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Benefits of vitamin E supplementation to Norplant users — in vitro and in vivo studies
Toxicology 148 (2000) 173 – 178 www.elsevier.com/locate/toxicol
Benefits of vitamin E supplementation to Norplant users — in vitro and in vivo studies S.B. Subakir, E. Setiadi, B. Affandi, S. Pringgoutomo, H.-J. Freisleben * Faculty of Medicine, Uni6ersity of Indonesia, Salemba Raya No. 4, Jakarta 10430, Indonesia
Abstract Norplant subcutaneous implantation is a contraceptive method used in Indonesia. Endometrial bleeding is one major reason to discontinue the use of Norplant. Angiogenic response in the endometrium of Norplant users was found to be lower than in women with normal menstrual cycle. This disturbance in the angiogenic process may be caused by an imbalance of pro- and antioxidant processes in the endometrium of Norplant users. The aim of this study is to investigate the effect of vitamin E on the endometrial angiogenic activity and to assess the efficacy of vitamin E supplementation in treating endometrial bleeding in Norplant users. Subjects for this study were selected from Norplant users with an exposure of at least 3 months, with endometrial bleeding and recruited on the basis of fully informed consent. TBA reaction was used to measure degradation products of lipid peroxidation. The endometrial angiogenic response was assayed according to Folkman et al. (Folkman et al., 1989. Nature 239, 58 – 61). Samples from endometrial biopsies were incubated in vitro with vitamin E or placebo before angiogenic measurement. For in vivo supplementation, vitamin E 200 mg/day, or placebo for 10 days/month were given to the subjects with double blind randomisation. The results showed that the blood levels of TBA-reactive substances were significantly higher in Norplant users than in controls. In the endometrium from Norplant users with bleeding problems, in vitro supplementation of vitamin E resulted in a significantly higher angiogenic score than placebo. Although a highly significant reduction of bleeding days in both groups, vitamin E and placebo, was seen during the 2 months of the study, the number of bleeding days was significantly lower in women treated with vitamin E than with placebo. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Angiogenesis; Endometrial bleeding; Lipid peroxides; Norplant; Vitamin E
1. Introduction The mechanism of endometrial bleeding in levonorgestrel (LNG) subdermal implant (NorAbbre6iations: FCS, fetal calf serum; IUD, intra uterine device; LNG, levonorgestrel; MDA, malonaldehyde; TBA, thiobarbituric acid; WHO, world health organization. * Corresponding author. Tel.: + 62-21-330335.
plant) is not clear. Prolonged bleeding in some women receiving LNG implant may be caused by disturbances in endometrial regeneration. This assumption is supported by reduction of endometrial angiogenic activity in Norplant users (Subakir et al., 1996). Production of epoxides in the endometrium is increased in progestogen contraceptive users (White et al., 1997), mediating release of oxygen
0300-483X/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 0 ) 0 0 2 0 8 - 0
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radicals (Rice-Evans and Cooke, 1990), which in turn may attack highly vulnerable polyunsaturated fatty acids in phospholipid membranes by hydrogen abstraction. This results in carbon centred lipid radicals and by addition of oxygen, lipid peroxides. Once lipid peroxidation is initiated, a chain reaction will be propagated, which can be highly destructive if not tightly controlled by antioxidants. The oxidant – antioxidant imbalance in the endometrium of Norplant users may cause disturbance in endometrial angiogenic processes. Vitamin E is the major physiological lipid-soluble chain breaking antioxidant, preventing lipid peroxidation and also modulating the metabolism of arachidonic acid (Packer and Landvik, 1989). Supplementation of vitamin E serves as an effective method of preventing membrane damage caused by oxygen radicals (Halliwell et al., 1992). The aims of this study are to investigate the effect of in vitro vitamin E incubation on endometrial angiogenic activity and to assess the possible effectiveness of vitamin E supplementation in treating endometrial bleeding in Norplant users.
