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Genotoxic effects of estradiol-17β on human lymphocyte chromosomes
Mutation Research 466 Ž2000. 109–115 www.elsevier.comrlocatergentox Community address: www.elsevier.comrlocatermutres
Genotoxic effects of estradiol-17b on human lymphocyte chromosomes Md. Equebal Ahmad, G.G.H.A. Shadab, Afsahul Hoda, Mohammad Afzal
)
Section of Genetics, Department of Zoology, Aligarh Muslim UniÕersity, Aligarh 202002, India Received 22 July 1999; received in revised form 27 October 1999; accepted 14 December 1999
Abstract The cytogenetic effect of a hormonal steroid, estradiol-17b, was assessed in peripheral blood human lymphocyte culture. Sister chromatid exchanges ŽSCE. and chromosome aberrations ŽCA. were scored as genetic end points. Significant induction of CA was observed at 25 mgrml and 50 mgrml concentrations of estradiol-17b in the absence of microsomal activation. The drug was effective in all treatments in the presence of rat liver S 9 microsomal fraction ŽS 9 mix. and exhibited increased frequency of chromosomal aberrations. The drug was effective in increasing the SCE frequency which was found to be maximum at the dose of 50 mgrml concentration Ži.e., 4.34 " 1.22. both with and without metabolic activation. It was found that estradiol-17b itself and possibly its metabolites are potent mutagens beyond a particular dose in human lymphocytes. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Estradiol-17b; Chromosomal aberration; Sister chromatid exchange; Lymphocyte
1. Introduction Sex hormonal steroids are used as medicine for human for a large variety of conditions, apart from their uses as oral contraceptive agents, viz., in the treatment of dysmenorrhea, endometriosis, dysfunctional uterine bleeding, climacterics and in the prevention of postmenopausal osteoporosis. They diffuse through the cell membrane and after conjugating with specific receptor proteins in the cytoplasm, pass through the nuclear pore to form steroid–DNA complexes. This complex alters the
) Corresponding author. Fax: q91-571-409081; e-mail: [email protected]@amu.up.nic.in
gene expression of many cellular processes including enzyme synthesis w1x. Estradiol also binds to isolated DNA w2,3x and induces fragmentation w4x. Several sex steroids have been studied for their teratogenic w5x, carcinogenic w6x, mutagenic and clastogenic w7x potentialities. Estrogens have been identified as mitotic inhibitors. It was suggested that the structural specificity is required for mitotic inhibition w8x. Estrogens are mitotic poisons, which at high concentration, cause metaphase arrest, abnormal cell division and CA w7x. Estrogens also induce the formation of endogenous DNA adducts both in human and animals and can ultimately lead to cancer development w9,10x. Contraceptive steroids induce DNA repair synthesis in rat hepatocytes cultures, indicating DNA damage w11x. These drugs can also have a tumor inducing potential in rat liver in addition to its tumor
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Md.E. Ahmad et al.r Mutation Research 466 (2000) 109–115
promoting activity w12x. Estradiol is carcinogenic in experimental animals. Administration of estrogens leads to an increase in endometrial cancer in women, and estradiol can be expected to have a similar effect w13x. Esrtradiol-17b is one of the sex hormones, and occurs in two forms alpha and beta. Beta estradiol has the greatest physiological activity of naturally occurring estrogens. The alpha form is relatively inactive. Estradiol was found to induce very low frequencies of hyperploidies in human synovial cells in vitro w14x and various numerical aberrations in pig oocytes in vitro w15x. In human embryonic fibroblasts and kidney epithelial cells, estradiol was reported to induce structural chromosomal aberration w16x. Simultaneous addition of estradiol, progesterone and human chorionic gonadotrophin ŽHCG. increased the SCE frequency in human lymphocyte culture, but their separate administration did not induce SCEs w17x. The hormone is negative in the Ames test w6x and does not induce point mutations in mammalian cells in vitro w18x. In the present study, we investigated the genotoxic effect of estradiol-17b on human chromosomes in lymphocytes cultures in vitro.
