ToxSci Advance Access originally published online on June 30, 2007
Toxicological Sciences 2007 99(1):58-69; doi:10.1093/toxsci/kfm168
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Diethylnitrosamine Initiation Does Not Alter Clofibric Acid–Induced Hepatocarcinogenesis in the Rat
* Department of Drug Safety Evaluation, sanofi aventis R&D, Centre de Recherche de Vitry/Alfortville-Evry, 94403 Vitry sur Seine, France
INSERM, U567, Paris, France
14 Rossett Park Road, Harrogate, North Yorkshire HG2 9NP, United Kingdom
AstraZeneca, Safety Assessment, Macclesfield, SK10 4TG Cheshire, United Kingdom
1 To whom correspondence should be addressed at sanofi aventis R&D, Centre de Recherche de Vitry/Alfortville-Evry, Drug Safety Evaluation, 13 quai Jules Guesde, 94403 Vitry sur Seine, France. Fax: +33-1-58-93-81-97. E-mail: Eric.Boitier{at}sanofi-aventis.com.
Received April 5, 2007; accepted May 22, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Clofibric acid (CLO) is a nongenotoxic hepatocarcinogen in rodents that causes altered hepatocellular foci and/or neoplasms. Initiation by DNA-damaging agents such as diethylnitrosamine (DEN) accelerates focus and tumor appearance and could therefore significantly contribute to shortening of the regulatory 2-year rodent carcinogenicity bioassays. However, it is crucial to evaluate the histological and molecular impact of initiation with DEN on hepatocarcinogenesis promoted by CLO. Male F344 rats were given a single nonnecrogenic injection of DEN (0 or 30 mg/kg) followed by Control diet or CLO (5000 ppm) in diet for up to 20 months. Histopathology and gene expression profiling were performed in liver tumors and surrounding nontumoral liver tissues. The molecular signature of DEN was characterized and its histopathological and immunohistopathological effects on focus and tumor types were also determined. Although foci and tumors appeared earlier in the DEN + CLO–treated group compared to the group treated with CLO alone, DEN had little impact on gene expression in nontumoral tissues since the gene expression profiles were highly similar between Control and DEN-treated rats, and DEN + CLO- and CLO-treated rats. Finally, tumors obtained from DEN + CLO and CLO-treated groups displayed highly correlated gene expression profiles (r > 0.83, independently of the time-point). The pathways involved in tumor development revealed by Gene Ontology functional analysis are similar when driven either by spontaneous initiation or by a chemically induced initiation step. Our work described here may contribute to the design optimization of shorter preclinical tests for the evaluation of the nongenotoxic hepatocarcinogenic potential of drugs under development.
Key Words: peroxisome proliferators; hepatocarcinogenesis; initiation; promotion; toxicogenomics; Clofibrate; diethylnitrosamine.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Nongenotoxic hepatocarcinogenesis is a slow, multistep process, which can be accelerated by chemical or physical initiation of liver cells (Scherer and Emmelot, 1975
). Although the concept of initiation is well understood, its molecular drivers remain unclear and there is still debate on the relative contributions of chemically induced initiation, oncogene activation, tumor suppressor gene inactivation, and how these render cells more sensitive to tumor promoters. This lack of clear understanding probably underpins the persistent absence of accepted short-term tests for carcinogenic potential that could replace the 2-year rodent carcinogenicity studies required for the registration of pharmaceutical agents. Different exploratory approaches are available today for shortening this evaluation: models such as the medium-term rat liver bioassay (Ito et al., 1988) or the use of gene expression profiles for early biomarker determination (Michel et al., 2005). In this paper, we aim to clarify the influence of the DNA-damaging initiating agent diethylnitrosamine (DEN) on the development of nongenotoxic hepatocarcinogenesis, particularly in terms of focus development and of the molecular mechanisms involved in order to evaluate whether short-term carcinogenicity bioassays could be established as potential replacement in vivo models for the current regulatory prerequisites.
