Histochemical profile of mouse hepatocellular adenomas and carcinomas induced by a single dose of diethylnitrosamine.

In continuation of earlier studies on murine neoplastic liver lesions, we characterized by histochemical methods the phenotype of hepatocellular adenomas and carcinomas induced by single injections of diethylnitrosamine (1.25, 2.5, or 5.0 micrograms/g of body weight) in 15-day-old C57BL/6 x male C3H F1 mice. The hepatocellular adenomas were composed predominantly of basophilic cells but stored excessive amounts of fat and glycogen in large portions of the tumors. Irrespective of the carcinogenic dose, the adenomas showed a consistent histochemical pattern. Glycogen synthase and phosphorylase were highly active in the hepatocytes that stored glycogen. In cells poor in, or free of, this polysaccharide, these enzymes were only moderately active or even inactive. In glycogen-storing parts of the adenomas, the activity of adenylate cyclase was reduced compared with normal liver parenchyma, but in fat-storing portions it was elevated. In a few adenomas, uniform increase in adenylate cyclase activity could be encountered. The levels of ATPase, acid phosphatase, and glucose-6-phosphatase were either increased or decreased. Glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase showed an increased activity in all adenomas compared with preneoplastic foci, which in turn exhibited a higher glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase activity than the surrounding parenchyma or the liver of untreated controls. The hepatocellular carcinomas showed remarkable histochemical changes compared with adenomas. The levels of fat and glycogen and the activities of glycogen synthase, phosphorylase, and in most cases also that of glucose-6-phosphate dehydrogenase, were reduced significantly. In contrast, adenylate cyclase, glucose-6-phosphatase, glyceraldehyde-3-phosphate dehydrogenase, and also alkaline phosphatase showed a striking elevation in developing carcinomas. Similar, although more pronounced, histochemical changes were seen in the advanced hepatocellular carcinomas. These observations indicated that progression from adenomas to hepatocellular carcinomas was associated with a change in the activity of several enzymes involved in cell membrane function, glycogen metabolism, the oxidative pentose phosphate pathway, and glycolysis.     

Early effects of a single intrarectal dose of 1,2-dimethylhydrazine in mice.

The early morphological and biochemical effects of intrarectally administered 1,2-dimethylhydrazine dihydrochloride on mouse colon were studied. Using [3H]thymidine autoradiography, it was found that 1,2-dimethylhydrazine dihydrochloride, 250 mg/kg decreased the number of prelabeled DNA-synthesizing cells in the distal colon as early as 30 min after instillation. During the interval from 24 hr to 2 weeks, however, the opposite effect was seen; incorporation of [3H]thymidine increased 3- to 5-fold over controls. At lower doses (0.25 to 25 mg/kg), a similar trend was observed. Histological examination showed no dramatic changes in cell structure or in tissue architecture. No changes were seen in labeling indices in the proximal colon. In the liver, cellular alterations were seen at concentrations of 25 to 250 mg/kg, particularly in the centrolobular region. These changes were evident at 2 hr and disappeared by 4 hr. The kidney was unaffected by 1,2-dimethylhydrazine dihydrochloride at any concentration. Our results suggest that enzymes capable of activating 1,2-dimethylhydrazine dihydrochloride are located within the mucosal cells of the distal colon.     

Threshold dose dependence in phenobarbital promotion of rat hepatocarcinogenesis initiated by diethylnitrosamine.

The dose-response of phenobarbital (PB) promotion of hepatocarcinogenesis in rats was investigated. Male F344 rats were given 1, 4, 16, 75, 300 or 1200 p.p.m. PB solutions given ad libitum as their drinking water for 39 weeks following initiation with a single i.p. injection of diethylnitrosamine (DEN) (100 mg/kg). At week 40, the incidence of hepatic tumors was increased clearly in the DEN + PB groups given 300 p.p.m. PB or above, as compared to that in the group given DEN only. Linear dose-response curves for numbers and sizes of enzyme-altered hepatic foci (gamma-glutamyl-transpeptidase or placental glutathione S-transferase positive foci) were obtained in the dose range 16-1200 p.p.m. PB. The minimum promoting dose level of PB for enzyme-altered foci, estimated from dose-response curves by the Logit model, was calculated to be 15-23 p.p.m. Thus while dose dependence was demonstrated over a large range, a threshold was evident at low doses.     

