Nicotine, Cotinine, and Anabasine Inhibit Aromatase in Human Trophoblast in Vitro
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Nicotine, cotinine, and anabasine inhibit aromatase in human trophoblast in vitro. R L Barbieri, … , J Gochberg, K J Ryan J Clin Invest. 1986;77(6):1727-1733. https://doi.org/10.1172/JCI112494. Research Article Epidemiologic studies suggest that women who smoke have lower endogenous estrogen than nonsmokers. To explore the possible link between cigarette smoking and decreased endogenous estrogens, we have examined the effects of constituents of tobacco on estrogen production in human choriocarcinoma cells and term placental microsomes. In choriocarcinoma cell cultures, nicotine, cotinine (a major metabolite of nicotine), and anabasine (a minor component of cigarette tobacco) all inhibited androstenedione conversion to estrogen in a dose-dependent fashion. Removal of nicotine, cotinine, and anabasine from the culture medium resulted in the complete reversal of the inhibition of aromatase. In the choriocarcinoma cell cultures, a supraphysiologic concentration of androstenedione (73 microM) in the culture medium blocked the inhibition of aromatase caused by nicotine, cotinine, and anabasine. In preparations of term placental microsomes, nicotine, cotinine, and anabasine inhibited the conversion of testosterone to estrogen. Kinetic analysis demonstrated the inhibition to be competitive with respect to the substrate. These findings suggest that some nicotinic alkaloids directly inhibit aromatase. This mechanism may explain, in part, the decreased estrogen observed in women who smoke. Find the latest version: https://jci.me/112494/pdf Nicotine, Cotinine, and Anabasine Inhibit Aromatase in Human Trophoblast In Vitro Robert L. Barbieri, Jan Gochberg, and Kenneth J. Ryan Laboratory of Human Reproduction and Reproductive Biology, Department of Obstetrics and Gynecology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115 Abstract constituents of tobacco on estrogen production in human cho- nocarcinoma cells and term placental microsomes. Epidemiologic studies suggest that women who smoke have lower endogenous estrogen than nonsmokers. To explore the possible Methods link between cigarette smoking and decreased endogenous es- Reagents. All radioactive materials were obtained from New England trogens, we have examined the effects of constituents of tobacco Nuclear (Boston, MA). RPMI 1640 and newborn calf serum were ob- on estrogen production in human choriocarcinoma cells and term tained from Gibco (Grand Island, NY). All other reagents were obtained placental microsomes. from Sigma Chemical Co. (St. Louis, MO). In choriocarcinoma cell cultures, nicotine, cotinine (a major Jar choriocarcinoma cell cultures. Jar choriocarcinoma cells were metabolite of nicotine), and anabasine (a minor component of obtained from Dr. R. A. Pattillo, Department of Obstetrics and Gyne- cigarette tobacco) all inhibited androstenedione conversion to cology, The Medical College of Wisconsin, Milwaukee, WI (9). The feeder estrogen in a dose-dependent fashion. Removal of nicotine, co- cell lines were cultured in plastic flasks in RPMI 1640 with 12% calf tinine, and anabasine from the culture medium resulted in the serum, 40 mM glutamine, 100 U/ml penicillin, and 100 gg/ml strep- tomycin. the cell number doubled every complete reversal of the inhibition of.aromatase. In the chorio- Under these conditions, 2 d. Replicate cultures for experimentation were obtained by trypsinizing carcinoma cell cultures, a supraphysiologic concentration of an- the cells in the feeder flasks and replating these cells at a density of -2.0 drostenedione (73 ,M) in the culture medium blocked the in- x 105 cells/well in a multiwell plate with a well diameter of 16 mm. The hibition of aromatase caused by nicotine, cotinine, and anabasine. cells were allowed to explant in the new wells for 16 h before experi- In preparations of term placental microsomes, nicotine, co- mentation. To begin all experiments the culture media were replaced tinine, and anabasine inhibited the conversion of testosterone to with RPMI 1640 without serum, and the experimental agents were then estrogen. Kinetic analysis demonstrated the inhibition to be added in a vehicle consisting of RPMI 1640. At the end of the experi- competitive with respect to the substrate. These findings suggest mental culture period the media were frozen, and the cells trypsinized that some nicotinic alkaloids directly inhibit aromatase. This and counted in a hemocytometer. mechanism may explain, in part, the decreased estrogen observed Assessment of estrogen production in Jar choriocarcinoma in women who smoke. cell cultures The media was extracted two times with 7 vol of ether. The ether extract Introduction was dried under a stream of N2, redissolved