Dose-dependent effects of 2-acetylaminofluorene on hepatic foci development and cell proliferation in rats

Dose-dependent development of pre-neoplastic liver cell foci induced by 2-acetylaminofluorene (2-AAF) was investigated in relation to cell-proliferative activity. Male F344 rats were initially given a single i.p. injection of diethylnitrosamine (DEN, 200 mg/kg) and starting 2 weeks later received diets containing 2-AAF at dose levels of 150, 100, 60, 45, 35 or 30 p.p.m., 500 p.p.m. phenobarbital (PB) or basal diet as a control for 6 weeks. Two-thirds partial hepatectomy (PH) was performed at week 3. The rats were sequentially killed from weeks 0 to 16 and liver sections were analysed by double staining for both BrdU incorporation and glutathione S-transferase placental form (GST-P) expression. 2-AAF increased numbers and areas of GST-P positive (GST-P+) foci in a dose-dependent manner, especially after PH. Proliferation of hepatocytes, as indicated by BrdU labelling indices (LI), was higher in GST-P+ foci than in surrounding hepatocytes in all 2-AAF-treated groups, even after cessation of carcinogen administration. Proliferative response of hepatocytes to PH was delayed in rats treated with the highest dose of 2-AAF in both foci and in surrounding areas possibly due to the 2-AAF toxicity. In the PB treated group, the results were similar to those for the lower dose 2-AAF-treated groups. It is concluded that the development of GST-P+ foci and cell proliferation in GST-P+ foci are directly related to 2-AAF treatment in a dose-dependent manner and the present assay system is reliable for detection of carcinogenicity of chemicals even at low doses.     

Phenobarbital at low dose exerts hormesis in rat hepatocarcinogenesis by reducing oxidative DNA damage,altering cell proliferation,apoptosis and gene expression

Our recent research indicated that phenobarbital (PB) may inhibit the development of N-diethylnitrosamine (DEN)-initiated pre-neoplastic lesions at low doses in a rat liver medium-term bioassay (Ito test), while high doses exhibit promoting activity. This raises the question of whether treatment with low doses of PB might reduce cancer risk. For clarification, male 6-week-old F344 rats were treated with PB at doses of 0, 2, 15 and 500 p.p.m. in the diet for 10 or 33 weeks after initiation of hepatocarcinogenesis with DEN. In a second, short-term experiment, animals were given PB at doses of 2, 4, 15, 60 and 500 p.p.m. for 8 days. Formation of glutathione S-transferase placental form (GST-P) positive foci and liver tumors was inhibited at 2 p.p.m. Generation of oxidative DNA damage marker, 8-hydroxy-2'-deoxyguanosine (8-OHdG), cellular proliferation within the areas of GST-P positive foci and apoptosis in background liver parenchyma were suppressed. Suppression of 8-OHdG formation by PB at low dose might be related to the enhanced mRNA expression of 8-OHdG repair enzyme, oxoguanine glycosylase 1 (Ogg1). Moreover, as detected by cDNA microarray analysis, PB treatment at low dose enhanced mRNA expression of glutamic acid decarboxylase (GAD65), an enzyme involved in the synthesis of gamma-aminobutyric acid (GABA), and suppressed MAP kinase p38 and other intracellular kinases gene expression. On the contrary, when PB was applied at a high dose, GST-P positive foci numbers and areas, tumor multiplicity, hydroxyl radicals and 8-OHdG levels were greatly elevated with the increase in CYP2B1/2 and CYP3A2 mRNA, protein, activity and gene expression of GST, nuclear tyrosine phosphatase, NADPH- cytochrome P-450 reductase and guanine nucleotide binding protein G(O) alpha subunit. These results indicate that PB exhibits hormetic effect on rat hepatocarcinogenesis initiated with DEN by differentially altering cell proliferation, apoptosis and oxidative DNA damage at high and low doses.     

