Nicotine, cotinine, and anabasine inhibit aromatase in human trophoblast in vitro.

In choriocarcinoma cell cultures, a supraphysiologic concentration of androstenedione (73 μM) blocks inhibition of aromatase caused by nicotine, cotinine, and anabasine.

“In the chorio- carcinoma cell cultures, a supraphysiologic concentration of an- drostenedione (73 ,M) in the culture medium blocked the in- hibition of aromatase caused by nicotine, cotinine, and anabasine.”

• Also stated in: § Effects ofnicotine, cotinine, and anabasine on estrogen accumulation in choriocarcinoma cell cultures · plain

  “when the culture medium contained 73 1tM androstenedione, 30 ,M nicotine produced no inhibition of estradiol accumulation (Fig. 4).”

Aqueous extracts of cigarette smoke inhibit androstenedione conversion to estradiol in a dose-dependent manner in choriocarcinoma cell cultures.

“Aqueous extracts ofsmoke inhibited androstenedione conversion to estradiol in a dose-dependent manner (Fig. I A).”

Nicotine inhibits estradiol accumulation at the first timepoint examined, 1 h.

“Examination of the timecourse of nicotine inhibition of estradiol accumulation demonstrated that nicotine inhibited es- tradiol accumulation at the first timepoint examined, 1 h (Fig. 3).”

At concentrations up to 150 μM, nicotine, cotinine, and anabasine do not alter cell number at the end of an 8-h incubation.

“At con- centrations up to 150 ,uM, nicotine, cotinine, and anabasine did not alter cell number at the end of an 8-h incubation.”

At concentrations in the range 0.3-2 mM, carbamylcholine, succinylcholine, hexamethonium, and decamethonium do not inhibit estradiol accumulation in choriocarcinoma cells.

“In contrast, at concentrations in the range offoot_2 mM, carbamylcholine, succinylcholine, hexamethonium, and decamethonium did not inhibit estradiol accumulation (Table III).”

When culture medium contains 73 μM androstenedione, 30 μM nicotine produces no inhibition of estradiol accumulation.

“In contrast, when the culture medium contained 73 1tM androstenedione, 30 ,M nicotine produced no inhibition of estradiol accumulation (Fig. 4).”

Nicotine inhibits estradiol accumulation in cell-free preparations in a manner comparable to intact cells.

“Nicotine in- hibited the accumulation of estradiol in a manner comparable to that observed in incubations of intact cells (data not shown).”

Apparent Ki values for cotinine inhibition of aromatization in placental microsomes from three women were 15, 36, and 47 μM (mean 32.7±9.38 μM).

“In these three separate experiments the Kis for cotinine inhibition of aroma- tization were 15, 36, and 47 MuM (32.7±9.38 MM, mean±SEM) (Fig. 6).”

Dialyzed smoke preparations (1,000 mol wt cutoff) inhibit androstenedione conversion to estradiol in a dose-dependent manner.

“Dialyzed smoke preparations (1,000 mol wt effective pore size) inhibited androstenedione conversion to estradiol in a dose- dependent manner (Fig. 1 B).”

In choriocarcinoma cell cultures, nicotine, cotinine, and anabasine inhibit androstenedione conversion to estrogen in a dose-dependent manner.

“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.”

• Also stated in: § Effects ofnicotine, cotinine, and anabasine on estrogen accumulation in choriocarcinoma cell cultures · plain

  “The addition of nicotine, cotinine, or anabasine to the chorio- carcinoma cell cultures inhibited estradiol accumulation in the media (Fig. 2).”

Removal of nicotine, cotinine, and anabasine from the culture medium completely reverses inhibition of aromatase in choriocarcinoma cells.

“Removal of nicotine, co- tinine, and anabasine from the culture medium resulted in the complete reversal of the inhibition of.aromatase.”

• Also stated in: § Effects ofnicotine, cotinine, and anabasine on estrogen accumulation in choriocarcinoma cell cultures · plain

  “Removal of nicotine, cotinine, and anabasine from the culture medium resulted in the complete reversal of the inhibition of estradiol accumulation and the return to normal rates of estradiol accu- mulation.”

Nicotine at concentrations as high as 100 μM does not significantly alter placental microsomal 17β-hydroxysteroid dehydrogenase activity.

