Histogenesis of dieldrin and DDT-induced hepatocellular carcinoma in Balb/c mice

Male, Balb/c mice were fed diets containing dieldrin (10 ppm) and DDT (100-175 ppm) for 75 weeks. Control and treated mice were serially killed and their livers analyzed by histological and histochemical procedures after 2, 4, 8, 16, 36, 52 and 75 weeks of exposure. Mice administered both chlorinated hydrocarbons initially responded with centrolobular hepatocytomegaly. The cells were characterized by decreased glucose-6-phosphatase and succinate dehydrogenase activity. At later periods 52 through 75 weeks, foci of phenotypically-altered hepatocytes were noted. The cells of these lesions were basophilic or clear-staining in hematoxylin and eosin-stained sections and displayed increased gamma glutamyl transpeptidase activity. In mice preloaded with iron dextran, cells of foci were negative for iron when the surrounding parenchyma was siderotic. Hepatocellular adenomas (HA) and carcinomas (HPC) were composed of cells with increased gamma glutamyl transpeptidase and glucose-6-phosphate dehydrogenase and decreased glucose-6-phosphatase and succinate dehydrogenase activity. In iron loaded mice, the cells of HA and HPC did not stain for iron in otherwise siderotic surroundings. Both hepatocellular foci and adenomas may be potential precursors of mouse hepatocellular carcinomas.     

Activity of glucose-6-phosphatase in regenerating tubular epithelium in rat kidney after necrosis induced with mercuric chloride: a light and electronmicroscopical study.

The activity of glucose-6-phosphatase was investigated in the renal proximal tubules of male albino rats after necrosis induced with mercuric chloride. Between the 3rd and 14th days an active zone could be observed in the inner cortex, which in the control animals was inactive. As regeneration progressed, the number of regenerating active tubules in the cortex increased, and in the 4th week activity approached that of the controls. Ultrastructurally, the enzyme was localized in the endoplasmic reticulum and nuclear envelope and sometimes even in necrotic cells. It appeared again in the regenerating cells on the 4th day. Our histochemical results complement the published biochemical results on the analysis of tissue homogenates localizing the changes of enzyme activity. From this comparison we conclude that glucose-6-phosphatase may take part in either glucose degradation or gluconeogenesis, but whereas gluconeogenesis also occurs in the first part of the proximal tubules in the normal kidney, the enzyme may play a role in glucose consumption only under pathological conditions.     

Activity of glucose-6-phosphatase in regenerating tubular epithelium in rat kidney after necrosis induced with mercuric chloride: a light and electronmicroscopical study

The activity of glucose-6-phosphatase was investigated in the renal proximal tubules of male albino rats after necrosis induced with mercuric chloride. Between the 3rd and 14th days an active zone could be observed in the inner cortex, which in the control animals was inactive. As regeneration progressed, the number of regenerating active tubules in the cortex increased, and in the 4th week activity approached that of the controls. Ultrastructurally, the enzyme was localized in the endoplasmic reticulum and nuclear envelope and sometimes even in necrotic cells. It appeared again in the regenerating cells on the 4th day. Our histochemical results complement the published biochemical results on the analysis of tissue homogenates localizing the changes of enzyme activity. From this comparison we conclude that glucose-6-phosphatase may take part in either glucose degradation or gluconeogenesis, but whereas gluconeogenesis also occurs in the first part of the proximal tubules in the normal kidney, the enzyme may play a role in glucose consumption only under pathological conditions.     

Effects of the peroxisome proliferator di(2-ethylhexyl)phthalate on enzymes in rat liver and on carcinogen-induced liver altered foci in comparison to the promoter phenobarbital

The livers of rats given either the peroxisome proliferating hepatocarcinogen di(2-ethylhexyl)phthalate (DEHP) following initiation by 2-acetylaminofluorene (AAF) or the neoplasm promoter phenobarbital (PB) were studied for changes in 8 histochemical properties. Male F344 rats were fed 200 ppm AAF for 7 weeks to induce hepatocellular altered foci, and were then fed diets containing either no chemical, 12,000 ppm DEHP or 500 ppm PB for 24 weeks. In hepatocytes, DEHP increased alkaline phosphatase activity throughout the lobule, but reduced gamma-glutamyltransferase (GGT) activity in periportal hepatocytes. PB, in contrast, increased GGT activity in periportal hepatocytes. In foci that were induced by AAF, DEHP reduced the histochemical activity of GGT and did not increase the number, mean volume or volume % of foci detected by deficiencies in iron storage, glucose-6-phosphatase, adenosine triphosphatase or fibronectin. PB enhanced the expression of all 8 phenotypic abnormalities in foci such that either more profiles were detected or the area of foci was increased.     

