A reevaluation of estrogen status in postmenopausal women who smoke

A reevaluation of estrogen status in postmenopausal women who smoke Denise Lee Cassidenti, MD, Malcolm C. Pike, PhD, Ariel G. Vijod, MS, Frank Z. Stanczyk, PhD, and Rogerio A. Lobo, MD

Los Angeles, California Hypoestrogenism in postmenopausal smokers has been suggested as the mechanism for the observed decreased risk of endometrial cancer and increased risk of osteoporosis. We have prospectively studied a well-matched group of smokers and nonsmokers and have evaluated their estrogen levels and compared them with existing data from the literature. We conclude that increased adrenal activity resulting in increased androgens , mainly androstenedione, is seen in postmenopausal smokers but that estrogen levels are not decreased. We hypothesize that in nonusers, unlike in users of estrogen, smoking is not associated with changes in estrogen levels and that other mechanisms must be responsible for the epidemiologic observations seen. (AM J OBSTET GVNECOL 1992;166:1444-8.)

Key words: Smoking, postmenopausal women , hormonal status

Recent studies have shown that postmenopausal women who smoke have a decreased risk of endometrial cancer and an increased risk of osteoporosis. 1. 2 It has been assu med that the mechanism for these observations is profound hypoestrogenism, with levels of estrogen being lower in postmenopausal women who smoke. Our recent study" and data from others':" suggest th at among users of oral estrogen hepatic metabolism of estrogen is inc reased with smoking, which results in lower estrogen levels.>" However, we hypothesize here that smoking in nonusers of estrogen does not re sult in lower estrogen levels , and therefore other mechanisms for the epidemiologic findings need to be entertained. To this end, we have prospectivel y studied a well-matched population of postmenopausal wom en not taking estrogen who were either smokers or nonsmokers and have evaluated their hormonal status. We have also compar ed and combined ou r data with the findings of other investiga tors in the literature.

Material and methods Subjects. Thirty-eight postmenopausal women agreed to participate in thi s study in r espo nse to indi vidual letters sent to members of a retirement community in the Los Angeles area. All women had been postmenopausal for ~5 year s. None had received hormonal replacement therapy, prednisone, cortisone, or From the Division of R eproductive Endocrinology, Department of Obstetrics and Gynecology, and the Department of Preventive M edicine, University of Southern California School of M edicine. Supp orted by grant N o. CA 49432 from the Na tiona l Cancer Institute, Na tional Institut es of H ealth, B ethesda, M aryland. Receioedfor publi cation May 14,1 991 ; accepted Novem ber 7,1 991 . R eprint requests: R ogeno A . Lobo, MD, Department of Obstetrics and Gynecology, Women 's H ospital, R oom 1M2, LA C + USC M edical Center, 1240 N. Mission R oad, Los Angeles, CA 90033. 6/1/34907 1444

anticoagulants for 6 months before the study. All patients were healthy and had no prior history of cancer or chronic illness . Seventeen women were nonsmokers , and 21 were chronic smokers who reported smoking an average of 19.9 cigarettes per da y (Table 1). Protocol. Volunteers were studied in the morning after they had eaten their usual breakfast. Smokers did not smoke on the morning of the study before baseline testing (approximately 8 AM). Blood samples were drawn at baseline and 3 hours later. During these 3 hours smokers were instructed to smoke five of their usual brand of cigarettes. Sera obtained wer e separated and stored at - 20 0 until assayed. Values for tar and nicotine content on brands of cigarettes were obtained from the United States Federal Trade Commission Report." Assays. All samples were assayed for estrone, estradiol, the estrogen conjugate estrone glucuronide, androstenedione, dehydroepiandrosterone sulfate (DH EAS), cortisol, sex hormone-binding globulin (SHBG) , and non-SHBG-bound estradi ol. Estrone, estradiol, androstenedione , non-SHBGbound estradiol, and SHBG levels were measured in serum by previously valid ated assays.": " Estrone and androstenedione were measured by radioimmunoassay (RIA) after diethyl ether extraction and Celite chromatography. Estradiol was measured by RIA after extraction with ethyl acetate /hexane (4 : 6, vol/vol), by means of an iodinated estrone kit fr om Pantex (San ta Mon ica, Calif.), Tritiated estradiol was ad d ed to th e aliquot of serum in which non-SHBG-bound estradiol was measured . All labeled and unlabeled estradiol bound to SHBG was precipitated. The remaining labeled unbound and non-SHBG-bound estra d iol in the supernatant was counted and expressed as a percent of the total tritiated estradiol added. The resulting per-

