Selective pressure during tumor promotion by phenobarbital leads to clonal outgrowth of beta-catenin-mutated mouse liver tumors.

Tumor promoters are non-mutagenic chemicals which increase the probability of cancer by accelerating the clonal expansion of cells transformed during tumor initiation. Phenobarbital (PB) is an antiepileptic drug which promotes hepatocarcinogenesis in rodents when administered subsequent to an initiating carcinogen like diethylnitrosamine (DEN). Here we have investigated the prevalence and patterns of mutations in two genes, Ha-ras and beta-catenin, both known mutational targets in mouse hepatocarcinogenesis. Liver tumors were generated by a single administration of DEN to 6 week old mice followed by feeding of PB (0.05%) containing or control diet for 39 weeks. Mutations at Ha-ras codon 61 were screened by allele-specific oligonucleotide hybridization; beta-catenin mutations were detected by direct sequencing of PCR products spanning exon 2. In tumors from mice treated with DEN alone, the prevalence of Ha-ras mutations was approximately 30% (6/20), while no beta-catenin mutations (0/13) were detectable in tumors of this treatment group. By contrast, Ha-ras mutations were undetectable in tumors from mice treated with DEN/PB (0/32), while approximately 80% (37/46) of tumors from this group showed beta-catenin mutations. These results demonstrate that PB strongly affects the prevalence of mutations in the two cancer-related genes, presumably by positive and negative selection for cells harboring the respective mutation.     

Activation of beta-catenin provides proliferative and invasive advantages in c-myc/TGF-alpha hepatocarcinogenesis promoted by phenobarbital

Previously, we have found that phenobarbital (PB) enhanced cell survival and facilitated tumor growth in our c-myc/transforming growth factor (TGF)-alpha transgenic mouse model of liver cancer. Given that PB selectively promoted initiated cells harboring beta-catenin mutations during chemically induced hepatocarcinogenesis and that Wnt/beta-catenin signaling is involved in both anti-apoptotic and proliferative processes, we now have extended our analysis to investigate whether promotion by PB affects the occurrence of beta-catenin mutations in c-myc/TGF-alpha-driven tumors. The frequency of beta-catenin activation as judged by somatic mutations and/or nuclear localization was significantly increased in hepatocellular carcinomas (HCCs) from c-myc/TGF-alpha mice treated with PB (15/28; 53.6%) as compared with that in control HCCs (2/28; 7.1%). Furthermore, an intact beta-catenin locus was detected in all neoplasms following PB treatment, whereas 57.1% (16/28) of malignant tumors from c-myc/TGF-alpha untreated mice displayed loss of heterozygosity at the beta-catenin locus. Strikingly, in the majority of PB-treated HCCs beta-catenin nuclear localization was limited to small cells with high nuclear/cytoplasmic ratio forming an invasion front (NAinv). beta-Catenin NAinv cells showed cytoplasmic redistribution of E-cadherin associated with intense mucin 1 and matrilysin immunostaining, suggesting their invasive phenotype. All beta-catenin-positive HCCs displayed increased proliferation and tumor size, but no difference in the apoptotic rate when compared with beta-catenin negative tumors. These findings show that PB treatment positively selects for a cell population displaying activation of beta-catenin in c-myc/TGF-alpha HCCs. beta-Catenin activation confers additional growth and invasive advantages in a model of liver cancer already accelerated by synergistic activity of the c-myc and TGF-alpha transgenes.     

