Mycopathologia 119: 115-125, 1992. 9 1992 KluwerAcademic Publishers. Printedin the Netherlands.
Light and electron microscopic studies on experimental nocardia-toxicosis in mice Kiyoshi Terao 1, Emiko Ito ~, Motoko Oarada', Misako Ohkusu t, Katsukiyo Yazawa t, Yuzuru Mikami 1 & Kazuei Igarashi2 1Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1, ln~',ana, Chuo-ku Chiba 260, Japan; 2Faculty of Pharmaceutical Science, Chiba University, 1-33. Yayoieho, Inage-ku Chiba, 263, Japan Received 29 August 1991; accepted in revised form 18 February 1992
Key words: Nocardia otitidiscaviarum, exotoxin poisoning, RER, autophagic vacuole, nocardiosis, fatty liver
Abstract
An exotoxin (HS-6) produced by Nocardia otitidiscaviarum isolated from certain lesions of cutaneous nocardiosis of a male 82-year-old patient induced severe injuries in the pancreas, liver, stomach, small intestine, heart, thymus and kidney of male ICR mice. Mice given Nocardia-free preparation of HS-6 at a dose of 1 mg/kg of body weight developed several autophagic vacuoles in the pancreas and liver within 20 min after the i.p. injection. Thereafter, the autophagic vacuoles increased in number and size with time. About 24 hr after the administration of HS-6, the liver showed marked accumulation of fat droplets in the cytoplasm of the hepatocytes. Although they contained abundant autophagic vacuoles in the regions of RER, there were no lipomatoses in the acinar cells of the pancreas, those of the chief cells and smooth muscle cells of the stomach, Paneth cells, goblet cells, smooth muscle cells of the small intestine, and plasma cells in the digestive tract. Biochemical examinations revealed that HS-6 had no significant effect on the protein synthesis of reticulocytes. Inoculation of the Nocardia into the mouse peritoneal cavities caused marked granulomatoses in the pancreas, liver and regional lymph nodes, but did not develop autophagic vacuoles in RER regions of these organs.
Introduction
Nocardia is a gram-positive, aerobic, filamentous bacterium of the order Actinomycetales. Since the first report of E. Nocard in 1888, it is now well recognized that Nocardia may be either a primary or an opportunistic pathogen [1]. Nocardiosis may occur in either normal individuals or in immuno-compromised patients [2-4]. Especially,
opportunistic infections due to Nocardia species are being recognized with increasing frequency in hospitals all over the world. The host-parasite relationship of actinomycetes involves a variety of mechanisms that include chemical agents produced by the microorganisms, cellular processes and defence mechanisms of the host. It would be of considerable interest if it were determined that certain toxins produced by actinomycetes actually
116 CH 3 i 0 CH3 CH3
H
C 30 I
'
l
u
~
C
H
I CH3 L'H3UE'Id3 CH3
viarurn strain IFM 0273 and purified as described previously [5]. 3
3
Fig. 1. Chemical structure of HS-6.
play some important role(s) in actinomycosis. In this regard, however, it has been stated that actinomycetes do not appear to synthesize specific exotoxins related to pathogenesis and disease production, although they produce a variety of antibiotics [1]. Recently, we found that Nocardia otitidiscaviarum strain IFM 0273 isolated from the cutaneous nocardiosis of a male 82-year-old patient in Japan produced a toxic substance against cultured cells [5]. The chemical structure of this toxin was found to belong to the 1 6 - m e m b e r e d macrocyclic group with a molecular formula of C43H68012 (Fig. 1). On the biological activities of this compound, however, no information has been available except that it has an antifungal activity [6]. In this study we determined the pathomorphological changes induced by this toxin in various organs of male ICR mice.
