P. Venkatachalam

ACE and MTHFR gene polymorphisms in unexplained recurrent pregnancy loss.

Aim:  To assess the association between polymorphisms in angiotensin converting enzyme and methylene tetrahydrofolate reductase genes and recurrent pregnancy loss by a case‐control study in South Indian women.

Bleomycin, neocarzinostatin and ionising radiation-induced bystander effects in normal diploid human lung fibroblasts, bone marrow mesenchymal stem cells, lung adenocarcinoma cells and peripheral blood lymphocytes

Purpose: To determine whether the bystander effects induced by chemotherapeutic agents are similar to those induced by ionising radiation and to analyse the cell dependency, if any, in different human cell types such as normal lung fibroblasts (WI-38), human bone marrow mesenchymal stem cells (hBMSC), lung adenocarcinoma (A-549, NCI-H23) and peripheral blood lymphocytes (PBL). Materials and methods: The cells mentioned above were exposed to two different concentrations of bleomycin (BLM) and neocarzinostatin (NCS) and to X-irradiation. Co-culture methodology was adopted to study the in vitro bystander effects. DNA damage was measured using a micronucleus (MN) assay as an endpoint to study the bystander response. High performance liquid chromatography (HPLC) was performed to rule out any residual activity of BLM and NCS. To further investigate if this bystander response is mediated through reactive oxygen species (ROS), the bystander cells were pretreated with dimethyl sulphoxide (DMSO), an ROS scavenger, and co-cultured with cells exposed to BLM. Results: Bystander response was observed in all five types of human cells (WI-38, hBMSC, NCI-H23, A-549 and PBL) co-cultured with exposed cells. While all cell types showed a bystander response, undifferentiated hBMSC and PBL showed a higher magnitude of bystander response. A reduction in the MN frequency was observed in co-cultured hBMSC and PBL pretreated with DMSO. Conclusion: These results suggest that the chemotherapeutic agents, BLM and NCS, induce bystander response which is similar to that induced by radiation. Furthermore, it is observed that the bystander effect is independent of the cell type studied. Our results further support the involvement of ROS in mediating the bystander response induced by BLM.

The effect of growth architecture on the induction and decay of bleomycin and X-ray-induced bystander response and genomic instability in lung adenocarcinoma cells and blood lymphocytes

Abstract Purpose: Cancer patients treated with radiomimetic drug bleomycin (BLM) have shown incidence of 7% second malignancy. Studies regarding BLM-induced genomic instability in bystander cells are scarce, and experiments with cells grown on three-dimensional (3D) cultures to mimic the in-vivo condition have never been attempted. Materials and methods: A549 and NCI-H23 (human lung adenocarcinoma) cells were grown as 3D cultures using Cytomatrix™, exposed to BLM or X-radiation and co-cultured with their respective unexposed cells. The DNA damage in direct and bystander cells were assessed by the induction of micronuclei (MN) or phosphorylated serine-15 residue in protein 53 (p53ser-15), a reflection of DNA damage, and by up-regulation of protein 21 (p21Waf1). The persistence of DNA damage was measured using MN assay and fluorescence in situ hybridization (FISH) in cancer cells and human peripheral blood lymphocytes (PBL) respectively. Results: BLM or X-irradiation induced DNA damage in both A549 and NCI-H23 cells and their respective bystander cells grown in 2D or 3D cultures. Further persistence of these damages in bystander PBL at delayed times indicated genomic instability in these cells. Conclusion: BLM-induced genomic instability in the progeny of bystander cells and their significance in therapy-induced second malignancy may not be eliminated completely.

Human brain glioblastoma cells do not induce but do respond to the bleomycin-induced bystander response from lung adenocarcinoma cells

To determine whether the bleomycin (BLM)-induced bystander response occurs in human brain glioblastoma (BMG-1) cells, the BMG-1 cells were exposed to two different concentrations of BLM. The co-culture methodology was adopted to study the in vitro bystander effects. DNA damage was measured using the micronucleus (MN) and γ-H2AX assays. Cytotoxicity was measured using the trypan blue assay. Cell cycle kinetics was analyzed using flow cytometry. The overall results did not show any significant increase in either genotoxicity or cytotoxicity or a delay in the cell cycle kinetics in BMG-1 bystander cells co-cultured with BLM-exposed cells, suggesting that BLM did not induce a bystander response in the BMG-1 cells. Furthermore, the MN results of the BLM-exposed BMG-1 cells co-cultured with unexposed bystander human lung adenocarcinoma (A549 and NCI-H460) cells and vice versa suggested that the BMG-1 cells do not secrete bystander signals but do respond to those signals. Analyzing the underlying mechanism and pathways involved in preventing the cells from secreting bystander signals will provide new insights that can be applied to inhibit these mechanisms in other cell types, thereby preventing and controlling the bystander response and genomic instability and increasing the therapeutic gain in chemotherapy.

