Identification of genetic loci controlling hepatocarcinogenesis on rat chromosomes 7 and 10.

Neoplastic liver nodules and hepatocellular carcinomas (HCCs) were induced, by "resistant hepatocyte" model, 32 and 70 weeks after initiation with diethylnitrosamine, respectively, in F344 Brown Norway (BN), and (BNxF344)F1 rats. Nodule number/liver (N) did not significantly differ among rat strains, whereas nodule mean volume (V) and nodule volume fraction (VF) were higher in susceptible F344 than in resistant BN and BFF1 strains and were predictive of subsequent development of HCCs. Genomic scanning of 157 backcross BFF1xF344 rats with 190 polymorphic microsatellites, and linkage analysis, revealed two quantitative trait loci (QTL) on chromosomes 7 and 10, which showed significant linkage with VF, and two QTL on chromosomes 4 and 8, which showed suggestive linkage with V and VF. On the basis of phenotypic patterns of homozygous and heterozygous backcross progeny and of allelic distribution pattern, QTL on chromosomes 10, 8, and 4 were tentatively identified as resistance loci, and QTL on chromosome 7 was identified as susceptibility locus for rat hepatocarcinogenesis. An analysis of interactions allowed us to identify additional putative QTL on chromosomes 5 and 8 and suggested an additive effect of loci on chromosomes 10, 8, and 4 for VF and V. These data are the first to identify chromosomal regions containing putative susceptibility/resistance loci for rat hepatocarcinogenesis, which seems to be highly complex in terms of the number of genetic factors involved.     

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.     

Modulation of diethylnitrosarnine-initiated placental glutathione S-transferase positive preneoplastic and neoplastic lesions by clofibrate, a hepatic peroxisome proliferator

The effect of clofibrate (CF) on proliferation of diethylnitrosamine(DEN)-initiated glutathione S-transferase placental form (GST-P)-positivepreneoplastic and neoplastic lesions was studied in male F344rats. Animals were given a single i.p. injection of 200 mg/kgbody weight of DEN, and then from 2 weeks later were given adiet containing 0.3% CF (group 1), or no supplement (group 2)until week 64. Group 3 received an injection of 0.9% NaCl insteadof DEN and then a diet containing 0.3% CF, like group 1. Animalsin all groups were subjected to partial hepatectomy at week3 and killed at weeks 8, 20, 32, 49 or 64. The results showedthat development of GST-P-positive lesions was significantlyless in group 1 than in group 2 from week 8 (P < 0.05). However,in group 1, morphologically distinguishable GSTP-negative preneoplasticlesions increased from week 20 (P < 0.05), and the totalnumber of GST-P-positive and -negative lesions was significantlygreater than that in group 2 from week 32 (P < 0.05). Theinduction of hepatocellular carcinoma (HCC) was greater in group1 than in group 2 from week 49. All the HCCs induced in group2 were GST-P-positive, whereas 38.9% (7/18) of those in group1 were GST-P-negative. In group 3, only a few GST-P-positiveand/or -negative preneoplastic lesions developed by week 64.These results suggest that CF has tumor-promoting activity,and that GST-P-positive cells induced by DEN changed to GST-P-negativecells on subsequent treatment with CF.     

The degradation of cell cycle regulators by SKP2/CKS1 ubiquitin ligase is genetically controlled in rodent liver cancer and contributes to determine the susceptibility to the disease

Previous work showed a genetic control of cell cycle deregulation during hepatocarcinogenesis. We now evaluated in preneoplastic lesions, dysplastic nodules and hepatocellular carcinoma (HCC), chemically induced in genetically susceptible F344 and resistant Brown Norway (BN) rats, the role of cell cycle regulating proteins in the determination of a phenotype susceptible to HCC development. p21^WAF1, p27^KIP1, p57^KIP2 and p130 mRNA levels increased in fast growing lesions of F344 rats. Lower/no increases occurred in slowly growing lesions of BN rats. A similar behavior of RassF1A mRNA was previously found in the 2 rat strains. However, p21^WAF1, p27^KIP1, p57^KIP, p130 and RassF1A proteins exhibited no change/low increase in the lesions of F344 rats and consistent rise in dysplastic nodules and HCC of BN rats. Increase in Cks1‐Skp2 ligase and ubiquitination of cell cycle regulators occurred in F344 but not in BN rat lesions, indicating that posttranslational modifications of cell cycle regulators are under genetic control and contribute to determine a phenotype susceptible to HCC. Moreover, proliferation index of 60 human HCCs was inversely correlated with protein levels but not with mRNA levels of P21^WAF1, P27^KIP1, P57^KIP2 and P130, indicating a control of human HCC proliferation by posttranslational modifications of cell cycle regulators.     

