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Effects of ethylene glycol on the ultrastructure of hepatocytes
Exp Toxic Patho11995; 47: 359-365 Gustav Fischer Verlag Jena
Department of Internal Medicine, Hospital of the Ministry of Internal Affairs, L6dt, Poland Department of Histology and Embryology, Military Medical Academy, L6dt, Poland
Effects of ethylene glycol on the ultrastructure of hepatocytes H. GIERMAZIAK,
s. ORKISZ
With 7 figures Received: May 20,1994; Revised: December 20,1994; Accepted: January 19, 1995 Address for correspondence: Dr. hab. med. STANISLAW ORKISZ, Department of Histology and Embryology, Medical Military Academy, Plac Hallera 1,90-647 Loot, Poland. Key words: Ethylene glycol; Hepatocytes, ultrastructure; Liver, ethylene glycol.
List of abbreviations SER RER EG L M Gly MVB N Chr PG NB
Smooth Endoplasmic Reticulum Rough Endoplasmic Reticulum Ethylene Glycol Lipid droplets Mitochondria Glycogen Multivesicular Bodies Nucleus Chromatin Perichromatin Granules Nuclear Bodies
Summary The ultrastructure of rat hepatocytes after acute experimental ethylene glycol poisoning was examined. On the 1st, 5th and 14th days after poisoning the material from the centrolobular zone (zone III) was collected. Proliferation and enlargement of SER at the early period of poisoning and evidence of mitochondrial damage both at the early and late time after ethylene glycol intoxication were found. In the liver extensive capillary deposits surrounded by membranes were seen, filled with flocculent material of middle electron density. The results show destruction of the cytoplasmic organelles' especially mitochondria, on the 1st and 5th days after ethylene glycol intoxication, and symptoms of damage removing together with regeneration on the 15th day of the experiment.
Introduction Ethylene glycol (EG) is a strong poison producing one ofthe most severe metabolic acidosis in the body (5, 7,9, 15,25, 27). Acute EG poisoning is responsible for the de-
velopment of injuries rather well known and described in literature: lesions in the central and peripheral nervous systems (5), acute renal failure (7, 23), ischaemic and degenerative lesions in the respiratory and circulatory systems (2, 25). Ethylene glycol becomes metabolized mainly in the liver and, as in the case of most hepatotoxic agents, its toxic properties are due to the action of its metabolites. Its oxidative transformations involve oxidation of glycol aldehyde with participation of alcohol dehydrogenase (3, 9, 15, 25). At the next stage glycol aldehyde is oxidized to glycolic acid and finally to glyoxalate with participation of lactate dehydrogenase or glycolic acid oxidase (22). In the studies which have hitherto been conducted, markedly less consideration was given to ultrastructural alterations of parenchymal cells of the liver, although numerous biochemical investigations have disclosed an increased activity of microsomal enzymes (13, 15, 29) and breaking up of oxidative phosphorylation processes in hepatocytes (15, 18). Thus, the aim of the present studies has been to assess subcellular alterations of hepatocytes in the course of acute intoxication with ethylene glycol.
Material and methods The experiments were performed on three-month-old male Wistar rats of 200-250 g body weight bred in compliance with the recommendations of the ICLA (11). The animals were divided into four experimental groups of five rats each. Group 1 included control animals without EO intoxication examined synchronously with rats after EO poisoning. In group 2 rat hepatocytes were assessed after 24 h following acute EO intoxication, group 3 included the material collected from rats 5 days after intoxication, and group 4 comprised animals whose livers were assessed after 14 days following EO poisoning. Ethylene glycol was administrated to the animals through a stomach tube in a single dose of Exp Toxic Patho147 (1995) 5
359
5.0 mg/kg body (i. e. a dose higher by 15 % than DLso) calculated with the Lichtfield-Wilcoxon method (17). 0.6-1 mm3 liver sections were collected for examinations from 4 different localisations of the left lateral lobe immediately after killing the animal. From each experimental animal 4 samples of liver were examined. The samples were fixed in 1.6 % glutaraldehyde in phosphate buffer at pH 7.4 at 4 0 C for 2 hours and were post-fixed with 1 % osmium tetroxide. After dehydration the samples were embedded in Epon. Semithin sections stained with toluidine blue were examined using light microscopy. Five tissue specimens were taken from each sample. Ultrathin sections obtained with a diamond knife of an LKB IV Ultratom were subjected to routine staining with lead citrate and uranyl acetate, and examined using a Philips EM 300 electron microscope.
