Polyclonal anti-thymocyte globulins for the prophylaxis of graft-versus-host disease after allogeneic stem cell or bone marrow transplantation in adults.

BACKGROUND: Allogeneic haematopoietic stem cell transplantation (SCT) is an established treatment for many malignant and non-malignant haematological disorders. Graft-versus-host disease (GVHD), a condition frequently occurring after an allogeneic SCT, is the result of host tissues being attacked by donor immune cells. It affects more than half of the patients after transplant either as acute and or chronic GVHD. One strategy for the prevention of GVHD is the administration of anti-thymocyte globulins (ATGs), a set of polyclonal antibodies directed against a variety of immune cell epitopes, leading to immunosuppression and immunomodulation. OBJECTIVES: To assess the effect of ATG used for the prevention of GVHD in patients undergoing allogeneic SCT with regard to overall survival, incidence and severity of acute and chronic GVHD, incidence of relapse, non-relapse mortality, graft failure and adverse events. SEARCH METHODS: For this update we searched the CENTRAL, MEDLINE, Embase, trial registers and conference proceedings on the 18th November 2022 along with reference checking and contacting study authors to identify additional studies. We did not apply language restrictions. SELECTION CRITERIA: We included randomised controlled trials (RCTs) investigating the impact of ATG on GVHD prophylaxis in adults suffering from haematological diseases and undergoing allogeneic SCT. The selection criteria were modified from the previous version of this review. Paediatric studies and studies where patients aged < 18 years constituted more than 20 % of the total number were excluded. Treatment arms had to differ only in the addition of ATG to the standard GVHD prophylaxis regimen. DATA COLLECTION AND ANALYSIS: We used standard methodological procedures expected by the Cochrane Collaboration for data collection, extraction and analyses. MAIN RESULTS: For this update we included seven new RCTs, leading to a total of ten studies investigating 1413 participants. All patients had a haematological condition which warranted an allogeneic SCT. The risk of bias was estimated as low for seven and unclear for three studies. ATG probably has little or no influence on overall survival (HR (hazard ratio) 0.93 (95 % confidence interval (CI) 0.77 to 1.13, nine studies, n = 1249, moderate-certainty evidence)). Estimated absolute effect: 430 surviving people per 1000 people not receiving ATG compared to 456 people surviving per 1000 people receiving the intervention (95 % CI 385 to 522 per 1000 people). ATG results in a reduction in acute GVHD II to IV with relative risk (RR) 0.68 (95 % CI 0.60 to 0.79, 10 studies, n = 1413, high-certainty evidence). Estimated absolute effect: 418 acute GVHD II to IV per 1000 people not receiving ATG compared to 285 per 1000 people receiving the intervention (95 % CI 251 to 331 per 1000 people). Addition of ATG results in a reduction of overall chronic GvHD with a RR of 0.53 (95 % CI 0.45 to 0.61, eight studies, n = 1273, high-certainty evidence). Estimated absolute effect: 506 chronic GVHD per 1000 people not receiving ATG compared to 268 per 1000 people receiving the intervention (95 % CI 228 to 369 per 1000 people). Further data on severe acute GVHD and extensive chronic GVHD are available in the manuscript. ATG probably slightly increases the incidence of relapse with a RR of 1.21 (95 % CI 0.99 to 1.49, eight studies,  n =1315, moderate-certainty evidence). Non relapse mortality is probably slightly or not affected by ATG with an HR of 0.86 (95 % CI 0.67 to 1.11, nine studies, n=1370, moderate-certainty evidence).   ATG prophylaxis may result in no increase in graft failure with a RR of 1.55 (95 % CI 0.54 to 4.44, eight studies, n = 1240, low-certainty evidence).  Adverse events could not be analysed due to the serious heterogeneity in the reporting between the studies, which limited comparability (moderate-certainty evidence) and are reported in a descriptive manner.   Subgroup analyses on ATG types, doses and donor type are available in the manuscript. AUTHORS' CONCLUSIONS: This systematic review suggests that the addition of ATG during allogeneic SCT probably has little or no influence on overall survival. ATG results in a reduction in the incidence and severity of acute and chronic GvHD. ATG intervention probably slightly increases the incidence of relapse and probably does not affect the non relapse mortality. Graft failure may not be affected by ATG prophylaxis. Analysis of data on adverse events was reported in a narrative manner. A limitation for the analysis was the imprecision in reporting between the studies thereby reducing the confidence in the certainty of evidence.