Chromosome mapping of multiple loci affecting the genetic predisposition to rat liver carcinogenesis

Previous studies on (BNxF344)F1 (BFF1) rat model of genetic predisposition to hepatocarcinogenesis led to the identification, in BFF1xF344 backcross progeny, of two hepatocarcinogenesis susceptibility (Hcs) and three resistance (Hcr) loci affecting the progression of neoplastic liver nodules. To evaluate the presence of other hepatocarcinogenesis-related loci in the BFF1 genome, nodule induction by resistant hepatocyte model in 116 male BFF2 rats 32 weeks after initiation with diethylnitrosamine was subjected to quantitative trait loci analysis. The rats were typed with 179 genetic markers, and linkage analysis identified three loci on chromosomes 1, 16, and 6, in significant linkage with nodule mean volume (V), volume fraction, and number, respectively, and two loci on chromosomes 4 and 8 in suggestive linkage with V. These loci were differently positioned with respect to Hcs and Hcr loci mapped previously in backcross rats. On the basis of phenotypic and allele distribution patterns of BFF2 rats, loci on chromosomes 1 and 16 were identified as Hcs3 and Hcs4, and loci on chromosomes 4, 8, and 6 as Hcr4, Hcr5, and Hcr6. Additive interactions occurred between Hcs3 and Hcs4, and Hcr4 and a locus on chromosome 3 with less than suggestive linkage with V. All of the loci were in chromosomal regions syntenic to mouse and/or human chromosomal segments showing allelic gain or loss in hepatocellular carcinomas. These data indicate that inheritance of predisposition to rat liver tumor is characterized by the interplay of several genetic factors and suggest some possible mechanisms of polygenic control of human liver cancer.     

The presence of alpha 2u-globulin is necessary for d-limonene promotion of male rat kidney tumors.

In a 2-year carcinogenesis bioassay, d-limonene (dL) induced kidney tumors in male F344 rats, but not in female F344 rats or either sex of mice, d-Limonene-1,2-oxide, a metabolite of dL, has been shown to bind reversibly the male rat-specific urinary protein, alpha2u-globulin (alpha 2u-G), lysosomal degradation than alpha 2u-G alone. This reduced degradation of alpha 2u-G-chemical complex leads to an accumulation of this protein in the proximal convoluted tubules of the male rat kidney and to the morphological changes characteristic for alpha 2u-globulin nephropathy. The only male rat strain known to be resistant to this renal disease is the alpha 2u-G deficient NCI-Black-Reiter (NBR) rat. The objectives of this study were to determine whether or not dL causes sustained increases in cell proliferation and has promoting activity for renal adenomas in male rats and if the male rat-specific urinary protein, alpha 2u-G, is required. In a 32-week initiation-promotion assay, male F344 and NBR rats were treated with either 0 or 500 ppm N-ethyl-N-hydroxyethylnitrosamine (EHEN) in the drinking water for 2 weeks. Experimental groups of 31 to 38 rats then received 0 or 150 mg d-limonene/kg/day in corn oil for 30 weeks by p.o. gavage 5 days/week. Cell proliferation in the proximal tubules was assessed via 5-bromo-2'-deoxyuridine-filled osmotic mini-pumps and immunohistochemistry after 7 weeks (2 weeks EHEN + 5 weeks dL) and at the end of the study (2 weeks EHEN + 30 weeks dL). Preneoplastic and neoplastic lesions were quantified in perfusion-fixed kidneys. A 5-fold increase in the labeling index of P2-cells was found after 5 weeks and 30 weeks of promotion in all dL-treated F344 rats, whereas no difference between treatment groups was detected in NBR rats. No increase in tumors or preneoplastic lesions was detected in dL-treated NBR rats, whereas a 10-fold increase in renal adenomas and atypical hyperplasias was found in the EHEN-dL-treated F344 rats compared with F344 rats treated with EHEN-corn oil. d-Limonene treatment alone caused a significant increase in the number of atypical tubules and atypical hyperplasias in F344 rats when compared with the F344 vehicle control. On the other hand, a significantly lower incidence of liver tumors was found in EHEN-dL-treated F344 rats compared with F344 rats treated with EHEN-corn oil, suggesting a chemopreventative effect of dL on EHEN-induced liver carcinogenesis in F344 rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ras-driven proliferation and apoptosis signaling during rat liver carcinogenesis is under genetic control

