Effects of N-nitrosomorpholine and phenobarbital on UDPglucuronyltransferase in putative preneoplastic foci of rat liver

Effects of initiators and promoters of hepatocarcinogenesison UDP-glucuronyltransferase and arylhydrocarbon hydroxylasewere investigated in foci of altered hepatocytes. A single admhktrationof N-nitmmorpholine (75 mg/kg, 24 h after partial hepatectomy)was used for initiation and chronic administration of phenobarbital(0.1% in tap water) for promotion. Histological evidence indicatedthat ATPase-negative, y-glutamyltranspeptidase-positive, andUDP-glucuronyltransferase-positive foci were highly correlated.Based on this evidence ATPase-negative foci were used as a guideto monitor early lesions and to microdissect lyophilized fociand extrafocal tissue. It was found that treatment with N-nitrosomorpholineled to a permanent increase of UDP-glucuronyltransferase activityin foci tissue (3- to 5-fold, detected 180 and 330 days afterinitiation). In contrast, arylhydrocarbon hydroxylase activitywas decreased by 50%. Administration of phenobarbital furtherincreased UDP-glucuronyltransferase activity in focal tissue(up to 9-fold, compared with control liver). However, this furtherincrease of enzyme activity by phenobarbital was reversible.The results suggest that (i) initiation by chemical carcinogensleads to permanent alterations of drug metabolizing enzymes,consistent with increased toxin-resistance of initiated hepatocytes,and (ii) chronic administration of phenobarbital markedly enhancesgene expression of UDP-glucuronyltransferase in initiated hepatocytes.     

The clonal nature of carcinogen-induced altered foci of gamma-glutamyl transpeptidase expression in rat liver

The clonality of tumors has been convincingly established. Because it is generally accepted that tumor formation involves a number of steps, it is important to determine which if any of the precursors of tumors are clonal. A series of chimeric rats produced between congenic strains by morulae aggregation were used to establish the cellular composition of foci of gamma-glutamyl transpeptidase (gamma-GTP; E.C. 2.3.2.2) expression in liver following initiation with N-nitrosodiethylamine and promotion with phenobarbital. The chimeras were produced between congenic rat strains (PVG and PVG-RT1a) genetically distinguished by alleles of the major histocompatibility complex (MHC). Monoclonal antibodies directed to distinctive class I MHC alloantigens were used to detect patterns of mosaicism in the animals. The parental genotypes present in most visceral tissues could be easily distinguished by our method. Analysis of 499 enzyme-altered foci revealed that 474 were comprised solely of either PVG-RT1a or PVG cells. Some apparent mixture of cells from the two lineages was observed in 25 lesions, most of which were very small. The observed pattern of distortion of normal patch distribution clearly indicated the expanding and clonal nature of these lesions.     

Influence of phenobarbital on glycogen metabolism of rat liver pretreated with N-nitrosomorpholine

Sprague-Dawley rats were treated with N-nitrosomorpholine. (NNM)alone (7 weeks, 120 mg/I in drinking water), with NNM followedby phenobarbital (PB) (750 mg/l for 6 weeks) or PB alone. Thelivers from these animals were investigated for glycogen contentand activities of glucose-6- phosphatase, glucose-6-phosphatedehydrogenase, glycogen phosphorylase and glycogen synthetase.The following parameters proved to be significantly alteredin the livers of rats treated with either NNM or PB or bothcompared with untreated controls: glycogen content was increasedand the activities of glucose-6-phosphalase and glycogen synthetasewere decreased. Although these data show some similarities inchanges of glycogen metabolism of livers treated with NNM orPB, earlier histochemical investigations revealed importantdifferences in the distribution of these alterations withinthe liver parenchyma.     

