Thiols can either enhance or suppress DNA damage induction by catecholestrogens.

The estrogen metabolites catecholestrogens (or hydroxyestrogens) are involved in carcinogenesis and the development of resistance to methotrexate. This induction of drug resistance correlates with the relative efficiency of catecholestrogens in the generation of reactive oxygen species (ROS) and the induction of DNA strand breaks. Although antioxidants can neutralize ROS, the generation of these reactive species by catecholestrogens can be enhanced by electron donors like NADH. Therefore, this study was undertaken to determine the ability of different thiol agents (GSH, NAC, DTT, DHLA) to either inhibit or enhance the level of DNA damage induced by the H(2)O(2) generating system 4-hydroxyestradiol/Cu(II). Our results show that GSH, DTT, and DHLA inhibited the induction of the 4-hydroxyestradiol/Cu(II)-mediated DNA damage, with GSH showing the best potential. In contrast, the GSH precursor NAC at low concentrations was able to enhance the level of oxidative damage, as observed with NADH. NAC can reduce Cu(II) to Cu(I) producing the radical NAC&z.rad;, which can generate the superoxide anion. However, the importance of this pathway appears to be relatively minor since the addition of NAC to the 4-hydroxyestradiol/Cu(II) system generates about 15 times more DNA strand breaks than NAC and Cu(II) alone. We suggest that NAC can perpetuate the redox cycle between the quinone and the semiquinone forms of the catecholestrogens, thereby enhancing the production of ROS. In conclusion, this study demonstrates the crucial importance of the choice of antioxidant as potential therapy against the negative biological effects of estrogens.

Oestrogen metabolism in lymphangioleiomyomatosis: catechol-O-methyltransferase pathway is not involved.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is an uncommon lung disease for which no effective method of treatment has been found. The predilection of LAM for premenopausal women has led to the assumption that hormonal factors play an important role in the pathogenesis of this disease. The aim of this study was to determine if women with LAM manifest alterations in the catechol-O-methyltransferase (COMT) pathway which is essential for preventing the generation of oestrogen derived reactive oxygen species (ROS). METHODS: Blood samples were collected from 15 women with LAM and compared with appropriate controls. The distribution of high and low activity alleles of COMT was determined with a PCR based RFLP assay. The enzymatic activity of COMT was measured in each sample and the potential presence of a circulating inhibitor of COMT was determined. Since an alteration in the COMT pathway could increase the oxidative stress, the plasma concentration of malondialdehyde (MDA), a secondary product generated from lipid peroxidation, has been used as an internal marker. RESULTS: The distribution of high and low activity alleles of COMT (named COMT(HH), COMT(LL), and COMT(HL)) was similar in the two groups with proportions of 40%, 7%, and 53%, respectively, in the women with LAM and 38%, 6%, and 56% in the control subjects. The mean (SD) COMT activity was 24.2 (12.3) pmol/min/mg protein in women with LAM and 24.1 (6.3) pmol/min/mg protein in the control group. Incubation of plasma from women in the two groups with a preparation of commercial COMT showed that no detectable COMT inhibitor was present. The plasma concentration of MDA in the women with LAM was also not significantly different from control subjects. CONCLUSIONS: This study shows that there are no significant alterations in the COMT pathway of women with LAM. It is therefore unlikely that alterations in oestrogen mediated cell signalling pathways are mediated by oxidants derived from an excess of catecholoestrogens in LAM.

DNA damage induced by catecholestrogens in the presence of copper (II): generation of reactive oxygen species and enhancement by NADH.

Certain estrogen metabolites are involved in carcinogenesis and the development of resistance to methotrexate (MTX). In this study, we determined whether these well-established biological effects correlate with the relative efficiency of several estrogen metabolites to induce DNA strand breaks in the presence of copper, and investigated the potential enhancing effect of reduced nicotinamide adenine dinucleotide (NADH). DNA strand breaks induced by estradiol metabolites were measured by the conversion of supercoiled phage phiX-174 RF1 DNA to open circular and linear forms. The most active catecholestrogens were the 4-hydroxy derivatives, which produced about 2.5 times more DNA double strand breaks than the 2-hydroxy derivatives, while estradiol and 16alpha-hydroxyestrone were inactive. In addition, our results show that 4-hydroxyestradiol (4-OHE2) at physiological concentrations was capable of exhibiting DNA cleaving activity. The formation of these catecholestrogen-induced DNA strand breaks was associated with the utilization of oxygen and the generation of H2O2, because catalase inhibited the DNA cleaving activity of 4-OHE2. Interestingly, we also observed that NADH enhanced the induction of DNA strands breaks by 4-OHE2/Cu(II), probably by perpetuating the redox cycle between the quinone and the semiquinone forms of the catecholestrogen. In conclusion, this study demonstrated that the relative efficiency of 2-, and 4-hydroxyestrogen in carcinogenesis and for the enhancement of MTX resistance correlates with their relative capability to induce DNA strand breaks. In order to inhibit these estrogen-mediated biological effects, it may be important to develop different strategies to block the production of reactive oxygen species by the catecholestrogen-redox cycle.

Induction by estrogens of methotrexate resistance in MCF-7 breast cancer cells.

Development of drug resistance is a major factor that limits the effectiveness of chemotherapy treatments. In this study, we determined whether estradiol or its metabolites 2-, 4- and 16alpha-hydroxyestrone could enhance the development of methotrexate resistance in the breast carcinoma cell line, MCF-7. Cells were incubated with the estrogens at a concentration of 10(-8) M for 12 cell doublings and enhancement of methotrexate resistance was measured with the Luria-Delbrück assay. The most efficient estrogens were the 4-hydroxyestrone and 16alpha-hydroxyestrone, which both stimulated methotrexate resistance by 88-fold as compared with the control without estrogen. 2-Hydroxyestrone had an enhancement factor of 33-fold, whereas estradiol showed a slight effect with an enhancement factor of 3.2-fold. To determine whether the estrogen receptor was involved in the development of resistance, expression of the pS2 gene, which contains an estrogen-responsive element, was measured. Both estradiol and 16alpha-hydroxyestrone stimulated expression of the pS2 gene. In contrast, 2- and 4-hydroxyestrone did not increase the level of pS2 mRNA. This suggests that tumors classified as estrogen receptor negative could also develop methotrexate resistance as the result of exposure to estrogens. The status of the tumor suppressor gene p53 was analyzed in methotrexate sensitive and resistant clones. In all the methotrexate resistant clones analyzed, the western blots indicated that the p53 protein was still present and transcriptionally competent, as measured by its capacity to stimulate transcription of the p21waf1/cip1 gene following UVB irradiation. However, the basal level of p53 was higher in resistant clones and addition of 2- or 4-hydroxyestrone increased p53 to levels equivalent to those observed following UVB irradiation. However, this induction of p53 accumulation by estrogens failed to stimulate the transcription of p21waf1/cip1, which indicates that a transcriptionally inactive form of p53 accumulated in methotrexate resistant cells.