Shiv Kumar Giri

Derivation, Characterization and Differentiation of Buffalo (Bubalus bubalis) Amniotic Fluid Derived Stem Cells

Amniotic fluid cells (AFCs) are obtained from amnion for pre-natal analysis and can be cultured in vitro. Heterogeneous amniotic fluid (AF) contains various cell types, and it is believed that some of these cells possess the stem cell properties. The aim of this study was to characterize these cells by phenotypical and genotypical means in buffalo. The differentiation potential of amniotic fluid stem (AFS) cells was carried out by converting these cells into neurons. The AFCs were cultured without feeder cells in DMEM containing 16% foetal bovine serum, 1% penicillin/streptomycin and 1%l-glutamine in 5% CO(2) at 38.5 ± 0.5 °C in a CO(2) incubator. After 6 days of culture, different types of cells viz., star shaped (62.7%), spherical without nucleus (1.9%), spherical with nucleus (26.4%), pentagonal (0.4%) and free floating/rounded cells (8.3%) were observed. Most of the cells started anchorage-dependent growth after day 7 of the culture. Expression of Oct-4, Sox-2, Nanog, alkaline phosphatase, 18s rRNA, stem cell factor, cyclin A, Nestin and FGF-5 was observed from the AFS cells in different passages with PCR amplicon of 314, 277, 317, 180, 162, 216, 421, 307 and 210 bp, respectively. During the differentiation step, at day 6, neuron-like cells could be clearly identified and confirmed with Nestin-specific RT-PCR. The cells were found to have a normal karyotype at different passages. These results may contribute towards establishing non-embryonic pluripotent stem cells for various therapeutic and reproductive biotechnological applications in the species.

Isolation, culturing and characterization of feeder-independent amniotic fluid stem cells in buffalo (Bubalus bubalis).

Heterogeneous amniotic fluid contains various cell types. The aim of this study was to characterize and differentiate some of the key stemness attributes of the amniotic fluid-derived cells in buffalo (Bubalus bubalis). The amniotic fluid (AF) cells were cultured without feeder cells, in DMEM containing 15% FBS, 1% non-essential amino acids, 1% penicillin/streptomycin/ampicillin, 1% vitamin solution, and 1% l-glutamine in 5% CO(2) in humidified air at 38.5±0.5 °C. After 6 days of culture different types of cells viz., star shaped (62.7%), spherical without nucleus (1.9%), spherical with nucleus (26.4%), pentagonal (0.4%), and free floating/rounded cells (8.3%) were observed. Most of the cells started anchorage-dependent growth after day 7 of the culture. Expression of alkaline phosphatase (AP) and Oct-4, Nestin and FGF-5 were observed from the AF cells at different passages. Using species-specific primers, a PCR amplicon of 200, 296 and 210 bp were observed for Oct-4, Nestin and FGF-5, respectively. The cells were found to have a normal karyotype at different passages. These results may contribute towards establishing non-embryonic pluripotent stem cells for various therapeutic and reproductive biotechnological applications in the species.

CYP1A1 Gene Polymorphisms: Modulator of Genetic Damage in Coal-Tar Workers

AIM It is well known that polycyclic aromatic hydrocarbons (PAHs) such as benzo (a) pyrene have carcinogenic properties and may cause many types of cancers in human populations. Genetic susceptibility might be due to variation in genes encoding for carcinogen metabolizing enzymes, such as cytochrome P-450 (CYP450). Our study aimed to investigate the effect of genetic polymorphisms of CYP1A1 (m1 and m2) on genetic damage in 115 coal-tar workers exposed to PAHs in their work place. METHODS Genetic polymorphisms of CYP1A1 were determined by the PCR-RFLP method. Comet and buccal micronucleus assays were used to evaluate genetic damage among 115 coal tar workers and 105 control subjects. RESULTS Both CYP1A1 m1 and CYP1A1 m2 heterozygous and homozygous (wt/mt+mt/mt) variants individually as well as synergistically showed significant association (P<0.05) with genetic damage as measured by tail moment (TM) and buccal micronuclei (BMN) frequencies in control and exposed subjects. CONCLUSION In our study we found significant association of CYP1A1 m1 and m2 heterozygous (wt/mt) +homozygous (mt/mt) variants with genetic damage suggesting that these polymorphisms may modulate the effects of PAH exposure in occupational settings.

Effect of genetic polymorphism of GSTM1 and GSTT1 genotypes on cytogenetic biomarkers among coaltar workers

Chromosomal aberrations (CAs) in peripheral blood lymphocytes and micronuclei (MN) in exfoliated buccal cells have been used for decades as cytogenetic biomarkers to investigate genotoxicity among occupationally or environmentally exposed population. In our study, we investigated the association of increased cytogenetic damage with genetic polymorphism in glutathione-S transferase genotypes among occupationally exposed 115 coaltar workers and 105 unexposed controls. We found higher mean value of chromosome aberrations (chromatid type-2.01±1.76; chromosomal type-2.22±1.73) and buccal micronuclei (BMN-7.10±1.56) in exposed subjects when compared to referents (chromatid type-0.82±.51; chromosomal type-0.87±.54; BMN-5.09±2.88). We observed that individuals having null genotype of GSTM1 and GSTT1 have significantly higher frequency of CAs and MN. Despite of small sample size, our findings suggest a significant association between polymorphism of glutathione-S transferase genotypes and cytogenetic biomarkers which are considered as early effects of genotoxic carcinogens.

