Basic and clinical genetic studies on male infertility in Iran during 2000-2016: a review

The male factor contributes to 50% of infertility. The cause of male infertility is idiopathic and could be congenital or acquired. Among different factors which are involved in idiopathic male infertility, genetic factors are the most prevalent causes of the disease. Considering, the high prevalence of male infertility in Iran and the importance of genetic factors in the accession of it, in this article we reviewed the various studies which have been published during the last 17 yr on the genetic basis of male infertility in Iran. To do this, the PubMed and Scientific information database [SID] were regarded for the most relevant papers published in the last 17 yr referring to the genetics of male factor infertility using the keywords ''genetics'', "cytogenetic", ''male infertility", and "Iranian population". Literatures showed that among the Iranian infertile men Yq microdeletion and chromosomal aberrations are two main factors that intervene in the genetics of male infertility. Also, protamine deficiency [especially P2] is shown to have an influence on fertilization rate and pregnancy outcomes. The highest rate of sperm DNA damages has been found among the asthenospermia patients. In several papers, the relation between other important factors such as single gene mutations and polymorphisms with male infertility has also been reported. Recognition of the genetic factors that influence the fertility of Iranian men will shed light on the creation of guidelines for the diagnosis, consultation, and treatment of the patients

In vitro radiosensitizing effects of temozolomide on U87MG cell lines of human glioblastoma multiforme

Background: Glioma is the most common primary brain tumor with poor prognosis. Temozolomide [TMZ] has been used with irradiation [IR] to treat gliomas. The aim of the present study was to evaluate the cytotoxic and radiosensitizing effect of TMZ when combined with high-dose and high-dose rate of gamma irradiation in vitro

Methods: Two 'U87MG' cell lines and skin fibroblast were cultured and assigned to five groups for 24, 48, and 72 hours. The groups were namely, TMZ group [2000 micro M/L], IR group [5 Gy], TMZ plus IR group, control group, and control solvent group. MTT assay was applied to evaluate cell viability. Data were analyzed with SPSS 21.0 software using one-way ANOVA and Kruskal-Wallis test. P<0.05 were considered statistically significant

Results: The slope of growth curve U87MG cells in semilogarithmic scale was equal to 27.36+/-0.89 hours. The viability of cells was determined for different TMZ and IR treatment groups. In terms of cell viability, there were no significant differences between the control and control solvent groups [P=0.35] and between treated group by IR [5 Gy] alone and TMZ [2000 micro M/ml] alone [P=0.15]. Data obtained for the cell viability of combined TMZ plus IR in both cell lines compared to TMZ or IR treated group alone showed a significant difference [P=0.002]

Conclusion: The evaluation of cells viability showed that TMZ in combination with IR on glioma cells led to a significant radiosensitivity compared to IR alone

Effects of gamma radiation on fetal development in mice

Background: Many cancer patients receive radiotherapy which may lead to serious damages to the ovary storage and the matrix muscle state. Some of these patients may admit to infertility clinics for having pregnancy and on the other hand hormonal administration for superovulation induction is a routine procedure in assisted reproduction technology [ART] clinics

Objective: This study aimed to investigate fertility and fetuses of hormone treated super ovulated female mice who had received whole-body gamma irradiation before mating.Materials and Methods: Female mice were randomly categorized into a control group and 3 experimental groups including: Group I [Irradiation], Group II [Superovulation], and Group III [Superovulation and Irradiation]. In hormone treated groups, mice were injected with different doses of 59Tpregnant mare's serum gonadotropin59T [PMSG] followed with human chorionic gonadotropin [HCG]. Irradiation was done using a Co-60 gamma ray generator with doses of 2 and 4 Gy. Number of fetuses counted and the fetus's weight, head circumference, birth height, the number of live healthy fetuses, the number of fetuses with detected anomalies in the body, the sum of resorption and arrested fetuses were all recorded as outcome of treatments

