Regulation of microsomal, xenobiotic epoxide hydrolase messenger RNA in persistent hepatocyte nodules and hepatomas induced by chemical carcinogens

We have utilized a DNA clone complementary to epoxide hydrolase mRNA as a probe to examine the level of the mRNA in persistent hepatocyte nodules and hepatomas induced by the Solt-Farber chemical carcinogenesis procedure. Epoxide hydrolase mRNA is increased 14-fold in nodules as compared to the level in normal liver. When rats with liver nodules were administered phenobarbital, an inducer of epoxide hydrolase mRNA in normal animals, a superinduction in epoxide hydrolase mRNA was observed in the nodules (22-fold) as compared to normal liver. Surprisingly, nodule induction in conjunction with phenobarbital administration also produced marked elevation in epoxide hydrolase mRNA levels in the tissue surrounding the nodules. Using HpaII and MspI to assess the degree of methylation of CCGG sites, we have found that the epoxide hydrolase gene is hypomethylated in nodules and hepatomas compared to the gene in normal liver tissue. Phenobarbital treatment alone increased epoxide hydrolase mRNA levels but did not result in hypomethylation of the epoxide hydrolase gene. These data further support the observation that hypomethylation of specific gene sequences occurs during chemical carcinogenesis and is correlated with an elevation in the steady state level of epoxide hydrolase mRNA in persistent hepatocyte nodules.     

Association between growth stimulation by phenobarbital and expression of cytochromes P450 1A1, 1A2, 2B1/2 and 3A1 in hepatic hyperplastic nodules in male F344 rats.

This study was conducted to examine relationships between phenobarbital (PB) treatment, specific cytochrome P450 gene expression patterns and growth rates of hepatic hyperplastic nodules. Nodules were induced in 8 week old male F344 rats by a Solt-Farber resistance protocol. Six weeks after diethylnitrosamine (DEN) initiation, subgroups of rats were either kept on control chow diet or transferred to a chow diet containing 0.05% PB, then killed 2 weeks later. [3H]Thymidine was delivered continuously via osmotic minipump during the final 3 days of the experimental to label dividing cells. PB treatment resulted in a 89% increase in the number of persistent gamma-glutamyl transpeptidase (GTT) nodules per cm2 section, a 278% increase in the area of persistent GGT nodules per cm2 section, and a 116% increase in the average area per persistent nodule. PB increased the number of [3H]thymidine-labeled persistent GGT nodules but did not significantly change the labeling index (LI) distribution pattern or the average LI. A slight but uniform increase in CYP1A2 expression (relative to surrounding, non-nodular tissue) was observed in 50% (23/46) and 59% (60/102) of persistent nodules in control and PB-treated animals respectively. In contrast, for nodules undergoing remodeling, CYP1A2 expression was elevated in only 9% (2/22) and 0% (0/24) in control and PB groups respectively. In the PB group, CYP2B1/2 was underexpressed in 53% (54/102) of persistent GGT nodules and in 0% (0/24) of the remodeling nodules. Comparing LI among the persistent GGT nodules, those that displayed simultaneous increases in CYP1A2 and decreases in CYP2B1/2 had the highest LI, and were followed in level by those expressing either increases in CYP1A2 or decreases in CYP2B1/2. Nodules that expressed both CYP1A2 and 2B1/2 in a manner similar to the surrounding tissue had the lowest LI. Thus, these data suggest that expression of specific forms of cytochrome P450 may be an important factor in determining other phenotypic characteristics, e.g. rate of cell proliferation and GGT expression, within specific nodules.     

