excluded. Whether cholestasis inhibits the binding of phalloidin by the liver, as antamanide a nontoxic cyclic peptide of Amanitaphalloides does in vivo [10] and in vitro [11], should be of importance in explaining the protective role of bile-duct ligation against phalloidin poisoning. We thank Profs. Th. Wieland and W. Hasselbach for encouragement. Phalloidin was kindly provided by Prof. Th. Wieland. The excellent assistance of Miss S. Ganninger is acknowledged.
Received October 12, 1978
Naturwissenschaften 65 (1978) 9 by Springer-Verlag 1978
bution was more rarely found. The HBsAg and HBcAg were found in focal areas in the tissue which did not overlap in frozen sections of the liver simultaneously incubated with both antibodies.
Elution of HBsAg from liver tissue by dispersion into single cells. Primary hepatocyte monolayer cultures were prepared from liver biopsies obtained during the recovery phase from two chimpanzees experimentally infected with HBV [8]. The suspension medium volume for the disclosure of the tissue represented an approximately 340fold dilution of the original tissue volume. It was tested for HBsAg by radioimmunoassay (RIA) after sedimentation of
The Liver as a Barrier Against Antigens in Viral Hepatitis Infection P.R. Lorenz* Forschungslaboratorien der Behringwerke AG, D-3550 Marburg Present knowledge of the pathogenesis of hepatitis type B indicates that the disease is caused by damage to the hepatocyte following viral infection [1, 2]. Other observations show that the hepatitis type B virus (HBV) may not be damaging the liver cell. Intact hepatocytes overloaded with hepatitis type B surface antigen (HBsAg), the surface component of HBV, have been fomad in patients under immunosuppression [2, 3]. Electron microscopical studies on the intracellular distribution of HBsAg have shown that the antigen was maiNy present in the proliferative degranulated endoplasmic reticulum. Hepatitis type B core antigen (HBcAg), the inner component of HBV, predominated in the nuclei and was occasionally found associated with HBsAg in the cysternae of the endoplasmic reticulum [4, 5]. The degranulated endoplasmic reticulum is the hepatocellular instrument of 'biotransformation' of products of foreign origin or resulting * Present address: Battelle Institute e.V. D-6000 Frankfurt 90 662
from decay of cellular substances in preparation for their elimination [1, 6]. This may indicate that the hepatitis type B antigen (HBAg) is brought into the liver via the blood stream after multiplying elsewhere. Experimental data were therefore sought on the intrahepatic distribution of HBsAg and HBcAg, and on the infectiousness of liver-cell-associated HBAg by in vitro cultivation of HBAg-positive hepatocytes obtained from experimentally infected chimpanzees. The distribution of IIBAg in the liver. Perimembranous distribution of HBsAg on hepatocytes has been described [5, 71. HBcAg, on the other hand, is typically associated with the nuclei of the cells [4]. This distribution pattern was confirmed by analysis of cryostatic sections of a human HBAg-positive autopsy liver. Figure t shows the distribution of HBsAg and HBcAg as revealed by direct immunofluorescence staining. T w o HBsAg distribution patterns were found. Perimembranous (periceUular) HBsAg deposition wedominated, while intracytoplasmic distri-
Fig. 1. HBsAg (a) and HBcAg (b) distribution in a frozen section of a human autopsy liver of a patient with chronic aggressive hepatitis type B. (a) Incubation ~Jth FITC-conjugated anti-HBs gamma globulin raised in a rabbit and diluted 20fold in phosphate buffer. Specificity of anti-HBs was determined by neutra~ lization with purified HBsAg prior to staining. Arrow indicates perimcmbranous (pericellular) distribution, triangle intracytoplasmic distribution. (b) Incubation with FITC-conju~ gated human anti~HBc gamma globulin diluted 80fold in phosphate buffer. Specificity of anti-HBc was determined by competition against an excess of non-labelled human antiHBc gamma globulin from a different donor. Magnification 250 x
