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Enhanced Sister-Chromatid Exchange Rate in Human Lymphocytes Exposed to 17β Estradiol in Vitro
Archives of Medical Research 33 (2002) 148–151
ORIGINAL ARTICLE
Enhanced Sister-Chromatid Exchange Rate in Human Lymphocytes Exposed to 17 Estradiol in Vitro Ninoslav Djelic and Dijana Djelic Department of Biology, Faculty of Veterinary Medicine, University of Belgrade, Belgrade, Serbia, Yugoslavia Received for publication March 9, 2001; accepted August 31, 2001 (01/039).
Background. Epidemiologic data and animal experiments strongly implicate that steroid hormones are involved in the process of malignant transformation due to their capability to stimulate mitotic division and/or elevate the level of mutations in susceptible cells. Methods. The objectives of this investigation were to evaluate the effects of 17 estradiol in sister-chromatid exchange (SCE) test on cultured human peripheral blood lymphocytes. The lowest concentration of 17 estradiol used in this experiment (1010 M) was calculated as comparable with the physiologic blood level of 17 estradiol in women. Three experimental concentrations corresponded to minimal (7 108 M), average (3.5 106 M), and maximal (7 106 M) therapeutic doses in human medicine. In addition, the highest concentrations exceed maximal therapeutic dose 10-fold (7 105 M) and 30fold (2.1 104 M), respectively. Results. The obtained results indicate that estradiol significantly elevates SCE per cell frequency at all concentrations applied except at the lowest one. However, estradiol has not influenced mitotic activity of cultured human lymphocytes significantly. Conclusions. It can be concluded that 17 estradiol expressed genotoxic effects and therefore might represent a human health risk. © 2002 IMSS. Published by Elsevier Science Inc. Key Words: Estradiol, Genotoxicity, Sister-chromatid exchange, Cancer, Lymphocyte.
Introduction Genotoxicologic characterization of hormones and investigations of their relevance to chemically induced mutagenesis represent a very complex field of study. The main problem in understanding the mechanisms of hormonal mutagenesis results from the fact that hormones are compounds normally present in human and animal bodies. Therefore, it is unlikely to expect that natural selection in the process of evolution would have allowed the presence of agents capable of disturbing genetic integrity (1). On the other hand, a low level of mutations is compatible with survival and to a greater degree contributes to changes of population genetic structure in long-term periods. Mutations may
Address reprint requests to: Ninoslav Djelic, Ph.D., Department of Biology, Faculty of Veterinary Medicine, University of Belgrade, Bul. JNA #18, Belgrade, 11000, Serbia, Yugoslavia. Phone and fax: (381) (11) 685-936; E-mail: [email protected]
change one form of gene into another. Afterward, natural selection favors mutant alleles that have average beneficial effect to better adaptation to environmental changes. Epidemiologic data and animal experiments strongly implicate the influence of sexual steroids in the development of certain forms of cancer (2). Although somewhat simplified, a three-stage model of carcinogenesis (initiation-promotion-progression) implies multistage formation of malignant tumors. It has been revealed that steroid hormones may act as complete carcinogens (3–5) capable not only of stimulating mitosis (tumor promoters) but also of elevating mutation rate in susceptible cells (tumor initiators). These findings shed new light on analysis of the relevance of hormones in malignant transformation. The objective of the present study was to evaluate sisterchromatid exchange (SCE) rate in cultured human lymphocytes exposed to 17 estradiol. Experimental concentrations of estradiol were calculated to correspond to a wide range of concentrations in human blood-physiologic level, therapeutic range, and up to 30-fold maximal therapeutic concentra-
0188-4409/02 $–see front matter. Copyright © 2002 IMSS. Published by Elsevier Science Inc. PII S0188-4409(01)00 3 5 5 - 1
Sister-Chromatid Exchange and Estradiol
