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.     

Histochemical studies of hepatocellular carcinomas in the rat induced by aflatoxin

A histochemical study has been done on a group of 18 hepatocarcinomas induced by aflatoxin. One hundred per cent, incidence of hepatocarcinomas is induced by feeding 5 p.p.m. aflatoxin for 6 weeks. The carcinomas were trabecular hepatocarcinomas with a mixed adenomatous pattern and showed considerable variation in histochemical reactions throughout the lesions. There was a patchy distribution of glycogen and glucose-6-phosphate and the membrane ATPase was present along much of the canalicular border of the cells but with an abnormal and tortuous pattern. Aniline hydroxylase was present in varying amounts in both trabecular and adenomatous carcinomas. It is concluded that the histological variants of hepatocarcinoma are all derived from hepatocytes, but no unique changes were observed related to the progress involved in malignant neoplasia. The observations form a basis for comparison with early lesions seen prior to the recognition of carcinoma.     

Histochemical and biochemical changes in skeletal muscles of rhabdomyolysis-sensitive racehorses following exertion. III: Elevated activity of various antioxidant enzymes

In this communication, the results of a histochemical and biochemical enzyme study on gluteus medius muscle of horses, sensitive to exertional myopathy, during attacks of rhabdomyolysis are presented. For the biochemical study the biopsy specimens investigated were selected by means of histological and enzyme histochemical staining methods. Dissected specimens were used which contained groups of muscle fibres with a high or low activity of glucose-6-phosphate dehydrogenase. The activity of glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, glutathione reductase, glutathione peroxidase, superoxide dismutase, and catalase was measured microbiochemically in these dissected specimens. A rise in activity of glucose-6-phosphate dehydrogenase in pathologically changed muscle fibres was always found to be coupled with a significant rise in activity of phosphogluconate dehydrogenase, glutathione reductase, and glutathione peroxidase. In these muscle fibres, the activity of superoxide dismutase and catalase was not significantly increased. On the basis of the combined histochemical and biochemical findings it is concluded that the application of the histochemical method for the demonstration of glucose-6-phosphate dehydrogenase activity can be highly recommended for the study of antioxidant enzymes in skeletal muscles with neuromuscular defects.     

二乙基亚硝胺诱发的变异肝细胞组化表型的研究

The histochemical changes of gamma-glutamyltransferase (gamma-GT), adenosine triphosphatase (ATPase), glucose-6-phosphatase (G-6-Pase) and ornithine carbamyltransferase (OCT) were studied in diethylnitrosamine (DEN) -induced and enzyme-altered liver cell lesions (Solt-Farber model) in rats. The number of altered liver cell foci tended to decrease after ceasation of 2-acetylaminofluorene (2-AAF); nevertheless, the number and size of the nodules increased rapidly within 20 weeks. The histochemical changes of most of the altered liver cell foci were focused on one or two kinds of enzyme activity (mostly gamma-GT and ATPase); while most of the nodules presented 3 or 4 kinds of histochemical changes, including OCT and G-6-Pase. It is concluded that some of those altered nodules of multi-enzyme changes might develop continuously to become tumors.     

Elevated activity of several antioxidant enzymes in neuromuscular diseases. A histochemical and biochemical study

In the present study the activity of glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase was measured in dissected specimens from muscle biopsies of patients with various neuromuscular diseases and from controls. The biopsy specimens investigated were selected by means of histological and enzyme histochemical staining methods. Specimens were used which contained muscle fibres with a high or low activity of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase and which were free from inflammatory infiltrates. A rise in activity of glucose-6-phosphate dehydrogenase in pathologically changed muscle fibres was always found to be coupled with a significant rise in activity of phosphogluconate dehydrogenase, glutathione peroxidase and glutathione reductase. In these muscle fibres the activity of superoxide dismutase and catalase was not significantly changed. On the basis of the histochemical and biochemical findings it is concluded that the application of the histochemical method for the demonstration of glucose-6-phosphate dehydrogenase activity can be highly recommended for the study of antioxidant enzymes in skeletal muscles with neuromuscular diseases.     

