Non-genotoxic Carcinogens Research Articles

Dioxin Induces Genomic Instability in Mouse Embryonic Fibroblasts

Ionizing radiation and certain other exposures have been shown to induce genomic instability (GI), i.e., delayed genetic damage observed many cell generations later in the progeny of the exposed cells. The aim of this study was to investigate induction of GI by a nongenotoxic carcinogen, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Mouse embryonic fibroblasts (C3H10T1/2) were exposed to 1, 10 or 100 nM TCDD for 2 days. Micronuclei (MN) and expression of selected cancer-related genes were assayed both immediately and at a delayed point in time (8 days). For comparison, similar experiments were done with cadmium, a known genotoxic agent. TCDD treatment induced an elevated frequency of MN at 8 days, but not directly after the exposure. TCDD-induced alterations in gene expression were also mostly delayed, with more changes observed at 8 days than at 2 days. Exposure to cadmium produced an opposite pattern of responses, with pronounced effects immediately after exposure but no increase in MN and few gene expression changes at 8 days. Although all responses to TCDD alone were delayed, menadione-induced DNA damage (measured by the Comet assay), was found to be increased directly after a 2-day TCDD exposure, indicating that the stability of the genome was compromised already at this time point. The results suggested a flat dose-response relationship consistent with dose-response data reported for radiation-induced GI. These findings indicate that TCDD, although not directly genotoxic, induces GI, which is associated with impaired DNA damage response.

Cell Transformation Assays: Are we Barking up the Wrong Tree?

There has been a current resurgence of interest in the use of cell transformation for predicting carcinogenicity, which is based mainly on rodent carcinogenicity data. In view of this renewed interest, this paper critically reviews the published literature concerning the ability of the available assays to detect IARC Group 1 agents (known human carcinogens) and Group 2A agents (probable human carcinogens). The predictivity of the available assays for human and rodent non-genotoxic carcinogens (NGCs), in comparison with standard and supplementary in vitro and in vivo genotoxicity tests, is also discussed. The principal finding is that a surprising number of human carcinogens have not been tested for cell transformation across the three main assays (SHE, Balb/c 3T3 and C3H10T1/2), confounding comparative assessment of these methods for detecting human carcinogens. This issue is not being addressed in the ongoing validation studies for the first two of these assays, despite the lack of any serious logistical issues associated with the use of most of these chemicals. In addition, there seem to be no plans for using exogenous bio-transformation systems for the metabolic activation of pro-carcinogens, as recommended in an ECVAM workshop held in 1999. To address these important issues, it is strongly recommended that consideration be given to the inclusion of more human carcinogens and an exogenous source of xenobiotic metabolism, such as an S9 fraction, in ongoing and future validation studies. While cell transformation systems detect a high level of NGCs, it is considered premature to rely only on this endpoint for screening for such chemicals, as recently suggested. This is particularly important, in view of the fact that there is still doubt as to the relevance of morphological transformation to tumorigenesis in vivo, and the wide diversity of potential mechanisms by which NGCs are known to act. Recent progress with regard to increasing the objectivity of scoring the transformed phenotype, and prospects for developing human cell-based transformation assays, are reviewed.

Hemangiosarcoma in Mice Administered Pregabalin: Analysis of Genotoxicity, Tumor Incidence, and Tumor Genetics

Pregabalin, (S)-3-(aminomethyl)-5-methylhexanoic acid, binds with high affinity to the α(2)δ subunit of voltage-gated calcium channels and exerts analgesic, anxiolytic, and antiseizure activities. Two-year carcinogenicity studies were completed in B6C3F1 and CD-1 mice and two separate studies in Wistar rats. Doses in mice were 200, 1000, and 5000 mg/kg/day, with systemic exposures (AUC(0-24 h)) up to 31 times the mean exposure in humans, given the maximum recommended clinical dose. In rats, doses were 50, 150, and 450 mg/kg/day in males and 100, 300, and 900 mg/kg/day in females; systemic exposures up to 24 times were achieved in clinical trials. In both strains of mice, pregabalin treatment was associated with an increased incidence of hemangiosarcoma primarily in liver, spleen, and bone marrow. The incidence of hemangiosarcoma was higher in B6C3F1 mice than in CD-1 mice, consistent with its spontaneous incidence. Pregabalin did not increase the incidence of any other tumor type in rats and was not genotoxic, based on an extensive battery of in vivo and in vitro tests in bacterial and mammalian systems. Thus, pregabalin is a single-species, single tumor-type, nongenotoxic mouse carcinogen. Hemangiosarcomas occurring in mice treated with pregabalin were genotypically distinct from hemangiosarcomas induced by genotoxic carcinogens in humans with respect to ras and p53 mutation patterns and were similar to spontaneous tumors. Furthermore, there was a strong association between pregabalin treatment and bone marrow changes in these studies in mice, suggesting a possible link between the effects observed in bone marrow and the increase in tumor incidence in pregabalin-treated mice.

