Enzyme histochemical and morphological phenotype of amphophilic foci and amphophilic/tigroid cell adenomas in rat liver after combined treatment with dehydroepiandrosterone and N-nitrosomorpholine

Male Sprague-Dawley rats were investigated after N-nitroso-morpholine(NNM) treatment with concomitant and subsequent administrationof dehydroepiandrosterone (DHEA) for development of pre-neoplasticand neoplastic liver lesions. In addition to clear, acidophilic,mixed cell and basophilic foci, a hitherto undescribed lesiontype demonstrating a unique morphological and histochemicalphenotype was observed in animals receiving both NNM and DHEA.The cells of the majority of these lesions for which we proposethe designation amphophilic foci were characterized by increasedgranular acidophilia and randomly scattered cytoplasmic hasophilia.Histochemically, reduced glycogen content and elevated activityof glucose-6-phosphate dehydrogenase (G6PDH) glyceraldehyde-3-phosphatedehydrogenase (GAPDH), acid phosphatase (AP), succinate dehydrogenase(SDH) and catalase (CAT) were evident. The lack of -glutamyltranspeptidase (GGT) or glutathione S-transferase placentalform (GST-P) in foci of this type allowed clear differentiationfrom other NNM-induced focal lesions while suggesting certainsimilarities to pre-neoplastic cells induced by hypolipidemicagents. Similar enzyme histochemical patterns were characteristicfor foci and later appearing nodules (adenomas) com posed ofamphophilic/tigroid cells the basophilic material of which wasIncreased and frequently arranged in long striped bands. DHEAtreatment, while not itself inducing any preneoplastic foci,was thus associated with altered phenotypic expression of fociand adenomas generated by NNM.     

Modifying influence of dehydroepiandrosterone or butylated hydroxytoluene treatment on initiation and development stages of azaserine-induced acinar pancreatic preneoplastic lesions in the rat

Dietary administration of dehydroepiandrosterone (DHEA) (0.6%) or butylated hydroxytoluene (BHT) (1%) during or subsequent to two i.p. injections of azaserine (30 mg/kg body wt) resulted in significant alteration of yield of preneoplastic lesions in both pancreas and liver. While concomitant application appeared not to have any effect on subsequent development of glutathione S-transferase P form (GST-P) positive hepatocellular lesions in either case, BHT and to a lesser extent also DHEA reduced initiation of pancreatic acinar carcinogenesis. Both BHT and DHEA were associated with significant increase in GST-A form positive pancreatic foci when administered after cessation of carcinogen treatment while clearly inhibiting liver lesion development. The results point to a marked differential in the response of the liver and pancreas to external stimulus with regard to preneoplastic focal lesions while demonstrating significant second stage promotion of pancreatic acinar carcinogenesis by BHT and DHEA.     

Phenotypic modulation of hepatocarcinogenesis and reduction in N-nitrosomorpholine-induced hemangiosarcoma and adrenal lesion development in Sprague-Dawley rats by dehydroepiandrosterone

Dietary administration of 0.25% dehydroepiandrosterone (DHEA)during and subsequent to 7 weeks treatment with N-nitrosomorpholine(NNM) resulted in significantly reduced development of adrenalcortical lesions and hemangiosarcomasns in the liver. In addition,distinct phenotypic modulation of hepatocellular tumours wasobserved after combined hormone and carcinogen treatment. Thusthe neoplasms were characterized by a higher degree of differentiation,lower mitotic rate and reduced potential for metastasis as comparedto tumours observed after NNM alone. The data clearly indicatethat DHEA exerts an inhibitory effect on both adrenal and hepatocarcinogenesissimilar to that earlier reported for neoplasia in lung, thyroid,colon and skin     

The influence of subsequent dehydroepiandrosterone,diaminopropane, phenobarbital,butylated hydroxyanisole and butylated hydroxytoluene treatment on the development of preneoplastic and neoplastic lesions in the rat initiated with di-hydroxy-di-N-propyl nitrosamine