The way we teach general pathology in Oslo, Norway

Hepatocellular carcinoma was induced in rats by administering aflatoxin B₁ (AFB₁) for 6 weeks. Malignant tumours were preceded by foci and nodules of altered hepatocytes of three histological types, composed of basophilic, eosinophilic, and vacuolated cells. In addition, there were areas of altered hepatocytes that were considered as hyperplastic. Lectins were used as histochemical markers to compare the expression of membrane glycoproteins in hepatocellular carcinomas and hepatic nodules with non-nodular or control hepatocytes. There were marked changes in the lectin-binding patterns of the hepatocellular carcinoma cells and the eosinophilic nodules. The lectin-binding patterns of basophilic nodules, vacuolated nodules, and hyperplastic areas were similar to non-nodular or untreated hepatocytes. The similarity in the lectin-binding changes of the eosinophilic nodules and hepatocellular carcinomas suggests that the eosinophilic nodules may be an early stage in the development of carcinoma.     

Two populations of cells with differing proliferative capacities in atypical acinar cell foci induced by 4-hydroxyaminoquinoline-1-oxide in the rat pancreas

A single intravenous injection of 4-hydroxyaminoquinoline-1-oxide in male Wistar rats at a dose of 6 mg. per kg. of body weight induced atypical acinar cell foci in 100 per cent of the animals. Atypical acinar cell foci could be classified histologically as basophilic and acidophilic foci and acidophilic nodules. Cells in baseophilic foci were large, contained an irregular nucleus and a markedly basophilic cytoplasm with a few to small number of zymogen granules (zymogen-poor cells). By transmission electron microscopy, these cells showed markedly irregular plasma membranes, a well-developed rough endoplasmic reticulum and a few zymogen granules. Cells in acidophilic foci and nodules contained an intensely eosinophilic granular cytoplasm (zymogen-rich cells) and a large oval to round nucleus. By transmission electron microscopy, the cells showed zymogen-rich cytoplasm and irregular lateral plasma membranes. Mitotic activity was completely absent or very rarely observed in normal pancreas or basophilic foci, in contrast to acidophilic foci and nodules in which a mean value of 2.75 +/- 1.27 per 1000 cells was found. Autoradiography confirmed these differences between the proliferative capacity of cells in basophilic foci (1 +/- 1 labeled nuclei per 1000 cells) and acidophilic foci (23.2 +/- 3.15 labeled nuclei per 100 cells). These studies indicate that 4-hydroxyaminoquinoline-1-oxide induces two types of atypical acinar cell foci with different morphologic features and proliferative capacity.     

家兔实验性胃溃疡期间甲状腺的组织学和组织化学变化(Ⅱ.滤泡旁细胞)

用成年雄性家兔49只,分为正常组、溃疡组和盐水组。正常组是未经处理的正常家兔;溃疡组为在无菌条件下,打开动物腹腔,用少量冰醋酸注入胃粘膜下层,造成实验性胃溃疡;盐水组模拟手术,用生理盐水注入胃粘膜下层。手术后1～28天,分批取甲状腺,进行组织学和组织化学的观察。实验结果表明,各组甲状腺滤泡旁细胞没有明显的组织学变化。正常家兔甲状腺滤泡旁细胞的乙酰胆碱酯酶、非特异性酯酶和酸性磷酸酶反应很强,乙酰胆碱酯酶有一定的特异性;硫胺素焦磷酸酶反应很弱;琥珀酸脱氢酶、乳酸脱氢酶和葡萄糖-6-磷酸脱氢酶反应弱阳性。盐水组的甲状腺滤泡旁细胞在手术后1～7天,某些组织化学产生一定的变化;在手术后14～28天恢复正常。溃疡组的甲状腺滤泡旁细胞在手术后1～3天,各组织化学反应与盐水组相似;手术后7～28天,许多组织化学反应均在一定程度上比盐水组的反应增强。本文讨论了以上组织化学变化的意义,提示在实验性胃溃疡期间,甲状腺滤泡旁细胞可能参与了调节活动。     

Hyperproliferative hepatocellular alterations after intraportal transplantation of thyroid follicles