Fast growth and deregulation of G1 and S phases characterize preneoplastic and neoplastic liver lesions of genetically susceptible F344 rats, whereas a G1-S block in lesions of resistant BN rats explains their low progression capacity. However, signal transduction pathways responsible for the different propensity of lesions from the 2 rat strains to evolve to malignancy remain unknown. Here, we comparatively investigated the role of Ras/Erk pathway inhibitors, involved in growth restraint and cell death, in the acquisition of a phenotype resistant or susceptible to hepatocarcinogenesis. Moderate activation of Ras, Raf-1 and Mek proteins was paralleled in both rat models by strong induction of Dab2 and Rkip inhibitors. Levels of Dusp1, a specific ERK inhibitor, increased only in BN rat lesions, leading to modest ERK activation, whereas a progressive Dusp1 decline occurred in corresponding lesions from F344 rats and was accompanied by elevated ERK activation. Furthermore, a gradual increase of Rassf1A/Nore1A/Mst1-driven apoptosis was detected in both rat strains, with highest levels in BN hepatocellular carcinoma (HCC), whereas loss of Dab2IP, a protein implicated in ASK1-dependent cell death, occurred only in F344 rat HCC, resulting in significantly higher apoptosis in BN than F344 HCC. Taken together, our results indicate a control of the Ras/Erk pathway and the pro-apoptotic Rassf1A/Nore1A and Dab2IP/Ask1 pathways by HCC susceptibility genes. Dusp1 possesses a prominent role in the acquisition of the phenotype resistant to HCC by BN rats, whereas late activation of RassF1A/Nore1A and Dab2IP/Ask1 axes is implicated in the highest apoptosis characteristic of BN HCC.     

Stearoyl-CoA desaturase 1 (Scd1) gene overexpression is associated with genetic predisposition to hepatocarcinogenesis in mice and rats

The stearoyl-CoA desaturase 1 (Scd1) gene is involved in the synthesis and regulation of unsaturated fatty acids. Its expression is increased by several treatments/conditions that are associated with hepatocarcinogenesis (peroxisome proliferators, iron overload, dichloroacetic acid). We found that the Scd1 gene is differentially expressed, showing >10-fold higher mRNA levels in the normal liver tissue of C3H/He mice, which are genetically susceptible to hepatocarcinogenesis, than of BALB/c mice, which are resistant. Similarly, Scd1 mRNA expression was approximately 4-fold higher in the normal liver of F344 rats, which are susceptible to hepatocarcinogenesis, than in Brown Norway (BN) rats, which are resistant. The chromosomal location of the Scd1 locus, both in mice and rats, excludes Scd1 candidacy as a hepatocellular tumor-modifier gene, as the Scd1 locus did not show allele-specific effects in a BALB/cxC3H/He intercross or in a BNxF344 backcross and intercross. No Scd1 coding polymorphisms were detected in the mouse and the rat strains showing elevated Scd1 expression. These results suggest that the Scd1 gene represents a downstream target of hepatocellular tumor-modifier loci in two rodent species.     

