BACKGROUND
di--phthalate  is a commonly used plasticizer in polyvinylchloride  formulations which have a number of applications, especially in food packaging, medical devices or cosmetics.
phthalates are not chemically bound to pvc and can migrate from pvc-containing products to the environment, resulting in significant environmental contamination and human exposure  <cit> .
dehp experiments have revealed toxicities including carcinogenesis and endocrine-disrupting effects, but no genotoxicity has been recorded  <cit> .
dehp is capable of disturbing the reproductive process by mimicking or antagonizing steroid hormone action  <cit>  and its effects on testosterone, luteinizing hormone or estrogen-like activity have been reported  <cit> .
dehp has been shown to decrease free testosterone levels in humans after occupational exposure  <cit>  and thyroid hormone levels in adult men otherwise exposed  <cit> .
dehp has been classified as a peroxisomal proliferator and as a non-genotoxic carcinogen in animals  <cit> .
experimental studies using rodents and in vitro assays showed that dehp and its active metabolite mehp -phthalate) can interact with nuclear receptors like pparα  <cit>  or pparγ  <cit> .
oxidative stress, as a result of peroxisome proliferation, and dna damage have been described in the human prostate adenocarcinoma cell line lncap  <cit>  and the mouse leydig tumor cell line ma- <dig>  <cit>  exposed to high concentrations of dehp .
peroxisome proliferation is one of the mechanisms that produce liver tumors in rats or mice, but this mechanism was not judged to be relevant in humans  <cit> .
the liver is not the sole target for dehp carcinogenicity: testicular tumors  <cit>  and pancreatic acinar adenomas have also been reported  <cit> .
other studies have pointed out that peroxisome proliferation is not a necessarily pathway in the carcinogenicity of dehp  <cit>  and more liver tumors occurred in pparα-null mice than in wild type animals  <cit> .
transcriptional changes independent of pparα were also found in rats and mice exposed to dehp  <cit> .
several non-pparα mechanisms were addressed: activation of p <dig> mitogen-activated protein kinase not involved in peroxisome proliferations  <cit> ; stimulation of growth regulatory pathways, mitogen-activated protein kinase, extracellular signal-regulated kinase and p <dig> phosphorylation  <cit> .
other mechanisms related to non-genotoxic carcinogenicity, like inhibition of gap junctional intercellular communication  <cit>  or inhibition of apoptosis, were reported.
apoptosis was shown to be suppressed by dehp through different pathways.
an interference with the cytokine tgf-β <dig>   <cit>  or with tnf-α  has been described  <cit> .
an increased level of bcl- <dig> and negative regulation of c-myc expression has been related to inhibition of apoptosis in syrian hamster embryo cells treated with  <dig> μm of dehp  <cit> .
several authors have demonstrated that dehp and its active metabolite mehp induce morphological transformation of she cells  <cit> , indicating the carcinogenic potency of the two chemicals.
although phthalate toxicity has been extensively investigated over the past  <dig> years, the mechanisms of dehp carcinogenicity have not been elucidated.
it was recently stated by the international agency for research on cancer  that ppar-independent mechanisms of dehp carcinogenesis are necessary to be studied .
the choice of cellular models and methodologies is critical to the study of the phenomenon of carcinogenesis.
syrian hamster embryo cells are a relevant model for mechanistic studies of chemical carcinogenicity.
she cells, unlike mouse and rat cells, are less responsive to peroxisomal proliferation and, in this respect, more similar to human cells.
she cells are normal, diploid, genetically stable and primary cells which are metabolically competent for procarcinogen activation.
therefore they are used to study mechanisms of in vitro carcinogenesis  <cit> .
she cells are obtained from embryos after removal of the differentiated tissues, and the population is mainly composed of epithelial and fibroblastic cells  <cit> .
she cells from colonies having been morphologically transformed after short exposure to chemical carcinogens induced tumours when transplanted back into hamsters  <cit> .
this validated the model and the cell transformation criteria for in vitro carcinogenicity.
recently, the she cell transformation assay has been recommended by oecd in  <dig> as in vitro method of screening chemical carcinogens on the basis of its performances to detect non-genotoxic as well as genotoxic carcinogens .
the aim of this work was to use a global transcriptomic approach to understand the molecular mechanisms of cell transformation induced by dehp in she cells.
the objectives were to identify changes in gene expression occurring in the early steps of cell transformation as well as pathway disturbances that may trigger a carcinogenic process.
a characterization of the genes expressed in she cells at dehp concentrations inducing cell transformation may give information on ppar-independent mechanisms and alternative pathways of dehp carcinogenicity.
the transcriptomic changes induced by dehp in she cells were analyzed in the first hours of exposure.
we focused secondly on changes of cytoskeleton-related genes underlying morphological transformation in she cells.
indeed, cell transformation is expressed by the alteration of cell morphology, a disorganized pattern of colony growth and the acquisition of anchorage-independent growth which is predictive of their ability to induce tumors when injected into syngenic animals  <cit> .
despite the central role of the actin cytoskeleton throughout the life cycle, little is known about the gene expression changes involved in deregulation of its dynamic in the first stages of tumorigenesis.
cytoskeleton defects in relation to cancer have been mostly studied in the late stages of cell invasion and metastasis.
differential display was chosen to identify differentially-expressed genes in she cells and to explore the entire genome.
the mrna differential display described by liang and pardee  <cit>  is a powerful approach for transcriptomic analysis.
this methodology has become popular as a tool for non-model organisms because of lack of requirement of previous genomic information about the species of interest.
as the genome of hamster is partly characterized so far, differential display appeared quite appropriate to study dehp dose-dependent effects in she cells.
we applied the current methodology that uses a combination of  <dig> "anchored" oligo-dt primers  and  <dig> "arbitrary" primers of 13-mers.
the  <dig> primer combination allowed us to obtain a level of 95% gene coverage  <cit> .
dd was applied to cells exposed for  <dig> hrs to dehp.
genes corresponding to differentially-expressed fragments were characterized.
differential display allowed us to screen a set of differentially-expressed fragments in treated cells, among which  <dig> genes were identified as targeted by a  <dig> hr-dehp exposure.
these genes were involved in such functions as transcription signalling pathways, cytoskeleton regulation, apoptosis, metabolism.
as differential display is a semi-quantitative method, the expression changes of the genes we were interested in, were checked by qpcr using hamster specific primers.
qpcr was applied to rnas not only from  <dig> hr-treated cells, but also from cells treated for  <dig> hrs in order to study the cell response in the meantime.
we particularly focused on changes of cytoskeleton-related genes underlying morphological transformation in she cells.
the objective was to explain from a mechanistic point of view the gene expression changes after dehp exposure.
to the best of our knowledge, this exercise has never been done previously.