tion. Thus, the investigation should contribute to a better understanding of genetic risk under a wide range of estradiol concentrations. Materials and Methods Human peripheral blood lymphocyte cultures were set up according to a slight modification of the protocol described by Evans and O’Riordan (6). Heparinized whole blood samples (0.8 mL) obtained from two healthy men 35 years of age were added to vials with 9.2 mL of Parker 199 medium containing 30% of inactivated calf serum (Serva, Heidelberg, Germany) and 0.04 mg/mL of phytohemagglutinin (Murex Diagnostics, Ltd., Dartford, UK). With the aim of providing successive visualization of SCEs, 5-bromo-2deoxyuridine (BrdUrd, Sigma Chemical Co., St. Louis, MO, USA, final concentration 25 M) was added 1 h after culture initiation. Two cultures per donor were incubated in complete darkness for 72 h at 37 C. To determine experimental concentrations of 17 estradiol that can be comparable to the blood level of estradioltreated women, we consulted textbooks on pharmacology (7,8). Additionally, considering characteristic 17 estradiol pharmacokinetics data (9), the expected amount of bloodstream estradiol following single-dose injection of 17 estradiol was divided by 5,000 mL (average blood volume in adult women). Thus, the concentration of 1010 M was calculated to correspond to the physiologic level of 17 estradiol in human blood. The concentration of 7 108 M is comparable to the blood level following minimal therapeutic dose of 17 estradiol, 3.5 106 M to the average, and 7 106 M to the maximal therapeutic dose in human medicine. Finally, 7 105 M and 2.1 104 M correspond to 10-fold and 30-fold maximal therapeutic doses, respectively. Exactly 47 h and 30 min after the beginning of incubation, 17 estradiol (CAS no. 50-28-2, Sigma) dissolved in dimethylsulfoxide (DMSO, Sigma) was added to the cultivation vials in amounts to obtain final experimental concentrations of 1010 M, 7 108 M, 3.5 106 M, 7 106 M, 7 105 M, and 2.1 104 M. The solvent (DMSO) was used as the negative control, whereas 106 M N-methyl-Nnitro-N-nitrosoguanidine (MNNG, Sigma) was the positive control. Two hours prior to harvesting, colcemid (CIBA) was added to the cultures to achieve a final concentration of 0.5 g/mL. After hypotonic treatment (0.075 M KCl) followed by three repetitive cycles of fixation in methanol/acetic acid solution (3:1, v/v), centrifugation, and resuspension, the cell suspension was dropped onto chilled, grease-free microscopic slides, air-dried, aged, and then differentially stained for the inspection of SCE rate according to fluorescence plus Giemsa (FPG) procedure (10). For each donor, 60 well-spread mitoses were scored, and the values obtained were calculated as SCEs per cell.
149
Experimental data were analyzed using one-way analysis of variance (ANOVA) to determine whether any treatment significantly differed from the controls and/or each other. Significant differences between the controls and/or treated samples were confirmed by Fisher’s least significant difference and Student’s t tests. Results In addition to the genotoxicologic examination, it is possible to obtain valuable data concerning mitotic activity on the same microscopic slides used for cytogenetic analysis. Values of mitotic index (MI, percentage of cells in mitosis) as a parameter used to determine possible cytotoxic or mitogenic effects are presented in Table 1. After treatment with negative control (DMSO), 5.34% of analyzed cells were found in mitosis. Positive control lowered MI to 3.54% (p 0.01). The experimental values of MI in cultures treated with 17 estradiol are in the range of 4.1–6.5%. Evidently, estradiol has not caused significant departures in MI values (p 0.05), compared to controls. With an aim to ascertain genotoxic effects, we analyzed sister-chromatid exchanges (SCEs) in human lymphocytes treated with estradiol in vitro (Figure 1). The results obtained are presented in Table 1. ANOVA statistical analysis showed significant variation among tested groups (untreated, negative and positive controls, and six experimental concentrations of 17 estradiol) (F 108.679; p 1.42 10132). Comparisons of the negative control and treated groups, as well as negative control and positive control, revealed that only the concentration of estradiol comparable to physiologic level has not influenced SCE rate significantly. Control value of SCE per cell frequency obtained after analysis of metaphase spreads in cultures treated with negative control amounts to 5.73 0.35. Treatment with positive control (MNNG) significantly (p 0.001) elevated SCE rate to 13.77 1.06. As for estradiol, SCE rate did not change significantly only at the lowest concentration used Table 1. Influence of 17 estradiol on SCE per-cell frequency in cultures of human peripheral blood lymphocytes
Treatment Untreated Negative control Positive control Estradiol, 1010 M Estradiol, 7 108 M Estradiol, 3.5 106 M Estradiol, 7 106 M Estradiol, 7 105 M Estradiol, 2.1 104 M
MI
SCE range
Mean value/SCE
SD
Xk
5.98 5.34 3.54b 4.91 5.17 6.29 6.48 4.37 4.08
310 311 729 410 412 512 411 312 515
5.87 5.73 13.77c 6.20 6.90a 7.03a 6.83a 7.37b 8.17c
1.80 2.00 5.60 1.46 2.03 1.59 1.77 2.09 2.24
100 97.61 234.58 105.62 117.55 119.76 116.35 125.55 139.18
MI, mitotic index; ap 0.05; bp 0.01; cp 0.001 (ANOVA, Fisher’s least significant difference, and Student’s t tests); SD, standard deviation; Xk, percentage of mean value of SCE of untreated culture.
