Induction of sister chromatid exchanges and cell division delays by clomiphene citrate in human lymphocytes

Abstract

Objective:

Clomiphene citrate (CC) is a selective estrogen receptor modulator and is used for the treatment of in vitro fertilization, intracytoplasmic sperm injection, intrauterine insemination, and so on. In this study, sister chromatid exchanges (SCEs) and cell cycle delays were analyzed to investigate genotoxicity and cytotoxicity of CC in peripheral blood lymphocytes of healthy women.

Methods:

Human peripheral blood lymphocytes obtained from two donors were used to detect genotoxicity and cytotoxicity of CC. Lymphocytes were treated with various concentrations (0.40, 0.80, 1.60, and 3.20 µg/ml) of CC. A negative (distilled water) and a positive control (mitomycin-C = 0.20 µg/ml) were also used simultaneously with test substance-treated cultures. SCEs and cell division delays were measured from 25 cells and 100 cells perdonor, respectively.

Results:

CC significantly increased the mean SCE value at all concentrations compared with the negative control. This increase was found to be dose dependent (r = 0.83) and at the highest concentration, nearly two times higher increase was observed than the negative control. However, replication index was not affected by the CC treatment.

Conclusion:

The present study shows that CC is genotoxic for human lymphocytes in vitro. Further investigations, especially in vivo are now needed in different test organisms to clarify the genotoxic activity of CC, which should also help to better understand genotoxic mechanism of this ovulation-stimulating drug.

Introduction

Ovulatory disorders are common causes of subfertility and infertility in women of reproductive age. Aromatase inhibitors, gonadotropin-releasing hormone analogs, clomiphene citrate (CC), and so on are used for the treatment of these disorders. CC is a selective estrogen receptor (ER) modulator and first choice for the treatment of ovulatory disorders because of its low cost and ease of use. It is primarily used for enhancing follicular development and inducing ovulation in women undergoing in vitro fertilization, intracytoplasmic sperm injection, and intrauterine insemination. ^{1 –3} It increases serum estradiol levels during the follicular phase of menstrual cycles of induced ovulation. ⁴ Induction of ovulation is most frequently used to restore ovulation in anovulatory patients with the aim of inducing unifollicular growth and release of a mature oocyte. ⁵ However, increased risk of endometrial, ovarian, and breast cancers have been reported in patients undergoing fertility treatments (gonadotropins and CC) in many studies. ^{6 –10} Likewise, opposite results about the relations between fertility treatments and such cancers have been reported in some other studies. ^{11 –17} Cancer is an uncontrolled somatic cell proliferation and results from mutations in special genes that control cell growth. Cytogenetic abnormalities are a characteristic attribute of cancer cells. ¹⁸ Chromosomal aberrations that are known important cytogenetic abnormalities have been found in all major tumor types which are determined by conventional and molecular cytogenetics. ¹⁸ There are some variety of techniques that are used for the prediction of the cancer risk of chemicals. Among them, genotoxicity tests in human lymphocytes are very important, because these are used for hazard identification with respect to DNA damage. These damages in cells can be responsible for the tumorogenesis. ^19,20 Sister chromatid exchange (SCE) is one of the test methods and is the process whereby, during DNA replication, two sister chromatids break and rejoin with one another, physically exchanging regions of the parental strands in the duplicated chromosomes. ²¹ These exchanges presumably involve DNA breakage reunion, and SCE analysis affords an excellent opportunity for cytological detection of DNA interchange. ²²

Information about the CC-induced genotoxicity is scanty. It was found to be positive in some bacterial ^23,24 and eukaryotic test systems. ^3,25,26 Since numerous data regarding the safety of a chemical are required, we aimed to assess further genotoxic and cytotoxic effects of ovulation-stimulating drug CC in cultured human lymphocytes using SCEs and replication index (RI).

Material and methods

Chemicals and reagents

Test substance CC (CAS no.: 298-02-2) was obtained from Fluka (Germany). Chromosome medium B was obtained from Biochrom (Cambridge, UK). Mitomycin-C (MMC; CAS no.: 200-008-6), bromodeoxyuridine (CAS no.: 59-14-3), and sodium chloride (CAS no.: 7647-14-5) were obtained from Sigma (St Louis, Missouri, USA).

SCE assay

Peripheral blood samples were obtained from two healthy nonsmoking (aged 24–26 years) female donors using a heparinized syringe and was added to 2.5 ml chromosome medium B supplemented with 10 µg/ml 5-bromo-2-deoxyuridine, incubated at 37°C for 72 h. Duplicate cultures were used at each concentration. CC was added after 48 h of culture initiation. Human lymphocytes were exposed to four concentrations (0.40, 0.80, 1.60, and 3.20 µg/ml) of CC which were equivalent to CC doses of 25, 50, 100, and 200 mg/day in average 60 kg females. A negative control (distilled water) and a positive control (MMC = 0.20 µg/ml) were also used simultaneously with CC-treated cultures. To arrest the cell cycle at metaphase stage, colchicine (0.06 mg/ml) was added to each culture during the last 2 h. The cultured lymphocytes were harvested by centrifugation at 1200 r/min for 10 min, resuspended in a hypotonic solution (potassium chloride, 0.075 M) for 30 min at 37°C, and then fixed in cold methanol acetic acid (3:1) for 20 min at room temperature. This fixation step was repeated three times. The slides were stained with fluorescence plus Giemsa method according to Speit and Haupter’s method, ²⁷ but with some modifications. ²⁸ The SCEs were scored from 25 cells/donor.