RESULTS
identification of dehp-responsive genes using differential display
the differential display technique was used to identify genes differentially expressed in she cells, after  <dig> hrs of treatment with dehp.
an illustration of differentially-expressed fragments is given in figure  <dig> which shows gels obtained after the dd protocol and highlights fragments regulated more than 2-fold by dehp.
using  <dig> anchored primers and  <dig> arbitrary primers,  <dig> differentially expressed fragments were identified .
among these transcripts,  <dig>  showed homology to known genes in the refseq database  of genbank, while  <dig>  had no homology or homology to hypothetical proteins.
the sequences of the fragments obtained by dd have been deposited in the genbank dbest database .
these  <dig> fragments corresponded to  <dig> genes that are listed in table  <dig> with their accession numbers and the tblastx expected .
these genes were classed according to  <dig> biological functions with reference to the go process database.
these functions included signal transduction and transcription, cytoskeleton regulation, xenobiotic metabolism, apoptosis, lipidogenesis, protein conformation or transport and cell cycle.
the regulation of the cytoskeleton was one of the most impacted pathways.
indeed,  <dig> genes involved in this function were differentially expressed after dehp exposure.
ten genes were up-regulated, and  <dig> were down-regulated.
a6101
g3801
a3501
a3502
c3501
g3502
1e-106
2e-21
7e-114
c6603
c6604
9e-08
c0101
c1501
1e-14
c7103
g4101
c6301
a3901
g3601
the dehp effect is identified by  for  <dig> -fold over-expression,  for 10-fold over-expression,  for  <dig> -fold under-expression and  for  <dig> -fold under-expression for at least one dose of dehp.
the  <dig> differential display fragments showing no match after comparison with the refseq database were not listed in the table.
id  represents the internal reference of the sequence used before the identification of differential display sequence.
transcription and signal transduction is another biological process targeted by dehp treatment.
we found  <dig> up-regulated genes, among which  <dig> were up-regulated more than 10-fold .
heat- shock response related genes  and the genes involved in promoter methylation  were up-regulated.
on the other hand,  <dig> genes were down-regulated .
xenobiotic metabolism genes such as cytochromes and glutathione s-transferases were also found to be differentially expressed, indicating a mobilization of cellular defence and detoxication systems.
an up-regulation of cyp1b <dig> and cyp2e <dig> was registered, whereas cyp2f <dig> was found to be down-regulated.
concerning gst, the pi family was over-expressed while the theta and mu families were down-regulated.
differential display results confirmed the down-regulation of c-myc and showed down-regulation of p <dig>  a down-regulation of pro-apoptotic genes  and an over-expression of anti-apoptotic genes  were also observed.
expression of genes involved in the regulation of the cytoskeleton by qpcr
the mrna level of the  <dig> genes involved in the regulation of the cytoskeleton that were identified as differentially expressed after  <dig> hrs was confirmed by qpcr.
the expression of these genes was also studied after exposure to dehp for  <dig> hrs.
out of the  <dig> genes, four  were significantly up-regulated by dehp treatment after  <dig> hrs of exposure  and one  was significantly down-regulated.
a clear dose-response relationship was observed for these  <dig> genes.
after  <dig> hrs, these changes were confirmed for  <dig> genes .
nevertheless, the down- and up-regulation was more pronounced after  <dig> hrs than after  <dig> hrs of dehp exposure, for nrp <dig> and kif <dig> respectively.
for instance, in cells exposed to  <dig> μm of dehp, kif <dig> was up-regulated 17-fold at  <dig> hrs versus 3-fold at  <dig> hrs.
after  <dig> hrs,  <dig> other genes were significantly up-regulated  by a factor ranging from  <dig>  to  <dig>  with a dose-related effect .
eight other genes were significantly down-regulated , with an expression ratio between  <dig>  and  <dig>  .
all these genes were down-regulated in a dose-dependent manner, except for cdh <dig>  enah, ctnnbip <dig>  lrrc8a and snx <dig>  a threshold was observed with the latter genes .
ctnnbip <dig> was significantly down-regulated only for the lowest dose of dehp .
although they had been identified as differentially expressed in dd, five genes  were not shown to be significantly over- or under-expressed by qpcr.
yet the expression profiles of these genes indicated a dose-related increase for tubb2b, β-actin and pleckha <dig> but below the qpcr  <dig> -fold threshold.
as for thy <dig> and nid <dig>  the dose-related decrease was inferior to  <dig> .
expression of apoptosis-related genes, ppars and cyp <dig> genes after dehp treatment
the expression level of bcl- <dig> and c-myc mrna was used as controls of dehp effects.
an increased level of bcl- <dig> after  <dig> hrs of exposure and a decreased level of c-myc after  <dig> hrs  were observed according to qpcr, as expected.
p <dig> was down-regulated in a dose- and time-dependent manner; a significant decrease of the mrna level was found after  <dig> hrs at  <dig> μm dehp.
none of the ppar genes was identified as being differentially expressed by dd after dehp exposure.
in order to check these results, we measured the mrna level of pparα, ppar β/δ and ppar γ, by qpcr using hamster specific primers.
no change in the expression of these genes was observed by qpcr after  <dig> or  <dig> hrs of exposure with dehp in our study conditions .
the same verification was carried out for cyp <dig> genes.
neither differential display nor qpcr allowed us to identify significant expression changes compared to the control.
discussion
the ddrt-pcr  technique was used in the present study to identify the differential mrna expression patterns between control and dehp-treated she cells.
indeed, this technique is still a method of choice for non-sequenced or partially-sequenced organisms and is able to identify nonabundant, rare or novel transcripts  <cit> .
using differential display, we found  <dig> genes whose expression was altered by dehp treatment .
the concentrations studied  were in the range of concentrations that induced a morphological transformation of she cells, i.e.
concentrations up to  <dig> μm for mikalsen et al.
<cit>  and in the range  <dig> μm- <dig> μm for cruciani et al.
