Toxicogenomics

oftewel

de zoektocht naar alternatieven voor de huidige

proefdiermodellen voor het bepalen van veiligheid

van chemische stoffen

Dept. of Health Risk Analysis & Toxicology

Maastricht University, the Netherlands

Jos Kleinjans

Vakgroep Toxicogenomics

Current protocol for toxicity testing

• Short-Term Tests for Genetic Toxicity

– Bacterial Reverse Mutation Test

– In vitro Mammalian Chromosomal Aberration Test

– In vitro Mouse Lymphoma TK +/- Gene Mutation Assay

– In vivo Mammalian Erythrocyte Micronucleus Test

• Acute Oral Toxicity Tests

• Short Term Toxicity Studies

– Short-Term Toxicity Studies with Rodents (3 dose levels, 2-4 weeks)

• Subchronic Toxicity Studies – Subchronic Toxicity Studies with Rodents (3 dose levels, 90 days)

• Carcinogenicity Studies with Rodents

• Combined Chronic Toxicity/Carcinogenicity Studies with Rodents

• In Utero Exposure Phase for Addition to Carcinogenicity Studies with Rodents

• Reproduction and Developmental Toxicity Studies – Multi-generation Reproduction Studies (3 dose levels, life time)

– Stand alone/multi-generation Developmental Toxicity Studies

• Neurotoxicity Studies

• Metabolism and Pharmacokinetic Studies

• Immunotoxicity Studies

Current demands on animal testing

in toxic risk assessment

Type of toxicological

research

Dutch %

n=63,305

EU %

n=781,866

% of

rodents

Acute lethal toxicity 11,1 6,7 37,9

Other acute toxicity 11,2 12,3 71,3

Subacute toxicity 10,8 89,4

Subchronic/chronic toxicity 5,3 13,8 90,0

Carcinogenicity 2,0 3,0 100,0

Mutagenicity 3,5 3,1 99,5

Reproductive toxicity 11,7 10,6 84,5

Teratogenicity 31,2 4, 8 50,2

Others 13,3 19,5 38,9

EU’s regulatory authorities

• Pharmaceuticals

– European Medicines Agency

• Food ingredients

– European Food Safety Authority

• Cosmetics

– Scientific Committee on Cosmetic Products

and Non-Food Products

• Industrial Chemicals

– European Chemical Agency

Increasing demands on chemical risk assessment:

• High failure rate of new drug candidates due to unmanageable toxicity, accounting for approximately 30% of this attrition

• The EU REACH program on industrial chemicals – Registration, Evaluation and Authorization of Chemicals:

– Existing and new substances should in the future be subject to the same procedure under a single system.

– Large amounts of additional tests required before 2018 • 30,000 existing chemicals already placed on the market since before 1981 and sold

at > 1 tonne per year

• EU-wide ban on animal use in cosmetics development – Council Directive 92/32/EEC (7th Amendment): testing ban prohibiting animal

testing of cosmetic products in the EU.

• Future EU regulations on food chemicals

The problem of high attrition rates of new

drug candidates

I. Kola & J. Landis. Nature Reviews/Drug Discovery 2004: 3: 711-715

~ 30 % due to human toxicity

The TeGenero case (NJEM, 2006: 354: 1869-1871)

• A t 8 a.m. on Monday, March 13, 2006, eight healthy young men entered a trial of a drug under development by the small German immunotherapeutics company TeGenero. Six of the volunteers were assigned to receive active drug, and two were to receive placebo.

• The six volunteers were to be the first humans to receive TGN1412, a humanized monoclonal antibody designed as an agonist of the CD28 receptor on T lymphocytes, which stimulates the production and activation of T lymphocytes.

• It was hoped that this product would benefit patients with B-cell chronic lymphocytic leukemia or autoimmune diseases such as multiple sclerosis or rheumatoid arthritis.

• However, after receiving injections of TGN1412, the six volunteers became desperately ill, had multiple-organ failure, and were transferred to an intensive care unit2 with what has been described as a cytokine release syndrome.