To characterize the histopathological and molecular influence of initiation on subsequent tumor promotion, Fisher F344 rats were given a single ip dose of DEN, an initiator that binds to DNA and induces mutations (den Engelse et al., 1983). DEN was given at a nonnecrogenic dose (30 mg/kg) (Scherer and Emmelot, 1975), therefore only exhibiting initiating properties. The glutathione-S-transferase-pi form (GST-p) is a highly sensitive liver focus biomarker that is overexpressed following DEN administration, allowing to evaluate the initiation efficiency (Moore et al., 1987). Clofibric acid (CLO), the primary metabolite of the hypolipidemic drug Clofibrate and its pharmacologically active form belongs to the class of peroxisome proliferators (PP), which have been shown to be nongenotoxic hepatocarcinogenic tumor promoters in the rodent (Mochizuki et al., 1982; Reddy and Qureshi, 1979) via the activation of the nuclear receptor/transcription factor peroxisome proliferator–activated receptor-
(Peters et al., 1997). This justifies the use of transcriptional profiling for studying the hepatocarcinogenic process induced by PP. As di(2-ethylhexyl)phthalate and Wyeth-14,643 but not Clofibrate were shown to significantly increase the mutation frequency in liver (Boerrigter, 2004), CLO was selected as a pure tumor promoter.
Together with the pathological evaluation, a global molecular snapshot of CLO-induced gene expression modulation was performed by gene expression profiling. Livers from control and DEN-treated rats presented highly correlated gene expression profiles, as were those obtained from DEN + CLO- or CLO-treated rats. Finally, tumors obtained from DEN + CLO and CLO-treated groups also exhibited molecular signatures that were strongly correlated in terms of genes modulated and functional categories involved. These results show that chemically induced initiation accelerates the molecular mechanisms of tumor promotion without modifying it.
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MATERIALS AND METHODS |
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Study design.
Details of the chemicals used and of the study design have been described elsewhere (Michel et al., 2005
). Briefly, 175 Fisher F344 rats were divided into four groups: after 1 week on basal diet, rats belonging to the DEN and DEN + CLO groups underwent ip injection of 30 mg/kg DEN, those from the Control and CLO groups received NaCl 0.9%. Twelve days after injection, rats belonging to the DEN + CLO and CLO groups received a diet containing 5000 ppm of CLO for up to 12 or 20 months, respectively. DEN and Control rats were fed on normal diet for up to 12 or 20 months, respectively. Series of five rats from each group were necropsied on days 18, 46, 102, 264, 377, and, for the CLO and Control groups, also 524, and 607 days after the injection of saline. They were fasted approximately 16 h before being anesthetized by ip injection of Pentobarbital and culled by exsanguination. Livers were immediately excised under sterile conditions. They were examined macroscopically and visible tumors were excised and stored at – 80°C for further RNA extraction and downstream gene expression profiling. The number of tumors obtained per time-point is detailed in Figure 1. Portions of nontumoral liver from all animals were collected in 10% neutral buffer formalin and embedded in paraffin for further Hematoxylin & Eosin (H&E) staining, histopathological examination, and immunohistological evaluation. Other portions were flash-frozen in liquid nitrogen for total RNA extraction. Animal experiments were conducted in accord with accepted standards (local and national regulations) of humane animal care.
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Evaluation of preneoplastic and neoplastic changes.
Liver foci, hepatocellular adenomas, and carcinomas were identified according to published criteria (Goodman et al., 1994
; Mohr, 1997). Liver foci were distinguished from adenomas by the absence of pronounced compression of the surrounding tissue and retention of lobular architecture. Foci were classified as tigroid, eosinophilic, clear cell, or basophilic, according to the predominant staining characteristics of the hepatocytes. The incidence of preneoplastic lesions was recorded using a linear semiquantitative size grade from 1 to 5 for each replicate of the group. The number and size of each type of focus were used to calculate a quantifier (Table 1).