WY-14,643-mediated promotion of hepatocarcinogenesis in connexin32-wild-type and connexin32-null mice

Connexin32 (Cx32) is the major gap junction forming protein in liver and lack of functional Cx32 enhances hepatocarcinogenesis. Many tumour-promoting agents block gap junctional intercellular communication, which may favour clonal expansion of neoplastic cells. We recently demonstrated that liver tumourigenesis is accelerated in Cx32-wild-type but not in Cx32-null mice by the model tumour promoter phenobarbital (PB). In the present study, male Cx32-wild-type and Cx32-null mice were treated with a single injection of 90 micro g/g body wt of N-nitrosodiethylamine (DEN) at 6 weeks of age and were subsequently kept on a diet containing the peroxisome proliferator [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (Wy-14,643) or on control diet. Thirty-eight weeks after DEN treatment, mice were killed, frozen liver sections were prepared and (pre)-neoplastic lesions were identified by alterations in glucose-6-phosphatase (G-6-Pase) and glutamine synthetase (GS) staining. G-6-Pase-deficient lesions were generally small in size and were observed in all groups of mice. Large focal pre-neoplastic and benign neoplastic lesions, however, which demonstrated increased rather than decreased activity in G-6-Pase were exclusively present in DEN/Wy-14,643-treated mice. G-6-Pase-positive lesions were strongly promoted by Wy-14,643, both in Cx32-wild-type and Cx32-null mice without significant difference in response between mice of the two genotypes. This contrasts G-6-Pase-negative lesions and lesions over-expressing GS, which were both increased by WY-14,643 treatment in number and size in Cx32-wild-type but not in Cx32-null mice. GS-positive lesions from WY-14,643-treated mice harboured beta-catenin mutations, a hallmark of lesions selected during promotion by PB, while G-6-Pase-positive lesions, which displayed negative or diffuse GS staining, did not show beta-catenin mutations. Our results demonstrate significant differences between mouse liver lesions of differing phenotype and genotype in their response towards selection by Wy-14,643 during the promotional phase of hepatocarcinogenesis.     

Tumor formation in liver of conditional β-catenin-deficient mice exposed to a diethylnitrosamine/phenobarbital tumor promotion regimen

The antiepileptic drug phenobarbital (PB) is a potent tumor promoter in mouse liver, where it stimulates the selective outgrowth of tumor populations harboring activating mutations in Ctnnb1, encoding β-catenin. A tumor initiation-promotion study was conducted in mice with conditional hepatocyte-specific knockout (KO) of Ctnnb1 and in Ctnnb1 wild-type controls. Mice received a single injection of N-nitrosodiethylamine (DEN) at the age of 6 weeks followed by continuous administration of PB given in the diet (0.05%) for 27 weeks. Metabolic activation of DEN in hepatocytes from both Ctnnb1 wild-type and KO mice was demonstrated. PB strongly enhanced liver tumor formation in Ctnnb1 wild-type mice, and 90% of the PB-promoted tumors were Ctnnb1-mutated. A similar increase in carcinogenic response was seen when using glucose-6-phosphatase and glutamine synthetase as tumor markers. The prevalence of tumors in Ctnnb1 KO mice was ～7-fold higher than in wild-type mice, suggesting an enhancing effect of the gene KO on liver tumor development. However, in strong contrast to wild-type mice, PB did not promote tumor formation in the Ctnnb1 KO mice. Livers of KO mice, particularly from the PB treatment group, demonstrated fibrosis and massive infiltration of immune cells, an effect not seen in wild-type mice. In summary, our data demonstrate that (i) liver tumor promotion by PB requires functional β-catenin signaling and (ii) absence of β-catenin enhances carcinogen-induced hepatocarcinogenesis and induces a pre-cirrhotic phenotype in mouse liver.     

Suppression of beta-catenin mutation by dietary exposure of auraptene, a citrus antioxidant, in N,N-diethylnitrosamine-induced hepatocellular carcinomas in rats