Results
Control group The ultrastructure of the centrolobular zone in hepatocytes of the control rats did not differ from the norm. In the nuclei nucleoli could be seen with small clumps of heterochromatin around them and under the nuclear envelope. In the cytoplasm, mitochondria were mostly oval and possessed short narrow cristae, a narrow external compartment and a matrix of medium electron density. The smooth endoplasmic reticulum (SER) showed small agglomerations of tiny vesicles, among which few glycogen rosettes were noted. Small agglomerations of rough
Fig. 1. Intact hepatocyte. Characteristic nucleic structure (Chr - chromatin). The structure of mitochondria (M), RER and SER is normal. Magn. x 7500. Fig. 2. Nuclear body (NB) in hepatocyte one day after EG intoxication. In higher magnification (top right comer) NB of a fibrillar capsule (arrows) and multigranular core inside is composed. Chr - chromation. Magn. x 13500. 360
Exp Toxic Pathol 47 (1995) 5
Fig. 3. Hepatocyte nucleus one day after intoxication. Note numerous perichromatin granules (PO - arrows) at periphery of condensed chromatine (Chr). Magn. x 17500 Fig. 4. The cytoplasm of hepatocyte one day after EO intoxication, with predominated irregular shape vacuoles of SER. Gly - glycogen, L -lipid droplet, M - mitochondria. Magn. x 44000. endoplasmic reticulum (RER) with parallel membrane system were found mainly near the nucleus (fig. 1). At the biliary pole of hepatocytes single lysosomes and peroxisomes were found. In the peripherolobular hepatocytes there were less mitochondria, but of greater size. In this zone, also SER vesicles were in smaller quantities. The Oolgi apparatus had more cisterns and vesicles as compared to the centrolobular zone.
Second group - 24 hours after EG administration In hepatic centrolobular nuclei the presence of socalled nuclear bodies could be disclosed very often. Most
frequently they were complex nuclear bodies made up of a delicate capsule and a granular core (fig. 2). Moreover, consideration was given to numerous perichromatin granules (PO) on the borders of condensed chromatin which most often mingled into agglomerations (fig. 3). In the cytoplasm of rat hepatocytes of this experimental group, vesicles of SER usually merging into structures of irregular shape predominated (fig. 4). In the fields of SER there were also visible numerous glycogen rosettes. Mitochondria revealed only rarefied structure of the mitochondrial matrix. Near the biliary pole of hepatocytes single irregularly shaped lysosomes and peroxisomes could be distinguished. In hepatocytes of the peripheral zone the above mentioned changes were much less visible. Exp Toxic Pathol 47 (1995) 5
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Fig. 5. Five days after EG administration. Swollen mitochondria (M) with empty spaces in matrix. Disordered membranes RER and various forms of widened vesicles SER are visible. Magn. x 62000. Fig. 6. Liver sinusoids 5 days after intoxication filled with large deposits, surrounded by membranes (asterisk). Magn. x 28000.
Third group - 5 days after EO administration On the 5th day after EG intoxication, the most conspicuous ultrastructural alterations could be observed in centrolobular hepatocytes and they concerned mainly mitochondria and SER. Mitochondria were markedly swollen and the swelling was associated with evident lacy contours of the external mitochondrial membranes. In the matrix, apart from numerous areas of reduced density, empty spaces were observed (fig. 5). Profiles of SER, although less numerous as compared with the previous experimental group, were distinctly widened. Also cisterns of the Golgi apparatus were very often extened and widened. Consideration was also given to numerous 362
Exp Toxic Pathol 47 (1995) 5
peroxisomes with distinct crystalline tubular cores. They were localized in the proximity of mitochondrial agglomerations. Very characteristic subcellular alterations were also observed within capillary vessels. Endothelial cells contained vesicles of various types and sizes. The vascular lumen was often filled with large structures, surrounded by membranes, containing flocculent material with medium electron density (fig. 6).
