Long-term dehydroepiandrosterone and 16{alpha}-fluoro-5-androsten-17-one administration enhances DNA synthesis and induces expression of c-fos and c-Ha-ras in a selected population of preneoplastic lesions in liver of diethylnitrosamine-initiated rats

Maria M. Simile, Maria R. De Miglio, Diego Calvisi, Maria R. Muroni, Maddalena Frau, Giuseppina Asara, Lucia Daino, Luca Deiana, Rosa M. Pascale and Francesco Feo1,

Dipartimento di Scienze Biomediche, Sezione di Patologia Sperimentale e Oncologia, Via P.Manzella 4, 07100 Sassari, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Dehydroepiandrosterone (DHEA) inhibits glucose 6-phosphate dehydrogenase (G6PD) activity and growth of preneoplastic lesions in various tissues, but its administration may also enhance tumorigenesis by genotoxic carcinogens. We have investigated in single preneoplastic liver lesions, induced in diethylnitrosamine-initiated rats by the resistant hepatocyte protocol, the mechanisms underlying these opposite DHEA effects. Administration of DHEA (0.45% in the diet) for 10 and 26 weeks and of its analog 16{alpha}-fluoro-5-androsten-17-one (FA, 0.25%) for 10 weeks, starting 4 weeks after initiation, induced an apparent decrease in the number of glutathione S-transferase (placental) (GST-P)-positive lesions and an increase in lesion volume. DHEA administration for 38 weeks enhanced hepatocellular carcinoma multiplicity. Depending on the rise in the number of slowly growing, remodeling GST-P-positive lesions induced by DHEA and FA, overall DNA synthesis decreased slightly in these lesions at 14 weeks, but increased in uniform lesions. Labeling index (LI) in single uniform lesions at 14 weeks ranged between very low (not different from normal liver) to high (>10-fold normal liver). DHEA and FA induced broad increases in lesions with a high LI, which showed a higher number of cells overexpressing c-Ha-ras and/or c-fos than those with a lower LI. High G6PD activity was inhibited by DHEA and FA in only ~50% of preneoplastic lesions. These data indicate selection in rats subjected to long-term DHEA and FA treatments of a subpopulation of GST-P-positive cells with high growth and progression potentials. Overall effects of these compounds depends on the relative numbers of lesions in which inhibition of DNA synthesis can counteract their transforming effect.

Abbreviations: ABC, avidin–biotin–peroxidase complex; BrdU, 2-bromo-3'-deoxyuridine; DHEA, dehydroepiandrosterone; FA, 16{alpha}-fluoro-5-androsten-17-one; G6PD, glucose 6-phosphate dehydrogenase; GST-P, glutathione S-transferase (placental); HCC, hepatocellular carcinoma; LI, labeling index; PBS, phosphate-buffered saline; RH, resistant hepatocyte; TK, Tukey–Kramer.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several studies have shown chemoprevention by dehydroepiandrosterone (DHEA) and its synthetic analog 16{alpha}-fluoro-5-androsten-17-one (FA) of tumors induced in different tissues by chemicals or radiation (reviewed in 1,2). Administration of 0.6% DHEA in the diet to rats treated with chemical carcinogens may result in inhibition of DNA synthesis in early preneoplastic (35) and neoplastic (6) liver lesions. When DHEA is administered after the development of large nodules (persistent, neoplastic nodules and adenomas) there occurs a decrease in volume, without a reduction in nodule incidence (7). Dietary administration of 0.25% DHEA to rats results in a phenotypic shift of hepatocellular carcinomas (HCCs) induced by N-nitrosomorpholine to more differentiated forms (3). This seems to depend on changes in cellular lineages involved in tumor development, with a decrease in glutathione S-transferase (placental) (GST-P)-positive and mixed cell lesions and an increase in amphophilic lesions, which are precursors of hepatocellular adenomas and carcinomas, which usually appear less malignant than those induced by N-nitrosomorpholine (8). However, long-term treatment with DHEA induces liver adenomas and carcinomas in normal rats (911) and enhances lung (3), kidney (12), pancreas (13,14) and liver (1517) carcinogenesis induced by genotoxic carcinogens.

The anticarcinogenic effect of DHEA has been attributed to inhibition of glucose 6-phosphate dehydrogenase (G6PD), a rate limiting enzyme of the hexose monophosphate pathway, with a consequent decrease in liver content of ribose 5-phosphate, a nucleotide precursor (4,5), as well as decreased mevalonate biosynthesis (18), which could result in decreased isoprenylation of p21RAS. However, this latter effect has only been described in in vitro cells with relatively high DHEA concentrations (19). On the other hand, production of reactive hydroxy radicals as a consequence of (20) or independently of (21,22) peroxisome proliferation has been suggested to account for the tumorigenic effect of DHEA. Reactive oxygen species can stimulate cell growth during liver tumorigenesis by inducing up-regulation of the Ras/MAPK pathway (23,24), c-fos and c-jun (2527) and NF-{kappa}B (28,29). DHEA increases the mutagenicity of N-nitrosobis(2-hydroxypropyl)amine and N-nitrosobis(2-hydroxypropyl)(2-oxopropyl)amine (30) and can act as a complete hepatocarcinogen in rainbow trout, in which it does not induce peroxisome proliferation, even in the absence of initiating chemical carcinogens (17). The genotoxicity of DHEA for this animal is strongly supported by the mutation of c-Ki-ras in liver tumors induced by the hormone. FA has been reported to be a more potent inhibitor of G6PD activity (31) and a more powerful and less toxic chemopreventive agent than DHEA (3133). It lacks DHEA hormonal activity (32), thus suggesting that tumor chemoprevention by these compounds could be attributed to a reduced availability of pentose phosphates for DNA synthesis more than to hormonal activity. To our knowledge, a carcinogenic effect of FA on rat liver has not yet been studied. FA does not induce liver tumors in non-initiated rainbow trout and increases HCC incidence only very slightly, without influencing multiplicity and size, in aflatoxin B1-initiated trout (34). Clinical trials with this product have been proposed (35).