<cit> .
we measured the mrna level of genes involved in the regulation of the cytoskeleton using qpcr.
this focus is justified by the fact that the modifications of cytoskeleton organization are early events in cell neoplastic process  <cit>  and can be recorded in she cells after  <dig> days of exposure to carcinogenic agents in cell transformation assays.
morphological transformation affects a few percentage of the mixed population of she cells and all cell types  <cit> .
from the present work, we can assume that the differentially-expressed genes measured in the first  <dig> hrs of exposure reflect the first targets of dehp in the entire she cell population.
the transcriptomic changes which were recorded correspond to the integrated mean of the cell responses significantly different in the exposed populations , without consideration of cell specificity and sensitivity to dehp.
these significant expression changes in genes involved in cytoskeleton regulation, can be seen as early indicators of disturbances that will lead to cell transformation further in a few percentage of the most susceptible cells of the she population.
the role of the cytoskeleton has been extensively studied in relation to invasion and metastasis, but little is known of its implication in the first stages of carcinogenesis.
the identification of genomic changes associated with the triggering of cell transformation is useful from a mechanistic point of view and may be valuable in screening.
effects on cytoskeleton-related genes
dehp was shown to affect several functions related to the cytoskeleton.
the genes involved in cytoskeleton regulation and identified by differential display are listed in table  <dig>  to summarize, dehp affects actin polymerisation and stabilization, as well as cell-to-cell and cell-to-matrix adhesion processes.
the expression of genes involved in organelle transport, in cytoskeleton remodelling, or adhesion in response to external factors was also modified by dehp.
these results are in line with the recent findings of posnack et al.
<cit>  who identified disturbances in mechanical adhesion function and protein trafficking in rats cardiomyocytes exposed to dehp.
kif23*
cdh3*
kif23*
col1a1*
crip1*
calml3*
dclk1*
cttnbp2*
plekha5
tubb2b
thbs*
flrt2*
cdh3*
has2*
enah*
lrcc8a*
snx6*
ctnnbip1*
nid2
this table summarizes the genes studied using qpcr.
indicates significant effects of dehp  at concentration specified in brackets.
a trend for up- or down-regulation was found for the other genes .
actin polymerization and stabilization
to summarize the basic process, actin polymerization requires the arp2/ <dig> complex that needs to be stabilized by enable homolog  and is regulated by coronins.
enah is involved in the dynamic reorganization of the actin cytoskeleton, and stimulates nucleation and polymerization  <cit> .
coronins act on f-actin binding and bundling activities, but are able to inhibit the activity of arp2/ <dig> complex  <cit> .
actin polymers also require cortactin, which stabilizes nucleation sites for actin branching and elongation  <cit> .
crip <dig> facilitates actin filament bundling and stabilizes actin interaction with α-actinin too  <cit> .
linkage of actin polymers to adherens junctions, mainly composed of the transmembrane proteins cadherins, is insured through binding to α-catenin and β-catenin  <cit> .
based on the gene expression data generated, we have tried to synthesize the effects of dehp on actin organisation and cell adhesion specifically .
a 5- and 24-hrs exposure to dehp over-expressed coronin 1c , resulting in f-actin disassembly  <cit> .
disorganization was amplified by under-expression of enah involved in actin nucleation and polymerization, and expression of cttnbp <dig> that counteracts cortactin which is known to stabilize the actin network.
on the other hand, the binding of actin filaments to cadherins through catenin links appears to be reinforced owing to under-expression of ctnnbip <dig>  and over-expression of crip <dig>  which intensifies fixation to actinin.
globally, the effects of dehp on actin cytoskeleton disturb actin polymerization while intensifying binding on actinin and catenins.
posnack et al.
<cit>  explored dehp effects on rats cardiomyocytes in a range of concentrations two and three orders of magnitude higher than here.
they found an over-expression of actinin, α-catenin and n-cadherin in a concentration-dependent manner.
cell-cell and cell-matrix adhesion
cell-cell adhesion and cell-matrix adhesion were also affected by dehp treatment.
the decrease in the p-cadherin  mrna level after  <dig> hrs of exposure indicates that dehp weakened cell-cell contact, after a transient increase at  <dig> hrs of exposure for all doses tested.
weakening of cell-matrix adhesion may result from a decrease in the hyaluronan synthase  <dig>  mrna level  <cit>  and in thrombospondin , an adhesive protein that interacts with fibronectin, laminin, integrins and collagen  <cit> .
loss of cell adhesion may also be explained by over-expression of coro1c because this gene negatively regulates cell-matrix adhesion through focal adhesion kinase -mediated signalling  <cit> .
also, under-expression of enah, which is known to be involved in the control of cellular adhesion by the recruitment of proteins containing sh3- domain  <cit> , contributes to the loss of cell-cell adhesion.
in addition, dehp may lessen extracellular matrix adhesion by reducing the expression level of a number of transmembrane proteins involved in cell-matrix connections: fibronectin leucine rich  <dig>  and leucine rich repeat 8a   <cit> , nidogen  <dig>  which connects laminin- <dig> to the matrix  <cit> , and thy- <dig>  which mediates fibroblastic adhesion  <cit>  and is thbs <dig> expression-dependent  <cit> .
on the other hand, dehp effects reinforce the extra-cellular matrix through an over-expression of col1a <dig> increasing collagen.
this effect may be seen as a compensatory reaction to the weakening of cell-to-matrix link proteins by dehp.
sobarzo et al.
<cit>  demonstrated an up-regulation of n-cadherin and α-catenin in rat testis after  <dig> and  <dig> days of dehp treatment, suggesting also a deregulation of cell adhesion molecules in seminiferous tubules.
dehp decreases the response to external factors, such as the vascular endothelial growth factor  or the epidermal growth factor  through under-expression of neuropilin  <dig>  and sorting nexin  <dig>  respectively.
nrp <dig> is a membrane receptor capable of binding vegf and semaphorins, therefore its under-expression may inhibit cell adhesion and migration via the loss of integrins  <cit> .
snx <dig> is able to interact with egf receptor and transforming growth factor -β receptor  <cit> .
under-expression of snx <dig> and thbs <dig> may lead to decreased interaction with latent tgf binding protein  in the upstream of the tgf-β pathway contributing to the repression of the tgf-β signaling pathway  <cit> .
under-expression of tgf-β is known to decrease apoptosis in rodent hepatocytes treated with peroxisome proliferators   <cit> .
organelle transport and cytoskeleton remodelling
dehp also interferes with functions of microtubules .