Increased yield in GST-P-positive liver pre-neoplastic foci induced by dena or enu in rats pre-treated with estradiol or tamoxifen

We investigated the mechanisms by which partial hepatec-tomy (PH) increases the ability of chemical hepatocarcinogens to induce pre-neoplastic liver foci. Comparison of the effects of pre-treatment with PH, estradiol (E2) or tamoxifen (TAM) on the yield in glutathione-S-transferase(GST-P)-positive pre-neoplastic foci in rat liver induced by subsequent treatment with ethylnitrosourea (ENU) or diethylnitrosamine (DENA) showed that pre-treatment with E2 increased the yield in foci induced by subsequent treatment with ENU or DENA, as compared with that in animals not pre-treated, the increase being of similar magnitude with either carcinogen. Compared with that of PH, the effect of the hormone was much more pronounced than would be expected from the relative mito-genic effect of the hormonal and surgical pre-treatments if the mitotic rate were the cause. On the other hand, the average volume of pre-neoplastic liver lesions in rats treated with ENU or DENA was 2.5 to 5.0 times higher than in rats not pre-treated whenever PH was included in the pre-treatment, whereas it was not affected by any other pre-treatment.     

Lack of peroxisomal enzyme inducibility in rat hepatic preneoplastic lesions induced by mutagenic carcinogens: contrasted expression of glutathione S-transferase P form and enoyl CoA hydratase

While glutathione S-transferase P form (GST-P), a reliable markerfor preneoplastic lesions induced by mutagenic hepatocarcinogens,is generally not expressed in rat liver foci, hyperplastic nodulesand hepatomas induced by peroxisome proliferators (PPs), suchlesions can be detected due to their peroxisomal enzyme-negativenature. For comparative purposes we examined the inducibilityof enoyl CoA hydratase (ECH), a key peroxisomal enzyme, in rathepatic preneoplastic lesions induced by mutagenic carcinogens.Clofibrate (CF) was therefore administered for 2 or 4 weeksfollowing performance of the Solt—Farber protocol usingdiethylnitrosamine and 2-acetylaminofluorene. Immunohistochemicalexamination revealed no or only very weak expression of ECHwithin the induced foci in clear contrast to the strong stainingof surrounding parenchyma. ECH expression was thus diametricallyopposed to that of GST-P which was found only in foci. AlthoughECH was completely lacking in GST-P-strongly positive foci,it was expressed in GST-P-negative hepatocytes inside some fociotherwise positive for GST-P. CF administration resulted ina significant decrease in the numbers and areas of foci exhibitingstrongly positive or positive GST-P staining; this being reflectedin a lowering of GST-P protein levels. Furthermore, in primarycultured rat hepatocytes, clofibric acid as well as dexamethasonesuppressed the expression of both GST-P and the oncogene, c-jun.These results taken together suggest that possible interactionof the PP receptor with JUN might be involved in loss of ECHexpression in GST-P-strongly positive foci.     

Resistance of DRH strain rats to chemical carcinogenesis of liver: genetic analysis of later progression stage.

The inbred DRH rats are highly resistant to the induction of hepatocellular carcinoma (HCC) by feeding of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). Previously, we found that two quantitative trait loci (QTLs), Drh1 and Drh2, significantly reduced the number, size and area of glutathione S-transferase-placental form (GST-P)-positive foci and GST-P mRNA levels in (F344xDRH)F(2) rat livers induced by feeding 3'-Me-DAB for 8 weeks. It is unclear, however, whether these QTLs affecting pre-neoplastic lesions are also the determinants of the later stage hepatocarcinogenesis, and whether there are any additional QTLs affecting hepatocarcinogenesis in the progression stage. To answer these questions, we analyzed QTL parameters for liver tumors in 99 (F344xDRH)F(2) rats induced by feeding 3'-Me-DAB for 20 weeks. The QTL parameters examined were GST-P mRNA, ornithine decarboxylase activity, and the number and total area of HCC/nodules macroscopically detectable on the liver surface. In composite interval mapping, we observed two major QTL peaks overlapping on the map positions of Drh1 on rat chromosome 1 (RNO1) and Drh2 on RNO4, respectively. The newly mapped QTL on RNO1 affected the GST-P mRNA level at 20 weeks of 3'-Me-DAB feeding, but did not affect the number and size of tumors. The primary effect of Drh1 is, therefore, to inhibit GST-P induction and to prevent enzyme altered foci (EAF) formation. On the other hand, the QTLs on RNO4, co-mapped to Drh2, affected all parameters of liver tumors examined except for the level of GST-P mRNA. The latter QTLs influenced not only the induction of GST-P and formation of EAF but also the progression of tumors in the later stage of hepatocarcinogenesis. The GST-P induction is differentially controlled by stages of hepatocarcinogenesis and the DRH resistance to carcinogenesis is principally attributed to the QTLs on RNO4 out of two resistance QTLs identified in the pre-neoplastic stage.     

