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.

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.

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.

Bioactive compound loaded stable silver nanoparticle synthesis from microwave irradiated aqueous extracellular leaf extracts of Naringi crenulata and its wound healing activity in experimental rat model.

An efficient and eco-friendly protocol for the synthesis of bioactive silver nanoparticles was developed using Naringi crenulata leaf extracts via microwave irradiation method. Silver nanoparticles were synthesized by treating N. crenulata leaf extracts with 1mM of aqueous silver nitrate solution. An effective bioactive compound such as alkaloids, phenols, saponins and quinines present in the N. crenulata reduces the Ag(+) into Ag(0). The synthesized silver nanoparticles were monitored by UV-vis spectrophotometer and further characterized by X-ray diffraction (XRD), Fourier Transform Infra Red (FTIR), Energy-dispersive X-ray spectroscopy (EDX) and field emission scanning electron microscopy (FESEM). UV-vis spectroscopy showed maximum absorbance at 390nm due to surface plasmon resonance of AgNPs. From FESEM results, an average crystal size of the synthesized nanoparticle was 72-98nm. FT-IR results showed sharp absorption peaks and they were assigned to phosphine, alkyl halides and sulfonate groups. Silver nanoparticles synthesized were generally found to be spherical and cubic shape. Topical application of ointment prepared from silver nanoparticles of N. crenulata were formulated and evaluated in vivo using the excision wound healing model on Wistar albino rats. The measurement of the wound areas was performed on 3rd, 6th, 9th, 12th and 15th days and the percentage of wound closures was calculated accordingly. By the 15th day, the ointment base containing 5% (w/w) of silver nanoparticles showed 100% wound healing activity compared with that of the reference as well as control bases. The results strongly suggested that the batch C ointment containing silver nanaoparticles synthesized from the leaf extracts of N. crenulata was found to be very effective in wound repair and encourages harnessing the potentials of the plant biomolecules loaded silver nanoparticle in the treatment of tropical diseases including wound healing.

Raman spectroscopic investigation of the chemopreventive response of naringenin and its nanoparticles in DMBA-induced oral carcinogenesis.

Raman spectroscopy is a vibrational spectroscopic technique that can be used to optically probe the biomolecular changes associated with tumor progression. The aim of the present study is to investigate the biomolecular changes in chemopreventive response of prepared naringenin-loaded nanoparticles (NARNPs) relative to efficacy of free naringenin (NAR) during 7,12-dimethyl benz(a)anthracene (DMBA)-induced oral carcinogenesis by Fourier Transform Raman (FT-Raman) spectroscopy. Oral squamous cell carcinoma (OSCC) was developed in the buccal pouch of golden Syrian hamsters by painting with 0.5% DMBA in liquid paraffin three times a week for 14weeks. Raman spectra differed significantly between the control and tumor tissues, with tumors showing higher percentage signals for nucleic acids, phenylalanine and tryptophan and a lower in the percentage of phospholipids. Moreover, oral administration of free NAR and NARNPs significantly increased phospholipids and decreased the levels of tryptophan, phenylalanine and nucleic acid contents. On a comparative basis, NARNPs was found to have a more potent antitumor effect than free NAR in completely preventing the formation of squamous cell carcinoma and in improving the biochemical status to a normal range in DMBA-induced oral carcinogenesis. The present study further suggest that Raman spectroscopy could be a valuable tool for rapid and sensitive detection of specific biomolecular changes in response to chemopreventive agents.

Chemopreventive efficacy of piperine in 7,12-dimethyl benz [a] anthracene (DMBA)-induced hamster buccal pouch carcinogenesis: an FT-IR study.

The present study is designed to investigate the effect of piperine in modifying the carcinogenic process, as well as biochemical alterations at the molecular level during DMBA-induced hamster buccal pouch carcinogenesis by FT-IR spectroscopy. Specific changes were noticed in the FT-IR spectral features of DMBA-induced hamster buccal pouch carcinoma. These alterations include structural changes of proteins and possible increase of its content, an increase in the nuclear-to-cytoplasm ratio, an increase in the relative amount of DNA, an enhancement in the phosphorylation of proteins, a loss of hydrogen bonding of the C-OH groups in the amino acid residues of proteins and diminished lipid peroxidation which were accompanied by a significant reduction in the relative amount of lipids compared to untreated control animals. Administration of piperine significantly increased the levels of lipid peroxidation and decreased the levels of proteins and nucleic acid content that were found to increase in oral cancer bearing animals. In conclusion, the results of the present study suggest that piperine may exert its chemopreventive effect by modulating the biochemical changes at the molecular level during DMBA-induced hamster buccal pouch carcinogenesis which can be detected using FT-IR spectroscopic technique.