Volume 166 Number 5

centage was multiplied by th e serum estradiol to dete r min e non-SGHB-bound estradiol in picograms /millilite r. SHBG was quantitated by determining its binding affinity to dihydrotestosterone in 0.1 ml of serum with the charcoal technique, whi ch u ses the temperature-d ependent properties of protein s. T he interassay coefficie n t of variation d et ermined in pooled serum was 13 % fo r es tro ne, 13% fo r estradiol, 12% for androste nedi o ne, 10% for non-SHBG-bound estradiol, a nd 13% for SHBG. Estrone glucuronide was me asured in an aliquot of 0.6 ml of ser u m combined with a n internal standard of approximately 1000 disintegrations /min tritiated estradiol glucuronide (Amersham Laboratories, Buckin ghamshire, England; specific activity 12.6 Ci/mmol) and dissolved in 0.1 ml of 0.2 m ol/ L sodium phosphate buffer, pH 6.8, to monitor procedural losses, then incu ba ted at 37° C for 30 minu tes. Two milliliters phosphate buffer is ad d ed to the aqu eo us la yer after extraction to remove unconjugated ste roids. To each sample 500 U of J3-glucuronidase (typ e Vll-A from Escherichia coli, Sigma C he m ical Co m pany , St. Louis) in 0.1 ml of phosphate buffer was ad d ed and in cubated 16 to 18 hours at 37 ° C . The h ydrolyzed estrogens were ext racted twice with 5 ml et hy l ace ta te / hexa ne (4: 6, vol/vol), After the organic solvents were evaporated , the extract was reconstituted and the estrone was measure d as previously described . All va lues a re corrected for molecular weight. The se nsitivity of the assay, defined as the smallest a mou nt of estrone glucuronide pe r RIA tube that reduces the number of counts pe r m inute of tritiated estrone gluc u ronid e bound at zero mass by 2 SD, was 3.9 pg pe r tube or 45 pg/ml. Accuracy of the RIA was established as follows. Known qu antities of authentic estrone glu curonide was added to aliq uo ts of pooled serum . Lin ear regression analysis of the estrone glucuronide m ea sured in pooled serum versu s th e amount of conjugated ste roid added was d one. A co r relatio n coe fficie nt of 0.99 was obtained. The specificity of the enzym e was 0.27% for co nj u ga ted sul fat es. The interassay coefficie nt of var iatio n for estrone glucu ro nid e was 10%. Se rum DHEAS was d etermined by RIA as previously d escribed.!"" Cortisol was measured by RIA after its extraction from serum (0.0 1 m l) with ethyl aceta te / hex ane (l: 1, vol/vol). The a nt ise r um was raised ag ainst co rtisol-3-(O-car boxymethy l)oxim e-bovine serum albu mi n (ICN/ RSL, Irvine, Ca lif.) and was used in conj u nctio n with tritiat ed cortisol (Du l' o n t NEN Research Pr oducts, Boston ; specific ac tivity 45 Ci/mmol). The antibo dy- bou n d co r tiso l was separated from the unbo u nd with dextran-coat ed charcoal. T he interassay coe fficien t o f variation fo r DHEAS and cortisol were 16.4 % and 12.1%, respectiv ely. T he ratio, androstenedione / estrone, was calculated as a measure of aromatase activity.

Smoking and hormonal status 1445

Table I. Characteristics of volu n teers population Nonsmokers

No. Age (yr) Weight (kg)

Height (m) Cigarettes (No.1 day) Tar (mg/ day) Nicotine (mg/ day)

17 72.3 :!: 2.3 62.9 :!: 9.3 1.59 :!: 0.04

o

o o

Smokers 68.8 64.1 1.60 19.9 204 14.9

21 :!: 3.9 ± 7.1 ± 0.07 ± 8.3 :!: 155 :!: I I.l

*Values are mean ± SD.