Quantitative stereological evaluation of four histochemical markers of altered foci in multistage hepatocarcinogenesis in the rat

Female F344/N rats dosed with diethylnitrosamine (DEN) 24 hafter partial hepatectomy were treated with the promoting agents,phenobarbital (PB) or 3,4,7,8-tetrachlorodibenzo-p-dioxin (TCDD),or the peroxisome proliferating agent, WY 14,643, for 6 months.Another group was subjected to the Solt-Farber protocol. Alteredhepatic foci (AHF) were analyzed by quantitative stereologyfrom frozen serial sections stained for gamma-gtutamyl transferase(GGT), canalicular adenosine triphosphatase (ATPase), glucose-6-phosphatase(G6Pase) and the placental isozyme of glutathione S-transferase(PGST). PGST scored more foci in all groups than GGT and ATPase.PGST marked greater focal volume than GGT or ATPase, and PGSTmarked focal volume equal to or greater than G6Pase in ratstreated with PB, TCDD or the Solt-Farber protocol. However,after treatment with WY 14,643, GGT and PGST marked much lessfocal volume than ATPase or G6Pase, and PGST scored fewer focithan G6Pase. Numerical estimations of foci scored by those markerson the basis of area of the entire tissue section (per cm²)were relatively different from those values determined by quantitativestereology. While these results confirm earlier studies, theydemonstrate the importance of quantitative stereologic analysisof AHF during multistage hepatocarcinogenesis.     

Effect of coadministration of phenobarbital sodium on N-nitrosodiethylamine-induced gamma-glutamyltransferase-positive foci and hepatocellular carcinoma in rats

The effect of concurrent administration of phenobarbital on the hepatocarcinogenicity of N-nitrosodiethylamine (diethylnitrosamine; DENA) in rats was investigated by determination of the incidence of gamma-glutamyltransferase (gamma-glutamyltranspeptidase) (GGT)-positive foci and liver tumors. Male outbred Sprague-Dawley rats received either a weekly oral dose of DENA (0.08 mol/kg), phenobarbital sodium (500 ppm) in their drinking water, or DENA and phenobarbital sodium concurrently. After 16 weeks, only the animals treated concurrently with DENA and phenobarbital sodium had GGT-positive foci (3.65 foci/cm2). At 30 weeks, the group treated with DENA and phenobarbital sodium exhibited more foci (23.6 foci/cm2) compared to the group that received only DENA (3.08 foci/cm2). The average size of foci in both of the DENA-treated groups was the same. The tumors in the group that received DENA plus phenobarbital sodium showed a greater incidence of GGT activity compared to the tumors in the DENA group. Under the conditions of this study the incidence of GGT-positive foci did not predict the incidence of hepatocellular carcinomas.     

Inhibition of phenobarbital and dietary choline deficiency promoted preneoplastic lesions in rat liver by environmental contaminant di(2-ethylhexyl)phthalate

The effect of di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer and environmental contaminant, on the emergence of gamma-glutamyltranspeptidase positive (GGT+) preneoplastic foci in the liver of rats fed promoting diets was studied. GGT+ foci were initiated in the liver of Sprague--Dawley male rats with a single dose of diethylnitrosamine (DEN) following partial hepatectomy. One series of control rats received saline vehicle alone. Promotion of foci was commenced by feeding: (1) a choline-deficient diet (CD); (2) a choline-supplemented diet (CS) containing 0.06% phenobarbital (CS + PHB); or (3) a CD diet containing 0.06% phenobarbital (CD + PHB). In the absence of initiation by DEN, dietary treatments did not increase the number of GGT+ foci. In rats receiving DEN, each promoting regimen effectively increased the number of GGT+ foci above levels in control rats fed only the choline-supplemented diet. Inclusion of the plasticizer at a level of 2% in each of the dietary promotion treatments, however, effectively inhibited the appearance of the foci.     

Hepatic foci of cellular and enzymatic alteration and nodules in rats treated with clofibrate or diethylnitrosamine followed by phenobarbital: their rate of onset and their reversibility