• Preclinical testing, including tests in rabbits and monkeys that used doses up to 500 times as high as the doses received by the first group of volunteers, reportedly showed no signs of toxicity.

Understanding REACH • REACH is an EU regulation to improve the protection of human health

and the environment from the risks that can be posed by chemicals, while enhancing the competitiveness of the EU chemicals industry. It also promotes alternative methods for the hazard assessment of substances in order to reduce the number of tests on animals.

• In principle, REACH applies to all chemical substances; not only those used in industrial processes but also in our day-to-day lives. Therefore, the regulation has an impact on most companies across the EU.

• REACH places the burden of proof on companies. To comply with the regulation, companies must identify and manage the risks linked to the substances they manufacture and market in the EU. They have to demonstrate to ECHA how the substance can be safely used, and they must communicate the risk management measures to the users.

• If the risks cannot be managed, authorities can restrict the use of substances in different ways. In the long run, the most hazardous substances should be substituted with less dangerous ones.

• REACH stands for Registration, Evaluation, Authorisation and Restriction of Chemicals. It entered into force on 1 June 2007.

The problem posed by REACH

• Existing and new industrial substances should in the future, following the phasing in of existing substances until 2018, be subject to the same procedure under a single system.

• According to the European Chemicals Bureau, only 14% of the highest production volume chemicals have a publicly available basic set of hazard data (as defined, but not necessarily required, in existing EU law); 65% has less than base-set and 21% has nothing

• Large amounts of additional tests required

• 30,000 existing chemicals already placed on the market since before 1981 and sold at > 1 tonne per year

• 50,000 to 120,000 intermediates

• Questions on false positive findings

• 50% of all chronically used human pharmaceuticals induce tumors in rodents, but only 20 human pharmaceutical carcinogens have been confirmed by epidemiologic studies

REACH Timeline

12

Nature 460, 1080-1081 (2009): “Some 65,000 companies made

more than 2.7 million pre-registrations for in excess of

140,000 substances”

• Claimed costs of REACH/questions on feasibiity

• time consuming: using all of Europe’s CRO capacity, REACH will take 11-40 years

• estimated to cost 2,5 – 6,5 billion Euros – Chemical industry: “Unemployment”.

“Deindustrialization of Europe”

– WWF: “Only 0,1% of the European chemical industry’s annual sales revenues”

• large ethical implications for animal welfare: REACH will use 8 – 20 million animals

• Claimed benefits of REACH

• benefits to human health amount up to €260 billion by 2020 in Europe

Ethical considerations

• Aristoteles

Hierarchical attitude towards nature which places humans above, and even separate from, nature.

Humans are different from the other animals because they not only sense but can make choices as well.

God expects, even demands, that we be stewards of His creation.

• Immanuel Kant

“The heart of a man can be judged by his treatment of animals."

• Mahamta Ghandi

“The greatness of a nation and its moral progress can be judged by the way its animals are treated"

• In their 1959 book "The Principles of Humane Experimental Technique", William Russel and Rex Burch presented the 3R principle referring to Replacement, Refinement and Reduction of animal testing.

Accepted by the European Partnership for Alternative Approaches to Animal Testing

Amendment to the latest consolidated version of

the REACH legislation REACH Regulation

1907/2006 :

• The Commission aims to reduce the number of animals used for testing, by half

• (39) The Commission, Member States, industry and other stakeholders should continue to contribute to the promotion of alternative test methods on an international and national level including computer supported methodologies, in vitro methodologies, such as appropriate, those based on toxicogenomics, and other relevant methodologies. The Community's strategy to promote alternative test methods is a priority and the Commission should ensure that within its future Research Framework Programmes and initiatives such as the Community Action Plan on the Protection and Welfare of Animals 2006-2010 this remains a priority topic. Participation of stakeholders and initiatives involving all interested parties should be sought.

Central dogma of gene function

Genome analysis:

dual labeling and microarray hybridisation

Composition of DNA microarrays

Every spot is a unique sequence / gene.