An understanding of cellular and molecular mechanisms leading to the prevalence of DHEA inhibitory or carcinogenic effects may be relevant to cancer prevention, taking into account the suggested or current use of DHEA and of its fluorinated derivatives for the treatment of a number of pathological conditions, such as atherosclerosis, hypertension, obesity, non-insulin-dependent diabetes, asbestosis, psychological disturbances and systemic lupus erythematosus (reviewed in ref. 1). The hepatocarcinogenicity of DHEA (911,1517), in apparent contrast to its antiproliferative action on preneoplastic liver lesions (35,7), suggests that as a consequence of DHEA genotoxicity, long-term treatment may result in the selection of a subpopulation of liver lesions resistant to the inhibitory effects of DHEA in which the activation of signal transduction pathways induces rapid evolution to HCC (3639). In the present work we attempted to identify these lesions by evaluating DNA synthesis and expression of the c-Ha-ras and c-fos genes in single liver lesions in the early stages of hepatocarcinogenesis induced in diethylnitrosamine-initiated rats by the resistant hepatocyte (RH) protocol (40). We have also quantified the changes induced in this subpopulation by prolonged treatments with DHEA and FA and attempted to correlate them with overall effects of these compounds on liver carcinogenesis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and treatments
Male Fisher 344 rats (160–180 g body wt at the beginning of the experiment) were housed individually in suspended wire-bottomed cages in a room with constant temperature (22°C) and humidity (55%) and with a 12 h light (6 a.m.–6 p.m.) and dark (6 p.m–6 a.m.) cycle. The rats had free access to food and water throughout the study. The basal diet was a standard diet, G.L.P. (Mucedola srl, Settimo Milanese, Italy). The DHEA- and FA-containing diets were prepared by adding 0.45% DHEA or 0.25% FA to powdered standard diet and pellets were prepared from the humidified diet, dried at 22°C and used within 24 h. Five untreated rats were used as a source of normal liver. The other rats were initiated with a single i.p. dose (150 mg/kg) of diethylnitrosamine. They were subjected, 2 weeks later, to a selection treatment consisting of 15 days feeding with a hyperproteic diet containing 0.02% 2-acetylaminofluorene, with partial hepatectomy at the mid-point of this treatment (RH protocol; 40). At the end of selection (week 4) the rats were divided into three groups (70 rats each at the beginning of the experiment; Figure 1Go). Group 1 (carcinogen-exposed control) did not receive any other treatment; groups 2 and 3 were fed the DHEA- or FA-containing diet, respectively. The DHEA-containing diet was given for 10, 26 or 38 weeks and the FA diet for 10 weeks. The rats treated for 38 weeks with DHEA received standard diet for an additional 10 weeks before death. All other DHEA-treated and FA-treated rats were killed at the end of these treatments. The body weight was measured once a week and the diet intake every day. Surviving rats (48, 45, and 38 for groups 1–3, respectively) were killed by bleeding through the abdominal aorta under ether anesthesia 14, 30 and 48 weeks after initiation. Livers were resected, cut into 2–3 mm slices and nodules with a diameter >3 mm were collected as described (7). All animals received humane care and the study protocols were in compliance with our institution's guidelines for use of laboratory animals.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1. Study design. Male F344 rats, initiated with diethylnitrosamine (DENA), were fed a hyperproteic diet containing 0.02% 2-acetylamino- fluorene (AAF) for 2 weeks, with partial hepatectomy (PH) at the mid-point of this feeding. Then the rats received a standard diet (group 1) or a standard diet containing 0.45% DHEA up to 38 weeks (group 2) or 0.25% FA up to 14 weeks (group 3). Upward arrows represent time points at which rats were killed for analysis.

 
Enzyme assay
G6PD activity of single nodules (14 weeks after initiation) was determined at 37°C in 30 000 g supernatants of homogenates in physiological saline containing 0.66 M EDTA and 0.05% Triton X-100 as previously published (5). Data are expressed as units (U) per mg protein. One unit of enzyme activity corresponds to 1 nmol NADPH produced/min. Protein was determined according to Lowry et al. (41) using bovine serum albumin as the standard.

Histology and immunohistochemistry
Isolated nodules and small pieces of liver from each lobe (of both normal rats and rats subjected to the RH protocol) were fixed in methacarn and processed for embedding in paraffin, cut into 5 µm thick slices, dewaxed in xylene, washed with alcohol and stained with hematoxylin/eosin. Some serial slides were soaked, after dewaxing and washing in alcohol, in absolute methanol containing 0.3% hydrogen peroxide for 45 min at room temperature to remove endogenous peroxidase activity. After rehydration in phosphate-buffered saline (PBS), the slides were microwaved for 20 min with 0.01 M sodium citrate buffer, pH 6, and then processed for GST-P immunohistochemistry as already published (5) or for oncogene immunohistochemistry using a standard avidin–biotin–peroxidase complex (ABC) method with a Vectastain ABC Kit (Vector Laboratories, Burlingame, CA). In brief, microwaved slices were washed in PBS, pH 7.4, and non-specific binding was blocked with 5% normal goat serum. Incubation was overnight at 4°C with the following primary rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA): c-Ha-ras (SC-520, 10 µg/ml) and c-fos (SC-7203, 40 µg/ml). After being washed with PBS the slices were incubated for 45 min with biotinylated secondary antibody, followed by ABC for 60 min according to the manufacturer's instructions. After washing in PBS the slides were visualized for peroxidase with 0.05% 3.1,3'-diaminobenzamide tetrahydrochloride containing 0.005% H2O2 in 50 mM Tris–HCl buffer, pH 7.6. Finally, the slides were lightly counterstained with hematoxylin. As controls, known positive tissue sections were used, and for negative controls exposure to the primary antibody was omitted.

For examination of G6PD-positive lesions small pieces of liver were frozen in isopentane at –140°C, embedded in O.C.T. compound (Tissue-Tek; Miles, Elkhart, IN) and cut in a cryostat into 5 µm sections which were mounted on glass slides for G6PD histochemistry as described (4) or for c-fos immunostaining. Remodeling lesions were identified as areas lacking uniformity for GST-P immunostaining (42). Other lesions were identified and classified according to Squire and Levitt (43). To determine labeling index (LI), the rats received three i.p. injections of 5 mg/100 g body wt 2-bromo-3'-deoxyuridine (BrdU) 21, 13 and 5 h before death. BrdU incorporation into nuclei was determined immunohistochemically with a `cell proliferation kit' (Amersham International, Amersham, UK).

