Characterization of an Acute Molecular Marker of Nongenotoxic

Development and Evaluation of a Genomic Signature for the Prediction and Mechanistic Assessment of Nongenotoxic Hepatocarcinogens in the Rat. Mark R...
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APRIL 2005 VOLUME 18, NUMBER 4 © Copyright 2005 by the American Chemical Society

Chemical Profiles Characterization of an Acute Molecular Marker of Nongenotoxic Rodent Hepatocarcinogenesis by Gene Expression Profiling in a Long Term Clofibric Acid Study Ce´cile Michel,*,†,‡ Ruth A. Roberts,§ Chantal Desdouets,† Kevin R. Isaacs, and Eric Boitier‡ Faculte´ de Me´ decine Necker-Enfants Malades, INSERM U370, 156 rue de Vaugirard, 75730 Paris Cedex 15, France, Drug Safety Evaluation, Sanofi Aventis, Centre de Recherche de Vitry/Alfortville, 13 quai Jules Guesde, 94403 Vitry sur Seine Cedex, France, and Safety Assessment, Astra Zeneca R&D, Macclesfield, Cheshire SK10 4TG, United Kingdom Received October 25, 2004

Evaluation of the nongenotoxic potential early during the development of a drug presents a major challenge. Recently, two genes were identified as potential molecular markers of rodent hepatic carcinogenesis: transforming growth factor-β stimulated clone 22 (TSC-22) and NAD(P)H cytochrome P450 oxidoreductase (CYP-R) (1). They were identified after comparing the gene expression profiles obtained from the livers of Sprague-Dawley rats treated with different genotoxic and nongenotoxic compounds in a 5 day repeat dose in vivo study. To assess the potential of these two genes as acute markers of carcinogenesis, we investigated their modulation during a long-term nongenotoxic study in the rat using a classic initiation-promotion regime. Clofibric acid (CLO), which belongs to the broad class of chemicals known as peroxisome proliferators, was used as a nongenotoxic hepatocarcinogen. Male F344 rats were given a single nonnecrogenic injection of diethylnitrosamine (0 or 30 mg/kg) and fed a diet containing none or 5000 ppm CLO for up to 20 months. Necropsies of five rats per groups were performed at 18, 46, 102, 264, 377, 447 (control, DEN, and DEN + CLO rats), 524, and 608 days (for the CLO and control rats). Gross macroscopic and microscopic evaluation and gene expression profiling (on Affymetrix microarrays) were performed in peritumoral and tumoral liver tissues. Bioanalysis of the liver gene expression data revealed that TSC-22 was strongly down-regulated early in the study. Its underexpression was maintained throughout the study but disappeared upon CLO withdrawal. These modulations were confirmed by real-time polymerase chain reaction. However, CYP-R gene expression was not significantly altered in our study. Taken together, our results showed that TSC-22, but not CYP-R, has the potential to be an acute early molecular marker for nongenotoxic hepatocarcinogenesis in rodents.

Introduction The molecular events leading to the slow transformation of normal hepatocytes to altered hepatocellular foci * To whom correspondence should be addressed. Tel: +33.1.58.93.35.76. Fax: +33.1.58.93.81.97. E-mail: cecile_michel@ hotmail.fr.

and/or hepatocellular neoplasms in nongenotoxic hepatocarcinogenesis are still unclear, and no acute molecular markers for such nongenotoxic hepatocarcinogenic potentials (in comparison to the in vitro tests available to † ‡ §

INSERM U370. Sanofi Aventis. AstraZeneca R&D.