Fourth group - 14 days after EO administration The rarefied hepatocytic cytoplasm revealed very numerous but tiny vesicles of SER and fields of glycogen rosettes. However, the most characteristic lesion of hepa-
Fig. 7. Fourteen days after EG intoxication. Note very remarkable lesions of mitochondria (M): swelling of internal compartment with fragmentation of cristae. Vacuoles and myelin figures within matrix. SER Smooth Endoplasmic Reticulum, MVB - Multivesicular Body. Magn. x 40800. tocytic substructure of the centrolobular zone was mito- occurring in acute EG poisoning must be related to the chondriosis. The whole cytoplasm of hepatocytes was action of metabolites and free radicals. In the nuclei of most often filled with mitochondria, which were charac- hepatocytes these changes consisted in the appearance of terized by various sizes and polymorphous signs of da- numerous so called nuclear bodies. Although biogenesis mage. These injuries were mainly associated with swell- and function ofNB are not as yet fully known (4,6,21), ing of the internal compartment, and this was usually ac- their appearance, especially complex nuclear bodies discompanied by fragmentation of cristae. Within the matrix closed in the present studies, must be related to disturbed there were often distinguishable vacuoles and myelin fi- synthesis of RNA and of protein. In the earlier biochegures (fig. 7). Apart from numerous peroxisomes en- mical investigations ZIMMERMAN (29) showed that EG countered among mitochondria, the cytoplasm revealed . metabolites cause - among others - inhibition of the synformations related to focal degradation of structures and thesis of RNA and of protein. LAMPE et al. (16) and to intracellular digestion: autophagolysosomes, multi- JOANNIDES et al. (13) are also of the opinion that active, vesicular bodies as well as myelin figures and dense metabolites originated under conditions of cytochromes P-450 - induced catalysis may act on nucleic acids and bodies. alter their chemical and biological properties. This suggestion has been confirmed by our own morphological Discussion observations. They have disclosed not only the presence of complex forms of nuclear bodies in the course of acute Ethylene glycol is a basic component of commonly used EG intoxication but also accumulation of perichromatin nonfreezing fluids, readily absorbed from the alimentary granules (PG) in the nuclei. The presence of agglomeratract. Damages have been described, caused by GE, in tions of PG in the nuclei, as is evident from earlier stuheart, liver, kidneys (2, 5, 9, 15, 23, 25, 29). The path- dies, among others OfPUVION AND MOYNE (21), has been ways of biochemical EG transformations initiated by its related to the inhibition of transport of pre m-RNA to the oxydation to glycolaldehyde and then - through glycolic cytoplasm. According to the results of recent biochemiacid - to glyoxylate, are quite well know (23, 27). Beside cal and immunocytochemical studies of various authors, its toxic effects in cells, connected with metabolic acido- the biogenesis of NB must be related to disturbed transsis, in recent years attention has been paid to free oxygen scription and maturation both of pre m-RNA and of pre rradicals participating in the development of injuries of RNA (4, 6). These observations together with the results cellular substructure (20, 28). Since oxidative transfor- of our own morphological studies of the nuclei in acute mations of EG take place in the liver, an ultrastructural EG intoxication appear to be proof that ethylene glycol assessment of hepatocytes in the course of acute experi- exerts an effect on the metabolism of nucleic acids in the mental EG intoxications seemed to be worthy of interest. nucleus. The most characteristic alterations in the ultraOur own studies have disclosed characteristic ultrastruc- structure of the cytoplasm of hepatocytes of the centrolotural alterations both in the nuclei and in the cytoplasm of bular zone were found in SER and in mitochondria. Marhepatocytes. Many of these submicroscopic changes ked proliferation and merging of SER vesicles observed Exp Toxic Pathol47 (1995) 5
363
in our own studies must be related to the induction of cytochromes of the P-450 family constituting an integral part of the membranes of SER where they form a multicomponent enzymatic - lipid complex defined by the name of microsomal monooxygenases. This was proved by biochemical methods by many authors (16, 20, 28). IMAZU et al. (12) disclosed a 17 % rise in the cytochrome P-450 content in homogenates of hepatic cells in the course of EG poisoning. A morphological manifestation of the increased amount and activity of cytochrome P450 was proliferation of SER with their coupling into irregularly shaped figures apparent especially on the 1st and 5th days after intoxication. Ultrastructural injuries of mitochondria, subtle on the 1st day of EG poisoning and evident on the 5th day, were most strongly expressed after 14 days of the experiment with well distinguishable destruction of the membranes. Weare of the opinion that