“Nicotine, at concentrations as high as 100 MM, did not signifi- cantly alter placental microsomal 17j3-hydroxysteroid dehydrogenase activity (Table V).”

Addition of nicotine to placental microsomal preparations elicits a type II cytochrome P-450 difference spectrum with a broad Soret minimum between 390 and 400 nm and a Soret maximum at 428 nm.

“Addition of nicotine to placental microsomal preparations elicited a type II cytochrome P-450 difference spec- trum with a broad Soret minimum between 390 and 400 nm and a Soret maximum at 428 nm (Fig. 7).”

50% inhibition of aromatase by aqueous smoke extract occurs at a dose between 1 and 5 μl.

“50% inhibition ofaromatase was observed at a smoke extract dose between 1 and 5 Ml (1 Ml of aqueous smoke solution represents 0.1% of a cigarette equivalent).”

Kinetic analysis demonstrates that inhibition of placental microsomal aromatase by nicotine, cotinine, and anabasine is competitive with respect to testosterone substrate.

“Kinetic analysis demonstrated the inhibition to be competitive with respect to the substrate.”

• Also stated in: § Effects ofnicotine, anabasine, and cotinine on aromatase activity in preparations ofterm placental microsomes · boosted

  “Dixon plot analysis demonstrated that nicotine, cotinine, and anabasine inhibition of aromatase was competitive with respect to the testosterone substrate (Figs. 5 and 6).”

Progesterone and estradiol are not rapidly metabolized by choriocarcinoma cells.

“Progesterone and estradiol were not rapidly metabolized by the choriocarcinoma cells.”

At concentrations up to 30 μM, nicotine, cotinine, and anabasine do not inhibit progesterone accumulation.

“At concentrations up to 30 uM these three compounds did not inhibit progesterone accumulation (Fig. 2).”

The concentration of androstenedione in culture medium strongly influences the degree of inhibition of estradiol accumulation produced by nicotine.

“In the choriocarcinoma cell cultures, the concentration of androstenedione present in the culture medium strongly influenced the degree of inhibition of estradiol accumulation pro- duced by nicotine.”

In term placental microsomes, nicotine, cotinine, and anabasine inhibit conversion of testosterone to estrogen.

“In preparations of term placental microsomes, nicotine, co- tinine, and anabasine inhibited the conversion of testosterone to estrogen.”

• Also stated in: § Effects ofnicotine, anabasine, and cotinine on aromatase activity in preparations ofterm placental microsomes · plain

  “In all preparations of placental microsomes examined, nicotine, cotinine, and anabasine inhibited the conversion of [ 1(,2,3-3H]testosterone to 3H20.”

Nicotine also inhibits estradiol accumulation in cell-free preparations of choriocarcinoma cells.

“In addition to inhibiting estradiol accumulation in intact choriocarcinoma cell cultures, nicotine also inhibited estradiol accumulation in cell-free preparations ofchoriocarcinoma cells.”

At the highest dose tested (25 μl), dialyzed smoke preparation inhibits aromatization by 40%.

“At the highest dose tested, 25 ,l, aromatization was inhibited by 40%.”

At all nicotine concentrations tested, the estradiol to estrone ratio exceeds 6:1.

“At all nicotine concentrations tested, the estradiol to estrone ratio exceeded 6:1.”

When culture medium contains 11 nM androstenedione, 30 μM nicotine produces approximately 50% inhibition of estradiol accumulation.

“when the culture medium con- tained 11 nM androstenedione, 30,M nicotine produced an -50% inhibition of estradiol accumulation.”

150 μM concentrations of anabasine and nicotine result in a decrease in progesterone accumulation of approximately 15%.

“150-!LM concentrations of both anabasine and nicotine resulted in a decrease in progesterone accumulation of -15% (Fig. 2).”

Removal of aqueous extract of cigarette smoke from culture medium completely reverses inhibition of aromatase.

“Removal of the aqueous extract ofcigarette smoke from the culture medium resulted in the complete reversal ofthe inhibition of aromatase, and the return of normal rates of aromatization.”

Androstenedione at concentrations as low as 2 nM results in significant accumulation of estradiol in choriocarcinoma cell cultures.

“Androstenedione in concentrations as low as 2 nM resulted in the significant accumulation of estradiol.”

Dixon plot analysis demonstrates that nicotine, cotinine, and anabasine inhibition of aromatase is competitive with respect to testosterone substrate.

