Can “omic” data assist in environmental monitoring and risk assessment of chemicals and particles?

Kevin ChipmanThe University of Birmingham, UK

The early days and now

• Too much hype at first regarding the immediate potential of “omics”...now a rebound

• Early problems around platform compatability-now largely resolved

• Insufficient datapoints and complexity of early work (mainly due to costs)- now largely resolved

• Many early analyses were not sufficiently objective and interpretation was flawed- informatics and pathway knowledge now starting to resolve

How omics may help to address the needs

• Need for improved predictivity for risk assessment• Related to this we need to improve understanding of modes of

action and to derive diagnostic and predictive biomarkers• The omic technologies have the ability to aid both of these areas• Contribute to “weight of evidence” in toxicity assessment

– Identify possible mode(s) of action– Identify and assess impacts on susceptible populations and

life stages – Improve assessments for mixtures– Dose-response assessment – Exposure assessment– Improving interspecies extrapolations

International Workshop to identify hurdles Vancouver 2008 :

Article in press. Env. Health Perspect.2009

• Regulatory bodies are already receptive e.g. US-FDA Critical Path Initiative which encourages innovation

• Reports of the National Research Council (USA) :

1. “Toxicology testing in the 21st century” shows the potential and the need to incorporate omics into safety assessment

2. Committee on application of toxicogenomic technologies and predictive toxicology and risk assessment

Transcriptomics / Proteomics /Metabolomics BIOINFORMATICS

Networks of responses to toxicants provide a profile of response

reflecting the global status of tissue

Establish fingerprints characteristic and predictive

of specific toxicities

Identify compensatory, non-toxicity responses

Derive focussed (custom/ biomarker) expression arrays,

reporter gene assays etc.

Define the “systems toxicology”of individuals and predict health

status

Help to understand

MECHANISM of toxicity

RISK ASSESSMENT

for populations

Relevant to environmental standard setting : can help to validate and monitor

Toxicogenomics in non-model organisms•Lack of genomic data

•Microarray studies do not have to be limited to a few genetic model organisms

•cDNA clones can be derived from conventional or subtracted EST libraries, eg. suppressive, subtractive hybridisation (SSH)

•Automatic, practical annotation solutions for cDNA clones are available, eg. Blast2GO, Partigene

•High throughput DNA sequencing (eg. 454, Solexa) can now allow swift design of oligonucleotide arrays for non-model species (e.g. Craft and Chipman Mussel programme)

•Non-pollutant environmental influences and inter-individual variation.

•Gene expression profiling should include laboratory exposures with the aim of identifying ‘predictive gene sets’

•Clear experimental design and sufficient replication are essential

•Inter-individual variation can inform on the population structure

• Now some examples of the power of the omics

Note: already successes

e.g. Mamoprint in medicine

e.g. Distinguishing between genotoxic and nongenotoxic carcinogens

Flounder cDNA Microarrays as Tools for the Identification of

Expression Changes in Gene Sets Predictive of Exposure to

Pollutants.

Tim Williams, Steven George, Amer Diab, Margaret Brown, John Craft, Ioanna

Katsiadaki, Fleur Geoghegan, Brett Lyons, Victoria Sabine, Fernando Ortega, Francesco

Falciani and Kevin Chipman

1:1 ratio

X-axis: Cadmium Stage 2 (Default Interpretation) : Treatm...Y-axis: Cadmium Stage 2 (Default Interpretation) : Treatm...

Colored by: Cadmium Stage 2, Default Interpretation (Trea...Gene List: Good Cd 1 (10664)

100 1000 1e4

100

1000

1e4

Treatment f Cadmium d01 (control)

HSP30Bclones

Apparent Induced Genes

ApparentRepressedGenes

2-fold up

2-fold downExample scatter plotof Cd-treated flounderat day 1 vs saline.

Treated fish show many changes in liver gene expressionWhich genes and which pathways are altered e.g. by Cd (pro-oxidant)??