this intensification of the destructive action of EG observed in the course of our experiment should be related to progressive uncoupling of oxidative phosphorylation disclosed by various authors (15, 18). It must be presumed that the features of lesioned mitochondria, most strongly expressed in the 3rd zone, are also connected with vascularization conditions within hepatic lobules (14). And finally we must consider the problem of the presence of peroxisomes, particularly abundant in the proximity of mitochondria. Although, no direct binding of peroxisomes to other organelles has been show in the studies which have been conducted so far, but many authors had regard to spatial relationships of this organelle with SER (1, 19) or to the appearance of peroxisomes in the immediate proximity of lipid droplets (8). These authors ascribe this fact to their participation in the fat metabolism. In turn, BAUMGART (1) suggests a permanent exchange of intermediate products in the biosynthesis of composite lipids. Our own studies disclosing the presence of numerous peroxisomes within agglomerations of mitochondria are approximate to the observations reported by GORGAS (10), although the functional significance of this relation is as yet obscure. The presence of peroxisomes with crystalline tubular cores may be related to the activity of free oxygen radicals released from hydrogen peroxide and from non-hemie iron demonstrated by KUKIELKA and CEDERBAUM (15), in the course of EG intoxication. The donor of free radicals is, and this was confirmed by documentary evidence, one of the oxidizing enzymes of peroxisomes (19, 26). Their large number observed especially on the 5th day after EG intoxication confirms the results of the biochemical studies of the above mentioned authors. However, the development and widening of Golgi apparatus cisterns, conspicuous particularly on the 5th day of acute EG poisoning, can be related to the activity of the enzyme TPP-ase because its substrate thiamine pyrophosphate (TPP) participates, according to the report of SCHAGGER and JAGOW (26) in the metabolism of the intermediate metabolite glyoxalate. And, finally, it must be said that the role of crystalloids, surrounded by membranes, filling, the hepa364
Exp Toxic Pathol47 (1995) 5
tic vessels in the development of ultrastructural lesions in hepatocytes remains an open question. The intracapillary structure we have described has identical ultrastructure to that of calcium oxalate in the renal vessels described earlier, having essential significance in the development of acute renal failure (7). The above observations seem to indicate that deposition of calcium oxalate in the course of acute EG poisoning concerns many organs and, therefore, it has more universal character.
References 1.
BAUMGART E, STEGMEIER KM, SCHMIDT FR, et al.: Proliferation of peroxisomes in pericentral hepatocytes of rat liver administration of a new hypocholesterolemic agent. Lab Invest 1987,56: 554-564. 2. BIELNIK K, SZRAM S: Ultrastructural signs of myocardial due to acute experimental intoxication with ethylene glycol. Patol Pol 1992; 43: 157-159. 3. BLAIR AH, VALLEE BL: Some catalytic properties of human liver alcohol dehydrogenase. Biochemistry 1966;5: 2026-2034. 4. BRASCH K, OCHS R: Nuclear bodies (NBs): a newly "rediscovered" organelle. Exp Cell Res 1992; 202: 211-223. 5. BURKHARD KK, KULIG KW: The other alcohols. Methanol, ethylene glycol and isopropanol. Emerg Med Clin North Ann 1990; 8: 913-928. 6. CARMO-FoNESCA M, PEPPERKOK E, et al.: Transcription dependent localization of the Ul, U2, U4IU6 and US sn RNPs in Coiled Bodies. J Cell BioI 1992; 117: 1-14. 7. CIECIURA L, KIDAW AZ, ORKISZ S, et al.: Ultrastructural appearances of nephron damage in acute poisoning with ethylene glycol. Proc EDTA 1983; 20: 236-240. 8. FAHIMI HD, SIES H: Morphogenesis of peroxisomes in lipid - synthesizing epithelia. In: Peroxisome in biology and medicine. Springer Berlin - Heidelberg 1987,3. 9. GESSNER PK, PARKE DV, EILIAMS RT: Studies in detoxycation. The metabolism of 14/C labelled ethylene glycol. Biochem J 1961; 79: 482-489. 10. GORGAS K: Peroxisomes in sebaceous glands. V. Complex peroxisomes in the mouse preputial gland: serial sectioning and three-dimensional reconstruction studies. Anat Embryo11984; 169: 261-270. 11 . ICLA BULLETIN: Recommendation for the specification of the animals, the husbandry and techniques used in animal experimentation. 1978; 42: 4-6. 12. IMAZU K, FUJIHIRO K, NINONE N, et al.: Effects of ethylene glycol on drug metabolizing enzymes in rat liver. Sanyo Ika Daigaku Zasshi 1991; 13: 13-18. 13. JOANNIDES C, LUM P, PARK D: Cytochrome P-448 and the activation of toxins, chemicals and carcinogenesis. Xenobiotica 1983; 14: 119-137. 14. KAMINSKI M: The cells, tissues and organs - their structure and function. In: Some problems of hepatology. Sil Med Acad Katowice 1992 pp. 7-24. 15. KUKIELKA E, CEDERBAUM AJ: Oxidation of ethylene glycol to formaldehyde by rat liver microsomes. Role of cytochrome P-450 and reactive oxygen species. Drug Metab Dispos 1991; 19: 1108-1115.