The SEURAT-1 approach towards animal free human safety assessment.

SEURAT-1 is a European public-private research consortium that is working towards animal-free testing of chemical compounds and the highest level of consumer protection. A research strategy was formulated based on the guiding principle to adopt a toxicological mode-of-action framework to describe how any substance may adversely affect human health.The proof of the initiative will be in demonstrating the applicability of the concepts on which SEURAT-1 is built on three levels:(i) Theoretical prototypes for adverse outcome pathways are formulated based on knowledge already available in the scientific literature on investigating the toxicological mode-of-actions leading to adverse outcomes (addressing mainly liver toxicity);(ii)adverse outcome pathway descriptions are used as a guide for the formulation of case studies to further elucidate the theoretical model and to develop integrated testing strategies for the prediction of certain toxicological effects (i.e., those related to the adverse outcome pathway descriptions);(iii) further case studies target the application of knowledge gained within SEURAT-1 in the context of safety assessment. The ultimate goal would be to perform ab initio predictions based on a complete understanding of toxicological mechanisms. In the near-term, it is more realistic that data from innovative testing methods will support read-across arguments. Both scenarios are addressed with case studies for improved safety assessment. A conceptual framework for a rational integrated assessment strategy emerged from designing the case studies and is discussed in the context of international developments focusing on alternative approaches for evaluating chemicals using the new 21st century tools for toxicity testing.

Pharmacokinetics explain in vivo/in vitro discrepancies of carcinogen-induced gene expression alterations in rat liver and cultivated hepatocytes.

Cultivated hepatocytes represent a well-established in vitro system. However, the applicability of hepatocytes in toxicogenomics is still controversially discussed. Recently, an in vivo/in vitro discrepancy has been described, whereby the non-genotoxic rat liver carcinogen methapyrilene alters the expression of the metabolizing genes SULT1A1 and ABAT, as well as the DNA damage response gene GADD34 in vitro, but not in vivo. If the collagen sandwich cultures of hepatocytes really produce false-positive data, this would compromise its application in toxicogenomics. To revisit the putative in vivo/in vitro discrepancy, we first analyzed and modeled methapyrilene concentrations in the portal vein of rats. The relatively short half-life of 2.8 h implies a rapid decrease in orally administered methapyrilene in vivo below concentrations that can cause gene expression alterations. This corresponded to the time-dependent alteration levels of GADD34, ABAT and SULT1A1 RNA in the liver: RNA levels are altered 1, 6 and 12 h after methapyrilene administration, but return to control levels after 24 and 72 h. In contrast, methapyrilene concentrations in the culture medium supernatant of primary rat hepatocyte cultures decreased slowly. This explains why GADD34, ABAT and SULT1A1 were still deregulated after 24 h exposure in vitro, but not in vivo. It should also be considered that the earliest analyzed time point in the previous in vivo studies was 24 h after methapyrilene administration. In conclusion, previously observed in vitro/in vivo discrepancy can be explained by different pharmacokinetics present in vitro and in vivo. When the in vivo half-life is short, levels of some initially altered genes may have returned to control levels already 24 h after administration.

Comparison of genotoxicant-modified transcriptomic responses in conventional and epigenetically stabilized primary rat hepatocytes with in vivo rat liver data.

The concept of mechanistic toxicogenomics implies that compound-induced changes in gene expression profiles provide valuable information about their mode of action. A growing number of research groups have presented evidence that whole-genome gene expression profiling techniques might be used as tools for in vivo and in vitro generation of gene signatures and elucidation of molecular mechanisms after exposure to toxic compounds. An important issue to be investigated is the in vivo relevance of in vitro-obtained data. In the current study, we compare the gene expression profiles generated in vitro, after exposing conventional and epigenetically stabilized primary rat hepatocytes to well-known genotoxic hepatocarcinogens (aflatoxin B1, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 2-nitrofluorene) with those derived in vivo after oral exposure of rats to these compounds. Similar statistical tools were applied on both sets of data. The major molecular pathways affected in the in vivo setting were DNA damage, detoxification and cell survival response, as previously described. In the conventional hepatocyte cultures, two of the three genotoxicants showed quite similar responses as in vivo with respect to these pathways. The third compound (2-nitrofluorene) revealed in vitro response which was not observed in vivo. In the epigenetically stabilized hepatocytes, in contrast to what was expected, the responses were less relevant for the in vivo situation. This study highlights the importance of in vitro/in vivo comparison of data that are generated using in vitro models and shows that conventional primary rat hepatocyte cultures represent an appropriate in vitro model to retrieve mechanistic information on the exposure to genotoxicants.

