Alternative methods in toxicology and ecotoxicology: the use of transcriptomics to define chemical profile

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1 Alternative methods in toxicology and ecotoxicology: the use of transcriptomics to define chemical profile Annamaria Colacci Environmental Protection and Health Prevention Agency

2 Environmental Carcinogenesis and Risk Assessment Center Experimental Toxicology Area Alternative methods to animal testing to screen carcinogens as single molecules or complex mixtures In vitro cell transformation (CE Reg 440/2008) Toxicogenomics Area Microarray technology (transcriptomics) CGH Risk assessment

3 BALB/c 3T3 Transformation Test International experience good degree of concordance with the in vivo rodent carcinogenesis tests (IARC/NCI/EPA Working Group 1989, 1993, OECD Working Group 2006) proposed use as a screening test for the carcinogenic potential of compounds that have no evidence of genotoxicity the experimental protocol is currently undergoing a prevalidation study which is coordinated by ECVAM Our experience (since 1988) 32 Single compounds Solvents Pesticides Pharmcological molecules Algal biotoxins 11 Complex mixtures Extracts from environmental media (soil, water, air) Complex mixtures to be introduced into the environment (fuels) Ionizing radiation Asbestos replacement fibers

4 Transformation test: BALB/c 3T3 model Cytotoxicity test - 48hr 0 48 hr 96 hr days carcinogen END POINT Cell seeding (250 cells) treatment 48 hours Medium change Fix and stain CLONAL EFFICIENCY Transformation test - 48hr 0 48 hr 96 hr days carcinogen Cell seeding (3 x 10 4 cells) Treatment 48 hours Medium change (twice a week) Fix and stain END POINT TRANSFORMATION FREQUENCY

5 Toxicological profiling Cytotoxicity toxic activity of the assayed compound/mixture (acute effects) Dose-response effects in order to choose the working doses for further experiments Transformation test potential carcinogenicity of the tested chemical to be compared with a positive control Dose response relationships Estimating a dose-response curve Data extrapolation for a quantitative assessment of the risk and/or identification of a threshold dose

6 Toxicological profile of the polycrystalline oxide fibers (PCF) in comparison with refractory ceramic fibers (RCF) and crocidolite Cells were exposed to RFC and PCF ( µg/cm 2 ) for 48 hrs to assess the effects on cloning efficiency % growth * * Cytotoxicity RCF PCF fiber concentration (µg/cm 2 ) Cells were exposed to RFC and PCF ( µg/cm 2 ) to estimate the occurrence of transformed foci TF (x 10-4 ) RCF PCF Cell transformation * µg/cm 2

7 % growth Toxicological profile of the polycrystalline oxide fibers (PCF) in comparison with refractory ceramic fibers (RCF) and crocidolite * Cytotoxicity * * fibers/cm 2 (x 10 3 ) RCF PCF CROCIDOLITE Evaluation of the dose-response relationship Identification of the critical parameters for the adverse effects of fibers Dose (Number of fibers) Dimensions Physical-chemical composition Reactivity of the surface Evaluation of the molecular mechanisms related to the biological effects of the fibers Screening of the effects of new man-made fibers Opportunity of comparative studies with natural fibers TF (x 10-4 ) Cell transformation * 0 RCF PCF CROCIDOLITE * * fibers/cm 2 (x 10 3 ) Some parameters, such as durability and biopersistence, which are essential for the pathogenesis of fibers, are strictly dependent on the microenvironment of the lung

8 Ionizing radiation exposure in BALB/c 3T3 cells Exponentially growing BALB/c 3T3 cells were γ-irradiated ( Gy) by exposure to 137 Cs radiation source Mean of colonies ± E.S. 0 (IBL 437C; 0.66 MeV; Gy Dose-rate 221 cgy/min) Cell transformation * Cytotoxicity * T.F. x Gy

9 From biological endpoints to genomic endpoints: the use of toxicogenomics approach 0.25, 0.75 and 3 Gy doses were selected because of their biological different behaviour: 0.25 Gy irradiation produced an increase in cellular proliferation without inducing foci formation 0.75 Gy irradiation was able to produce an increment of cellular growth rate and of foci occurrence, but not in a statistically significant way 3 Gy irradiation had an extremely toxic effect on clonal efficiency and a strong transforming activity

10 TOXICOGENOMICS In recent years, completion of the sequencing of the human genome as well as the genomes of dozens of other organisms and subsequent development of tools for comprehensive gene analysis have revolutionized biology. These new technologies, referred as genomics, have integrated biologic sciences with information sciences and engineering.

11 Toxicogenomics: an approach to investigate the gene-environment interaction Toxicogenomics is defined as the application of genomic technologies to toxicology, to study the adverse effects of environmental and pharmaceutical chemicals to define the toxicological profile of chemicals Fingerprints to evaluate the risk from exposure to understand the mechanism of action of chemicals to highlight individual susceptibilities to identify biomarkes of exposure and risk

12 Overview of the Toxicogenomic Technologies Toxicogenomic technologies comprise several different technology platforms for analysis of genomes, transcripts, proteins, and metabolites. Produce a large quantity of information and the comprehensive nature of this information, is much greater than what traditional experiments generate. The advancement of computing power and techniques enable these large amounts of information to be synthesized from different sources and experiments and to be analyzed in novel ways.