The comparative modifying potential of dehydroepiandrosterone (DHEA), diaminopropane (DAP), phenobarbital (PB), butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on the development of lesions initiated by dihydroxy-di-n-propyl nitrosamine (DHPN) in F344 rats were investigated. DHEA, BHA and BHT were all associated with significant reduction in numbers of glutathione-S-transferase P form (GST-P) positive foci in the liver whereas PB brought about their enhanced development. BHT and PB exerted promoting activity on the incidence of thyroid adenomas while DAP similarly increased lung adenoma formation. The results illustrate the advantages to be gained from two stage experiments using broad spectrum carcinogen initiation for comparative analysis of ‘modifiers’ of the neoplastic process and suggest that studies of enzyme alteration within putative preneoplastic lesions may be directly relevant to elucidation of mechanisms underlying such modification.     

Modulation of Dihydroxy-di-n-propylnitrosamine-induced Liver Lesion Development in Opisthorchis-infected Syrian Hamsters by Praziquantel Treatment in Association with Butylated Hydroxyanisole or Dehydroepiandrosterone Administration

The effects of praziquantel coupled with dehydroepiandrosterone (DHEA) or butylated hydroxyanisole (BHA) administration 16 weeks subsequent to dihydroxy-di- n -propylnitrosamine (DHPN) treatment and infection with Opisthorchis viverrini (OV) on lesion development in the liver of Syrian hamsters were investigated. Animals were given 80 OV metacercariae and then two i.p. injections of DHPN (500 mg/kg body weight) 4 and 5 weeks thereafter. At week 16, groups received praziquantel (250 mg/kg, i.g.) and were placed on normal diet or diet supplemented with BHA (1%) or DHEA (0.6%) until they were killed at week 24. Histopathological assessment revealed that, whereas antihelminthic treatment alone resulted in a clear reduction in hepatocellular lesion development, effects on cholangiocellular lesions were equivocal. BHA and DHEA, in contrast, were both associated with a significant reduction in frequency of cholangiofibrosis and cholangiocellular carcinoma. The former chemical, however, increased the numbers of liver nodules while the hormone brought about a decrease as well as a shift in the phenotype of the lesions. The results thus indicate that although cholangiocellular lesion development may, unlike generation of hepatocellular nodules, be to a certain extent independent of the continued presence of parasite, it can be influenced by exogenous treatments.     

Early lesions induced by DHPN in Syrian golden hamsters: influence of concomitant Opisthorchisinfestation, dehydroepiandrosterone or butylated hydroxyanisole administration

The effects of concomitant Opisthorchis infestation and dehydroepiandrosterone(DHEA) or butylated hydroxyanisole (BHA) administration on dihydroxy-di-n-propylnitrosamine(DHPN) induction of preneoplastic lesions were investigatedin Syrian golden hamsters. Whereas parasite infection was primarilyassociated with first-order ductular proliferation in the liverand a secondary appearance of cholangiofibrotic lesions, DHEAtreatment brought about increased carcinogen toxicity and enhancedgeneration of glutathione-S-transferase P (GST-P)-positive hepatocellularfoci, liver cysts and focal proliferative changes in the pancreas.BHA also exerted an enhancing influence on pancreatic but notliver carcinogenesis. The results suggest that whereas alterationof DHPN metabolism by DHEA and BHA treatment effected changesat the initiation level, opisthorchiasis principally exertedan enhancing influence subsequent to carcinogen withdrawal.     