2. Materials and methods 2.1. Chromosomal aberration analysis Metaphase chromosome analysis for the detection of CAs was performed according to conventional technique w19x. Lymphocyte cultures were set up by adding 0.5 ml of heparinized whole blood, from two adult and healthy male donors Žwho were not occupationally exposed to mutagens. to 4.5 ml of TC-199 medium ŽFlow Laboratories. supplemented with 15% fetal calf serum ŽGibco., antibiotics ŽPenicillin and Streptomycin 100 IUrml each; Hoechst. and L-glutamine Ž1 mM; Gibco.. Lymphocytes were stimulated to divide by adding 0.1 ml of phytohaemagglutinin-M ŽPHA-M; Microlab.. The cultures were incubated at 378C and 5% CO 2 for 72 h in the dark. Estrtadiol-17b ŽWyeth Lab, India., at a final concentration of 10, 25 and 50 mgrml was added for 24, 48 and 72 h of duration by adding the steroid after 0, 24 and 48 h in reverse order after the initiation of cultures. Dimethylsulphoxide ŽDMSO 5 mgrml; SRL Bombay, India. was added to the
simultaneously kept cultures for 72 h of incubation as negative control. And for positive control, cyclophosphamide ŽCP 1 = 10y7 M. was added for 24, 48 and 72 h of incubation. In the metabolic activation experiments, 6 to 30 h old cultures were given treatment with 10, 25 and 50 mgrml concentrations of the drug in the presence of rat liver S 9 microsomal fraction. Liver S 9 fraction was prepared from healthy albino rats induced with phenobarbital and it was stored in liquid nitrogen until use. S 9 mix was freshly prepared for use. The cells were collected after centrifugation and the pellets were washed twice in the pre-warmed medium to remove the chemical and the S 9 mix. The parallel cultures receiving the same doses of the drug for similar treatment duration but without S 9 mix were also simultaneously set for comparison. Colchicine Ž0.20 mgrml; Microlab. was added to the cultures prior to harvesting. The cells were collected by centrifugation Ž10 min; 1200 rpm., hypotonic treatment Ž0.075 M KCl. was given for 10–12 min at 378C and the cells recollected by centrifugation were fixed in methanol: acetic acid Ž3:1.. Preparation of slides, staining and scanning was done under code. A total of 300 well-spread metaphases were analyzed per treatment per duration for all types of chromatid and chromosome type of aberrations. In case of cultures with metabolic activation, only 200 wellspread metaphases were analyzed. 2.2. Sister chromatid exchange analysis Analysis of SCEs was carried out following the fluorescent plus Giemsa techniques w20,21x. The cells in the cultures were exposed to 5-bromo-2-deoxyuridine ŽBrdU 2 mgrml; Sigma. after 24 h of the initiation of cultures. The test compounds were added together with the BrdU. To minimize photolysis of BrdU another 48 h of cultures were maintained in the dark. For SCE analysis in the presence of metabolic activation system, the drug along with S 9 mix was added into the culture after 48 h of its initiation and re-incubated at 378C. After 90 min of this pulse treatment the cells were spun down and the supernatant discarded. The cells were washed twice to remove the traces of the steroid and the liver metabolites. Finally, the cells pellets were re-suspended in
Md.E. Ahmad et al.r Mutation Research 466 (2000) 109–115
fresh medium supplemented with fetal calf serum, PHA-M and BrdU, and put to culture for another 24 h in the dark at 378C. For comparison, parallel cultures without S 9 mix were also set simultaneously. Hoechst 33258 stain Ž0.5 mgrml; Sigma. was used along with 10% Giemsa stain for differential staining of the sister chromatids. The slides were coded prior to scoring, and 50 well-spread metaphase cells were scanned per concentration and the number of exchanges scored.
3. Results 3.1. Chromosomal aberrations The chromosomal aberration frequencies in human lymphocytes induced by 10 to 50 mgrml estradiol-17b are presented in Table 1. At 10 mgrml of dose, the drug was not effective at any exposure time. At 25 mgrml and 50 mgrml of concentration, the drug affects both the aberrant metaphases as well as chromosomal aberration frequency. At 25 mgrml,
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the values for 24, 48 and 72 h of incubations were 18.20%, 18.80% and 48.60%, respectively, while at 50 mgrml the values were 19.33%, 21.00% and 54.34% at 24, 48 and 72 h of incubations, respectively, which were highly significant at p - 0.05 compared to the normal control value. The drug was effective in all treatments and an increased frequency of aberrant metaphases as well as chromosomal aberrations were observed when treated with S 9 mix ŽTable 2.. These values were 9.60%, 20.00% and 18.00% at 10, 25 and 50 mgrml of concentrations, respectively. These data were highly significant at p - 0.05 when compared to normal control Ž3.5%.. The percentage of chromosomal aberration was 61.00% for CP, which was highly significant. 3.2. Sister chromatid exchanges Data on the mean frequency of SCEs for cells after the treatment of estradiol-17b are presented in Table 3. The enhanced SCE rate was observed and at
Table 1 Chromosomal aberrations in human lymphocytes after estradiol-17b treatment Treatment Žmgrml.
Estradiol-17b 10
25
50
Control Normal DMSO Ž5 mgrml. Žyve control. CP Ž1 = 10y7 M. Žqve control
Duration Žh.
Metaphase scanned
Percent aberration metaphase
Types of aberration Ž%.
Aberrationr cell " SE
Including gap
Excluding gap
Chromatid
Chromosome
Total
24 48 72 24 48 72 24 48 72
300 300 300 300 300 300 300 300 300
7.33 5.00 8.30 11.34U 26.67U 39.70U 16.00U 23.00U 39.67U
3.70 2.70 3.70 11.34U 20.00U 39.00U 15.00U 20.00U 38.33U
3.70 2.70 3.00 12.50 11.50 21.30 13.33 12.67 23.67
0.00 0.00 1.50 5.70 7.30 27.30 6.00 8.33 30.67
3.70 2.70 4.50 18.20U 18.80U 48.60U 19.33U 21.00U 54.34U
0.04 " 0.01 0.03 " 0.00 0.05 " 0.01 0.18 " 0.02 0.19 " 0.02 0.49 " 0.04 0.19 " 0.03 0.21 " 0.03 0.54 " 0.05
72 72
300 300
2.30 3.30
1.60 1.00
1.30 2.00
1.00 1.00
2.30 3.00
0.02 " 0.01 0.03 " 0.01
24 48 72
300 300 300
23.00U 39.00U 40.00U
20.00U 35.00U 34.00U
24.00 44.00 45.00
6.00 24.00 21.00
30.00U 68.00U 66.00U
0.30 " 0.03 0.68 " 0.09 0.66 " 0.09
CP: Cyclophosphamide; DMSO: dimethyl sulphoxide; SE: standard error. U Significant at p - 0.05.
