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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

Phenotypic end points of estrogenic response in the uterus of mice treated with saline vehicle (V), E2, E3, DES, or PPT for 24 (A–C) or 72 hr (D–I). (A) Representative photomicrographs showing cell proliferation 24 hr after treatment, indicated by incorporation of EdU (green); blue indicates Hoescht staining of DNA. Uterine epithelial cells show active DNA synthesis, and basal EdU incorporation is present in vehicle-treated mice; bar = 0.1 mm. (B) Percentage of EdU-positive cells in uterine tissue 24 hr after injection (mean ± SE; n = 5–9 mice/group). **p < 0.01 compared with V by one-way ANOVA with multiple comparisons and uncorrected Fisher’s least significant difference (LSD). (C) Uterine weight 24 hr after injection (mean ± SE; n = 3–5 mice/group). *p < 0.05, ***p < 0.001, and ****p < 0.0001, compared with V by one-way ANOVA, with multiple comparisons, with Tukey’s multiple test correction. (D) Uterine weight in the 72 hr (3 day) bioassay, with uteri collected 24 hr after the last of three daily injections (mean ± SE; n = 4–7 mice/group). **p < 0.001 for PPT compared with DES or E2; and ****p < 0.0001 compared with V, by one-way ANOVA, with multiple comparisons, with Tukey’s multiple test correction. (E) Luminal epithelial cell height (LEH) in the 72‑hr bioassay (mean ± SE; n = 4–7 mice/group). ***p <0.001, and ****p < 0.0001 compared with V; +p < 0.0001 compared with E2 or DES; and #p < 0.001 compared with E2 by one-way ANOVA, with multiple comparisons, with Tukey’s multiple test correction. (F) Level of RNA for the apoptosis inhibitor Birc1a measured by RT‑PCR in uteri from the 72‑hr bioassay (mean ± SE; n = 4–7 mice/group).***p < 0.001, and ****p < 0.0001 compared with V by one-way ANOVA with multiple comparisons and uncorrected Fisher’s LSD. (G) Ltf transcript measured by RT-PCR in uteri from the 72‑hr bioassay (mean ± SE; n = 4–7 mice/group). **p < 0.01, ***p < 0.001, and **** p < 0.0001 compared with V; and +p < 0.01 for PPT compared with E2 by one-way ANOVA, with multiple comparisons, with Tukey’s multiple test correction. (H) Representative photomicrographs showing apoptotic cells in uteri from the 72‑hr bioassay as indicated by the TUNEL assay; bar = 0.1 mm. Each arrowhead points to a TUNEL-positive cell. (I) Percent TUNEL-positive luminal epithelial cells (mean ± SE; n = 4–10 mice/group.
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f2: Phenotypic end points of estrogenic response in the uterus of mice treated with saline vehicle (V), E2, E3, DES, or PPT for 24 (A–C) or 72 hr (D–I). (A) Representative photomicrographs showing cell proliferation 24 hr after treatment, indicated by incorporation of EdU (green); blue indicates Hoescht staining of DNA. Uterine epithelial cells show active DNA synthesis, and basal EdU incorporation is present in vehicle-treated mice; bar = 0.1 mm. (B) Percentage of EdU-positive cells in uterine tissue 24 hr after injection (mean ± SE; n = 5–9 mice/group). **p < 0.01 compared with V by one-way ANOVA with multiple comparisons and uncorrected Fisher’s least significant difference (LSD). (C) Uterine weight 24 hr after injection (mean ± SE; n = 3–5 mice/group). *p < 0.05, ***p < 0.001, and ****p < 0.0001, compared with V by one-way ANOVA, with multiple comparisons, with Tukey’s multiple test correction. (D) Uterine weight in the 72 hr (3 day) bioassay, with uteri collected 24 hr after the last of three daily injections (mean ± SE; n = 4–7 mice/group). **p < 0.001 for PPT compared with DES or E2; and ****p < 0.0001 compared with V, by one-way ANOVA, with multiple comparisons, with Tukey’s multiple test correction. (E) Luminal epithelial cell height (LEH) in the 72‑hr bioassay (mean ± SE; n = 4–7 mice/group). ***p <0.001, and ****p < 0.0001 compared with V; +p < 0.0001 compared with E2 or DES; and #p < 0.001 compared with E2 by one-way ANOVA, with multiple comparisons, with Tukey’s multiple test correction. (F) Level of RNA for the apoptosis inhibitor Birc1a measured by RT‑PCR in uteri from the 72‑hr bioassay (mean ± SE; n = 4–7 mice/group).***p < 0.001, and ****p < 0.0001 compared with V by one-way ANOVA with multiple comparisons and uncorrected Fisher’s LSD. (G) Ltf transcript measured by RT-PCR in uteri from the 72‑hr bioassay (mean ± SE; n = 4–7 mice/group). **p < 0.01, ***p < 0.001, and **** p < 0.0001 compared with V; and +p < 0.01 for PPT compared with E2 by one-way ANOVA, with multiple comparisons, with Tukey’s multiple test correction. (H) Representative photomicrographs showing apoptotic cells in uteri from the 72‑hr bioassay as indicated by the TUNEL assay; bar = 0.1 mm. Each arrowhead points to a TUNEL-positive cell. (I) Percent TUNEL-positive luminal epithelial cells (mean ± SE; n = 4–10 mice/group.

