Resistance of DRH strain rats to chemical carcinogenesis of liver: genetic analysis of later progression stage
Ying Yan,
Zhao-Zhu Zeng1,
Shin Higashi1,
Ayumi Denda2,
Yoichi Konishi2,
Shunzo Onishi3,
Hikaru Ueno,
Ken Higashi1 and
Hiroshi Hiai1,4
Department of Biochemistry, School of Medicine, University of Occupational and Environmental Health, Yahatanishi-Ku, Kitakyushu 807-8555, Japan,
1 Department of Pathology and Biology of Diseases, Kyoto University Graduate School of Medicine, Yoshida-Konoe-Cho, Sakyo-Ku, Kyoto 606-8501, Japan,
2 Department of Oncology, Cancer Institute, Nara Medical University, Kashihara, Nara 634-8521, Japan and
3 Division of Pathology, Laboratory Center, Tane General Hospital, Nishi-Ku, Osaka 550-0024, Japan
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Abstract
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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)F2 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)F2 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.
Abbreviations: AFP, alpha-fetoprotein; ARE, antioxidant response element; EAF, enzyme altered foci; ERE, electrophile response element; GGT, gamma-glutamyltranspeptidase; Ghrhr, growth hormone releasing hormone receptor; GST-P, glutathione S-transferase-placental form; HCC, hepatocellular carcinoma: LOD, logarithm of odds; 3'-Me-DAB, 3'-methyl-4-dimethylaminoazobenzene; ODC, ornithine decarboxylase; PCR, polymerase chain reaction; QTL, quantitative trait locus; RNO, rat chromosome: Xhsl, X-ray hypersensitivity in LEC rat.
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Introduction
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Chemical hepatocarcinogenesis occurs as a multi-step process (13). Administration of most hepatocarcinogens to rats leads to the development of enzyme-altered foci (EAF) in their livers within 46 weeks. The number and size of EAF in the liver have been regarded as excellent quantitative parameters for the carcinogen-initiated pre-cancerous lesions (36). Tumor promotion, the second step of carcinogenesis, accelerates the growth of cells within these foci and consequently increases the risk of secondary genetic alterations (79). The cells within these foci may undergo further mutational changes and new cell clones with a more malignant phenotype may be selected during the progression step, resulting in the development of hepatocellular carcinomas (HCCs). These mutational changes leading to the ultimate progression to HCC, however, occur infrequently, as HCC develops from only a very small fraction of EAF, probably at a rate on the order of 104.
To dissect the molecular mechanisms involved in the steps leading to progression, genetic analysis of each step should be highly informative. The DRH rat is an inbred strain established by inbreeding closed colony Donryu rats for >20 generations under continuous feeding of a diet containing 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). The DRH rats are highly resistant to chemical-induced liver carcinogenesis (10,11). Glutathione S-transferase-placental form (GST-P) is one of the most reliable markers for visualizing the largest number of EAF, i.e. 90% of heterogenous EAF can be detected by GST-P staining alone (9). The DRH rats show remarkably fewer and smaller GST-P-positive foci than susceptible Donryu or F344 rats upon induction by 3'-Me-DAB (12). In a previous study, the resistance of DRH rats to pre-neoplastic liver lesions was studied by analyzing (F344xDRH)F2 male rats fed 3'-Me-DAB for 8 weeks (13). F344 rats were used as a carcinogen-susceptible strain in the genetic analysis as the parental Donryu rats were not an inbred strain. Using number, size and area of GST-P-positive EAF, as well as mRNA levels of GST-P, as quantitative parameters, we identified at least two highly significant clusters of quantitative trait locus (QTLs) on RNO1 and RNO4. These clusters of resistant QTLs were designated as Drh1 and Drh2, respectively (13).
In the present study, we asked whether these QTLs affecting pre-neoplastic lesions are also the determinants of the later stage of hepatocarcinogenesis as well, and whether there are any additional QTLs affecting hepatocarcinogenesis in the progression stage. (F344xDRH)F2 rats were fed 3'-Me-DAB for 20 weeks, and the quantitative parameters GST-P mRNA, ornithine decarboxylase (ODC) activity, number, size and total area of HCC nodules were examined and analyzed. We concluded that Drh1 is inhibitory to GST-P induction in both the pre-neoplastic and neoplastic stages in a dose-dependent manner and that Drh1 inhibited the growth of EAF in the pre-neoplastic stage but did not affect the progression to HCC. On the other hand, the QTLs in Drh2 cluster play a role in the expansion of the GST-P-positive foci as well as in the development of HCC.