Comparison of hepatocarcinogen-induced gene expression profiles in conventional primary rat hepatocytes with in vivo rat liver

At present, substantial efforts are focused on the development of in vitro assays coupled with "omics" technologies for the identification of carcinogenic substances as an alternative to the classical 2-year rodent carcinogenicity bioassay. A prerequisite for the eventual regulatory acceptance of such assays, however, is the in vivo relevance of the observed in vitro findings. In the current study, hepatocarcinogen-induced gene expression profiles generated after the exposure of conventional cultures of primary rat hepatocytes to three non-genotoxic carcinogens (methapyrilene hydrochloride, piperonyl butoxide, and Wy-14643), three genotoxic carcinogens (aflatoxin B1, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and 2-nitrofluorene), and two non-carcinogens (nifedipine and clonidine) are compared with previously obtained in vivo data after oral administration for up to 14 days of the same hepatocarcinogens to rats. In addition to the comparison of deregulated genes and functions per compound between in vivo and in vitro models, the major discriminating cellular pathways found in vivo in livers of exposed rats were examined for deregulation in vitro. Further, in vivo-derived gene signatures for the identification of genotoxic versus non-genotoxic carcinogens are used to classify in vitro-tested hepatocarcinogens and non-carcinogens. In the primary hepatocyte cultures, two out of the three tested genotoxic carcinogens mimicked the in vivo-relevant DNA damage response and were correctly assessed. Exposure to the non-genotoxic hepatocarcinogens, however, triggered a relatively weak response in the in vitro system, with no clear similarities to in vivo. This study contributes to the further optimization of toxicogenomics predictive tools when applied in in vitro settings.

A Linear Prediction Rule Based on Ensemble Classifiers for Non-Genotoxic Carcinogenicity

This article presents a novel approach for developing a linear prediction rule to predict the non-genotoxic carcinogenicity potential of a new compound in the drug development pipeline. We construct the approach using data from 24-hour microarray experiments on rats treated with the compound. This method was developed to address an actual problem that we were presented with by scientists in mechanistic toxicology. Short-term, preclinical assays for non-genotoxic carcinogenicity, a toxicity commonly observed in long-term rodent carcinogenicity studies, have proven difficult to develop. A quick and early preclinical assay, such as this, is of particular interest and urgency. The linear prediction rule is derived using an Ensemble Linear Discriminant classifier. This is a hybrid approach which leverages the advantages of ensemble approaches for addressing over-fitting (a problem endemic to microarray data), and that of LDA for interpretability. In a limited comparison with some other classifiers, including random forest, we show that our approach has good predictive performance in addition to being interpretable.

Epigenetics – relevance to drug safety science

AbstractEpigenetics describes the study of heritable changes in gene expression that occur in the absence of a change to the DNA sequence. Specific patterns of epigenetic signatures can be stably transmitted through mitosis and cell division and form the molecular basis for developmental stage- and cell type-specific gene expression. Associations have been observed that endogenous and exogenous stimuli can change the epigenetic control of both somatic and stem cell differentiation and thus influence phenotypic behaviours and/or disease progression. In relation to drug safety, DNA methylation changes have been identified in many stages of tumour development following exposure to non-genotoxic carcinogens. However, it is not clear whether DNA methylation changes cause cancer, or arise as a consequence of the transformed state. Toxic agents could act at different levels, by directly modifying the epigenome or indirectly by altering signalling pathways. These alterations in chromatin structure may or may not be heritable but are probably reversible. That said, there is currently insufficient data to support inclusion of epigenetic profiling into pre-clinical evaluation studies. Several international collaborations aim to generate data to determine whether epigenetic modifications are causal links in disease and/or tumour progression. It will only be when an understanding of chemical mode-of-action is required that evaluation of epigenetic changes might be considered. The current toxicological testing battery is expected to identify any potential adverse effects regardless of the mechanism, epigenetic or otherwise. It is recommended that toxicologists keep a close watch of new developments in this field, in particular identification of early epigenetic markers for non-genotoxic carcinogenicity. Scientific collaborations between academia and industry will help to understand inter-individual variations in response to drug and toxin exposure to be able to distinguish between adverse and non-adverse epigenetic changes.

