Blunt cell growth potential in carcinogen resistant inbred DRH rats.

A carcinogen-resistant inbred strain DRH/Sea has been developed from the Crj:Donryu strain. The rats had a very low incidence of liver tumors when they were fed diets containing a hepatocarcinogen such as 3'-methyl-4-dimethylamino-azobenzene (3'-Me-DAB). Despite using 3'-Me-DAB during the stage of selection, the DRH/Sea rats developed normally, reproduced and did not have any spontaneous tumor in the lung, liver or uterus at over 1 year of age. Although their growth curves were similar to the Crj:Donryu rats, the progression of polyploidization in the liver was significantly delayed when compared with Crj:Donryu rats. Mitogenic changes that occurred in the liver caused by either 3'-Me-DAB or lead nitrate were less significant in the DRH rats than in Crj:Donryu rats. Furthermore, the growth rate of cultured fibroblasts derived from the DRH rats was slower than that of Crj:Donryu rats. These results, together with our previous results, suggest that slow growth potential is present under certain conditions in DRH rats. These findings may explain partly the meaning of the different susceptibility to hepatocarcinogens.     

Hepatic gap junctions in the hepatocarcinogen-resistant DRH rat

Although the gap junction or connexin (Cx) is considered to be a tumor-suppressor, it is also required for tumor promotion. Therefore, we examined hepatic gap junctions in hepatocarcinogen-resistant (DRH) rats. Specifically, we investigated gap junction structure and Cx32 expression during normal conditions and in response to a hepatocarcinogen, 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB). On a basal diet without 3'-MeDAB, hepatic gap junctions and Cx32 protein expression were greater in DRH rats than in control Donryu rats, as evidenced by morphometry, immunohistochemistry and immunoblotting. On a diet containing 3'-MeDAB, gap junctions and expressed Cx32 were increased significantly in Donryu rats, but not in DRH rats. In this condition, Donryu rats lost weight but DRH rats increased relative liver weight. After 3'-MeDAB treatment, cathepsin D expression in hepatocytes was significantly increased only in Donryu rats, indicating that DRH rats were less susceptible to 3'-MeDAB. The abundance of mitogen-activated protein kinase, some constituent of which might be associated with the degree of Cx protein phosphorylation, was reduced to a greater extent in Donryu than in DRH rats after 3'-MeDAB treatment. The resistance of DRH rats to carcinogenesis may be due partially to their stabilized gap junctions, which could coordinate metabolic coupling to evade 3'-MeDAB toxicity.     

Inheritance of resistance to promotion of preneoplastic liver lesions in Copenhagen rats

Previously, we have shown that Copenhagen (Cop) rats are highly resistant to the induction of putative preneoplastic, glutathione S-transferase 7-7- (GST 7-7) positive liver lesions following treatment with a modified resistant hepatocyte (RH) protocol. The objective of this study was to determine if resistance is inherited in a dominant or recessive manner and to derive an estimate of the number of genetic loci involved. We crossed male and female Cop rats with F344 rats to produce F1 offspring. Backcross rats were generated using female F1 rats and either Cop or F344 males, resulting in B1c and B1f generations, respectively. The male rats from all these crosses were initiated with diethylnitrosamine (200 mg/kg) at 7 to 8 weeks of age and were promoted 3 weeks later with the RH protocol (2-acetylaminofluorene and a two-thirds partial hepatectomy). The rats were sacrificed 3 weeks after the partial hepatectomy and their livers were sectioned and stained for GST 7-7-positive lesions. The susceptibility of F1 rats was in between Cop and F344 rats, having 21.7% +/- 2.0% (mean +/- SEM) of their liver volume occupied by lesions versus 4.2% +/- 0.8% for Cop and 53.0% +/- 5.8% for F344 rats. As expected, B1c rats had a volume of liver occupied by lesions that was in between the F1 and Cop rats at 13.5% +/- 1.6%. Surprisingly, B1f rats were similar to B1c rats in their resistance (9.1% +/- 2.1%). These results point to a complex, polygenic inheritance pattern that can be explained by a minimum of four loci, one of which shows recessive epistasis.     