The histologic appearance and cytochemical characteristics of foci of hepatic cellular alteration, hepatic nodules, and hepatocellular carcinomas occurring in male Sprague-Dawley rats treated with the hypolipidemic agent clofibrate (CAS: 637-07-0), with phenobarbital (CAS: 50-06-6), or with diethylnitrosamine [(DENA) CAS: 55-18-5] followed by phenobarbital were studied after treatment periods from 1 month to 2 years. Rats treated with clofibrate revealed foci of cellular alteration that were more often basophilic and occurred slightly sooner (wk 42) than those in untreated controls (wk 60). Of 36 rats that had received 68 or more weeks of continuous clofibrate, 19 had hepatic nodules. Of the 11 nodules examined cytochemically, none was gamma-glutamyltransferase (gamma-GT) positive and 2 were positive to glucose-6-phosphate dehydrogenase (G-6-PD) under oxygen. In rats withdrawn from clofibrate for 16-18 weeks after 68-95 weeks of clofibrate, 0 of 14 had nodules. In several of these rats zones of hepatic scarring were observed, suggesting the reversibility of the nodules. Phenobarbital alone had little effect on the incidence of foci of cellular alteration, although the number of gamma-GT-positive foci was increased. DENA followed by phenobarbital led to the early appearance of foci of cellular alteration (from wk 4), of nodules (from wk 13), and of hepatocellular carcinomas (from wk 26). gamma-GT activity was raised in most of these nodules and carcinomas, while G-6-PD activity was raised in only 3 of 9 nodules but in all 9 carcinomas examined. DENA-phenobarbital given for 13 or 26 weeks followed by withdrawal of phenobarbital for 28 and 26 weeks, respectively, produced an essentially similar pattern of lesions. In view of the growing recognition of the nonspecificity gamma-GT as a marker of carcinogen-initiated foci, the value of G-6-PD (under oxygen) as a marker merits further investigation.     

Induction of altered hepatic foci in rats by the administration of hypolipidemic peroxisome proliferators alone or following a single dose of diethylnitrosamine

The effect of feeding hypolipidemic peroxisome proliferators on the induction of altered hepatic foci (AHF) in Fischer rats was studied in order to determine whether such agents can induce or promote the development of AHF. In the first study, rats were fed ciprofibrate (10 mg/kg/day) for 1 yr. AHF, neoplastic nodules, and hepatocellular carcinomas were induced. The presence of putative gamma-glutamyltranspeptidase (GGT) activity was numerically the most common marker, although it was absent in larger foci and nodules. A deficiency in canalicular ATPase and glucose-6-phosphatase provided the best markers for the larger foci and nodules. In the second study, rats were subjected to partial hepatectomy, and half of the animals were then intubated with diethylnitrosamine (10 mg/kg). One wk later, rats were fed Wy-14,643 at concentrations of 0, 0.05, and 0.1% in the diet for 6 mo. At 6 mo, the number and volume of foci were increased by the feeding of Wy-14,643 after partial hepatectomy alone and were greatly increased when Wy-14,643 was fed after partial hepatectomy/diethylnitrosamine administration. Canalicular adenosine triphosphatase and glucose-6-phosphatase deficiencies were the most common markers of AHF, and AHF of these phenotypes occupied practically all of the focal volume. The larger AHF did not express GGT, and those foci exhibiting GGT were much less common and occupied very little volume. The absence of the GGT protein itself, as opposed to an inhibition of GGT activity, was verified by immunohistochemical staining using an antibody to GGT. These studies show that hypolipidemic peroxisome proliferators can stimulate an increase in AHF following a single dose of diethylnitrosamine and a mitotic stimulus, and they thus can act as promoters in two-stage liver carcinogenesis. GGT is a poor marker for identifying AHF induced by peroxisome proliferators during the early, premalignant phase of hepatocarcinogenesis.     

Dose-response relationship of phenobarbital promotion of diethylnitrosamine initiated tumors in rat liver

