increased frequencies of micronuclei formation and sister chromatid exchanges in mice. However in the Ames Salmonella assay, both without and with $9 mix, ...
Genetic Toxicology
ELSEVIER
Mutation Research 345 (1995) 87-95
Genotoxicity evaluation of estradiol Varinderpal S. Dhillon *, Ipninder K. Dhillon Department of Human Genetics, Guru Nanak Dec UniversiO', Amritsar 143 005, India Received 31 January 1995; revised 19 July 1995; accepted 20 July 1995
Abstract
Genotoxic evaluation of estradiol, a commonly used estrogen, was undertaken using various short-term in vitro and in vivo assays. The clastogenic potential of estradiol was evident from the chromosome aberrations and sister chromatid exchanges induced by it both without and with metabolic activation in cultured human lymphocytes and also from the increased frequencies of micronuclei formation and sister chromatid exchanges in mice. However in the Ames Salmonella assay, both without and with $9 mix, and also in the host-mediated assay, estradiol was unable to cause a significant increase/decrease in the His + revertants/plate. Keywords: Ames test; Human lymphocyte; Sister chromatid exchange (SCE); Micronucleus; Chromosomal aberration; $9 mix; Estradiol
1. Introduction
Hormonal drugs occupy an important place in the chemotherapeutic treatment of a number of metabolic, homeostatic and sexual disorders (Rubin, 1990). Estrogens also fall under this category and are extensively used to treat many types of sexual disorders in both sexes and are an important component of oral contraceptives. Estrogens are converted to catechol estrogens and these, or the resulting quinones, interact with DNA, or which, through redox cycling, produce oxidative damage to DNA and hence cause mutations (Rubin, 1990). Estrogens like diethylstilbestrol and phorbolesters have been reported to promote tumor development in experimental animals (Lang and Redmann, 1979; Blumberg, 1980). In rodents, an increased frequency of
pituitary and mammary cancers and neoplastic changes in the liver were frequently observed after estrogen, progestagen and estrogen-pregestagen treatment (Lehmann et al., 1989). Some other studies also indicate the potentiality of the estrogens to act as promoters as well as initiators of cancer (Klein, 1979; Bansal et al., 1983). Use of estrogens was associated with the development of several types of cancer in humans and induced various kinds of tumors in rodents (Liehr, 1990). Bearing in mind the extensive use of estrogens and their suspected mutagenic and carcinogenic potential, the present investigation was undertaken to assess the genotoxic potential of estradiol through various short-term in vitro and in vivo assays.
2. Materials and methods * Corresponding author at: 462 Basant Avenue, Amritsar 143 001, India.
Estradiol (l,3,5(10)-estratriene-3,17/3-diol; CAS no. 50-28-2; Organon India Limited, Calcutta) is an
0165-1218/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 5 - 1 2 1 8 ( 9 5 ) 0 0 0 4 5 - 3
88
V.S. Dhillon, L K. Dhillon / Mutation Research 345 (1995) 87- 95
endogenous estrogen and its common therapeutic dose ranges from 0.02 to 2.0 mg/day. 2. I. Ames Salmonella / $9 assay The revised standard plate incorporation procedure of Maron and Ames (1983) was followed with slight modifications to evaluate the mutagenicity of estradiol without $9 mix. The modified preincubation method of Yahagi et al. (1975) was followed to assess the effects of the drug after metabolization with $9 mix. Concentrations of estradiol ranging from 1.0 to 1 X 10 4 /~g/plate were assayed without $9 mix, while concentrations ranging from 1.0 to 1.0 X 103 /zg/plate could only be tested due to the procedural limitations (precipitation and toxicity of drug at higher concentration with $9 mix). Liver $9 fraction was prepared from healthy albino rats induced with phenobarbitone and was stored in liquid nitrogen until use (Bali et al., 1990). Dimethyl sulfoxide (DMSO, 50 and 500 /zl/plate) was used as negative control. Positive controls included sodium azide (i.5 /xg/plate) for