The perinatal development of glucose-6-phosphatase activity distribution pattern in rat liver. A microdensitometrical study

The ontogenetic development of the intralobular distribution pattern of glucose-6-phosphatase activity in the rat liver is described in terms of histochemical changes determined with microdensitometry. A newly developed cerium-lead technique was employed and compared with the common lead technique optimized by Teutsch (1978a). The cerium technique has advantages, meets the prerequisites for quantitative determinations and yields results comparable to biochemically obtained data from microdissected tissue. The first signs of a heterogeneous distribution pattern of glucose-6-phosphatase activity are observed on the 3rd d after birth, and differences between periportal and centrolobular areas are largest around 10th and 15th d. At 30th d after birth, the adult pattern is complete with a centrolobular glucose-6-phosphatase activity of 67% of the periportal value.     

Glucose-6-phosphatase and age: biochemical and histochemical studies

Glucose-6-phosphatase catalyzes the final reactions in both gluconeogenesis and glycolysis. It occurs mainly in glycogenic tissues, such as the liver, where it plays an important role in the synthesis of glucose, a carbohydrate essential for tissue functioning. The effect of age on liver glucose-6-phosphatase activity was evaluated in male Wistar rats treated with mixed function oxidase system (MFO) inducers. The rats were divided into the following age groups: 0.5, 1, 2, 4, 8, 12, 20 and 28 months of age. Glucose-6-phosphatase activity was evaluated biochemically and histochemically. Biochemical glucose-6-phosphatase activity increased up to the 20th month of rat life and then decreased rapidly. A similar tendency was observed in inducer-treated groups, though only dexamethasone stimulated this enzyme activity in all age groups studied. Histochemical glucose-6-phosphatase activity was strongest in the periportal zones. Glucose-6-phosphatase activity decreased significantly at month 8 and then it increased significantly until month 20. In the oldest age group, glucose-6-phosphatase activity decreased again. On histochemical analysis, the inducers used variably affected glucose-6-phosphatase activity.     

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.     

Morphologic and histochemical analysis of the newt (Notophthalmus viridescens) liver

Architectural arrangement, ultrastructure, and selected histochemical properties of the newt (Notophthalmus viridescens) liver were examined. Although hematopoietic tissue (1-4 cells thick) invested the liver, direct vascular communication between this tissue and hepatic parenchyma was not observed. The liver was intensely positive when stained with Oil-red-O and periodic acid-Schiff reagent and connective tissue was limited to large vascular channels and the capsule. A distinctive polarity was observed in the hepatic vascular system when lobes were viewed in cross section. Dorsally, portal venules accompanied arterioles and branches of the biliary system, while tributaries of hepatic veins were observed ventrally. Following perfusion fixation, hepatocytes appeared as sheets of cells 1-5 cells thick; however, lobules as defined in adult mammalian liver were absent. Hepatocytes contained abundant smooth endoplasmic reticulum, mitochondria, electron-dense lysosomes, patches of granular endoplasmic reticulum, and lipid droplets. Continuous endothelial cells lined sinusoids and exhibited fenestrae organized into structures similar to sieve plates observed in mammalian liver. Variable numbers of melanin-containing macrophages and subendothelial macrophages were observed; however, Kupffer cells and lipid containing perisinusoidal fat-storing cells were not seen. Patterns of reaction product for glucose-6-phosphatase (G-6-Pase), glucose-6-phosphate dehydrogenase (G-6-PDH), and succinic dehydrogenase (SDH) were localized in the newt liver. All enzymes exhibited a uniform distribution pattern; however, small punctate regions of intensely positive G-6-PDH cells were noted within hepatic parenchyma. Cells comprising the hematopoietic tissue were intensely positive for G-6-Pase, G-6-PHD, and negative for SDH.     