2. Methods and materials Subjects were selected from Norplant users with an exposure of Norplant between 3 and 12 months. All subjects were healthy, age between 18 and 40 years, and recruited to the study on the basis of fully informed consent. The blood levels of lipid peroxides were measured from 36 Norplant users with bleeding problems, seven without bleeding problems, and 25 controls. Control subjects were chosen from women not using hormonal contraceptives or IUD for at least 3 months. Menstrual record data were analysed using the current WHO terminology and definition. Lipid peroxides were evaluated as malonaldehyde (MDA) reaction with thiobarbituric acid (TBA). The TBA reaction products were measured spectrofluorometrically at an excitation wavelength of 515 nm and emission at 532 nm.
2.1. Endothelial cell culture Endothelial cells from human umbilical cord vein were isolated and cultured according to modified Jaffe et al., (1973). Endothelial cells were harvested using collagenase type Ia (Sigma Chemical Co.). The culture medium was M199 (Gibco Laboratories) containing 20% FCS (Sigma Chemical Co.), 0.20 mg/ml gentamycin (Schering), and 1.75 mg/ml fungizone (Flow Laboratories). After confluent, the primary endothelial cell culture was used in the angiogenic assay.
2.2. Endometrial angiogenis assay Endometrial biopsies were taken using a microhysteroscope. Endometrial samples were incubated with RPMI (Flow Laboratories) as controls or vitamin E 20 mg/l RPMI for 60 min prior to angiogenic measurement. Endometrial angiogenic response was assayed using an endothelial cell migration assay. Explants were cultured utilising a three-dimensional collagen matrix containing suspended endothelial cells (Folkman et al., 1989; modified by Rogers et al., 1992). Endothelial cells were suspended in NCTC (Flow Laboratories) with FCS 20%, gentamycin and fungizone. The medium was adjusted with 0.1 M NaOH to reach a pH of 7.4 after the addition of chilled vitrogen 100 (Collagen Corporation). Explants (approximately 500 mm in diameter) were cultured for 96 h and observed at 24-h intervals using an inverse phase-contrast microscope. The angiogenic score ranged from 0 to 4: score 0 represents no endothelial cell migration, score 1 represents elongation and some orientation toward explant, score 2 represents minimal spokewheel response, score 3 represents medium spokewheel response, and score 4 represents extreme spokewheel formation.
2.3. Clinical trial Subjects were Norplant users with bleeding problems. Vitamin E 200 mg, daily (n= 38) or placebo (n= 34) for 10 days were given to subjects under double blind randomisation and repeated after 30 days. Subjects were kept on
S.B. Subakir et al. / Toxicology 148 (2000) 173–178
menstrual diary card. Assessment of treatment effectiveness was based on the analysis of menstrual diaries using the reference period method of analysis.
2.4. Statistical analysis Differences in endometrial score between different groups were analysed using the Mann – Whitney test. Spearman’s rank correlation was used to calculate the correlation between endometrial angiogenic activity and blood levels of lipid peroxides. Differences of bleeding days between control group (placebo) and treatment group (vitamin E) were analysed using Student’s t-test.
3. Results and discussion Blood levels of lipid peroxides from Norplant users with bleeding problems were significantly higher than in the control group (X9 S.D.: 8.06 92.74 vs. 5.64 91.91 mmol/l, P 5 0.01; Fig. 1). The increased concentration of lipid peroxides may be caused by elevated radical chain reactions in lipid membranes.
175
Lipid peroxide concentrations did not clearly correlate with endometrial angiogenic activity (Fig. 2, controls), however, higher lipid peroxide concentrations tend to have a lower score, especially in Norplant users with bleeding problems (Fig. 2). Furthermore, comparison of Norplant users with angiogenic score 0 and score 1.5 in Fig. 2 shows a significant difference of 45% in their levels of lipid peroxides. Although there may not be a clear correlation between lipid peroxide concentrations and endometrial angiogenic scores in general, lower angiogenic scores appear to correlate with higher lipid peroxide concentrations in Norplant users with bleeding problems. Recently, White et al. (1997) observed an increased epoxide formation via prostaglandin synthesis, which correlated with ruptures of blood vessels in the endometrium of levonorgestrel contraceptive users. In our study, addition of vitamin E/RPMI (in vitro) to the endometrial explants from Norplant users with bleeding problems resulted in a significantly higher angiogenic score than addition of RPMI without vitamin E (z= −2.24, 2 tailed, P 5 0.05; Fig. 3). In both groups, the angiogenic score ranged from 0 to 2.
