Resistant Hepatocyte Research Articles

Receptor‐Mediated Targeting of Toxin Results in Selection of Genetically Protected Hepatocytes Without Bystander Toxicity

AIM To target G418 specifically to liver cells via asialoglycoprotein receptors (AsGR) for selection of stably transfected neomycin resistant hepatocytes (NR+) without collateral damage. METHODS: A novel G418 conjugate was synthesized by peptide coupling to a galactose-terminal asialoorosomucoid (AsOR). Uptake studies were done by labeled-ligand assays, and toxicity by MTT assays on Huh7 (AsGR+, NR−), GFP-Huh7 (AsGR+, NR+), and 3T3 (AsGR−, NR−) cell lines. RESULTS: Uptake of conjugate (7–8 moles) of G418 per AsOR was the same as for AsOR alone. Conjugate inhibited growth of Huh7 cells by 84.3%. This inhibition was blocked by excess free AsOR. Incubation of 3T3 cells with free G418, but not conjugated G418 resulted in toxicity. GFP-Huh cells were not susceptible either to free or conjugated G418. Co-cultures of GFP-Huh7 and 3T3 in G418 alone resulted in only inhibition of 3T3 cells. In contrast, co-cultures with AsOR-G418 resulted in no significant toxicity to either cell type. CONCLUSIONS: G418-protein conjugates retained inhibitory activity targeted against AsGR (+), NR (−) cells. G418 modification to form a conjugate did not block its activity. Lack of effect of G418 conjugate on ASGR (−) cells suggests that the effects were specific, and not due to release of free G418 in the medium. The presence of NR gene in AsGR (+) cells protected those cells, and the effect without collateral damage to co-cultured AsGR (−) cells. Supported by Lopata Chair in Hepatitis Research and R01-DK042182.

Mapping a Sex Hormone–Sensitive Gene Determining Female Resistance to Liver Carcinogenesis in a Congenic F344.BN-Hcs4 Rat

Hepatocellular carcinoma (HCC) is prevalent in human and rodent males. Hepatocarcinogenesis is controlled by various genes in susceptible F344 and resistant Brown Norway (BN) rats. B alleles at Hcs4 locus, on RNO16, control neoplastic nodule volume. We constructed the F344.BN-Hcs4 recombinant congenic strain (RCS) by introgressing a 4.41-cM portion of Hcs4 from BN strain in an isogenic F344 background. Preneoplastic and neoplastic lesions were induced by the "resistant hepatocyte" protocol. Eight weeks after initiation, lesion volume and positivity for proliferating cell nuclear antigen (PCNA) were much higher in lesions of F344 than BN rats of both sexes. These variables were lower in females than in males. Lesion volume and PCNA values of male RCS were similar to those of F344 rats, but in females corresponded to those of BN females. Carcinomatous nodules and HCC developed at 32 and 60 weeks, respectively, in male F344 and congenics and, rarely, in F344 females. BN and congenic females developed only eosinophilic/clear cells nodules. Gonadectomy of congenic males, followed by beta-estradiol administration, caused a decrease in Ar expression, an increase in Er-alpha expression, and development of preneoplastic lesions comparable to those from BN females. Administration of testosterone to gonadectomized females led to Ar increase and development of preneoplastic lesions as in F344 males. This indicates a role of homozygous B alleles at Hcs4 in the determination of phenotypic patterns of female RCS and presence at Hcs4 locus of a high penetrance gene(s), activated by estrogens and inhibited/unaffected by testosterone, conferring resistance to females in which the B alleles provide higher resistance.

Gene Expression Profile Related to the Progression of Preneoplastic Nodules toward Hepatocellular Carcinoma in Rats

In this study, we investigated the time course gene expression profile of preneoplastic nodules and hepatocellular carcinomas (HCC) to define the genes implicated in cancer progression in a resistant hepatocyte model. Tissues that included early nodules (1 month, ENT-1), persistent nodules (5 months, ENT-5), dissected HCC (12 months), and normal livers (NIL) from adult rats were analyzed by cDNA arrays including 1185 rat genes. Differential genes were derived in each type of sample (n = 3) by statistical analysis. The relationship between samples was described in a Venn diagram for 290 genes. From these, 72 genes were shared between tissues with nodules and HCC. In addition, 35 genes with statistical significance only in HCC and with extreme ratios were identified. Differential expression of 11 genes was confirmed by comparative reverse transcription-polymerase chain reaction, whereas that of 2 genes was confirmed by immunohistochemistry. Members involved in cytochrome P450 and second-phase metabolism were downregulated, whereas genes involved in glutathione metabolism were upregulated, implicating a possible role of glutathione and oxidative regulation. We provide a gene expression profile related to the progression of nodules into HCC, which contributes to the understanding of liver cancer development and offers the prospect for chemoprevention strategies or early treatment of HCC.