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Liver sections from animals euthanized on days 46 and 377 were used for immunohistochemical characterization of GST-p (Biotrin, Dublin, Ireland). Briefly, sections were deparaffinized and blocked for endogenous peroxidase activity. Sections were incubated with an anti-GST-p antibody (Dako France SAS, Trappes, France) diluted at 1:300 for 1 h at room temperature, then incubated with peroxidase-conjugated goat antirabbit immunoglobulin. Slides were then incubated with peroxidase antiperoxidase conjugate, reacted with 3',3-diaminobenzidine tetrahydrochloride and 0.2% hematoxylin counter-stained. GST-p–positive foci were counted and the number of cells per focus was evaluated. For each time-point, data were pooled for the five rats from each group.
Total RNA extraction and processing.
The standard Affymetrix protocol used has been described elsewhere (Michel et al., 2005).
Gene expression data analysis.
Reproducibility and similarity of the DNA chips were evaluated on normalized intensity data with the in-house developed analysis GECKO platform (Theilhaber et al., 2004). First, the 1000 genes that varied most between conditions were taken for a principal component analysis (PCA), and the first three dimensions (these account for most of the variance between treatment groups) were inspected graphically to verify that chips of the same treatment group clustered together. All microarray processing and downstream statistical analysis were performed in Rosetta Resolver version 4 (Rosetta Biosoftware, Seattle, WA). Data for each chip were automatically background-corrected via a chip-specific error model producing expression profiles. These individual scan data processing step were then followed by additional procedures to correct interchip variability performed via the standard analysis pipelines implemented in Resolver (normalization). Mean intensity ratio profiles were calculated for a treatment group over a baseline using the five replicates per group (except for the tumor samples).
In order to evaluate the influence of the DEN-induced initiation step on the hepatocarcinogenic process, ratio analyses were performed:
- (1) For nontumor liver tissues, significantly modulated genes were selected by a two-factor ANOVA (treatment and time, p < 1.10–5) on at least three time-points for CLO versus Control, and DEN + CLO versus DEN. Significantly modulated genes obtained in the two analyses were compared and used for downstream functional pathway mapping (see below).
- (2) For tumor tissues:
- Significantly modulated genes were selected by a two-factor ANOVA (treatment and time, p < 1.10–5) on at least two time-points for CLO tumors versus CLO nontumors and DEN + CLO tumors versus DEN + CLO nontumors. Results of the two analyses were compared.
- Functional pathway mapping of tumors was carried out on genes significantly modulated by DEN + CLO or CLO treatment on day 377 (early tumors, with similar adenoma phenotype).
- (3) To understand the molecular mechanisms involved in DEN initiation, signature of DEN-modulated genes was determined by a two-factor ANOVA (treatment and time, p < 1.10–5) on at least three time-points for DEN versus Control.
- (2) For tumor tissues:
Clustering was then performed on log-transformed ratios, using the union of the gene lists identified by the two ANOVA analyses, either for comparison 1 or comparison 2a by an agglomerative algorithm (Toronen et al., 1999), using a Euclidean distance metric, and visualized as heat-maps. Green shades indicate a transcriptional downregulation of a gene, and red an upregulation, whereas black represents no modulation. The intensity of the shades represents the magnitude of the change in gene expression (Chiang et al., 2001). Pearson's correlation coefficients were calculated on the basis of linear regression between pairs of mean intensity ratio profiles representative of the various study conditions.
Gene lists were analyzed for biological processes with an in-house Gene Ontology (GO) annotation tool (Muller et al., 2005). The complete data set can be accessed at the following web site: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE2216.
Quantitative real-time PCR.