16. LAMPE J, BUTSCHAM G, SCHELER W: Cytochrome P450 dependent biotransformation of drugs and other xenobiotics. In: RUCKPAUL KH, REIN (Eds.): Cytochrome P-450. Academie Verlag Berlin 1984 pp. 277-366. 17. LICHTFIELD JT, WILCOXON J: A simplified method of evaluating dose effect experiments. J Phannacol Exp Therapy 1949; 96: 99-103. 18. LIEBER CS: Biochemical mechanism of alcohol- induced hepatic injury. Alcohol and Metab 1991; 91: 283-290. 19. LITWIN JA: Peroxisomes in animal cells. Progr BioI Cell 1989; 1: 19-60. 20. LUTZ W, SULKOWSKI W: Bioactivation xenobiotics with P-448 cytochrome and their participation in cancerogenesis. Progr Exper Med Hyg 1989; 43: 5-6. 21. PuVION E, MOYNE G: In situ localization of RNA structures. In: BUSCH H (Ed.): The cell nucleus vol VIII, Acad Press New York 1981 pp. 59-115. 22. RICHARDSON KE, TOLBERT NE: Oxidation of glyoxalic acid to oxalic acid by glycolic acid oxidase. J BioI Chern 1961; 236: 1280-1284. 23. ROBINSON M, POND CL, LAURIE RD, et al.: Subacute
24. 25.
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subchronic toxicity of ethylene glycol administrated in drinking water to Spraque - Dawley rats. Drug Chern Toxical 1990; 13: 43-70. RODRICKS JV, TURNBULL DT: Interspecies differences in peroxisomes and peroxisome proliferation. Toxicol Industr Health 1987; 3: 197-212. SALDINO R, SHANNON M: Accidental and intentional poisonings with ethylene glycol in infancy: diagnostic clues and management. Pediatr Emerg Cave 1991; 7: 93-96. SCHAAGER M, JAGOW G: Blue negative electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 1991; 199: 223-226. WIENER HJ, RICHARDSON KR: The metabolism and diethylenglycol toxicity of ethylene glycol. Res Commun Substance Abuse 1988; 9: 77-87. ZANG CS, Tu YY, Koop DR, et al.: Metabolism of nitrosamines by purified rabbit liver cytochrome P-450 isozymes. Cancer Res 1985; 45: 1140-1145. ZIMMERMAN H: Hepatotoxicity. In: CROFTS (ed.): The adverse effects of drugs and other chemicals on the liver. Appelton - Century - New York 1978.
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Department of Internal Medicine, Hospital of the Ministry of Internal Affairs, L6dt, Poland Department of Histology and Embryology, Military Medical Academy, L6dt, Poland
Effects of ethylene glycol on the ultrastructure of hepatocytes H. GIERMAZIAK,
s. ORKISZ
With 7 figures Received: May 20,1994; Revised: December 20,1994; Accepted: January 19, 1995 Address for correspondence: Dr. hab. med. STANISLAW ORKISZ, Department of Histology and Embryology, Medical Military Academy, Plac Hallera 1,90-647 Loot, Poland. Key words: Ethylene glycol; Hepatocytes, ultrastructure; Liver, ethylene glycol.
List of abbreviations SER RER EG L M Gly MVB N Chr PG NB
Smooth Endoplasmic Reticulum Rough Endoplasmic Reticulum Ethylene Glycol Lipid droplets Mitochondria Glycogen Multivesicular Bodies Nucleus Chromatin Perichromatin Granules Nuclear Bodies
Summary The ultrastructure of rat hepatocytes after acute experimental ethylene glycol poisoning was examined. On the 1st, 5th and 14th days after poisoning the material from the centrolobular zone (zone III) was collected. Proliferation and enlargement of SER at the early period of poisoning and evidence of mitochondrial damage both at the early and late time after ethylene glycol intoxication were found. In the liver extensive capillary deposits surrounded by membranes were seen, filled with flocculent material of middle electron density. The results show destruction of the cytoplasmic organelles' especially mitochondria, on the 1st and 5th days after ethylene glycol intoxication, and symptoms of damage removing together with regeneration on the 15th day of the experiment.