Capn10, a candidate gene responsible for type 2 diabetes mellitus in the OLETF rat

The rat strain Otsuka Long-Evans Tokushima Fatty (OLETF) is an animal model for type 2 diabetes mellitus. Nidd8/of has been identified as one of 14 quantitative trait loci (QTLs) involved in the diabetes by a whole genome search in 160 F2 progenies obtained by mating the OLETF and F344 rats. Comparative mapping between human and rat indicated that the Nidd8/of genomic region, near D9rat21 on rat chromosome 9, contains the calpain10 (Capn10) gene, which is putative type 2 diabetes-susceptibility gene in humans. In this study, we found no difference in Capn10 mRNA expression in the heart, liver, skeletal muscle and pancreas between OLETF and F344 rats at 5 and 10 weeks of age. However, we found a single nucleotide polymorphism (SNP) (A/A genotype in OLETF and G/G genotype in F344 and LETO rats) at the base 583 downstream from the translation start site in the rat Capn10 cDNA sequence. This SNP was deduced to substitute serine (OLETF) for glycine (F344 and LETO) at the 195 amino acid residue within the protease domain of rat Capn10. Because serine is generally not interchangeable with glycine in respect of the protein structure and function, it was deduced that the A/A genotype in OLETF is not a 'safe' mutation. This non-conservative amino acid substitution might be associated with susceptibility to type 2 diabetes in OLETF rats.     

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 beta-galactosidase gene. After labeling rats were given phenobarbital for 10 months and sacrificed. The presence of beta-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 beta-galactosidase positive hepatocytes were never detected in preneoplastic foci. Finally, the size of the beta-galactosidase positive 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 beta-galactosidase positive cells clusters demonstrate that phenobarbital does not induce hepatocyte proliferation in rats.     

Fine mapping of radiation susceptibility and gene expression analysis of LEC congenic rat lines

LEC rats constitute an animal model of high susceptibility to X-rays. We developed congenic LEC rat lines (recipient strain, Fischer 344 (F344)) and performed genome-wide genotyping to identify radiation susceptibility genes. We mapped seven positional candidate genes, Bmp10, Gpr73, Gp9, Cnbp, Copg, Rab7, and Rpn1, to an approximately 1.2-Mb region located between loci D4Got85 and D4Got148 on chromosome 4. None of the seven genes has been reported to be associated with radiation susceptibility. Comparison of the coding sequences for these seven genes in F344 and LEC rats showed no changes in deduced amino acid sequences. We determined gene expression differences in Gp73, Gp9, and Cnbp as well as strain-specific variations in upstream sequences of these genes. Our results suggest that radiation susceptibility in the LEC rat is primarily attributable to one of the genes within this approximately 1.2-Mb region; however, expression analysis gave no clear indication as to which gene is responsible.     

Streptococcal cell wall-induced arthritis and adjuvant arthritis in F344----Lewis and in Lewis----F344 bone marrow chimeras

Streptococcal cell wall (SCW)-induced arthritis and adjuvant arthritis (AA) are rat models for chronic, erosive polyarthritis. Both models can be induced in susceptible Lewis rats, whereas F344 rats are resistant. In AA as well as in SCW arthritis, antigen-specific T lymphocytes have been demonstrated to be crucial for chronic disease. In this communication we describe our studies to probe the cellular mechanism responsible for the difference in susceptibility of Lewis and F344, using bone marrow chimeras. By transplanting bone marrow cells from F344 into lethally irradiated Lewis recipients, Lewis rats were rendered resistant to SCW arthritis induction. F344 rats reconstituted with Lewis bone marrow, i.e., Lewis----F344 chimeras, develop an arthritis upon SCW injection. For AA comparable results were obtained. These data suggest that both resistance and susceptibility to bacterium-induced chronic arthritis are mediated by hemopoietic/immune cells and that the recipiental environment does not influence the susceptibility to chronic joint inflammation.     

Effect of common Brassica vegetables (Brussels sprouts and red cabbage) on the development of preneoplastic lesions induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in liver and colon of Fischer 344 rats

