Geranylgeraniol and ß-ionone inhibit hepatic preneoplastic lesions, cell proliferation, total plasma cholesterol and DNA damage during the initial phases of hepatocarcinogenesis, but only the former inhibits NF-
B activation
Roseli de Moura Espíndola,
Rogério Pietro Mazzantini,
Thomas Prates Ong,
Aline de Conti,
Renato Heidor and
Fernando Salvador Moreno *
Laboratory of Diet, Nutrition and Cancer, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil
* To whom correspondence should be addressed: Departamento de Alimentos e Nutrição Experimental, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Bloco 14, Av. Prof. Lineu Prestes 580, 05508-900, São Paulo, SP, Brazil. Tel: 55 11 3091 3630; Fax: 55 11 3815 4410; Email: RMORENO{at}USP.BR
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Abstract
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Chemopreventive activities of the isoprenoids geranylgeraniol (GGO) and ß-ionone (BI) were evaluated during initial phases of hepatocarcinogenesis. Rats received 8 or 16 mg/100 g body wt GGO (GGO8 and GGO16 groups) or BI (BI8 and BI16 groups), or only corn oil (CO group, controls) daily for 7 weeks. Incidence (%) and the mean number of visible hepatocyte nodules/animal were inhibited in the GGO8 (64% and 21 ± 40), GGO16 (33% and 3 ± 5), BI8 (50% and 13 ± 34) and BI16 (42% and 9 ± 19) groups compared with the CO group (100% and 34 ± 51) (P < 0.05, except for the GGO8 group). Number/cm2 liver section, mean area (mm2) and % liver section area occupied by persistent hepatic placental glutathione S-transferase positive preneoplastic lesions (PNL) were reduced in the GGO8 (11 ± 9; 0.26 ± 0.35; 2.7 ± 3.0), GGO16 (6 ± 6; 0.18 ± 0.16; 0.9 ± 0.9), BI8 (9 ± 5; 0.13 ± 0.20; 1.1 ± 1.2) and BI16 (8 ± 6; 0.08 ± 0.09; 0.6 ± 0.4) groups compared with the CO group (26 ± 18; 0.29 ± 0.34; 7.0 ± 5.5) (P < 0.05). GGO16 and BI16 groups showed smaller visible hepatocyte nodules, reduced PNL cell proliferation and total plasma cholesterol levels compared with the CO group (P < 0.05), but did not show any differences (P > 0.05) in PNL apoptosis. DNA damage expressed as comet length (µm) was reduced in the GGO8 (96.7 ± 1.5), GGO16 (94.2 ± 1.5), BI8 (97.1 ± 1.1) and BI16 (95.1 ± 1.5) groups compared with the CO group (102.1 ± 1.7) (P < 0.05). In comparison with normal animals, the CO group animals showed increased (P < 0.05) nuclear levels of nuclear factor kappa B (NF-
B) p65 subunit in hepatic cells, which were decreased (P < 0.05) in the GGO16 group animals. Anticarcinogenic actions of these isoprenoids seem to follow a doseresponse relationship. Results indicate that GGO and BI could be represented as promising chemopreventive agents against hepatocarcinogenesis. Inhibition of cell proliferation and DNA damage seems to be important for the anticarcinogenic actions of isoprenoids, while the inhibition of NF-
B activation seems to be specifically related to GGO actions.
Abbreviations: 2-AAF, 2-acetylaminofluorene; AI, apoptosis index; BI, ß-ionone; BI8, ß-ionone (8 mg/100 g body wt); BI16, ß-ionone (16 mg/100 g body wt); BrdU, 5-bromo-2'-deoxyuridine; body wt, body weight; CO, corn oil; DMSO, dimethyl sulfoxide; GGO, geranylgeraniol; GGO8, geranylgeraniol (8 mg/100 g body wt); GGO16, geranylgeraniol (16 mg/100 g body wt); GST-P, placental glutathione S-transferase; H&E, hematoxylineosin; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; LI, labeling index; N, normal; NF-
B, nuclear factor kappa B; PBS, phosphate-buffered saline; PNL, preneoplastic lesions; RH, resistant hepatocyte
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Introduction
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Several epidemiologic studies have shown consistently that high intake of fruits and vegetables presents a protective action against different types of cancer (1). Isoprenoids, widely distributed in fruits and vegetables, are a class of substances with >22000 constituents that originate from the mevalonate pathway. In vivo and in vitro protecting actions against some types of cancer were described for ß-ionone (BI) (2) (Figure 1A), a cyclic isoprenoid. Although cell culture studies utilizing diverse cancer cell lines have demonstrated the pronounced effects of geranylgeraniol (GGO) (Figure 1B), an acyclic isoprenoid, on the induction of apoptosis (35), the results of its evaluation in vivo were inconclusive (6).
