Single Cell Gel Electrophoresis Analysis Research Articles

Hesperidin Potentiates the Chemosensitivity of HT-29 Colon Cancer Cells to 5‐Fluorouracil

Aim: Hesperidin is an important flavonoid compound of citrus fruits. It has the potential to be evaluated as a cytotoxic agent against a variety of malignant human cancer cells, especially in colon, pancreatic, breast, and other cancer types. The practical application of combination therapies is commonly used to eliminate or reduce drug resistance. Materials and Methods: In this study, hesperidin (0.001-300µM) and 5-fluorouracil (5-FU) (1-300 µM), a cytotoxic chemotherapy drug used for treating cancer, were applied both individually and in combination (100 µM) on the HT-29 cells (human colon cancer cell line). The effects of these compounds on HT-29 cell viability were revealed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method, and the genotoxic effects on HT-29 cells were revealed using single-cell gel electrophoresis analysis. Results: The data obtained showed that the combination of 5-FU and hesperidin importantly decreased HT-29 cell viability compared to the group treated with 5-FU alone. These findings indicate that the combined application of these compounds is more effective than the individual administration of 5-FU or hesperidin alone. Conclusion: The research suggests that hesperidin might possess the ability to counter drug resistance in cancer cells, offering promising prospects when used alongside current anti-cancer medications. Nevertheless, to validate these findings, more extensive research and clinical trials are imperative.

Response of Zea mays to multimetal contaminated soils: a multibiomarker approach.

Heavy metals present in mine tailings pollute agroecosystems, put the integrity of the environment at risk and become a major route of exposure to humans. The present study was carried out in Taxco, Guerrero, Mexico, where millions of tons of mine tailings have been deposited. Soils from this region are used for agricultural activities. Maize (Zea mays) was selected as a test plant, because it is one of the most common and important cereal crops in Mexico and worldwide. Thirteen metals were selected and their bioaccumulation in roots, leaves and fruits were measured in plants cultivated in soils contaminated with mine tailings and those cultivated in non-contaminated soils. The effect of metal bioaccumulation on: macro and micromorphology, size, biomass, coloration leaf patterns and on DNA damage levels in different structures were determined. The bioaccumulation pattern was: root > leaf > fruit, being only Mn and Cr bioaccumulated in all three structures and V in the roots and leaves. A significant effect of metal bioaccumulation on 50% of the size and leaf shape and 55% of the biomass characters in Z. mays exposed plants was detected. Regarding micromorphological characters, a significant effect of metal bioaccumulation on 67% of the leaf characters and on 100% of the color basal leaf characters was noted. The effect of metal bioaccumulation on the induction of DNA damage (leaf > fruit > root) was detected employing single cell gel electrophoresis analysis. An approach, in which multi endpoints are used is necessary to estimate the extent of the detrimental effects of metal pollution on agroecosystem integrity contaminated with mine tailings.

Characterization of placenta-derived mesenchymal stem cells cultured in autologous human cord blood serum

Human placenta-derived mesenchymal stem cells (P-MSCs) have drawn increasing attention in the field of stem cell research due to their potential in clinical applications as well as their rich and easy to procure cell source. While studies demonstrating the potential of P-MSCs for therapeutic transplantations have been documented, a clinically compliant procedure for P-MSC expansion in vitro has yet to be established. To this end, previous studies have demonstrated that MSCs of bone marrow and cord blood origins cultured in human cord blood serum (hCBS) are comparable to those cultured in fetal bovine serum (FBS), indicating that hCBS may be an alternative to FBS for the development of in vitro cell expansion procedures free of animal components. However, stem cells from origins other than bone marrow or cord blood, particularly from human placental tissues, which have demonstrated a good potential for clinical applications, have not been characterized under similar conditions. In this study, in an attempt to define a clinically compliant protocol for P-MSC expansion in vitro, we examined the effects of human hCBS as a replacement for FBS on cell proliferation capacity, differentiation potential, MSC-specific phenotypic expression and the genetic stability of P-MSCs in cultures. P-MSCs expanded in vitro in autologous hCBS maintained the capacity of self‑renewal and expressed surface antigens characteristic of bone marrow-derived mesenchymal stem cells. Under differentiation conditions, the P-MSCs expanded in hCBS developed into adipogenic, osteogenic and neurogenic cell phenotypes. Chromosomal karyotyping and single cell gel electrophoresis analysis demonstrated that P-MSCs cultured in autologous hCBS were genetically stable. These results suggest that autologous hCBS may be used as an alternative to FBS for the in vitro expansion of P-MSCs for clinical applications.

