Effect of various doses of gestogens on micronuclei frequency in human peripheral blood lymphocytes of pregnant women

Olivera Milosevic-Ðordevic1,4, Darko Grujicic1, Dragoslav Marinkovic2, Slobodan Arsenijevic3 and Smilja Bankovic3

1 Faculty of Science, Institute of Biology, Department of Genetics, University of Kragujevac, Radoja Domanovica 12, P.O. Box 60, 34000 Serbia, Kragujevac, 2 Faculty of Biology, Belgrade, and 3 Clinic of Gynecology, Kragujevac, Yugoslavia 4 To

whom To whom correspondence should be addressed. e-mail: olivera@knez.uis.kg.ac.yu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Since gestogens, in the form of hormonal substitution therapy, have been proposed to have a role in the prevention of threatened spontaneous abortions during the first three months of pregnancy, we decided to evaluate possible genotoxic effects of these preparations. METHODS: A total of 30 pregnant women, with a diagnosis of threatened spontaneous abortions, received the gestogen therapy in the first 3 months, and a sample of 30 pregnant women without indication for hormonal therapy were included as the control group. For investigation of mutagenic effects of gestogens in vivo the cytokinesis block (CB) micronucleus (MN) test was applied. RESULTS: Average MN frequency in the control group was 6.79 ± 0.69 MN/1000 cells. The second analysed group included 12 patients with threatened spontaneous abortions, who received gestogen therapy in doses of 100–750 mg. Average MN frequency in these patients before therapy was 11.83 ± 1.33 MN/1000 cells, and after therapy it was 16.50 ± 1.32 MN/1000 cells (P < 0.001). The third analysed group comprised nine patients, who received gestogen therapy in doses of 750–2000 mg. Average MN frequency in these patients before therapy was 15.67 ± 3.00 MN/1000 cells, and after therapy was 23.89 ± 2.49 MN/1000 cells (P < 0.001). The fourth analysed sample comprised nine patients, treated with gestogen doses of 2000–8400 mg. The average MN frequency in these patients before therapy was 11.89 ± 1.63 MN/1000 cells, and after therapy was 21.22 ± 2.80 MN/1000 cells (P < 0.001). CONCLUSIONS: The increase of therapeutic gestogen doses was followed by an increase of average MN frequency. The greatest rise of MN frequency (1.8-fold) was observed in the group of patients who were treated with the highest gestogen doses (2000–8400 mg). The smallest increase (1.4-fold) of MN frequency was found in the group of patients whose therapeutic doses were the lowest (100–750 mg).

Key words: genotoxicity/gestogens/micronuclei/peripheral blood lymphocytes/pregnancy


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Progestins include the naturally occurring hormone progesterone, which is rarely used therapeutically, as well as a number of frequently used synthetic compounds that have progestational activity. These synthetic agents are frequently used in the treatment of dysmenorrhoea, endometriosis, hirsutism, uterine bleeding and during the first trimester of pregnancy for imminent or habitual abortions (Williams and Stancel, 1996Go).

Natural progesterone is spontaneously produced in great abundance during normal pregnancy, and is devoid of any disturbing teratogenic, metabolic, or haemodynamic effects. This is contrary to certain artificial progestogens (Benedeti, 1983Go).

Meta-analysis has reported that exogenous progesterone supplementation after conception does not improve pregnancy outcome (Goldstein et al., 1989Go).

A group of authors studied the teratogenic effect of synthetic progesterones. Reports of abnormalities in infants due to the exposure to progestogens in utero have included hipospadias (Czeizel, 1985Go), other genito-urinary anomalies (Evans et al., 1980Go), tetralogy of Fallot (Heinonen et al., 1977Go) and fetal masculinization (Wilkins, 1960Go).

Certain authors consider that artificial steroid hormones are genotoxic, directly involved in the change of DNA structure (Schluster et al., 1976Go), while others suppose that these hormones are acting indirectly by oxidation of reactive metabolites formed during their metabolism (Liehr, 1990Go).

The detection of micronuclei (MN) has been used as a rapid and sensitive screening system for evaluating genotoxic risk in populations. MN in cells undergoing mitosis are the result of structural chromosomal aberrations, or disturbed function of the mitotic spindle. Some authors consider that MN frequencies in peripheral blood lymphocytes of humans provide an effective estimation of the effects of physical and chemical factors (Fenech and Morley, 1985Go). This test allows an analysis of a large number of lymphoblasts per sample, which is important for a more realistic picture of the cell’s cytogenetic damage, as well as for the discovery of genotoxic effects in tested preparations (Di Giorgio et al., 1994Go; Bonassi et al., 1995Go; Fenech, 1997Go).