150
Djelic and Djelic/ Archives of Medical Research 33 (2002) 148–151
Figure 1. Metaphase spread with differentially stained sister-chromatids (FPG technique) from lymphocytes treated with 2.1 104 M of 17 estradiol.
in these experiments. However, even the concentration that corresponds to minimal therapeutic dose in human medicine (7 108 M) induced significant (p 0.05) increase of SCE per-cell frequency (SCE rate 6.90 0.39; 20% more than the negative control). Concentrations comparable to average and maximal therapeutic doses elevated SCE percell frequency by 22.7% and 19.2%, respectively (p 0.05). Following treatment of cultured lymphocytes with concentrations comparable to 10-fold maximal therapeutic dose, SCE rate was elevated by 28.6% (p 0.01). Finally, at the highest concentration of estradiol (2.1 104 M) SCE rate of 8.17 0.4 was 42.6% higher (p 0.001) in comparison to the negative control. These findings indicate that estradiol is capable of increasing SCE per-cell frequency significantly in cultured human lymphocytes at concentrations that correspond to estradiol level in blood of treated patients.
Discussion Undoubtedly, hormones are at present considered as possible endogenous mutagens. Among all hormones, the beststudied group is sexual steroids, probably because the earliest endocrinologic and epidemiologic studies suggested a possible role of estrogens in carcinogenesis (11,12). In the present study, a wide range of 17 estradiol concentrations was used to test the capability of this hormone to induce sister-chromatid exchanges (SCEs). An experimental concentration similar to physiologic level of 17 estradiol in women (1010 M) has not elevated SCE per-cell frequency compared to both untreated cultures and cultures treated with negative control (the solvent, DMSO). In light of this experimental data, there is no concern that, at physiologic range, 17 estradiol may be directly or indirectly involved in DNA damage leading to cytogenetically detectable SCEs. It should be noticed, however, that a concentra-
tion of 7 108 M estradiol, calculated to reflect blood concentration following minimal therapeutic dose in human medicine, expressed some genotoxic effects. The same level of statistical significance was found after treatment with concentrations comparable to minimal and maximal therapeutic doses of 17 estradiol. Finally, when concentrations exceeded 10-fold maximal therapeutic dose the average level of SCEs per cell was 7.37 0.45 (p 0.01). The highest concentration used (2.1 104 M) exhibited a stronger effect on SCE rate elevation (p 0.001). The results of the present study provide some insight into responsiveness of human lymphocytes to 17 estradiol measured by SCE per-cell frequency as genotoxic endpoint. It is particularly interesting that even the minimal therapeutic dose in human medicine might be expected to cause DNA damage. However, in cultures treated with concentrations corresponding to minimal therapeutic dose, SCE per-cell frequency was elevated 17.5% more than untreated cultures. Although significant (p 0.05), this enhancement of SCE rate is relatively low because positive control (MNNG) caused 135.6% elevation of SCE per-cell frequency. Therefore, 17 estradiol acted as a potentially weak mutagen. Experimental findings obtained in this study are mainly in accordance with previous investigations of genotoxic effects of estrogens. Standard genotoxicologic test protocols revealed mutagenic effects even in some bacterial systems (13), gene mutation test on Chinese hamster V79 cells (14), various cytogenetically analyzed cell lines (15–18), leukocytes of women on oral contraceptives (19), and postmenopausal patients (20,21). Interestingly, Ivett and Tice (22) observed that nonsteroid estrogen diethylstilbestrol (DES) induces chromosome aberrations in bone marrow cells of in vivo treated mice, though SCE per-cell level remained unchanged. It is noteworthy that results of SCE in vivo test are difficult to compare with those obtained in vitro, because effect of mutagen may depend on type of cell line used. To determine possible cytotoxic, cytostatic, or mitogenic effects of 17 estradiol, we analyzed the mitotic index (MI) for each experimental concentration as well as for controls. The results showed that 17 estradiol did not cause significant departures of MI values in relation to the negative control. As expected, positive control (MNNG) decreased MI by 34% compared to negative control, possibly due to arrest of mitosis because of repair of genetic material. Additionally, in cells with a relatively high level of genetic damage cytotoxic effects occur. Finally, the question remains of what current knowledge exists concerning molecular mechanisms of estrogen mutagenesis. Although it is quite obvious that estrogens induce mutations, standard genotoxicologic tests usually reveal the specific change but not the mechanism of the appearance of estrogen mutagenesis. Development of modern techniques has partially answered this question. One of the most frequently used techniques is 32P-postlabeling (23). Use of 32P-
Sister-Chromatid Exchange and Estradiol
postlabeling is convenient because this method does not require prior knowledge of adduct structures. Following chronic treatment of male Syrian hamsters with diethylstilbestrol, Roy et al. (24) discovered chemically modified nucleotide 8-hydroxydeoxyguanosine. The most convincing explanation for the appearance of DNA adducts is that at elevated concentrations, metabolic reactions of estrogens leading to formation of reactive free radicals may become predominant biochemical activity, overshadowing their hormonal effects (25,26). The susceptibility of the target cell to create favorable conditions for the appearance of DNA covalent modifications depends on the level of enzymatic and nonenzymatic defense systems involved in protection from oxidant stress (27,28). Acknowledgments This investigation was supported by the Serbian Ministry for Science and Technology, grant #12M18.
References 1. Djelic N. Cytogenetic effects of estradiol, thyroxine, insulin and epinephrine on human lymphocytes in vitro (PhD thesis). Belgrade, Serbia, Yugoslavia: Faculty of Biology, University of Belgrade;1997. 2. Henderson BE, Ross RK, Pike MC, Casagrande JT. Endogenous hormones as a major factor in human cancer. Cancer Res 1982;42:3232– 3239. 3. Liehr JG. Genotoxic effects of estrogens. Mutat Res 1990;238:269– 276. 4. Bhat HK, Han X, Gladek A, Liehr J. Regulation of the formation of the major diethylstilbestrol-DNA adduct and some evidence of its structure. Carcinogenesis 1994;15:2137–2142. 5. Winter M, Liehr J. Possible mechanism of induction of benign prostatic hyperplasia by estradiol and dihydrotestosterone in dogs. Toxicol Appl Pharmacol 1996;136:211–219. 6. Evans HJ, O’Riordan ML. Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. Mutat Res 1975;31:135–148. 7. Goldfein A. The gonadal hormones and inhibitors. In: Kartzung BG, editor. Basic and clinical pharmacology. 6th ed. East Norwalk, CT, USA: Appleton and Lange;1995. pp. 608–615. 8. Reynolds JEF, editor. The extra pharmacopoeia. 31st ed. London: Royal Pharmaceutical Society;1996. pp. 1501–1502. 9. Stumpf PG. Pharmacokinetics of estrogen. Obstet Gynecol 1990;75: 9S–14S. 10. Perry P, Wolff S. New Giemsa method for the differential staining of sister chromatids. Nature 1974;251:156–158.