Portion of the complementary DNA synthesized during the Affymetrix labeling process was diluted 1/150 into RNAse-free water. The following genes, most statistically modulated by DEN (p < 1.10–11), were tested relative to rat ß2-microglobulin: the chaperonin containing t complex polypeptide 1 (TCP1), epoxide hydrolase 1 (EPHX1), cyclin G1 (CCNG1), O(6)-methylguanine-DNA methyltransferase (MGMT), aldehyde dehydrogenase 1A1 (ALDH1A1), and p-glycoprotein 1 (MDR1). Their primers were purchased as Assays-on-Demand (respectively, Rn00562030, Rn00563349, Rn00563907, Rn00563462, Rn00755490, and Rn00561753, Applied Biosystems, Foster City, CA). Quantitative real-time polymerization chain reaction (qRT-PCR) was performed using the ABI 7700 Sequence Detection System (Applied Biosystems) according to the manufacturer's instructions. The Taqman Universal PCR Master Mix was used. Amplification reactions were carried out using the following temperature profile: 50°C, 2 min; 95°C, 10 min (95°C, 15 s; 60°C, 1 min) for 40 cycles. Fluorescence emission was detected for each PCR cycle and the threshold cycle (Ct) values were determined. Induction or repression of a gene in a DEN-treated sample relative to Control was calculated as follows:
Fold change = log2(2–(ct treated – ct ctrl) Gene of interest/ 2– (ct treated – ct ctrl) ß2-microglobulin). Values were reported as an average of duplicate analyses.
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RESULTS |
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DEN Accelerates the Appearance of Foci and Tumors
The results presented in Table 1 show the influence of DEN initiation on the kinetics of appearance of each type of focus and of neoplastic lesion. In Controls, tigroid foci were the first type to appear on day 264 of the study and their incidence increased with time. They represented the most abundant focus type in this group. DEN injection led to an earlier appearance of all types of focus, with tigroid foci appearing on day 102. In addition, clear cell and basophilic foci appeared on day 264, eosinophilic foci appeared on day 377, instead of day 524 in Controls, respectively. No tumors were observed in either the Control or DEN groups within the study course (607 days).
CLO treatment had no influence on tigroid foci, as they were seen on day 264 in both the CLO and Control groups with similar incidence. Clear cell and eosinophilic foci appeared 264 and 377 days, respectively, after the beginning of treatment with CLO. Basophilic foci appeared on day 524 in Control and CLO-treated groups but their size was increased after CLO treatment and they were the most prominent focus type on day 607.
The influence of DEN initiation on tigroid foci was inflected by CLO promotion as they appeared later and in fewer number in the DEN + CLO group compared to the DEN group, although more prominent than in the Control or CLO groups. DEN and CLO had a synergistic effect on eosinophilic, clear cell and basophilic foci as they appeared earlier and were more prominent in the DEN + CLO group compared to all the other groups. A higher incidence and an earlier onset of hepatocellular neoplasms were also noted in the DEN + CLO group as compared to the CLO group (day 264 vs. day 377).
Observation of the number and size of GST-p–positive foci enabled the evaluation of the efficiency of the initiation step and to monitor the fate of this neoplastic marker throughout the hepatocarcinogenic process (Fig. 2). Control animals developed a few GST-p–positive foci on day 377, likely corresponding to those observed by H&E staining. Both number and size of foci increased over time for all other groups. On day 46, 13 GST-p–positive foci were observed in DEN animals. Their number and size increased with time (see Fig. 2b). No GST-p–positive foci were observed on day 46 in the CLO and DEN + CLO groups, but 15 and 35 foci were counted on day 377, respectively. Figure 2a shows that some foci of the DEN + CLO group stained for GST-p, whereas others did not. There was no significant difference in terms of number of GST-p–positive foci between DEN and DEN + CLO groups on day 377.
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As a conclusion, in the two-step carcinogenesis protocol, DEN injection accelerated the appearance of foci and tumors without detectably modifying their types (though more GST-p–positive foci were found in the DEN + CLO group than after a pure CLO promotion).
