Different physiology of interferon-α/-γ in models of liver regeneration in the rat.

Liver regeneration may take place after liver injury through replication of hepatocytes or hepatic progenitor cells called oval cells. Interferons (IFN) are natural cytokines with pleiotrophic effects including antiviral and antiproliferative actions. No data are yet available on the physiology and cellular source of natural IFNs during liver regeneration. To address this issue, we have analyzed the levels and biologic activities of IFN-α/IFN-γ in two models of partial hepatectomy. After 2/3rd partial hepatectomy (PH), hepatic levels of IFN-α and IFN-γ declined transiently in contrast to a transient increase of the IFN-γ serum level. After administration of 2-acetylaminofluorene and partial hepatectomy (AAF/PH model), however, both IFN-α and IFN-γ expression were up-regulated in regenerating livers. Again, the IFN-γ serum level was transiently increased. Whereas hepatic IFN-γ was up-regulated early (day 1-5), but not significantly, in the AAF/PH model, IFN-α was significantly up-regulated at later time points in parallel to the peak of oval cell proliferation (days 7-9). Biological activity of IFN-α was shown by activation of IFN-α-specific signal transduction and induction of IFN-α specific-gene expression. We found a significant infiltration of the liver with inflammatory monocyte-like mononuclear phagocytes (MNP) concomitant to the frequency of oval cells. We localized IFN-α production only in MNPs, but not in oval cells. These events were not observed in normal liver regeneration after standard PH. We conclude that IFN-γ functions as an acute-phase cytokine in both models of liver regeneration and may constitute a systemic component of liver regeneration. IFN-α was increased only in the AAF/PH model, and was associated with proliferation of oval cells. However, oval cells seem not to be the source of IFN-α. Instead, inflammatory MNP infiltrating AAF/PH-treated livers produce IFN-α. These inflammatory MNPs may be involved in the regulation of the oval cell compartment through local expression of cytokines, including IFN-α.

Global gene expression profiling reveals a key role of CD44 in hepatic oval-cell reaction after 2-AAF/CCl4 injury in rodents.

Liver progenitors, so-called oval cells, proliferate remarkably from periportal areas after severe liver injury when hepatocyte regeneration is compromised. These cells invade far into the liver parenchyma. Molecular mechanisms underlying these behaviors of oval cells remain poorly understood. In this study, we treated rats with 2-acetylaminofluorene/carbon tetrachloride to induce hepatic oval cells. By expression microarray analysis, we investigated global gene expression profiles in liver tissue, with an emphasis on adhesion molecules, extracellular matrix proteins, matrix metalloproteinases (MMPs), growth factors/cytokines, and receptors that might contribute to the distinct behaviors of oval cells. Genes upregulated at least twofold were selected. We then performed immunostaining to verify the microarray results and identified expression of MMP-7 and CD44 in oval cells. Staining of cytokeratin (CK)-19, an oval-cell marker, was similar between oval cells located next to periportal areas and those located far within the parenchyma. In contrast, CD44 staining was more intense in the parenchyma than in periportal areas, suggesting a role of CD44 in oval-cell invasion. Moreover, newly differentiated CK-19+ hepatocytes within foci did not show CD44 staining, suggesting that CD44 is related to the undifferentiated oval-cell phenotype. We then investigated oval-cell reactivity in CD44-deficient mice fed an oval cell-inducing diet of 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Results showed significantly reduced oval-cell reactivity in CD44-deficient mice. Thus, oval cells express MMP-7 and CD44, and CD44 appears to play critical roles in the proliferation, invasion, and differentiation of hepatic oval cells in rodents.

The tumor-promoting activity of 2-acetylaminofluorene is associated with disruption of the p53 signaling pathway and the balance between apoptosis and cell proliferation.