Growth hormone modifies the growth rate of enzyme-altered hepatic foci in male rats treated according to the resistant hepatocyte model

Male and female Wistar rats were given an initiating i.p. injectionof diethylnitrosamine (DEN; 200 mg/kg body wt). Two weeks laterthe rats were given a diet containing 0.02% (w/w) 2-acetylaminofluorene(2-AAF) for 2 weeks. In the middle of the 2-AAF treatment a70% partial hepatectomy (PH) was performed. In order to identifythe pituitary hormone responsible for the previously observedsex difference (male > female) in and influence of ectopicpituitary grafts on focal growth during 2-AAF/PH selection ofenzyme-altered foci, male rats were treated with a continuousinfusion of bovine growth hormone (bGH; 6 µg/h) or ovineprolactin (oPrl; 6 µg/h) by way of osmotic minipumps.Hormonal treatment was started 1 week after initiation and wasfinished 1 week after the 2-AAF selection period. All rats werekilled 6 weeks after initiation and liver sections were stainedfor -ghitamyttransferase. The number of foci/cm² as well asthe area per focus and area ratio (mm² foci/cm² liver section)were calculated. Whereas no significant differences in the numberof foci /cm² were observed between the different groups of rats,bGH treatment of male rats decreased both the area/focus andthe area ratio down to the female level. No significant effectswere seen following oPrl administration when compared with controlmales. In vitro studies of subcellular preparations from theliver lobes obtained at PH showed that the sexually differentiatedN-hydroxy-2-AAF sulfotransferase activity (male > female)in male rats was ‘feminized’, i.e. decreased, bybGH administration, but not by infusion of oPrl. The presentinvestigation strengthens the view of growth hormone as an importantdeterminant of sex differences in chemical carcinogenesis inrat liver, possibly via an influence on carcinogen metabolism.     

Positive influence of dietary deoxycholic acid on development of pre-neoplastic lesions initated by N-methyl-Nnitrosourea in rat liver

The effect of deoxycholic acid (DCA) treatment subsequent toinitiation of F344 male rats with N-methyl-N-nitrosourea (MNU),a wide spectrum carcinogen inducing tumors in many organs, wasinvestigated. Rats were initially given four doses of MNU (50mg/kg) i.p. within a 2-week period combined with a two-thirdspartial hepatectomy performed at day 7 and then placed on basaldiet containing DCA at concentrations of 0.313, 0.125, 0.050and 0.020% for 21 weeks prior to final sacrifice. All organsstudied were carefully examined histologkally and histochemicallyfor development of neoplastic and pre-neoplastic lesions. DCAenhanced the induction of glutathione S-transferase positive(GST-P+) liver cell foci in a dose-related manner. Furthermoregroups of rats given DCA without prior MNU administration alsodeveloped dosedependent numbers of pre-neoplastic liver lesions.In addition, increased numbers of small intestine tumors wereapparent in DCA-treated animals although the difference wasnot significant. Induction of tumors in the thyroids, Zymbalglands, skin and peripheral nerves was not affected. The resultsindicate that DCA is a strong promoter of hepatocarcinogenesiswith possible complete carcinogenkity in the liver and promotionpotential for tumor development in the small intestine.     

Inhibition of 7,12-dimethylbenz[a]anthracene-initiated and 12-O-tetradecanoylphorbol-13-acetate-promoted skin papilloma formation in mice by dehydroepiandrosterone and two synthetic analogs

Previous work has demonstrated that the adrenal steroid, dehydroepiandrosterone(3-ß-hydroxy-5-androsten-17-one, DHEA), has broadspectrum tumor chemopreventive activity in laboratory mice andrats, inhibiting the development of spontaneous breast cancerand chemically induced tumors of the lung, colon, skin, thyroidand liver. DHEA treatment produces specific side-effects, includingestrogenic and androgenic action and an increase in liver weight,which could limit its use as a cancer chemopreventive drug.It is now shown that oral administration of the synthetic steroid16-fluoro-5-androsten-17-one, which lacks the side-effects ofDHEA treatment, to CD-1 mice inhibits 7,12-dimethyl- benz[a]anthracene-initiatedand 12-O-tetradecanoylphorbol- 13-acetate-promoted skin papillomaformation at both the initiation and promotion stage. The syntheticsteroid is more potent as an inhibitor of papilloma formationthan comparably admInistered DHIEA.     

