carcinogenic mechanism of tce and its metabolites

Carcinogenic Mechanism of TCE and Its Metabolites, Dichloroacetic Acid and Trichloroacetic Acid: DNA Methylation.

Michael A. Pereira Professor of Medicine

Division of Hematology and Oncology Ohio State University

College of Medicine and Public Health 300 West Tenth Avenue, 1148B CHRI

Columbus, OH 43210 Phone # (614)-293-6864

Fax: (614)-293-3333 e-mail: pereira-1@medctr.osu.edu

Two Types of Alterations in DNA Methylation found during Carcinogenesis

1. DNA hypomethylation.

2. Hypermethylation of tumor suppressor genes.

DNA Hypomethylation

1.

2. methylation.

3. a. decreased binding of methylated DNA-binding proteins, b. alterations in the binding and /or recruitment of

modifications, c.

genes), d. chromosomal instability, and e. hypermethylation of tumor suppressor genes.

Found early in all solid tumors; even in normal appearing tissue at risk of cancer and in precancerous lesions (foci). There can be a 30-60% decrease in total extent of DNA

DNA hypomethylation is important because it can result in

transcription factors and enzymes involved in histone

increased histone acetylation (increased expression of

Normal Tissue

Tumor

DNA Methylation in Normal Tissue and Tumors

= 5-Methylcytosine

MeBP HDAC

H3-K9Me

MeBP HDAC

H3-K9Me

H3-K9Ac H3-K9AcH3-K9Ac

XX

= Cytosine

Exon 2 Exon 1

Exon 2 Exon 1

Hypermethylation of Tumor Suppressor Genes

1.

in tumors. 2.

in precancerous lesions (foci). 3.

expression of tumor suppressor genes in cancer.

CpG islands in exon-1 and its upstream promoter region that are unmethylated in normal tissue become hypermethylated

Usually found at a later stage than DNA hypomethylation, although can be found in normal tissue at risk of cancer and

A major mechanism for the down-regulation of the mRNA

Normal Tissue

Tumor

Methylation of Tumor Suppressor Genes in Normal Tissue and Tumors

= 5-Methylcytosine

X

MeBP HDAC

H3-K9Me

H3-K9Ac

= Cytosine

Exon 2 Exon 1

Exon 2 Exon 1

DNA Hypomethylation induced by DCA, TCA and TCE: Carcinogenic Mechanism

1.

2. DNA hypomethylation.

promoted tumors upon cessation of exposure.

3.

DCA, TCA and TCE induce DNA hypomethylation within days. Other nongenotoxic mouse liver carcinogens also induce DNA hypomethylation, i.e. THM, Wy-14,643, 2,4-dichlorophenoxy-acetic acid, dibutyl phthalate, gemfibrozil and phenobarbital.

Mouse liver tumors induced/promoted by DCA and TCA contain Upon cessation of exposure, DNA

hypomethylation in DCA- but not in TCA-promoted liver tumors is reversible correlating with the regression of DCA but not TCA-

Methionine prevents DNA hypomethylation induced by DCA TCA, TCE and Wy-14,643. Chloroform prevents DCA but not TCA-induced DNA hypomethylation in the liver.

DNA Hypomethylation induced by DCA, TCA and TCE: Carcinogenic Mechanism

4. tumors and foci. tumors. chloroform correlated with their prevention of DNA hypomethylation.

5.

TCA-promoted kidney tumors.

6.

7.

Methionine and chloroform prevent DCA-induced mouse liver Chloroform does not prevent TCA induced liver

Thus, prevention of liver tumors by methionine and

Chloroform increases DCA but not TCA-induced DNA hypomethylation in mouse kidney and increases only DC-but not

DNA hypomethylation could be used as a biomarker for the effect of route of administration and for cancer chemoprevention.

DNA hypomethylation decreases the extent to which histone H3 is acetylated.

