Arch. Toxicol. 35, 69-73 (1976) 9 by Springer-Verlag 1976
Increased Penetration of Amanitine into Hepatocytes when Conjugated with Albumin E. Bonetti, M. Derenzini, and L. Flume Istituto di Patologia generale, Universit~ di Bologna, Bologna, Italy Received July 20, 1975 Abstract. The amount of amanitine able to cause the specific nuclear lesions in mouse hepatocytes is 10 times lower when amanitine is administered conjugated to albumin instead of being injected as free toxin. Key words: Amanitine -- Amanitine-albumin conjugate -- Hepatic damage. Zusammenfassung. Die Amanitinmenge, die spezifische Kernschfiden in den Leberzellen von M~iusen zu verursachen vermag, ist zehnmal geringer, wenn Amanitin an Albumin gebunden verabreicht wird. Schlfisselwtrter: Amanitin -- Amanitin-albumin Konjugate -- Leberschaden.
The toxic peptides amanitines (Wieland, 1968; Fiume and Wieland, 1970) cause necrosis of hepatocytes and of the cells in the proximal convoluted tubules of the kidney in mice (Fiume and Laschi~ 1965). In these cells the first ultrastructural changes are found in nuclei, where a breakup of nucleoli and a condensation of chromatin can be observed 1 5 - 3 0 min after injection of the toxin (Fiume and Laschi, 1965; Fiume et al., 1969; Marinozzi and Fiume, 1971). Conjugation with albumin greatly increased the toxicity of amanitines (Cessi and Fiume, 1969; Derenzini et al., 1973). Mice injected with a lethal dose of an amanitine-albumin conjugate die of a hepatic necrosis morphologically different from that observed following ceamanitine administration (Derenzini et al., 1973). When only 1 or 2 LD~0 of the conjugate were injected, the nuclear lesions characteristic of amanitine poisoning were found in the liver only in sinusoidal cells. Damage and disappearance of these cells cause nonspecific changes in hepatocytes which culminate in cellular necrosis (Derenzini et al., 1973). The great sensitivity of sinusoidal cells to conjugate is very likely due to high protein uptake displayed by these cells (Kruse and McMaster, 1949). This conclusion is supported by the finding that conjugation to albumin gives amanitine a selective toxicity for those cells which take up proteins at high degree, such as macrophages cultured in vitro (Barbanti-Brodano and Flume, 1973; Fiume and Barbanti-Brodano, 1974) and the proximal tubule cells of the rat kidney (Bonetti et al., 1974).
70
E. Bonetti et aL
The present experiments were performed to establish whether doses o f conjugate h i g h e r t h a n 2 LDs0 p r o d u c e t h e n u c l e a r lesions t y p i c a l o f a m a n i t i n e in h e p a t o c y t e s too. S i n c e t h e lesions w e r e o b s e r v e d , w e c o m p a r e d t h e l o w e s t d o s e s o f b o t h free cea m a n i t i n e a n d a m a n i t i n e b o u n d to a l b u m i n w h i c h w e r e able to p r o d u c e t h e specific n u c l e a r c h a n g e s in h e p a t o c y t e s . Materials
and
Methods
The amanitine-albumin conjugate (AMA-RSA) was prepared as described by Derenzini et al., (1973) with the difference that rabbit serum albumin (RSA) instead of bovine albumin was used. The molar ratio of amanitine to albumin in the conjugate was calculated according to Derenzini et al. (1973). SDS polyacrylamide gel electrophoresis of AMA-RSA was performed as described (Derenzini et al., 1973). The LDs0 for Swiss albino male mice weighing 28-30 g was determined according to Litchfield and Wilcoxon (1949). cr-amanitine and AMA-RSA in 0.9% NaC1 were administered by intraperitoneal injection. For all doses injected the volume of the solution was 0.1 ml per 10 g body weight. For electron-microscopic observations, mice were killed 1, 8 and 16 hrs after injection. Two mice were killed for each dose and time interval. Liver samples were immediately fixed in either (a) 4% formaldehyde and 2.5% glutaraldehyde, or (b) 1% osmium tetroxide. The fixatives were dissolved in 0.I M Sorensen buffer, pH 7.2. All the samples were embedded in an Araldite Epon mixture according to Mollenhauer (1964). Ultrathin sections were double stained with uranium and lead. Semithin sections were stained with Azur II and methylene blue (Richardson et al., 1960). Results