Experimental animals. Male ICR mice 4 weeks of age weighing 20 to 23g were obtained from Charles River Japan Inc (Tokyo). For examination of sequential morphological changes induced by HS-6, a total of 49 mice were divided into 4 groups. Group 1: Four mice receiving physiological saline served as control. Group 2: Three mice each were injected with HS-6 i.p. at concentrations of 0.5, 1, 1.5, 2 and 4 m g / k g of body weight. The mice were sacrificed by dislocation of the cervical spine 24 hr after the injections. Group 3: Three mice each were given HS6 at doses of 2, 4 and 7 mg/kg orally by intubation with stomach tubes. The mice were sacrificed in a similar manner to group 2. Group 4: A total of 21 mice were given HS-6 i.p. at a dose of 1 mg/kg, and then 3 mice each were killed at 20, 40 min and 1, 1.5, 4, 6, and 24 hr after the injections. For the thiobarbituric acid (TBA) reaction a total of 18 mice were divided into 3 groups. Group 1: Six mice given physiological saline i.p. served as control. Group 2: Six mice were given HS-6 at a dose of 0.735 mg/kg body weight i.p. Group 3: Six mice received HS-6 at a dose of l m g / k g i.p. Three mice from each group were sacrificed at 6 and at 24 hr, respectively, following the injections, and immediately after sacrifice, both the pancreas and liver were homogenized.
Material and methods
Cultures of Nocardia. N.otitidiscaviarum strain IFM 0273 was isolated from certain cutaneous lesions of a male 82-year-old patient and grown in Sabraud dextrose agar (Difco, Detroit, MI). Culture material from a slant was inoculated into a 10-ml Erlenmyer shake flask containing 5 ml of brain heart infusion broth (Difco) and eight 4mm glass beads intended to reduce aggregation of the culture cells. The culture was grown for 4 days on a rotary shaker (5.8-cm stroke) at 32 ~
Inoculation experiment. The cultured N. otitidiscaviarum strain IFM 0273 was diluted with 5% mucin solution (Wako Pure Chemical Inc., Osaka, Japan) to give a 108 colony forming unit (CFU/ml). One-half ml of the diluted culture broth was inoculated intraperitoneally into 3 male ddY mice (4 weeks old), and 8 days after the inoculation the mice were sacrificed by cervical dislocation. All mice were autopsied and several organs were examined light and electron microscopically.
Nocardia toxins. Nocardia toxin (HS-6) used in the present studies was isolated from N. otitidisca-
Morphological examination. The animals were autopsied and all internal organs were examined
117 macroscopically. These organs were then fixed in 10% neutral formalin and embedded in paraffin. The slides for light microscopy were stained with hematoxylin and eosin, or with periodic acid Schiff (PAS). For electron microscopy of the sequential changes of tissues induced by HS-6, pieces of the heart, pancreas, liver, stomach, small intestine, kidney and thymus were fixed in a cold 2% paraformaldehyde-glutaraldehyde solution buffered at pH 7.4 with 0.1 M cacodylate for 12 hr, and then postfixed in buffered 1% OsO4 at room temperature for 2 hr. For the mice inoculated with Nocardia, pieces of the pancreas and liver were prepared for electron microscopy. They were dehydrated in a series of graded concentrations of ethanol, embedded in Epon 812, and then cut with a diamond knife on a Porter II ultratome. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Hitachi H 700 H transmission electron microscope. Semi-thin sections of Epon-embedded specimens were stained with an alkaline solution of toluidine blue and used for a preliminary search for injured tissues by light microscopy.
Quantitative analysis of autophagic vacuoles induced by HS-6. For electron microscopy, 20 sections from each experimental group were chosen and photographed at random at • 4,800 magnification. The areas of autophagic vacuoles were measured with an automatic image processor analyzer (Nikon Luzex IID, Nikon, Tokyo). In order to clarify the pathogenesis of the autophagic vacuoles induced by HS-6, the effects of HS-6 on protein synthesis and on the production of lipid peroxidation in the pancreas and liver were determined.
