Xenobiotics Modulate the p53 Response to DNA Damage in Preneoplastic Enzyme-Altered Foci in Rat Liver; Effects of Diethylnitrosamine and Phenobarbital

Niklas Finnberg*, Ulla Stenius*,1 and Johan Högberg{dagger}

* Occupational Toxicology Group, Institute of Environmental Medicine, Karolinska Institutet, Box 210, S-171 77 Stockholm, Sweden; and {dagger} National Institute of Working Life, S-171 84 Solna, Sweden

Received July 16, 1999; accepted October 26, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Enzyme-altered foci (EAF) develop in rat liver in response to carcinogen treatment. Our hypothesis is that EAF adapt to genotoxic stimuli by lowering their expression of p53 and that such decreased p53 expression confers a growth advantage on the hepatocytes present in EAF. After a single neonatal dose of diethylnitrosamine (DEN), rats were treated with either 2 – 12 additional doses of DEN or phenobarbital (PB) for 3 – 14 months. Twenty-four hours prior to sacrifice, all rats also received a challenging dose of DEN. The numbers of p53-positive hepatocytes (demonstrating immunohistological staining in the nucleus) in EAF and surrounding tissue were subsequently determined. In DEN-treated rats, p53 expression was attenuated in EAF compared to surrounding tissue. The longer the period of treatment and the larger the size of the EAF, the fewer the p53-positive hepatocytes/mm2 were observed in these lesions. These data were confirmed by Western blot analysis. PB-treated rats did not demonstrate this effect seen in DEN-treated rats. In this case, the expression of p53 was not related to size of EAF or length of treatment. Many EAF in PB-treated animals contained very large numbers of p53-positive cells. Upon staining for terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling (the TUNEL procedure), many apoptotic hepatocytes were also seen in EAF. These data indicate that the p53 response to DNA damage can be modulated by xenobiotics. This can be explained as an adaptive alteration in the p53 response.

Key Words: p53; preneoplastic enzyme-altered foci; diethylnitrosamine; phenobarbital; adaptation; genotoxicity; dose response; carcinogenesis; risk assessment.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Quantitative risk assessment of chemical carcinogens is often hampered by a lack of specific mechanistic information (Davis and Farland, 1998Go). Conventionally, carcinogens are often assigned to broad categories of substances with a presumed common mode of action, e.g., "genotoxic" and "non-genotoxic" carcinogens (Ames et al., 1993Go). However, such categorization is usually based on in vitro tests or short-term animal experiments, models that provide relatively limited information. The development of a tumor is a multi-step process that takes place over an extended period of time (Vogelstein and Kinzler, 1993Go), and in cancer tests, the tested chemical is usually administered for 2 years. During this extended time period, a test substance may induce many types of primary and adaptive responses in a complex series of events. One simple example of this is that a genotoxic compound may evoke tumor-promoting effects at the same time it induces DNA damage.

Phenobarbital (PB) and diethylnitrosamine (DEN) belong to the relatively small group of carcinogens that have been thoroughly investigated. Both PB and DEN induce hepatocellular carcinomas, which is one of the most common endpoints in cancer tests. PB is a non-genotoxic carcinogen and has often been used as a tumor promoter (Peraino et al., 1971Go). The details of its molecular mechanism of action are still largely unknown, but PB may affect cell proliferation (Mansbach et al., 1996Go) or decrease rates of apoptosis (Osanai et al. 1997Go; Schulte-Hermann et al., 1981Go, 1990Go).

DEN is considered to be a genotoxic carcinogen (Lewis et al., 1997Go), since it is positive in tests for genotoxicity and can induce many types of DNA damage by alkylation and other mechanisms (Nakae et al., 1997Go). It is often assumed that DEN initiates and propagates tumor development primarily by inducing DNA alterations that lead to mutations (Peto et al., 1991Go). Indeed, indicative mutations in the ras gene have been observed in mouse liver tumors arising in response to DEN treatment (Stowers et al., 1988Go). However, like many other genotoxic compounds, DEN may also induce cytotoxicity and apoptosis. A progressive increase in replication of hepatocytes has been observed in response to increasing doses of DEN (Deal et al., 1989Go).

Carcinogenesis in rodent liver is characterized by initiation and clonal growth of enzyme-altered foci (EAF) (Pitot, 1990Go). These lesions can be regarded as precursors of tumors. Ample data indicate that EAF formation is initiated by inheritable genetic changes (Dragan, et al., 1994Go). However, the altered phenotypes of EAF confer resistance to many types of toxic stress and it has been suggested that EAF are adaptive alterations (Farber, 1992Go).

