Ataxia Telengectesia Protein Influences Bleomycin-Induced DNA Damage in Human Fibroblast Cells.

Human cancer is caused mainly by exposure to genotoxic chemicals; therefore, cellular defence mechanisms against genotoxic stress are crucial. Genetic factors are essential to maintaining genome stability and play a vital role in overcoming this by repairing the genome damage caused by any agent in order to prevent chromosomal instability. To examine the influence of the genetic makeup in specific ataxia-telangiectasia (ATM), we have examined non-cancerous fibroblast cell lines (HLF, AG1522 and L6) and cells with ATM mutated deficiency (GM4405). Cell lines were exposed in vitro to bleomycin (0, 40 and 80 µg/mL). The induced DNA damages were measured using endpoints including the micronucleus assay (MN) to measure chromosome damage and gamma-H2AX (γ-H2AX) assay to measure DNA damage/repair foci formation. An increase in DNA damage were observed in bleomycin-treated cells compared to unexposed controls (p < 0.05). A concentration-dependent increase of MN and γ-H2AX foci was observed and the sensitivity differed among the cell lines as follows: GM4405 > HLF > AG1522 > L6 for MN frequency and HLF > AG1522 > GM4405 > L6 for γ-H2AX foci. These findings suggest that the genetic makeup of the cellular genome would play an essential role in repairing bleomycin-induced DNA damage. Signalling of DNA damage, and the genes responsible for the repair process, could contribute to the differential susceptibility of different tissues to carcinomas induced by environmental mutagens.

Embedded silicon gratings for high-efficiency light-chip coupling to thin film silicon nitride waveguides.

Thin film silicon nitride (<150 nm) waveguide has emerged as a dominant ultra-low-loss platform for many loss-critical applications. While thin-film silicon nitride propagation loss is a crucial characteristic, coupling light between an optical fiber and the waveguide is still challenging. While the larger mode size of the decoupled thin waveguide offers better coupling than a highly-confined waveguide, the coupling efficiency is still sub-optimal. The poor diffraction efficiency of such thin films limits the scope of implementing standalone surface gratings. We demonstrate an efficient way to couple into thin film silicon nitride waveguides using amorphous silicon strip gratings. The high contrast gratings provide an efficient means to boost the directionality from thin films leading to an enhanced coupling performance. In addition, we incorporate a bottom reflector to further improve the coupling. We present an optimal design for uniform strip gratings with a maximum coupling efficiency of -1.7 dB/coupler. We achieved a maximum coupling efficiency of -0.28 dB/coupler by engineering the scattering strength along the grating through apodization. We have experimentally shown the highest coupling efficiency reported yet of -2.22 dB/coupler and -1.84 dB/coupler for uniform and apodized grating couplers in the C-L band. We present a detailed design strategy, simulation, fabrication and characterization data on the effect of various parameters on the coupling efficiency.

Guided mode resonance aided polarization insensitive in-plane spectral filters for an on-chip spectrometer.

We demonstrate an on-chip in-plane polarization independent multi-spectral color filter in the visible to near-infrared wavelength band. We experimentally show a four-channel transmission and in-plane spectral filter characteristics spanning a 400-nm spectral range. Engineered 2D guided mode resonance structures in a silicon nitride-on-sapphire substrate are used to realize the filters. The in-plane color filters could provide the necessary impetus for developing robust integrated photonic platforms for on-chip devices and applications.

Chromosome length ratio as a biomarker of DNA damage in cells exposed to high dose ionizing radiation.

The premature chromosome condensation (PCC) assay is considered as complementary bio-dosimetry tool for chromosome aberration assay and the PCC assay can be used to estimate high dose exposure. Though the PCC ring is considered as prospective biomarker, chromosome length ratio (ratio of longest and shortest chromosome length in PCC spreads) of chemically induced PCC is shown to be very good indicator of ionizing radiation. In view of this, an in-vitro study has been performed using PCC assay to suggest chromosome length ratio (LR) as potential bio-dosimeter induced by high dose ionizing radiation. Blood samples were collected from healthy subjects (n = 3) after prior consent and irradiated to ten different doses ranging between 0 and 20 Gy using 6 MV LINAC X-rays with dose rate of 5.6 Gy/min. Irradiated lymphocytes were cultured and calyculin induced PCC spreads were prepared. PCC spreads were captured using image analysis system and chromosome lengths were measured using open-source ImageJ software. For each dose, LR for 50 chromosome spreads were computed and mean LR value was calculated. LR varies between 6.0 ± 0.08 and 23.6 ± 0.55 for the dose range between 2 and 20 Gy. The dose response curve for LR was observed to be linear with y = 1.02x + 3.36, R2 = 0.97. Linear dose response relationship obtained in the present study confirms the prospective use of LR measurement. This study is first of its kind to examine chromosome length ratio as a biomarker of DNA damage in cells exposed to high dose X-ray exposure.

