Comparative Hepatic Gene Expression Elicited by o,p’-DDT in the Rat and Mouse

INTRODUCTION

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.

INTRODUCTION AND OBJECTIVES

OBJECTIVES:

Comprehensively assess the temporal gene expression changes elicited by o,p’-DDT in the mouse liver

Compare the gene expression profile with o,p’-DDT-treated rat liver

EXPERIMENTAL DESIGN

Figure 1. Study design

(A) Animal treatment

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.

(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.

2 4 8 12 18 24 72

Dosing Time (h)0 24 48

Sacrifice Time (h)

ClCl

ClClCl

ClCl

ClClCl(A)

(B)

o,p’-DDT (300 mg/kg)

SPECIES DIFFERENCE IN DHEA METABOLISM

SUMMARY: Novel Endocrine Disruption Pathway?

CONCLUSION

o,p’-DDT elicited relatively similar gene expression profile in the liver between rats and mice (Fig. 4B)

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)

SUMMARY OF MICROARRAY RESULTS

Figure 2. Number of active genes

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 controls. Annotated genes were used for 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.

Figure 6. Perturbation of DHEA metabolism in the mouse

(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 two-way ANOVA with Tukey’s post hoc test.

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.

Naoki Kiyosawa 1,2, Joshua C. Kwekel 1, Lyle D. Burgoon 1, Timothy R. Zacharewski 1 1 Department of Biochemistry and Molecular Biology and National Food Safety & Toxicology Center,

Michigan State University, East Lansing, MI, 488242 Medicinal Safety Research Labs., Daiichi-Sankyo Co., Ltd., Fukuroi, Shizuoka 437-0065, Japan

2V

2T

4V

4T

8V

8T

12V

12T

18V

18T

24V

24T

72V

72T

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: kiyosawa@msu.edu (N.K.), tzachare@msu.edu (T.R.Z.)

SPECIES-COMPARISON: Gene expression profiles

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).

(A) (B)

(C)

Cyp17a1

-1.0 -0.5 0.5 1.0

-1.0

-0.5

-0.0

0.5

1.0

-1.0 -0.5 0.5 1.0

-1.0

-0.5

-0.0

0.5

1.0

CASCADDASDAD

Fold Change

P(1

)t

Correlation of gene expression

Cor

rela

tion

of

gen

e ex

pres

sion

(A) Steroidogenic pathway

PXR/CAR- AND DNA DAMAGE-RELATED GENES

Figure 5. Heat map for microarray data

PXR/CAR-regulated and DNA damage-responsive genes were up-regulated in the mouse liver.

Table 1. Microarray results. Genes associated with DNA damage or PXR/CAR following o,p’-DDT treatment in the mouse liver are presented.

Highlighted: p1(t)>0.999

2 4 8 12 18 24 72

PXR/CAR-regulatedCyp2b9 1.8 1.9 2.4 4.1 3.8 5.0 2.2Cyp2b10 2.2 2.1 2.7 11.4 12.5 18.4 4.3Cyp2b13 1.5 1.4 1.5 3.8 3.5 4.1 2.0Gsta2 1.5 0.8 2.2 3.5 7.0 6.6 2.9Gstm2 1.0 1.1 2.2 2.1 2.4 3.2 1.9Abcc2 1.1 1.1 1.4 2.1 2.2 2.7 2.6Abcc3 1.3 1.5 1.8 2.7 2.4 3.8 2.6Abcc4 1.1 0.9 2.1 3.7 4.1 3.5 6.1Ces2 0.9 1.6 2.5 2.5 3.4 4.6 3.0

DNA damage-inducibleGadd45a 3.0 12.6 13.5 8.5 3.2 1.4 2.3Gadd45b 6.8 11.8 8.3 7.9 4.3 8.5 3.9Cdkn1a 2.5 15.4 3.4 2.3 0.8 1.5 1.0

Gene SymbolFold change (h)

Cyp2b9Cyp2b10Cyp2b13Abcc3Gsta2Gstm2Ces2Abcc2Abcc4

Gadd45aGadd45bCdkn1

2 4 8 12 18 24 72

Time (h)

Up-regulation

Down-regulation

PX

R/C

AR

-reg

ulat

edD

NA

da

mag

e

No change

QRT-PCR VERIFICATION

Figure 3. Verification of mouse microarray results by QRT-PCR

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.