Effects of rat strain, diet composition, and phenobarbital on hepatic gamma-glutamyl transpeptidase histochemistry and on the induction of altered hepatocyte foci and hepatic tumors by diethylnitrosamine

To extend our ongoing characterization of modulatory influences on hepatic tumorigenesis, we examined effects of rat strain (Sprague-Dawley versus Fischer), diet composition (semipurified diet versus standard nonpurified laboratory chow), and dietary phenobarbital on the production of gamma-glutamyl transpeptidase (GGT)-positive hepatocyte foci and hepatic tumors initiated by diethylnitrosamine. In addition to GGT-positive foci, we observed, under certain conditions, the appearance of extensive hepatic GGT staining not associated with focal lesions. This elevated nonfocal GGT was found in rats of both strains fed the nonpurified rather than the purified diet, but the level of staining was higher in Fischer than in Sprague-Dawley rats. Enhancement of this nonfocal staining by dietary phenobarbital appeared insignificant. By comparison, frequencies of GGT-positive foci were generally higher in rats fed the semipurified rather than the nonpurified diet, and the frequencies of GGT-positive foci were invariably higher in Sprague-Dawley than in Fischer rats. Moreover, dietary phenobarbital generally enhanced focus production. Assessments of focus and tumor yields among these experimental groups showed that differences in focus frequencies did not correspond closely to differences in subsequent tumor formation. These results document the need to consider the influences of diet and rat strain on experimental end points in designing protocols for hepatocarcinogenesis studies, especially those involving GGT histochemistry. The data also raise questions about the mechanistic relevance of GGT induction to hepatocarcinogenesis and support our prior evidence against the putative lineal relationship between foci and tumors.     

Effect of chronic phenobarbital administration on the gamma-glutamyl transpeptidase activity of hyperplastic liver lesions induced in rats by the Solt/Farber initiation: selection process of hepatocacinogenesis

A chronic 8 to 11 week administration of the hepatic tumor promoterphenobarbital (0.05% in drinking water) to rats previously subjectedto the initiation:selection process of Solt and Farber was foundto further increase the gamma-glutamyl transpeptidase activityof individual hyperplastic liver nodules of 4.0–10.0 mmin diameter over comparably sized nodules form control livers.Those rats which received 11 weeks of the chronic phenobarbitaltreatment also showed a significant increase in their liverwet weights. In addition, random tissue samples of non-nodularliver taken from the 11 week phenobarbital-treated rats exhibiteda gamma-glutamyl transpeptidase mean specific activity whichwas {small tilde}3 times higher than that of control non-nodularliver samples. In contrast, there was a 1.9-fold increase inthe mean % gamma-glutamyl transpeptidase-positive area (cm²),as determined histochemically, in cryostat sections made fromnon-nodular samples of the 11 week phenobarbital-treated ratswhen compared with that of control liver sections. Interruptionof the chronic phenobarbital administration at 8 weeks followedby 3 weeks of control treatment resulted in a reversal of thegammaglutamyl transpeptidase activity response shown by thehyperplastic liver nodules and non-nodular liver tissue samples.Thus, phenobarbital can quantitatively modulate gamma-glutamyltranspeptidase activity in carcinogeninduced hyperplastic liverlesions in the rat during the early stages of hepatocarcinogenesis.     

Expression of gamma-glutamyl transpeptidase in adult rat liver cells after transformation with SV40 virus

By the use of histochemical techniques we have shown that the transformation of rat hepatocytes with simian virus 40 resulted in a large majority of cells producing gamma-glutamyl transpeptidase (gamma GT). Using a temperature-sensitive mutant of this virus we have shown that the percentage of cells exhibiting this enzyme in a temperature-sensitive SV40 mutant is much higher in cells grown at the permissive temperature of 33 degrees C than at the nonpermissive temperature of 40.5 degrees C. We thus conclude that, the correlation between gamma GT expression and chemical or spontaneous transformation of hepatocytes, previously described by other authors, can now be extended to viral transformation and that the enzyme activity is dependent on the expression of a transformed cell phenotype.     

Promoting effect of 4-dimethylaminoazobenzene on enzyme altered foci induced in rat liver by N-nitrosodiethanolamine