The dose-response relationship was determined in rats for the enhancement by phenobarbital of diethylnitrosamine (DENA)-initiated neoplastic nodules and hepatocellular carcinomas. Male Sprague-Dawley rats received a single oral dose of either 80 mg/kg DENA or water. Seven days later, the animals were divided into groups that started to receive 0, 62.5, 125, 250, 500 or 1000 ppm sodium phenobarbital in the drinking water. Animals from each group were killed at 48 and 70 weeks after the DENA. No significant difference was observed in the low response of neoplastic nodules among the DENA-initiated groups. The incidence of DENA-initiated hepatocellular carcinoma was enhanced at 70 weeks by 250, 500 and 1000 ppm sodium phenobarbital but not by 62.5 or 125 ppm sodium phenobarbital. Equal enhancement of the incidence of hepatocellular carcinomas was obtained with 250, 500 and 1000 ppm sodium phenobarbital. In non-DENA-initiated rats, phenobarbital did not induce neoplastic nodules or hepatocellular carcinomas. Our results suggest that a daily dose of at least 250 ppm sodium phenobarbital is required in order for it to exert tumor promoting activity.     

Differential effects of dehydroepiandrosterone and deoxyribonucleosides on DNA synthesis and de novo cholesterogenesis in hepatocarcinogenesis in rats.

Previous studies from our laboratory have shown that dehydroepiandrosterone (DHEA), an inhibitor of glucose-6-phosphate dehydrogenase (G6PD), prevents the development of gamma-glutamyltranspeptidase (GGT)-positive foci in the early stages of hepatocarcinogenesis in rats. Since high rates of DNA and cholesterol (CH) synthesis are observed during promotion of carcinogenesis, and mevalonate (MVA), or some other intermediates of CH synthesis, could be mediators of DNA synthesis, we investigated the effect of DHEA on CH synthesis in rat liver during the development of GGT-positive foci. Hepatocarcinogenesis was induced by diethylnitrosamine in female Wistar rats by the Solt-Farber protocol (initiation/selection) with and without phenobarbital treatment. A 15 day treatment with DHEA (0.6% in the diet), started after selection, caused a great fall in labeling and mitotic indices of GGT-positive foci, which was prevented by the simultaneous administration of a mixture of four deoxyribonucleosides (DRNs) of adenine, guanine, cytosine and thymine or four ribonucleosides (RNs) of adenine, guanine, cytosine and uridine, but not by the corresponding bases. DHEA greatly inhibited G6PD activity and the production of ribulose-5-phosphate, without affecting NADPH levels, due to the compensatory increase in malic enzyme and isocitric dehydrogenase activities. Serum lecithin/cholesterol acyltransferase activity underwent a reduction in conditions allowing a rapid growth of GGT-positive tissue (absence of DHEA or presence of DHEA plus DRNs or RNs). Liver slices isolated from DHEA-treated rats showed a rise in CH content, coupled with a 80% fall in the incorporation of labeled acetate, but not of labeled MVA, into CH. A 25 day treatment of rats subjected to initiation/selection, started after the appearance of persistent nodules, caused a 36 and 78% fall in the incorporation, in vivo, of 3H2O into nodular and surrounding liver CH respectively. DRN did not counteract DHEA-induced inhibition on CH synthesis. Thus DHEA inhibits the CH biosynthetic pathway before MVA synthesis, in conditions (presence of DHEA plus DRN/RN) allowing rapid growth of preneoplastic lesions. Therefore, the development of these lesions does not need the synthesis of large amounts of CH and CH metabolites. Thus, the antipromotion effect of DHEA may depend on a decreased availability of pentose phosphates for DNA synthesis.     

Phenobarbital enhances the formation of reactive oxygen in neoplastic rat liver nodules

The effect of treatment of rats with the liver monooxygenase inducer phenobarbital on the formation of reactive oxygen in neoplastic liver nodules and the surrounding normal tissue was investigated. Liver nodules were induced by treatment of rats with diethylnitrosamine (single i.p. injection of 0.15 mumol/kg body weight on day 1 after birth) followed by chronic administration of phenobarbital-sodium (PB; 0.05% in diet) after weaning. Groups of rats were kept on PB until sacrifice or were withdrawn from the promoter 3-6 weeks prior to killing. Emission of chemiluminescence was used as a sensitive means to detect the formation of reactive oxygen in microsomal preparations from the various tissues incubated with NADPH and the chemiluminigenic detector lucigenin. In addition, a 2-dimensional photon counting system has been developed that permits the analysis of the spatial distribution of lucigenin-chemiluminigenic signals over liver tissue sections incubated with reduced phosphopyridine dinucleotides. In general, we observed increased levels of reactive oxygen formation in liver nodules when compared with the normal liver tissue. Highest levels were seen in nodules that stemmed from PB-induced rats. Studies on the expression and activity of cytochrome P-450 in liver nodules as well as experiments with specific inhibitors point towards a participation of the liver monooxygenase system in reactive oxygen formation, although additional metabolic pathways seem to be involved as well. The observed increases in reactive oxygen in liver nodules of PB-treated rats might be related to the promoting activity of this drug.     