T h e m o l a r r a t i o o f a m a n i t i n e t o a l b u m i n in t h e c o n j u g a t e w a s f o u n d t o be 2.3. S D S p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f A M A - R S A s h o w e d t h a t o n l y a small p r o p o r t i o n (about 20%) of the conjugate had a molecular weight corresponding to t h e m o n o m e r i c f o r m o f a l b u m i n . T h e bulk o f the c o n j u g a t e is m a d e u p b y m o l e c u l a r
Table 1. Doses of amanitine (~g/10 g body weight) which produce nuclear lesions in sinusoidal cells and hepatocytes Doses
~r-amanitine Nonconjugated
Amanitine Conjugated to albumin
Sinusoidal cells
Hepatocytes
8 hrs
1 hr
8 hrs
-
+
_+
1
-
+
+
1.50
-
+
+
-
+ +
0.50 0.75
0.015 0.029 0.058 0.073 0.087 0.102 0.116
(0.5) . (1) (2) (2.5) (3) (3.5) (4)
. _+ + + +
.
16 hrs.
.
- , _+, and + respectively indicate that the nuclear lesions were observed in no cell, in some cells, in all the cells. a In parentheses the corresponding doses of conjugate.
Increased Penetration of Amanitine into Hepatocytes
71
Fig. 1. Nucleus of mouse hepatocyte 8 hrs after AMA-RSA injection (3.5 ug per 10 g body weight). 4 fragments are visible. Fixation: aldehyde. Staining: uranyl acetate and lead citrate, x 40,000
species corresponding to high molecular weight polymers of albumin. The presence of the polymers is due to the formation of covalent bounds among the albumin molecules in presence of E C D I (Derenzini et al., 1973). The intraperitoneal LDs0 of the conjugate (AMA-RSA) was found to be 1 p~g per 10 g body weight. Since the molar ratio amanitine albumin in this conjugate was 2.3, I pxg of it contained 0.029 ~xg of amanitine. The effects of ce-amanitine and AMA-RSA on the nuclear ultrastructure of sinusoidal cells and hepatocytes are reported in Table 1. When 1--3 LDs0 of conjugate were injected, the nuclear lesions typical of amanitine poisoning were observed only in sinusoidal cells in agreement with previous observations (Derenzini et al., 1973). 3.5 pxg of AMA-RSA per 10 g body weight (= 3.5 LDs0) or higher doses produced the specific nuclear lesions not only in sinusoidal cells, but also in the hepatocytes (Fig. 1). The lowest dose of free ce-amanitine which caused nuclear lesions in all the hepatocytes was 1 ~xg per I0 g body weight. The lesions caused by ce-amanitine in hepatocytes appeared earlier than those induced by AMA-RSA. o~-amanitine did not bring about changes in sinusoidal cells. Discussion
The present results show that the amount of toxin able to cause the specific nuclear lesions in hepatocytes is 10 times lower when amanitine is administered conjugated to albumin instead of being injected as free toxin.
72
E. Bonetti et aL
This result could be explained by admitting that mouse hepatocytes take up proteins to a high degree. When albumin tagged with a dye (Kruse and McMaster, 1949) or with fluorescein (Schiller et al., 1953) is administered to animals it is not observed in hepatocytes. But such experiments cannot rule out a high albumin uptake by hepatocytes (for discussion see Holtzer and Holtzer, 1960). A n o t h e r possible explanation m a y be the following. After injection o f small amounts of heat-aggregated albumin in mouse the protein is found almost entirely in the liver (Benacerraf et al., 1957). The bulk o f A M A R S A is m a d e up by high molecular weight polymers of albumin (see above). If the behavior o f our conjugate is similar to that of heat-aggregated albumin and if the hepatocytes do not participate in the uptake of the conjugate, A M A - R S A should be concentrated in liver sinusoidal cells. Since proteins after penetration into reticuloendothelial cells are digested by lysosomal enzymes (Unanue, 1972), it is possible that amanitine is slowly released as free toxin from sinusoidal cells and in small amounts but for a relatively long time it comes into contact with and enters the adjacent hepatocytes. On the contrary, after injection o f free toxin the disappearance of o~amanitine from the blood is very rapid, no more toxin being detectable by 4 - 5 hrs in the dog and mouse (Faulstich and Fauser, 1973; F i u m e et al., 1975). W h a t e v e r the real mechanism is (and both could be operative) our results suggest that in some instances conjugation o f a substance to a protein could be a means o f obtaining a higher concentration of the substance in hepatocytes than in other cells.
