Labeling of hepatic glycogen after short- and long-term stimulation of glycogen synthesis in rats injected with 3H-galactose

The effects of short-and longterm stimulation of glycogen synthesis elicited by dexamethasone were studied by light (LM) and electron (EM) microscopic radioautography (RAG) and biochemical analysis. Adrenalectomized rats were fasted overnight and pretreated for short- (3 hr) or long-term (14 hr) periods with dexamethasone prior to intravenous injection of tracer doses of ³H-galactose. Analysis of LM-RAGs from short-term rats revealed that about equal percentages (44%) of hepatocytes became heavily or lightly labeled cells increased slightly 6 hr after labeling, and unlabeled glycogen became apparent in some hepatocytes. The percentage of heavily labeled cells had decreased somewhat 12 hr after labeling, and more unlabeled glycogen was evident. In the long-term rats 1 hr after labeling, a higher percentage of heavily labeled cells (76%) was observed compared to short-term rats, and most glycogen was labeled. In spite of the high amount of labeling seen initially, the percentage of heavily labeled hepatocytes had decreased considerably to 55% by 12 hr after injection; and sparsely labeled and unlabeled glycogen was prevalent. The EM-RAGs of both short- and long-term rats were similar. Silver grains were associated with glycogen patches 1 hr after labeling; 12 hr after labeling, the glycogen patches had enlarged; and label, where present, was dispersed over the enlarged glycogen clumps. Analysis of DPM/mg tissue corroborated the observed decrease in label 12 hr after adminstration in the long-term animals. The loss of label observed 12 hr after injection in the long-term pretreated rats suggests that turnover of glycogen occurred during this interval despite the net accumulation of glycogen that was visible morphologically and evident from biochemical measurement.     

Comparison of 3H-galactose and 3H-glucose as precursors of hepatic glycogen in control-fed rats

Labeling of hepatic glycogen derived from 3H-galactose and 3H-glucose was compared shortly after intravenous injection in control-fed rats. The rats were allowed to accumulate 5-8% glycogen prior to receiving label. Fifteen minutes to 2 hours after labeling, liver was excised and processed for routine light (LM) and electron microscopic (EM) radioautography (RAG) or biochemical analysis. After injection of 3H-galactose, LM-RAGs revealed that the percentage of heavily labeled hepatocytes increased from 37% after 15 minutes to 68% after 1 hour but showed no further increase after 2 hours. alpha-Amylase treatment removed most glycogen and incorporated label; thus few silver grains were observed, indicating little incorporation of label except into glycogen. EM-RAGs demonstrated that most label occurred where glycogen was located. Biochemical analysis showed initially a high blood level of label that rapidly plateaued at a reduced level by 5 minutes. Concomitantly, glycogen labeling determined by liquid scintillation counting reflected the increases observed in the RAGs. After injection of 3H-glucose, LM-RAGs revealed that only 12% of the hepatocytes were heavily labeled at 1 hour and 20% at 2 hours. In tissue treated with alpha-amylase, glycogen was depleted and label was close to background level at each interval observed. EM-RAGs showed most grains associated with glycogen deposits. Biochemically, blood levels of label persisted at a high level for 30 minutes and tissue levels increased slowly over the 2-hour period. This study shows that incorporation from 3H-galactose was more rapid than incorporation of 3H-glucose; however, label derived from both carbohydrates appeared to be incorporated mainly into glycogen.     

