Ethinyl Estradiol

Adult (3 months old) female Sprague–Dawley rats were monitored for estrous cycles with daily vaginal smears. Only rats with at least two regular 4- to 5-day estrous cycles were included in the studies. The females were caged with proven males, and pregnancy was verified by observation of a sperm plug, which was designated as day 0 of pregnancy. Dams were randomly assigned to the following drug conditions: LNG (20 μg levonorgestrel), EE (10 μg ethinyl estradiol), or LNG/EE (20 μg LNG and 10 μg EE combination), and VEH rats received the same volume of vehicle. Drugs were suspended in 5% ethanol in organic sesame oil, and 0.1 ml were given daily through subcutaneous injection at the nape starting from day 1 until pup delivery for ~21 days. The same individuals handled the rats for injections, and experimenters were naive to drug conditions. The male and female offspring were separated from the dams on day 21 and fed until 9–10 weeks old for further experiments. Parts of 9- to 10-week-old offspring were then used for behavior tests, including autism-like and anxiety-like behavior testing. After that, the offspring were sacrificed, and the different brain tissues, including the amygdala, hypothalamus, and hippocampus, were isolated, flash frozen in dry ice, and then stored in a −80 °C freezer for the analysis of gene expression, SOD2 activity, superoxide anion release, DNA damage, mitochondrial function, and fatty acid metabolism. In addition, other parts of the offspring were used for the isolation of primary amygdala neurons.

The serum testosterone level of mice was determined using a competitive binding Testosterone Parameter Assay Kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions with testosterone as the standard. The minimum detectable dose of testosterone for the ELISA kit was approximately 0.03 ng/mL. The following compounds were tested for their cross-reactivity (testosterone cross-reactivity was set as 100%): DHT (2.6%), AD (<0.1%), 17β-estradiol (<0.1%), and progesterone (<0.1%). On the other hand, the serum estradiol level of mice was determined using a competitive binding Estradiol Parameter Assay Kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions with 17β-estradiol as the standard. The minimum detectable dose of estradiol was approximately 5 pg/mL. The following compounds were tested for cross-reactivity (17β-estradiol cross-reactivity was set as 100%): estrone (0.26%), estriol (0.86%), 17α-ethinylestradiol (<0.1%), and progesterone (<0.1%).

Denitrifying strain GDN1 (500 mL, initial OD_600 nm = 0.2) was incubated at 37°C and pH 6.0 with testosterone (1 mM) and nitrate (10 mM) in a chemically defined medium as described elsewhere^{31 (link)}. 17α-Ethinylestradiol (final concentration = 50 μM), which cannot be utilized by strain GDN1, was added to the bacterial culture to serve as an internal control. Cultural samples (5 mL each) were withdrawn every 8 h (0 ~ 48 h). The pH of the cultural samples (1 mL) was adjusted to pH < 2 using 5 M HCl. The acid-treated cultures were extracted three times with the same volume of ethyl acetate to recover the residual testosterone and its derivatives from the aqueous phase. The testosterone-derived intermediates extracted from the cultural samples were identified and quantified using UPLC – HRMS. The protein and nitrate contents of strain GDN1 cultures were determined as described below. The remaining cultural samples (1 mL) were extracted with ethyl acetate three times. After the solvent was completely evaporated, the residue was re-dissolved in 10 μL of dimethyl sulfoxide (DMSO) to determine its androgenic activity (see below).

Strain GDN1 was aerobically grown in a phosphate-buffered shake-flask (300 ml in 1 L-Erlenmeyer flask) containing 1 mM testosterone. 17α-Ethinylestradiol (50 μM; indigestible by strain GDN1) was added as an internal control. Nitrate was omitted from the medium. In 1 L of distilled water, the medium contained the following: 0.29 g testosterone, 2.0 g NH₄Cl, 0.5 g MgSO₄7 H₂O, and 0.1 g CaCl₂2 H₂O. After autoclaving, sterile 50 ml KH₂PO₄-K₂HPO₄ buffer solution (1 M, pH 6.0), vitamins (1 mL/L)^{54 (link)}, EDTA-chelated mixture of trace elements (1 mL/L)^{55 (link)}, and selenite and tungstate solution (1 mL/L)^{56 (link)} were added. The aerobic culture was incubated at 37°C in an orbital shaker (180 rpm). The cultural samples (5 mL) were withdrawn every 6 h (0 ~ 30 h). The testosterone-derived intermediates extracted from the cultural samples were identified and quantified using UPLC – HRMS The androgenic activity of the cultural samples was determined as described later.