Naturwissenschaften 65 (1978) 9 by Springer-Verlag 1978
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Fig. 2. HBsAg elimination. Liver-biopsy cylinders (0.005 ml) were washed 2 times in an icecold heparin solution [14], rinsed once in icecold glycine-glucose buffer pH 8.3, 300 mOsmol [15], then stirred for 30 min in 1.7 ml of this buffer at 35 ~ The resulting turbid suspension was cleared by centrifugation, and the supernatant was tested by RIA for HBsAg. Results are expressed in terms of RIA indices=counts of the sample/counts of an HBsAg-negative control serum. (a) Elimination by chimpanzee No. 1, (b) by chimpanzee No. 2 the cells. Plasma samples obtained at the same time were tested in parallel. One of the chimpanzees (No. 1) showed no histopathological damage of the liver. The other animal (No. 2) developed a mild persistent hepatitis [8]. Chimpanzee No. 1 eliminated the HBsAg approximately three months earlier from its liver than from the plasma. Chimpanzee No. 2 did not clear the HBsAg earlier from its liver than from the plasma (Fig. 2). These results may suggest that perimembranouslydistributed HBsAg may be eluted from tissue by dispersion into single cells, and that the elimination pattern from the liver may be characteristic for a histopathological disease pattern when compared with the results of parallel plasma-HBsAg tests. The infectivity o f liver-cell-associated H B A g . Apart from a few successes of preliminary nature [9], the serial passage of hepatitis viruses has not been achieved in liver cell and organ cultures [10]. It was therefore attempted to infect various cell lines by long-term co-cultivation ( 1 4 days before the culture transfer) with HBAgpositive primary hepatocyte cultures in order to investigate the possibility that the hepatocyte-associated antigen may be infectious for these cells. Such hepatocyte cultures were obtained from the experimentally infected chimpanzees referred to above. The cells were sedimented from the suspensions, the supernatants of which were used for HBsAg determinations. Similar cultures were also studied for their potential of multiplying the HBAg in vitro. The hepatocytes did not divide in the cultures and remained unchanged for observation periods up to eight months, which
was determined in a pilot study using human fetal liver cells and cells prepared from liver tissue of Cynomolgous monkeys. Figure 3 shows the HBsAg associated with cells of a culture of primary chimpanzee hepatocytes as traced by the direct immunofluorescence method and by phase contrast. Such cells were co-cultivated with WI-38 cells, with a human fetal lung cell line, and with a permanent cell line of epithelial-like chimpanzee liver cells (chimpanzee liver 60/4, Paul-Ehrlich-Institut, D-6000 Frankfurt, Germany). Culture supernatants were tested for HBsAg by RIA, and Leighton slide subcultures were investigated for HBsAg and HBcAg by the direct immunofluorescence method as described. Only the WI-38 cells did not survive the co-cultivation for longer than three culture transfers. No indication of infection or of multiplication of the antigens was found during observation periods varying between 16 days and 6 weeks, covering between 2 and 9 culture transfers, of all the described cell types. It has not been possible to find experimental evidence proving that HBV multiplies in the liver. All data demonstrating the presence of the HBAg in and around the hepatocytes would agree with the hypothesis that the HBV may be carried to the liver via the portal vein or the hepatic artery after its reproduction in another organ. Furthermore, the hepatocellular damage following the infection of humans and primates with HBV does not represent a typical viral cytopathic effect [6]. Similar cell damage may ensue from influences completely unrelated to hepatitis virus such as anoxia caused by amyloid deposition, the trapping of cofactors as in galac-