A proposed mechanism for the inhibition of the development of neoplasia by isoprenoids such as GGO and BI is based on their ability to post-transcriptionally inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, thus, reducing the synthesis of cholesterol and the intermediaries of the mevalonate pathway such as farnesyl and geranylgeranyl pyrophosphates (2). These mevalonate derivatives are important for protein farnesylation and geranylgeranylation processes of certain proto-oncogenes (2). In comparison with normal cells, preneoplastic and neoplastic liver lesions present a loss in the transcriptional downregulation mechanism of HMG-CoA reductase and cholesterologenesis by sterols (7). However, this enzyme retains a sensitivity to the isoprenoid-mediated inhibition in these lesions (2). Thus, isoprenoids could prove to be potential chemopreventive agents against hepatocarcinogenesis.
The objective of this study was to evaluate the chemopreventive effects of GGO and BI during the initiation and promotion phases of chemical hepatocarcinogenesis in rats submitted to the resistant hepatocyte (RH) model. Parameters evaluated included preneoplastic lesion (PNL) development, hepatic cell proliferation, apoptosis, DNA damage, activation of nuclear factor kappa B (NF-
B) (8), and plasma cholesterol concentration, an indirect measure of HMG-CoA reductase activity.
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Materials and methods
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Chemicals
GGO was kindly provided by Eisai Co. (Tokyo, Japan). BI, diethylnitrosamine (DEN), 2-acetylaminofluorene (2-AAF), 5-bromo-2'-deoxyuridine (BrdU), dimethyl sulfoxide (DMSO), 3,3'-diaminobenzidine and low melting point agarose were purchased from Sigma (St Louis, USA). The commercial diet was purchased from Purina (Campinas, Brazil). Corn oil (CO) was obtained from Mazola® (São Paulo, Brazil). Polyclonal anti-placental glutathione S-transferase (GST-P) rabbit antibody was purchased from Medical and Biological Laboratories Co. (Tokyo, Japan). Polyclonal anti-BrdU rat antibody, secondary biotinylated antibody and streptavidinbiotinperoxidase complex (StrepABComplex/HRP Duet, mouse/rabbit) were purchased from Dako (Glostrup, Denmark). The total plasma cholesterol kit was purchased from BioSystems (Barcelona, Spain). N-PERTM and BCA protein assay kit were purchased from Pierce (Rockford, USA). Polyclonal anti-p65 antibody and secondary antibody conjugated to horse radish peroxidase were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Nitrocellulose membrane (HybondTM-C extra) and ECL chemoluminescence kit were purchased from Amersham Biosciences (Piscataway, USA). All the chemicals were of the highest available quality.
Animals and experimental protocol
Male Wistar rats from the colony of the Faculty of Pharmaceutical Sciences, initially weighing 4045 g, maintained in cages having 4 animals each, at a constant temperature (22°C), with 12 h lightdark cycle and receiving water and commercial diet ad libitum, were used.
Figure 2 illustrates the experimental protocol design. At the end of a 7 day acclimatization period, with the exception of 12 Wistar rats not submitted to any experimental procedure [normal (N) group], 60 animals were randomly divided into five experimental groups. The rats in the groups GGO8 and GGO16 were treated with GGO (8 and 16 mg/100 g body wt, respectively), and those of BI8 and BI16 groups were treated with BI (8 and 16 mg/100 g body wt, respectively). As both isoprenoids were dissolved in CO (0.25 ml/100 g body wt), animals receiving only CO (0.25 ml/100 g body wt) were used as controls (CO group). All treatments were conducted by gavage, for 7 consecutive weeks every day. Rats were submitted to the RH model of hepatocarcinogenesis as follows (9):2 weeks after the beginning of the different treatments, the animals received an intraperitoneal (i.p.) dose of DEN (20 mg/100 g body wt) for initiation. After 2 weeks, the animals received six intragastric doses of 2-AAF (3 mg/100 g body wt), the first four doses on 4 consecutive days before partial (2/3) hepatectomy and the remaining two doses (2.5 mg/100 g body wt) on day 2 and 4 thereafter. All animals were euthanized by light ether anesthesia and exsanguination 5 weeks after DEN administration. One hour before being killed, the rats received a single i.p. injection of BrdU (10 mg/100 g body wt) dissolved in DMSO and saline (1:3 v/v). The study was conducted in accordance with NIH guidelines for the care and use of laboratory animals.
