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Species-specific regulation of PXR/CAR/ER-target genes in the mouse and rat liver elicited by o, p'-DDT.

Kiyosawa N, Kwekel JC, Burgoon LD, Dere E, Williams KJ, Tashiro C, Chittim B, Zacharewski TR - BMC Genomics (2008)

Bottom Line: In addition, o, p'-DDT induced Gadd45a, Gadd45b and Cdkn1, suggesting DNA damage may be an additional risk factor.Although DDT is known to cause rodent hepatic tumors, the marked species differences in PXR/CAR structure, expression patterns and ligand preference as well as significant species-specific differences in steroidogenesis, especially CYP17A1 expression and activity, confound the extrapolation of these results to humans.Nevertheless, the identification of potential modes of action as well as species-specific responses may assist in the selection and further development of more appropriate models for assessing the toxicity of DDT to humans and wildlife.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, USA. kiyosawa.naoki.wr@daiichisankyo.co.jp

ABSTRACT

Background: Dichlorodiphenyltrichloroethane (DDT) is a persistent estrogenic organochlorine pesticide that is a rodent hepatic tumor promoter, with inconclusive carcinogenicity in humans. We have previously reported that o, p'-DDT elicits primarily PXR/CAR-mediated activity, rather than ER-mediated hepatic responses, and suggested that CAR-mediated effects, as opposed to ER-mediated effects, may be more important in tumor promotion in the rat liver. To further characterize species-specific hepatic responses, gene expression analysis, with complementary histopathology and tissue level analyses were investigated in immature, ovariectomized C57BL/6 mice treated with 300 mg/kg o, p'-DDT, and compared to Sprague-Dawley rat data.

Results: Rats and mice exhibited negligible histopathology with rapid o, p'-DDT metabolism. Gene expression profiles were also similar, exhibiting PXR/CAR regulation with the characteristic induction of Cyp2b10 and Cyp3a11. However, PXR-specific target genes such as Apoa4 or Insig2 exhibited more pronounced induction compared to CAR-specific genes in the mouse. In addition, mouse Car mRNA levels decreased, possibly contributing to the preferential activation of mouse PXR. ER-regulated genes Cyp17a1 and Cyp7b1 were also induced, suggesting o, p'-DDT also elicits ER-mediated gene expression in the mouse, while ER-mediated effects were negligible in the rat, possibly due to the inhibitory effects of CAR on ER activities. In addition, o, p'-DDT induced Gadd45a, Gadd45b and Cdkn1, suggesting DNA damage may be an additional risk factor. Furthermore, elevated blood DHEA-S levels at 12 h after treatment in the mouse may also contribute to the endocrine-related effects of o, p'-DDT.

Conclusion: Although DDT is known to cause rodent hepatic tumors, the marked species differences in PXR/CAR structure, expression patterns and ligand preference as well as significant species-specific differences in steroidogenesis, especially CYP17A1 expression and activity, confound the extrapolation of these results to humans. Nevertheless, the identification of potential modes of action as well as species-specific responses may assist in the selection and further development of more appropriate models for assessing the toxicity of DDT to humans and wildlife.

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Microarray and QRT-PCR results. (A) Identification of differentially regulated genes. (B) Number of differentially expressed genes following o, p'-DDT treatment in the mouse liver. Differentially expressed genes were selected based on a p1(t) ≥ 0.999 at two or more time points and an absolute fold change ≥ 1.5 at one or more time points relative to time-matched vehicle controls. All differentially expressed genes are listed in Additional file 3. (C) Verification of microarray results by QRT-PCR. QRT-PCR results relative to time-matched vehicle controls are shown as bar and presented as mean ± SE. Microarray results are represented as lines. The dashed line indicates the expression level of the time-matched vehicle control. The asterisk (*) indicates a significant (p < 0.05) difference from the time-matched vehicle controls for QRT-PCR, n = 5. (D) Hepatic expression of PXR/CAR-target genes in o, p'-DDT-treated mouse. A heat map of o, p'-DDT elicited microarray expression profiles for selected PXR-, CAR-specific and PXR/CAR-shared target genes identified in the literature [20-23]. While some CAR-regulated genes such as Cyp1a1, Fmo5, Sult1d1 or Abcc2 were moderately induced, several PXR-target genes, including ApoA4, Ces2, Gstm2 or Insig2, exhibited strong induction. However, other PXR-target genes such as Hmgcs1 and Hmgcs2 were down-regulated.
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Figure 2: Microarray and QRT-PCR results. (A) Identification of differentially regulated genes. (B) Number of differentially expressed genes following o, p'-DDT treatment in the mouse liver. Differentially expressed genes were selected based on a p1(t) ≥ 0.999 at two or more time points and an absolute fold change ≥ 1.5 at one or more time points relative to time-matched vehicle controls. All differentially expressed genes are listed in Additional file 3. (C) Verification of microarray results by QRT-PCR. QRT-PCR results relative to time-matched vehicle controls are shown as bar and presented as mean ± SE. Microarray results are represented as lines. The dashed line indicates the expression level of the time-matched vehicle control. The asterisk (*) indicates a significant (p < 0.05) difference from the time-matched vehicle controls for QRT-PCR, n = 5. (D) Hepatic expression of PXR/CAR-target genes in o, p'-DDT-treated mouse. A heat map of o, p'-DDT elicited microarray expression profiles for selected PXR-, CAR-specific and PXR/CAR-shared target genes identified in the literature [20-23]. While some CAR-regulated genes such as Cyp1a1, Fmo5, Sult1d1 or Abcc2 were moderately induced, several PXR-target genes, including ApoA4, Ces2, Gstm2 or Insig2, exhibited strong induction. However, other PXR-target genes such as Hmgcs1 and Hmgcs2 were down-regulated.