Results: In the group I and group II, increased radiation and hormone dose led to a decrease in the number of survived fetuses [45 in 2 Gy vs. 29 in 4 Gy for irradiated group] as well as from 76 in 10 units into 48 in 15 units. In the group III, a higher dose of hormone in the presence of a 2 Gy irradiation boosted the slink rate; i.e. the number of aborted fetuses reached 21 cases while applying the dose of 15 Iu, whereas 6 cases of abortion were reported applying the hormone with a lower dose. Among different parameters studied, there was a significant difference in parameters of weight and height in the mouse fetuses [p=0.01]

Conclusion: The data indicated that use of ovarian stimulating hormones in mice that received pre mating gamma irradiation did not significantly increase the pregnancy rates

Effects of arbutin on radiation-induced micronuclei in mice bone marrow cells and its definite dose reduction factor

Background: Interactions of free radicals from ionizing radiation with DNA can induce DNA damage and lead to mutagenesis and carsinogenesis. With respect to radiation damage to human, it is important to protect humans from side effects induced by ionizing radiation. In the present study,the effects of arbutin were investigated by using the micronucleus test for anti-clastogenic activity, to calculate the ratio of polychromatic erythrocyte to polychromatic erythrocyte plus normochromatic erythrocyte [PCE/PCE+NCE] in order to show cell proliferation activity

Methods: Arbutin [50, 100, and 200 mg/kg] was intraperitoneally [ip] administered to NMRI mice two hours before gamma radiation at 2 and 4 gray [Gy]. The frequency of micronuclei in 1000 PCEs [MnPCEs] and the ratio of PCE/PCE+NCE were calculated for each sample. Data were statistically evaluated using one-way ANOVA,Tukey HSD test, and t-test

Results: The findings indicated that gamma radiation at 2 and 4 Gy extremely increased the frequencies of MnPCE [P<0.001] while reducing PCE/PCE+NCE [P<0.001] compared to the control group. All three doses of arbutin before irradiation significantly reduced the frequencies of MnPCEs and increased the ratio of PCE/PCE+NCE in mice bone marrow compared to the non-drug-treated irradiated control [P<0.001]. All three doses of arbutin had no toxicity effect on bone marrow cells. The calculated dose reduction factor [DRF] showed DRF=1.93 for 2Gy and DRF-2.22 for 4 Gy

Conclusion: Our results demonstrated that arbutin gives significant protection to rat bone against the clastogenic and cytotoxic effects of gamma irradiation

Effects of famotidine and vitamin C on low dose radiation-induced micronuclei in mice bone marrow cells

The radioprotective effects of vitamin C and famotidine were investigated using the micronucleus test for anticlastogenic and cell proliferation activity. Various doses of vitamin C and famotidine were administered intraperitoneally 2 h before 2Gy gamma irradiation to NMRI adult male mice. Frequency of micronuclei in 1000 PCEs [MnPCEs] were scored for each sample. Cell proliferation ratio [PCE/PCE+NCE] was also calculated. Data were statistically evaluated using one-way ANOVA and Tukey's HSD test. The results indicated that gamma irradiation alone caused a significant increase in the MnPCEs and reduced the cell proliferation ratio. Administration of various doses of famotidine and vitamin C before gamma irradiation reduced MnPCEs and therefore clastogenic effects of radiation. Famotidine didn't change cell proliferation compared to the irradiation group but vitamin C significantly improved and increased cell proliferation to the control group's level. The dose reduction factor [DRF] calculated, shows a DRF=2 for famotidine and a DRF=1.7 for vitamin C which is indicative of a high radioprotective property of these compounds. The way in which these compounds reduced the clastogenic effects of radiation might be via antioxidant property and free radical scavenging mechanism

Evaluation of embryo quality after concurrent use of ovarian stimulating hormones and gamma irradiation

Background: Radiotherapy has many side effects on fertilization in young women. Radiation can lead to ovarian failure in women who underwent abdomen or pelvic radiotherapy