In an effort to understand the primary toxic effects caused by carcinogens, as well as adaptations to these toxic effects, we have developed models for investigating EAF. We have focused primarily on altered expression of genes regulating the cell cycle (Lennartsson et al., 1998Go; Martens et al., 1996Go; Stenius and Högberg, 1995Go). For example, we have shown that EAF hepatocytes demonstrate an attenuated response with respect to expression of wt-p53, a tumor suppressor involved in the surveillance of radiation- or chemical-induced DNA damage (el-Deiry et al., 1994Go; Kastan et al., 1991Go). We employed DEN in these studies and found that EAF cells both in vitro (Stenius and Högberg, 1995Go) as well as in situ (Lennartsson et al., 1998Go) showed this alteration. We concluded that an attenuated p53 response might lead to inadequate control of the cell cycle, thus conferring a growth advantage and facilitating clonal expansion of EAF hepatocytes. Relaxed cell-cycle control may also increase mutation rates and predispose cells to malignant conversion (Lennartsson et al., 1998Go).

In the present study, we compare the effects of DEN and PB on p53 expression in EAF arising in the livers of rats who have received an initiating dose of DEN at birth. After weaning, rats were administered either 2–12 weekly injections of DEN or drinking water containing PB for 3–14 months. All rats were challenged with DEN 24 h prior to sacrifice. The size of this challenging dose was chosen so that p53 expression was induced in the nucleus of a large fraction of the non-EAF hepatocytes. Our major question was whether p53 expression, in response to DNA damage in EAF hepatocytes, can be modulated by toxic substances administered as tumor promoters.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and husbandry.
Pregnant Sprague-Dawley rats were obtained from Alab (Sollentuna, Sweden) 5 days before parturition. Animals were housed in cages with sawdust-covered floors, in a controlled environment, with a 12-h light/dark cycle and uniform temperature and humidity. Rats received diet (RM3, Special Diets Service, Witham, England) and water ad libitum throughout the experiment. Experiments performed were sanctioned by the Swedish National Board for Laboratory Animals (CFN; Stockholm, Sweden).

Treatment of donor animals.
Twenty-four h after birth, female Sprague-Dawley rats were injected intraperitoneally (ip) with DEN (0.30 mmol/kg body wt). At 3 weeks-of-age, the animals were weaned and were treated thereafter either with phenobarbital (500 ppm in the drinking water) (Peraino et al., 1984Go) for approximately 3 or 14 months (PB-rats) or DEN (0.30 mmol/kg body wt, ip) once weekly (Lennartsson et al., 1998Go) for 2 weeks or 10–12 weeks (DEN-rats). DEN-rats and PB-rats were then challenged with 0.60 and 1.20 mmol DEN/kg body weight, respectively, 24 h prior to sacrifice (Lennartsson et al., 1998Go).

Immunohistological staining.
Livers were perfused with 3.7% buffered formaldehyde at 37°C for 1.5 h and thereafter placed in formalin for 24 h (Martens and Stenius, 1999Go; Peraino et al., 1984Go). Prior to staining, the slides were placed in citrate buffer and treated in a microwave oven (5 x 5 min, with a cooling period of 5 min. after each treatment).

Sections from PB-rats and DEN-rats were double-stained by overnight incubation with polyclonal antibodies for glutathione S-transferase pi (GST-P) and monoclonal antibodies raised against p53 (Ab-3, 1/100 dilution) (Calbiochem). Secondary alkaline phosphatase-conjugated anti-rabbit antibodies (1/100) were then applied to the slides and incubation continued for 1.5 h. New Fuchsin (Dako, Sweden) was employed to detect GST-P, while p53 was visualized using the EnVision +TM peroxidase kit (Dako, Denmark) with 3-diaminobenzidine tetrahydrochloride (DAB) as substrate. Sections from control animals were stained only for p53, using 1/100 dilution of antibodies as described above. Visualization of p53 was made using an EnVision+TM peroxidase kit (Dako, Denmark) with DAB as substrate.

Other sections from PB-rats were also double-stained for GST-P and terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling (TUNEL). These slides were washed 3x in TBS after paraffin sectioning, and endogenous peroxidases were blocked by treatment with 3% H2O2 in methanol for 30 min. The slides were incubated (1 h) in blocking reagent (2.5% BSA in TBS) prior to overnight incubation with primary antibodies toward GST-P. Sections were subsequently covered with secondary alkaline phosphatase-conjugated anti-rabbit antibodies at a dilution of 1:100 for 1.5 h. GST-P was visualized using New Fuchsin (Dako, Sweden) and thereafter apoptotic nuclei were visualized with terminal deoxynucleotidyl transferase using the TdT FragELTM apoptosis kit (TUNEL staining, Calbiochem).