Inhibition of protein synthesis. The inhibitory effects of the toxins on cell-free globin synthesis in a rabbit reticulocyte cell-free system were examined essentially as described previously [7]. The reaction mixture (0.05 ml) consisted of 15 mM Hepes-KOH buffer (pH 7.6), 15 ~M hemin, 100 mM potassium acetate, 0.5 mM spermidine, 2 mM dithiothreitol, 0.2 mM glucose-6-phos-
phate, 1.5 mM ATP, 0.3 mM GTP, 8 mM creatine phosphate, 7.5 #,g creatine kinase, 5 txg rat liver tRNA, 0.6 ~xgglobin mRNA, 15 ~1 nucleasetreated reticulocyte lysate, 0.1 txCi [~4C] leucine and 30 txM concentrations of nineteen other amino acids. After incubation at 37 ~ for 30 min, a sample of 0.04 ml of each reaction mixture was placed on a paper disk (24mm diameter) and the radioactivity of the hot-trichloroacetic-acidinsoluble material was counted in a liquid scintillation counter. Measurement of thiobarbituric acid (TBA) reactants: TBA reactants in tissue homogenates (pancreas and liver) were measured following the methods of Ohkawa et al. [8]. Ten percent (w/v) tissue homogenate was mixed with sodium dodecyl sulfate, acetate buffer (pH 3.5), and an aqueous solution of thiobarbituric acid. After heating at 95 ~ for 60 min, red pigment products were extracted with n-butanol/pyridine and estimated by absorbance at 532 nm with a Shimadzu spectrophotometer (Shimadzu Seisakusho, Co., Kyoto, Japan).
Results
Sequential morphological changes induced by HS-6 Mice given HS-6 at doses of 2 mg/kg or more of body weight died within 4 hr. For the morphological observations, the optimum concentration of the toxin given i.p. was l m g / k g of body weight. The pathomorphological changes induced by oral administration via a stomach tube were essentially the same as those induced i.p., although the severity of the latter was about 7 times greater than the former (A comparison of the occurrence of autophagic vacuoles induced by the toxin at the same dose level in i.p. and oral administration). Therefore, we would like to describe the results of mice given HS-6 i.p. The most prominent histopathological changes were seen in the pancreas and liver. Electron
118 microscopically, a small number of autophagic vacuoles appeared in the vicinity of the Golgi apparatus of pancreatic acinar cells at 20 min postinjection. By 90min, the pancreatic acinar cells showed considerable dilation of the stacks of R E R (Fig. 2a). The autophagic vacuoles appeared mainly in the apical region and increased in number and size rather suddenly. R E R located at the basal region of the cytoplasm was not affected. The pancreatic ductules and Langerhans' islet cells were less sensitive to HS-6 than the acinar cells. By 3 hr, normal stacks of R E R membranes were scarcely seen in the apical region. The membranes of R E R cisternae had partially disappeared and ribosomes were detached from the membranes. Many autophagic vacuoles were fused and had irregular contours. The electron density of zymogen granules was reduced markedly (Fig. 2b). By 6 hr, light microscopical observation revealed that the pancreatic acinar cells had marked vacuolation and that all zymogen granules had disappeared. Electron microscopically, the autophagic vacuoles were distributed not only at the apical but also at the basal regions of acinar cells. There were no discernible morphologic changes in the nucleoli of pancreatic acinar cells. Figure 3 shows the time-course changes of the total areas of autophagic vacuoles at various intervals after the i.p. administration of HS-6 at a dose of l m g / k g . The areas increased at a logarithmic rate up to 6 hr and then reached a plateau. Twenty-four hr later, the cytoplasm of most pancreatic acinar cells was occupied by bizarre vacuoles containing remnants of R E R and electron dense debris. The ultrastructure of the liver also showed autophagic vacuoles in the cytoplasm of the hepatocytes and Kupffer cells by 20 min after the injection of the toxin. The usual content of these autophagic vacuoles was rough surfaced endoplasmic reticulum, but occasionaly mitochondria were also observed. Macroscopically, the livers were swollen and yellowish in color within several hr after the administration of HS-6 at a dose of I mg/kg of body weight. Histopathologically,