176. An Inhibitor of Fumarylacetoacetate Hydrolase (Fah) for Selection of Transplanted Hepatocytes and Gene Correction In Vivo

Mice deficient in the tyrosine catabolic enzyme Fah are an excellent model for liver repopulation by selection. Fah positive cells from several different origins can engraft and correct the phenotype of a Fah-mutant liver. Non-cell based gene therapy leading to the in situ correction of the Fah-deficiency also works and repopulation levels of >90% are routinely achieved. Thus, hepatic Fah deficiency provides a powerful in vivo selection environment for cells genetically resistant to the enzyme deficiency. In order to mimic the same selective pressure in animals not genetically deficient in Fah, we developed a small molecule inhibitor of Fah. 4-[(2-carboxyethyl)- hydroxyphosphinyl]-3-oxobutyrate (CEHPOBA) has a nanomolar Ki for Fah and was injected intraperitoneally into C57/BL6 mice at a concentration of 1|[mu]|moles per gram body weight. In a first round of experiments, the duration of Fah inhibition in vivo was tested. We found that a once-daily injection resulted in a lasting enzyme repression. Next, we tested if transplanted hepatocytes could be selected under continuous CEHPOBA administration. As shown previously, hepatocytes mutant for the homogentisic acid dioxygenase (Hgd) an enzyme upstream of Fah in tyrosine catabolism are able to repopulate the livers of mice with Fah-deficiency. We therefore transplanted 500,000 sex-mismatched Hgd-mutant hepatocytes into Fah+ C57/BL6 mice and concomitantly started CEHPOBA treatment. Treated animals appeared healthy and displayed no major side-effects. After 4 weeks of daily CEHPOBA injection, the livers were harvested and analyzed by semi-quantitative PCR and histology for the presence of Y-chromosome. 1|[ndash]|10% of total hepatocytes were Y-chromosome positive. Transplanted, Y-chromosome positive hepatocytes displaying Fah wild type formed nodules proving clonal expansion and ongoing selection of the genetically favored hepatocytes. Our results show that a significant selection of genetically resistant hepatocytes can be achieved by CEHPOBA administration in vivo. This proof-of-principle opens the door to a whole new field of experimental and therapeutic options for cell transplantation and gene correction of liver diseases in vivo. CEHPOBA resistance occurs in cells lacking enzymes upstream of Fah in tyrosine metabolism and could therefore be achieved by shRNAs targeting these genes. This strategy is currently under investigation.

Role of HSP90, CDC37, and CRM1 as modulators of P16INK4A activity in rat liver carcinogenesis and human liver cancer†

Current evidence indicates that neoplastic nodules induced in liver of Brown Norway (BN) rats genetically resistant to hepatocarcinogenesis are not prone to evolve into hepatocellular carcinoma. We show that BN rats subjected to diethylnitrosamine/2-acetylaminofluorene/partial hepatectomy treatment with a "resistant hepatocyte" protocol displayed higher number of glutathione-S-transferase 7-7(+) hepatocytes when compared with susceptible Fisher 344 (F344) rats, both during and at the end of 2-acetylaminofluorene treatment. However, DNA synthesis declined in BN but not F344 rats after completion of reparative growth. Upregulation of p16(INK4A), Hsp90, and Cdc37 genes; an increase in Cdc37-Cdk4 complexes; and a decrease in p16(INK4A)-Cdk4 complexes occurred in preneoplastic liver, nodules, and hepatocellular carcinoma of F344 rats. These parameters did not change significantly in BN rats. E2f4 was equally expressed in the lesions of both strains, but Crm1 expression and levels of E2f4-Crm1 complex were higher in F344 rats. Marked upregulation of P16(INK4A) was associated with moderate overexpression of HSP90, CDC37, E2F4, and CRM1 in human hepatocellular carcinomas with a better prognosis. In contrast, strong induction of HSP90, CDC37, and E2F4 was paralleled by P16(INK4A) downregulation and high levels of HSP90-CDK4 and CDC37-CDK4 complexes in hepatocellular carcinomas with poorer prognosis. CDC37 downregulation by small interfering RNA inhibited in vitro growth of HepG2 cells. In conclusion, our findings underline the role of Hsp90/Cdc37 and E2f4/Crm1 systems in the acquisition of a susceptible or resistant carcinogenic phenotype. The results also suggest that protection by CDC37 and CRM1 against growth restraint by P16(INK4A) influences the prognosis of human hepatocellular carcinoma.