Analysis of immunohistologically stained liver sections.
Stained liver sections were analyzed microscopically. The minimum section area analyzed per liver was 49.9 mm2 and all GST-P-positive EAF within a section with a size >=0.00084 mm2 were analyzed. Calculation of EAF parameters was performed using software based on the procedure of Pugh et al. (1983). In addition, twenty randomly selected GST-P-negative areas (non-EAF tissue), with a total area of at least 1.88 mm2 per rat were analyzed. Expression of p53 was quantitated by counting p53-positive hepatocytes in EAF and non-EAF areas. The data for the EAF were divided into 8 groups based on the size of the EAF.

Immunopreciptation and Western blot.
Livers from rats given 11 weekly doses of DEN (0.30 mmol/kg body wt), and a challenging dose of DEN (1.50 mmol/kg body wt) 24 h prior to sacrifice were used. Visible lesions and tissue free from visible lesions were dissected and homogenized in ice-cold 0.25 M sucrose. Livers from untreated rats and rats treated with DEN (1.20 mmol/kg body wt) were also isolated and homogenized in a similar way. Liver homogenates were subjected to nuclear fractioning at 600 g in a Beckmann centrifuge. Pellets and supernatants were suspended in IPB-7 (20 mM triethanolamine, 0.7 M NaCl, 0.5% NP-40, 0.2% DOC, 1 mM PMSF, and 0.1 mg/ml trypsin inhibitor II-T) and p53 was immunopreciptated essentially as described by van Gijssel et al. (1997), using monoclonal pAb 122 antibodies (Boehringer Mannheim, Germany) and protein A-Sepharose (Pharmacia Biotech Products). Precipitates were subjected to SDS-PAGE and proteins were subsequently transferred to a PVDF membrane (BioRad). Membranes were probed with CM-1 polyclonal antibodies (Novocastra Laboratories, England) followed by incubation with HRP-conjugated secondary antibodies (p217, DAKO, Denmark). Visualization of p53 was achieved by using the ECL procedure (Amersham).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GST-P was used as a marker for EAF and liver sections were double-stained for GST-P and p53. In the livers of all rats treated with the challenging dose of DEN 24 h prior to sacrifice, p53-positive hepatocytes were seen. The staining for p53 was strictly nuclear and hepatocytes with positive nuclei were more abundant in perivenous and mid-zonal areas. No staining was observed when primary antibodies were omitted. In EAF-bearing animals that did not receive a challenging dose of DEN, there was also no staining for p53 (data not shown).

In PB-rats, the distribution of p53 in EAF and in non-EAF tissue was similar. Figure 1AGo shows a lesion in a zone of p53-positive hepatocytes, in the liver of such a rat. Many p53-positive nuclei can be seen in both EAF and non-EAF tissue. In the centrilobular area, the number of p53-positive cells was fewer than in the mid-zonal area. This distribution in non-EAF tissue in PB rats was relatively frequent. The reason for this is not known. Figure 1BGo shows part of an EAF in higher magnification.



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FIG. 1. (A) A p53-positive EAF from the liver of a PB-rat (3 months). This liver section was double-stained for GST-P and p53. The GST-P-positive EAF (red cytoplasm) is seen to contain a large number of p53-positive hepatocytes (brown nuclei) and some fragmented cells. A central vein and many p53-positive hepatocytes (mainly in the mid-zonal area) can be seen in non-EAF tissue. (B) Border line (in higher magnification) between non-EAF tissue and a p53-positive EAF from the liver of a PB-rat (3 months). (C) EAF tissue from a PB-rat (14 months) double-stained for TUNEL and GST-P. Many TUNEL-positive hepatocytes (brown nuclei or nuclear fragments) are seen. (D) EAF from a DEN-rat (2 weeks) does not stain for p53. The section was also stained for GST-P. A central vein and several p53-positive hepatocytes (brown nuclei) can be seen in non-EAF tissue.

 
Many EAF from PB-rats contained dead cells, and liver sections from these rats were also double-stained for GST-P and TUNEL, which revealed that many EAF exhibited a high frequency of TUNEL-positive hepatocytes, i.e., apoptotic cells (Gavrieli et al., 1992Go). Figure 1CGo depicts a lesion with many such cells. However, the TUNEL-positive cells were few compared to the p53-positive cells in EAF from PB-rats.