Fig. 1. Blood levels of lipid peroxides in control group and Norplant users. Blood levels of lipid peroxides were measured as thiobarbituric acid reactive substances (TBARS), especially malonaldehyde (MDA), in controls with normal pregnancies (n = 23), in Norplant users with bleeding problems (n= 36), and in Norplant users without bleeding problems (n = 7).
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Fig. 2. Blood levels of lipid peroxides and endometrial angiogenic activity in Norplant users with bleeding problems and controls. Angiogenic activity was determined from endometrial explants, the score ranged from 0 to 2.
Fig. 3. Effect of in vitro supplementation of vitamin E 20 mg/l RPMI (vitamin E) and RPMI without vitamin E (placebo) on the in vitro angiogenic activity in cultured explants from the endometrium of Norplant users with bleeding problems. Endometrial angiogenic score ranged from 0 to 2.
Thus, in vitro incubation showed that the antioxidant vitamin E can increase endothelial cell migratory signal production and angiogenic activity in endometrial explants from Norplant users with bleeding problems. Other investigators found that vitamin E can inhibit free radical formation and can protect endothelial cell cultures from cell
damage (Matsuo and Kaneko, 1990; Niki, 1990; Faruqi et al., 1994). It is concluded that in Norplant users with bleeding problems, higher lipid peroxide levels correlate with lower endometrial angiogenic scores and it is suggested that vitamin E supplementation may be effective to increase endome-
S.B. Subakir et al. / Toxicology 148 (2000) 173–178
177
Fig. 4. Effect of supplementation with 200 mg/day vitamin E (vitamin E) on the number of bleeding days in Norplant users with bleeding problems vs. those receiving placebo only (placebo). Vitamin E and placebo were administered double blinded for 10 days (cycle 1) and administration for another 10 days repeated after 30 days (cycle 2).
trial angiogenic activity and to reduce bleeding problems in Norplant users. This needs to be confirmed in more extended clinical trials. In our preliminary clinical study, before treatment, the vitamin E group and the placebo group had no significant difference in the number of bleeding days (18.1 91.0 vs. 20 9 1.13). In the initial 30 days from the start of treatment, this number fell to 7.791.4 days in the vitamin E group and to 12.1 9 1.3 days in the placebo group (Fig. 4). A highly significant effect is evident of both placebo and vitamin E to reduce the number of bleeding days, however, the number was also significantly lower in women treated with vitamin E as compared to placebo and this difference was maintained also in the second cycle of treatment (Fig. 4).
Acknowledgements This study was supported by WHO (HRP 91409) and Department of Education and Culture of the Republic of Indonesia.