C - Protein Synthesis

-Chaperonin containing TCP-1, subunit 6A-Chaperonin containing TCP-1, delta-Chaperonin subunit 7-Heat shock protein Hsp70 (*not statistically significant)-Hsp70 binding protein-Hsp70/Hsp90 organising protein SIP1/XST1-DnaJ (Hsp40) homolog, subfamily C member 1 -Nucleophosmin 1-DnaJ (Hsp40) homolog, subfamily C, member 8-Heat shock protein gp96-Protein disulfide isomerase related protein -ER-resident chaperone calreticulin-Heat shock protein Hsp90 beta-Heat shock cognate Hsc71-Heat shock protein HSP 90 alpha-Low molecular weight heat shock protein Hsp30B-DnaJ (Hsp40) homolog subfamily B, member 1-MGC65804, similar to HSP90 co-chaperone P23-CAG07414, containing DnaJ domain-T-complex polypeptide 1

A - Chaperones

PfIL295A08 (Chaperonin containing TCP1, subunit 6A (zeta 1))

PfIL252A10 (chaperonin containing TCP-1 delta)

PfIL009H04 (chaperonin subunit 7)

PfH70-g1 (heat shock protein 70)

PfIL294C08 (Hsp70 binding protein)

Contig417 (stress-induced-phosphoprotein 1 (Hsp70/Hsp90-organizi...

PfIL236A07 (DnaJ (Hsp40) homolog, subfamily C, member 1)

PfIL232G03 (Nucleophosmin 1)

PfIL228B06 (DnaJ (Hsp40) homolog, subfamily C, member 8)

PfIL255E12 (heat shock protein gp96)

Contig204 (Protein disulfide isomerase related protein)

Contig298 (ER-resident chaperone calreticulin)

Contig775 (heat shock protein 90 beta)

Contig768 (Heat shock cognate 71 kDa protein)

Contig490 (heat shock protein HSP 90 alpha)

Contig426 (low molecular weight heat shock protein Hsp30B)

Contig1015 (DnaJ (Hsp40) homolog, subfamily B, member 1)

PfIL273G12 (Unknown (protein for MGC:65804))

PfIL209A02 (unnamed protein product CAG07414)

Contig85 (T-complex polypeptide 1)

Day after Cd treatment01 02 04 08 16

-PolyA binding protein (P. platessa)-40S ribosomal protein S3-40S ribosomal protein S3a (P. americanus)-60S ribosomal protein L3-Translation initiation factor 4E binding protein 3-40S ribosomal protein S14-40S ribosomal protein S27-2 -Density regulated protein-40S ribosomal protein S18-40S ribosomal protein S16-60S ribosomal protein L7a-40S ribosomal protein S2-40S ribosomal protein S5-60S ribosomal protein L13A-40S ribosomal protein S3a-Translation initiation factor 1-delta-60S ribosomal protein L18-40S ribosomal protein Sa-Similar to translation initiation factor 3 subunit 9-60S ribosomal protein L17-40S ribosomal protein S12-Translation initiation factor 1-beta 2-40S ribosomal protein S10-40S ribosomal protein S21-60S ribosomal protein L10-60S ribosomal protein L5-60S ribosomal protein L12-Similar to S-phase kinase associated protein 1A b-G1 to S phase transition 1-Siah-interacting protein-NHP2-like 1-ADP-ribosylation factor-like 6 interacting protein-Pescadillo-Density regulated protein-60S ribosomal protein L39-60S ribosomal protein L19-60S ribosomal protein L24-Translation initiation factor 3 /P42-Translation initiation factor 4A

Day after Cd treatment01 02 04 08 16

B - Oxidative Stress-Paraoxonase 2-Catalase-MAP kinase interacting serine/threonine kinase 2-Glutathione reductase (*not statistically significant)-Selenoprotein M-Glutaredoxin-Selenide water dikinase 2-Selenium binding protein 1-Plasma glutathione peroxidase precursor-Ferritin H-1-Ferritin M-Cu/Zn Superoxide dismutase-Peroxiredoxin (Thioredoxin peroxidase)-Carbonyl reductase 1

PfPARA-o2 (Paraoxonase 2)

PfIL265A03 (Catalase)

Contig456 (MAP kinase-interacting serine/threonine kinase 2)

PfGR-1 (Glutathione reductase)

Contig691 (selenoprotein M)

Contig406 (Glutaredoxin)

PfIL256D11 (selenide water dikinase 2)

Contig1002 (Selenium binding protein 1)

Contig658 (Plasma glutathione peroxidase precursor)

Contig419 (Ferritin H-1)

Contig416 (Ferritin, middle subunit (Ferritin M))

Contig123 (copper/zinc superoxide dismutase)

Contig459 (Peroxiredoxin (Thioredoxin peroxidase) (NKeF))

Contig444 (Carbonyl reductase 1 (20 beta hydroxysteroid dehydrogen...