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11. Adams J. Enzymatic synthesis of steroid sulfates. II. Presence of steroid sulfokinase in human mammary carcinoma extracts. J Clin Endocrinol 1964;24:988–996. 12. Huggins C. Endocrine-induced regression of cancers. Science 1967; 156:1050–1054. 13. van Aswegen CH, Nieuwoudt LB, van Rensburg HGJ, Steyn PL, du Plessis DJ. Estradiol and catecholestrogens as possible genotoxic carcinogens. Clin Physiol Biochem 1989;7:34–39. 14. Kong LY, Szaniszlo P, Albrecht T, Liehr JG. Frequency and molecular analysis of hprt mutations induced by estradiol in Chinese hamster V79 cells. Int J Oncol 2000;17:1141–1149. 15. Sawada M, Ishidate M Jr. Colchicine-like effect of diethylstilbestrol (DES) on mammalian cells in vitro. Mutat Res 1989;57:175–182. 16. Kochhar TS. Inducibility of chromosome aberrations by steroid hormones in cultured Chinese hamster ovary cells. Toxicol Lett 1985;29: 201–206. 17. Kochhar TS. Steroid hormones enhanced sister-chromatid exchange in cultured CHO cells. Experientia 1988;44:62–63. 18. Bukvic N, Susca F, Burvic D, Fanelli M, Guanti G. 17-alpha-ethynilestradiol and norgestrel in combination induce micronucleus increases and aneuploidy in human lymphocyte and fibroblast cultures. Teratog Carcinog Mutagen 2000;20:147–159. 19. Goh KO. Chromosomal breaks in women taking birth control pills. USAEC-ORAU Res Report 1967;106:97–104. 20. Sahin FI, Sahin I, Ergun MA, Saracoglu OF. Effects of estrogen and alendronate on sister chromatid exchange (SCE) frequencies in postmenopausal osteoporosis patients. Int J Gynaecol Obstet 2000;71:49– 52. 21. Kayikcioglu F, Gunes M, Baltaci V, Kocak M, Alpas I, Haberal A. Sister-chromatid exchange frequencies in postmenopausal hormone replacement patients. Mutat Res 2000;452:37–39. 22. Ivett JL, Tice RR. Diethylstilbestrol-diphosphate induces chromosomal aberrations but not sister chromatid exchanges in murine bone marrow cells in vivo. Environ Mutagen 1981;3:445–452. 23. Randerath K, Randerath E. 32P-postlabeling methods for DNA adduct detection: overview and critical evaluation. Drug Metab Rev 1994;26: 67–85. 24. Roy D, Floyd RA, Liehr JG. Elevated 8-hydroxydeoxyguanosine levels in DNA of diethylstilbestrol-treated Syrian hamsters: covalent DNA damage by free radicals generated by redox cycling of diethylstilbestrol. Cancer Res 1991;51:3882–3885. 25. Liehr JG, Roy D. Free radical generation by redox cycling of estrogens. Free Radic Biol Med 1990;8:415–423. 26. Liehr JG. Is estradiol a genotoxic mutagenic carcinogen? Endocr Rev 2000;21:40–54. 27. Roy D, Liehr JG. Changes in activities of free radical detoxifying enzymes in kidneys of male Syrian hamsters treated with estradiol. Cancer Res 1989;49:1475–1480. 28. Karbownik M, Reiter RJ, Cabrera J, García JJ. Comparison of the protective effect of melatonin with other antioxidants in the hamster kidney model of estradiol-induced DNA damage. Mutat Res 2001;474: 87–92.
ORIGINAL ARTICLE
Enhanced Sister-Chromatid Exchange Rate in Human Lymphocytes Exposed to 17 Estradiol in Vitro Ninoslav Djelic and Dijana Djelic Department of Biology, Faculty of Veterinary Medicine, University of Belgrade, Belgrade, Serbia, Yugoslavia Received for publication March 9, 2001; accepted August 31, 2001 (01/039).
Background. Epidemiologic data and animal experiments strongly implicate that steroid hormones are involved in the process of malignant transformation due to their capability to stimulate mitotic division and/or elevate the level of mutations in susceptible cells. Methods. The objectives of this investigation were to evaluate the effects of 17 estradiol in sister-chromatid exchange (SCE) test on cultured human peripheral blood lymphocytes. The lowest concentration of 17 estradiol used in this experiment (1010 M) was calculated as comparable with the physiologic blood level of 17 estradiol in women. Three experimental concentrations corresponded to minimal (7 108 M), average (3.5 106 M), and maximal (7 106 M) therapeutic doses in human medicine. In addition, the highest concentrations exceed maximal therapeutic dose 10-fold (7 105 M) and 30fold (2.1 104 M), respectively. Results. The obtained results indicate that estradiol significantly elevates SCE per cell frequency at all concentrations applied except at the lowest one. However, estradiol has not influenced mitotic activity of cultured human lymphocytes significantly. Conclusions. It can be concluded that 17 estradiol expressed genotoxic effects and therefore might represent a human health risk. © 2002 IMSS. Published by Elsevier Science Inc. Key Words: Estradiol, Genotoxicity, Sister-chromatid exchange, Cancer, Lymphocyte.