Carcinogen-specific mutational and epigenetic alterations in INK4A, INK4B and p53 tumour-suppressor genes drive induced senescence bypass in normal diploid mammalian cells

Immortalization (senescence bypass) is a critical rate-limiting step in the malignant transformation of mammalian somatic cells. Human cells must breach at least two distinct senescence barriers to permit unfettered clonal evolution during cancer development: (1) stress- or oncogene-induced premature senescence (SIPS/OIS), mediated via the p16-Rb and/or ARF-p53-p21 tumour-suppressive pathways, and (2) replicative senescence triggered by telomere shortening. In contrast, because their telomerase is constitutively active, cells from small rodents possess only the SIPS/OIS barrier, and are therefore useful for studying SIPS/OIS bypass in isolation. Dermal fibroblasts from the Syrian hamster (SHD cells) are exceptionally resistant to spontaneous SIPS bypass, but it can be readily induced following exposure to a wide range of chemical and physical carcinogens. Here we show that a spectrum of carcinogen-specific mutational and epigenetic alterations involving the INK4A (p16), p53 and INK4B (p15) genes are associated with induced SIPS bypass. With ionizing radiation, immortalization is invariably accompanied by efficient biallelic deletion of the complete INK4/CDKN2 locus. In comparison, SHD cells immortalized by the powerful polycyclic hydrocarbon carcinogen benzo(a)pyrene display transversion point mutations in the DNA-binding domain of p53 coupled with INK4 alterations such as loss of expression of p15. Epimutational silencing of p16 is the primary event associated with immortalization by nickel, a human non-genotoxic carcinogen. As SIPS/OIS bypass is a prerequisite for the immortalization of normal diploid human epithelial cells, our results with the SHD model will provide a basis for delineating combinations of key molecular changes underpinning this important event in human carcinogenesis.

Application of in vitro cell transformation assays in regulatory toxicology for pharmaceuticals, chemicals, food products and cosmetics

Two year rodent bioassays play a key role in the assessment of carcinogenic potential of chemicals to humans. The seventh amendment to the European Cosmetics Directive will ban in 2013 the marketing of cosmetic and personal care products that contain ingredients that have been tested in animal models. Thus 2-year rodent bioassays will not be available for cosmetics/personal care products. Furthermore, for large testing programs like REACH, in vivo carcinogenicity testing is impractical. Alternative ways to carcinogenicity assessment are urgently required. In terms of standardization and validation, the most advanced in vitro tests for carcinogenicity are the cell transformation assays (CTAs). Although CTAs do not mimic the whole carcinogenesis process in vivo, they represent a valuable support in identifying transforming potential of chemicals. CTAs have been shown to detect genotoxic as well as non-genotoxic carcinogens and are helpful in the determination of thresholds for genotoxic and non-genotoxic carcinogens. The extensive review on CTAs by the OECD (OECD (2007) Environmental Health and Safety Publications, Series on Testing and Assessment, No. 31) and the proven within- and between-laboratories reproducibility of the SHE CTAs justifies broader use of these methods to assess carcinogenic potential of chemicals.

Transcriptomic fingerprints in human peripheral blood mononuclear cells indicative of genotoxic and non-genotoxic carcinogenic exposure

For evaluating genotoxic exposure in human populations a number of biomarkers has been successfully applied over the last 30 years to determine early biological effects due to exposure to carcinogens. Despite their success, these early biological effect markers provide limited mechanistic insight, and do not allow detection of exposure to non-genotoxic carcinogens. Gene expression profiling forms a promising tool for the development of new biomarkers in blood cells to overcome these limitations. The aim of our research was to identify novel genomics-based candidate markers for genotoxic and non-genotoxic carcinogen exposure in human peripheral blood cells (PBMC). Whole genome gene expression changes were investigated following 20h of in vitro exposure to a high and low concentration of eight genotoxic and three non-genotoxic carcinogenic compounds using whole genome microarrays. Per condition, PBMC of five independent donors were exposed, all in the presence of human liver S9. Sets of genes, as well as biological pathways indicative of genotoxic exposure and of non-genotoxic carcinogenic exposure were identified. Furthermore, networks were built using the genotoxic and non-genotoxic gene sets, showing the majority of the genes to be interlinked and revealing distinctive transcription factors for both classes. The identification of these potential candidate marker genes might contribute to the development of genomic based biomarkers of carcinogen exposure.