Introduction Ethylene glycol (EG) is a strong poison producing one ofthe most severe metabolic acidosis in the body (5, 7,9, 15,25, 27). Acute EG poisoning is responsible for the de-
velopment of injuries rather well known and described in literature: lesions in the central and peripheral nervous systems (5), acute renal failure (7, 23), ischaemic and degenerative lesions in the respiratory and circulatory systems (2, 25). Ethylene glycol becomes metabolized mainly in the liver and, as in the case of most hepatotoxic agents, its toxic properties are due to the action of its metabolites. Its oxidative transformations involve oxidation of glycol aldehyde with participation of alcohol dehydrogenase (3, 9, 15, 25). At the next stage glycol aldehyde is oxidized to glycolic acid and finally to glyoxalate with participation of lactate dehydrogenase or glycolic acid oxidase (22). In the studies which have hitherto been conducted, markedly less consideration was given to ultrastructural alterations of parenchymal cells of the liver, although numerous biochemical investigations have disclosed an increased activity of microsomal enzymes (13, 15, 29) and breaking up of oxidative phosphorylation processes in hepatocytes (15, 18). Thus, the aim of the present studies has been to assess subcellular alterations of hepatocytes in the course of acute intoxication with ethylene glycol.
Material and methods The experiments were performed on three-month-old male Wistar rats of 200-250 g body weight bred in compliance with the recommendations of the ICLA (11). The animals were divided into four experimental groups of five rats each. Group 1 included control animals without EO intoxication examined synchronously with rats after EO poisoning. In group 2 rat hepatocytes were assessed after 24 h following acute EO intoxication, group 3 included the material collected from rats 5 days after intoxication, and group 4 comprised animals whose livers were assessed after 14 days following EO poisoning. Ethylene glycol was administrated to the animals through a stomach tube in a single dose of Exp Toxic Patho147 (1995) 5
359
5.0 mg/kg body (i. e. a dose higher by 15 % than DLso) calculated with the Lichtfield-Wilcoxon method (17). 0.6-1 mm3 liver sections were collected for examinations from 4 different localisations of the left lateral lobe immediately after killing the animal. From each experimental animal 4 samples of liver were examined. The samples were fixed in 1.6 % glutaraldehyde in phosphate buffer at pH 7.4 at 4 0 C for 2 hours and were post-fixed with 1 % osmium tetroxide. After dehydration the samples were embedded in Epon. Semithin sections stained with toluidine blue were examined using light microscopy. Five tissue specimens were taken from each sample. Ultrathin sections obtained with a diamond knife of an LKB IV Ultratom were subjected to routine staining with lead citrate and uranyl acetate, and examined using a Philips EM 300 electron microscope.
Results
Control group The ultrastructure of the centrolobular zone in hepatocytes of the control rats did not differ from the norm. In the nuclei nucleoli could be seen with small clumps of heterochromatin around them and under the nuclear envelope. In the cytoplasm, mitochondria were mostly oval and possessed short narrow cristae, a narrow external compartment and a matrix of medium electron density. The smooth endoplasmic reticulum (SER) showed small agglomerations of tiny vesicles, among which few glycogen rosettes were noted. Small agglomerations of rough
Fig. 1. Intact hepatocyte. Characteristic nucleic structure (Chr - chromatin). The structure of mitochondria (M), RER and SER is normal. Magn. x 7500. Fig. 2. Nuclear body (NB) in hepatocyte one day after EG intoxication. In higher magnification (top right comer) NB of a fibrillar capsule (arrows) and multigranular core inside is composed. Chr - chromation. Magn. x 13500. 360
Exp Toxic Pathol 47 (1995) 5
Fig. 3. Hepatocyte nucleus one day after intoxication. Note numerous perichromatin granules (PO - arrows) at periphery of condensed chromatine (Chr). Magn. x 17500 Fig. 4. The cytoplasm of hepatocyte one day after EO intoxication, with predominated irregular shape vacuoles of SER. Gly - glycogen, L -lipid droplet, M - mitochondria. Magn. x 44000. endoplasmic reticulum (RER) with parallel membrane system were found mainly near the nucleus (fig. 1). At the biliary pole of hepatocytes single lysosomes and peroxisomes were found. In the peripherolobular hepatocytes there were less mitochondria, but of greater size. In this zone, also SER vesicles were in smaller quantities. The Oolgi apparatus had more cisterns and vesicles as compared to the centrolobular zone.