10.1021/tx049705v CCC: $30.25 © 2005 American Chemical Society Published on Web 04/02/2005

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detect the genotoxic potentials) are available for drugs under development. For this reason, two year rodent carcinogenesis studies are required prior to registration of many new pharmaceutical agents intended for chronic or intermittent use over 6 months of duration. These studies are often planned late in the development process and require a large quantity of the active pharmaceutical ingredient and a large number of animals. They are the most expensive studies performed for preclinical safety assessment and are often rate limiting for development. It is therefore of paramount importance to identify and characterize early hepatocarcinogenic biomarkers that could be used in short term in vivo studies to screen candidate compounds. The transcriptome hypothetically comprises in excess of 30 000 gene specific transcripts (2), each of them being a potential tool for evaluating the epigenetic events taking place during nongenotoxic hepatocarcinogenesis. The application of DNA microarrays for a number of prototypical toxicants has shown its ability to delineate mechanisms of toxicity (3, 4), and they hold great promise for identifying predictive molecular markers of toxicity. Recently, Kramer et al. described two genes as potential molecular marker candidates for rodent carcinogenicity: transforming growth factor-β stimulated clone 22 (TSC22)1 and NAD(P)H cytochrome P450 oxidoreductase (CYP-R) (1). They were selected by performing gene profiling using cDNA microarrays against fluorescentlabeled probes generated from liver mRNA obtained from Sprague-Dawley rats treated with nongenotoxic carcinogens (bemitradine, clofibrate, doxylamine, methapyrilene, and phenobarbital), genotoxic carcinogens (2acetylaminofluorene and probably tamoxifen), a nongenotoxic toxicant (4-acetylaminofluorene), or a mitogen (isoniazid) in a 5 day repeat dose in vivo study. In this paper, we evaluate how these two genes are modulated in a longterm clofibric acid (CLO)-induced nongenotoxic hepatocarcinogenesis study where Fisher 344 rats were treated for up to 608 days with 5000 ppm of CLO. The hepatocarcinogenic effect of CLO treatment was evaluated by classical histopathology. We show that TSC-22 gene expression, unlike CYP-R, is strongly modulated in the long-term hepatocarcinogenic process. We describe its putative biological involvement in such a process and discuss the characteristics required for a gene to be considered an acute molecular marker. Finally, we evaluate the potential of TSC-22 to be one of those markers.

Experimental Procedures Chemicals and Animals. CLO [2-(p-chlorophenoxy)-2-methylpropionic acid] (CAS no. 882-09-7) (purity 97%) and DEN (CAS no. 55-18-5) were purchased from Sigma-Aldrich Chimie (Saint Quentin Fallavier, France). A total of 175 7 week old Fisher F344 male rats (Iffa-Credo, L’Arbresle, France) were 1 Abbrevations: ACMS, R-amino-β-carboxymuconate--semialdehyde; AFP, R-fetoprotein; CLO, clofibric acid; CT, threshold cycle; CYPR, NAD(P)H cytochrome P450 oxidoreductase; DEN, diethylnitrosamine; Dusp1, dual specificity phosphatase 1; EST, expressed sequence tag; FDR, false discovery rate; FGF, fibroblast growth factor; FGFR4, fibroblast growth factor receptor 4; γGT, γ-glutamyltranspeptidase; GST-p, glutathione S transferase placental form; HSD11B1, 11-βhydroxysteroid dehydrogenase; KCNN, small conductance calcium activated potassium channel; MAP, mitogen activated protein; PP, peroxisome proliferator; PPARR, peroxisome proliferator activated receptor R; RIP140, receptor interacting protein 140; RT-PCR, realtime polymerase chain reaction; SOM, self-organizing map; TSC-22, transforming growth factor-β stimulated clone 22; TGFβ1, transforming growth factor β 1.

Michel et al.

