Preneoplastic Foci Research Articles

Cocarcinogenic Effects of Islet Hormones and N-Nitrosomorpholine in Hepatocarcinogenesis after Intrahepatic Transplantation of Pancreatic Islets in Streptozotocin-Diabetic Rats

In contrast to high local insulin levels obtained after low-number transplantation (n = 350) of islets of Langerhans into the livers of diabetic rats, low insulin levels after high-number transplantation (n = 1,000) do not suffice to induce hepatocarcinogenesis. Herein, we investigated the possible cocarcinogenic potential of high and, in particular, low insulin levels, combining this in vivo model with a chemical model of hepatocarcinogenesis after administration of N-nitrosomorpholine (NNM). In three main experiments, different schemes of single or continuous NNM administration were combined with different transplantation procedures in diabetic or nondiabetic animals, i.e., low-number and high-number islet transplantation, transplantation of polystyrene particles, and sham transplantation. Animals were sacrificed between 3 and 53 weeks after transplantation procedures. Evidence for the cocarcinogenic effects of NNM and insulin was provided in each main experiment. NNM treatment after low-number islet transplantation resulted in an increase in the number of preneoplastic hepatocellular foci, and a significant increase in the number and an earlier appearance of hepatocellular adenomas and carcinomas compared with controls. Most intriguing was the increase in preneoplastic foci after combined NNM treatment and high-number islet transplantation, proving that insulin, even in lower doses, has at least cocarcinogenic effects on the downstream hepatocytes and thus promotes an otherwise initiated hepatocarcinogenic process. Conclusively, intrahepatic transplantation of pancreatic islets acts as a strong cocarcinogenic factor together with NNM in streptozotocin-diabetic rats.

Enzymatic detection of precursor cell populations of preneoplastic foci positive for γ‐glutamyltranspeptidase in rat liver

An improved staining method for gamma-glutamyltranspeptidase (GGT) was developed using Vibratome-prepared microslices. Microscopic precursor cell populations of preneoplastic foci positive for the marker enzyme were detectable sequentially in rat liver by tracing back from 5 to 1 week after carcinogen injection in a hepatocarcinogenesis model. Mirror-image comparisons of serial sections stained for GGT activity and immunocytochemically stained for GST-P (glutathione S-transferase P-form) revealed that GGT expression was confined within GST-P(+) cell populations (GST-P(+) minifoci), which are induced in the periportal area (zone 1) of the liver. GGT expression level differed from one minifocus to another, and the larger the GST-P(+) focus, the stronger was the GGT expression in it, indicating that GST-P(+)/GGT(-) phenotypes are convertible into proliferating GST-P(+)/GGT(+) ones. Our results suggest that there are at least 2 closely related precursors, GST-P(+)/GGT(-) and GST-P(+)/GGT(+) phenotypes, of preneoplastic foci in rat chemical hepatocarcinogenesis.

Uroporphyria and hepatic carcinogenesis induced by polychlorinated biphenyls–iron interaction: Absence in the Cyp1a2(− /− ) knockout mouse

Aryl hydrocarbon receptor ligands, such as polychlorinated biphenyls (PCBs), cause inhibition of the heme biosynthesis enzyme, uroporphyrinogen decarboxylase; this leads to uroporphyria and hepatic tumors, which are markedly enhanced by iron overload in C57BL/10 and C57BL/6 strains of mice. Cyp1a2(− /− ) knockout mice were used to compare the effects of CYP1A2 expression on uroporphyria and liver carcinogenesis. PCBs in the diet (100 ppm) of Cyp1a2(+/+) wild-type mice caused hepatic uroporphyria, which was strongly increased by iron–dextran (800 mg Fe/kg). In contrast, uroporphyria was not detected in Cyp1a2(− /− ) knockout mice, although expression of CYP1A1 and CYP2B10 was greatly induced. After 57 weeks on this diet, hepatic preneoplastic foci and tumors were seen in the Cyp1a2(+/+) mice; numbers and severity were enhanced by iron. No foci or tumors were detected in Cyp1a2(− /− ) mice, although evidence for other forms of liver injury was observed. Our findings suggest a link not only between CYP1A2, iron metabolism, and the induction of uroporphyria by PCBs, but also with subsequent hepatocarcinogenesis.