The aromatic amine 2-acetylaminofluore (2-AAF) is a powerful complete genotoxic rat liver carcinogen that induces tumors without any additional interventions. While the tumor-initiating genotoxic activity of 2-AAF is well established, its tumor-promotion activity is far less understood. It is believed that the tumor-promoting property of 2-AAF is associated with selective enhancement of cell replication and sustained suppression of apoptosis in initiated cells. In the present study, we investigated the underlying mechanisms of tumor-promoting events induced by 2-AAF-exposure. Male Sprague-Dawley rats were fed NIH-31 diet containing 0.02% of 2-AAF for 12 and 24 weeks, and the expression pattern of genes associated with the p53-signaling pathway and microRNA genes was determined in the livers of control and 2-AAF-fed rats. The results indicate that the tumor-promoting property of 2-AAF during hepatocarcinogenesis is associated predominantly with the up-regulation of anti-apoptotic growth-related genes and down-regulation of expression of pro-apoptotic genes. This disrupts the balance between cell proliferation and apoptosis, which leads to consequential unrestricted cell proliferation, especially of initiated cells. Also, the long-term-administration of 2-AAF resulted in disruption of regulatory miR-34a-p53 feed-back loop that mediates apoptosis. This was evidenced by an increased expression of miR-34a in response to genotoxic effects of 2-AAF in the absence of p53 up-regulation, and loss of regulatory control of mir-34a on SIRT1 function. Additionally, the livers of 2-AAF-exposed rats were characterized by the substantial deregulation of expression of miR-18, miR-21, miR-182, and miR-200 family, microRNAs involved in control of apoptosis/cell proliferation and cell-cell contact pathways, two major pathways disrupted during the promotion stage of hepatocarcinogenesis.

Low selection of preneoplastic hepatocytes after treatment with the 2-acetylaminofluorene diet-partial hepatectomy regimen in the liver of hepatocarcinogenesis-resistant DRH strain rats.

In hepatocarcinogenesis-resistant DRH rats, preneoplastic hepatocytic lesions are smaller than those of usual rats during carcinogenesis. When preneoplastic hepatocytes from DRH and Donryu (original strain of DRH) were reciprocally transplanted into the livers of DRH and Donryu treated with 2-acetylaminofluorene (2-AAF) diet/two-thirds hepatectomy (PH), the Donryu cells formed small colonies within the DRH liver, whereas the DRH cells formed large colonies within the Donryu liver. The DRH liver showed less degree of oval cell proliferation after treatment with 2-AAF and PH, and DRH hepatocytes were more resistant to the growth-inhibitory effect of 2-AAF after PH. Furthermore, DRH hepatocytes were generally resistant to cytotoxicity of hepatotoxins. The tissue environment of the DRH liver, therefore, is less effective for selective growth of preneoplastic hepatocytes during the carcinogen treatment, which is probably a major cause of the hepatocarcinogenesis-resistance in DRH rats.

2-AAF-induced tumor development in nucleotide excision repair-deficient mice is associated with a defect in global genome repair but not with transcription coupled repair.

The nucleotide excision repair (NER) pathway comprises two sub-pathways, transcription coupled repair (TCR) and global genome repair (GGR). To establish the importance of these separate sub-pathways in tumor suppression, we exposed mice deficient for either TCR (Csb), GGR (Xpc) or both (Xpa) to 300 ppm 2-acetylaminofluorene (in feed, ad libitum) in a unique comparative exposure experiment. We found that cancer proneness was directly linked to a defect in the GGR pathway of NER as both Xpa and Xpc mice developed significantly more liver tumors upon 2-AAF exposure than wild type or Csb mice. In contrast, a defect in TCR appeared to act tumor suppressive, leading to a lower hepatocellular tumor response in Xpa mice (tumor incidence of 25%) as compared to Xpc mice (53% tumor-bearing mice). The link between deficient GGR and tumor proneness was most pronounced in the liver, but this phenomenon was also found in the urinary bladder. As tumor induction by 2-AAF appeared almost exclusively dependent on a defect in GGR, we examined whether gene mutation induction in the non-transcribed lacZ locus could reliably predict tumor risk. Interestingly, however, short-term 2-AAF exposure induced lacZ mutant levels in Csb mice almost as high as those found in Xpa or Xpc mice. This indicates that lacZ mutant frequencies are not correlated with a specific DNA repair defect and eventual tumor outcome, at least not in the experimental design presented here.

Early induction of TGF-beta1 through a fasting-re-feeding regimen promotes liver carcinogenesis by a sub-initiating dose of diethylnitrosamine.

