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Reproductive Endocrinology |
Departments of Obstetrics and Gynecology and Pathology II (K.O.), Faculty of Health Sciences, University Hospital, Linkoping; and the Department of Physiology and Pharmacology, Karolinska Institute (U.U.), Stockholm, Sweden
Address all correspondence and requests for reprints to: Charlotta Dabrosin, M.D., Department of Obstetrics and Gynecology, Faculty of Health Sciences, University Hospital, S-581 85 Linkoping, Sweden.
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Abstract |
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Introduction |
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Microdialysis has proved to be a good technique for sampling the extracellular fluid and has previously been used to study metabolism in both animals and humans (4). However, it has never been used in human breast tissue.
Oxidative damage to DNA and alterations in the antioxidative defense systems have been suggested to be important in carcinogenesis (5). Oxy-radicals are highly reactive compounds that can damage DNA bases and cause mutations that lead to malignant transformation (6). Glutathione (GSH) is the most abundant thiol in cells and acts as a major antioxidant in addition to other biological functions. During oxidative stress with initial depletion of GSH, increased synthesis of GSH has been found, because the rate-limiting step of GSH synthesis is feedback inhibited by GSH (7). In breast tumors, the GSH concentration is more than twice that found in normal breast tissue (8), and GSH seems to be important in modulating the sensitivity of breast carcinomas to chemotherapeutic drugs (9).
Microdialysis has been used in several animal experiments to study the extracellular levels of GSH compared with the intracellular levels of GSH in tissue homogenate (10, 11, 12). These studies showed that the extracellular levels are much lower (micromoles per L) than the intracellular levels (millimoles per L).
The menstrual cycle is associated with several metabolic and hormonal variations. Whether the GSH levels in plasma or those in individual organs change during the menstrual cycle is not known.
The aim of this study was to examine the in vivo levels of GSH in normal breast tissue and sc fat during the menstrual cycle with the microdialysis technique.
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Subjects and Methods |
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Six healthy women, aged 23–32 yr, participated in the study. All women were free of medication, had a history of regular menstrual cycles (cycle length, 28–32 days), and had never been pregnant. The breasts were normal on clinical examination.
The study was approved by the local ethical committee, and all women gave their informed consents.
Procedure
Microdialysis was performed twice during the menstrual cycle: early in the follicular phase and in the midluteal phase.
Blood samples were drawn on each occasion to determine estradiol, progesterone, and PRL levels (AutoDELFIA, Wallac Oy, Åbo, Finland). After administration of 0.5 mL mepivacaine (5 mg/mL) intracutaneously, two microdialysis catheters were inserted, one into the sc fat of the abdomen and the other into the breast tissue. After 3 h of equilibration, two 60-min fractions were collected into 10 µL 0.1 mol/L perchloric acid and stored at -70 C for subsequent analysis of GSH. The GSH levels given in Results are the mean value from the two fractions. In one woman, samples were collected hourly for 8 h alternately in Ringer’s solution or perchloric acid.
Microdialysis device
The principle of microdialysis has previously been described in detail (4). We used microdialysis catheter CMA 60 (CMA/Microdialysis AB, Stockholm, Sweden). This consists of a tubular dialysis membrane (30 x 0.52 mm; 20,000 relative atomic mass cut-off) glued to the end of a double lumen tube (20 x 0.8 mm). The catheter was connected to a microinfusion pump (CMA 106, CMA/Microdialysis AB) and perfused with Ringer’s solution (0.3 µL/min). The perfusate is a reflection of the chemical composition in the tissue due to the diffusion of substances over the membrane.
GSH assay
Separation of GSH was performed on a Kromasil 100–5C18 reverse phase column (250 x 4.6; Hichrom, Reading, UK). The mobile phase, supplied by a ConstaMetric III pump (LDC/Milion Roy, Riviera Beach, FL), consisted of 0.1 mol/L NaH2PO4, 0.1 mmol/L ethylenediamine tetraacetate, 0.2 mmol/L n-octyl sodium sulfate (pH 2.5), and 5% methanol (13). The flow rate was 0.5 mL/min, 10 µL sample were injected, and the retention time for GSH was 15 min. GSH was detected using a BAS LC-4B Amperometric Detector (Bioanalytical Systems, West Lafayette, IN) equipped with an Au electrode at +0.150 V. GSH concentrations (micromoles per L) were calculated from standard curves obtained daily before the analysis. The intraassay variation was 1.2 ± 0.4%. The detection limit was 1 x 10-12 mol GSH.