numerous fine fat droplets were seen in the hepatocytes throughout the hepatic lobules. A marked accumulation of fat droplets was also observed in Kupffer cells. The fat droplets appeared in glycogen 10 areas about 6 hr after the administration of HS-6. By 24 hr, the cytoplasm of almost all hepatocytes as well as those of the endothelial lining cells of the sinusoids were filled with autophagic vacuoles (Fig. 4). No discernible morphogical changes were seen in the nucleoli of all hepatocytes even after 24hr. The endothelial lining cells were occasionally swollen and desquamated from the wall of the sinusoids. Therefore, microvilli of the hepatocytes facing the sinusoids were often flattened or had disappeared. Bile capillaries between the injured hepatocytes were extremely dilated. Then, by 24 hr, the fat droplets had increased considerably in number and size. There were autophagic vacuoles in the regions of R E R in various cells such as Paneth cells (Fig. 5a), smooth muscle cells (Fig. 5b) and plasma cells (Fig. 5c) in the small intestine as well as in chief cells (Fig. 5d) and smooth muscle cells of the stomach. Those in the smooth muscle cells of the stomach and small intestine appeared from the initial stage (within 90 min). The left and right ventricles of the heart were dilated. Histopathological examinations showed multiple single cell necroses in both ventricles and the septum (Fig. 6a). Electron microscopically, the density of these necrotizing cells increased and contracted markedly, although the fine structure of myofilaments was preserved relatively well. In contrast, mitochondria in these necrotizing muscle cells were swollen and the arrangement of cristae was irregular. Most cardiac muscle cells around the necrotizing cells were slightly swollen, but the cristae arrangement showed no discernible changes. No autophagic vacuoles were seen in the cardiac muscle cells (Fig. 6b). The kidneys were pale brown and swollen 24 hr after the administration of HS-6 at a dose of l mg/kg. Histopathologically, abundant PASpositive, eosinophilic fine granules were seen in the cytoplasm of the proximal convoluted tubules and in that of the distal tubules. Electron micro-
119
Fig. 2. Electron micrographs of the pancreas of mice treated with HS-6 at a dose of 1 mg/kg body weight i.p.a. Ninety min after the injection. Many autophagic vacuoles (A) are present in the apical portion of the pancreatic acinar cells. RER in basal regions are not changed. Note that the configuration of nucleoli in the acinar cell is well preserved. N: nucleus, RER: rough surfaced endoplasmic reticulum, x6,800; b. Three hr after the treatment. Numerous autophagic vacuoles (A) in the apical region. The cysternae of most RER are dilated. Electron-density of zymogen granules (Z) is reduced markedly, x ll,000.
120 (pm ~ 15C
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2
100
t3~ r~ 0
so
Light and electron microscopic studies on experimental nocardia-toxicosis in mice Kiyoshi Terao 1, Emiko Ito ~, Motoko Oarada', Misako Ohkusu t, Katsukiyo Yazawa t, Yuzuru Mikami 1 & Kazuei Igarashi2 1Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1, ln~',ana, Chuo-ku Chiba 260, Japan; 2Faculty of Pharmaceutical Science, Chiba University, 1-33. Yayoieho, Inage-ku Chiba, 263, Japan Received 29 August 1991; accepted in revised form 18 February 1992
Key words: Nocardia otitidiscaviarum, exotoxin poisoning, RER, autophagic vacuole, nocardiosis, fatty liver
Abstract
An exotoxin (HS-6) produced by Nocardia otitidiscaviarum isolated from certain lesions of cutaneous nocardiosis of a male 82-year-old patient induced severe injuries in the pancreas, liver, stomach, small intestine, heart, thymus and kidney of male ICR mice. Mice given Nocardia-free preparation of HS-6 at a dose of 1 mg/kg of body weight developed several autophagic vacuoles in the pancreas and liver within 20 min after the i.p. injection. Thereafter, the autophagic vacuoles increased in number and size with time. About 24 hr after the administration of HS-6, the liver showed marked accumulation of fat droplets in the cytoplasm of the hepatocytes. Although they contained abundant autophagic vacuoles in the regions of RER, there were no lipomatoses in the acinar cells of the pancreas, those of the chief cells and smooth muscle cells of the stomach, Paneth cells, goblet cells, smooth muscle cells of the small intestine, and plasma cells in the digestive tract. Biochemical examinations revealed that HS-6 had no significant effect on the protein synthesis of reticulocytes. Inoculation of the Nocardia into the mouse peritoneal cavities caused marked granulomatoses in the pancreas, liver and regional lymph nodes, but did not develop autophagic vacuoles in RER regions of these organs.