Visible hepatocyte nodules counting
The rats were killed and the liver was removed from each animal, weighed and examined grossly on the surface and in 3 mm cross sections for the presence of visible hepatocyte nodules of varied sizes and a whitish or yellowish color, distinct from the hepatic parenchyma. These visible hepatocyte nodules were classified into three categories according to their diameter: <1 mm,
1 and
3 mm, and >3 mm.
Histopathologic examination
Representative fragments of each liver lobe were fixed in Carnoy's solution (60% methanol, 30% chloroform and 10% glacial acetic acid) for
24 h and embedded in paraffin. Five micrometer sections were hematoxylineosin (H&E) stained for histopathologic examination, which was conducted by an experienced pathologist. Hepatic PNL were classified as mixed (acidophilic and vacuolated or basophilic and vacuolated) foci or nodules according to the criteria established in the literature (10). Hepatocyte foci were considered PNL smaller than one hepatic lobule, whereas hepatocyte nodules comprised spherical PNL larger than one or more hepatic lobules (11).
Immunohistochemistry for GST-P and BrdU
Histological sections of the liver samples were also examined using immunohistochemical reactions in order to detect PNL (foci/nodules) positive for GST-P or hepatocytes positive for BrdU, according to the method described by Hsu et al. (12). After the removal of paraffin, the endogenous peroxidase was blocked by 3% hydrogen peroxide in phosphate-buffered saline (PBS) for 5 min. Thereafter, the sections were incubated overnight at 4°C with primary anti-GST-P rabbit antibody at a 1:1000 dilution or primary anti-BrdU rat antibody at a 1:60 dilution, in 1% BSA. Finally, the sections were incubated for 1 h with secondary biotinylated antibody, thereafter applying the streptavidinbiotinperoxidase complex. Peroxidase binding sites were detected by incubation with 3,3'-diaminobenzidine (0.5%) and hydrogen peroxide (0.1%) dissolved in PBS for
2 min at room temperature. Sections were counterstained with hematoxylin.
Hepatocyte foci and nodules that stained in a uniform and non-uniform manner for GST-P were classified as the persistent and remodelingtype, respectively (13) (Figure 3A and B). They were measured by theKS-300 program (Kontron Elektronic, Munich, Germany) using a Nikon (Microphot-FXA, Tokyo, Japan) photomicroscope connected with a microcomputer. Data were expressed as GST-P positive PNL number (n/cm2) and area (mm2), and % liver section occupied by these PNL.
In order to evaluate the BrdU labeling index (LI) 1000 hepatocytes were analyzed in each animal, of which 500 were in PNL (foci/nodules) areas and 500 in the surrounding normal tissue (14), using a light microscope (Carl Zeiss, Göttingen, Germany). BrdU LI was expressed as the number of BrdU-positive hepatocyte nuclei x100/total number of analyzed hepatocytes.
Apoptosis evaluation
Apoptosis was evaluated in H&E stained liver sections by the identification of hepatocytes and corpuscles undergoing apoptosis, according to the criteria established by Goldsworthy et al. (15). Compact cells with condensed chromatin at the nuclear periphery and with intense acidophilic cytoplasm were considered to be hepatocytes undergoing apoptosis. Corpuscles undergoing apoptosis, in turn, were characterized by acidophilic bodies, fragmentation or lack of chromatin accompanied by cytoplasmic condensation and/or fragmentation. In order to evaluate the apoptosis index (AI), 1000 hepatocytes were analyzed in each animal, of which 500 were in PNL (foci/nodules) areas and 500 in the surrounding normal tissue (14), using a light microscope (Carl Zeiss). AI was expressed as the number of hepatocytes and corpuscles undergoing apoptosis x100/total number of analyzed hepatocytes.
Total plasma cholesterol concentration
The rats were killed and blood was collected by a puncture of the abdominal aorta at the time of death. The blood was placed in centrifuge tubes containing 5 mg EDTA immediately after collection and centrifuged at 3500 g at 4°C for 10 min. Total plasma cholesterol concentration was determined using an enzymatic-spectrophotometric technique. Analysis was performed at 500 nm with a Model U 110 (Hitachi, Tokyo, Japan) spectrophotometer.