Introduction Genotoxicologic characterization of hormones and investigations of their relevance to chemically induced mutagenesis represent a very complex field of study. The main problem in understanding the mechanisms of hormonal mutagenesis results from the fact that hormones are compounds normally present in human and animal bodies. Therefore, it is unlikely to expect that natural selection in the process of evolution would have allowed the presence of agents capable of disturbing genetic integrity (1). On the other hand, a low level of mutations is compatible with survival and to a greater degree contributes to changes of population genetic structure in long-term periods. Mutations may
Address reprint requests to: Ninoslav Djelic, Ph.D., Department of Biology, Faculty of Veterinary Medicine, University of Belgrade, Bul. JNA #18, Belgrade, 11000, Serbia, Yugoslavia. Phone and fax: (381) (11) 685-936; E-mail: [email protected]
change one form of gene into another. Afterward, natural selection favors mutant alleles that have average beneficial effect to better adaptation to environmental changes. Epidemiologic data and animal experiments strongly implicate the influence of sexual steroids in the development of certain forms of cancer (2). Although somewhat simplified, a three-stage model of carcinogenesis (initiation-promotion-progression) implies multistage formation of malignant tumors. It has been revealed that steroid hormones may act as complete carcinogens (3–5) capable not only of stimulating mitosis (tumor promoters) but also of elevating mutation rate in susceptible cells (tumor initiators). These findings shed new light on analysis of the relevance of hormones in malignant transformation. The objective of the present study was to evaluate sisterchromatid exchange (SCE) rate in cultured human lymphocytes exposed to 17 estradiol. Experimental concentrations of estradiol were calculated to correspond to a wide range of concentrations in human blood-physiologic level, therapeutic range, and up to 30-fold maximal therapeutic concentra-
0188-4409/02 $–see front matter. Copyright © 2002 IMSS. Published by Elsevier Science Inc. PII S0188-4409(01)00 3 5 5 - 1
Sister-Chromatid Exchange and Estradiol
tion. Thus, the investigation should contribute to a better understanding of genetic risk under a wide range of estradiol concentrations. Materials and Methods Human peripheral blood lymphocyte cultures were set up according to a slight modification of the protocol described by Evans and O’Riordan (6). Heparinized whole blood samples (0.8 mL) obtained from two healthy men 35 years of age were added to vials with 9.2 mL of Parker 199 medium containing 30% of inactivated calf serum (Serva, Heidelberg, Germany) and 0.04 mg/mL of phytohemagglutinin (Murex Diagnostics, Ltd., Dartford, UK). With the aim of providing successive visualization of SCEs, 5-bromo-2deoxyuridine (BrdUrd, Sigma Chemical Co., St. Louis, MO, USA, final concentration 25 M) was added 1 h after culture initiation. Two cultures per donor were incubated in complete darkness for 72 h at 37 C. To determine experimental concentrations of 17 estradiol that can be comparable to the blood level of estradioltreated women, we consulted textbooks on pharmacology (7,8). Additionally, considering characteristic 17 estradiol pharmacokinetics data (9), the expected amount of bloodstream estradiol following single-dose injection of 17 estradiol was divided by 5,000 mL (average blood volume in adult women). Thus, the concentration of 1010 M was calculated to correspond to the physiologic level of 17 estradiol in human blood. The concentration of 7 108 M is comparable to the blood level following minimal therapeutic dose of 17 estradiol, 3.5 106 M to the average, and 7 106 M to the maximal therapeutic dose in human medicine. Finally, 7 105 M and 2.1 104 M correspond to 10-fold and 30-fold maximal therapeutic doses, respectively. Exactly 47 h and 30 min after the beginning of incubation, 17 estradiol (CAS no. 50-28-2, Sigma) dissolved in dimethylsulfoxide (DMSO, Sigma) was added to the cultivation vials in amounts to obtain final experimental concentrations of 1010 M, 7 108 M, 3.5 106 M, 7 106 M, 7 105 M, and 2.1 104 M. The solvent (DMSO) was used as the negative control, whereas 106 M N-methyl-Nnitro-N-nitrosoguanidine (MNNG, Sigma) was the positive control. Two hours prior to harvesting, colcemid (CIBA) was added to the cultures to achieve a final concentration of 0.5 g/mL. After hypotonic treatment (0.075 M KCl) followed by three repetitive cycles of fixation in methanol/acetic acid solution (3:1, v/v), centrifugation, and resuspension, the cell suspension was dropped onto chilled, grease-free microscopic slides, air-dried, aged, and then differentially stained for the inspection of SCE rate according to fluorescence plus Giemsa (FPG) procedure (10). For each donor, 60 well-spread mitoses were scored, and the values obtained were calculated as SCEs per cell.