DEN + CLO and CLO Groups Exhibit Highly Correlated Liver Gene Expression Patterns
We performed a general comparison of the liver gene expression profiles obtained from the four study groups. Figure 3 represents a qualitative comparison of the raw gene expression data for each replicate of the four groups from the nontumoral or tumoral liver tissues for the 46-day and 377-day time-points. The scatter plot obtained from the PCA shows that the gene expression profiles from Control replicates cluster by time-point, suggesting good data reproducibility, as for the other groups. Three main clusters could be distinguished in this scatter plot. Whatever the time-point, gene expression profiles from the Controls clustered together with the DEN-treated animals (green circle). Second, CLO and DEN + CLO–treated animals clustered together (orange circle). Third, tumor samples clustered away (red circle) from the nontumoral liver tissues and showed a broader spread, suggesting a higher variability between the various tumor samples. The similarity of the raw gene expression data between the DEN + CLO and the CLO groups was consistent throughout the study (data not shown). This similarity led us to compare in further details the gene expression modulations observed during the hepatocarcinogenic process after a spontaneous or a chemically induced initiation.
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Genes Significantly Modulated in the DEN + CLO and the CLO Groups are Similar
This part focuses on identifying the effect of a chemically induced initiation on the CLO-induced transcriptional regulation during the hepatocarcinogenic process. Statistically modulated genes identified in the CLO-treated nontumoral groups (CLO vs. Control and DEN + CLO vs. DEN pairs) throughout the study were compared. In the CLO group (vs. Control), 495 genes were selected as significantly modulated, whereas 553 genes were identified in the DEN + CLO group (vs. DEN). Table 2 showed that 391 genes were in common between the two groups. The heat-map in Figure 4a showed that a majority of the differentially expressed genes exhibited a similar trend of modulation between the two CLO-treated groups. Since the GeneChip used contains 15,923 genes and the ANOVA p value cut-off was 1.10–5, less than one false positive was expected in each data set. This similarity was quantified by the calculation of Pearson correlation coefficients on the significantly modulated expression ratios obtained from the different sample comparisons: for a given time-point, the correlation coefficient was above 0.90 when comparing (CLO vs. Control) and (DEN + CLO vs. DEN) profiles, whereas this coefficient slightly decreased down to 0.71 when comparing different time-points. In order to place these complex lists of modulated genes in the context of underlying biological responses, we performed a statistical analysis of the significantly overexpressed (enriched) GO biological processes so that the molecular pathways affected could be identified. As shown in Figure 5a the unsupervised GO mapping analysis of the differentially regulated genes revealed that there were a majority of GO biological processes in common between the CLO and DEN + CLO nontumoral groups, namely lipid/fatty acid metabolism, fatty acid beta-oxidation, acyl-CoA metabolism, tricarboxylic acid cycle, mitochondrial electron transport, lipoprotein metabolism, lipid transport, steroid metabolism/biosynthesis, hormone metabolism, purine nucleotide metabolism, vitamin metabolism, amino acid metabolism/biosynthesis, sulfur metabolism, aromatic compound metabolism, translation, and complement activation. Four additional GO biological processes were unique to CLO-treated nontumoral liver tissues: hexose metabolism, regulation of cellular biosynthesis, amine biosynthesis, and heterocycle metabolism. DEN + CLO–treated nontumoral liver tissues exhibited two unique GO biological processes: tricarboxylic acid cycle/intermediate metabolism and cholesterol metabolism. However, all the GO biological processes could be clustered in four main functional categories: lipid and energy metabolism, nucleotide and vitamin metabolism, protein biosynthesis, and complement activation. When analyzed at this functional level, CLO and DEN + CLO groups displayed strictly identical molecular pathways.