Md.E. Ahmad et al.r Mutation Research 466 (2000) 109–115
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Table 2 Chromosomal aberrations in human lymphocytes after estradiol-17b treatment with and without metabolic activation Treatment Žmgrml.
Estradiol-17b 10 25 50
Control Normal DMSO Ž5 mgrml. Žyve control. CP Ž1 = 10y7 M. Žqve control.
Metabolic activation
Metaphase scanned
Percent aberration metaphase
Types of aberration Ž%.
Including gap
Excluding gap
Chromatid
Chromosome
Aberrationr cell " SE Total
yS 9 qS 9 yS 9 qS 9 yS 9 qS 9
300 200 300 200 300 200
8.70U 10.50U 11.30U 16.00U 10.00U 14.00U
7.90U 9.00U 11.30U 15.00U 9.30U 13.00U
6.60 8.60 12.50 15.00 8.30 13.50
0.60 1.00 5.60 5.00 4.00 4.50
7.20U 9.60U 18.10U 20.00U 12.30U 18.00U
0.07 " 0.02 0.09 " 0.02 0.18 " 0.02 0.20 " 0.01 0.12 " 0.02 0.18 " 0.02
yS 9 qS 9 yS 9
300 200 300
2.30 4.50 3.30
1.70 3.00 1.70
1.30 3.50 2.00
0.30 0.00 0.00
1.60 3.50 2.00
0.02 " 0.01 0.04 " 0.01 0.02 " 0.00
qS 9
200
4.50
3.50
3.50
0.00
3.50
0.04 " 0.01
yS 9 qS 9
300 200
6.30U 40.50U
4.67U 39.50U
10.00 37.50
8.67 23.50
18.67U 61.00U
0.19 " 0.03 0.61 " 0.07
CP: Cyclophosphamide; DMSO: dimethyl sulphoxide; SE: standard error. U Significant at p - 0.05.
50 mgrml of concentration the maximum frequency of SCE per cell was found to be 4.34 " 1.22. This value was highly significant compared to the mean SCE value of normal control, i.e., 1.74 " 0.14.
In the presence of S 9 mix, both doses Ž25 and 50 mgrml. enhanced the SCE frequency significantly, i.e., 4.58 " 0.41 and 6.08 " 1.31, respectively ŽTable 4.. Both these values were significantly higher than
Table 3 Sister chromatid exchange ŽSCE. in human lymphocytes after estradiol-17b exposure in vitro Treatment Žmgrml.
Duration Žh.
No. of metaphases
Total SCE
Range
SCEsr cell " SE
Estradiol-17b 10 25 50
48 48 48
50 50 50
76 171 217
0–6 1–9 2–11
1.52 " 0.14 3.42 " 0.52U 4.34 " 1.22U
48 48
50 50
87 96
0–5 0–6
1.74 " 0.14 1.92 " 0.12
48
50
340
2–11
6.80 " 1.29U
Controls Normal DMSO Ž5 mgrml. Žyve control. CP Ž1 = 10y7 M. Žqve control.
CP: Cyclophosphamide; DMSO: dimethylsulphoxide; SE: standard error. U Significant at p - 0.05.
Md.E. Ahmad et al.r Mutation Research 466 (2000) 109–115
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Table 4 Sister chromatid exchange ŽSCE. in human lymphocytes after estradiol-17b with metabolic activation Treatment Žmgrml.
Duration Žh.
No. of metaphases
Total SCE
Range
SCEsr cell " SE
Estradiol-17b 10 25 50
48 48 48
50 50 50
97 229 304
0–5 1–11 2–12
1.94 " 0.50 4.58 " 0.41U 6.08 " 1.31U
48 48
50 50
78 89
0–5 0–6
1.56 " 0.12 1.78 " 0.11
48
50
380
4–12
7.60 " 1.34U
Controls Normal DMSO Ž5 mgrml. Žyve control. CP Ž1 = 10y7 M. Žqve control.
CP: Cyclophosphamide; DMSO: dimethylsulphoxide; SE: standard error. U Significant at p - 0.05.
the mean SCE value of 1.56 " 0.12 observed in normal controls Ž p - 0.05..