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.

Transcriptomic alterations induced by Ochratoxin A in rat and human renal proximal tubular in vitro models and comparison to a rat in vivo model.

Ochratoxin A (OTA) is a widely studied compound due to its role in renal toxicity and carcinogenicity. However, there is still no consensus on the exact mechanisms of toxicity or carcinogenicity. In the current study, we analysed the effect of OTA on three human renal proximal tubular models (human primary, RPTEC/TERT1 and HK-2 cells) and two rat renal proximal tubular models (rat primary and NRK-52E cells). Global transcriptomics analysis at two exposure times was performed to generate a set of 756 OTA sensitive genes. This gene set was then compared in more detail across all models and additionally to a rat in vivo renal cortex model. The results demonstrate a well-conserved response across all models. OTA resulted in deregulation of a number of pathways including cytoskeleton, nucleosome regulation, translation, transcription, ubiquitination and cell cycle pathways. Interestingly, the oxidative stress activated Nrf2 pathway was not enriched. These results point to an epigenetic action of OTA, perhaps initiated by actin binding as the actin remodelling gene, advillin was the highest up-regulated in all models. The largest model differences were observed between the human and the rat in vitro models. However, since the human in vitro models were more similar to the rat in vivo model, it is more likely that these differences are model-specific rather than species-specific per se. This study demonstrates the usefulness of in vitro cell culture models combined with transcriptomic analysis for the investigation of mechanisms of toxicity and carcinogenicity. In addition, these results provide further evidence supporting a non-genotoxic mechanism of OTA-induced carcinogenicity.

An elastic network model to identify characteristic stress response genes.

Exposing eukaryotic cells to a toxic compound and subsequent gene expression profiling may allow the prediction of selected toxic effects based on changes in gene expression. This objective is complicated by the observation that compounds with different modes of toxicity cause similar changes in gene expression and that a global stress response affects many genes. We developed an elastic network model of global stress response with nodes representing genes which are connected by edges of graded coexpression. The expression of only few genes have to be known to model the global stress response of all but a few atypical responder genes. Those required genes and the atypical response genes are shown to be good biomarker for tox predictions. In total, 138 experiments and 13 different compounds were used to train models for different toxicity classes. The deduced biomarkers were shown to be biologically plausible. A neural network was trained to predict the toxic effects of compounds from profiling experiments. On a validation data set of 189 experiments with 16 different compounds the accuracy of the predictions was assessed: 14 out of 16 compounds have been classified correctly. Derivation of model based biomarkers through the elastic network approach can naturally be extended to other areas beyond toxicology since subtle signals against a broad response background are common in biological studies.

Assessment of a robust model protocol with accelerated throughput for a human recombinant full length estrogen receptor-alpha binding assay: protocol optimization and intralaboratory assay performance as initial steps towards validation.

Despite about two decades of research in the field of endocrine active compounds, still no validated human recombinant (hr) estrogen receptor-alpha (ERalpha) binding assay is available, although hr-ERalpha is available from several sources. In a joint effort, US EPA and Bayer Schering Pharma with funding from the EU-sponsored 6th framework project, ReProTect, developed a model protocol for such a binding assay. Important features of this assay are the use of a full length hr-ERalpha and performance in a 96-well plate format. A full length hr-ERalpha was chosen, as it was considered to provide the most accurate and human-relevant results, whereas truncated receptors could perform differently. Besides three reference compounds [17beta-estradiol, norethynodrel, dibutylphthalate] nine test compounds with different affinities for the ERalpha [diethylstilbestrol (DES), ethynylestradiol, meso-hexestrol, equol, genistein, o,p'-DDT, nonylphenol, n-butylparaben, and corticosterone] were used to explore the performance of the assay. Three independent experiments per compound were performed on different days, and dilutions of test compounds from deep-frozen stocks, solutions of radiolabeled ligand and receptor preparation were freshly prepared for each experiment. The ERalpha binding properties of reference and test compounds were well detected. As expected dibutylphthalate and corticosterone were non-binders in this assay. In terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using a human recombinant ERalpha ligand binding domain. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.5. Our data demonstrate that the assay was robust and reliably ranked compounds with strong, weak, and no affinity for the ERalpha with high accuracy. It avoids the manipulation and use of animals, i.e., the preparation of uterine cytosol as receptor source from ovariectomized rats, as a recombinant protein is used and thus contributes to the 3R concept (reduce, replace, and refine). Furthermore, in contrast to other assays, this assay could be adjusted to an intermediate/high throughput format. On the whole, this assay is a promising candidate for further validation.