References Barbanti-Brodano, G., Fiume, L.: Selective killing of macrophages by amanitine-albumin conjugates. Nature (Lond.) New Biol. 244, 281 (1973) Benacerraf, B., Biozzi, G., Halpern, B. N., Stiffel, C., Mouton, D.: Phagocytosis of heat-denatured human serum albumin labelled with 13~Iand its use as a means of investigating liver blood flow. Brit. J. exp. Path. 38, 35 (1957) Bonetti, E., Derenzini, M., Fiume, L.: Lesions in the cells of proximal convoluted tubules in rat kidney induced by amanitine-albumin conjugate. Virchows Arch., Abt. B Cell Path. 16, 71 (1974) Cessi, 'C., Fiume, L.: Increased toxicity of ~-amanitine when bound to a protein. Toxicon 6, 309 (1969) Derenzini, M., Fiume, L., Marinozzi, V., Mattioli, A., Montanaro, L., Sperti, S.: Pathogenesis of liver necrosis produced by amanitine-albumin conjugates. Lab. Invest. 29, 150 (1973) Faulstich, H., Fauser, U.: Untersuchungen zur Frage der H~imodialysebei der Knollenblfitterpilzvergiftung. Dtsch. med. Wschr. 98, 2258 (1973) Fiume, L., Barbanti-Brodano, G.: Selective toxicity of amanitine-albumin conjugates for macrophages. Experientia (Basel) 30, 76 (1974) Fiume, L., Busi, C., Campadelli-Fiume, G., Franceschi, C.: Production of antibodies to amanitines as the basis for their radioimmunoassay. Experientia (Basel) 31, 1233 (1975) Flume, L., Laschi, R.: Lesioni ultrastrutturali prodotte nelle cellule parenchimali epatiche dalla falloidina e dalla ~-amanitina. Sperimentale 115, 288 (1965) Fiume, L., Marinozzi, V., Nardi, F.: The effects of amanitine poisoning on mouse kidney. Brit. J. exp. Path. 50, 270 (1969) Flume, L., Wieland, T.: Amanitines. Chemistry and action. FEBS Letters 8, 1 (1970) Holtzer, H., Holtzer, S.: In vitro uptake of fluorescein labelled plasma proteins. C. R. Lab. Carlsberg 31, 373 (1960) Kruse, H., McMaster, P. D.: The distribution and storage of blue antigenic azoproteins in the tissue of mice. J. exp. Med. 90, 425 (1949)
Increased Penetration of Amanitine into Hepatocytes
73
Litchfield, J. T., Jr., Wilcoxon, F. A.: A simplified method of evaluating dose-effect experiments. J. Pharmacol. exp. Ther. 96, 99 (1949) Marinozzi, V., Fiume, L.: Effects of oL-amanitine on mouse and rat liver cell nuclei. Exp. Cell Res. 67, 311 (1971) Mollenhauer, H.: Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111 (1964) Richardson, K. C., Jarett, L., Finke, E. M.." Embedding in epoxiresins for ultrathin sectioning in electron microscopy. Stain Technol. 35, 313 (1960) Schiller, A. A., Schayer, R. W., Hess, E. L.: Fluorescein-conjugated bovine albumin. Physical and biological properties. J. gen. Physiol. 36, 489 (1953) Unanue, E. R.: The regulatory role of macrophages in antigenic stimulation. Advanc. Immunol. 15, 95 (1972) Wieland, T.: Poisonous principles of mushrooms of the genus Amanita. Science 159, 946 (1968) Dr. L. Fiume Istituto di Patologia generale Via San Giacomo 14 1-40126 Bologna, Italy
Increased Penetration of Amanitine into Hepatocytes when Conjugated with Albumin E. Bonetti, M. Derenzini, and L. Flume Istituto di Patologia generale, Universit~ di Bologna, Bologna, Italy Received July 20, 1975 Abstract. The amount of amanitine able to cause the specific nuclear lesions in mouse hepatocytes is 10 times lower when amanitine is administered conjugated to albumin instead of being injected as free toxin. Key words: Amanitine -- Amanitine-albumin conjugate -- Hepatic damage. Zusammenfassung. Die Amanitinmenge, die spezifische Kernschfiden in den Leberzellen von M~iusen zu verursachen vermag, ist zehnmal geringer, wenn Amanitin an Albumin gebunden verabreicht wird. Schlfisselwtrter: Amanitin -- Amanitin-albumin Konjugate -- Leberschaden.