4. Discussion The results of the present investigation demonstrate that the use of estradiol-17b is associated with clastogenicity. A significant increase in the incidence of chromosomal abnormalities per cell as well as an increase in the number of abnormal cells were observed when compared with normal control. At 10 mgrml of concentration, the results were insignificant for all treatment duration Ž24, 48 and 72 h.. However, at 25 mgrml concentration a significant increase in aberration frequency was observed. But the maximal effect was observed at the highest concentration, an observation in line with the majority of clastogens. Of the various chromosomal aberrations induced by estradiol-17b, chromatid and chromosome fragments and breaks were more frequent than other types of aberrations, such as ring and bridge formation, acentric fragments, etc. The increased clastogenicity by estradiol-17b at the longest treatment duration as compared to the shorter treatment duration could be attributable to the inherent ability of the human lymphocytes to metabolize certain compounds per se w22x. The common effects of steroid hormone on chromosomes are stickiness in diakinesis and metaphase, and bridges
and sometimes lagging chromosomes in the anaphase w23x. Similar observations were made in Anovlar Ža steroid. treated plant chromosomes w24x. Chromatid breaks were seen more frequently than chromosome breaks in the metaphase plates analyzed. This indicates that the drug introduces replication errors or breaks replicated chromatids. Estradiol-17b induced reversible mitotic delay in HeLa cells, which were localized at the beginning of mitosis. The duration of mitotic lag was dose dependent w25x. The present data on the time–response analysis revealed a significant increase in chromosomal aberration at 72 h of treatment w26x. In the presence of S 9 microsomal activation system, the clastogenic effects of estradiol-17b were more prominent. In the absence of S 9 mix, the chromosomal aberrations observed were also significant but slightly less. The time–response analysis in the presence of S 9 revealed significant increase at 48 h of incubation and decreased later on w27x. The maximum frequency of chromosomal aberration was found at 48 h long treatment on mouse bone marrow cells, while it decreased later on and gave no significant result. This suggests that the drug andror its metabolites were active during this Ž48th h. period. The decline in the frequency of aberrations observed at the later time intervals may be due to some or all of the following reasons: Ži. repairs of damaged genetic material, Žii. elimination of the drug and its metabolites, Žiii. elimination of cellsrchromosomes with damaged genetic material, Živ. or due to cell
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Md.E. Ahmad et al.r Mutation Research 466 (2000) 109–115
cycle delay and cell killing effect, and Žv. inactivation of drug or its metabolites. The results obtained from the SCE analyses indicated the drug has strong SCE inducing potential. The frequency of SCE further increases if the drug is treated in the presence of S 9 mix. There exists a relationship between increased SCE rates and altered mutation frequency w28x. Therefore, it can be concluded that estradiol-17b is capable of attacking the genetic material directly in its unmetabolized as well as metabolized state. Because of the apparent relationship among chromosome abnormalities, mutagenesis and carcinogenesis w29x, in vivo and in vitro screening for chromosome aberrations is an established tool in the evaluation of the potential hazards to the genetic material due to environmental agents. The theoretical possibility of genetic damage during post-natal life will apply directly to the health of the drug user. Any mutational effect on adult somatic cells could lead to cancer w30x. People should, therefore, be careful and aware of the potential hazards of such drugs, especially if they are continuously exposed to for such long periods.
Acknowledgements Thanks are due to Dr. Md. Asim Azfer, Lecturer, Department of Zoology, for his valuable suggestions and to the Chairman of the Department of Zoology for his kind help and providing necessary facilities to complete this work.
References w1x T. Thomas, T.J. Thomas, Estradiol control of ornithine decarboxylase mRNA, enzyme activity and polyamine levels in MCF-7 breast cancer cells: therapeutic implications, Breast Cancer Res. Treat. 29 Ž1993. 189–201. w2x G.M. Blackburn, A.J. Flavell, M.H. Thompson, Oxidative and photochemical linkage of diethylstilbestrol to DNA in vitro, Cancer Res. 34 Ž1974. 2015–2019. w3x P.O.P. Ts’O Lu, Interaction of nucleic acids: 1. Physical aromatic hydrocarbons to nucleic acids, Proc. Natl. Acad. Sci. USA 51 Ž1964. 17–24. w4x K. Yamafuji, S. Lijama, K. Shinohara, Mode of action of steroid hormones on deoxyribonucleic acid, Enzymologia 40 Ž1971. 259–264.