References Faruqi, R., de la Motte, C., Dicorleto, P.E., 1994. Alfa-tocopherol inhibits agonist-induced monocytic cell adhesion to cultured human endothelial cells. J.Clin. Invest. 94, 592 – 600. Folkman, J., Watson, K., Ingber, D., Hanahan, D., 1989. Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature 239, 58 – 61. Halliwell, B., Gutteridge, J.M.C., Cross, E., 1992. Review article: free radicals, antioxidants and human disease: where are we now? J. Lab. Clin. Med. 119, 598 – 620. Jaffe, E.A., Nachman, R.L., Becker, C.G., Minick, C.R., 1973. Culture of human endothelial cells derived from umbelical vein. J. Clin. Invest. 52, 2745 – 2756. Matsuo, M., Kaneko, T., 1990. Protective effect of vitamin E on linoleic acid hydroperoxide-induced injury to human umbilical vein endothelial cells. In: Kelvin, J.A.D. (Ed.), Oxidative Damage and Repair: Chemical, Biological and Medical Aspects. Pergamon, Oxford, pp. 584 – 588. Niki, E., 1990. Antioxidant compounds. In: Kelvin, J.A.D. (Ed.), Oxidative Damage and Repair: Chemical, Biological and Medical Aspects. Pergamon, Oxford, pp. 57 – 64. Packer, L., Landvik, S., 1989. Vitamin E: introduction to biochemistry and health benefits. In: Diplock, A.T., Machlin, J.L., Pryor, W.A. (Eds.), Vitamin E, Biochemistry and Health Implications. New York Academy of Sciences, New York, pp. 1 – 6. Rice-Evans, C., Cooke, B., 1990. Oxygen radicals, bleeding and tissue injury. In: d’Arcangues, C., Fraser, I.S., New-
178
S.B. Subakir et al. / Toxicology 148 (2000) 173–178
ton, J.R., Odlind, V. (Eds.), Contraception and Mechanisms of Endometrial Bleeding. Cambridge University Press, Cambridge, pp. 411–430. Rogers, P.A.W., Abberton, K.M., Susil, B., 1992. Endothelial cell migratory signal production produced by human endometrium during menstrual cycle. Hum. Reprod. 7, 1061 – 1068. Subakir, S.B., Hadisaputra, W., Siregar, B., Soeria Santoso, D.I., Cornain, S., Affandi, B., 1996. Reduced endothelial
cell migratory signal production by endometrial explants from women using Norplant contraception. Hum. Reprod. 10, 2579 – 2583. White, J.O., Sulivan, M.H.F., Patel, L., Croxtail, J.D., d’Arcangues, C., Belsey, E.M., Elder, M.G., 1997. Prostaglandin production in human endometrium following continuous exposure to low-dose levonorgestrel release from a vaginal ring. Contraception 43, 401 – 412.
.
Benefits of vitamin E supplementation to Norplant users — in vitro and in vivo studies S.B. Subakir, E. Setiadi, B. Affandi, S. Pringgoutomo, H.-J. Freisleben * Faculty of Medicine, Uni6ersity of Indonesia, Salemba Raya No. 4, Jakarta 10430, Indonesia
Abstract Norplant subcutaneous implantation is a contraceptive method used in Indonesia. Endometrial bleeding is one major reason to discontinue the use of Norplant. Angiogenic response in the endometrium of Norplant users was found to be lower than in women with normal menstrual cycle. This disturbance in the angiogenic process may be caused by an imbalance of pro- and antioxidant processes in the endometrium of Norplant users. The aim of this study is to investigate the effect of vitamin E on the endometrial angiogenic activity and to assess the efficacy of vitamin E supplementation in treating endometrial bleeding in Norplant users. Subjects for this study were selected from Norplant users with an exposure of at least 3 months, with endometrial bleeding and recruited on the basis of fully informed consent. TBA reaction was used to measure degradation products of lipid peroxidation. The endometrial angiogenic response was assayed according to Folkman et al. (Folkman et al., 1989. Nature 239, 58 – 61). Samples from endometrial biopsies were incubated in vitro with vitamin E or placebo before angiogenic measurement. For in vivo supplementation, vitamin E 200 mg/day, or placebo for 10 days/month were given to the subjects with double blind randomisation. The results showed that the blood levels of TBA-reactive substances were significantly higher in Norplant users than in controls. In the endometrium from Norplant users with bleeding problems, in vitro supplementation of vitamin E resulted in a significantly higher angiogenic score than placebo. Although a highly significant reduction of bleeding days in both groups, vitamin E and placebo, was seen during the 2 months of the study, the number of bleeding days was significantly lower in women treated with vitamin E than with placebo. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Angiogenesis; Endometrial bleeding; Lipid peroxides; Norplant; Vitamin E
1. Introduction The mechanism of endometrial bleeding in levonorgestrel (LNG) subdermal implant (NorAbbre6iations: FCS, fetal calf serum; IUD, intra uterine device; LNG, levonorgestrel; MDA, malonaldehyde; TBA, thiobarbituric acid; WHO, world health organization. * Corresponding author. Tel.: + 62-21-330335.