Dicentric assay: Inter-laboratory comparison in Indian laboratories for routine and triage applications

An Inter-Laboratory Comparison (ILC) study on Dicentric Chromosome Assay (DCA) was carried out between two Indian biodosimetry labs. Human peripheral blood samples exposed to 10 different doses of X-rays up to 5Gy were shared between the labs to generate calibration data. Validation of calibration curves was done by dose estimation of coded samples exposed to X- or gamma radiation. Reliability of the DCA data for triage application was evaluated by scoring 20, 50 and 100 metaphases in the dose range of 0.5-3.0Gy. No significant difference was observed between labs regarding the established calibration data as well as the DCA triage dose assessments. Scoring of 20 metaphases (MP) was adequate to detect radiation exposure of >2Gy whereas 50 MP were sufficient to determine exposures of 0.5Gy. Both labs performed the DCA in a reliable manner and made the first step in setting up a biodosimetry network in India.

Mean frequency and relative fluorescence intensity measurement of γ-H2AX foci dose response in PBL exposed to γ-irradiation: An inter- and intra-laboratory comparison and its relevance for radiation triage

Measurement of γ‐H2AX protein changes in the peripheral blood lymphocytes (PBL) of individuals exposed to ionizing radiation is a simple, sensitive, and rapid assay for radiation triage and early marker of dose estimation. The qualitative and quantitative measurements of the protein changes were examined using flow cytometry and microscopy. Whole blood and isolated lymphocytes were exposed in vitro between 0.1 and 5 Gy doses of 60Co γ‐radiation at a dose rate of 1 Gy/min. Radiation induced γ‐H2AX foci frequency (n = 3) and relative fluorescence intensity (n = 7) in PBL was measured at 0.5 and 2 hrs postexposure. The observed dose response for γ‐H2AX foci frequency at both time points, for whole blood and isolated lymphocytes did not show any significant (P > 0.05) differences. However, when compared with γ‐H2AX foci frequency scored manually (microscopy), the semiautomated analysis (captured images) showed a better correlation (r2 = 0.918) than that obtained with automated (Metafer) scoring (r2 = 0.690). It is noteworthy to mention that, the γ‐H2AX foci frequency quantified using microscopy showed a dose dependent increase up to 2 Gy and the relative fluorescence intensity (RFI) measured with flow cytometry revealed an increase up to 5 Gy in the PBL exposed in vitro. Moreover, a better correlation was observed between the γ‐H2AX foci frequency obtained by manual scoring and RFI (r2 = 0.910). Kinetic studies showed that the γ‐H2AX foci remain more or less unchanged up to 4 hrs and reduces gradually over 48 hrs of postexposure at 37°C. Further, inter and intra‐laboratory comparisons showed consistency in the scoring of γ‐H2AX foci frequency by manual and semiautomated scoring. The overall results suggest that measurement of γ‐H2AX (microscopy and flow cytometry) should be employed within 4 to 6 hrs for a reliable dosimetry either by sharing the work load between the laboratories or investing more manpower; however, triage can be possible even up to 48 hrs of postirradiation.

Modifications of Bleomycin Induced Cytogenetic Damages by 2-Deoxy-D-Glucose on Normal and Tumor Cells

Abstract The radiomimetic drug bleomycin induced chromosomal aberrations (CA) and micronuclei (MN) were studied in peripheral blood lymphocytes (PBL), as reference to normal cells and Glioma (BMG -1) as reference to tumor cells, with and without exposure to 2-deoxy D-glucose (2-DG, an analogue of glucose). Treatment with bleomycin increased both CA and MN frequency in a dose dependent manner in both cell types. The frequencies of CA are 2 fold higher than MN for a given concentration of bleomycin. Exposure to bleomycin predominantly induced exchange type chromosome aberrations. While in the presence of 2-DG the aberrations induced by bleomycin reduced significantly in PBL, the same was increased significantly in BMG cells (P<0.001) showing a protective effect and sensitizing effect on normal and tumor cells respectively. The dose modulatory factor (protection) for different concentration of bleomycin exposure varied between 0.38 and 0.72 for CA and 0.1 and 0.84 for MN in PBL. In the case of BMG-1 cells, the modulatory factor (sensitization) varied between 1.42 and 2.59 for CA, 1.25 and 1.66 for MN at different concentration of bleomycin exposure. The modulatory effect of 2-DG was also evidenced from the coefficients obtained for the dose-response curves of the aberrations studied. The paper discusses the types of aberrations induced by bleomycin and the mechanism involved for differential modifications of cytogenetic damage by 2-DG in normal (PBL) and tumor (Glioma) cells.