“Beter dan een kristallen bol - Microarray

voorspelt het gedrag van een borsttumor”

• Onderzoekers van het Nederlands Kanker Instituut en het Erasmus Medisch Centrum hebben een grote hoeveelheid borsttumoren onderzocht

• Het vergelijken van de borsttumoren van patiënten jonger dan 55 jaar die bij diagnose een kleine tumor hadden (tot 5 cm diameter) leverde in Amsterdam een set van 70 genen op die heel goed gebruikt kunnen worden om te voorspellen of de tumor zich wel of niet gaat uitzaaien.

• In Rotterdam werd in materiaal van patiënten van alle leeftijden en met zowel grote als kleine tumoren een genexpressieprofiel van 76 genen gevonden, dat eveneens een hoge voorspellende waarde heeft.

• De genexpressieprofielen kunnen gebruikt worden om bij nieuwe patiënten het tumorweefsel te analyseren.

– Nadat de chirurg de tumor heeft verwijderd wordt een klein stukje hiervan bevroren, en naar een speciaal laboratorium gestuurd.

– Hier wordt de activiteit van de genen in de tumor vergeleken met het genexpressieprofiel. Het resultaat geeft aan of de patiënt een hoog of een laag risico heeft op uitzaaiingen.

– Afhankelijk van de uitslag kunnen de arts en de patiënt bepalen of een chemokuur of een hormoonbehandeling nodig is.

Animal

in vivo

Animal cells

in vitro

Human

in vivo

Human cells

in vitro

Development of alternatives to animal toxicity models:

in vitro-in vivo and inter-species extrapolation in

validating toxicogenomics-based predictive screens

?

Currently available ‘omics technologies for toxicology

Messenger RNA synthesis

Cellular functions/activities

DNA template

Protein synthesis

transcription

translation

Transcriptomics

Proteomics

Metabolomics

• a prospective study of more

than 600 patients from 22 US

tertiary care centers found

that acetaminophen-related

liver damage is the leading

cause of acute liver failure in

the country

• about half of such cases

involved unintentional

overdose

• toxic mechanism related to

generation of oidative stress

upon APAP metabolism

Biological pathways affected by APAP in mouse and rat liver. Heat map of

toxicological phenotypes (liver necrosis, serum ALT activity) and gene expression from

integrated mouse and rat data sets

APAP- induced gene expressions in vivo in rodents

Beyer RP et al. Toxicol. Sci. 2007

Prediction accuracies for rat liver toxicity vs. sub-toxicity by APAP

with different predictive methods as applied to blood

Bushel PR et al. PNAS, 2007

Differentially expressed genes in

blood that discriminate between

exposure to subtoxic/nontoxic or

toxic dose of APAP. Pictured is a

subgroup of genes involved in

immune response and inflammation.

Applying the human orthologue (66

genes) of the 270 rat blood gene

predictor for hepatotoxicity to blood

from APAP overdose victims versus

controls allows clear separation

Bushel PR et al. PNAS, 2007

ASAT Innovation Programme

Applying ‘omics technologies (transcriptomics and metabolomics) to a case study on human

volunteers

X X

ASAT Innovation Programme

Classic clinical liver toxicity tests

• Alkaline phosphatase (ALP)

• Gamma glutamyl transpeptidase (GGT)

• Alanine transaminase (ALT)

• Lactate dehydrogenase (LDH)

• Total bilirubin (Tbil)

• Albumin (Alb)

No significant changes after any APAP dose

ASAT Innovation Programme

Stringently selected, differentially expressed genes

vs T=0 at FDR < 10% and fold change of 1,2

(SAM analyses)

t = 1 t = 7 t = 25 Time course

0,5 g APAP/24 h

0 0 15 5

2 g APAP/24h

177 83 1811 1630

4 g APAP/24h

n.a. n.a. 1212 n.a.

ASAT Innovation Programme

Cell processes found with MetaCore

R1 2g, R2 0.5g and R3 4g

ASAT Innovation Programme

ASAT Innovation Programme

Metabolomics in volunteers, upon administration of 0,5-4 g APAP/24 hr –

metabolomics data analysis

• Serum

– Polar metabolites using NMR

– (Semi-polar) metabolites using LC-TOF-MS

• Urine:

– Polar metabolites using NMR

– (Semi-polar) metabolites using LC-TOF-MS

– Identification of unknown metabolites

ASAT Innovation Programme

Dose dependency of metabolites in urine

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Average ratio (6 persons): MS signal 4g:0.5g

Average ratio (6 persons): MS signal 2g:0.5g

Stand.