Influence of GSTM1 and GSTT1 genotypes and confounding factors on the frequency of sister chromatid exchange and micronucleus among road construction workers.

In the present study, we have investigated the influence of polymorphism of GSTM1 and GSTT1 genes and confounding factors such as age, sex, exposure duration and consumption habits on cytogenetic biomarkers. Frequency of sister chromatid exchanges (SCEs), high frequency cell (HFC) and cytokinesis blocked micronuclei (CBMN) were evaluated in peripheral blood lymphocytes of 115 occupationally exposed road construction workers and 105 unexposed individuals. The distribution of null and positive genotypes of glutathione-S transferase gene was evaluated by multiplex PCR among control and exposed subjects. An increased frequency of CBMN (7.03±2.08); SCE (6.95±1.76) and HFC (6.28±1.69) were found in exposed subjects when compared to referent (CBMN - 3.35±1.10; SCE - 4.13±1.30 and HFC - 3.98±1.56). These results were found statistically significant at p<0.05. When the effect of confounding factors on the frequency of studied biomarkers was evaluated, a strong positive interaction was found. The individuals having GSTM1 and GSTT1 null genotypes had higher frequency of CBMN, SCE and HFC. The association between GSTM1 and GSTT1 genotypes and studied biomarkers was found statistically significant at p<0.05. Our findings suggest that individuals having null type of GST are more susceptible to cytogenetic damage by occupational exposure regardless of confounding factors. There is a significant effect of polymorphism of these genes on cytogenetic biomarkers which are considered as early effects of genotoxic carcinogens.

Expression of Transcriptional Factor Genes ( Oct-4 , Nanog , and Sox-2 ) and Embryonic Stem Cell-Like Characters in Placental Membrane of Buffalo ( Bubalus bubalis )

The aim of the study was to assess the expression of transcriptional factor genes and embryonic stem cell-like characters in the placental membrane of buffalo (Bubalus bubalis). Along with the placenta, amniotic fluid, maternal peripheral blood, and umbilical cord blood samples were taken for the future study. The isolation and culture of cells from the placental membrane was followed by the determination of RT-PCR-based markers (Oct-4, Nanog, Sox-2, alkaline phosphatase, stem cell factor, and Nestin) of these cells. Placental membrane cells also positively expressed alkaline phosphatase staining. We isolated adherent cells from trypsin–EDTA-digested placentas and examined these cells for morphology, surface markers, and differentiation potential and found that they expressed several stem cell markers. They also showed neurogenic and adipogenic differentiation potentials under appropriate guided conditions. We suggest that placenta-derived cells have multilineage differentiation potential similar to mesenchymal stem cells in terms of morphology and cell-surface antigen expression. The placenta may prove to be a useful source of mesenchymal stem cells.

Polymorphic Variation of CYP1A1 and CYP1B1 Genes in a Haryana Population

Cytochrome P450 (CYP) 1A1 and CYP1B1 are important phase I xenobiotic metabolizing enzymes involved in the metabolism of numbers of toxins, endogenous hormones, and pharmaceutical drugs. Polymorphisms in these phase I genes can alter enzyme activity and are known to be associated with cancer susceptibility related to environmental toxins and hormone exposure. Their genotypes may also display ethnicity-dependent population frequencies. The present study was aimed to determine the frequencies of commonly known functional polymorphisms of CYP1A1 and CYP1B1 genes in a Haryana state population of North India. The allelic frequency of CYP1A1 polymorphism m1 (MspI) was 29.65% and m2 (Ile462Val) was 24.85%. The frequency of CYP1B1 polymorphism m1 (Val432Leu) was 45.85% and m2 (Asn453Ser) was 16.2%. We observed inter- and intra-ethnic variation in the frequency distribution of these polymorphisms. Analysis of polymorphisms in these genes might help in predicting the risk of cancer. Our results emphasize the need for more such studies in high-risk populations.

Association of GSTM1 and GSTT1 polymorphisms with DNA damage in coal-tar workers

DNA damage was evaluated by alkaline comet assay in peripheral blood lymphocytes of 115 coal-tar workers occupationally exposed to polycyclic aromatic hydrocarbons (PAHs) and 105 control subjects. The effect of polymorphisms of glutathione S-transferase (GST) genotypes on the DNA damage was assessed. The mean tail moment (TM) value in the coal-tar workers was significantly higher as compared to the control subjects (12.06 ± 0.55 versus 0.44 ± 0.31; P<0.05). No significant association (P>0.05) between the GSTT1 and GSTM1 genotypes and the TM values was found, however highest mean rank TM value was reported in GSTM1 null and GSTT1 null genotypes in both control and exposed subjects. Our results suggest that there is increased DNA damage in coal-tar workers due to PAHs exposure. Polymorphisms in GSTM1 and GSTT1 genes do not show significant effect (P>0.05) on DNA damage.