Cytoplasmic Phospholipase A2 Levels Correlate with Apoptosis in Human Colon Tumorigenesis

Colon cancers often display perturbations in arachidonic acid metabolism, with elevated levels of cyclooxygenase (COX)-2 expression and prostaglandin E(2) (PGE(2)) production frequently observed. Whereas COX-2 and PGE(2) are associated with cancer cell survival and tumor angiogenesis, arachidonic acid itself is a strong apoptotic signal that may facilitate cancer cell death. To further explore how cancer cells exploit the progrowth actions of prostaglandins while suppressing the proapoptotic actions of intracellular arachidonic acid, we determined the cytoplasmic phospholipase A(2) (cPLA(2)) and COX-2 expression levels in a panel of human colon tumors by immunohistochemistry. Although high levels of cPLA(2) and COX-2 expression are predicted to facilitate maximal prostaglandin production, tumors frequently displayed a high-COX-2/low-cPLA(2) phenotype. The least represented phenotype was the high expression of cPLA(2), a characteristic predicted to generate the highest levels of intracellular arachidonic acid. The potential proapoptotic role of cPLA(2) was supported by a higher frequency of terminal deoxynucleotidyl transferase-mediated nick end labeling staining in cPLA(2)-positive tumors. Moreover, analysis of preneoplastic aberrant crypt foci from high-risk patients suggests that acquisition of the high-COX-2/low-cPLA(2) phenotype may arise at an early stage of colon carcinogenesis. We additionally inhibited cPLA(2) in HT-29 cells using antisense oligonucleotides. Our results indicate that cPLA(2) plays an important role in tumor necrosis factor alpha-induced apoptosis in human colon cancer cells. Our data further support the model in which colon cancer growth is favored when intracellular arachidonic acid levels are suppressed by inhibition of cPLA(2) or by a high-COX-2/low-cPLA(2) phenotype.

Downregulation of apoptosis revealed by laser microdissection and cDNA microarray analysis of related genes in rat liver preneoplastic lesions

Regulation of cell proliferation and apoptosis plays a key role in tumor development. To elucidate mechanisms underlying hepatocarcinogenesis, patterns of gene expression, including apoptosis-related genes, were compared between glutathione S-transferase placental form (GST-P)-positive preneoplastic foci and surrounding tissue in the rat. Lesions were induced with a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg bw) and then 100 ppm 2-acetylaminofluorene (2-AAF)-containing diet combined with two-thirds partial hepatectomy. Frozen sections of the livers were applied for immunohistochemical staining of GST-P, and both positive foci and surrounding negative areas were harvested by laser microdissection. Total RNAs were extracted and amplified with T7 polymerase to allow gene expression analysis by cDNA microarray assays. In the GST-P-positive foci, altered levels were observed for many genes, mostly related to metabolism or catalysis, with increased expression of testosterone-repressive prostate message-2 (TRPM-2), which is reported to act as a protective factor against apoptosis, and decreased expression of thymus-expressed acidic protein (TEAP), which is considered to promote apoptosis. The results indicate that rat liver preneoplastic lesions might be protected against apoptosis and that the approach adopted is useful for clarification of mechanisms underlying hepatocarcinogenesis.

Apoptosis in Stages of Mouse Hepatocarcinogenesis: Failure to Counterbalance Cell Proliferation and to Account for Strain Differences in Tumor Susceptibility

C3H/He and B6C3F1 show much higher liver cancer susceptibility than C57BL/6J mice. We studied the hypothesis that this difference might result from failure of apoptosis. Hepatocarcinogenesis was induced by a single dose of N-nitrosodiethylamine (NDEA), followed by phenobarbital (PB) for up to 90 weeks. We observed (1) earlier appearance of putative preneoplastic foci (PPF), hepatocellular adenoma (HCA), and carcinoma (HCC) in C3H/He than in C57Bl/6J mice and (2) an increase of hepatocellular DNA synthesis in C3H/He and C57Bl/6J mice, compared to normal liver, via PPF and HCA to HCC. PB enhanced DNA synthesis and growth of PPF, in the C3H/He strain only, and of HCA and HCC of both strains. Apoptoses were rare in unaltered livers as well as in preneoplastic lesions, but tended to increase in HCA and HCC of both strains. PB lowered apoptotic activity in PPF of C3H/He mice, but enhanced it in HCA and HCC of C57Bl/6J mice at late stages. In conclusion, the strain difference in growth rates of PPF and tumors is largely determined by higher rates of cell proliferation in C3H/He mice, with and without promotion by PB. Moreover, in C57Bl/6J mice the promoting effect of PB was restricted to HCA and HCC and was not seen in PPF. Apoptosis was generally low and was not a major cause of the strain difference in tumor susceptibility. In contrast with rat liver, inhibition of apoptosis appears to be a minor determinant of tumor promotion in mice.