We previously reported that a sub-necrogenic dose (20 mg/kg) of diethylnitrosamine (DENA) can induce the development of liver cancer when rats undergo a fasting-re-feeding regimen. The present study was undertaken to establish whether fasting followed by re-feeding builds up mechanisms able to trigger liver fibrosis, eventually leading to cirrhosis and cancer. Adult male rats, for fasted 4 days, were given 20 mg/kg of DENA after 1 day of re-feeding; in parallel, consistently fed animals receiving 20 mg/kg (sub-necrogenic) or 200 mg/kg (necrogenic dose) of DENA were used as negative and positive controls, respectively. All three groups were then subjected to the 2-acetylaminofluorene/carbon tetrachloride promoting regimen. Fasting induced moderate apoptosis in liver tissue, as evidenced by increased levels of transforming growth factor-beta1 (TGF-beta1) and Bax proteins and by a dramatic drop in the level of Bcl-2. Subsequent re-feeding caused all changes to revert except TGF-beta1 up-regulation. Histological findings of inflammation and fibrosis were consistently associated with increased production of TGF-beta1, the inflammatory cytokine with the most pronounced profibrogenic action. Thus, up-regulation of TGF-beta1 expression appears as a major mechanism by which the fasting-re-feeding regimen predisposes to initiation and promotion of liver carcinogenesis in rats. Avoiding fasting-re-feeding could be considered in the nutritional status of patients with liver fibrosis.

Association between cage shelf level and spontaneous and induced neoplasms in mice.

Data from a chronic feeding study with 2-acetylaminofluorene [(2-AAF) CAS: 53-96-3; N-fluoren-2-yl-acetamide] done on 20,880 female BALB/c mice were analyzed for associations between cage shelf level and occurrence of induced and spontaneous neoplasms. Each cage was maintained on a rack at a given shelf level throughout the experiment, allowing analysis of data by shelf level. Differences in the crude incidence of 2-AAF-induced liver and bladder neoplasms appeared to be shelf related, but these differences were small and disappeared when shelf-level analyses of time-to-tumor onset distributions were performed. There was evidence for a shelf-level influence on 5 of the 6 major spontaneous neoplasms noted. Time to onset of uterine polyps and reticulum cell sarcomas was significantly delayed on the top shelf of five of six animal rooms. Also, there was a significant delay in onset of lymphomas, adrenocortical adenomas, and lung alveolar cell tumors on the top shelf when data were combined from all six animal rooms, but these delays on the top shelf were significant in no more than two of six animal rooms when rooms were analyzed separately. There was no indication of any shelf-level influence on the development of harderian gland adenomas. In conclusion, shelf level is an environmental factor that should be considered in the design and analysis of carcinogenesis studies.

Kupffer cell sarcoma in rats after exposure to small doses of dimethylnitrosamine and N-2-acetylaminofluorene during hepatic regeneration.

During experiments to study the evolution of hepatocellular carcinoma in adult male Wistar rats by exposing regenerating livers to the action of small doses of dimethylnitrosamine (DMN) or N-2-acetylaminofluorene (AAF), several primary sarcomas of the liver were incidentally observed. The morphology and behavior of the tumors suggest their origin from Kupffer's cells. Kupffer cell sarcomas occurred more frequently when 70% hepatectomy was used as the regenerative stimulus. None of the 36 animals treated with AAF alone and 2 of the 38 rats given DMN only had this tumor.

Enhancement of rat hepatocellular-altered foci by the liver tumor promoter phenobarbital: evidence that foci are precursors of neoplasms and that the promoter acts on carcinogen-induced lesions.

Altered foci resistant to iron accumulation were induced by N-2-fluorenylacetamide in livers made siderotic by feeding 8-hydroxyquinoline and ferrous gluconate to inbred F344 rats. Following cessation of carcinogen feeding, most foci reverted to iron accumulation and could no longer be detected by 24 weeks, but some persisted for this interval. Futhermore, in 2 groups, 33 and 50% of rats developed liver tumors by 24 weeks after removal of the carcinogen. The addition of phenobarbital to the diet after cessation of carcinogen feeding enhanced persistence of altered foci and increased the incidence of liver tumors to 78--89%.

Quantitative kinetics of development of N-2-fluorenylacetamide-induced, altered (hyperplastic) hepatocellular foci resistant to iron accumulation and of their reversion or persistence following removal of carcinogen.

For study of the behavior of previously described carcinogen-induced foci of hepatocytes refractory to iron accumulation, iron deposition was induced in all hepatocytes of rat livers by feeding 8-hydroxyquinoline and ferrous gluconate to the animals for 3 months. The kinetics of development of the altered foci of hepatocytes free of stainable iron was then quantified for each lobe of liver for as long as 13 weeks of feeding of the carcinogen N-2-fluorenylacetamide and for 6 months following cessation of carcinogen exposure. The foci increased slowly in number and size for the first 12 weeks of carcinogen administration and then grew rapidly between weeks 12 and 13. At week 13, they corresponded to altered foci that have been observed in sections stained with hematoxylin and eosin, and they also displayed abnormal glycogen storage. After the carcinogen was discontinued, the foci disappeared by a reversion back to iron storage while temporarily retaining the nuclear abnormalities of altered cells. Over 95% of altered foci at this stage underwent reversion by 6 months following removal of the carcinogen. More advanced lesions developed in the lobes that displayed the greatest incidence and persistence of foci.