Female Wistar rats were treated sequentially with 4-dimethyl-aminoazobenzene(4-DAB) and N-nitrosodiethanolamine (NDEOL) for periods of 6weeks. One group received first 4-DAB (0.06% in the diet) andNDEOL (2000 p.p.m. in the drinking water) thereafter, whilethe second group was treated in the reversed sequence; controlgroups received the single agent alone. The extent of foci negativefor adenosine-triphosphatase (ATPase) or positive for -glutamyl-transpep-tidase(-GT)-activity was quantitated in liver as a means to assesscarcinogenic efficacy. A very low response was obtained in ratstreated first with 4-DAB and then with NDEOL whereas a strongincrease in number and especially in size of foci was observedwhen 4-DAB was given after NDEOL. The response in this lattergroup was clearly over-additive. Treatment of rats with eithercarcinogen alone resulted in similar pattern of increases inthe volumetric fraction of liver occupied by ATPase-deficientfoci. A differential behaviour, however, was observed with respectto islet size. NDEOL produced large numbers of small foci whereaswith 4-DAB only few foci were obtained which grew rapidly inthe presence of the carcinogen. These findings are consistentwith the hypothesis that 4-DAB, besides acting as an initiator,has very strong promoting activity as was to be expected fromthe characteristic relationship between carcinogen dose andtime of liver tumour induction.     

Inhibitory effect of dietary iron deficiency on the induction of putative preneoplastic foci in rat liver initiated with diethylnitrosamine and promoted by phenobarbital.

The effects of dietary iron deficiency on induction of putative preneoplastic, gamma-glutamyltransferase (GGT)-positive hepatocyte focal lesions in the liver of rats treated with diethylnitrosamine (DEN) followed by phenobarbital (PB) were investigated. Male Fischer 344 rats of 4 weeks old were placed on an iron deficient (ID) diet containing less than 5 p.p.m. of iron or an iron supplemented (IS) diet containing 180 p.p.m. of iron throughout experimental period of 12 weeks. Both groups of rats were administered 200 mg kg-1 body weight of DEN by a single intraperitoneal injection at Week 4 followed by PB mixed into each diet at a concentration of 0.05% from Week 6 to the final sacrifice at Week 12 when induction of GGT-positive foci was quantitatively analysed. On the ID and IS diets, respective numbers of GGT-positive foci were 6.3 and 14.2 cm-2. The sizes of foci were not altered by the iron content of the diet. The present results indicate that iron plays a role in the development of preneoplastic foci in the livers of rats initiated with DEN and promoted by PB especially in the initiation phase.     

The dose dependence and sequential appearance of putative preneoplastic populations induced in the rat liver by stop experiments with N-nitrosomorpholine

Different doses of N-nitrosomorpholine (NNM) (80, 120, 160,200 mg/l drinking water) were administered for various periodsof time (1, 4, 7, 14, 20 weeks) to male Sprague-Daw-ley ratsin order to investigate the dose dependence and sequential appearanceof preneoplastic and neoplastic lesions in the liver. With alldose levels studied a sequence was established leading fromdear cell and acidophilic cell glycogen storage foci throughmixed cell foci and neoplastic nodules to hepatocellular carcinomas.The first appearance and frequency of the different lesionsinvestigated proved dependent on dose of carcinogen administered.Morphometric analysis demonstrated that the rare small clearcell foci apparent after 7 weeks treatment with NNM increasein number and size with progression through the sequence tocarcinomas without further administration of carcinogen. Noevidence was found to suggest that pronounced focal populationswere reversible upon cessation of NNM-treatment under the presentexperimental conditions.     

c-fos gene expression in rat liver is induced by phenobarbital

In the present study we investigated c-fos expression in rat livers, that was initiated with the three arylamines, 2-acetylaminofluorene, 2-acetylaminophenanthrene and trans-4-acetylaminostilbene. The tumor promoter phenobarbital was applied chronically for 26, 52 and 100 weeks. Gene expression, determined by the mRNA level, and FOS protein were increased after 52 weeks of treatment in arylamine initiated as well as in phenobarbital only treated animals. Expression of c-fos seems to be a phenobarbital induced effect that is independent of additional initiator treatments. This finding was supported by immunohistochemical studies demonstrating increased FOS levels to be localized around the central vein. The results indicate that phenobarbital, a widely used tumor promoter, induces c-fos expression. In addition, we demonstrated enhanced FOS in GST-P-positive foci and in tumors.     