Long term phenobarbital administration does not promote the multiplication of hepatocytes replicating after single cyproterone acetate administration

Cyproterone acetate (CPA) is a synthetic antiandrogenic compound which is widely used in clinic but suspected to be hepatocarcinogenic. CPA is also mitogenic in rat liver. Using genetic labeling of dividing cells, we examined whether hepatocytes dividing in response to acute CPA administration could give rise to preneoplastic foci after administration of a tumor promoter: phenobarbital. CPA was administered orally to rats and dividing hepatocytes were genetically labeled using retroviral vectors carrying the β-galactosidase gene. After labeling rats were given phenobarbital for 10 months and sacrificed. The presence of β-galactosidase labeled hepatocytes as well as preneoplastic hepatocytes was assessed by immunohistochemistry. Genetic labeling of hepatocytes was obtained in all animals. At the end of phenobarbital administration, no hepatic tumors were observed. Preneoplastic foci were not increased in treated animals as compared to control rats. Moreover β-galactosidase positive hepatocytes were never detected in preneoplastic foci. Finally, the size of the β-galactosidase postive clusters was smaller in treated animals as compared to control rats. We conclude that acute CPA administration is not carcinogenic in rat liver and does not initiate preneoplastic hepatocytes capable to give rise to foci after phenobarbital promotion. Therefore the mitogenic property of CPA is distinct from its putative carcinogenic activity. Finally, analysis of the size of β-galactosidase positive cells clusters demonstrate that phenobarbital does not induce hepatocyte proliferation in rats.

Overexpression of fatty acid synthase in chemically and hormonally induced hepatocarcinogenesis of the rat

Fatty acid synthase (FAS) is the key enzyme of de novo fatty acid synthesis and has been shown to be involved in carcinogenesis of numerous human malignancies, including breast, colorectal, and prostate carcinomas, often associated with a worse prognosis. Although FAS is mainly expressed in the liver, an implication of FAS in hepatocarcinogenesis, has not yet been investigated. FAS expression is stimulated by insulin and glucose, and insulin is also the primary trigger of hepatocarcinogenesis in an endocrine experimental model, which is induced by low-number transplantation of islets of Langerhans into the livers of diabetic rats. We therefore investigated, whether FAS is implicated in hepatocarcinogenesis in this model and in comparison to chemically induced hepatocarcinogenesis after N-nitrosomorpholine (NNM) treatment in diabetic and normoglycemic rats. Preneoplastic clear-cell foci of altered hepatocytes (FAH), harvested after laser-microdissection of kryostat sections, showed an overexpression of FAS messenger RNA in gene expression profiles, done by array-hybridization, and in quantitative RT-PCR (Light-Cycler). Virtually, all (96–98%) of the subsequently investigated FAH and the glycogenotic hepatocellular adenomas and carcinomas showed an additional strong FAS protein overexpression. In the NNM-model, FAS protein was also overexpressed in the vast majority (87%) of glycogenotic FAH and neoplasms, in particular in the diabetic animals. Also two spontaneous glycogenotic FAH in control animals displayed strong FAS overexpression. Basophilic lesions and neoplasms, which occasionally develop out of the primary clear-cell FAH at later stages of carcinogenesis, however, lost FAS overexpression. In conclusion, FAS overexpression is an early phenonemon in spontaneous, hormonally and chemically induced rat hepatocarcinogenesis, demonstrable in early clear-cell (glycogenotic) FAH and hepatocellular neoplasms. FAS overexpression can be attributed to the local hyperinsulinemia in the transplantation model and belongs to cellular and metabolic alterations in the chemical model, which are induced by an insulinomimetic but yet unknown mechanism.

