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.     

Enhancement of low-dose N-2-fluorenylacetamide-induced hyperplastic liver nodules by pre-existing cirrhosis

The potentiating effect of pre-existing cirrhosis on the fonnationof hyperplastic liver nodules and/or foci was investigated byfeeding a low dose (0.008%) of 2-N-fluor-enylacetamide (FAA)in a choline-deficient (CD) diet for 32 weeks to cirrhotic andnon-cirrhotic rats. Liver cirrhosis was induced by feeding therats a CD diet for the preceding 36 weeks. The number and areaof -glutamyltranspeptidase-positive hyperplastic liver nodulesand/or foci were significantly larger in cirrhotic rats exposedto low-dose FAA than in non-cirrhotic rats similarly treated.Hyperplastic liver nodules and/or foci were not observed inrats continuously fed a CD diet alone for 68 weeks (controlcirrhotic rats). The results suggest that cirrhotic liver mightalter the metabolic response to FAA, even at low doses, andlead to enhanced induction of hyperplastic liver nodules and/orfoci.     

The regulation of serine dehydratase and glucose-6-phosphatase in hyperplastic nodules of rat liver during diethylnitrosamine and N-2-fluorenylacetamide feeding.

Changes in the levels of serine dehydratase and glucose-6-phosphatase induced by dietary stimuli or starvation in hyperplastic nodules of rat liver during diethylnitrosamine or N-2-fluorenylacetamide feeding were studied by immuno- and enzyme histochemical methods. The study was performed during carcinogenesis through a combined method of enzyme histochemistry and radioautography. Serine dehydratase was observed diffusely in the cytoplasm of the original hepatocytes in the periportal zone and was induced markedly during diethynitrosamine feeding but only slightly during N-2-fluorenylacetamide feeding. The enzyme was deficient and not inducible in hyperplastic nodules during their developing phase. Later during the feeding period, however, there was an elevation of the level of serine dehydratase and its inducibility with time in the majority of the nodules. A good correlation was observed between serine dehydratase and glucose-6-phosphatase in their elevated levels and response to enviornmental stimuli. There was a minor group of hyperplastic nodules in which the deficiencies of these enzymes persisted and enzyme induction was not observed. A greater number of hyperplastic nodules with persistent enzyme deficiency was seen during diethylnitrosamine carcinogenesis. These results provide further information about the changing biological nature of hyperplastic nodules with respect to their metabolic adaptability and enzyme levels during hepatocarcinogenesis.     

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.     

Effect of neonatal castration on liver tumor induction by N-2-fluorenylacetamide in suckling BALB/c mice

BALB/c mice were castrated at 2 days of age and control animalswere sham-operated. Untreated male and female mice were alsoincluded for comparison. One-half of the mice in each groupwere fed on alternate days with 1.5% N-2-fluorenylacetamidesuspended in 1% gelatine by stomach tube beginning at 1 weekof age for a total of 14 feedings. The experiment was terminatedwhen the mice reached one year old. Approximately 30% of themale mice, but none of the females, developed liver tumors ingroups fed carcinogen. This male predominance in the incidenceof hepatocellular carcinomas was completely abolished when theanimals were castrated neonatally. No lesions were observedin the liver of mice treated with gelatine suspension aloneexcept one with neoplastic nodule. Although it has been observedthat liver tumors are produced more readily in younger thanin older mice, the present investigation shows that male hormonalenvironment during early life is more important than the ageon the development of liver tumors initiated by carcinogen.     

Microsomal metabolism of the carcinogen, N-2-fluorenylacetamide, by the mammary gland and liver of female rats. II. Glucuronidation of ring- and N-hydroxylated metabolites of N-2-fluorenylacetamide

We determined UDP-glucuronyltransferse (UDP-GT) activities ofhepatic and mammary gland microsomes of female rats with p-nitrophenoland the ring- and N-hydroxylated metabolites of N-2-fluorenylacetamlde(2-FAA) and the effects of hepatic inducers of UDP-GT's on theseglucuronidations. Pre-treatment of non-lactating (NL)and lactating(L) rats with ß-naphthoflavone (ß-NF) significantlyincreased glucuronidations, of p-nitrophenil, aphenolic metabolitesof 2-FAA, especially of 5-hydroxy 2-FAA and also of N-hydrosy-2-FAAby hepatic microsomes. Pre-treatment of L rats with ß-NFor 3-methyl cholanthrene (3-MC) significantly incresed glucuronidationsof these compounds by mammary gland microsomes suggesting thatboth liver and mammary gland of L rats possess similar UDP-GTactivities. In NL rats, UDP-GT activities of mammary microsomestoward phenols were greater than in L rats, and except for thatof 5- and 7- hydroxy-2-FAA, were not inducible with ß-NF.The data obtained with L rats, the greater magnitude of stimulationof the hepatic UDP-GT of NL rats by ß-NF than by phenobarbital, and the lack of effect of the latter on UDP-GT ofmammary microsomes suggested that the phenolic metab olitesof 2-FAA and N-hydroxy-2-FAA share chiefly the characteristicsof substrates for group 1 UDP-GT activities (i.e., those induciblewith ß-NF or 3-MC). Neither inducer increased glucuronidationof 9-hydroxy-2-FAA, a relatively poor substrate for UDP-GT ofmammary or hepatic micro somes. In contrast to hepatic microsomeswhich formed considerable amounts of the glucuronide of N-hydroxy-2-FAA,mammary gland microsomes glucuronidated this substrate to aminor extent only. This suggested that glu curonide of N-hydroxy-2-FAAmay play a role in systemic, but not in local mammary tumorigenesisby N-hydroxy-2-FAA.     

Differential regulation of two microsomal epoxide hydrolases in hyperplastic nodules from rat liver

Two microsomal epoxide hydrolases of the rat liver were foundto be differentially regulated in hyperplastic nodules. Whilstthe activity for substrates of the well-known microsomal epoxidehydrolase with a broad substrate specificity (EHb), benzo[a]pyrene4,5-oxide and androstene oxide (16,17-epoxyandrosten-3-one),was greatly (5-fold) increased in the nodule microsomes andmoderately (2-fold) increased in the surrounding tissue, thatfor the substrate of the novel microsomal epoxide hydrolase,cholesterol 5, 6-oxide (EH_ch) remained unchanged. Since bothenzymes convert endogenous steroid epoxides but with distinctstructural features, this differential regulation may indicatea role of endogenous steroid epoxide(s) of a defined structureduring hepatocarcinogenesis. Alternatively, this differentialregulation may serve as a marker during hepatocarcinogenesis.     

Concanavalin A agglutination of cells from primary hepatocellular carcinomas and hepatic nodules induced by N-2-fluorenylacetamide.

A previous study demonstrated that cells of transplantable hepatocellular carcinomas were agglutinated by the plant lectin concanavalin A, while normal hepatocytes were not. In the present experiments, 95% or more of cells obtained from primary hepatocellular carcinomas which resulted from exposure of rats to N-2-fluorenylacetamide were agglutinated by this lectin. Exposure to this carcinogen also produces grossly visible foci of morphologically and biochemically altered hepatocytes which have been termed hepatic (hyperplastic; premalignant, neoplastic) nodules. Although these hepatocyte aggregates are generally accepted as precursors of the hepatocellular carcinomas, no agglutination was detected when their cells were exposed to concanavalin A. These results indicate that concanavalin A agglutinability is not acquired as a result of tumor transplantation. Furthermore, they suggest that significant alterations must occur in the cells of hepatic nodules prior to the manifestation of malignant behavior.