p65 western blot analysis
Nuclear protein extracts were prepared from liver samples of the experimental rats, previously stored at 78°C, using the N-PERTM reagent. Protein concentration was determined using the BCA protein assay kit. Samples of nuclear protein extracts (50 µg) were separated by electrophoresis in 10% denaturing polyacrylamide gel (SDSPAGE), in 1x Trisglycine buffer, according to the method by Laemmli (16). The proteins were then transferred from the gel onto a nitrocellulose membrane. Nitrocellulose membrane blockade was performed with PBS containing 5% powdered milk overnight at 4°C. After washing with PBS buffer containing 0.1% Tween-20, it was incubated for 2 h at room temperature with the primary anti-p65 antibody (1:1000) in PBS buffer containing 0.5% powdered milk, followed by incubation with the secondary antibody conjugated to horse radish peroxidase. The membrane was developed using the ECL chemiluminescence kit. The membrane was then exposed to an X-ray film resulting in
65 kDa bands, corresponding to the expected molecular weight of p65. In order to quantify band intensities, the BIO-RAD densitometer (Imaging Densitometer, Model GS-700) with a specific software (Molecular Analyst) was used. Control of the relative amount of the protein of interest was made by previous staining of the nitrocellulose membrane with Coomassie blue (17).
Hepatic DNA strand breakage (single cell electrophoresiscomet assay)
DNA strand breakage in samples of the right liver lobe of animals, previously stored at 78°C, was assessed using the comet assay (18). The tissues were gently homogenized in PBS at 4°C and filtered. The isolated cells were then added to a 0.8% low melting point agarose solution in PBS buffer, homogenized and immobilized on a microscope slide, which had previously received a layer of 0.5% low melting point agarose. Thereafter, these slides were transferred to the lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Trizma Base, 10% DMSO, 1% N-laurylsarcosine, pH adjusted to 10 and addition of 1% Triton X-100). After 1 h the slides were washed three times for 20 min with water. The slides were placed in a horizontal electrophoresis unit containing the running buffer (300 mM NaOH and 1 mM EDTA, pH >13) where they remained immersed for 20 min at 5°C. Electrophoresis was performed for 20 min at 0.9 V/cm at 5°C. The resulting comets were neutralized with 0.4 M Tris three times for 5 min, fixed and stained according to the method described by Nadin et al. (19), where silver is used instead of ethidium bromide. Cells extracted from the livers of rats not submitted to the RH model (N group), treated with or without hydrogen peroxide (10% final concentration, 15 min at room temperature and under sonication), were used as positive and negative controls, respectively (20). Length of the comets was evaluated using the previously described image analysis system. One hundred nucleoids per animal were randomly analyzed (50 images/slide). Coded slides were scored blindly. The viability of the liver cells was indirectly determined by analyzing the comet images after electrophoresis (21,22). The comet image was considered to be from a non-viable cell when it presented a cloudy appearance or a very small head and a tail like a balloon (necrotic or apoptotic cells). The viability of the cell suspension was considered acceptable when the frequency of such images was <2% (21,22).
Statistical analysis
Sigma Stat 2.0 (Jandel, San Rafael) program was used for the statistical analysis. Fisher's exact test was used when indicated. One-way ANOVA and Student's t-tests were used when the results presented a normal distribution and, in case this did not occur, KruskallWallis and MannWhitney's tests were used. A level of significance of P < 0.05 was applied to all cases.
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Results
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Body and liver weights
Body and liver weight values of rats treated for 7 consecutive weeks with GGO [8 mg/100 g body wt (GGO8 group) or 16 mg/100 g body wt (GGO16 group)], BI [8 mg/100 g body wt (BI8 group) or 16 mg/100 g body wt (BI16 group)], or only CO (0.25 ml/100 g body wt; controls, CO group) and submitted to the RH model of hepatocarcinogenesis are shown in Table I. No statistically significant differences (P > 0.05) were observed between the different experimental groups with respect to the final body weights and relative liver weights. The GGO8, GGO16 and BI16 groups, but not the GGO8 group, presented a higher (P < 0.05) gain in body weight compared with the CO group. All isoprenoid-treated groups presented higher (P < 0.05) absolute liver weights compared with the CO group. These results indicate that GGO and BI did not present toxicity at the used doses.
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Table I. Body and liver weights of rats treated with CO, GGO or BI and submitted to the RH model of hepatocarcinogenesis
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Incidence, mean number and size distribution of visible hepatocyte nodules
Table II presents the data on incidence, mean number/rat and size distribution of visible hepatocyte nodules. When compared with the CO group, the animals of GGO16, BI8 and BI16 groups presented a lower incidence (P < 0.05) and mean number (P < 0.05) of hepatocyte nodules. Similar results were observed in the animals of GGO8 group, although in this case, the difference did not reach a statistical significance (P > 0.05). Only the animals of GGO16 and BI16 groups presented smaller (P < 0.05) lesions compared with the CO group. These inhibitory actions of GGO and BI on visible hepatocyte nodules tended to be more pronounced at the higher dose (16 mg/100 g body wt), suggesting a doseresponse relationship.
