Putative Preneoplastic Foci Research Articles

Inhibition of dietary fat promoted development of (pre)neoplastic lesions in exocrine pancreas of rats and hamsters by supplemental selenium and β-carotene

The modulating effects of selenium and β-carotene on the development of putative preneoplastic foci in exocrine pancreas were investigated in rats and hamsters, treated with azaserine and N-nitrosobis(2-oxopropyl)amine (BOP), respectively. The animals were fed a semipurified diet high in saturated fat (20% lard) either supplemented or not with β-carotene or selenium. A separate group maintained on a diet low in saturated fat (5% lard) was incorporated as an extra control group. The animals were given their diets 12 days after the last treatment with carcinogen. Four months postinitiation, the pancreata were quantitatively examined for the number and size of putative (pre)neoplastic lesions. Rats as well as hamsters maintained on a low-fat diet had significantly less putative preneoplastic pancreatic lesions as compared to animals fed a diet containing 20% lard. Selenium and β-caroten both inhibited the growth of basophilic foci in rat pancreas. In hamster pancreas, β-carotene and selenium caused a significant decrease in the number of early ductal complexes. The number of carcinomas was decreased in the β-carotene group and increased in the group fed a diet high in selenium.

Inhibition of dietary fat-promoted development of (pre)neoplastic lesions in exocrine pancreas of rats and hamsters by supplemental vitamins A, C and E

The effects of vitamins A, C and E on the development of putative preneoplastic foci in exocrine pancreas were investigated in azaserinetreated rats and N-nitrosobis(2-oxoproypy)amine-treated hamsters. The animals were fed a semipurified diet high in saturated fat (20% lard) either or not supplemented with vitamin A, vitamin C or vitamin E. A separate group maintained on a diet low in saturated fat (5% lard) was incorporated as extra controls. The animals were given their diets 12 days after the last treatment with carcinogen. At 4 months postinitiation, the pancreata were quantitatively examined for both the number and size of early, putative preneoplastic lesions and the presence of neoplastic lesions. Rats as well as hamsters maintained on 5% lard exhibited a significantly lower number of putative preneoplastic pancreatic lesions than animals fed a diet containing 20% lard. Growth of acidophilic but not of basophilic foci was inhibited in rats of the high vitamin A and C group, whereas vitamin E exerted an inhibitory effect on growth of basophilic but not of acidophilic foci. In hamsters maintained on a diet high in vitamins A or C, the number of early ductular lesions was significantly decreased, whereas the number of (micro)carcinomas was increased. Vitamin E did not have any modulating effect on development of ductal lesions in hamster pancreas.

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 activity of NADPH-producing enzymes and the development of enzyme-altered foci has been investigated in the liver of female Wistar rats subjected to an initiating treatment (a necrogenic dose of diethylnitrosamine) followed, 15 days later, by a selection treatment [a 15-day feeding of a diet containing 0.03% 2-acetylaminofluorene (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 the activity of NADPH-producing enzymes was also studied in normal rats fed, for 15 days, a diet containing 0.6% DHEA and in their pair-fed controls. DHEA caused a 43-58% inhibition of glucose-6-phosphate dehydrogenase (G6PD) and, respectively, 338-420% and 21-24% increases in malic enzyme (ME) and isocitric dehydrogenase activities in all rat groups. This was 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 activity of hexose monophosphate shunt (HMS), which was not stimulated by methylene blue, an exogenous oxidizing agent that markedly stimulated HMS activity in control hepatocytes. DHEA caused a great fall in the percentage of liver occupied by gamma-glutamyltranspeptidase (GGT)-positive foci, in the rats subjected to the initiation-selection treatments. PB enhanced the development of these foci, an effect which was completely overcome by DHEA. In addition, focal cells no longer expressed a G6PD activity higher than that of surrounding liver in DHEA-treated rats, but exhibited a high histochemical reaction for ME. DHEA also caused a great fall in labelling index of GGT-positive foci. Starting at the end of 2-AAF feeding, a mixture of ribonucleosides (RNs) of adenine, cytosine, guanine and uracil and of deoxyribonucleosides (DRNs) of adenine, cytosine, guanine and thymine were injected i.p. every 8 h for 12 days to the rats subjected to the initiation-selection treatments plus PB. Rats were killed 3 days after the end of RN and DRN treatments. These treatments completely overcome the DHEA effect on the development of GGT-positive foci and DNA synthesis by the focal cells, without affecting G6PD activity of both whole liver and putative preneoplastic foci. Experiments with labeled nucleosides revealed that RNs and DRNs produced derivatives that were incorporated into liver DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