Neoplasms of urinary bladders of hamsters treated with 2-acetylaminofluorene and indole.

Tested in hamsters was the effect of neonatal treatment with 2-acetylaminofluorene (AAF) followed by prolonged feeding in combination with indole on the development of bladder tumors. Neonatal males and females were given intraperitoneal injections of AAF, 5 mg/100 g body weight, 3 times weekly until weaning. They were then fed a synthetic diet containing 0.06% AAF and 1.6% indole. The 26 hamsters surviving 10-12 months developed transitional cell carcinomas of the bladder, all but 2 of which were invasive. No hamsters had malignant tumors in the liver. Seven hamsters developed peliosis of the spleen.

Effects of route of administration on tissue distribution of DNA adducts in mice: comparison of 7H-dibenzo(c,g)carbazole, benzo(a)pyrene, and 2-acetylaminofluorene.

The environmental pollutant 7H-dibenzo(c,g)carbazole (DBC) has been shown to be a potent carcinogen in various mouse tissues, but displays an unusual degree of hepatocarcinogenicity. We have previously reported that in accord with this activity, mouse liver is the target organ for DBC-DNA binding, with total levels being up to 2700 times greater than in extrahepatic tissues after s.c. administration. To elaborate on this finding, we have directly compared the tissue distribution of DNA damage by three diverse aromatic carcinogens, DBC, benzo(a)pyrene (BP), and 2-acetylaminofluorene (AAF). Following a single topical, p.o., or s.c. administration of 80 mumol/kg of test compound to male BALB/c mice, a 32P-postlabeling assay showed the total number of DBC adducts in liver DNA to be 11-138 times that in kidney, lung, or skin DNA. The degree of hepatic adduction varied as a function of the route of administration, with the highest occurring after topical application and the lowest after s.c. injection. The tissue preference for AAF and BP adducts varied with the route of administration and was much less than for DBC adducts, except that topical application of BP gave DNA adduct levels in skin that were 91-218 times greater than in other tissues. For a given tissue and route of administration, DNA adduction by DBC was 1.7- to 950-fold greater than that by BP and AAF, except in skin where the level of DNA adducts from BP was 3 to 4 times that from DBC. We conclude that (a) DBC exhibits an exceptional and unique preference for liver DNA adduction after different routes of administration; (b) DBC is more potent overall than BP or AAF in causing tissue DNA damage; and (c) for each of the three carcinogens, the route of exposure is a much less important factor than the nature of the carcinogen in determining the tissue distribution of covalent DNA damage.

Enzyme deviation patterns in primary rat hepatomas induced by sequential administration of two chemically different carcinogens.

Enzyme deviation patterns were examined in primary rat hepatomas induced by short-term sequential administration of two chemical carcinogens from among 2-fluorenylacetamide (FAA), diethylnitrosamine (DENA), and 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) or by FAA or 3'-Me-DAB followed by phenobarbital as a promoter. The purpose was to discern how the patterns are influenced by different administration schedules of carcinogens and which of the two carcinogens in the sequence affects the pattern more. Biochemical differentiation of hyperplastic hepatic nodules and hepatomas was determined by simultaneous assays of activities and isozyme composition of glucose-adenosine triphosphate phosphotransferase, pyruvate kinase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, and gamma-glutamyltransferase with consideration of histological classification of nodules and tumors. Poorly differentiated hepatomas were predominantly induced by 3'-Me-DAB followed by FAA or DENA except for hepatomas induced by 3'-Me-DAB followed by phenobarbital, which were mainly well and moderately differentiated; well and moderately differentiated hepatomas were predominantly induced by FAA followed by 3'-Me-DAB or phenobarbital. The degree of enzyme deviation of the hepatomas induced by DENA as the first carcinogen was intermediate between those of hepatomas induced by FAA or 3'-Me-DAB, although the degree tended to increase with increased dose or term of DENA. These results indicate that deviations of some enzymes, such as pyruvate kinase and fructose-1,6-bisphosphatase, as well as histological differentiation of the primary hepatomas are more strongly influenced by the first carcinogen than by the second under our administration schedules and that the degree of enzyme deviation shown by hepatomas produced by a particular carcinogen treatment regimen principally related to the potential of that regimen to induce the more anaplastic tumors.