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Table II. Quantification of visible hepatocyte nodules of rats treated with CO, GGO or BI and submitted to the RH model of hepatocarcinogenesis
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Morphometric quantification of GST-P positive PNL
GST-P is a marker of foci and nodules that can demonstrate more numbers and larger sizes of these putative PNL than other markers in various rat liver carcinogenesis studies (23). A typical and critical property of hepatocyte foci and nodules is their capability of expressing one of two options: spontaneously remodeling to a normal appearing liver by the majority (9598%) or persistence with cell proliferation and evolution to cancer by a small minority (25%) (24). Persistence of nodules could indicate a block in remodeling by differentiation (24) or apoptosis (25) and appears to be linked to an enhanced evolution of hepatocellular carcinomas (26).
Table III shows the values obtained by morphometric quantification of the number and mean area of total (persistent + remodeling), persistent or remodeling GST-P positive PNL, as well as a percentage of the histological section area occupied by these PNL. In comparison with the CO group, the GGO16 group, but not the GGO8, BI8 and BI16 groups, presented a lower (P < 0.05) number of total (persistent + remodeling) GST-P positive PNL. In comparison with the CO group, the GGO8, GGO16, BI8 and BI16 groups presented a lower (P < 0.05) number of persistent GST-P positive PNL but not (P > 0.05) of the remodeling ones. All isoprenoid-treated groups presented total (persistent + remodeling) and persistent GST-P positive PNL with smaller (P < 0.05) sizes when compared with the CO group. BI16 group, but not the other isoprenoid-treated groups, presented smaller (P < 0.05) remodeling GST-P positive PNL when compared with the CO group. Moreover, the GGO16, BI8 and BI16 groups presented total (persistent + remodeling) and persistent GST-P positive PNL that occupied a smaller (P < 0.05) area of the liver section when compared with the CO group. GGO8 group persistent GST-P positive PNL, but not the total (persistent + remodeling) ones, occupied a smaller (P < 0.05) area of the liver section when compared with the CO group. There were no differences (P > 0.05) between the CO group and the isoprenoid-treated groups with respect to the liver section area occupied by remodeling GST-P positive PNL. Altogether, these results with respect to the GGO and BI inhibitory effects on persistent GST-P positive PNL tended to be more pronounced at the higher dose (16 mg/100 g body wt), suggesting again a doseresponse relationship.
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Table III. Morphometric analysis of GST-P positive PNL of rats treated with CO, GGO or BI and submitted to the RH model of hepatocarcinogenesis
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Liver histopathologic examination
Histopathologic examination of H&E stained liver sections revealed the presence of mixed (acidophilic and vacuolated or basophilic and vacuolated) hepatocyte foci and nodules (10) in the CO group. The GGO8 and GGO16 groups presented a mostly mixed (acidophilic and vacuolated or basophilic and vacuolated) hepatocyte foci (10), whereas the BI8 and BI16 groups presented mostly mixed (acidophilic and vacuolated) hepatocyte foci. In addition, the CO group presented a slight to intense proliferation of oval cells. The GGO8, GGO16, BI8 and BI16 groups presented a slight to moderate proliferation of these cells.
Cell proliferation and apoptosis
Figure 4 shows BrdU LI of normal liver tissue and areas surrounding PNL, as well as the PNL areas themselves at the end of the 7 week experiment. BrdU LI of PNL surrounding normal tissue areas in the CO group was higher (P < 0.05) than the BrDU LI of the N group. The increase in the normal tissue area LI in the CO group is likely to be related to the fact that these animals were subjected to a 2/3 partial hepatectomy while they were on 2-AAF, a mitoinhibitor. As the mitoinhibitory effects of 2-AAF decrease, the LI in the suppressed liver begins to increase. Moreover, BrdU LI of PNL areas was higher (P < 0.05) than BrdU LI of the respective surrounding normal tissue areas in the CO group. These results agree with the finding that cell proliferation increases during hepatocarcinogenesis (27). In comparison with the CO group, the GGO16 and BI16 groups presented smaller BrdU LI in normal tissue areas surrounding PNL (P < 0.05) and in PNL areas (P < 0.05). There were no differences (P > 0.05) between the CO group and the GGO8 and BI8 groups with respect to BrdU LI in normal tissue areas surrounding PNL and in PNL areas. The GGO and BI inhibitory actions on cell proliferation occurred only at the higher dose (16 mg/100 g body wt), suggesting a doseresponse relationship as well.