Harman and Norharman Suppressed but NaNO2 Enhanced the Development of Preneoplastic Liver Cell Foci in 2-Amino-3,8-Dimethylimidazo[4,5-f]Quinoxaline (MeIQx)-Treated Rats

The modifying potentials of two heterocyclic amines, harman and norharman, as well as sodium nitrite (NaNO2), on 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced rat liver carcinogenesis were investigated using a medium-term liver bioassay system. Harman (500 ppm in diet), norharman (500 ppm in diet) or NaNO2 (0.1% in drinking water) were given with or without MeIQx (300 ppm in diet) for 6 weeks after initiation with a single dose of dimethylnitrosamine (DEN). The appearance of MeIQx-induced glutathione S-transferase placental form positive foci in the liver was significantly decreased in the harman and norharman cases (p< 0.001), but it was significantly increased by NaNO2 (p< 0.001). These chemicals, however, did not modify DEN-liver carcinogenesis in the absence of MeIQx. Neither harman nor norharman affected mRNA expression of CYP1A1 and 1A2 in the liver, with or without MeIQx administration, whereas NaNO2 significantly enhanced CYP1A1 mRNA levels together with MeIQx.

The gender-specific risk to liver toxicity and cancer of flounder ( Platichthys flesus ( L.)) at the German Wadden Sea coast

Flatfish living in coastal areas are chronically exposed to a wide range of toxic and (pro)carcinogenic compounds derived from agriculture and industry. Flounder ( Platichthys flesus ( L.)) is the main target species for monitoring health effects of contamination in North Sea and Baltic Sea since the species is abundant, benthic, and inhabits shallow waters such as the Wadden Seas and estuaries along salinity gradients into fresh water. Chemical analysis in the same livers as investigated for histopathology in the present study showed positive correlation between accumulation of certain organochlorines in liver and the extend of liver injury. Toxipathic liver changes including neoplasms in female and male flounder were analysed by macroscopic and light microscopic diagnosis during a five-year survey on the basis of internationally accepted criteria agreed upon during the European BEQUALM intercalibration of liver histopathology of flatfish. Hepatocellular carcinogenesis of wild flounder principally showed sequential changes similar to experimental chemical carcinogenesis in other fish species and mammals. These ranged from early foci of altered hepatocytes (vacuolated/clear/eosinophilic, basophilic cells) and the development of adenomas. With progression to hepatocellular carcinomas, livers of wild flounder entered a multistage phase of carcinogenesis comprising of early foci, hepatocellular adenomas and carcinomas, as observed in human liver cancers. Female flounder had three-fold higher frequencies of macroscopically visible neoplasms than males of the same age classes. Histopathological diagnosis showed that hepatocellular alterations in male flounder never developed further than stages of basophilic foci and adenomas, and never into malignancies. In females, tumors of hepatocellular origin clearly dominated, occurred alone and together with cancers of bile duct epithelial cells and endothelial cells (cholangio-carcinomas, angiosarcomas). Because mutations of relevant genes could not be identified in the liver cancers that were analysed in the present study, we conclude that epigenetic events initiate carcinogenesis. Therefore, we conclude that mitogenic carcinogens found in the Elbe estuary play a dominant role for cancer development in this flounder population. Furthermore, the lower availability of reducing power (NADPH) needed for detoxification reactions in females compared to males, together with annual upregulation of the highly potent promotor 17-β-estradiol during sexual maturation, promote progression of preneoplastic foci of initiated cells to malignant cancers in females.

Chemopreventive Effect of Indole-3-Carbinol on Induction of Preneoplastic Altered Hepatic Foci

: Indole-3-carbinol (I3C) is a cleavage product of glucobrassicanin, a natural compound present in a wide variety of plant food substances including members of the family Cruciferae. I3C is known to possess cancer-chemopreventive potential in various animal models. The present study reveals the protective effect of I3C on the development of diethylnitrosamine (DEN)-initiated and 2-acetylaminofluorene (AAF)-promoted preneoplastic, altered hepatic foci (AHF) in Wistar rats. I3C was given at dose levels of 0.5 and 1 mg/kg body weight for five consecutive days along with DEN and AAF. AHF were scored and analyzed by quantitative stereology using the Image Analysis System from frozen liver sections stained for positive and negative biological markers of AHF, that is, glutathione S-transferase (GST-P), γ-glutamyl transpeptidase (GGT), glucose-6-phosphatase (G6Pase), adenosine triphosphatase (ATPase), and alkaline phosphatase (AlkPase). Results revealed the chemopreventive effect of I3C on the DEN-initiated AHF in Wistar rats. The expression of G6Pase, ATPase, and AlkPase was restored in the I3C-supplemented animal. Similarly the induced expression GST-P and GGT also decreased in the animals with I3C administration. The recovery of altered levels of these biomarkers was of comparatively higher magnitude in the animals given a higher dose of I3C (1 mg/kg body weight) in comparison with the animals given 0.5 mg/kg body weight dose of I3C, although no dose-dependence pattern was recorded in I3C-supplemented groups. These results thus suggest the chemopreventive effect of I3C in rat hepatocarcinogenesis by suppressing DEN- and AAF-induced AHF development.