In previous papers (Grujicic, 1999Go; Grujicic et al., 1999Go), we showed that gestogens (progesterone and its derivatives) induced a significant increase of MN frequency in female patients who maintained their pregnancies by taking these preparations, as well as in their newborns. With respect to the wide application of gestogens in gynaecological practice, as well as the presence of controversies in literature about the possible role of steroids or hormonal contraceptives in induction of chromosomal changes in mammalian cells, the main aim of this study was to determine dose-dependent genotoxic effect of these preparations, using the cytokinesis block micronucleus (CB MN) test.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
For investigation of mutagenic effects of gestogens in vivo, the CB MN test was applied to a sample of 30 patients with a diagnosis of threatened spontaneous abortions, who received gestogen therapy in the first 3 months. Patients received one of the following models for hormonal substitution therapy: gestormone (17-{alpha}-allylestr-4-en-17 {beta}-ol) 2x5 mg/day, gravibinone (Hydroxyprogesteronum capronicum) 250mg i.m on day 3 and progesterone (20-{alpha}- dihydroprogesterone) 250mg i.m. on day 3.

The analyses of MN frequency in peripheral blood lymphocytes of patient were carried out before and after therapy. A control sample consisted of 30 women between weeks 7–8 of pregnancy who were without indication for hormonal therapy.

In our cytogenetic study a classic method for cultivation of human peripheral blood lymphocytes was used (Edwards, 1962Go; Frland, 1962Go) together with some modifications which were necessary for applying the CB MN test (Fenech and Morley, 1985Go).

For the cell cultures 77% Parker 199 (Institute Torlak, Belgrade, Yugoslavia, 20% fetal calf serum (Sigma) and 3% phytohaema glutinin (INEP, Belgrade, Yugoslavia) were used. Under sterile conditions, 20 drops of whole heparinized blood were put into sterile bottles with 5 ml of medium.

Cell cultures were incubated at 37°C, for a total of 72 h. Cytochalasin B at a final concentration of 4 µg/ml was added after 48 h from the start of incubation.

Cell cultures were centrifuged at 78 g for 10 min and the cell suspension was washed in 0.9% saline and treated with hypotonic solution (0.56% KCl). Cell suspension was fixed for 2x15 min in a fixation solution 3:1 (methanol:acetic acid).

The material obtained was spread onto specially prepared, dry, cold, and lamp-dried slides. After 5–7 days the slides were stained with 2% Giemsa (Alfapanon, Novi Sad, Yugoslavia).

The MN frequency was determined by the analysis of 1000 binucleated cells per person. The obtained results were compared using the Student’s t-test.


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 Materials and methods
 Results
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 References
 
The results of the analyses of MN frequency in binucleated cytochalasin B-stimulated lymphoblasts in all four tested samples are presented in Table I and in Figure 1.


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Table I. Effect of different doses of gestogens on micronucleus frequency in control and treated sample of patients
 


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Figure 1. The effect of different doses of gestogens on micronucleus (MN) frequency before and after therapy. *Statistically significantly different (P < 0.001; Student’s t-test).

 
The A group included patients who received gestogen therapy in doses of 100–750 mg. The average MN frequency in these patients after the applied therapy increased 1.4-fold. Correlation (Spearman’s) between received gestogen doses and MN frequency in this group of patients after the therapy was the highest (r = 0.60; P = 0.04).

The B group included nine patients who received gestogen therapy in doses of 750–2000 mg. The average MN frequency in this group of patients after therapy increased 1.5-fold. There was no significant correlation between received gestogen doses and MN frequency in this group of patients after therapy.

The C group included nine patients treated with gestogen doses of 2000–8400 mg. In this group of patients the average MN frequency after the applied therapy increased 1.8-fold. We found positive correlation, although not significant, between received gestogen doses and MN frequency (r = 0.51; P = 0.16) in this investigated group after therapy.

Summarizing the results for all four analysed groups, we observed that, prior to receiving therapy, patients with an indication for hormonal therapy (groups A, B and C) had ~2-fold higher MN frequency, in comparison with those in the control group of pregnant women.