Quantification of liver lesions
The livers were resected and the superficial gross nodules and HCCs were isolated. The livers were then rapidly cut into 2–3 mm slices and nodules with a diameter >1 mm were identified and counted. Morphometric analysis of microscopic lesions was carried out using a Leica Quantimet 500 image analyzer (Leica, Cambridge, UK). The number of lesions per cm3 (N) and the mean volume of lesions (V) and the volume fraction (VF, the percentage of liver volume occupied by GST-P-positive lesions, calculated by the Delesse method) were determined according to Pugh et al. (44). Transections with a radius >35 µm could be reliably identified and were included in the analysis. Immunohistochemical analyses were performed in a blind fashion without knowledge of the rat group by two pathologists. The staining intensity for gene immunohistochemistry was scored as: not detectable, low, moderate or intense. The extent of staining was scored as: 0, low (<10% scattered stained cells), moderate (10–50%) or high (>50%). To quantitate S phase cells 2000–5000 preneoplastic hepatocytes per liver were counted and the percentage of GST-P-positive hepatocytes that incorporated BrdU was determined. The extent of DNA synthesizing cells in single lesions was scored as: 0, very low (values in the range of normal liver values), low (up to 3-fold higher than normal liver values), moderate (3- to 10-fold higher than normal liver values) or high (>10-fold normal liver values).

Statistical analysis
Data are expressed as means ± SD. Data relative to body and liver weights, number and volume of liver lesions, remodeling and LI (total nodule averages) and G6PD activity were analyzed by ANOVA and multiple comparisons were performed by the Tukey–Kramer (TK) test. The distribution of LI among single lesions was analyzed by non-parametric ANOVA (Kruskal–Wallis test) and multiple comparisons were made by Dunn's test. Quantitation of DNA synthesizing cells, gene expression in single lesions and tumor yield were analyzed by Fisher's exact test. GraphPad InStat 3 (www.graphpad.com) was used for statistical evaluation of the results.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nodule development
Food intake did not differ and ranged between 12.5 and 13.4 g/100 g body wt/day in all rat groups, but a significant decrease in body weight occurred 10, 26 and 38 weeks after starting DHEA treatment. This treatment caused 31, 20 and 26% increases in liver weight after 10, 26 and 38 weeks, respectively (at least P < 0.05; mean values of control liver 6.96 ± 1.6, 9.1 ± 0.56 and 13.6 ± 2.1 at 14, 30 and 48 weeks, respectively). In the rats treated for 10 weeks with FA an insignificant 10% rise in liver weight occurred.

Fourteen and 30 weeks after initiation persistent nodules were present in the liver of all rat groups. The majority of them consisted of clear/eosinophilic cell lesions larger than a liver lobule and compressing surrounding parenchyma. Figure 2Go shows 38–41% decreases in nodule number/cm3 liver at 14 weeks in rats treated with DHEA or FA with respect to carcinogen-exposed rats not treated with DHEA/FA (group 1, control). At 30 weeks the number of lesions/cm3 decreased in all rat groups, probably as a consequence of phenotypic reversion (remodeling; see below). However, it was 45% lower in rats treated with DHEA for 26 weeks (group 2) than in the control. These changes were associated with a great rise in nodule volume, with respect to control, at both 14 and 30 weeks in rats fed DHEA and at 14 weeks in FA-treated rats. Volume fraction decreased at 14 weeks from 70% in controls to 30 and 26% in DHEA- and FA-treated rats. At 30 weeks volume fraction decreased to 40% in control, but rose to 62% in the DHEA group.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 2. Effect of DHEA and FA on the number, mean volume and volume fraction of GST-P-positive lesions in rat liver. Persistent nodules were induced by the RH protocol in diethylnitrosamine-initiated rats. Treatment with DHEA (0.45% in the diet) or FA (0.25%) was started 4 weeks after initiation. Carcinogen-exposed rats without DHEA/FA (group 1) acted as controls. The rats were killed at 14 weeks, at the end of a 10 week treatment with DHEA or FA, or at 30 weeks, at the end of a 26 week treatment with DHEA. {blacksquare}, Without DHEA/FA (control); {blacksquare}, DHEA-treated; {blacksquare}, FA-treated. Data are means ± SD, n = 7 for each time point. Number (TK test): DHEA versus control, 14 weeks, P < 0.01 and 30 weeks, P < 0.05; FA versus control, P < 0.05. Volume (TK test): DHEA versus control, P < 0.001 at 14 and 30 weeks; FA versus control, P < 0.001. Volume fraction (TK test): DHEA versus control, P < 0.001 at 14 and 30 weeks; FA versus control, P < 0.001.

 
Since phenotypic reversion could be at least in part responsible for the decrease in number of GST-P-positive lesions, the percentage of positive lesions with a non-uniform pattern of GST-P immunostaining was evaluated at 14 weeks. The percentage of remodeling lesions was 26.3 ± 8.16 in carcinogen-exposed controls, as against 48.3 ± 7.9 and 40.1 ± 5.2 in the DHEA- and FA-treated rats (means ± SD, n = 7; TK test: DHEA versus control, P < 0.001; FA versus control, P < 0.01).

Forty-eight weeks after initiation visible nodules (2–25 mm diameter) were present on the liver surface and in transections of liver from carcinogen-exposed rats treated with DHEA (groups 1 and 2). Relatively few (no more than 300/cm3) microscopic liver lesions were present at this time. They were mostly clear/eosinophilic cell nodules in both DHEA-treated and untreated rats. Larger lesions, visible with the naked eye, were in general persistent nodules (neoplastic nodules and adenomas) or well-differentiated HCCs. No differences in the microscopic pattern of lesions were seen between DHEA-treated and untreated rats nor were differences in tumor incidence between these rat groups found, but the multiplicity of well-differentiated and total HCCs was significantly higher in DHEA-treated than in control rats (Table IGo). The increase in multiplicity of moderately differentiated HCCs was small and insignificant.