Chemopreventive N-(4-hydroxyphenyl)retinamide (fenretinide) targets deregulated NF- B and Mat1A genes in the early stages of rat liver carcinogenesis

Cell-cycle deregulation is an early event of hepatocarcinogenesis. We evaluated the role of changes in activity of nuclear factor kappaB (NF-kappaB) and some related pathways in this alteration, and the interference of N-(4-hydroxyphenyl)retinamide (HPR), a retinoid chemopreventive for various cancer types, with these molecular mechanisms and the evolution of preneoplastic liver to cancer. Male F344 rats, initiated according to the 'resistant hepatocyte' model of liver carcinogenesis, received weekly 840 nmol of liposomal HPR (SL-HPR)/100 g body wt or empty liposomes, between 5 and 25 weeks after initiation. Inhibition of DNA synthesis and induction of apoptosis occurred in pre-cancerous lesions, 7-147 days after starting SL-HPR, and a decrease in carcinoma incidence and multiplicity was observed 25 weeks after arresting treatment. An increase in NF-kappaB expression and binding activity, and under-expression of the inhibitor kappaB-alpha (IkappaB-alpha) were found in preneoplastic liver and neoplastic nodules, 5 and 25 weeks after initiation, respectively. These lesions also showed low expression of Mat1A and low activity of methionine adenosyltransferase I/III, whose reaction product, S-adenosyl-l-methionine, enhances IkappaB-alpha expression. SL-HPR prevented these changes and induced a decrease in expression of iNos, c-myc, cyclin D1 and Vegf-A genes, that were over-expressed in preneoplastic liver and nodules, and a decrease in Bcl-2/Bax, Bcl-2/Bad and Bcl-xL/Bax mRNA ratios with respect to the lesions of control rats. Liposomes alone did not influence the parameters tested. These results indicate that signal transduction pathways controlled by NF-kappaB, nitric oxide and S-adenosyl-l-methionine are deregulated in pre-cancerous lesions. Recovery from these alterations by SL-HPR is associated with chemoprevention of hepatocarcinogenesis. Overall, these studies elucidate some molecular changes, in early stages of hepatocarcinogenesis, and underline their pathogenetic role. Moreover, they demonstrate a partially new mechanism of HPR chemopreventive effect and indicate the potential clinical relevance of this compound for prevention of hepatocellular carcinoma.