kif <dig>  which encodes a kinesin protein, was highly over-expressed after  <dig> hrs and  <dig> hrs of dehp exposure.
kif <dig> has been shown to transport membranous organelles and protein complexes from cell nucleus to cell periphery in a microtubule- and atp-dependent manner.
doublecortin-like kinase  is a microtubule-associated protein encoding a ca2+/calmodulin-dependent kinase.
its activities on binding and microtubule polymerization facilitate cell motility by remodelling the microtubule cytoskeleton  <cit> .
over-expression of dclk at  <dig> hrs of dehp treatment is in line with an increased trend in β-tubulin .
calmoduline-like  <dig>  was over-expressed after  <dig> hrs of dehp exposure.
calmodulin  is a calcium-binding protein that translates the ca2+ signal into a wide variety of cellular processes, including the regulation of cytoskeleton remodelling acting with caldesmon  <cit>  or with wnt pathway  <cit> .
calml <dig> is a cam family member protein which increases cell motility by stabilizing and increasing myosin- <dig> for cell migration  <cit> .
other genes involved in signal transduction pathways and cytoskeleton regulation
we measured an over-expression level of phosphatidylinositol 3-kinase r <dig>  using differential display and qpcr.
pi3k is a key signalling molecule in the pip <dig> signalling transduction pathway and in actin reorganization and cell adhesion  <cit>  and is able to regulate the synthesis of collagen i  <cit> .
an activation of pi3k is also associated with a phosphorylation-dependent activation of akt which contributes to tumorigenesis and metastasis  <cit> .
the over-expression of pi3kr <dig> can be related to the under-expression of ctnnbip <dig> which interacts with β-catenin.
in addition to the function of β-catenin in the actin cytoskeleton, its role in the regulation of akt pathway activation  <cit>  or in wnt pathway regulation  <cit>  is advanced.
this protein forms part of a complex that captures growth and proliferation signals from the cell surface and is then activated to stimulate the expression of genes involved in cell proliferation.
it would be worth studying β-catenin-dependent transcription in relation to carcinogenicity.
dehp effects in the she model compared to rats and mice
while the expression of cyp1b <dig> and cyp2e <dig> was up-regulated and cyp2f <dig> under-expressed, no change in expression level of cyp <dig> genes was found using dd and qpcr after dehp exposure in our experimental conditions.
cyp <dig> genes are said to be involved in peroxisome proliferation  <cit> .
eveillard et al.
<cit>  who studied the involvement of dehp in lipidogenesis in rats, found a slight increase in the pparα level after  <dig> days of oral exposure to dehp .
they registered a significant increase in cyp <dig> levels after  <dig> days  <cit>  and after  <dig> days  <cit>  of exposure.
on the other hand, we found no increased mrna level of cyp <dig> and ppar genes in dehp-treated she cells.
this underlines that the genes expression changes noted in the present study are independent of ppars induction.
eveillard et al.
<cit>  found that induced expression of cyp2b <dig> by dehp was also independent of pparα induction but car dependent.
no change in car expression was registered in she cells, which may explain why no change in cyp2b <dig> was noted.
our results are consistent with the study of ren et al.
<cit>  who identified dehp regulated genes independent of pparα and car in rats and mice.
in our study, lipogenesis and xenobiotic metabolism pathways were impacted by dehp, but not in a major prior way.
this may be explained by the lower sensitivity of the hamster model compared with rats and mice to peroxisome proliferators  <cit> .
indeed, the syrian hamster model presents an intermediate response between rats or mice and humans who are known to be non-responsive to pp induction  <cit> .
the hamster model, like humans, is less responsive to pp induction than rats and mice, which is an advantage for mechanistic studies of pp effects and for screening human chemical carcinogens.
on the other hand, three genes  and  <dig> gene isoforms  were commonly found in our study and those carried out by eveillard et al.
<cit> , suggesting a pattern of response specific to dehp.
takashima et al.
<cit>  also found similar responses in dehp-treated mice.
up-regulation of rab1b, a ras oncogene family member involved in cellular signal transduction or survival, was found in the latter study and in the present one.
β-tubulin was clearly over-expressed in mice, a trend which was noted in our study.
some gene isoforms of cadherin, nidogen, cyp <dig> family genes or lim domain were also impacted in the liver of mice exposed to dehp  <cit> .
dehp effects on transcription factors
other genes identified by differential display and involved in transcription and signal transduction pathways or apoptosis were also targeted by dehp.
a significant under-expression of p <dig> was found after  <dig> hrs of dehp exposure using differential display and qpcr.
this under-expression is in line with the anti- apoptotic effects of dehp.
we confirmed the over-expression of bcl- <dig> after  <dig> hrs and the under-expression of c-myc after  <dig> hrs, events reported in a previous study on dehp treated she cells in conditions similar to the present ones  <cit> .
map kinases such as mapk <dig>  mapk <dig> and mapk <dig> were targeted by dehp.
further investigations of map kinase pathways could be relevant due to their involvement in activities of transcription factors.
the g protein-coupled estrogen receptor  was found to be over-expressed in differential display.
gper can be activated by estrogen-like compounds and its effect on cytoskeleton architecture has been reported  <cit> .
because of its implication in the regulation of mapk  <cit>  or tgf-β pathways  <cit> , it would be worthwhile to investigate gper further.
performances of dd
the confirmation of differentially-expressed genes by qpcr showed that the expression levels of more than 75% of genes identified by dd were confirmed by qpcr.
a comparative table of the sensitivity of dd versus qpcr is given in additional file  <dig>  qpcr is more likely to quantify subtle changes in the expression level of mrnas at different concentrations while dd seems to be more sensitive but is less discriminating.
to summarize, 35% of the genes identified as differentially expressed in dd gave the same response at the same dehp concentrations with qpcr while 40% were detected by dd at a lower dehp concentration than with qpcr.
CONCLUSIONS
transcriptional responses of she cells to dehp were studied in conditions inducing the cell neoplastic transformation, in order to identify gene expression changes in relation with effects of this non-genotoxic carcinogen.
functions impacted by dehp were found to be ppar-independent.
effects on cytoskeleton related genes indicated disturbances on actin polymerization and stabilization, cell-cell and cell-matrix adhesion and protein trafficking.
this is the first study that elucidates the genomic changes of dehp on the organization of the cytoskeleton.
whether the expression changes of cytoskeleton-related genes identified here such as coro1c, nrp <dig>  kif <dig>  are specific to dehp or to cell transforming agents more generally would require further studies.
to answer, the gene sets identified as significantly over- or under-expressed in this study must be explored on other non-genotoxic carcinogens to identify biomarkers predictive of early events in the multistep carcinogenic process.
early disturbances in the expression of cytoskeleton-related genes should be considered good candidates.