View this table:
[in this window]
[in a new window]
 
Table I. Incidence and multiplicity of persistent nodules and carcinomas in rats treated with DHEA and their controlsa
 
DNA synthesis
Fourteen weeks after initiation LI values of GST-P-positive lesions were ~22% lower in the DHEA and FA rat groups than in carcinogen-exposed controls (P < 0.001). DNA synthesis decreased by 28–31% in remodeling lesions in all rat groups. The LI of lesions with a uniform pattern (non-remodeling lesions) exhibited 42.8 and 47.6% increases in the DHEA and FA groups, respectively, with respect to the carcinogen-exposed control (Figure 3Go). Thus, the overall decrease in LI in these latter groups was largely influenced by a rise in remodeling lesions exhibiting relatively low DNA synthesis. The differences among groups in the LI of uniform lesions, however, cannot account for the 6- to 7-fold rise in lesion volume in DHEA- and FA-treated rats. The possibility was thus explored that treatments with DHEA and FA resulted in an expansion of a subpopulation of GST-P-positive lesions with elevated DNA synthesis.



View larger version (43K):
[in this window]
[in a new window]
 
Fig. 3. Effect of DHEA and FA on DNA synthesis in GST-P-positive lesions in rat liver. Preneoplastic lesions were induced by the RH protocol in diethylnitrosamine-initiated rats. Treatment with DHEA (0.45% in the diet) or FA (0.25%) was started 4 weeks after initiation and was continued up to death (week 14). Carcinogen-exposed rats without DHEA/FA (group 1) acted as controls. {blacksquare}, Total lesions; {blacksquare}, uniform lesions; {blacksquare}, remodeling lesions. Data are means ± SD of the percentage of BrdU-incorporating cells, n = 7. Total lesions (TK test): DHEA and FA versus control, P < 0.001; uniform versus remodeling, P < 0.001 for control (C), DHEA and FA. Uniform lesions (TK test): DHEA and FA versus control, P < 0.001. Remodeling lesions (TK test): DHEA and FA versus control, P < 0.05.

 
The results in Figure 4Go indicate that single GST-P-positive lesions with uniform pattern show great heterogeneity with respect to DNA synthesis values in all rat groups. Median values were 0.5, 1.85 and 3.45 for carcinogen-exposed control, DHEA- and FA-treated rats, respectively (control versus DHEA and FA groups, P < 0.0001; DHEA versus FA, P < 0.01). The broad scattered distribution of LI values in single lesions in all rat groups revealed the existence of different populations of lesions (Figure 4Go and Table IIGo). A number of lesions showed LI values in the range of normal liver (very low group, VL) or undergoing a relatively low increase (up to 3-fold, low group, L). It could be calculated, on the basis of data in Table IGo, that these slowly growing lesions represented ~71% of total liver lesions in carcinogen-exposed rats without DHEA/FA and only 52 and 33% in the DHEA- and FA-treated groups, respectively. In contrast, the percentage of lesions with a moderate/high LI (up to 10-fold/>10-fold normal liver values) was 51 and 67% in the DHEA and FA groups, against only 29% in the carcinogen-exposed controls.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 4. Effect of DHEA and FA on the distribution diagrams of LI in single GST-P-positive lesions. Preneoplastic lesions were induced by the RH protocol in diethylnitrosamine-initiated rats. Treatment with DHEA (0.45% in the diet) or FA (0.25%) was started 4 weeks after initiation and was continued up to death (week 14). Carcinogen-exposed rats without DHEA/FA (group 1) acted as controls. The abscissa indicates the LI of each of the GST-P-positive lesions developing in a total of seven rats for the control and DHEA groups and in 14 rats for the FA group. Median values: 0.5, 1.85 and 3.45 for the control, DHEA and FA groups, respectively. KS test: P < 0.0001 for all groups. Dunn's test: DHEA and FA versus control, P < 0.001; FA versus DHEA, P < 0.01.

 

View this table:
[in this window]
[in a new window]
 
Table II. Quantitation of DNA synthesizing cells in persistent liver nodules in rats treated with DHEA and FA and their controlsa
 
Gene expression
Rapid growth of preneoplastic and neoplastic liver lesions is associated with overexpression of genes related to the MAPK cascade, leading to overproduction of the transcriptional factors c-Fos and c-Jun (45). Ras brings Raf, the first kinase of the MAPK cascade, to the cell membrane and leads to sequential phosphorylation of the other cascade members. Thus, expression of the c-Ha-ras and c-fos genes was studied in single liver lesions 14 weeks after initiation by immunostaining of gene products (Figure 5Go and Table IIIGo). The analysis of 116 and 99 lesions in the carcinogen-exposed control (group 1) and DHEA-treated rats (group 2), respectively, showed c-fos expression in 26–28% of lesions and c-fos plus c-Ha-ras expression in ~22 and 30% of lesions in both groups. Only one lesion from a control rat exhibited c-Ha-ras expression without c-fos expression. Lesions positive for gene expression showed moderate/intense immunostaining for both genes, without any appreciable difference among groups. The distribution of total lesions positive for c-fos and c-fos plus c-Ha-ras among different LI subgroups (from VL to H) did not exhibit any significant difference between carcinogen-exposed control and DHEA-treated rats (Table IIIGo). However, the percentage of lesions with >50% positive cells (subgroup H) was constantly higher in DHEA-treated than in control rats (Table IIIGo). The behavior of c-fos and c-Ha-ras expression in FA-treated rats (Figure 5Go) did not differ from that of DHEA-treated rats and the results are not included in Table IIIGo.



View larger version (86K):
[in this window]
[in a new window]
 
Fig. 5. Effect of DHEA and FA on expression of c-fos and c-Ha-ras in GST-P-positive liver lesions. Preneoplastic lesions were induced by the RH protocol in diethylnitrosamine-initiated rats. Treatment with DHEA (0.45% in the diet) or FA (0.25%) was started 4 weeks after initiation and was continued up to death (week 14). Carcinogen-exposed rats without DHEA/FA (group 1) acted as controls. Note that there are at least two GST-P-positive lesions in the FA group, only one of which expressed c-fos and c-Ha-ras. Magnification x10.