w5x N.J. Joshi, L.M. Ambani, S.R. Munshi, Evaluation of teratogenic potential of a combination of norethisterone and ethinyl estradiol in rats, Indian J. Exp. Biol. 21 Ž1983. 591–596. w6x R. Lang, U. Redmann, Non-mutagenicity of some sex hormones in the Ames Salmonellarmicrosomes mutagenicity test, Mutat. Res. 67 Ž1979. 361–365. w7x W.J. Wheeler, L.M. Cherry, T. Downs, T.C. Hsu, Mitotic inhibition and aneuploidy induction by naturally occurring and synthetic estrogens in chinese hamster cells in vitro, Mutat. Res. 171 Ž1986. 31–41. w8x P.N. Rao, J. Engelberg, Structural specificity of estrogens in the induction of mitotic chromatid non-disjunction in HeLa cells, Exp. Cell Res. 48 Ž1967. 71–81. w9x J.G. Liehr, T.A. Avitts, E. Randerath, K. Randerath, Estrogen-induced indigenous DNA adduction: possible mechanism of hormonal cancer, Proc. Natl. Acad. Sci. USA 83 Ž1986. 5301–5305. w10x J.G. Liehr, Genotoxic effects of estrogens, Mutat. Res. 238 Ž1990. 269–276. w11x I. Neuman, D. Thierau, U. Andrae, H. Grein, L.R. Shwarz, Cyproterone acetate induces DNA damage in cultured rat hepatocytes and preferentially stimulates DNA synthesis in glutamyltranspeptidase-positive cells, Carcinogenesis 13 Ž1992. 373–387. w12x T. Wolff, Steroids hormones as DNA damaging agents. Abstract 30th anniversary of the Czech Society of medical genetics of association of medical societies, in: J.E. Purkyne ŽEd.., Genetics in Medicine, Prague, Czech Republic, September 2–5, 1993, pp. 26. w13x IARC ŽInternational Agency for Research on Cancer., Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Sex Hormones ŽII. ŽIARC-Monographs., Vol. 21, 1979. w14x R.J. Lycette, S. Whyte, C.J. Chapman, Aneuploid effect of estradiol on cultured human synovial cells, N. Z. Med. J. 74 Ž1970. 114–117. w15x R.W. McGaughey, The culture of pig oocytes in minimal medium, and the influence of progesterone and estradiol-17b on meiotic maturation, Endocrinology 100 Ž1977. 39–45. w16x J.A. Serova, Y.Y. Kerkis, Cytogenetic effect of some steroid hormones and change in the activity of lysosomal enzymes in vitro, Sov. Genet. 10 Ž1974. 387–392. w17x T. Sharma, B.C. Das, Higher incidence of spontaneous sister chromatid exchanges and X-ray induced chromosome aberrations in peripheral blood lymphocytes during pregnancy, Mutat. Res. 174 Ž1986. 27–33. w18x C. Drevon, C. Piccoli, R. Montesano, Mutagenicity assays of estrogenic hormones in mammalian cells, Mutat. Res. 89 Ž1981. 83–90. w19x P.S. Moorehead, P.C. Nowell, W.J. Mellman, D.M. Battips, D.A. Hungerford, Chromosome preparations of leukocytes cultured from human peripheral blood, Exp. Cell Res. 20 Ž1960. 613–616. w20x P. Perry, S. Wolff, New Giemsa method for the differential staining of sister chromatids, Nature 251 Ž1974. 156–158. w21x S.A. Latt, J.W. Allen, W.E. Rogers, L.A. Juergens, In vitro and in vivo analysis of sister chromatid exchange formation,
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in: B.J. Kilbey, M. Legator, W. Nichols, C. Ramel ŽEds.., Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, 1977, pp. 275–291. O. Iskander, Vijayalaxmi, The enhancement of the effect of aflatoxin B1 by metabolic activation with rat liver microsomes on human lymphocyte assayed with the micronucleus test, Mutat. Res. 91 Ž1981. 63–66. H. Hakeem, S. Amer, Meiotic effects of water extracts of some contraceptive tablets, Naturwissenschaften 53 Ž1965. 184. Kabriaty, S. Mazrooei, Further investigations on the cytological effects of some contraceptives, Mutat. Res. 135 Ž1984. 181–188. P.N. Rao, Estradiol-induced mitotic delay in HeLa cells: reversal by calcium chloride and putrescine, Exp. Cell Res. 57 Ž1969. 230–234.
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w26x S.K. Shyama, A.A. Rahiman, K.K. Vijayalaxmi, Genotoxic effect of Anovlar 21, an oral contraceptive, on mouse bone marrow, Mutat. Res. 260 Ž1991. 47–53. w27x N. Banduhn, G. Obe, Mutagenicity of methyl 2-benzimidazole carbamate, diethylstilbestrol and estradiol: structural chromosomal aberrations, sister chromatid exchanges, Cmitoses, polyploidies and micronuclei, Mutat. Res. 156 Ž1985. 199–218. w28x A.V. Carrano, L.H. Thompson, P.A. Lindi, J.L. Minkler, Sister chromatid exchange as an indicator of mutagenesis, Nature 271 Ž1978. 551–553. w29x A.A. Sandberg, The Chromosomes in Human Cancer and Leukemia, Elsevier, New York, 1980. w30x N.S. Weiss, D.R. Szekely, D.F. Austin, Increasing incidence of endometrial cancer in the United States, N. Engl. J. Med. 294 Ž1976. 1259–1261.
Genotoxic effects of estradiol-17b on human lymphocyte chromosomes Md. Equebal Ahmad, G.G.H.A. Shadab, Afsahul Hoda, Mohammad Afzal
)
Section of Genetics, Department of Zoology, Aligarh Muslim UniÕersity, Aligarh 202002, India Received 22 July 1999; received in revised form 27 October 1999; accepted 14 December 1999
Abstract The cytogenetic effect of a hormonal steroid, estradiol-17b, was assessed in peripheral blood human lymphocyte culture. Sister chromatid exchanges ŽSCE. and chromosome aberrations ŽCA. were scored as genetic end points. Significant induction of CA was observed at 25 mgrml and 50 mgrml concentrations of estradiol-17b in the absence of microsomal activation. The drug was effective in all treatments in the presence of rat liver S 9 microsomal fraction ŽS 9 mix. and exhibited increased frequency of chromosomal aberrations. The drug was effective in increasing the SCE frequency which was found to be maximum at the dose of 50 mgrml concentration Ži.e., 4.34 " 1.22. both with and without metabolic activation. It was found that estradiol-17b itself and possibly its metabolites are potent mutagens beyond a particular dose in human lymphocytes. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Estradiol-17b; Chromosomal aberration; Sister chromatid exchange; Lymphocyte
1. Introduction Sex hormonal steroids are used as medicine for human for a large variety of conditions, apart from their uses as oral contraceptive agents, viz., in the treatment of dysmenorrhea, endometriosis, dysfunctional uterine bleeding, climacterics and in the prevention of postmenopausal osteoporosis. They diffuse through the cell membrane and after conjugating with specific receptor proteins in the cytoplasm, pass through the nuclear pore to form steroid–DNA complexes. This complex alters the
) Corresponding author. Fax: q91-571-409081; e-mail: [email protected]@amu.up.nic.in