Figure 1DGo shows an EAF from a DEN-rat. As can be seen, in this case there was a marked difference with respect to p53 distribution in EAF and non-EAF tissue. No positive nuclei can be seen in EAF tissue, whereas the frequency of stained hepatocytes in the surrounding tissue is high. Similar results indicating attenuated expression of p53 in EAF hepatocytes in response to a genotoxic challenge had been reported earlier (Lennartsson et al., 1998Go).

Data obtained from 6 PB- and 6 DEN-rats are documented in Tables 1–5GoGoGoGoGo. None of the lesions had an area >5.21 mm2. Table 1Go shows that livers from 3 months PB-rats contained GST-P-positive EAF of comparable size to those seen in 2 weeks DEN-rats. The 14 months PB-rats contained EAF of comparable size to those seen in 10–12 weeks DEN-rats. In the latter group, there were many more EAF than in any other group of rats.


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TABLE 1 GST-P-Positive EAF in DEN- and PB-Rats
 

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TABLE 2 p53 Expression in Non-EAF and EAF Liver Tissue
 

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TABLE 3 p53 Expression in EAF of Different Sizes in DEN- and PB-Rats
 

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TABLE 4 p53 Expression in EAF of Different Sizes in DEN- and PB-Rats
 

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TABLE 5 Summary of the Data Concerning p53 Expression in EAF of Different Sizes
 
Quantitative data regarding p53 expression are presented in Table 2Go. Two of the 3-month PB-rats showed a higher frequency of p53-positive hepatocytes in the EAF-tissue than in the non-EAF tissue. However, the mean ratio between these two p53 parameters was close to unity (0.93 ± 0.21) in this group. The corresponding mean ratio was 7.6 ± 4.0 for the 2-week DEN-rats. This ratio was significantly larger than mean ratio for PB-rats. We also counted the number of p53-positive EAF (containing <75 p53-positive cells/mm2 [see Lennartsson et al., 1998]). The majority of all EAF in 3-month PB-rats were p53-positive. This was not the case in 2-week DEN-rats.

Differences between PB- and DEN-rats became more pronounced with prolonged treatment (Table 2Go). In the 14-month PB-rats, the mean ratio was still close to unity (1.1 ± 0.8). In the 10–12-week DEN-rats the corresponding ratio was 26 ± 5.8. The ratio for DEN-rats (10–12 weeks) was significantly larger than the ratio for PB-rats (14 months). The ratio for DEN-rats (10–12 weeks) was also significantly larger than the ratio for DEN-rats (2 weeks). In 14-month PB-rats, 71% of all EAF were p53-positive (<75 p53-positive cells/mm2) while the corresponding value for DEN-rats was 8.8 % (Table 2Go). It may also be mentioned that with higher challenging doses of DEN (up to 1.8 mM/kg body weight) given to DEN-rats, there was still an attenuated p53 response in EAF (Lennartsson et al., 1998Go). In PB-rats given lower challenging doses (0.6 mM/kg body weight), very few p53-positive cells were seen in non-EAF or in EAF tissue (data not shown).

The EAF were also divided into groups of different sizes, designated size category a, b, c, and d in Table 3Go. The ratio (the number of p53-positive hepatocytes/mm2 in non-EAF tissue to the number of p53-positive hepatocytes/mm2 EAF-tissue) spanned from 0.36 to 1.2 and varied, independent of size, in 3-month PB-rats. Corresponding ratios for the 2-week DEN-rats varied from 1.8 to 61. The largest size category always showed the highest ratio. However, among the smaller EAF, there was no evident trend.

In 14-month PB-rats (Table 4Go), there were small or no differences in the number of p53-positive hepatocytes/mm2 present in EAF in groups of different sizes (here designated size categories 1, 2, 3, or 4). The ratio p53-positive hepatocytes in EAF tissue relative to non-EAF tissue for EAF of different sizes varied between 0.11 and 5.8 in PB-rats with no apparent trend. In the case of DEN-rats, there was an overall trend to have fewer p53-positive hepatocytes/mm2 in larger EAF. With two exceptions (i.e., size category b for rats near 10 and 11), lower counts were obtained with increasing EAF size. For size category 4, the ratios of p53-positive cells in EAF relative to non-EAF tissue for the three 10–12-week DEN-rats varied between 31 and 530.