Naturwissenschaften 65 (1978) 9 by Springer-Verlag 1978
Fig. 3. (a) Perimembranous association of HBsAg with primary hepatocytes of an experimentally infected chimpanzee, traced as for Fig. 1. Staining was done 1 day after the initiation of the culture. Medium E [16] without serum was used as nutrient solution. The presence of serum tended to interfere with the adhesion of the cells on the culture-flask surface and to stimulate overgrowth of fibroblast-like cells. Similar hepatocyte cultures were used for the co-cultivation experiments described in the text. (b) Phase contrast picture of the same culture. Magnification 1200x tosamine poisoning, the formation of free radicals as in CC14 intoxication, or covalent binding of protein due to chemical poisoning [1, 2]. The common terminal pathway for the cellular damage inflicted by these causes is a cell-membrane defect [21. Rather than by HBV multiplication in the liver, the liver damage in viral hepatitis may be caused by the involvement of the immunological defenses. Insufficient or reduced function of the reticuloendothelial cells of this organ may lead to persistence of the antigens in the liver resulting in increased access of HBAg to the general circulation and particularly the spleen [2, 11]. The increased induction of cellular and humoral immune reactions directed against the antigen-covered or -loaded hepatocytes resulting in hepatocellular damage would be the consequence [2, 12]. Such hepatocytotoxic immune reactions have been described for a number of antigens completely unrelated to HBV [13]. 663
When it is assumed that the HBV does not multiply in the liver, the question where it arises remains to be answered. Standard virological technics have not been helpful in explaining the pathogenesis of viral hepatitis. The isolation of the HBAg-specific hereditary substance (mRNA) during the incubation period of hepatitis type B in experimentally infected laboratory animals would identify the organ of primary synthesis of HBV. Received May 16 and September 15, 1978 1. Altmann, H.-W., Klinge, O., in: Spezielle Pathologic, Bd. 1 (eds. F. B/ichner, E. Grundmann). Miinchen: Urban & Schwarzenberg 1974 2. Popper, H. : Am. J. Pathol. 81, 609 (1975) 3. Kendrey, G., et al.: Kiln. Wschr. 53, 1081 (1975) 4. Huang, S.-N.: Am. J. Med. Sci. 270, 131 (1975) 5. Roos, C.M., Feltkamp-Vroom, T.M., Helder, A.W.: J. Pathol. 118, 1 (1976) 6. Schaffner, F., in: The Liver and Its Diseases (eds. F. Schaffner, S. Sherlock, C.M. Leevy). Stuttgart: Thieme 1974 7. Alberti, A., et al. : Clin. Exp. Immunol. 25, 396 (1976); Arnold, W., etal.: Klin.
Wschr. 53, 231 (1975); Murphy, B.L., et al. : Intervirology 6, 207 (1976) 8. Thomssen, R., et al.: Zbl. Bakt. Hyg. I. Abt. Orig. A 235, 242 (1976) 9. Panouse-Perrin, J., Courouc6-Pauty, A.M., Rachman, F.: Develop. Biol. Stand. 30, 211 (1975); Watanabe, M., et al. : Brit. J. Exp. Pathol. 57, 211 (1976) 10. Buynak, E.B., etal.: JAMA 235, 2832 (1976); Zuckerman, A.J., in: Hepatitis Associated Antigens and Viruses (ed. A.J. Zuckerman). Amsterdam: North-Holland 1972; Zuckerman, A.J.: Am. J. Med. Sci. 270, 197 (1975) 11. Sherlock, S. : Proc. Roy. Soc. Med. 70, 851 (1977); Thomas, H.C. : ibid. 70, 521 (1977) 12. Newble, D.I., et al. : Clin. Exp. Immunol. 20, 17 (1975); Paronetto., F., Vernace, S. : ibid. 19, 99 (1975) 13. Aubertin, A.-M., et al. : Nature 265, 456 (1977); Eckhardt, R., Heinisch, M., Meyer zum Btischenfelde, K.H.: Scand. J. Gastroent. 11, 49 (1976); News. Hepatitis and herpes viruses: Brit. Med. J. 3, 484 (1974); Rabin, B.S., Rogers, S.: Am. J. Pathol. 84, 201 (1976) 14. Bonney, R.J., Walker, P.R., Potter, V.R. : Biochem. J. 136, 947 (1973) 15. McLimans, W.F., in: Axenic Mammalian Cell Reactions, p. 307 (ed. G.L. Tritsch). New York: Dekker 1969 16. Williams, G.M., Gunn, J.M.: Exp. Cell Res. 89, 139 (1974)
