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Development of phenotypic and transcriptional biomarkers to evaluate relative activity of potentially estrogenic chemicals in ovariectomized mice.

Hewitt SC, Winuthayanon W, Pockette B, Kerns RT, Foley JF, Flagler N, Ney E, Suksamrarn A, Piyachaturawat P, Bushel PR, Korach KS - Environ. Health Perspect. (2015)

Bottom Line: Microarray analysis using tools to recognize patterns of response have been utilized in the cancer field to develop biomarker panels of transcripts for diagnosis and selection of treatments most likely to be effective.Biological effects elicited by long- versus short-acting estrogens greatly affect the risks associated with exposures; therefore, we sought to develop tools to predict the ability of chemicals to maintain estrogenic responses.The end points used are relevant to uterine tissue, but the resulting classification of the compounds is important for other sensitive tissues and species.

View Article: PubMed Central - PubMed

Affiliation: Receptor Biology, Reproductive and Developmental Biology Laboratory.

ABSTRACT

Background: Concerns regarding potential endocrine-disrupting chemicals (EDCs) have led to a need for methods to evaluate candidate estrogenic chemicals. Our previous evaluations of two such EDCs revealed a response similar to that of estradiol (E2) at 2 hr, but a less robust response at 24 hr, similar to the short-acting estrogen estriol (E3).

Objectives: Microarray analysis using tools to recognize patterns of response have been utilized in the cancer field to develop biomarker panels of transcripts for diagnosis and selection of treatments most likely to be effective. Biological effects elicited by long- versus short-acting estrogens greatly affect the risks associated with exposures; therefore, we sought to develop tools to predict the ability of chemicals to maintain estrogenic responses.

Methods: We used biological end points in uterine tissue and a signature pattern-recognizing tool that identified coexpressed transcripts to develop and test a panel of transcripts in order to classify potentially estrogenic compounds using an in vivo system. The end points used are relevant to uterine tissue, but the resulting classification of the compounds is important for other sensitive tissues and species.

Results: We evaluated biological and transcriptional end points with proven short- and long-acting estrogens and verified the use of our approach using a phytoestrogen. With our model, we were able to classify the diarylheptanoid D3 as a short-acting estrogen.

Conclusions: We have developed a panel of transcripts as biomarkers which, together with biological end points, might be used to screen and evaluate potentially estrogenic chemicals and infer mode of activity.

No MeSH data available.


Related in: MedlinePlus

Effects of saline vehicle (V), E2, and D3 after treatment for 24‑hr (B–D) or 72 hr (E–G). (A) Structure of D3. (B) Representative photomicrographs showing EdU incorporation in uterine epithelial cells 24 hr after treatment bar = 0.1 mm. (C) Percentage of EdU-positive cells in uterine epithelial cells 24 hr after treatment (mean ± SE; n = 5–7 mice/group). ***p < 0.001 compared with V by one-way ANOVA with uncorrected Fisher’s least significant difference (LSD). (D) Uterine weight (mean ± SE) at 24 hr after treatment (top; n = 9 mice/group) or 72 hr (24 hr after the last of three daily injections; bottom; n = 5 mice/group). ***p < 0.001, and ****p < 0.0001 compared with V, and +p < 0.01 compared with D3 by one-way ANOVA with uncorrected Fisher’s LSD. (E) Representative photomicrographs showing apoptotic cells in uteri collected at 72 hr (24 hr after the last of three daily injections) as determined by the TUNEL assay (bar = 0.2 mm; the arrowhead points to a TUNEL-positive cell. (F) Percentage of TUNEL-positive cells in uteri collected at 72 hr (24 hr after the last of three daily injections; mean ± SE; n = 8 mice/group). ***p < 0.001 by unpaired t-test. (G) LEH in uteri collected at 72 hr (24 hr after the last of three daily injections; mean ± SE; n = 8 mice/group). ****p < 0.0001 compared with V, and +p < 0.001 compared with E2 by one-way ANOVA with Tukey’s multiple comparison test.
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f3: Effects of saline vehicle (V), E2, and D3 after treatment for 24‑hr (B–D) or 72 hr (E–G). (A) Structure of D3. (B) Representative photomicrographs showing EdU incorporation in uterine epithelial cells 24 hr after treatment bar = 0.1 mm. (C) Percentage of EdU-positive cells in uterine epithelial cells 24 hr after treatment (mean ± SE; n = 5–7 mice/group). ***p < 0.001 compared with V by one-way ANOVA with uncorrected Fisher’s least significant difference (LSD). (D) Uterine weight (mean ± SE) at 24 hr after treatment (top; n = 9 mice/group) or 72 hr (24 hr after the last of three daily injections; bottom; n = 5 mice/group). ***p < 0.001, and ****p < 0.0001 compared with V, and +p < 0.01 compared with D3 by one-way ANOVA with uncorrected Fisher’s LSD. (E) Representative photomicrographs showing apoptotic cells in uteri collected at 72 hr (24 hr after the last of three daily injections) as determined by the TUNEL assay (bar = 0.2 mm; the arrowhead points to a TUNEL-positive cell. (F) Percentage of TUNEL-positive cells in uteri collected at 72 hr (24 hr after the last of three daily injections; mean ± SE; n = 8 mice/group). ***p < 0.001 by unpaired t-test. (G) LEH in uteri collected at 72 hr (24 hr after the last of three daily injections; mean ± SE; n = 8 mice/group). ****p < 0.0001 compared with V, and +p < 0.001 compared with E2 by one-way ANOVA with Tukey’s multiple comparison test.