Modelling the genesis and treatment of cancer: the potential role of physiologically based pharmacodynamics.

Physiologically based modelling of pharmacodynamics/toxicodynamics requires an a priori knowledge on the underlying mechanisms causing toxicity or causing the disease. In the context of cancer, the objective of the expert meeting was to discuss the molecular understanding of the disease, modelling approaches used so far to describe the process, preclinical models of cancer treatment and to evaluate modelling approaches developed based on improved knowledge. Molecular events in cancerogenesis can be detected using 'omics' technology, a tool applied in experimental carcinogenesis, but also for diagnostics and prognosis. The molecular understanding forms the basis for new drugs, for example targeting protein kinases specifically expressed in cancer. At present, empirical preclinical models of tumour growth are in great use as the development of physiological models is cost and resource intensive. Although a major challenge in PKPD modelling in oncology patients is the complexity of the system, based in part on preclinical models, successful models have been constructed describing the mechanism of action and providing a tool to establish levels of biomarker associated with efficacy and assisting in defining biologically effective dose range selection for first dose in man. To follow the concentration in the tumour compartment enables to link kinetics and dynamics. In order to obtain a reliable model of tumour growth dynamics and drug effects, specific aspects of the modelling of the concentration-effect relationship in cancer treatment that need to be accounted for include: the physiological/circadian rhythms of the cell cycle; the treatment with combinations and the need to optimally choose appropriate combinations of the multiple agents to study; and the schedule dependence of the response in the clinical situation.

Assessment of a recombinant androgen receptor binding assay: initial steps towards validation.

Despite more than a decade of research in the field of endocrine active compounds with affinity for the androgen receptor (AR), still no validated recombinant AR binding assay is available, although recombinant AR can be obtained from several sources. With funding from the European Union (EU)-sponsored 6th framework project, ReProTect, we developed a model protocol for such an assay based on a simple AR binding assay recently developed at our institution. Important features of the protocol were the use of a rat recombinant fusion protein to thioredoxin containing both the hinge region and ligand binding domain (LBD) of the rat AR (which is identical to the human AR-LBD) and performance in a 96-well plate format. Besides two reference compounds [dihydrotestosterone (DHT), androstenedione] ten test compounds with different affinities for the AR [levonorgestrel, progesterone, prochloraz, 17alpha-methyltestosterone, flutamide, norethynodrel, o,p'-DDT, dibutylphthalate, vinclozolin, linuron] were used to explore the performance of the assay. At least three independent experiments per compound were performed. The AR binding properties of reference and test compounds were well detected, in terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using recombinant AR preparations. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.6. Our data demonstrate that the assay reliably ranked compounds with strong, weak, and no/marginal affinity for the AR with high accuracy. It avoids the manipulation and use of animals, as a recombinant protein is used and thus contributes to the 3R concept. On the whole, this assay is a promising candidate for further validation.

Short-term dynamic culture of rat testicular fragments as a model to assess effects on steroidogenesis--potential use and limitations.

Short-term dynamic culture of rat testicular fragments was evaluated as a model to assess effects on steroidogenesis. A total of 11 compounds differentially affecting testosterone synthesis (aminoglutethimide, ketoconazole, danazol, flutamide, diethylstilbestrol, genistein, butylparaben, nonoxynol-9, dimethoate, RU 486, and cadmium chloride) were used to explore the performance of the assay. Testosterone secretion into the medium and testosterone retained in tissue fragments was determined as a measure of steroidogenesis. Three independent experiments per compound were performed. The known in vitro inhibitory properties of most compounds could be detected. Whenever significant inhibition of testosterone synthesis was observed, low effect concentrations for a given compound differed frequently only by a factor of

Molecular characterization of preneoplastic lesions provides insight on the development of renal tumors.

