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Total liver RNA was extracted by the guanidium thiocyanate-phenol-chloroform method. cDNA was synthesized for 20 minutes at 50°C from 1 µg of RNA with Moloney murine leukemia virus reverse transcriptase. The transcribed cDNA was amplified by 30 cycles of PCR for ERα (5′-CGG CTG CCA CTT ACC TGG GAG CTC TCA GAT-3′ and 5′-GGG GAG CCT GGG AGC TCT CAG AT-3′), ERβ (5′-TCT CTG AGA GCA TCATGT CC-3′ and 5′-CAG CCT GGC CGT CAC TGT GA-3′), and hypoxanthine phosphoribosyltransferase (5′-GTA ATG ATC AGT CAA CGG GGG AC-3′ and 5′-CCA GCA AGC TTG CAA CCT TAA CCA-3′).[@b24-ceg-8-175],[@b30-ceg-8-175],[@b31-ceg-8-175]

Surgeries (PHs, orchiectomies, and OVXs)
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Liver resection of the left and median lobes was performed following midventral laparotomy between 8 am and 11 am under isoflurane anesthesia as previously described.[@b23-ceg-8-175],[@b32-ceg-8-175] The bilateral OVX procedure was performed as follows: the mice were anesthetized with peritoneal injections of pentobarbital, one central lateral incision was made in the skin, and two lateral incisions were made in the muscle layer, and the ovaries were extracted through the incision and excised after ligation.

Animal study compliance
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All experiments were performed in accordance with the ethical guidelines for animal care of the National Center for Geriatrics and Gerontology (NCGG). The experimental protocols were approved by the Animal Care Committee of the NCGG. All the surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize the suffering of the animals. E2 injections were decreased from five repetitions to a single injection to decrease the suffering of the sacrificed and operated mice.

Hep G2 cell culture
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Hep G2 cells from a human liver carcinoma were maintained in α-minimal essential medium (11900-073; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal calf serum (FCS, CC3008-504, Ce