Genetic signatures of high- and low-risk aberrant crypt foci in a mouse model of sporadic colon cancer.

To determine whether cancer risk is related to histopathological features of preneoplastic aberrant crypt foci (ACF), gene expression analysis was performed on ACF from two mouse strains with differing tumor sensitivity to the colonotropic carcinogen, azoxymethane. ACF from sensitive A/J mice were considered at high risk, whereas ACF from resistant AKR/J mice were considered at low risk for tumorigenesis. A/J and AKR/J mice received weekly injections of azoxymethane (10 mg/kg body weight), and frozen colon sections were prepared 6 weeks later. Immunohistochemistry was performed using biomarkers associated with colon cancer, including adenomatous polyposis coli, beta-catenin, p53, c-myc, cyclin D1, and proliferating cell nuclear antigen. Hyperplastic ACF, dysplastic ACF, microadenomas, adjacent normal-appearing epithelium, and vehicle-treated colons were laser captured, and RNA was linearly amplified (LCM-LA) and subjected to cDNA microarray-based expression analysis. Patterns of gene expression were identified using adaptive centroid algorithm. ACF from low- and high-risk colons were not discriminated by immunohistochemistry, with the exception of membrane staining of beta-catenin. To develop genetic signatures that predict cancer risk, LCM-LA RNA from ACF was hybridized to cDNA arrays. Of 4896 interrogated genes, 220 clustered into six broad clusters. A total of 226 and 202 genes was consistently altered in lesions from A/J and AKR/J mice, respectively. Although many alterations were common to both strains, expression profiles stratified high- and low- risk lesions. These data demonstrate that ACF with distinct tumorigenic potential have distinguishing molecular features. In addition to providing insight into colon cancer promotion, our data identify potential biomarkers for determining colon cancer risk in humans.

Absence of carcinogenic and anticarcinogenic effects of annatto in the rat liver medium-term assay

Annatto (Bixa orellana L.) is a natural food colorant extensively used in many processed foods, especially dairy products. The lower cost of production and the low toxicity, make annatto a very attractive and convenient pigment in substitution to the many synthetic colorants. In the present study we investigate the carcinogenic and anticarcinogenic effects of dietary annatto in Wistar rat liver using the preneoplastic glutathione S-transferase (GST-P) foci and DNA damage biomarkers. Annatto, containing 5% bixin, was administered in the diet at concentrations of 20, 200, and 1000 ppm (0.07; 0.80 and 4.23 bixin/kg body wt/day, respectively), continuously during 2 weeks before, or 8 weeks after DEN treatment (200 mg/kg body wt, i.p.), to evaluate its effect on the liver-carcinogenesis medium-term bioassay. The comet assay was used to investigate the modifying potential of annatto on DEN (20 mg/kg body wt)-induced DNA damage. The results showed that annatto was neither genotoxic nor carcinogenic at the highest concentration tested (1000 ppm). No protective effects were also observed in both GST-P foci development and comet assays. In conclusion, in such experimental conditions, annatto shows no hepatocarcinogenic effect or modifying potential against DEN-induced DNA damage and preneoplastic foci in the rat liver.