Mentions: Biological end points. We compared proliferation of uterine epithelial cells—a hallmark of estrogenic response—in ovariectomized mice treated with E2, E3, DES (a synthetic estrogen), or PPT [a synthetic estrogen receptor α (ERα)–selective agonist]. Treatment with all of the estrogens resulted in an increase in epithelial cells in S phase, as reflected by incorporation of the thymidine triphosphate analog EdU (Figure 2A,B). At the 24-hr time point, uterine wet weights were increased by E2, E3, or DES (p < 0.05), but not significantly by PPT (Figure 2C). However, after 3 days of treatment, uterine weight was not significantly increased by E3; however, E2 or DES treatment led to a robust uterine weight increase (p < 0.0001), whereas PPT caused an increase (p < 0.001) that was significantly lower than the increase induced by E2 or DES (p < 0.001) (Figure 2D). In the 72-hr bioassay, E2, E3, DES, and PPT treatment resulted in increased epithelial thickness and cell height (p < 0.001; Figure 2E); however, PPT and E3 increased the epithelial thickness significantly less than did E2 (p < 0.001 vs. E2; Figure 2E). Three days of E3 treatment did not induce transcripts of either the apoptosis inhibitor Birc1a (baculoviral IAP repeat-containing protein 1a) or the epithelial cell secretory protein Ltf (lactoferrin), whereas E2, DES, and PPT induced both of these (Figure 2F,G). In addition, E3 and PPT exhibited significantly more TUNEL-positive epithelial cells than E2 or DES (p < 0.05; Figure 2H,I), indicating increased apoptosis.


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)