Ploidy and karyotype of hepatocytes isolated from enzyme-altered foci in two different protocols of multistage hepatocarcinogenesis in the rat

The ploidy and karyotypes of hepatocytes isolated from the livers of rats subjected to the protocols of Peraino et al. and of Solt and Farber were determined by the examination of such cells in primary culture. A study of 100 or more metaphases from each of five rats on each protocol revealed that 75-80% of gamma-glutamyltranspeptidase-positive (GGT+) hepatocytes isolated from livers of rats in either protocol were diploid, whereas only 23-33% of GGT- cells were diploid. Fifty percent or more of the karyotypes of hepatocytes from livers of rats receiving the Solt-Farber protocol exhibited one or more chromosomal breaks, whereas hepatocytes from livers of rats subjected to the Peraino protocol showed no increase in chromosomal breakage over that in normal controls. These studies demonstrate that the majority of GGT+ cells from altered hepatic foci are diploid and that the greater toxicity of the Solt-Farber protocol over that of Peraino is correlated with marked chromosomal breakage of GGT+ hepatocytes.     

Changes in gamma-glutamyltransferase activity during N-2-fluorenylacetamide-induced rat hepatocarcinogenesis

Changes in gamma-glutamyltransferase (GGT) activity throughout the course of rat hepatocarcinogenesis induced by brief dietary exposure to N-2-fluorenylacetamide (2-FAA) followed by promotion with dietary phenobarbital (PB) were studied. By examination of changes in total GGT activity and its histochemical localization, the effects of initiator and promoter on this enzyme can be clearly differentiated. Both 2-FAA and PB increase total GGT activity in grossly normal liver, PB causing a tenfold greater increase than that caused by 2-FAA. Although the elevation of GGT activity in livers of rats sequentially treated with 2-FAA-PB was not additive or synergistic, the course of increase was distinct from that of either 2-FAA- or PB-caused changes in activity, suggesting that 2-FAA and PB interact to alter the GGT phenotype of sequentially treated animals. GGT activity in neoplasms induced by either 2-FAA or 2-FAA-PB was highly variable, i.e., from nearly basal levels to those 170-fold greater than basal. Lesions induced by 2-FAA without PB promotion had elevated GGT, indicating that PB is not required to produce GGT-positive neoplasms by this protocol. Histochemically, changes in GGT activity occurred in both hepatocytes and nonhepatocyte cell populations in a characteristic, treatment-dependent manner, well-correlated with total GGT activity. Treatment with 2-FAA, especially that with 2-FAA-PB, induced primarily focal and nodular GGT activity patterns. PB alone produced no GGT-positive foci, but it did cause GGT-positive ductular proliferation. Continued PB exposure produced a GGT activity pattern which clearly defined the interlobular regions of the liver; no similar staining pattern was seen in either 2-FAA-treated or 2-FAA-PB-treated livers. These results indicate that an initiator and one of its promoters, combined and individually, alter GGT activity in a characteristic manner over the course of hepatocarcinogenesis. Moreover, the effects of the promoter on GGT activity are dependent on whether or not prior exposure to initiator has occurred.     