Kidneys are the second most frequent site for chemically induced cancers in rats. However, there is still limited information on direct effects of carcinogens on pathways involved in the development of kidney tumors. Since transformed tumor cells have different characteristics than their cell of origin, it was hypothesized that healthy tissue and progressing stages of preneoplastic lesions are differentially influenced by chemical carcinogens. To elucidate this question, TSC2(-/-) Eker rats were gavaged with genotoxic aristolochic acid or nongenotoxic ochratoxin A for 3 and 6 months, respectively. Histopathology and cell proliferation analysis demonstrated a compound- and sex-specific onset of preneoplastic lesions. In contrast, comparable gene expression profiles of laser-microdissected preneoplastic lesions from carcinogen-treated and control rats, including reduced expression of genes involved in carcinogen uptake and metabolism, point to a compound-independent lesion progression. Gene expression profiles and additional immunostaining suggested that clonal expansion of renal lesions appears primarily driven by disturbed mammalian target of rapamycin complex 1 and mammalian target of rapamycin complex 2 pathway regulation. Finally, prolonged carcinogen exposure resulted in only marginal gene expression changes in tubules with normal morphology, indicating that some tubules may have adapted to the treatment. Taken together, these findings indicate that the final outcome of in vivo carcinogenicity studies is primarily determined by time-restricted initial events, while lesion progression may be a compound-independent process, involving deregulated mTOR signaling in the Eker rat model.

Inter-laboratory comparison of human renal proximal tubule (HK-2) transcriptome alterations due to Cyclosporine A exposure and medium exhaustion.

There is an acknowledged need to promote and further develop in vitro techniques in order to achieve the goal of improved risk assessment of chemicals and pharmaceuticals to humans. The EU 6th framework project "PREDICTOMICS" was established in order to contribute to the further development of in vitro toxicology, with a particular focus on emerging techniques including toxicogenomics. DNA microarray technology is being used more frequently in the in vitro field, however, only very few studies have assessed the reproducibility of this technique with respect to in vitro toxicology. To this end we conducted an interlaboratory comparison to test the reproducibility of transcriptomic changes induced by the immunosuppressive agent, Cyclosporine A (CsA) on the human renal proximal tubular cell line, HK-2 cell. Four European laboratories took part in this study. Under standardised conditions, each laboratory treated HK-2 cells with 5microM CsA for 12 and 48h. RNA was isolated and hybridised to Affymetrix HGU-133 plus two arrays at three different sites. Analysis of the transcription profiles demonstrated that one laboratory clustered away from the other laboratories, potentially due to an inclusion of a trypsinisation step by this laboratory. Once the genes responsible for this separate clustering were removed all laboratories showed similar expression profiles. There was a major impact of time since feed, due to medium exhaustion in the 48h arrays compared to the 12h arrays, regardless of CsA treatment. Biological processes including general vesicle transport, amino acid metabolism, amino acid transport and amino acid biosynthesis were over-represented due to time since feed, while cell cycle, DNA replication, mitosis and DNA metabolism were under-represented. CsA responsive genes were involved in cell cycle, the p53 pathway and Wnt signaling. Additionally there was an overlap of differentially expressed genes due to CsA and medium exhaustion which is most likely due to CsA induced glycolysis. The glucose deprivation dependent genes HspA5 and GP96 and the Hsp70 chaperones DNAJ/Hsp40, DNAJ/HspB9, DNAJ/HspC3 DNAJ/HspC10 were induced by both CsA and medium exhaustion. We conclude that under standardised conditions the application of Affymetrix DNA microarrays to in vitro toxiciological studies are satisfactorily reproducible. However, confounding factors such as medium exhaustion must also be considered in such analyses.

Application of toxicogenomics to study mechanisms of genotoxicity and carcinogenicity.