Construction of dose response curve for 6 MV LINAC X-rays using Premature Chromosome Condensation assay for radiation dosimetry.

Quantification of chromosomal aberrations in the exposed personnel blood samples is considered as a 'gold standard' and sensitive biomarker in biological dosimetry. Despite technological developments, culture of cells for 48-52 h remains an unmet need in case of triage biodosimetry. Moreover, it is difficult to get sufficient number of metaphase spreads for scoring after high doses of exposures. The technique which causes condensation of chromatin before mitosis using biological or chemical agent is named as Premature Chromosome Condensation (PCC) assay. This assay is considered as an alternative to chromosome aberration assay, particularly at high acute doses of low and high LET radiation. To establish the PCC assay, blood samples were collected from healthy non-smoking individuals (n = 3) and exposed to various doses (0-20 Gy) of 6 MV X-rays at a dose rate of 5.6 Gy/min, using a high energy Linear accelerator (LINAC). Irradiated blood samples were subjected to Calyculin-A induced PCC. About 500 cells or more than 100 Ring Chromosomes (RC) were scored at each dose. Dicentric chromosomes (DC) and acentric fragments were also scored at each dose; the number of chromosomal aberrations in G1, M, G2/M and M/A phase of cell cycle were recorded and the frequency was used to construct the dose response curve. A dose dependent increase in RC and DC frequency were observed with a slope of 0.049 ± 0.002 and 0.30 ± 0.02 respectively. This study is first of its kind to construct a dose response curve for LINAC X-rays using a PCC assay.

Primary and secondary bystander effect and genomic instability in cells exposed to high and low linear energy transfer radiations.

Purpose: Non-Targeted effects (NTE), such as bystander effect (BE) and genomic instability (GI) challenge central dogma of radiation biology. Moreover, there is a need to understand its universality in different type of cells and radiation quality.Materials and method: To study BE (primary and secondary) and GI Human adult dermal fibroblast (HADF) and peripheral blood lymphocytes (PBL) were exposed to low fluence of 241Am alpha (α) particle and 6 MV X-ray. The BE was carried out by means of co-culture methodology after exposing the cells to both types of radiation and damage was measured using micronucleus assay (MN) and chromosomal aberration assay (CA) in the p1 cells while the GI was followed up in their progeny.Results: A dose-dependent increase in DNA damages (MN and CA) was observed in directly irradiated and bystander cells. The magnitude of BE was higher (6 fold) in cells co-cultured with the α-irradiated cells than that of with X-irradiated cells. Cross exposure of both cell types confirms that radiation induced BE is cell type dependent. In addition, induced DNA damage persisted for a longer population doubling in α-particle irradiated cells.Conclusion: This work adds evidence to secondary bystander response generated from primary bystander normal cells and its dependence to radiation quality.

Direct and bystander effects in human blood lymphocytes exposed to 241Am alpha particles and the relative biological effectiveness using chromosomal aberration and micronucleus assay.

Purpose: It is important to understand the significance of alpha (α) radiation-induced bystander effects (RIBE) and its relative biological effectiveness (RBE); this is because the phenomenon is not universal and the mechanism is unclear and because the RBE is widely varying and projected to be very high. Materials and methods: Isolated lymphocytes from healthy volunteers (n = 10) were exposed to either low fluence α-particles (241Am), γ-rays (60Co), or X-rays (225 kVp and 6 MV). Co-culture methodology was employed to investigate bystander effects (BEs). Chromosomal aberrations (CA) and micronucleus (MN) formation were used to study the BE and calculated RBE. Results: Lymphocytes directly exposed to the types of radiation used showed a dose-dependent increase in the frequency of CA and MN; dose independent increases in the frequency of these chromosomal damages in co-cultured bystander cells, implies that all three types of radiation-induced a BE. The calculated RBE at the level of 5% induced aberrations varied between 9 and 20. Conclusion: The magnitude of low fluence α-particle induced RIBE is higher than in low LET (linear energy transfer) radiation. The RBE also varies depending upon the endpoints used and adds up to targeted effects. Since the endpoint of CA is considered as an important and early marker of risk prediction, the RIBE and RBE using CA as a marker are relevant for radiation protection purposes.