Evidence of two separate mechanisms for the decrease in aryl sulfotransferase activity in rat liver during early stages of 2-acetylaminofluorene–induced hepatocarcinogenesis

Enzymatic and immunohistochemical experiments were conducted to evaluate the mechanistic basis for the downregulation of the important detoxication/bioactivation enzyme aryl sulfotransferase IV (AST IV) during 2- acetylaminofluorene (2AAF)–induced hepatocarcinogenesis. To distinguish between possible genotoxic and cytotoxic actions of 2AAF, three different dietary protocols were used in these experiments: group 1 received 2AAF for 12 wk, group 2 received 2AAF for 3 or 6 wk and then a control diet lacking xenobiotics for 3 or 6 wk, and group 3 received 2AAF for 3 or 6 wk and then phenobarbital for 3 or 6 wk. When hepatic AST IV activity was assessed, N-hydroxy-2AAF sulfotransferase activity was found to decrease 80–90% in response to 2AAF feeding, but activity recovered to essentially normal levels in the livers of rats subsequently placed on either control diets or diets with phenobarbital, suggesting a reversible cytotoxic mechanism for loss of AST IV activity. However, when liver sections from the rats were evaluated immunohistochemically, two distinct patterns were detected for the downregulation of AST IV expression was observed throughout the liver and among most but not all newly developed nodules. In tissue sections from rats initially fed 2AAF and then placed on a control diet (group 2) or a diet with phenobarbital (group 3), the nodules continued to show low levels of AST IV expression, while expression in the areas surrounding nodules returned to the normal, high levels. In addition, among those rats fed 2AAF for just 3 wk and then control diet or diet containing phenobarbital for 6 wk, only rats fed phenobarbital developed altered foci that stained weakly for AST IV expression. These results show that there were two kinds of 2AAF-mediated decrease in hepatic AST IV activity: a general overall loss of AST IV expression dependent on administration of 2AAF and reversible upon removal of 2AAF from the diet and a loss of AST IV expression among newly developed liver foci and nodules that persisted in the absence of 2AAF administration and appeared to be a property of 2AAF-induced subpopulations of cells. These patterns may correspond, respectively, to cytotoxic and genotoxic mechanisms of 2AAF action. © 1994 Wiley-Liss, Inc.     

Induction of 8-hydroxydeoxyguanosine but not initiation of carcinogenesis by redox enzyme modulations with or without menadione in rat liver.

Inducibility of oxidative stress in rat liver in vivo by menadione-associated redox cycling activation under redox enzyme modulating conditions was examined by monitoring hepatocyte injury and 8-hydroxydeoxyguanosine (8-OHdG) levels of liver DNA. In addition, the treatment-associated liver tumor initiating activity was assessed in terms of development of gamma-glutamyl-transpeptidase (GGT)- and glutathione S-transferase placental form (GST-P)-positive foci and hyperplastic nodules. With or without following menadione treatment (50 mg/kg, i.g.), redox enzyme modulations of increased cytochrome P450 reductase activity induced by phenobarbital (PB)-Na (100 mg/kg, i.p. for 5 days), inhibition of DT-diaphorase by dicumarol (25 mg/kg, i.p.) and depletion of glutathione by phorone (200 mg/kg, i.p.), with or without further supplement of iron EDTA-Na-Fe(III) (70 mg/kg, i.p.), caused both substantial hepatocyte necrosis and 8-OHdG production in Fischer 344 male rats. Subsequent feeding with a 0.05% PB diet for 64 weeks resulted in slightly increased development of GGT-positive foci but not GST-P positive lesions or hyperplastic nodules, suggesting a lack of tumor-initiating activity of the oxidative DNA damage associated with redox enzyme modulations with or without menadione.     