Enzymatic detection of precursor cell populations of preneoplastic foci positive for gamma-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.     

Development of glutathione S-transferase placental form (GST-P) stained foci during hamster buccal pouch mucosa carcinogenesis.

The expression of the placental form of glutathione S-transferase (GST-P) using anti-rat liver GST-P antibody was investigated in hamster buccal pouch mucosa (HBPM) treated with 0.5% dimethylbenz[a]anthracene (DMBA) biweekly for 12 weeks. This preliminary study showed that the anti-rat liver GST-P antibody is applicable to the HBPM model and that DMBA treatment induced GST-P positive foci. These foci are randomly distributed and frequently involved the hyperplastic and dysplastic segments of the epithelium, as well as squamous cell carcinoma. Further study is needed to explore the kinetics of these GST-P positive foci.     

Effects of pinocembrin on the initiation and promotion stages of rat hepatocarcinogenesis

Pinocembrin (5, 7-dihydroxyflavanone) is a flavanone extracted from the rhizome of Boesenbergia pandurata. Our previous studies demonstrated that pinocembrin had no toxicity or mutagenicity in rats. We here evaluated its effects on the initiation and promotion stages in diethylnitrosamine-induced rat hepatocarcinogenesis, using short- and medium-term carcinogenicity tests. Micronucleated hepatocytes and liver glutathione-S-transferase placental form foci were used as end point markers. Pinocembrin was neither mutagenic nor carcinogenic in rat liver, and neither inhibited nor prevented micronucleus formation as well as GST-P positive foci formation induced by diethylnitrosamine. Interestingly, pinocembrin slightly increased the number of GST-P positive foci when given prior to diethylnitrosamine injection.     

Existence of a no effect level for MeIQx hepatocarcinogenicity on a background of thioacetamide-induced liver damage in rats

As exposure to heterocyclic amines might increase the risk of liver cancer, we investigated the carcinogenic potential of MeIQx under conditions of liver damage caused by TAA. Male, 6-week-old F344 rats (n = 280) were divided into 14 groups; groups 1-7 received TAA (0.03% in drinking water) and groups 8-14 received water for the first 12 weeks. Thereafter, the animals received MeIQx at doses from 0, 0.001, 0.01, 0.1, 1, 10 to 100 p.p.m. (groups 1-7 and 8-14, respectively) in pellet basal diet for 16 weeks. All survivors were killed at week 28 for assessment of numbers and areas of GST-P positive foci, considered to be pre-neoplastic lesions of the liver. Values were increased significantly in all the groups receiving TAA-->MeIQx compared to MeIQx alone (P < 0.01). Numbers of GST-P positive foci were significantly increased in groups 7 and 14 (treated with 100 p.p.m. MeIQx) as compared to 0 p.p.m.-MeIQx (groups 1 and 8) (P < 0.01), along with areas in group 14 compared to group 8 (P < 0.01). However, with the maximum likelihood method, the data for numbers of GST-P positive foci (groups 1-7 and groups 8-14) fitted the hockey stick regression model, representing no differences from groups 1-5 and from groups 8-13, despite a linear dose-dependent increase of MeIQx-DNA adducts from 0.1 to 100 p.p.m. We conclude that there is a no effect level for MeIQx hepatocarcinogenicity, even on a background of TAA-induced liver damage.     