Objective: This study helps us to investigate ovarian response of NMRI female mice to ovarian stimulating hormones [PMSG, HCG] after whole-body gamma irradiation

Materials and Methods: 45 pregnant mice were divided into two groups of control and experimental. The experimental group was classified into three sub-groups: Irradiation group [2 or 4Gy],Superovulation group [10 or 15IU],and superovulation and gamma-radiation group [2Gy and 10IU, 2Gy and 15IU, 4Gy and 10IU,4Gy and 15IU]. Female mice were killed and embryos were removed from oviduct .The number of embryos cells counted and the quality of them was evaluated in each group. Kruskal-Wallis test and Mann-Whitney test were used to analyze the data

Results: There was a significant difference in the number of 2-4 cells grade D embryos in 2Gy and 15IU group compared with control and 2Gy groups [p=0.01], and the number of embryos in 4Gy group was more than in 10IU and 15IU [p=0.03] and 2Gy and 15IU groups [p=0.01]. It was more significantly embryos in 4Gy and 15IU group compared to 2Gy and 15IU group [p=0.01].In addition There were no significant differences in the number of 2-4 cells grades A, B and C embryos and also number of 4-8 cells grades A, B and C, D embryos in groups

Conclusion: The concurrent use of ovulation stimulating hormones and gamma rays ameliorates this problem of drastic decrease in number of living embryos due to whole-body irradiation

Cytogenetic alterations in preimplantation mice embryos following male mouse gonadal gamma-irradiation: comparison of two methods for reproductive toxicity screening

Genome instability is a main cause of chromosomal alterations in both somatic and germ cells when exposed to environmental, physical and chemical genotoxicants. Germ cells especially spermatozoa are more vulnerable to suffering from DNA damaging agents during spermatogenesis and also more potent in transmitting genome instability to next generation. To investigate the effects of gamma-rays on inducing abnormalities manifested as numerical Chromosome Aberrations [CA] and Micronucleus [MN] in preimplantation embryos, adult male NMRI mice were irradiated with 4 Gy of gamma-rays. They were then mated at weekly intervals with super ovulated, nonirradiated female mice in 6 successive weeks. About 68 hr post coitous, four to eight cell embryos were retrieved and fixed on slides using standard methods in order to screen for CA and MN. In embryos generated from irradiated mice, the frequency of aneuploidy and MN increased dramatically at all post-irradiation sampling times as compared to the control [p<0.01]. The frequency of embryos expressed MN was much higher than chromosomally abnormal embryos, although the trend of MN formation was similar to chromosomal abnormalities seen in corresponding sampling times. Irradiation of sperms at any stages of spermatogenesis may lead to stable chromosomal abnormalities affecting pairing and disjunction of chromosomes in successive preimplantation embryos that are expressed as MN. Although chromosome analysis of embryos showed various types of chromosomal abnormalities, MN assay provide a simpler and faster technique for investigating the genotoxicity of agents affecting embryos at preimplantation stages

Variable number tandem repeat [VNTR] genotyping of hydatidiform mole in Iranian patients

Classification of molar gestation into Complete Hydatidiform Mole [CHM] and Partial Hydatidiform Mole [PHM] is done according to clinical, ultrasonographic, histologic and genetic criteria. However, making a distinction between CHM and PHM using histologic criteria alone may be difficult and several studies have shown that misclassifications are frequent, even for experienced pathologists. CHM is the most common precursor to choriocarcinoma and heterozygous moles carry an increased predisposition to transformation. Formalin-fixed, paraffin-embedded tissue sections of patients as well as peripheral blood of patients and their partners' were collected in EDTA tubes. Tissue samples were obtained by curettage. Histological evaluation was performed on routine section stained with Hematoxylin and Eosin. Variable Number Tandem Repeats [VNTRs] genotyping was performed for 30 cases in two groups of CHM [n=21] and PHM [n=9], with Polymerase Chain Reaction [PCR] amplification of 2 different polymorphic loci, namely the Col2A1 and D1S80. The results of DNA analysis by VNTR genotyping showed that in 16 cases of CHM, amplification of the VNTR polymorphic loci showed androgenetic mono-spermic moles [homozygote] and in 5 cases of CHM androgenetic dispermic moles [heterozygote] in molar tissue. In cases of PHM, 6 samples were triploid dispermic and 3 samples were diploid biparental. This study confirmed that VNTR genotyping can identify the parental source of polymorphic alleles in hydatidiform mole. Compared to STR genotyping, VNTR genotyping was performed by PCR amplification of several minisatellite markers of DNA. This method significantly requires less time and is cost-effective