Alteration of beta-catenin expression in hepatocellular carcinomas (HCCs) may play an important role in tumor progression by stimulating cell proliferation. We have previously reported that auraptene (AUR), an antioxidant agent isolated from citrus fruit, effectively inhibits chemically induced hepatocarcinogenesis in rats. In this study, we investigated the molecular mechanism of the inhibitory effects of dietary supplementation with AUR on N,N-diethylnitrosamine (DEN)-initiated hepatocarcinogenesis. Male F344 rats initiated with DEN were fed the AUR-containing diet during either the initiation stage (initiation feeding for 7 weeks) or post-initiation phase (post-initiation feeding for 25 weeks) of hepatocarcinogenesis. Liver tumors >5 mm in diameter were used for the analysis of beta-catenin gene mutation and beta-catenin protein expression. After PCR amplification of exon 2 of the beta-catenin gene, the products were sequenced directly. Mutations in the beta-catenin gene were detected in 8 of 24 HCCs (33.3%) in the DEN alone group, 7 of 15 HCCs (46.7%) in the DEN + AUR (initiation feeding) group, and 0 of 8 HCCs (0%) in the DENright curved arrow AUR (post-initiation feeding) group. No mutations of beta-catenin gene were detected in liver cell adenomas of any group. These results demonstrate that AUR exposure in post-initiation period suppresses the occurrence of HCCs with beta-catenin mutation, presumably by negative selection for neoplastic cells harboring the mutation.     

SHORT COMMUNICATION: Phenobarbital selectively promotes initiated cells with reduced TGF{beta} receptor levels

Phenobarbital (PB) is a potent tumor promoter in rodent liver.In this study we investigated whether PB selectively promotesa population of initiated cells with reduced levels of transforminggrowth factor-ß (TGFß) receptors types I,II and III. Liver tumors were induced in male Fischer F344 ratsby diethylnitrosamine (DEN). Following induction the animalswere divided into PB-treated (DEN/PB) and untreated groups (DEN).After 3 months of treatment half of the PB-treated rats wereremoved from PB for the final month (DEN/PB/OFF). At 4 months,the livers from rats in the three treatment groups were removed,tumors excised and frozen with matched surrounding normal tissue.The mRNA levels for the TGFß receptors types I-IIIwere significantly decreased in tumor tissue from DEN/PB ratswhen compared with surrounding normal liver tissue or tumorsfrom age-matched untreated controls. In tumors from DEN/PB/OFFrats the TGFß receptor types I-III were also significantlyreduced compared with controls and not different to tumors fromDEN/PB rats. There was no difference in the mRNA levels forthe TGFß receptors in tumors from rats exposed toDEN alone, when compared with the surrounding normal tissue.These results demonstrate that PB selectively promotes initiatedcells with reduced levels of TGFß types I-III receptorsand suggests a mechanistic role for TGFß in PB-inducedliver tumor promotion.     

Hepatocarcinogenesis in mice with a conditional knockout of the hepatocyte growth factor receptor c‐Met

The receptor for the hepatocyte growth factor/scatter factor (HGF/SF), c‐Met, plays a role in tumour promotion, progression and metastasis. In this study, we analysed chemically induced hepatocarcinogenesis in mice lacking a functional HGF receptor in their liver. Control and c‐Met deficient mice were injected with a single dose of N‐nitrosodiethylamine (DEN, 90 μg/g b.wt.) at 6 weeks of age and mice were subsequently kept on a phenobarbital (PB) containing diet (0.05%) for 35 weeks or on control diet. At the end of the experiment, the carcinogenic response in liver of the animals was monitored. Conditional c‐met knockout (KO) mice showed a higher prevalence of macroscopically visible liver tumours and of glutamine synthetase positive and glucose‐6‐phosphatase deficient lesions in liver. Tumour promotion by PB led to significant increases in the number of preneoplastic and neoplastic lesions in liver of both wild‐type and c‐met knockout mice, with only minor differences in response. Our results indicate that a defect in c‐Met‐mediated signaling increases chemically induced tumour initiation in liver but does not significantly affect PB‐mediated tumour promotion. © 2008 Wiley‐Liss, Inc.     