Growth of pancreatic foci and development of pancreatic cancer with a single dose of azaserine in the rat

Studies were undertaken to characterize the growth of the azaserine-inducedputative preneoplastic lesions in rats and to determine if asingle dose of azaserine would be carcinogenic. Male Lewis ratswere given a single i.p. injection of 30 mg L-azaserine/kg bodyweight at 7 weeks of age. A purified diet was fed throughoutthe study. Rats (10–12 per group) were autopsied at 6,9, 12, 15 and 18 months postinitiation, and pancreases werequantitatively evaluated to characterize the growth of acidophilicand basophilic foci and nodules (henceforth called foci), andthe incidence of neoplasms. All azaserine-treated rats had foci,and at all times approximately equal numbers of acidophilicand basophilic foci were present in the pancreas. The numberof basophilic foci increased with time, and while their sizealso increased, this change was small compared with the increasein size of the acidophilic foci. Conversely, all acidophillcfoci appeared to be present by 6–9 months, and their sizegreatly increased with time. The data suggest that virtuallyall foci persist rather than regress or remodel. At 9 monthsthe incidence of carcinoma in situ was 30% and by 18 monthsthere was a 100% incidence of pancreatic cancers (58% carcinomain situ and 42% carcinoma).     

Inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase activity and gene expression by dehydrocpiandrosterone in preneoplastic liver nodules

Previous work has demonstrated that dehydropeiandros-terone(DHEA) strongly inhibits growth and de novo cholesterol (CH)biosynthesis in preneoplastic rat liver. Administration of amixture of 4 ribo- or deoxyribo-nucleosides of adenine, guanine,cytosine and uracil/thym-ine, prevents growth inhibition butnot inhibition of CH synthesis. The purpose of this paper wasto identify the site of inhibition of CH synthesis by DHEA.Persistent nodules (PNs) were induced, in diethylnitrosamine-initiatedmale F344 rats, by ‘resistant hepatocyte’ protocol.Fifteen weeks after initiation, nodule bearing rats and normalcontrols received a diet containing 0.6% DHEA for 3 weeks. Theywere then killed. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR)activity and mRNA levels were 18- and 14-fold higher, respectivelyin nodules than in normal liver. DHEA strongly inhibited HMGRactivity in both tissues in vivo, but had a slight effect onHMGR activity, when added in vitro to the reaction mixture fordetermination of this activity. In vivo DHEA treatment causeda 65% decrease in the level of HMGR mRNA in PNs, which, however,does not seem to completely account for the decrease in HMGRactivity (83%). Low density lipoprotein receptor (LDL-R) mRNAlevel underwent a slight decrease in PNs, with respect to controlliver, which did not lead to a significant decrease in ¹²⁵I-LDLbinding to LDL-R. DHEA treatment caused 30% and 24% increasesin LDL-R expression and ¹²⁵I-LDL binding, respectively, in nodules.These observations indicate that in addition to HMGR gene expression,increased influx of LDL into preneoplastic cells may contributeto the deregulation of mevalonate synthesis by DHEA. The observationthat HMGR activity and gene expression were still 3- to 5-foldhigher in PNs of DHEA-treated rats than in control liver, andprevious findings of preneoplastic liver cell growth in thepresence of relatively low CH synthesis, suggest that even relativelylow levels of mevalonate are sufficient for the growth of preneoplasticliver cells.     