Chromosome abnormalities and viability of vitrified eight-cell mouse embryos at presence of two different cryoprotectants at different storage durations

Experiments were conducted to find the differences between post-thaw viability and chromosome aberrations in eight-cell mouse embryos at presence of dimethyl sulfoxide [DMSO] and 1, 2-propanediol [PROH] as croprotectants in different storage durations. In this case-control study, a total number of 720 mouse embryos from about 250 NMRI mice were vitrified with 30% PROH or DMSO; each diluted with a solution containing 30% ficol plus 0.5 M sucrose. Embryos were exposed to the solutions for 0.5 minute at 25[degree sign] followed by cooling in liquid nitrogen, then after appropriate storage duration, they were rapidly warmed. Besides, there were 100 mouse embryos for each cryoprotectant group [totally 200 embryos] as control. Embryo survival was assessed by in vitro development, and chromosome abnormalities were analyzed by Giemsa staining. The proportion of mitotic abnormalities in PROH/DMSO vitrified embryos was significantly higher than unfrozen control group. This was confirmed also by a reduced viability of the embryos as judged by a culture at the blastocyst stage [p<0.05 in all test groups]. It can be deduced that long term cryopreservation may result in chromosomal abnormalities and/or low viability

Helicobacterpylori-induced genotoxic damage in human b lymphocytes

Helicobacter pylori [H. pylori] is recognized as the causative agent of peptic and duodenal ulcers, gastric adenocarcinoma, and low-grade mucosa-associated lymphoid tissue [MALT] lymphoma. In the present study, we investigate the genotoxic damage of lysates of H, pylori in human B lymphocytes. Human B lymphocytes were treated with 0, 10, 20, and 30 microg/mL of total protein concentration of lysates obtained from H. pylori isolates from dyspeptic patients. Direct H. pylori-induced DNA damage was investigated by the in vitro cytokinesis-block micronucleus assay which detects chromosomal fragments and maldistributed whole chromosomes. The total mean micronuclei number [tMMN] observed per 1000 binucleus B cells significantly correlated with increasing protein concentration of H pylori lysates [r[2]=0.994, p=0.006]. The highest tMMN [3.81-fold] was observed in cells treated with 30 microg/mL of H, pylori lysate [12.43 +/- 1.91] when compared with the control [3.26 +/- 0.48]. This study provides evidence of the direct effect of H, pylori in chromosomal breakage of human B lymphocytes, which might lead to the development of abnormal B cells. Long-term infection by H. pylori has been implicated in epithelial cell damage as a result of continuous induction of the immune system by bacterial antigens. However, the results of this study propose that persistent H. pylori infection could also directly damage B lymphocyte DNA from which gastric MALT lymphoma arises

rates of premature chromosome condensation and embryo development after injection of irradiated sperms into hamster oocytes

