Centro Tematico Regionale Cancerogenesi Ambientale e Valutazione del Rischio
Alternative methods in toxicology and
ecotoxicology:
the use of transcriptomics to
define chemical profile
Annamaria Colacci
Environmental Protection and Health
Prevention Agency
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
Centro Tematico Regionale Cancerogenesi Ambientale e Valutazione del Rischio
BALB/c 3T3 Transformation Test
International experiencegood 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 pre-validation study which is coordinated by ECVAM Our experience (since 1988)
32 Single compounds
Solvents Pesticides Pharmcological molecules Algal biotoxins11 Complex mixtures
Extracts from environmental media (soil, water, air)
Complex mixtures to be introduced into the
environment (fuels)
Ionizing radiation
Asbestos replacement
Transformation test: BALB/c 3T3 model
END POINT Fix and stain
Medium change (twice a week) Treatment 48 hours Cell seeding (3 x 104cells) 96 hr 48 hr 0 28-35 days - 48hr carcinogen Transformation test CLONAL EFFICIENCY END POINT Cytotoxicity test
Fix and stain
Medium change treatment 48 hours Cell seeding (250 cells) 96 hr 48 hr 0 10-12 days - 48hr carcinogen
Centro Tematico Regionale Cancerogenesi Ambientale e Valutazione del Rischio
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
Toxicological profile of the polycrystalline oxide
fibers (PCF) in comparison with refractory ceramic
fibers (RCF) and crocidolite
Cells were exposed to RFC and PCF (3.75-20 µg/cm2) for 48 hrs to assess the effects on cloning efficiency Cytotoxicity ** ** * ** ** 0 1 2 3 0 7 .5 1 0 1 5 T F ( x 1 0 -4 ) RC F P C F ** ** ** * ** ** ** ** * 0 20 40 60 80 100 120 0 3.75 7.5 10 15 20 fiber concentration (µg/cm2) % g ro w th RCF PCF Cell transformation
Cells were exposed to RFC and PCF (3.75-10 µg/cm2) to estimate the occurrence of transformed
Centro Tematico Regionale Cancerogenesi Ambientale e Valutazione del Rischio
Toxicological profile of the polycrystalline oxide fibers (PCF) in comparison with refractory ceramic fibers (RCF) and
crocidolite ** ** ** * * ** ** ** ** * ** ** ** ** 0 20 40 60 80 100 120 0 5 10 15 20 fibers/cm2 (x 103) % g ro w th RCF PCF CROCIDOLITE 0 * ** * ** ** ** ** ** * 0 2 4 6 8 10 0 2 4 6 8 10 12 fibers/cm2 (x 103) T F ( x 1 0 -4 ) RCF PCF CROCIDOLITE
Cytotoxicity Cell transformation
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
Some parameters, such as
durability and biopersistence, which are essential for the pathogenesis of fibers, are strictly dependent on the microenvironment of the lung
Ionizing radiation exposure in BALB/c 3T3
cells
Exponentially growing BALB/c
3T3 cells were γ-irradiated
(0.25-6 Gy) by exposure to 137Cs radiation source (IBL 437C; 0.66 MeV; Dose-rate 221 cGy/min) ** ** ** 0 2 4 6 8 10 12 14 16 0 0.25 0.75 1.5 2 3 T .F . x 1 0 -4 ** * ** * 0 5 10 15 20 25 30 35 40 45 50 0 0.25 0.75 1.5 2 3 Gy M e a n o f c o lo n ie s ± E .S . Cytotoxicity Cell transformation
Centro Tematico Regionale Cancerogenesi Ambientale e Valutazione del Rischio
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
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
Centro Tematico Regionale Cancerogenesi Ambientale e Valutazione del Rischio
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
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.
Centro Tematico Regionale Cancerogenesi Ambientale e Valutazione del Rischio
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).
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
Centro Tematico Regionale Cancerogenesi Ambientale e Valutazione del Rischio
Transcriptomics… at our lab
Since 2001
Morandi et al. (2006) A cDNA-microarray analysis of
camptothecin resistance in glioblastoma cell lines. Cancer Lett., 231, 74-86
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: 1312-1315
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):500-509
Perdichizzi et al (2010) Biomarkers identification of ionizing
Lab MATER @ ER-EPA
O
Centro Tematico Regionale Cancerogenesi Ambientale e Valutazione del Rischio
Not exposed
cells (control) Exposed cells
RNA RNA
ibridation
scanner
RNA purification and quality control (Agilent
Bioanalyser 2100)
↓RNA labeling
(direct labeling→→→→linear amplification
Hybridyzation of cy3- and cy5-labeled samples to 60-mer oligo microarray
↓ ↓ ↓ ↓ Washing ↓ ↓ ↓ ↓
Drying under nitrogen ↓
↓ ↓ ↓
Scan (Agilent scanner)
Centro Tematico Regionale Cancerogenesi Ambientale e Valutazione del Rischio
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
histones higher levels after 0.25 Gy irradiation indicate an active S-phase compared to 0.75 and 3 Gy irradiation.
-0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 H 3 f3 a H 3 f3 b H is t1 h 1 d is t1 h 2 a c is t1 h 2 b c H is t1 h 4 i H is t2 h 4 M e a n L o g2 R a ti o 0.25 Gy 0.75 Gy 3 Gy
Centro Tematico Regionale Cancerogenesi Ambientale e Valutazione del Rischio
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
0.142 ppm 0.5µM (≈≈≈≈ LMR)
NT
0.014 ppm 0.05µM 1.4 ppm 5µM 4.26 ppm 15µM (≈≈≈≈ GI50) A reference design, including two technicalreplicates for each point, was used
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)
Centro Tematico Regionale Cancerogenesi Ambientale e Valutazione del Rischio
The term ‘
ecotoxicogenomics’ describes the
ecotoxicogenomics
integration of genomics into ecotoxicology
.
OECD SERIES ON TESTING AND ASSESSMENT, 29-Apr-2005.
Number 50, REPORT OF THE OECD/IPCS WORKSHOP ON
TOXICOGENOMICS, Kyoto, 13-15 October 2004
The Population and Molecular Stress Responses of
Daphnia magna
• Slide for studying the gene profile (3000 genes)
• Response to cadmium
Environ Sci Technol. 2008 Mar, Linking molecular and population stress responses in Daphnia magna exposed to cadmium.
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
Centro Tematico Regionale Cancerogenesi Ambientale e Valutazione del Rischio
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 3747 60 mer-probes
371 probes from V. cholerae (specificity control)
11.2 mg/l FB1+5.6 FB2 (3:1) 16 mg/l, FB1 in triplicates 16 mg/l, FB2 Gene Spring GX: 1-way ANOVA FDR p<0.05 PCA FB1 FB2 DMSO FB1+FB2
The use of a
Vibrio fischeri
slide to
Centro Tematico Regionale Cancerogenesi Ambientale e Valutazione del Rischio Elena Morandi Maria Grazia Mascolo Stefania Perdichizzi Francesca Rotondo Paola Silingardi Monica Vaccari Annamaria Colacci