Modifying influence of dehydroepiandrosterone on the development of dihydroxy-di-n-propylnitrosamine-initiated lesions in the thyroid, lung and liver of F344 rats

Following initial treatment of F344 rats with dihydroxy-di-n-propyinitrosamine,exposure to dehydroepiandrosterone (DHEA) administered in thediet at a concentration of 0.6% brought about significant decreasein weight gain, independent of food consumption, and inhibitedthe development of thyroid tumors and hepatocyte-altered enzymefoci. In addition to inducing a diffuse increase in glucose-6-phosphatasedehydrogenase (G6PD) and gammaglutamyl transpeptidase in theliver. DHEA treatment was associated with development of smallnumbers of basophilic hepatocellular foci which differed markedlyin enzyme phenotype from the clear cell (glycogen storing) lesionspredominating in the carcinogen-treated animals maintained onbasal diet. The results are consistent with the concept thatDHEA-modification of neoplastic development, as reported earlierin a number of different organs and here in the liver and thyroid,may be in some way partly mediated by changed expression ofthe key enzyme of the pentose phosphate pathway, G6PD, and relatedmetabolic systems. Heterogeneity in the quality of initiatedhepatocytes with regard to capacity for inhibition or promotionindicated by the present data point to the existence of morethan one pathway to tumour development in the rat liver.     

Phenotypic instability in focal and nodular lesions induced in a short term system in the rat liver

A comparative morphologic, morphometric and enzyme histochemicalinvestigation of lesions induced by short-term application ofN-nitrosomorpholine (NNM) and subsequent so-called ‘selectionpressure’ was carried out in order to assess the characteristicsof the numbers of induced putative preneoplastic populationsand to cast light on reversibility associated with this model.The glycogen storage foci, mixed cell foci and neoplastic nodulesobserved after ‘selection pressure’ were in principlesimilar to those seen after stop experiments, although alterationsin morphology and enzyme phenotype of individual cells wereusually far more pronounced after short-term induction. It wasestablished that 75% of the lesions were no longer visible 11weeks after withdrawal of induction stimuli and that a largeproportion of these remaining demonstrated heterogeneity inmorphological and histochemical markers indicative of reversionto normal phenotype. After a further 10 weeks a slight increasein number of foci associated with decrease in size and enhancedhomogeneity in phenotypic markers was established. The behaviourof foci and nodules undergoing reversion was considered withrespect to changes in basophilia and glycogen storage and activityof the enzymes glucose-6-phosphate dehydrogenase, glucose-6-phosphatase,glyceraldehyde 3-phosphate dehydrogenase, glycogen phosphorylaseand synthase, acid phosphatase and -glutamyl transpeptidaseand correlated with location of altered cellular populationswithin the liver functional acinus.     

Expression of peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase enzyme and its mRNA in peroxisome proliferator-induced liver tumors

We have examined ciprofibrate and dehydroepiandrosterone (DHEA)-inducedhepatic lesions for the peroxisomal ß-oxidation systemenzyme peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase(PBE) and its mRNA using SDS-polyacrylamide gel electrophoresis,antibodies and cDNA probe. All 12 neoplastic nodules and ninehepatocellular carcinomas (HCCs) that were analyzed for PBEmRNA by in situ hybridization showed an intense signal comparableto the adjacent non-neoplastic liver. SDS-polyacrylamide gelelectrophoresis of postnuclear fractions of six HCC and adjacentliver tissue showed a marked increase in an 80 kDa polypeptide.Immunoblot and Northern blot analysis showed a marked increasein PBE enzyme and PBE mRNA respectively in HCC and adjacentnon-neoplastic liver tissue. In control livers (animals nottreated with peroxisome proliferators), the levels of PBE enzymeand mRNA were very low or undetectable. The results of thisstudy clearly indicate that peroxisomal proliferator (PP)-inducedliver lesions express peroxisomal enzymes to the same extentas adjacent liver and that these enzymes are not useful markersfor identification of PP-induced lesions.     