TA 100 and TA1535 strains, 4-nitrophenylenediamine (20 /xg/plate) for TA97a and TA98 strain and methylmethane sulfonate (MMS, 1.0 /xl/plate) for TA100 strain and 2-aminofluorene (20 /~g/plate) for TA97a, TA98 and TAI00 strains of Salmonella. His + revertant colonies were scored with electronic colony counter after 48 h of incubation at 37°C. 2.2. In uitro chromosomal aberration assay without metabolic actiuation Lymphocyte culturing was carried out on the blood samples obtained from young and healthy male donors (20-25 years age). Whole blood cultures were set up by adding 0.8 ml of blood to 10.0 ml of RPMI-1640 medium (Flow Laboratories) supplemented with 10% fetal calf serum (Microlab) and 0.1 ml phytohemagglutinin (Microlab) and were incubated for 72 h at 37°C. Three concentrations of estradiol (1, l0 and 1 0 0 / z g / m l dissolved in DMSO) were added to different cultures separately for 24, 48 and 72 h. MMS (1 X l 0 - 7 M) and DMSO (5 /zl/ml were added to the simultaneously kept cultures, as positive and negative controls, respectively. Colcemid (0.01 /~g/ml; Gibco) was added to the cultures 2 h prior to harvesting. The slides were pre-
pared, stained and scanned under code as described previously (Bali et al., 1990). At least 50-100 well spread metaphases were scanned per treatment for chromatid and chromosome type aberrations. Student's two tailed t-test was employed to ascertain statistical significance. 2.3. In uitro chromosomal aberration assay with and without metabolic actiL'ation In the metabolic activation experiments, 30-h-old cultures were given a 6-h treatment with different concentrations of estradiol in the presence of $9 mix. The cells were collected after centrifugation and the pellet was washed twice with prewarmed unsupplemented RPMI-1640 medium to remove the drug and $9 mix. Parallel cultures receiving the same dose of the estradiol for the same treatment duration but without $9 mix were also simultaneously set up for comparison. DMSO (5 /~l/ml) and cyclophosphamide (CP; 5 x 10 7 M) were used as negative and positive controls respectively in these metabolic activation experiments. Student's two tailed t-test was employed to ascertain statistical significance. 2.4. In uitro sister chromatid exchange analysis without metabolic actiuation Sister chromatid exchange (SCE) analysis was performed following the procedure of Perry and Wolff (1974) with slight modifications. BrdU (10 /~g/ml; Sigma) was added 24 h of initiation of the cultures. Three doses of estradiol (1, 10 and 100 /~g/ml) were added to each culture for 24 and 48 h. Positive (MMS, 1.1 /zl/ml) and negative (DMSO, 5 /zl/ml) controls were also simultaneously studied for each treatment duration. The cultures were incubated at 37°C for 72 h, harvested and slides were prepared. At least one hundred M~ cells with 46 chromosomes from each test were studied on the differentially stained coded slides. Student's two tailed t-test was employed to ascertain statistical significance. 2.5. In L'itro sister chromatid exchange analysis with and without metabolic actit'ation Estradiol and $9 mix was added to the cultures after 48 h of initiation. The cultures were reincubated
V.S. Dhillon, L K. Dhillon / Mutation Research 345 (1995) 87-95
at 37°C in the dark. After 90 rain of this pulse treatment, the cells were collected after centrifugation, washed twice with the prewarmed unsupplemented RPMI-1640 medium to remove the traces of estradiol and $9 mix. Finally, the cell pellet was resuspended in fresh prewarmed media supplemented with fetal calf serum, PHA and BrdU and the cultures were reincubated for 24 h in dark at 37°C. In order to compare the effects of the drug with and without $9 mix, the parallel cultures receiving same concentration of the drug, but without $9 mix, were also set up. The cultures were then harvested and slides were prepared and stained differentially. At least one hundred M 2 cells with 46 chromosomes from each test were scanned under code. Student's two tailed t-test was employed to ascertain statistical significance.