Comparison of different initiation protocols in the resistant hepatocyte model

Several models in rat liver have been developed to study multistage carcinogenesis, including the Solt-Farber resistant hepatocyte model. In this model, initiation consists of either a necrogenic dose of a hepatocarcinogen or a non-necrogenic dose in conjunction with partial hepatectomy (PH). As an alternative to PH, we investigated two different procedures: fasting for 96 h followed by refeeding, or the use of one-day-old neonates. Male Fisher 344 rats were injected p.o. with diethylnitrosamine (DEN) (0, 20, or 100 mg/kg) 24 h after refeeding or PH (controls received DEN alone with no proliferative stimulus). For the neonatal group, male and female Fisher 344 rats were treated with DEN (0 or 20 mg/kg, i.p.) at one day of age. All initiated animals were treated at the same age (11 weeks) with the following selection agents: three daily doses of 2-acetylaminofluorene (AAF) (30 mg/kg), followed by a single dose of carbon tetrachloride (2 ml/kg), followed by three additional daily treatments of AAF (30 mg/kg). Rats were euthanized 2 weeks after the last AAF injection. The PH, neonatal male, and neonatal female groups receiving DEN developed more gamma-glutamyl transpeptidase (GGT)-positive foci per cubic centimeter and foci per liver as compared to untreated rats receiving the same proliferative stimulus, whereas the fasting/refeeding group and the group receiving no proliferative stimulus did not. All DEN-treated groups receiving one of the proliferative stimuli had more foci per cubic centimeter than the DEN-treated group receiving no proliferative stimulus. The volume fractions of GGT-positive foci in the PH/DEN and neonatal male/DEN groups were higher than those of both the DEN-treated group receiving no proliferative stimulus and the groups receiving the same proliferative stimulus without DEN. In neonatal females-receiving DEN, the volume fraction was not different from either neonatal females not receiving DEN or DEN-treated rats receiving no proliferative stimulus. The volume fraction in the fasting/refeeding group was increased when DEN was administered at 100 mg/kg but not at 20 mg/kg. We conclude that the use of male neonatal rats can replace the PH in the Solt-Farber protocol. Fasting/refeeding was also effective at increasing the volume fraction of GGT-positive foci, but only at the higher dose of DEN.

Squalene Does Not Exhibit a Chemopreventive Activity and Increases Plasma Cholesterol in a Wistar Rat Hepatocarcinogenesis Model

: The eventual chemopreventive effect of squalene (SQ), a triterpene present in olive oil, was evaluated when administered to Wistar rats during a period comprising the initiation and selection/promotion of the "resistant hepatocyte" (RH) model of hepatocarcinogenesis. During 8 consecutive wk, animals received by gavage SQ (100 or 150 mg/100 g body weight) dissolved in corn oil (CO) daily. Animals treated with only CO and submitted to the RH model were used as controls. Treatments with SQ did not result in inhibition of macroscopically visible hepatocyte nodules (P > 0.05) or of hepatic placental glutathione S-transferase- positive preneoplastic lesions (PNL; P > 0.05). Hepatic cell proliferation and apoptosis indexes were not different (P > 0.05) among the different experimental groups, both regarding PNL and surrounding normal tissue areas. There were no significant differences (P > 0.05) among comets presented by rats treated with the two SQ doses or with CO. On the other hand, SQ increased total plasma cholesterol levels when administered at both doses (P < 0.05). This indicates that the isoprenoid was absorbed. Thus, SQ did not present chemopreventive activity during hepatocarcinogenesis and had a hypercholesterolemic effect, suggesting caution when considering its use in chemoprevention of cancer.

Selenium prevents tumor development in a rat model for chemical carcinogenesis.

Previous studies in animals and humans have shown that selenium compounds can prevent cancer development. In this work we studied the tumor preventive effect of selenium supplementation, administrated as selenite, in the initiation, promotion and progression phases in a synchronized rat model for chemically induced hepatocarcinogenesis, the resistant hepatocyte model. Selenite in supra-nutritional but subtoxic doses (1 and 5 p.p.m.) was administrated to the animals through the drinking water. Such supplementation during the initiation phase did not have a tumor preventive effect. However, selenite treatment during the promotion phase decreased the volume fraction of pre-neoplastic liver nodules from 38% in control animals to 25 (1 p.p.m.) and 14% (5 p.p.m.) in the selenite-supplemented groups. In addition the cell proliferation within the nodules decreased from 42% in the control to 22 (1 p.p.m.) and 17% (5 p.p.m.). Immunohistochemical staining for the selenoenzyme thioredoxin reductase 1 revealed an increased expression of the enzyme in liver nodules compared with the surrounding tissue. The activity was reduced to 50% in liver homogenates from selenium-treated animals but the activity of the selenoenzyme glutathione peroxidase was essentially unaltered. Selenite treatment (5 p.p.m.) during the progression phase resulted in a significantly lower volume fraction of liver tumors (14 compared with 26%) along with a decrease in cell proliferation within the tumors (34 compared with 63%). Taken together our data indicate that the carcinogenetic process may be prevented by selenium supplementation both during the promotion and the progression phase.

Polygenic control of hepatocarcinogenesis in Copenhagen x F344 rats.