Day after Cd treatment01 02 04 08 16

-Translocon associated protein gamma-ADP ribosylation factor 5-SEC22, vesicle trafficking protein-like 1B-TMED 7-TIMM 23 homolog-Clathrin, heavy polypeptide-TRAP-like protein precursor-Protective protein for beta galactosidase-ADP ribosylation factor-like 6 interacting protein-Adaptor-related protein complex 3 sigma 1 -Ran nuclear GTPase-Syntaxin 5a-GTP-binding protein-like 1

Day after Cd treatment01 02 04 08 16 D - Protein Transport

-zgc:56219/Ubiquitin conjugating enzyme E2Q -Proteasome subunit alpha type 4 -Proteosome subunit alpha type 6-Proteosome alpha 1subunit isoform 2-Proteasome 26S subunit 12-26S proteasome regulatory subunit S10b-Proteasome subunit N3-Proteasome 26S, regulatory subunit 6-Proteasome beta subunit C5-Proteasome delta-26S proteasome regulatory subunit 8-Proteasome subunit beta type 3-Ubiquitin conjugating enzyme E2 variant 2

E - Protein degradationDay after Cd treatment01 02 04 08 16

-Alpha-tubulin-Dynein light chain 2-Syndecan 2-Actin related protein 3 homolog-Cysteine and glycine rich protein 2-Thymosin beta 4-Nucleolar and spindle associated protein 1 /ANKT-Annexin max 3-Beta-actin-Microtubule based motor protein FKIF2-Adducin 3 gamma

Day after Cd treatment01 02 04 08 16 F Cytoskeleton

-Similar to Programmed Cell Death 6-Cytochrome c-Reticulon 1-Thioredoxin-like 1-Ethylmalonic enaphalopathy 1-APG 16L beta-Anticoagulant protein C precursor-p8 / Candidate Of Metastasis 1-Thymidine phosphorylase precursor-COMM-domain-containing 3 / BUP-Integral membrane protein 2B-Similar to DIABLO-Survivin 1

Day after Cd treatment01 02 04 08 16 G - Apoptosis

-NHP2-like 1-G1 to S phase transition 1-Chaperonin subunit 7-Ran nuclear GTPase-GTP-binding protein like 1-Centromere/kinetochore protein zw10 homolog-Septin 5-Cyclin H

Day after Cd treatment01 02 04 08 16 H - Cell Cycle

-Alpha-1-microglobulin precursor-IgM heavy chain constant region-Interleukin 8-MHC II invariant chain-Melanoma ubiquitous mutated protein MUM1-Similar to small inducible cytokine-Small inducible cytokine-T-lymphocyte antigen receptor beta chain constant region-Similar to complement component C8 gamma-Similar to integral membrane protein 2A-Similar to TNF 13b-Src family associated phosphoprotein 2-TNF ligand superfamily member 14-Cysteine rich protein 1-Class 1 helical cytokine receptor 26-Immunoglobulin light chain L2-C-type lectin domain 1-Complement component C9-Macrophage asialoglycoprotein binding protein 1-Plasma protease C1 inhibitor precursor

Day after Cd treatment01 02 04 08 16 I - Immune and Inflammation

-Glutathione-S-transferase theta 3-Microsomal glutathione-S-transferase 3-Cytochrome P450 CYP2K6-Glutathione-S-transferase A-Cytochrome P450 CYP2F2-Metallothionein (* not statistically significant)-Microsomal glutathione-S-transferase 1-Cytochrome P450 CYP1A-Vitellogenin A-Choriogenin L (* not statistically significant)-Vitellogenin A

Day after Cd treatment01 02 04 08 16 J - Biomarkers

Cadmium treatment(Williams et al EST 2006)Single intraperitoneal injection of flounder with a low dose of cadmium (0.05 mg/kg) resulted in hepatic gene expression changes related to -

Chaperones Oxidative stressProtein synthesis Protein transportProtein degradation CytoskeletonApoptosis Cell cycleImmune InflammationBiomarkers

C - Protein Synthesis

-Chaperonin containing TCP-1, subunit 6A-Chaperonin containing TCP-1, delta-Chaperonin subunit 7-Heat shock protein Hsp70 (*not statistically significant)-Hsp70 binding protein-Hsp70/Hsp90 organising protein SIP1/XST1-DnaJ (Hsp40) homolog, subfamily C member 1 -Nucleophosmin 1-DnaJ (Hsp40) homolog, subfamily C, member 8-Heat shock protein gp96-Protein disulfide isomerase related protein -ER-resident chaperone calreticulin-Heat shock protein Hsp90 beta-Heat shock cognate Hsc71-Heat shock protein HSP 90 alpha-Low molecular weight heat shock protein Hsp30B-DnaJ (Hsp40) homolog subfamily B, member 1-MGC65804, similar to HSP90 co-chaperone P23-CAG07414, containing DnaJ domain-T-complex polypeptide 1