Promotion of liver cancer development by brief exposure to dietary 2-acetylaminofluorene plus partial hepatectomy or carbon tetrachloride.

Adult male Fischer rats were exposed to a necrogenic dose (200 mg/kg) of diethylnitrosamine or to nonnecrogenic doses of N-methyl-N-nitrosourea, 1,2-dimethylhydrazine, or benzo(a)pyrene following partial hepatectomy or sham hepatectomy. This treatment by itself led to no hepatocellular carcinomas by 8 to 18 months, except in animals given N-methyl-N-nitrosourea, which showed a 30% incidence by 12 months. With each treatment regimen, exposure to dietary 2-acetylaminofluorene for 2 weeks coupled with partial hepatectomy or the administration of a necrogenic dose of CCl4, was associated with an incidence of 68 to 94% of cancer at 8, 12, or 18 months, depending upon the initiating carcinogen used. Appropriate controls showed either no hepatocellular carcinoma or a much lower incidence. It is concluded that the 2-week exposure to dietary 2-acetylaminofluorene plus partial hepatectomy or the administration of CCl4 has a strong promoting effect on liver carcinogenesis with four different chemical carcinogens.

Nacroautoradiographic assays in pregnant mice and their fetuses given N-acetyl-9-[14C]-2-aminofluorene and p-[14C]-dimethylaminoazobenzene.

The distribution of radioactivity from N-acetyl-9-[114C]-2-aminofluorene and p-[14C]-dimethylaminoazobenzene administered i.v. or p.o. to 18-day-pregnant mice was observed by means of autoradiography of longitudinal section of the whole mouse. Radioactive substances derived from these carcinogens passed through the placenta and were distributed in the fetal organs including the kidney and intestine. Column, thin-layer, and paper chromatography revealed the presence, in while fetuses as well as in maternal livers, of N-acety-9-[14C]-2-aminofluorene. N-hydroxy-N-acetyl-9-[14C]-2-aminofluorene, ring-hydroxy-N-acetyl-9-[14C]-2-aminofluorene, and p-[14C]-dimethylaminoazobenzene. These results establish that N-acetyl-9-[14C]-2-aminofluorene and [14C]-p-dimethylaminoazobenzene are transported, metabolized, and excreted in the mouse fetus.

Modulation by butylated hydroxytoluene of liver and bladder carcinogenesis induced by chronic low-level exposure to 2-acetylaminofluorene.

The modulating effect of five dose levels of butylated hydroxytoluene (BHT) on liver and bladder carcinogenesis induced in rats by concurrent exposure to 2-acetylaminofluorene (AAF) was investigated. AAF at a low dose of 50 ppm was fed simultaneously with concentrations of 100, 300, 1000, 3000, or 6000 ppm BHT in the diet to male F344 rats for up to 76 weeks. By 12 weeks, AAF alone induced altered hepatocellular foci, identified by iron storage deficiency and gamma-glutamyltranspeptidase activity. At subsequent time points of 24, 36, and 48 weeks, the number of foci progressively increased, and at the end of the study, the incidence of liver neoplasms was 100%, a new finding with such a low dose of AAF. Simultaneous feeding of BHT inhibited the induction of liver altered foci by AAF in a dose-related manner and reduced the incidence of hepatocellular carcinomas and the number of liver neoplasms per animal. Feeding of 6000 ppm BHT, but not of lower doses, together with AAF resulted in an increase in the incidence and multiplicity of bladder neoplasms, and 3000 ppm increased nodular hyperplasia of the bladder. These results suggest that the chemoprevention by BHT of cancer resulting from low-level long-term carcinogen exposure may be achieved at doses that do not produce adverse effects.

Comparisons of thresholds for carcinogenicity on linear and logarithmic dosage scales.