Mechanism of TCE, DCA and TCA Carcinogenic Activity

DNA Hypomethylation Field Cancerization: Increased risk

of one of the first alterations required for cancer to develop.

Increased histone acetylation and binding of transcription factors to increase the expression of protooncogenes

suppressor genes: Decreased expression

Chromosomal instability: LOH, breaks, rearrangements and exchanges

of cancer due to the accomplishment

Hypermethylation of tumor

DNA Hypomethylation in DCA and TCA-induced Mouse Liver Tumors

1. In contrast, TCA tumors do not regress.

2. Promotion by dichloroacetic

Cancer Letters, 102: 133-141.

Pereira, M. A. (1996). Carcinogenic activity of dichloroacetic

Fundam. Appl. Toxicol., 31: 192-199.

Dichloroacetic Acid and Trichloroacetic Acid on DNA Methylation in Liver and Tumors of Female B6C3F1 Mice. Toxicol. Sciences, 43: 139-144.

DCA tumors regress after termination of exposure.

Pereira, M. A. and Phelps, J.B. (1996). acid and trichloroacetic acid of methylnitrosurea-induce cancer in the liver of female B6C3F1 mice.

acid and trichloroacetic acid in the liver of female B6C3F1 mice.

Tao, L., Kramer, P.M., Ge, R., and Pereira, M.A. (1998) Effect of

0

1

2

3

4

5

Non-InvolvedLiver

Adenoma

Control

*

Adenoma-Recovery

DNA M ethylation in DCA-Treated M ice

DN

A M

eth

yla

tio

n (

% 5

-Me

C)

0

1

2

3

4

* *

*

*

Control

Non-InvolvedLiver

Adenoma

Adenoma-Recovery

Carcinoma

Carcinoma-Recovery

DNA Methylation in TCA-Treated Mice

DN

A M

eth

ylat

ion

(%

5-M

eC)

Correlation between the Ability of DCA and TCA to induce DNA Hypomethylation and Their Ability to induce

Mouse Liver Tumors: Prevention by Methionine

(2004). Prevention by

Hypomethylation in Mice. Toxicological Sciences. 77:243-248.

Dichloroacetic Acid and Trichloroacetic Acid on DNA Methylation and Cell Proliferation in B6C3F1 Mice. J. Biochem. Molecul. Toxicol. 15: 100-106.

(2000) Hypomethylation Jun and c-Myc Protooncogenes and Increased DNA

Promoted Mouse Liver Tumors. Cancer Letters 158: 185-193.

on the Methylation and Expression of c-Jun and c-Myc Protooncogenes in Female Toxicol. Sci. 54: 399-407.

Pereira, M.A., Wang, W., Kramer, P.M., and Tao, L. Methionine of Dichloroacetic Acid-induced Liver Cancer and DNA

Ge, R., Yang, S., Kramer, P.M., Tao, L. and Pereira, M.A. (2001) The Effect of

Tao, L., Yang, S., Xie, M., Kramer, P.M. and Pereira, M.A. and Overexpression of c-Methyltransferase Activity in Dichloroacetic Acid and Trichloroacetic Acid-

Tao, L., Ge, R., Xie, M., Kramer, P.M. and Pereira, M. A. (2000) Effect of Trichloroethylene and Its Metabolites, Dichloroacetic Acid and Trichloroacetic Acid

B6C3F1 Mouse Liver: Prevention by Methionine.

0 300 0 30 100 300 0 30 100 300 0 30 100 300(mg/kg)

Control DCA TCA

3.2kb 1.5kb

c-Jun

0.5kb

1.0kb 2.2kb

Dose-Response for the Effect of Methionine on DCA, TCA and TCE-induced Hypomethylation of the c-Jun and c-Myc Genes

Methionine TCE

c-Myc

Protocol for the Effect of Methionine on DCA-induced Liver Tumor in Female B6C3F1 Mice

3.2gm/l DCA + 0, 4.0 or 8.0 gm/kg Methionine

0 8 Wks 44 Wks

/kg)

0 1 2 3 4 5 6 7 8 9

Les

ion

/Mo

use

0

1

2

3

4

5

Foci Adenomas

*

*

*

Effect of Methionine on DCA-induced Foci and Adenomas

Methionine Added to the AIN-76A Diet (gm

/

0

2

4

6

8

10

12

14

DCA * * *

* * *

Effect of Methionine on the Liver Body Weight Ratio L

iver

/Bo

dy

Wei

gh

t X

100

Cont. Meth.

