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PURPOSE: Amcenestrant (oral selective estrogen receptor degrader) demonstrated promising safety and efficacy in earlier clinical studies for endocrine-resistant, estrogen receptor-positive/human epidermal growth factor receptor 2-negative (ER+/HER2-) advanced breast cancer (aBC). PATIENTS AND METHODS: In AMEERA-3 (ClinicalTrials.gov identifier: NCT04059484), an open-label, worldwide phase II trial, patients with ER+/HER2- aBC who progressed in the (neo)adjuvant or advanced settings after not more than two previous lines of endocrine therapy (ET) were randomly assigned 1:1 to amcenestrant or single-agent endocrine treatment of physician's choice (TPC), stratified by the presence/absence of visceral metastases, previous/no treatment with cyclin-dependent kinase 4/6 inhibitor, and Eastern Cooperative Oncology Group performance status (0/1). The primary end point was progression-free survival (PFS) by independent central review, compared using a stratified log-rank test (one-sided type I error rate of 2.5%). RESULTS: Between October 22, 2019, and February 15, 2021, 290 patients were randomly assigned to amcenestrant (n = 143) or TPC (n = 147). PFS was numerically similar between amcenestrant and TPC (median PFS [mPFS], 3.6 v 3.7 months; stratified hazard ratio [HR], 1.051 [95% CI, 0.789 to 1.4]; one-sided P = .643). Among patients with baseline mutated ESR1; (n = 120 of 280), amcenestrant numerically prolonged PFS versus TPC (mPFS, 3.7 v 2.0 months; stratified HR, 0.9 [95% CI, 0.565 to 1.435]). Overall survival data were immature but numerically similar between groups (HR, 0.913; 95% CI, 0.595 to 1.403). In amcenestrant versus TPC groups, treatment-emergent adverse events (any grade) occurred in 82.5% versus 76.2% of patients and grade ≥3 events occurred in 21.7% versus 15.6%. CONCLUSION: AMEERA-3 did not meet its primary objective of improved PFS with amcenestrant versus TPC although a numerical improvement in PFS was observed in patients with baseline ESR1 mutation. Efficacy and safety with amcenestrant were consistent with the standard of care for second-/third-line ET for ER+/HER2- aBC.
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Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/patología , Receptores de Estrógenos/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Receptor ErbB-2/metabolismoRESUMEN
Circulating microRNAs are biomarkers reported to be stable and translational across species. MicroRNA-122 (miR-122) is a hepatocyte-specific microRNA biomarker for drug-induced liver injury (DILI). We developed a single molecule, dynamic chemical labeling (DCL) assay to directly detect miR-122 in blood. The DCL assay specifically measured miR-122 directly from 10 µL of serum or plasma without any extraction steps, with a limit of detection of 1.32 pM that enabled the identification of DILI. Testing of 192 human serum samples showed that DCL accurately identified patients at risk of DILI after acetaminophen overdose (area under ROC curve 0.98 (95% CI; 0.96-1), P < 0.0001). The DCL assay also identified liver injury in rats and dogs. The use of specific captured beads had the additional benefit of stabilizing miR-122 after sample collection, with no signal loss after 14 days at room temperature, in contrast to PCR that showed significant loss of signal. RNA sequencing demonstrated the presence of multiple miR-122 isomiRs in the serum of patients with DILI that were at low concentration or not present in healthy individuals. Sample degradation over time produced more isomiRs, particularly rapidly with DILI. PCR was inaccurate when analyzing miR-122 isomiRs, whereas the DCL assay demonstrated accurate quantification. We conclude that the DCL assay can accurately measure miR-122 to diagnose liver injury in humans and other species and can overcome microRNA stability and isomiR challenges.