A - Chaperones

PfIL295A08 (Chaperonin containing TCP1, subunit 6A (zeta 1))PfIL252A10 (chaperonin containing TCP-1 delta)PfIL009H04 (chaperonin subunit 7)PfH70-g1 (heat shock protein 70)PfIL294C08 (Hsp70 binding protein)Contig417 (stress-induced-phosphoprotein 1 (Hsp70/Hsp90-organizi...PfIL236A07 (DnaJ (Hsp40) homolog, subfamily C, member 1)PfIL232G03 (Nucleophosmin 1)PfIL228B06 (DnaJ (Hsp40) homolog, subfamily C, member 8)PfIL255E12 (heat shock protein gp96)Contig204 (Protein disulfide isomerase related protein)Contig298 (ER-resident chaperone calreticulin)Contig775 (heat shock protein 90 beta)Contig768 (Heat shock cognate 71 kDa protein)Contig490 (heat shock protein HSP 90 alpha)Contig426 (low molecular weight heat shock protein Hsp30B)Contig1015 (DnaJ (Hsp40) homolog, subfamily B, member 1)PfIL273G12 (Unknown (protein for MGC:65804))PfIL209A02 (unnamed protein product CAG07414)Contig85 (T-complex polypeptide 1)

Day after Cd treatment01 02 04 08 16

-PolyA binding protein (P. platessa)-40S ribosomal protein S3-40S ribosomal protein S3a (P. americanus)-60S ribosomal protein L3-Translation initiation factor 4E binding protein 3-40S ribosomal protein S14-40S ribosomal protein S27-2 -Density regulated protein-40S ribosomal protein S18-40S ribosomal protein S16-60S ribosomal protein L7a-40S ribosomal protein S2-40S ribosomal protein S5-60S ribosomal protein L13A-40S ribosomal protein S3a-Translation initiation factor 1-delta-60S ribosomal protein L18-40S ribosomal protein Sa-Similar to translation initiation factor 3 subunit 9-60S ribosomal protein L17-40S ribosomal protein S12-Translation initiation factor 1-beta 2-40S ribosomal protein S10-40S ribosomal protein S21-60S ribosomal protein L10-60S ribosomal protein L5-60S ribosomal protein L12-Similar to S-phase kinase associated protein 1A b-G1 to S phase transition 1-Siah-interacting protein-NHP2-like 1-ADP-ribosylation factor-like 6 interacting protein-Pescadillo-Density regulated protein-60S ribosomal protein L39-60S ribosomal protein L19-60S ribosomal protein L24-Translation initiation factor 3 /P42-Translation initiation factor 4A

PfIL233E09 (40S ribosomal protein S3)

Pa003 (Ribosomal Protein S3A)

Contig557 (60S ribosomal protein L3)

PfIL288F02 (eukaryotic translation initiation factor 4E binding protein 3)

Contig800 (40S ribosomal protein S14)

Contig795 (40S ribosomal protein S27-2)

Contig770 (40S ribosomal protein S18)

Contig600 (40S ribosomal protein S16)

Contig748 (60S ribosomal protein L7a)

Contig684 (40S ribosomal protein S2)

Contig642 (40S ribosomal protein S5)

Contig392 (60S ribosomal protein L13A)

Contig576 (40S ribosomal protein S3a)

Contig474 (Eukaryotic translation elongation factor 1-delta)

Contig582 (60S ribosomal protein L18)

Contig48 (40S ribosomal protein Sa)

Contig346 (cDNA clone hab41f08.x1 similar to TIF3 subunit 9)

Contig786 (60S Ribosomal protein L17)

Contig2 (40S ribosomal protein S12)

Contig661 (Eukaryotic translation elongation factor 1 beta 2)

Contig618 (40S ribosomal protein S10)

Contig577 (40S ribosomal protein S21)

PfIL318H11 (60S ribosomal protein L10)

Contig602 (60S ribosomal protein L5)

Contig527 (60S ribosomal protein L12)

PfIL211H04 (similar to S-phase kinase-associated protein 1A isoform b)

PfIL277F10 (G1 to S phase transition 1; hm:zehn1143)

PfIL257A11 (Siah-interacting protein (Sip-prov))

Contig317 (NHP2 non-histone chromosome protein 2-like 1)

PfIL203B10 (ADP-ribosylation factor-like 6 interacting protein)

PfIL289C07 (Pescadillo)

PfCF1H9 (Density-regulated protein; smooth muscle cell associated protei...