DCA+4gMeth. DCA+8gMeth.

8 Wks 48 Wks

Cont. Methionine DCA DCA+4g Met. DCA+8g Met.

a a a

Effect of Methionine on DCA-induced Peroxisome Proliferation

2

2 O 2 /

min

/mg

pro

tein

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Lau

royl

-Co

A O

xid

ase

Act

ivit

y (n

mo

l H

Effect of Methionine on DCA-induced Increase

0

20

40

60

80

100

120

140

Cont.

DCA

4 Wks 8 Wks

a b

a

b b

b

b

in Hepatic Glycogen G

lyco

ge

n (

mg

/gm

Liv

er) 8gMeth.

DCA+4gMet. DCA+8gMet.

2 4 6

DNA (µg)

1 : 0

1 : 1

1 : 2.5

1 : 5.0

1 : 10

5-MeC AntibodyStaining

MethyleneBlue

Staining

5-MeC Cytosine2 4 6

DNA (µg)

1 : 0

1 : 1

1 : 2.5

1 : 5.0

1 : 10

Blocking of 5-MeC Monoclonal Antibody by 5-MeC and Cytosine

Control 8gm Meth. DCA DCA+4gMet. DCA+8gMet.

a a

b

c

b

2 0

50

100

150

200

250

300

Effect of Methionine on DCA-induced DNA Hypomethylation D

NA

Me

thyl

ati

on

(O

.D.)

Summary: Effect of Methionine on DCA-induced DNA Hypomethylation and Mouse Liver Tumors

DCA-induced Mouse Liver Tumors.

a. liver weight, b. peroxisomes, and c. only limitedly reduced glycogen accumulation.

adenomas.

1. Methionine prevented DCA-induced DNA hypomethylation and

2. Methionine did not prevent DCA induction of :

3. Methionine appeared to slow the progression of foci to

Effect of Chloroform on the Carcinogenic Activity of DCA and TCA

1. Chloroform, DCA and TCA are mouse liver carcinogens found in drinking water.

2. Possible interaction: additive, synergism, and inhibition. 3. Parameters determined:

a. cell proliferation. b. hypomethylation of the c-myc gene. c. expression of the mRNA of the c-myc gene. d. tumor response in liver and kidney.

Effect of Chloroform on Dichloroacetic Acid and Trichloroacetic Acid-Induced Hypomethylation and Expression of the c-Myc Gene and on Their Promotion of Liver and Kidney Tumors in Mice. Carcinogenesis 22:1511-1519.

Pereira, M.A., Kramer, P. M., Conran, P.B. and Tao, L. (2001).

Protocol for the Effect of Chloroform on DCA and TCA-induced DNA Hypomethylation

0, 400, 800 or 1600 mg/l Chloroform in Drinking Water

0 13 14 15 16 17 Days 500 mg/kg DCA or TCA

Chloroform 0 400 800 1,600 (mg/l)

Myc Gene in Mouse Liver

— 2.2 kb

— 1.0 kb

— 0.5 kb

Effect of Chloroform on the Methylation of c-

Chloroform 0 400 800 1,600 (mg/l) +500 mg/kg DCA

Hypomethylation of c-Myc Gene in Mouse Liver

— 2.2 kb

— 1.0 kb

— 0.5 kb

Effect of Chloroform on DCA-Induced

0 800

0

100

200

300

400

0.5kb Band 1.0kb Band 2.2kb Band

* *

* *

*

*

Effect of Choroform on DCA-Induced Hypomethylation of the c-Myc Gene

Chloroform (mg/l)