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Acetaminofén/efectos adversos , MicroARNs/sangre , Acetaminofén/administración & dosificación , Adolescente , Adulto , Animales , Biomarcadores/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas , Perros , Hepatocitos/efectos de los fármacos , Humanos , Masculino , MicroARNs/genética , Ratas , Adulto JovenRESUMEN
OBJECTIVE: Preclinical studies suggest a role for lysophosphatidic acid (LPA) in the pathogenesis of systemic sclerosis (SSc). We undertook this study to assess SAR100842, a potent selective oral antagonist of the LPA1 receptor, for safety, biomarkers, and clinical efficacy in patients with diffuse cutaneous SSc (dcSSc). METHODS: An 8-week double-blind, randomized, placebo-controlled study followed by a 16-week open-label extension with SAR100842 was performed in patients with early dcSSc who had a baseline modified Rodnan skin thickness score (MRSS) of at least 15. The primary end point was safety during the double-blind phase of the trial. Exploratory end points included the identification of an LPA-induced gene signature in patients' skin. RESULTS: Seventeen of 32 patients were randomly assigned to receive placebo and 15 to receive SAR100842; 30 patients participated in the open-label extension study. The most frequent adverse events reported for SAR100842 during the blinded phase were headache, diarrhea, nausea, and falling, and the safety profile was acceptable during the open-label extension. At week 8, the reduction in MRSS was numerically greater in the SAR100842 group than in the placebo group (mean ± SD change -3.57 ± 4.18 versus -2.76 ± 4.85; treatment effect -1.2 [95% confidence interval -4.37, 2.02]; P = 0.46). A greater reduction of LPA-related genes was observed in skin samples from the SAR100842 group at week 8, indicating LPA1 target engagement. CONCLUSION: SAR100842, a selective orally available LPA1 receptor antagonist, was well tolerated in patients with dcSSc. The MRSS improved during the study although the difference was not significant, and additional gene signature analysis suggested target engagement. These results need to be confirmed in a larger controlled trial.
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Benzamidas/uso terapéutico , Indenos/uso terapéutico , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Esclerodermia Difusa/tratamiento farmacológico , Adulto , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esclerodermia Difusa/patología , Índice de Severidad de la Enfermedad , Piel/patología , Resultado del TratamientoRESUMEN
BACKGROUND: Circulating microRNAs are undergoing exploratory use as safety biomarkers in drug development. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is one common approach used to quantitate levels of microRNAs in samples that includes the use of a standard curve of calibrators fit to a regression model. Guidelines are needed for setting assay quantitation thresholds that are appropriate for this method and to biomarker pre-validation. RESULTS: In this report, we develop two workflows for determining a lower limit of quantitation (LLOQ) for RT-qPCR assays of microRNAs in exploratory studies. One workflow is based on an error threshold calculated by a logistic model of the calibration curve data. The second workflow is based on a threshold set by the sample blank, which is the no template control for RT-qPCR. The two workflows are used to set lower thresholds of reportable microRNA levels for an example dataset in which miR-208a levels in biofluids are quantitated in a cardiac injury model. LLOQ thresholds set by either workflow are effective in filtering out microRNA values with large uncertainty estimates. CONCLUSIONS: Two workflows for LLOQ determinations are presented in this report that provide methods that are easy to implement in investigational studies of microRNA safety biomarkers and offer choices in levels of conservatism in setting lower limits of acceptable values that facilitate interpretation of results.
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Límite de Detección , MicroARNs/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , Calibración , Marcadores Genéticos , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/estadística & datos numéricos , Flujo de TrabajoRESUMEN
Advanced sequencing technologies like next-generation sequencing (NGS) not only detect microRNAs (miRNAs) in biological samples but also facilitate de novo identification of miRNAs. Using an Ion Torrent's Ion Proton System, here we described miRNAs sequencing of urine samples collected from Macaca fascicularis (Cynomolgus monkey) to investigate miRNAs as potential novel biomarkers of nephrotoxicity in this species. Urinary miRNA sequencing methodologies described here include (a) urinary exosomal RNA isolation, (b) sequencing library preparation, (c) sequencing template preparation, and (d) template library sequencing using Ion Proton System. The sequencing method presented in this study serves as a valuable resource in the identification of novel urinary miRNAs in M. fascicularis.
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Biomarcadores/orina , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , MicroARNs/orina , Animales , Perfilación de la Expresión Génica/métodos , Riñón/metabolismo , Macaca fascicularis , Análisis de Secuencia de ARNRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0142708.].