in isooctane, and applied to celite chromatography columns. Using isooctane and ethyl acetate as the mobile phase, progesterone, androstenedione, dihydrotestosterone, Recent epidemiologic evidence suggests that women who smoke testosterone, estrone, and estradiol were sequentially eluted from the have decreased endogenous estrogens (1). For example, endo- column (10, 1). Steroids were then measured using radioimmunoassays metrial cancer, which is thought to be an estrogen-dependent employing specific antibodies. Nicotine, cotinine, and anabasine did not process, occurs less frequently in women who smoke than in interact with the antibodies used in the radioimmunoassays. Tritiated nonsmokers (2). MacMahon et al. (3) have reported that in nor- steroids were added to the samples just before ether extraction to assess mally cycling women who smoke, urinary estrogens in the luteal recovery. phase of the cycle are lower than in nonsmokers. In pregnancy, Placental microsomal preparations. Human term placentas were ob- women who smoke have lower estrogens than nonsmokers (4). tained at cesarean section. Placental microsomes were prepared as pre- viously described (12). Microsomal protein concentration was measured Circumstantial evidence that women who smoke have disturbed using the method of Bradford ( 13). endogenous estrogen production and/or metabolism is provided by epidemiologic studies which demonstrate that women who Assessment ofaromatase activity in human term smoke have an earlier age of menopause (5, 6) and are at greater placental microsomes risk for postmenopausal osteoporosis (7, 8) than nonsmokers. Aromatase activity was assessed in the placental microsomes by measuring To explore the possible link between cigarette smoking and de- the amount of 3H2O formed due to the release of 3H during the aro- creased endogenous estrogens, we have examined the effects of matization of [1 1,,2,B-3H]testosterone (14). The reaction mixture consisted of varying concentrations of [1I ,2,3-3H]testosterone (48.3 Ci/mmol, sp act), 0.15 M NaCl, 10 mM P04 buffer, pH 7.4, at 37°C, 500MuM NADPH, Address reprint requests to Dr. Barbieri, Laboratory of Human Repro- 50 mM glucose-6-phosphate, and 10 U/ml of glucose-6-phosphate de- duction, Harvard Medical School, 45 Shattuck St., Boston, MA 02115. hydrogenase, in a final volume of 0.5 ml. The reaction was started by Receivedfor publication 10 December 1985. adding the placental microsomes. The reactions were carried out in room air at 37°C in triplicate for 3 and 6 min to assess linearity of reaction J. Clin. Invest. velocity. The reaction was terminated by adding 0.6 ml of dextran-coated © The American Society for Clinical Investigation, Inc. charcoal. The charcoal-treated samples were incubated for 30 min and 0021-9738/86/06/1727/07 $ 1.00 then centrifuged at 3,000 g for 10 min. The quantity of 3H20 in the Volume 77, June 1986, 1727-1733 aqueous supernatant was assessed by liquid scintillation counting. Nicotine, Cotinine, and Anabasine Inhibit Aromatase 1727 Assessment of 1 7f-hydroxysteroid dehydrogenase activity 2.5 X I05 cells/well). In serum-free choriocarcinoma cell cultures, in human term placental microsomes estradiol and estrone were not detectable (<0.05 pmol/ml) unless 17#-Hydroxysteroid dehydrogenase was assayed as previously described androgen substrate (androstenedione or testosterone) was added (15). In brief, the reaction mixture consisted of 0.15 M KCI, 50 mM to the medium. Androstenedione in concentrations as low as 2 Tris, pH 7.4, at 37°C, 500 AM NADPH, 50 mM glucose-6-phosphate, nM resulted in the significant accumulation of estradiol. Max- 5 U glucose-6-phosphate dehydrogenase, 5 mM MgC12, -0.5 MCi of imal estradiol accumulation occurred at androstenedione con- [6,7-3H]estrone (45 Ci/mmol, sp act), 10 ,g placental microsomal protein, centrations between 400 nM and 1 AM. In women, androstene- and various concentrations of nicotine in a final volume of 0.5 ml. The dione concentrations in the circulation ranged between 0.3 nM reaction mixture and the microsomal protein were preincubated sepa- rately at 37°C. The reaction was started by the addition of 0.1 ml of and 11 nM. Therefore, in most incubations the androstenedione microsomal protein to the reaction mixture. Samples were incubated in concentration used was 11 nM (19). Progesterone and estradiol duplicate for 3 and 6 min to verify linearity of reaction velocity. After were not rapidly metabolized by the choriocarcinoma cells. Ad- incubation, the reaction was stopped by the addition of 0.5 ml methanol. dition of 1 MCi of [1,2-3H]progesterone or [6,7-3H]estradiol to An aliquot of the reaction mixture was applied to silica gel thin-layer 2 X I05 choriocarcinoma cells for 24 h resulted in the conversion plates and product (estradiol) and substrate (estrone)