400 1200 1600

Op

tica

l Den

sity

(O

D)

Chloroform 0 400 800 1,600 (mg/l) +500 mg/kg TCA

Hypomethylation of c-Myc Gene in Mouse Liver

— 2.2 kb

— 1.0 kb

— 0.5 kb

Effect of Chloroform on TCA-Induced

0 800 1600

0

100

200

300

400

0.5kb Band 1.0kb Band 2.2kb Band

Effect of Chloroform on TCA-Induced Hypomethylation of the c-Myc Gene

Chloroform (mg/l)

400 1200

Op

tica

l Den

sity

(O

D)

Summary of Effect of Chloroform on DCA and TCA-induced Hypomethylation

1. DCA and TCA decreased the methylation of the c-myc gene and increased it’s expression.

3. the c-myc gene.

4.

methylation of the gene.

2. Although chloroform decreased the methylation of c-myc, it prevented DCA but TCA induced decrease in the methylation of c-myc.

Chloroform prevented DCA but not TCA increase in the expression of

DCA and TCA decreased the methylation of c-myc in the kidney and chloroform enhanced the ability of DCA but not TCA to decrease the

Protocol for the Effect of Chloroform on the Tumor Promoting Activity of DCA and TCA in Mouse Liver

Chloroform with 3.0gm/l DCA or 4.0 gm/l TCA in the Drinking Water

MNU

Day 15 Sacrifice at Week 52

Start Exposure at Week 7

Effect of Chloroform on Liver to Body Weight Ratio in DCA and TCA-treated Female and Male B6C3F1 Mice

Chloroform (mg/liter)

0 200 400 600 800 1000 1600

Liv

er W

eig

ht/

Bo

dy

Wei

gh

t X

100

2

4

6

8

10

2

4

6

8

10

Females-DCA Females-TCA Males-DCA Males-TCA

1200 1400

Effect of Chloroform on the Yield of Foci of Altered Hepatocytes in Female Mouse Liver

Chloroform (mg/liter)

0 800 1200 1600

Fo

ci o

f A

lter

ed H

epat

ocy

tes/

Mo

use

0

1

2

3

4

5

6

7

8

DCA TCA

*

* 400

Effect of Chloroform on the Yield of Tumors in Female Mouse Liver

Chloroform (mg/liter)

0 400 800

Tu

mo

rs/M

ou

se

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

DCA TCA

*

* 1200 1600

Effect of Chloroform on the Yield of Foci of Altered Hepatocytes in Male Mouse Liver

Chloroform (mg/liter)

0 800 1600

Fo

ci o

f A

lter

ed H

epat

ocy

tes/

Mo

use

0.0

0.5

1.0

1.5

2.0

2.5

3.0

DCA TCA

*

400 1200 0.0

0.5

1.0

1.5

2.0

2.5

3.0

Effect of Chloroform on the Yield of Tumors in Male Mouse Liver

Chloroform (mg/liter)

0 800

Tu

mo

rs/M

ou

se

0

1

2

3

4

5

6

0

1

2

3

4

5

6

DCA TCA

*

400 1200 1600

Effect of Chloroform on the Yield of Kidney Tumors in Male Mice

Chloroform (mg/liter)

0 400 800 1200 1600

Kid

ney

Tu

mo

rs/M

ou

se

0.0

0.5

1.0

1.5

2.0

2.5

0.0

0.5

1.0

1.5

2.0

2.5

DCA *TCA

Summary of the Effect of Chloroform on the Carcinogenic Activity of DCA and TCA

1. Chloroform prevented the carcinogenic activity of DCA but not TCA in the liver: Correlated with Its prevention of DCA but not TCA-induced DNA Hypomethylation.