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Extracellular microRNAs (miRNAs) represent a promising new source of toxicity biomarkers that are sensitive indicators of site of tissue injury. In order to establish reliable approaches for use in biomarker validation studies, the HESI technical committee on genomics initiated a multi-site study to assess sources of variance associated with quantitating levels of cardiac injury induced miRNAs in biofluids using RT-qPCR. Samples were generated at a central site using a model of acute cardiac injury induced in male Wistar rats by 0.5 mg/kg isoproterenol. Biofluid samples were sent to 11 sites for measurement of 3 cardiac enriched miRNAs (miR-1-3p, miR-208a-3p, and miR-499-5p) and 1 miRNA abundant in blood (miR-16-5p) or urine (miR-192-5p) by absolute quantification using calibration curves of synthetic miRNAs. The samples included serum and plasma prepared from blood collected at 4 h, urine collected from 6 to 24 h, and plasma prepared from blood collected at 24 h post subcutaneous injection. A 3 parameter logistic model was utilized to fit the calibration curve data and estimate levels of miRNAs in biofluid samples by inverse prediction. Most sites observed increased circulating levels of miR-1-3p and miR-208a-3p at 4 and 24 h after isoproterenol treatment, with no difference seen between serum and plasma. The biological differences in miRNA levels and sample type dominated as sources of variance, along with outlying performance by a few sites. The standard protocol established in this study was successfully implemented across multiple sites and provides a benchmark method for further improvements in quantitative assays for circulating miRNAs.
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Lesiones Cardíacas/metabolismo , MicroARNs/sangre , MicroARNs/orina , Animales , Biomarcadores/sangre , Biomarcadores/orina , Lesiones Cardíacas/inducido químicamente , Isoproterenol/toxicidad , Masculino , Plasma/química , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Suero/químicaRESUMEN
Most studies to evaluate kidney safety biomarkers have been performed in rats. This study was conducted in Cynomolgus monkeys in order to evaluate the potential usefulness of novel biomarkers of nephrotoxicity in this species. Groups of 3 males were given daily intramuscular injections of gentamicin, a nephrotoxic agent known to produce lesions in proximal tubules, at dose-levels of 10, 25, or 50mg/kg/day for 10days. Blood and 16-h urine samples were collected on Days -7, -3, 2, 4, 7, and at the end of the dosing period. Several novel kidney safety biomarkers were evaluated, with single- and multiplex immunoassays and in immunoprecipitation-LC/MS assays, in parallel to histopathology and conventional clinical pathology parameters. Treatment with gentamicin induced a dose-dependent increase in kidney tubular cell degeneration/necrosis, ranging from minimal to mild severity at 10mg/kg/day, moderate at 25mg/kg/day, and to severe at 50mg/kg/day. The results showed that the novel urinary biomarkers, microalbumin, α1-microglobulin, clusterin, and osteopontin, together with the more traditional clinical pathology parameters, urinary total protein and N-acetyl-ß-D-glucosaminidase (NAG), were more sensitive than blood urea nitrogen (BUN) and serum creatinine (sCr) to detect kidney injury in the monkeys given 10mg/kg/day gentamicin for 10days, a dose leading to an exposure which is slightly higher than the desired therapeutic exposure in clinics. Therefore, these urinary biomarkers represent non-invasive biomarkers of proximal tubule injury in Cynomolgus monkeys which may be potentially useful in humans.
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Antibacterianos/toxicidad , Gentamicinas/toxicidad , Enfermedades Renales/inducido químicamente , Enfermedades Renales/orina , Acetilglucosaminidasa/orina , Alanina Transaminasa/sangre , alfa-Globulinas/orina , Animales , Antibacterianos/sangre , Antibacterianos/farmacocinética , Aspartato Aminotransferasas/sangre , Biomarcadores/sangre , Biomarcadores/orina , Glucemia/análisis , Nitrógeno de la Urea Sanguínea , Clusterina/orina , Creatina/sangre , Creatina/orina , Gentamicinas/sangre , Gentamicinas/farmacocinética , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/sangre , Enfermedades Renales/patología , Macaca fascicularis , Masculino , Necrosis/inducido químicamente , Osteopontina/orina , Albúmina Sérica/análisisRESUMEN
MicroRNAs (miRNAs) present in tissues and biofluids are emerging as sensitive and specific safety biomarkers. MiRNAs have not been thoroughly described in M. fascicularis, an animal model used in pharmaceutical industry especially in drug safety evaluation. Here we investigated the miRNAs in M. fascicularis. For Macaca mulatta, a closely related species of M. fascicularis, 619 stem-loop precursor miRNAs (pre-miRNAs) and 914 mature miRNAs are available in miRBase version 21. Using M. mulatta miRNAs as a reference list and homology search tools, we identified 604 pre-miRNAs and 913 mature miRNAs in the genome of M. fascicularis. In order to validate the miRNAs identified by homology search we attempted to sequence miRNAs expressed in kidney cortex from M. fascicularis. MiRNAs expressed in kidney cortex may indeed be released in urine upon kidney cortex damage and be potentially used to monitor drug induced kidney injury. Hence small RNA sequencing libraries were prepared using kidney cortex tissues obtained from three naive M. fascicularis and sequenced. Analysis of sequencing data indicated that 432 out of 913 mature miRNAs were expressed in kidney cortex tissues. Assigning these 432 miRNAs to pre-miRNAs revealed that 273 were expressed from both the -5p and -3p arms of 150 pre-miRNAs and 159 miRNAs expressed from either the -5p or -3p arm of 176 pre-miRNAs. Mapping sequencing reads to pre-miRNAs also facilitated the detection of twenty-two new miRNAs. To substantiate miRNAs identified by small RNA sequencing, 313 miRNAs were examined by RT-qPCR. Expression of 262 miRNAs in kidney cortex tissues ware confirmed by TaqMan microRNA RT-qPCR assays. Analysis of kidney cortex miRNA targeted genes suggested that they play important role in kidney development and function. Data presented in this study may serve as a valuable resource to assess the renal safety biomarker potential of miRNAs in Cynomolgus monkeys.
