2005 Superfund Basic Research Program Annual Meeting

Chromate and Polycyclic Aromatic Hydrocarbon Interactions

(Transcriptional Repression by Chromium-Chromatin Interactions)

Alvaro PugaDepartment of Environmental Health

University of Cincinnati

Why study mixtures of PAHs and metals?

As, Cr, B(a)P and PCBs are in USEPA top 20 hazardous substance priority list

9,727 Superfund sites with PAH contamination;2,539 have also Cr; 2,274 have also As

1,200 sites in National Priority List; 40% have Cr/As and PAHs.

Is there enough time in the combined lifetimesOf all biological scientists to study all mixtures?

275 CERCLA compounds

37,675 binary combinations3,428,425 ternary combinations

233,132,900 quaternary combinations…

175,000 biological scientists (Latest NSF estimates)

To study complex mixtures we need a different experimental paradigm

For binary mixtures:

Cross-talking signaling pathways

How does one member of the mixturemodify the effect of the other?

Ah Receptor-Dependent Transactivation and Target Gene Expression in the presence of Cr

Cytosol

Nucleus

TCDD AHR

B[a]P

TCDD

MAPK pathways

?

AIP

AhRECYP1A1

AHR

Hsp90p23 ARNT

C Cr?

Mouse Hepa-1 cells

Cr inhibits inducible gene expression

• Cr-DNA crosslinks• Cr-DNA adducts

Zhitkovich (2005) Chem.Res.Tox. 18:3-11

• Disruption of transcriptional complexes

• MT, PEPCK, but not actinHamilton et al (1989) Mol.Carcinog. 2:274-286

• Blocks binding of p300 to NFkB p65Shumilla et al (1999) J.Biol.Chem. 274:36207-36212

• Chromium Speciation and Distribution

• Transrepression of AHR-inducible Genes by Chromium

• Epigenetics of Chromium-Chromatin Interactions

Chromium speciation by HPLC/ICP-MS

Cr(VI)

e-

Cr(V)

e-

Cr(IV)

e-

Cr(III)

K2CrO4

K2Cr2O7

a) t = 0 b) t = 1 h

Chromium distribution to nuclei and cytoplasm

ICP-MS

nucleicytoplasm

K2Cr2O7

nucleicytoplasm

K2CrO4Cell Fraction

ng C

r per

100

mm

pla

te0

50

100

150

200

250

51Cr Uptake

0

2

4

6

8

nuclei cytoplasmCell Fraction

dpm

per 1

00 m

m p

late

(X10

7 )

DMSOBaP

As, Cd and Cr affect AHR-dependent gene expression differently in Hepa-1 cells

The effect of Chromium on AHR-dependent gene expression is transcriptional

CYP1A1 GSTa GSTp

Rel

ativ

e m

RN

A le

vel

0

1

2

3

4

5 DMSOBaPCr+BaPBaP+Cr, nuclei

Rel

ativ

e m

RN

A le

vel

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 5 25 50

BaP + + + +

µM Cr

Cr represses only inducible gene expression

B[a]P-induced CYP1A1 mRNA in Human HepG2 cells

0

20

40

60

80

100

120

DMSO Cr BaP Cr+BaP

Fold

indu

ctio

n

*

+

00.20.40.60.8

11.21.41.6

DMSO Cr BaP Cr+BaP

Fold

indu

ctio

n

00.20.40.60.8

11.21.4

DMSO Cr BaP Cr+BaP

Fold

indu

ctio

n

BaP: Benzo[a]pyrene (5 µM, 8h)Cr: chromium (50 µM, 9h) Cr+BaP: Cr (50 µM, 1h) + BaP (5 µM, 8h)

* p<0.01 Vs DMSO, + p<0.01 Vs BaP

CYP1A1

CYP1A2 CYP1B1

p1647LUCin Hepa-1 cells

Cr is less effective if added after AHR activation

DMSO Cr -2 h -0.5 h 0 h 0.5 h 2 h

Rel

ativ

e lu

cife

rase

act

ivity

0.0

0.2

0.4

0.6

0.8

1.0

+ TCDD

* **

* *

*

*

+ BaP

Cr affects the expression profiles of BaP-induced genes

1.00

10.00

100.00

Control BaP Cr+BaP BaP+Cr

Treatment

Fold

Cha

nge

from

Con

trol

AB025408

AF006482

AF016294

AJ297743AK002894

AK007683

BC003982

L06047

L24118

M32032

NM009992NM025797

U12785

U12961

U23921

U38261

U40796

U75215U85498

U96441

X91825

EsteraseTP dyphosphohydrolaseE74-like factor 3

Torsin 1 B

P gluc oxygenase

TNF-related

RIKEN cDNA 1110035H23

GST alpha 4

TNF alpha induced

Selenium binding protein 1

CYP1A1

RIKEN-NM025797ALDH3A1

NQO1

Osmotic stress protein

SOD3

Excision repair comp.grp5

Amino acid transporter

GCLC

Numb-like

Small proline-rich protein

Profiles of Other BaP-Induced Genes

0

0.5

1.0

1.5

2.0

2.5

Control BaP Cr+BaP BaP+Cr

Treatment

Fold

Cha

nge

from

Con

trol

AB041604AB041657AB049653AB055070AF012923AF124725AF232709AK003562AK004305AK004431AK005192AK005691AK008611AK009025AK010334AK010545AK012430AK013765BC003237BC004041BC004667D49439NM010590NM023665NM023685NM025498NM025964NM025982NM026373NM026494NM029385NM030564U21960Z11911