2. Chloroform did not affect the kidney tumor promoting activity of TCA while enhancing the activity of DCA.

3. In the kidney, Chloroform enhanced DCA but not TCA-induced DNA hypomethylation that correlated with its enhancement of DCA but not TCA-promotion of kidney tumors.

DNA hypomethylation

B6C3F1 Mouse Liver. Toxicol. Sci. 58: 243-252.

Effect of Route of Administration on THMs Induction of

Coffin, J.C., Ge, R. Yang, S., Kramer, P.M., Tao, L., and Pereira, M.A. (2000) Effect of Trihalomethanes on Cell Proliferation and DNA Methylation in Female

3 2 2 3

c-

CHCl CHBrCl CHClBr CHBr

1.09 2.18 0.92 1.83 0.48 1.44 0.79 1.98

2.7kb

2.2kb

1.0kb

0.5kb

0.2kb

Effect of the THM Administered by Gavage on the Methylation of the myc Promoter

Controls

Pos. Oil

CHCl3 2 2 3CHBrCl CHClBr CHBr

2.7kb

2.2kb

1.0kb

0.5kb

0.2kb

Effect of the THM Administered in the Drinking Water on the Methylation of the c-myc Promoter

Controls

Pos. Water

Effect of Chloroform on the Methylation of the c-Myc Gene in Mouse Liver

CHCl3

0.0 1.0 2.0 3.0

ten

of

Ban

d 3

0

25

50

75

100

125

150

175

200

225

250

Gavage

(mmol/kg)

0.5 1.5 2.5

Insi

ty

Drinking Water

Hypermethylation of Tumor Suppressor Genes in DCA and TCA-induced Mouse Liver Tumors

1. α and p16INK4AHypermethylation of estrogen receptor-gene in DCA and TCA-induced mouse liver tumors.

Methylation of the ER-α Gene in DCA-inducedMouse Liver Tumors

117 156 187 195 225 233 252 280 301 310 320 360 381 383 390 398 417 Normal Live 6AA-1 6AB-1 M 6AB-2 M 5-48-B 5-49-B-2 M 5-49-B-3 5-51-B 5-50-L-2 5-50-L-3

DCA-induced Liver Tumor 3-29-L-1 M M M M M 3-30-N-2 M M M M M M M M M M3-30-N-4 M M M M M M M M M M MM1-30R-1 M M M M M M M M M3-32-N M M M M M M M M3-33-L M M M M M M

DCA-treated Non-involved Liver 3-29-L 3-32-N 3-33-L 3-35-R-1 3-35-R-2 M

117 156 187 195 225 233 252 280 301 310 320 360 381 383 390 398 417

Methylation of the ER-α Gene in TCA-inducedMouse Liver Tumors

117 156 187 195 225 233 252 280 301 310 320 360 381 383 390 398 417 Normal Live 6AA-1 6AB-1 M 6AB-2 M 5-48-B 5-49-B-2 M 5-49-B-3 5-51-B 5-50-L-2 5-50-L-3

TCA-induced Liver Tumor 245-2 M M M M M 270-1 M M M M M M287-1 M M M M M 264-2 M M M M M M M 14-1 M M M M M M M M14-2 M M M M M M14-3 M M M M M M M M 142-1 M M M M M 142-2 M M M M M M M M

TCA-treated Non-involved Liver 171-1 173-1 M M M 221-2 M 221-3

117 156 187 195 225 233 252 280 301 310 320 360 381 383 390 398 417

Methylation of the p16 Gene in DCA-induced Mouse Liver Tumors

26 47 67 106 119 130 141 193 222 287 299 389 397 405 420 422 431 437 468 491 494 505 516 537 545 552 557 567 571 578

Normal Liver 6AA M M 6AB 5-48-B M 5-49-B M

DCA-induced Liver Tumor 3-29-L M M M 3-30-N M M1-30-R M M M 3-32-N M M3-33-L

DCA-treated Non-involved Liver 3-29-L M M 3-32-N M M 3-33-L M M M 3-35-R

26 47 67 106 119 130 141 193 222 287 299 389 397 405 420 422 431 437 468 491 494 505 516 537 545 552 557 567 571 578