Some of these data are summarized in Table 5Go. The mean values for p53-positive cells/mm2 in EAF tissue and ratios for all the PB- and DEN-rats are presented. The mean ratios for 3-month PB-rats showed small variations over the 4 EAF size categories compared to DEN-rats. Corresponding data for 2 weeks for DEN-rats show that the largest EAF size category (0.026–0.38 mm2) has the highest ratio. The fraction of the area, as well as the percentage of the total foci area, are also presented for each size group. The largest size group for PB-rats (3-month) accounted for 69.5% of the total foci area. The corresponding value for DEN-rats (2-week) was 28.0%. In 14-month PB-rats, the p53 parameter ratio was close to unity for EAF of all sizes, suggesting that there is no difference in p53 expression by hepatocytes localized in EAF and non-EAF tissue in these animals. In DEN-rats (10–12 wk), the largest EAF category demonstrated a mean ratio of 210. The largest EAF category accounted for 59.7% of the total EAF area fraction for PB-rats (14-mo) and the corresponding value for DEN-rats was 31.9%. Overlapping sizes among EAF from DEN-rats (2-week) and from DEN-rats (10–12-week) had similar ratios.

Western blot analysis supported the immunohistological data; p53 was readily detected in immunoprecipitated material from the nuclear fraction of the macroscopically lesion-free tissue after a dose of DEN 24 h prior to sacrifice. Two bands were detected with the used antibody (CM-1). In preliminary experiments, we have used an antibody specific to phosphorylated (Ser 15) p53 and this antibody detected one band, suggesting that the two bands reflect phosphorylated and unphosphorylated p53, respectively; p53 was not detected in the nuclear fraction isolated from 2 samples (each sample from one rat) of several macroscopic EAF (Fig. 2AGo). In control experiments, p53 was not detected in liver tissue samples from untreated rats. However, a single dose of DEN given 24 h prior to sacrifice induced p53 accumulation (Fig. 2BGo).



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FIG. 2. Western blot analysis of immunopreciptated p53 from liver tissues; lack of p53 response in EAF-lesions from DEN-rats confirms immunohistological data. (A) Two DEN-rats (10–12-weeks) received a challenging dose of DEN 24 h prior to sacrifice. Several small macroscopic lesions and lesion free tissue was dissected from each rat, fractionated into nuclear and cytoplasmic fractions, and further treated as described in Material and Methods. (B) Two control (lesion-free) rats were used. One rat received DEN (1.20 mmol/kg body wt) and the other saline 24 h before sacrifice. Liver tissue was prepared as described above.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The data presented here suggest that cellular expression of p53 in response to DNA damage can be modulated by long-term exposure to xenobiotics. Two patterns were observed in EAF hepatocytes: an attenuated response in DEN-treated rats and an apparently unaltered response, in PB-treated rats, compared to surrounding tissue. The altered response seen in DEN-rats may reflect an adaptation to the genotoxic effect of this xenobiotic.

We have previously described an attenuated p53 response in EAF hepatocytes in DEN-rats and discussed the possible growth advantage and increased mutation rate conferred by such attenuation (Lennartsson et al., 1998Go; Stenius and Högberg, 1995Go). Here we present additional evidence in this connection. Thus, after 2 weeks of DEN treatment, many lesions exhibited an attenuated p53 response, and we documented a development over time. Furthermore, DEN-rats exhibited larger EAF than in the previous study. Many lesions in rats receiving DEN for 10–12 weeks had probably reached the point when conversion to a tumor was imminent. Nevertheless, we did not find a single large lesion that expressed high levels of p53. Instead, we found a similar, but stronger trend resembling that seen in the previous study: the larger the size of the lesion, the fewer p53-positive hepatocytes/mm2 were present. An attenuated or lack of response was also documented by employing Western blot analysis. It can thus be concluded that the attenuated p53 response may have facilitated selective expansion of p53-non-responsive cells throughout the promotion phase in DEN-rats.

There was no apparent relationship between the level of p53 expression and the size of EAF in PB-rats. The variations in ratios were mainly confined to variations between individual rats. This finding suggests that p53 expression is not related to the rate of volume growth of EAF in such rats. Furthermore, the mean ratio between non-EAF and EAF tissue in PB-rats was close to unity. In light of the possibility that alterations in drug metabolism may provide protection for EAF hepatocytes, this later observation is somewhat surprising. Based on in vitro data, we have calculated that DNA damage is 3–4-fold more common in non-EAF hepatocytes than in EAF hepatocytes isolated from DEN-rats (Stenius and Högberg, 1995Go). To what extent EAF cells in PB-rats are protected from the challenging dose of DEN is not known.