 

View this table:
[in this window]
[in a new window]
 
Table III. Quantitation of lesions expressing c-fos and c-Ha-rasa
 
G6PD activity
Figure 6Go shows the presence of GST-P-positive lesions expressing high G6PD activity and LI in liver of carcinogen-exposed control rats treated with DHEA and FA 14 weeks after initiation. G6PD activity was low or absent in some lesions of DHEA- and FA-treated rats, which also exhibited low LI. c-fos expression was absent in these lesions and was instead seen in lesions with high G6PD activity (not shown). This behavior was constantly observed in several sections from six rats per treatment group. Moreover, it was confirmed by biochemical determination of G6PD activity in single isolated nodules (Figure 7Go). As expected (2,4,5), nodules showed 2.9-fold higher G6PD activity than normal rat liver (sp. act. normal liver 48.72 ± 6.31, n = 4) which was partially inhibited by DHEA and FA. However, G6PD activity in single nodules of DHEA- and FA-treated rats exhibited greater variations than that of control rats: ~50% of nodules showed activity in the range of that of lesions developing in rats which did not receive these compounds.



View larger version (46K):
[in this window]
[in a new window]
 
Fig. 6. Effect of DHEA and FA on G6PD activity and LI of single GST-P-positive lesions. Preneoplastic lesions were induced by the RH protocol in diethylnitrosamine-initiated rats. Treatment with DHEA (0.45% in the diet) or FA (0.25%) was started 4 weeks after initiation and was continued up to death (week 14). Carcinogen-exposed rats without DHEA/FA (group 1) acted as controls. The LI of single nodules are included in the Table. Magnification x5.

 


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 7. Effect of DHEA and FA on G6PD activity of isolated preneoplastic nodules. Preneoplastic lesions were induced by the RH protocol in diethylnitrosamine-initiated rats. Treatment with DHEA (0.45% in the diet) or FA (0.25%) was started 4 weeks after initiation and was continued up to death (week 14). Carcinogen-exposed rats without DHEA/FA (group 1) acted as controls. Data are means ± SD, n = 11 (first bar of each group), or G6PD activity of single nodules. Specific activity of normal rat liver: 48.02 ± 6.3. TK test: DHEA and FA versus control, P < 0.05.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our results show a high heterogeneity of putative preneoplastic lesions induced in rat liver by the RH protocol with respect to DNA synthesis and expression of c-fos and c-Ha-ras. A good correlation between oncogene expression and LI was found: in general, the higher the number of S phase hepatocytes in each lesion, the higher the number of cells expressing c-fos and c-Ha-ras. The majority of lesions exhibited poor growth capacity coupled with phenotypic reversion, at least in some lesions. However, a consistent subpopulation of liver lesions (29%) was identified in which a moderate/high LI and active signal transduction pathway, involving c-fos and c-Ha-ras, indicate a tendency to progress to full malignancy.

Long-term treatment with DHEA and FA results in an apparent decrease in nodule number, but a marked increase in volume of remaining nodules. Large and coalescent liver lesions, especially in DHEA- and FA-treated rats, could include or obscure smaller lesions, resulting in an underestimation of lesion number during the conversion from two to three dimensions. Number and volume of lesions may also be influenced by an increase in liver weight secondary to hypertrophy, as well as to the presence of great amounts of nodular tissue (4,46) in the DHEA group at 14 weeks. Nonetheless, some decrease in lesion number follows stimulation of remodeling by DHEA and FA, with consequent progressive disappearance of GST-P-positive lesions (36,47). An increase in the number of remodeling lesions exhibiting low DNA synthesis is probably responsible for a small decrease in LI of total liver lesions, even in the presence of stimulation of DNA synthesis in uniform lesions. It clearly appears that DHEA and FA induce an increase in the relative proportion of lesions resistant to their inhibitory effects exhibiting moderate/high LI and c-fos and c-Ha-ras overexpression. This results in rapid progression of a consistent number of lesions, as documented by a rise in tumor multiplicity at 48 weeks, at least in DHEA-treated rats.

Several studies have pointed to the mechanisms of DHEA effects in carcinogenesis. Although small DHEA doses exhibit an antioxidant effect (48,49), high doses, in the range of those used in the present study, induce production of hydroxyl radicals, even in the absence of peroxisome proliferation (21,22), strongly suggesting that production of reactive oxygen species during DHEA treatment could enhance tumorigenesis by inducing DNA damage (45). Nonetheless, short-term treatments with DHEA inhibit cell proliferation, indicating the prevalence of restraint of nucleic acid synthesis (35). However, continuous production of reactive oxygen species during long-term treatment in the early stages of tumorigenesis may induce accumulation of genomic damage (17,30) and activation of c-Ha-ras- and c-fos-dependent signal transduction pathways (2329). This was indeed observed in a subpopulation of initiated (GST-P-positive) hepatocytes in which DNA synthesis was not affected by DHEA. Although the genotoxicity of FA has not yet been tested, the mechanistic similarity with DHEA, at least with respect to inhibition of DNA synthesis and gene expression, suggests that these compounds may share analogous effects on rat liver carcinogenesis.