Effects of glucagon on hepatic glycogen and smooth endoplasmic reticulum

The action of glucagon on hepatic glycogen and smooth endoplasmic reticulum (SER) was studied in samples of liver taken sequentially from anesthetized rats. The physiological state of each animal was assessed by continuously monitoring aortic blood pressure and blood lactate/pyruvate ratios. High hepatic glycogen levels were established by using 10–12 hr fasted control-fed rats infused continuously with glucose. In rats receiving glucose only, hepatic glycogen levels remained above 5.0% during the 4-hr period of glucose administration. Centrilobular hepatocytes displayed an abundance of glycogen which often appeared dispersed with elements of SER between the glycogen particles. Periportal cells had dense clumps of glycogen with few vesicles of SER restricted to the periphery of the glycogen masses. The addition of glucagon to the glucose infusate caused a marked stimulation of glycogenolysis. In these rats, the hepatic glycogen level (x?±SE) was 6.71±.15% 1 hr after glucose and declined after initiation of glucagon infusion as follows: 5.86±.29% (15 min), 4.89±.26% (1 hr), 2.16±.40% (2 hr), and 1.66±.29% (3 hr). The fine structure of hepatocytes showed a dramatic response to the administration of glucagon. The glycogen regions of the cells were noticeably decreased in size and number of glycogen granules 3 hr after initiation of glucagon infusion, and SER was abundant in both periportal and centrilobular hepatocytes. The interpretation offered is that glucagon induces the formation of new SER membranes which participate in glycogen breakdown and/or glucose release from hepatocytes.     

Action of metabolic hormones on the fine structure of rat liver cells III. Effects of adrenalectomy and administration of cortisone

Electron microscopic studies were made of hepatocytes from sham-operated rats, adrenalectomized animals fasted 15 hours, and adrenalectomized rats fasted 15 hours but given a single I.P. injection (10 mg) of cortisone acetate. The objective of this work was to define the earliest morphological response of hepatocytes to injection of a glucocorticoid and to provide additional information on the mechanism of hormone action at the cellular level. Hepatocytes from fasted, adrenalectomized rats contained no glycogen particles and very little smooth endoplasmic reticulum (SER). In addition the rough endoplasmic reticulum was disorganized and showed fewer ribosomes and polysomes than found in liver cells from sham-operated rats. Two hours after glucocorticoid injection glycogen particles were seen in numerous centrilobular cells and some periportal hepatocytes. Elements of SER were associated with the glycogen particles. By 4 hours after hormone injection abundant glycogen was found in all hepatocytes. Centrilobular cells showed dispersed glycogen with extensive tubules of SER associated with the glycogen particles. Periportal hepatocytes accumulated glycogen as dense masses scattered throughout the cytosome. SER occurred mainly at the edges of the glycogen masses. Midlobular cells showed glycogen patterns intermediate between periportal and centrilobular cells; masses of dispersed glycogen with abundant SER occurred within and around the glycogen areas of the cells. Glucocorticoid stimulation also caused cisternae of RER to align in parallel arrays, and more ribosomes and polysomes appeared on membranes of RER than in similar cells from adrenalectomized rats. The interpretation is offered that the glucocorticoid-stimulated proliferation of SER is the morphological expression of induced microsomal enzyme synthesis (glucose-6-phosphatase) known to occur under these hormonal conditions.     

Fine structure of hepatocytes from fasted and fed rats

The fine structure of hepatocytes from rats maintained on a controlled feeding schedule are described. Liver samples were processed for electron microscopy, histochemistry and chemical determinations of glycogen at precise time-intervals following a 30-hour fast and a 2-hour meal. Hepatocytes from 30-hour-fasted rats with extremely low hepatic glycogen levels were devoid of glycogen particles. Centrilobular cells showed areas of the cytoplasm rich in vesicles of smooth endoplasmic reticulum (SER) while periportal hepatocytes contained less extensive regions of SER. Soon after feeding the fasted rats, glycogen particles appeared in regions of the cell rich in SER. Centrilobular hepatocytes contained numerous glycogen areas which were infiltrated with tubules of SER, while periportal cells showed dense glycogen deposits with SER restricted to the periphery of the masses of glycogen. Throughout glycogen deposition each glycogen particle was closely associated with membranes of SER until maximum glycogen deposition was achieved 12 hours after initiation of feeding. At this point SER was reduced to the lowest amounts of the time-periods studied. During stages of glycogen depletion SER proliferated and reached the highest concentration measured in this study. Tubules of SER were present throughout the glycogen masses of centrilobular hepatocytes, whereas in periportal cells the organelle was restricted to the periphery of the glycogen masses. It is concluded that SER is associated with glycogen particles in rat hepatocytes during both deposition and depletion of glycogen.     

