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Comparative Hepatic Gene Expression Elicited by o,p’-DDT
in the Rat and Mouse
Naoki Kiyosawa
1,
Joshua C. Kwekel
Lyle D. Burgoon
1,
Timothy R. Zacharewski
1
Department of Biochemistry and Molecular Biology and National Food Safety & Toxicology Center,
Michigan State University, East Lansing, MI, 48824
2 Medicinal Safety Research Labs., Daiichi-Sankyo Co., Ltd., Fukuroi, Shizuoka 437-0065, Japan
Technical grade dichlorodiphenyltrichloroethane (DDT), a mixture of p,p’-DDT (65 – 80%),
o,p’-DDT (15 – 21%), p,p’-TDE (~ 4%), is an agricultural pesticide and malarial vector control
agent that has been designated a potential human hepatocarcinogen. We previously reported
that o,p’-DDT elicits PXR/CAR-, not ER-, mediated response in immature, ovariectomized rat
liver (Kiyosawa et al. 2008, Toxicol Sci 101(2):350-63), suggesting that tumor promotion
effects caused by o,p’-DDT in rodents may be derived from CAR but not ER. The objective of
the present study is to investigate the molecular response in immature, ovaeriectomized mouse
liver against o,p’-DDT exposure by toxicogenomic technique. Comparative toxicogenomic
analysis was also conducted using rat microarray data sets reported previously.
Relative expression (fold)
QRT-PCR VERIFICATION
5050
45
Cyp2b10
4040
Microarray
1.8
1.8
*
1.6
1.4
PCR
CAR
1.2
1.2
1
* *
*
0.8
0.6
0.6
* *
2h
4h
8h
0.4
*
12h
18h
24h
0.2
00
72h
18
2h
4h
14
1
12 *
12
0.8
0.8
18h
24h
72h
0.6
0.4
0.4
*
0.2
*
6
0.6
0.6
44
12h
18h
24h
72h
18
24
88h 12
72
OBJECTIVES:
44
(A) Steroidogenic pathway
Cholesterol
00
2h
4h
8h
12h
18h
24h
72h
3.5
*
Gclm
2h
4h
8h
12h
18h
24h
72h
33 Hmox1
2.5
9
1.5
Cyp17a1
11
0.5
12h
18h
24h
72h
18
24
88h 12
72
4
4h
Time (h)
SD
H
3b
Cyp17a1
17OH Progesterone
22h
4
4h
Time (h)
12h
18h
24h
72h
18
24
8 12
72
8h
Time (h)
Cyp17a1
QRT-PCR was performed using the same RNA samples obtained from the mouse liver treated
with o,p’-DDT. * p<0.05 for QRT-PCR data by two-way ANOVA with Tukey’s post hoc test.
17bHSD
(B) Hepatic Cyp17a1 expression
3
Rat
Mouse *
3
2.5
*
7
66
44
Figure 1. Study design
INTRODUCTION
AND
OBJECTIVES
(A)
Cl
o,p’-DDT (300 mg/kg)
Cl
(A) Animal treatment
Cl
Dosing Time (h)
24
Cl
48
2 4 8 12 18 24
Sacrifice Time (h)
72
One or three daily oral doses of o,p’-DDT (300
mg/kg b.w.) or sesame oil vehicle was
administered to immature, ovariectomized
C57BL/6 mice. Each treatment group consisted
of five animals. Liver was harvested at 2, 4, 8,
12, 18, 24 or 72 hrs. The dosage level was the
same as our rat DDT study reported previously.