Irradiation is one of the major causes of induced sperm DNA damage. Various studies suggested a relation between sperm DNA damage and fertilization rate after intra-cytoplasmic sperm injection [ICSI]. In this study, fertilization rate and premature chromosome condensation [PCC] formation after ICSI of hamster oocytes with irradiated sperms from normal and oligosperm individuals was investigated. Human sperms were classified according to counts to normal and oligosperm. Ten samples were used for each group. Golden hamster oocytes were retrieved after super ovulation by PMSG and HCG injection. From retrieved oocytes, 468 were in metaphase II. Control and 4 Gy gamma irradiated sperms were then injected into oocytes. After pronuclei formation in injected oocytes and formation of 8 cells embryos, slides were prepared using Tarkowskie's standard air-drying technique. The frequency of embryos and PCC were analyzed using 1000x microscope after staining in 5% Giemsa. The extent of embryo development in oocytes injected by irradiated sperms was lower than those injected by non-irradiated sperms [p=0.0001]. The frequency of PCC in failed fertilized oocytes was significantly higher in oligosperms [46%] compared with normal ones [0%], but there was no significant difference between irradiated and non-irradiated samples in each group [p=0.12]. The results showed that irradiation of sperms might influence the fertilization outcome possibly due to sperm DNA damage. One possible cause of precluding oocytes from fertilization in oligosperm individuals might be the formation of PCC.

Variation of ATM gene expression in peripheral blood cells of sporadic breast carcinomas in Iranian patients

The ataxia telangiectasia mutated gene [ATM], candidate for breast cancer susceptibility gene, encode a 350-kDa protein belongs to the core components of DNA-damage response machinery. Female AT carriers have at least 5-fold increase risk for breast cancer. Reduction in ATM expression is shown in multiple studies in breast tissues. We aimed to perform a research to measure the ATM mRNA expression in peripheral blood cells in breast cancer patients. Peripheral blood sample from 40 newly diagnosed, histologically confirmed female breast cancer patients was collected before surgery. Total RNA was isolated from blood cells using the RNX-Plus solution and reverse transcribed into cDNA. Real-time PCR was performed using the 2-delta delta CT method to calculate relative changes in gene expression by REST software. The Relative Quantitation [RQ] mean was 1.27 with the min. and max. equal to 0.20 and 3.34, respectively. Calculation of patient frequencies in different groups revealed that 17.5% had reduced expression lower than two fold decreases and 15% high expression more than two fold increases, but according to REST software there was no up-regulation or down-regulation compared to normal females. The findings of multiple studies consistent with this study indicate that the ATM gene may play an important role in breast cancer development and progression, and ATM expression is down-regulated in breast cancer tissues. Although, some of the results do not support a suppressor role for ATM in the development of sporadic breast cancer, 17.5% of our patients had under expression of ATM mRNA less than two fold relative to control

Sex chromosome aneuploidy in cytogenetic findings of referral patients from south of Iran

Chromosome abnormality [CA] including Sex chromosomes abnormality [SCAs] is one of the most important causes of disordered sexual development and infertility. SCAs formed by numerical or structural alteration in X and Y chromosomes, are the most frequently CA encountered at both prenatal diagnosis and at birth. This study describes cytogenetic findings of cases suspected with CA referred for cytogenetic study. Blood samples of 4151 patients referred for cytogenetic analysis were cultured for chromosome preparation. Karyotypes were prepared for all samples and G-Banded chromosomes were analyzed using x100 objective lens. Sex chromosome aneuploidy cases were analyzed and categorized in two groups of Turners and Klinefelter's syndrome [KFS]. Out of 230 [5.54%] cases with chromosomally abnormal karyotype, 122 [30%] cases suspected of sexual disorder showed SCA including 46% Turner's syndrome, 46% KFS and the remaining other sex chromosome abnormalities. The frequency of classic and mosaic form of Turner's syndrome was 33% and 67%, this was 55% and 45% for KFS, respectively. This study shows a relatively high sex chromosome abnormality in this region and provides cytogenetic data to assist clinicians and genetic counselors to determine the priority of requesting cytogenetic study. Differences between results from various reports can be due to different genetic background or ethnicity

Cytogenetic and FMS-like tyrosine kinase 3 mutation analyses in acute promyelocytic leukemia patients