Over-expression of 3-hydroxy-3- methylglutaryl-coenzyme A reductase 1 (hmgr1) gene under super-promoter for enhanced latex biosynthesis in rubber tree (Hevea brasiliensis Muell. Arg.).

Natural rubber (cis-1, 4-polyisoprene) is being produced from bark laticifer cells of Hevea brasiliensis and the popular high latex yielding Indian rubber clones are easily prone to onset of tapping panel dryness syndrome (TPD) which is considered as a physiological syndrome affecting latex production either partially or completely. This report describes an efficient protocol for development of transgenic rubber plants by over-expression of 3-hydroxy 3-methylglutaryl Co-enzyme A reductase 1 (hmgr1) gene which is considered as rate limiting factor for latex biosynthesis via Agrobacterium-mediated transformation. The pBIB plasmid vector containing hmgr1 gene cloned under the control of a super-promoter was used for genetic transformation using embryogenic callus. Putatively transgenic cell lines were obtained on selection medium and produced plantlets with 44% regeneration efficiency. Transgene integration was confirmed by PCR amplification of 1.8 kb hmgr1 and 0.6 kb hpt genes from all putatively transformed callus lines as well as transgenic plants. Southern blot analysis showed the stable integration and presence of transgene in the transgenic plants. Over expression of hmgr1 transgene was determined by Northern blot hybridization, semi-quantitative PCR and real-time PCR (qRT-PCR) analysis. Accumulation of hmgr1 mRNA transcripts was more abundant in transgenic plants than control. Increased level of photosynthetic pigments, protein contents and HMGR enzyme activity was also noticed in transgenic plants over control. Interestingly, the latex yield was significantly enhanced in all transgenic plants compared to the control. The qRT-PCR results exhibit that the hmgr1 mRNA transcript levels was 160-fold more abundance in transgenic plants over untransformed control. These results altogether suggest that there is a positive correlation between latex yield and accumulation of mRNA transcripts level as well as HMGR enzyme activity in transgenic rubber plants. It is presumed that there is a possibility for enhanced level of latex biosynthesis in transgenic plants as the level of mRNA transcripts and HMGR enzyme activity is directly correlated with latex yield in rubber tree. Further, the present results clearly suggest that the quantification of HMGR enzyme activity in young seedlings will be highly beneficial for early selection of high latex yielding plants in rubber breeding programs.

DNA damage and the bystander response in tumor and normal cells exposed to X-rays.

Monolayer and suspension cultures of tumor (BMG-1, CCRF-CEM), normal (AG1522, HADF, lymphocytes) and ATM-mutant (GM4405) human cells were exposed to X-rays at doses used in radiotherapy (high dose and high dose-rate) or radiological imaging (low dose and low dose-rate). Radiation-induced DNA damage, its persistence, and possible bystander effects were evaluated, based on DNA damage markers (γ-H2AX, p53ser15) and cell-cycle-specific cyclins (cyclin B1 and cyclin D1). Dose-dependent DNA damage and a dose-independent bystander response were seen after exposure to high dose and high dose-rate radiation. The level of induced damage (expression of p53ser15, γ-H2AX) depended on ATM status. However, low dose and dose-rate exposures neither increased expression of marker proteins nor induced a bystander response, except in the CCRF-CEM cells. Bystander effects after high-dose irradiation may contribute to stochastic and deterministic effects. Precautions to protect unexposed regions or to inhibit transmission of DNA damage signaling might reduce radiation risks.

Green engineered biomolecule-capped silver and copper nanohybrids using Prosopis cineraria leaf extract: Enhanced antibacterial activity against microbial pathogens of public health relevance and cytotoxicity on human breast cancer cells (MCF-7).