Statistics
Differences were assessed by t test and Wilcoxon’s signed rank test for paired observations.
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Results |
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GSH levels increased late in the menstrual cycle in both tissues, and
the differences between the follicular and luteal phases were
statistically significant (P < 0.02 and
P < 0.01, respectively, with paired t test;
P < 0.05 with Wilcoxon’s signed rank test; Fig. 1
). In the woman who was followed for 8 h, GSH
levels were not detectable in the samples collected in Ringer’s
solution. In perchloric acid, GSH levels were stable; in the first
sample, collected less than 1 h after implantation of the
microdialysis catheter, the GSH concentration was 6.8 µmol/L. Two to
8 h after implantation, GSH levels ranged between 5.58–5.84
µmol/L, a variability of less than 5%.
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Discussion |
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GSH levels varied during the menstrual cycle in both adipose and breast tissues, with higher levels late in the cycle. This could reflect an overall change in GSH levels during the menstrual cycle. Alterations in liver metabolism due to the hormonal changes could be one explanation. Another cause of the variability of the oligopeptide GSH could be differences in amino acid metabolism during the menstrual cycle. The rise could also indicate increased oxidative stress when the levels of estradiol and progesterone are high. This could be important in tissues that are dependent on sex hormones for their proliferation and apoptosis, as oxygen radicals and antioxidants such as GSH have been suggested to be generators of both events. In the breast, the proliferation and apoptotic rates reach their maxima late in the menstrual cycle (14).
Several genes that are critical in the regulation of apoptosis have been defined, one of these being the bcl-2 protooncogene (15). The protein encoded by the bcl-2 gene has been identified in breast cancer, preinvasive breast lesions, normal breast epithelium, and normal endometrium (16, 17, 18, 19). In normal breast tissue and endometrium, bcl-2 expression shows a cyclic variation correlated with the menstrual cycle, indicating that the regulation could be hormone dependent (17, 19). Several studies also suggest that its expression may be controlled by oxy-radicals and antioxidants (20, 21, 22). It has been suggested that the bcl-2 protein creates oxy-radicals that induce the cell to produce antioxidants such as GSH (20), and in human T cells apoptosis is modulated by GSH (23). However, there are probably other stimuli that cause apoptosis independent of oxidative stress (24). Yet, a possible explanation, among others, for the GSH rise late in the menstrual cycle could be high concentrations of sex hormones leading to increased expression of the Bcl-2 protein, which produces oxy-radicals, resulting in increased synthesis of GSH.
In summary, this study of human breast tissue in vivo suggests that GSH levels are influenced by sex hormones, which may be important in the carcinogenesis of the breast. Whether the observed GSH variation during the menstrual cycle indicates a true increase in oxidative stress or a hormone-dependent sensitivity to oxidative stress or other events during the menstrual cycle remains to be elucidated.
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Footnotes |
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Received September 17, 1996.
Revised December 12, 1996.
Accepted February 13, 1997.
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This article has been cited by other articles:
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  C. Dabrosin Positive Correlation between Estradiol and Vascular Endothelial Growth Factor but not Fibroblast Growth Factor-2 in Normal Human Breast Tissue In vivo Clin. Cancer Res., November 15, 2005; 11(22): 8036 - 8041. [Abstract] [Full Text] [PDF]
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 C. Dabrosin Increased extracellular local levels of estradiol in normal breast in vivo during the luteal phase of the menstrual cycle J. Endocrinol., October 1, 2005; 187(1): 103 - 108. [Abstract] [Full Text] [PDF]
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 S. Garvin and C. Dabrosin Tamoxifen Inhibits Secretion of Vascular Endothelial Growth Factor in Breast Cancer in Vivo Cancer Res., December 15, 2003; 63(24): 8742 - 8748. [Abstract] [Full Text] [PDF]
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 C. Dabrosin Variability of Vascular Endothelial Growth Factor in Normal Human Breast Tissue in Vivo during the Menstrual Cycle J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2695 - 2698. [Abstract] [Full Text] [PDF]
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), and b) breast tissue (•), early in
the follicular phase (days 3–5) and in the midluteal phase (days
21–26) in six healthy women. The extracellular fluid was sampled using
the microdialysis technique, stored at -70 C, and subsequently
analyzed by high performance liquid chromatography. GSH levels
increased in the midluteal phase in both sc fat and breast tissue
(P < 0.05, by Wilcoxon’s signed rank test;
P < 0.02, by paired t test).