The secondary genetic changes other than the promyelocytic leukemia-retinoic acid receptor [PML-RARA] fusion gene may contribute to the acute promyelocytic leukemogenesis. Chromosomal alterations and mutation of FLT3 [FMS-like tyrosine kinase 3] tyrosine kinase receptor are the frequent genetic alterations in acute myeloid leukemia. However, the prognostic significance of FLT3 mutations in acute promyelocytic leukemia [APL] is not firmly established. In this study, the chromosomal abnormalities were analyzed by bone marrow cytogenetic in 45 APL patients and FLT3 internal tandem duplications [ITD] screening by fragment length analysis and FLT3 D835 mutation by melting curve analysis were screened in 23 APL samples. Cytogenetic study showed 14.3% trisomy 8 and 17.1% chromosomal abnormalities other than t[15;17]. About 13% of the patients had FLT3 ITD, and 26% had D835 point mutation. FLT3 ITD mutation was associated with higher white blood cell count at presentation and poor prognosis. The PML-RARA translocation alone may not be sufficient to induce leukemia. Therefore, we assume that FLT3 mutations and the other genetic and chromosomal alterations may cooperate with PML-RARA in the development of APL disease

Impact of DNA damage on the frequency of sperm chromosomal aneuploidy in normal and subfertile men

Various frequencies of sperm aneuploidy are reported in sperms of subfertile patients compared to normal individuals. Moreover, sperm DNA damage is shown to be associated with male infertility. In this study, the rate of DNA damage and frequencies of aneuploidy in sperms of subfertile patients was investigated. Semen samples were obtained from healthy normal and subfertile [oligozoospermia, asthenozoospermia, and oligoasthenozoospermia] men. The frequency of aneuploidy was assessed using primed in situ labeling [PRINS] analysis with specific primers for chromosomes 18, 21, X, and Y. Sperm DNA damage was assessed using alkaline comet assay. The mean frequencies of disomy for the patients were significantly higher than normal for all chromosomes [P<0.01]. The extent of DNA damage in sperms of subfertiles was significantly higher than in normal individuals [P<0.001]. The obtained results indicated that higher rate of DNA damages led to higher frequency of chromosomal disomy except for asthenozoospermia samples which exhibited higher rate of DNA damage and lower frequency of chromosomal disomy. These results demonstrate that men with oligozoospermia and oligoasthenozoospermia have an elevated risk for chromosome abnormalities in their sperm, particularly sex chromosomes. DNA damage might be involved in the process of malsegregation of chromosomes

Radiosensitivity and repair kinetics of gamma-irradiated leukocytes from sporadic prostate cancer patients and healthy individuals assessed by alkaline comet assay

Impaired DNA repair mechanism is one of the main causes of tumor genesis. Study of intrinsic radiosensitivity of cancer patients in a non-target tissue [e.g. peripheral blood] might show the extent of DNA repair deficiency of cells in affected individuals and might be used a predictor of cancer predisposition. Initial radiation-induced DNA damage [ratio of Tail DN A/Head DNA], dose-response curves and kinetics of DNA repair in leukocytes from healthy volunteers and prostate cancer patients were assessed using alkaline comet assay after exposure to [60]Co gamma rays. Results showed that higher levels of baseline and gamma rays induced DNA damage in leukocytes of prostate cancer cases than in controls. A similar dose response was obtained for both groups. After a repair time of 24 h following in vitro irradiation, samples from the healthy individuals showed no residual DNA damage in their leukocytes, whereas prostate cancer patients revealed more than 20%. Although similar initial radiosensitivity was observed for both groups, the repair kinetics of radiation induced DNA damage of leukocytes from prostate cancer cases and healthy subjects were statistically different. These results support the hypothesis that men affected by prostate cancer may have a constitutional genomic instability

Chromosomal variation in three human-mouse hybridoma cell lines after various passaging intervals as assessed with two different staining methods