Statistical analyses. All hormonal measurements we re transformed to logarithmic values to approximate normal distributions for stati stical analysis. Group valu es are reported as geometric mean concentrations. St andard sta tistical methods of analysis of va ria nce (and covaria nce) were used in the a na lysis of th e results. All p values quoted are two-sid ed . Results

The cha racteristics of th e postmenopausal women sam pled are shown in T abl e I. The mean age was 72.3 years for th e nonsmokers and 68 .8 year s among smokers; th is was not statisticall y sign ificant. The groups were well matched for height an d weig ht. Smokers aver aged 19.9 cigarettes per day with an average tar of 204 mg per day a nd an averag e nicot ine of 14.9 mg per da y. The ge o me tric mean ho rmone levels o f the nonsmoker s and sm oker s are given in T able II. When sm ok ers a re compared with nonsm okers, estrone was 13 % lower at baseline and 6 % lower after smoking. These changes, however, were not statistically significant. Estrone glucuronide values were similar in smokers an d nonsmokers, being on ly 5% an d 4% greater be fo re a nd after smokin g. Estradi ol cha n ges were also not significant. They were 5 % grea ter an d 1% less befo re a nd aft er smoking. SH BG was 22 % lower at 8 AM and I 1% lower at 11 AM in smoke rs, but this also was no t statistically sig n ifica nt. Bioavailabl e non -SHBGbound es tradiol was not significa ntly d ifferent in smoke rs and nonsmokers. Androstenedione wa s 28 % hi gher a t 8 AM in smo kers (P = 0.1 3) and 52% hi gh e r at 11 A M (afte r the smokers had sm o ked five cigarettes over a 3-h our period , p = 0.011 ). No ns m oke rs sh owed a d ecrease in androstenedi o ne over time (492 p g /ml at 8 AM vs 404 pg/ml at 11 AM , P = 0.1 3); th e d ecline was much less in smokers (628 vs 61 4 pg/ml). DHEAS was 42 % hi gh er at 8 AM a nd 62 % highe r at II AM in sm o ke rs than in nonsm ok er s, but these results did not ac hie ve statistical significance. Cortisol was 3% lower at 8 AM in smokers than in nonsmokers but 19% hi gher at 11 AM (after smokin g). This also was not statisticall y significant. As expected , co r tisol showed a

1446 Cassidenti et al.

May 1992

Am J Obstet Gynecol

Table II. Serum hormone levels in postmenopausal nonsmokers and smokers* Baseline (AM)

After smoking (11 AM)

(Smokers nonsmokers) /

(Smokers nonsmokers) /

nonsmokersi

nonsmokersi

Nonsmokers No . Estrone (pg/ml) Estrone glucuronide (pg/ml) Estradiol (pg/ml) SHBG (nmol/L) Non-SHBGbound estradiol (pg/ml) Androstenedione (pg/ml) DHEAS (lJ.g/dl) Cortisol (lJ.g/ dl) Androstenedione/estrone

pt

(%)

17 38.7 182

21 33 .7 191

-13 +5

0.30 0.62

17 34.1 190

21 32.1 198

-6 +4

0.71 0.64

15.1 158 4.48

15.8 123 5.96

+5 -22 +33

0.69 0.20 0.12

14.6 145 4.87

14.5 129 5.39

-1 -11 + 11

0.95 0.56 0.51

492

628

+28

0.13

404

614

+52

0.011

+42 -3 +47

0.20 0.75 0.026

+62 + 19 + 61

0.10 0.16 0.009

59.3 16.1 12.7

84.5 15.7 18.7

Nonsmokers

54.8 11.7 11.9

Smokers

pt

Smokers

88.7 13.9 19.1

( %)

*Results shown are geometric means. tComparison is difference between smokers and nonsmokers as percent of nonsmokers. :j:Two-sided statistical significance levels .

marked decrease over a 3-hour period in nonsmokers P = 0.017) ; the decline was much less in smokers (15.7 vs 13.9 tJ.g /dl). Smokers showed a 47 % higher androstenedione/estrone ratio at baseline (p = 0.026) and a 61 % higher ratio after smoking (P = 0.009). The results shown in Table II remained essentially unchanged after adjustment for age, age at menopause, weight, height, and alcohol consumption. (16.1 vs 11.7 tJ.g / dl,