“Dixon plot analysis demonstrated that nicotine, cotinine, and anabasine inhibition of aromatase was competitive with respect to the testosterone substrate (Figs. 5 and 6).”

In all preparations of placental microsomes examined, nicotine, cotinine, and anabasine inhibit conversion of [1β,2β-3H]testosterone to 3H2O.

“In all preparations of placental microsomes examined, nicotine, cotinine, and anabasine inhibited the conversion of [ 1(,2,3-3H]testosterone to 3H20.”

Addition of 18 nM androstenedione to 200,000 choriocarcinoma cells for 18 h results in detection of 12.6 nM estradiol, 1.26 nM estrone, 25.3 nM progesterone, and <0.2 nM testosterone and dihydrotestosterone in media.

“For example, the addition of 18 nM androstenedione to 200,000 choriocarcinoma cells for 18 h resulted in the detection of 12.6 nM estradiol, 1.26 nM estrone, 25.3 nM progesterone, and <0.2 nM testosterone and dihydrotestosterone in the media at the end of the incubation (Table I).”

Cholinergic agents hexamethonium, decamethonium, acetylcholine, succinylcholine, carbamylcholine, and muscarine do not inhibit aromatase in placental microsomes.

“In contrast to nicotine, cotinine, and anabasine, the cholin- ergic agents-hexamethonium, decamethonium, acetylcholine, succinylcholine, carbamylcholine, and muscarine-did not in- hibit aromatase in the placental microsomes (Table IV).”

Apparent Ki values for nicotine inhibition of aromatization in placental microsomes from five women were 15, 17, 25, 37, and 73 μM (mean 33±10.6 μM).

“In these five separate experiments the apparent enzymatic inhibition constants (K,)' for nicotine inhibition of aromatization were 15, 17, 25, 37, and 73 ,M (33±10.6MuM, mean±SEM) (Fig. 5).”

PBS vehicle up to 30 μl does not affect estradiol accumulation.

“The PBS vehicle, in doses up to 30 ,l, did not affect estradiol accumu- lation.”

In serum-free choriocarcinoma cell cultures, estradiol and estrone are not detectable unless androgen substrate is added.

“In serum-free choriocarcinoma cell cultures, estradiol and estrone were not detectable (<0.05 pmol/ml) unless androgen substrate (androstenedione or testosterone) was added to the medium.”

Maximal estradiol accumulation occurs at androstenedione concentrations between 400 nM and 1 μM.

“Maximal estradiol accumulation occurred at androstenedione concentrations between 400 nM and 1 AM.”

Nicotine inhibition of estradiol accumulation is linear over the first 8 h of incubation.

“Nicotine inhibition ofestradiol accumulation was linear over the first 8 h of incubation (Fig. 3).”

Apparent Ki values for anabasine inhibition of aromatase in placental microsomes from two women were 2 and 3 μM.

“In these two experiments the apparent Kis for anabasine inhibition of aromatase were 2 and 3 MM.”

Lobeline at 100 μM produces 30% inhibition of placental microsomal aromatase.

“Lobe- line, at concentrations of 100 MM, produced a 30% inhibition of placental microsomal aromatase.”

Approximately 10% of the aromatase inhibitory activity of aqueous extracts of cigarette smoke is present in the >1,000-mol-wt fraction.

“Therefore, -10% of the aromatase inhibitory activity of aqueous extracts of cigarette smoke was present in the > I,000-mol-wt fraction.”

In pregnancy, women who smoke have lower estrogens than nonsmokers.

“In pregnancy, women who smoke have lower estrogens than nonsmokers (4).”

• cites [4] 4. Mochizuki, M., T. Maruo, K. Masuko, and T. Ohtsu. 1984. Effects of smoking on fetoplacental-maternal system during pregnancy. Am. J. Obstet. Gynecol. 149:413-420. sourceNot checked — no full text of the source

In nonpregnant women who smoke, plasma nicotine and cotinine concentrations are in the range of 0.25 and 2 μM, respectively.

“In nonpregnant women who smoke, plasma nicotine and cotinine concentrations are in the range of 0.25 and 2 ,M, re- spectively (22)(23)(24).”

• cites (22)(23)(24)Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

This mechanism may explain, in part, the decreased estrogen observed in women who smoke.