Aim of the present study was the investigation of effects of juices from commonly consumed Brassica vegetables (two cultivars of Brussels sprouts and two cultivars of red cabbage) on formation and development of preneoplastic lesions in colons (aberrant crypt foci, ACF) and livers (glutathione-S-transferase placental form, GST-P+) in male F344 rats. The foci were induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a widespread carcinogenic heterocyclic aromatic amine which is found in fried meats. Recently, we reported on pronounced protective effects in the two-organ foci model when the vegetable juices were given during the carcinogen treatment but several findings by other groups indicated that breakdown products of glucosinolates contained in Brassica vegetables cause tumour promotion in various organs of laboratory rodents. In the present study, the animals received the juices in the drinking water (5%) over a period of 20 days after treatment with IQ (100 mg/kg bw on 10 alternate days). To increase the foci yield (which facilitates the detection of modifying effects), the animals were fed with a modified (high fat, fibre free) AIN-76 diet. With exception of the sprout variety "Cyrus", all juices lowered the number of GST-P+ foci as well as the foci area in the liver, but none of these effects was statistically significant. In the colon, none of the juices had an impact on crypt multiplicity (number of crypts/focus), whereas the number of ACF was decreased; only with the sprout variety Maximus the protective effect was significant (reduction 49%). The present findings show that administration of vegetable juices to the animals after the carcinogen does not increase the number and size of IQ-induced preneoplastic lesions in liver and colon.     

Quantitative trait loci determining weight reduction of testes and pituitary by diethylstilbesterol in LEXF and FXLE recombinant inbred strain rats.

Increasing exposure to environmental endocrine disruptor, xeno-estrogen, is a serious hazard to male reproductive activity. To explore possible genetic control in susceptibility to xeno-estrogen, the weight reduction of testes induced by the continuous administration of a synthetic estrogen, diethylstilbesterol, were investigated by quantitative trait analysis in LEXF and FXLE recombinant inbred strain rats, consisting of 21 independent strains, 9 of their substrains, parental F344/Stm and LE/Stm strains, and (F344 x LE)F1. For the weight of testes, one highly significant quantitative trait locus (QTL) and one significant QTL were mapped on chromosomes 7 and 1, respectively. The QTL on chromosome 7 is closely associated with c-myc. Pituitary weight and serum prolactin were also variable among recombinant inbred strains, but no QTL was detected for them in this study.     

Altered transferrin gene expression in preneoplastic and neoplastic liver lesions induced in rats with N-nitrosomorpholine

The expression of the gene for the iron transport protein transferrin was found to be altered in preneoplastic and neoplastic lesions induced in the rat liver by N-nitrosomorpholine. The total RNA of ten hepatocellular carcinomas (HCC) was investigated by Northern blot analysis using a cDNA-probe comprising 150 bp of the 3′ region and compared with the total hepatic RNA in untreated rats. Seven hepatocellular carcinomas showed slight or pronounced reduction in transferrin expression. In situ hybridization of two additional hepatocellular carcinomas revealed marked reduction in the mRNA level for the transferrin gene compared with the surrounding tissue. In contrast, the majority of early preneoplastic lesions storing excess glycogen and tigroid cell foci expressed increased levels of transferrin mRNA. The loss of glycogen in mixed cell foci, which represent a later stage of hepatocarcinogenesis, was usually accompanied by a decrease in transferrin mRNA suggesting a close relationship between this change in gene expression and cellular dedifferentiation emerging during hepatocarcinogenesis.     

Altered transferrin gene expression in preneoplastic and neoplastic liver lesions induced in rats with N-nitrosomorpholine.

The expression of the gene for the iron transport protein transferrin was found to be altered in preneoplastic and neoplastic lesions induced in the rat liver by N-nitrosomorpholine. The total RNA of ten hepatocellular carcinomas (HCC) was investigated by Northern blot analysis using a cDNA-probe comprising 150 bp of the 3' region and compared with the total hepatic RNA in untreated rats. Seven hepatocellular carcinomas showed slight or pronounced reduction in transferrin expression. In situ hybridization of two additional hepatocellular carcinomas revealed marked reduction in the mRNA level for the transferrin gene compared with the surrounding tissue. In contrast, the majority of early preneoplastic lesions storing excess glycogen and tigroid cell foci expressed increased levels of transferrin mRNA. The loss of glycogen in mixed cell foci, which represent a later stage of hepatocarcinogenesis, was usually accompanied by a decrease in transferrin mRNA suggesting a close relationship between this change in gene expression and cellular dedifferentiation emerging during hepatocarcinogenesis.     