Mentions: Gene expression was assessed using the 4 × 44 K Mouse Agilent array containing approximately 44,000 oligonucleotide probes, representing 34,204 annotated genes including approximately 21,000 unique genes. Model-based t-values that compared treated and vehicle responses on a per time-point basis followed by Empirical Bayes analysis identified 1,206 differentially expressed genes across the time course based on a p1(t) > 0.999 for a minimum of two time points and absolute fold change ≥ 1.5-fold compared to time-matched vehicle control for at least one time point (Fig. 2A). Overall, 859 genes were induced while 382 genes were repressed. Changes in gene expression ranged from 36.4-fold induction for Cyp2c55 gene to -13.0-fold repression for gene Car3. The 12 h time point exhibited the greatest number of differentially expressed genes (Fig. 2B). All of the gene expression data as well as the identified differentially regulated genes are provided in Additional files 2 and 3, respectively.


Species-specific regulation of PXR/CAR/ER-target genes in the mouse and rat liver elicited by o, p'-DDT.

Kiyosawa N, Kwekel JC, Burgoon LD, Dere E, Williams KJ, Tashiro C, Chittim B, Zacharewski TR - BMC Genomics (2008)

Microarray and QRT-PCR results. (A) Identification of differentially regulated genes. (B) Number of differentially expressed genes following o, p'-DDT treatment in the mouse liver. Differentially expressed genes were selected based on a p1(t) ≥ 0.999 at two or more time points and an absolute fold change ≥ 1.5 at one or more time points relative to time-matched vehicle controls. All differentially expressed genes are listed in Additional file 3. (C) Verification of microarray results by QRT-PCR. QRT-PCR results relative to time-matched vehicle controls are shown as bar and presented as mean ± SE. Microarray results are represented as lines. The dashed line indicates the expression level of the time-matched vehicle control. The asterisk (*) indicates a significant (p < 0.05) difference from the time-matched vehicle controls for QRT-PCR, n = 5. (D) Hepatic expression of PXR/CAR-target genes in o, p'-DDT-treated mouse. A heat map of o, p'-DDT elicited microarray expression profiles for selected PXR-, CAR-specific and PXR/CAR-shared target genes identified in the literature [20-23]. While some CAR-regulated genes such as Cyp1a1, Fmo5, Sult1d1 or Abcc2 were moderately induced, several PXR-target genes, including ApoA4, Ces2, Gstm2 or Insig2, exhibited strong induction. However, other PXR-target genes such as Hmgcs1 and Hmgcs2 were down-regulated.
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Related In: Results  -  Collection