The thyroid hormone 3,5,3'-triiodo-L-thyronine (T3) is a strong direct hepatocyte mitogen in vivo. The effects of T3 resemble those of peroxisome proliferators, which are known to induce hepatocellular tumors in rats. With the aim of studying long-term local effects of thyroid hormones on liver parenchyma, small pieces of thyroid tissue were transplanted via the portal veins into the livers of thyroidectomized male Lewis rats. At 1 week, 3 weeks, 3 months, and 18 months after transplantation, the transplants were found to proliferate, to synthesize thyroglobulin, and to release thyroxine and T3. At 3 and 18 months after transplantation, the hepatocytes of the liver acini downstream of the transplanted follicles showed an increase in cytoplasmic basophilia, a loss of glycogen, an enlargement and hyperchromasia of their nuclei, and a strong increase in cell turnover compared with unaltered liver acini. The altered hepatocytes exhibited an increase in the activities of glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, malic enzyme, mitochondrial glycerol-3-phosphate dehydrogenase, cytochrome-c-oxidase, and acid phosphatase; the activities of glycogen synthase and glycogen phosphorylase were strongly decreased. The hepatocytic alterations downstream of the transplanted follicles could be explained by effects of T3. On the other hand, they resembled alterations characteristic of amphophilic preneoplastic liver foci observed in different models of hepatocarcinogenesis.     

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.     

Dose-dependent induction of preneoplastic lesions by the tobacco-specific nitrosamine carcinogen NNK in the in ovo carcinogenicity assessment (IOCA) assay

The potential of the carcinogenic tobacco specific nitrosamine 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-1-butanone (NNK) to induce preneoplastic hepatocellular altered foci (HAF) was tested in the in ovo carcinogenicity assessment (IOCA) assay. Single doses of NNK over a dose range from 0.1 mg to 6 mg were injected into fertilized turkey eggs prior to incubation for 24 days. The livers were investigated by histological, histochemical and morphometric methods. Mortality was increased for eggs exposed to 6 mg. In this group, the whole livers were severely altered, showing pronounced changes of nucleus size and signs of cell death. At the dose of 2 mg various types of foci of altered hepatocytes (HAF) were observed. Basophilic cell foci of the solid or tubular type were most frequent. The NNK-induced HAF were very similar to the preneoplastic lesions that occur in the livers of mammals during hepatocarcinogenesis which are regarded as early indicators of carcinogenesis. The similarity to the HAF in rodents included histochemically detectable alterations like decreased activities of glucose-6-phosphatase, adenosine triphosphatase and glycogen phosphorylase. At doses of 1 mg or below, no HAF were detected. At all dose levels an increased occurrence of enlarged hepatocytes with enlarged nuclei and prominent nucleoli (karyomegalic hepatocytes) were observed. The increase in karyomegalic hepatocytes was also statistically significant at the low dose of 0.1 mg/kg NNK but the dose–effect curve for their induction was clearly non-linear. Induction of HAF and karyomegalic hepatocytes in ovo is a simple (one dose), rapid (24 days) and inexpensive (no animal purchase or housing) experimental approach for studies on chemically induced hepatocarcinogenesis.     

Altered liver acini induced in diabetic rats by portal vein islet isografts resemble preneoplastic hepatic foci in their enzymic pattern.

As demonstrated previously, liver acini draining the blood from intraportally transplanted pancreatic islets in streptozotocin-diabetic rats are altered in various respects. The hepatocytes in these acini store glycogen and/or fat, and they show an increase in proliferation as well as in apoptotic activity. Thus, they are phenotypically similar to carcinogen-induced preneoplastic liver foci (glycogen-storing foci and sometimes also mixed cell foci). By means of catalytic enzyme histochemistry or immunohistochemistry, we investigated the activity of key enzymes of alternative pathways of carbohydrate metabolism and some additional marker enzymes (well known from studies on preneoplastic hepatic foci) in the altered liver acini surrounding the islet isografts. In addition, the expression of glucose transporter proteins 1 and 2 (GLUT-1 and GLUT-2) were investigated immunohistochemically. The activities of hexokinase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and glucose-6-phosphate dehydrogenase were increased, whereas the activities of glycogen phosphorylase, adenylate cyclase, glucose-6-phosphatase, and membrane-bound adenosine triphosphatase were decreased in the altered liver acini. The expression of GLUT-2 was also decreased. GLUT-1 and glutathione S-transferase placental form were not expressed, and the activities of glycogen synthase and gamma-glutamyl-transferase remained unchanged. All changes of the enzyme activities were in line with the well known effects of insulin and resembled alterations characteristic of preneoplastic liver foci observed in different models of hepatocarcinogenesis. It remains to be clarified in long-term experiments whether or not these foci represent preneoplastic lesions and may proceed to neoplasia.     

