Variability of glutathione during the menstrual cycle—due to estrogen effects on hepatocytes?

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Abstract

Oxidative stress and alterations in the antioxidative defense system may be involved in carcinogenesis. We have previously shown that the levels of glutathione (GSH) in vivo in both breast tissue and subcutaneous fat were higher in the luteal phase compared with the follicular phase, suggesting an overall increase in GSH. This result was confirmed in the present study. Moreover, we exposed normal breast tissue in vivo, breast epithelial cells in vitro, and hepatocytes in culture to ovarian hormones. We found that local perfusion with estradiol, using microdialysis, in normal human breast tissue did not alter the local GSH levels in vivo. In vitro, treatment with estradiol and progesterone of normal human breast epithelial cells did not alter GSH levels. However, levels of GSH in hepatocytes were after 8 h estradiol exposure initially decreased, 76.6 ± 5% of control cells, p < .05, whereas 20 h exposure more than doubled GSH, 209 ± 26% compared with control cells, p < .01. Progesterone had no additional effect. Exposure of hepatocytes to estradiol increased the cellular content of γ-glutamylcysteine synthetase, the rate-limiting enzyme in GSH synthesis. In conclusion we suggest that estradiol affects the GSH homeostasis mainly by effects on hepatocytes, whereas local production in the breast is unaffected by estradiol.

Introduction

Although there is strong epidemiological evidence that exposure to ovarian hormones influences the risk of breast cancer, the mechanisms by which these hormones promote cancer formation remain poorly understood [1], [2]. Oxidative damage to DNA and alterations in the antioxidant defense system are suggested to be key factors in carcinogenesis [3], [4]. Estrogen has been shown to contribute to mammary carcinogenesis and continued cancer growth by increasing cell division and production of growth factors and by stimulating angiogenesis [5], [6]. However, other mechanisms involving estrogen may also contribute to cancer development. Estrogen may act as a chemical carcinogen through its metabolism to quinones and subsequent redox cycling, causing oxidative damage [7]. Moreover, estrogen-dependent modulation of the endogenous antioxidant system has been shown [8], [9].

Glutathione (GSH) is the most abundant thiol in cells and acts as a major antioxidant in addition to other biological functions [10], [11], [12], [13]. It keeps proteins and enzymes in reduced states and regulates protein activity via GSH transferases and thioredoxin [11], [12]. GSH is used as an electron donor for glutathione peroxidase during reduction of peroxides [10], and GSH can also react directly with an oxygen radical as a free radical scavenger [10], [14], [15]. Synthesis of GSH occurs in two regulated enzymatically catalyzed steps [13]. The rate-limiting enzyme, γ-glutamylcysteine synthetase (glutamate·cysteine ligase), is a heterodimer made up of a catalytic (heavy, 73 kDa) and a regulatory (light, 30 kDa) subunit. Expression of those genes are regulated by many conditions, including oxidative stress, activators of phase II detoxifying enzymes, antioxidants, and hormones [10], [13].

In breast tumors, the GSH levels are more than twice that found in normal breast tissue [16]. Moreover, GSH seems to be important in modulating the sensitivity of breast carcinomas to chemotherapeutic drugs [17]. We have previously found that the levels of GSH increased in subcutaneous fat and breast tissue in vivo late in the menstrual cycle when the ovarian hormones were elevated [9]. Whether this was a systemic effect only or to what extent a local production of GSH in the breast contributed to the increase seen in breast tissue is unclear.

In the present study we used microdialysis to expose normal human breast tissue in vivo to estradiol with simultaneous monitoring of GSH. Moreover, GSH was measured in normal breast epithelial cells in culture and hepatocytes after treatment with sex steroids.

Section snippets

Subjects

Twelve healthy women ages 22–29 years participated in the study. All were free of medication and had been off contraceptive pills for at least 3 months before the investigation. All women had a history of regular menstrual cycles (cycle length 28–32 days). Nine women were investigated in early follicular phase (days 1–3) in the menstrual cycle and three women were investigated early and late in the menstrual cycle, confirmed by serum estradiol and progesterone concentrations measured by

GSH increased late in the menstrual cycle in vivo

All women had regular menstrual cycles during the investigation. Serum levels of estradiol increased from 92 ± 10 pmol/l in the follicular phase to 340 ± 71 pmol/l in the luteal phase, p < .05. Ovulation was confirmed by increased progesterone levels in the luteal phase, 0.9 ± 0.4 to 20 ± 5 nmol/l, p < .05. There were no subsequent complications after the microdialysis experiments. To exclude the possibility that the catheter position influenced the results of GSH in the breast two

Discussion

In this study we show that GSH varied during the menstrual cycle in vivo both in breast tissue and in subcutaneous fat. Moreover, we show that estradiol had only minor effects on the local production of GSH in breast tissue in vitro and in vivo. However, the production of GSH by hepatocytes was highly influenced by estradiol. This suggests that estradiol may cause alterations in GSH homeostasis in the liver, which in turn may explain the detected overall increase in GSH late in the menstrual

Acknowledgements

This study was supported by grants from the Swedish Cancer Foundation, IVAX, and the Åke Wiberg and Percy Falk Research Foundations.

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