Promoting effects of phenobarbital and 3'-methyl-4-dimethylaminoazobenzene on the appearance of gamma-glutamyltranspeptidase positive foci in rat liver pretreated with varying doses of diethylnitrosamine

The promotion potential of phenobarbital (PB) and 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) on liver carcinogenesis after initiation with various doses of diethylnitrosamine (DEN) was assessed using an in vivo short term system. Male F344 rats were pretreated with a single intraperitoneal injection of varying doses of DEN (0, 6, 12, 25, 50, 100 or 200 mg/kg body wt), and 2 weeks later were treated with 0.05% PB or 0.06% 3'-Me-DAB for 6 weeks. All animals were subjected to partial hepatectomy 3 weeks after the DEN treatment. Quantitation of gamma-glutamyltranspeptidase-positive (gamma-GT+) foci revealed a DEN dose-dependent response. Magnitude of promotion by PB and more pronounced by 3'-Me-DAB was, in contrast, strongest at the lower doses of DEN. The results suggest that quantitative differences with regard to initiation level may exist, influencing the promotability of initiated cells.     

Loss of adenylate cyclase activity in preneoplastic and neoplastic lesions induced in rat liver by N-nitrosomorpholine

Adenylate cyclase (AC) activity was demonstrated histochemicallyusing adenylate-(ß, -methylene)diphosphate as substratein cryostat sections of livers from 45 rats treated for 7–10weeks with N-nitrosomorpholine (NNM) (120 mg/1 drinking water)and from nine untreated control rats. The enzyme patterns ofnormal tissue, preneoplastic and neoplastic lesions were characterizedand correlated with the morphologically defined stages of tumourdevelopment in the liver. Light microscopically, the enzymeactivity of normal tissue was restricted to the plasma membrane,and was most pronounced along the bile canaliculi of the hepatocytes.In glycogen storage foci and mixed cell foci induced by NNMno, or only very weak, AC activity was visible. In the cellsof neoplastic nodules and hepatocellular carcinomas AC activitywas also clearly reduced. However, in small parts of the plasmamembrane which lined lumina resembling normal bile canaliculiand in cytoplasmic vesicles closely associated with these structures,some AC activity was occasionally detected by light and electronmicroscopy. Whereas the tissue of normal appearance surroundingthe lesions showed a marked increase in AC activity in the presenceof glucagon, forskolin and cholera toxin. AC activity in thepreneoplastic and neoplastic liver lesions could not, or couldonly weakly, be stimulated by this treatment. As demonstratedin serial sections of the foci, the reduction in AC activitycorresponded to changes in the activity of other enzymes studiedearlier in the same model. Thus the reduction in AC activitywas accompanied by a decrease in the activity of glucose-6-phosphataseand glycogen phosphorylase, and by an increase in the activityof glucose-6-phosphate dehydrogenase. The results support theconcept that the focal changes in the activity of many enzymes(including those of carbohydratemetabolism) during hepatocarcinogenesisare the consequence of aberrations in superordinate regulatorymechanisms of cell metabolism.     

Effect of varying the concentration of phenobarbital and its duration of treatment on the evolution of carcinogen induced enzyme-altered foci in rat liver

The present study was aimed to investigate whether the promoting activity of phenobarbital in rodent liver is related to its daily dose level and duration of treatment or rather to its total dose administered. For this purpose groups of female Wistar rats were treated for 5 consecutive days with an initiating dose of 10 mg/kg body weight N-nitrosodiethylamine. Subsequently, rats were given phenobarbital-sodium (PB) in their drinking water at concentrations of 20, 50, 100 and 200 mg/l for varying lengths of time, such that the total dose of xenobiotic was very similar throughout the different treatment groups ranging from approximately 950 to 1100 mg/kg body weight. The number and volume fraction of lesions negative for the marker enzyme adenosine triphosphatase in liver were subsequently scored as a means to determine the carcinogenic response in this organ. Slight promoting effects of PB were only seen at the lowest concentration of 20 mg/l, whereas no significant effects were observed at 50 and 100 mg/l. At the highest concentration of 200 mg/l an inhibition of carcinogenic response was obtained. Although the effects seen in this study were only moderate, our data favour the idea that the promoting effects of PB depend on the actual concentration of the compound and the duration of treatment rather than on the total dose administered.     