13 Use of transcriptomics The utility of transcriptional profiling as a sensitive method to classify cells, identify the response to pharmacological treatment and determine good or poor prognosis has been demonstrated 60 cell lines from diverse tumor tissues were classified based solely on the observed patterns of gene expression which corresponded to the tissue of origin from which the cell lines originated. (Ross et al. 2000). 70,000 antitumor drugs were then tested against the 60 cell lines for mrna expression profile to derive sensitivity to therapy, on the basis of molecular characteristics of a patient tumour. (Scherf et al. 2000) A 70-gene prognosis profile was established using microarray analysis to evaluate patient s prognosis for breast cancer (van de Vijver, 2002).

14 Transcriptomics and biomarkers of effect Transcriptomics begins to be applied to the screening of chemical compounds for identifying the chemical hazard or the mechanisms of action. It involves: measuring gene changes in response to specific doses of an administered test compound at specific time points evaluating molecular mechanisms of toxic action by sorting transcripts changes into groups of selected biological processes studying the degree of conservation of biologic response pathways across species that are responsible for toxicity

15 Transcriptomics at our lab Since 2001 Morandi et al. (2006) A cdna-microarray analysis of camptothecin resistance in glioblastoma cell lines. Cancer Lett., 231, Maffei et al. (2007) Angiopoietin-2 expression in B-cell chronic lymphocytic leukemia: association with clinical outcome and immunoglobulin heavy-chain mutational status. Leukemia, 21: Morandi et al.(2008) Gene expression time-series analysis of camptothecin effects in U87-MG and DBTRG-05 glioblastoma cell lines Mol Cancer. 7: 66 Morandi et al (2009) Gene expression changes in medical workers exposed to radiation Radiat Res. 72(4): Perdichizzi et al (2010) Biomarkers identification of ionizing radiation exposure in BALB/c 3T3 model (in preparation)

16 Lab ER-EPA O 3 : 5µg/m 3

17 Dual-colour gene expression analysis Not exposed cells (control) Exposed cells RNA RNA RNA purification and quality control (Agilent Bioanalyser 2100) ibridation RNA labeling (direct labeling linear amplification Hybridyzation of cy3- and cy5-labeled samples to 60-mer oligo microarray Washing Centro Tematico Regionale Cancerogenesi scanner Ambientale e Valutazione del Rischio Scan (Agilent scanner) Drying under nitrogen

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19 Microarray analysis of irradiated BALB/c 3T3 cells Two different analysis were performed: in order to highlight the gene expression profile specific for cells exposed to 0.25 Gy (NT vs 0.25 Gy) in order to achieve information about differences among the three doses (0.25 vs 0.75 vs 3 Gy)

20 histones higher levels after 0.25 Gy irradiation indicate an active S-phase compared to 0.75 and 3 Gy irradiation Gy 0.75 Gy 3 Gy MeanLog 2 Ratio H3f3a H3f3b Hist1h1d Hist1h2ac Hist1h2bc Hist1h4i Hist2h4

21 Identification of possible endocrine disruptors by toxicogenomics approach Which genes are transcriptionally modulated by the fungicide penconazol in T47D cells? We tested four concentrations based on the sensitivity of the cellular model to penconazol A reference design, including two technical replicates for each point, was used ppm 0.05µM ppm 0.5µM ( LMR) 1.4 ppm 5µM 4.26 ppm 15µM ( GI50) NT

22 Penconazol data-set analysis Gene Spring GX Base-line to median of all samples Dose interpretation 1 way Anova, p<0.05; Benjamini-Hochberg correction 1874 modulated gene (preliminary results)

23 OECD SERIES ON TESTING AND ASSESSMENT, 29-Apr Number 50, REPORT OF THE OECD/IPCS WORKSHOP ON TOXICOGENOMICS, Kyoto, October 2004 The term ecotoxicogenomics describes the integration of genomics into ecotoxicology. The Population and Molecular Stress Responses of Daphnia magna Slide for studying the gene profile (3000 genes) Response to cadmium Environ Sci Technol Mar, Linking molecular and population stress responses in Daphnia magna exposed to cadmium.

24 From ecotoxicology to ecotoxicogenomics Microtox toxicity test system is a standardised toxicity test system which is rapid, sensitive, reproducible, ecologically relevant and cost effective. It accurately measure toxicity from water and other environmental samples, but it is also used to test the toxicity of single compounds It is based upon a bioluminescent marine bacterium (Vibrio fischeri) as the test organism. The bacteria are exposed to a range of concentrations of the material being tested. The change in light output and concentration of the toxicant produce a dose / response relationship. The results are normalised and the EC50 (concentration producing a 50% reduction in light) is calculated Excellent correlation with the toxicity values for fish, crustaceans and algae for a wide range of organic and inorganic chemicals

25 The design of a Vibrio fischeri slide By using the sequences from V. fischeri strain ES114, from the squid Euprymna scolopes, we were able to design a microarray slide with V. fischeri genome 8 x 15K slide 3 replicates of the entire genome mer-probes 371 probes from V. cholerae (specificity control)

26 The use of a Vibrio fischeri slide to discriminate toxicological profiles 11.2 mg/l FB1+5.6 FB2 (3:1) 16 mg/l, FB1 in triplicates 16 mg/l, FB2 FB1+FB2 Gene Spring GX: 1-way ANOVA FDR p<0.05 PCA FB1 FB2 DMSO

27 Annamaria Colacci Monica Vaccari Paola Silingardi Maria Grazia Mascolo Francesca Rotondo Elena Morandi Stefania Perdichizzi

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