Lack of Promoting Effects of Phenobarbital at Low Dose on Diethyl-nitrosamine-induced Hepatocarcinogenesis in TGF-a Transgenic Mice

Phenobarbital (PB), a rodent non-genotoxic carcinogen, showed hormesis, biphasic effects on rat livercarcinogenesis. To test the hypothesis that the hormesis earlier observed for PB induced hepatocarcinogenesis mightalso exist in the TGF-α transgenic mice model, one which is highly susceptible to carcinogenesis, the carcinogenic orpromotion effects of a wide range of phenobarbital (PB) concentrations were investigated. Two weeks after a singlei.p. dose of 5 mg /kg bw of diethylnitrosamine (DEN) to 15 day old mice, animals were treated with diet containingPB at doses of 0, 2, 15 or 500 ppm. The incidence and multiplicity of tumors, including hepatocellular adenomas andcarcinomas, were significantly increased by the high dose of PB, but no significant difference among the groupsreceiving 2 and 15 ppm for liver tumors when compared to DEN alone group. The proliferating cell nuclear antigenindices for liver tumors and surrounding hepatocytes in high dose PB treated mice were significantly increased, butno change was noted at the lower doses. The total cytochrome P450 content in the liver was also elevated by 500 ppmof PB, while hepatic 8-OHdG levels demonstrated no significant change. In conclusion, PB at high dose enhancesDEN-induced hepatocarcinogenesis in TGF-α transgenic mice, but low doses lack any significant effects. One possiblemechanism of phenobarbital carcinogenicity might be influenced by cytochrome P450 system exhibiting a strongpromoting activity for liver of mice.     

The inhibition by methionine and choline of liver carcinoma formation in male C3H mice dosed with diethylnitrosamine and fed phenobarbital

The ability of the dietary methyl donors methionine and choline to inhibit the carcinogenic and tumor-promoting effects of phenobarbital (PB) in the livers of male weanling C3H mice was examined. The mice were fed a commercial rodent diet with or without 0.05% PB. Thirty animals from each set received the diet with either: (1) no dietary supplementation, (2) an additional 1.0% choline chloride, (3) 1.5% DL-methionine or (4) both 1.5% DL-methionine and 1.0% choline chloride. Additional groups of 30 animals with the same eight dietary and PB-treatment regimens described above were given a single initiating dose of 150 mg diethylnitrosamine (DENA)/kg body wt dissolved in saline, or the saline solution only, 1 week prior to the start of PB feeding. The 16 treatment groups were fed their respective diets for 12 months. Statistical trend analysis showed that increasing levels of supplemental methyl donors gave highly significant protection in PB-treated mice (P less than 0.01). The incidence of liver carcinomas in the four dietary groups not receiving PB or DENA varied from 0 to 7%. The PB-treated animals not receiving an initiating dose of DENA developed hepatocellular carcinomas (HCCs) at incidences of 79% in group 1 animals, 74% in group 2 animals, 60% in group 3 animals, and 31% in group 2 animals respectively. Thus, incidence of HCCs in group 4 was significantly lower than in groups 1, 2 or 3 (P less than 0.01). However, the total incidence of liver tumors (adenomas plus carcinomas) was about the same in all DENA or PB-treated groups. Thus, dietary supplementation with methyl donors increased the proportion of animals bearing liver adenomas as their most advanced hepatic lesion in PB-treated mice. In DENA-treated mice fed PB, dietary supplementation with methionine and choline protected against the formation of liver carcinomas (P less than 0.02); however, methionine and choline had no significant effect on liver tumor formation in mice fed the PB-free diets. Methionine and choline supplementation gave significant protection against HCC metastases in the lungs of the tumor-bearing mice in groups initiated with DENA followed by PB promotion. These results support the hypothesis that PB exerts it tumorigenic activity in mice at least in part through a physiological insufficiency of labile methyl groups.     