The natural history and dose-response characteristics of enzyme-altered foci in rat liver following phenobarbital and diethylnitrosamine administration

Enzyme-altered foci (EAF) were induced in the Liver of femalerats by 70? partial hepatectomy (PH), followed by a single intragastricadministration of diethylnitrosamine (DEN) at a dose of 10 mg/kg.The stability and response of these foci to various doses ofthe hepatic promoting agent, phenobarbital (PB), were studied.The number of yglutamyltranspeptidasepositive (GGT⁺) EAF resultingfrom PH/DEN followed by PB (0.05%) administration for 1 week,2 weeks, 1 month, 2 months, 3 months or 4 months did not significantlychange when the administration of the promoting agent was followedby a 6-month period of a diet containing no PB. These data demonstratethe stability of the fwi induced by the PH/DEN/PB regimen andindicate that the increased number of foci resulting from PBpromotion in the absence of overt hepatic necrosis are not reversibleon removal of the promoting stimulus. Chronic administrationof dose levels of PB below 0.001% in the diet failed to demonstratean increase in the number of EAF over the number in the controlanimals not promoted with PB. A linear increase in the numberof EAF was observed when rats were chronically fed doses ofPB ranging between 0.001% and 0.05% in the diet, whereas dietconcentrations of PB > 0.05% did not result in any furtherincrease in the number of EM. The number of EAF resulting fromPH/DEN followed by 0.05% PB in the diet increased during thefirst 3–4 months of promotion. Thereafter, the numberof foci did not change despite the continued administrationof PB for as long as 8 months. These data suggest the presenceof an apparent threshold (no effect level) for promotion byPB and demonstrate the presence of a maximal response of EAFto this promoting agent after initiation by a single dose ofDEN.     

Dietary orotic acid enhances the incidence of {gamma}-glutamyltransferase positive foci in rat liver induced by chemical carcinogens

Feeding male Fischer F-344 rats for 5 weeks a diet containing1% orotic acid, a precursor for pyrimidine nucleotide biosynthesis,resulted in an increased incidence of -glutamyltrans-ferase(EC 2.3.2.2 [EC] ) positive foci induced by chemical carcinogens including1,2-dimethylhydrazine, diethylnitrosamine, benzo[a]pyrene, andaflatoxin B₁. This unique effect of orotic acid can be accentuatedby supplying a liver cell proliferative stimulus. The enzymealtered hepatocytes have a higher labelling index (4.4%) comparedwith that of the hepatocytes in the surrounding liver (0.26%).The effect of orotic acid on the increased incidence of focicannot be attributed to either the induction of liver cell proliferationor the imposition of a preferential inhibitory effect on theproliferation of normal hepatocytes while permitting the carcinogen-modifiedhepatocytes to respond to an endogenous or exogenous liver cellproliferative stimulus and grow to form foci. Orotic acid alsodid not behave like some of the promoters of liver carcinogenesissuch as phenobarbital and polychlorinated biphenyls in thatit did not induce either the phase I or phase II componentsof hepatic drug metabolizing enzyme systems. Some of the possiblemechanisms by which orotic acid enhances the incidence of -glutamyltransferasepositive foci by carcinogens are discussed.     

The metabolism of N-nitrosomorpholine by rat liver microsomes and its oxidation by the Fenton system

The metabolism of N-nitrosomorpholine by rat liver microsomesgave acetaldehyde, formaldehyde, glyoxal and N-nitroso-2-hydroxymorpholine.Oxidation of N-nitroso-morpholine by Fenton's reagent gave acetaldehyde,glycol-aldehyde, glyoxal, (2-hydroxyethoxy)acetaldehyde andN-nitroso-2-hydroxymorpholine. N-Nitroso-3-hydroxymor-pholinewas synthesised. In water the new compound gave mainly acetaldehyde,with glycolaldehyde, (2-hydroxy-ethoxy)acetaldehyde and glyoxal.These observations indicated the probability of 3-hydroxylationin the biological and chemical oxidations. N-Nitroso-3-morpholonebehaved similarly in water to the 3-hydroxy compound, and gavemainly acetaldehyde, with glycollk acid, (2-hydroxyethoxy)aceticacid and glyoxal. N-Nitroso-3-morpholone and N-nitroso-3-hydroxymorpholinereacted with 3, 4-dichlorobenzenethiol. The action of lightor alkali on N-nitrosomorpholine gave glyoxal and labile glyoxal-yieldingcompounds.