Sequential hepatic histologic and histochemical changes produced by diethylnitrosamine in the rhesus monkey.

Six young adult male rhesus monkeys were given diethylnitrosamine ip for 3-5 years. Liver biospies were done monthly. After 6 months, biopsy specimens showed individual hepatocytes and small foci of hepatocytes that were intensely positive for glycogen. During the second and later years, larger foci of such cells developed. In sections stained with hematoxylin and eosin, the glycogen-containing hepatocytes generally appeared unusually clear. Some hepatocytes, however, had eosinophilic or basophilic cytoplasm. Nuclear enlargement and atypic developed, particularly outside the foci. The hepatocytes within most foci were uniform in their histochemical features: glycogen was elevated, glucose-6-phosphatase was decreased, and ATPase activity was present not only along the bile canalicular surface but also along the enire cell membrane. After 3-5 years, neoplastic nodules and hepatocarcinomas developed in 5 of 6 animals. Two nodules and particularly the heptocarcinomas differed from the foci in one of more histochemical parameters. The findings suggested that the glycogen-containing, histochemically altered cells of the foci in one or more histochemical parameters. The findings suggested that the glycogen-containing, histochemically altered cells of the foci may be the first step in the development of neoplasia; further steps toward malignancy appeared to be frequently associated with additional alterations, such as loss of sinusoidal ATPase and re-formation of glucose-6-phosphatase.

Irreversibility of liver tumors in C3H mice.

The present study was designed to test the possibility that spontaneous regression of hepatocellular tumors might be observed in mice. This problem was studied by sequential liver biopsies in C3H male mice that had been treated with dieldrin (CAS: 60-57-1) as well as in animals treated with N-diethylnitrosamine (CAS: 55-18-5) and in untreated control mice. Adenomas were seen in some animals at the second laparotomy when there had been no tumor at the first laparotomy. In a few mice there was histologic progression from adenoma to carcinoma. A change in predominant cell type in adenomas from clear to basophilic or eosinophilic was also observed in some cases. Additional hepatocellular carcinomas were observed in some animals necropsied at 2 years of age. These observations suggest that spontaneous hepatic tumors and tumors in mice treated with either complete carcinogens or nongenotoxic compounds have a strong tendency to progress. Tumor regression in mice appears to be unusual. No consistent relationship of histologic grade of hepatocarcinoma to the type of chemical employed was observed.

Carcinogenicity of dietary aflatoxin M1 in male Fischer rats compared to aflatoxin B1.

Aflatoxin M1 (AFM), an hydroxy metabolite of the potent carcinogenic mycotoxin aflatoxin B1 (AFB) is frequently found in milk and other dairy products. Sufficient amounts of AFM were produced to study the carcinogenicity of this compound. AFM was fed to male Fischer rats starting at 7 weeks up to 21 months of age. Agar-based semisynthetic diets contained 0.0, 0.5, 5.0, and 50.0 micrograms/kg of AFM or 50 micrograms/kg of AFB. Hepatocellular carcinomas were detected in two of 37 rats and neoplastic nodules were found in six of 37 rats fed 50 micrograms/kg AFM between 19 and 21 months. No nodules or carcinomas were observed in the lower AFM dose groups. Nineteen of 20 rats fed a diet containing 50 micrograms/kg of AFB developed hepatocellular carcinomas by 19 months of age. Carcinogenic potency of the aflatoxins was reflected by morphometric quantitation of foci detected in hematoxylin and eosin stained sections. Three rats fed the diet containing 50 micrograms/kg AFM developed intestinal carcinomas. None were observed in other groups. Under the conditions of this experiment AFM was found to be a weak hepatic carcinogen compared to AFB and to possess intestinal carcinogenicity.

Serum epoxide hydrolase (preneoplastic antigen) in human and experimental liver injury.