Histochemical studies on the early proliferative lesion induced in the rat liver by aflatoxin

Male inbred Fischer rats were fed a diet containing 5 p.p.m. aflatoxin for 1, 3, 4½ and 6 weeks at which times groups were killed for histological and histochemical study. Aflatoxin produced a scattered individual cell necrosis of parenchymal cells by 1 week. At 3 weeks small basophilic proliferative foci were seen which increased in size and abundance to 6 weeks. These foci showed starvation-resistant glycogen, variable depletion of glucose-6-phosphatase, succinic dehydrogenase, aniline hydrogenase, membrane ATPase and acid phosphatase. At 6 weeks the foci showed the presence of gamma glutamyl transpeptidase and glucose-6-phosphate dehydrogenase. The basophilic foci were not preceded by other focal histological and histochemical change. The basophilic proliferative lesions are observed when an irreversible change has been induced in the liver. The role of such lesions in the histogenesis of hepatocellular carcinoma is discussed.     

Islet graft-induced changes of beta-hydroxybutyrate dehydrogenase and glucose-6-phosphatase activity in liver cells of diabetic recipient rats.

It is known that the liver is a favourable site for implantation of pancreatic islets since the grafted islets remain metabolically intact and provide long-term normoglycemia in diabetic animals. However, the long-term effects exerted by the grafted tissue on the host organ are not well defined. We therefore investigated by light and electron microscopy the effects of syngeneic islets on the host organ after intraportal transplantation into the liver of streptozotocin (STZ)-induced diabetic LEW.1W rats. In addition, tissue sections of graft-bearing liver were stained by enzyme histochemical methods for beta-hydroxybutyrate dehydrogenase (HBDH) and glucose-6-phosphatase (G6Pase). At 12 weeks after transplantation, the changes seen in the hepatocytes surrounding the grafted islets were hyperproliferation and accumulation of glycogen. Hepatocytes adjacent to the implanted islets displayed increased HBDH activity, whereas G6Pase activity was variable, either decreased or increased. Increased HBDH activity was also observed in the periportal region and in liver cells extending to the central veins. The results demonstrate that intraportal islet grafts, in addition to normalizing glucose homeostasis, exert remarkable effects on the liver parenchyma of experimentally diabetic recipient rats.     

On histochemical enzyme changes in association with canalicular activity of alkaline phosphatase in human liver.

56 human liver biopsy specimens with insignificant or no histological changes, but with abnormally strong canalicular alkaline phosphatase activity, were studied histochemically for other enzyme changes. In comparison with normal specimens, more extensive and increased canalicular activity of gamma-glutamyl transferase, and increase of canalicular leucine aminopeptidase, was found, while the sinusoidal activity of the latter enzyme was decreased. Staining for adenosine triphosphatase regularly desclosed the normal pattern of sinusoidal and canalicular activity. The lysosomal enzymes, acid phosphatase and beta-glucuronidase, stained more intensely than ordinarily, while the reactions for enzymes present in the cytosol (lactic dehydrogenase), in the mitochondria (succinic dehydrogenase, imonoamine oxidase) and in the endoplasmic reticulum (glucose-6-phosphatase) were normal.     

Effect of Nilzan and albendazole on the absorptive surfaces of Haemonchus contortus (Nematoda)--a histoenzymic study

Haemonchus contortus, incubated in 10 micrograms/ml and 50 micrograms/ml concentrations of Nilzan and albendazole in Tyrode solution were stained for histoenzymatic demonstration of various phosphatases, oxido-reductases and esterases. The intestine showed major alterations after drug treatments. The alkaline phosphatases (AkPase), adenosine triphosphatase (ATPase), glucose-6-phosphatase, succinic dehydrogenase (SDH), glutamate dehydrogenase (GDH), reduced nicotinamide adenine dinucleotide phosphate diaphorase and reduced nicotinamide adenine dinucleotide diaphorase showed a decreased activity in intestine after Nilzan treatment, whereas lactic dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G-6-PD) and monoamine oxidase resisted increased reaction. The albendazole treatment resulted in altered distribution pattern of the AkPase, ATPase, SDH, and GDH; while LDH, G-6-PD, and non-specific esterases exhibited slightly enhanced activity in the epithelium. The functional significance of these changes has been fully discussed.     