The toxic peptides amanitines (Wieland, 1968; Fiume and Wieland, 1970) cause necrosis of hepatocytes and of the cells in the proximal convoluted tubules of the kidney in mice (Fiume and Laschi~ 1965). In these cells the first ultrastructural changes are found in nuclei, where a breakup of nucleoli and a condensation of chromatin can be observed 1 5 - 3 0 min after injection of the toxin (Fiume and Laschi, 1965; Fiume et al., 1969; Marinozzi and Fiume, 1971). Conjugation with albumin greatly increased the toxicity of amanitines (Cessi and Fiume, 1969; Derenzini et al., 1973). Mice injected with a lethal dose of an amanitine-albumin conjugate die of a hepatic necrosis morphologically different from that observed following ceamanitine administration (Derenzini et al., 1973). When only 1 or 2 LD~0 of the conjugate were injected, the nuclear lesions characteristic of amanitine poisoning were found in the liver only in sinusoidal cells. Damage and disappearance of these cells cause nonspecific changes in hepatocytes which culminate in cellular necrosis (Derenzini et al., 1973). The great sensitivity of sinusoidal cells to conjugate is very likely due to high protein uptake displayed by these cells (Kruse and McMaster, 1949). This conclusion is supported by the finding that conjugation to albumin gives amanitine a selective toxicity for those cells which take up proteins at high degree, such as macrophages cultured in vitro (Barbanti-Brodano and Flume, 1973; Fiume and Barbanti-Brodano, 1974) and the proximal tubule cells of the rat kidney (Bonetti et al., 1974).
70
E. Bonetti et aL
The present experiments were performed to establish whether doses o f conjugate h i g h e r t h a n 2 LDs0 p r o d u c e t h e n u c l e a r lesions t y p i c a l o f a m a n i t i n e in h e p a t o c y t e s too. S i n c e t h e lesions w e r e o b s e r v e d , w e c o m p a r e d t h e l o w e s t d o s e s o f b o t h free cea m a n i t i n e a n d a m a n i t i n e b o u n d to a l b u m i n w h i c h w e r e able to p r o d u c e t h e specific n u c l e a r c h a n g e s in h e p a t o c y t e s . Materials
and
Methods
The amanitine-albumin conjugate (AMA-RSA) was prepared as described by Derenzini et al., (1973) with the difference that rabbit serum albumin (RSA) instead of bovine albumin was used. The molar ratio of amanitine to albumin in the conjugate was calculated according to Derenzini et al. (1973). SDS polyacrylamide gel electrophoresis of AMA-RSA was performed as described (Derenzini et al., 1973). The LDs0 for Swiss albino male mice weighing 28-30 g was determined according to Litchfield and Wilcoxon (1949). cr-amanitine and AMA-RSA in 0.9% NaC1 were administered by intraperitoneal injection. For all doses injected the volume of the solution was 0.1 ml per 10 g body weight. For electron-microscopic observations, mice were killed 1, 8 and 16 hrs after injection. Two mice were killed for each dose and time interval. Liver samples were immediately fixed in either (a) 4% formaldehyde and 2.5% glutaraldehyde, or (b) 1% osmium tetroxide. The fixatives were dissolved in 0.I M Sorensen buffer, pH 7.2. All the samples were embedded in an Araldite Epon mixture according to Mollenhauer (1964). Ultrathin sections were double stained with uranium and lead. Semithin sections were stained with Azur II and methylene blue (Richardson et al., 1960). Results
T h e m o l a r r a t i o o f a m a n i t i n e t o a l b u m i n in t h e c o n j u g a t e w a s f o u n d t o be 2.3. S D S p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f A M A - R S A s h o w e d t h a t o n l y a small p r o p o r t i o n (about 20%) of the conjugate had a molecular weight corresponding to t h e m o n o m e r i c f o r m o f a l b u m i n . T h e bulk o f the c o n j u g a t e is m a d e u p b y m o l e c u l a r
Table 1. Doses of amanitine (~g/10 g body weight) which produce nuclear lesions in sinusoidal cells and hepatocytes Doses
~r-amanitine Nonconjugated
Amanitine Conjugated to albumin
Sinusoidal cells
Hepatocytes
8 hrs
1 hr
8 hrs
-
+
_+
1
-
+
+
1.50
-
+
+
-
+ +
0.50 0.75
0.015 0.029 0.058 0.073 0.087 0.102 0.116
(0.5) . (1) (2) (2.5) (3) (3.5) (4)
. _+ + + +
.