Introduction
Nocardia is a gram-positive, aerobic, filamentous bacterium of the order Actinomycetales. Since the first report of E. Nocard in 1888, it is now well recognized that Nocardia may be either a primary or an opportunistic pathogen [1]. Nocardiosis may occur in either normal individuals or in immuno-compromised patients [2-4]. Especially,
opportunistic infections due to Nocardia species are being recognized with increasing frequency in hospitals all over the world. The host-parasite relationship of actinomycetes involves a variety of mechanisms that include chemical agents produced by the microorganisms, cellular processes and defence mechanisms of the host. It would be of considerable interest if it were determined that certain toxins produced by actinomycetes actually
116 CH 3 i 0 CH3 CH3
H
C 30 I
'
l
u
~
C
H
I CH3 L'H3UE'Id3 CH3
viarurn strain IFM 0273 and purified as described previously [5]. 3
3
Fig. 1. Chemical structure of HS-6.
play some important role(s) in actinomycosis. In this regard, however, it has been stated that actinomycetes do not appear to synthesize specific exotoxins related to pathogenesis and disease production, although they produce a variety of antibiotics [1]. Recently, we found that Nocardia otitidiscaviarum strain IFM 0273 isolated from the cutaneous nocardiosis of a male 82-year-old patient in Japan produced a toxic substance against cultured cells [5]. The chemical structure of this toxin was found to belong to the 1 6 - m e m b e r e d macrocyclic group with a molecular formula of C43H68012 (Fig. 1). On the biological activities of this compound, however, no information has been available except that it has an antifungal activity [6]. In this study we determined the pathomorphological changes induced by this toxin in various organs of male ICR mice.
Experimental animals. Male ICR mice 4 weeks of age weighing 20 to 23g were obtained from Charles River Japan Inc (Tokyo). For examination of sequential morphological changes induced by HS-6, a total of 49 mice were divided into 4 groups. Group 1: Four mice receiving physiological saline served as control. Group 2: Three mice each were injected with HS-6 i.p. at concentrations of 0.5, 1, 1.5, 2 and 4 m g / k g of body weight. The mice were sacrificed by dislocation of the cervical spine 24 hr after the injections. Group 3: Three mice each were given HS6 at doses of 2, 4 and 7 mg/kg orally by intubation with stomach tubes. The mice were sacrificed in a similar manner to group 2. Group 4: A total of 21 mice were given HS-6 i.p. at a dose of 1 mg/kg, and then 3 mice each were killed at 20, 40 min and 1, 1.5, 4, 6, and 24 hr after the injections. For the thiobarbituric acid (TBA) reaction a total of 18 mice were divided into 3 groups. Group 1: Six mice given physiological saline i.p. served as control. Group 2: Six mice were given HS-6 at a dose of 0.735 mg/kg body weight i.p. Group 3: Six mice received HS-6 at a dose of l m g / k g i.p. Three mice from each group were sacrificed at 6 and at 24 hr, respectively, following the injections, and immediately after sacrifice, both the pancreas and liver were homogenized.
Material and methods
Cultures of Nocardia. N.otitidiscaviarum strain IFM 0273 was isolated from certain cutaneous lesions of a male 82-year-old patient and grown in Sabraud dextrose agar (Difco, Detroit, MI). Culture material from a slant was inoculated into a 10-ml Erlenmyer shake flask containing 5 ml of brain heart infusion broth (Difco) and eight 4mm glass beads intended to reduce aggregation of the culture cells. The culture was grown for 4 days on a rotary shaker (5.8-cm stroke) at 32 ~
Inoculation experiment. The cultured N. otitidiscaviarum strain IFM 0273 was diluted with 5% mucin solution (Wako Pure Chemical Inc., Osaka, Japan) to give a 108 colony forming unit (CFU/ml). One-half ml of the diluted culture broth was inoculated intraperitoneally into 3 male ddY mice (4 weeks old), and 8 days after the inoculation the mice were sacrificed by cervical dislocation. All mice were autopsied and several organs were examined light and electron microscopically.