Rapid alterations induced by insulin in hepatocyte ultrastructure and glycogen levels

The speed with which insulin alters hepatocyte ultrastructure and glycogen levels in insulin-deficient rats has been studied. Insulin deficiency was induced with alloxan, followed by insulin treatment with regular and NPH insulin. Rats were killed at various times after the insulin injection, blood samples were obtained, plasma glucose levels were determined, and liver samples were prepared for electron microscopy and glycogen determinations. Plasma glucose levels in insulin-deficient rats declined to normal values by 4 hours post insulin, returning to insulin-deficient levels by 8 hours post insulin. Hepatic glycogen was considerably reduced in the insulin-deficient rats. By 1 hour post insulin hepatic glycogen increased, reached maximal levels by 8 hours, then declined to insulin-deficient levels by 36 hours. The ultrastructural appearance of both centrilobular and periportal hepatocytes from insulin-deficient rats showed abundant vesicular smooth endoplasmic reticulum (SER), decreased rough endoplasmic reticulum (RER), and enlarged RER intracisternal spaces. One-half hour post insulin, centrilobular hepatocytes were unchanged. In periportal hepatocytes, however, vesicular SER was no longer visible, the RER intracisternal spaces appeared normal, and the amount of RER had increased. By 1 hour post insulin the centrilobular hepatocytes showed similar ultrastructural changes. These changes became more pronounced in the next few hours and remained through 24 hours. By 36 hours both centrilobular and periportal hepatocytes appeared similar to those in the insulin-deficient rat. These results demonstrate the rapid and lobular-specific effects insulin has on the hepatocyte.     

The effects of insulin replacement and withdrawal on hepatic ultrastructure and biochemistry

This study examines the early hepatic biochemical and ultra-structural responses to insulin replacement in streptozotocin-diabetic rats and insulin withdrawal from insulin-maintained diabetic rats. Insulin administration rapidly lowered plasma glucose and the elevated glucose-6-phosphatase (G-6-Pase) specific activity of the diabetic rats. However, hepatic glycogen did not increase until after 3 hr of insulin treatment. Hepatic ultrastructure responded to insulin replacement after the decline in glucose and G-6-Pase. This was seen in periportal hepatocytes as a reduction in the close association between smooth endoplasmic reticulum (SER) and glycogen particles in the diabetic animals. The treated rats showed hepatic SER restricted to the periphery of glycogen masses, as is characteristic of these cells from normal rats, in many cells by 6 hr and all cells by 18 hr. Insulin withdrawal from insulin-treated diabetic rats elicited nearly a total reversal of the above events. Plasma insulin declined to a value half that of the normal rats by 6 hr after withdrawal; concurrently, plasma glucose rose sharply to hyperglycemic values as hepatic glycogen content dropped. Following the rise in plasma glucose and fall in glycogen content, G-6-Pase specific activity increased and by 16 hr reached the high values characteristic of the diabetic animal. Hepatic ultra-structure was also changed as evidenced by an intrusion of elements of the SER into the dense glycogen masses; the result was dispersed glycogen closely associated with SER as seen in the diabetic animal. It is concluded that the hepatic response to insulin replacement in diabetic animals and diabetic onset in insulin-withdrawn animals is rapid and occurs through defined stages.     