The average MN frequency after therapy in groups A, B and C was statistically significantly higher (P < 0.001), in comparison with average MN frequencies in the same patients before therapy (~1.5-fold), and also with those of the control group. In comparison with the control group of healthy, pregnant women, average MN frequency in the investigated groups was increased after therapy (~3-fold).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since chromosomal aberrations are basic in MN genesis, many authors consider that MN frequency is a valid indicator for frequency of chromosomal aberrations (Countryman and Heddle, 1976Go; Fenech and Morley, 1985Go; Fenech, 1997Go). Our previous paper showed that at the beginning, and even before therapy, pregnant women with an indication for hormonal therapy had ~2-fold higher MN frequency, in comparison with the control group of pregnant women (Grujicic et al, 1999Go). The obtained result could be compared with the results of Trkova et al. (2000Go), in that patients with reproductive failure have increased MN frequency in comparison with those in the sample of fertile (control) women.

In a number of published studies on MN it has been established that higher frequencies were found in the populations exposed to genotoxic agents (Augusto et al., 1997Go; Bukvic et al., 2000Go).

The obtained results confirmed that those gestogens showed a genotoxic effect on human peripheral blood lymphocytes. A number of authors (Klopper et al., 1974Go; Heras et al., 1982Go; Lukic and Barjaktarovic, 1987Go; Liehr, 1990Go; Ahmed et al., 2000Go) obtained the same results while investigating the genotoxic effect of steroid hormones by applying other cytogenetic methods, such as the sister chromatide exchange.

Our results showed that the average MN frequency in the investigated groups of patients increased (A group) with the increase of gestogen therapeutic doses in later applications, and the plateau was established (for B and C groups), regardless of the intensity of the applied therapy.

We observed contradictory results in group B of patients. The average MN frequency after treatment (23.89 ± 2.49 MN/1000 cells) was greater in comparison with all other groups while the number of binucleated cells that contained MNs was the lowest (1.2-fold higher than the number of MNs in the cells before therapy).

Bukvic et al. (2000Go) reported that the highest hormone concentrations increased the number of highly damaged cells and a reduced mitotic index.

The cited therapeutic doses for therapy in patients induced remarkable changes in genetic material, which we detected as the greatest average MN frequency. The cells with damaged genetic material held the cell cycle in the G1-phase and prevented the cell from entering the S-phase, until all damages were repaired. This was the signal for p53-mediated cell cycle checkpoint, preventing proliferation of micronucleated cells (Schwardz et al., 1997Go), which is why the cell cycle was slower, and present in a smaller percentage of cells in mitosis. Mitosis was late and we found an increased MN frequency and smaller number of binucleated cells with MNs; their number was smaller than in other groups.

A series of recently published studies reported that steroid hormones induced dose-dependent micronuclei increases and aneuploidy (Ahmed et al., 2000Go; Bukvic et al., 2000Go). Gestogens as steroid hormones affect target cells by binding their steroid receptors (Prendergast et al., 1996Go; Klotzbucher et al., 1997Go). Then hormone-receptor (H-R) complex interacts directly with DNA chromatin. Consequently, H-R complexes act until target cells achieve free receptors while the dose-dependent increase of MN frequency appears with the increase of therapeutic gestogen doses. This conclusion confirms a positive and statistically significant correlation (r = 0.60; P < 0.05) between the applied gestogen doses and MN frequency in group A patients after the therapy.

Continuous increase of hormone concentration leads to saturation of steroid receptors and therefore, a plateau is established (group B).We believe that the plateau in the rise of MN frequency in investigated groups after therapy was due to saturation of progesterone receptors by permanent increase of total therapeutic gestogen doses.

After the increase of therapeutic gestogen dose >2000 mg, isomeric receptor forms were generated and new H-R complexes that affected chromatin appeared. Therefore, in the group C patients we observed the greatest increase of MN frequency (1.8-fold) after therapy in comparison with MN frequency before therapy.

The dose of gestogens of 750–2000 mg was critical for maintaining the cell cycle and for activation of the repair system, as well as for formation of isomeric forms of receptors.

Continuous rise of therapeutic gestogen dose (from 2000 to 8400 mg), established a positive correlation between dose and MN frequency after therapy and formation of isomeric forms of receptors.

Despite controversies in the literature, our results obtained by CB MN test, indicate clearly that gestogens, as a class of steroid hormones, contribute to chromosomal changes in peripheral blood lymphocytes. We observed that gestogens as hormonal substitution therapy induced a significant increase (P < 0.001) in MN frequencies after therapeutic treatment, without regard to therapeutic doses.

This paper provides evidence that synthetic progesterones are genotoxic and should not be used during pregnancy.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on April 29, 2002; resubmitted on July 12, 2002; accepted on October 17, 2002.