Modulation of altered hepatic foci induction by diallyl sulphide in Wistar rats

Diallyl sulphide (DAS) is a sulphur-containing volatile compound present in garlic (Allium sativum). It has been shown to inhibit a number of chemically induced forms of cancer in experimental animals. The present study demonstrates the inhibitory effect of DAS on the development of diethylnitrosamine (DEN) initiated and 2-acetyl-aminofluorene (2-AAF) promoted preneoplastic altered hepatic foci (AHF) in Wistar rats. AHF were scored and analysed by quantitative stereology using the Image Analysis system from frozen liver sections stained for biological markers, namely glutathione S-transferase, placental form (GST-P), gamma-glutamyl transpeptidase (GGT), adenosine triphosphatase (ATPase), glucose-6-phosphatase (G6 Pase) and alkaline phosphatase (AlkPase). DAS-supplemented rats were found to restore the near-normal levels of enzymes GST-P and GGT when exposed to DEN and 2-AAF. DAS administration following DEN and 2-AAF exposure led to the restoration of enzymic activity of ATPase, G6 Pase and AlkPase, as evident by number and area of the foci. These findings suggest the protective role of DAS in rat hepatocarcinogenesis, by suppressing DEN- and 2-AAF-induced AHF development.

Interferon alpha-induced apoptosis on rat preneoplastic liver is mediated by hepatocytic transforming growth factor beta(1).

In previous work we showed that interferon alfa-2b (IFN-alpha2b) increases apoptosis on rat hepatic preneoplastic foci. The aim of this study was to determine if transforming growth factor beta1 (TGF-beta1) was involved in the programmed cell death on the foci. Animals were divided into 6 groups: subjected to a 2-phase model (diethylnitrosamine plus 2-acetylaminofluorene) of preneoplasia development (group 1); treated with IFN-alpha2b during the 2 phases (group 2); treated with IFN-alpha2b during initiation with diethylnitrosamine (group 3); treated with IFN-alpha2b during 2-acetylaminofluorene administration (group 4); subjected only to an initiation stage (group 5); and treated with IFN-alpha2b during the initiation period (group 6). Serum TGF-beta1 levels were increased in IFN-alpha2b-treated rats. Immunohistochemical studies showed that IFN-alpha2b significantly increased the quantity of TGF-beta1-positive hepatocytes in groups 2 to 4. Phosphorylated-Smads-2/3 (p-Smads-2/3) proteins in liver nuclear extracts were significantly elevated. To determine the source of TGF-beta1, isolated hepatocytes, Kupffer cells, and peritoneal macrophages from animals in groups 1 and 5 were cultured with or without IFN-alpha2b. IFN-alpha2b stimulus induced several-fold increases of TGF-beta1 secretion from hepatocytes. Neither Kupffer cells nor peritoneal macrophages secreted detectable TGF-beta1 levels when they were treated with IFN-alpha2b. IFN-alpha2b-stimulated cultured hepatocytes from preneoplastic livers showed enhanced apoptosis, measured by fluorescence microscopy and caspase-3 activity. They presented higher nuclear accumulation of p-Smads-2/3, indicating increased TGF-beta1 signaling. When anti-TGF-beta1 was added to the culture media, TGF-beta1 activation and apoptosis induced by IFN-alpha2b were blocked. In conclusion, IFN-alpha2b-induced production of TGF-beta1 by hepatocytes from preneoplastic liver is involved in the apoptotic elimination of altered hepatic foci.

Role of peroxisome proliferator-activated receptor-alpha (PPARalpha) in bezafibrate-induced hepatocarcinogenesis and cholestasis.

Prolonged administration of peroxisome proliferators to rodents typically leads to hepatocarcinogenesis. Peroxisome proliferator-activated receptor-alpha (PPARalpha) is required to mediate alterations in PPARalpha target gene expression, repress apoptosis, enhance replicative DNA synthesis, oxidative stress to DNA and hepatocarcinogenesis induced by the relatively specific PPARalpha agonist, Wy-14,643. Interestingly, administration of the less specific PPARalpha agonist, bezafibrate, leads to a modest induction of PPARalpha target genes in the absence of PPARalpha expression. In these studies, the role of PPARalpha in modulating hepatocarcinogenesis induced by long-term feeding of 0.5% bezafibrate was examined in wild-type (+/+) and PPARalpha-null (-/-) mice. The average liver weight was significantly higher in (+/+) and (-/-) mice fed bezafibrate than controls, but this effect was considerably less in (-/-) mice as compared with similarly treated (+/+) mice. Increased levels of mRNA encoding cell cycle regulatory proteins and DNA repair enzymes were found in (+/+) mice fed bezafibrate, and this effect was not found in (-/-) mice. In mice fed bezafibrate for 1 year, preneoplastic foci, adenomas and a hepatocellular carcinoma were found in (+/+) mice, while only a single microscopic adenoma was found in one (-/-) mouse. This effect was observed in both Sv/129 and C57BL/6N strains of mice, although only preneoplastic foci were observed in the latter strain. Interestingly, hepatic cholestasis was observed in 100% of the bezafibrate-fed (-/-) mice, and this was accompanied by significantly elevated hepatic expression of mRNA encoding bile salt export pump and lower expression of mRNA encoding cytochrome P450 7A1, consistent with enhanced activation of the bile acid receptor, farnesoid X receptor. Results from these studies demonstrate that the PPARalpha is required to mediate hepatocarcinogenesis induced by bezafibrate, and that PPARalpha protects against potential cholestasis.