In EAF from PB-rats, many TUNEL-positive hepatocytes were seen. This suggests that the p53 response was functional in these lesions, although other regulators of apoptosis exist. As mentioned in Results, there was no p53 expression detected in the livers of rats not receiving the challenging dose of DEN. This indicates that the data presented in Tables 2–5GoGoGoGo reflect wild-type p53 responses to DNA damage. It may also be argued that the altered p53 response of EAF in DEN-rats cannot be explained by mutations (Lennartsson et al., 1998Go; Stenius and Högberg 1995Go). This argument is based on the high frequency of altered EAF, the low frequency of p53 mutations found in rat liver tumors (Barbin et al., 1997Go; Masui et al., 1997Go), and the finding that the p53 response was decreased (i.e., we did not see an increased expression, as is usually seen in mutated cells; Harris, 1996) in EAF.

A down-regulation of the p53 response in EAF is consistent with a genotoxic mode of action for DEN. We interpret our findings as suggesting that DEN promotes EAF development by inducing cell death or a stop in the cell cycle in surrounding hepatocytes and by inducing a protective adaptation to such effects, i.e., down-regulation of p53 expression, in EAF hepatocytes. PB is not genotoxic and our data suggested that p53 does not play a central role in primary defenses against PB toxicity. The question can be raised whether the development of EAF with an attenuated p53 expression is a specific adaptive response to genotoxic stress in the liver.

The data presented here thus suggest that, in DEN-rats, the accumulation of requisite mutations within EAF that subsequently develop into tumors may not only be a direct consequence of, e.g., DEN-induced adducts. A decrease in the level of p53 expression and relaxed control of the cell cycle might also be of critical importance for this accumulation. Two recent studies on p53-(+/-)-knockout mice support the notion that a low level of p53 expression, rather than mutations in the second allele, is a critical factor in tumor development. Spontaneous (Venkatachalam et al., 1998Go), as well as nitrosamine-induced (Ozaki et al., 1998Go) tumors in these animals often retain the wild-type allele for p53, suggesting that the level of p53 protein is critical for tumor formation.

It is reasonable to assume that sufficiently low levels of DEN may not down-regulate p53 expression. On the other hand, at high doses, it might become a dominating mechanism for accumulating mutations, and it can be expected to give rise to non-linearities in the dose-response curve for tumors. In a recent dose-response study (Williams et al., 1996Go) the experimental design was similar to that employed here (i.e., weekly injections of DEN) and, indeed, in that study, non-linearities in tumor response were seen at approximately the dose levels used in our present study. A marked increase in cytotoxicity and cell proliferation was observed at this dose level (Williams, 1996Go). Further studies may reveal whether this non-linearity in a more direct way can be linked to an attenuated p53 response.

Gonzales et al. (1998) suggest that an attenuated p53 response is an important feature in connection with PB-induced hepatocarcinogenesis. They used primary cultures of mouse hepatocytes and observed an attenuated p53 response after a 23-h PB-treatment of the cultures. Some of our data may support this finding, i.e., the relatively low response in non-EAF tissue in PB-rats (as compared to the response in DEN-rats; see Table 2Go) and the low expression of p53 in many centrilobular areas in PB-rats (as shown in Fig. 1AGo). Such an effect may facilitate EAF initiation. However, our data emphasize the importance of p53 expression in preneoplastic lesions in the rat and data concerning this parameter tend to contradict the hypothesis by Gonzales et al., provided the species differences can be excluded.

The mechanism leading to the attenuated p53 response in DEN-rats remains to be elucidated. In preliminary immunohistochemical studies, we have observed that MDM2 (c.f. Wu et al., 1993 and Momand et al. 1992) is overexpressed in EAF in rats used in this study. However, both PB-rats and DEN-rats were equally affected, so this isolated finding does not explain the differences in p53 expression. More promising is the observation that p53 can be induced in EAF hepatocytes from DEN-rats by HIF-1{alpha} stabilization (to be published elsewhere). This suggests that a selective down-regulation of the DNA damage-signaling pathway attenuated the p53 response in DEN-rats.


    ACKNOWLEDGMENTS
 
This work was supported financially by the National Institute for Working Life, Sweden.


    NOTES
 
1 To whom correspondence should be addressed. E-mail: ulla.stenius{at}imm.ki.se. Back


    REFERENCES
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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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