Reports of an increase in a serum epoxide hydrolase (sEH), immunochemically related to microsomal EH in humans and rats with hepatocellular carcinoma (HCC), suggested its use as a serum marker for this disease. We have now measured sEH levels (as either immunochemically determined content or enzyme activity) in a number of human and experimental models of liver disease. sEH was elevated above the normal range in at least 50% of individuals with HCC, including: 3 of 6 northern Californians; 4 of 7 Koreans with hepatitis B-associated HCC; hepatitis B-associated HCC in woodchucks; and male rats receiving chronic treatment with aflatoxin B1 or ciprofibrate. sEH was rarely elevated in other forms of chronic liver disease. Only 2 of 9 Koreans with hepatitis B-associated cirrhosis, 1 of 8 carriers, but none with chronic active hepatitis or infection with no apparent liver disease had elevated sEH. In addition, no elevations were found in woodchucks with noncancerous viral hepatitis. In aflatoxin B1- and M1-treated rats sEH was not elevated in those with only hyperplastic foci or hepatocellular adenomas, and in two rat initiation-promotion protocols sEH was elevated only in those rats which received the entire set of treatments. sEH was also increased during acute hepatotoxicity in rats treated with CCl4 or 1,2-dibromo-3-chloropropane. The mechanism of increase in sEH during hepatocarcinogenesis appears to be different from that of other markers of HCC, for in the Korean patients, there was no correlation between sEH concentrations and those of alpha-fetoprotein or ferritin, nor was there a correlation with alpha-fetoprotein concentrations in the aflatoxin-treated rats. Furthermore, the increase in sEH does not correlate with induction of microsomal EH in the liver of experimental animals. Studies to date indicate that sEH is selective for HCC and severe hepatonecrotic injury, and may be of some use in the diagnosis of HCC, particularly as a complement to other serum markers.

Buerger's disease involving the celiac artery.

A young male with Buerger's disease who had previously required a left leg amputation died in renal failure and sepsis. Postmortem examination revealed an obliterative lesion of the celiac artery, which resulted in hepatic, splenic, and pancreatic infarctions. Celiac artery involvement represents an unusual manifestation of thromboangiitis obliterans.

Colorectal cancer and noncancer patients have similar labeling indices by microscopy and computed image analysis.

The labeling index (LI), a microscopic measurement of proliferative activity in colonic crypts, is proposed as an indicator of colonic cancer risk. Computed image analysis of proliferative regions is less labor intensive and more objective than is direct microscopy but has not been validated for labeling indices by direct comparison. The authors compared colonic crypt proliferation in 26 cancer and 13 noncancer patients by using Ki-67 monoclonal antibody (McAb) labeling of flat mucosa obtained from surgically removed, frozen specimens. In cancer patients, the mucosa specimen was excised 10 cm away from the tumor, and the LI was determined microscopically for the whole crypt, the upper two thirds, and the upper one third of 15 crypts. Nuclear antigen levels of 15 whole crypts were determined by using the CAS-200 computed image analyzer (Cell Analysis Systems, Elmhurst, IL). Cancer and noncancer specimens were compared as were microscopically determined LI and stained nuclei specimens by using image analysis. No statistically significant difference in proliferative activity of whole crypts, or the upper two thirds of crypts, was observed between cancer specimens and noncancer specimens from using either technique. However, a significant correlation existed between microscopic analysis and computed image analysis of labeled nuclei. Computed image analysis using Ki-67 McAb labeling can be used instead of microscopy to determine crypt LI, but neither method can be used to distinguish cancer specimens from noncancer specimens.

Characterization of monoclonal antihuman small bowel and colon specific mucin antibodies.

Three human small bowel and colon mucosal specific monoclonal antibodies with distinct morphologic and electrophoretic characteristics were generated by fusion of immunized Balb/c spleen cells and murine plasmacytoma cells. Morphologic specificity by indirect immunofluorescence (IIF) revealed three antibody binding patterns corresponding to villus surface (TP-NG-43), goblet cell apical granules (TP-NG-2), and a combined surface/goblet cell apical granule antibody (TP-NG-20). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) produced three distinct electrophoretic migration patterns. These antibodies reacted with very high molecular weight determinants: TP-NG-2, one band greater than 400 kD; TP-NG-20, two bands corresponding to 370-400 kD; and TP-NG-43, two bands in the 350-400-kD range with smaller bands in the 50-94-kD range. Cross-reactivity with various other human organ systems was evaluated by indirect immunofluorescence and SDS-PAGE electrophoresis with Western blotting. By IIF, all three monoclonal antibodies reacted very strongly with components of gastric mucosa. Weak cross-reactivity was seen with colon, rectum and mucin-producing adenocarcinoma of the colon. No cross-reactivity was observed by IIF with other mucin-containing and non-mucin-containing tissues. However, cross-reactivity with gastric mucin was not detected by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Antibody reactivity with mucin was confirmed by purifying various regional gastrointestinal mucins and by subsequent testing by ELISA. Monoclonal antibody affinity columns were prepared and evaluated. The utility of these methods will allow for further definition of important goblet cell mucin glycoprotein characteristics and isolation of mucin subpopulations.

Effect of total parenteral nutrition on hepatic histology.