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Materials and methods
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Chemicals
3'-Me-DAB was purchased from Tokyo Kasei Kogyo (Tokyo). Rabbit anti-rat GST-P antibody was obtained from Medical and Biological Lab. (Nagoya). [5-C14]L-Ornithine (sp. act. 1.85 GBq/µmol) was obtained from Muromachi Chemical (Tokyo).
Animals
The origin of DRH rats was described in detail previously (12). Male DRH, F344 rats and (F344xDRH)F1 rats were purchased from SEAC Yoshitomi (Fukuoka). They were fed on commercial rat chow (EM, Oriental Yeast, Tokyo) and given tap water ad libitum, and were used after 1 week of acclimatization. (F344xDRH)F2 rats were generated in our laboratory by inter-crossing male and female (F344xDRH)F1 rats.
Animal treatments
A preliminary experiment to evaluate the effect of 3'-Me-DAB on pre-cancerous and cancerous stage was carried out. To compare the number and size of GST-P-positive foci in the pre-cancerous stage, eight F344 rats and 10 DRH rats were fed diet containing 0.06% 3'-Me-DAB from 5 weeks of age and killed under ether anesthesia 8 weeks later. Count and measurement of GST-P-positive foci were carried out as described previously (12). For the experiments of hepatocarcinogenesis using individual strains, 11 F344 rats, five DRH rats and 12 (F344xDRH)F1 rats were similarly fed 3'-Me-DAB for 20 weeks. Livers and sera were used for biochemical and histological studies. For genetic analysis, 99 (F344xDRH)F2 rats were fed a diet containing 3'-Me-DAB for 20 weeks under the same conditions as above. Macroscopically detectable neoplastic nodules and/or tumors on the entire surface of the livers of individual rats were counted and their sizes were determined using slide calipers. Pieces from non-tumorous regions of individual rat livers were used to determine either GST-P mRNA levels or ODC activities and stored at 70°C until use.
Histopathological analysis
The livers from rats fed 3'-Me-DAB for 20 weeks, gross neoplastic liver nodules and HCC were routinely stained with hematoxylin and eosin for histological examination and were diagnosed according to the criteria described by Squire and Levitt (14).
ODC activity
ODC (EC 4.1.1.17) activity was assayed in non-tumor regions of the liver according to the method described by Djurhuus (15). Briefly, dithiothreitol (5 mM), pyridoxal phosphate (0.2 mM), sodium phosphate (50 mM, pH 7.2), L-ornithine (0.2 mM), [5-C14]L-ornithine (0.16 µCi, a total of 0.48 mM L-ornithine in the incubation mixture) and cell extract in a final volume of 100 µl were incubated at 37°C for 60 min. The reactions were stopped by transfer of the reaction tubes to melting ice, followed by application of the 40 µl of the reaction mixture to dry p81 cation-exchange papers (2 cm diameter, in duplicate). The papers were washed with 2x5 l of 0.1 M NH3, and dried in counting vials, and the radioactivity of the putrescine formed was counted in an Aloka LSC 3500 scintillation counter.
Northern blot analysis
Total RNA of either non-tumor- or tumor-containing regions of individual livers were prepared as described previously (16). GST-P mRNA levels were analyzed using a 32P-labeled probe for GST-P, were quantified using a BAS 2000 Bioimaging analyzer (Fuji Photofilm, Tokyo) after the hybridization reaction (13). In a previous study (13), it was found that the GST-P mRNA level and percent liver area occupied by GST-P-positive foci determined by immunohistochemistry are highly and significantly correlated (r = 0.763) after 8 weeks of 3'-Me-DAB administration (13).
Genetic analysis
Genomic DNAs from individual rat kidneys were prepared by the guanidine isothiocyanatephenolchloroform protocol (16) with slight modification. The DNA was used as templates for microsatellite length polymorphism assays as described previously (13). Metaphor agarose (Takara, Kyoto) was used for electrophoresis of PCR products to obtain better resolution of the bands (17). All primers for microsatellite analysis were purchased from Research Genetics (Huntsville, AL). In the preliminary genome scan, linkage was evaluated by the non-parametric KruskalWallis test. According to the criteria of Lander and Kruglyak (18), linkage in the F2 generation was taken as significant when P was <5.2x105 (LOD score 4.3) and was suggestive when P was <1.6x103 (LOD score 2.8). Genotype data were analyzed using either the MapMaker/QTL program or Cartographer QTL software (13). Phenotypic differences between the three genotype (F/F, F/D and D/D) were analyzed by the unpaired Student's t-test.