 di--phthalate is a commonly used plasticizer in polyvinylchloride formulations which have a number of applications, especially in food packaging, medical devices or cosmetics. phthalates are not chemically bound to pvc and can migrate from pvc-containing products to the environment, resulting in significant environmental contamination and human exposure. dehp experiments have revealed toxicities including carcinogenesis and endocrine-disrupting effects, but no genotoxicity has been recorded. dehp is capable of disturbing the reproductive process by mimicking or antagonizing steroid hormone action and its effects on testosterone, luteinizing hormone or estrogen-like activity have been reported. dehp has been shown to decrease free testosterone levels in humans after occupational exposure and thyroid hormone levels in adult men otherwise exposed. dehp has been classified as a peroxisomal proliferator and as a non-genotoxic carcinogen in animals. experimental studies using rodents and in vitro assays showed that dehp and its active metabolite mehp -phthalate) can interact with nuclear receptors like pparα or pparγ. oxidative stress, as a result of peroxisome proliferation, and dna damage have been described in the human prostate adenocarcinoma cell line lncap and the mouse leydig tumor cell line ma- exposed to high concentrations of dehp. peroxisome proliferation is one of the mechanisms that produce liver tumors in rats or mice, but this mechanism was not judged to be relevant in humans. the liver is not the sole target for dehp carcinogenicity: testicular tumors and pancreatic acinar adenomas have also been reported. other studies have pointed out that peroxisome proliferation is not a necessarily pathway in the carcinogenicity of dehp and more liver tumors occurred in pparα-null mice than in wild type animals. transcriptional changes independent of pparα were also found in rats and mice exposed to dehp. several non-pparα mechanisms were addressed: activation of p mitogen-activated protein kinase not involved in peroxisome proliferations; stimulation of growth regulatory pathways, mitogen-activated protein kinase, extracellular signal-regulated kinase and p phosphorylation. other mechanisms related to non-genotoxic carcinogenicity, like inhibition of gap junctional intercellular communication or inhibition of apoptosis, were reported. apoptosis was shown to be suppressed by dehp through different pathways. an interference with the cytokine tgf-β or with tnf-α has been described. an increased level of bcl- and negative regulation of c-myc expression has been related to inhibition of apoptosis in syrian hamster embryo cells treated with μm of dehp. several authors have demonstrated that dehp and its active metabolite mehp induce morphological transformation of she cells, indicating the carcinogenic potency of the two chemicals. although phthalate toxicity has been extensively investigated over the past years, the mechanisms of dehp carcinogenicity have not been elucidated. it was recently stated by the international agency for research on cancer that ppar-independent mechanisms of dehp carcinogenesis are necessary to be studied. the choice of cellular models and methodologies is critical to the study of the phenomenon of carcinogenesis. syrian hamster embryo cells are a relevant model for mechanistic studies of chemical carcinogenicity. she cells, unlike mouse and rat cells, are less responsive to peroxisomal proliferation and, in this respect, more similar to human cells. she cells are normal, diploid, genetically stable and primary cells which are metabolically competent for procarcinogen activation. therefore they are used to study mechanisms of in vitro carcinogenesis. she cells are obtained from embryos after removal of the differentiated tissues, and the population is mainly composed of epithelial and fibroblastic cells. she cells from colonies having been morphologically transformed after short exposure to chemical carcinogens induced tumours when transplanted back into hamsters. this validated the model and the cell transformation criteria for in vitro carcinogenicity. recently, the she cell transformation assay has been recommended by oecd in as in vitro method of screening chemical carcinogens on the basis of its performances to detect non-genotoxic as well as genotoxic carcinogens. the aim of this work was to use a global transcriptomic approach to understand the molecular mechanisms of cell transformation induced by dehp in she cells. the objectives were to identify changes in gene expression occurring in the early steps of cell transformation as well as pathway disturbances that may trigger a carcinogenic process. a characterization of the genes expressed in she cells at dehp concentrations inducing cell transformation may give information on ppar-independent mechanisms and alternative pathways of dehp carcinogenicity. the transcriptomic changes induced by dehp in she cells were analyzed in the first hours of exposure. we focused secondly on changes of cytoskeleton-related genes underlying morphological transformation in she cells. indeed, cell transformation is expressed by the alteration of cell morphology, a disorganized pattern of colony growth and the acquisition of anchorage-independent growth which is predictive of their ability to induce tumors when injected into syngenic animals. despite the central role of the actin cytoskeleton throughout the life cycle, little is known about the gene expression changes involved in deregulation of its dynamic in the first stages of tumorigenesis. cytoskeleton defects in relation to cancer have been mostly studied in the late stages of cell invasion and metastasis. differential display was chosen to identify differentially-expressed genes in she cells and to explore the entire genome. the mrna differential display described by liang and pardee is a powerful approach for transcriptomic analysis. this methodology has become popular as a tool for non-model organisms because of lack of requirement of previous genomic information about the species of interest. as the genome of hamster is partly characterized so far, differential display appeared quite appropriate to study dehp dose-dependent effects in she cells. we applied the current methodology that uses a combination of "anchored" oligo-dt primers and "arbitrary" primers of 13-mers. the primer combination allowed us to obtain a level of 95% gene coverage. dd was applied to cells exposed for hrs to dehp. genes corresponding to differentially-expressed fragments were characterized. differential display allowed us to screen a set of differentially-expressed fragments in treated cells, among which genes were identified as targeted by a hr-dehp exposure. these genes were involved in such functions as transcription signalling pathways, cytoskeleton regulation, apoptosis, metabolism. as differential display is a semi-quantitative method, the expression changes of the genes we were interested in, were checked by qpcr using hamster specific primers. qpcr was applied to rnas not only from hr-treated cells, but also from cells treated for hrs in order to study the cell response in the meantime. we particularly focused on changes of cytoskeleton-related genes underlying morphological transformation in she cells. the objective was to explain from a mechanistic point of view the gene expression changes after dehp exposure. to the best of our knowledge, this exercise has never been done previously.  