Depletion of HIF-1α by Inducible Cre/loxP Increases the Sensitivity of Cultured Murine Hepatocytes to Ionizing Radiation in Hypoxia.

The transcription factor hypoxia-inducible factor (HIF) is the main oxygen sensor which regulates adaptation to cellular hypoxia. The aim of this study was to establish cultured murine hepatocyte derived cells (mHDC) as an in vitro model and to analyze the role of HIF-1α in apoptosis induction, DNA damage repair and sensitivity to ionizing radiation (IR). We have crossed C57/BL6 mice that bear loxP sites flanking exon 2 of Hif1a with mice which carry tamoxifen-inducible global Cre expression. From the offspring, we have established transduced hepatocyte cultures which are permanently HIF-1α deficient after tamoxifen treatment. We demonstrated that the cells produce albumin, acetylcholine esterase, and the cytokeratins 8 and 18 which functionally characterizes them as hepatocytes. In moderate hypoxia, HIF-1α deficiency increased IR-induced apoptosis and significantly reduced the surviving fraction of mHDC as compared to HIF-1α expressing cells in colony formation assays. Furthermore, HIF-1α knockout cells displayed increased IR-induced DNA damage as demonstrated by increased generation and persistence of γH2AX foci. HIF-1α deficient cells showed delayed DNA repair after IR in hypoxia in neutral comet assays which may indicate that non-homologous end joining (NHEJ) repair capacity was affected. Overall, our data suggest that HIF-1α inactivation increases radiation sensitivity of mHDC cells.

The biological effects of vanadyl curcumin and vanadyl diacetylcurcumin complexes: the effect on structure, function and oxidative stability of the peroxidase enzyme, antibacterial activity and cytotoxic effect.

Curcumin has multiple pharmacological effects, but it has poor stability. Complexation of curcumin with metals improves its stability. Here, the effects of vanadyl curcumin and vanadyl diacetylcurcumin on the function and structure of horseradish peroxidase enzyme were evaluated by spectroscopic techniques. Cytotoxic effect of the complexes was also assessed on MCF-7 breast cancer, bladder and LNCaP prostate carcinoma cell line. The results showed that the complexes improve catalytic activity of HRP, and also increase its tolerance against the oxidative condition. The result also indicated that the affinity of HRP for hydrogen peroxide substrate decreases, while the affinity increases for phenol substrate. Circular dichroism and fluorescence spectroscopies showed that compactness of the enzyme structure around the catalytic heme group and the distance between the heme group and tryptophan residue decreases after the binding. The antibacterial and cytotoxic results indicated that the complexes have anticancer potential, but they have no considerable antibacterial activity.

Investigating the effect of gallium curcumin and gallium diacetylcurcumin complexes on the structure, function and oxidative stability of the peroxidase enzyme and their anticancer and antibacterial activities.

Curcumin has a wide spectrum of biological and pharmacological activities including anti-inflammatory, antioxidant, antiproliferative, antimicrobial and anticancer activities. Complexation of curcumin with metals has gained attention in recent years for improvement of its stability. In this study, the effect of gallium curcumin and gallium diacetylcurcumin on the structure, function and oxidative stability of horseradish peroxidase (HRP) enzyme were evaluated by spectroscopic techniques. In addition to the enzymatic investigation, the cytotoxic effect of the complexes was assessed on bladder, MCF-7 breast cancer and LNCaP prostate carcinoma cell lines by MTT assay. Furthermore, antibacterial activity of the complexes against S. aureus and E. coli was explored by dilution test method. The results showed that the complexes improve activity of HRP and also increase its tolerance against the oxidative condition. After addition of the complexes, affinity of HRP for hydrogen peroxide substrate decreases, while the affinity increases for phenol substrate. Circular dichroism, intrinsic and synchronous fluorescence spectra showed that the enzyme structure around the catalytic heme group becomes less compact and also the distance between the heme group and tryptophan residues increases due to binding of the complexes to HRP. On the whole, it can be concluded that the change in the enzyme structure upon binding to the gallium curcumin and gallium diacetylcurcumin complexes results in an increase in the antioxidant efficiency and activity of the peroxidise enzyme. The result of anticancer and antibacterial activities suggested that the complexes exhibit the potential for cancer treatment, but they have no significant antibacterial activity.

Optimization of a new polymeric chromium (III) membrane electrode based on methyl violet by using experimental design.

Plackett-Burman and Box-Behnken designs were applied as experimental design strategies to screen and optimize the influence of membrane ingredients on the electrode performance. A new poly(vinyl chloride) membrane sensor for Cr(III) based on methyl violet as an ionophore was planned. The major variables to find a model for achieving the best Nernstian slope as response were: PVC, plasticizers, methyl violet, KpClTPB, pH, conditioning time and internal solution concentration. Plackett-Burman design was used to screen the main factors and Box-Behnken response surface was led to find a model for optimizing the response. The optimized membrane electrode shows a Nernstian slope for chromium (III) ions over a wide linear range from 1.99x10(-6) to 3.16x10(-2)molL(-1) and a slope of 19.5+/-0.1mVdecade(-1) of activity. It would be successfully applied in the pH range from 3.5 to 6.5 with detection limit of 1.77x10(-6)molL(-1) (0.092mgL(-1)). The response time of the sensor is about 8s and the membrane can be used for more than 6 weeks without any deviation. The relative standard deviations (R.S.D.) for six replicate the measurements of 1.0x10(-4) and 1.0x10(-3)molL(-1) of Cr(III) were 3.2 and 3%, respectively. The electrode revealed comparatively good selectivity with respect to many cations including alkali earth, transition and heavy metal ions. The electrode was successfully used as an indicator in the potentiometric titration of Cr(III) with EDTA and was also applied to the direct determination chromium (III) content of spiked water and soil samples.