Cop and CFF1 rats exhibit resistance to hepatocarcinogenesis, associated with high rates of remodeling of neoplastic lesions. We have mapped hepatocarcinogenesis susceptibility, resistance and remodeling loci affecting the number, volume and volume fraction of neoplastic nodules induced by the "resistant hepatocyte" model in male CFF2 rats. Three loci in significant linkage with the number or volume of nonremodeling lesions were identified on chromosomes 1, 4 and 18. Suggestive linkage with number or volume fraction of total, nonremodeling or remodeling lesions was found for 7 loci on chromosomes 1, 2, 13, 14 and 15. All of these loci showed significant allele-specific effects on the phenotypic traits. We also detected by analysis of variance 19 2-way interactions inducing phenotypic effects not predictable on the basis of the sum of separate effects. These novel epistatic loci were in significant linkage with the number and/or volume of total, nonremodeling or remodeling nodules. These data indicate that susceptibility to hepatocarcinogenesis in Cop rats is controlled by a complex array of genes with several gene-gene interactions and that different genetic mechanisms control remodeling and nonremodeling liver nodules. Frequent deregulation in human liver cancer of genes positioned in chromosomal segments syntenic to rat susceptibility/resistance loci suggests some similarities between the genetic mechanisms involved in hepatocarcinogenesis in rats and humans.

Initiating activity of 4-chlorobiphenyl metabolites in the resistant hepatocyte model.

We recently reported that several mono- to tetrachlorinated biphenyls have initiating activity in the livers of Fischer 344 rats. In the present study, we investigated the metabolic activation of one of those compounds, 4-chlorobiphenyl (PCB 3). Monohydroxy (400 micromol/kg), dihydroxy (200 micromol/kg), and quinone (100 micromol/kg) metabolites of PCB 3 were evaluated for their initiating activity. Fischer 344 male rats were fasted for 4 days; 24 h after feeding again, they were injected (ip) with metabolites, vehicle, or diethylnitrosamine (DEN, 20 or 40 mg/kg). All animals were treated with selection agents as follows: three daily p.o. doses of 2-acetylaminofluorene (2-AAF, 30 mg/kg), followed by a single p.o. dose of carbon tetrachloride (2 ml/kg) and three additional daily treatments of 2-AAF. Rats were killed 2 weeks after the last 2-AAF intubation. Livers were evaluated for changes in morphology, and the number and volume of gamma-glutamyl transpeptidase-positive foci were measured. Of the metabolites tested, only one monohydroxy and one quinoid metabolite showed initiating activity. The metabolic activation of PCB 3, therefore, proceeds via parahydroxylation and oxidation to the ortho 3,4-quinone, the ultimate carcinogen. This is the first report to demonstrate that specific PCB metabolites possess initiating activity in the rodent liver in vivo. The results support the conclusion that 4-OH PCB 3 and 3,4-BQ PCB 3 act as proximate and ultimate carcinogenic metabolites resulting from the bioactivation of PCB 3 in rat liver.

Chemoprotective effect of caffeic acid phenethyl ester on promotion in a medium-term rat hepatocarcinogenesis assay.

Caffeic acid phenethyl ester (CAPE), a natural honeybee product exhibits a spectrum of biological activities including anti-microbial, anti-inflammatory, antioxidant and anti-tumoral actions. CAPE is also chemopreventive against intestinal, colon and skin cancer. Our aim was to extend the study of its chemoprotective features to the promotion of hepatocarcinogenesis. Male Wistar rats were subjected to a protocol under a modified promotion regimen of the resistant hepatocyte model. The altered hepatic foci (AHF) were quantitatively analyzed by histochemistry and image processing. When given during promotion, CAPE (20 mg/kg) decreased the expression of number and area gamma-glutamyl transpeptidase (GGT) positive AHF by 91% and 97%, respectively. When GGT expression was analyzed by RT-PCR, CAPE drastically decreased and prevented expression of almost all GGT transcripts at this stage of the carcinogenic process. Glutathione S-transferase placental form (GST-P), another protein marker for preneoplastic lesions was measured by Western blot and a decrease of 82% was observed. Additionally, we evaluated the effect of CAPE on the expression of nuclear factor NF-kappaB and found an 85% decrease in nuclear localization of the p65 subunit of NF-kappaB; however, their repressor, IkappaBalpha was not modified. Our results showed that CAPE given during promotion in hepatocarcinogenesis protects against induction of GGT-positive AHF, GST-P protein, GGT mRNA expression and translocation of p65. This phenomenon was independent of IkappaBalpha degradation.