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Fig. 4. Quantification of BrdU LI of normal Wistar rats and animals treated with CO, GGO or BI and submitted to the RH model of hepatocarcinogenesis. Values are means ± SEM, n = 6 (N group), 9 (CO group), 11 (GGO8 group) and 12 (GGO16, BI8 and B16 groups). Statistics by MannWhitney's test: statistically significant differences (P < 0.05), awhen compared with the N group, bwhen compared with the respective surrounding normal tissue area, cwhen compared with the CO group (controls) surrounding normal tissue area, dwhen compared with the CO group (controls) PNL area.
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Figure 5 shows the AI of normal liver tissue and areas surrounding PNL, as well as the PNL areas themselves at the end of the 7 week experiment. AI of PNL surrounding normal tissue areas in the CO group was higher (P < 0.05) than the AI of the N group. AI of PNL areas was higher (P < 0.05) than AI of the respective surrounding normal tissue areas in the CO, GGO8, GGO16, BI8 and BI16 groups. These results agree with the finding that apoptosis also increases during hepatocarcinogenesis (28). In comparison with the CO group, the BI16 group presented smaller AI, both in the normal tissue areas surrounding PNL (P < 0.05) and in PNL areas (P < 0.05). There were no differences between the CO group and the GGO8, GGO16 and BI8 groups with respect to AI, both in the normal tissue areas surrounding PNL (P > 0.05) and in PNL areas (P > 0.05).

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Fig. 5. Quantification of AI of normal Wistar rats and animals treated with CO, GGO or BI and submitted to the RH model of hepatocarcinogenesis. Values are means ± SEM, n = 6 (N group), 9 (CO group), 11 (GGO8 group) and 12 (GGO16, BI8 and B16 groups). Statistics by MannWhitney's test: statistically significant differences (P < 0.05), awhen compared with the N group, bwhen compared with the respective surrounding normal tissue area, cwhen compared with the CO group (controls) surrounding normal tissue area, dwhen compared with the CO group (controls) PNL area.
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Total plasma cholesterol
Figure 6 shows the total plasma cholesterol concentration values of all experimental groups at the end of the 7 week experiment. It can be observed that the CO group presented higher (P < 0.05) total plasma cholesterol concentrations compared with the N group. Compared with the CO group, the GGO16 and BI16 groups presented smaller (P < 0.05) total plasma cholesterol concentrations. There were no differences (P > 0.05) between the CO group and the GGO8 and BI8 groups with respect to the total plasma cholesterol concentrations. The inhibitory effects of GGO and BI on total plasma cholesterol concentrations occurred only at the higher dose (16 mg/100 g body wt), suggesting a doseresponse relationship as well.

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Fig. 6. Total plasma cholesterol concentrations of normal Wistar rats and animals treated with CO, GGO or BI and submitted to the RH model of hepatocarcinogenesis. Values are means ± SEM, n = 12 (N group), 9 (CO group), 11 (GGO8 group) and 12 (GGO16, BI8 and B16 groups). Statistics by Student's t test: statistically significant differences (P < 0.05), awhen compared with the N group, bwhen compared with the CO group (controls).
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Nuclear p65 western blot analysis
Figure 7 shows a p65 immunoblot performed with nuclear extracts of normal rat livers and entire livers (nodules + non-nodular surrounding tissues) of animals from the CO, GGO8, GGO16, BI8 and BI16 groups. In comparison with the N group, the CO group presented a 50% increase (P < 0.05) of p65 nuclear levels in hepatic cells. On the other hand, the GGO16 group presented a 50% reduction (P < 0.05) in the nuclear levels of p65 compared with the CO group. No reductions (P > 0.05) in p65 nuclear levels were observed in the other isoprenoid-treated groups compared with the CO group. This inhibitory action of GGO occurred only at the higher dose (16 mg/100 g body wt), suggesting a doseresponse relationship as well.

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Fig. 7. (A) p65 Western blot analysis performed with nuclear proteins extracted from normal Wistar rats and animals treated with CO, GGO or BI and submitted to the RH model of hepatocarcinogenesis. Representative samples from two animals of each group; a total of 6 (N and CO groups) and 4 (all isoprenoid-treated groups) animals were analyzed. (B) Membrane coomassie blue staining for protein equal loading control. (C) Quantification of p65 nuclear presence in liver samples of normal Wistar rats and animals treated with CO, GGO or BI and submitted to the RH model of hepatocarcinogenesis. Values are means ± SEM, n = 6 (N and CO groups) and 4 (all isoprenoid-treated groups). Statistics by Student's t-test: statistically significant differences (P < 0.05), awhen compared with the N group, bwhen compared with the CO group (controls).