Inhibition by phenobarbital and lack of effect of amobarbital on the development of liver tumors induced by N-nitrosodiethylamine in juvenile B6C3F1 mice

The effects of phenobarbital (PB) and amobarbital (AB) on the rate of development of hepatocarcinogenesis induced by N-nitrosodiethylamine (DEN) were studied in mice. Groups of 40 B6C3F1 male mice were injected i.p. at 15 days of age with 5 micrograms DEN/g body wt. Beginning at 4 weeks of age, DEN treated groups were given either normal drinking water or water containing either 0.05% PB or AB for up to 36 weeks. DEN alone induced multiple focal hepatic lesions including hepatocellular foci, hepatocellular adenomas and trabecular carcinomas. Subsequent exposure to PB had a suppressing effect on DEN-induced hepatocarcinogenesis. Hepatocellular foci in PB-exposed mice were significantly smaller in size (area) and fewer in number throughout the study. Also, PB treatment either prolonged the latency period or significantly slowed the rate at which hepatocellular tumors developed in these mice. No such effects were seen in AB-exposed mice; AB neither inhibited nor promoted the development of focal hepatic lesions in DEN-pretreated mice. Possible mechanisms responsible for the inhibition of DEN-induced hepatocarcinogenesis include the feminizing effects of perinatal administration of PB.     

Pentachlorophenol (PCP) produces liver oxidative stress and promotes but does not initiate hepatocarcinogenesis in B6C3F1 mice.

To elucidate the mechanism of hepatocarcinogenesis of pentachlorophenol (PCP) in mice, critical effects related to carcinogenicity were studied in the livers of B6C3F1 male mice administered PCP at concentrations of 600 and 1200 p.p.m. in the diet for 8 weeks. Oxidative stress was assessed by measurements of 8-oxodeoxyguanosine (8-oxodG) in the liver nuclear DNA and hepatocyte cell proliferation was quantified by bromodeoxyuridine incorporation. Also, initiation and promotion were assessed in a two-stage hepatocarcinogenesis model in which one group of mice was given PCP at concentrations of 600 and 1200 p.p.m. as initiator for the first 13 weeks with subsequent administration of phenobarbital (PB) as promoter at a concentration of 500 p.p.m. in the drinking water for 29 weeks. A second group was initiated with diethylnitrosamine (DEN) at 20 p.p.m. in the drinking water for the first 13 weeks followed after a 4 week recovery interval by PCP at concentrations of 300 and 600 p.p.m. in the diet for 25 weeks. Significant elevations in 8-oxodG levels and cell proliferation were observed in a dose-dependent manner. Incidences and multiplicities of hepatocellular tumors in mice treated with PCP after DEN initiation were increased compared with those in mice given initiation only. In contrast, in mice given PCP as initiator followed by PB no enhancement of neoplastic lesions occurred. These findings are interpreted to demonstrate that PCP exerts a promoting action, but not an initiating effect on liver carcinogenesis and that the promoting action is related to oxidative stress and compensatory hepatocellular proliferation.     

Beta-catenin (Ctnnb1) gene mutations in diethylnitrosamine (DEN)-induced liver tumors in male F344 rats.

Alterations in multiple phosphorylation sites on exon 3 of the beta-catenin gene have recently been implicated in hepatocarcinogenesis in humans as well as mice. To identify genetic alterations which could be involved in the chemical-induced hepatocarcinogenesis of rats, we analyzed the status of the sites in the beta-catenin gene (Ctnnb1) of liver neoplasms induced by diethylnitrosamine (DEN) in male F344 rats, using the polymerase chain reaction-single strand conformation polymorphism method. In the present investigation, we examined 35 hepatocellular neoplasms (28 adenomas and 7 carcinomas) for the expression of mutations in the region of the beta-catenin gene. Point mutation at codon 32, 35, 37 or 41, which has been reported in human and mouse liver cell carcinomas and/or other cancers, was recognized in eleven (31%) out of 35 lesions (8 adenomas and 3 carcinomas). Our results indicate that Ctnnb1 mutations may contribute to hepatocarcinogenesis in rats. Our finding that Ctnnb1 mutation was present in adenomas as well as carcinomas also suggests that the mutation is a relatively early event in DEN-induced hepatocarcinogenesis in rats.     