Localization of glycogen synthase activity in liver of fasted normal and adrenalectomized rats after injection of dexamethasone

Hepatic glycogen synthase activity was localized in normal and adrenalectomized (ADX) rats after fasting overnight and in fasted ADX rats after injection of dexamethasone (DEX) 2–8 h prior to sacrifice to stimulate glycogen synthesis. Cryostat sections were incubated in medium containing substrate to demonstrate glycogen synthase activity as indicated by glycogen synthesized during incubation. Sections from fasted normal rats showed limited dispersed glycogen synthase activity in both periportal and centrilobular regions. In contrast, activity for glycogen synthase in hepatocytes from fasted ADX rats appeared as large aggregates in random hepatocytes throughtout the lobule. Two hours after injection of DEX the reaction product appeared as aggregates in some hepatocytes, but other cells revealed dispersed enzyme activity. Glycogen synthase activity was evident in more hepatocytes after 4 h treatment with DEX and after 8 h virtually all hepatocytes contained abundant reaction product. The results suggest that synthase activity becomes concentrated in limited regions of selected hepatocytes in fasted ADX rats. DEX stimulation of glycogen synthesis for 4–8 h results in increased enzyme activity. © 1993 Wiley-Liss, Inc.     

Glycogen metabolism in cultured chick hepatocytes: A morphological study

Ultrastructural and autoradiographic observations of cultured chick hepatocytes under the following conditins are described: Induction of glycogen synthesis with glucose alone and glucose plus insulin, and glucagon-induced glucogen glucogen breakdown. Profiles of hepatocytes cultured in medium containig 10 mM glucose showed typical cellular organelles and occasionally a few glycogen granules. After incubation of hepatocytes with ³H-glucose, silver grains were found over these sparse glycogen granules, indicating a low level of glycogen synthesis by a few cells. After addition of 75 mM glucose for 1 hr about 3% of the profiles of cells showed glycogen, and by 24 hr half of the hepatocytes had glycogen. Addition ofinsulin plus glucose induced glycogen synthesis in 82% of the cells after 6 hr. and by 24 hr almost every cellular profile showed glyocgen particles. Morphologically, glycogen accumulation was similar whether the cells were stimulated by high glucose or by glucose plus insulin: glycogen granules appeared in restricted regions of the cytoplasm, which were rich in smooth endoplasmic reticulum (SER), and peroxisomes were found close to the newly deposited glycogen particles. At maximum glycogen accumulation the association of SER and peroxisomes with glycogen was less obvious. Glycogenolysis induced by incubation of glycogen-rich hepatocytes with glucagon resulted in proliferation of SER in the glycogen regions of the cells. These observations are compatible with the concept of regions in the hepatocyte cytoplasm specialized for glycogen metabolism. Possible roles for SER and peroxisomes found near glycogen particles and other organelles in hepatic glycogen metabolism are discussed.     

Scanning microdensitometry of glycogen zonation in the livers of rats adapted to a controlled feeding schedule and to 30, 60, or 90% casein diets

The effect of diet composition on diurnal changes in glycogen zonation patterns in rat liver was investigated in individually-caged male Sprague-Dawley rats adapted to the 2 + 22 controlled feeding and lighting schedule and to diets containing 30% casein/55% carbohydrates, 60% casein/25% carbohydrates, or 90% casein (30 rats/dietary group). Three rats from each dietary group were killed at the following times relative to the onset of feeding (0 min):?60, ?30, 0, 15, 30, 45, 60, 90, 120, and 180 min. Glycogen in cryostat sections from the median and right lateral lobes of the liver was fixed and stained by standard techniques. The optical density of glycogen at points along the path between the central and portal veins of a given lobule was determined, and lobular glycogen gradients of replicate animals were integrated to form a composite lobular glycogen distribution profile. In the period from ?60 to 0 min, liver glycogen levels were similar for rats on any of the diets, and the glycogen concentration was similar in periportal (P), midlobular (M), and centrilobular (C) hepatocytes. During the 0- to 45-min period, diet-related glycogen depletion occurred (90 > 60 ? 30% casein) by asymmetrical glycogen loss (P > M ? C hepatocytes) from the liver lobules. Similar food intake curves occurred for all diets. During the 45- to 180-min period, asymmetrical glycogen accumulation began in lobular parenchymal cells (P > M ? C hepatocytes), and the rate of accumulation was related to dietary composition (30 > 60 ? 90% casein). The differential responses of parenchymal cells within liver lobules to physiological stimuli resulted in glycogen distributional changes that were rapid and of large magnitude. Our results are consistent with the hypothesis that periportal and midlobular hepatocytes are more metabolically responsive and active than centrilobular hepatocytes.     