Figure 1. Experimental design of the study. acclimatized for 17 days before examination for selection of healthy animals. The animals were housed individually under standard conditions of temperature (22 ( 2 °C) and humidity (55 ( 15%) with a 12 h light-dark cycle. Rats received rodent powdered diet UAR A04C (UAR, Epinay sur Orge, France) and filtered drinking water ad libitum. All animal procedures were conducted in accordance with the requirements EEC guidelines (1986) (5) and U.S. federal guidelines (1985). Experimental Design (See Figure 1). F344 rats were divided into four groups: vehicle control, DEN, DEN + CLO, and CLO. After 1 week of basal diet, rats belonging to the groups DEN and DEN + CLO underwent intraperitoneal injection of DEN (30 mg/kg body weight) dissolved in 9‰ NaCl. The two other groups were injected with 9‰ NaCl alone. At 30 mg/kg, DEN is nonnecrogenic (6), thus only exhibiting initiating properties. Twelve days after injection, the diet from rats belonging to the groups DEN + CLO and CLO was changed for a diet containing 5000 ppm CLO for up to 377 and 608 days, respectively. A series of five rats from each group were necropsied at days 18, 46, 102, 264, 377, and 447 (10 week reverse phase from day 377: no more CLO in the diet for rats belonging to the DEN + CLO group) and, for the CLO and control groups, 524 and 608 days after the injection of DEN or saline. From day 524, half of the CLO-treated rats were kept on the basal diet (12 week reverse phase) until day 608. Necropsy. Rats were fasted approximately 16 h before being anaesthetized by i.p. injection of pentobarbital (51 mg/kg body weight) and culled by exsanguination. Livers were immediately excised and weighed under sterile conditions. They were examined macroscopically, and visible lesions were excised and cut in half for histopathological examination and further RNA extraction. Portions of nontumoral liver from all animals were collected in 10% neutral buffer formalin and embedded in paraffin for further hematoxylin and eosin staining and histopathological examination. Other portions were flash-frozen in liquid nitrogen for total RNA extraction. Animals found dead during the study (three controls at days 242, 412, and 608 and two CLO-treated animals at days 542 and 595) were discarded without further evaluation. Evaluation of Preneoplastic and Neoplastic Lesions. Liver foci, hepatocellular adenomas, and hepatocellular carcinomas were identified according to published literature (7, 8). Liver foci of altered hepatocytes were distinguished from adenomas by the absence of pronounced compression of the surrounding tissue and preservation of lobular architecture. The incidence of preneoplastic and neoplastic lesions was evaluated on approximately 1 cm2 sections of each lobe for each replicate of the groups. The importance of the preneoplastic lesions was evaluated by a linear semiquantitative severity grade from 1 to 5 for each replicate of the groups to accommodate both the number and the size of foci in the sections. The mean grade was calculated as follows: mean grade ) sum n1f5(grade n × incidence of this grade n). In this paper, foci types were not specified (manuscript in preparation), and the mean grades of tigroid, eosinophilic, basophilic, and clear cell foci were summed

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Table 1. Selected in Vivo Observations and Histopathological Findings in Liver days after beginning of the study control

DEN

DEN + CLO

CLO

liver weight (g) tumors foci liver weight (g) tumors foci liver weight (g) tumors foci liver weight (g) tumors foci

mean SD mean SD 0 0 mean SD 0 0 mean SD

18 8.3 0.51 0 0 8.7 0.42 0 0 12.5 0.93 0 0 11.4 0.58 0 0

46 8.9 0.45 0 0 9.1 0.31 0 0.2 14.1 0.88 0 0 14.3 1.42 0 0

102 9.6 0.22 0 0 9.4 0.37 0 2 14.9 1.12 2/5A and 1/5C 1.8 14.5 0.69 0 0

264 10.8 0.36 0 0.2 10.9 1.10 0 2.4 15.9 0.48 3/5A 4.2 15.1 0.93 0 0.6

377 10.5 0.47 0 0.6 10.8 0.35 3.1 15.5 1.15 1/5A and 1/5C 4.2 16.4 1.06 2/5A 2.6

447 10.5 1.02 0 1.3 10.9 0.85

524

608

11.4 2.39 0 2.8

11.9a

17.9 4.31 2/5A and 2/5C 3.4

16.4 2.91 3/6C 4.7

608rev

2.29 0 1.9

9.5 0.81 8.5a 0.39 0 3.8

a Liver weights obtained after CLO withdrawal are statistically different than those of the control group at the same time point for a p value < 0.05.