Dose and time dependence of the cellular phenotype in rat hepatic preneoplasia and neoplasia induced in stop experiments by oral exposure to N-nitrosomorpholine

The dose and time dependence of the cellular phenotype In preneoplasticand neoplastic liver lesions was evaluated quantitatively ingroups of male Sprague–Dawley rats exposed for 7 weeksto 0, 12 and 24 mg/kg body wt of N-nitrosomorpholine (NNM) andstudied at different time points up to 80 weeks after withdrawalof NNM (stop model). NNM-treated rats showed a dose- and time-dependentincrease in the total number and volume of preneoplastic fociof altered hepatocytes (FAH) and in the incidence of hepatocellularadenomas (HCA) and carcinomas (HCC) at both dose levels, comparedwith the untreated controls. After stopping treatment with 12mg/kg body wt, the well-known sequence of cellular changes leadingfrom glycogenotic clear and dear/acidophilic cell foci to mixedand diffusely basophllic cell populations poor in glycogen wasfound. In contrast, at the higher NNM dose level (24 mg/kg)predominantly mixed and diffusely basophilic cell foci appearedimmediately after cessation of treatment, but their number rapidlydedined up to 13 weeks after withdrawal. At the same time, therewas a reciprocal increase in the number of the less alteredclear/acidophilic cell foci, indicating an early reversion-linkedphenotypic instability of FAH However, in spite of this reversionhigher numbers of mixed and diffusely basophilic cell foci wereretained after treatment with 24 compared to 12 mg/kg of NNMat all time points studied, and there was even a slow additionalincrease in the number of these types of FAH 20 weeks afterwithdrawal of NNM At both dose levels, the volume fraction ofthe persistent mixed cell foci correlated positively with theincidence of HCA and HCC, suggesting that this phenotype ofFAH represents a direct precursor of the neoplastic lesions.Tigroid cell foci, which appeared most frequently after treatmentwith the lower dose of NNM, were not integrated into the predominantsequence of cellular changes leading to HCC, but they may representan intermediate stage in a side lineage of this sequence, endowedwith the potential to progress at least to HCA. Our resultsshow that reversion-linked phenotypic instability of FAH occursmainly after high dose treatment, possibly resulting from rapidadaptive cellular responses to the primary carcinogenic lesion(s)which may be fixed by genetic or epigenetic mechanisms. In contrast,progression-linked phenotypic instability of FAH is a slowprocessdeveloping in a dose- and time-dependent manner at all doselevels leading to hepatic neoplasia.     

Lack of peroxisomal enzyme inducibility in rat hepatic preneoplastic lesions induced by mutagenic carcinogens: contrasted expression of glutathione S-transferase P form and enoyl CoA hydratase

While glutathione S-transferase P form (GST-P), a reliable markerfor preneoplastic lesions induced by mutagenic hepatocarcinogens,is generally not expressed in rat liver foci, hyperplastic nodulesand hepatomas induced by peroxisome proliferators (PPs), suchlesions can be detected due to their peroxisomal enzyme-negativenature. For comparative purposes we examined the inducibilityof enoyl CoA hydratase (ECH), a key peroxisomal enzyme, in rathepatic preneoplastic lesions induced by mutagenic carcinogens.Clofibrate (CF) was therefore administered for 2 or 4 weeksfollowing performance of the Solt—Farber protocol usingdiethylnitrosamine and 2-acetylaminofluorene. Immunohistochemicalexamination revealed no or only very weak expression of ECHwithin the induced foci in clear contrast to the strong stainingof surrounding parenchyma. ECH expression was thus diametricallyopposed to that of GST-P which was found only in foci. AlthoughECH was completely lacking in GST-P-strongly positive foci,it was expressed in GST-P-negative hepatocytes inside some fociotherwise positive for GST-P. CF administration resulted ina significant decrease in the numbers and areas of foci exhibitingstrongly positive or positive GST-P staining; this being reflectedin a lowering of GST-P protein levels. Furthermore, in primarycultured rat hepatocytes, clofibric acid as well as dexamethasonesuppressed the expression of both GST-P and the oncogene, c-jun.These results taken together suggest that possible interactionof the PP receptor with JUN might be involved in loss of ECHexpression in GST-P-strongly positive foci.     