This research focused on green engineering and characterization of silver (PcAgNPs) and copper nanoparticles (PcCuNPs) using Prosopis cineraria (Pc) leaf extract prepared by using microwave irradiation. We studied their enhanced antimicrobial activity on human pathogens as well as cytotoxicity on breast cancer cells (MCF-7). Biofabricated silver and copper nanoparticles exhibited UV-Visible absorbance peaks at 420 nm and 575 nm, confirming the bioreduction and stabilization of nanoparticles. Nanoparticles were characterized by FTIR, XRD, FESEM, and EDX analysis. FTIR results indicated the presence of alcohols, alkanes, aromatics, phenols, ethers, benzene, amines and amides that were possibly involved in the reduction and capping of silver and copper ions. XRD analysis was performed to confirm the crystalline nature of the silver and copper nanoparticles. FESEM analysis suggested that the nanoparticles were hexagonal or spherical in shape with size ranging from 20 to 44.49 nm and 18.9-32.09 nm for AgNPs and CuNPs, respectively. EDX analysis confirmed the presence of silver and copper elemental signals in the nanoparticles. The bioengineered silver and copper nanohybrids showed enhanced antimicrobial activity against Gram-positive and Gram-negative MDR human pathogens. MTT assay results indicated that CuNPs show potential cytotoxic effect followed by AgNPs against MCF-7 cancer cell line. IC50 were 65.27 µg/ml, 37.02 µg/ml and 197.3 µg/ml for PcAgNPs, PcCuNPs and P. cineraria leaf extracts, respectively, treated MCF-7 cells. The present investigation highlighted an effective protocol for microwave-assisted synthesis of biomolecule-loaded silver and copper nanoparticles with enhanced antibacterial and anticancer activity. Results strongly suggested that bioengineered AgNPs and CuNPs could be used as potential tools against microbial pathogens and cancer cells.

Reprint of: Silver and titanium dioxide nanoparticle toxicity in plants: A review of current research.

Nanoparticles (NPs) have become widely used in recent years for many manufacturing and medical processes. Recent literature suggests that many metallic nanomaterials including those of silver (Ag) and titanium dioxide (TiO2) cause significant toxic effects in animal cell culture and animal models, however, toxicity studies using plant species are limited. This review examines current progress in the understanding of the effect of silver and titanium dioxide nanoparticles on plant species. There are many facets to this ongoing environmental problem. This review addresses the effects of NPs on oxidative stress-related gene expression, genotoxicity, seed germination, and root elongation. It is largely accepted that NP exposure results in the cellular generation of reactive oxygen species (ROS), leading to both positive and negative effects on plant growth. However, factors such as NP size, shape, surface coating and concentration vary greatly among studies resulting in conflicting reports of the effect at times. In addition, plant species tend to differ in their reaction to NP exposure, with some showing positive effects of NP augmentation while many others showing detrimental effects. Seed germination studies have shown to be less effective in gauging phytotoxicity, while root elongation studies have shown more promise. Given the large increase in nanomaterial applications in consumer products, agriculture and energy sectors, it is critical to understand their role in the environment and their effects on plant life. A closer look at nanomaterial-driven ecotoxicity is needed. Ecosystem-level studies are required to indicate how these nanomaterials transfer at the critical trophic levels affecting human health and biota.

Zinc oxide nanoparticles (ZnONPs) alleviate heavy metal-induced toxicity in Leucaena leucocephala seedlings: A physiochemical analysis.

The present study describes the role of zinc oxide nanoparticles (ZnONPs) in reversing oxidative stress symptoms induced by heavy metal (Cd and Pb) exposure in Leucaena leucocephala (Lam.) de Wit. Seedling growth was significantly enhanced with the augmentation of ZnONPs following Cd and Pb exposure. Heavy metal accumulations were recorded as 1253.1 mg Cd per kg DW and 1026.8 mg Pb per kg DW for the respective treatments. Results demonstrated that ZnONPs augmentation caused an increase in photosynthetic pigment and total soluble protein contents while a significant decrease in malondialdehyde (MDA-lipid peroxidation) content in leaves. Antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX) were, in turn, elevated in heavy metal-exposed leaves amended with ZnONPs. The ameliorating effect of ZnO nanoparticles on oxidative stress induced toxicity was also confirmed by the reduced MDA content and the elevated level of antioxidative enzyme activities in leaf tissues of L. leucocephala seedlings. Further, addition of ZnONPs in combination with Cd and Pb metals induced distinct genomic alterations such as presence of new DNA bands and/or absence of normal bands in the RAPD pattern of the exposed plants. This study uniquely suggests a potential role of zinc oxide nanoparticles in the remediation of heavy metal contaminated media.

Enhanced plant growth promoting role of phycomolecules coated zinc oxide nanoparticles with P supplementation in cotton (Gossypium hirsutum L.).