gene expression of many cellular processes including enzyme synthesis w1x. Estradiol also binds to isolated DNA w2,3x and induces fragmentation w4x. Several sex steroids have been studied for their teratogenic w5x, carcinogenic w6x, mutagenic and clastogenic w7x potentialities. Estrogens have been identified as mitotic inhibitors. It was suggested that the structural specificity is required for mitotic inhibition w8x. Estrogens are mitotic poisons, which at high concentration, cause metaphase arrest, abnormal cell division and CA w7x. Estrogens also induce the formation of endogenous DNA adducts both in human and animals and can ultimately lead to cancer development w9,10x. Contraceptive steroids induce DNA repair synthesis in rat hepatocytes cultures, indicating DNA damage w11x. These drugs can also have a tumor inducing potential in rat liver in addition to its tumor
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Md.E. Ahmad et al.r Mutation Research 466 (2000) 109–115
promoting activity w12x. Estradiol is carcinogenic in experimental animals. Administration of estrogens leads to an increase in endometrial cancer in women, and estradiol can be expected to have a similar effect w13x. Esrtradiol-17b is one of the sex hormones, and occurs in two forms alpha and beta. Beta estradiol has the greatest physiological activity of naturally occurring estrogens. The alpha form is relatively inactive. Estradiol was found to induce very low frequencies of hyperploidies in human synovial cells in vitro w14x and various numerical aberrations in pig oocytes in vitro w15x. In human embryonic fibroblasts and kidney epithelial cells, estradiol was reported to induce structural chromosomal aberration w16x. Simultaneous addition of estradiol, progesterone and human chorionic gonadotrophin ŽHCG. increased the SCE frequency in human lymphocyte culture, but their separate administration did not induce SCEs w17x. The hormone is negative in the Ames test w6x and does not induce point mutations in mammalian cells in vitro w18x. In the present study, we investigated the genotoxic effect of estradiol-17b on human chromosomes in lymphocytes cultures in vitro.
2. Materials and methods 2.1. Chromosomal aberration analysis Metaphase chromosome analysis for the detection of CAs was performed according to conventional technique w19x. Lymphocyte cultures were set up by adding 0.5 ml of heparinized whole blood, from two adult and healthy male donors Žwho were not occupationally exposed to mutagens. to 4.5 ml of TC-199 medium ŽFlow Laboratories. supplemented with 15% fetal calf serum ŽGibco., antibiotics ŽPenicillin and Streptomycin 100 IUrml each; Hoechst. and L-glutamine Ž1 mM; Gibco.. Lymphocytes were stimulated to divide by adding 0.1 ml of phytohaemagglutinin-M ŽPHA-M; Microlab.. The cultures were incubated at 378C and 5% CO 2 for 72 h in the dark. Estrtadiol-17b ŽWyeth Lab, India., at a final concentration of 10, 25 and 50 mgrml was added for 24, 48 and 72 h of duration by adding the steroid after 0, 24 and 48 h in reverse order after the initiation of cultures. Dimethylsulphoxide ŽDMSO 5 mgrml; SRL Bombay, India. was added to the
simultaneously kept cultures for 72 h of incubation as negative control. And for positive control, cyclophosphamide ŽCP 1 = 10y7 M. was added for 24, 48 and 72 h of incubation. In the metabolic activation experiments, 6 to 30 h old cultures were given treatment with 10, 25 and 50 mgrml concentrations of the drug in the presence of rat liver S 9 microsomal fraction. Liver S 9 fraction was prepared from healthy albino rats induced with phenobarbital and it was stored in liquid nitrogen until use. S 9 mix was freshly prepared for use. The cells were collected after centrifugation and the pellets were washed twice in the pre-warmed medium to remove the chemical and the S 9 mix. The parallel cultures receiving the same doses of the drug for similar treatment duration but without S 9 mix were also simultaneously set for comparison. Colchicine Ž0.20 mgrml; Microlab. was added to the cultures prior to harvesting. The cells were collected by centrifugation Ž10 min; 1200 rpm., hypotonic treatment Ž0.075 M KCl. was given for 10–12 min at 378C and the cells recollected by centrifugation were fixed in methanol: acetic acid Ž3:1.. Preparation of slides, staining and scanning was done under code. A total of 300 well-spread metaphases were analyzed per treatment per duration for all types of chromatid and chromosome type of aberrations. In case of cultures with metabolic activation, only 200 wellspread metaphases were analyzed. 2.2. Sister chromatid exchange analysis Analysis of SCEs was carried out following the fluorescent plus Giemsa techniques w20,21x. The cells in the cultures were exposed to 5-bromo-2-deoxyuridine ŽBrdU 2 mgrml; Sigma. after 24 h of the initiation of cultures. The test compounds were added together with the BrdU. To minimize photolysis of BrdU another 48 h of cultures were maintained in the dark. For SCE analysis in the presence of metabolic activation system, the drug along with S 9 mix was added into the culture after 48 h of its initiation and re-incubated at 378C. After 90 min of this pulse treatment the cells were spun down and the supernatant discarded. The cells were washed twice to remove the traces of the steroid and the liver metabolites. Finally, the cells pellets were re-suspended in
Md.E. Ahmad et al.r Mutation Research 466 (2000) 109–115
fresh medium supplemented with fetal calf serum, PHA-M and BrdU, and put to culture for another 24 h in the dark at 378C. For comparison, parallel cultures without S 9 mix were also set simultaneously. Hoechst 33258 stain Ž0.5 mgrml; Sigma. was used along with 10% Giemsa stain for differential staining of the sister chromatids. The slides were coded prior to scoring, and 50 well-spread metaphase cells were scanned per concentration and the number of exchanges scored.