16 hrs.
.
- , _+, and + respectively indicate that the nuclear lesions were observed in no cell, in some cells, in all the cells. a In parentheses the corresponding doses of conjugate.
Increased Penetration of Amanitine into Hepatocytes
71
Fig. 1. Nucleus of mouse hepatocyte 8 hrs after AMA-RSA injection (3.5 ug per 10 g body weight). 4 fragments are visible. Fixation: aldehyde. Staining: uranyl acetate and lead citrate, x 40,000
species corresponding to high molecular weight polymers of albumin. The presence of the polymers is due to the formation of covalent bounds among the albumin molecules in presence of E C D I (Derenzini et al., 1973). The intraperitoneal LDs0 of the conjugate (AMA-RSA) was found to be 1 p~g per 10 g body weight. Since the molar ratio amanitine albumin in this conjugate was 2.3, I pxg of it contained 0.029 ~xg of amanitine. The effects of ce-amanitine and AMA-RSA on the nuclear ultrastructure of sinusoidal cells and hepatocytes are reported in Table 1. When 1--3 LDs0 of conjugate were injected, the nuclear lesions typical of amanitine poisoning were observed only in sinusoidal cells in agreement with previous observations (Derenzini et al., 1973). 3.5 pxg of AMA-RSA per 10 g body weight (= 3.5 LDs0) or higher doses produced the specific nuclear lesions not only in sinusoidal cells, but also in the hepatocytes (Fig. 1). The lowest dose of free ce-amanitine which caused nuclear lesions in all the hepatocytes was 1 ~xg per I0 g body weight. The lesions caused by ce-amanitine in hepatocytes appeared earlier than those induced by AMA-RSA. o~-amanitine did not bring about changes in sinusoidal cells. Discussion
The present results show that the amount of toxin able to cause the specific nuclear lesions in hepatocytes is 10 times lower when amanitine is administered conjugated to albumin instead of being injected as free toxin.
72
E. Bonetti et aL
This result could be explained by admitting that mouse hepatocytes take up proteins to a high degree. When albumin tagged with a dye (Kruse and McMaster, 1949) or with fluorescein (Schiller et al., 1953) is administered to animals it is not observed in hepatocytes. But such experiments cannot rule out a high albumin uptake by hepatocytes (for discussion see Holtzer and Holtzer, 1960). A n o t h e r possible explanation m a y be the following. After injection o f small amounts of heat-aggregated albumin in mouse the protein is found almost entirely in the liver (Benacerraf et al., 1957). The bulk o f A M A R S A is m a d e up by high molecular weight polymers of albumin (see above). If the behavior o f our conjugate is similar to that of heat-aggregated albumin and if the hepatocytes do not participate in the uptake of the conjugate, A M A - R S A should be concentrated in liver sinusoidal cells. Since proteins after penetration into reticuloendothelial cells are digested by lysosomal enzymes (Unanue, 1972), it is possible that amanitine is slowly released as free toxin from sinusoidal cells and in small amounts but for a relatively long time it comes into contact with and enters the adjacent hepatocytes. On the contrary, after injection o f free toxin the disappearance of o~amanitine from the blood is very rapid, no more toxin being detectable by 4 - 5 hrs in the dog and mouse (Faulstich and Fauser, 1973; F i u m e et al., 1975). W h a t e v e r the real mechanism is (and both could be operative) our results suggest that in some instances conjugation o f a substance to a protein could be a means o f obtaining a higher concentration of the substance in hepatocytes than in other cells.