to give a general mean grade. Neoplastic lesions were classified as adenomas or carcinomas, and the incidence of each type of lesion was recorded as a proportion of animals bearing that lesion. RNA Extraction and Processing. The total RNA was extracted from rat liver using the QIAGEN Rneasy Maxi kit (QIAGEN, Valencia, United States). The quality of total RNA was checked on the Agilent Biotechnologies 2100 Bioanalyzer (Agilent Technologies, Massy, France), and quantitation was performed using an Uvikon 860 Spectrophotometer (Secomam, Domont, France) at λ ) 260 nm. Fifteen micrograms of total RNA samples was labeled using standard Affymetrix protocols and then hybridized on Affymetrix rat RAE230A GeneChips [15 923 full-length cDNA + expressed sequence tag (EST), Affymetrix, Santa Clara, United States]. The arrays were scanned using the GeneArray scanner (Affymetrix), and the scanned image was quantitatively analyzed using the computer software Microarray Suite (MAS) 5.0 (Affymetrix). Gene Expression Data Analysis. A total of 176 samples were hybridized onto a microarray at least once (those not satisfying our quality control criteria were relabeled and rehybridized), and 162 were used for the analysis (not all of the time points were used). Gene expression data were analyzed using an analysis platform developed in-house (GECKO 2, ref 9). The GECKO software first enables global normalization across the various GeneChips, using a reference chip from the control group (the 75th percentile of intensities of genes on the control chip, which are called “present” by the Affymetrix MAS 5.0 algorithm, was used as the reference intensity for normalization). Two separate analyses were performed to select the statistically modulated genes. A P-FOLD algorithm (10) was used to calculate the ratios of the 15 923 genes (CLO vs control and DEN + CLO vs DEN ratios for each time point and for peritumoral and tumoral tissues) and to assign a “p value” to each gene. Ratios were transformed as follows: y ) log2(x) allowing ratios belonging to [0,1] to become negative. This simple analysis allowed an easy comparison with real-time polymerase chain reaction (RT-PCR) results. However, in the text, results are directly expressed as fold changes, with ratios belonging to [0,1] transformed as y ) -1/x. Modulated genes were also selected by a two-factor ANOVA (treatment and time), and p values were corrected for multiple testing by using a false discovery rate (FDR) selection criterion equal to 0.05. The algorithm also converted expression values to ratios for ratio-based analyses. A tool implemented in the GECKO platform allows for statistical contrast calculations, where the contrasts consisted of pairwise comparisons of intensities of the five replicates per group, at each time point separately for the selected genes. For each contrast, p values and overall expression ratios were computed. After log base 2 transformation of ratios, clustering was then performed by the self-organizing map (SOM) algorithm (11) in GECKO and

visualized as heat maps in Spotfire (Spotfire, Somerville, United States). The complete data set can be accessed at the web site http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc)GSE2216. Real Time Polymerase Chain Reaction. A portion of the cDNA synthesized in the Affymetrix labeling process was diluted 1/150 into RNAse-free water. TSC-22 was tested relative to rat β-2-microglobulin. Primers were purchased as Assays-onDemand (Applied Biosystems, Foster City, United States, respectively, Rn00560865 and Rn00564852). RT-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 during PCR amplification (Applied Biosystems) in a 20 µL reaction volume. Amplification reactions were carried out using the following temperature profiles: 50 °C, 2 min; 95 °C, 10 min; 95 °C, 15 s; and 60 °C, 1 min for 40 cycles. Fluorescence emission was detected for each PCR cycle, and the threshold cycle (CT) values were determined. The CT value was defined as the actual PCR cycle when the fluorescence signal increased above the background threshold. Induction or repression of a gene in a treated sample relative to control was calculated as follows:

fold change ) log2 (2-[CTtreated-CTctrl] TSC22/2-[CTtreated-CTctrl] β-2-microglobulin) Values were reported as an average of duplicate analyses.