Induction of DNA strand breaks is critical to predict the cytotoxicity of gemtuzumab ozogamicin against leukemic cells

Gemtuzumab ozogamicin (GO) consists of the CD33 antibody linked to calicheamicin. The binding of GO to the CD33 antigen on leukemic cells results in internalization followed by the release of calicheamicin, thereby inducing DNA strand breaks. We hypothesized that the induction of DNA strand breaks would be a surrogate marker of GO cytotoxcity. Here, two GO-resistant variants (HL/GO-CSA [225-fold], HL/GO [200-fold]) were established by serially incubating human leukemia HL-60 cells with GO with or without a P-glycoprotein (P-gp) inhibitor, cyclosporine A, respectively. The CD33 positivity was reduced in both variants. The HL/GO-CSA cells showed an increased multidrug resistance protein-1 (MRP1) transcript, and an MRP1 inhibitor partially reversed GO resistance. The HL/GO cells had neither P-gp nor MRP1 overexpression. Microarray analysis and Western blotting indicated elevated levels of DNA repair-associated proteins in both variants. Two other leukemic subclones, showing either P-gp or MRP1 overexpression, were also GO-resistant. Using single cell gel electrophoresis analysis, it was determined that GO-induced DNA strand breaks increased dose-dependently in HL-60 cells, whereas the number of breaks was reduced in the GO-resistant cell lines. The induction of DNA strand breaks was correlated with GO sensitivity among these cell lines. The CD33 positivity and the expression levels of transporters were not proportional to drug sensitivity. Using primary leukemic cells, the induction of DNA strand breaks appeared to be associated with GO sensitivity. Thus, GO-induced DNA strand breaks as the final output of the mechanism of action would be critical to predict GO cytotoxicity.

The behavior of Bupleurum scorzonerifolium as a parent of somatic hybrid introgressed lines is associated with UV resistance of its chromosomes

Protoplasts of Bupleurum scorzonerifolium irradiated with 380 μW/cm2 UV for 5 min were fused by the PEG-mediated method with untreated protoplasts of Arabidopsis thaliana. The fusion products were cultured in the P5 liquid medium for single hybrid cell clone formation. As a total, 81 independent putative hybrid clones (cell lines) were obtained, and seventeen of them were identified as somatic hybrids by chromosome counting, GISH, RAPD, and SSR analyses. More than 80 B. scorzonerifolium-like green introgressed plants and leaves were regenerated from 49 somatic hybrid cell lines, which contained chromatin and DNA characteristic of A. thaliana. To assess the UV tolerance of both parents with chromatin exclusion and introgression, their protoplasts were UV-irradiated (380 μW/cm2 for 0 and 5 min), and the protoplasts of A. thaliana were more sensitive to UV than those of B. scorzonerifolium as judged by Single Cell Gel Electrophoresis analysis. The possible relationship between UV resistance of B. scorzonerifolium and A. thaliana chromosome elimination and the formation of somatic introgressed hybrid plants is discussed.

Hypoglycemic effects of crude polysaccharide from Purslane.

The effects of crude polysaccharide from Purslane (CPP) on body weight (bw), blood glucose, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), triglyceride (TG) and serum insulin levels were studied in diabetes mellitus mice. CPP treatment (200, 400 mg/kg bw) for 28 days resulted in a significant decrease in the concentrations of fasting blood glucose (FBG), TC and TG. Furthermore, CPP significantly increased the concentration of HDL-c, body weight and serum insulin level in the mice. In addition, according to acute toxicity studies and single cell gel electrophoresis analysis, CPP did not produce any physical or behavioral signs of toxicity. More significantly, our data demonstrated CPP exhibited the best effects at the dose of 400 mg/kg bw. The above results suggest that CPP can control blood glucose and modulate the metabolism of glucose and blood lipids in diabetes mellitus mice, so we conclude that CPP should be evaluated as a candidate for future studies on diabetes mellitus.