Nocardia toxins. Nocardia toxin (HS-6) used in the present studies was isolated from N. otitidisca-
Morphological examination. The animals were autopsied and all internal organs were examined
117 macroscopically. These organs were then fixed in 10% neutral formalin and embedded in paraffin. The slides for light microscopy were stained with hematoxylin and eosin, or with periodic acid Schiff (PAS). For electron microscopy of the sequential changes of tissues induced by HS-6, pieces of the heart, pancreas, liver, stomach, small intestine, kidney and thymus were fixed in a cold 2% paraformaldehyde-glutaraldehyde solution buffered at pH 7.4 with 0.1 M cacodylate for 12 hr, and then postfixed in buffered 1% OsO4 at room temperature for 2 hr. For the mice inoculated with Nocardia, pieces of the pancreas and liver were prepared for electron microscopy. They were dehydrated in a series of graded concentrations of ethanol, embedded in Epon 812, and then cut with a diamond knife on a Porter II ultratome. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Hitachi H 700 H transmission electron microscope. Semi-thin sections of Epon-embedded specimens were stained with an alkaline solution of toluidine blue and used for a preliminary search for injured tissues by light microscopy.
Quantitative analysis of autophagic vacuoles induced by HS-6. For electron microscopy, 20 sections from each experimental group were chosen and photographed at random at • 4,800 magnification. The areas of autophagic vacuoles were measured with an automatic image processor analyzer (Nikon Luzex IID, Nikon, Tokyo). In order to clarify the pathogenesis of the autophagic vacuoles induced by HS-6, the effects of HS-6 on protein synthesis and on the production of lipid peroxidation in the pancreas and liver were determined.
Inhibition of protein synthesis. The inhibitory effects of the toxins on cell-free globin synthesis in a rabbit reticulocyte cell-free system were examined essentially as described previously [7]. The reaction mixture (0.05 ml) consisted of 15 mM Hepes-KOH buffer (pH 7.6), 15 ~M hemin, 100 mM potassium acetate, 0.5 mM spermidine, 2 mM dithiothreitol, 0.2 mM glucose-6-phos-
phate, 1.5 mM ATP, 0.3 mM GTP, 8 mM creatine phosphate, 7.5 #,g creatine kinase, 5 txg rat liver tRNA, 0.6 ~xgglobin mRNA, 15 ~1 nucleasetreated reticulocyte lysate, 0.1 txCi [~4C] leucine and 30 txM concentrations of nineteen other amino acids. After incubation at 37 ~ for 30 min, a sample of 0.04 ml of each reaction mixture was placed on a paper disk (24mm diameter) and the radioactivity of the hot-trichloroacetic-acidinsoluble material was counted in a liquid scintillation counter. Measurement of thiobarbituric acid (TBA) reactants: TBA reactants in tissue homogenates (pancreas and liver) were measured following the methods of Ohkawa et al. [8]. Ten percent (w/v) tissue homogenate was mixed with sodium dodecyl sulfate, acetate buffer (pH 3.5), and an aqueous solution of thiobarbituric acid. After heating at 95 ~ for 60 min, red pigment products were extracted with n-butanol/pyridine and estimated by absorbance at 532 nm with a Shimadzu spectrophotometer (Shimadzu Seisakusho, Co., Kyoto, Japan).