Inhibitory Effects of Lutein and Lycopene on Placental Glutathione S-Transferase-Positive Preneoplastic Lesions and DNA Strand Breakage Induced in Wistar Rats by the Resistant Hepatocyte Model of Hepatocarcinogenesis

Inhibitory effects of lutein (LUT) and lycopene (LYC) on hepatic preneoplastic lesions (PNLs) and DNA strand breakage induced in Wistar rats by the resistant hepatocyte (RH) model of hepatocarcinogenesis were investigated. Animals received by gavage during 8 consecutive weeks on alternate days 70 mg/kg body weight of LUT or LYC. Rats treated with only corn oil and submitted to this model were used as controls. At the end of the experiment, treatment of the animals with LUT or LYC resulted in an increase in the respective liver carotenoid concentrations (P < 0.05). Moreover, it tended to reduce the incidence, total number, and multiplicity of hepatocyte nodules compared with the control group, although the differences did not reach statistical significance. Animals treated with LUT or LYC presented also a lower number of hepatic placental glutathione S-transferase-positive (GST-P) PNLs (P < 0.05), which were smaller (P < 0.05) and occupied a smaller area of the liver section (P < 0.05). Finally, hepatic DNA strand breakage evaluated by the comet assay was lower (P < 0.05) in carotenoid-treated animals when compared with the control group. Therefore, the results indicate that LUT and LYC represent promising chemopreventive agents during hepatocarcinogenesis and whose anticarcinogenic actions could be related to a protection against DNA instability.

Polychlorinated biphenyls as initiators in liver carcinogenesis: resistant hepatocyte model

A modified Solt-Farber protocol was established to investigate the potential initiating activity of lower chlorinated polychlorinated biphenyl (PCB) congeners in rat liver. Two different studies were conducted in male Fisher 344 rats. PCBs investigated were PCB3, PCB12, PCB38, and PCB77 in study 1 and PCB15, PCB52, PCB77, and the combination of PCB52 and PCB77 in study 2. Rats were subjected to partial hepatectomy followed by a single dose of the suspected initiating agent, diethylnitrosamine, or vehicle. Two weeks later all groups received selection treatment consisting of three daily doses of 2-acetylaminofluorene (2-AAF) and then a single dose of carbon tetrachloride, followed by three additional daily treatments of 2-AAF via gavage. Rats were killed 2 weeks after the last treatment of 2-AAF, and the number and volume of γ-glutamyltranspeptidase (GGT)-positive foci were determined. Among the PCBs tested, PCB3, PCB15, PCB52, and PCB77 significantly increased the number of GGT-positive foci per cm 3 of liver and per liver. Only PCB3 and PCB15 increased the volume fraction of GGT-positive foci. Histopathologic analysis of hematoxylin- and eosin-stained liver sections showed that rats with significantly increased GGT-positive foci also had extensive cellular alteration. This effect was not seen in nonselection groups. We conclude that, under the conditions and time courses of these experiments, several PCBs have initiating activity in male Fischer 344 rats.

The chemopreventive activities of farnesol and geraniol in rats submitted to the resistant hepatocyte model of hepatocarcinogenesis involve inhibition of cell proliferation

The chemopreventive activities of farnesol and geraniol in rats submitted to the resistant hepatocyte model of hepatocarcinogenesis involve inhibition of cell proliferation

Inhibitory Effects of β-Carotene and Vitamin A During the Progression Phase of Hepatocarcinogenesis Involve Inhibition of Cell Proliferation but Not Alterations in DNA Methylation