Regulation of normal cell cycle progression by flavin-containing oxidases.

Mechanisms underlying the role of reactive oxygen species (ROS) generated by flavin-containing oxidases in regulating cell cycle progression were examined in human and rodent fibroblasts. Incubation of confluent cell cultures with nontoxic/nonclastogenic concentrations of the flavoprotein inhibitor, diphenyleneiodonium (DPI), reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase activity and basal ROS levels, but increased proteolysis of cyclin D1, p21(Waf1) and phospho-p38(MAPK). When these cells were allowed to proliferate by subculture in DPI-free medium, an extensive G(1) delay was observed with concomitant activation of p53/p21(Waf1) signaling and reduced phosphorylation of mitogen-activated kinases. Compensation for decreased oxidant generation by simultaneous exposure to DPI and nontoxic doses of the ROS generators, gamma-radiation or t-butyl-hydroperoxide, attenuated the G(1) delay. Whereas the DPI-induced G(1) checkpoint was completely dependent on PHOX91, ATM and WAF1, it was only partially dependent on P53. Interestingly, G(1) to S progression was not affected when another flavin-containing enzyme, nitric oxide synthase, was inhibited nor was it associated with changes in mitochondrial membrane potential. Proliferating cells treated with DPI also experienced a significant but attenuated delay in G(2). We propose that ATM performs a critical function in mediating normal cellular proliferation that is regulated by nonphagocytic NAD(P)H oxidase enzymes activity, which may serve as a novel target for arresting cancer cells in G(1).

XRCC1 and XPD gene polymorphisms in a South Indian population.

DNA repair systems play an important role in maintaining the integrity of the human genome. Deficiency in the repair capacity due to either mutations or inherited polymorphisms in DNA repair genes may contribute to variations in the DNA repair capacity and subsequently susceptibility to cancer. The interindividual variability as well as ethnic differences in DNA repair polymorphisms, stress the importance to establish genotype profiles unique to a population. Hence the present study aimed to determine the frequencies of XRCC1 and XPD gene polymorphisms in 255 healthy random unrelated individuals from South India. DNA was isolated from the peripheral blood sample of these individuals and the XRCC1 and XPD genotypes were determined by PCR- RFLP with Msp1 and Pst1 enzymes respectively. The XRCC1 genotype frequencies revealed 36% Arg/Arg, 47% Arg/Gln and 17% Gln/Gln with Gln allele frequency of 0.41. Analysis of XPD genotypes revealed 51% Lys/Lys, 41% Lys/Gln and 8% Gln/Gln with Gln allele frequency of 0.29. No significant difference in the distribution of genotypes was seen based on gender. Comparison of the frequencies of XRCC1 and XPD polymorphisms observed in the present study with other populations revealed a distinctive nature of the South Indian population. An understanding of DNA repair gene polymorphisms might not only enable risk assessment of humans exposed to environmental carcinogens but also response to therapy, which target the DNA repair pathway.

Molecular cloning and characterization of the rubber elongation factor gene and its promoter sequence from rubber tree (Hevea brasiliensis): A gene involved in rubber biosynthesis.