Three structurally homologous isothiocyanates exert “Janus” characteristics in human HepG2 cells

In this study, we used the single cell gel electrophoresis (SCGE) assay and the micronucleus (MN) test to investigate the DNA damaging effects and the antigenotoxic potencies of three structurally related ITCs in human HepG2 cells. The results show that all three ITCs possess the characteristic of a "Janus" compound, i.e., they exert both significant genotoxicity and antigenotoxicity, depending on the concentrations used in the test systems applied. Regression line analysis of the results derived by SCGE analysis showed genotoxic potency of the ITCs in the following order: 3-methylthiopropyl ITC (MTPITC) > 4-methylthiobutyl ITC (MTBITC) > 5-methylthiopentyl ITC (MTPeITC); however, this order in genotoxic potency was not confirmed by MN analysis. Additionally, the MN test showed significant mutagenicity of the test substances at higher concentrations when compared with the SCGE assay. Twenty-four hour-treatment of the cells with the ITCs, followed by a 1-hr recovery period, showed significant DNA repair in the SCGE assay at a concentration > or =10 microM MTPITC, > or =3 microM MTBITC, and > or =0.1 microM MTPeITC, respectively. In antigenotoxicity studies, the most effective concentration of MTPITC and MTPeITC toward B(a)P-induced DNA damage was 0.1 muM in both test systems. MTBITC suppressed MN formation in B(a)P-treated cells to the background level at a concentration of 1 muM. The ambivalent character of the ITCs under studymust be further clarified, especially in the possiblecontext of high dose therapeutic applications.

Biomonitoring of primary aluminium industry workers: detection of micronuclei and repairable DNA lesions by alkaline SCGE

The genetic effects of occupational exposure to low polycyclic aromatic hydrocarbon (PAH) concentrations were investigated in primary aluminium industry workers. The study subjects were employed in a plant that uses pre-baked anode cells, and has relatively low PAH contamination. Forty-two male workers belonging to different job categories (anode fabrication, baking, rodding, electrolysis, maintenance), together with 16 male local residents with no occupational exposure to PAHs were selected for the analysis of micronuclei and DNA lesions in peripheral lymphocytes. The incidence of micronuclei determined in 1000 cytokinesis-blocked cells in each subject was not significantly different between workers and controls (8.5±5.4‰ versus 9.7±4.9‰, respectively), nor between smokers and non-smokers (8.3±5.8‰ versus 9.2±5.1‰), but was significantly ( P<0.05) related to the subjects’ age. Also the analysis of DNA damage in unstimulated and mitogen-stimulated lymphocytes by single cell gel electrophoresis (SCGE) did not show significant differences between the studied groups (average tail moment values were 0.53±0.53 and 0.49±0.45 μm in exposed subjects and controls, respectively). However, when lymphocytes were cultured in the presence of cytosine arabinoside (Ara-C, 1 μg/ml for 16 h) the SCGE analysis revealed a significant ( P=0.018) difference in tail moment values between aluminium workers and controls (1.73±1.05 μm versus 0.93±0.88 μm, respectively). This difference may highlight an excess of relatively stable DNA lesions, that do not affect strand integrity, and are expressed as intermediates of excision repair in stimulated cells, when gap refilling is inhibited by cytosine arabinoside (Ara-C).

Single cell gel electrophoresis analysis of genomic damage induced by ethyl methanesulfonate in cultured tobacco cells

A procedure for employing cultured tobacco cells (line TX1) in the SCGE assay was developed. The effect on DNA migration was studied in control and EMS-treated cells at different stages in their growth curve. The experimental parameters of treatment time and the unwinding time were analyzed in TX1 cells. With EMS in a concentration range from 0 to 30 mM the average median (±S.E.) tail moments ranged from 1.84±0.36 μm for the negative controls and increased in a direct concentration dependent manner to 44.47±1.90 for cells treated with 30 mM EMS. Nuclei isolated from TX1 cells and treated with EMS had a similar sensitivity as TX1 cells after EMS treatment. The plant cells express similar concentration–response curves for EMS as reported with mammalian (CHO) cells. This plant cell SCGE assay may prove to be a useful tool for the study of agricultural chemicals in specific plant cell types, to compare the response of mutagens in plant and animal cells and for basic research in genetic toxicology and DNA repair in plants.