The influence of phenobarbital and butylated hydroxytoluene on the ploidy rate in rat hepatocarcinogenesis.

The effect of the 'promoters' phenobarbital (PB) and butylated hydroxytoluene (BHT) on the ploidy changes during hepatocarcinogenesis in rats was compared in a densitometric analysis of Feulgen-stained nuclei on paraffin-embedded tissue slices. The triphasic Gerlans protocol for liver-cancer induction was applied. Initiation with a single dose of diethylnitrosamine (DEN), and selection with 2-acetylamino-fluorene (2-AAF) combined with a proliferative stimulus (CCl4 administration), was followed by a treatment with PB or BHT for periods up to 22 weeks. Control animals received no treatment after the initiation and selection procedure. Despite intra- and inter-individual variations, an increase in the amount of 2N nuclei is found in the putative preneoplastic lesions of animals that received initiation and selection (I-S) and 3 weeks basal diet (BD). When the diet is supplemented with PB (after I-S), the increase of diploid nuclei starts earlier. At the time carcinoma arise (22 weeks PB treatment) a decrease in the frequency of 2N nuclei is found. BHT-treated animals which develop no carcinoma within the considered timespan, show a clear increased amount of 2N nuclei in the precancerous lesions only after 14 weeks treatment. It seems that there is a positive correlation between the outgrowth of putative preneoplastic foci and nodules in rat liver and an increase of diploid nuclei in these lesions. PB, as promoter used after initiation and selection, speeds up the development of carcinoma in rat liver, and therefore also the shift to diploidization in these rats starts earlier in comparison with I-S-treated rats. Although BHT does not promote liver carcinogenesis, an increase of diploid nuclei is also observed here during lesion formation. It may, therefore, be concluded that the phenomenon of diploidization is closely linked to and probably necessary for preneoplastic development, but that it is not an absolute indicator for neoplastic transformation.

Persistent effect of a low dose of preadministered diethylnitrosamine on the induction of enzyme-altered foci in rat liver.

The effect of a low dose of preadministered diethylnitrosamine (DEN) on the induction of enzyme-altered foci in the livers of male full-grown Fischer 344 rats was studied. As a pretreatment, DEN at a dose of 10 mg/kg body wt was injected i.p. At various times after DEN pretreatment a complete initiation, consisting of administration of the same dose of DEN by the same route in rats subjected to partial hepatectomy (PH), was performed, followed by application of selection pressure. Enzyme-altered foci stained with gamma-glutamyltranspeptidase (gamma-GTP) and glutathione S-transferase placental form (GST-P) were then assayed. Decreases in the numbers and areas of foci in the rats which received saline + PH 14 or 28 days after DEN pretreatment were observed in comparison with rats which received saline + PH immediately after DEN. On the other hand, the numbers and areas of foci were not decreased in rats which received the complete initiation, consisting of DEN + PH, at various times after DEN pretreatment when compared with rats which received these at the same time as the DEN pretreatment. This persistent effect of DEN pretreatment on the complete initiation lasted up to 182 days after the time of DEN pretreatment. In this experiment, GST-P was found to be a more sensitive marker for the detection of putative preneoplastic liver-cell foci than gamma-GTP.

Transformation sensitivity in early S-phase and clonogenic potential are target-cell characteristics in liver carcinogenesis by N-methyl-N-nitrosourea.