Hevea rubber tree (Hevea brasiliensis) is the only plant species being cultivated for commercial production of rubber in the world. In order to meet ever increasing rubber demand, it is a prerequisite to identify and characterize a key gene involved in rubber biosynthesis and over-expression of rubber biosynthesis gene will eventually lead to enhance the latex (rubber) production in transgenic Hevea plants. Rubber elongation factor (REF) is a major protein located on the surface of large rubber particles in latex and is involved which is involved in rubber biosynthesis in H. brasiliensis. We report here cloning and characterization of REF gene as well as its 5' promoter region from Hevea. REF gene (1367bp) has three exons interrupted by two introns and encoded a 138 amino acid peptide containing an open reading frame of 414bp with a calculated MW of 14,700Da. Nucleotide sequence analysis showed that 1.3kb genomic DNA showed 100% homology to REF cDNA from Hevea. Southern blot hybridization of genomic DNA with REF gene probe revealed that REF gene is encoded by a small gene family consisting of two members. RNA blot analysis indicated that REF transcript is highly expressed in high yielding clone than in low yielder. The cloned 5' promoter region has a putative TATA element at -150 and CAAT box at -221 position. To identify the regulatory role of REF promoter, chimaeric fusion between REF promoter sequence and the ß-glucuronidase (GUS) coding, uidA gene was constructed and used to transform tobacco and Arabidopsis. Expression of the uidA reporter gene was detected histochemically in the transformed tobacco plants where, GUS activity was detected in the leaf and petiole of transformed plants. The stable integration of REF:uidA fusion into the tobacco genome was further confirmed by PCR amplification and Southern blot analysis. A histochemical study of stable transformants demonstrated that the 5' upstream region of REF can drive strong GUS gene expression specifically in the vascular tissues (xylem and phloem) of leaf, stem and midribs of transgenic Arabidopsis. GUS staining revealed that REF:GUS expression was also induced by wounding. The results suggested that the cloned REF promoter is capable of directing gene expression. Our ultimate goal is to produce transgenic Hevea plants with enhanced latex yield by over expression of REF protein.

Comparison of inter- and intra-chromosomal aberrations in blood samples exposed to different dose rates of gamma radiation.

Peripheral blood samples obtained from a normal healthy volunteer were exposed in vitro to gamma radiation with various doses at different dose rates of 1.0, 0.1 and 0.0014 Gy min(-1). The exposed samples were analysed for different chromosomal aberrations such as dicentrics (DC), centric rings (CR) and double-minutes (DM). The ratio of DC chromosomes (inter) to the total number of centric rings (CR) and double-minutes (DM) (CR + DM = intra) were analysed for all the three dose rates. The study showed that the frequency of inter-arm chromosomal aberrations was more then three times higher than that observed with intra-arm chromosomal aberrations in samples exposed at a dose rate of 1.0 and 0.1 Gy min (-1). However, the frequency of inter- and intra-arm chromosomal aberrations were almost same (ratio 1:1) in samples exposed at a dose rate of 0.0014 Gy min(-1). This paper discusses the usefulness of the ratio of inter- and intra-arm chromosome aberration in finding out whether the sample was exposed to high or low dose rate radiation.

Comparison of chronic exposures received by radiation workers using different biological end-points with the doses recorded by TLD.

The frequency of different biological end-points such as translocation, dicentrics (DC) and micronuclei (MN) was studied in 14 radiation workers and 21 non-radiation workers. The average frequencies for different types of aberrations were significantly higher in radiation workers compared to those of respective aberrations in non-radiation workers. Out of 14 radiation workers, eight subjects showed a dose above the detection limit as per translocation and seven subjects as per DC frequency and no patient showed a dose above the detection limit as per MN frequency. Regression analysis carried out between the recorded doses according to Thermo Luminescence Dosimeter (TLD) and the dose estimated as per translocation frequency gave a correlation coefficient of 0.32, whereas that obtained with TLD dose and the dose estimated as per DC was 0.81. When the correlation was made between the TLD dose, which was above 0.15 Gy (the detection limit for translocation), and the dose estimated as per translocation frequency in these subjects, a correlation coefficient of 0.98 was found. A similar analysis between the TLD dose above 0.5 Gy (the detection limit for DC) and the dose estimated as per DC frequency in these subjects, a correlation coefficient of 0.26 resulted. This paper discusses the reasons for the poor correlation obtained between TLD dose and dose estimated as per DC and MN frequency.

Comparison of UV-induced unscheduled DNA synthesis in lymphocytes exposed to low doses of ionising radiation in vivo and in vitro.