Micr odeletions of AZFc Region in Infertile Men with Azoospermia and Oligoasthenoteratozoospermia

Abstract Considerable progress has been made to study the pathophysiology of male infertility in the last few years. About 10-15% of genetic causes such as chromosomal abnormality, single gene and multifactorial mutations may be a causative factor. Submicroscopic microdeletions on chromosome Y is considered to be the most frequent genetic defects associated with impaired spermatogenesis. Therefore, the aim of this present study was to assess pregnancy outcome of intracytoplasmic sperm injection (ICSI) in infertile men from 285 study subjects; fertile controls (n=110) and cases (n=175) with sperm count less than 5 millions/ml [azoospermia (n=150) and oligoasthenoteratozoospermia (n=25)]. Microdeletions located on chromosome Y-sY84, sY86 (AZFa), sY127, sY134 (AZFb), and sY254, sY255 (AZFc) were confirmed with Polymerase Chain Reaction (PCR). A 12.56% was observed in infertile men in one or more STS with a percentage of 1.14, 2.28, 9.14 for AZFa, b and c respectively. The azoospermia factor region c (AZFc) loci showed an increase in frequency when compared to AZFb and a regions. Intracytoplasmic Sperm Injection (ICSI) could be offered or an alternative method to patients with AZFc deletion.

Protective Effect of 2-Deoxy-D-Glucose on Chemotherapeutic Drugs Induced Damages on Peripheral Blood Lymphocytes Exposed in-Vitro

Abstract The effect of 2-deoxy-D-glucose (2-DG), an antimetabolite of glucose was studied in peripheral blood lymphocytes (PBL) exposed to radiomimetic drug bleomycin and an alkylating agent mitomycin-C. The PBL were exposed to 2-DG (5 mM), 30 minutes pretreatment and with Bleomycin (10 to 80 μg/ml) and Mitomycin-C (2 to12 μg/ml) for three hours. The drug as well as 2-DG was removed by washing the cells with HBSS buffer. Then the cells were cultured for 48 hours to study chromosomal aberrations (CA), Translocations (TL) and 72 hours for micronuclei (MN) and Sister Chromatid Exchanges (SCE). Exposures of PBL to Bleomycin and Mitomycin-C showed, a concentration dependent increase in the aberration frequencies, both in the presence and absence of 2-DG. While, the regression analysis showed, that the presence of 2-DG reduced bleomycin induced TL, CA frequencies and Mitomycin-C induced CA and MN frequencies significantly (P<0.001) when compared to PBL treated with the drugs alone, Bleomycin induced MN frequencies and Mitomycin-C induced SCE’s reduction were not significant. The difference could be attributed to the mechanism of the action of drugs on the cells. Furthermore the alteration in the cell cycle kinetics, suggest that the presence of 2-DG during drug exposure, alter the cellular environment and delay the cell proliferation and provide sufficient time to repair the damages, could resulted in the reduced aberration frequencies.

A Study of Lactate Dehydrogenase (LDH) Isoenzyme is a Biochemical Tumour Marker in Cervical Carcinoma Patients

Abstract To establish Lactate dehydrogenase (LDH) isoenzyme is a biochemical tumor marker in cervical carcinoma patients for diagnosis and treatment monitoring of the disease. The Serum and cervical tissue LDH isoenzyme was analyzed qualitatively by Poly Acrylamide Disc Gel Electrophoresis method. The total LDH activity was estimated quantitatively by the method of UV spectrophotometry. 50 untreated cervical carcinoma patients were taken up and compared with age matched healthy females (controls). Out of 50 patients, 5 patients were histologically classified as well differentiated squamous cell carcinoma (Grade-I), 23 were moderately differentiated squamous cell carcinoma (Grade-II) and 22 were poorly differentiated squamous cell carcinoma (Grade-III). Grade-I patients did not show any change, in the serum and cervical tissue LDH isoenzyme fractions compared to the controls but variation in electrophoretic mobility have been observed. Grade-II patients showed two new additional isoenzyme fractions in LDH2 and LDH3 locations, where as Grade-III patients showed only three LDH isoenzyme fractions. The total LDH activity in both serum and cervix tissue of normal individuals are 0.458±0.0796 units/ml; and 0.680±0.0218 units/mg. Grade-I patients did not show any change in total LDH activity (serum: 0.384±0.0404 units/ml; cervical tissue: 0.589±0.0292 units/mg; P<0.01). Grade-III patients showed much lower values (serum: 0.284±0.0109 units/ml; cervical tissue: 0.327±0.043 units/mg; P<0.001), where as Grade-II patients showed significant increase in total LDH activity (serum: 0.878±0.0531 units/ml; cervical tissue: 1.296±0.0813 units/mg; P<0.001) respectively. The results suggest that LDH isoenzyme is useful biochemical tumor marker for diagnosis and to assess the grade of malignancy.