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Corteza Renal/metabolismo , Macaca fascicularis/genética , MicroARNs/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Análisis de Secuencia de ARN/métodos , Animales , Biomarcadores/orina , Perfilación de la Expresión Génica/métodos , Genoma/genética , Humanos , MicroARNs/orina , Precursores del ARN/genéticaRESUMEN
Gentamicin is an aminoglycoside antibiotic, which induces renal tubular necrosis in rats. In the context of the European InnoMed PredTox project, transcriptomic and proteomic studies were performed to provide new insights into the molecular mechanisms of gentamicin-induced nephrotoxicity. Male Wistar rats were treated with 25 and 75 mg/kg/day subcutaneously for 1, 3 and 14 days. Histopathology observations showed mild tubular degeneration/necrosis and regeneration and moderate mononuclear cell infiltrate after long-term treatment. Transcriptomic data indicated a strong treatment-related gene expression modulation in kidney and blood cells at the high dose after 14 days of treatment, with the regulation of 463 and 3241 genes, respectively. Of note, the induction of NF-kappa B pathway via the p38 MAPK cascade in the kidney, together with the activation of T-cell receptor signaling in blood cells were suggestive of inflammatory processes in relation with the recruitment of mononuclear cells in the kidney. Proteomic results showed a regulation of 163 proteins in kidney at the high dose after 14 days of treatment. These protein modulations were suggestive of a mitochondrial dysfunction with impairment of cellular energy production, induction of oxidative stress, an effect on protein biosynthesis and on cellular assembly and organization. Proteomic results also provided clues for potential nephrotoxicity biomarkers such as AGAT and PRBP4 which were strongly modulated in the kidney. Transcriptomic and proteomic data turned out to be complementary and their integration gave a more comprehensive insight into the putative mode of nephrotoxicity of gentamicin which was in accordance with histopathological findings.
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Antibacterianos/toxicidad , Perfilación de la Expresión Génica , Gentamicinas/toxicidad , Riñón/efectos de los fármacos , Proteómica , Animales , Biomarcadores , Riñón/metabolismo , Masculino , Ratas , Ratas WistarRESUMEN
Evaluating the risk of chemical carcinogenesis has long been a challenge owing to the protracted nature of the pathology and the limited translatability of animal models. Although numerous short-term in vitro and in vivo assays have been developed, they have failed to reliably predict the carcinogenicity of nongenotoxic compounds. Extending upon previous microarray work (Fielden, M. R., Nie, A., McMillian, M., Elangbam, C. S., Trela, B. A., Yang, Y., Dunn, R. T., II, Dragan, Y., Fransson-Stehen, R., Bogdanffy, M., et al. (2008). Interlaboratory evaluation of genomic signatures for predicting carcinogenicity in the rat. Toxicol. Sci. 103, 28-34), we have developed and extensively evaluated a quantitative PCR-based signature to predict the potential for nongenotoxic compounds to induce liver tumors in the rat as a first step in the safety assessment of potential nongenotoxic carcinogens. The training set was derived from liver RNA from rats treated with 72 compounds and used to develop a 22-gene signature on the TaqMan array platform, providing an economical and standardized assay protocol. Independent testing on over 900 diverse samples (66 compounds) confirmed the interlaboratory precision of the assay and its ability to predict known nongenotoxic hepatocarcinogens (NGHCs). When tested under different experimental designs, strains, time points, dose setting criteria, and other preanalytical processes, the signature sensitivity and specificity was estimated to be 67% (95% confidence interval [CI] = 38-88%) and 59% (95% CI = 44-72%), respectively, with an area under the receiver operating characteristic curve of 0.65 (95% CI = 0.46-0.83%). Compounds were best classified using expression data from short-term repeat dose studies; however, the prognostic expression changes appeared to be preserved after longer term treatment. Exploratory evaluations also revealed that different modes of action for nongenotoxic and genotoxic compounds can be discriminated based on the expression of specific genes. These results support a potential early preclinical testing paradigm to catalyze broader understanding of putative NGHCs.