Development of ultrastructural heterogeneity among hepatocytes in the mouse

Ultrastructural stereological analyses of periportal and centrilobular hepatocytes of newborn, 5- and 10-day-old, and adult male ddY mice were carried out to study the postnatal development of the morphologic heterogeneity among hepatocytes. In newborn animals, the periportal and centrilobular cells did not differ in the volume densities of the smooth and rough endoplasmic reticulum; in the volume and numerical densities of the mitochondria, lysosomes, peroxisomes, and lipid droplets; or in the shape (the axial ratio) of the mitochondria. In 5-day-old animals, the volume densities of the mitochondria and rough endoplasmic reticulum were greater in periportal cells than centrilobular cells, and the volume density of the smooth endoplasmic reticulum was greater in centrilobular cells than periportal cells. In 10-day-old animals, a further difference was seen in the numerical density of the mitochondria, which was greater in centribular cells than periportal cells. Adult hepatocytes showed also a difference in the axial ratio of the mitochondria, which was greater in centrilobular than periportal cells; there was no difference in the volume density of the rough endoplasmic reticulum. When the data were expressed as volume and number per hepatocyte, the patterns of sublobular distributions of these organelles differed from the patterns seen in the volume and numerical density data, mainly in adult animals. This difference was caused by the marked increase in hepatocyte volume between 10 days of age and adulthood, especially in centrilobular cells. The results show that, in general, the ultrastructural heterogeneity among hepatocytes, evident in adult animals, is not present in newborn animals but arises during postnatal development, and suggest the occurrence of a lobular gradient in postnatal development of hepatocyte functions.     

Alterations in hepatic fine structure after chronic exposure of rats to dexamethasone

The objective of this study was to determine the effects of chronic dexamethasone (DEX) administration on hepatic ultrastructure and to correlate these changes with plasma lipoprotein levels. Electron microscopic studies were made of hepatocytes from male rats killed 1, 3 and 5 days after DEX (2 mg, twice per day) administration. Three days after treatment plasma lipoprotein levels were highest and hepatocytes contained regions of the cytosome rich in elements of the smooth endoplasmic reticulum (SER). Osmiophilic particles were present in the tubules and vesicles of the SER, in the saccules and vacuoles of the Golgi complex, in secretory vesicles near the cell surface and in the space of Disse. DEX treatments also caused hepatocytes to accumulate tightly packed masses of β-particles of glycogen in some regions of the cell while other areas displayed dispersed glycogen particles that were associated with the SER. These observations are consistent with the hypothesis that glucocorticoids 1. cause an elevation of plasma lipoprotein levels by in creasing hepatic synthesis and secretion of VLDL, which involves the sequential participation of the ER, the Golgi complex and exocytosis of VLDL-containing vacuoles into the space of Disse, and 2. produce a change in the nature of the association of glycogen particles with the SER membranes in response to the physiological state of the animal.     

Alterations in hepatic fine structure after chronic exposure of rats to dexamethasone.