Histochemical characterization of focal hepatic lesions induced by single diethylnitrosamine treatment in infant mice

Focal hepatocellular lesions, induced in our infant mouse system (15-day-old B6C3F1 mice) by a single carcinogenic dose of diethylnitrosamine (2.5 or 5.0 micrograms/g body weight), were characterized histochemically using toluidine blue, periodic acid-Schiff, glycogen phosphorylase, glycogen synthetase, glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, ATPase, gamma-glutamyl transpeptidase, and acid phosphatase. Animals were killed 5, 12, 18, and 24 weeks following diethylnitrosamine treatment. The first focal lesions were observed in mice killed at 12 weeks. All foci showed patchy cytoplasmic basophilia and a slight decrease in the glycogen content. The early foci (12 weeks) showed no change in the levels of glycogen phosphorylase and glycogen synthetase, a strong reduction of glucose-6-phosphatase, and a high increase in glucose-6-phosphate dehydrogenase. In addition, 56% of foci in males and 86% of foci in females showed a slight rise in glyceraldehyde-3-phosphate dehydrogenase, and 12% of foci in males and 17% of foci in females had a lower acid phosphatase. The level of cytoplasmic ATPase was slightly decreased in 22% of foci. By 24 weeks, a decrease in the activity of cytoplasmic ATPase was observed in 84 and 100% of foci in males and females, respectively. The increase in the membrane ATPase was observed in 65% of foci in males and 7% of foci in females. By that time, the decrease in acid phosphatase was observed in 78% of foci in males and 37% of foci in females. The gamma-glutamyl transpeptidase failed to show any increase in its activity, indicating that this enzyme was not a "marker" of the hepatocellular lesions developing under the experimental conditions. Strong decrease in glucose-6-phosphatase in association with a manifest increase in glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase activities indicated a shift from gluconeogenesis to glycolysis. Since this metabolic shift occurred concurrently with an increase in the labeling indices and focal size, it appears that these changes act in concert, representing expression of the acquired functional and replicating potential of the focal cell population.     

A morphologic classification and incidence of spontaneous ovarian neoplasms in three strains of mice

Ovarian tumors were reviewed and classified in 5,404 control inbred BALB/c, C57BL/6, and C3H mice. In descending order of frequency, the most common types in the C3H mice were the tubular mesothelioma (adenoma), thecoma, luteoma, granulosa cell tumor, and cystadenoma. In descending order the most common types in the BALB/c mice were the luteoma, tubular mesothelioma, granulosa cell tumor, thecoma, and cystadenoma. The tubular mesothelioma was the only ovarian tumor of significance in the C57BL/6 mice. Most of the ovarian tumors were rare before 1 year of age, but they increased with age after 1 year.     

Carcinogenic effects of N-ethyl-N-hydroxyethylnitrosamine and its metabolites in rats and mice.

N-ethyl-N-hydroxyethylnitrosamine (EHEN), a member of the nitrosamine class of carcinogens induces renal cancer. However, since very little is known about the metabolic products of EHEN and their effects, these were investigated in rats and mice. EHEN, N-ethyl-N-formylmethylnitrosamine (EFMN) and N-ethyl-N-carboxymethyl-nitrosamine (ECMN) were administered in the drinking water for 2 weeks and the animals were then maintained until sacrifice at week 32. The urine of the rats was collected over the 2-week exposure period and analyzed by HPLC. The results showed that EHEN but not EFMN or ECMN induces tumors in the kidneys of rats. In mice the lungs were targeted not only by the parent compound but also by both metabolites. The findings suggest that the kidney is the most susceptible organ to EHEN effects in the rat while the lung is the most susceptible organ in mice. These results are consistent with inter-species variation in the metabolism of xenobiotics.     