Early high expression of IP-10 in F344 rats resistant to Sendai virus-induced airway injury

Weanling F344 and BN rats differ markedly in their susceptibility to Sendai virus-induced airway injury. Early gene expression that controls their differences in susceptibility remains poorly understood. In this study we combined suppressive subtractive hybridization and cDNA library array hybridization to identify genes differentially expressed in virus-susceptible BN and virus-resistant F344 rats during the first 3 days after inoculation. Differential expression of selected clones was further verified by quantitative RT-PCR. Seven virus-induced gene segments were identified. Of them, interferon-gamma-inducible protein 10 (IP-10), Mx1, and guanylate-binding protein-2 mRNA abundance in infected F344 rats was 201.5, 188.2, and 281.7% higher, respectively, than that of infected BN rats at 2 days after inoculation. In situ hybridization indicated that virus-induced IP-10 was expressed mainly in airway epithelial cells of F344 rats. Sendai virus infection can directly induce IP-10 expression in rat tracheal epithelial cells in vitro. IP-10 early high expression might contribute to the resistance to virus-induced airway disease in F344 rats by promoting Th1 responses and increasing antiviral activity.     

CCAAT enhancer-binding protein alpha suppresses the rat placental glutathione S-transferase gene in normal liver

The rat placental glutathione S-transferase (GST-P), an isozyme of glutathione S-transferase, is not expressed in normal liver but is highly induced at an early stage of chemical hepatocarcinogenesis and in hepatomas. Recently, we reported that the NF-E2 p45-related factor 2 (Nrf2)/MafK heterodimer binds to GST-P enhancer 1 (GPE1), a strong enhancer of the GST-P gene, and activates this gene in preneoplastic lesions and hepatomas. In addition to the positive regulation during hepatocarcinogenesis, negative regulatory mechanisms might work to repress GST-P in normal liver, but this remains to be clarified. In this work, we identify the CCAAT enhancer-binding protein alpha (C/EBPalpha) as a negative regulator that binds to GPE1 and suppresses GST-P expression in normal liver. C/EBPalpha binds to part of the GPE1 sequence, and the binding of Nrf2/MafK and C/EBPalpha to GPE1 is mutually exclusive. In a transient-transfection analysis, C/EBPalpha activated GPE1 in F9 embryonal carcinoma cells but strongly inhibited GPE1 activity in hepatoma cells. The expression of C/EBPalpha was specifically suppressed in GST-P-positive preneoplastic foci in the livers of carcinogentreated rats. A chromatin immunoprecipitation analysis showed that C/EBPalpha bound to GPE1 in the normal liver in vivo but did not bind in preneoplastic hepatocytes. Introduction of the C/EBPalpha gene fused with the estrogen receptor ligand-binding domain into hepatoma cells, and subsequent activation by beta-estradiol led to the suppression of endogenous GST-P expression. These results indicate that C/EBPalpha is a negative regulator of GST-P gene expression in normal liver.     

The non-MHC quantitative trait locus Cia5 contains three major arthritis genes that differentially regulate disease severity, pannus formation, and joint damage in collagen- and pristane-induced arthritis

Cia5 is a locus on rat chromosome 10 which regulates the severity of collagen- and pristane-induced arthritis (CIA and PIA). To refine the region toward positional identification, Cia5 subcongenic strains were generated and studied in PIA and CIA. The protective effect of the telomeric locus Cia5a was confirmed in both models. A second arthritis severity locus (Cia5d) was identified within the most centromeric portion of Cia5. DA.F344(Cia5d) rats had a significantly lower median arthritis severity index in PIA, but not in CIA, compared with DA. On histologic analyses DA.F344(Cia5a) and DA.F344(Cia5d) congenics with PIA preserved a nearly normal joint architecture compared with DA, including significant reduction in synovial hyperplasia, pannus, angiogenesis, inflammatory infiltration, bone and cartilage erosions. Cia5 and Cia5a synovial levels of IL-1beta mRNA were reduced. Although both DA.F344(Cia5) and DA.F344(Cia5a) rats were protected in CIA, the arthritis scores of DA.F344(Cia5) were significantly higher than those of DA.F344(Cia5a), suggesting the existence of a third locus where F344-derived alleles centromeric from Cia5a contribute to increased arthritis severity. The existence of the third locus was further supported by higher levels of autoantibodies against rat type II collagen in DA.F344(Cia5) congenics compared with DA.F344(Cia5a). Our results determined that Cia5 contains three major arthritis severity regulatory loci regulating central events in the pathogenesis of arthritis, and differentially influencing CIA and PIA. These loci are syntenic to regions on human chromosomes 17q and 5q implicated in the susceptibility to rheumatoid arthritis, suggesting that the identification of these genes will be relevant to human disease.     