Protection of Rats Against PhaHoidin by Ligation of Bile Duct B. Agostini Abteilung Physiologic, Max-Planck-Institut ftir medizinische Forschung, D-6900 Heidelberg A.K. Walli and H. Wieland Klinisch-Chemisches Labor, Medizinische Universit/itsklinik, Heidelberg Parenteral administration of a lethal dose of phalloidiu, one of the main toxic components of the fungus Amanita phalloides [1], causes death of adult rats within a few hours. A rapid increase of actin filaments and endocytotic vacuolization within the liver cells, and a massive accumulation of blood in the liver are observed [2]. Furthermore, phalloidin diminishes bile flow in isolated perfused liver [3]. Daily administration of a sublethal dose of phalloidin leads to an increase in the microfilaments, dilation of the bile eanaliculi with a loss of microvilli and a decrease in bile flow [4, 5]. These changes resemble the morphological alterations seen after extrahepatic cholestasis induced by bile-duct ligation [6]. Since rats survive this daily ad664
ministration of phalloidin, despite the alteration to the bile secretory system, we investigated whether cholestasis brought about by bile-duct ligation could protect against the toxic effect of phalloidin. Male Wistar strain rats weighing 180-200 g were used. Under Evipan natrium (Bayer, Leverkusen, BRD) anaesthesia a midline abdominal incision was made and the common bile duct ligated. The animals were divided into six groups of five rats and 1, 2, 7, 14, 21 and 28 days after bile-duct ligation three rats in each group were injected intraperitoneally with 3 mg phalloidin per kg body weight, while two rats in each group served as a control of cholestasis. This amount of phalloidin is about twice the lethal dosage for adult rats.
Blood and liver were removed under Evipan natrium anaesthesia 24 h after phalloidin administration. The activity of alkaline phosphatase and 7-glutamyl transpeptidase (7-GT), the bilirubin content in serum and examination of liver by light and electron microscopy were taken as indices of cholestasis. As shown in Table 1, up to 21 days after bile-duct ligation the mortality rate was zero. However, at 28 days after bile-duct ligation rats became sensitive to phalloidin toxicity and the mortality rate rose to 66%. As was expected, bile-duct ligation led to a severalfold increase in bilirubin levels and in the activity of alkaline phosphatase and y-GT in serum [7, 8]. During the first 2 weeks of cholestasis, phalloidin administration caused a further slight rise in the activity of alkaline phosphatase with a more pronounced rise in that of 7-GT. Light and electron microscopy of cholestatic nonpoisoned livers revealed characteristic alterations associated with bileduct ligation [6]. No definite morphological alterations that could be attributed to phalloidin poisoning were observed in rats which received phalloidin in the first week after bile-duct ligation. In contrast, typical phalloidin-indnced proliferation of actin filaments, vacuolization of hepatocytes, and accumulation of blood in the liver were found in rats which died from phalloidin 28 days after cholestasis. The increased amount of bile acids [7] in the liver following extrahepatic cholestasis may play a protective role against phalloidin poisoning. It has recently been shown that bile acids prevent the formation of phalloidin-induced protrusions in isolated hepatocyte [9]. However, in these in vitro experiments the unspecific effect on the cell membrane of the bile acids associated with their action as detergent cannot be excluded. Whether cholestasis inhibits the binding of phalloidin by the liver, as antamanide a nontoxic cyclic peptide of Amanitaphalloides does in vivo [10] and in vitro [11], should be of importance in explaining the protective role of bile-duct ligation against phalloidin poisoning. We thank Profs. Th. Wieland and W. Hasselbach for encouragement. Phalloidin was kindly provided by Prof. Th. Wieland. The excellent assistance of Miss S. Ganninger is acknowledged.
Received October 12, 1978
Naturwissenschaften 65 (1978) 9 by Springer-Verlag 1978