Production of monoclonal antibodies to preneoplastic liver cell populations induced by chemical carcinogens in rats and to transplantable Morris hepatomas

Monoclonal antibodies (moabs) to neoplastic and preneoplastic liver cells in rats have been selected to follow cellular changes in the livers during chemical carcinogenesis. The moabs were induced by immunizations of BALB/c mice with four partially purified liver cell preparations: 1) oval cells induced in male Fischer rats fed 0.05% N-2-acetylaminofluorene in a choline deficient diet: 2) preneoplastic gamma-glutamyltranspeptidase positive hepatocytes induced by i.p. injection of diethylnitrosamine into male Fischer rats followed by 0.02% N-2-acetylaminofluorene and partial hepatectomy (Solt-Farber model): 3) sharply dissected neoplastic nodules induced in male Fischer rats by five 2-week cycles of 0.05% N-2-acetylaminofluorene diet: and 4) Morris hepatomas 7777 and 5123 passaged in male Buffalo rats. The hybridomas were screened by enzyme linked immunosorbent assay or by indirect immunofluorescence on composite cryostat sections of fetal and adult rat liver, liver containing neoplastic nodules, and Morris hepatoma 7777. Positive clones were limit diluted and partially characterized by indirect immunofluorescence on cryostat sections of other preneoplastic and neoplastic rat livers as well as normal rat tissues. Two moabs to oval cells, two moabs to hepatocytes, and one moab to hepatomas have been selected for further study.     

Utilization of circulating glutathione by nodular and cancerous intact rat liver.

The relationship of the rate of extraction of circulating glutathione (GSH) to the level of activity of gamma-glutamyltransferase (GGT) of hepatocytes of nodular and of cancer-bearing livers was studied in rats perfused in situ via the portal vein. Fischer adult male rats with many nodules (10 rats) or few (nine rats) liver nodules and four rats with hepatomas were compared as to their ability to remove GSH (10 microM) from the perfusate. The rate of extraction of infused GSH was directly proportional to the numbers of GGT(+)-hepatocytes in the liver tissue, inhibitable completely by adding the GGT inhibitor serine borate at 6-8 mM in the perfusate, and significantly enhanced in all rats by adding the gamma-glutamyl acceptor glycyl-glycine to the perfusate. These results suggest that nodules and cancers are able to remove GSH much more efficiently from the circulation than the surrounding liver tissue and that their enhanced GSH utilization is directly dependent on their GGT activity, which is present at much higher levels than in the surrounding tissues. The increases in GGT activity in nodule hepatocytes and enhanced ability to utilize GSH could be critical factors in the response to resistance selection of chemical hepatocarcinogenesis.     

Inhibitory effect of vanadium on rat liver carcinogenesis initiated with diethylnitrosamine and promoted by phenobarbital.

The chemoprotective effect of vanadium, a dietary micronutrient, against chemically induced hepatocarcinogenesis in rats was investigated. Initiation was performed by a single intraperitoneal injection of diethylnitrosamine (DENA; 200 mg kg-1) followed by promotion with phenobarbital (0.05%) in the diet. Supplementary vanadium (0.5 p.p.m.) in the drinking water was provided ad libitum throughout the experiment, before the initiation or during the promotion period. At the end of the study (20 weeks), vanadium supplementation throughout the experiment reduced the incidence (P < 0.01), total number and multiplicity (P < 0.001) and altered the size distribution of visible persistent nodules (PNs) as compared with DENA control animals. Mean nodular volume (P < 0.05) and nodular volume as a percentage of liver volume (P < 0.01) were also attenuated following long-term vanadium treatment. It also caused a large decrease in the number (P < 0.001) and surface area (P < 0.01) of gamma-glutamyltranspeptidase (GGT)-positive hepatocyte foci and in the labelling index (P < 0.001) of focal cells, coupled with increased (P < 0.01) remodelling. The activity of GGT, measured quantitatively, was found to be significantly less in the PNs (P < 0.001) and non-nodular surrounding parenchyma (P < 0.01) of vanadium-supplemented rats. The anticarcinogenic effect of vanadium was also reflected in the histopathological analysis of liver sections that showed a well-maintained hepatocellular architecture as compared with DENA control. Similar results were observed when vanadium was given only before the initiation. However, supplementation of vanadium during the promotion period did not result in significant alterations of these parameters. Our results, thus, strongly suggest that vanadium may have a unique anti-tumour potential which is primarily exerted on the initiation phase and only secondarily on the promotion stage.     