The objective of this study was to determine the effects of chronic dexamethasone (DEX) administration on hepatic ultrastructure and to correlate these changes with plasma lipoprotein levels. Electron microscopic studies were made of hepatocytes from male rats killed 1, 3 and 5 days after DEX (2 mg, twice per day) administration. Three days after treatment plasma lipoprotein levels were highest and hepatocytes contained regions of the cytosome rich in elements of the smooth endoplasmic reticulum (SER). Osmiophilic particles were present in the tubules and vesicles of the SER, in the saccules and vacuoles of the Golgi complex, in secretory vesicles near the cell surface and in the space of Disse. DEX treatments also caused hepatocytes to accumulate tightly packed masses of beta-particles of glycogen in some regions of the cell while other areas displayed dispersed glycogen particles that were associated with the SER. These observations are consistent with the hypothesis that glucocorticoids 1. cause an elevation of plasma lipoprotein levels by increasing hepatic synthesis and secretion of VLDL, which involves the sequential participation of the ER, the Golgi complex and exocytosis of VLDL-containing vacuoles into the space of Disse, and 2. produce a change in the nature of the association of glycogen particles with the SER membranes in response to the physiological state of the animal.     

Action of metabolic hormones on the fine structure of liver cells. II. Effects of hypophysectomy and chronic administration of somatotropin

Electron microscopic studies of hepatocytes from sham-operated, hypophysectomized, and hypophysectomized rats treated with somatotropin were undertaken in order to obtain morphological information on the mechanism of hormone action at the cellular level. Fed and fasted animals from each of the above groups were studied. Hepatocytes from fed rats of the three groups showed similar characteristics: periportal cells contained large dense masses of glycogen throughout the cytosome with relatively little smooth endoplasmic reticulum; mid-lobular cells displayed small dense masses of glycogen with few associated elements of smooth endoplasmic reticulum; centrilobular hepatocytes showed dispersed glycogen and abundant smooth endoplasmic reticulum. Hepatocytes from hypophysectomized rats fasted fifteen hours showed significant alterations in structure when compared with liver cells from sham-operated animals. Differences noted were: decrease in mid-lobular cell size and amount of smooth endoplasmic reticulum; decrease in glycogen, number of ribosomes and polysomes per hepatocyte; disorganization of the rough endoplasmic reticulum; and swelling of mitochondria and increase in number per cytoplsmic volume. Periportal hepatocytes from fasted hypophysectomized rats treated with somatotropin for ten days contain numerous small masses of glycogen with elements of smooth endoplasmic reticulum at the periphery of the glycogen masses. Mid-lobular and centrilobular hepatocytes contain masses of dispersed glycogen with an abundance of smooth endoplasmic reticulum associated with the glycogen particles.     

Cocaine hepatotoxicity in mice: histologic and enzymatic studies

The severity of hepatotoxicity in CF-1 mice given 5 daily doses of 5, 10, and 20 mg cocaine/kg body weight and sacrificed 24 hr after the last injection appeared to be dose-dependent. Using light microscopy, the hallmark of cocaine early toxicity was manifested by pallor and ballooning of the hepatocytes in the midzonal and then in the centrilobular areas. Degeneration and necrosis of the liver cells in the same zones were encountered while the hepatocytes in the periportal areas remained intact. When examined under the electron microscope, such pallor and ballooning of the hepatocytes appeared to to be due to dilation of the rough endoplasmic reticulum (RER) profiles which often revealed a significant loss of their ribosomes. Dilation of the smooth endoplasmic reticulum (SER) was also common and moderate proliferation of peroxisomes was frequently seen. In the degenerating hepatocytes, the 2 forms of endoplasmic reticulum were difficult to recognize and the peroxisomes appeared sparse. Cocaine treatment elevated the level of glutamic pyruvic transaminase and glutamic oxaloacetic transaminase in a dose-dependent manner. Although hepatic cytochrome P450 was slightly increased in the low dose groups, a reduction in the enzyme activity was noticed in the group treated with 20 mg cocaine/kg. However, hepatic reduced glutathione content manifested a significant increase in the group which received 20 mg cocaine/kg.     