We evaluated the effect of total parenteral nutrition (TPN) on abnormalities of hepatic histology. Liver biopsies of 93 patients who were concurrently receiving TPN were compared with a control group of 35 patients. The control patients were matched for extent of preexisting liver disease and degree of illness. The liver biopsy specimens were blindly graded on 19 histopathologic findings, including fatty change, portal inflammation, and cholestasis. Twenty-seven clinical variables, such as preexisting liver disease, the presence or absence of sepsis or shock, and number of days receiving TPN before biopsy, were recorded. Individual and partial correlations were established between the clinical variables and histopathologic findings. The control and TPN groups proved to have been closely matched regarding the extent of risk factors for hepatic histopathologic features. Positive correlations were repeatedly found between abnormal hepatic histologic features and preexisting liver disease, abdominal sepsis, renal failure, and blood transfusion but not with the administration of TPN. We conclude that clinical phenomena, such as existing liver disease, renal failure, and abdominal sepsis, rather than administration of TPN, had a predominant effect on histopathologic features in this group of patients.

Sclerosing mesenteritis seen clinically as pancreatic pseudotumor: two cases and a review.

Sclerosing mesenteritis is an uncommon nonneoplastic inflammatory process in the mesentery that is seen as a pseudotumor, usually involving the small bowel mesentery, the mesenteric fat, and less commonly, the mesentery of the large bowel. We report two cases of sclerosing mesenteritis and review the literature on this rare disease. Both patients had pain, profound weight loss, and a mass on computed tomography (CT) scan of the abdomen. The provisional diagnosis was pancreatic neoplasm on the basis of clinical presentation and imaging studies. The diagnosis of sclerosing mesenteritis was established by histologic findings in biopsy material obtained at laparotomy in both cases. Interval histologic studies in one patient who had a high CA 19-9 level, progressive biliary ductal and partial duodenal compression, revealed a transitional histologic pattern from predominant inflammation and fat necrosis to predominant fibrosis. This may explain the varied descriptive terms used in the literature to describe this entity.

What is new in epithelioid hemangioendothelioma of the liver?

An unusual hepatic vascular neoplasm has been delineated in recent years. It has characteristic morphologic features consisting of multiple nodules with relatively acellular centers which may be focally calcified. More peripherally there is a cellular zone containing elongated or plump tumor cells embedded in a fibromyxoid stroma. At the outer edge of the nodules, and particularly in vessels, the tumor cells may assume an epithelioid appearance. The tumor cells exhibit focally positive staining for Factor VIII related antigen and Weibel Palade bodies may be seen on electron microscopy. The tumor is malignant, but may have an indolent course over many years. It may be associated with hepatic cirrhosis and pulmonary osteoarthropathy. Other organs, particularly the lung and soft tissues are frequently involved.

Gastric tumors in patients with pulmonary chondroma or extra-adrenal paraganglioma: an ultrastructural study.

The histogenesis of a distinctive metastasizing intramural gastric tumor found in patients with extra-adrenal paragangliomas, pulmonary chondromas, or both, is unknown. By light microscopy, it has appeared to be of smooth-muscle derivation, and it has been interpreted as being epithelioid leiomyosarcoma. For further clues to its nature, we studied three examples by electron microscopy (two obtained from patients with extra-adrenal paragangliomas and one from a patient with pulmonary chondroma). Ultrastructurally, they were characterized by interdigitating cytoplasmic processes, plasma membrane-associated dense patches, an incomplete basement membrane, junctional complexes, randomly oriented filaments without periodicity, cytoplasmic dense bodies, pinocytotic vesicles, clustering of mitochondria, and rare cilia-features of normal smooth muscle or of smooth-muscle tumors with classic histologic patterns. Thus, the findings suggest that the three tumors we studied are of smooth-muscle derivation.

Tumors of the pancreas with osteoclast-like and pleomorphic giant cells: an immunohistochemical and ploidy study.

