Study of the action of tamoxifen on the mammary gland epithelium of premenopausal patients by lysosome quantification

Tamoxifen is an antiestrogen drug widely utilized for the adjuvant hormonal treatment of breast carcinoma. Its use in the primary prophylaxis of this disease is currently being proposed. Although the drug has few side effects, its precise action on breast tissue that has not undergone neoplastic transformation has not been fully elucidated. This prospective, randomized study assessed the estrogen activity of tamoxifen on the mammary gland epithelium of premenopausal patients using a quantitative analysis of mammary epithelium Iysosomes identified by the cytochemical technique of GOMORI for acid phosphatase and by light microscopy. Tamoxifen significantly increased the number of Iysosomes only during the secretory phase of the menstrual cycle. We concluded that the early effect of the drug on normal mammary tissue is synergistic with the effect of estrogen during the premenopausal period.


INTRODUCTION T
amoxifen is considered to be an antiestrogen nonsteroid drug that inhibits the growth of breast carcinoma, thus representing the.drug of choice for hormonal treatment of this disease.t Recent studies have shown a lower incidence of primary cancer and cyclic pain in the contralateral breast of patients with carcinoma treated with tamoxifen.t -3 Thus, it is of great interest to know how the drug acts upon normal breasts, to the end of it being used as a primary prophylaxis of breast carcinoma in women at high-risk for developing this disease. 3 Studies relating the mamlTIary gland to lysosomes have shown a larger number of these organelIes in breast tUlTIOr ceIls compared to the normal adjacent parenchyma. 7• X Lysosomes are important markers of estrogen-mediated ceIl proliferation in the breast parenchyma.7.X GIRÃO et aI.,9 studying the numerical variation of lysosomes in the mammary epithelium during the menstrual cycle, observed a significantly greater amount of lysosomes during the first phase than in the second phase, demonstrating that the number of lysosomes refiects estrogen activity.
The objecti ve of this study was to assess the estrogen activity of talTIoxifen on the epitheliulTI of the malTImary gland of premenopausal WOlTIen by lysosome qua~tification.

Tamoxifen
is the most wielely-used elrug for chemoprophylaxis of breast carcinoma.However, its action on the norlnal mammary lobule before and after menopause is still unknown. 14-ló  It is difficult to create an experimental modeI that will.reproduce the complex endocrine interaction of the hUlnan breast, and there are obvious ethicaI restrictions with re.spect to taking biopsies froln the nOrInal breast.
Tamoxifen significantly increased the number of lysosomes in the mammary epithelium during the second phase of the menstrual cycle (Figs. 1 and 2).
During tamoxifen administration, the number of lysosomes remained stable in both phases of the menstrual cycle (Table 1).
Progesterone leveIs were significantly higher in group B patients during the Iuteal phase of the menstrual cycle (Table 2).

MATERIALS ANO METHOOS
To evaluate quantitatively the lysosomes of the human malnmary gland epithelium in the proliferative and secretory phase of the menstrual cycle during tamoxifen administration, and to compare the values obtained with those detected in untreated women during these same phases.
The study was carried out on 33 women aged 15-37 with fibroadenomas ] -3 cm in diameter which were removed and submitted to biopsy.Normal parenchyma samples were obtained from breast tissue situated at least 2 cm from the fibroadenoma at the time of surgical exeresis under local anesthesia.AlI patients gave informed written OISCUSSION consent and the study was approved by the Ethics Committee of Hospital São Paulo.
The patients were divided randomly into two groups: Group A (controI) consisted of 16 patients, and Group B consisted of 17 patients who took tamoxifen (20 mg/day) for 30 days before surgery.
All patients had normal menstrual cycles.The cycle phase was characterized on the basis of the date of last menstruation and measurement of plasma progesterone.Patients with endocrine disease, pregnant patients or those who had taken hormones during the prior 12 months were excluded from the study, as were also patients whose biopsy did not confirm the presence of a fibroadenoma in the nodule or normality of the adjacent parenchyma.
The chüice of menstrual cycle phase for exeresis of the nodule was aleatory for both groups, but the dates were fixed for a period between the 7th and 10th day during the fir~t phase, and between the 21 st and 23rd day during the second.
Lysosolnes were identified by the cytochemical method of aciel phosphatase detection, lO anel.counts were carrieel out using the PRICOLI" anelMARTINS 12 methoels.Counts were Tamoxifen adlninistered before menopause first causes an increase in serum estrogen and progesterone leveIs, possibly initialing the proliferation of .the maITImary epithelium.21 The increased number of lysosolnes observed during the second phase of the cycle in women taking tamoxifen confirms the estrogen agonist effect of the drug on premenopausal WOITIen.
On the other hand, the increased concentration of active serum progesterone acti vates the enzyme 17beta-hydroxysteroid oxidoreductase, which favors the conversion of estrone to estradiol in breast tissue, which in turns increases estrogen activity.22 Recent studies using ITIolecular techniques have demonstrated alterations.in the gene expression of the estradiol receptors which ITIayexplain the anti proliferati ve action of taInoxifen despite the agonist estrogen acti vity.23.24 The explanation of the antiproliferative effect on the breast dllring long-tenn treatnlent lnay be clown-reglllation The tissue fragment utilized in the present study was obtained froIn patients with fibroadenoma who did not respond to tamoxifen treatment, after authorization by the MedicaI Ethics Committee of Hospital São Paulo.There are numerous.theories attelnpting to explain the antiproliferative action ofthis drug on neoplastic tisslle.The major lnechanisln of action, however, is thought to be the combination of talTIoxifen with nuclear estrogen receptor protein (ERP), which produces a G] block and decreases production of growthstimulating proteins such as transforming growth factor alpha (TGF-a), and stimulation of growthinhibiting proteins like transfonning growth factor beta (TGF-B).17-19 TaITIOxifen is predominantly a tumoristatic agent; it doubles the leveIs of sex horlnone-binding globulin (SHBG) and may therefore exert its antiestrogen effect by Inaki ng it unavailable to breast cells.\  of estrogen receptors, reduction ofTGF-a and increase of TGF-B.Future studies on patients using talnoxifen for long periods of tilne are needed to better evaluate the endocrine effect of the drug on normal breast tissue, thus pennitting a Inore rational indication of the drug for treatlnent of benign diseases and for cancer chelnoprophylaxis.

CONCLUSION
We conclude that talnoxifen significantly increased the nUlnber of lysosomes in the Inammary epithelium in the second phase of the lnenstrual cycle, with leveIs similar to those detected in the proliferative phase for both groups.Thus, the drug appears to have a synergistic effect with estrogen during the prelnenopausal period.

Figure 1 -
Figure 1 -Photomicrograph of a histological section of human mammary gland epithelium carrieel out in a double-blind manner on during the secretory phase from a patient NOT treated with tamoxifen (Gomori method 25 reticulated areas per 400x field for acid phosphatase.Magnification.:t 1.440 X).

Figure 2 -
Photomicrograph of a histological section of human mammary gland epithelium intracellular lysoSOITIeS indirectly during the secretory phase from a patient treated with tamoxifen (Gomori method for measures estrogen activity on target acid phosphatase.Magnification.:t 1.4440 X).

Table 1
Number of Iysosomes in the breast epithelium of patients from Group A (control) and Group B (tamoxifen) during the proliferative and secretory phases of the cell cycle.