identification of dehp-responsive genes using differential display the differential display technique was used to identify genes differentially expressed in she cells, after hrs of treatment with dehp. an illustration of differentially-expressed fragments is given in figure which shows gels obtained after the dd protocol and highlights fragments regulated more than 2-fold by dehp. using anchored primers and arbitrary primers, differentially expressed fragments were identified. among these transcripts, showed homology to known genes in the refseq database of genbank, while had no homology or homology to hypothetical proteins. the sequences of the fragments obtained by dd have been deposited in the genbank dbest database. these fragments corresponded to genes that are listed in table with their accession numbers and the tblastx expected. these genes were classed according to biological functions with reference to the go process database. these functions included signal transduction and transcription, cytoskeleton regulation, xenobiotic metabolism, apoptosis, lipidogenesis, protein conformation or transport and cell cycle. the regulation of the cytoskeleton was one of the most impacted pathways. indeed, genes involved in this function were differentially expressed after dehp exposure. ten genes were up-regulated, and were down-regulated. a6101 g3801 a3501 a3502 c3501 g3502 1e-106 2e-21 7e-114 c6603 c6604 9e-08 c0101 c1501 1e-14 c7103 g4101 c6301 a3901 g3601 the dehp effect is identified by for -fold over-expression, for 10-fold over-expression, for -fold under-expression and for -fold under-expression for at least one dose of dehp. the differential display fragments showing no match after comparison with the refseq database were not listed in the table. id represents the internal reference of the sequence used before the identification of differential display sequence. transcription and signal transduction is another biological process targeted by dehp treatment. we found up-regulated genes, among which were up-regulated more than 10-fold. heat- shock response related genes and the genes involved in promoter methylation were up-regulated. on the other hand, genes were down-regulated. xenobiotic metabolism genes such as cytochromes and glutathione s-transferases were also found to be differentially expressed, indicating a mobilization of cellular defence and detoxication systems. an up-regulation of cyp1b and cyp2e was registered, whereas cyp2f was found to be down-regulated. concerning gst, the pi family was over-expressed while the theta and mu families were down-regulated. differential display results confirmed the down-regulation of c-myc and showed down-regulation of p a down-regulation of pro-apoptotic genes and an over-expression of anti-apoptotic genes were also observed. expression of genes involved in the regulation of the cytoskeleton by qpcr the mrna level of the genes involved in the regulation of the cytoskeleton that were identified as differentially expressed after hrs was confirmed by qpcr. the expression of these genes was also studied after exposure to dehp for hrs. out of the genes, four were significantly up-regulated by dehp treatment after hrs of exposure and one was significantly down-regulated. a clear dose-response relationship was observed for these genes. after hrs, these changes were confirmed for genes. nevertheless, the down- and up-regulation was more pronounced after hrs than after hrs of dehp exposure, for nrp and kif respectively. for instance, in cells exposed to μm of dehp, kif was up-regulated 17-fold at hrs versus 3-fold at hrs. after hrs, other genes were significantly up-regulated by a factor ranging from to with a dose-related effect. eight other genes were significantly down-regulated, with an expression ratio between and. all these genes were down-regulated in a dose-dependent manner, except for cdh enah, ctnnbip lrrc8a and snx a threshold was observed with the latter genes. ctnnbip was significantly down-regulated only for the lowest dose of dehp. although they had been identified as differentially expressed in dd, five genes were not shown to be significantly over- or under-expressed by qpcr. yet the expression profiles of these genes indicated a dose-related increase for tubb2b, β-actin and pleckha but below the qpcr -fold threshold. as for thy and nid the dose-related decrease was inferior to. expression of apoptosis-related genes, ppars and cyp genes after dehp treatment the expression level of bcl- and c-myc mrna was used as controls of dehp effects. an increased level of bcl- after hrs of exposure and a decreased level of c-myc after hrs were observed according to qpcr, as expected. p was down-regulated in a dose- and time-dependent manner; a significant decrease of the mrna level was found after hrs at μm dehp. none of the ppar genes was identified as being differentially expressed by dd after dehp exposure. in order to check these results, we measured the mrna level of pparα, ppar β/δ and ppar γ, by qpcr using hamster specific primers. no change in the expression of these genes was observed by qpcr after or hrs of exposure with dehp in our study conditions. the same verification was carried out for cyp genes. neither differential display nor qpcr allowed us to identify significant expression changes compared to the control. discussion the ddrt-pcr technique was used in the present study to identify the differential mrna expression patterns between control and dehp-treated she cells. indeed, this technique is still a method of choice for non-sequenced or partially-sequenced organisms and is able to identify nonabundant, rare or novel transcripts. using differential display, we found genes whose expression was altered by dehp treatment. the concentrations studied were in the range of concentrations that induced a morphological transformation of she cells, i.e. concentrations up to μm for mikalsen et al. and in the range μm- μm for cruciani et al.. we measured the mrna level of genes involved in the regulation of the cytoskeleton using qpcr. this focus is justified by the fact that the modifications of cytoskeleton organization are early events in cell neoplastic process and can be recorded in she cells after days of exposure to carcinogenic agents in cell transformation assays. morphological transformation affects a few percentage of the mixed population of she cells and all cell types. from the present work, we can assume that the differentially-expressed genes measured in the first hrs of exposure reflect the first targets of dehp in the entire she cell population. the transcriptomic changes which were recorded correspond to the integrated mean of the cell responses significantly different in the exposed populations, without consideration of cell specificity and sensitivity to dehp. these significant expression changes in genes involved in cytoskeleton regulation, can be seen as early indicators of disturbances that will lead to cell transformation further in a few percentage of the most susceptible cells of the she population. the role of the cytoskeleton has been extensively studied in relation to invasion and metastasis, but little is known of its implication in the first stages of carcinogenesis. the identification of genomic changes associated with the triggering of cell transformation is useful from a mechanistic point of view and may be valuable in screening. effects on cytoskeleton-related genes dehp was shown to affect several functions related to the cytoskeleton. the genes involved in cytoskeleton regulation and identified by differential display are listed in table to summarize, dehp affects actin polymerisation and stabilization, as well as cell-to-cell and cell-to-matrix adhesion processes. the expression of genes involved in organelle transport, in cytoskeleton remodelling, or adhesion in response to external factors was also modified by dehp. these results are in line with the recent findings of posnack et al. who identified disturbances in mechanical adhesion function and protein trafficking in rats cardiomyocytes exposed to