Changes in the transformation sensitivity of hepatocytes during the cell cycle were in relation to clonogenicity investigated in partially resected rat liver. The hourly rate of the G1-S transit was measured in a control group by means of tritium-labelled thymidine (3H-TdR). At defined periods after partial hepatectomy the animals were injected with a single dose of N-methyl-N-nitrosourea (MNU) (25 mg/kg) and subsequently exposed to phenobarbital (0.05% in the diet) for 80 days. ATPase-deficient cell populations which arise by clonal growth (Rabes et al., 1982) were determined in the liver 90 days after MNU treatment and served as a marker for the initiating action of the carcinogen. The number of foci appeared to be related to the hourly rate of influx of hepatocytes into DNA synthesis at the time of MNU administration. Few foci were observed after MNU exposure in G1 and their frequency increased steeply when MNU treatment coincided with the first peak of cellular G1-S transit. As the rate of influx into S-phase decreased, so did the number of foci, despite the proportion of cells in S-phase still being at a maximum. The possibility of a gradient of clonogenicity existing within the cells of the liver lobule is envisaged. It is suggested that proliferative cells of the peri-portal region, which begin DNA synthesis early after partial hepatectomy, have the greatest ability to form clones of ATPase-deficient putative pre-neoplastic foci after initiation by a carcinogen and might thus represent, if exposed to carcinogen in early S-phase, the effective target cell population for the induction of hepatocellular carcinomas.

Modulation of calcium by the carcinogenic process in the liver induced by a choline-deficient diet.

A diet devoid of choline and low in methionine (CD), without any added carcinogen, has been shown to induce 100% preneoplastic nodules and more than 50% cancer in the rat liver. Attempts to understand the mechanism by which a CD diet induces liver cell cancer revealed that like chemical carcinogens, a CD diet also appears to cause alterations in DNA, perhaps mediated by free radicals. Indeed, a CD diet induces nuclear lipid peroxidation prior to the changes in DNA. The CD diet induced DNA alterations coupled with continuing liver cell proliferation may account for the induction of initiated hepatocytes by the CD diet. To gain insight into the nature of free radicals generated by the CD diet, experiments were designed to determine whether agents that modulate free radical effects influence the CD diet induced changes in the liver. We investigated the effect of Ca2+ in the modulation of CD diet induced alterations in the liver. The results show that extra Ca2+ when added to the CD diet prevented some of the early changes due to choline deficiency, such as nuclear lipid peroxidation and DNA damage, but had little or no effect on the triglyceride accumulation in the liver. Also, the same CD diet with extra Ca2+, when used as a promoter after initiation by diethylnitrosamine, decreased the number and size of early putative preneoplastic foci and nodules.

Histochemical and immunohistochemical detection of putative preneoplastic liver foci in women after long-term use of oral contraceptives.

Localized areas with altered enzyme patterns were observed in liver tissue surrounding focal nodular hyperplasia in women after long-term use of oral contraceptives. These localized lesions were of three different types. Type I lesions were characterized by glycogen storage, a reduction in ATPase and an increase in gamma-glutamyltranspeptidase (gamma-GT) and UDP-glucuronyltransferase (UDP-GT) detected immunohistochemically. Type II lesions, which were morphologically very similar to small hyperplastic nodules, showed only a decreased ATPase reaction. Type III lesions showed an increase in gamma-GT (detected histochemically) and a slight reduction in ATPase. The results indicated that in human liver from patients given oral contraceptives long-term, localized lesions with altered enzyme patterns may occur which are very similar to those observed in animal models during experimental hepatic carcinogenesis.

DNA cytophotometry of hepatocytes in individual ATPase-deficient putative preneoplastic foci in rat liver

DNA cytophotometry of hepatocytes in individual ATPase-deficient putative preneoplastic foci in rat liver

Pyruvate kinase isoenzymes in altered foci and carcinoma of rat liver.