In an attempt to understand and ascertain the stimulatory effects of low-dose ionising radiation, a study was conducted to compare the changes in the UV-induced repair capacity of human blood cells exposed to low conditioning doses of ionising radiation under in vivo and in vitro conditions. A significant increase in the rate of UV induced Unscheduled DNA synthesis (UDS) in lymphocytes pre-exposed to low doses of ionising radiation was observed both under in vitro and in vivo conditions. There was also a significant correlation between the adapting dose and net UDS in lymphocytes of radiation workers implying that the triggering action of the adaptation process is dose dependent. However, on comparing the extent of UV-induced UDS of the in vivo and in vitro exposures, significantly higher rates of UDS were observed in the lymphocytes of radiation workers when compared to a corresponding in vitro adapting dose. We postulate that the response in vivo is much more pronounced due to cell repopulating events and extra cellular secretory factors like hormones etc,.

Estimation of dose in cancer patients treated with fractionated radiotherapy using translocation, dicentrics and micronuclei frequency in peripheral blood lymphocytes.

The frequency of translocation, dicentrics (DC) and micronuclei (MN) was studied in blood samples exposed in vitro to (60)Co gamma radiation and cervical cancer patients undergoing fractionated radiotherapy. The samples exposed under in vitro condition showed that the frequency of translocation and dicentric followed Poisson distribution ('u' varied between -0.04 and +1.41 for translocation and between -0.09 and +1.81 for DC) and that obtained with MN follow over dispersion ('u' varied between +2.04 and +9.28). However, the cervical cancer patients undergoing radiotherapy showed over dispersion for all these three aberrations (DC, MN and translocation). The frequencies of aberrations obtained in cancer patients were found to be lower than those obtained for in vitro exposure for doses more than 2 Gy equivalent whole body dose (EWBD). The dose-response curves were constructed using the frequencies of DC, MN and translocation as a function of EWBD. Doses as measured from the dose response curves were compared with the estimated dose in order to check whether the measured and estimated doses agree. The percent variation between the doses measured from aberration frequencies and that of the estimated dose was lower with translocation (10.8+/-7.41%) compared to those obtained with DC (38. 08+/-31.85%) and MN (47.19+/-31.80%).

Higher frequency of dicentrics and micronuclei in peripheral blood lymphocytes of cancer patients.

The presence of dicentric chromosome (DC) and micronuclei (MN) frequency in the peripheral blood lymphocytes of 25 cancer patients prior to chemo and radiotherapy and 21 healthy volunteers were studied. The overall DC and MN showed significantly higher frequency compared to those obtained in normal healthy volunteers (p<0.0001). However, among 25 patients only 15 showed a higher frequency of DC aberration, nine patients showed the presence of minutes (M) and seven patients showed chromatid breaks (ChB). The reasons for the higher frequency of aberration observed in these cancer patients are discussed in this paper.

Influence of in vitro low-level gamma-radiation on the UV-induced DNA repair capacity of human lymphocytes--analysed by unscheduled DNA synthesis (UDS) and comet assay.

Unscheduled DNA synthesis (UDS) induced by ultraviolet radiation (UV) was studied in human lymphocytes after exposing blood samples in vitro to doses ranging between 1 and 10 mGy gamma-radiation, by way of measuring tritiated thymidine (3H-TdR) uptake in the DNA of these lymphocytes. The results indicate that samples pre-exposed to gamma-ray doses ranging between 2.5 and 4 mGy show higher UDS levels compared with those pre-exposed to doses of less than 2.5 or more than 4 mGy. These results were verified by studying the rate of removal of UV-induced photoproducts using the comet assay. The reason for the increase in DNA repair capacity in this dose range is discussed in comparison with earlier reports on this phenomenon. The DNA repair capacity with respect to inter-individual variability and age is also analysed. The study implies that the comet assay is a simple and sensitive visual method to track nucleotide excision repair and hence can be used to estimate UV-induced DNA repair in the place of the more reliable yet cumbersome and time-consuming, grain-counting autoradiographic technique.

Analysis of radiation dose-response curve obtained with cytokinesis block micronucleus assay.

The frequency of micronuclei and acentrics obtained with different doses of 60Co gamma radiation was examined. When compared to acentric frequency the micronuclei frequency was found to be higher at about 115% for doses below 1 Gy. However, it dropped to about 65% as the dose was increased to 4 Gy. This paper discusses the causes for the reduced frequency of micronuclei at higher doses by taking into account the possibility of their being masked from view by the daughter nuclei in the binucleated cell.