Comparative tumor initiating activities of N-nitrosomethylbenzylamine and N-nitrosodimethylamine in rat liver

The tumor initiating activities of nitrosomethylbenzylamine(NMBzA), an esophageal carcinogen, and nitrosodimethylamine(NDMA), a hepatocarcinogen, were compared in rat liver usingmodifications of the initiation assays of Tsuda et al. (CancerRes., 40, 1157, 1980) and Pitot et al. (Nature, 271, 456, 1978).Equimolar doses of NMBzA or NDMA (33.5 µmol/kg) were injectedi.p. into male Sprague-Dawley rats 18 h after partial hepatectomy.Following selection, foci of preneoplastic hepatocytes weredetected by histochemical staining for / glutamyltranspeptidase.Nitrosomethylamylamine (NMAmA), 115 µmol/kg), also anesophageal carcinogen, was tested in the assay of Tsuda et al.,and nitrosodiethylamine (NDEA, 33.5 µmol/kg), a hepaticand esophageal carcinogen, was tested in the assay of Pitotet al. Both NDMA and NDEA induced significant increases in thenumber of preneoplastic foci above background. In contrast,neither NMBzA nor NMAmA increased the number of foci above background.Microsomes from regenerating liver had a lower capacity to metabolizeboth NDMA and NMBzA compared to microsomes from intact liver,but the decrease in activity was similar for both compounds.Neither NDMA nor NMBzA significantly inhibited the first waveof DNA synthesis in vivo in the regenerating liver. The resultsdemonstrate that in contrast to NDMA and NDEA, NMBzA and NMAmAlack tumor initiating activity in the liver.     

Enhancement of DHPN induced hepatocellular, cholangiocellular and pancreatic carcinogenesis by Opisthorchis viverrini infestation in Syrian golden hamsters

Infection with 100 Opirthorchis viverrini (OP) metacercariaeprior to two injections of dihydroxy-di-n-propyl nitrosamine(DHPN) (1000 mg/kg body weight) brought about significant enhancementof resultant preneoplastic lesion development in Syrian hamsterliver and pancreas tissue. Thus combined treatment with carcinogenand parasite was associated with pancreatic atypical (dysplastic)foci, hepatocellular nodules, cholangiofibrosis and cholangiocarcinomas.No such lesions were observed in carcinogen alone, parasitealone or untreated control groups. In addition, parasite inducedhyperplastic gall bladder epithelium was found to include areasof putative preneoplastic cells only in the DHPN-OP combinedgroup. The results strongly suggest that pancreatitis and biliarycirrhosis associated with liver fluke infestation are responsiblefor the observed enhancement of carcinogenesis, and that theresultant increased proliferation plays a major role in tumorigenesis.     

Rapid growth of preneoplastic lesions in hepatocarcinogen-Sensitive C3H/HeJ male mice relative to C57BL/6J male mice

We have previously shown that C3H/HeJ male mice are {small tilde}20-fold more susceptible to the induction of liver tumors byN-ethyl-N-nitrosourea (ENU) than are C57BL/6J male mice andthat this difference in sensitivity is largely determined bya single genetic locus (Hcs, hepatocarcinogen sensitivity).In order to determine whether the Hcs locus affects initiationor promotion of hepatocarcinogenesis, we studied the developmentof putatively preneoplastic hepatic lesions that are deficientin glucose-6-phosphatase (G6Pase) in mice treated at 12 daysof age with ENU. In ENU-treated male mice of both strains, thenumber and size of G6Pase-deficient hepatic foci increased overtime between 12 and 24 weeks of age. However, the rate of growthof the lesions was 1.7 times faster for C3H/HeJ male mice (volumedoubling time 2.0 ± 0.1 weeks) than for C57BL/6J mice(3.4 ± 0.4 weeks). Although the number and size of G6Pase-deficientfoci induced by ENU treatment of female C3H/HeJ and C57BL/6Jmice were smaller than for foci in similarly treated male mice,there was no significant difference between the growth ratesof the foci in female C3H/HeJ and C57BL/6J mice. Thus, the phenotypiceffect of the Hcs locus appears to be dependent on promotionof liver tumor induction by the male hormonal environment. Inagreement with studies on the growth rate of the foci in malemice, the [₃H]thymidine labeling index of G6Pase-deficient hepatocytesin C3H/HeJ males (12%) was 1.5-fold higher than in C57BL/6Jmale mice (8.0%) at 20 weeks and 1.2-fold higher at 28 weeks(11% versus 9.5%). The labeling index of histochemically normalhepato cytes in C3H/HeJ male mice (0.38%) was 2.6-fold higherthan in C57BL/6J mice (0.15%) The Hcs locus may affect the promotionphase of hepatocarcinogenesis in male mice by increasing theproliferative rate of both normal and preneoplastic hepatocytes.     