149
Experimental data were analyzed using one-way analysis of variance (ANOVA) to determine whether any treatment significantly differed from the controls and/or each other. Significant differences between the controls and/or treated samples were confirmed by Fisher’s least significant difference and Student’s t tests. Results In addition to the genotoxicologic examination, it is possible to obtain valuable data concerning mitotic activity on the same microscopic slides used for cytogenetic analysis. Values of mitotic index (MI, percentage of cells in mitosis) as a parameter used to determine possible cytotoxic or mitogenic effects are presented in Table 1. After treatment with negative control (DMSO), 5.34% of analyzed cells were found in mitosis. Positive control lowered MI to 3.54% (p 0.01). The experimental values of MI in cultures treated with 17 estradiol are in the range of 4.1–6.5%. Evidently, estradiol has not caused significant departures in MI values (p 0.05), compared to controls. With an aim to ascertain genotoxic effects, we analyzed sister-chromatid exchanges (SCEs) in human lymphocytes treated with estradiol in vitro (Figure 1). The results obtained are presented in Table 1. ANOVA statistical analysis showed significant variation among tested groups (untreated, negative and positive controls, and six experimental concentrations of 17 estradiol) (F 108.679; p 1.42 10132). Comparisons of the negative control and treated groups, as well as negative control and positive control, revealed that only the concentration of estradiol comparable to physiologic level has not influenced SCE rate significantly. Control value of SCE per cell frequency obtained after analysis of metaphase spreads in cultures treated with negative control amounts to 5.73 0.35. Treatment with positive control (MNNG) significantly (p 0.001) elevated SCE rate to 13.77 1.06. As for estradiol, SCE rate did not change significantly only at the lowest concentration used Table 1. Influence of 17 estradiol on SCE per-cell frequency in cultures of human peripheral blood lymphocytes
Treatment Untreated Negative control Positive control Estradiol, 1010 M Estradiol, 7 108 M Estradiol, 3.5 106 M Estradiol, 7 106 M Estradiol, 7 105 M Estradiol, 2.1 104 M
MI
SCE range
Mean value/SCE
SD
Xk
5.98 5.34 3.54b 4.91 5.17 6.29 6.48 4.37 4.08
310 311 729 410 412 512 411 312 515
5.87 5.73 13.77c 6.20 6.90a 7.03a 6.83a 7.37b 8.17c
1.80 2.00 5.60 1.46 2.03 1.59 1.77 2.09 2.24
100 97.61 234.58 105.62 117.55 119.76 116.35 125.55 139.18
MI, mitotic index; ap 0.05; bp 0.01; cp 0.001 (ANOVA, Fisher’s least significant difference, and Student’s t tests); SD, standard deviation; Xk, percentage of mean value of SCE of untreated culture.
150
Djelic and Djelic/ Archives of Medical Research 33 (2002) 148–151
Figure 1. Metaphase spread with differentially stained sister-chromatids (FPG technique) from lymphocytes treated with 2.1 104 M of 17 estradiol.
in these experiments. However, even the concentration that corresponds to minimal therapeutic dose in human medicine (7 108 M) induced significant (p 0.05) increase of SCE per-cell frequency (SCE rate 6.90 0.39; 20% more than the negative control). Concentrations comparable to average and maximal therapeutic doses elevated SCE percell frequency by 22.7% and 19.2%, respectively (p 0.05). Following treatment of cultured lymphocytes with concentrations comparable to 10-fold maximal therapeutic dose, SCE rate was elevated by 28.6% (p 0.01). Finally, at the highest concentration of estradiol (2.1 104 M) SCE rate of 8.17 0.4 was 42.6% higher (p 0.001) in comparison to the negative control. These findings indicate that estradiol is capable of increasing SCE per-cell frequency significantly in cultured human lymphocytes at concentrations that correspond to estradiol level in blood of treated patients.