Results Characterization of the CLO-Induced Hepatocarcinogenic Process. The results given in Table 1 summarized the progression of liver weight and the number of tumors and foci as a result of CLO pharmacological and hepatocarcinogenic actions during the CLO study and the recovery period. Liver weights during the time of the study were noticably similar between control and DEN groups, increasing slowly during the lifespan of the rats (+25 and +30% between day 18 and day 377 for the DEN and control groups, respectively). The liver weight of the CLO and DEN + CLO groups increased in a similar fashion but exhibited values more than 40% higher than DEN and control groups from day 18 onward. The rapid increase in liver weight occurred at the beginning of the CLO treatment (visible at 18 days). These increased liver weights were associated with CLOrelated microscopic findings (data not shown). They were similar in both CLO-treated groups and consisted of diffuse hepatocellular hypertrophy characterized by enlarged hepatocytes with eosinophilic cytoplasm. After the

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first month of CLO treatment, the normal increase in the liver weight during the lifespan took place (+23 and +43% between day 18 and day 377 for DEN + CLO and CLO groups, respectively). After stopping the treatment with CLO, the liver weight of the CLO-treated groups came rapidly back to a lower liver weight than their respective controls (9.5 and 10.9 g for the DEN + CLO recovery and DEN liver weights, respectively, and 8.5 and 11.9 g for the CLO recovery liver weights statistically smaller than the mean of the respective controls). Foci appeared spontaneously in control rats of around 11 months of age (day 264). Their incidence increased during the study to reach a mean grade of ∼1.9-2.8. Injection of the DNA damaging agent DEN led to an earlier onset and higher incidence of foci, which appeared 6 months earlier than in the control group and reached a quantity of 3.1 at day 447. In both CLO-free groups, no neoplastic lesions were observed. In CLO-treated groups, a higher incidence of hepatocellular foci and an earlier onset of neoplasms (adenomas and carcinomas) were observed in the DEN + CLO group as compared to the CLO group, respectively, 1.8 foci vs 0.6, three rats out of five bearing hepatocellular neoplasms vs none at day 264. CLO withdrawal had a pronounced effect in the CLO group: The number of foci (incidence and severity) decreased and no neoplasms persisted microscopically, but one was identified in the group at the macroscopic level (used for molecular evaluation). CLO withdrawal in the DEN + CLO group had less of an impact. Gene Expression Modulation of TSC-22 and CYP-R as a Function of the Carcinogenic Process. We were interested in evaluating if the two genes selected by Kramer et al. were significantly modulated in tumoral and nontumoral liver tissues during a long-term carcinogenic process. TSC-22 expression data were analyzed and selected as a significantly modulated gene using two different statistical methods. However, the ratios obtained for the two qualifiers corresponding to CYP-R (NADPH-cytochrome P450 reductase on the Affymetrix RAE-230A gene chip) were not statistically selected by any of the two methods. Figure 2 represents the modulation of TSC-22 in the course of a CLO-induced long-term hepatocarcinogenic process. The upper part (a) of the figure defines its modulation as determined by Affymetrix gene expression profiling. TSC-22 was highly downregulated as an acute effect of CLO treatment irrespective of chemically induced initiation: -4- and -4.72-fold in CLO vs control and DEN + CLO vs DEN ratios, respectively, 5 days after initiation of CLO treatment. This down-regulation was minimized afterward to reach -1.7-fold at 9 months of treatment for both CLO-treated groups. After CLO treatment arrest (recovery), the CLO effect on TSC-22 expression was either reversed (+1.36fold in the CLO recovery group vs control) or abolished (no modulation between DEN + CLO recovery group vs DEN). In tumors, TSC-22 was also down-regulated, although the effect of the recovery period was less clear. TSC-22 was more strongly down-regulated in the DEN + CLO tumors at day 447 after recovery than in the DEN + CLO tumors at day 377 and was slightly less downregulated in the CLO tumor obtained macroscopically at day 608 after CLO withdrawal (initiated at day 524). TSC-22 gene expression modulation was validated by RT-PCR as shown in the lower part (b) of the figure. The modulation was similar to that obtained by gene profiling although the down-regulation was slightly stronger: -5.2-

Michel et al.