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.     

Effect of aging on metabolic zonation in rat liver: acinar distribution of GSH metabolism.

The effect of age on the glutathione antioxidant system and its acinar distribution in rat liver was studied. GSH/GSSG ratio in blood and liver was lower in old than in young rats. Hepatic glutathione peroxidase and glutathione S-transferase activities were higher in old than in young rats, whereas hepatic gamma-glutamyl transpeptidase activity was lower in old than in young rats. Glutathione reductase and glucose-6-phosphate dehydrogenase activities did not change with age in rat liver. Total glutathione levels and glutathione peroxidase activity were higher in periportal than in perivenous areas of young rats, but this heterogeneous distribution did not occur in old rats. No change with age was found in hepatic zonation of glutathione reductase and glucose-6-phosphate dehydrogenase.     

The fructose induces "glycogenosis". III. Histochemical and morphometrical studies of glycogen metabolism in mouse liver after fructose overload (author's transl)]

Introduction: After feeding fructose for 7 days rat liver cells show an accumulation of glycogen, a high activity of glucose-6-phosphatase combined with a SER- and RER-reduction. This result was reviewed by mouse liver cells using histochemical and morphometrical methods. Material and Methods: 60% fructose in drinking water was given mice as only nutritional source. Controls had free access to Altromin-R-standard diet and drinking water. Glycogen and glycogen metabolizing enzymes are demonstrated in the course of an 1-14 days fructose diet. After a 7 days diet liver tissue was analysed morphometrically. Results and discussion: Feeding of fructose leads to a high glycogen content, combined with a high activity of glycogen-phosphorylase and glucose-6-phosphatase in the liver parenchyma of mouse. Glycogen-synthetase activity falls to a low level. The SER and RER and the peroxisomes are reduced. The single volume of the hepatic nucleus is decreased and the hepatocellular chondrioma is transformed in a smaller number of larger mitochondria. Compared with the rate the analysed organelles and enzymes of mouse liver show only slight quantitative differences. The increase of glucose-6-phosphatase and simultaneous reduction of endoplasmic reticulum-membranes is illustrated by the dynamic structure of endoplasmic reticulum-membranes, which adapt to metabolic changes. The variable turnover of different parts of endoplasmic reticulum-membranes seems to be very important.     

Enzyme patterns in human hepatocellular carcinoma.

Characteristic enzyme alterations have been demonstrated during the stages of experimental hepatocarcinogenesis in rats. The activity of gamma-glutamyl transpeptidase (GGTPase) in hyperplastic and neoplastic hepatocytes is usually increased, whereas that of canalicular adenosine triphosphatase (ATPase) and of glucose-6-phosphatase (G6Pase) is more variable. The activities of these marker enzymes were studied by histochemical techniques in 10 human hepatocellular carcinomas (HCCs), 1 liver cell adenoma, and 1 cholangiocarcinoma of liver. In 9 cases, the nontumorous liver was also examined. All HCCs, but not the liver cell adenoma, displayed enzyme patterns that differed from normal. GGTPase activity was markedly increased in 8 HCCs, whereas the activities of G6Pase and ATPase were lost in 6 and 8 HCCs, respectively. These enzyme alterations occurred as 5 of 7 possible combinations, resulting in significant heterogeneity of enzyme phenotypes, similar to that in experimental hepatocarcinogenesis.     

Cis-diamminedichloroplatinum (II)-induced acute renal failure in the rat: enzyme histochemical studies