2.6. In uico micronucleus assay For micronucleus studies, the standard procedures of Schmid (1975) and Zambrano et al. (1982) were followed. Swiss albino male mice (Mus musculus L., 20-30 g, 8-12 weeks old) were procured from Disease Free Small Animal House, Haryana Agricultural University, Hissar and grouped in different cages (5 animals/group). Three different doses (1 x 10 2, 1 X 10 3 and 1 × 10 4 /xg/kg body weight) of estradiol were injected (single i.p. injection) to 3 separate groups of mice, respectively. DMSO (0.1 ml/animal) as negative control and N-methyl-Nnitro-N'-nitrosoguanidine (MNNG, 4 X 10 4 /xg/kg body weight) and ethylmethane sulfonate (EMS; 2.5 X 10 4 /xg/kg body weight) were injected to the two other groups of mice as positive controls, respectively. After 30 h, the animals were sacrificed and bone marrow smears were prepared. The slides were stained and scanned under code as per the method described previously (Bali et al., 1990). One thousand polychromatic erythrocytes (PCEs) were scored per animal. The 2 × 2 contingency X~ test with Yate's correction was used to ascertain statistical significance.
2.7. In uico sister chromatid exchange (SCE) analysis The fluorescent plus Giemsa technique of Wolff and Perry (1974) and Latt et al. (1977) was followed
89
with slight modifications for SCE analysis. Swiss albino male mice (Mus musculus L., 20-30 g, 8-12 weeks old) were grouped in different cages (5 animals/group). 5-Bromo-2-deoxyuridine in tablet form (BrdU; 1.6 g / k g body weight; Sigma) was implanted subcutaneously in the neck region of each mice under mild anesthesia. Three different doses of estradiol (1 X 10 2, 1 X 10 3 and 1 X 10 4 /xg/kg body weight), 0.1 ml of DMSO as negative control and 1.2 X 10 4 /~g/kg body weight of MNNG as positive control were injected to different groups of mice. Twenty-four hours later, colchicine (6.0 m g / k g body weight; for 2 h) was given as a single i.p. injection. Animals were then sacrificed and bone marrow smears were prepared. The slides were stained and scanned under code as per the method described previously (Bali et al., 1990). At least 25 second-division metaphases were scanned per animal. Student's two tailed t-test was used to ascertain statistical significance between treated and control groups.
2.8. Host-mediated assay The methods of Connor and Legator (1984) and Mohn et al. (1984) were followed for host-mediated assay. Animals were grouped in different cages (5 groups; 3 animals/group, i.e., one negative control group, three treatment groups and one positive control group). DMSO (5 m l / k g body weight) and 2-aminofluorene (2-AF, 20 m g / k g body weight) were injected to two groups of mice as negative and positive controls, respectively. Two injections of each chemical were given to each mouse with an interval of 12 h. After 4 h, 1.5 ml culture of each tester strain of Salmonella typhimurium was injected into the tail vein of the host animal. After a 2-h incubation of bacteria inside the host animal, the mice were killed and the livers were removed, minced, washed in ice-cold normal saline and then homogenized. Two hundred and fifty micoliters of upper homogenate was added to 2 ml top agar in a test tube, mixed and spread onto minimal glucose agar plates. Four replicates from each liver homogenate were made. The plates were incubated for 48 h at 37°C. His + revertant colonies wele counted using electronic colony counter and the results were analyzed.
V.S. Dhillon, 1.K. Dhillon / Mutation Research 345 (1995) 87-95
90
Table I Frequency of c h r o m o s o m a l aberrtions induced by estradiol in h u m a n l y m p h o c y t e s without metabolic activation Drug/
Dose
Treatment
Cells
% Aberrant metaphases
% Aberrations
chemical
(/xg/ml)
duration(h)
scored
Including
Excluding
Total
Chromatid
Chromosomal
cell _+ S.E.