dehp. kif23* cdh3* kif23* col1a1* crip1* calml3* dclk1* cttnbp2* plekha5 tubb2b thbs* flrt2* cdh3* has2* enah* lrcc8a* snx6* ctnnbip1* nid2 this table summarizes the genes studied using qpcr. indicates significant effects of dehp at concentration specified in brackets. a trend for up- or down-regulation was found for the other genes. actin polymerization and stabilization to summarize the basic process, actin polymerization requires the arp2/ complex that needs to be stabilized by enable homolog and is regulated by coronins. enah is involved in the dynamic reorganization of the actin cytoskeleton, and stimulates nucleation and polymerization. coronins act on f-actin binding and bundling activities, but are able to inhibit the activity of arp2/ complex. actin polymers also require cortactin, which stabilizes nucleation sites for actin branching and elongation. crip facilitates actin filament bundling and stabilizes actin interaction with α-actinin too. linkage of actin polymers to adherens junctions, mainly composed of the transmembrane proteins cadherins, is insured through binding to α-catenin and β-catenin. based on the gene expression data generated, we have tried to synthesize the effects of dehp on actin organisation and cell adhesion specifically. a 5- and 24-hrs exposure to dehp over-expressed coronin 1c, resulting in f-actin disassembly. disorganization was amplified by under-expression of enah involved in actin nucleation and polymerization, and expression of cttnbp that counteracts cortactin which is known to stabilize the actin network. on the other hand, the binding of actin filaments to cadherins through catenin links appears to be reinforced owing to under-expression of ctnnbip and over-expression of crip which intensifies fixation to actinin. globally, the effects of dehp on actin cytoskeleton disturb actin polymerization while intensifying binding on actinin and catenins. posnack et al. explored dehp effects on rats cardiomyocytes in a range of concentrations two and three orders of magnitude higher than here. they found an over-expression of actinin, α-catenin and n-cadherin in a concentration-dependent manner. cell-cell and cell-matrix adhesion cell-cell adhesion and cell-matrix adhesion were also affected by dehp treatment. the decrease in the p-cadherin mrna level after hrs of exposure indicates that dehp weakened cell-cell contact, after a transient increase at hrs of exposure for all doses tested. weakening of cell-matrix adhesion may result from a decrease in the hyaluronan synthase mrna level and in thrombospondin, an adhesive protein that interacts with fibronectin, laminin, integrins and collagen. loss of cell adhesion may also be explained by over-expression of coro1c because this gene negatively regulates cell-matrix adhesion through focal adhesion kinase -mediated signalling. also, under-expression of enah, which is known to be involved in the control of cellular adhesion by the recruitment of proteins containing sh3- domain, contributes to the loss of cell-cell adhesion. in addition, dehp may lessen extracellular matrix adhesion by reducing the expression level of a number of transmembrane proteins involved in cell-matrix connections: fibronectin leucine rich and leucine rich repeat 8a, nidogen which connects laminin- to the matrix, and thy- which mediates fibroblastic adhesion and is thbs expression-dependent. on the other hand, dehp effects reinforce the extra-cellular matrix through an over-expression of col1a increasing collagen. this effect may be seen as a compensatory reaction to the weakening of cell-to-matrix link proteins by dehp. sobarzo et al. demonstrated an up-regulation of n-cadherin and α-catenin in rat testis after and days of dehp treatment, suggesting also a deregulation of cell adhesion molecules in seminiferous tubules. dehp decreases the response to external factors, such as the vascular endothelial growth factor or the epidermal growth factor through under-expression of neuropilin and sorting nexin respectively. nrp is a membrane receptor capable of binding vegf and semaphorins, therefore its under-expression may inhibit cell adhesion and migration via the loss of integrins. snx is able to interact with egf receptor and transforming growth factor -β receptor. under-expression of snx and thbs may lead to decreased interaction with latent tgf binding protein in the upstream of the tgf-β pathway contributing to the repression of the tgf-β signaling pathway. under-expression of tgf-β is known to decrease apoptosis in rodent hepatocytes treated with peroxisome proliferators. organelle transport and cytoskeleton remodelling dehp also interferes with functions of microtubules. kif which encodes a kinesin protein, was highly over-expressed after hrs and hrs of dehp exposure. kif has been shown to transport membranous organelles and protein complexes from cell nucleus to cell periphery in a microtubule- and atp-dependent manner. doublecortin-like kinase is a microtubule-associated protein encoding a ca2+/calmodulin-dependent kinase. its activities on binding and microtubule polymerization facilitate cell motility by remodelling the microtubule cytoskeleton. over-expression of dclk at hrs of dehp treatment is in line with an increased trend in β-tubulin. calmoduline-like was over-expressed after hrs of dehp exposure. calmodulin is a calcium-binding protein that translates the ca2+ signal into a wide variety of cellular processes, including the regulation of cytoskeleton remodelling acting with caldesmon or with wnt pathway. calml is a cam family member protein which increases cell motility by stabilizing and increasing myosin- for cell migration. other genes involved in signal transduction pathways and cytoskeleton regulation we measured an over-expression level of phosphatidylinositol 3-kinase r using differential display and qpcr. pi3k is a key signalling molecule in the pip signalling transduction pathway and in actin reorganization and cell adhesion and is able to regulate the synthesis of collagen i. an activation of pi3k is also associated with a phosphorylation-dependent activation of akt which contributes to tumorigenesis and metastasis. the over-expression of pi3kr can be related to the under-expression of ctnnbip which interacts with β-catenin. in addition to the function of β-catenin in the actin cytoskeleton, its role in the regulation of akt pathway activation or in wnt pathway regulation is advanced. this protein forms part of a complex that captures growth and proliferation signals from the cell surface and is then activated to stimulate the expression of genes involved in cell proliferation. it would be worth studying β-catenin-dependent transcription in relation to carcinogenicity. dehp effects in the she model compared to rats and mice while the expression of cyp1b and cyp2e was up-regulated and cyp2f under-expressed, no change in expression level of cyp genes was found using dd and qpcr after dehp exposure in our experimental conditions. cyp genes are said to be involved in peroxisome proliferation. eveillard et al. who studied the involvement of dehp in lipidogenesis in rats, found a slight increase in the pparα level after days of oral exposure to dehp. they registered a significant increase in cyp levels after days and after days of exposure. on the other hand, we found no increased mrna level of cyp and ppar genes in dehp-treated she cells. this underlines that the genes expression changes noted in the present study are independent of ppars induction. eveillard et al. found that induced expression of cyp2b by dehp was also independent of pparα induction but car dependent. no change in car expression was registered in she cells, which may explain why no change in cyp2b was noted. our results are consistent with the study of ren et al. who