The telomerase activities in several organs and strains of rats with ageing

Telomerase activity is known to be implicated both in cell immortalization and carcinogenesis. Telomerase activity has not been detected in most human somatic tissues. However, we previously confirmed that the activity is present both in methylazoxymethanol acetate-induced rat colonic adenocarcinoma and non-treated colonic mucosa, presumably indicating the tissue-specific activity of the enzyme in rats. To determine the standard activity of rat telomerase in various organs in relation to differences in sex, age and strain, we examined the activity by using the telomeric repeat amplification protocol (TRAP) assay. The testis, liver, and colon mucosa showed the activity. The brain had very low or negative activity in 5-week-old male rats of the F344, SD, Wistar, Donryu or ACI strains. Age (5-week-old and 9-month-old) or sex difference for the activity was not apparent in rats of these strains. In general, telomerase activity in the fetal brain, liver and kidney was stronger than in the adult organ. The telomerase activity of each organ was different from that of human. This difference may indicate that the rat has a specific mechanism for maintaining the telomeric repeats of the chromosome even in somatic tissues. The basic information resulting from this study may be useful for the study of the role of telomerase in tumorigenesis in animal experiment models.     

Decreased density of beta1-adrenergic receptors in preneoplastic and neoplastic liver lesions of F344 rats

There is some evidence that rodent hepatocarcinogenesis is accompanied by changes in the adrenergic responsiveness of liver cells to catecholamines. In this study, immunohistochemical expression of beta1-adrenergic receptors (beta1-ARs) has been examined in spontaneous and chemically induced preneoplastic and neoplastic liver lesions of female and male Fischer 344 rats. An antibody specific for beta1-AR subtype was used. The study was carried out on archival formalin-fixed and paraffin-embedded livers from rats used in a previous study of hepatocarcinogenesis. One control group given distilled water by gavage, and two experimental groups, one initiated with a single dose of diethylnitrosamine (DEN) and one initiated with DEN and continuously treated with phenobarbital (PB) were examined. Rats were sacrificed after 2, 4, 8 and 21 months of experimentation. All types of liver putative preneoplastic lesions examined (basophilic, glycogen-retaining, or mixed cell foci) show a lower density of beta1-ARs than the surrounding normal liver parenchyma, either in control and in DEN-treated or DEN+PB-treated rats. No immunostaining is detectable in several altered cell foci. Hepatocellular adenomas and hepatocellular carcinomas also show a very low density of beta1-ARs, extensive areas completely devoid of beta1-ARs being mingled with areas showing a weak immunostaining.     

Factors affecting production of transgenic rats by ICSI-mediated DNA transfer: effects of sonication and freeze-thawing of spermatozoa, rat strains for sperm and oocyte donors, and different constructs of exogenous DNA

Factors affecting the efficiency of producing transgenic rats by intracytoplasmic sperm injection (ICSI)-mediated DNA transfer were investigated. Epididymal spermatozoa from Sprague-Dawley (SD) rats were sonicated and/or frozen-thawed for cutting the tail and membrane disruption. The sperm heads were exposed for 1 min to different concentrations (0.02-2.5 microg/ml) of 3.0 kb enhanced green fluorescent protein (EGFP) DNA solution, and then microinjected into the denuded F1 hybrid (Donryu x LEW) rat oocytes. The optimal concentration of EGFP DNA solution was 0.1 microg/ml, as determined by the in vitro developmental competence into morulae/blastocysts of the ICSI oocytes and the EGFP expression of the resultant embryos. The efficiency of producing transgenic rat offspring (per transferred zygote) was 2.8%, 1.6%, and 3.3% in the oocytes injected with sonicated, frozen-thawed, and sonicated + frozen-thawed sperm heads, respectively. The founder transgenic rats carrying the EGFP gene transmitted their transgenes to their progeny according to the Mendelian fashion, suggesting the stable incorporation of the transgenes into the rat genomes. Four rat strains (F344, LEW, Donryu, and SD) were compared for their suitability as sperm/oocyte donors for the production of transgenic rats by ICSI with sonicated, frozen-thawed and solution of EGFP DNA-exposed sperm heads. The efficiency of producing transgenic rats in the SD strain (8.2%) was higher than that in the LEW strain (0.9%), while those in the F344 and Donryu strains (4.3%-4.4%) were intermediate. One plasmid DNA (Fyn, 5.0 kb) and two BAC DNA (BAC/Fyn, 208 kb; Svet1/IRES-Cre, 186 kb) were successfully introduced into the SD rat genomes via ICSI, with the producing efficiencies of 2.8%, 0.9%, and 2.4%, respectively.     

