Effect of phenacetin and N-alkylacetanilides on N-2-fluorenylacetamide hepatocarcinogenesis

Phenacetin, N-methylacetanilide and N-ethylacetanilide prevented N-2-fluorenylacetamide hepatocarcinogenesis in F344 strain rats. Phenacetin was most effective followed by N-ethylacetanilide. Phenacetin was not carcinogenic when fed at 0.8% level for 16 weeks followed by control diet for another 10 weeks.     

Nucleolar organizer regions in soft tissue sarcomas

Argyrophilic proteins of nucleolar organizer regions (AgNORs) were studied in a series of 65 sarcomas of the soft tissues and 2 cases of fibrohistiocytic tumors of intermediate malignancy. The numbers of AgNORs per nucleus and the size of AgNORs were determined and compared with pathomorphologic parameters such as grading of malignancy, cellularity and tumor diameter. The main finding of this study was that AgNOR counts showed significant differences between low-grade malignant (G 1) and high-grade malignant (G 3) tumors predominantly based on a correlation with the frequency of mitosis. All the other criteria of tumor grading (nuclear pleomorphism, differentiation, amount of necrosis), tumor diameter and cellularity showed no differences with regard to AgNOR counts. Though it was observed that tumor giant cells possessed predominantly coarse granular AgNORs, no correlation was found between AgNOR size and any of the parameters investigated. In spite of a clear statistically significant result gained by an evaluation of all cases, a heterogeneity of AgNORs in relation to mitotic activity within a few histological tumor types could be observed, making it difficult to suggest the determination of AgNORs as a parameter with general validity for all soft tissue sarcomas.     

NUCLEOLAR ORGANIZER REGIONS IN CUTANEOUS T CELL LYMPHOMAS

The present work studied the application of AgNOR count to differential diagnosis between cutaneous T cell lymphoma (CTCL) and cutaneous pseudolymphoma (CPL). Paraffin sections from 50 mycosis fungoides (22 MFI-Premycotic stage, 24 MF Ⅰ infiltrative stage and 4 MF Ⅲ - tumor stage), 2 nonepidermotropic cutaneous T cell lymphoma (NECTCL) and 9 CPL were investigated. In each case, 200 cells randomly selected were examined using a × 100 oil immersion lens. The mean number, standard deviation and standard error of the mean of AgNOR counts were as follows: MFⅠ 1.17±0.09, SEM = 0.01; MⅡ 1.17±0.01, SEM = 0.01; MF Ⅲ. 3.55±0.87, SEM = 0.43; NECTCL 4.5±0.28, SEM -0.199; CPL 1.17±0.1, SEM ± 0.03. The results revealed a highly significant difference between CTCL (MFⅢ NECTCL) and CPL (t = 4.75, P<0.001), tumor stage (MF Ⅲ) and pretumor stage (MFI, MF Ⅱ) of mycosis fungoides (t = 4.75, P<0.001). Thus. AgNOR count is valuable in differential diagnosis.     

-Fetoprotein and hepatocarcinogenesis in rats fed 3'-methyl-4-(dimethylamino)azobenzene or N-2-fluorenylacetamide

Serum α-fetoprotein (af) of rats during hepatocarcinogenesis by 3′-methyl-4-(dimethylamino)azobenzene (3′-Me-DAB) or N-2-Fluorenylacetamide (FAA) was checked by the micro-Ouchterlony method throughout the course of the study. The serum af appeared in two phases, early from the 3rd to 5th week and late during the development of large carcinomas. The frequency of early appearance of serum af varied considerably according to the difference in age, sex, and strain of the animals or the difference in type or dose of the carcinogen. In general, 3′-Me-DAB was a much stronger carcinogen than FAA in producing early serum af. A good correlation was observed between the serum af level and the intensity of oval cell proliferation in the liver at this stage. The cell composing the nodular hyperplasia in 3′-Me-DAB or the area of hyperplasia in FAA carcinogenesis, which replaced the major part of the liver in the next stage and had been considered the most possible forerunner of the carcinoma, did not seem to produce af. The serum af in the late phase was produced by the carcinoma. The majority of the 3′-Me-DAB-induced carcinomas belonged to the mixed type carcinoma. This type of carcinoma was strongly af-producing with the required minimal size of 10 mm in diameter for positive serum af. Most of the FAA-induced carcinomas were of the trabecular type. This type of carcinoma was relatively weak in af-production and the serum af was negative until a trabecular carcinoma became larger than 20 mm in diameter. The appearance of early serum af was distinct evidence of a fair progress of hepatocarcinogenesis, and a positive correlation was found between the degree of this phenomenon and the frequency of development of a carcinoma producing large amounts of af. However, the appearance of early serum af was not an absolute requisite for later development of carcinomas.     