gaps
gaps
300 300 300 300 300 300 300 300 300
5 5 7 6 8 10 8 12 14
4 5 6 6 8 9 8 11 13
7 9 11 I0 12 14 13 16 18
5 6 8 6 9 10 8 10 13
2 3 3 4 3 4 5 6 5
0.07 0.09 0.1l 0.10 0.12 0.14 0.13 0.16 0.18
500
2
2
3
2
0
0.03 _+ 0.01
24 48 72
500 500 500
3 4 3
3 3 2
4 4 3
3 2 3
I 2 0
0.04 ± 0.01 0.04 -+ 0.01 0.03 -+ 0.01
24 48 72
300 300 300
17 19 21
15 19 19
20 22 25
15 14 19
5 8 6
0.20 _+ 0.05 0.22 _+ 0.05 0.25 _+ 0.05
1.0
Estradiol
10.0
100.0
24 48 72 24 48 72 24 48 72
Untreated
Aberration/
± 0.02 ± 0.02 _+ 0.03 ± 0.03 _+ 0.03 _+ 0.03 _+ 0.03 _+ 0.04 _+ 0.04
a ~ ~ ~ ~ h
Negatit:e control DMSO
5.0
Positice control MMS
1 × l0 -7 M
D M S O , dimethyl sulfoxide; MMS, methylmethane sulfonate. ~,b Significant at " P < 0.05 and b p < 0.01.
Table 2 Frequency of c h r o m o s o m a l aberrations induced by estradiol in h u m a n l y m p h o c y t e s with and without metabolic activation Drug/
Dose
Treatment
$9
Cells
% Aberrant metaphases
% Aberrations
chemical
(/.Lg/ml)
duration(h)
mix
scored
Including
Excluding
Total
gaps
gaps
1.0
Estradiol
10.0 100.0
Untreated
-
Chromatid
Aberration/ Chromo-
cell ± S.E.
somal
6 6 6 6 6 6
+ + +
300 300 300 300 300 300
4 6 7 10 8 15
3 5 5 8 8 13
5 6 6 10 8 13
4 5 4 8 6 10
1 1 2 2 2 3
0.05 ± 0.01 0.07 _+ 0.02 0.07 _+ 0.02 0.10_+0.03 a 0.08 _+ 0.02 0.13_+0.03 b
6
+
500 500
2 3
2 2
3 3
3 2
0 1
0.03 _+ 0.01 0.03 _+ 0.01
6 6
+
500 500
3 4
3 4
4 5
3 4
1 1
0.04 _+ 0.01 0.05 _+ 0.02
6 6
+
500 500
10 13
9 13
18 24
14 16
4 8
0.18_+0.04 0.24_+0.05
Negatit'e control DMSO
5.0
Positice control CP
5 × l0 7 M
D M S O , dimethyl sulfoxide; CP, c y c l o p h o s p h a m i d e . a.b Significant at a p < 0.05 and b p < 0.01 (as c o m p a r e d to negative control).
V.S. Dhillon, 1.K. Dhillon /Mutation Research 345 (1995) 87-95
91
3. Results
Table 4 Frequency of sister chromatid exchange (SCE) induced by estradiol in human lymphocytes after 90 minute pulse treatment both with and without metabolic activation
3.1. In vitro assays
Drug/ chemical
Dose
Estradiol
1.0
Estradiol was unable to induce any significant dose-related increase/decrease in the mean number of His + revertants/plate both with and without $9 mix. However, at the highest doses (5 X 103 and l × 10 4 p~g/plate), there was a decrease in the number of His + revertants which was more evident in TA98 and TA100 strains (data not shown as it represents negative results). A significant increase in the aberration frequencies has been observed at all doses and at all treatment durations (except at dose 1.0 tzg/ml; 24 h and 48 h treatment; at dose 10.0 /xg/ml; 24 h treatment; Table 1) without metabolic activation. However, 6 h treatment of the drug in the presence of $9 mix induced a significant increase in aberration frequencies at the highest doses (10.0 and 100.0 / z g / m l ) as compared to the results obtained without metabolic activation (Table 2). In human lymphocyte cultures, both chromatid and chromosomal type aberrations were observed. However, the frequency of chromatid-type aberrations was more than chromosomal type. Estradiol also caused doseand duration-related significant increases in the frequencies of the sister chromatid exchange in human
(tzg/ml)
10.0 100.0
$9 Cells SCE/metaphase mix scanned (Mean_+ S.E.) -
150
5.1 _+0.42
+
150
7.8_+0.49 b
-+
150 150
5.9_+0.52 ~ 8.5_+0.58 h
--
150
6.8_+0.76 b
+
150
9.8_+0.89 h
NegatiL~e control DMSO
4.0
-
300
4.2+0.21
+
300
4.5+0.31
-
300
12.2-+0.85
+
300
14.7-+0.90
Positit,e control CP
5 . 0 × 10 7 M
Untreated
-
-
400
3.7+0.21
+
400
3.8+0.24
D M S O , dimethyl sulfoxide; CP, cyclophosphamide. ,,b Significant at " P < 0.01 and h P < 0 . 0 0 1 (as
compared to
negative control).