identified dehp regulated genes independent of pparα and car in rats and mice. in our study, lipogenesis and xenobiotic metabolism pathways were impacted by dehp, but not in a major prior way. this may be explained by the lower sensitivity of the hamster model compared with rats and mice to peroxisome proliferators. indeed, the syrian hamster model presents an intermediate response between rats or mice and humans who are known to be non-responsive to pp induction. the hamster model, like humans, is less responsive to pp induction than rats and mice, which is an advantage for mechanistic studies of pp effects and for screening human chemical carcinogens. on the other hand, three genes and gene isoforms were commonly found in our study and those carried out by eveillard et al., suggesting a pattern of response specific to dehp. takashima et al. also found similar responses in dehp-treated mice. up-regulation of rab1b, a ras oncogene family member involved in cellular signal transduction or survival, was found in the latter study and in the present one. β-tubulin was clearly over-expressed in mice, a trend which was noted in our study. some gene isoforms of cadherin, nidogen, cyp family genes or lim domain were also impacted in the liver of mice exposed to dehp. dehp effects on transcription factors other genes identified by differential display and involved in transcription and signal transduction pathways or apoptosis were also targeted by dehp. a significant under-expression of p was found after hrs of dehp exposure using differential display and qpcr. this under-expression is in line with the anti- apoptotic effects of dehp. we confirmed the over-expression of bcl- after hrs and the under-expression of c-myc after hrs, events reported in a previous study on dehp treated she cells in conditions similar to the present ones. map kinases such as mapk mapk and mapk were targeted by dehp. further investigations of map kinase pathways could be relevant due to their involvement in activities of transcription factors. the g protein-coupled estrogen receptor was found to be over-expressed in differential display. gper can be activated by estrogen-like compounds and its effect on cytoskeleton architecture has been reported. because of its implication in the regulation of mapk or tgf-β pathways, it would be worthwhile to investigate gper further. performances of dd the confirmation of differentially-expressed genes by qpcr showed that the expression levels of more than 75% of genes identified by dd were confirmed by qpcr. a comparative table of the sensitivity of dd versus qpcr is given in additional file qpcr is more likely to quantify subtle changes in the expression level of mrnas at different concentrations while dd seems to be more sensitive but is less discriminating. to summarize, 35% of the genes identified as differentially expressed in dd gave the same response at the same dehp concentrations with qpcr while 40% were detected by dd at a lower dehp concentration than with qpcr.  transcriptional responses of she cells to dehp were studied in conditions inducing the cell neoplastic transformation, in order to identify gene expression changes in relation with effects of this non-genotoxic carcinogen. functions impacted by dehp were found to be ppar-independent. effects on cytoskeleton related genes indicated disturbances on actin polymerization and stabilization, cell-cell and cell-matrix adhesion and protein trafficking. this is the first study that elucidates the genomic changes of dehp on the organization of the cytoskeleton. whether the expression changes of cytoskeleton-related genes identified here such as coro1c, nrp kif are specific to dehp or to cell transforming agents more generally would require further studies. to answer, the gene sets identified as significantly over- or under-expressed in this study must be explored on other non-genotoxic carcinogens to identify biomarkers predictive of early events in the multistep carcinogenic process. early disturbances in the expression of cytoskeleton-related genes should be considered good candidates. 
the latter genes may be candidates as biomarkers predictive of early events in the multistep carcinogenic process. the aim of this study was to identify genes whose level of expression is altered by dehp by using a global wide-genome approach in syrian hamster embryo cells, a model similar to human cells regarding their responses to this type of carcinogen. this is the first study that elucidates the genomic changes of dehp involved in the organization of the cytoskeleton. with mrna differential display, we analysed the transcriptional regulation of she cells exposed to, and μm of dehp for hrs, conditions which induced neoplastic transformation of these cells. the genes of interest were involved in many biological pathways, including signal transduction, regulation of the cytoskeleton, xenobiotic metabolism, apoptosis, lipidogenesis, protein conformation, transport and cell cycle. the gene expression changes identified in the present study were ppar-independent. this study identified a set of genes whose expression is altered by dehp exposure in mammalian embryo cells. gene expression profiling showed differentially-expressed fragments corresponding to genes after tblastx comparisons, up-regulated and down-regulated. we then focused particularly on genes involved in the regulation of the cytoskeleton, one of the processes occurring during carcinogenesis and in the early steps of neoplastic transformation. di--phthalate is a commonly used plasticizer in polyvinylchloride formulations and a potentially non-genotoxic carcinogen. the up-regulated genes were involved in the regulation of the actin cytoskeleton and this would suggest a role of cellular plasticity in the mechanism of chemical carcinogenesis.
 di--phthalate is a commonly used plasticizer in polyvinylchloride formulations and a potentially non-genotoxic carcinogen. the aim of this study was to identify genes whose level of expression is altered by dehp by using a global wide-genome approach in syrian hamster embryo cells, a model similar to human cells regarding their responses to this type of carcinogen. with mrna differential display, we analysed the transcriptional regulation of she cells exposed to, and μm of dehp for hrs, conditions which induced neoplastic transformation of these cells. a real-time quantitative polymerase chain reaction was used to confirm differential expression of genes identified by dd.  gene expression profiling showed differentially-expressed fragments corresponding to genes after tblastx comparisons, up-regulated and down-regulated. the genes of interest were involved in many biological pathways, including signal transduction, regulation of the cytoskeleton, xenobiotic metabolism, apoptosis, lipidogenesis, protein conformation, transport and cell cycle. we then focused particularly on genes involved in the regulation of the cytoskeleton, one of the processes occurring during carcinogenesis and in the early steps of neoplastic transformation. twenty one cytoskeleton-related genes were studied by qpcr. the down-regulated genes were involved in focal adhesion or cell junction. the up-regulated genes were involved in the regulation of the actin cytoskeleton and this would suggest a role of cellular plasticity in the mechanism of chemical carcinogenesis. the gene expression changes identified in the present study were ppar-independent.  this study identified a set of genes whose expression is altered by dehp exposure in mammalian embryo cells. this is the first study that elucidates the genomic changes of dehp involved in the organization of the cytoskeleton. the latter genes may be candidates as biomarkers predictive of early events in the multistep carcinogenic process. 