Phenotypic end points of estrogenic response in the uterus of mice treated with saline vehicle (V), E2, E3, DES, or PPT for 24 (A–C) or 72 hr (D–I). (A) Representative photomicrographs showing cell proliferation 24 hr after treatment, indicated by incorporation of EdU (green); blue indicates Hoescht staining of DNA. Uterine epithelial cells show active DNA synthesis, and basal EdU incorporation is present in vehicle-treated mice; bar = 0.1 mm. (B) Percentage of EdU-positive cells in uterine tissue 24 hr after injection (mean ± SE; n = 5–9 mice/group). **p < 0.01 compared with V by one-way ANOVA with multiple comparisons and uncorrected Fisher’s least significant difference (LSD). (C) Uterine weight 24 hr after injection (mean ± SE; n = 3–5 mice/group). *p < 0.05, ***p < 0.001, and ****p < 0.0001, compared with V by one-way ANOVA, with multiple comparisons, with Tukey’s multiple test correction. (D) Uterine weight in the 72 hr (3 day) bioassay, with uteri collected 24 hr after the last of three daily injections (mean ± SE; n = 4–7 mice/group). **p < 0.001 for PPT compared with DES or E2; and ****p < 0.0001 compared with V, by one-way ANOVA, with multiple comparisons, with Tukey’s multiple test correction. (E) Luminal epithelial cell height (LEH) in the 72‑hr bioassay (mean ± SE; n = 4–7 mice/group). ***p <0.001, and ****p < 0.0001 compared with V; +p < 0.0001 compared with E2 or DES; and #p < 0.001 compared with E2 by one-way ANOVA, with multiple comparisons, with Tukey’s multiple test correction. (F) Level of RNA for the apoptosis inhibitor Birc1a measured by RT‑PCR in uteri from the 72‑hr bioassay (mean ± SE; n = 4–7 mice/group).***p < 0.001, and ****p < 0.0001 compared with V by one-way ANOVA with multiple comparisons and uncorrected Fisher’s LSD. (G) Ltf transcript measured by RT-PCR in uteri from the 72‑hr bioassay (mean ± SE; n = 4–7 mice/group). **p < 0.01, ***p < 0.001, and **** p < 0.0001 compared with V; and +p < 0.01 for PPT compared with E2 by one-way ANOVA, with multiple comparisons, with Tukey’s multiple test correction. (H) Representative photomicrographs showing apoptotic cells in uteri from the 72‑hr bioassay as indicated by the TUNEL assay; bar = 0.1 mm. Each arrowhead points to a TUNEL-positive cell. (I) Percent TUNEL-positive luminal epithelial cells (mean ± SE; n = 4–10 mice/group.
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f2: Phenotypic end points of estrogenic response in the uterus of mice treated with saline vehicle (V), E2, E3, DES, or PPT for 24 (A–C) or 72 hr (D–I). (A) Representative photomicrographs showing cell proliferation 24 hr after treatment, indicated by incorporation of EdU (green); blue indicates Hoescht staining of DNA. Uterine epithelial cells show active DNA synthesis, and basal EdU incorporation is present in vehicle-treated mice; bar = 0.1 mm. (B) Percentage of EdU-positive cells in uterine tissue 24 hr after injection (mean ± SE; n = 5–9 mice/group). **p < 0.01 compared with V by one-way ANOVA with multiple comparisons and uncorrected Fisher’s least significant difference (LSD). (C) Uterine weight 24 hr after injection (mean ± SE; n = 3–5 mice/group). *p < 0.05, ***p < 0.001, and ****p < 0.0001, compared with V by one-way ANOVA, with multiple comparisons, with Tukey’s multiple test correction. (D) Uterine weight in the 72 hr (3 day) bioassay, with uteri collected 24 hr after the last of three daily injections (mean ± SE; n = 4–7 mice/group). **p < 0.001 for PPT compared with DES or E2; and ****p < 0.0001 compared with V, by one-way ANOVA, with multiple comparisons, with Tukey’s multiple test correction. (E) Luminal epithelial cell height (LEH) in the 72‑hr bioassay (mean ± SE; n = 4–7 mice/group). ***p <0.001, and ****p < 0.0001 compared with V; +p < 0.0001 compared with E2 or DES; and #p < 0.001 compared with E2 by one-way ANOVA, with multiple comparisons, with Tukey’s multiple test correction. (F) Level of RNA for the apoptosis inhibitor Birc1a measured by RT‑PCR in uteri from the 72‑hr bioassay (mean ± SE; n = 4–7 mice/group).***p < 0.001, and ****p < 0.0001 compared with V by one-way ANOVA with multiple comparisons and uncorrected Fisher’s LSD. (G) Ltf transcript measured by RT-PCR in uteri from the 72‑hr bioassay (mean ± SE; n = 4–7 mice/group). **p < 0.01, ***p < 0.001, and **** p < 0.0001 compared with V; and +p < 0.01 for PPT compared with E2 by one-way ANOVA, with multiple comparisons, with Tukey’s multiple test correction. (H) Representative photomicrographs showing apoptotic cells in uteri from the 72‑hr bioassay as indicated by the TUNEL assay; bar = 0.1 mm. Each arrowhead points to a TUNEL-positive cell. (I) Percent TUNEL-positive luminal epithelial cells (mean ± SE; n = 4–10 mice/group.
Mentions: Biological end points. We compared proliferation of uterine epithelial cells—a hallmark of estrogenic response—in ovariectomized mice treated with E2, E3, DES (a synthetic estrogen), or PPT [a synthetic estrogen receptor α (ERα)–selective agonist]. Treatment with all of the estrogens resulted in an increase in epithelial cells in S phase, as reflected by incorporation of the thymidine triphosphate analog EdU (Figure 2A,B). At the 24-hr time point, uterine wet weights were increased by E2, E3, or DES (p < 0.05), but not significantly by PPT (Figure 2C). However, after 3 days of treatment, uterine weight was not significantly increased by E3; however, E2 or DES treatment led to a robust uterine weight increase (p < 0.0001), whereas PPT caused an increase (p < 0.001) that was significantly lower than the increase induced by E2 or DES (p < 0.001) (Figure 2D). In the 72-hr bioassay, E2, E3, DES, and PPT treatment resulted in increased epithelial thickness and cell height (p < 0.001; Figure 2E); however, PPT and E3 increased the epithelial thickness significantly less than did E2 (p < 0.001 vs. E2; Figure 2E). Three days of E3 treatment did not induce transcripts of either the apoptosis inhibitor Birc1a (baculoviral IAP repeat-containing protein 1a) or the epithelial cell secretory protein Ltf (lactoferrin), whereas E2, DES, and PPT induced both of these (Figure 2F,G). In addition, E3 and PPT exhibited significantly more TUNEL-positive epithelial cells than E2 or DES (p < 0.05; Figure 2H,I), indicating increased apoptosis.

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