Cell kinetics

A total of 200 cells (100 cells from each donor) were scored for the RI, which was calculated according to the following formula: RI = [(1 × M₁) + (2 × M₂) + (3 × M₃)]/N, where M ₁, M ₂, and M ₃ represent the number of cells undergoing first, second, and third mitoses, respectively, and N represents the total number of metaphases scored. ²⁹

Statistical analyses

The frequency of SCEs in the treated cultures was compared with their respective controls using the Student’s t test. For each group, the mean ± SE was calculated. Dose-dependent relationships were determined from the regression and correlation coefficients. The RI results were analyzed using the z test.

Results

Table 1 shows the results of the genotoxic and cytotoxic evaluations of CC in human lymphocyte cultures using the SCE and RI assays. CC significantly increased the mean SCE value at all concentrations compared with the negative control. This increase was found to be dose dependent (r = 0.83). At the highest concentration, nearly two times increase compared with the negative control was observed. Minimum 1 and maximum 14 SCEs were detected in treated cultures. RI, an indicator of cell cycle delay, was not affected by the CC treatments.

Table 1.

Effects of CC on SCE and RI in cultured human lymphocytes.

Test substance	Treatment		Min–max SCE	SCE/cell ± SE	M 1	M 2	M 3	RI ± SE
	Period (h)	Dose (µg/ml)
Control	24	0.00	0–6	3.29 ± 1.48	44	54	102	2.29 ± 0.06
MMC	24	0.20	10–36	29.02 ± 8.88	68	59	73	2.03 ± 0.06
CC	24	0.40	1–13	4.92 ± 2.83a	35	64	101	2.33 ± 0.05
		0.80	2–10	5.27 ± 2.10a	43	60	97	2.27 ± 0.06
		1.60	2–8	5.55 ± 1.49a	39	69	92	2.27 ± 0.05
		3.20	3–14	6.15 ± 1.98a	43	84	73	2.15 ± 0.05

CC: clomiphene citrate; SCE: sister chromatid exchange; RI: replication index; MMC: mitomycin-C; M ₁: first mitosis; M ₂: second mitosis; M ₃: third mitosis.

^a p < 0.05: significantly different from the control (t test).

Discussion

CC is one of the most important antiestrogens used for ovulation induction. It is a nonsteroidal triphenylethylene derivative that exhibits both estrogen agonist and antagonist properties. ³⁰ It stimulates the secretion of gonadotropins from the pituitary gland by blocking the negative feedback effect of estradiol and this leads to follicle selection and estrogen production with the final occurrence of a mid-cycle luteinizing hormone surge. ^{31 –33} Structural similarity to estrogen also allows CC to bind to ERs throughout the reproductive system. CC binds nuclear ER for an extended period of time and ultimately depletes ER concentrations by interfering with the normal process of ER replenishment. ³⁰ CC treatment was found to increase gonadotropin-releasing hormone pulse frequency. ³³ Selective ER modulators are structurally diverse nonsteroidal compounds that bind to ERs and produce estrogen agonist effects in some tissues and estrogen antagonist effects in others. ³⁴ Furthermore, some epidemiological studies have linked the use of these drugs with an increased incidence of various cancers. ^{7 –10,35}

In this study, CC significantly increased the mean SCE value at all concentrations compared with the negative control. This increase was found to be dose dependent (r = 0.83). SCE is a natural molecular process exchanging genetic material between two identical sister chromatids. Single-strand breaks are prominent DNA intermediates that promote SCE. ²¹ Since SCEs represent recombinogenic events arising at DNA lesions, they became a widely used end point in studying the mutagenic and clastogenic effects of different agents.

Genotoxicity of CC has been poorly investigated in eukaryotic systems and in some bacterial systems. Ohnishi et al. reported that clomid, contains CC, caused DNA strand breaks, but few SOS responses such as mutation, induction of prophage, and expression of the umuC + gene were induced in Escherichia coli. ²³ Arriaga-Alba et al. reported that CC induces frameshift mutations in the Salmonella typhimurium TA97, TA100, and TA1538 strains with S9 but not base pair substitution mutations with neither the standard nor the preincubation method. ²⁴ CC-induced genolethal DNA damages on the E. coli PolA−/PolA+ with S9 on the preincubation method or without S9 on the disk diffusion one. London et al. reported that CC treatment resulted in a decrease in the number of ovulated oocytes and a significant (p < .05) increase in hyperploidy at 100 mg/kg in vivo in mice. In vitro, 5.0 μg/ml of CC significantly (p < .05) increased hyperploidy and reduced the proportion of metaphase I oocytes in mice. ³ Duran et al. investigated the potential genotoxicity of CC in rat reticulocytes by micronucleus assay. CC with 0.16, 0.32, and 0.64 mg/kg doses were administrated to rats intraperitoneally for 5 days. The authors have concluded that the micronucleus genotoxicity assay suggests a dose-dependent CC effect on genomic instability in bone marrow stem cells of rats in vivo. ²⁵ Yılmaz et al. have also reported increased frequency of in vitro chromosomal aberrations, micronuclei, and DNA damage in human lymphocytes. However, 1.40, 2.80, 5.60, and 11.20 μg/plate concentrations of CC did not induce base pair substitutions and frameshift mutations in TA98 and TA100 strains of S. typhimurium. ²⁶

Conclusion

In conclusion, the present study shows that CC is genotoxic for human lymphocytes in vitro confirming the results obtained by Ohnishi et al., Arriaga-Alba et al., London et al., Duran et al., and partly of Yilmaz et al. ^3,23,24,25 Further investigations, especially in vivo, are now needed in different test organisms to clarify the genotoxic activity of CC, which should also help to better understand the genotoxic mechanism of this ovulation-stimulating drug. ²⁶