Correlation of c-myc overexpression and amplification with progression of preneoplastic liver lesions to malignancy in the poorly susceptible Wistar rat strain.

Persistent liver nodules (PNs) and hepatocellular carcinomas (HCCs) induced in F344 rats by the resistant hepatocyte (RH) model exhibit c-myc overexpression and amplification. The role of these changes in progression of PN was investigated in nodules with different propensities to evolve to HCC in resistant Wistar rats and, for comparison, in susceptible F344 rats. Initiation of rats with diethylnitrosamine was followed by selection with 2-acetylaminofluorene (AAF) plus partial hepatectomy (RH groups). Two additional Wistar rat groups received a second AAF treatment without (RH+AAF) and with a necrogenic dose of CCl4 (RH+AAF/CCl4) 15 d after selection. The number to liver ratio and volume of glutathione-s-transferase placental form-positive lesions were lower in the Wistar than the F344 RH groups 9 and 32 wk after initiation and increased after a second AAF cycle treatment with and without CCl4. DNA synthesis in glutathione-s-transferase placental form-positive lesions was low in Wistar RH group at 9 wk and was stimulated by additional AAF treatments. HCCs developed at 57-60 wk in F344 RH, Wistar RH+AAF, and RH+AAF/CCl4 rats. Tumor incidence and multiplicity were lower in RH+AAF rats than in RH+AAF/CCl4 and F344 rats. At 32 wk, PN exhibited c-myc overexpression that increased from RH to RH+AAF rats and to RH+AAF/CCl4 Wistar rats. This was associated with c-myc amplification in Wistar RH+AAF/CCl4 rats. These results showed correlation of c-myc overexpression and amplification with nodule propensity to progress to HCC in poorly susceptible Wistar rats and suggested a possible genetic mechanism for susceptibility to hepatocarcinogenesis. The experimental system used in this work may be a valuable tool for studies on molecular mechanisms underlying liver growth and tumorigenesis supported by c-myc overexpression.     

DNA adduct formation, cell proliferation and aberrant crypt focus formation induced by PhIP in male and female rat colon with relevance to carcinogenesis

2-Amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP) inducescolon tumors in male, but not female, F344 rats, We investigatedthe mechanisms leading to this difference by measuring the levelof PhlP-DNA adducts, the enhancement of cell proliferation andaberrant crypt focus (ACF) formation in colon mucosa. PhIP wasadministered in the diet at a level of 0.04% to both male andfemale F344 rats for 1–8 weeks. The level of DNA adductsin the colon mucosa was measured using the ³²P-postlabelingmethod. Four major PhlP-DNA adducts were detected in fairlyconstant proportions in all the animals examined. The levelof PhlP-DNA adducts in male and female rats was the same, indicatingno direct correlation between adduct levels and carcinogenesis.Labeling indices (LIs) were determined by measuring BrdU incorporationin rats after feeding with a PhIP diet for 4, 8 and 12 weeks.After 8 weeks administration the LI had increased 1.5-fold inthe colon of the male rats, but no increase was observed inthe female rats. ACF formation was examined after feeding witha PhIP diet for 14 weeks. The number of aberrant crypt fociwas 6.6 ± 1.5 per rat in males and 1.9 ± 0.5 perrat in females. Thus differences in colon tumor developmentin male and female rats takes place at an early stage(s). Ourresults suggest that, in addition to DNA adduct formation, enhancedproliferation contribites to the formation of ACFs, which arepremalignant lesions of the colon.     

The Edpm5 locus prevents the 'angiogenic switch' in an estrogen-induced rat pituitary tumor