Depletion of hepatocyte nuclear estrogen receptor expression is associated with promotion of tamoxifen induced GST-P foci to tumours in rat liver

The expression of hepatocyte nuclear estrogen receptor (ER) in putative preneoplastic foci, adenomas and carcinomas, induced by the rat liver carcinogen tamoxifen, has been examined immunohistologically. ER staining of normal rat liver shows between 30-50% of hepatocyte nuclei to be positive, depending on fixation. Depletion of ER was defined as <10% of cells in foci or tumours staining for nuclear ER. A proportion of all but the smallest glutathione-S-transferase, placental form (GST- P) expressing foci had depleted expression of nuclear ER. The percentage of GST-P expressing foci with depletion of nuclear ER increased with the size of the foci. The liver adenomas and carcinomas induced by tamoxifen showed a high incidence (90%) of depletion of ER. This suggests that abnormal expression of the ER is associated with the promotion of putative preneoplastic foci to adenomas and carcinomas in tamoxifen exposed rat livers. Dysfunction of the ER could contribute to selective continued stimulation of initiated cells that would be consistent with a role for modification of the ER in target cells and the promotion stage of liver cancer. Liver tumours induced by other carcinogens in both sexes of rat were also found to have a high incidence of ER depletion, indicating that this could be a general regulatory mechanism for rat liver tumour promotion, irrespective of the possible estrogen like action of individual carcinogens.      

Alpha-benzene hexachloride exerts hormesis in preneoplastic lesion formation of rat hepatocarcinogenesis with the possible role for hepatic detoxifying enzymes

Recently there has been a shift in the prevailing paradigm regarding the dose dependence of carcinogen action with increasing acceptance of hormesis phenomenon, although underlying mechanisms remain to be established. To ascertain whether alpha-benzene hexachloride (alpha-BHC) might act by hormesis, rats were initiated with diethylnitrosamine and then alpha-BHC ranging from 0.01 to 500 ppm was administered in the diet for 10 weeks. The highest concentration of alpha-BHC significantly increased the number and area of glutathione S-transferase placental form (GST-P) positive foci, preneoplastic lesions in the liver, but its low dose, 0.05 ppm, caused significant reduction, showing a J-shape dose-response curve. The proliferating cell nuclear antigen positive index for GST-P positive foci in the low dose-treated group was significantly reduced. The dose response curves of CYP450 content, NADPH-P450 reductase activity and 8-hydroxydeoxyguanosine formation revealed the same pattern as GST-P positive foci data. The response curves of CYP2B1 and 3A2 in their activities, protein and mRNA expression showed a threshold but CYP2C11 activity exhibited an inverted J-shape. These results might suggest the possibility of hormesis of alpha-BHC at early stages of rat hepatocarcinogenesis. The possible mechanism involves induction of detoxifying enzymes at low dose, influencing free radical production and oxidative stress, and consequently pathological change in the liver.     

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

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

Anomalous elevation of glutathione S-transferase P-form (GST-P) in the elementary process of epigenetic initiation of chemical hepatocarcinogenesis in rats

The molecular mechanism of the specific expression of glutathione S-transferase P-form (GST-P) in the rat hepatic preneoplastic foci and "GST-P-positive" single cells requires elucidation. Immunochemical and stereological analyses revealed that the enzyme level in preneoplastic foci was 150-250-fold (6.7 +/- 2.4 mg/g liver and 0.29 +/- 0.1 mM subunits) higher than in normal cells. GST-P content in the single cells was higher than in preneoplastic foci, as determined by densitometry. In addition, the single cells were larger in cell diameter and area, corresponding to 2-3-fold increase in cell volume, relative to normal cells, but showed a significant shrinkage of their nuclei. Prior to the induction of single cells in the liver by diethylnitrosamine (DEN), microsomes were severely damaged as reflected by the low yield (approximately 60% that of untreated controls) after 2 h of DEN injection. Considering that GST-P is mainly a binding protein for GSH conjugates of endogenous carcinogens, together with our findings of morphological expansion, low viability of single cells and microsomal damage, our results suggest anomalous elevation of the ligand counterparts to lethal levels in preneoplastic cells, especially in single cells. We propose that the epigenetic mechanism rather than the genetic mechanism could account for GST-P induction in hepatocytes.     