Comparisons on a linear and the Rozman logarithmic scale for dosage versus carcinogenicity in rodents are presented for methyl eugenol (ME), nitrosodiethylamine (NDEA), ethyl carbamate (EC) and 2-acetylaminofluorene (AAF). Each of these chemicals has been shown to be carcinogenic in experimental animals and, in addition, humans are regularly exposed to at least three of these compounds (ME, NDEA, EC) in foods. Although the source of adducts from AAF is not known, the aminofluorene (AF) adduct is present in humans. Plotted on the same graphs are either some doses from common foods (ME, NDEA, EC) or adducts (AF) on human haemoglobin, for perspective, with their thresholds for carcinogenesis in animals. Use of a linear scale when comparing doses administered to animals in studies of carcinogenicity with doses of those same chemicals to which humans are exposed does not provide useful, comparative information. On the other hand, the Rozman logarithmic scale for dose allows one to put these relative doses in perspective. It is also evident that forcing a linear extrapolation through the zero, zero origin does not agree with the experimental data. Further analyses for goodness of fit for these dose responses reveal that the dose response for three of these compounds (ME, NDEA, EC) appears to be linear with the logarithm of the dose. However, AAF appears to be linear with the logarithm of the dose for bladder, but not for liver. It is suggested that the high background incidence of tumours in the BALB/c StCrlfC3Hf/Nctr mouse liver may confound the interpretation of dose response from AAF carcinogenesis in mouse liver.

Induction of an altered lipid phenotype by two cancer promoting treatments in rat liver.

Changes in lipid metabolism have been associated with tumor promotion in rat liver. Similarities and differences of lipid parameters were investigated using the mycotoxin fumonisin B1 (FB1) and the 2-acetylaminofluorene/partial hepatectomy (AAF/PH) treatments as cancer promoters in rat liver. A typical lipid phenotype was observed, including increased membranal phosphatidylethanolamine (PE) and cholesterol content, increased levels of C16:0 and monounsaturated fatty acids in PE and phosphatidylcholine (PC), as well as a decrease in C18:0 and long-chained polyunsaturated fatty acids in the PC fraction. The observed lipid changes, which likely resulted in changes in membrane structure and fluidity, may represent a growth stimulus exerted by the cancer promoters that could provide initiated cells with a selective growth advantage. This study provided insight into complex lipid profiles induced by two different cancer promoting treatments and their potential role in the development of hepatocyte nodules, which can be used to identify targets for the development of chemopreventive strategies against cancer promotion in the liver.

Changes in rat liver N-hydroxy-2-acetylaminofluorene aryl sulfotransferase activity at early and late stages of hepatocarcinogenesis resulting from dietary administration of 2-acetylaminofluorene.

The ability of 2-acetylaminofluorene (AAF) to mediate a loss in N-hydroxy-AAF (N-OH-AAF) aryl sulfotransferase activity when fed to male Sprague-Dawley rats was examined at early and late stages of hepatocarcinogenesis. Administration of 0.05% AAF in the diet for 1 week caused liver N-OH-AAF aryl sulfotransferase activity to decrease to 15 +/- 5% of that for liver from non-carcinogen-fed rats, and the activity remained low throughout 19 weeks of AAF feeding. When rats were fed AAF diet for 3 weeks, then placed on a control diet, liver N-OH-AAF aryl sulfotransferase activity returned to normal levels within 3 weeks. In contrast, when rats were fed AAF for 19 weeks, then placed on control diet for an additional 10 weeks, little or no recovery of N-OH-AAF aryl sulfotransferase activity was observed in cytosols from whole livers or isolated hyperplastic nodules, respectively. These findings suggest two types of AAF-mediated decreases in sulfotransferase activity: (a) a decrease observed early in the initial stages of AAF feeding which returns to normal levels when AAF is removed from diet, and (b) a persistent decrease in activity following long term AAF administration.

High sensitivity of the LEC rat liver to the carcinogenic effect of diethylnitrosamine.

The initiation sensitivity of the liver of the LEC (Long-Evans with a cinnamon-like coat color) rat, a new mutant strain with a high incidence of spontaneous liver tumors, was studied by treatment with low doses of diethylnitrosamine (DEN) coupled with modified Solt and Farber's selection. LEC and control LEA (Long-Evans with an agouti coat color) rats received i.p. injections of 10 mg/kg of DEN, then selected by feeding with a diet containing 0.02% 2-acetylaminofluorene (AAF) for 7 days combined with partial hepatectomy (PH). The numbers of placental glutathione S-transferase (GST-P)-positive foci in the livers of LEC rats were 10 times higher than those in LEA rats. These results suggested a high sensitivity of the LEC rat liver to the carcinogenic effect of DEN. The association between initiation sensitivity and spontaneous liver-tumor development and the possible usefulness of the LEC rat for in vivo short-term tests of hepatocarcinogens are discussed.