Induction of the Estrogenic Marker Calbindn-D₉k by Octamethylcyclotetrasiloxane

Abstract

Interrupting the hormonal balance of an organism by interfering with hormones and their target receptors gives rise to various problems such as developmental disorders. Collectively, these reagents are known as endocrine disruptors (EDs). Cyclic volatile methyl siloxanes (cVMSs) are a group of silicone polymers that including octamethylcyclotetrasiloxane (D4). In the present study, we examined the estrogenicity of D4 through in vitro and in vivo assays that employed calcium-binding protein 9K (calbindin-D9k; CaBP-9K) as a biomarker. For in vitro investigation, GH3 rat pituitary cells were exposed to vehicle, 17β-estradiol (E2), or D4 with/without ICI 182 780 (ICI). CaBP-9K and progesterone receptor (PR) both were up-regulated by E2 and D4 which were completely blocked by ICI. Transcription of estrogen receptor α (ER α) was decreased by E2 and D4 but increased by ICI. D4 was also administered to immature female rats for an uterotrophic (UT) assay and detection of CaBP-9K. Ethinyl estradiol (EE) or D4 was administered subcutaneously with or without ICI. Although uterine weight was not significant altered by D4, an effect thought to be due to cytochrome P450 (CYP), it induced CaBP-9K and PR gene expression. Based on these results we reveal that D4 has estrogenic potential proven under in vitro and in vivo experimental conditions.

Keywords: GH3 cell; calbindin-D9k; endocrine disruptors; octamethylcyclotetrasiloxane; uterotrophic assay.

Figure 1

CaBP-9K expression in GH3 cells at the mRNA (a); and protein (b) levels. Results presented in the bar graph are divided according to chemical administered and subdivided according to the presence or absence of ICI 182 780. Ve, vehicle; E2, 17β-estradiol; D4, octamethylcyclotetrasiloxane. ^a p < 0.05 vs. vehicle; ^b p < 0.05 vs. without ICI. Data are presented as the mean ± SD.

Figure 1

CaBP-9K expression in GH3 cells at the mRNA (a); and protein (b) levels. Results presented in the bar graph are divided according to chemical administered and subdivided according to the presence or absence of ICI 182 780. Ve, vehicle; E2, 17β-estradiol; D4, octamethylcyclotetrasiloxane. ^a p < 0.05 vs. vehicle; ^b p < 0.05 vs. without ICI. Data are presented as the mean ± SD.

Figure 2

PR expression in GH3 cells at the mRNA (a); and protein (b) levels; ER α expression in GH3 cells at the mRNA (c); and protein (d) levels. Results presented in the bar graph are divided according to chemical administered and subdivided according to the presence or absence of ICI 182 780 (shown as ICI). ^a p < 0.05 versus vehicle; ^b p < 0.05 versus without ICI. Data are presented as the mean ± SD.

Figure 3

Liver/body weight ratio (a); and CYP2B1 mRNA expression (b) in immature female SD rats. Data presented in the bar graph are divided according to chemical administered and subdivided according to the presence or absence of ICI 182 780 (shown as ICI). ^a p < 0.05 vs. vehicle; ^b p < 0.05 vs. without ICI. Data are presented as the mean ± SD.

Figure 4

Uterus/body weight ratio measured by the UT assay in immature female SD rats. Results in the graph are divided according to the chemical administered and subdivided according to the presence/absence of ICI 182 780 (shown as ICI). ^a p < 0.05 vs. vehicle; ^b p < 0.05 vs. without ICI. Data are presented as the mean ± SD.

Figure 5

CaBP-9K expression at the mRNA (a); and protein (b) levels in immature female SD rats. Data presented in the graph are divided according to chemical administered and subdivided according to the presence or absence of ICI 182 780 (shown as ICI). ^a p < 0.05 vs. vehicle, ^b p < 0.05 vs. without ICI. Data are presented as the mean ± SD.

Figure 5

CaBP-9K expression at the mRNA (a); and protein (b) levels in immature female SD rats. Data presented in the graph are divided according to chemical administered and subdivided according to the presence or absence of ICI 182 780 (shown as ICI). ^a p < 0.05 vs. vehicle, ^b p < 0.05 vs. without ICI. Data are presented as the mean ± SD.

Figure 6

PR expression at the mRNA (a); and protein (b); levels in immature female SD rats. ER α expression in immature female SD rats at the mRNA (c); and protein (d) levels. Findings presented in the bar graph are divided according to chemical administered and subdivided according to the presence or absence of ICI 182 780 (shown as ICI). ^a p < 0.05 versus vehicle, ^b p < 0.05 versus without ICI. Data are presented as the mean ± SD.