Discussion Undoubtedly, hormones are at present considered as possible endogenous mutagens. Among all hormones, the beststudied group is sexual steroids, probably because the earliest endocrinologic and epidemiologic studies suggested a possible role of estrogens in carcinogenesis (11,12). In the present study, a wide range of 17 estradiol concentrations was used to test the capability of this hormone to induce sister-chromatid exchanges (SCEs). An experimental concentration similar to physiologic level of 17 estradiol in women (1010 M) has not elevated SCE per-cell frequency compared to both untreated cultures and cultures treated with negative control (the solvent, DMSO). In light of this experimental data, there is no concern that, at physiologic range, 17 estradiol may be directly or indirectly involved in DNA damage leading to cytogenetically detectable SCEs. It should be noticed, however, that a concentra-
tion of 7 108 M estradiol, calculated to reflect blood concentration following minimal therapeutic dose in human medicine, expressed some genotoxic effects. The same level of statistical significance was found after treatment with concentrations comparable to minimal and maximal therapeutic doses of 17 estradiol. Finally, when concentrations exceeded 10-fold maximal therapeutic dose the average level of SCEs per cell was 7.37 0.45 (p 0.01). The highest concentration used (2.1 104 M) exhibited a stronger effect on SCE rate elevation (p 0.001). The results of the present study provide some insight into responsiveness of human lymphocytes to 17 estradiol measured by SCE per-cell frequency as genotoxic endpoint. It is particularly interesting that even the minimal therapeutic dose in human medicine might be expected to cause DNA damage. However, in cultures treated with concentrations corresponding to minimal therapeutic dose, SCE per-cell frequency was elevated 17.5% more than untreated cultures. Although significant (p 0.05), this enhancement of SCE rate is relatively low because positive control (MNNG) caused 135.6% elevation of SCE per-cell frequency. Therefore, 17 estradiol acted as a potentially weak mutagen. Experimental findings obtained in this study are mainly in accordance with previous investigations of genotoxic effects of estrogens. Standard genotoxicologic test protocols revealed mutagenic effects even in some bacterial systems (13), gene mutation test on Chinese hamster V79 cells (14), various cytogenetically analyzed cell lines (15–18), leukocytes of women on oral contraceptives (19), and postmenopausal patients (20,21). Interestingly, Ivett and Tice (22) observed that nonsteroid estrogen diethylstilbestrol (DES) induces chromosome aberrations in bone marrow cells of in vivo treated mice, though SCE per-cell level remained unchanged. It is noteworthy that results of SCE in vivo test are difficult to compare with those obtained in vitro, because effect of mutagen may depend on type of cell line used. To determine possible cytotoxic, cytostatic, or mitogenic effects of 17 estradiol, we analyzed the mitotic index (MI) for each experimental concentration as well as for controls. The results showed that 17 estradiol did not cause significant departures of MI values in relation to the negative control. As expected, positive control (MNNG) decreased MI by 34% compared to negative control, possibly due to arrest of mitosis because of repair of genetic material. Additionally, in cells with a relatively high level of genetic damage cytotoxic effects occur. Finally, the question remains of what current knowledge exists concerning molecular mechanisms of estrogen mutagenesis. Although it is quite obvious that estrogens induce mutations, standard genotoxicologic tests usually reveal the specific change but not the mechanism of the appearance of estrogen mutagenesis. Development of modern techniques has partially answered this question. One of the most frequently used techniques is 32P-postlabeling (23). Use of 32P-
Sister-Chromatid Exchange and Estradiol
postlabeling is convenient because this method does not require prior knowledge of adduct structures. Following chronic treatment of male Syrian hamsters with diethylstilbestrol, Roy et al. (24) discovered chemically modified nucleotide 8-hydroxydeoxyguanosine. The most convincing explanation for the appearance of DNA adducts is that at elevated concentrations, metabolic reactions of estrogens leading to formation of reactive free radicals may become predominant biochemical activity, overshadowing their hormonal effects (25,26). The susceptibility of the target cell to create favorable conditions for the appearance of DNA covalent modifications depends on the level of enzymatic and nonenzymatic defense systems involved in protection from oxidant stress (27,28). Acknowledgments This investigation was supported by the Serbian Ministry for Science and Technology, grant #12M18.
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