Pyruvate kinase (PK) isoenzymes, rate limiting for the last steps of glycolysis, were studied in normal rat liver, putative preneoplastic foci, neoplastic nodules and hepatocellular carcinoma. These lesions were produced by an initiation-promotion protocol: treatment with a single dose of N-nitrosomorpholine (NNM) was followed by feeding diets containing phenobarbital (PB) or alpha-hexachlorocyclohexane (alpha-HCH), or basal diet. PK was demonstrated (i) by immunocytochemistry on histological sections with antibodies specifically directed against the L and M2 isoenzymes, (ii) by electrophoretic separation of isoenzymes in homogenates from liver and larger tumors, and (iii) by electrophoretic separation of isoenzymes in parenchymal and stromal cells isolated from liver and tumors. Immunocytochemistry showed decreases of L-PK (L-PK-) in hepatocytes of most of the foci, nodules and carcinomas. Most L-PK- foci showed increases in gamma-glutamyltransferase (gamma-GT) and epoxide hydrolase (EH). PB or alpha-HCH treatment further decreased expression of L-PK in foci, but not in normal liver. Cells and foci with enhanced L-PK (L-PK+) were also found after carcinogen treatment. These did not show increases of gamma-GT or EH or any distinct morphological alterations with the exception of some which were basophilic ('tigroid') in H and E stained sections. No L-PK+ tumors were found. We could not demonstrate the M2-type PK in parenchymal cells of liver or any of the lesions described above. This isoenzyme was restricted to stromal cells in normal rat liver and in all stages of carcinogenesis as shown by immunohistology and by electrophoresis of preparations from isolated cell populations. However, stromal cells from hepatocellular carcinomas exhibited a 3-fold increase of M2-PK compared with stromal cells from normal liver. These results do not support an isoenzyme shift from L to M2-PK in the course of malignant transformation of hepatocytes as suggested previously.

Immunohistochemical differentiation of gamma-glutamyltranspeptidase in focal lesions and in zone I of rat liver after treatment with chemical carcinogens.

gamma-Glutamyltranspeptidase (gamma-GT) is known to be increased in putative pre-neoplastic foci but also in the periportal zone I of rat liver under a variety of circumstances not directly related to carcinogenesis. To be able to distinguish between these two instances gamma-GT was studied by enzyme determination in micro-dissections obtained from the two locations and by both histochemical and immunohistochemical staining in serial sections. Altered hepatic foci and alterations in zone I were produced in three models of hepatocarcinogenesis: initiation by N-nitrosomorpholine and tumor promotion by phenobarbital, continuous administration of 2-acetylaminofluorene and continuous administration of methapyrilene hydrochloride. In micro-dissections gamma-GT activity was similarly increased in focal lesions and in zone I after feeding methapyrilene. Histochemically detectable gamma-GT, stained according to Rutenburg et al. (23), was observed both in zone I and in focal lesions. Focal lesions were also ATPase negative and UDP-glucuronyltransferase positive in all three models. gamma-GT in focal lesions could be selectively detected by immunohistochemical staining using antibodies to the rat kidney enzyme and an indirect peroxidase reaction. These findings suggest immunochemical differences between gamma-GT in focal lesions and in zone I.

Correlations between ploidy and initiation probability determined by DNA cytophotometry in individual altered hepatic foci.

Single cell DNA cytophotometry of ATPase-deficient putative preneoplastic foci in rat liver, induced by a single dose of N-methyl-N-nitrosourea (25 mg/kg or 50 mg/kg) and subsequent phenobarbital feeding (0.05% in the diet), revealed three different types: the majority of foci consisted of an almost exclusive diploid cell population, others showed a preferential tetraploid pattern and a few large foci contained a mixture of di-, tetra- and octoploid hepatocytes. The results of this selective measurement of individual preneoplastic foci in Feulgen-stained 20-microns-thick cryostat sections indicate that not only diploid hepatocytes are a target for the transforming action of a carcinogen, but also tetraploid cells, and that both initiated cell types are capable of expanding as a homogeneous clone. However, clonal homogeneity appears to be lost when foci enlarge. A few preneoplastic foci, heterogeneous with respect to DNA content and endowed with an increased proliferative potential, may represent cell populations at high risk of further development into malignancy.

Modulation of putative preneoplastic foci in exocrine pancreas of rats and hamsters. I. Interaction of dietary fat and ethanol.