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Carcinógenos/toxicidad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Hepáticas Experimentales/inducido químicamente , Hígado/efectos de los fármacos , Modelos Genéticos , Animales , Carcinógenos/clasificación , Marcadores Genéticos , Genómica , Hígado/metabolismo , Neoplasias Hepáticas Experimentales/genética , Masculino , Valor Predictivo de las Pruebas , Ratas , Ratas Sprague-DawleyRESUMEN
The main goal of the present work was to better understand the molecular mechanisms underlying liver hypertrophy (LH), a recurrent finding observed following acute or repeated drug administration to animals, using transcriptomic technologies together with the results from conventional toxicology methods. Administration of 5 terminated proprietary drug candidates from participating companies involved in the EU Innomed PredTox Project or the reference hepatotoxicant troglitazone to rats for up to a 14-day duration induced LH as the main liver phenotypic toxicity outcome. The integrated analysis of transcriptomic liver expression data across studies turned out to be the most informative approach for the generation of mechanistic models of LH. In response to a xenobiotic stimulus, a marked increase in the expression of xenobiotic metabolizing enzymes (XME) was observed in a subset of 4 studies. Accumulation of these newly-synthesized proteins within the smooth endoplasmic reticulum (SER) would suggest proliferation of this organelle, which most likely is the main molecular process underlying the LH observed in XME studies. In another subset of 2 studies (including troglitazone), a marked up-regulation of genes involved in peroxisomal fatty acid ß-oxidation was noted, associated with induction of genes involved in peroxisome proliferation. Therefore, an increase in peroxisome abundance would be the main mechanism underlying LH noted in this second study subset. Together, the use of transcript profiling provides a means to generate putative mechanistic models underlying the pathogenesis of liver hypertrophy, to distinguish between subtle variations in subcellular organelle proliferation and creates opportunities for improved mechanism-based risk assessment.
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Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Cromanos/toxicidad , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes/fisiología , Tiazolidinedionas/toxicidad , Animales , Hipertrofia , Masculino , Proteómica/métodos , Ratas , Ratas Wistar , TroglitazonaRESUMEN
In this publication, we report the outcome of the integrated EU Framework 6 PROJECT: Predictive Toxicology (PredTox), including methodological aspects and overall conclusions. Specific details including data analysis and interpretation are reported in separate articles in this issue. The project, partly funded by the EU, was carried out by a consortium of 15 pharmaceutical companies, 2 SMEs, and 3 universities. The effects of 16 test compounds were characterized using conventional toxicological parameters and "omics" technologies. The three major observed toxicities, liver hypertrophy, bile duct necrosis and/or cholestasis, and kidney proximal tubular damage were analyzed in detail. The combined approach of "omics" and conventional toxicology proved a useful tool for mechanistic investigations and the identification of putative biomarkers. In our hands and in combination with histopathological assessment, target organ transcriptomics was the most prolific approach for the generation of mechanistic hypotheses. Proteomics approaches were relatively time-consuming and required careful standardization. NMR-based metabolomics detected metabolite changes accompanying histopathological findings, providing limited additional mechanistic information. Conversely, targeted metabolite profiling with LC/GC-MS was very useful for the investigation of bile duct necrosis/cholestasis. In general, both proteomics and metabolomics were supportive of other findings. Thus, the outcome of this program indicates that "omics" technologies can help toxicologists to make better informed decisions during exploratory toxicological studies. The data support that hypothesis on mode of action and discovery of putative biomarkers are tangible outcomes of integrated "omics" analysis. Qualification of biomarkers remains challenging, in particular in terms of identification, mechanistic anchoring, appropriate specificity, and sensitivity.