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Hepatic DNA strand breakage
In this study, we opted for staining the formed comets with silver nitrate because of the advantages of the method, for e.g. to allow the permanent record of the experiment and independent verification of the results, as well as to avoid problems associated with fluorescence such as decay. In addition, staining with silver allows the comets to be analyzed using a simple light microscope instead of expensive and complex equipment such as fluorescence microscopes (20,29).
Table IV shows the comet length values of normal rat livers challenged with or without hydrogen peroxide as well as the entire rat livers (nodules + non-nodular surrounding tissues) of the CO, GGO8, GGO16, BI8 and BI16 groups. Normal rat liver samples challenged with hydrogen peroxide (positive comet assay controls) showed comets with increased lengths in comparison with those observed in the unchallenged ones (negative comet assay controls, P < 0.05). The CO group animals presented comets with increased lengths compared with those observed in the N group (normal rats, P < 0.05). All the isoprenoid-treated groups presented smaller comets (P < 0.05) than those observed in the CO group.
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Table IV. Lengths of comets of normal rat livers challenged witha or withoutb hydrogen peroxide (HP), as well as the livers of rats treated with CO, GGO or BI and submitted to the RH model of hepatocarcinogenesis
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Discussion
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In the present study, GGO and BI inhibited visible hepatocyte nodules and persistent GST-P positive PNL at both doses (8 and 16 mg/100 g body wt) in Wistar rats submitted to the RH model. To our knowledge, these are the first reports on the specific chemopreventive activity of GGO and BI on hepatocarcinogenesis.
Treatment of rats with GGO at doses (0.08 and 0.16% in the diet) (6) similar to those used in our study (8 and 16 mg/100 g body wt) and before the initiation with azoxymethane yielded mixed effects: at the lower dose, the isoprenoid inhibited the incidence of aberrant crypt foci, whereas at the higher dose it slightly enhanced the incidence of these PNL. On the other hand, our results show inhibitory effects on visible hepatocyte nodules and persistent GST-P PNL when GGO was administered at both doses during the initiation and promotion phases of hepatocarcinogenesis. Moreover, at the higher dose GGO tended to be more effective, suggesting a doseresponse relationship. BI presented similar inhibitory effects in the present study. Protective BI actions were also reported in a study of female SpragueDawley rats where treatment with the isoprenoid for 2 weeks before initiation with 7,12-dimethylbenz[a]anthracene and 22 weeks afterward, resulted in an inhibition in the incidence of mammary tumor and multiplicity and in increase of tumor latency (30).
In addition to being well-characterized and particularly adapted to compare the effects of compounds potentially able to modulate an ongoing carcinogenic process, one of the most positive features of the RH model is its ability to distinguish the few persistent nodules from the large remodeling ones and thus, allow studies of the nodule to cancer sequence (31). Interestingly, in our study GGO and BI nearly did not present inhibitory actions on remodeling GST-P positive PNL. On the other hand, these isoprenoids markedly inhibited the persistent ones considered to be the precursors of hepatocellular carcinomas (26). This reinforces the protective actions of GGO and BI during hepatocarcinogenesis and indicates that these protective actions by the isoprenoids occur specifically on the relevant PNL with respect to chemoprevention.
In the present study, GGO and BI were administered continuously for 7 consecutive weeks starting 2 weeks before initiation with DEN and in a period comprising promotion with 2-AAF and partial hepatectomy. BI has been shown to inhibit rat liver microsomal enzymes and it was suggested that it could antagonize the effects of procarcinogens (32). On the other hand, GGO did not inhibit rabbit liver CYP2E1 (33). Thus, BI but not GGO protective actions in our study could be eventually related to the inhibition of DEN and 2-AAF metabolism.
Tumor growth suppression by BI and other isoprenoids is attributed to both the suppression of cell division and the induction of apoptosis (2). BI has been shown to inhibit the proliferation of murine melanoma cells in vitro and in vivo (34) and induce apoptosis in leukemia, breast and colon adenocarcinoma cells (35). GGO has been shown principally to be a potent inducer of apoptosis in leukemia (3), lung cancer (4) and hepatoma (5) cells. It was suggested that apoptosis induction by GGO could be relevant for its chemopreventive activities during hepatocarcinogenesis (5).
Inhibition of cell proliferation was observed in the present study both in PNL and in the surrounding normal tissues after treatment with GGO or BI at the dose of 16 mg/100 g body wt, but not of 8 mg/100 g body wt. Similarly, in a previous study by our group, it was observed that geraniol and farnesol, when administered at a dose of 25 mg/100 g body wt during the initiation and promotion phases of the RH model, also inhibited cell proliferation in hepatic PNL (36). However, GGO and BI did not induce apoptosis either in the PNL area or in the normal surrounding area at both doses (8 and 16 mg/100 g body wt). This suggests that apoptosis induction does not seem to be involved with the isoprenoid chemopreventive activities observed in our study.