Edpm5 is one member of a group of quantitative trait loci that are responsible for the difference in susceptibility to estrogen-induced prolactinoma between the Fischer 344 (F344) and Brown Norway (BN) strains. Upon chronic estrogen treatment F344 rats develop large, hemorrhagic and invasive pituitary tumors, which exhibit both tumor angiogenesis and neoplasia. In contrast, BN rats do not develop a tumor despite an estrogen-induced increase in lactotroph density. To investigate the role of Edpm5 in the development of these tumors, we have generated a novel congenic rat strain F344.BN-Edpm5BN by introgressing the segment of rat chromosome bearing Edpm5 from BN into the F344 strain background. Phenotypic differences between F344 and F344.BN-Edpm5BN must be due to a gene(s) within the chromosomal interval encompassing Edpm5. Through use of these strains, we find that Edpm5 specifically regulates the switch to angiogenic phenotype, independent of neoplasia. The F344.BN-Edpm5BN rats developed tumors, which exhibited significant growth, 7-fold greater mass than the pituitary of untreated rats, and neoplasia indistinguishable from that of the F344 strain. However, the F344.BN-Edpm5BN rat tumor had a non-angiogenic phenotype. After chronic estrogen treatment, there was no increase in microvessel count over untreated controls in F344.BN-Edpm5BN tumors, whereas F344 rat tumors showed a significant increase (P < 0.0005). The ultrastructural morphology of the pituitary blood vessels also did not show significant angiogenesis associated changes in F344.BN-Edpm5BN rat pituitary tumors. In contrast the parental strain F344 had pronounced angiogenic activity. The F344.BN-Edpm5BN strain also fails to express VEGF at the high levels seen in the F344 rat pituitary after estrogen treatment. Hence at least one gene that has a large impact, directly or indirectly, on the switch to angiogenic phenotype must reside within the chromosomal interval that is the Edpm5 quantitative trait locus.     

Genetic properties for the suppression of development of putative preneoplastic glutathione S-transferase placental form-positive foci in the liver of carcinogen-resistant DRH strain rats

The post-initiation stage of hepatocarcinogenesis was investigated in carcinogen-resistant inbred DRH rats and the parental strain, carcinogen-sensitive Donryu rats. Male rats at 5 weeks of age from both strains were treated with N-nitrosodiethylamine (200 mg/kg i.p.) followed by feeding with a diet containing 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB) from 2 weeks later and were then subjected to partial hepatectomy at 1 week later. At 8 weeks after the start of treatment, the mean area occupied by glutathione S-transferase placental form (GST-P)-positive lesions was about 30% in Donryu rats but less than 4% in DRH rats despite the presence of comparable numbers of foci in the livers of both strains. These observations suggested that clonal expansion of GST-P-positive foci in DRH rat liver was significantly suppressed under these conditions. Furthermore, this genetic property was dominantly inherited in the Fl rats by crosses of DRH and carcinogen-sensitive inbred F344 rats; that is, the induction of GST-P mRNA in the livers of F344×DRH Fl rats was dominantly suppressed after administration of 3′-Me-DAB for 8 weeks as compared with parental F344 rats under the same conditions. We compared the intrinsic properties related to growth potential of liver cells between adult DRH and Donryu rats. DRH rat liver showed retarded and/or reduced DNA synthesis after partial hepatectomy or a single i.v. injection of lead nitrate and lower activity of telomerase induced by 3′-Me-DAB administration for 1 week, as compared with the Donryu rat liver. The intrinsic properties observed in this study may be related, at least in part, to the low incidence of liver tumors induced by hepatocarcinogens in DRH rats.     

Low-dose carcinogenicity of a heterocyclic amine, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline: relevance to risk assessment

Male, 21-day-old, F344 rats were administered 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in the diet at various low doses and a high dose, 100 ppm for 16 weeks. Quantitative values for glutathione-S-transferase placental form (GST-P)-positive foci in their livers were similar among the 0, 0.001, 0.01, 0.1 and 1 ppm MeIQx group while 10 ppm MeIQx administration slightly and 100 ppm MeIQx significantly increased their numbers. These results indicate that MeIQx has a no-observed effect level for induction of preneoplastic lesions in rats. Transplacental and trans-breast milk exposure to low doses of MeIQx also did not exert carcinogenic potential in F344 rats and 20% of calorie restriction clearly inhibited development of GST-P-positive foci. The results are of direct significance to human risk assessment.     