Correlations between different phenotypic parameters were evaluated by correlation analysis with StatView-J4.11 software (Abacus Concepts, Berkeley, CA) on a Macintosh personal computer.
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Results
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HCC development in DRH, F344 and their F1 rats induced by 3'-Med-DAB
Table I
shows data of preliminary experiments to evaluate several phenotypic parameters in F344, DRH and their F1 rats fed 3'-Me-DAB for 8 (Table IA
) or 20 weeks (Table IB
). At 8 weeks, the number and size of GST-P-positive foci, as well as percentage of liver area occupied by foci, were significantly larger in F344 than in DRH rats. Especially the difference in size was much more conspicuous than that in number. At this stage, however, no macroscopic neoplastic nodule was observed even in susceptible F344 rats. At 20 weeks, all 11 F344 rats developed macroscopically evident liver tumors but the five DRH rats did not develop any tumors. Seven of 12 (F344xDRH)F1 rats developed almost equivalent number of nodules/tumors as the F344 rats, but they were far smaller in size than those in the F344 rats (Table IB
). GST-P mRNA was detected in both tumor and non-tumor liver tissues of F344 rats but was scarcely detected in DRH rat livers. In F1 rat livers, the level of GST-P mRNA in the tumor region was as high as that in F344 rat liver, but that in non-tumor region was far lower. ODC activity was higher in F344 than in DRH or F1 rats. Serum alpha-fetoprotein (AFP) levels were significantly lower in tumor-bearing (F344xDRH)F1 rats than in F344 rats (Table I
), indicating the effect of a DRH dominant/semi-dominant resistance gene on tumor progression. These observations indicate that DRH had a powerful semi-dominant resistance to HCC, especially to the growth of HCC as represented by tumor area and serum AFP level.
Genetic parameters of HCC in (F344xDRH)F2
To study the genetic control of hepatocarcinogenesis by 3'-Me-DAB, we analyzed four quantitative parameters possibly relevant to HCC induction as observed at 20 weeks of 3'-Me-DAB feeding. They were: (i) GST-P mRNA levels in non-tumor regions of the liver (Figure 1A
); (ii) the number of macroscopically detectable neoplastic liver nodules and/or tumors on the whole liver surface (Figure 1B
); (iii) ODC activity in non-tumor regions of the liver (Figure 1C
); and (iv) the total area of nodules/tumors in individual livers (Figure 1D
).

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Fig. 1. Distribution of phenotypic parameters in F2 rats after 20 weeks of 3'-Me-DAB administration. (A) GST-P mRNA levels in non-tumor region of the liver. (B) The number of macroscopically detectable neoplastic nodules detectable on the liver surface. (C) ODC activity in non-tumor region of the liver. (D) The total area of the nodules in individual liver. Bars in each panel represent means and SD for each parameter in DRH, F344 and F1 rats given 3'-Me-DAB for 20 weeks.
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Table II
shows the correlations among these parameters in the F2 rats fed 3'-Me-DAB for 20 weeks. As the total area of tumor and the mean size of tumor were highly correlated (r = 0.914), we employed only the total area of tumor in individual F2 rats for genetic analysis. Total area and numbers of tumor were not correlated (r = 0.0010), suggesting the hitness carcinogen and growth of tumor were under distinct genetic control. The ODC activity was significantly correlated with both the total area (r = 0.663) and the mean size (r = 0.702) of the tumor. Therefore, it is an excellent parameter for increased proliferation of hepatocytes (19,20). Interestingly, neither GST-P mRNA levels nor number of HCCs were significantly correlated with any of the other parameters, at least as determined by the correlation coefficient. The increased induction of GST-P certainly paralelled with the formation of EAF, but the chance of its malignant transformation did not directly correlate with the enzyme level as discussed later.
QTL analysis of F2 rats
A preliminary genome-wide screening was carried out using 22 (F344xDRH)F2 rats with macroscopically evident HCC and 19 resistant F2 rats (nine rats without any lesions and 10 with one or two barely detectable small-sized tumors). This screening revealed possible linkages on RNO1, RNO4 and RNO13 for at least some of the parameters examined in this study. Subsequently, all 99 (F344xDRH)F2 rats were genotyped for all available loci polymorphic between DRH and F344 rats in these chromosomal regions. We used 18 microsatellite loci on RNO1, 17 loci on RNO4 and 15 loci on RNO13, respectively. The average distance between marker loci was 6.5 cM; the longest were between D1Mgh8 and D1Rat66 (19.7 cM) and between D13Rat3 and D13Rat85 (20.8 cM).