Second group - 24 hours after EG administration In hepatic centrolobular nuclei the presence of socalled nuclear bodies could be disclosed very often. Most
frequently they were complex nuclear bodies made up of a delicate capsule and a granular core (fig. 2). Moreover, consideration was given to numerous perichromatin granules (PO) on the borders of condensed chromatin which most often mingled into agglomerations (fig. 3). In the cytoplasm of rat hepatocytes of this experimental group, vesicles of SER usually merging into structures of irregular shape predominated (fig. 4). In the fields of SER there were also visible numerous glycogen rosettes. Mitochondria revealed only rarefied structure of the mitochondrial matrix. Near the biliary pole of hepatocytes single irregularly shaped lysosomes and peroxisomes could be distinguished. In hepatocytes of the peripheral zone the above mentioned changes were much less visible. Exp Toxic Pathol 47 (1995) 5
361
Fig. 5. Five days after EG administration. Swollen mitochondria (M) with empty spaces in matrix. Disordered membranes RER and various forms of widened vesicles SER are visible. Magn. x 62000. Fig. 6. Liver sinusoids 5 days after intoxication filled with large deposits, surrounded by membranes (asterisk). Magn. x 28000.
Third group - 5 days after EO administration On the 5th day after EG intoxication, the most conspicuous ultrastructural alterations could be observed in centrolobular hepatocytes and they concerned mainly mitochondria and SER. Mitochondria were markedly swollen and the swelling was associated with evident lacy contours of the external mitochondrial membranes. In the matrix, apart from numerous areas of reduced density, empty spaces were observed (fig. 5). Profiles of SER, although less numerous as compared with the previous experimental group, were distinctly widened. Also cisterns of the Golgi apparatus were very often extened and widened. Consideration was also given to numerous 362
Exp Toxic Pathol 47 (1995) 5
peroxisomes with distinct crystalline tubular cores. They were localized in the proximity of mitochondrial agglomerations. Very characteristic subcellular alterations were also observed within capillary vessels. Endothelial cells contained vesicles of various types and sizes. The vascular lumen was often filled with large structures, surrounded by membranes, containing flocculent material with medium electron density (fig. 6).
Fourth group - 14 days after EO administration The rarefied hepatocytic cytoplasm revealed very numerous but tiny vesicles of SER and fields of glycogen rosettes. However, the most characteristic lesion of hepa-
Fig. 7. Fourteen days after EG intoxication. Note very remarkable lesions of mitochondria (M): swelling of internal compartment with fragmentation of cristae. Vacuoles and myelin figures within matrix. SER Smooth Endoplasmic Reticulum, MVB - Multivesicular Body. Magn. x 40800. tocytic substructure of the centrolobular zone was mito- occurring in acute EG poisoning must be related to the chondriosis. The whole cytoplasm of hepatocytes was action of metabolites and free radicals. In the nuclei of most often filled with mitochondria, which were charac- hepatocytes these changes consisted in the appearance of terized by various sizes and polymorphous signs of da- numerous so called nuclear bodies. Although biogenesis mage. These injuries were mainly associated with swell- and function ofNB are not as yet fully known (4,6,21), ing of the internal compartment, and this was usually ac- their appearance, especially complex nuclear bodies discompanied by fragmentation of cristae. Within the matrix closed in the present studies, must be related to disturbed there were often distinguishable vacuoles and myelin fi- synthesis of RNA and of protein. In the earlier biochegures (fig. 7). Apart from numerous peroxisomes en- mical investigations ZIMMERMAN (29) showed that EG countered among mitochondria, the cytoplasm revealed . metabolites cause - among others - inhibition of the synformations related to focal degradation of structures and thesis of RNA and of protein. LAMPE et al. (16) and to intracellular digestion: autophagolysosomes, multi- JOANNIDES et al. (13) are also of the opinion that active, vesicular bodies as well as myelin figures and dense metabolites originated under conditions of cytochromes P-450 - induced catalysis may act on nucleic acids and bodies. alter their chemical and biological properties. This suggestion has been confirmed by our own morphological Discussion observations. They have disclosed not only the presence of complex forms of nuclear bodies in the course of acute Ethylene glycol is a basic component of commonly used EG intoxication but also accumulation of perichromatin nonfreezing fluids, readily absorbed from the alimentary granules (PG) in the nuclei. The presence of agglomeratract. Damages have been described, caused by GE, in tions of PG in the nuclei, as is evident from earlier stuheart, liver, kidneys (2, 5, 9, 15, 23, 25, 29). The path- dies, among others OfPUVION AND MOYNE (21), has been ways of biochemical EG transformations initiated by its related to the inhibition of transport