Synthesis and in vitro characterization of the genotoxic, mutagenic and cell-transforming potential of nitrosylated heme

Data from epidemiological studies suggest that consumption of red and processed meat is a factor contributing to colorectal carcinogenesis. Red meat contains high amounts of heme, which in turn can be converted to its nitrosylated form, NO-heme, when adding nitrite-containing curing salt to meat. NO-heme might contribute to colorectal cancer formation by causing gene mutations and could thereby be responsible for the association of (processed) red meat consumption with intestinal cancer. Up to now, neither in vitro nor in vivo studies characterizing the mutagenic and cell transforming potential of NO-heme have been published due to the fact that the pure compound is not readily available. Therefore, in the present study, an already existing synthesis protocol was modified to yield, for the first time, purified NO-heme. Thereafter, newly synthesized NO-heme was chemically characterized and used in various in vitro approaches at dietary concentrations to determine whether it can lead to DNA damage and malignant cell transformation. While NO-heme led to a significant dose-dependent increase in the number of DNA strand breaks in the comet assay and was mutagenic in the HPRT assay, this compound tested negative in the Ames test and failed to induce malignant cell transformation in the BALB/c 3T3 cell transformation assay. Interestingly, the non-nitrosylated heme control showed similar effects, but was additionally able to induce malignant transformation in BALB/c 3T3 murine fibroblasts. Taken together, these results suggest that it is the heme molecule rather than the NO moiety which is involved in driving red meat-associated carcinogenesis. © 2020, The Author(s)

Bioactive Phenolic Compounds from Lingonberry (Vaccinium vitis-idaea L.): Extraction, Chemical Characterization, Fractionation and Cellular Antioxidant Activity

Lingonberries contain high contents of bioactive compounds such as chlorogenic acids and anthocyanins. In addition to radical scavenging and antioxidant activities, these compounds can protect cells from DNA damage. For this reason, lingonberries might be well suited for nutraceuticals or natural biomedicines. To assess these applications, the present study characterized and identified the most effective extract, only consisting of anthocyanins, copigments or a mixture of both, obtained from a lingonberry juice concentrate. An extract was generated by using a XAD-7 column followed by fractionation into anthocyanins and copigments using adsorptive membrane chromatography. After identification of main polyphenols by HPLC–photodiode array–electrospray ionization–tandem mass spectrometry, free radical scavenging activity was analyzed by electron spin resonance spectroscopy using 2,2-diphenyl-1-picrylhydrazyl and galvinoxyl radicals. Furthermore, cyclic voltammetry analyses and the Trolox equivalent antioxidant capacity (TEAC) assay were applied. Finally, the reactive oxygen species (ROS) reducing effects of the lingonberry extract and its fractions were evaluated in HepG2 cells. While the combination of anthocyanins and copigments possessed the highest antioxidant activities, all samples (XAD-7 extract, anthocyanin and copigment fraction) protected cells from oxidative stress. Thus, synergistic effects between phenolic compounds may be responsible for the high antioxidant potential of lingonberries, enabling their use as nutraceuticals

Probing the Nanoscale Viscoelasticity of Intracellular Fluids in Living Cells

We have used fluorescence correlation spectroscopy to determine the anomalous diffusion properties of fluorescently tagged gold beads in the cytoplasm and the nucleus of living cells. From the extracted mean-square displacement v(τ) ∼ τα, we have determined the complex shear modulus G(ω) ∼ ωα for both compartments. Without treatment, all tested cell lines showed a strong viscoelastic behavior of the cytoplasm and the nucleoplasm, highlighting the crowdedness of these intracellular fluids. We also found a similar viscoelastic response in frog egg extract, which tended toward a solely viscous behavior upon dilution. When cells were osmotically stressed, the diffusion became less anomalous and the viscoelastic response changed. In particular, the anomality changed from α ≈ 0.55 to α ≈ 0.66, which indicates that the Zimm model for polymer solutions under varying solvent conditions is a good empirical description of the material properties of the cytoplasm and the nucleoplasm. Since osmotic stress may eventually trigger cell death, we propose, on the basis of our observations, that intracellular fluids are maintained in a state similar to crowded polymer solutions under good solvent conditions to keep the cell viable