Reversal by ribo- and deoxyribonucleosides of dehydroepiandrosterone-induced inhibition of enzyme altered foci in the liver of rats subjected to the initiation-selection process of experimental carcinogenesis

The effect of dehydroepiandrosterone (DHEA) on the activityof NADPH-producing enzymes and the development of enzyme-alteredfoci has been investigated in the liver of female Wistar ratssubjected to an initiating treatment (a necrogenic dose of diethylnitrosaimine)followed, 15 days later, by a selection treatment [a 15-dayfeeding of a diet containing 0.03% 2-acetylamlnofluorene (2-AAF),with a partial hepatectomy at the midpoint of this feeding].At the end of the selection treatment all rat groups received,for 15 days, a basal diet containing, when indicated, 0.05%phenobarbital (PB) and/or 0.6% DHEA. The effect of DHEA on theactivity of NADPH producing enzymes was also studied in normalrats fed, for 15 days, a diet containing 0.6% DHEA and in theirpair-fed controls. DHEA caused a 43–58% inhibition ofglucose-6-phosphate dehydrogenase (G6PD) and, respectively,338–420% and 21–24% increases in malic enzyme (ME)and isocitric dehydrogenase activities in all rat groups. Thiswas coupled with a great fall in the production of ribulose-5-phosphate,while no change in NADP⁺/NADPH ratio occurred. Hepatocytes,isolated from DHEA-treated rats, exhibited a very low activityof hexose monophosphate shunt (HMS), which was not stimulatedby methylene blue, an exogenous oxidizing agent that markedlystimulated HMS activity in control hepatocytes. DHEA causeda great fall in the percentage of liver occupied by -glutamyltranspeptidase(GGT)-positive foci, in the rats subjected to the initiation- selection treatments. PB enhanced the development of thesefoci, an effect which was completely overcome by DHEA. In addition,focal cells no longer expressed a G6PD activity higher thanthat of surrounding liver in DHEA-treated rats, but exhibiteda high histochemical reaction for ME. DHEA also caused a greatfall in labelling index of GGT-positive foci. Starting at theend of 2-AAF feeding, a mixture of ribonucleosides (RNs) ofadenine, cytosine, guanine and uracil and of deoxyribonucleosides(DRNs) of adenine, cytosine, guanine and thymine were injectedi.p. every 8 h for 12 days to the rats subjected to the initiation- selection treatments plus PB. Rats were killed 3 days afterthe end of RN and DRN treatments. These treatments completelyovercome the DHEA effect on the development of GGT-positivefoci and DNA synthesis by the focal cells, without affectingG6PD activity of both whole liver and putative preneoplasticfoci. Experiments with labeled nucleosides revealed that RNsand DRNs produced derivatives that were incorporated into liverDNA. These data indicate that liver of DHEA-treated rats produceenough NADPH for reduction of RNs to DRNs and growth. The antipromotingeffect of DHEA could depend on a relative deficiency of nudeosidesfor DNA synthesis, caused by a great fall in pentose phosphateproduction.