A Simple and Sensitive Microtiter Plate Estrogen Bioassay Based on Stimulation of Alkaline Phosphatase in Ishikawa Cells: Estrogenic Action of Δ⁵Adrenal Steroids*

Abstract

We have developed an estrogen bioassay using the Ishikawa human endometrial adenocarcinoma cell line growing in 96-well microtiter plates. Alkaline phosphatase enzyme activity (AlkP) in these cells is markedly stimulated by estrogens, and this enzyme can be easily quantified in situ using a chromogenic substrate. These cells are very sensitive to estrogens; estradiol induces AlkP at levels as low as 10(-12) M. Antiestrogens completely block the action of estradiol. Various estrogens stimulate AlkP with potencies comparable to those achieved in vivo. The induction of AlkP is specific for estrogens; no other type of steroid, including androgens, progestins, mineralocorticoids, or glucocorticoids produce this effect. The stimulation of AlkP in Ishikawa cells is specific for estrogens, is highly reproducible and sensitive, and permits large numbers of samples to be assayed with ease. We have used this assay to investigate the estrogenic action of the adrenal delta 5-3 beta-hydroxysteroids. While pregnenolone is inactive, dehydroepiandrosterone and its sulfate ester induce AlkP slightly. However, the C19 steroid, 5-androstene-3 beta, 17 beta-diol is considerably more estrogenic in this assay, although it stimulates Ishikawa AlkP with a potency of 1/30,000 that of estradiol. The stimulation by 5-androstene-3 beta,17 beta-diol is inhibited by antiestrogens, but it is not blocked by the delta 5-3 beta-hydroxysteroid isomerase/dehydrogenase inhibitor, cyanoketone, or by the aromatase inhibitor, 4-hydroxy-androstenedione. Thus, neither conversion to a delta 4-3-ketone nor aromatization is required for the action of this unusual estrogen.

Estrogen actions on mitochondria—Physiological and pathological implications

“…At high levels of excitotoxic stimulation, however, estrogens prevented both cytosolic and mitochondrial influx of Ca 2+ (Wang et al, 2001;Brinton, 2003, Nilsen et al, 2002;Wang et al, 2006), presumable providing a protection from neurotoxic Ca 2+ influx. Comparable effects on mitochondrial stability and function have been reported by Morin et al (2002) who studied the ability of 17 α-estradiol, an isomer of 17 β-estradiol that is equipotent as a cytoprotectant yet at least 200-fold less active than as a hormone (Littlefield et al, 1990) to maintain respiratory coupling after imposed ischemia reoxygenation.…”

Alteration of maternal Hoxa10 expression by in vivo gene transfection affects implantation

“…Ishikawa cells, a well differentiated human endometrial adenocarcinoma cell line 11,12,26,27 (a generous gift from R Hochberg, Yale University, New Haven, CT, USA) were grown to 80% confluence in Minimum Essential Media with Earles Salts and l-glutamine (MEM) (Life Technologies), 10% fetal bovine serum, 1% sodium pyruvate, 1% penicillin and 1% streptomycin. A 25 cm 2 cellular monolayer was transfected with 3 ml of a DNA/liposome complex for 5 h, washed in PBS and maintained in MEM as above, for an additional 19 h. For RNA extraction, cells were lysed in 1 ml Trizol (Life Technologies).…”

Determination of Estrogenic Substances in the Water of Muko River Using in Vitro Assays, and the Degradation of Natural Estrogens by Aquatic Bacteria

“…In many countries, water pollution has been caused by both domestic and industrial chemicals, and many estrogenic substances have been loaded into rivers through sewage treatment plants. 1) In the present study, three types of in vitro assay [E-Screen, [2][3][4] Ishikawa cell-alkaline phosphatase (Ishikawa cell-ALP), 5,6) and yeast estrogen screen (YES) assay 7) ] were used to examine the estrogenic substances in an aquatic environment. The human breast cancer cell T-47D has an estrogen receptor and proliferates in the presence of estrogenic substances in an E-Screen assay.…”