The inhibitory effects of β-Carotene and vitamin A administered to rats in the progression phase of the resistant hepatocyte model of hepatocarcinogenesis were investigated. β-Carotene- and vitamin A-treated animals tended to present with a lower incidence of hepatic cancers than controls at sacrifice. Vitamin A, but not β-Carotene, administration also tended to reduce the total number of persistent hepatocyte nodules. Histological examination of sections stained with hematoxylin and eosin confirmed these results. This suggests that both compounds exhibit inhibitory effects during conversion of persistent nodules to cancers, whereas only the retinoid is also capable of inhibiting the evolution of persistent nodules or causing them to regress. Moreover, β-carotene- and vitamin A-treated animals showed lower hepatic bromodeoxyuridine labeling indexes in neoplastic lesions as well as in adjacent normal tissues than controls, suggesting an inhibitory action of these substances on cell proliferation. However, neither β-carotene nor vitamin A administration resulted in substantial alterations in the CCGG sequence methylation pattern of hydroxymethylglutaryl coenzyme A reductase, c-myc, and c-Ha-ras genes, the products of which are related to cell proliferation and carcinogenesis. Therefore, these inhibitory effects of β-carotene and vitamin A on progression of hepatocarcinogenesis do not seem to be related to DNA methylation.

Chemoprevention of hepatocarcinogenesis: S-adenosyl- l-methionine

Accumulation of genetic changes characterizes the progression of cells, initiated by carcinogens, to full malignancy. Various epigenetic mechanisms, such as high polyamine synthesis, aberrant DNA methylation, and production of reactive oxygen species, may favor this process by stimulating growth and inducing DNA damage. We observed a decrease in S-adenosyl- l-methionine (SAM) content in the liver, associated with DNA hypomethylation in rat liver, during the development of preneoplastic foci, and in neoplastic nodules and hepatocellular carcinomas, induced in diethylnitrosamine-initiated rats by “resistant hepatocyte” (RH) protocol. Reconstitution of the methyl donor level in the liver by SAM administration inhibits growth and induces phenotypic reversion and apoptosis of preneoplastic cells. A 6-month SAM treatment results in a sharp and persistent decrease in development of neoplastic nodules, suggesting a long duration of SAM chemopreventive effect. Various observations support the suggestion of a role of DNA methylation in chemoprevention by SAM: (1) Exogenous SAM reconstitutes the SAM pool in preneoplastic and neoplastic liver lesions. (2) DNA methylation is positively correlated with SAM: S-adenosylhomocysteine (SAH) ratio in these lesions. (3) 5-Azacytidine, a DNA methyltransferase inhibitor, inhibits chemoprevention by SAM. (4) c-Ha- ras, c-Ki- ras, and c- myc are hypomethylated and overexpressed in preneoplastic liver. Their expression is inversely correlated with SAM:SAH ratio in SAM-treated rats. (5) S-Adenosyl- l-methionine treatment results in overall DNA methylation and partial methylation of these genes. Other possible mechanisms of SAM treatment include inhibition of polyamine synthesis, linked to partial transformation of SAM into 5′-methylthioadenosine (MTA), and antioxidant and antifibrogenic activities of both SAM and MTA.

Cancer. Plants provide prevention.

Research ArticleOpen AccessCancer. Plants provide prevention. Julia R Barrett Julia R Barrett Search for more papers by this author Published:1 April 2002https://doi.org/10.1289/ehp.110-a180Cited by:3AboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InReddit FiguresReferencesRelatedDetailsCited by McQuistan T, Simonich M, Pratt M, Pereira C, Hendricks J, Dashwood R, Williams D and Bailey G (2012) Cancer chemoprevention by dietary chlorophylls: A 12,000-animal dose–dose matrix biomarker and tumor study, Food and Chemical Toxicology, 10.1016/j.fct.2011.10.065, 50:2, (341-352), Online publication date: 1-Feb-2012. Kim H, Kim H and Choi H (2008) Pre- and post-initiation modulating effects of green tea ingestion on rat hepatocarcinogenesis, Nutrition Research and Practice, 10.4162/nrp.2008.2.4.234, 2:4, (234), . Toledo L, Ong T, Pinho A, Jordao A, Vanucchi H and Salvador Moreno F (2003) Inhibitory Effects of Lutein and Lycopene on Placental Glutathione S-Transferase-Positive Preneoplastic Lesions and DNA Strand Breakage Induced in Wistar Rats by the Resistant Hepatocyte Model of Hepatocarcinogenesis, Nutrition and Cancer, 10.1207/s15327914nc4701_8, 47:1, (62-69), Online publication date: 1-Sep-2003. Vol. 110, No. 4 April 2002Metrics About Article Metrics Publication History Originally published1 April 2002Published in print1 April 2002 Financial disclosuresPDF download License information EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted. Note to readers with disabilities EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact [email protected]. Our staff will work with you to assess and meet your accessibility needs within 3 working days.