Resistance to the promotion of glutathione S-transferase 7-7-positive 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 protocol. The objective of the current study was to establish the time course for the development of resistance and examine potential resistance mechanisms in Cop rats using F344 rats as susceptible controls. Male Cop and F344 rats (n = 25), 7-8 weeks of age, were initiated with diethylnitrosamine (200 mg/kg) and promoted 3 weeks later with four doses of 2-acetylaminofluorene (20 mg/kg) and a 2/3 partial hepatectomy (PH). Groups of rats from each strain were killed on days 2, 4, 7, 14 and 21 post-PH, 2 h after receiving bromodeoxyuridine. Cop livers contained similar numbers of GST 7-7-positive lesions to F344 livers on days 2 and 4 post-PH. The percent volume of liver occupied by these lesions did not differ between the strains on days 2, 4 and 7 post-PH. On day 14, however, approximately 29% of the liver volume in F344 rats was occupied by lesions, whereas in Cop rats this was significantly less (approximately 9%, P < 0.001). On day 21, lesions occupied approximately 58% of F344 rat livers and only approximately 6% of Cop livers. Despite these differences, the labeling index of hepatocytes was not significantly different between the strains at any time point, either within lesions or within surrounding normal liver. Furthermore, the apoptotic indices were not different between the strains at any time. However, differences were found in the extent of lesion remodeling (redifferentiation) and in the pattern of oval cell response following PH in Cop livers. By day 14 post-PH, approximately 76% of Cop liver lesions showed evidence of remodeling, compared with only approximately 14% of F344 lesions. The oval cell response to PH was equivalent in the two strains up to day 4 post-PH but by day 7, in F344 livers there was extensive migration of these cells into the liver parenchyma, whereas in Cop livers, the response remained localized to the portal regions. These results suggest that Cop resistance occurs at the promotion stage and not the initiation stage of carcinogenesis. Resistance appears not to be due to a lower proliferation rate nor to a higher apoptotic rate within Cop lesions. Precocious remodeling and/or a diminished oval cell response, however, may contribute to the resistance of Cop rats to the growth of GST 7-7-positive hepatic lesions.     

High susceptibility of the ACI and spontaneously hypertensive rat (SHR) strains to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) prostate carcinogenesis

Carcinogenic responses in the prostate to 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP) were compared among seven rat strains (F344, ACI, Spontaneously Hypertensive Rat (SHR), Sprague-Dawley (SD), Wistar, Lewis and Brown Norway (BN)). Ten-week-old animals of each strain were given PhIP at 400 ppm in the diet for 20 weeks then maintained until week 54. The final survival rates were 92, 92, 83, 75, 67, 42 and 42%, respectively, and the SHR strain showed the highest sensitivity with regard to development of prostatic intraepithelial neoplasias (PINs) in the ventral prostate. With regard to the induction of adenocarcinomas of the ventral prostate, the ACI strain was most sensitive, whereas Lewis and F344 rats were relatively resistant. No adenocarcinomas were found in the dorsolateral or anterior prostate or seminal vesicles in any of the strains. The levels of serum testosterone and estrogen, PhIP-DNA adducts and cell kinetics did not correlate with the development of ventral prostatic lesions and thus other factors are presumably responsible for the variations in susceptibility. The present data indicate that ACI and SHR rats are appropriate strains for experimental investigation of PhIP-induced prostate carcinogenesis.     

Resistance of Copenhagen rats to hepatocarcinogenesis does not involve T-cell immunity