The effect of dietary fat and ethanol and their interactions on the development of putative, preneoplastic foci in exocrine pancreas was investigated in rats and hamsters. Rats were given a single i.p. injection of 30 mg azaserine per kg body wt at 19 days of age. Hamsters were injected s.c., with 20 mg N-nitrosobis(2-oxopropyl)amine (BOP)/kg body wt at 6 and 7 weeks of age. The animals were fed a low fat (LF) control diet (5% corn oil) or a high fat (HF) diet (25% corn oil). Ethanol was provided in drinking water at a 15% (w/v) concentration. The animals were given the respective diets and ethanol after the treatment with carcinogen. At 4 months post-initiation, the pancreata were quantitatively examined for the number and size of preneoplastic foci. In rats, acidophilic as well as basophilic foci were subject to modulation by HF and ethanol. The results point to a specific promoting effect of unsaturated fat on the growth potential of azaserine-induced acidophilic acinar cell foci in rat pancreas. There was no evidence of an interaction between HF and ethanol as far as acidophilic foci are concerned. Evaluation of the number and size of the basophilic foci demonstrated an enhancing effect of ethanol on the modulation of pancreatic carcinogenesis by fat, pointing to a possible interaction between these two lifestyle factors. This suggestion was supported by the finding that six out of 20 rats in the HF with ethanol group exhibited a carcinoma in situ, whereas in the HF and in the ethanol group such an advanced lesion was found in one animal only. Unlike in rats, ethanol had no modulating effect on number and growth of putative, preneoplastic lesions in hamsters, either in combination with LF or in combination with HF. A HF diet, however, caused a significant increase in number as well as an increase in percentage of pancreatic tissue occupied by early lesions induced in hamster pancreas by BOP.

The effects of dietary fat and selenium on the development of preneoplastic lesions in rat liver.

In this study, the effects of dietary fat and selenium on the development of putative preneoplastic foci induced by aflatoxin B1 were examined. Rat hepatic gamma-glutamyl transpeptidase (GGT) activity, selenium-dependent glutathione peroxidase activity, glutathione, and total lipid were analyzed 3 and 13 weeks after the start of dietary and carcinogen treatment. Results indicate that high dietary fat has an enhancing effect on the development of preneoplastic foci. A high-fat, selenium-deficient diet resulted in an increase in foci development, but the increase was not significant. An interaction between aflatoxin and the two nutrients was also observed in liver glutathione content and GGT activity at Week 3.

Programmed cell death (apoptosis) in rat liver and its role in the growth of putative preneoplastic foci

Programmed cell death (apoptosis) in rat liver and its role in the growth of putative preneoplastic foci

Increased UDP-glucuronyltransferase in putative preneoplastic foci of human liver after long-term use of oral contraceptives.

Previous research by the authors had suggested that uridine-diphosphate-glucuronyl-transferase (UDP-GT) is a useful preneoplastic marker in chemical carcinogenesis. Recently the authors report that they found typical clear cell foci in a macroscopically normal liver surrounding focal nodular hyperplasia with a 6 cm diameter in a 27-year old woman who had been using oral contraceptives (OCs) containing ethinyl-estradiol and lynestrenol for 9 years. These foci were further characterized by a reduction of canalicular and cytoplasmic ATPase activity an increased glycogen content and a positive immunohistochemical reaction for UDP-GT. OC users develop 2 basic types of benign liver tumors: hepatic adenoma and focal nodular hyperplasia. Hepatic adenoma appears to be caused by OCs whereas the relationship between OC use and focal nodular hyperplasia is less clear. The tumorigenic action of OCs has been ascribed to a promotor action on liver cells; however there is no evidence that OCs are initiators of liver tumors. The case reported shows 2 manifestations of toxic lesions promoted by OC use: the development of focal nodular hyperplasia and enzyme-altered foci comparable to those seen in experimental liver carcinogenesis. Further studies are needed to get more information about the preneoplastic potential of these foci in humans. Since enzyme-altered foci could not be identified in the liver tissue of healthy women these foci may be of prognostic significance in longterm OC users.