Figure 2. Gene expression modulation of TSC-22 as a function of time by gene expression profiling and RT-PCR. The reverse period is represented by a dashed line. The CLO vs control ratios (-×-) are represented in orange. The expression ratios obtained from the tumors of the CLO group as compared to controls (-4-) are represented in red. The DEN + CLO vs DEN ratios (-0-) are represented in orange. The expression ratios obtained from the tumors of the CLO group as compared to the controls (-]-) are represented in blue.

and -5.9-fold in CLO vs control and DEN + CLO vs DEN ratios, respectively, 5 days after the beginning of treatment with CLO. In tumors, TSC-22 was also downregulated but less than that observed in the gene profiling results. No exact concordance between the values of the modulations was obtained, but the shapes of the modulation curves could be superimposed. Genes Clustering with TSC-22 in the Course of CLO-Induced Hepatocarcinogenic Process. We were interested in defining the biological significance of TSC22 modulation as a function of the hepatocarcinogenic process. Hierarchical clustering allows the clustering of genes that are similarly modulated. One of the dogmas is that these clustered genes should have at least a biological link or even be modulated via the same nuclear receptors. Figure 3 represents the cluster of genes whose modulation was similar to that of TSC-22 as determined by the SOM algorithm. R-Amino-β-carboxymuconate--semialdehyde (ACMS) dehydrogenase is the only enzyme known to process the

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Figure 3. Heat map visualization of the differentially regulated genes of the TSC-22 cluster as a function of the treatment time in the different groups: DEN, DEN + CLO, and CLO vs control. Two-way ANOVA was performed against the treatment, and the time point and statistically modulated genes were selected for a FDR of 0.05. Genes were clustered using a SOM algorithm. The fold changes obtained from the contrast performed between CLO vs control and DEN + CLO vs DEN were color coded. The green color indicates a transcriptional down-regulation of the gene by CLO treatment, and the red color indicates an up-regulation, whereas the black color represents no modulation. The color intensity represents the magnitude of the change in gene expression from log2 (0.25) ) -2 to log2 (4) ) 2 (51).

ACMS to acetyl CoA and thus to prevent the accumulation of quinolinate (12), a potent endogenous excitotoxin. 11-β-Hydroxysteroid dehydrogenase (HSD11B1) is a primary regulator of tissue specific glucocorticoid bioavailability (13). FGFR4 (also called tyrosine kinase related to fibroblast growth receptor 4) is specifically expressed in mature hepatocytes and binds FGF1 in liver parenchymal cells (14). The small conductance calcium activated potassium channel (KCNN2) responds to changes in intracellular calcium concentration and couples calcium metabolism to potassium flux and membrane excitability (15). Dual specificity phosphatase 1 (Dusp1) was first described as a mRNA, highly inducible by oxidative stress and heat shock (16), isolated by differential screening of a library made from normal human skin fibroblasts stressed by hydrogen peroxide. As with TSC-22, those genes were down-regulated throughout the carcinogenic process and in tumors whatever the time point. Following CLO withdrawal, their down-regulation was abolished, except for ACMSD and HSD11B1 in the CLO group where the downregulation was minimized. Some of them are even slightly up-regulated in this CLO group (KCNN2, Dusp1, and the EST XP_342321).

Discussion Recently, Kramer et al. identified potential acute molecular markers of carcinogenicity, namely, TSC-22 and CYP-R in short-term toxicity studies in rats. Here, we present an in-depth evaluation of how the two genes were modulated in the course of a long-term hepatocarcinogenesis study in CLO-treated rats. This study has been performed to better understand the mechanisms of nongenotoxic hepatocarcinogenesis and to characterize early markers of this carcinogenic process. The transcriptomic analysis and pharmacological action of CLO were clearly representative of a peroxisome proliferator (PP) (data not shown). Indeed, genes involved in peroxisomal proliferation such as peroxisomal bifunctional enzyme, peroxisomal 3-oxoacyl-coenzyme A thiolase, stearoyl-coenzyme A desaturase, and fatty acid binding protein were highly up-regulated as reported by others (17, 18). Rats treated with CLO developed hepatic tumors and the pathological findings (manuscript in preparation)