In vivo studies on the effects of DHEA on G6PD activity have given contrasting results, showing a marked reduction or no change (reviewed in ref. 2). Overall reduction in G6PD activity in preneoplastic rat liver has been attributed to phenotypic shift leading to a decrease in glycogenotic (clear cell) lesions, with high G6PD activity, and an increase in amphophilic lesions, with low G6PD activity (50). Phenotypic shift has not been evaluated in this model, but several neoplastic nodules 14 and 30 weeks after initiation consist of clear cells and still exhibit high G6PD activity in DHEA- and FA-treated rats. These lesions may be pushed to progress to malignancy during long-term DHEA treatment, whereas reduced DNA synthesis in the lesions in which DHEA treatment inhibits G6PD activity hinders further evolution to malignancy. A correlation between G6PD activity and growth rate has been documented in preneoplastic and neoplastic rat liver lesions induced by N-nitrosomorpholine (51). At present the mechanisms responsible for resistance of some preneoplastic lesions to G6PD inhibitors are unknown. The great heterogeneity of preneoplastic liver lesions found in the present study could explain variations in their response to environmental conditions. For instance, these lesions exhibit a large range of susceptibility to the mitoinhibitory effect of orotic acid (52). It should be noted in this respect that, due to the genotoxicity of DHEA (17,30) and probably of FA, different sets of initiated cells could be present in livers of rats subjected to the RH protocol plus these co-carcinogens, i.e. hepatocytes initiated by diethylnitrosamine, diethylnitrosamine + DHEA (or FA) or DHEA (or FA) alone. These cells probably exhibit different growth and progression potentials, as well as different DHEA and FA metabolism, production of hydroxyl radicals and expression of the G6PD gene. Further research is necessary to clarify this point.

In conclusion, our results indicate a mechanism of rapid evolution to malignancy of preneoplastic liver lesions in DHEA-treated rats and suggest a similar effect for FA. They show that the transforming effect of long-term administration of these compounds depends on expansion of a subpopulation of early preneoplastic cells in which activation of c-Ha-ras- and c-fos-dependent signal transduction pathways and cell proliferation cannot be counteracted by inhibition of pentose phosphate synthesis. Contrasting results on the in vivo effects of DHEA and FA on G6PD activity and tumorigenicity (cf. ref. 2) could be consequent on the relative proportions of lesions responding differently to these compounds according to the carcinogen used, the experimental model and the developmental stage of the lesions. Due to the possibility that `endogeneously' initiated cells or liver cells initiated by silent viral infection or small (food-borne) carcinogen doses (cf. ref. 45) are pushed to malignant transformation by long-term DHEA administration, the therapeutic use of this compound and its derivatives (1), especially when alternative therapies are available, should be reconsidered.