Methylation of the p16 Gene in TCA-induced Mouse Liver Tumor

26 47 67 106 119 130 141 193 222 287 299 389 397 405 420 422 431 437 468 491 494 505 516 537 545 552 557 567 571 578

Normal Liver 6AA M M 6AB 5-48-B M 5-49-B M

TCA-induced Liver Tumor 245 M M270 M M 287 M 14-2 M M 142-1 M M M 264-1 M M M

TCA-treated Non-involved Liver 171-1 M 173-1 221-1 M M M

26 47 67 106 119 130 141 193 222 287 299 389 397 405 420 422 431 437 468 491 494 505 516 537 545 552 557 567 571 578

Mechanism of TCE, DCA and TCA Carcinogenic Activity

DNA Hypomethylation Field Cancerization: Increased risk

of one of the first alterations required for cancer to develop.

Increased expression of protooncogenes

suppressor genes: Decreased expression

Chromosomal instability: LOH, breaks, rearrangements and exchanges

of cancer due to the accomplishment

Hypermethylation of tumor

CONCLUSIONS

1. The mechanism of DCA and TCA carcinogenic activity could be explained by their ability to induce DNA hypomethylation and thus increase the risk of cancer.

2. Hypermethylation of tumor suppressor genes is involved in DCA and TCA carcinogenic activity.

Co-Investigators Dr. Lianhui Tao

Post-Doctoral Fellows Dr. Wei Wang Dr. Siming Yang

Graduate Students

Long Li

Research Associates Ms. Paula M. Kramer

Support by 2 grants from the US EPA.

Fadel S. Alyaqoub Rungrung Ge

Epithelium Early

Adenoma Late

Adenoma Cancer

Multistage Model of Microsatellite Unstable Colon Carcinogenesis including Hereditary Nonpolyposis Colorectal Cancer (HNPCC)

Mutations/loss/ hypermethylation of MMR genes

Mutations in APC or β-catenin or loss of APC (5q).

DNA Hypomethylation

Frameshift mutations in TGFβRII, BAX, E2F4, IGF-IIR, TCF-4, etc.

Hypermethylation of Estrogen Receptor-α, p16, THBS1, CACNA1G, etc.

Normal Aberrant

crypt foci, ACF

200 bp 300 bp 400 bp500 bp

c-myc 393 bp

249 bp

1600

800

400 0 (mg/l)1600

800

400 0

1600

800

400 0

DCA TCA

Effect of Chloroform on DCA- and TCA-Induced Expression of the mRNA of the c-Myc Gene: RT-PCR Analysis

100 bp

HPRT

Chloroform

Vehicle Control

Gene

O.D

. R

atio

of

c-m

yc/H

PR

T

0

1

/l

b a

c

Saline DCA TCA

Effect of Chloroform on DCA and TCA-Induced Expression of the mRNA of the c-Myc

CHCl3-0 mgCHCl3-400 mg/l CHCl3-800 mg/l CHCl3-1600 mg/l

Effect of PCNA-Labeling Index

PC

NA

-L

abel

ing

Ind

ex (

%)

0.0

0.5

1.0

1.5

2.0

2.5

Control DCA TCA

1600 ppm Chloroform

* *

*

*

Chloroform on DCA and TCA-induced Cell Proliferation:

800 ppm Chloroform 400 ppm Chloroform

0 ppm Chloroform

S-Adenosylmethionine

(SAM) S-Adenosylhomocysteine

(SAH)

5-MethylcytosineCytosine

DNA Methylation

DNA methyltransferase

Effect of BDCM on the Methylation of the c-Myc Gene in Mouse Liver

BDCM (mmol/kg)

0

25

50

75

100

125

150

175

200

225

250

Drinking Water

0.0 0.5 1.0 1.5 2.0

Inte

nsi

ty o

f B

and

3

Gavage