Acute toxic effects of paraquat on ultrastructure of rat liver

Paraquat was administrated to pathogen-free male rats orally, and the livers were studied by light and electron microscope at intervals of 6 hours to 5 days. Congestion and hepatocellular injury (degeneration and/or fatty metamorphosis) were seen by light microscope. Electron microscope showed that degranulation of RER, proliferation of SER, decreasing of glycogen particles and mitochondrial swelling occurred in the cytoplasm of the hepatocytes within 2 layers around the central vein at 6 hours. After 12 hours the liver cells throughout the centrolobular area were injured. Degranulation of RER, proliferation of SER, and decreasing of glycogen particles became prominent, and mitochondria showed swelling and transformation. In the midzonal and periportal areas, numerous lipid droplets were seen in the cytoplasm of the hepatocytes. From the result of ultrastructural findings, it is considered that detoxication and biotransformation of paraquat occur in the hepatocytes within 2 layers around the central vein at an early stage, and spread to the hepatocytes throughout centrolobular area later.     

New insights into functional aspects of liver morphology

The liver is structurally and functionally complex and has been considered second only to brain in its complexity. Many mysteries still exist in this heterogeneous tissue whose functional unit of the lobule has continued to stump morphologists for over 300 years. The primary lobule, proposed by Matsumoto in 1979, has been gaining acceptance as the functional unit of the liver over other conceptual views because it's based on vessel architecture and includes the classic lobule as a secondary feature. Although hepatocytes comprise almost 80% of the liver, there are at least another dozen cell types, many of which provide "cross-talk" and play important functional roles in the normal and diseased liver. The distribution and functional roles of all cells in the liver must be carefully considered in both the analysis and interpretation of research data, particularly data in the area of genomics and "phenotypic anchoring" of gene expression results. Discoveries regarding the functional heterogeneity of the various liver cell types, including hepatocytes, hepatic stellate cells, sinusoidal endothelia, and Kupffer cells, are providing new insights into our understanding of the development, prevention and treatment of liver disease. For example, functional differences along zonal patterns (centrilobular or periportal) have been demonstrated for sinusoidal endothelium, Kupffer cells, and hepatocytes and can explain the gradients and manifestations of disease observed within lobules. Intralobular gradients of bile uptake, glycogen depletion, glutamine synthetase, and carboxylesterase by hepatocytes; widened fenestrations in centrilobular sinusoidal lining cells; and differences in the components of centrilobular extracellular matrix or function of Kupffer cells have been demonstrated. Awareness of the complexities and heterogeneity of the liver will add to a greater understanding of liver function and disease processes that lead to toxicity, cancer, and other diseases.     

Chlordecone-induced potentiation of carbon tetrachloride hepatotoxicity: a light and electron microscopic study

Previous studies have shown that a chlorinated pesticide, chlordecone (Kepone), greatly potentiates carbon tetrachloride (CCl4) hepatotoxicity and lethality (Curtis, L.R., Williams, W.L., and Mehendale, H.M. (1979). Toxicol. Appl. Pharmacol. 51, 283-293; Curtis, L.R., and Mehendale, H.M. (1980). Drug Metab. Dispos. 8, 23-27). The present study describes sequential morphologic changes which occurred in livers of rats given a "nontoxic" level of chlordecone (10 ppm for 15 days) followed by a single injection of CCl4 (0.1 ml/kg). The hepatic alterations were examined 1 to 36 hr after exposure of the rats to CCl4. Those changes were compared to hepatic alterations which occurred in rats that received the same dose of chlordecone (10 ppm for 15 days) or a single injection of CClr (0.1 ml/kg) alone. The only change noted in livers from rats that received chlordecone alone was focal increase in smooth endoplasmic reticulum (SER) of hepatocytes at 24 hr and continuing throughout the time course of the experiment. Livers from animals that received CCl4 alone showed morphologic changes at 6 hr consisting of glycogen loss, increase in SER, and dilatation of rough endoplasmic reticulum (RER) in pericentral hepatocytes. Accumulation of small lipid droplets was also noted in midzonal hepatocytes. After 6 hr, there was no further increase in severity of injury. At 12 hr recovery was noticeable and, by 36 hr, livers from the CCl4 group appeared normal. Prior administration of chlordecone greatly potentiated pathologic changes in livers of animals that received CCl4. By 4 hr, there was total loss of glycogen in hepatocytes throughout the entire lobule. Small lipid droplets were present in pericentral, midzonal and periportal hepatocytes. Hepatocytes with extremely dilated RER were randomly scattered throughout the entire lobule. At 6 hr, there was further accumulation of lipid in the form of large droplets in hepatocytes. Focal, necrotic cells surrounded by polymorphonuclear leukocytes were randomly distributed throughout the lobule. The number of necrotic foci had progressively increased at the 12- and 24-hr intervals. By 36 hr, confluent areas of necrosis in pericentral and midzonal areas were observed in livers of some animals. This study indicates that although the combination of chlordecone and CCl4 produces much greater hepatic injury resembling damage due to a massive dose of CCl4, histologically, some differences in the progression and distribution of hepatocellular damage within the lobular architecture of the liver are evident.     