lymphocyte cultures both without and with $9 mix (Tables 3 and 4). 3.2. In vi~'o assays
The highest doses of the estradiol (1 × 103 and 1 × 10 4 /.~g/kg body weight) caused a significant
Table 3 Frequency of sister chromatid exchange (SCE) induced by estradiol in human lymphocytes after 24 and 48 h treatments without metabolic activation Drug/chemical
Dose(/xg/ml)
Cells scanned
SCE/metaphase (mean + S.E.) 24 h
Estradiol
48 h
1.0 10.0
150 150
5.9 _+ 0.30 ~ 6.8 + 0.56 b
6.2 _+ 0.39 ~ 7.3 + 0.59 b
100.0
150
8.2 _+ 0.61 b
8.7 _+ 0.62 ~
4.0
300
4.2 + 0.20
4.3 + 0.21
300 300
12.4 ± 0.82 11.8 ± 0.72
13.7 ± 0.89 12.8 ± 0.82
400
3.8 + 0.26
Negatire control DMSO
Positive control MMS CP
Untreated
1.1 5.0 x 10 -7 M -
D M S O , dimethyl sulfoxide; MMS, methylmethane ~.b Significant at ~ P < 0.01 and b p < 0.001.
sulfonate; CP, cyclophosphamide.
V.S. Dhillon, I.K. Dhillon / Mumtion Research 345 (1995) 87-95
92
Table 5 Micronucleated (MN) PCEs in mice bone marrow alter estradiol treatment Drug/ chemical
Dose ( / z g / k g b.wt.)
Estradiol
I × t02 I × 10 ~ I × 104
5,000 5,000 5,000
0.1 m l / animal 4 × 10~ 2.5 × 10~
10,000
Controls DMSO MNNG EMS
No of PCEs analyzed
5,000 5,000
Mean MNPCEs/ 1000 PCEs _+ S.D.
Range
Mean P C E / NCE ratio
3.99 _+ 1.0 5.25_+0.8 ~ 7.10_+ 1.3 b
2 5 4-7 5-10
0.89 0.92 1.09
1.4 _+ 0.75
1-3
0.92
8.6 _+ 2.7 9.2 _+ 2.9
6-12 6-14
1.20 1.27
PCE, polychromatic erythrocytes; NCE, normochromatic erythrocytes; DMSO, dimethyl sulfoxide; MNNG, N-methyl-N'-nitro-Nnitrosoguanidine; EMS, ethylmethane sulfonate; 1000 PCEs scanned/animal; 5 animals/treatment. a.b Significant at ~ P < 0.05 and b p < 0.01.
increase in the number of micronucleated PCEs and sister chromatid exchanges as compared to the negative controls (Tables 5 and 6).
3.3. Host-mediated assay
4. Discussion
The results obtained in the host-mediated assay did not show any significant increase/decrease in the mean number of His + revertants/plate, hence indicating the non-mutagenicity of both estradiol and
Table 6 Sister chromatid exchange (SCE) in mouse bone marrow after estradiol treatment )rug/ "heroical
Dose (/xg/kg b.wt.)
No. of metaphases analyzed
Mean S C E / metaphase _+S.D.
Range
=~stradiol
I × 10 -~ I)