Rat cDNA microarray
11
22
0.5
1
2
2h
(B)
4V
2T
8V
4T
8T
12V
12T
18V
18T
24V
24T
72V
72T
34,222 genes
Rat active genes
Common homologues
430 genes
p1(t)>0.99
|Fold change|>1.5
Mouse active genes
4,497
274
genes
1,480 genes
p1(t)>0.99
|Fold change|>1.5
367
genes
106 genes
 Correlation analysis (Fig. 3B)
(C)
Number of active genes
-0.5
0.5
1.0
Up
Down
900
800
800
700
600
600
500
400
400
300
200
200
100
2
2h
4
4h
8
8h
12
12h
18
18h
Time (h)
24 72h
72
24h
Empirical Bayes analysis of microarray data
identified differentially expressed genes (active
genes) following o,p’-DDT treatment at each
time point relative to time matched vehicle
Down
Annotated genes were used for
Upcontrols.
further analysis. Genes with absolute fold
change value greater than 1.5 at one or more
time points and p1(t) value greater than 0.999
were selected. Number of active genes. Black
and white indicate the proportion of up- and
down-regulated genes, respectively.
Supported by National Institute of General Medical Sciences (GM075838);
U.S. Environmental Protection Agency (RD83184701).
T.R.Z. is partially supported by the Michigan Agricultural Experiment Station.
Contact: [email protected] (N.K.), [email protected] (T.R.Z.)
12h
18h
24h
00
72h
22h
4
4h
12h 18
18h 24
24h 72
72h
88h 12
Time (h)
Androstenedione
SD
H
3b
(C) Blood DHEA-S level
3bHSD
Testosterone
SD
H
3b
Cyp19a1
Androstenediol
Estradiol
0.16
Mouse
*
0.16
0.12
0.12
0.08
0.08
0.04
0.04
2
4
8 12 18 24 72
Time (h)
0
Rat
2
Vehicle
o,p’-DDT
4
8 12 18 24 72
Time (h)
Figure 6. Perturbation of DHEA metabolism in the mouse
SUMMARY: Novel Endocrine Disruption Pathway?
-0.5
CAS
CAD
DAS
DAD
Cyp17a1
o,p’-DDT
exposure
-1.0
Figure 2. Number of active genes
1000
1000
00
-0.0
-1.0
Figure 4. Correlation analysis
(A) Active genes overlapping in both rat and mouse.
The 106 homologous genes that were active following
o,p’-DDT treatment in both mouse and rat.
(B) Correlation analysis between rats and mice
treated with o,p’-DDT. The Pearson’s correlation
coefficients were calculated for both gene expression
level and statistical significance. Each plot represents
individual genes, and the plots are colored based on
the similarity of expression patterns.
(C) Definition of CAS, CAD, DAS and DAD).
SUMMARY OF MICROARRAY RESULTS
8 12 18 24 72
Time (h)
8h
0.5
Perturbation of
sex hormone
homeostasis
Liver
DNA damage Cyp17a1
CAR activation induction
?
Cell proliferation
Tumor promotion
Endocrine system
Elevation in
DHEA-S level
Direct binding
to ER
ER-expressing tissues
(e.g. uterus)
ER-mediated response
Cell proliferation
Uterotrophic response
Mouse-specific response
Figure 7. Summary of molecular response following o,p’-DDT treatment in the mouse.
CAR activation and DNA damage may be risk factors for tumor promotion in the liver. In
addition, alteration of steroid hormone profile by elevation of blood DHEA level may be a
risk factor for perturbation of endocrine system.
PXR/CAR- AND DNA DAMAGE-RELATED GENES
Table 1. Microarray results. Genes associated
with DNA damage or PXR/CAR following o,p’DDT treatment in the mouse liver are presented.