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0.05 was required and a difference between the numbers of observed and expected genes of at least 4.As shown in Figure 4a, DEN + CLO and CLO liver tumors also presented a similar trend of gene expression modulation throughout the study, although a higher intragroup data variability was noticed compared to the nontumoral liver tissues. When comparing profiles of statistically modulated genes between DEN + CLO tumors (vs. DEN + CLO nontumors) and CLO tumors (vs. CLO nontumors) at any time-point, correlations were bigger than 0.83 (Fig. 4b). Table 2 summarizes the number of genes in common between the CLO and DEN + CLO tumors (233), the number of genes unique to CLO tumors (218) and the number of genes unique to DEN + CLO tumors (601) on day 377. The statistical GO mapping analysis of the genes significantly modulated in the tumors revealed only two GO biological processes in common between DEN + CLO and CLO liver tumors: fatty acid metabolism, and nucleobase, nucleoside, nucleotide, and nucleic acid transport. The nine GO biological processes specific to CLO tumors were cholesterol metabolism, phospholipid metabolism, hexose metabolism, electron transport, nitrogen compound metabolism, RNA processing, translation, and protein import into nucleus. As for the DEN + CLO tumors, 15 GO biological processes were unique: response to drug, cellular carbohydrate metabolism, generation of precursor metabolites and energy, amino acid and derivative metabolism, amine biosynthesis, aromatic compound metabolism, rRNA processing, regulation of translation, translation initiation, protein folding, protein import, nuclear transport, nucleo-cytoplasmic transport, nucleotide metabolism, and DNA repair. When integrating the GO biological processes at a higher level, five main functional categories were identified, three of which were in common between CLO and DEN + CLO tumors: lipid and energy metabolism, protein biosynthesis, and nucleo-cytoplasmic trafficking and nucleotide metabolism. These three categories encompassed 91% of the GO biological processes identified across CLO and DEN + CLO tumors. The two remaining functional categories, namely response to drug and DNA repair, were unique to DEN + CLO tumors. Taken together, these results indicate that CLO and DEN + CLO tumors share similar treatment-impacted molecular pathways.
DEN Modulates the Expression of a Restricted Number of Genes
The similarity of the results obtained with our approach when comparing the effect of spontaneous and chemically induced initiation on CLO promotion raised the question of the sensitivity of our analysis to evaluate the impact of a single injection of DEN on transcription regulation. The six most-regulated genes in the DEN versus Control analysis were EPHX1, ALDH1A1, MGMT, MDR1, and cyclin G1: upregulated; TCP1: downregulated. Their modulation rapidly returned to baseline on day 18 (Fig. 6a). Their DEN-induced expression modulation was confirmed by qRT-PCR, except for TCP1 (Fig. 6b). Some of them were also modulated throughout CLO treatment (Fig. 6a): e.g., cyclin G1, MGMT, and MDR1 were slightly but increasingly downregulated throughout the carcinogenic process. ALDH1A1 was highly upregulated during the first 3 months of CLO treatment, following or not DEN injection.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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The majority of published data on the initiation–promotion paradigm are more than 10 years old. Although initiation mechanisms are well known, the events leading to the promotion of these initiated cells remain unclear and no molecular data have been provided to validate the concept. Oncogene activation or tumor suppressor gene inactivation was postulated to render cells more sensitive to tumor promoters. However, creation and use of transgenic mice for these genes has complicated the picture and most of these models are insensitive to nongenotoxic promoter chemicals (Gulezian et al., 2000
). Here, we present an analysis of the histological and molecular liver changes occurring during preneoplastic and neoplastic events that take place when promotion is induced by CLO administration after an initiation step, either spontaneous or induced by the DNA-damaging agent DEN. We characterized the molecular signature of the initiation step and evaluated its influence on CLO-promoted tumors in terms of histological changes and molecular pathways modulated. This allowed us to show that, although DEN initiation favored the incidence and the early onset of hepatocellular foci and tumors in rats, it did not modify the molecular processes taking place in the liver, both in the nontumoral and tumoral tissues, under the hepatocarcinogenic effect of CLO.