were in accordance with those published in the literature (19). As expected, hepatocarcinogenic development was faster when initiation was induced by the DNA damaging agent DEN (20-22). Finally, discontinuation of the promoting regimen was followed by a decreased incidence of preneoplastic and neoplastic lesions during a “reversal phase” as previously described by others (23). An acute molecular marker for carcinogenicity should be modulated soon after carcinogen application and be linked to the tumorigenic and not a toxic, adaptative effect of the carcinogen. Unlike Kramer’s results, our study showed that CYP-R was not modulated significantly as its expression was low and its modulation too small to be distinguished from background levels. Our data demonstrated, however, that TSC-22 was strongly down-regulated during the acute phase of the treatment with CLO, as found by Kramer et al. It is interesting to note that our results, 5 days after the beginning of the treatment, corroborate those described by Kramer et al., although different strains of rats (Fisher vs SpragueDawley) and different gene profiling platforms were used (Affymetrix instead of Incyte Pharmaceuticals). In addition, we showed that TSC-22 was significantly downregulated, as demonstrated by gene profiling and RTPCR, throughout the treatment period with CLO up to the induction of tumors by this nongenotoxic hepatocarcinogen. In tumors, the results obtained by gene profiling and RT-PCR were less concordant. In RT-PCR, the expression modulation is compared to a reference gene but it remains difficult to find a gene whose expression is not modulated by any of the parameters involved in the study, especially when working with pathological states such as tumors. So, differences observed between the two techniques in tumor gene expression modulation may be due to the variation of β-2-microglobulin expression, which was slightly down-regulated in tumors as compared to peritumoral samples (data not shown). This difficulty has been noticed by others (24). One of the features of a marker for a given mode of action or toxicity is that all of the effectors from this class should systematically modulate its expression. TSC-22 has been shown to be down-regulated by both nongenotoxic (1, 25) and genotoxic carcinogens (1). These results support the idea that this marker would not only be

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useful for nongenotoxic carcinogens but also for genotoxic carcinogens. Indeed, TSC-22 was not down-regulated by the few noncarcinogenic molecules tested so far such as the mitogen isoniazid or the nongenotoxic toxicant 4-acetylaminofluorene (1), and its modulation was dependent on the presence of a carcinogen. In peritumoral tissues, TSC22 down-regulation was abolished by CLO withdrawal. In tumors, the effect of the recovery period was less clear. These results could indicate that TSC-22 down-regulation is physiologically linked to the carcinogenic state. Indeed, TSC-22 was found to be down-regulated in human salivary gland tumors as compared to tumor-free salivary glands (26). TSC-22 coded for a leucine zipper protein able to homoor heterodimerize and then have transcription factor activity (27). This transcription factor was shown to be highly expressed during mouse development (28) and likely in a transforming growth factor β 1 (TGFβ1)independent manner. Its demonstrated role during embryogenesis led to comparison of this potential marker to others used during hepatocarcinogenesis, such as GST-p (gluthatione S transferase placental form) (29), γGT (γ-glutamyltranspeptidase), or AFP (R-fetoprotein) (30), all playing a role during carcinogenesis and development. None of these markers, however, could be used to evaluate PP carcinogenicity (31). On the contrary, TSC-22 is acutely down-regulated by carcinogens and seems to work with a broad range of carcinogens, PPs included. We showed that TSC-22 is down-regulated soon after treatment with a carcinogen and throughout the hepatocarcinogenic process induced by CLO. However, without knowledge of the exact function of the gene, it remains difficult to determine whether the modulation observed could be attributed to a primary effect related to its pharmacology or a secondary effect caused by an alteration in cellular condition. It has been shown that PPs suppress both spontaneous and TGFβ1-induced apoptosis (32, 33) and that TGFβ1 could induce apoptosis via induction of TSC-22 (34): Overexpression of TSC-22 has been clearly linked to enhancement of apoptosis (35), and we suggest that CLO could inhibit apoptosis by directly inhibiting TSC-22 expression. This direct interaction would explain why PPs also suppress TGFβ1-induced apoptosis. The apparent regression of hyperplasia (foci) following carcinogen withdrawal, observed particularly in the CLO-treated group, seemed to be accompanied by a reversion of the down-regulation of TSC-22, probably allowing an increase of apoptosis, as suggested by others (36). Taken together, these data suggest that TCS-22 down-regulation plays an important biological role during tumor development. It has been shown to be induced by the anticancer drug vesnarinone in the human salivary gland cancer cell line TYS (37). The down-regulation of ACMS dehydrogenase by a phthalate ester was reported by Fukuwatari et al. (12), which supports our results by showing that this effect is not specific for phthalate esters but can probably be extended to all PPs. It was shown that HSD11B1 deficiency produced an improved metabolic profile characterized by an increased lipid catabolism, insulin sensitivity, and reduced intracellular glucocorticoid concentration (13). FGFR4 signaling has been shown to play a role in liver cell growth and structure together with an inhibiting action on cholesterol and bile acid metabolism.