3. Results 3.1. Chromosomal aberrations The chromosomal aberration frequencies in human lymphocytes induced by 10 to 50 mgrml estradiol-17b are presented in Table 1. At 10 mgrml of dose, the drug was not effective at any exposure time. At 25 mgrml and 50 mgrml of concentration, the drug affects both the aberrant metaphases as well as chromosomal aberration frequency. At 25 mgrml,
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the values for 24, 48 and 72 h of incubations were 18.20%, 18.80% and 48.60%, respectively, while at 50 mgrml the values were 19.33%, 21.00% and 54.34% at 24, 48 and 72 h of incubations, respectively, which were highly significant at p - 0.05 compared to the normal control value. The drug was effective in all treatments and an increased frequency of aberrant metaphases as well as chromosomal aberrations were observed when treated with S 9 mix ŽTable 2.. These values were 9.60%, 20.00% and 18.00% at 10, 25 and 50 mgrml of concentrations, respectively. These data were highly significant at p - 0.05 when compared to normal control Ž3.5%.. The percentage of chromosomal aberration was 61.00% for CP, which was highly significant. 3.2. Sister chromatid exchanges Data on the mean frequency of SCEs for cells after the treatment of estradiol-17b are presented in Table 3. The enhanced SCE rate was observed and at
Table 1 Chromosomal aberrations in human lymphocytes after estradiol-17b treatment Treatment Žmgrml.
Estradiol-17b 10
25
50
Control Normal DMSO Ž5 mgrml. Žyve control. CP Ž1 = 10y7 M. Žqve control
Duration Žh.
Metaphase scanned
Percent aberration metaphase
Types of aberration Ž%.
Aberrationr cell " SE
Including gap
Excluding gap
Chromatid
Chromosome
Total
24 48 72 24 48 72 24 48 72
300 300 300 300 300 300 300 300 300
7.33 5.00 8.30 11.34U 26.67U 39.70U 16.00U 23.00U 39.67U
3.70 2.70 3.70 11.34U 20.00U 39.00U 15.00U 20.00U 38.33U
3.70 2.70 3.00 12.50 11.50 21.30 13.33 12.67 23.67
0.00 0.00 1.50 5.70 7.30 27.30 6.00 8.33 30.67
3.70 2.70 4.50 18.20U 18.80U 48.60U 19.33U 21.00U 54.34U
0.04 " 0.01 0.03 " 0.00 0.05 " 0.01 0.18 " 0.02 0.19 " 0.02 0.49 " 0.04 0.19 " 0.03 0.21 " 0.03 0.54 " 0.05
72 72
300 300
2.30 3.30
1.60 1.00
1.30 2.00
1.00 1.00
2.30 3.00
0.02 " 0.01 0.03 " 0.01
24 48 72
300 300 300
23.00U 39.00U 40.00U
20.00U 35.00U 34.00U
24.00 44.00 45.00
6.00 24.00 21.00
30.00U 68.00U 66.00U
0.30 " 0.03 0.68 " 0.09 0.66 " 0.09
CP: Cyclophosphamide; DMSO: dimethyl sulphoxide; SE: standard error. U Significant at p - 0.05.
Md.E. Ahmad et al.r Mutation Research 466 (2000) 109–115
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Table 2 Chromosomal aberrations in human lymphocytes after estradiol-17b treatment with and without metabolic activation Treatment Žmgrml.
Estradiol-17b 10 25 50
Control Normal DMSO Ž5 mgrml. Žyve control. CP Ž1 = 10y7 M. Žqve control.
Metabolic activation
Metaphase scanned
Percent aberration metaphase
Types of aberration Ž%.
Including gap
Excluding gap
Chromatid
Chromosome
Aberrationr cell " SE Total
yS 9 qS 9 yS 9 qS 9 yS 9 qS 9
300 200 300 200 300 200
8.70U 10.50U 11.30U 16.00U 10.00U 14.00U
7.90U 9.00U 11.30U 15.00U 9.30U 13.00U
6.60 8.60 12.50 15.00 8.30 13.50
0.60 1.00 5.60 5.00 4.00 4.50
7.20U 9.60U 18.10U 20.00U 12.30U 18.00U
0.07 " 0.02 0.09 " 0.02 0.18 " 0.02 0.20 " 0.01 0.12 " 0.02 0.18 " 0.02
yS 9 qS 9 yS 9
300 200 300
2.30 4.50 3.30
1.70 3.00 1.70
1.30 3.50 2.00
0.30 0.00 0.00
1.60 3.50 2.00
0.02 " 0.01 0.04 " 0.01 0.02 " 0.00
qS 9
200
4.50
3.50
3.50
0.00
3.50
0.04 " 0.01
yS 9 qS 9
300 200
6.30U 40.50U
4.67U 39.50U
10.00 37.50
8.67 23.50
18.67U 61.00U
0.19 " 0.03 0.61 " 0.07
CP: Cyclophosphamide; DMSO: dimethyl sulphoxide; SE: standard error. U Significant at p - 0.05.
50 mgrml of concentration the maximum frequency of SCE per cell was found to be 4.34 " 1.22. This value was highly significant compared to the mean SCE value of normal control, i.e., 1.74 " 0.14.