PfIL265G02 (60S ribosomal protein L39)

Contig542 (60S ribosomal protein L19)

PfIL011D06 (60S ribosomal protein L24)

PfG6D-l2 (TIF3 / P42)

Contig292 (Eukaryotic translation initiation factor 4A)

Day after Cd treatment01 02 04 08 16

B - Oxidative Stress-Paraoxonase 2-Catalase-MAP kinase interacting serine/threonine kinase 2-Glutathione reductase (*not statistically significant)-Selenoprotein M-Glutaredoxin-Selenide water dikinase 2-Selenium binding protein 1-Plasma glutathione peroxidase precursor-Ferritin H-1-Ferritin M-Cu/Zn Superoxide dismutase-Peroxiredoxin (Thioredoxin peroxidase)-Carbonyl reductase 1

PfPARA-o2 (Paraoxonase 2)

PfIL265A03 (Catalase)

Contig456 (MAP kinase-interacting serine/threonine kinase 2)

PfGR-1 (Glutathione reductase)

Contig691 (selenoprotein M)

Contig406 (Glutaredoxin)

PfIL256D11 (selenide water dikinase 2)

Contig1002 (Selenium binding protein 1)

Contig658 (Plasma glutathione peroxidase precursor)

Contig419 (Ferritin H-1)

Contig416 (Ferritin, middle subunit (Ferritin M))

Contig123 (copper/zinc superoxide dismutase)

Contig459 (Peroxiredoxin (Thioredoxin peroxidase) (NKeF))

Contig444 (Carbonyl reductase 1 (20 beta hydroxysteroid dehydrogen...

Day after Cd treatment01 02 04 08 16

-Translocon associated protein gamma-ADP ribosylation factor 5-SEC22, vesicle trafficking protein-like 1B-TMED 7-TIMM 23 homolog-Clathrin, heavy polypeptide-TRAP-like protein precursor-Protective protein for beta galactosidase-ADP ribosylation factor-like 6 interacting protein-Adaptor-related protein complex 3 sigma 1 -Ran nuclear GTPase-Syntaxin 5a-GTP-binding protein-like 1

Day after Cd treatment01 02 04 08 16

Contig369 (translocon-associated protein gamma)

PfIL294D10 (ADP-ribosylation factor 5)

PfIL230G07 (SEC22, vesicle trafficking protein-like 1B)

PfIL245F05 (transmembrane emp24 protein transport domain contai...

PfIL288A03 (Translocase of inner mitochondrial membrane 23 homol...

PfIL312A07 (Clathrin, heavy polypeptide (Hc))

PfIL309B02 (TRAP-like protein precursor)

PfIL223C05 (Protective protein for beta-galactosidase)

PfIL203B10 (ADP-ribosylation factor-like 6 interacting protein)

PfIL256F09 (adaptor-related protein complex 3, sigma 1 subunit)

PfCF2C3 (Ran protein - member of Ras superfamily, nuclear GTP-ase.)

PfIL206D01 (syntaxin 5a)

PfIL306C03 (GTP-binding protein like 1)

D - Protein Transport

-zgc:56219/Ubiquitin conjugating enzyme E2Q -Proteasome subunit alpha type 4 -Proteosome subunit alpha type 6-Proteosome alpha 1subunit isoform 2-Proteasome 26S subunit 12-26S proteasome regulatory subunit S10b-Proteasome subunit N3-Proteasome 26S, regulatory subunit 6-Proteasome beta subunit C5-Proteasome delta-26S proteasome regulatory subunit 8-Proteasome subunit beta type 3-Ubiquitin conjugating enzyme E2 variant 2

E - Protein degradationPfIL315E08 (zgc:56219)

PfIL272D08 (proteasome (prosome, macropain) subunit, alpha type 4)

PfIL314H06 (proteasome (prosome, macropain) subunit, alpha type 6)

PfIL258H08 (proteasome alpha 1 subunit isoform 2)

Contig443 (proteasome (prosome, macropain) 26S subunit, non-ATPase, 12)

Contig1004 (26S protease regulatory subunit S10B)

Contig254 (proteasome subunit N3)

PfIL308H05 (proteasome, 26S, non-ATPase regulatory subunit 6)

PfIL273D04 (Proteasome beta-subunit C5 (Proteasome (Prosome, macropain) sub...