plant) is not clear. Prolonged bleeding in some women receiving LNG implant may be caused by disturbances in endometrial regeneration. This assumption is supported by reduction of endometrial angiogenic activity in Norplant users (Subakir et al., 1996). Production of epoxides in the endometrium is increased in progestogen contraceptive users (White et al., 1997), mediating release of oxygen
0300-483X/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 0 ) 0 0 2 0 8 - 0
174
S.B. Subakir et al. / Toxicology 148 (2000) 173–178
radicals (Rice-Evans and Cooke, 1990), which in turn may attack highly vulnerable polyunsaturated fatty acids in phospholipid membranes by hydrogen abstraction. This results in carbon centred lipid radicals and by addition of oxygen, lipid peroxides. Once lipid peroxidation is initiated, a chain reaction will be propagated, which can be highly destructive if not tightly controlled by antioxidants. The oxidant – antioxidant imbalance in the endometrium of Norplant users may cause disturbance in endometrial angiogenic processes. Vitamin E is the major physiological lipid-soluble chain breaking antioxidant, preventing lipid peroxidation and also modulating the metabolism of arachidonic acid (Packer and Landvik, 1989). Supplementation of vitamin E serves as an effective method of preventing membrane damage caused by oxygen radicals (Halliwell et al., 1992). The aims of this study are to investigate the effect of in vitro vitamin E incubation on endometrial angiogenic activity and to assess the possible effectiveness of vitamin E supplementation in treating endometrial bleeding in Norplant users.
2. Methods and materials Subjects were selected from Norplant users with an exposure of Norplant between 3 and 12 months. All subjects were healthy, age between 18 and 40 years, and recruited to the study on the basis of fully informed consent. The blood levels of lipid peroxides were measured from 36 Norplant users with bleeding problems, seven without bleeding problems, and 25 controls. Control subjects were chosen from women not using hormonal contraceptives or IUD for at least 3 months. Menstrual record data were analysed using the current WHO terminology and definition. Lipid peroxides were evaluated as malonaldehyde (MDA) reaction with thiobarbituric acid (TBA). The TBA reaction products were measured spectrofluorometrically at an excitation wavelength of 515 nm and emission at 532 nm.
2.1. Endothelial cell culture Endothelial cells from human umbilical cord vein were isolated and cultured according to modified Jaffe et al., (1973). Endothelial cells were harvested using collagenase type Ia (Sigma Chemical Co.). The culture medium was M199 (Gibco Laboratories) containing 20% FCS (Sigma Chemical Co.), 0.20 mg/ml gentamycin (Schering), and 1.75 mg/ml fungizone (Flow Laboratories). After confluent, the primary endothelial cell culture was used in the angiogenic assay.
2.2. Endometrial angiogenis assay Endometrial biopsies were taken using a microhysteroscope. Endometrial samples were incubated with RPMI (Flow Laboratories) as controls or vitamin E 20 mg/l RPMI for 60 min prior to angiogenic measurement. Endometrial angiogenic response was assayed using an endothelial cell migration assay. Explants were cultured utilising a three-dimensional collagen matrix containing suspended endothelial cells (Folkman et al., 1989; modified by Rogers et al., 1992). Endothelial cells were suspended in NCTC (Flow Laboratories) with FCS 20%, gentamycin and fungizone. The medium was adjusted with 0.1 M NaOH to reach a pH of 7.4 after the addition of chilled vitrogen 100 (Collagen Corporation). Explants (approximately 500 mm in diameter) were cultured for 96 h and observed at 24-h intervals using an inverse phase-contrast microscope. The angiogenic score ranged from 0 to 4: score 0 represents no endothelial cell migration, score 1 represents elongation and some orientation toward explant, score 2 represents minimal spokewheel response, score 3 represents medium spokewheel response, and score 4 represents extreme spokewheel formation.