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Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/metabolismo , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Unión Europea , Riñón/metabolismo , Riñón/patología , Hígado/metabolismo , Hígado/patología , Toxicología/métodos , Animales , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/diagnóstico , Riñón/efectos de los fármacos , Hígado/efectos de los fármacos , Masculino , Metabolómica/métodos , Metabolómica/tendencias , Necrosis , Valor Predictivo de las Pruebas , Proteómica/métodos , Proteómica/tendencias , Ratas , Ratas Wistar , Toxicología/tendenciasRESUMEN
The genotoxicity testing battery is highly sensitive for detection of chemical carcinogens. However, it features a low specificity and provides only limited mechanistic information required for risk assessment of positive findings. This is especially important in case of positive findings in the in vitro chromosome damage assays, because chromosome damage may be also induced secondarily to cell death. An increasing body of evidence indicates that toxicogenomic analysis of cellular stress responses provides an insight into mechanisms of action of genotoxicants. To evaluate the utility of such a toxicogenomic analysis we evaluated gene expression profiles of TK6 cells treated with four model genotoxic agents using a targeted high density real-time PCR approach in a multilaboratory project coordinated by the Health and Environmental Sciences Institute Committee on the Application of Genomics in Mechanism-based Risk Assessment. We show that this gene profiling technology produced reproducible data across laboratories allowing us to conclude that expression analysis of a relevant gene set is capable of distinguishing compounds that cause DNA adducts or double strand breaks from those that interfere with mitotic spindle function or that cause chromosome damage as a consequence of cytotoxicity. Furthermore, our data suggest that the gene expression profiles at early time points are most likely to provide information relevant to mechanisms of genotoxic damage and that larger gene expression arrays will likely provide richer information for differentiating molecular mechanisms of action of genotoxicants. Although more compounds need to be tested to identify a robust molecular signature, this study confirms the potential of toxicogenomic analysis for investigation of genotoxic mechanisms.
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Daño del ADN , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Laboratorios , Pruebas de Mutagenicidad/métodos , Mutágenos/toxicidad , Reacción en Cadena de la Polimerasa , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Aberraciones Cromosómicas/inducido químicamente , Cisplatino/toxicidad , Análisis por Conglomerados , Aductos de ADN/metabolismo , Roturas del ADN de Doble Cadena , Relación Dosis-Respuesta a Droga , Etopósido/toxicidad , Perfilación de la Expresión Génica/normas , Humanos , Laboratorios/normas , Pruebas de Mutagenicidad/normas , Variaciones Dependientes del Observador , Paclitaxel/toxicidad , Reacción en Cadena de la Polimerasa/normas , Reproducibilidad de los Resultados , Medición de Riesgo , Cloruro de Sodio/toxicidad , Huso Acromático/efectos de los fármacos , Factores de TiempoRESUMEN
Clofibric acid (CLO) is a nongenotoxic hepatocarcinogen in rodents that causes altered hepatocellular foci and/or neoplasms. Initiation by DNA-damaging agents such as diethylnitrosamine (DEN) accelerates focus and tumor appearance and could therefore significantly contribute to shortening of the regulatory 2-year rodent carcinogenicity bioassays. However, it is crucial to evaluate the histological and molecular impact of initiation with DEN on hepatocarcinogenesis promoted by CLO. Male F344 rats were given a single nonnecrogenic injection of DEN (0 or 30 mg/kg) followed by Control diet or CLO (5000 ppm) in diet for up to 20 months. Histopathology and gene expression profiling were performed in liver tumors and surrounding nontumoral liver tissues. The molecular signature of DEN was characterized and its histopathological and immunohistopathological effects on focus and tumor types were also determined. Although foci and tumors appeared earlier in the DEN+CLO-treated group compared to the group treated with CLO alone, DEN had little impact on gene expression in nontumoral tissues since the gene expression profiles were highly similar between Control and DEN-treated rats, and DEN+CLO- and CLO-treated rats. Finally, tumors obtained from DEN+CLO and CLO-treated groups displayed highly correlated gene expression profiles (r>0.83, independently of the time-point). The pathways involved in tumor development revealed by Gene Ontology functional analysis are similar when driven either by spontaneous initiation or by a chemically induced initiation step. Our work described here may contribute to the design optimization of shorter preclinical tests for the evaluation of the nongenotoxic hepatocarcinogenic potential of drugs under development.