Contig347 (Proteasome delta)

Contig714 (26S protease regulatory subunit 8)

Contig579 (Proteasome subunit beta type 3)

PfIL277B08 (ubiquitin-conjugating enzyme E2 variant 2 (Ube2v2))

Day after Cd treatment01 02 04 08 16

-Alpha-tubulin-Dynein light chain 2-Syndecan 2-Actin related protein 3 homolog-Cysteine and glycine rich protein 2-Thymosin beta 4-Nucleolar and spindle associated protein 1 /ANKT-Annexin max 3-Beta-actin-Microtubule based motor protein FKIF2-Adducin 3 gamma

Day after Cd treatment01 02 04 08 16

Contig305 (alpha tubulin)

Contig626 (Dynein light chain 2, cytoplasmic)

PfIL316A06 (Syndecan 2)

PfIL240E02 (ARP3 actin-related protein 3 homolog)

Contig475 (cysteine and glycine-rich protein 2)

Contig789 (Thymosin beta-4)

Contig98 (nucleolar and spindle associated protein 1; nucleolar protein ANKT)

PfIL207C10 (annexin max3)

PfIL242A06 (pfBF2D7, beta actin)

PfIL242G01 (microtubule-based motor protein (FKIF2))

PfIL231A02 (adducin 3 (gamma);)

F Cytoskeleton

-Similar to Programmed Cell Death 6-Cytochrome c-Reticulon 1-Thioredoxin-like 1-Ethylmalonic enaphalopathy 1-APG 16L beta-Anticoagulant protein C precursor-p8 / Candidate Of Metastasis 1-Thymidine phosphorylase precursor-COMM-domain-containing 3 / BUP-Integral membrane protein 2B-Similar to DIABLO-Survivin 1

PfIL300G11 (similar to programmed cell death 6)

Contig465 (Cytochrome c)

PfIL255H02 (Reticulon 1)

Contig1023 (Thioredoxin-like 1)

Contig779 (Ethylmalonic encephalopathy 1)

PfIL236H02 (APG16L beta)

Contig717 (anticoagulant protein C precursor (PROC))Contig196 (p8 protein (candidate of metastasis 1))

Contig605 (Thymidine phosphorylase precursor)

Contig376 (COMM domain containing 3; BUP protein;)

PfIL224F06 (Integral membrane protein 2B)

Contig269 (similar to direct IAP binding protein with low PI)

PfIL209H11 (Survivin 1)

Day after Cd treatment01 02 04 08 16 G - Apoptosis

-NHP2-like 1-G1 to S phase transition 1-Chaperonin subunit 7-Ran nuclear GTPase-GTP-binding protein like 1-Centromere/kinetochore protein zw10 homolog-Septin 5-Cyclin H

Day after Cd treatment01 02 04 08 16

Contig317 (NHP2 non-histone chromosome protein 2-like 1)

PfIL277F10 (G1 to S phase transition 1; hm:zehn1143)

PfIL009H04 (chaperonin subunit 7)

PfCF2C3 (Ran protein - member of Ras superfamily, nuclear GTP-ase.)

PfIL306C03 (GTP-binding protein like 1)

PfIL233F02 (centromere/kinetochore protein zw10 homolog)

PfIL235F11 (Septin 5)

PfIL282H07 (Cyclin H)

H - Cell Cycle

Contig596 (alpha-1-microglobulin/bikunin precursor)

Contig405 (IgM heavy chain constant region)

Contig197 (interleukin 8)

Contig773 (MHC II invariant chain)

PfIL291G12 (melanoma ubiquitous mutated protein MUM1)

PfIL249C03 (Similar to Small inducible cytokine)

Contig281 (Small inducible cytokine)

PfIL263G01 (T-lymphocyte antigen receptor beta-chain constant region 2)

PfIL248D01 (Similar to complement component C8 gamma)

PfIL263E02 (clone WA8-6 sim to integral membrane protein 2A)

PfIL140C08 (cDNA clone JFConA894F, Sim to TNF 13b)

PfIL295H08 (src family associated phosphoprotein 2)

PfIL273F10 (tumor necrosis factor ligand superfamily, member 14)

Contig707 (Cysteine-rich protein 1)

PfIL259D05 (class I helical cytokine receptor number 26)

Contig80 (immunoglobulin light chain L2)

Contig471 (C-type lectin 1)

Contig529 (complement component C9)