This report focuses on application of zinc oxide nanoparticles (ZnONPs) carrying phycomolecule ligands as a novel plant growth promoter aimed at increasing the crop productivity. The present investigation examined the effect of ZnONPs on plant growth characteristics, and associated biochemical changes in cotton (Gossypium hirsutum L.) following growth in a range of concentrations (25-200 mg L-l ZnONPs) in combination with 100 mM P in a hydroponic system. Treated plants registered an increase in growth and total biomass by 130.6% and 131%, respectively, over control. Results demonstrated a significant increase in the level of chlorophyll a (141.6%), b (134.7%), carotenoids (138.6%), and total soluble protein contents (179.4%); at the same time, a significant reduction (68%) in the level of malondialdehyde (MDA) in leaves with respect to control. Interestingly, a significant increase in superoxide dismutase (SOD, 264.2%), and peroxidase (POX, 182.8%) enzyme activities followed by a decrease in the catalase (CAT) activity, in response to above treatments. These results suggest that bioengineered ZnONPs interact with meristematic cells triggering biochemical pathways conducive to an accumulation of biomass. Further investigations will map out the mode of action involved in growth promotion.

Silver and titanium dioxide nanoparticle toxicity in plants: A review of current research.

Nanoparticles (NPs) have become widely used in recent years for many manufacturing and medical processes. Recent literature suggests that many metallic nanomaterials including those of silver (Ag) and titanium dioxide (TiO2) cause significant toxic effects in animal cell culture and animal models, however, toxicity studies using plant species are limited. This review examines current progress in the understanding of the effect of silver and titanium dioxide nanoparticles on plant species. There are many facets to this ongoing environmental problem. This review addresses the effects of NPs on oxidative stress-related gene expression, genotoxicity, seed germination, and root elongation. It is largely accepted that NP exposure results in the cellular generation of reactive oxygen species (ROS), leading to both positive and negative effects on plant growth. However, factors such as NP size, shape, surface coating and concentration vary greatly among studies resulting in conflicting reports of the effect at times. In addition, plant species tend to differ in their reaction to NP exposure, with some showing positive effects of NP augmentation while many others showing detrimental effects. Seed germination studies have shown to be less effective in gauging phytotoxicity, while root elongation studies have shown more promise. Given the large increase in nanomaterial applications in consumer products, agriculture and energy sectors, it is critical to understand their role in the environment and their effects on plant life. A closer look at nanomaterial-driven ecotoxicity is needed. Ecosystem-level studies are required to indicate how these nanomaterials transfer at the critical trophic levels affecting human health and biota.

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.

Impact assessment of mercury accumulation and biochemical and molecular response of Mentha arvensis: a potential hyperaccumulator plant.

The present study was focused on examining the effect of Hg oxidative stress induced physiochemical and genetic changes in M. arvensis seedlings. The growth rate of Hg treated seedlings was decreased to 56.1% and 41.5% in roots and shoots, respectively, compared to the control. Accumulation of Hg level in both roots and shoots was increased with increasing the concentration of Hg. Superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activities were found to be increased with increasing the Hg concentration up to 20 mg/L; however, it was decreased at 25 mg/L Hg concentration. The POX enzyme activity was positively correlated with Hg dose. The changes occurring in the random amplification of ploymorphic DNA (RAPD) profiles generated from Hg treated seedlings included variations in band intensity, disappearance of bands, and appearance of new bands compared with the control seedlings. It was concluded that DNA polymorphisms observed with RAPD profile could be used as molecular marker for the evaluation of heavy metal induced genotoxic effects in plant species. The present results strongly suggested that Mentha arvensis could be used as a potential phytoremediator plant in mercury polluted environment.

Influence of plant growth regulators (PGRs) and various additives on in vitro plant propagation of Bambusa arundinacea (Retz.) Wild: A recalcitrant bamboo species.

An efficient micropropagation protocol for high frequency plant regeneration was developed using nodal explants derived in vitro seedlings of Bambusa arundinacea which is an important multipurpose and edible bamboo species and recalcitrant to tissue culture. The nodal explants excised from 20-day-old seedlings were cultured on Murashige and Skoog (MS) medium fortified with various concentrations of 6-benzyl amino purine (BAP) and kinetin (KIN) (0.5-5.0 mg/l) alone and/or in combination with 0.5 mg/l of different auxins [indole-3-butyric acid (IBA) α-naphthalene acetic acid (NAA) and indole-3-acetic acid (IAA)] for shoot bud induction. The combination of BAP (3.0 mg/l) and IBA (0.5 mg/l) was found to be the best for the highest percent of shoot bud initiation (87.2%), with 24.2 shoots/explant. The highest frequency (95.2%) of shoot bud multiplication with maximum number of shoots (90.5 shoots/culture) was noticed on medium containing 4% coconut water with 4% sucrose. The regenerated shoot buds were cultured on MS medium supplemented with various concentrations of auxins alone and/or in combination with AgNO3 (0.5-4.0 mg/l) for in vitro rooting. Maximum percent of rooting (85%) was noticed on MS medium augmented with 3.0 mg/l IBA and 2.0 mg/l AgNO3 after 14 days of culture. Well rooted plantlets obtained were established in the field with 92% survival rate. The present plant regeneration protocol could be used for large scale propagation and ex-situ conservation of this important bamboo species in the near future.