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Figure 2: Microarray and QRT-PCR results. (A) Identification of differentially regulated genes. (B) Number of differentially expressed genes following o, p'-DDT treatment in the mouse liver. Differentially expressed genes were selected based on a p1(t) ≥ 0.999 at two or more time points and an absolute fold change ≥ 1.5 at one or more time points relative to time-matched vehicle controls. All differentially expressed genes are listed in Additional file 3. (C) Verification of microarray results by QRT-PCR. QRT-PCR results relative to time-matched vehicle controls are shown as bar and presented as mean ± SE. Microarray results are represented as lines. The dashed line indicates the expression level of the time-matched vehicle control. The asterisk (*) indicates a significant (p < 0.05) difference from the time-matched vehicle controls for QRT-PCR, n = 5. (D) Hepatic expression of PXR/CAR-target genes in o, p'-DDT-treated mouse. A heat map of o, p'-DDT elicited microarray expression profiles for selected PXR-, CAR-specific and PXR/CAR-shared target genes identified in the literature [20-23]. While some CAR-regulated genes such as Cyp1a1, Fmo5, Sult1d1 or Abcc2 were moderately induced, several PXR-target genes, including ApoA4, Ces2, Gstm2 or Insig2, exhibited strong induction. However, other PXR-target genes such as Hmgcs1 and Hmgcs2 were down-regulated.
Mentions: Gene expression was assessed using the 4 × 44 K Mouse Agilent array containing approximately 44,000 oligonucleotide probes, representing 34,204 annotated genes including approximately 21,000 unique genes. Model-based t-values that compared treated and vehicle responses on a per time-point basis followed by Empirical Bayes analysis identified 1,206 differentially expressed genes across the time course based on a p1(t) > 0.999 for a minimum of two time points and absolute fold change ≥ 1.5-fold compared to time-matched vehicle control for at least one time point (Fig. 2A). Overall, 859 genes were induced while 382 genes were repressed. Changes in gene expression ranged from 36.4-fold induction for Cyp2c55 gene to -13.0-fold repression for gene Car3. The 12 h time point exhibited the greatest number of differentially expressed genes (Fig. 2B). All of the gene expression data as well as the identified differentially regulated genes are provided in Additional files 2 and 3, respectively.

Bottom Line: In addition, o, p'-DDT induced Gadd45a, Gadd45b and Cdkn1, suggesting DNA damage may be an additional risk factor.Although DDT is known to cause rodent hepatic tumors, the marked species differences in PXR/CAR structure, expression patterns and ligand preference as well as significant species-specific differences in steroidogenesis, especially CYP17A1 expression and activity, confound the extrapolation of these results to humans.Nevertheless, the identification of potential modes of action as well as species-specific responses may assist in the selection and further development of more appropriate models for assessing the toxicity of DDT to humans and wildlife.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, USA. kiyosawa.naoki.wr@daiichisankyo.co.jp

ABSTRACT

Background: Dichlorodiphenyltrichloroethane (DDT) is a persistent estrogenic organochlorine pesticide that is a rodent hepatic tumor promoter, with inconclusive carcinogenicity in humans. We have previously reported that o, p'-DDT elicits primarily PXR/CAR-mediated activity, rather than ER-mediated hepatic responses, and suggested that CAR-mediated effects, as opposed to ER-mediated effects, may be more important in tumor promotion in the rat liver. To further characterize species-specific hepatic responses, gene expression analysis, with complementary histopathology and tissue level analyses were investigated in immature, ovariectomized C57BL/6 mice treated with 300 mg/kg o, p'-DDT, and compared to Sprague-Dawley rat data.

Results: Rats and mice exhibited negligible histopathology with rapid o, p'-DDT metabolism. Gene expression profiles were also similar, exhibiting PXR/CAR regulation with the characteristic induction of Cyp2b10 and Cyp3a11. However, PXR-specific target genes such as Apoa4 or Insig2 exhibited more pronounced induction compared to CAR-specific genes in the mouse. In addition, mouse Car mRNA levels decreased, possibly contributing to the preferential activation of mouse PXR. ER-regulated genes Cyp17a1 and Cyp7b1 were also induced, suggesting o, p'-DDT also elicits ER-mediated gene expression in the mouse, while ER-mediated effects were negligible in the rat, possibly due to the inhibitory effects of CAR on ER activities. In addition, o, p'-DDT induced Gadd45a, Gadd45b and Cdkn1, suggesting DNA damage may be an additional risk factor. Furthermore, elevated blood DHEA-S levels at 12 h after treatment in the mouse may also contribute to the endocrine-related effects of o, p'-DDT.

Conclusion: Although DDT is known to cause rodent hepatic tumors, the marked species differences in PXR/CAR structure, expression patterns and ligand preference as well as significant species-specific differences in steroidogenesis, especially CYP17A1 expression and activity, confound the extrapolation of these results to humans. Nevertheless, the identification of potential modes of action as well as species-specific responses may assist in the selection and further development of more appropriate models for assessing the toxicity of DDT to humans and wildlife.

Show MeSH
Related in: MedlinePlus