PfIL267A02 (Macrophage asialoglycoprotein-binding protein 1)

PfIL264F04 (Plasma protease C1 inhibitor precursor)

-Alpha-1-microglobulin precursor-IgM heavy chain constant region-Interleukin 8-MHC II invariant chain-Melanoma ubiquitous mutated protein MUM1-Similar to small inducible cytokine-Small inducible cytokine-T-lymphocyte antigen receptor beta chain constant region-Similar to complement component C8 gamma-Similar to integral membrane protein 2A-Similar to TNF 13b-Src family associated phosphoprotein 2-TNF ligand superfamily member 14-Cysteine rich protein 1-Class 1 helical cytokine receptor 26-Immunoglobulin light chain L2-C-type lectin domain 1-Complement component C9-Macrophage asialoglycoprotein binding protein 1-Plasma protease C1 inhibitor precursor

Day after Cd treatment01 02 04 08 16 I - Immune and Inflammation

PfIL260C08 (Glutathione S-transferase, theta 3)

Contig531 (microsomal glutathione S-transferase 3)

Contig723 (Cytochrome P450 monooxygenase CYP2K6)

Contig367 (glutathione S-transferase)

Contig218 (Cytochrome P450 2F2)

Contig22 (metallothionein)

PfIL254F12 (Microsomal glutathione S-transferase 1)

Contig501 (cytochrome P450 1A CYP1A)

Contig458 (Vitellogenin)

Contig401 (choriogenin L)

Contig1030 (Vitellogenin)

-Glutathione-S-transferase theta 3-Microsomal glutathione-S-transferase 3-Cytochrome P450 CYP2K6-Glutathione-S-transferase A-Cytochrome P450 CYP2F2-Metallothionein (* not statistically significant)-Microsomal glutathione-S-transferase 1-Cytochrome P450 CYP1A-Vitellogenin A-Choriogenin L (* not statistically significant)-Vitellogenin A

Day after Cd treatment01 02 04 08 16 J - Biomarkers

HMG-CoA synthase (down 6 fold)

HMG-CoA reductase(down 5 fold)

Isopentenyl-diphosphate delta isomerase (down 5 fold)

Farnesyl diphosphate synthase (down 2.5 fold)

Mevalonate kinase (down 1.5 fold)

Mouse model of Wilson’s disease (ATP7B -/-)

Huster et al., 2007 JBC

Stickleback Exposure to 128g/L Cu

Cu exposure of Stickleback shows similar hepatic expression changes in cholesterol biosynthesis pathway genes to Wilson’s disease, a copper accumulation disorder

Tyne(Heavy industrial)Howden, Team

Alde(rural)

Outer Elbe(CuxhavenHelgoland)

Elbe Harbour(industrial, harbour, canal

Brunsbuttel)

FLOUNDER FIELD SITESQ. If fish provided “blind” could genomics identify sampling location and if so are the gene patterns reflective of pollutant exposure e.g. oxidative stress??

Predicting Site Membership by genetic algorithm GALGO (NERC Project)

Oxidative StressCatalaseSuperoxide dismutaseChaperonesCalreticulinHaem biosynthesisCoproporphyrinogen oxidase

Phase IAldehyde dehydrogenaseAlcohol dehydrogenaseCYP1ACYP2FCYP3ACYP8B

Examples of genes induced at polluted sites

Artificial chromosome

Population andfitness valueattached

new population

crossover

mutation

Create Initial Populationof Chromosomes

with random genes

Evaluate all chromosomesusing the fitness function

Generate new popultation:Reproduce chromosomes

proportionally to its fitness

Random crossover betweenchromosomes pairs

Random mutatations on new population

if some fitness >= goal

SELECTno

yes

Artificial chromosome

Population andfitness valueattached

new population

crossover

mutation

Create Initial Populationof Chromosomes

with random genes

Evaluate all chromosomesusing the fitness function

Generate new popultation:Reproduce chromosomes

proportionally to its fitness

Random crossover betweenchromosomes pairs

Random mutatations on new population

if some fitness >= goal

SELECTno

yes

Stage 1

Stage 2

Stage 4

Stage 5

Stage 3

Stage 6

Stag

e 7

Phase 2UDPGTGST

Proliferation markerPCNAProtein degradationProteasome subunits

Trevino V. & Falciani F. Bioinformatics. 2006

1;22 :1154-6.

Aroclor

Lindane

PFOA

Cadmium

TBHP

3 MC

Time course chemical treatments

Could a subset of combined stress-response genes help to classify the environmental samples?