Hepatocellular carcinoma as a complex polygenic disease. Interpretive analysis of recent developments on genetic predisposition

The different frequency of hepatocellular carcinoma (HCC) in humans at risk suggests a polygenic predisposition. However, detection of genetic variants is difficult in genetically heterogeneous human population. Studies on mouse and rat models identified 7 hepatocarcinogenesis susceptibility (Hcs) and 2 resistance (Hcr) loci in mice, and 7 Hcs and 9 Hcr loci in rats, controlling multiplicity and size of neoplastic liver lesions. Six liver neoplastic nodule remodeling (Lnnr) loci control number and volume of re-differentiating lesions in rat. A Hcs locus, with high phenotypic effects, and various epistatic gene-gene interactions were identified in rats, suggesting a genetic model of predisposition to hepatocarcinogenesis with different subset of low-penetrance genes, at play in different subsets of population, and a major locus. This model is in keeping with human HCC epidemiology. Several putative modifier genes in rodents, deregulated in HCC, are located in chromosomal segments syntenic to sites of chromosomal aberrations in humans, suggesting possible location of predisposing loci. Resistance to HCC is associated with lower genomic instability and downregulation of cell cycle key genes in preneoplastic and neoplastic lesions. p16(INK4A) upregulation occurs in susceptible and resistant rat lesions. p16(INK4A)-induced growth restraint was circumvented by Hsp90/Cdc37 chaperons and E2f4 nuclear export by Crm1 in susceptible, but not in resistant rats and human HCCs with better prognosis. Thus, protective mechanisms seem to be modulated by HCC modifiers, and differences in their efficiency influence the susceptibility to hepatocarcinogenesis and probably the prognosis of human HCC.     

Induction by butylated hydroxytoluene of rat liver gamma-glutamyl transpeptidase activity in comparison to expression in carcinogen-induced altered lesions

Butylated hydroxytoluene (BHT) at concentrations of 300-6000 ppm in the diet caused a dose-dependent increase in gamma-glutamyl transpeptidase (GGT) activity in normal F344 male rat liver at 18 weeks. However, the activities of glutathione S-transferases (GSTs) of rat liver cytosol were enhanced only at concentrations of 3000 or 6000 ppm BHT. Histochemically, the enhanced GGT activity was localized to hepatocytes surrounding the portal areas. Autoradiographic measurements of DNA synthesis showed that dietary BHT did not increase the level of cell proliferation and the GGT-positive hepatocytes did not exhibit different rates of DNA synthesis from those of GGT-negative cells. Feeding of the liver carcinogen N-2-fluorenylacetamide (FAA) induced foci and nodules of GGT-positive altered cells which exhibited higher rates of DNA synthesis than those of surrounding GGT-negative hepatocytes. Following iron loading, the periportal GGT-positive hepatocytes produced by BHT accumulated cellular iron, whereas the cells in FAA-induced lesions excluded iron. These results suggest that dietary BHT induces GGT activity in periportal hepatocytes without proliferation of the cells and that induction does not represent fetal expression or a preneoplastic alteration.     

Immunohistochemical and biochemical detection of uridine-diphosphate-glucuronyltransferase (UDP-GT) activity in putative preneoplastic liver foci

Preneoplastic liver foci were produced in female Wistar rats by the administration of 2-acetylaminofluorene (0.03% w/w) in the diet for 174 days. Increased UDP-glucuronyltransferase (UDP-GT) could be visualized immunohistochemically in the same focal areas which were ATPase-negative and gamma-glutamyltranspeptidase-positive. Immunohistochemical detection was possible using rabbit anti-UDP-GT and peroxidase-labeled swine anti-rabbit immunoglobulins. The results of immunohistochemistry were substantiated by enzyme determination in microdissected material. UDP-GT activity was 5-fold higher in focal areas in comparison with the surrounding liver tissue. Increased UDP-GT activity in conjunction with the altered pattern of other drug-metabolizing enzymes is consistent with increased resistance of preneoplastic cells to the cytotoxicity of carcinogens. Immunohistochemical detection of UDP-GT may provide a new marker for preneoplastic lesions which, in conjunction with other markers, may prove useful in analyzing the various stages of liver carcinogenesis and the remodeling of preneoplastic lesions after cessation of carcinogenic stimuli.