Possible tumor development from double positive foci for TGF-alpha and GST-P observed in early stages on rat hepatocarcinogenesis

Expression of TGF-alpha during promotion of neoplastic development from GST-P-positive foci in rat chemical hepatocarcinogenesis was investigated. One-hundred male F344 rats were given a single intraperitoneal injection of DEN (200 mg/kg bodyweight) and subjected to two-thirds partial hepatectomy at week 3. Commencing 2 weeks from the start, PB at doses of 0 or 500 p.p.m. was fed to the rats for 46 weeks. Groups of 10 rats were killed at weeks 4, 8, 16, 32, 48 and their livers were immunohistochemically examined for expression of GST-P and TGF-alpha. TGF-alpha-positive foci and single positive cells were observed from week 4, partially overlapping with GST-P-positive foci but being much fewer. Numbers of TGF-alpha-positive lesions did not increase from weeks 4-48, but their areas showed increment at weeks 32 and 48, especially with PB administration. Almost all of the tumors observed at weeks 16, 32 and 48 were positive for TGF-alpha (98%). In addition, epidermal growth factor receptor overexpression was observed in most TGF-alpha-positive lesions (foci and tumors). The proliferating cell nuclear antigen labeling index in double positive foci for GST-P and TGF-alpha was significantly higher than that in TGF-alpha-negative foci. In conclusion, TGF-alpha may be closely related with promotion from altered foci to neoplasms in rat hepatocarcinogenesis. Our data suggest that double positive foci for GST-P and TGF-alpha in the early stages of rat hepatocarcinogenesis may develop into tumors with promotion.     

Lack of tumorigenesis in the mouse liver after adenovirus-mediated expression of a dominant stable mutant of beta-catenin

Mutations in the glycogen synthase kinase 3beta (GSK3beta) phosphorylation sites of the beta-catenin gene exon 3 are found in 20-30% of human primary hepatocellular carcinoma (HCC), whereas mutations in the APC or AXIN genes are found in other HCC populations. These data strongly suggest that the Wnt signaling pathway is involved in hepatocarcinogenesis. To determine the role of beta-catenin in intestinal tumorigenesis, we earlier constructed a mutant mouse strain Catnb(lox(ex3)), in which exon 3 of the beta-catenin gene was sandwiched by loxP sequences. By genetic crosses of these mice with the Fabpl-cre transgenic mice that express the cre gene controlled by the fatty acid binding protein gene promoter, we introduced the beta-catenin stabilizing mutation into the small intestine and liver. Although numerous polyps were formed in the small intestine, we did not find any neoplastic (i.e., dysplastic) foci in the liver, and the mice died in 5 weeks after birth because of acute liver damage accompanying mitochondrial swelling. When a recombinant adenovirus that expresses the cre gene from a human cytomegalovirus early gene promoter was constructed and inoculated at a high multiplicity (10(9) plaque-forming units/mouse), the Catnb(lox(ex3)) mice showed marked hepatomegaly, with similar mitochondrial swelling in the hepatocytes, and died within 3 weeks after infection. On the other hand, when inoculated at lower multiplicities of infection (10(7) and 10(8) plaque-forming units/mouse, respectively), the Catnb(lox(ex3)) mice survived >6 months without any neoplastic foci in the liver, although the nuclear localization of beta-catenin was found in some hepatocytes even after 6 months. These results suggest that, in contrast to intestinal polyposis, the Wnt pathway activation by stabilized beta-catenin is not sufficient for hepatocarcinogenesis, but additional mutations or epigenetic changes may be required.     

Enhancement of murine hepatocarcinogenesis by all-trans-retinoic acid and two synthetic retinamides