Figure 2
shows the results of a composite interval mapping of parameters for pre-neoplastic lesions observed at 8 weeks (13) and GST-P mRNA at 20 weeks (shown in Figure 2A and B
) and parameters for neoplastic nodules/HCC (Figure 2C and D
). A LOD score of 4.3 was taken as the threshold of significance for F2 inter-cross (18). Two major clusters of QTLs on RNO1 and RNO4 were observed at map position essentially the same as those of Drh1 and Drh2, identified previously as genes related to resistance to pre-neoplastic liver lesions (13). There were also other minor peaks of QTLs on RNO1 and RNO13, which will be addressed below. On the distal segment of RNO1 (Figure 2A
), there were two peaks of QTLs for GST-P mRNA levels at 20 weeks of 3'-Me-DAB treatment. The major peak, with LOD score 8.1, was mapped between D1Wox10 and D1Rat302, which corresponded to the location of Drh1b observed in our previous study (13). A minor peak with LOD score 3.1 was found between D1Mit28 and D1Wox10, which corresponded to the location of Drh1a. On RNO4, it is noteworthy that the remarkable QTL for GST-P mRNA seen at 8 weeks disappeared at 20 weeks, and instead, a weak peak with LOD score <3.0 was seen close to D4Mgh6 (Figure 2B
). As seen in Table III
, GST-P was expressed even at 20 weeks, but it was affected by a host gene on RNO1 but not by that on RNO4. As seen in Figure 2C
, there was no significant tumor-related QTL corresponding to Drh1. On the proximal segment of RNO1, another QTL for total area of tumors was found around D1Mit9, between D1Mgh2 and D1Mgh30, peak LOD score 3.3 (data not shown). This peak was seen only at 20 weeks and, thus, seems specific to tumor growth, but its presence was suggestive but not statistically significant.

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Fig. 2. QTL analysis by composite interval mapping using the Cartographer software. The horizontal line at LOD score 4.3 denotes the threshold level of significance for F2 (18). Bar = 10 cM; (A) RNO1 and (B) RNO4. Phenotypic parameters for EAF. LOG GST-P mRNA in the F2 rats fed 3'-Me-DAB for 20 weeks (bold solid line) and for 8 weeks (- - -); size of EAF (transformed to square root for calculation) at 8 weeks (); and number of EAF (square root) at 8 weeks (. . .). Data for the F2 rats fed 3'-Me-DAB for 8 weeks are cited from our previous work (13) for ease of comparison by permission. (C) RNO1 and (D) RNO4. Phenotypic parameters for neoplastic nodules/HCC. Number of nodules/tumors at 20 weeks (. . .), area of nodules/tumors () and ODC activity in non-tumor region at 20 weeks (- - -). Parameters in (C) and (D) are transformed to square root.
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A second cluster of QTLs was located on a middle segment of RNO4 (Figure 2D
), between D4Rat15 and D4Mgh7. In this chromosome segment, peaks of QTLs for number of tumors, total area of tumors and ODC activity were clustered. The peak for number of tumors was observed between D4Rat23 and D4Rat37, with a peak LOD score 4.3. The LOD plot for total area of tumors formed two peaks between D4Mgh32 and D4Rat48, the proximal one with LOD score 3.9 and the distal one, 4.8, respectively. The plot for ODC activity formed a peak with LOD score 4.4 between D4Rat48 and D4Mgh7.
There was another minor QTL for number of tumors on RNO13, between D13Rat23 and D13Rat85, but its LOD score did not reach the level of significance (data not shown).
Phenotype and genotype relationship in F2 population
The relationship between phenotype and genotype at D1Wox10, D1Rat302, D4Wox22, D4Rat37 and D4Mgh6 was shown in Table III
. These loci did not represent the peak position of LOD blot, but located closely to the peaks. For GST-P mRNA, semi-dominant resistance of the DRH allele was observed at D1Wox10 and D1Rat302. For ODC, dominant resistance of the DRH allele was seen at D4Mgh6. At D4Wox22, the DRH allele gave recessive resistance to number of tumors. For tumor size, it was difficult to see the DRH gene effect, because occasional extremely large tumors obscured statistical comparison.
