carcinogenic mechanism of tce and its metabolites
TRANSCRIPT
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: [email protected]
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