Nonclonal growth of preneoplastic cells positive for glutathione S-transferase P-form in the rat liver

We investigated the process of induction of preneoplastic cells positive for glutathione S-transferase P-form (GST-P) in the rat liver. AAF (2-Acetylaminofluorene) mixed with normal rat chow at high concentration (0.04%) induced 517 000 ± 86,000 GST-P(+) single hepatocytes/g liver after 2 weeks followed by induction of a few foci and nodules after 4-6 weeks. Overproduction of GST-P(+) single hepatocytes was dose- and time-dependent, and the induction kinetics were typical of first-order consecutive reaction, by which induction of the positive cells was nongenetic. Quantitative analysis indicated that the estimated numbers of cells in foci and nodules at 4-6 weeks after exposure to AAF ranged from 2.7 × 10(4) (2(14.7)) to 3.6 × 10(6) (2(21.7)) cells, and 2.0 × 10(4) (2(14.3)) to 2.7 × 10(6) (2(21.4)) cells, respectively, when analyzed by using two equations. According to the initiated cell theory of Farber, foci and nodules are formed through sequential cell division of 14 to 21-times or more within a short time period. The rapid growth exceeded the rate of cell division, indicating that the growth of preneoplastic cells is based on a nonclonal penetration mechanism.     

Positive influence of dietary deoxycholic acid on development of pre-neoplastic lesions initated by N-methyl-Nnitrosourea in rat liver

The effect of deoxycholic acid (DCA) treatment subsequent toinitiation of F344 male rats with N-methyl-N-nitrosourea (MNU),a wide spectrum carcinogen inducing tumors in many organs, wasinvestigated. Rats were initially given four doses of MNU (50mg/kg) i.p. within a 2-week period combined with a two-thirdspartial hepatectomy performed at day 7 and then placed on basaldiet containing DCA at concentrations of 0.313, 0.125, 0.050and 0.020% for 21 weeks prior to final sacrifice. All organsstudied were carefully examined histologkally and histochemicallyfor development of neoplastic and pre-neoplastic lesions. DCAenhanced the induction of glutathione S-transferase positive(GST-P+) liver cell foci in a dose-related manner. Furthermoregroups of rats given DCA without prior MNU administration alsodeveloped dosedependent numbers of pre-neoplastic liver lesions.In addition, increased numbers of small intestine tumors wereapparent in DCA-treated animals although the difference wasnot significant. Induction of tumors in the thyroids, Zymbalglands, skin and peripheral nerves was not affected. The resultsindicate that DCA is a strong promoter of hepatocarcinogenesiswith possible complete carcinogenkity in the liver and promotionpotential for tumor development in the small intestine.     

Role of diethylnitrosamine, 2-acetylaminofluorene and partial hepatectomy in the expression of glutathione-S-transferase-P and gamma-glutamyltranspeptidase in the early steps of rat liver carcinogenesis.

Three factors involved in the Solt and Farber model of rat liver carcinogenesis were studied alone and in various combinations: diethylnitrosamine (DEN) initiating dose, 2-acetylaminofluorene (2-AAF) feeding and partial hepatectomy. The administration of DEN alone (200 mg/kg) was able to switch on glutathione-S-transferase, placental type (GST-P) expression 3 weeks later at a low level (85 U/micrograms protein) which was stable for 10 weeks in the absence of histopathological lesions. During the same time, gamma-glutamyl transpeptidase (GGT) activity presented 2 waves of increase. The feeding of 0.03% 2-AAF for 2 weeks appeared as a determinant factor in the expression of GST-P protein as well as GGT induction (15- and 7-fold versus DEN alone, respectively). The addition of partial hepatectomy enhanced again GST-P expression (1.5-fold) and GGT induction (2-fold). However, GST-P foci increased in size, not in number while GGT foci increased both in size and in number. These data indicated that 2-AAF was a crucial component of the selection procedure since partial hepatectomy alone, with or without DEN initiation was inefficient in promoting GST-P expression. Therefore, 2-AAF would be able to promote the growth of GST-P-positive cells initiated by DEN, a mechanism likely responsible for its tumor-promoting effect.     