Specific genotoxic events such as gene mutations and/or chromosome damage are considered hallmarks of cancer. The genotoxicity testing battery enables relatively simple, rapid and inexpensive hazard identification, namely by assessing a chemical's ability to cause genetic damage in cells. In addition, the 2-year rodent carcinogenicity bioassay provides an assessment of a risk associated with the chemical to develop cancer in animals. Although the link between genotoxicity and carcinogenicity is well documented, this relationship is complicated due to the impact of non-genotoxic mechanisms of carcinogenesis and by character of the in vitro genotoxicity assays and specific endpoints making the interpretation of test results in light of human risk and relevance difficult. In particular, the specificity of test results has been questioned. Therefore, the development of novel scientific approaches bridging genotoxicity and carcinogenicity testing via understanding underlying mechanisms is extremely important for facilitating cancer risk assessment. In this respect, toxicogenomics approaches are considered promising as these have the potential of providing generic insight in molecular pathway responses. The goal of this report thus is to review recent progress in the development and application of toxicogenomics to the derivation of genomic biomarkers associated with mechanisms of genotoxicity and carcinogenesis. Furthermore, the potential for application of genomic approaches to hazard identification and risk assessment is explored.

The carcinoGENOMICS project: critical selection of model compounds for the development of omics-based in vitro carcinogenicity screening assays.

Recent changes in the European legislation of chemical-related substances have forced the scientific community to speed up the search for alternative methods that could partly or fully replace animal experimentation. The Sixth Framework Program project carcinoGENOMICS was specifically raised to develop omics-based in vitro screens for testing the carcinogenic potential of chemical compounds in a pan-European context. This paper provides an in-depth analysis of the complexity of choosing suitable reference compounds used for creating and fine-tuning the in vitro carcinogenicity assays. First, a number of solid criteria for the selection of the model compounds are defined. Secondly, the strategy followed, including resources consulted, is described and the selected compounds are briefly illustrated. Finally, limitations and problems encountered during the selection procedure are discussed. Since selecting an appropriate set of chemicals is a frequent impediment in the early stages of similar research projects, the information provided in this paper might be extremely valuable.

Prediction of a carcinogenic potential of rat hepatocarcinogens using toxicogenomics analysis of short-term in vivo studies.

The carcinogenic potential of chemicals is currently evaluated with rodent life-time bioassays, which are time consuming, and expensive with respect to cost, number of animals and amount of compound required. Since the results of these 2-year bioassays are not known until quite late during development of new chemical entities, and since the short-term test battery to test for genotoxicity, a characteristic of genotoxic carcinogens, is hampered by low specificity, the identification of early biomarkers for carcinogenicity would be a big step forward. Using gene expression profiles from the livers of rats treated up to 14 days with genotoxic and non-genotoxic carcinogens we previously identified characteristic gene expression profiles for these two groups of carcinogens. We have now added expression profiles from further hepatocarcinogens and from non-carcinogens the latter serving as control profiles. We used these profiles to extract biomarkers discriminating genotoxic from non-genotoxic carcinogens and to calculate classifiers based on the support vector machine (SVM) algorithm. These classifiers then predicted a set of independent validation compound profiles with up to 88% accuracy, depending on the marker gene set. We would like to present this study as proof of the concept that a classification of carcinogens based on short-term studies may be feasible.

Carcinogen-specific gene expression profiles in short-term treated Eker and wild-type rats indicative of pathways involved in renal tumorigenesis.

Eker rats heterozygous for a dominant germline mutation in the tuberous sclerosis 2 (Tsc2) tumor suppressor gene were used as a model to study renal carcinogenesis. Eker and corresponding wild-type rats were exposed to genotoxic aristolochic acid (AA) or non-genotoxic ochratoxin A (OTA) to elucidate early carcinogen-specific gene expression changes and to test whether Eker rats are more sensitive to carcinogen-induced changes in gene expression. Male Eker and wild-type rats were gavaged daily with AA (10 mg/kg body weight) or OTA (210 microg/kg body weight). After 1, 3, 7, and 14 days of exposure, renal histopathology, tubular cell proliferation, and Affymetrix gene expression profiles from renal cortex/outer medulla were analyzed. AA-treated Eker and wild-type rats were qualitatively comparable in all variables assessed, suggesting a Tsc2-independent mechanism of action. OTA treatment resulted in slightly increased cortical pathology and significantly elevated cell proliferation in both strains, although Eker rats were more sensitive. Deregulated genes involved in the phosphatidylinositol 3-kinase-AKT-Tsc2-mammalian target of rapamycin signaling, among other important genes prominent in tumorigenesis, in conjunction with the enhanced cell proliferation and presence of preneoplastic lesions suggested involvement of Tsc2 in OTA-mediated toxicity and carcinogenicity, especially as deregulation of genes involved in this pathway was more prominent in the Tsc2 mutant Eker rat.