Enzyme histochemical techniques were utilized to examine the progression and extent of proximal tubular injury during the development of cis-diamminedichloroplatinum (II) (CDDP)-induced acute renal failure. Acute renal failure was induced in male rats by the intraperitoneal administration of 10 mg CDDP/kg body weight. At 6, 24, 48, 72, and 96 hr following treatment, renal function was assessed and tissue was collected for renal morphologic and enzyme histochemical studies. The enzymes examined were gamma-glutamyl transpeptidase, alkaline phosphatase, sodium-potassium ATPase (nitrophenyl phosphatase), acid phosphatase, glucose-6-phosphatase, succinic dehydrogenase, alpha-glycerophosphate dehydrogenase, and lactic dehydrogenase. By 24 hr, the activity of acid phosphatase was reduced throughout the proximal tubule, with the greatest decrease occurring in the P3 segment of the proximal tubule located in the outer stripe of the outer medulla. Changes in the histochemical staining of the remaining enzymes were not consistently observed until 48 or, in some cases, 72 hr. These alterations involved all portions of the proximal tubule with the most severe changes involving P3. The results of the enzyme histochemical studies along with the morphologic findings indicating that the initiation of CDDP-induced acute renal failure, first apparent at 48 hr in this model, is associated with cell injury throughout the proximal tubule. The majority of the histochemical changes did not become apparent until late in the course of tubular injury. This suggests that most of the changes in enzyme activity represent nonspecific effects of CDDP-induced tubular injury, as opposed to direct enzyme inhibition by the drug.     

Histochemical changes in developing mouse liver after administration of phenobarbital

The effects of phenobarbital on development of some microsomal enzymes and neutral lipid were studied in mouse liver during a period of rapid cell differentiation. Specific alterations attributable to phenobarbital were identified in developing liver and used as markers for the onset of certain aspects of cell maturation. This investigation was designed to determine (1) the earliest developmental age at which fetal liver can react to phenobarbital and (2) the nature of the alterations in developing and adult liver. Intraperitoneal injections of phenobarbital were given for three days to 14, 17 and 19 day pregnant mice. Histochemical techniques were used to localize neutral lipid and the activities of NADPH diaphorase, glucose-6-phosphatase, and glucose-6-phosphate dehydrogenase. Total cell lipid was measured in livers from 17- and 19-day pregnant mice and livers from their fetuses. The density of reaction product for NADPH diaphorase and glucose-6-phosphate dehydrogenase increased while glucose-6-phosphatase decreased in adult liver. Similar changes were seen in livers from day-19 fetal animals but not in fetuses of younger ages. Neutral lipid droplets increased in size and number after phenobarbital in fetal mice of day 19 of gestation but not in mice of day 17 of gestation. It was concluded that liver from day-19 fetal mice had gained the competence, lacking in earlier ages, to respond to phenobarbital by increased production of some enzymes and neutral lipid.     

Examination of enzyme-altered foci with gamma-glutamyl transpeptidase, aldehyde dehydrogenase, glucose-6-phosphate dehydrogenase, and other markers in methacrylate-embedded liver

F344 Male rats weighting between 90 and 110 gm were given 90 ppm diethylnitrosamine in their drinking water for 5 weeks. Seven weeks after the administration of carcinogen was completed, the rats were sacrificed and sections of their livers were embedded in methacrylate. Serial sections 2 or 4 micron in thickness demonstrated the presence of gamma-glutamyl transpeptidase, acid phosphatase, adenosine triphosphatase, aldehyde dehydrogenase, alkaline phosphatase, alpha-naphthyl butyrate esterase, DT diaphorase, glucose-6-phosphate dehydrogenase, and 5'-nucleotidase activity and glycogen. The use of 4-micron sections of methacrylate-embedded tissue allows the evaluation of many more phenotypic markers in serial sections than is currently possible with frozen sections.     

Electron histochemical characteristics of laser necrosis

Linear or dot-shaped lesions were inflicted on rat liver with Nd:YAG laser, and fine structural alterations of hepatocytes were studied in the specimens processed for an endoplasmic reticulum (ER) marker glucose-6-phosphatase (GP). 5-7 s after irradiation a severe cell damage and GP inhibition occurred near the lesions, with less injured cells located laterally. 24 hr later the zone of the necrosis increased markedly. An autolytic decomposition in the newly formed necrotic area was much more pronounced as compared to the area of the initial necrosis. Phagocytic resorption of the intensively irradiated tissue was retarded this explaining some clinical observations on the long-term healing after Nd:YAG laser surgery. Based on our observations the so-called contact regimen of the irradiation is recommended due to the small size of the initial necrosis produced with this method. The various patterns of cell injuries including some changes in ER and enzyme GP as its marker are described in detail.