The main objective of this study was to investigate the status of chromosome stability in 3 human-mouse hybridoma cell lines over a period of time in various passages. Metaphase spreads from 3 human-mouse cell lines [HF2X653, SPMO-4 and F3B6] that had been cultured in 4 successive passages, from 1 to 4 weeks, were prepared and analyzed. Metaphase chromosomes stained in Giemsa and a fluorescent dye, Hoechst 33258, for differential staining. This staining was performed for differentiating human and mouse chromosomes. Numerical chromosome analysis showed that although in successive passages the total number of chromosomes in hybridoma cells remained unchanged, some changes occurred in the number of human and mouse metacentric and acrocentric chromosomes during different passages. These changes were detectable, using fluorescence staining method. Since one of the main uses of human-mouse hybridoma cells is producing monoclonal antibody, chromosomal instability in these cells causes the loss of human chromosomes coding the antibody of interest occasionally. Therefore, cytogenetical analysis and characterization of these cells, especially by using the appropriate ways of chromosomal identification, is essential prior to use

Increase of CXCR4 expression on expanded non-enriched cord blood CD34+cells using MSCs

A number of potential cell adhesion molecules, which mediate essential cell-to-cell or cell-to-matrix interactions, are expressed on the surface of CD34+ hematopoietic progenitor cells [HPCs], including integrins, CD44, and CXCR4. These molecules are essential for homing process. In this study, we compared the changes of expression of CD44 and CXCR4 on the CD34+ hematopoietic progenitor cells expanded on MSCs in the presence of cytokines. Cord blood CD34+ cells were expanded using human bone marrow mesenchymal stem cells and cytokines [TPO, SCF, FLt-3, IL-6, and IL-3], and then expression of CD44 and CXCR4 on CD34+ cells were evaluated by flow cytometric analysis. After 2 weeks of serum free culture of CD34+ cells in the presence of cytokines, the expression of CXCR4 on CD34+ cells was decreased 3.4 fold [p<0.05]. In contrast, the expression of CXCR4 on CD34+ cells expanded on hMSCs was increased [p<0.05]. The expression of CD44 on expanded CD34+ cells in both methods did not differ significantly. Our results indicated that co-culture of cord blood stem cells on hMSCs significantly increased CXCR4 expression on cord blood CD34+ cells

Induction of apoptosis and micronuclei by bleomycin sulfate in different cell cycle stages of human lymphocytes

Introduction: Bleomycin sulfate is a DNA damaging agent used in cancer chemotherapy. The effect of this drug on various cell cycle stages might be different, thus inducing different modes of death [apoptotic or mitotic death]. The aim of this investigation was to study the effects of bleomycin on human peripheral blood lymphocytes at various cell cycle stages by two different end points [induction of apoptosis or micronuclei]

Material and Methods: Human peripheral blood lymphocytes were treated with various doses of bleomycin at G0, G1, and G2 phases of the cell cycle and the percentages of apoptosis [AP] and micronuclei [MN] were determined. The peripheral lymphocytes were isolated by ficoll hypaque and suspended in RPMI-1640 containing 15 % fetal calf serum. The isolated lymphocytes were stimulated by phytohemagglutinin [PHA], cultured again inRPMI-1640, harvested after 64 hrs and 96 hrs, and stained with acridine orange and ethidium bromide to determine the percentage of apoptotic cells. MN assay was done according to the standard in vitro micronucleus assay

Results: The results showed that the percentages of apoptotic cells and MN at G2 stage were significantly higher than those of G0 and G1 stages. At higher doses, MN formation and apoptotic cells were increased; however with increasing time, the percentage of MN decreased while the percentage of apoptotic cells generally increased in all the cell cycle stages

Conclusion: The results indicate that bleomycin is a potent inducer of both micronuclei and apoptosis. The incidence of apoptotic cells following bleomycin treatment in G0 and G1 was much higher than the incidence of micro nucleated cells at the two sampling times. The percentage of AP cells following bleomycin treatment remained constant across cell cycle stages