Mentions: RT-PCR and validation of transcript panels. To validate our potential screening strategy, as shown in Table 1, we evaluated a diarylheptanoid, D3 (Figure 3A) (Suksamrarn et al. 2008), which has traditionally been used by women in Thailand to relieve postmenopausal symptoms. Studies by Winuthayanon et al. (2009, 2013) have suggested that this compound has estrogenic properties. As a test of this concept, we used biological response end points together with probes selected from each biomarker panel. Treatment with D3 resulted in the entry of uterine epithelial cells into S phase, and EdU incorporation and uterine weight was increased to a level comparable with E2 after 24 hr (Figure 3B–D). However, the uterine weight after 72 hr was lower than that reached by E2 (Figure 3D) and was not statistically significant (p = 0.054), as reported in earlier studies (Winuthayanon et al. 2009, 2013). The lower weight increase after 72 hr of treatment was reflected in increased TUNEL-positive cells (Figure 3E,F) as well as in an attenuated LEH increase (Figure 3G). These biological observations are consistent with short-acting estrogenic activity. Therefore, we evaluated transcriptional responses of selected biomarker panel transcripts after 2 or 24 hr of treatment (Figure 4A,B). Of the 10 transcripts from the 2-hr panel we tested, 8 showed similar responses with E2, E3, or D3, but D3 did not significantly induce two of the transcripts (Cdkn1a and Stat5a) and induced Nup50 less robustly than did E2 or E3 (Figure 4A). In the 24-hr panel, 2 transcripts (Ndufab1 and Gfm1) showed no regulation with any of the tested substances (Figure 4B). The remaining 7 transcripts either showed no response with D3 (Kifc2, Rorc, Sox4, Ccnb1, Nubp1, and Aurkb) or a response that was significant but was blunted compared with the E2 response (Ccnb2) (Figure 4B). Depending on the transcript, E3 exhibited responses similar to those of D3 (Kifc2, Rorc, and Sox4) but in some cases the response was between those of D3 and E2 (Aurkb and Ccnb2).


Development of phenotypic and transcriptional biomarkers to evaluate relative activity of potentially estrogenic chemicals in ovariectomized mice.

Hewitt SC, Winuthayanon W, Pockette B, Kerns RT, Foley JF, Flagler N, Ney E, Suksamrarn A, Piyachaturawat P, Bushel PR, Korach KS - Environ. Health Perspect. (2015)

Effects of saline vehicle (V), E2, and D3 after treatment for 24‑hr (B–D) or 72 hr (E–G). (A) Structure of D3. (B) Representative photomicrographs showing EdU incorporation in uterine epithelial cells 24 hr after treatment bar = 0.1 mm. (C) Percentage of EdU-positive cells in uterine epithelial cells 24 hr after treatment (mean ± SE; n = 5–7 mice/group). ***p < 0.001 compared with V by one-way ANOVA with uncorrected Fisher’s least significant difference (LSD). (D) Uterine weight (mean ± SE) at 24 hr after treatment (top; n = 9 mice/group) or 72 hr (24 hr after the last of three daily injections; bottom; n = 5 mice/group). ***p < 0.001, and ****p < 0.0001 compared with V, and +p < 0.01 compared with D3 by one-way ANOVA with uncorrected Fisher’s LSD. (E) Representative photomicrographs showing apoptotic cells in uteri collected at 72 hr (24 hr after the last of three daily injections) as determined by the TUNEL assay (bar = 0.2 mm; the arrowhead points to a TUNEL-positive cell. (F) Percentage of TUNEL-positive cells in uteri collected at 72 hr (24 hr after the last of three daily injections; mean ± SE; n = 8 mice/group). ***p < 0.001 by unpaired t-test. (G) LEH in uteri collected at 72 hr (24 hr after the last of three daily injections; mean ± SE; n = 8 mice/group). ****p < 0.0001 compared with V, and +p < 0.001 compared with E2 by one-way ANOVA with Tukey’s multiple comparison test.
© Copyright Policy - public-domain
Related In: Results  -  Collection