Nucleolar organizer regions in Spitz nevi and malignant melanomas

Nucleolar organizer regions (NOR) are loops of DNA that transcribe ribosomal RNA; they can be easily identified in formalin fixed paraffin embedded tissue using a silver (Ag) method. It has been suggested that the number of AgNOR per cell can differentiate between benign and malignant melanocytic lesions of skin. We have studied 29 Spitz nevi (SN) and 39 invasive malignant melanomas (MM) by the same silver method. SN showed between 1.0 and 1.6 AgNOR per cell with a mean of 1.2. MM counts ranged from 1.2 to 4.2 with a mean of 2.0. It is concluded that the AgNOR method cannot reliably differentiate SN from MM; however, a count of more than 2.0 AgNOR per cell would favor a diagnosis of MM rather than SN.     

DNA ligase activities during hepatocarcinogenesis induced by N-2-acetylaminofluorene

A progressive accumulation of DNA breaks has been reported tooccur in nuclear DNA obtained from putative premalignant hepaticlesions induced by carcinogens. To determine if this alterationresulted from a defect in the level of, or functional activityof DNA ligases, we compared these enzymes in normal rat liver,24-h regenerating liver, and hepatic nodules at intervals aftercessation of N-2-acetylaminofluorene (AAF) treatment. Nuclearextracts of hepatocytes were separated into soluble and chromatinfractions, and multiple forms of DNA ligase activity were obtainedby AcA34 gel filtration chromatography. The activities of thetwo largest species, DNA ligase la (480 kd) and DNA ligase Ib(240 kd), were present exclusively in soluble, nuclear fractionsand were increased 4-fold and 2-fold, respectively, in 24-hregenerating livers. In AAF-induced nodules, these species wereincreased 3-fold and 1.5-fold, respectively, above those ofnormal rat liver, somewhat higher than predicted from the rateof cell division. In all of the test tissues, these ligase speciesdemonstrated identical sensitivity to inhibition with 0.1 MNaCl or heating at 50°C. DNA ligase II (80 kd) was foundin both soluble nuclear fractions and chromatin at approximatelyidentical levels in all tissues tested. Ligase II from all tissuesalso demonstrated identical responses to salt and heat. Thesedata support the concept that DNA ligases la and Ib are relatedto DNA replication and suggest that ligase II may be a repairenzyme. The failure to detect significant alterations from expectedvalues in the hepatic nodules and the lack of alteration insensitivity to salt and heat indicate that the accumulationof DNA damage (presumably breaks) previously observed in carcinogen-inducedaltered hepatocytes is not due to an alteration in the levelor the biochemical properties of DNA ligase.     

Intrahepatic polyamine levels during rat liver carcinogenesis induced by N-2-fluorenylacetamide

During a period of 200 days, the chronological changes of polyaminelevels (putrescine, spermidine and spermine) were observed inthe liver of adult female Sprague Dawley rats submitted to hepatocarcinogenesisby N-2-fluorenylacetamide (FAA). Three groups of 70 rats eachwere used: (1) Control 1: normal diet; (2) Control 2: low proteinand low riboflavin diet; and (3) Experimental: 0.06% FAA addedto the diet. No significant differences were noted for tissuelevels of the three polyamines when the two control groups werecompared. In contrast, considerable variations of these moleculeswere observed as a function of time in the FAA treated group:(a) an early and constant rise was seen in putrescine, with3 maxima at days 10, 60 and 150. This last peak was the highest:25 ± 6 nmol/g (8 times the value for the controls atthis time), and coincided with the appearance of cancerous lesions.(b) While spermidine levels varied during the experiment, nosignificant differences were noted in comparison with the controlgroups. Mean levels (nmol/g) were: 535 ± 108 Control1; 552 ± 95 Control 2; 633 ± 160 FAA-treated group,(c) Spermine levels were significantly lowered, with 3 minimacorresponding to the putrescine maxima. The lowest minima wasobserved on day 60: 114 ± 67 nmol/g, i.e., 4 times lowerthan the controls. This work shows that polyamine metabolismis profoundly modified during chemical carcinogenesis, but thepossible effect of polyamines on tumorigenesis itself cannotbe assessed at this point since modifications of polyamine levelsare probably also associated with phenomna of liver necrosisand compensatory tissue proliferation observed during the experiment.     

Nucleolar organizer regions in precancerous and cancerous lesions of the bronchus

Using a silver staining technique, nucleolar organizer region-associated proteins (Ag-NOR) were studied in paraffin sections of five specimens of normal bronchial epithelium, eight of atypical squamous metaplasia, five of carcinoma in situ, and seven of microinvasive squamous cell carcinoma. The mean number of Ag-NOR in the nucleus were normal epithelium 1.2 +/- 0.1 (mean +/- SD), atypical squamous metaplasia (borderline lesion) 2.2 +/- 0.5, carcinoma in situ 3.8 +/- 0.6, and microinvasive squamous cell carcinoma 4.8 +/- 1.1. There was a highly significant difference between the Ag-NOR numbers in the atypical squamous metaplasia and those in the carcinoma in situ (P less than 0.01). The Ag-NOR staining is a useful technique for the differential diagnosis of difficult borderline lesions in the bronchial epithelium.     