2.3. Clinical trial Subjects were Norplant users with bleeding problems. Vitamin E 200 mg, daily (n= 38) or placebo (n= 34) for 10 days were given to subjects under double blind randomisation and repeated after 30 days. Subjects were kept on
S.B. Subakir et al. / Toxicology 148 (2000) 173–178
menstrual diary card. Assessment of treatment effectiveness was based on the analysis of menstrual diaries using the reference period method of analysis.
2.4. Statistical analysis Differences in endometrial score between different groups were analysed using the Mann – Whitney test. Spearman’s rank correlation was used to calculate the correlation between endometrial angiogenic activity and blood levels of lipid peroxides. Differences of bleeding days between control group (placebo) and treatment group (vitamin E) were analysed using Student’s t-test.
3. Results and discussion Blood levels of lipid peroxides from Norplant users with bleeding problems were significantly higher than in the control group (X9 S.D.: 8.06 92.74 vs. 5.64 91.91 mmol/l, P 5 0.01; Fig. 1). The increased concentration of lipid peroxides may be caused by elevated radical chain reactions in lipid membranes.
175
Lipid peroxide concentrations did not clearly correlate with endometrial angiogenic activity (Fig. 2, controls), however, higher lipid peroxide concentrations tend to have a lower score, especially in Norplant users with bleeding problems (Fig. 2). Furthermore, comparison of Norplant users with angiogenic score 0 and score 1.5 in Fig. 2 shows a significant difference of 45% in their levels of lipid peroxides. Although there may not be a clear correlation between lipid peroxide concentrations and endometrial angiogenic scores in general, lower angiogenic scores appear to correlate with higher lipid peroxide concentrations in Norplant users with bleeding problems. Recently, White et al. (1997) observed an increased epoxide formation via prostaglandin synthesis, which correlated with ruptures of blood vessels in the endometrium of levonorgestrel contraceptive users. In our study, addition of vitamin E/RPMI (in vitro) to the endometrial explants from Norplant users with bleeding problems resulted in a significantly higher angiogenic score than addition of RPMI without vitamin E (z= −2.24, 2 tailed, P 5 0.05; Fig. 3). In both groups, the angiogenic score ranged from 0 to 2.
Fig. 1. Blood levels of lipid peroxides in control group and Norplant users. Blood levels of lipid peroxides were measured as thiobarbituric acid reactive substances (TBARS), especially malonaldehyde (MDA), in controls with normal pregnancies (n = 23), in Norplant users with bleeding problems (n= 36), and in Norplant users without bleeding problems (n = 7).
176
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Fig. 2. Blood levels of lipid peroxides and endometrial angiogenic activity in Norplant users with bleeding problems and controls. Angiogenic activity was determined from endometrial explants, the score ranged from 0 to 2.
Fig. 3. Effect of in vitro supplementation of vitamin E 20 mg/l RPMI (vitamin E) and RPMI without vitamin E (placebo) on the in vitro angiogenic activity in cultured explants from the endometrium of Norplant users with bleeding problems. Endometrial angiogenic score ranged from 0 to 2.
Thus, in vitro incubation showed that the antioxidant vitamin E can increase endothelial cell migratory signal production and angiogenic activity in endometrial explants from Norplant users with bleeding problems. Other investigators found that vitamin E can inhibit free radical formation and can protect endothelial cell cultures from cell
damage (Matsuo and Kaneko, 1990; Niki, 1990; Faruqi et al., 1994). It is concluded that in Norplant users with bleeding problems, higher lipid peroxide levels correlate with lower endometrial angiogenic scores and it is suggested that vitamin E supplementation may be effective to increase endome-
S.B. Subakir et al. / Toxicology 148 (2000) 173–178
177
Fig. 4. Effect of supplementation with 200 mg/day vitamin E (vitamin E) on the number of bleeding days in Norplant users with bleeding problems vs. those receiving placebo only (placebo). Vitamin E and placebo were administered double blinded for 10 days (cycle 1) and administration for another 10 days repeated after 30 days (cycle 2).