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Alquilantes/toxicidad , Anticolesterolemiantes/toxicidad , Carcinógenos/toxicidad , Ácido Clofíbrico/toxicidad , Cocarcinogénesis , Dietilnitrosamina/toxicidad , Neoplasias Hepáticas Experimentales/inducido químicamente , Lesiones Precancerosas/inducido químicamente , Animales , Interacciones Farmacológicas , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/patología , Masculino , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Ratas , ToxicogenéticaRESUMEN
Based on the assumption that compounds having similar toxic modes of action induce specific gene expression changes, the toxicity of unknown compounds can be predicted after comparison of their molecular fingerprints with those obtained with compounds of known toxicity. These predictive models will therefore rely on the characterization of marker genes. Toxicogenomics (TGX) also provides mechanistic insight into the mode of toxicity, and can therefore be used as an adjunct to the standard battery of genotoxicity tests. Promising results, highlighting the ability of TGX to differentiate genotoxic from non-genotoxic carcinogens, as well as DNA-reactive from non-DNA reactive genotoxins, have been reported. Additional data suggested the possibility of ranking genotoxins according to the nature of their interactions with DNA. This new approach could contribute to the improvement of risk assessment. TGX could be applied as a follow-up testing strategy in case of positive in vitro genotoxicity findings, and could contribute to improve our ability to identify the molecular mechanism of action and to possibly better assess dose-response curves. TGX has been found to be less sensitive than the standard genotoxicity end-points, probably because it measures the whole cell population response, when compared with standard tests designed to detect rare events in a small number of cells. Further validation will be needed (1) to better link the profiles obtained with TGX to the established genotoxicity end-points, (2) to improve the gene annotation tools, and (3) to standardise study design and data analysis and to better evaluate the impact of variability between platforms and bioinformatics approaches.
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Toxicogenética/métodos , Toxicogenética/normas , Animales , Carcinógenos/toxicidad , Línea Celular , Expresión Génica/efectos de los fármacos , Ratones , Modelos Genéticos , Pruebas de Mutagenicidad/métodos , Pruebas de Mutagenicidad/normas , Mutágenos/toxicidad , Análisis de Secuencia por Matrices de Oligonucleótidos , Medición de Riesgo/métodos , Medición de Riesgo/normasRESUMEN
Gene expression profiling technology is expected to advance our understanding of genotoxic mechanisms involving direct or indirect interaction with DNA. We exposed human lymphoblastoid TK6 cells to 14 anticancer drugs (vincristine, paclitaxel, etoposide, daunorubicin, camptothecin, amsacrine, cytosine arabinoside, hydroxyurea, methotrexate, 5-fluorouracil, cisplatin, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU), and bleomycin) for 4-h and examined them immediately or after a 20-h recovery period. Cytotoxicity and genotoxicity, respectively, were evaluated by cell counting and by in vitro micronucleus assay at 24h. Effects on the cell cycle were determined by flow cytometry at 4 and 24h. Gene expression was profiled at both sampling times by using human Affymetrix U133A GeneChips (22K). Bioanalysis was done with Resolver/Rosetta software and an in-house annotation program. Cell cycle analysis and gene expression profiling allowed us to classify the drugs according to their mechanisms of action. The molecular signature is composed of 28 marker genes mainly involved in signal transduction and cell cycle pathways. Our results suggest that these marker genes could be used as a predictive model to classify genotoxins according to their direct or indirect interaction with DNA.
Asunto(s)
Antineoplásicos/toxicidad , Mutágenos/toxicidad , Ciclo Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Pruebas de Micronúcleos , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Timidina Quinasa/genéticaRESUMEN
Clofibrate is a peroxisome proliferator known to induce liver tumours in rats. A proteomics study was conducted to provide new insights into the molecular mechanisms of clofibrate-induced non-genotoxic hepatocarcinogenesis. Rats were treated with 250 mg/kg day clofibrate orally and sacrificed after 7 days. Proteins extracted from the liver were analysed by 2-DE using DIGE technology. The protein identification performed by MS showed that clofibrate induced up-regulation of 77 proteins and down-regulation of 27 proteins. The highest expression ratios corresponded to proteins involved in a series of biochemical pathways such as lipid metabolism, fatty acid metabolism, amino acid metabolism, protein metabolism, citric acid cycle, xenobiotic detoxification and oxidative stress. Proteins implicated in cell proliferation and apoptosis, such as prohibitin, 10-formyl tetrahydrofolate dehydrogenase, senescence marker protein-30, pyridoxine 5'-phosphate oxidase and vimentin, were also identified as being regulated. These results provide leads for further investigations into the molecular mechanisms of liver tumours induced by clofibrate. In addition, MS results showed that a series of regulated proteins were detected as several spots corresponding to different pI and/or M(r). Differential effects on those variants could result from specific PTM and could be a specific molecular signature of the clofibrate-induced protein expression modulation in rat liver.