In the presence of S 9 mix, both doses Ž25 and 50 mgrml. enhanced the SCE frequency significantly, i.e., 4.58 " 0.41 and 6.08 " 1.31, respectively ŽTable 4.. Both these values were significantly higher than
Table 3 Sister chromatid exchange ŽSCE. in human lymphocytes after estradiol-17b exposure in vitro Treatment Žmgrml.
Duration Žh.
No. of metaphases
Total SCE
Range
SCEsr cell " SE
Estradiol-17b 10 25 50
48 48 48
50 50 50
76 171 217
0–6 1–9 2–11
1.52 " 0.14 3.42 " 0.52U 4.34 " 1.22U
48 48
50 50
87 96
0–5 0–6
1.74 " 0.14 1.92 " 0.12
48
50
340
2–11
6.80 " 1.29U
Controls Normal DMSO Ž5 mgrml. Žyve control. CP Ž1 = 10y7 M. Žqve control.
CP: Cyclophosphamide; DMSO: dimethylsulphoxide; SE: standard error. U Significant at p - 0.05.
Md.E. Ahmad et al.r Mutation Research 466 (2000) 109–115
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Table 4 Sister chromatid exchange ŽSCE. in human lymphocytes after estradiol-17b with metabolic activation Treatment Žmgrml.
Duration Žh.
No. of metaphases
Total SCE
Range
SCEsr cell " SE
Estradiol-17b 10 25 50
48 48 48
50 50 50
97 229 304
0–5 1–11 2–12
1.94 " 0.50 4.58 " 0.41U 6.08 " 1.31U
48 48
50 50
78 89
0–5 0–6
1.56 " 0.12 1.78 " 0.11
48
50
380
4–12
7.60 " 1.34U
Controls Normal DMSO Ž5 mgrml. Žyve control. CP Ž1 = 10y7 M. Žqve control.
CP: Cyclophosphamide; DMSO: dimethylsulphoxide; SE: standard error. U Significant at p - 0.05.
the mean SCE value of 1.56 " 0.12 observed in normal controls Ž p - 0.05..
4. Discussion The results of the present investigation demonstrate that the use of estradiol-17b is associated with clastogenicity. A significant increase in the incidence of chromosomal abnormalities per cell as well as an increase in the number of abnormal cells were observed when compared with normal control. At 10 mgrml of concentration, the results were insignificant for all treatment duration Ž24, 48 and 72 h.. However, at 25 mgrml concentration a significant increase in aberration frequency was observed. But the maximal effect was observed at the highest concentration, an observation in line with the majority of clastogens. Of the various chromosomal aberrations induced by estradiol-17b, chromatid and chromosome fragments and breaks were more frequent than other types of aberrations, such as ring and bridge formation, acentric fragments, etc. The increased clastogenicity by estradiol-17b at the longest treatment duration as compared to the shorter treatment duration could be attributable to the inherent ability of the human lymphocytes to metabolize certain compounds per se w22x. The common effects of steroid hormone on chromosomes are stickiness in diakinesis and metaphase, and bridges
and sometimes lagging chromosomes in the anaphase w23x. Similar observations were made in Anovlar Ža steroid. treated plant chromosomes w24x. Chromatid breaks were seen more frequently than chromosome breaks in the metaphase plates analyzed. This indicates that the drug introduces replication errors or breaks replicated chromatids. Estradiol-17b induced reversible mitotic delay in HeLa cells, which were localized at the beginning of mitosis. The duration of mitotic lag was dose dependent w25x. The present data on the time–response analysis revealed a significant increase in chromosomal aberration at 72 h of treatment w26x. In the presence of S 9 microsomal activation system, the clastogenic effects of estradiol-17b were more prominent. In the absence of S 9 mix, the chromosomal aberrations observed were also significant but slightly less. The time–response analysis in the presence of S 9 revealed significant increase at 48 h of incubation and decreased later on w27x. The maximum frequency of chromosomal aberration was found at 48 h long treatment on mouse bone marrow cells, while it decreased later on and gave no significant result. This suggests that the drug andror its metabolites were active during this Ž48th h. period. The decline in the frequency of aberrations observed at the later time intervals may be due to some or all of the following reasons: Ži. repairs of damaged genetic material, Žii. elimination of the drug and its metabolites, Žiii. elimination of cellsrchromosomes with damaged genetic material, Živ. or due to cell
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Md.E. Ahmad et al.r Mutation Research 466 (2000) 109–115
cycle delay and cell killing effect, and Žv. inactivation of drug or its metabolites. The results obtained from the SCE analyses indicated the drug has strong SCE inducing potential. The frequency of SCE further increases if the drug is treated in the presence of S 9 mix. There exists a relationship between increased SCE rates and altered mutation frequency w28x. Therefore, it can be concluded that estradiol-17b is capable of attacking the genetic material directly in its unmetabolized as well as metabolized state. Because of the apparent relationship among chromosome abnormalities, mutagenesis and carcinogenesis w29x, in vivo and in vitro screening for chromosome aberrations is an established tool in the evaluation of the potential hazards to the genetic material due to environmental agents. The theoretical possibility of genetic damage during post-natal life will apply directly to the health of the drug user. Any mutational effect on adult somatic cells could lead to cancer w30x. People should, therefore, be careful and aware of the potential hazards of such drugs, especially if they are continuously exposed to for such long periods.
Acknowledgements Thanks are due to Dr. Md. Asim Azfer, Lecturer, Department of Zoology, for his valuable suggestions and to the Chairman of the Department of Zoology for his kind help and providing necessary facilities to complete this work.
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