“This mechanism may explain, in part, the decreased estrogen observed in women who smoke.”

• Also stated in: § Discussion · hedged

  “the in vitro findings reported here may have relevance in vivo.”

• AssumptionThe authors state this as an assumption: The aromatase enzyme system in human granulosa cells is similar to placental aromatase.
• AssumptionRests on an assumption: The concentrations of nicotine and cotinine in body fluids reflect their concentrations at the aromatase enzyme site in ovarian or placental tissues.

It is unlikely that nicotine inhibition of estradiol accumulation in choriocarcinoma cell cultures is due to a nonspecific detrimental effect on cell function.

“It is unlikely that the nicotine inhibition of estradiol accumulation observed in the choriocarcinoma cell cultures is due to a”

In women, circulating androstenedione concentrations range between 0.3 nM and 11 nM.

“In women, androstene- dione concentrations in the circulation ranged between 0.3 nM and 11 nM.”

• cites [19] 19. C. F. Chignell, editor. Appleton Century Crofts, NY. 303-350.Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Progesterone and estradiol accumulation in media correlate well with production.

“These results suggest that progesterone and estradiol accumulation in the media correlate well with production.”

Women who smoke have an earlier age of menopause than nonsmokers.

“women who smoke have an earlier age of menopause (5,6)”

• cites [5] 5. Jick, H., J. Porter, and A. S. Morrison. 1977. Relation between smoking and age of natural menopause. Lancet. i: 1354-1355.Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [6] 6. Willett, W., M. J. Stampfer, C. Bain, R. Lipnick, F. E. Speizer, B. Rosner, D. W. Cramer, and C. H. Hennekens. 1983. Cigarette smoking, relative weight and menopause. Am. J. Epidemiol. 117:651-658. sourceNot checked — no full text of the source

MacMahon et al. reported that normally cycling women who smoke have lower urinary estrogens in the luteal phase than nonsmokers.

“MacMahon et al. (3) have reported that in nor- mally cycling women who smoke, urinary estrogens in the luteal phase of the cycle are lower than in nonsmokers.”

• cites [3] 3. MacMahon, B., D. Trichopoulos, P. Cole, and J. Brown. 1982. Cigarette smoking and urinary estrogens. N. EngI. J. Med. 307:1062- 1065. sourceNot checked — no full text of the source

Nicotine concentrations in the hair of smokers have been reported to be 100 μM.

“Nicotine con- centrations in the hair of smokers has been reported to be 100 ,uM (27, 28).”

• cites [27] 27. Sasson, I. M., N. J. Haley, D. Hoffman, E. L. Wynder, D. Hellberg, and S. Nilsson. 1985. Cigarette smoking and neoplasia of the uterine cervix: smoke constituents in cervical mucus. N. Engl. J. Med. 312:315- 316. sourceNot checked — no full text of the source
• cites [28] 28. Ishiyama, I., T. Nagai, and S. Toshida. 1983. Detection of basic drugs from human hair. J. Forensic Sci. 28:380-385. sourceNot checked — no full text of the source

Some nicotinic alkaloids directly inhibit aromatase.

“These findings suggest that some nicotinic alkaloids directly inhibit aromatase.”

• Also stated in: § Discussion · hedged

  “the experiments examining the effects of nicotine on aromatase in term placental microsomes suggests that nicotine directly inhibits the choriocarcinoma cell aromatase enzyme system.”

Epidemiologic evidence suggests that women who smoke have decreased endogenous estrogens.

“Recent epidemiologic evidence suggests that women who smoke have decreased endogenous estrogens (1).”

• cites [1] 1. Baron, J. A. 1984. Smoking and estrogen-related disease. Am. J. Epidemiol. 119:9-22. sourceNot checked — no full text of the source

Experiments examining effects of nicotine on aromatase in term placental microsomes suggest that nicotine directly inhibits the choriocarcinoma cell aromatase enzyme system.

“However, the experiments examining the effects of nicotine on aromatase in term placental microsomes suggests that nicotine directly inhibits the choriocarcinoma cell aromatase enzyme system.”

Women who smoke are at greater risk for postmenopausal osteoporosis than nonsmokers.

“and are at greater risk for postmenopausal osteoporosis (7,8) than nonsmokers.”