Presence of alpha-fetoprotein-positive cells in hepatocellular foci and microcarcinomas induced by single injections of diethylnitrosamine in infant mice

Single injections of diethylnitrosamine (5 and 50 micrograms/g body weight) in male C57BL/6J X C3HeB/FeJ F1 mice when they were 15 days old resulted in the induction of RNA-rich hepatocellular foci and nodules that contained alpha-fetoprotein (AFP)-positive hepatocytes after 20 and 28 weeks. The focal lesions were composed of 1- to 2-cell-thick plates of hepatocytes or closely packed clusters of cells, but they did not show the histological patterns that are diagnostic of trabecular hepatocellular carcinoma. AFP-positive hepatocytes were found in almost one-fourth (14 of 60) of the foci and nodules in a serially sectioned block of liver from a mouse given one injection of 50 micrograms/g body weight diethylnitrosamine and killed at 28 weeks. In general, the presence or absence of AFP-positive cells correlated with the size of the foci and nodules. All six nodules with diameters greater than 1.5 mm contained AFP-positive cells, while all 12 foci smaller than 0.24 mm in diameter were negative for AFP. However, among the 42 foci that were intermediate in size, there were 8 AFP-positive foci, the sizes of which appeared rather randomly distributed among the negative foci. Reactive changes in hepatocytes could be ruled out as a cause of the induction of AFP because the foci first appeared many weeks after the administration of diethylnitrosamine in these mice. Since bile ductules or oval cells, which occasionally appeared in these foci, were lacking entirely in AFP and since ductules are absent from the early-appearing and smallest foci, we believe that in this model the AFP-positive foci arise only from hepatocytes. The presence of AFP in the focal lesions and in tumor thrombi that extended from them into hepatic vein branches supports the hypothesis that some foci undergo progression to invasive microcarcinomas and that these in turn are precursors of late-appearing (after 1 year) metastasizing trabecular hepatocellular carcinomas.     

杂色曲霉素和脱氧雪腐镰刀菌烯醇对小鼠致癌作用的研究

目的探讨杂色曲霉素(ST)对NIH小鼠的致癌作用,同时研究脱氧雪腐镰刀菌烯醇(DON)对ST致癌作用的影响。方法将180只NIH小鼠随机分为ST 3μg/kg组、ST30μg／kg组、STμg/kg DON1．5pμg／kg组、ST30μg／kg DON1．5μg／kg组、DON1．5μg/kg组和对照组等6组,每组30只。各实验组按要求分别灌喂不同剂量的真菌毒素,对照组灌喂同等容量的生理盐水,3次／周,共24周。实验第58周和第74周处死小鼠,观察各器官组织病变。结果对照组小鼠各器官组织均未见明显病理改变。ST和DON处理组均有部分小鼠发生肺腺癌和腺胃黏膜上皮异型增生。ST3μg／kg组、ST30μg/kg组、ST3μg／kg DON1．5μg／kg组、ST30μg/kg DON1．5μg/kg组和DON1．5μg／kg组肺癌的发生率分别为25．0％、41．7％、62．5％、69．2％和37．5％,腺胃黏膜上皮异型增生发生率分别为50．0％、58．3％、37．5％、53.8％和25．0％。结论经口灌喂ST和DON可诱发NIH小鼠肺癌和腺胃黏膜异型增生。ST加DON可明显提高肺癌的发生率。     

Persisting long-term effects of a single carcinogenic dose of methylnitrosourea on epidermal growth in mice

The cell proliferation in hairless mouse epidermis was studiedbefore tumor development following a single application of acarcinogenic dose (2 mg) of N-methyl-N-nitrosourea (MNU). Thenumber of basal and suprabasal cells, the mitotic index (MI)and the mitotic rate (MR) were scored in histological sections.The [³H labeling index (LI) and the mean grain count (MGC) werescored in histological sections or in nears of basal cells.Flow cytometric two-parameter analyses of cellular DNA and proteincontent were performed on isolated epidermal basal cells. Anincreased MR and a slight but consistent epidermal hyperplasiawere found. A 24-h study performed 25 weeks after MNU applicationshowed that the MR and MI were altered in a circadian stage-dependentmanner with considerably increased values around noon when thecircadian rhythms had their peaks, followed by normal valuesaround midnight. The LI was generally increased, but showeda normal circadian rhythm with high values at night and lowvalues during day. The MGC was reduced at night and in the morningwhen the LI values were high. The results show that a singlecarcinogenic dose of MNU caused alterations in the epidermalgrowth kinetics that persisted until tumor development. Thealtered growth parameters, however, had circadian rhythms thatwere in phase with control rhythms. Assuming a constant sizeof the proliferative compartment, the increased mitotic activityindicated a considerable shortening of the mean cell cycle time.     