Non-genotoxic carcinogen exposure induces defined changes in the 5-hydroxymethylome

BackgroundInduction and promotion of liver cancer by exposure to non-genotoxic carcinogens coincides with epigenetic perturbations, including specific changes in DNA methylation. Here we investigate the genome-wide dynamics of 5-hydroxymethylcytosine (5hmC) as a likely intermediate of 5-methylcytosine (5mC) demethylation in a DNA methylation reprogramming pathway. We use a rodent model of non-genotoxic carcinogen exposure using the drug phenobarbital.ResultsExposure to phenobarbital results in dynamic and reciprocal changes to the 5mC/5hmC patterns over the promoter regions of a cohort of genes that are transcriptionally upregulated. This reprogramming of 5mC/5hmC coincides with characteristic changes in the histone marks H3K4me2, H3K27me3 and H3K36me3. Quantitative analysis of phenobarbital-induced genes that are involved in xenobiotic metabolism reveals that both DNA modifications are lost at the transcription start site, while there is a reciprocal relationship between increasing levels of 5hmC and loss of 5mC at regions immediately adjacent to core promoters.ConclusionsCollectively, these experiments support the hypothesis that 5hmC is a potential intermediate in a demethylation pathway and reveal precise perturbations of the mouse liver DNA methylome and hydroxymethylome upon exposure to a rodent hepatocarcinogen.

Detection of genotoxic and non-genotoxic renal carcinogens in vitro in NRK-52E cells using a transcriptomics approach

There is a need to develop quick, cheap, sensitive and specific methods to detect the carcinogenic potential of chemicals. Currently there is no in vitro model system for reliable detection of non-genotoxic carcinogens (NGTX) and current tests for detection of genotoxic carcinogens (GTX) can have low specificity. A transcriptomics approach holds promise and a few studies have utilised this technique. However, the majority of these studies have examined liver carcinogens with little work on renal carcinogens which may act via renal-specific NGTX mechanisms. In this study the normal rat renal cell line (NRK-52E) was exposed to sub-toxic concentrations of selected rat renal carcinogens and non-carcinogens (NC) for 6 h, 24 h and 72 h. Renal carcinogens were classified based on their presumed mode of action into GTX and NGTX classes. A whole-genome transcriptomics approach was used to determined genes and pathways as potential signatures for GTX, NGTX and those common to both carcinogenic events in vitro. For some of the GTX compounds an S9 drug metabolising system was included to aid pro-carcinogen activation. Only three genes were commonly deregulated after carcinogen (GTX + NGTX) exposure, one Mdm2 with a detection rate of 67%, and p21 and Cd55 with a detection rate of 56%. However, examination of enriched pathways showed that 3 out of 4 NGTX carcinogens and 4 out of 5 GTX carcinogens were related to known pathways involved in carcinogenesis giving a detection rate of 78%. In contrast, none of the NC chemicals induced any of the above genes or well-established carcinogenic pathways. Additionally, five genes (Lingo1, Hmox1, Ssu72, Lyrm and Usp9x) were commonly altered with 3 out of 4 NGTX carcinogens but not with NC or GTX carcinogens. However, there was no clear separation of GTX and NGTX carcinogens using pathway analysis with several pathways being common to both classes. The findings presented here indicate that the NRK-52E cell line has the potential to detect carcinogenic chemicals, although a much larger number of chemicals need to be used to confirm these findings.

Alterations in Histone H4 Lysine 20 Methylation: Implications for Cancer Detection and Prevention

Cancer development and progression are associated with numerous genetic, epigenetic, and metabolic changes. A number of epigenetic aberrations have been characterized in cancer, including DNA methylation and various histone modification changes. One of the most unique and enigmatic epigenetic marks that is noticeably altered in several major human cancers is methylation of histone H4 lysine 20; however, there is insufficient knowledge of the underlying molecular mechanisms associated with this abberation. This review presents current evidence of the role of histone H4 lysine 20 methylation in normal and cancer cells and during tumorigenesis induced by genotoxic and nongenotoxic carcinogens. Additionally, it describes molecular mechanisms that may cause this alteration and highlights the significance of this epigenetic mark as an early indicator of carcinogenesis. Accumulating evidence suggests that dietary components may be significant regulators of the cellular epigenome, including histone methylation, by providing and maintaining the adequate levels of S-adenosyl-L-methionine, flavin adenine dinucleotide, α-ketoglutarate, and iron. Future research should elucidate the potential for modifying cellular metabolism through dietary intervention for timely regulation of the epigenome as means for the prevention of cancer development.