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

Effects of initiators and promoters of hepatocarcinogenesis on UDP-glucuronyltransferase and arylhydrocarbon hydroxylase were investigated in foci of altered hepatocytes. A single administration of N-nitrosomorpholine (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 indicated that ATPase-negative, gamma-glutamyltranspeptidase-positive, and UDP-glucuronyltransferase-positive foci were highly correlated. Based on this evidence ATPase-negative foci were used as a guide to monitor early lesions and to microdissect lyophilized foci and extra-focal tissue. It was found that treatment with N-nitrosomorpholine led to a permanent increase of UDP-glucuronyltransferase activity in foci tissue (3- to 5-fold, detected 180 and 330 days after initiation). In contrast, arylhydrocarbon hydroxylase activity was decreased by 50%. Administration of phenobarbital further increased UDP-glucuronyltransferase activity in focal tissue (up to 9-fold, compared with control liver). However, this further increase of enzyme activity by phenobarbital was reversible. The results suggest that (i) initiation by chemical carcinogens leads to permanent alterations of drug metabolizing enzymes, consistent with increased toxin-resistance of initiated hepatocytes, and (ii) chronic administration of phenobarbital markedly enhances gene expression of UDP-glucuronyltransferase in initiated hepatocytes.

Formation of O6-methylguanine in rat liver DNA by nitrosamines does not predict initiation of preneoplastic foci.

Nitrosodimethylamine (NDMA) at 26 mumol/kg, and nitrosomethylbenzylamine (NMBzA) at 33.5 mumol/kg were equally potent in producing 7-methylguanine and O6-methylguanine in rat liver DNA. These doses were used to compare the abilities of the two nitrosamines to initiate production of putative preneoplastic foci in rat liver. Whereas NMBzA resulted in no increase above the background level of foci (0.9 +/- 0.1 foci/cm2), NDMA produced 7.5 +/- 0.6 foci/cm2. Co-administration of NDMA and NMBzA produced no more foci than NDMA alone, even though the combined effect of the two nitrosamines on DNA methylation was additive. The results suggest that methylation of hepatic DNA by nitrosamines does not predict initiation of preneoplastic foci.

Controlled death (apoptosis) of normal and putative preneoplastic cells in rat liver following withdrawal of tumor promoters.

Numerous drugs, hormones and environmental pollutants induce liver growth by hypertrophy and/or hyperplasia, and promote preferential growth of putative preneoplastic foci in the liver. In the present study the regression of hyperplasia after cessation of inducer/promoter treatment was studied in normal liver and in liver foci. High doses of cyproterone acetate (CPA), a synthetic sex steroid, were administered to rats and produced a doubling of liver size; after cessation of treatment liver size declined, and 27% of the total liver DNA disappeared within 6 days. In histological sections from the involuting liver no necroses, but numerous apoptotic bodies (ABs) were found; retreatment with CPA interrupted the formation of ABs. These findings suggest that elimination of excess liver DNA after cessation of CPA treatment is due to controlled cell death by apoptosis. In a further series of experiments putative preneoplastic foci were produced by a single dose of N-nitrosomorpholine and subsequently stimulated to grow by 10 or 28 weeks of phenobarbital (PB) treatment. After withdrawal of PB numerous ABs were present in normal liver and in the foci; in both, retreatment with PB decreased the appearance of ABs. It appears that inhibition of cell death by PB may contribute to tumour promotion. Under all conditions tested more ABs were found in the foci than in non-focal parts of the liver, suggesting an enhanced cell turnover in foci. The apparent sensitivity of foci to mechanisms controlling cell death might eventually provide a means for elimination of preneoplastic lesions.

Immunocytochemical demonstration of a phenobarbital-inducible cytochrome P450 in putative preneoplastic foci of rat liver.

Putative preneoplastic foci of rat liver, so far believed to be deficient in monooxygenases, are shown to contain a cytochrome P450 isoenzyme inducible by phenobarbital. The isoenzyme is also present and appears catalytically active in liver tumors obtained after promotion with phenobarbital and alpha-hexachlorocyclohexane.