Differential Effects of Partial Hepatectomy and Carbon Tetrachloride Administration on Induction of Liver Cell Foci in a Model for Detection of Initiation Activity

Differential effects of partial hepatectomy (PH) and carbon tetrachloride (CC14) administration on induction of glutathione S‐transferase placental form (GST‐P)‐positive foci were investigated in a model for detection of initiation activity. Firstly, we surveyed cell proliferation kinetics and fluctuation in cytochrome P450 (CYP) mRNA levels by means of relative‐quantitative real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR) and CYP 2E1 apoprotein amount by immuno‐blotting (experiment I) after PH or CC14 administration. Next, to assess the interrelationships among cell proliferation, fluctuation of CYPs after PH or CC14 administration and induction of liver cell foci, the non‐hepatocarcinogen, 1,2‐dimethylhydrazine (DMH) was administered to 7‐week‐old male F344 rats and initiated populations were selected using the resistant hepatocyte model (experiment II). In experiment I, the values of all CYP isozyme mRNAs after PH or CC14 administration were drastically decreased at the 12‐h tune point. From 72 h, mRNAs for all CYP isozymes began increasing, with complete recovery after 7 days. The CYP 2E1 apoprotein content in the PH group fluctuated weakly, whereas in the CC14 group it had decreased rapidly after 12 h and was still low at the 48 h point. In experiment II, induction of GST‐P‐positive foci was related to cell kinetics in the PH group, with about a 6‐h time lag between tune for carcinogen administration giving greatest induction of GST‐P‐positive foci and peaks in bromodeoxyuridine (BrdU) labeling, presumably due to the necessity for bioactivation of DMH. With CC14 administration, induction of foci appeared dependent on the recovery of CYP 2E1. In conclusion, PH was able to induce cell proliferation with maintenance of CYP 2E1, therefore being advantageous for induction of liver cell foci in models to detect initiation activity.

Matrix metalloproteinases-2 and 9 do not play a role in the growth of preneoplastic liver lesions in F344 rats.

Matrix metalloproteinases- (MMPs) 2 and 9 (gelatinases A and B) have been implicated in tumor invasion and metastasis, and recent studies have shown increased levels of these enzymes during recovery from partial hepatectomy (PH) in rats. F344 rats are highly susceptible to the growth of glutathione S-transferase 7-7- (GST 7-7) positive preneoplastic liver lesions promoted using the modified resistant hepatocyte (RH) protocol. Since the RH protocol consists of 2-acetylaminofluorene (2-AAF) followed by a PH, we reasoned that MMP-2 and -9 might be critical for the growth of lesions. Using gelatin zymography, we examined the expression of these enzymes in the livers of F344 rats treated with the RH protocol and sacrificed on Days 2, 4, 7, 14, and 21 after 2-AAF/PH. We found increases in both pro- and active MMP-2 and -9 over baseline levels, with the highest levels occurring on Day 7 post-PH. Also, a 54-kDa band, likely to be proMMP-1, was elevated in a pattern similar to MMP-2 and -9. In contrast to F344 rats, identically treated Copenhagen rats that are highly resistant to promotion of liver lesion growth using the RH protocol had significantly lower levels of proMMP-1 and -2. To test the importance of these MMPs to the growth of liver lesions, F344 rats that had been initiated with diethylnitrosamine were treated using the RH protocol. They then received either the MMP inhibitor batimastat (30 mg/kg, intraperitoneally) or vehicle alone daily from Day 3 to 20 post-PH and were sacrificed on Day 21. There were no differences in the percentage of liver volume occupied by GST 7-7-positive lesions (19.1 +/- 4.84 vs 19.4 +/- 3.31, treated versus vehicle, mean +/- SEM) or liver weight as a percentage of body weight (4.11% +/- 0.15 vs 4.07% +/- 0.18, treated versus vehicle, mean +/- SEM) between the treated and control groups. Treatment of rats with batimastat clearly did not affect lesion growth or liver regeneration following the RH protocol. These results suggest that increases in gelatinase expression during the RH protocol are a result of the promotional stimulus rather than a mechanism by which 2-AAF/PH causes lesion growth.