Aging and cancerogenesis. IV. Interrelationships among age, immune response, and tumor incidence in several strains of mice

Immune reactivity of mice with various incidences of spontaneous tumors was measured at different points in their lifespan. Cytotoxic activity of immune sera from 3- and 12-month-old mice was high or moderately high in AKR/J, SJL/J, and A/HeJ mice in which high incidences of spontaneous tumors occur by the 12th month, and in SWR/J and C3HeB/-FeJ mice in which high incidences of spontaneous tumors reportedly occur after 18 months of age. A decrease in primary antibody response accompanied old age in 4 of the 6 strains tested (including randombred Swiss ICR/Ha), but not in AKR/J or A/HeJ mice. These facts, together with previous tests of cellular response in these mice, imply that under these experimental conditions little correspondence exists between humoral and cellular response. Further, host immunity may be only secondarily implicated in neoplastic formation.     

Carcinogenic effects of acrylamide in Sencar and A/J mice

Acrylamide structurally resembles vinyl carbamate, a proposed proximate carcinogenic form of ethyl carbamate. To test the hypothesis that acrylamide should possess carcinogenic properties, it was tested in the Salmonella-microsome assay for point mutation, as a skin tumor initiator in the Sencar mouse, and for its ability to induce lung adenomas in the A/J mouse. Acrylamide was found to be without activity as a mutagen in Salmonella strains TA 1535, TA 1537, TA 98, and TA 100 both in the presence and absence of rat liver microsomes using both the plate and liquid suspension assays. However, acrylamide was found to approximate ethyl carbamate in potency as a tumor initiator in the skin of the female Sencar mice. As with ethyl carbamate, acrylamide was more potent by systemic routes of administration relative to topical application. Acrylamide was also found to induce lung adenomas in male and female A/J mice using both the p.o. and i.p. routes of administration. Acrylamide was approximately one-seventh as potent as ethyl carbamate in the induction of lung adenomas. These data confirm the hypothesis that acrylamide possesses carcinogenic properties similar to ethyl carbamate.     

Myocardial injury induced by a single dose of adriamycin: an electron microscopic study.

Adriamycin cardiomyopathy has been studied under the electron microscope using myocardial ventircular cells of CRF mice, previously treated with 10 mg/kg body weight of the drug given in a single intravenous injection. Within 10 min myocardial cell nucleoli show a nucleolonema fragmentation, and during the following 3 hours they acquire the nucleolar segregation pattern. Fourteen hours after drug injection, nucleolar morphology again becomes normal, while areas of focal degeneration, characterized by damaged mitochondria and enlarged smooth reticulum cisternae, appear in the sarcoplasm. One to 3 days later the degeneration process involves the myofibrillar component, and after 50 days the great majority of myocardial ventricular cells is damaged. The early appearance and the functional significance of nucleolar segregation support the hypothesis that adriamycin cardiotoxicity might be dependent on its ability to bind to myocardial cell DNA. The consequent failure of RNA and protein synthesis, impairing the continuous renewal of myofibrillar and mitochondrial components of the cell, might explain the progressive myocardial damage.     

Comprehensive genetic and histopathologic study reveals three types of neuroblastoma tumors.

PURPOSE: To determine the relationship between multiple genetic features, tumor morphology, and prognosis in neuroblastoma. PATIENTS AND METHODS: The genetic alterations and morphologic features that underpin three histopathologic risk classifications were analyzed in 108 neuroblastoma patients. Tumors were subdivided into four groups based on the three most frequent and prognostically significant genetic alterations (17q gain, 1p deletion, and MYCN amplification), and all other genetic, morphologic, and clinical data were analyzed with respect to these groups. RESULTS: Our analyses identify three nonoverlapping tumor types with distinct genetic and morphologic features, defined here as types 1, 2, and 3. Type 1 tumors show none of the three significant genetic alterations and have good prognosis. Both type 2 (17q gain only or 17q gain and 1p del) and type 3 (17q gain, 1p del, and MYCN amplification) tumors progress. However, these tumor types are distinguished clinically by having significantly different median age at diagnosis and median progression-free survival (PFS). Multivariate analysis indicates that 17q gain is the only independent prognostic factor among all genetic, histopathologic, and clinical factors analyzed. Among histopathologic risk systems, the International Neuroblastoma Pathology Classification was the best predictor of PFS. CONCLUSION: Our results indicate that specific combinations of genetic changes in neuroblastoma tumors contribute to distinct morphologic and clinical features. Furthermore, the identification of two genetically and morphologically distinct types of progressing tumors suggests that possibilities for different therapeutic regimens should be investigated.