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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

(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

(3)

Centro Tematico Regionale Cancerogenesi Ambientale e Valutazione del Rischio

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 pre-validation 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

(4)

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

(5)

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

(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 (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

(7)

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

(8)

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

(9)

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

(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

(11)

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

(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)

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).

(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

(15)

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

(16)

Lab MATER @ ER-EPA

O

(17)

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)

(18)
(19)

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

(20)

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

(21)

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 technical

replicates for each point, was used

(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)

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.

(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

(25)

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)

(26)

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

(27)

Centro Tematico Regionale Cancerogenesi Ambientale e Valutazione del Rischio Elena Morandi Maria Grazia Mascolo Stefania Perdichizzi Francesca Rotondo Paola Silingardi Monica Vaccari Annamaria Colacci

(28)

http://www.arpa.emr.it/cancerogenesi/

VISIT US

@

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