BACKGROUND: Tumors of the pancreas with osteoclast-like giant cells are of uncertain histogenesis and aggressiveness. Their relationship, if any, to undifferentiated (anaplastic) carcinomas of the pancreas with pleomorphic giant cells is also not clear. METHODS: Eleven tumors with osteoclast-like giant cells were studied by immunohistochemistry for epithelial and mesenchymal markers, as well as for a proliferation marker (Ki67) and p53 protein expression. Cytometric image analysis for nuclear DNA content was also performed. K-ras mutations were investigated by DNA sequence analysis. RESULTS: Neoplastic, predominantly spindle-shaped cells and osteoclast-like giant cells were positive for mesenchymal markers CD68, LCA, and A1ACT. These spindle-shaped cells were also positive for human muscle actin. Spindle-shaped cells of seven tumors were also positive for epithelial markers carcinoembryonic antigen, epithelial membrane antigen, or keratin. Nine tumors contained a variable number of pleomorphic giant cells in addition to osteoclast-like giant cells. Pleomorphic giant cells were much less positive for mesenchymal markers than were osteoclast-like giant cells, but they were positive for some epithelial markers. A high percentage of spindle-shaped and pleomorphic giant cells were positive for Ki67. Diploid and aneuploid populations were present in varying proportions in both spindle cells and pleomorphic giant cells. The nuclei of osteoclast-like giant cells, however, were diploid and not proliferating. Spindle-shaped and pleomorphic giant cells were positive for p53 protein in 5 of 10 cases. Five of six tumors studied were positive for K-ras mutations. CONCLUSION: The distinction between tumors with osteoclast-like giant cells and undifferentiated carcinomas with pleomorphic giant cells is often not clear-cut. Both types of tumors have mesenchymal and epithelial characteristics in varying proportions and may arise from an undifferentiated pancreatic stem cell. Long-term survival of two patients suggests that some tumors with osteoclast-like giant cells may have a better prognosis than the usual pancreatic ductal adenocarcinoma.

Hepatic and metabolic effects of ethanol on rhesus monkeys.

Rhesus monkeys were tube-fed 100 calories per kg of a liquid diet based on casein in which 41% of the calories were derived from grain alcohol. The alcohol intake was 5.8 g per kg per day. Control diets contained isocaloric amounts of glucose. The protein content of the diet was 15% and fat supplied 21% of the calories. After 28 days the animals which had been fed ethanol developed hepatic fatty change and serum L.D.H. levels were elevated. The most striking electron microscopic changes in the alcohol animals were mitochondrial swelling, focal cytoplasmic degradation, and dilatation of the rough endoplasmic reticulum. In the monkeys which had received ethanol the metabolism of alcohol increased from 17.4 mg per 100 ml per hour to 26.6 mg and antipyrene half-life decreased from 61.0 minutes to 49.9 minutes. The carbohydrate animals showed no significant change in alcohol metabolism or antipyrene half life. The ethanol animals lost weight significantly while the carbohydrate animals gained significantly. The metabolic effects of alcohol thus were not reproduced by glucose. Administration of phenobarbital at 30 mg per kg for 5 days increased alcohol metabolism from 16.5 mg per hour to 22.5 mg per hour and shortened antipyrene half life from 76.5 minutes to 33.6 minutes. Alcohol and phenobarbital both induced enhanced drug metabolism but alcohol was a more powerful inducer of its own metabolism than phenobarbital. Phenobarbital on the other hand was a better inducer of antipyrene metabolism than alcohol.

Effect of selenium on aflatoxin hepatocarcinogenesis in the rat.

The effects of selenium (Na2SeO3) on aflatoxin B1 (AFB1)-induced hepatic neoplasia were studied in the rat. Putative preneoplastic foci and nodules composed of basophilic, eosinophilic, and clear cells developed early. Basophilic foci were seen first; in the later stages basophilic and eosinophilic nodules predominated. At each stage the AFB1 + Se groups showed fewer and smaller foci and nodules than the AFB1 - Se group. The number of foci in the AFB1 + 3 ppm Se group and their mean area were smaller than those in the 6 ppm Se + AFB1 group. At the end of the experiment hepatocellular carcinoma (HCC) was found in 11/18 rats (61%) of the AFB1 - Se group. HCC was not found in either of the groups given AFB1 + Se. We conclude that Se had an inhibitory effect on the initiation and promotion stages of AFB1-induced preneoplastic foci and nodules. Se also prevented progression of these nodules to HCC even after cessation of AFB1 administration. The inhibitory effect of Se at 3 ppm was greater than at 6 ppm. The 6 ppm Se group also showed evidence of toxicity.

A retrospective study of probable glucocorticoid-induced hepatopathy in dogs.

Hepatopathy associated with glucocorticoid therapy or with naturally occurring hyperadrenocorticism was detected retrospectively in hepatic biopsy specimens from 22 of 60 dogs, by histologic review. The hepatopathy was characterized by centrilobular vacuolization, perivacuolar glycogen accumulation within hepatocytes, and focal centrilobular necrosis. The predominant clinical findings were hepatomegaly, increased serum enzyme activity associated with hepatic disease, and increased bromsulphalein retention. The hepatopathy appeared to be reversible.