Effects of Cessation of Alcohol Exposure on Rat Hepatocarcinogenesis

Cessation of long-term alcohol exposure is reported to enhance rat hepatocarcinogenesis. The purpose of the ‍present study was to assess this possibility using glutathione-S transferase placental form (GST-P) positive foci as ‍end point lesions. All rats were treated with a single i.p. injection of diethylnitrosamine (DEN) (200 mg/kg body ‍weight) and then given a MF pellet diet for 2 weeks. Thereafter, the animals were maintained on: alcohol liquid diet ‍in which 36% of total calories were provided by alcohol (5% Al diet) for 6 weeks (group 1); control liquid diet (C ‍diet) for 6 weeks (group 2); 5% Al diet for 6 weeks and subsequently C diet for 4 weeks (group 3); 5% Al diet for 10 ‍weeks (group 4); or C diet for 10 weeks (group 5). All rats were subjected to two thirds partial hepatectomy at 3 ‍weeks after DEN injection. The number and area of GST-P positive foci per cm2 of liver tissue were slightly increased ‍in group 1 compared to the group 2 and significantly elevated in the group 4 compared to group 5. However, numbers ‍in group 3 were significantly lower in group 4 and similar to the group 5 values. PCNA positive cells in the GST-P ‍positive foci in the group 1 and group 4 were significantly increased as compared with respective controls (groups 2 ‍and 5, respectively), while indices in the group 3 were again similar to values for group 5. Cessation of short-term ‍alcohol administration thus had no promoting effects on development of GST-P foci, suggesting that the duration of ‍alcohol treatment may be important. The results also imply the existence of a cumulative exposure time or dose ‍threshold for alcohol if promoting effects of cessation are to be seen on rat hepatocarcinogenesis.     

3′-Me-DAB诱导大鼠肝癌发生过程中p53及相关基因mRNA的定量分析

背景与目的:有关肝癌中 p53基因突变及 p53蛋白表达异常已有报道 , 但其在 mRNA水平上的变化尚不清楚 . 为了解在肝癌发生、发展和预后过程中 p53、谷胱甘肽转硫酶 P( glutathione S-transferase P,GST-P) 、甲胎蛋白 ( α -fetoprotein,AFP) 和白蛋白 mRNA水平的变化 , 本研究定量分析癌前病变和癌灶中 GST-P、 AFP和白蛋白 mRNA的量 . 方法:在 3′ -甲基 -4-二甲胺偶氮苯 (3′ -methyl-4-dimethylaminoazobenzene,3′ -Me-DAB)诱发 F344大鼠肝癌过程中 , 用激光捕获显微取样仪 (LCM)准确获得大鼠肝脏中微小癌灶或癌前病变组织后 , 采用 LightCyclerTM V3 System 实时 (real-time)RT-PCR定量分析这些病灶组织中 mRNA水平 . 结果:在实验的第 6、 12和24周,癌前病变组织中p53mRNA量均显著高于正常组织(P≤0.001)。从第6周到第24周,癌前病变组织中p53mRNA逐渐降低(P<0.01)。癌组织中p53mRNA含量高于正常组织,低于同期癌前病变组织(P=0.028和0.0136)。第24周的癌前病变组织或癌组织中细胞核呈p53强染色。各实验期,癌前病变中GST-PmRNA量明显高于正常组织和癌组织(P<0.001)。癌组织中AFPmRNA的表达量显著高于癌前病变组织和正常组织(P<0.001),白蛋白mRNA的表达量显著低于这两种组织(P<0.01)。GST-P和AFP分别在癌前病变组织和癌组织中呈灶状强表达。结论：GST-P和AFPmRNA分别在癌前病灶组织和癌组织中强表达,是这些病变组织的重要标志物。p53mRNA在早期肝癌前病变和癌灶中显示高水平的表达,而较晚期p53蛋白升高。