• cites [7] 7. Daniell, H. W. 1976. Osteoporosis of the slender smoker. Arch. Intern. Med. 136:298-304. sourceNot checked — no full text of the source
• cites [8] 8. Williams, A. R., N. S. Weiss, C. L. Ure, J. Ballard, and J. R. Daling. 1982. Effect of weight, smoking and estrogen use on the risk of hip and forearm fractures in postmenopausal women. Obstet. Gynecol. 60:695-699.Not checked yet

  Not checked in the free run.

Nicotine may inhibit placental aromatase by reversibly altering the function of the active site(s) of the cytochrome P-450 component of the aromatase enzyme system.

“This finding, in conjunction with the spectral evidence that nicotine interacted directly with placental microsomal cytochrome P-450, suggests that nicotine may inhibit placental aromatase by reversibly altering the function of the active site(s) of the cytochrome P-450 component of the aromatase enzyme system.”

Nicotine inhibition of estradiol accumulation is due to a highly specific mechanism.

“Evidence to suggest that nicotine inhibition ofestradiol accumulation was due to a highly specific mechanism includes the following observations:”

Salivary nicotine concentrations of 1 μM have been reported.

“salivary nicotine concentrations of 1 gM (25)”

• cites [25] 25. Herning, R I., R. T. Jones, N. L. Benowitz, and A. H. Mines. 1983. How a cigarette is smoked determines blood nicotine levels. Clin. Pharmacol. Ther. 33:84-90. sourceNot checked — no full text of the source

Cervical mucus nicotine concentrations of 10 μM have been reported.

“cervical mucus nicotine concentrations of 10 tsM have been reported (26).”

• cites [26] 26. Russell, M. A. H., and C. Feyerabend. 1978. Cigarette smoking: a dependence on high-nicotine boli. Drug Metab. Rev. 8:29-57. sourceNot checked — no full text of the source

In the follicular phase of the menstrual cycle, the dominant ovarian follicle produces the majority of circulating estrogens.

“In the follicular phase ofthe menstrual cycle, the ovarian follicle that gains dominance produces the majority of circulating es- trogens (29).”

• cites [29] 29. Haley, N. J., and D. Hoffman. 1985. Analysis for nicotine and cotinine in hair to determine cigarette smoker status. Clin. Chem. 31: 1598-1600. sourceNot checked — no full text of the source

In pregnant women, cotinine concentration in the fetal circulation is 2-4-fold higher than in the maternal circulation.

“In pregnant women the cotinine concentration in the fetal circulation is 2-4-fold higher than the cotinine concen- tration in the maternal circulation (4).”

• cites [4] 4. Mochizuki, M., T. Maruo, K. Masuko, and T. Ohtsu. 1984. Effects of smoking on fetoplacental-maternal system during pregnancy. Am. J. Obstet. Gynecol. 149:413-420. sourceNot checked — no full text of the source

In postmenopausal women, circulating androstenedione concentrations less than 2 nM are common.

“In postmenopausal women, circulating androstenedione concentrations less than 2 nM are common (18).”

• cites [18] 18. Estabrook, R. W., J. Peterson, J. Baron, and A. Hildebrant. 1972. A spectrometric measurement of turbid suspensions of cytochromes as- sociated with drug metabolism. In Methods in Pharmacology, Vol. II.Not checked yet

  Not checked in the free run.

The concentrations of nicotine and cotinine reported in body fluids of women who smoke suggest that the in vitro findings may have relevance in vivo.

“However, the concentrations of nicotine and cotinine that have been re- ported to be present in the body fluids of women who smoke suggests that the in vitro findings reported here may have relevance in vivo.”

• AssumptionRests on an assumption: The concentrations of nicotine and cotinine in body fluids reflect their concentrations at the aromatase enzyme site in ovarian or placental tissues.

The aromatase enzyme system in the granulosa cell is probably exposed to the intrafollicular androstenedione concentration of 2.5 μM.

“The aromatase enzyme system in the granulosa cell is probably exposed to the intrafollicular concentration of androstenedione, 2.5 ,uM.”

In postmenopausal women, the ratio of nicotine plus cotinine to androstenedione at the peripheral aromatase site may be greater than 1,000:1.

“Therefore, in postmenopausal women, the ratio of nicotine plus cotinine to androstenedione at the peripheral aromatase site may be greater than 1,000 to 1.”