The sensitivity and heterogeneity of histochemical markers for altered foci involved in liver carcinogenesis.

Subcutaneous injection of iron dextran resulted in a hepatic siderosis within 2 weeks in rats, as previously reported for mice. Hepatic carcinomas as well as neoplastic nodules in rats were entirely or mainly free of stainable iron and, thus, could be readily identified histologically. In addition, early carcinogen-induced altered foci were resistant to iron accumulation. In rats fed 0.02% N-2-fluorenylacetamide (FAA) for 13 weeks, the number of iron-resistant foci identified following iron injection was the same as that observed with dietary iron overload. Histochemical investigation of enzymatic markers that have been used to identify foci in rats revealed that foci characterized by enzymatic reactions of positive gamma-glutamyl transpeptidase and decreased adenosine triphosphatase and glucose-6-phosphatase corresponded to those characterized by resistance to iron accumulation. However, in quantitative analysis of the early carcinogen-induced foci in rats given iron dextran following a diet containing 0.02% 2-FAA for 13 weeks, more lesions were detected by resistance to iron accumulation than by any of these other properties. There was considerable phenotypic heterogeneity among foci for the enzyme markers. It is concluded that resistance to iron accumulation is a more sensitive and reliable marker for early carcinogen-induced altered hepatocellular foci than is any other histochemical property.     

Histoenzymologic characteristics of the myocardium in sudden death in patients with alcoholic cardiomyopathy

Study of the activity of some myocardial enzymes in sudden death cases with alcoholic cardiomyopathy (ACMP) was made by quantitative histochemical methods. The decrease of dehydrogenase activity of succinate, lactate, beta-hydroxybutyrate, alpha-glycerophosphate and NAD-diaphorase was found in line with the increase of the activity of glucose-6-phosphate dehydrogenase, alcohol dehydrogenase and catalase versus control (myocardium of those who died of trauma). Disorders of major metabolic processes in the myocardium may occasionally lead to electrical instability resulting in ventricular fibrillation and sudden cardiac death. In almost 80% of sudden cardiac deaths in ACMP foci of acute myocardial ischemia are found, that can lead to ventricular fibrillation with lethal outcome.     

Cytochemical correlates of atherosclerosis-resistant and susceptible lesions of the normal rabbit aorta

Quantitative cytochemical and microfluorimetric techniques were employed to compare mural intermediary metabolism—endothelial macromolecular uptake changes in spontaneous aortic-arteriosclerotic lesions of normolipemic New Zealand White rabbits. Specifically, mural succinic (SDH), lactic (LDH), and glucose-6-phosphate (G-6-PDH) dehydrogenase activities and luminal surface uptake of fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) were measured in lesion sites abnormally resistant (calcified) and susceptible (proliferative) to dietary hypercholesterolemia. Calcified lesions exhibited severe (55–66%) diminution of SDH, LDH, and G-6-PDH activities within the involved inner mural zone and a comparable (68%) decline in luminal FITC-BSA uptake. Concomitant reductions in FITC-BSA uptake (30%) and marker enzymes of the predominant energy transducing pathways in arterial tissue, i.e., SDH (30%) and LDH (31%), were evidenced in proliferative foci, whereas G-6-PDH was augmented (52%) in comparison to nonlesioned aortic segments. These data lend additional support to the concept that endothelial uptake of plasma-borne macromolecules is coupled to oxidizable substrate requirements of inner avascular compartments of the arterial wall. It is postulated that diminished macromolecular transport in these degenerative lesions stems from reduced mural metabolic demands, and that pharmacologic reduction of vascular smooth muscle metabolism may depress uptake of sclerogenic macromolecules.     