0

1

2

3

4

5

6

7

2h 4h 8h 12h 18h 24h 72h

0

5

10

15

20

25

30

35

40

45

50

2h 4h 8h 12h 18h 24h 72h

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2h 4h 8h 12h 18h 24h 72h

0

0.5

1

1.5

2

2.5

2h 4h 8h 12h 18h 24h 72h

0

0.2

0.4

0.6

0.8

1

1.2

1.4

2h 4h 8h 12h 18h 24h 72h

0

2

4

6

8

10

12

14

16

18

2h 4h 8h 12h 18h 24h 72h

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2h 4h 8h 12h 18h 24h 72h

0

0.5

1

1.5

2

2.5

3

3.5

2h 4h 8h 12h 18h 24h 72h2 4 8 12 18 7224Time (h)

2 4 8 12 18 7224Time (h)

2 4 8 12 18 7224Time (h)

2 4 8 12 18 7224Time (h)

Cyp2b10 CAR

Srebf1

Cyp7b1

* *

* *

*

*

Gclm * Hmox1Gadd45b

** *

PXR* *

*

** *

**

**

0

0.4

0.8

1.2

0

10

20

30

4050

0

0.6

1.2

1.8

0

4

8

12

16

0

0.6

1.2

1.80

0.5

1.0

1.5

2.0

2.5

0

1

2

3

0

2

4

6Microarray

PCR

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

Mouse

(C) Blood DHEA-S level

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

Rat Vehicleo,p’-DDT

Blo

od D

HE

A-S

leve

l (n

mol

/L)

0

0.04

0.08

0.12

0.16

0

0.04

0.08

0.12

0.16

2 4 8 12 18 24 72 2 4 8 12 18 24 72Time (h) Time (h)

*

0

100

200

300

400

500

600

700

800

900

1000

2h 4h 8h 12h 18h 24h 72h

Down

Up

0

200

400

600

800

1000

2 4 8 12 18 24 72Time (h)

Num

ber

of a

ctiv

e ge

nes Up

Down

Rel

ativ

e ex

pres

sion

(fo

ld)

Cholesterol

Pregnenolone

17OH Pregnenolone

Dehydroepiandrosterone(DHEA)

Androstenediol

Cyp11a1

Cyp17a1

Cyp17a1

17bHSD

Progesterone

17OH Progesterone

Androstenedione

Testosterone

Estradiol

Cyp19a1

3bHSD

Cyp17a1

Cyp17a1

0

0.5

1

1.5

2

2.5

3

2h 4h 8h 12h 18h 24h 72h

0

1

2

3

4

5

6

7

8

9

10

2h 4h 8h 12h 18h 24h 72h

*

**

*

Mouse Rat

Rel

ativ

e C

yp17

a1

expr

essi

on (

fold

)

0

2

4

6

8

10

2 4 8 12 18 24 72

Time (h)

0

1

2

3

2 4 8 12 18 24 72

Time (h)

PCR

(B) Hepatic Cyp17a1 expressionMicroarray

Liver

Endocrine system

ER-expressing tissues (e.g. uterus)

Elevation in DHEA-S level

CAR activation

o,p’-DDT exposure

DNA damage

Cell proliferationTumor promotion Uterotrophic response

Cyp17a1 induction

?

Perturbation of sex hormone homeostasis

Mouse-specific response

Direct binding to ER

ER-mediated response

Cell proliferation

Rat cDNA microarray Mouse Agilent microarray

Rat active genes Mouse active genes

34,222 genes5,692 genes

430 genes 1,480 genes

p1(t)>0.99|Fold change|>1.5

p1(t)>0.99|Fold change|>1.5

106 genes Correlation analysis (Fig. 3B)

274 genes

367 genes

4,497Common homologues