Spontaneously initiated cells have been shown to be absent or rare in young Control Fisher 344 rats, but increase with age (Harada et al., 1989; Popp et al., 1985). We observed H&E and GST-p–stained foci, mainly tigroid of type, at later time-points in Controls. DEN increased the number of all focus type as compared to Controls, as described in the literature (Kraupp-Grasl et al., 1991), and specifically induced an earlier onset of eosinophilic and basophilic foci during CLO treatment, reinforcing the specific role of these foci during tumor development (Bannasch et al., 1985; Cattley and Popp, 1989; Grasl-Kraupp et al., 1993; Weber and Bannasch, 1994). Our results showing that basophilic foci appeared late upon CLO treatment were in line with what Kraupp-Grasl et al. (1990) reported in rats treated with nafenopin (another PP) for 13 months. Immunostaining for GST-p showed a significant increase of GST-p–positive foci after DEN initiation as previously described (Bralet et al., 2002). However, there was no statistically significant effect of CLO treatment on the number of GST-p–positive foci when comparing DEN + CLO and DEN groups, or CLO and Control groups, respectively. The few GST-p–positive foci found in the CLO group were in line with the findings of Rao et al. (1986), who found that 10% and 48% of foci obtained after a treatment with 0.1% Wy-14,643 and 0.025% ciprofibrate, respectively, were GST-p positive. Unlike what was shown by some authors (Grasl-Kraupp et al., 1993), our results showed that CLO did not lead to the disappearance of GST-p–positive cells. However, the only tumor present in an immunohistochemically stained liver portion (from the DEN + CLO group) was GST-p negative. This would be in favor of the specific promotion of GST-p–negative lesions by PP.
The use of a low nonnecrogenic dose of DEN in our study could explain why our results were not in agreement with other publications that showed CLO treatment induced mainly basophilic and GST-p–negative foci (Cattley et al., 1989; Grasl-Kraupp et al., 1993; Weber and Bannasch, 1994). Williams et al. (2000) recently showed that the molecular mechanisms observed with low or high exposures to the DNA-reactive carcinogens, 2-acetylaminofluorene, and DEN drastically differed. Indeed, contrary to previous assumptions, the authors demonstrated the existence of thresholds for the hepatocellular initiating effects of these carcinogens. They also attributed the exaggerated responses at high exposures to cytotoxicity and compensatory hepatocyte proliferation. These papers point out the importance of the protocol used with regard to the mechanism, and to the development of different types of preneoplastic and neoplastic lesions in rodent models. In our study, the injection of DEN was nonnecrogenic and was not followed by partial hepatectomy. The lack of regenerative cell proliferation following initiation could explain the low amount and late appearance of basophilic foci obtained after CLO promotion in our study.
We observed that CLO alone was sufficient to induce foci and further led to the appearance of tumors. As demonstrated by others, the hepatocarcinogenicity of CLO alone could be due to the promotion of either spontaneously initiated cells (Cattley et al., 1991) or foci initiated by CLO itself, although all the evaluations of the initiating properties of PP previously tested in two-stage models turned out negative (Cattley and Popp, 1989; Glauert and Clark, 1989).
In addition to histopathology, we evaluated the molecular mechanisms underlying the hepatocarcinogenic process induced by CLO, focusing particularly on the influence of the initiation step on the promotion process. Indeed, we showed that genes selected as statistically modulated by CLO treatment were similar in the CLO and the DEN + CLO nontumoral groups, i.e., after a spontaneous or a chemically induced initiation. The corresponding molecular signatures were strongly correlated. When integrated at a biological level, the genes from both groups could be mapped to identical functional categories. The most enriched functional category was lipid and energy metabolism and corresponded to the specific signature of PP (genes such as peroxisomal bifunctional enzyme, peroxisomal 3-oxoacyl-coenzyme A thiolase, stearoyl-coenzyme A desaturase and fatty acid-binding protein, see supplementary data), therefore confirming previous microarray data (Cherkaoui-Malki et al., 2001; Yamazaki et al., 2002).