Mosaicism in female hybrid hares heterozygous for glucose-6-phosphate dehydrogenase. VII. Evidence for selective advantage of one phenotype over the other in ditypic samples from aortas of hares fed cholesterol oxidation products

In a previous study the authors demonstrated monotypic foci, all of timidus type, in the atherosclerotic lesions and normal aortic tissue of glucose-6-phosphate dehydrogenase (G-6-PD) mosaic hares fed 25-hydroxycholesterol or triol in addition to a high-cholesterol diet. Other hares fed the high-cholesterol diet without the cholesterol oxidation product additives did not develop monotypic foci. In the current study the question asked was whether ditypic samples from lesions or aortic mediae of the same hares fed the additives would show a higher percentage of timidus type G-6-PD than the hares fed the cholesterol alone. The results showed a significantly higher percentage of timidus type in both lesions and mediae in the groups fed either 25-hydroxycholesterol or triol than in the group fed cholesterol alone. The observations suggest that the change from ditypism to monotypism reported earlier with these cholesterol oxidation products was the result of progressive elimination of the europaeus type to leave only the timidus G-6-PD. This is consistent with the "selective advantage of one phenotype over the other" hypothesis for explaining the observed monotypism as opposed to the "genetic transformation" hypothesis.     

Pathology of diethylnitrosamine toxicity in the fish Rivulus marmoratus

Rivulus marmoratus were exposed to 0, 10, 21, 45, 95, or 200 mg/liter diethylnitrosamine (DEN) for 6 weeks and examined 12 weeks after the end of exposure. Fatty change, hepatocellular glycogenosis, multiple basophilic foci, enlarged and distorted cells with or without an enlarged nucleus, and hyaline bodies and cytological alterations observed after exposure to DEN. Hemangiomas, cholangiomas, biliary cystadenomas, and glandular, trabecular and anaplastic hepatocellular carcinomas were observed at the 18th week. Only those fish exposed to 95 mg/liter DEN had cavernous hemangiomas and peliosis-like lesions, which could be a preneoplastic lesion preceding cavernous hemangiomas. Adenomatous hyperplasia of thyroid and granulomas were other chronic reactions caused by DEN toxicosis.     

Histoenzymological characteristics of the myocardium in sudden cardiac death

Histochemical study of enzymatic activity in the myocardium was performed in sudden cardiac death. Human hearts in which there were no macroscopic and histological focal or diffuse changes served as material. The following enzymes were studied in the anterior or posterior walls of the left ventricle or in the interventricular septum: succinate dehydrogenase, lactate dehydrogenase (LDH), beta-hydroxybutyrate dehydrogenase (OHBDH), alpha-glycerophosphate- and glucose-6-phosphate dehydrogenase, NAD-diaphorase and phosphorylase. Increased activity of OHBDH and LDH was found: 36,0 and 22,6% higher than in trauma and brain hemorrhage that served as control. These alterations seem to be connected with the increase of blood content of fatty acids, and lactate as a response to the catecholamine excess. Foci of an acute ischemia were found in the interventricular septum in 80% of cases in which phosphorylase was revealed. The appearance of the ischemic foci was obviously due to the coronary arteries contraction.     

Histochemical research on metabolic pathways of glucose in some species of Mollusca Gastropoda

The metabolic pathways of glucose were studied by histochemical reactions in some species of gastropods living in different habitats. The glycolytic pathway is histochemically indicated by positive results for glucose-6-phosphate isomerase, fructose-1,6-biphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and D-lactate dehydrogenase. The enzymes of the Krebs cycle gave different responses: isocitrate dehydrogenase and L-malate dehydrogenase were positive, whilst succinate dehydrogenase was constantly negative. Malate synthetase activity was also demonstrated. Despite L-glutamate dehydrogenase is undetectable, the presence of transaminase indicates the gluconeogenetic route. Phosphoglucomutase and glucose-6-phosphate phosphatase appear also positive. The metabolic meaning of our results were discussed.     

The ultrastructural features of aflatoxin B1-induced lesions in the rat liver.

Hepatocellular carcinoma was induced in rats by administering aflatoxin B1 (AFB1) for 6 weeks. Malignant tumours were preceded by foci and nodules of altered hepatocytes. The ultrastructural characteristics of the nodular lesions have been studied and compared with those of the hepatocellular carcinoma cells. Alterations in the endoplasmic reticulum, junctional complexes and nuclei were common to both the basophilic and eosinophilic nodular cells and the carcinoma cells. These most likely represent hyperplastic changes rather than malignant alterations. The eosinophilic nodules were distinguished from other lesions by the abundance of concentric, membranous whorls in the cytoplasm of nodular cells. These cytoplasmic structures were also present in some hepatocellular carcinoma cells. The observations provided further evidence suggesting that the eosinophilic nodule, rather than the basophilic nodule, may play a role in the development of malignancy in the rat liver.