Dev.

ASAT Innovation Programme

All

Person-Avg

3-10-17

ASAT Innovation Programme

Results on MX and TX integration

• 0.5g APAP - t1

– Mitochondria, transcription (increase time course)

• 0.5g APAP - t25

– Transcription

• 2g APAP - t1

– Inflammatory response, ribosome (GSTO1)

• 2g APAP - t25

– Protein ubiquitination (decrease time course)

• 4g APAP - t25

– Transcription (GSTT1)

ASAT Innovation Programme

Rat-man comparison of APAP discriminating genes

Bushel PR et al. PNAS, 2007

rat human

ASAT Innovation Programme

• Few genes from Bushel’s profile are differentially expressed upon APAP administration to human volunteers at any time point at any dose level • The behaviour of this subset of genes is more comparable to Bushel’s control group than to his APAP overdose group

ASAT Innovation Programme

A. Kienhuis, M. van de Poll, H. Wortelboer, M. van Herwijnen, R. Gottschalk, A. Boorsma,

J. Kleinjans, R. Stierum, C. DeJong, J. van Delft.

Inter-species and in vitro – in vivo comparison of acetaminophen-induced gene expression profiles.

Toxicol Sci 2009 Feb;107(2):544-52.

Comparing APAP-induced changes in MetaCore pathways (all doses) at t = 24h

in human in vitro vs human in vivo

Cell process # pathways

Immune response, cytokine,chemokine-mediated signaling

7

Apoptosis/cell cycle 5

G-protein coupled receptor protein signaling

11

Transcription/translation 2

Response to extracellular stimulus

6

Small GTP-ase mediated signal transduction

1

hepatocytes in vitro

PBMCs in vivo

Using DNA microarrays, gene expression data are derived from

exposure of model systems to known toxicants (Group A, B, and C

genes). These data are compared to a set of gene expression changes

elicited by a suspected toxicant. If the characteristics match, a putative

mechanism of action can be assigned to the unknown agent.

Toxicogenomics-based screens for toxic class prediction

IP PL

037712

40

The carcinoGENOMICS

project - main outcomes -

Jos Kleinjans

Dept. of Toxicogenomics Maastricht University, The Netherlands

IP PL 037712

41

1. Genotoxicity testing

1. In vitro (2 or 3 tests on mutagenicity and

clastogenicity)

2. In vivo for in vitro positives

2. Carcinogenicity testing

1. For in vivo GTX compounds

2. For compounds to which humans will be exposed (drugs, cosmetics, some occupation settings)

Current carcinogenicity testing strategy

IP PL 037712

42

Demands for better tests: examples for genotoxicity and carcinogenicity

For pharmaceuticals, the current test battery on genotoxicity (bacterial mutagenesis, in vitro mammalian mutagenesis, in vitro chromosome aberration analysis and an in vivo chromosome stability assay) has been assessed to predict rodent carcinogenicity correctly by not more than 38 % while simultaneously producing high percentages of false positives

(Snyder RD, Green JW. A review of the genotoxicity of marketed pharmaceuticals. Mutat Res. 2001, 488:151-69 )

A recent survey of over 700 chemicals demonstrated that even 75–95% of non-carcinogens gave positive (i.e. false positive) results in at least one test in the in vitro test battery

(Kirkland D et al. Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. Mutat Res. 584 (2005) 1–256)

The current rodent cancer bioassays provide inadequate data to estimate human cancer risk at low dose; accuracies of approximately 60 % are achieved

(Ames BN et al. Cancer prevention, rodent high-dose cancer tests, and risk assessment. Risk Analysis, 16: 613-617 (1996) )

50% of all chronically used human pharmaceuticals induce tumors in rodents, but only 20 human pharmaceutical carcinogens have been confirmed by epidemiologic studies

For the important class of non-genotoxic carcinogens, no suitable test model is available

These assays have not been modified substantially since the initiation of their use.