Dye-sensitized solar cell using sprayed ZnO nanocrystalline thin films on ITO as photoanode.

ZnO thin films had been successfully prepared by spray pyrolysis (SP) technique on ITO/Glass substrates at different substrate temperature in the range 250-400°C using Zinc acetylacetonate as precursor. The X-ray diffraction studies confirmed the hexagonal wurtzite structure with preferred orientation along (002) plane at substrate temperature 350°C and the crystallite size was found to vary from 18 to 47nm. The morphology of the films revealed the porous nature with the roughness value of 8-13nm. The transmittance value was found to vary from 60% to 85% in the visible region depending upon the substrate temperature and the band gap value for the film deposited at 350°C was 3.2eV. The obtained results revealed that the structures and properties of the films were greatly affected by substrate temperature. The near band edge emission observed at 398nm in PL spectra showed better crystallinity. The measured electrical resistivity for ZnO film was ∼3.5×10(-4)Ωcm at the optimized temperature 350°C and was of n-type semiconductor. The obtained porous nature with increased surface roughness of the film and good light absorbing nature of the dye paved way for implementation of quality ZnO in DSSCs fabrication. DSSC were assembled using the prepared ZnO film on ITO coated glass substrate as photoanode and its photocurrent - voltage performance was investigated.

Comparison of results of the manual and automated scoring of micronucleus frequencies in (60)Co-irradiated peripheral blood lymphocytes for triage dosimetry.

Scoring micronuclei in the peripheral blood lymphocytes of individuals exposed to ionizing radiation is a rapid biodosimetry assay. Peripheral blood lymphocytes from five individuals were exposed in vitro to 0-5Gy of (60)Co γ-radiation at a dose rate of 0.76Gy/min. The blood cultures were initiated with RPMI-1640 (80%) supplemented with FBS (20%), stimulated with mitogen and incubated at 37°C for 44h. At the 44th hour, cytochalasin-B (6µg/mL) was added, and the cultures were incubated for 28h more. The cells were harvested with a pre-chilled hypotonic solution (0.075M) and fixed with a Carnoy's solution (methanol/acetic acid 5:1). Giemsa- and propidium-iodide-stained cells affixed to slides for microscopy were scored manually and automatically with the micronucleus scoring software from MetaSystems. The micronucleus frequencies determined in the Giemsa-stained cells by manual and automated scoring were 23.6% different (P<0.0001) with an efficiency of 24.9%. Slides stained with propidium iodide are a better choice for automated scoring than Giemsa-stained ones.

Delayed mitogenic stimulation decreases DNA damage assessed by micronucleus assay in human peripheral blood lymphocytes after (60)co irradiation.

While contradictory reports are available on the yield of dicentric chromosomes (DC) in blood samples stored at different temperature and stimulated to enter into cell cycle, various times gap followed by exposure, limited information is available on the micronucleus (MN) assay. As scoring the micronuclei frequency from the blood lymphocytes of exposed individuals is an alternative to the gold standard DC assay for triage applications, we examined radiation induced MN yield in delayed mitogenic stimulation after irradiation of in vitro. Peripheral blood lymphocytes (PBL) were exposed to low LET ((60)Co) radiation dose (0.1 to 5Gy) and incubated at 37°C for 2, 6 and 24 hours. The MN frequency obtained in blood samples stimulated 2 hours post-irradiation showed a dose dependent increase and used to construct the dose-response curve. Further, the results also showed that blood samples stimulated twenty four hours of post-irradiation, a significant reduction (p<0.05) in MN frequencies were obtained when compared to that of blood samples stimulated two hours and six hours after post-irradiation (0.5, 1, 3 and 5Gy). The observed result suggests that the prolonged PBL storage without mitogenic stimulation could lead to interphase cell death and a delayed blood sampling could results in underestimation of dose in biological dosimetry.