Michel et al.

FGFR4 deficient mice have also been shown to exhibit an increase in liver mass (14, 38). Dusp1 is a mitogen activated protein (MAP) kinase phosphatase, whose expression has been shown to be decreased in ovarian tumors as compared with normal tissues (39). These coregulated genes could represent a direct effect of the pharmacological action of the drug (e.g., HSD11B1 and FGFR4) but could also play a role in the cancer development (e.g., Dusp1) observed in rodents after long-term exposure to CLO. Most of these genes clustering with TSC-22, however, have never been described to be modulated by PPs but have been shown to be sensitive to glucocorticoids. For example, dexamethasone has been shown to inhibit the formation of quinolate (40), maybe by enhancing ACMS dehydrogenase activity and fibroblast growth factor (FGF) (41-43), and KCNN (15) and Dusp1 (44-46) have been shown to be regulated by glucocorticoids. TSC-22 was shown to be modulated by growth factors such as glucocorticoid receptor agonists (47, 48). Thus, the modulations observed would not be directly affected by CLO but by its inhibition of the glucocorticoid receptor or pathway. CLO activation of peroxisome proliferator activated receptor R (PPARR), for example, could lead to the release of the nuclear receptor corepressor receptor interacting protein 140 (RIP140), leaving it free to inhibit the glucocorticoid receptor (49, 50). Another explanation would be a decrease of the cellular active glucocorticoid concentration due to HSD11B down-regulation, leading to a decrease of the other genes of the cluster. A study of the genes clustering with TSC-22 indicated that its modulation seems to be linked with the metabolic variations induced by PPs, as their pharmacological action. This modulation would be driven by an inhibition of glucocorticoids through unknown mechanisms. It was difficult to decide if TSC-22 repression was linked to the carcinogenic potential of CLO (cause) or if it was an adaptative response linked to the CLO induced stress (consequence), but it is clear that TSC-22 downregulation signals a change between proliferation and apoptosis. TSC-22 fills the criteria needed to make it an acute molecular marker at least for nongenotoxic hepatocarcinogenesis in the rats as it is early and strongly down-regulated by different nongenotoxic hepatocarcinogens and probably also by the genotoxic ones. In addition, it is not down-regulated by noncarcinogenic compounds or by a carcinogenic compound used at noncarcinogenic dose. In our study, the injection of a noncarcinogenic dose of DEN (no tumor in the DEN group) did not lead to a down-regulation of TSC-22. Further studies on its biological function(s) would be needed to validate its use in regulatory processes. As nongenotoxic rodent hepatocarcinogenesis is judged to be irrelevant for humans, it would be of great interest to evaluate TSC-22 expression in human cell lines treated with rodent hepatocarcinogens (e.g., PPs). This early marker could be easily assessed after short-term in vivo studies in the early phase of the development of a new drug candidate to evaluate its hepatocarcinogenic liability and could help in the prioritization of different lead compounds.

Acknowledgment. We greatly thank A. Benevaut for performing the long-term in vivo study and the General Toxicology team for their support in this purpose, particularly S. Trognon for preparing the diet. We thank Alexandre Secq for the histopathological preparations.

Acute Marker for Nongenotoxic Hepatocarcinogenesis

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