IP PL 037712

43

Major aim of carcinoGENOMICS is to develop in vitro methods for

assessing the carcinogenic potential of compounds, as an

alternative to current rodent bioassays for genotoxicity and

carcinogenicity.

KEY TERMS:

Metabolome and transcriptome profiling.

Major target organs: the liver, the lung, and the kidney.

Robust in vitro systems (rat/human).

Interindividual variability.

Exploring stem cell technology.

Well-defined set of model compounds.

Phenotypic markers for genotoxic and carcinogenic events.

Extensive biostatistics to identify predictive pathways.

In silico model of chemical carcinogenesis.

Dedicated high throughput technology

IP PL 037712

44

Human proximal tubular epithelial cells:

Human Primary Cells

HK-2 human cell line

- Human pamplona virus transformed

RPTEC/TERT1 human cell line

- transfected with human telomerase (hTERT) (~ telomerase

positive)

Rat proximal tubular epithelial cells:

NRK-52E cell line

Carcinogenomics WP 3 Kidney Models :

Initial Cell Models:

IP PL 037712

45

robust human proximal tubular epithelial cell model selected and optimized based on: Morphology and characteristics Barrier function Genetic stability Metabolic characterization Transcriptomic profiling

Primary cilia

The Human - RPTEC/TERT1 Kidney model M. Wieser et al. Am. J. Physiol. Am J Physiol Renal Physiol. 2008, 295:F1365-75.

RPTEC/hTERT1 cells contain high levels of renal osmolytes e.g. betaine

Metabolites: 1:Isoleucine, 2:Leucine, 3:Valine, 4:EtOH, 5:Threonine, 6:Lactate, 7:Alanine, 8:Lysine, 9:5-Oxoproline, 10:Glutamine, 11:Pyruvate, 12:DMSO, 13:Tyrosine, 14:Glucose, 15:Myo-inositol, 16:Histidine, 17:1-methylnicotinamide, 18:NAD+/NADP+, 19:Tryptophan, 20:Contaminant, 21:GSSG, 22:Beta-alanine, 23:GSH, 24:Aspartate, 25:Phosphocholine, 26:Glutamate, 27:Phenylalanine, 28:Glycerophosphocholine, 29:Betaine, 30:Glycine, 31:MeOH, 32:ATP/ADP/AMP, 33:Formate and 34:Phenol. (nb. vertical expansion in aromatic region of both spectra)

Ellis JK et al, Mol. BioSyst., 2011, 7, 247–257.

Misclassification rates of the RPTEC/TERT1 Kidney model

• Human RPTEC/TERT1 cells were treated for 6h, 24h, and 72h with 1 concentration

• Each treatment was performed in at least 3 replicates

• Each tox class (GTX, Non-GTX, Non Carcinogen) is represented by 10 compounds

• Lowest misclassification rates obtained using:

– All experiments

– 72 h experiments

Classifier construction in the RPTEC/TERT1 human in vitro model using the Consensus dB pathway finding tool

-> Classifier was

based on 149 pre-

defined human

pathways (ANOVA p-

value<0.05) and 30

chemicals

-> Additional blinded

compounds were

correctly classified

with respect to all

three toxicity classes

-> Pre-validation study

assessed high inter-

lab reproducibility

Conclusions and future directions

• Our society needs assays for chemical safety

– which better predict human toxicity than current animal

models

– which are less costly and time-consuming

– which are preferably non-animal based

• The toxicogenomics approach applied to dedicated

human cellular models in vitro appears promising

• Future work will focus on

– integrating the latest ‘omics platforms (e.g next generation

sequencing)

– upgrading current in vitro models (e.g. 3D models, stem

cell-derived models)

– translating in vitro results to human disease (e.g. by

connecting to – drug-treated - patient-derived ‘ omics data)

– interacting with the EU’s regulatory authorities

Dank U voor uw aandacht !!

Check:

www.toxicogenomics.nl

www.carcinogenomics.eu

www.watisgenomics.nl