Set of stress-related genes up & down regulated.

Merge all the IDs that were selected in each of representative models for each treatment: 98 IDs

Class Confusion ( 1 Models) [project]:knn-3K1LeuclideanD-0,1-loocv

0.8970.011

0.015

0

0.013

0.046

0.018

0

0.750

0.078

0

0

0.172

0

0

10

0

0

0

0

0.044

0

0.9560

0

0

0

0

0.016

0

0.844

0.104

0.037

0

0

0

0

0.13

0.8660.004

Alde

Elbe

Elbe H.

Heligolang

Tyne H

Tyne T

(NA)

X1

X2

X3

X4

X5

X6

X7

X8

X9

X1

0X

11

X1

2X

13

X1

4X

15

X1

6X

17

X1

8X

19

X2

0X

21

X2

2X

23

X2

4X

25

X2

6X

27

X2

8X

29

X3

0X

31

X3

2X

33

X3

4X

35

X3

6X

37

X3

8X

39

X4

0X

41

X4

2X

43

X4

4X

45

X4

6X

47

X4

8X

49

X5

0X

51

X5

2X

53

X5

4X

55

X5

6X

57

X5

8X

59

X6

0X

61

X6

2X

63

X6

4X

65

X6

6X

67

X6

8X

69

X7

0X

71

X7

2X

73

X7

4X

75

X7

6X

77

Alde29/29

Samples

0.8971

Elbe4/4

Samples

0.750.989

Elbe H.21/21

Samples

10.994

Heligolang5/5

Samples

0.9560.984

Tyne H10/10

Samples

0.8440.971

Tyne T8/8

Samples

0.8660.97

SensitSpecif

Pre

dic

ted

Cla

ss

Original Class (sorted)

Contig442: Glutamate carboxypeptidase (Darmin)

Contig665: Ependymin

Contig620: Retinol-binding protein II, cellular (CRBP-II)

NB This does not necessarily implicate these pollutants as being responsible but it helps to identify stress response differences at the sites

Conclude: A small number of stress response genes are predictive of site of origin !

Use of genetic algorithm analysis using combined stress responsive genes

Examples:

Modeling

We are using linkage networks (Dr Francesco Falciani) to integrate gene expression and metabolomics (Dr Mark Viant)with traditional measures.

Linkage shows where data are related.

This simplified example was generated using ARACNE and cytoscape, employing 50 selected nodes.

Interestingly traditional markers (in blue) (eg condition factor) are linked both to transcripts (purple) and to metabolites (red).

We can focus on particular areas to visualise which genes are linked, in terms of expression profiles. Here NF kappa B is centre of an extensive hub and linked to survivin (an anti-apoptotic gene) and vitellogenin.

NF kappa B

Survivin

Vitellogenin

Using class-prediction algorithms (eg GALGO) we identified the areas of the network containing genes and metabolites most predictive of (differentially polluted) sampling sites (red)

These overlap with an area of the network populated by genes related to metabolism and energy production (in green)

So, starting to see connectivity between components of the network and the field

Application of “open” technologies to the study of nanomaterials

• In ecotoxicology, genomics has a major value in assessing novel agents and also mixtures of contaminants for which we do not know appropriate end points or mechanisms. It provides a non-biased, global approach.

• A highly appropriate application therefore is the assessment of the effects of nanomaterials, the products and by-products of which enter the environment as mixtures with largely unknown effects.

Omics, monitoring and safety assessment– Elucidate mechanisms of toxicity (e.g distinguish genotoxic vs

nongenotoxic carcinogens)– Provide more informative batteries of biomarkers– Create practical assays e.g. real-time PCR, custom arrays, reporter

assays, ELISAs– Focus on PROCESSES disturbed rather than single gene products

– Characterise responses of sentinel species to ‘new’ pollutants– Assess the effects of mixtures– Inform on the basis of population susceptibility to toxicants– Provide detailed case studies of specific sites

A major challenge will be the ability to distinguish between adaptive vs toxic responses and the effective use of these markers in risk assessment. We need to discover patterns of change that are diagnostic and predictive

Challenges & Recommendations ResearchResearch– Needs:

• Linking genomic changes to adverse outcomes (AOP)

• Interpreting genomic information for risk assessment

• Training risk assessors and managers to interpret and understand genomics data in the context of a risk assessment

• Development of technical framework for analysis and acceptance criteria for “omic” information for scientific and regulatory purposes

Adapted from Bill Benson 2008