References Barbanti-Brodano, G., Fiume, L.: Selective killing of macrophages by amanitine-albumin conjugates. Nature (Lond.) New Biol. 244, 281 (1973) Benacerraf, B., Biozzi, G., Halpern, B. N., Stiffel, C., Mouton, D.: Phagocytosis of heat-denatured human serum albumin labelled with 13~Iand its use as a means of investigating liver blood flow. Brit. J. exp. Path. 38, 35 (1957) Bonetti, E., Derenzini, M., Fiume, L.: Lesions in the cells of proximal convoluted tubules in rat kidney induced by amanitine-albumin conjugate. Virchows Arch., Abt. B Cell Path. 16, 71 (1974) Cessi, 'C., Fiume, L.: Increased toxicity of ~-amanitine when bound to a protein. Toxicon 6, 309 (1969) Derenzini, M., Fiume, L., Marinozzi, V., Mattioli, A., Montanaro, L., Sperti, S.: Pathogenesis of liver necrosis produced by amanitine-albumin conjugates. Lab. Invest. 29, 150 (1973) Faulstich, H., Fauser, U.: Untersuchungen zur Frage der H~imodialysebei der Knollenblfitterpilzvergiftung. Dtsch. med. Wschr. 98, 2258 (1973) Fiume, L., Barbanti-Brodano, G.: Selective toxicity of amanitine-albumin conjugates for macrophages. Experientia (Basel) 30, 76 (1974) Fiume, L., Busi, C., Campadelli-Fiume, G., Franceschi, C.: Production of antibodies to amanitines as the basis for their radioimmunoassay. Experientia (Basel) 31, 1233 (1975) Flume, L., Laschi, R.: Lesioni ultrastrutturali prodotte nelle cellule parenchimali epatiche dalla falloidina e dalla ~-amanitina. Sperimentale 115, 288 (1965) Fiume, L., Marinozzi, V., Nardi, F.: The effects of amanitine poisoning on mouse kidney. Brit. J. exp. Path. 50, 270 (1969) Flume, L., Wieland, T.: Amanitines. Chemistry and action. FEBS Letters 8, 1 (1970) Holtzer, H., Holtzer, S.: In vitro uptake of fluorescein labelled plasma proteins. C. R. Lab. Carlsberg 31, 373 (1960) Kruse, H., McMaster, P. D.: The distribution and storage of blue antigenic azoproteins in the tissue of mice. J. exp. Med. 90, 425 (1949)
Increased Penetration of Amanitine into Hepatocytes
73
Litchfield, J. T., Jr., Wilcoxon, F. A.: A simplified method of evaluating dose-effect experiments. J. Pharmacol. exp. Ther. 96, 99 (1949) Marinozzi, V., Fiume, L.: Effects of oL-amanitine on mouse and rat liver cell nuclei. Exp. Cell Res. 67, 311 (1971) Mollenhauer, H.: Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111 (1964) Richardson, K. C., Jarett, L., Finke, E. M.." Embedding in epoxiresins for ultrathin sectioning in electron microscopy. Stain Technol. 35, 313 (1960) Schiller, A. A., Schayer, R. W., Hess, E. L.: Fluorescein-conjugated bovine albumin. Physical and biological properties. J. gen. Physiol. 36, 489 (1953) Unanue, E. R.: The regulatory role of macrophages in antigenic stimulation. Advanc. Immunol. 15, 95 (1972) Wieland, T.: Poisonous principles of mushrooms of the genus Amanita. Science 159, 946 (1968) Dr. L. Fiume Istituto di Patologia generale Via San Giacomo 14 1-40126 Bologna, Italy