Post-initiation modulating effects of allyl sulfides in rat hepatocarcinogenesis

Effects of administration of diallyl sulfide (DAS) and diallyl disulfide (DADS) on the promotion stage of hepatocarcinogenesis were investigated in rats using the Ito model. They were compared with those of phenobarbital (PB), a well-known liver promoter in rats. Initiation was induced by a single dose of N-nitrosodiethylamine (NDEA) and 3 weeks later, a partial hepatectomy was conducted. Two weeks after the NDEA injection, rats received either 0.05% allyl sulfides, PB or both in their diet for 8 weeks. Feeding with DAS increased the number of liver preneoplastic foci by 63% with respect to the untreated group. However, rats fed DAS showed a lower foci development than rats fed PB. The DADS group did not differ from control group for any of the measured morphometric parameters. Simultaneous administration of DADS with PB partially reduced the promotional activity of PB whereas DAS co-treatment did not modify PB properties. These findings confirm that DAS can act as a promoter in rat liver but exerts no co-promoting effect. Conversely, DADS was found to have promotion-inhibiting ability, suggesting that DADS has greater value than DAS as a chemopreventive agent.

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Regulatory mechanism of glutathione S-transferase P-form during chemical hepatocarcinogenesis: old wine in a new bottle

The expression of glutathione S-transferase P-form (GST-P) is markedly up-regulated in the initial phase of chemical hepatocarcinogenesis. It is unlikely that a specific genetic change is associated with this common response to a variety of carcinogens. Here, we describe how GST-P gene expression is induced by carcinogenic treatment, focusing on the changes in the network of liver-enriched transcription factors, including CCAAT/enhancer-binding proteins. Although the balance of positive and negative transcription factors regulates the expression of the GST-P gene, additional factors such as the altered regulation of growth control may certainly be necessary for these cells to develop into preneoplastic foci. Furthermore, our genetic analyses on the tumor susceptibility of (F344 x DRH)F2 rats support the hypothesis that the formation of GST-P-positive lesions is required but is not directly associated with final malignant transformation.

Induction of preneoplastic rat liver lesions with an attenuated p53 response by low doses of diethylnitrosamine.

Previous reports have documented an attenuated p53 response to DNA-damage in preneoplastic enzyme-altered foci (EAF). Data suggest that this alteration is an adaptation to genotoxic stress induced by carcinogens. Here, we have studied whether the altered p53 response in EAF can be related to acutely apoptotic or cytotoxic doses of the carcinogen diethylnitrosamine (DEN). Eight groups of rats received cumulative doses of 0.25, 0.5, 1.0 and 2.0 mmol DEN/kg, administered weekly for either 10 or 20 weeks. A ninth group received 3.0 mmol/kg for 10 weeks, which gave a supralinear EAF response. Twenty-four hours before sacrifice, all rats were given a challenge dose of DEN in order to induce a p53 response in hepatocytes. The numbers of p53-positive hepatocytes in EAF and in surrounding tissue were analyzed. Unexpectedly, all cumulative doses gave rise to p53-negative EAF and 20-week treatment gave larger EAF with fewer p53-positive hepatocytes than 10-week treatment. It was also observed that at the lowest doses, most EAF developed in midzonal areas. Similar results were obtained with aflatoxin B1. Single high doses of DEN induced p53 accumulation and apoptosis within 24 h, whereas lower doses did not. It is concluded that EAF with an attenuated p53 response can be induced by low doses of genotoxic compounds, not giving rise to acute apoptosis or necrosis. Instead, it is suggested that time is an important determinant for its development at low doses and that a delayed type of apoptosis might be important.