At the tumor level, the raw data were more variable within each group (Fig. 4). However, when focusing on the ratios, the gene expression profiles observed for the DEN + CLO and the CLO tumors were quantitatively similar (r > 0.9). This higher intragroup variability could be related to the drift of the tumor phenotype observed during the tumor progression, shifting from the adenoma type to the carcinoma type (see Fig. 1). The variability in the tumor phenotype together with the uneven number of tumors sampled and analyzed across the study decreased the power of the statistical analysis performed. Consequently, for the intergroup comparison of the molecular processes involved in tumor development, we decided to cross-analyze early tumors of the same phenotype (adenoma, see Table 1) and selected the day 377 time-point. Unlike what was observed with the nontumoral tissues, a statistical GO mapping analysis of the genes significantly modulated in the tumors revealed only a very few number of GO biological processes in common between DEN + CLO and CLO liver tumors. The higher number of genes found statistically modulated in the DEN + CLO tumors (Table 2) are in line with the higher number of statistically significant GO biological processes identified in the analysis when compared with those revealed in the CLO tumors. Here again, this difference may be due to the difference in the number of tumors used to generate the gene lists (3 and 2, respectively) impacting the statistical power of our analyses. However, when focusing on the functional categories, the three functional pathways in common between CLO and DEN + CLO tumors (lipid and energy metabolism, protein biosynthesis, and nucleo-cytoplasmic trafficking and nucleotide metabolism) encompassed 91% of the GO biological processes identified across CLO tumors, be after a spontaneous or a chemically induced initiation. Taken together, these functional categories indicated an increase in the protein turn-over and lipid metabolic state of the tumors, typical of these highly metabolically active tissues (Hruban, 1979; Seemann et al., 2006) and in line with what is described in the literature in response to PP treatment (Leonard et al., 2006). The two functional categories specific of the DEN + CLO tumors, namely, response to drug and DNA repair, led us to investigate the molecular effect of DEN and how the genes modulated could interfere with the CLO tumorigenic process.
Gene expression analysis linked to DEN injection led to the identification of six genes specifically and transiently modulated following DEN injection: EPHX1, ALDH1A1, MGMT, MDR1, cyclin G1, and TCP1. These genes, considered as the molecular signature of DEN initiation, have already been described in the literature to be modulated by alkylating agents such as DEN (except TCP1) within the scope of a 2-week toxicogenomics study in the rat up to 14 days after toxicogenomic evaluation (Ellinger-Ziegelbauer et al., 2004). DEN administration accelerated the appearance and increased the number of hepatocellular foci and tumors of rats treated with CLO (Cattley and Popp, 1989; Oesterle and Deml, 1983; Solt and Farber, 1976) but there was no evidence for long-term effect of DEN initiation on the gene expression level. Indeed, no DEN molecular effects could be identified when comparing DEN + CLO to CLO groups at the transcriptional level. Therefore, the mobilization of the DNA repair machinery in the tumors seems to be rather more an indication that the tumor cells undergo major gene/chromosome modifications as a function of the cancer progression and try to counteract these alterations.
This study demonstrates that initiation by a nonnecrogenic dose of DEN accelerates CLO-induced hepatocarcinogenesis, but without altering the histological profiles nor inducing any long-term gene expression changes in the focus. In particular, events linked to DEN-induced initiation and leading to the earlier appearance of carcinogenic lesions did not modify the long-term transcriptional modulations induced by CLO. These results need to be validated on microdissected foci, as it is probable that the effect of DEN is restricted to a few initiated cells. Gene expression profiling of microdissected basophilic foci (Michel et al., 2003) should be of particular interest to validate the molecular mechanisms of the hepatocarcinogenic process, to identify novel cancer early biomarkers and to confirm that nonnecrogenic doses of DEN can be used to boost the hepatocarcinogenic process without modifying the molecular events leading to CLO-induced cancer. Finally, our work could serve as a basis for the development of shorter-term bioassays for the evaluation of the nongenotoxic hepatocarcinogenic potential of drugs under development.
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SUPPLEMENTARY DATA |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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ACKNOWLEDGMENTS |
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We thank A. Benevaut for performing the long-term in vivo study and the General Toxicology team for their support, particularly S. Trognon for preparing the diet. We thank Alexandre Secq for the histopathological preparations. S. Nicolas is thanked for statistical analysis. We warmly thank J. Theilhaber and A. Müller for assistance in data analysis.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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