Results
Sequential morphological changes induced by HS-6 Mice given HS-6 at doses of 2 mg/kg or more of body weight died within 4 hr. For the morphological observations, the optimum concentration of the toxin given i.p. was l m g / k g of body weight. The pathomorphological changes induced by oral administration via a stomach tube were essentially the same as those induced i.p., although the severity of the latter was about 7 times greater than the former (A comparison of the occurrence of autophagic vacuoles induced by the toxin at the same dose level in i.p. and oral administration). Therefore, we would like to describe the results of mice given HS-6 i.p. The most prominent histopathological changes were seen in the pancreas and liver. Electron
118 microscopically, a small number of autophagic vacuoles appeared in the vicinity of the Golgi apparatus of pancreatic acinar cells at 20 min postinjection. By 90min, the pancreatic acinar cells showed considerable dilation of the stacks of R E R (Fig. 2a). The autophagic vacuoles appeared mainly in the apical region and increased in number and size rather suddenly. R E R located at the basal region of the cytoplasm was not affected. The pancreatic ductules and Langerhans' islet cells were less sensitive to HS-6 than the acinar cells. By 3 hr, normal stacks of R E R membranes were scarcely seen in the apical region. The membranes of R E R cisternae had partially disappeared and ribosomes were detached from the membranes. Many autophagic vacuoles were fused and had irregular contours. The electron density of zymogen granules was reduced markedly (Fig. 2b). By 6 hr, light microscopical observation revealed that the pancreatic acinar cells had marked vacuolation and that all zymogen granules had disappeared. Electron microscopically, the autophagic vacuoles were distributed not only at the apical but also at the basal regions of acinar cells. There were no discernible morphologic changes in the nucleoli of pancreatic acinar cells. Figure 3 shows the time-course changes of the total areas of autophagic vacuoles at various intervals after the i.p. administration of HS-6 at a dose of l m g / k g . The areas increased at a logarithmic rate up to 6 hr and then reached a plateau. Twenty-four hr later, the cytoplasm of most pancreatic acinar cells was occupied by bizarre vacuoles containing remnants of R E R and electron dense debris. The ultrastructure of the liver also showed autophagic vacuoles in the cytoplasm of the hepatocytes and Kupffer cells by 20 min after the injection of the toxin. The usual content of these autophagic vacuoles was rough surfaced endoplasmic reticulum, but occasionaly mitochondria were also observed. Macroscopically, the livers were swollen and yellowish in color within several hr after the administration of HS-6 at a dose of I mg/kg of body weight. Histopathologically,
numerous fine fat droplets were seen in the hepatocytes throughout the hepatic lobules. A marked accumulation of fat droplets was also observed in Kupffer cells. The fat droplets appeared in glycogen 10 areas about 6 hr after the administration of HS-6. By 24 hr, the cytoplasm of almost all hepatocytes as well as those of the endothelial lining cells of the sinusoids were filled with autophagic vacuoles (Fig. 4). No discernible morphogical changes were seen in the nucleoli of all hepatocytes even after 24hr. The endothelial lining cells were occasionally swollen and desquamated from the wall of the sinusoids. Therefore, microvilli of the hepatocytes facing the sinusoids were often flattened or had disappeared. Bile capillaries between the injured hepatocytes were extremely dilated. Then, by 24 hr, the fat droplets had increased considerably in number and size. There were autophagic vacuoles in the regions of R E R in various cells such as Paneth cells (Fig. 5a), smooth muscle cells (Fig. 5b) and plasma cells (Fig. 5c) in the small intestine as well as in chief cells (Fig. 5d) and smooth muscle cells of the stomach. Those in the smooth muscle cells of the stomach and small intestine appeared from the initial stage (within 90 min). The left and right ventricles of the heart were dilated. Histopathological examinations showed multiple single cell necroses in both ventricles and the septum (Fig. 6a). Electron microscopically, the density of these necrotizing cells increased and contracted markedly, although the fine structure of myofilaments was preserved relatively well. In contrast, mitochondria in these necrotizing muscle cells were swollen and the arrangement of cristae was irregular. Most cardiac muscle cells around the necrotizing cells were slightly swollen, but the cristae arrangement showed no discernible changes. No autophagic vacuoles were seen in the cardiac muscle cells (Fig. 6b). The kidneys were pale brown and swollen 24 hr after the administration of HS-6 at a dose of l mg/kg. Histopathologically, abundant PASpositive, eosinophilic fine granules were seen in the cytoplasm of the proximal convoluted tubules and in that of the distal tubules. Electron micro-
119
Fig. 2. Electron micrographs of the pancreas of mice treated with HS-6 at a dose of 1 mg/kg body weight i.p.a. Ninety min after the injection. Many autophagic vacuoles (A) are present in the apical portion of the pancreatic acinar cells. RER in basal regions are not changed. Note that the configuration of nucleoli in the acinar cell is well preserved. N: nucleus, RER: rough surfaced endoplasmic reticulum, x6,800; b. Three hr after the treatment. Numerous autophagic vacuoles (A) in the apical region. The cysternae of most RER are dilated. Electron-density of zymogen granules (Z) is reduced markedly, x ll,000.
120 (pm ~ 15C
o o 0~ >
2
100
t3~ r~ 0
so