Experimental paracetamol-induced hepatic necrosis: a light- and electron-microscope, and histochemical study.

Rats were killed at various time-intervals up to 48 hr after a single large dose of paracetamol (3 g per kg) and their livers examined by light and electron microscopy. In general, this revealed glycogen depletion, loss of ribosomes, and cytoplasmic matrix swelling commencing 3-6 hr after administration which in centrilobular hepatocytes progressed to frank coagulative necrosis at 12-24 hr. Midzonal cells showed more prominent aqueous swelling with besiculation of the endoplasmic reticulum, and in some cells, gross hydropic vacuolation.     

Covalent affinity labeling,radioautography, and immunocytochemistry localize the glucocorticoid receptor in rat testicular leydig cells

The presence and distribution of glucocorticoid receptors in the rat testis were examined by using 2 approaches: in vivo quantitative radioautography and immunocytochemistry. Radioautographic localization was made possible through the availability of a glucocorticoid receptor affinity label, dexamethasone 21-mesylate, which binds covalently to the glucocorticoid receptor, thereby preventing dissociation of the steroid-receptor complex. Adrenalectomized adult rats were injected with a tritiated (³H) form of this steroid into the testis and the tissue was processed for light-microscope radioautography. Silver grains were observed primarily over the Leydig cells of the interstitial space and to a lesser extent, over the cellular layers which make up the seminiferous epithelium, with no one cell type showing preferential labeling. To determine the specificity of the labeling, a 25- or 50-fold excess of unlabeled dexamethasone was injected simultaneously with the same dose of (³H)-dexamethasone 21-mesylate. In these control experiments, a marked reduction in label intensity was noted over the Leydig as well as tubular cells. Endocytic macrophages of the interstitium were non-specifically labeled, indicating uptake of the ligand possibly by fluid-phase endocytosis. A quantitative analysis of the label confirmed the presence of statistically significant numbers of specific binding sites for glucocorticoids in both Leydig cells and the cellular layers of the seminiferous epithelium; 86% of the label was found over Leydig cells, and only 14% over the cells of the seminiferous epithelium. These binding data were confirmed by light-microscope immunocytochemistry using a monoclonal antibody to the glucocorticoid receptor. Intense immunocytochemical staining was seen predominantly in the cytoplasm of the Leydig cells, whereas the cells of the seminiferous epithelium were weakly stained. Thus, the present data reveal a high glucocorticoid receptor content within Leydig cells and a lower level within cells of the seminiferous epithelium. Furthermore, the present experiments demonstrate for the first time the usefulness of dexamethasone 21-mesylate as an affinity label for the morphological localization of the glucocorticoid receptor. The localization of glucocorticoid receptors in Leydig cells suggest a role for this steroid in regulating the function of these cells.