    Notes
 
1 To whom correspondence should be addressed Email: feo{at}ssmain.uniss.it Back


    Acknowledgments
 
This work was supported by funds from the Associazione Italiana Ricerche sul Cancro, RAS (Assessorato della Sanità) and MURST (program 60%).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Gursoy,E., Hu,H., Cardounel,A., Regelson,W. and Kalimi,M. (2000) Biological effects of dehydroepiandrosterone: a review. In Kalimi,M. and Regelson,W. (eds), Dehydroepiandrosterone (DHEA). Biochemical, Physiological and Clinical Aspects. Walter de Gruyter, Berlin, Germany, pp. 1–29.
  2. Feo,F., Pascale,R.M., Simile,M.M. and De Miglio,M.R. (2000) Role of dehydroepiandrosterone in experimental and human hepatocarcinogenesis. In Kalimi,M. and Regelson,W. (eds), Dehydroepiandrosterone (DHEA). Biochemical, Physiological and Clinical Aspects. Walter de Gruyter, Berlin, Germany, pp. 215–236.
  3. Moore,M.A., Weber,E., Thornton,M. and Bannasch,P. (1988) Sex-dependent, tissue-specific opposing effects of dehydroepiandrosterone on initiation and modulation stages of liver and lung carcinogenesis induced by dihydroxy-di-n-propylnitrosamine in F344 rats. Carcinogenesis, 9, 1507–1509.[Abstract]
  4. Garcea,R., Daino,L., Pascale,R.M., Frassetto,S., Cozzolino,P., Ruggiu,M.E. and Feo,F. (1987) Inhibition by dehydroepiandrosterone of liver preneoplastic foci formation in rats after initiation-selection in experimental carcinogenesis. Toxicol. Pathol., 15, 164–168.[ISI]
  5. Garcea,R., Daino,L., Frassetto,S., Cozzolino,P., Ruggiu,M.E., Vannini,M.G., Pascale,R., Lenzerini,L., Simile,M.M., Puddu,M. and Feo,F. (1988) Reversal by ribo- and deoxyribonucleosides of dehydroepiandrosteroneinduced inhibition of enzyme altered foci in the liver of rats subjected to the initiation–selection process of experimental carcinogenesis. Carcinogenesis, 9, 931–938.[Abstract]
  6. Weber,E., Moore,M. and Bannasch,P. (1988) Phenotypic modulation of hepatocarcinogenesis and reduction in N-nitrosomorpholine-induced hemangiosarcoma and adrenal lesion development in Sprague–Dawley rats by dehydroepiandrosterone. Carcinogenesis, 9, 1191–1195.[Abstract]
  7. Simile,M.M., Pascale,R.M., De Miglio,M.R., Nufris,A., Daino,L., Seddaiu,M.A., Muroni,M.R., Rao,K.N. and Feo,F. (1995) Inhibition by dehydroepiandrosterone of growth and progression of persistent liver nodules in experimental rat liver carcinogenesis. Int. J. Cancer, 62, 210–215.[ISI][Medline]
  8. Bannasch,P., D'Introno,A., Leonetti,P., Metzeger,C., Klimek,F. and Mayer,D. (1998) Early aberrations of energy metabolism in carcinogenesis. In Bannasch,P., Kanduc, D., Papa,S. and Tager,G.M. (eds), Cell Growth and Oncogenesis. Birkhauser Verlag, Basel, Switzerland, pp. 191–212.
  9. Rao,M.S., Subbarao,V., Yeldandi,A.V. and Reddy,J.K. (1992) Hepatocarcinogenicity of dehydroepiandrosterone in the rat. Cancer Res., 52, 2977–2979.[Abstract]
  10. Hayashi,F., Tamura,H., Yamada,J., Kasai,H. and Suga,T. (1994) Characteristics of the hepatocarcinogenesis caused by dehydroepiandrosterone, a peroxisome proliferator, in male F-344 rats. Carcinogenesis, 15, 2215–2219.[Abstract]
  11. Metzger,C., Mayer,D., Hoffmann,H., Bocker,T., Hobe,G., Benner,A. and Bannasch,P. (1995) Sequential appearance and ultrastructure of amphophilic cell foci, adenomas, and carcinomas in the liver of male and female rats treated with dehydroepiandrosterone. Toxicol. Pathol., 23, 591–605.[ISI][Medline]
  12. Ogiu,T., Hard,G.C., Schwartz,A.G. and Magee,P.N. (1990) Investigation into the effect of DHEA on renal carcinogenesis induced in rat by single dose of DMN. Nutr. Cancer, 14, 57–67.[ISI][Medline]
  13. Tagliaferro,A.R., Davis,J.R., Truchon,S. and van Hamont,N. (1986) Effects of dehydroepiandrosterone acetate on metabolism, body weight and composition of male and female rats. J. Nutr., 116, 1977–1983.[ISI][Medline]
  14. Thornton,M., Moore,M.A. and Ito,N. (1989) Modifying influence of dehydroepiandrosterone or butylated hydroxytoluene treatment on initiation and development stages of azaserine-induced acinar pancreatic lesions in the rat. Carcinogenesis, 10, 407–410.[Abstract]
  15. Shibata,M.-A., Hasegawa,R., Imaida,K., Hagiwara,A., Ogawa,K., Hirose,M., Ito,N. and Shirai,T. (1995) Chemoprevention by dehydroepiandrosterone and indomethacin in a rat multiorgan carcinogenesis model. Cancer Res., 55, 4870–4874.[Abstract]
  16. Metzger,C., Bannasch,P. and Mayer,D. (1997) Enhancement and phenotypic modulation of N-nitrosomorpholine-induced hepatocarcinogenesis by dehydroepiandrosterone. Cancer Lett., 121, 125–131.[ISI][Medline]
  17. Orner,G.A., Mathews,C., Hendricks,J.D., Carpenter,H.M., Bailey,G.S. and Williams,D.E. (1995) Dehydroepiandrosterone is a complete hepatocarcinogen and potent tumor promoter in the absence of peroxisome proliferation in rainbow trout. Carcinogenesis, 16, 2893–2898.[Abstract]
  18. Pascale,R.M., Simile,M.M., De Miglio,M.R., Nufris,A., Seddaiu,M.A., Muroni,M.R., Danni,O., Rao,K.N. and Feo,F. (1995) Inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase activity and gene expression by dehydroepiandrosterone in preneoplastic liver nodules. Carcinogenesis, 16, 1537–1542[Abstract]
  19. Schulz,S., Klann,R.C., Schönfeld,S. and Nyce,J.W. (1992) Mechanism of cell growth inhibition and cell cycle arrest in human colonic adenocarcinoma cells by dehydroepiandrosterone: role of isoprenoid biosynthesis. Cancer Res., 52, 1372–1376.[Abstract]
  20. Rao,M.S., Reid,B., Ide,H., Subbaro,V. and Reddy,J.K. (1994) Dehydroepiandrosterone-induced peroxisome proliferation in the rat: evaluation of sex differences. Proc. Soc. Exp. Biol. Med., 207, 186–190.[Abstract]
  21. Swierczynski,J., Bannasch,P. and Mayer,D. (1996) Increase of lipid peroxidation in rat liver microsomes by dehydroepiandrosterone feeding. Biochim. Biophys. Acta, 1315, 193–198.[ISI][Medline]
  22. Swierczynski,J., Kochan,Z. and Mayer,D. (1997) Dietary {alpha}-tocopherol prevents dehydroepiandrosterone-induced lipid peroxidation in rat liver microsomes and mitochondria. Toxicol. Lett., 91, 129–136.[ISI][Medline]
  23. Guyton,K.Z., Liu,Y., Gorospe,M., Xu,Q. and Holbrook,N.J. (1996) Activation of mitogen-activated protein kinase by H2O2. Role in cell survival following oxidant injury. J. Biol. Chem., 271, 4138–4142.[Abstract/Free Full Text]
  24. Wilmer,W.A., Tan,L.C., Dicherson,J.A., Danne,M. and Rovin,B.H. (1997) Interleukin-1beta induction of mitogen-activated protein kinases in human mesangial cell. Role of oxidation. J. Biol. Chem., 272, 10877–10881.[Abstract/Free Full Text]
