Proteomics Complementation of the Rat Uterotrophic Assay for Estrogenic Endocrine Disruptors: A Roadmap of Advancing High Resolution Mass Spectrometry-Based Shotgun Survey to Targeted Biomarker Quantifications

The widely used rat uterotrophic assay to assess known and potential estrogenic compounds only considers uterine weight gain as endpoint measurement. To complement this method with an advanced technology that reveals molecular targets, we analyzed changes in protein expression using label-free quantitative proteomics by nanoflow liquid chromatography coupled with high-resolution mass spectrometry and tandem mass spectrometry from uterine protein extracts of ovariectomized rats after daily 17β-estradiol exposure for five days in comparison with those of vehicle-treated control animals. Our discovery-driven study revealed 165 uterine proteins significantly regulated by estrogen treatment and mapped by pathway analyses. Estrogen-regulated proteins represented cell death, survival and development, cellular growth and proliferation, and protein synthesis as top molecular and cellular functions, and a network found with the presence of nuclear estrogen receptor(s) as a prominent molecular node confirmed the relevance of our findings to hormone-associated events. An exploratory application of targeted proteomics to bisphenol A as a well-known example of an estrogenic endocrine disruptor is also presented. Overall, the results of this study have demonstrated the power of combining untargeted and targeted quantitative proteomic strategies to identify and verify candidate molecular markers for the evaluation of endocrine-disrupting chemicals to complement a conventional bioassay.

Estrogenic and anxiolytic effects of the decoction of stem bark of Khaya anthotheca (Welw.) C.DC (Meliaceae) in ovariectomised Wistar rats

<p><em>Khaya anthotheca</em> (Welw.) C.DC (Meliaceae) is a plant used in Cameroon to alleviate vaginal dryness in postmenopausal women and is also known to have anxiolytic properties.This work was designed to evaluate estrogen-like effects of this plant on primary estrogens targets of ovariectomized adult rats, as well as to evaluate its anxiolytic activities in the elevated plus-maze (EPM) test. In the 3-day uterotrophic assay, the extract increased (p &lt; 0.01) the size of the vaginal epithelia and stimulated the acini differentiation of the mammary gland. In the EPM test, the extract increased the percentage of number of entries (p &lt;0.05; p &lt;0.01) and the percentage of time spent (p &lt; 0.05) into open arms.Italso induced a decrease in percentage of number of entries (p &lt; 0.05; p &lt; 0.01) and the percentage of time spent (p &lt; 0.05) into closed arms. The extract also induced an increase of total arms entries (p &lt; 0.05; p &lt; 0.01) and rearing (p &lt; 0.05). Moreover, there was a decrease of defecation and grooming (p &lt; 0.05; p &lt; 0.01). These results suggest that <em>K. anthotheca</em> is endowed with estrogenic and anxiolytic properties, likely due to the presence of some estrogen-like compounds.</p>

Estrogenic properties of spices of the traditional Cameroonian dish “Nkui” in ovariectomized Wistar rats

Besides the basic role to flavor and color foods, several health benefits have been attributed to spices. The traditional Cameroonian food “Nkui” is prepared using several spices (: OVX female Wistar rats were randomly separated in several groups of five animals each and submitted to a 3-day uterotrophic assay (: These extracts increased the uterine wet weight, the uterine and vaginal epithelial heights, and the lumen and diameter of alveoli in the mammary glands. They also altered the estradiol-induced increase of uterine wet weight. The dichloromethane and methanol fractions of the ethanol extract exhibited estrogenic properties as well by increasing uterine and vaginal endpoints.: These results suggest that the spices of “Nkui” contain estrogenic phytoconstituents and this traditional food may be considered as functional.

Usefulness of immature golden hamster (Mesocricetus auratus) as a model for uterotrophic assay

The present study assessed the sensitivity of immature hamster uterotrophic assay to reference oestrogen agonists/antagonists in order to develop a sensitive model for evaluation of endocrine-active compounds in diets. After performing a baseline for control animals, the sensitivity of immature females (postnatal day 18) to reference compounds was evaluated in a three-day uterotrophic assay. The absolute and adjusted dry uterine weights, fold induction over control for absolute wet uterine weight, and wet uterine weight/body weight ratio (%) were used as endpoints. The significantly active doses for reference oestrogens were as follows: 0.6 μg/kg for 17α-ethinyloestradiol (s.c.); 1 μg/kg/day (s.c.) and 40 μg/kg (p.o.) for diethylstilboestrol; 40 mg/kg (s.c.) and 160 mg/kg (p.o.) for bisphenol A. Co-treatment with tamoxifen at a dose of 1 mg/kg significantly antagonised the uterotrophic effect induced by 1 μg/kg 17α-ethinyloestradiol, and showed the attenuated proliferative effect in histopathological examination. We found immature hamster uterotrophic assay as a sensitive model that could be a good alternative to the rat assay.