Previously, we have shown that Copenhagen (Cop) rats are highly resistant, compared with susceptible F344 rats, to the growth of glutathione S-transferase 7-7 (GST 7-7) positive preneoplastic liver lesions following treatment with a modified resistant hepatocyte (RH) protocol. Donryu rats, a strain with a level of susceptibility similar to F344, have a reduced T-cell response compared with the closely related, but highly resistant, DRH rat. Cop and DRH rats share several characteristics in their resistance to preneoplastic liver lesion growth and this study, therefore, was designed to examine whether T-cells play a role in Cop resistance. Cop rats were crossed with an athymic (nude) rat to produce F1s that were then interbred to produce F2 animals, some of which were nude with a partial Cop background. A comparison of the susceptibility of nude F2 animals and their euthymic (non-nude) littermates allowed us to determine what role, if any, T-cells play in Cop resistance. We treated 11 Cop, 11 F344, 19 nude F2s, and 18 non-nude F2s with diethylnitrosamine (DEN), followed 3 weeks later by a modified RH protocol. As expected, F344 rats were highly susceptible, having 41.9 +/- 3.3% (mean +/- SEM) of their liver section areas occupied by GST 7-7-positive lesions and Cop rats were highly resistant, having only 4.7 +/- 1.1% of their liver section areas occupied by lesions. Both nude and non-nude F2s were, like Cop rats, highly resistant (1.8 +/- 0.29 and 2.7 +/- 0.45%, respectively). These results show that T-cells are unnecessary for Cop rat resistance, or only play a minor role, and that the nude parental strain is also likely to be resistant to the growth of preneoplastic liver lesions.     

Decreased expression of Bcl-x protein during hepatocarcinogenesis induced exogenously and endogenously in rats.

Dysregulations of apoptosis have been widely recognized as important events in multi-stage carcinogenesis. Bcl-x, a member of the Bcl-2 family, is known to act as a regulator of apoptosis. The present study was conducted to assess the role of altered Bcl-x protein expression in exogenous and endogenous hepatocarcinogenesis in rats. In the short-term exogenous models, male Fischer 344 rats, 6 weeks old, were given a single intraperitoneal injection of diethylnitrosamine (DEN) at a dose of 200 mg / kg body weight, partially hepatectomized at the end of week 3, administered phenobarbital at a concentration of 0.05% from the end of week 2 for 6 weeks, and sacrificed. In the livers, glutathione S-transferase (GST-P)-positive, putative preneoplastic lesions were induced, and Bcl-x protein expression was decreased in 24.7% of such lesions. The incidence of GST-P-positive lesions with decreased Bcl-x increased depending on the size of the lesions; 18.9%, 32.4% and 86.5% in the lesions smaller than 0.03, between 0.03 and 0.3, and larger than 0.3 mm(2), respectively. In GST-P-positive lesions larger than 0.3 mm(2), both apoptosis induction and cell proliferation activity were enhanced when Bcl-x protein expression was decreased. In the long-term exogenous models, rats were given 10 mg / kg of DEN, partially hepatectomized 4 h after treatment, administered 0.5 mg / kg of colchicine at the end of days 1 and 3, subjected to a selection procedure, and sacrificed at the end of week 45. Hepatocellular carcinomas were induced with the decreased Bcl-x protein expression. In the endogenous model, rats were fed a choline-deficient, L-amino acid-defined diet for 16 or 80 weeks and sacrificed. Bcl-x protein expression was decreased both in GST-P-positive lesions and hepatocellular carcinoma. These results suggest that this decrease of Bcl-x protein might serve as an indicator of the advanced form of preneoplastic lesions, and that this decrease could also be associated with a potential to progress into carcinoma in both exogenous and endogenous hepatocarcinogenesis of rats.     

Strain Differences in N-Butyl-N-(4-hydroxybutyl)nitrosamine Bladder Carcinogenesis in Rats

Differences in susceptibility of the urinary bladder epithelium to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in various strains were examined. In experiment 1, 5 strains of male rats were given 0.025% BBN in the drinking water for 8 weeks followed by drinking water without BBN for 32 weeks. Analbuminemic rats (NAR) and ACI rats had high incidences of urinary bladder lesions (papillary or nodular hyperplasia, papilloma and carcinoma), F344 and Wistar rats had low incidences, and Sprague-Dawley (SD) rats showed an intermediate incidence. Carcinoma area was largest in NAR rats followed in decreasing order by SD, ACI and F344 rats. The extent of tumor invasion was higher in NAR and ACI rats than in SD rats. In experiment 2, the 5 strains of male rats were administered 0.025% BBN in the drinking water. Some rats from each group were killed after each of weeks 4 and 8. The urinary bladder of ACI and NAR rats given BBN had the most marked lesions observed by scanning electron microscopy, with less marked changes in SD rats. F344 and Wistar rats showed the weakest response. Cytochrome P-450 content of the liver in ACI rats treated with BBN for 4 weeks was significantly higher than those of the controls. Cytochrome P-450 and Cytochrome b ₅ contents of the control and BBN-treated rats were significantly higher in ACI and SD rats than in Wistar, F344 or NAR rats. These results indicate that there are strain differences in the urinary bladder response to BBN.     