It has been described that animals submitted to the RH model present a loss in the hepatic downregulation mechanism of HMG-CoA reductase and cholesterologenesis (7), as well as increases in hepatic nodule cholesterol (37). This could explain the higher plasma cholesterol levels observed in CO animals compared with the normal ones.
Treatment of birds, rats or mice with isoprenoids such as BI, limonene or geraniol resulted in the inhibition of HMGCoA reductase and a decrease in total plasma cholesterol concentration (38,39). GGO has been shown to markedly inhibit the HMGCoA reductase activity in vitro through stimulation of its degradation (40). In our experiment, treatment of rats with 16 mg/100 g body wt. GGO or BI also resulted in a significant decrease of total plasma cholesterol concentration. Taking into account that the reduction in total cholesterol concentrations in rats treated with isoprenoids would reflect an inhibition of HMGCoA reductase activity (41), we suggest that cell proliferation by GGO and BI in our experiment could be related to inhibition of this enzyme. This would result in a reduced synthesis of intermediaries of the mevalonate pathway such as farnesyl and geranylgeranyl pyrophosphates, essential for the prenylation of proteins such as those coded by proto-oncogenes such as ras (2). Interestingly, at the dose of 8 mg/100 g body wt. GGO and BI did not inhibit either total plasma cholesterol concentration or cell proliferation.
NF-
B is an important transcription factor that is aberrantly activated in several cancer cells (8), including human hepatocarcinoma cells (42). Independent of the etiology in human hepatocarcinogenesis, activation of NF-
B is an early event that probably contributes to cell transformation (43). Recently, it has been described that in the initial phases of the RH model, NF-
B is highly activated as shown by the increased levels of hepatic nuclear p65 levels, through western blot analysis (44). Similar results were also observed in our study and this activation of NF-
B seems to be a reflection of the carcinogenic process and not caused by DEN and 2-AAF administered several weeks. Since oxidative stress could be responsible for the activation of this transcription factor in the RH model as previously suggested (44), we decided to use the comet assay, which enables the evaluation of breaks in the DNA strands and oxidized bases owing to ROS action (45). Interestingly, as previously observed (20), DNA damage was increased in the livers of Wistar rats submitted to the RH model compared with the normal rats. This DNA damage seems to be a reflection of the carcinogenic process and not caused by DEN or 2-AAF given several weeks before the rats were killed. Agents with antioxidant activity have been shown to inhibit NF-
B activity, which by itself is considered to be a potential biomarker for oxidative stress (46). However, although DNA damage was inhibited in our study by treatments at both doses with GGO or BI, isoprenoids with no pronounced antioxidant activity (47,48), only treatment with GGO at the higher dose inhibited NF-
B activation. Thus, reduced DNA damage in isoprenoid-treated rats could be due to the induction of the repair system as shown for other chemopreventive agents (49) or simply be a reflection of an inhibited carcinogenic process. Mechanisms other than inhibition of oxidative stress could probably account for the inhibition of NF-
B activation by GGO. A possible mechanism could be the interference in the processing of ras, an important activator of this transcription factor (8) or inhibition of I
B phosphorylation and degradation, as well as inhibition of p65 phosphorylation, as previously shown for the cyclic isoprenoid ursolic acid (50). These possibilities remain to be elucidated.
Polyprenoic acid, also known as acyclic retinoid, has shown efficacy in the prevention of second primary hepatocellular carcinomas (51) and has increased the 5 year survival rate of patients with these tumors (52). Polyprenoic acid presents a very similar structure compared with geranylgeranoic acid, which is also a potential chemopreventive agent and a derivative of GGO (53). Thus, this reinforces the protective actions of GGO observed in our study and suggests that this isoprenoid could be relevant for the chemoprevention of liver cancer in humans.
Altogether, the results of the present study indicate that GGO and BI could be represented as promising chemopreventive agents against hepatocarcinogenesis. Furthermore, inhibition of cell proliferation and DNA damage seems to be important for the anticarcinogenic actions of isoprenoids, while inhibition of NF-
B activation seems to be specifically related to the protective actions of GGO.
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Acknowledgments
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The authors thank Associate Prof. Maria L.Z. Dagli for the histopathological examination and Silvania M.P. Neves for providing the care and maintenance of the animals. The authors are also indebted to Eisai Co. Ltd (Japan) for the donation of geranylgeraniol. This study was supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico).
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Received November 5, 2004;
revised February 1, 2005;
accepted February 6, 2005.