of pre m-RNA to the oxydation to glycolaldehyde and then - through glycolic cytoplasm. According to the results of recent biochemiacid - to glyoxylate, are quite well know (23, 27). Beside cal and immunocytochemical studies of various authors, its toxic effects in cells, connected with metabolic acido- the biogenesis of NB must be related to disturbed transsis, in recent years attention has been paid to free oxygen scription and maturation both of pre m-RNA and of pre rradicals participating in the development of injuries of RNA (4, 6). These observations together with the results cellular substructure (20, 28). Since oxidative transfor- of our own morphological studies of the nuclei in acute mations of EG take place in the liver, an ultrastructural EG intoxication appear to be proof that ethylene glycol assessment of hepatocytes in the course of acute experi- exerts an effect on the metabolism of nucleic acids in the mental EG intoxications seemed to be worthy of interest. nucleus. The most characteristic alterations in the ultraOur own studies have disclosed characteristic ultrastruc- structure of the cytoplasm of hepatocytes of the centrolotural alterations both in the nuclei and in the cytoplasm of bular zone were found in SER and in mitochondria. Marhepatocytes. Many of these submicroscopic changes ked proliferation and merging of SER vesicles observed Exp Toxic Pathol47 (1995) 5
363
in our own studies must be related to the induction of cytochromes of the P-450 family constituting an integral part of the membranes of SER where they form a multicomponent enzymatic - lipid complex defined by the name of microsomal monooxygenases. This was proved by biochemical methods by many authors (16, 20, 28). IMAZU et al. (12) disclosed a 17 % rise in the cytochrome P-450 content in homogenates of hepatic cells in the course of EG poisoning. A morphological manifestation of the increased amount and activity of cytochrome P450 was proliferation of SER with their coupling into irregularly shaped figures apparent especially on the 1st and 5th days after intoxication. Ultrastructural injuries of mitochondria, subtle on the 1st day of EG poisoning and evident on the 5th day, were most strongly expressed after 14 days of the experiment with well distinguishable destruction of the membranes. Weare of the opinion that this intensification of the destructive action of EG observed in the course of our experiment should be related to progressive uncoupling of oxidative phosphorylation disclosed by various authors (15, 18). It must be presumed that the features of lesioned mitochondria, most strongly expressed in the 3rd zone, are also connected with vascularization conditions within hepatic lobules (14). And finally we must consider the problem of the presence of peroxisomes, particularly abundant in the proximity of mitochondria. Although, no direct binding of peroxisomes to other organelles has been show in the studies which have been conducted so far, but many authors had regard to spatial relationships of this organelle with SER (1, 19) or to the appearance of peroxisomes in the immediate proximity of lipid droplets (8). These authors ascribe this fact to their participation in the fat metabolism. In turn, BAUMGART (1) suggests a permanent exchange of intermediate products in the biosynthesis of composite lipids. Our own studies disclosing the presence of numerous peroxisomes within agglomerations of mitochondria are approximate to the observations reported by GORGAS (10), although the functional significance of this relation is as yet obscure. The presence of peroxisomes with crystalline tubular cores may be related to the activity of free oxygen radicals released from hydrogen peroxide and from non-hemie iron demonstrated by KUKIELKA and CEDERBAUM (15), in the course of EG intoxication. The donor of free radicals is, and this was confirmed by documentary evidence, one of the oxidizing enzymes of peroxisomes (19, 26). Their large number observed especially on the 5th day after EG intoxication confirms the results of the biochemical studies of the above mentioned authors. However, the development and widening of Golgi apparatus cisterns, conspicuous particularly on the 5th day of acute EG poisoning, can be related to the activity of the enzyme TPP-ase because its substrate thiamine pyrophosphate (TPP) participates, according to the report of SCHAGGER and JAGOW (26) in the metabolism of the intermediate metabolite glyoxalate. And, finally, it must be said that the role of crystalloids, surrounded by membranes, filling, the hepa364
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tic vessels in the development of ultrastructural lesions in hepatocytes remains an open question. The intracapillary structure we have described has identical ultrastructure to that of calcium oxalate in the renal vessels described earlier, having essential significance in the development of acute renal failure (7). The above observations seem to indicate that deposition of calcium oxalate in the course of acute EG poisoning concerns many organs and, therefore, it has more universal character.
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