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Discussion
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Carcinogen-resistant DRH strain rats provide a unique model for genetic evaluation of the steps of chemical hepatocarcinogenesis. Among such steps, the first is generation of activated carcinogen metabolites, which are generally toxic to normal liver cells; the second, induction of detoxifying enzymes such as GST-P to circumvent the toxicity; the third, formation of DNA adducts that introduce mutations; and the fourth, enhanced growth of the initiated target cells. Some of these steps can be susceptible to host genetic composition. The phase I drug-metabolizing enzymes that activate most carcinogens exhibit high-substrate specificity. DRH rats are resistant not only to 3'-Me-DAB, but also to other hepatocarcinogens such as 2-acetylaminofluorene and N-nitrosodimethylamine (21,22). Furthermore, there is no apparent difference between DRH and susceptible strain rats in the amount of DNA adducts formed after 3'-Me-DAB treatment (21). This lack of chemical specificity as well as the formation of similar levels of DNA adducts rule out the direct involvement of phase I enzymes from mechanisms of resistance of DRH rats.
Feeding of 3'-Me-DAB seems not to pose a serious health hazard to DRH rats (21), indicating they have some efficient mechanism to escape from the toxicity of a variety of chemicals. Glutathione acts as a nucleophile that binds electrophiles, the products of metabolically activated carcinogens (23), directly or via GST-P (24,25). GST-P, thus converts reactive electrophiles to less toxic and more readily excretable products. GST-P and gamma-glutamyltranspeptidase (GGT), which replenish the glutathione content (24), are not simple markers of pre-neoplastic cells, but rather play a role in providing a selective advantage to hepatocytes to survive in the presence of carcinogens and to proliferate under such genotoxic conditions (3,7). From the map positions of resistance QTLs on RNO1, the GST-P gene is one of the likely candidates for Drh1 (13). The rat GST-P gene is located at the D1Wox36 locus, which exists between D1Rat181 and D1Rat71, and is within Drh1a (13). The second peak of LOD score, located between D1Wox10 and D1Rat302 corresponding to Drh1b, was much higher than that at Drh1a, suggesting that gene(s) other than GST-P may be involved to determine GST-P mRNA level. The promoter of the rat GST-P gene has a regulatory sequence known as electrophile response element (ERE)/antioxidant response element (ARE) (26). We compared the nucleotide sequences of the promoter region of rat GST-P gene (27), including the regulatory sequence (ERE/ARE). No polymorphism, however, was found among DRH, F344 and Donryu rats (28), although CG island methylation involving the 5' regulatory region of the GST-P gene may cause allelic loss events without any mutation (29). The QTL on RNO4 affecting GST-P mRNA and number as well as size of EAF which was so remarkable in pre-neoplastic stage, disappeared at 20 weeks, leaving a small non-significant peak of LOD plot at around D4Mgh6. Lack of correlation between size and number of tumors is an unexpected observation, irrespective the QTLs affecting these phenotypes are located on the same chromosome. During tumor progression, stochastic somatic events may occur and provide further complexities. Recent studies showed that induction of phase II detoxifying enzymes is regulated by a common mechanism, in which an NF-E2-related factor (Nrf2) is involved (30,31). Furthermore, it is reported that loss of expression of Nrf2 significantly enhances the susceptibility of mice to chemical carcinogenesis, suggesting that phase II enzyme induction could play a major role in protection against neoplasia and toxicity (32).