Comment

The data presented here suggest that in postmenopausal women who smoke there is no alteration in serum estrogen levels if they are not receiving hormonal replacement. Our data suggest that the effect of smoking on serum hormone levels in healthy postmenopausal women is an increase in adrenal androgens (androstenedione, DHEAS). This is evident at baseline (androstenedione, 28 % increase; DHEAS, 42 % increase) and to a greater extent later in the morning after smoking (androstenedione, 52 % increase; DHEAS, 62 % increase). There is evidence for an increased level of cortisol in smokers compared with nonsmokers. The normal diurnal decline of cortisol is blunted in the smoking group. There have been at least four other comparative studies of serum hormone levels in postmenopausal smokers and nonsrnokers.v"" Some details and results of these studies together with our results are given in Table III. All studies that have looked at serum androstenedione have found elevated levels in smokers (mean increase at 20 cigarettes per day of 41 %, P < 0.000 1,

Table III). This 41 % mean value may be too high, because the biggest effect was seen in the study of Friedman et al." and their smoking study subjects were close to menopause and 5 years younger than their nonsmoking subjects. This discrepancy will tend to exaggerate any difference between smokers and nonsmokers as androstenedione declines at menopause and with increasing age. When the results of Friedman et al. 7 are excluded, the mean increase in androstenedione in smokers is 37% (P = 0.0002). Friedman et al.? and Khaw et al.' also foun d, as we did, that DHEAS levels are elevated in smo kers; however, Key et al. 18 did not. There is no obvious reason for the discrepancy between these studies. The subjects described by Key et al. 18 tended to be samples later in the day than the subjects in the other studies, but we know of no reason why this should be of any importance. Key et al." did provide some evidence for adrenal activation in smokers; however, urinary l l-hydroxyketosteroids were increased. When the combined data are considered, the mean increase in DHEAS at 20 cigarettes per day is 18% (P = 0.059, Table Ill). The results of Friedman et al.? may again be considered suspect for the reasons given above, and when their results are excluded, the mean increase in DHEAS in smokers is 14% (P = 0.15). Cortisol was elevated in our smokers but only at 11 AM after smoking. The smokers reported by Friedman et al.' had elevated baseline levels. We have no explanation for this discrepancy. When all the available data are examined, changes in androstenedione, DHEAS, and cortisol all appear to be higher in smokers. When the res ults are taken together, there do es not

Smoking and hormonal status 1447

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Table III. Results of previous studies of serum hormone levels in postmenopausal nonsmokers and smokers Reference Friedman et

Time of sampling Nonsmokers No . Age (yr) Weight (kg) Smokers No. Age (yr) Weight (kg) Average No. of cigarettes/ day Estrone Estr adiol SHBG Androstenedione DHEAS Cortisol

a: (19 87)

Key et aU' (1 991)

Khato et al :" (1988)

Longcope and J ohnston'

Cassidenti

(19 88)

(1992)

(nonfasting)

±8 AM (fasting)

16 54 68

213t 57 24.0§

187 69 24.7§

29 53 62

17 72 63

9 49 69 25

48 57 24.4§ 15

46 66 23.9§ 12

12 51 58 15

21 69 64 20

+10% +8% +13% +25% +43%

-1 % -12 %

-13% +5% -22% +28% +42% - 3%

8 AM (fasting)

-2% +27 % +74% +88% +47%

PM

+5 % -5%

Combined two-sided

Combined two-sided

et al. M ean"

p

M ean t

p

-4% +5% -9% +37 % +14%

0.65 0.58 0.54 0.0002 0.15

8 AM (nonfasting)

+32%

-3% 0.66 0.16 + 11% -9% 0.54 +41 % < 0.000 1 0.059 +18% 0.006 + 16%

* At 20 cigarettes per da y. t At 20 cigarettes per da y but with results of Friedman et al ,? excluded . :j:For estradiol, the numbers of subjects were 40 and 36 for nonsmokers and smokers, respectively. §Kilograms per square meter.