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Show All Figures
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f3: Effects of saline vehicle (V), E2, and D3 after treatment for 24‑hr (B–D) or 72 hr (E–G). (A) Structure of D3. (B) Representative photomicrographs showing EdU incorporation in uterine epithelial cells 24 hr after treatment bar = 0.1 mm. (C) Percentage of EdU-positive cells in uterine epithelial cells 24 hr after treatment (mean ± SE; n = 5–7 mice/group). ***p < 0.001 compared with V by one-way ANOVA with uncorrected Fisher’s least significant difference (LSD). (D) Uterine weight (mean ± SE) at 24 hr after treatment (top; n = 9 mice/group) or 72 hr (24 hr after the last of three daily injections; bottom; n = 5 mice/group). ***p < 0.001, and ****p < 0.0001 compared with V, and +p < 0.01 compared with D3 by one-way ANOVA with uncorrected Fisher’s LSD. (E) Representative photomicrographs showing apoptotic cells in uteri collected at 72 hr (24 hr after the last of three daily injections) as determined by the TUNEL assay (bar = 0.2 mm; the arrowhead points to a TUNEL-positive cell. (F) Percentage of TUNEL-positive cells in uteri collected at 72 hr (24 hr after the last of three daily injections; mean ± SE; n = 8 mice/group). ***p < 0.001 by unpaired t-test. (G) LEH in uteri collected at 72 hr (24 hr after the last of three daily injections; mean ± SE; n = 8 mice/group). ****p < 0.0001 compared with V, and +p < 0.001 compared with E2 by one-way ANOVA with Tukey’s multiple comparison test.
Mentions: RT-PCR and validation of transcript panels. To validate our potential screening strategy, as shown in Table 1, we evaluated a diarylheptanoid, D3 (Figure 3A) (Suksamrarn et al. 2008), which has traditionally been used by women in Thailand to relieve postmenopausal symptoms. Studies by Winuthayanon et al. (2009, 2013) have suggested that this compound has estrogenic properties. As a test of this concept, we used biological response end points together with probes selected from each biomarker panel. Treatment with D3 resulted in the entry of uterine epithelial cells into S phase, and EdU incorporation and uterine weight was increased to a level comparable with E2 after 24 hr (Figure 3B–D). However, the uterine weight after 72 hr was lower than that reached by E2 (Figure 3D) and was not statistically significant (p = 0.054), as reported in earlier studies (Winuthayanon et al. 2009, 2013). The lower weight increase after 72 hr of treatment was reflected in increased TUNEL-positive cells (Figure 3E,F) as well as in an attenuated LEH increase (Figure 3G). These biological observations are consistent with short-acting estrogenic activity. Therefore, we evaluated transcriptional responses of selected biomarker panel transcripts after 2 or 24 hr of treatment (Figure 4A,B). Of the 10 transcripts from the 2-hr panel we tested, 8 showed similar responses with E2, E3, or D3, but D3 did not significantly induce two of the transcripts (Cdkn1a and Stat5a) and induced Nup50 less robustly than did E2 or E3 (Figure 4A). In the 24-hr panel, 2 transcripts (Ndufab1 and Gfm1) showed no regulation with any of the tested substances (Figure 4B). The remaining 7 transcripts either showed no response with D3 (Kifc2, Rorc, Sox4, Ccnb1, Nubp1, and Aurkb) or a response that was significant but was blunted compared with the E2 response (Ccnb2) (Figure 4B). Depending on the transcript, E3 exhibited responses similar to those of D3 (Kifc2, Rorc, and Sox4) but in some cases the response was between those of D3 and E2 (Aurkb and Ccnb2).

Bottom Line: Microarray analysis using tools to recognize patterns of response have been utilized in the cancer field to develop biomarker panels of transcripts for diagnosis and selection of treatments most likely to be effective.Biological effects elicited by long- versus short-acting estrogens greatly affect the risks associated with exposures; therefore, we sought to develop tools to predict the ability of chemicals to maintain estrogenic responses.The end points used are relevant to uterine tissue, but the resulting classification of the compounds is important for other sensitive tissues and species.

View Article: PubMed Central - PubMed

Affiliation: Receptor Biology, Reproductive and Developmental Biology Laboratory.

ABSTRACT

Background: Concerns regarding potential endocrine-disrupting chemicals (EDCs) have led to a need for methods to evaluate candidate estrogenic chemicals. Our previous evaluations of two such EDCs revealed a response similar to that of estradiol (E2) at 2 hr, but a less robust response at 24 hr, similar to the short-acting estrogen estriol (E3).

Objectives: Microarray analysis using tools to recognize patterns of response have been utilized in the cancer field to develop biomarker panels of transcripts for diagnosis and selection of treatments most likely to be effective. Biological effects elicited by long- versus short-acting estrogens greatly affect the risks associated with exposures; therefore, we sought to develop tools to predict the ability of chemicals to maintain estrogenic responses.

Methods: We used biological end points in uterine tissue and a signature pattern-recognizing tool that identified coexpressed transcripts to develop and test a panel of transcripts in order to classify potentially estrogenic compounds using an in vivo system. The end points used are relevant to uterine tissue, but the resulting classification of the compounds is important for other sensitive tissues and species.

Results: We evaluated biological and transcriptional end points with proven short- and long-acting estrogens and verified the use of our approach using a phytoestrogen. With our model, we were able to classify the diarylheptanoid D3 as a short-acting estrogen.

Conclusions: We have developed a panel of transcripts as biomarkers which, together with biological end points, might be used to screen and evaluate potentially estrogenic chemicals and infer mode of activity.

No MeSH data available.


Related in: MedlinePlus