Strain differences in N-butyl-N-(4-hydroxybutyl)nitrosamine bladder carcinogenesis in rats

Differences in susceptibility of the urinary bladder epithelium to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in various strains were examined. In experiment 1, 5 strains of male rats were given 0.025% BBN in the drinking water for 8 weeks followed by drinking water without BBN for 32 weeks. Analbuminemic rats (NAR) and ACI rats had high incidences of urinary bladder lesions (papillary or nodular hyperplasia, papilloma and carcinoma), F344 and Wistar rats had low incidences, and Sprague-Dawley (SD) rats showed an intermediate incidence. Carcinoma area was largest in NAR rats followed in decreasing order by SD, ACI and F344 rats. The extent of tumor invasion was higher in NAR and ACI rats than in SD rats. In experiment 2, the 5 strains of male rats were administered 0.025% BBN in the drinking water. Some rats from each group were killed after each of weeks 4 and 8. The urinary bladder of ACI and NAR rats given BBN had the most marked lesions observed by scanning electron microscopy, with less marked changes in SD rats. F344 and Wistar rats showed the weakest response. Cytochrome P-450 content of the liver in ACI rats treated with BBN for 4 weeks was significantly higher than those of controls. Cytochrome P-450 and cytochrome b5 contents of the control and BBN-treated rats were significantly higher in ACI and SD rats than in Wistar, F344 or NAR rats. These results indicate that there are strain differences in the urinary bladder response to BBN.     

Enhancement of glutathione S-transferase placental-form positive liver cell foci development by microcystin-LR in aflatoxin B1-initiated rats

The objective of this study was to elucidate whether microcystin-LR(MC-LR), a hepatotoxic blue-green algal toxin in drinking water,is carcinogenic or possesses the ability to modulate aflatoxinB₁ (AFB₁)-induced hepatocarcinogenicity. In a medium-term liverbioassay, male Fischer 344 rats were given a single i.p. injectionof diethylnitrosamine (DEN, 200 mg/kg) followed by an i.p. injectionof MC-LR for 6 weeks after 2 weeks of DEN treatment. To studythe synergism between AFB₁ and MC-LR, DEN-treated rats weregiven an i.p. injection of AFB₁ (0.5 mg/kg) dissolved in dimethylsulfoxide (DMSO) followed by MC-LR at 2 weeks after the treatment.In a separate experiment, the rats were first given AFB₁ (0.5mg/kg) and 2 weeks later an i.p. injection of 1 or 10 µg/kgof MC-LR twice a week for 6 weeks. Most rats were subjectedto a two-thirds partial hepatectomy (PH) at week 3 and werekilled under anesthesia at week 8. Liver sections were analyzedfor glutathione S-transferase placental form (GST-P) expression,and subjected to histopathological examination for phenotypicalteration of hepatocellular foci. In rats that did not receiveDEN, MC-LR did not cause a significant increase in the numbersof GST-P-positive foci, whereas AFB₁ induced a slight increasein GST-P-positive foci development. In rats given DEN, MC-LRenhanced the expression of GST-P-positive foci, as did AFB₁but no synergism was observed. Histopathological analysis revealedthat the area of eosinophilic foci, a biomarker for preneoplasticliver lesion, markedly increased because of MC-LR. In rats givenAFB₁ as an initiator, treatment with MC-LR resulted in a synergisticincrease in the development of GST-P-positive foci. These resultssuggest that the hepatocarcinogenicities of MC-LR and AFB₁ canbe predicted in experimental animals with a medium-term bioassay.Furthermore, tumor promoting activity of MC-LR was demonstratedin rats treated with AFB₁.