appear to be an y effect of smoking on estrone and estradiol in postmenopausal women who are not re ceiving oral estrogen. It has been suggested that catechol estrogen metabolism is enhanced in smokers" : however, if this was a significant finding, we would expe ct that it would have resulted in a difference in estrogen levels, an effect that has not been observed. That serum estrone gluc uronide and SHBG levels were also not increased in smoking women suggests th at th e enhancement in he patic activity , which occurs with smoking, occurs only in oral estrogen users. Our results, however, show that the androstenedione /estrone ratio distinguishes smokers from nonsmokers more effectively than androstenedione alone. This is consistent with experimental data of nicotineinhibiting aromatase acti vity.2
There is no evidence that cortisol has an influence on endometrial tissue, but there is some evidence that androgens can inhibit estrogen-mediated effects in the rat ut erus." In one study" it was shown that dih ydrotestosterone inhibited estrogen-mediated rat uterine growth, and this effect appeared to be due to a dihydrotestosterone-induced reduction in estrogen-induced ribonucleic acid transcription rather than to any in hibition of estrogen receptor synthesis (as is true of progesterone). T he only relevant epidemiologic d ata of androgen levels in endometrial cancer have been reported by Judd et al." They found no reduction in and rostened ione in endometrial canc er cases as would be expected if androstenedione antagonized the effect of estro ne in endometrial tissu e. Indeed, there is evidence that some patients with endometrial cancer have incr eased androgen levels, which may be related to hyperinsulinernia." The obese insulin-resistant patient ma y in fact have an additional risk factor because insulin ma y stimulate endometrial aromatase activit y." Although androgens may still be inhibitory to estrogen action in the endometrium of normal-weight wom en who smoke, there are probabl y other mechanisms to explain the epidemiologic observations. Reasons that smokers are at greater risk of osteoporosis may be due to the relative cortisol excess in smokers. Smoking itself ma y also have direct toxic (carbon monoxide, nicotine) effects on bone cells, thereby leading directly to osteoporosis. It is well known that smoking is toxic to other tissues; e .g., smokin g influ -

1448 Cassidenti et al.

ences oocyte atresia" and may impair sperm density, motility, and morphologic features." However, increased androstenedione levels would tend to be beneficial to bone. In conclusion, we suggest that estrogen levels are not decreased in nonusers of oral estrogen and therefore ma y not be implicated in the associated epidemiologic findings of a decrease in endometrial cancer and an increase in osteoporosis. Our findings and those of others reviewed here strongly suggest that there is increased adrenal activity in smoking women. This, as well as direct toxic effects of smoking, may be associated with the epidemiologic observations. More studies should be directed at these other mechanisms rather than at alterations in estrogen metabolism.

Addendum In our patients we more recently measured 24-hour urinary estriol and estrone to assess whether estrogen metabolism may be altered (lowered 16-hydroxylase activity, thus favoring 2-h ydroxylase), as suggested previously." The 42 % decrease in the estriol/estrone ratio described'" suggested enhancement of the conversion to catechol estrogens; however, we found only a 6% decrease. The ratio was 1.028 in nonsmokers and 0.963 in smokers (p = 0.37). REFERENCES I. Weiss NS. Cigarette smoking and the incidence of endometrial cancer. In : Wald N, Baron ], eds . Smoking and hormone-related disorders. Oxford : Oxford University Press, 1990:145-53 . 2. Law M. Smoking and osteoporosis. In: Wald N, Baronj , eds. Smoking and hormone-related disorders. Oxford: Oxford University Press, 1990:83-92. 3. Cassidenti DL, Vijod AG, Vijod MA, Stanczyk FZ, Lobo RA. Short-term effects of smoking on the pharmacokinetic profiles of micronized estradiol in postmenopausal women. AMj OBSTET GYNECOL 1990;163:1953-60. 4. jensen j , Christiansen C, Rodbro P. Cigarette smoking, serum estrogens, and bone loss during hormone-replacement therapy early after menopause. N Engl j Med 1985 ;313:973-5. 5. Jensen J , Christiansen C. Effects of smoking on serum lipoproteins and bone mineral content during postmenopausal hormone replacement therapy. AM J OBSTET CvNECOL 1988;159:820-5. 6. Khaw K, Taude S, Barrett-Conner E. Cigarette smoking and levels of adrenal androgens in postmenopausal women. N EnglJ Med 1988;318 :1705-9. 7. Friedman AJ, Ravnikar VA, Barbieri RL. Serum steroid hormone profiles in postmenopausal smokers and nonsmokers. Fertil Steril 1987;47 :398-401. 8. Longcope C, Johnston CC. Androgen and estrogen d ynamics in pre- and postmenopausal women : a comparison between smokers and nonsmokers. j Clin Endocrinol Metab 1988;67:379-83. 9. Federal Trade Commission Report. "Tar," nicotine and carbon monoxide of the smoke of 207 varieties of domestic cigarettes. Washington: Federal Trade Commission, 1985 Jan.

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