Monoclonal antibodies recognizing oval cells induced in the liver of rats by N-2-fluorenylacetamide or ethionine in a choline-deficient diet

In this paper, we describe the production of a panel of monoclonal antibodies which define antigens which distinguish between hepatocytes and oval cells. These antibodies were obtained from hybridomas constructed from the spleens of mice immunized by a novel protocol designed to suppress response to unwanted or immunodominant epitopes. Of the antibodies obtained, four, 258.7, 270.11, 258.34, and 270.38, were directed to antigens of morphologically defined oval cells, while two, 258.26 and 270.26, defined cytoplasmic antigens of hepatocytes. Examination of frozen sections of normal, regenerating adult and fetal liver and livers from rats fed 2-acetylaminofluorene or ethionine in a choline-deficient diet indicates that morphologically defined oval cells may in fact comprise a phenotypically complex set of cells composed of at least three antigenically distinct subpopulations. The patterns of expression of the antigens defined by these antibodies suggest two possible pathways of liver cell differentiation.     

Microsomal metabolism of the carcinogen, N-2-fluorenylacetamide, by the mammary gland and liver of female rats. I. Ring- and N-hydroxylations of N-2-fluorenylacetamide

We determined ring- and N-hydroxybtions of a systemic mammarygland cardnogen, N-2-fluorenylaeetamide (2-FAA), by microsomalfractions of liver and mammary gland of female rats and theeffects of in vivo and/or in vitro modifiers of these oxidations.Pretreatment of lactating rats with 3-methylcholanthrene (3-MC)or ß-naphthoflavone (ß-NF) and non-lactating(50-day old virgin) rats with ß-NF showed similareffects in that the formation of 3-, 5-, 7-, 9- and N-hydroxy-2-FAAby hepatic microsomes was increased manyfold and the formationof 1-hydroxy-2-FAA was induced. In mammary gland microsomes,the formation of 3-, 5- and 7-hydroxy-2-FAA was likewise increased,but of 9-hydroxy-2-FAA was unaffected. Only mammary microsomesof lactating rats had capacity for N-hydroxylation which wasincreased {small tilde}3 times by pretreatment of rats with3-MC or ß-NF. All of the induced increases of metabolitesof 2-FAA in hepatic and mammary microsomes were inhibited by0.1 mM -naphthoflavone (-NF) in vitro. Pretreatment of non-lactatingrats with phenobarbital increased only the formation of 7-hydroxy-2-FAAin hepatic microsomes which was further stimulated by -NF invitro. The latter also stimulated the formation of 7- and 9-hydroxy-2-FAA by hepatic microsomes of the uninduced rats, buthad no effects in mammary microsomes, in which 9-hydroxy-2-FAAwas a major metabolite. Hence, the data showed qualitative andquantitative differences between lactating and non-lactatingrats in metabolism of 2-FAA by mammary microsomes which mayresult from differences in the levels (e.g., of cytochrome P-450)and activities of microsomal enzymes determined herein. In hepaticmicrosomes of these rats, differences in quantities of metabolitesof 2-FAA (3-, 7-, 9- and N-hydroxy-2-FAA) were found in cornoil-treated rats only. The solvent (methanol or acetone) usedfor addition of 2-FAA to the incubation mixtures altered quantitativelythe metabolite profiles in hepatic and mammary microsomes of3-MC or ß-NF treated rats. The formations of 1- and3- or 5- and 7-hydroxy-2-FAA were greater in the presence ofacetone or methanol, respectively. The results of this studysuggest that the formation of phenolic and N-hydroxy metabolitesof 2-FAA in both hepatic and mammary microsomes of lactatingrats is catalyzed by similar form(s) of cytochrome P-450 inducedby pretreatment with 3-MC or ß-NF. Lack of inductionN-hydroxylation of 2-FAA in mammary microsomes of non-lactatingrats supports our earlier conclusion that the formation of aproximate metabolite in mammary tumorigenesis by 2-FAA is accomplishedin the liver.     

Inhibitory effect of hypothalamic lesions on liver tumor induction by N-2-fluorenylacetamide in male rats.

Bilateral electrolytic lesions were placed in the median eminence area of the hypothalamus in 12-week-old male Wistar rats. Sham-operated and untreated control rats were also included. Two weeks later, one-half of them were given 0.03% N-2-fluorenylacetamide incorporated into the diet for 16 weeks with adequate resting periods in between. The animals were killed 34 weeks after the last carcinogen feeding. The results show that lesions in the hypothalamus effectively inhibited liver tumor formation (0 of 16, 0%). In contrast, the incidence of hepatocellular carcinomas in sham-operated rats was 38.5% (5 of 13), and that of untreated controls was 42.9% (6 oactive thyroid glands, and shorter nasoanal lengths were observed in rats with lesions in the hypothalamus irrespective of carcinogen treatment. It is apparent from these data that lesions in the median eminence area of the hypothalamus inhibit the induction of liver carcinogenesis with N-2-fluorenylacetamide in male rats.