Hypothetical protein MNCb-5081Hypothetical protein, MNCb-2990Mitochondrial ribosomal protein L34Clone MGC:7904Wild-type p53-induced gene 1Apoptotic chromatin condensation Syntaxin 5CDC42 EFFECTOR PROTEIN 2SNAP25 INTERACTING PROTEIN 30Di-George syndrome related protein Translation factor SUI1homolog GC20Zinc finger protein 2RIKENUbiquitin-like protein SB132RIKEN-BBP-like protein 1Downstream of tyrosine kinase 5Endothelial cell growth factor 1Similar to ATP/GTP-binding proteinPeptidylprolyl isomerase F (cyclophilin F)Clone IMAGE:3498604TBP-associated factorAjubaRIKENSCAN-KRAB-zinc finger gene 1RIKEN-RIKEN-RIKEN-RIKEN-RIKENRIKEN-Hypothetical protein MGC7670Calcium modulating ligandGlucose-6-phosphate dehydrogenase

TCDD activates the AHR to a DNA-binding configuration in the presence of Cr

St

In the presence of Cr AHR binds to DNA but transcription is inhibited

Several co-regulators increase BaP-inducedgene expression and their effect is blocked by Cr

50000

100000

150000

200000

250000

0Bluescript SRC-1 SRC-2 p300 BRG-1

Luc/β-

gal

DMSOBaP BaP+25 µM CrBaP+50 µM Cr

Chromium interferes with co-activator recruitment and chromatin structure

-1200 -1000 -800 -600 -400 -200 +1

Endogenous Cyp1a1

Integrated AhRDtkLuc tk

NaB blocks the Inhibition by Cr of BaP-

induced gene expression …. But inhibition takes place at the level of the

immediate promoter

ChIP for AHR and ARNT

α-AHR α-ARNT

Fold

cha

nge

D Cr B Cr+B D Cr B Cr+B02468

1012

0.00.51.01.52.02.53.0

D Cr B Cr+B D Cr B Cr+Bα-AHR α-ARNT

D Cr B Cr+B D Cr B Cr+B

α-AHR

α-ARNT

A Enhancer Proximal promoter

-1200 -1000 -800 -600 -400 -200 +1

-1141 -784 +71-291Cyp1a1

ChIP for p300 and HDAC-1

Fold

cha

nge

0.00.51.01.52.02.5

D Cr B Cr+B D Cr B Cr+B0.0

0.5

1.0

1.5

2.0

2.5

D Cr B Cr+B D Cr B Cr+Bα-p300 α-HDAC1 α-p300 α-HDAC1

α-p300

α-HDAC1

Enhancer Proximal promoter

D Cr B Cr+B D Cr B Cr+B

B

-1200 -1000 -800 -600 -400 -200 +1

-1141 -784 +71-291Cyp1a1

Effect of Cr on AHR-inducible genes

HDAC-1

AHR ARNT

p300 TFIIsRNAPol II

HDAC-1

Ligand induction

HDAC-1AHR ARNT

Cr

Ligand inductionIn the presence of Cr

Cyp1a1

HDAC-1

HDAC-1

Cr crosslinks HDAC-1 to the Cyp1a1 promoter in Hepa-1 cells

0.0

0.5

1.0

1.5

2.0

DMSO BaP

αAH

R%

of t

otal

inpu

t

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7enhancerproximal promoter

αHD

AC

-1%

of t

otal

inpu

t

FHO + + - - + + - - + -EDTA + - + - + - + - + +

DMSO Cr BaP

CYP1A1 enhancer αHDAC-1

Input

FHO + + - - + + - - + -EDTA + - + - + - + - + +

DMSO Cr BaP

DMSO

BaP

αAHR

Input

CYP1A1 promoterInput

αHDAC-1

Input

αAHR

50 µM Cr, 2h; 5 µM BaP, 2hn=3

DNA demethylation by 5-azacytidine inhibits Cr effects

0

5

10

15

0

20

40

60

80

100DMSO Cr BaP Cr+BaP

20 1µM 5-AzaC

Rel

ativ

e C

YP1A

1 m

RN

AR

elat

ive

NQ

O1

mR

NA

5AzaC: 56h BaP: 5 µM, 8hCr: 50 µM, 9h Cr+BaP: Cr (1h) + BaP (8h)

Effect of Cr on AHR-inducible genes

HDAC-1

AHR ARNT

p300 TFIIsRNAPol II

HDAC-1

Ligand induction

HDAC-1AHR ARNT

Cr

Ligand inductionIn the presence of Cr

Cyp1a1

HDAC-1

HDAC-1

Conclusions and Questions

Cr inhibits BaP inducible gene expression bykeeping HDAC-1 bound to chromatin and derailing methylation/demethylation patterns in promoters

Does chromium affect epigenetic imprinting?

How generalized is this mechanism?

Kathy Tepperman Yu-Dan Wei Ming-ya Huang

(R01 ES10807, P42 ES04908)

Michael Schnekenburger

GSH depletion or replenishment does not affect significantly inhibition by Cr of the expression of an AHR reporter

-1200 -1000 -800 -600 -400 -200 +1

p1647Luc LucR

elat

ive

luci

fera

se a

ctiv

ity

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

DMSOTCDD Cr

Cr+TCDD

DMSOTCDD Cr

Cr+TCDD

DMSOTCDD Cr

Cr+TCDD

DMSO 20 µM BSO 5 mM NAC