Risk assessment of coumarin using the bench mark dose (BMD) approach: Children in Norway which regularly eat oatmeal porridge with cinnamon may exceed the TDI for coumarin with several folds

Coumarin is a naturally occurring flavouring substance in cinnamon and many other plants. It is known that coumarin can cause liver toxicity in several species, and it is considered a non-genotoxic carcinogen in rodents. By using the bench mark dose approach we re-assessed coumarin toxicity and established a new TDI for coumarin of 0.07mg/kgbw/day. Oral intake of coumarin is related to consumption of cinnamon-containing foods and food supplements. Cinnamon is a widely used spice in Norway, and can be used as topping on oatmeal porridge. Based on analyses of coumarin in Norwegian foods, intake calculations for children and adults were conducted, and a risk assessment of coumarin in the Norwegian population was performed. Intake estimates of coumarin show that small children eating oatmeal porridge several times a week sprinkled with cinnamon could have a coumarin intake of 1.63mg/kgbw/day and may exceeding the TDI with several folds. Adults drinking cinnamon-based tea and consuming cinnamon supplements also can exceed TDI. The coumarin intake could exceed the TDI by 7- to 20-fold in some intake scenarios. Such large daily exceedances of TDI, even for a limited time period of 1–2weeks, cause concern of adverse health effects.

The Syrian hamster embryo (SHE) assay (pH 6.7): mechanisms of cell transformation and application of vibrational spectroscopy to objectively score endpoint alterations

Using morphological transformation as an endpoint, the Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) is an in vitro system with a high sensitivity and specificity for testing the carcinogenic potential of test agents. Advantages of the assay are that SHE cells are metabolically competent, genetically stable and acquire spontaneous transformation with a low frequency; additionally, it detects both genotoxic and non-genotoxic carcinogens. However, in comparison with other short-term mammalian cell assays, it is time consuming, laborious and, most importantly, the visual scoring of morphological transformation might be subjective. In this review, we examine the background to the test and why it has the potential for use in safety risk assessment. Additionally, we propose a novel approach to objectively interrogate and classify SHE colonies using vibrational spectroscopy coupled to a mathematical framework for high-throughput screening. It is our view that this alternative approach has the potential to improve the sensitivity and specificity of the in vitro SHE assay.

The effect of four non-genotoxic carcinogens and four non-carcinogens on NRK-52E cells using a transcriptomics approach

The effect of four non-genotoxic carcinogens and four non-carcinogens on NRK-52E cells using a transcriptomics approach

ECVAM prevalidation study on in vitro cell transformation assays: General outline and conclusions of the study

The potential for a compound to induce carcinogenicity is a key consideration when ascertaining hazard and risk assessment of chemicals. Among the in vitro alternatives that have been developed for predicting carcinogenicity, in vitro cell transformation assays (CTAs) have been shown to involve a multistage process that closely models important stages of in vivo carcinogenesis and have the potential to detect both genotoxic and non-genotoxic carcinogens. These assays have been in use for decades and a substantial amount of data demonstrating their performance is available in the literature. However, for the standardised use of these assays for regulatory purposes, a formal evaluation of the assays, in particular focusing on development of standardised transferable protocols and further information on assay reproducibility, was considered important to serve as a basis for the drafting of generally accepted OECD test guidelines. To address this issue, a prevalidation study of the CTAs using the BALB/c 3T3 cell line, SHE cells at pH 6.7, and SHE cells at pH 7.0 was coordinated by the European Centre for the Validation of Alternative Methods (ECVAM) and focused on issues of standardisation of protocols, test method transferability and within- and between-laboratory reproducibility. The study resulted in the availability of standardised protocols that had undergone prevalidation [1,2]. The results of the ECVAM study demonstrated that for the BALB/c 3T3 method, some modifications to the protocol were needed to obtain reproducible results between laboratories, while the SHE pH 6.7 and the SHE pH 7.0 protocols are transferable between laboratories, and results are reproducible within- and between-laboratories. It is recommended that the BALB/c 3T3 and SHE protocols as instituted in this prevalidation study should be used in future applications of these respective transformation assays. To support their harmonised use and regulatory application, the development of an OECD test guideline for the SHE CTAs, based on the protocol published in this issue, is recommended. The development of an OECD test guideline for the BALB/c 3T3 CTA should likewise be further pursued upon the availability of additional supportive data and improvement of the statistical analysis.