Although retinoids have significant chemopreventive activity in many in vivo carcinogenesis models, their influence on experimental hepatocarcinogenesis has received only limited study. The present experiment was designed to determine the effects of three retinoids on hepatocarcinogenesis induced in female B6D2F1 mice by diethylnitrosamine (DEN). Beginning 1 week after a single i.p. dose of 0, 50 or 100 mg DEN per kg body wt, groups of 35 mice received dietary supplements of (per kg diet): 0.1 mmol all-trans-retinoic acid (RA), 0.5 or 1.0 mmol all-trans-ethyl retinamide (ER), 0.5 or 1.0 mmol 13-cis-ethyl retinamide (13-cis-ER), or vehicle only. In mice treated with 100 mg DEN/kg, all three retinoids significantly increased the incidence of liver tumors (benign + malignant) from the level seen in dietary controls; significant increases in the incidence of hepatocellular carcinoma were seen in groups fed ER. At the lower DEN dose, all three retinoids significantly increased the incidence of total liver tumors and of hepatocellular carcinomas. In mice not treated with DEN, liver tumors were seen only in mice fed ER or 13-cis-ER at 1.0 mmol/kg diet. These data indicate that RA, ER and 13-cis-ER can promote liver carcinogenesis in mice when administered at doses that inhibit cancer induction in other tissues. Because chronic exposure to many retinoids results in the deposition and accumulation of both parent compound and metabolites in the liver, these results should suggest caution in the design of clinical chemoprevention trials with this class of compounds.     

Liver regeneration and hepatocarcinogenesis in transforming growth factor-α-targeted mice

Transforming growth factor-α (TGFα), a member of the epidermal growth factor receptor ligand family, has been implicated in the regeneration and transformation of liver. Our recent development of mice that are homozygous for a disrupted TGFα gene allowed us to assess the requirement for this growth factor in these complex processes. We report here that although a 70% hepatectomy produced a significant increase in hepatic TGFα protein levels in wild-type mice, liver regeneration nevertheless proceeded normally in the absence of the growth factor. The hepatocyte labeling indices determined for homozygous targeted and wild-type mice at 36 and 48 h after hepatectomy were comparable, and the total liver DNA to body weight ratios 8 d after hepatectomy were essentially identical for the two genotypes. These results indicate that TGFα is not necessary for liver regeneration. To test its requirement in liver carcinogenesis, young mice were administered single doses of diethylnitrosamine (DEN) with or without subsequent chronic treatment with the promoting agent phenobarbital (PB). Both wild-type and homozygous mutant male mice treated with DEN or DEN plus PB developed multiple preneoplastic foci or tumors by 9 mo of age with relatively high incidence. However, while five of 88 tumors in wild-type mice attained a diameter greater than 5 mm and were classified as hepatocellular carcinomas, none of 132 tumors in livers of targeted mice reached this size. Furthermore, three of these large wild-type tumors expressed significantly elevated levels of TGFα protein compared with normal liver. These results indicate that TGFα is not required for early events in chemically induced hepatocarcinogenesis but suggest that it could be important in the progression from small preneoplastic foci to large tumors. © 1996 Wiley-Liss, Inc.     

Anti-carcinogenic action of phenobarbital given simultaneously with diethylnitrosamine in the rat

The present work has been planned in order to elucidate the effect of phenobarbital (PB: 15 mg per rat of ingested dose) on carcinogenesis when it is administered simultaneously with diethylnitrosamine (DEN: 10 mg/kg/day). Wistar rats (180 g) were treated by DEN alone or by DEN + PB during 2, 4 and 6 weeks according to our schedule for hepatocarcinogenesis. After the end of the treatment, the number and the size of induced PAS positive preneoplastic foci was significantly reduced when PB was given simultaneously with DEN for 4 and 6 weeks. The mitotic inhibition and the production of micronuclei normally observed after partial hepatectomy in DEN treated rats were also significantly decreased in DEN + PB treated rats. When the treatment last only 2 weeks, the presence of PB did not change significantly the last parameters. In DEN + PB treated rats, the survival was prolonged and the tumor incidence decreased as compared with the results obtained by DEN alone. It is concluded that PB, which promotes carcinogenesis when administered after the DEN treatment, reduces the carcinogen effect when given simultaneously with DEN. This ‘anti-carcinogen’ effect acts on the initiation as well as on the promotion of the precancerous lesions. Biochemical investigations are in progress to obtain more information about this ‘paradoxical’ PB effect.