  25. Amstad,P., Crawford,D., Muehlematter,D., Zbinden,I. and Larsson,R. (1990) Oxidants stress induces the proto-oncogenes c-fos and c-myc in mouse epidermal cells. Bull. Cancer, 77, 501–502.[ISI][Medline]
  26. Nose,K., Shibanuma,M., Kikichi,K., Kageyama,H., Sakiyama,S. and Kuroki,T. (1991) Transcriptional activation of early-response genes by hydrogen peroxide in a mouse osteoblastic cell line. Eur. J. Biochem., 291, 99–106.
  27. Abate,C., Patel,L., Rauscher,F.J. and Curran,T. (1990) Redox regulation of fos and jun DNA-binding activity in vitro. Science, 2149, 1157–1161.
  28. Schreck,R., Rieber,P. and Baeuerle,P.A. (1991) Reactive oxygen intermediates as apparently widely used messengers in the activation of NF-{kappa}B transcription factor and HIV-1. EMBO J., 10, 2247–2258.[Abstract]
  29. Toledano,M.B. and Leonard,W.J. (1991) Modulation of transcriptor factor NF-{kappa}B binding activity by oxidation-reduction in vitro. Proc. Natl Acad. Sci. USA, 88, 4328–4332.[Abstract]
  30. Lawson,T.A. (1991) Involvement of lauric acid hydroxylase in the activation of beta-substituted nitrosamines. Cancer Lett., 59, 177–182.[ISI][Medline]
  31. Schwartz,A.G., Lewbart,M.L. and Pashko,L.L. (1988) Novel dehydroepiandrosterone analogues with enhanced biological activity and reduced side-effects in mice and rats. Cancer Res., 48, 4817–4822.[Abstract]
  32. Schwartz,A.G. and Pashko,L.L. (1993) Cancer chemoprevention with the adrenocortical steroid dehydroepiandrosterone and structural analogs. J. Cell. Biochem., 17, 73–79.
  33. Perkins,S.N., Hursting,S.D., Haines,D.C., James,S.J., Miller,B.J. and Phang,J.M. (1997) Chemoprevention of spontaneous tumorigenesis in nullizygous p53-deficient mice by dehydroepiandrosterone and its analog 16{alpha}-fluoro-5-androsten-17-one. Carcinogenesis, 18, 989–994.[Abstract]
  34. Orner,G.A., Donohoe,R.M., Hendricks,J.D., Curtis,L.R. and Williams,D.E. (1996) Comparison of the enhancing effects of dehydroepiandrosterone with the structural analog 16-{alpha}-fluoro-5-androsten-17-one on aflatoxin B1 hepatocarcinogenesis in rainbow trout. Fundam. Appl. Toxicol., 34, 132–140.[ISI][Medline]
  35. Boone,C.W. and Kelloff,G.J. (1997) Biomarker End-points in Cancer Chemoprevention Trials, IARC Scientific Publications no. 142. IARC, Lyon, pp. 273–280.
  36. Farber,E. and Sarma,D.S.R. (1987) Hepatocarcinogenesis: a dynamic cellular perspective. Lab. Invest., 56, 4–22.[ISI][Medline]
  37. Dragani,T.A., Manenti,G. and Della Porta,G. (1991) Quantitative analysis of genetic susceptibility to liver and lung carcinogenesis in mice. Cancer Res., 51, 6299–6303.[Abstract]
  38. Pascale,R.M., Simile,M.M., De Miglio,M.R., Muroni,M.R., Gaspa,L., Dragani,T.A. and Feo,F. (1996) The BN rat strain carries dominant hepatocarcinogen resistance loci. Carcinogenesis, 17, 1765–1768.[Abstract]
  39. De Miglio,M.R., Simile,M.M., Muroni,M.R., Pusceddu,S., Calvisi,D., Carru,A., Seddaiu,M.A., Daino,L., Deiana,L., Pascale,R.M. and Feo,F. (1999) Correlation of c-myc overexpression and amplification with progression of preneoplastic liver lesions to malignancy in the poorly susceptible Wistar rat strain. Mol. Carcinog., 25, 21–29.[ISI][Medline]
  40. Solt,D.B., Medline,A. and Farber E. (1977) Rapid emergence of carcinogen-induced hyperplastic lesions in a new model for the sequential analysis of liver carcinogenesis. Am. J. Pathol., 88, 595–618.[ISI][Medline]
  41. Lowry,O.K., Rosebrough,N.J., Farr,A.L. and Randall,R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 293, 265–275.
  42. Enomoto,K. and Farber,E. (1982) Kinetics of phenotypic maturation of remodeling hyperplastic nodules during liver carcinogenesis. Cancer Res., 42, 2330–2335.[Abstract]
  43. Squire,R.A. and Levitt,M.H. (1975) Report of a workshop on classification of specific hepatocellular lesions in rats. Cancer Res., 35, 3214–3223[ISI][Medline]
  44. Pugh,T.D., King,J.H., Koen,H., Nychka,D., Chover,J., Wahba,G., He,Y.-H. and Goldfarb,G. (1983) Reliable stereological method for estimating the number of microscopic hepatocellular foci from their transections. Cancer Res., 43, 261–1268.
  45. Feo,F., Pascale,R.M., Simile,M.M., De Miglio,M.R., Muroni,M.R. and Calvisi,D.F. (2000) Genetic alterations in liver carcinogenesis: implications for new preventive and therapeutic strategies. Crit. Rev. Oncogen., 11, 19–62.[ISI][Medline]
  46. Mayer,D. (1998) Carcinogenic and anticarcinogenic effects of dehydroepiandrosterone in the liver of male and female rats. Aging Male, 1, 56–66.
  47. Garcea,R., Daino,L., Pascale,R., Simile,M.M., Puddu,M., Frassetto,S., Cozzolino,P., Seddaiu,M.A., Gaspa,L. and Feo,F. (1989) Inhibition of promotion and persistent nodule growth by S-adenosyl-L-methionine in rat liver carcinogenesis: role of remodeling and apoptosis. Cancer Res., 49, 1850–1856.[Abstract]
  48. Aragno,M., Brignardello,E., Tamagno,E., Gatto,V., Danni,O. and Boccuzzi,G. (1997) Dehydroepiandrosterone administration prevents the oxidative damage induced by actute hyperglycemia in rats. J. Endocrinol., 155, 233–240.[Abstract/Free Full Text]
  49. Boccuzzi,G., Aragno,M., Seccia,M., Brignardello,E., Tamagno,E., Albano,E., Danni,O. and Bellomo,G. (1997) Protective effect of dehydroepiandrosterone against copper-induced lipid peroxidation in the rat. Free Radic. Biol. Med., 22, 1289–1294.[ISI][Medline]
  50. Mayer,D., Metzger,C., Leonetti,P., Beier,K., Benner,A. and Bannasch,P. (1998) Differential expression of key enzymes of energy metabolism in preneoplastic and neoplastic rat liver lesions induced by N-nitrosomorpholine and dehydroepiandrosterone. Int. J. Cancer, 79, 232–240.[ISI][Medline]
  51. Baba,M., Yamamoto,R., Iishi,H., Tatsuta,M. and Wada,A. (1989) Role of glucose-6-phosphate dehydrogenase enhanced proliferation of pre-neoplastic and neoplastic cells in rat liver induced by N-nitrosomorpholine. Int. J. Cancer, 43, 892–895.[ISI][Medline]
  52. Sheikh,A., Yusuf,A., Laconi,E., Rao,P.M., Rajalakshmi,S. and Sarma,D.S.R. (1993) Effect of orotic acid on in vivo DNA synthesis in hepatocytes of normal rat liver and hepatic foci/nodules. Carcinogenesis, 14, 907–912.[Abstract]
Received August 31, 2000; revised October 20, 2000; accepted October 27, 2000.





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (2)
Request Permissions
Google Scholar
Articles by Simile, M. M.
Articles by Feo, F.
PubMed
PubMed Citation
Articles by Simile, M. M.
Articles by Feo, F.