Asunto(s)
Clofibrato/farmacología , Hígado/efectos de los fármacos , Proteínas/metabolismo , Proteómica/métodos , Secuencia de Aminoácidos , Animales , Cromatografía Liquida , Regulación hacia Abajo/efectos de los fármacos , Electroforesis en Gel Bidimensional , Regulación Enzimológica de la Expresión Génica , Concentración de Iones de Hidrógeno , Hígado/metabolismo , Hígado/patología , Extractos Hepáticos/metabolismo , Masculino , Espectrometría de Masas , Datos de Secuencia Molecular , Mapeo Peptídico , Proteínas/química , Proteínas/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tripsina/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The multi-lab International Life Sciences Institute (ILSI) project on the application of genomics to risk assessment offered the unique opportunity to investigate the influence of variability within and between laboratories on identifying biologically relevant gene expression changes. We assessed the gene expression profiles of mouse lymphoma L5178Y cells treated with hydroxyurea (HU) in three independent studies from two different laboratories, Sanofi Aventis and Procter and Gamble. Cells were dosed for 4 hr and harvested immediately at the end of the treatment or after a 20-hr recovery period. Cytotoxicity and genotoxicity were evaluated by standard assays. Statistical analysis of these data revealed that, while gene expression responses to HU treatment were markedly different at 4 hr vs. 24 hr, there was otherwise a consistent pattern of dose-response across the three studies. Therefore, the studies were merged and each time point was evaluated separately. At 4 hr, we identified 173 (P < 0.0001) dose-responsive genes with a common trend in all three studies. These were mainly associated with the cell cycle, DNA repair and DNA metabolism, and in particular, the intra-S and G2/M phase checkpoints. At 24 hr, we identified 434 dose-responsive genes common across studies. These genes were involved in lymphocyte-specific activities and the activation of apoptosis via the caspase cascade. Our results show that despite inter-laboratory variability, combining the three studies in a single statistical analysis identifies more significantly-modulated genes than in any of the individual studies, due to improved statistical sensitivity. The genes identified in our study provide information that is relevant to HU biology.
Asunto(s)
Proteínas de Ciclo Celular/genética , Técnicas de Laboratorio Clínico/normas , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hidroxiurea/toxicidad , Animales , Antineoplásicos/toxicidad , Proteínas de Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Técnicas de Laboratorio Clínico/estadística & datos numéricos , Relación Dosis-Respuesta a Droga , Leucemia L5178 , Ratones , Modelos Biológicos , Pruebas de Mutagenicidad , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacosRESUMEN
Evaluation of the nongenotoxic potential early during the development of a drug presents a major challenge. Recently, two genes were identified as potential molecular markers of rodent hepatic carcinogenesis: transforming growth factor-beta stimulated clone 22 (TSC-22) and NAD(P)H cytochrome P450 oxidoreductase (CYP-R) (1). They were identified after comparing the gene expression profiles obtained from the livers of Sprague-Dawley rats treated with different genotoxic and nongenotoxic compounds in a 5 day repeat dose in vivo study. To assess the potential of these two genes as acute markers of carcinogenesis, we investigated their modulation during a long-term nongenotoxic study in the rat using a classic initiation-promotion regime. Clofibric acid (CLO), which belongs to the broad class of chemicals known as peroxisome proliferators, was used as a nongenotoxic hepatocarcinogen. Male F344 rats were given a single nonnecrogenic injection of diethylnitrosamine (0 or 30 mg/kg) and fed a diet containing none or 5000 ppm CLO for up to 20 months. Necropsies of five rats per groups were performed at 18, 46, 102, 264, 377, 447 (control, DEN, and DEN + CLO rats), 524, and 608 days (for the CLO and control rats). Gross macroscopic and microscopic evaluation and gene expression profiling (on Affymetrix microarrays) were performed in peritumoral and tumoral liver tissues. Bioanalysis of the liver gene expression data revealed that TSC-22 was strongly down-regulated early in the study. Its underexpression was maintained throughout the study but disappeared upon CLO withdrawal. These modulations were confirmed by real-time polymerase chain reaction. However, CYP-R gene expression was not significantly altered in our study. Taken together, our results showed that TSC-22, but not CYP-R, has the potential to be an acute early molecular marker for nongenotoxic hepatocarcinogenesis in rodents.