Genetic analysis of two different stages of hepatocarcinogenesis revealed that the QTLs on RNO4 affect both EAF formation and progression to HCC. The map position of these QTLs was extensively overlapped with Drh2, a gene cluster affecting EAF size, number and GST-P mRNA level. In contrast, the QTL on RNO1 affected GST-P induction but none of HCC parameters. EAFs have been assumed to be pre-cancerous lesions (13,33,34). The number of EAF is much larger than the number of tumors, indicating that only a small fraction of EAF develop into HCC (35). EAF contained a large number of proliferating hepatocytes and its volume fraction in F344 rats at 8 weeks is estimated at 1000 times as much as that of DRH rats by a calculation from the ratio of percent liver area occupied by GST-P foci. Under continuous feeding of 3'-Me-DAB, the total mutational hits in proliferating hepatocytes within EAF should be much higher in susceptible strains, and they may facilitate the EAF cells to progress towards HCC. If the above assumption is literally correct, a reduced induction of EAF in DRH rats should be unfavorable to carcinogenesis. However, it is only partly fulfilled as the QTLs on RNO4 are involved in induction as well as growth of EAF at earlier stage and later determine the chance of their progression to HCC, but the QTL on RNO1 did not modify tumor progression. The fact that the GST-P mRNA level at 20 weeks of treatment did not correlate with size and number of tumors indicates that GST-P expression per se in the non-tumor region is not a requirement for tumor progression. Our work demonstrated that the regulation of GST-P induction is phase-dependent during 3'-Me-DAB-induced carcinogenesis and is under the control of multiple QTLs as the GST-P mRNA level is determined by both Drh1 and Drh2 at 8 weeks, but only by Drh1 at 20 weeks. Such findings lead to the hypothesis that progression of EAF to HCC may occur exclusively in a subset of EAF susceptible to Drh2 control. It has been suggested that EAFs are heterogenous in morphology (36) profile of enzyme expression (37,38) and cell-division rate (39) Especially Enzmann and Bannasch (36) claimed an ordered sequence in the shift of morphological types of EAF depending on the stages of hepatocarcinogenesis, from foci rich in glycogen through mixed and basophilic cell foci to neoplastic nodules and HCC. To date, however, there has been no direct evidence that each HCC is a progeny of certain subset of EAFs and even the possibility that HCC arises independently of EAF (40) has not been formally ruled out. To provide a clue to this problem, we are attempting to establish reciprocal congenic strains for Drh1 or Drh2 between DRH and F344 and also to analyze the heterogeneity of EAFs in the progression stage are on the way in our laboratory.
Studying crosses between F344 and BN rats, DeMiglio et al. (41) observed two significant QTLs on RNO7 and RNO10 affecting hepatocarcinogenesis and two suggestive QTLs on RNO4 and RNO8 for tumor mean volume and its volume fraction. One of them, Hcr2 on RNO4, is mapped between D4Mgh4 and D4Rat47 and overlapped with Drh2a between D4Mgh32 and D4Rat48. This suggests a possible coincidence of Hcr2 with a part of Drh2. One of the candidate genes for this locus is growth hormone releasing hormone receptor (Ghrhr) at D4Rat108. In several experimental cancers, treatment with antagonists of Ghrh causes a reduction in IGF-I and IGF-II, concomitant to the inhibition of tumor growth (42,43). Our preliminary RTPCR analysis showed that both Ghrh and Ghrhr mRNA were induced in several tumors induced by 3'-Me-DAB obtained in this study, but they were undetectable in non-tumor-bearing rat livers (data not shown). It is unknown whether the Ghrhr product directly affects tumor susceptibility as both DRH and F344 rats had equal significant levels of Ghrh and Ghrhr mRNA expression in their brain (data not shown).
Recently, Agui et al. (44) mapped a QTL, Xhsl, for X-ray hypersensitivity in LEC strain rats between D4Rat48 and D4Rat79. This region overlaps with Drh2 on RNO4. One of the possible candidate genes for Xhsl is Tgf
(gene encoding tumor growth factor alpha) mapped at D4Rat49. TGF
is a ligand of the EGF receptor and stimulates the growth of liver cells (45). The possible contribution of TGF
to the preferential growth of carcinogen-induced pre-neoplastic and neoplastic hepatocytes (46) and also its role in sustaining clonal expansion by promoter-dependent, chemically initiated rat hepatocytes (47) have been reported.
The present genetic analysis has introduced a novel insight to steps of hepatocarcinogenesis in rats, which is a combination of genetic predisposition and somatic mutational events. We could identify the QTLs functioning in different stages of carcinogenesis and propose a possible diversity in EAF induced by the carcinogen. This type study will provide a basis of understanding of genetic polymorphism determining cancer susceptibility among individuals.
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Notes
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4 To whom correspondence should be addressed Email: hiai{at}path1.med.kyoto-u.ac.jp 
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Acknowledgments
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We are grateful to Dr Shinzo Nishi for measuring serum AFP and to Dr Taneaki Higashi for discussions about the present study. This work was supported by a Grant in Aid for Cancer Research from the Ministry of Education, Culture, Science and Technology, Japan, and a grant from the Ministry of Health, Welfare and Labor, Japan. We are grateful for the generous financial support from the Eiko Norihara Memorial Fund.
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Received May 29, 2001;
revised September 12, 2001;
accepted October 24, 2001.