Lack of hepatocarcinogenic potential of acetaminophen in rats with liver damage associated with a choline-devoid diet

The potential carcinogenic activity of acetaminophen (paracetamol, APAP) was studied in male F344 rats with pre-existing liver damage induced by a choline-devoid (CD) diet. In a short-term experiment, APAP was administered by intragastric intubation as single doses of 0.5-1.5 g/kg body wt after 4 weeks feeding of CD diet had produced fatty livers in rats. Two-thirds partial hepatectomy was performed 4 h subsequent to the initiating treatment step. After a 2 week recovery period, all rats were subjected to the selection procedure of Cayama et al. and killed at week 9 of the experiment. Quantitative analysis of placental form glutathione S-transferase (GST-P)-positive liver lesion development did not reveal any enhancement by APAP, whereas administration of a non-necrogenic dose of diethylnitrosamine (20 mg/kg body wt) in the same protocol demonstrated significant promotion, confirming the utility of the model for detection of weak carcinogenicity of chemicals. In the second long-term experiment, APAP was fed at doses of 0.45 and 0.9% for 25 weeks following 27 weeks administration of CD diet which produced liver cirrhosis in the rats. Despite a slight enhancement of focal liver lesions positive for gamma-glutamyltranspeptidase (GGT), no significant promotion of GST-P-positive altered foci or nodules was observed. In contrast, continuous feeding of CD diet or 0.5% phenobarbital treatment after generation of cirrhosis with CD diet clearly enhanced the induction of both GST-P and GGT-positive liver lesions. Thus, these results indicate that APAP does not possess significant carcinogenic activity in damaged rat liver.     

Possible enhancing effect of the immunosuppressive agent, 6-mercaptopurine(6-MP) on focal lesion development in cirrhotic liver induced by carbon tetrachloride but not furfural in F344 rats.

The modifying effects of an immunosuppressive agent, 6-mercaptopurine (6-MP), on development of focal lesions in liver cirrhosis models induced by carbon tetrachloride (CCl4) or furfural were studied in male F344 rats. Feeding of 6-MP at 50 p.p.m. for 20 weeks to animals with pre-existing liver cirrhosis caused immunosuppression, and significantly enhanced the induction of gamma-glutamyltranspeptidase (GGT)-positive foci and nodules in the CCl4 but not furfural case. Glutathione S-transferase P (GST-P)-positive preneoplastic lesions were not affected. Moreover, phenobarbital (PB) also enhanced the induction of GGT-positive hepatocellular lesions only in the CCl4-induced liver cirrhosis model, no promotion influence being exerted after treatment with the non-carcinogenic furfural. This study, therefore, suggests that 6-MP can enhance the induction of one type of preneoplastic foci and nodules and that essential differences exist between focal lesions arising in cirrhotic livers caused by CCl4 as opposed to furfural.     

Phenobarbital promotes the development of adenocarcinomas in the accessory sex glands of MNU-inoculated L-W rats

Aged Lobund-Wistar (L-W) rats develop: (i) spontaneous and inducedmetastasizing adenocarcinomas in the prostate and seminal vesicle(P-SV) complex; and (ii) spontaneous hepatomas and hepatocarcinoinas.Within the time-frame of 14 months, similar adenocarcinomaswere induced in the P-SV complex in 70–90% of youngerL-W rats by a single i.v. inoculation of methylmtrosourea (MNU)which was followed by slow release s.c. implants of testosteroneproplonate (TP). Within the same time-frame, neither MNU norTP alone induced signillcant incidences of P-SV tumors; anduntreated control L-W rats were disease-free. Methylnitrosoareaor TP and combinations thereof did not induce liver tumors.However, when MNU-inoculated L-W rats were fed phenobarbital(PB), they developed (i) metastasizing adenocarcinomas in theP-SV complex and (ii) altered cellular foci and nodules in thelivers. Methylnitrosourea induced a high incidence of benignlung adenomas which progressed to lung cancers in numbers whichwere of marginal significance. Thus, dormant MNU-initiated cellsin the P-SV complex were activated by phenobarbital, to produceadenocarcinomas in that complex.