Fold change (h)
Gene Symbol
2
PXR/CAR-regulated
Cyp2b9
1.8
Cyp2b10
2.2
Cyp2b13
1.5
Gsta2
1.5
Gstm2
1.0
Abcc2
1.1
Abcc3
1.3
Abcc4
1.1
Ces2
0.9
4
1.9
2.1
1.4
0.8
1.1
1.1
1.5
0.9
1.6
8
2.4
2.7
1.5
2.2
2.2
1.4
1.8
2.1
2.5
DNA damage-inducible
Gadd45a
3.0 12.6 13.5
Gadd45b
6.8 11.8 8.3
Cdkn1a
2.5 15.4 3.4
12
4.1
11.4
3.8
3.5
2.1
2.1
2.7
3.7
2.5
8.5
7.9
2.3
18
3.8
12.5
3.5
7.0
2.4
2.2
2.4
4.1
3.4
3.2
4.3
0.8
24
5.0
18.4
4.1
6.6
3.2
2.7
3.8
3.5
4.6
1.4
8.5
1.5
72
2.2
4.3
2.0
2.9
1.9
2.6
2.6
6.1
3.0
2.3
3.9
1.0
Highlighted: p1(t)>0.999
Time (h)
2 4 8 12 18 24 72
PXR/CAR-regulated
2V
Correlation ofFold
gene
expression
Change
Mouse Agilent microarray
5,692 genes
4
4h
(A) Biosynthesis of steroid horomone. (B) Hepatic Cyp17a1 mRNA level following
o,p’-DDT treatment in the mouse or rat. (C) DHEA-S levels in mouse serum or rat
plasma. Species difference in hepatic Cyp17a1 was observed, which may related with
elevation in serum DHEA-S level observed exclusively in the mouse. * p<0.05 by twoway ANOVA with Tukey’s post hoc test.
1.0
(B) Microarray study design
Temporal gene expression patterns were
analyzed by Agilent whole genome microarray
(4X44K) using the reference design including
dye-swaps. Three samples per group were used
for labeling and examined. Arrow tails
represent Cy3 while arrow heads represent
Cy5. V and T indicate vehicle and treated
samples, respectively; numbers indicate time.
(B)
Cyp2b9
Cyp2b10
Cyp2b13
Abcc3
Gsta2
Gstm2
Ces2
Abcc2
Abcc4
DNA
damage
0
Cl
(A)
Correlation of gene
expression
P(1)t
EXPERIMENTAL DESIGN
PCR
1.5
3
0
SPECIES-COMPARISON: Gene expression profiles
Microarray
22
*
*
5
Cyp17a1
Dehydroepiandrosterone
(DHEA)
Figure 3. Verification of mouse microarray results by QRT-PCR
88
00
SD
H
3b
17OH Pregnenolone
00
22h
Cyp11a1
Progesterone
Pregnenolone
22
00
12h
18h
24h
72h
18
24
88h 12
72
*
1
0.2
44h
1010
22
0.4
22h
*
*
3
1
0.8
*
5
1.2
88
*
66 Cyp7b1
1.2
00
44h
7
1.4
2
00
0.5
0.5
00
1.8
1.8
1.6
* *
Time (h)
Compare the gene expression profile with o,p’-DDT-treated rat liver
12h
10
22h
Comprehensively assess the temporal gene expression changes elicited by o,p’-DDT in the
mouse liver
8h
1616 Gadd45b
Srebf1
1.2
1.2
2.5
2.5
PXR
2.02
**
1.5
1.5
1.01
1.4
35
3030
25
2020
15
1010
5
00
SPECIES DIFFERENCE IN DHEA METABOLISM
Relative Cyp17a1
expression (fold)
INTRODUCTION
Blood DHEA-S level
(nmol/L)
1
1,2,
Gadd45a
Gadd45b
Cdkn1
Upregulation
CONCLUSION
o,p’-DDT elicited relatively similar gene expression profile in the liver between rats and
mice (Fig. 4B)
No change
Downregulation
Figure 5. Heat map for microarray data
PXR/CAR-regulated and DNA damageresponsive genes were up-regulated in the
mouse liver.
o,p’-DDT elicited CAR activation in the mouse liver (Fig. 5 and Table 1) as well as rat
(Toxicol Sci 101(2):350-63), which may be associated with tumor promotion
Induction of Gadd45a, Gadd45b and Cdkn1 genes suggests DNA damage in the mouse
liver (Fig. 5 and Table 7)
Blood DHEA-S level was elevated in the mouse following o,p’-DDT treatment (Fig.
6C), which might be associated with hepatic Cyp17a1 induction (Fig. 6B)
Elevation in blood DHEA-S level may cause alteration of steroid hormone profile in the
mouse (Fig. 7)