Carcinogenic potency of perfluorooctane sulfonate (PFOS) on Syrian hamster embryo (SHE) cells

Perfluorooctane sulfonate (PFOS) is the degradation product of many fluoroderivatives and a widespread environmental contaminant. Its persistence, its long half-life in humans and its toxicity explain high concerns on human health side effects in future. PFOS is suspected to be a non-genotoxic carcinogen. In the present work, we assessed carcinogenic potential of PFOS by studying morphological transformation in Syrian hamster embryo (SHE) cells; cell transformation of SHE cells is an in vitro assay recommended by the Organization for Economic Cooperation and Development to detect carcinogens, genotoxic or not. Genotoxicity of PFOS and expression of PPARs genes in SHE cells were also measured. PFOS was shown to induce cell transformation (P<0.05) at non-cytotoxic concentrations (0.2 and 2μg/mL) (P≤0.01). No genotoxic effect was recorded in the range of PFOS concentrations tested (2×10(-4) to 50μg/mL) using the single-cell gel electrophoresis (comet) assay after 5 and 24h of exposure. The expression of PPARs genes was measured by qPCR within the first 24h and after 7days of PFOS treatment. Results indicated an increased expression of ppar-β/δ isoform as early as 24h. After 7days, the increase of ppar-β/δ mRNA was significant at the concentrations inducing cell transformation (0.2 and 2μg/mL), while overexpression of ppar-γ and ppar-α did not closely relate to effective concentrations. The results indicate that PFOS behave as a non-genotoxic carcinogen and impacted PPARs genes. Its cell transforming potential paralleled an increased expression of ppar-β/δ.

Identification of early target genes of aflatoxin B1 in human hepatocytes, inter-individual variability and comparison with other genotoxic compounds

Gene expression profiling has recently emerged as a promising approach to identify early target genes and discriminate genotoxic carcinogens from non-genotoxic carcinogens and non-carcinogens. However, early gene changes induced by genotoxic compounds in human liver remain largely unknown. Primary human hepatocytes and differentiated HepaRG cells were exposed to aflatoxin B1 (AFB1) that induces DNA damage following enzyme-mediated bioactivation. Gene expression profile changes induced by a 24 h exposure of these hepatocyte models to 0.05 and 0.25 μM AFB1 were analyzed by using oligonucleotide pangenomic microarrays. The main altered signaling pathway was the p53 pathway and related functions such as cell cycle, apoptosis and DNA repair. Direct involvement of the p53 protein in response to AFB1 was verified by using siRNA directed against p53. Among the 83 well-annotated genes commonly modulated in two pools of three human hepatocyte populations and HepaRG cells, several genes were identified as altered by AFB1 for the first time. In addition, a subset of 10 AFB1-altered genes, selected upon basis of their function or tumor suppressor role, was tested in four human hepatocyte populations and in response to other chemicals. Although they exhibited large variable inter-donor fold-changes, several of these genes, particularly FHIT, BCAS3 and SMYD3, were found to be altered by various direct and other indirect genotoxic compounds and unaffected by non-genotoxic compounds. Overall, this comprehensive analysis of early gene expression changes induced by AFB1 in human hepatocytes identified a gene subset that included several genes representing potential biomarkers of genotoxic compounds.

In Posidonia oceanica cadmium induces changes in DNA methylation and chromatin patterning.

In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 μM) and high (50 μM) doses of Cd, through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.

Transcriptomic effects of di-(2-ethylhexyl)-phthalate in Syrian hamster embryo cells: an important role of early cytoskeleton disturbances in carcinogenesis?

BackgroundDi-(2-ethylhexyl)-phthalate (DEHP) is a commonly used plasticizer in polyvinylchloride (PVC) 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 (SHE) cells, a model similar to human cells regarding their responses to this type of carcinogen. With mRNA Differential Display (DD), we analysed the transcriptional regulation of SHE cells exposed to 0, 12.5, 25 and 50 μM of DEHP for 24 hrs, conditions which induced neoplastic transformation of these cells. A real-time quantitative polymerase chain reaction (qPCR) was used to confirm differential expression of genes identified by DD.ResultsGene expression profiling showed 178 differentially-expressed fragments corresponding to 122 genes after tblastx comparisons, 79 up-regulated and 43 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.ConclusionThis 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.