trial angiogenic activity and to reduce bleeding problems in Norplant users. This needs to be confirmed in more extended clinical trials. In our preliminary clinical study, before treatment, the vitamin E group and the placebo group had no significant difference in the number of bleeding days (18.1 91.0 vs. 20 9 1.13). In the initial 30 days from the start of treatment, this number fell to 7.791.4 days in the vitamin E group and to 12.1 9 1.3 days in the placebo group (Fig. 4). A highly significant effect is evident of both placebo and vitamin E to reduce the number of bleeding days, however, the number was also significantly lower in women treated with vitamin E as compared to placebo and this difference was maintained also in the second cycle of treatment (Fig. 4).
Acknowledgements This study was supported by WHO (HRP 91409) and Department of Education and Culture of the Republic of Indonesia.
References Faruqi, R., de la Motte, C., Dicorleto, P.E., 1994. Alfa-tocopherol inhibits agonist-induced monocytic cell adhesion to cultured human endothelial cells. J.Clin. Invest. 94, 592 – 600. Folkman, J., Watson, K., Ingber, D., Hanahan, D., 1989. Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature 239, 58 – 61. Halliwell, B., Gutteridge, J.M.C., Cross, E., 1992. Review article: free radicals, antioxidants and human disease: where are we now? J. Lab. Clin. Med. 119, 598 – 620. Jaffe, E.A., Nachman, R.L., Becker, C.G., Minick, C.R., 1973. Culture of human endothelial cells derived from umbelical vein. J. Clin. Invest. 52, 2745 – 2756. Matsuo, M., Kaneko, T., 1990. Protective effect of vitamin E on linoleic acid hydroperoxide-induced injury to human umbilical vein endothelial cells. In: Kelvin, J.A.D. (Ed.), Oxidative Damage and Repair: Chemical, Biological and Medical Aspects. Pergamon, Oxford, pp. 584 – 588. Niki, E., 1990. Antioxidant compounds. In: Kelvin, J.A.D. (Ed.), Oxidative Damage and Repair: Chemical, Biological and Medical Aspects. Pergamon, Oxford, pp. 57 – 64. Packer, L., Landvik, S., 1989. Vitamin E: introduction to biochemistry and health benefits. In: Diplock, A.T., Machlin, J.L., Pryor, W.A. (Eds.), Vitamin E, Biochemistry and Health Implications. New York Academy of Sciences, New York, pp. 1 – 6. Rice-Evans, C., Cooke, B., 1990. Oxygen radicals, bleeding and tissue injury. In: d’Arcangues, C., Fraser, I.S., New-
178
S.B. Subakir et al. / Toxicology 148 (2000) 173–178
ton, J.R., Odlind, V. (Eds.), Contraception and Mechanisms of Endometrial Bleeding. Cambridge University Press, Cambridge, pp. 411–430. Rogers, P.A.W., Abberton, K.M., Susil, B., 1992. Endothelial cell migratory signal production produced by human endometrium during menstrual cycle. Hum. Reprod. 7, 1061 – 1068. Subakir, S.B., Hadisaputra, W., Siregar, B., Soeria Santoso, D.I., Cornain, S., Affandi, B., 1996. Reduced endothelial
cell migratory signal production by endometrial explants from women using Norplant contraception. Hum. Reprod. 10, 2579 – 2583. White, J.O., Sulivan, M.H.F., Patel, L., Croxtail, J.D., d’Arcangues, C., Belsey, E.M., Elder, M.G., 1997. Prostaglandin production in human endometrium following continuous exposure to low-dose levonorgestrel release from a vaginal ring. Contraception 43, 401 – 412.
.