In an effort to understand the mechanism of radical formation on heme proteins, the formation of radicals on hemoglobin was initiated by reaction with hydrogen peroxide in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The DMPO nitrone adducts were analyzed by mass spectrometry (MS) and immuno-spin trapping. The spin-trapped protein adducts were then subjected to tryptic digestion and MS analyses. When hemoglobin was reacted with hydrogen peroxide (H₂O₂) in the presence of DMPO, a DMPO nitrone adduct could be detected by immuno-spin trapping. To verify that DMPO adducts of the protein free radicals had been formed, the reaction mixtures were analyzed by flow injection electrospray ionization mass spectrometry (ESI/MS). The ESI mass spectrum of the hemoglobin/H₂O₂/DMPO sample shows one adduct each on both the α chain and the β chain of hemoglobin which corresponds in mass to the addition of one DMPO molecule. The nature of the radicals formed on hemoglobin was explored using proteolysis techniques followed by liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (MS/MS) analyses. The following sites of DMPO addition were identified on hemoglobin: Cys-93 of the β chain, and Tyr-42, Tyr-24, and His-20 of the α chain. Because of the pi-pi interaction of Tyr-24 and His-20, the unpaired electron is apparently delocalized on both the tyrosine and histidine residue (pi-pi stacked pair radical).

Inorganic arsenic is an environmental carcinogen that may act through multiple mechanisms including formation of methylated derivatives in vivo. Sodium arsenite (up to 5.0 microM) renders arsenic methylation-competent TRL1215 rat liver epithelial cells tumorigenic in nude mice at 18 weeks of exposure and arsenic methylation-deficient RWPE-1 human prostate epithelial cells tumorigenic at 30 weeks of exposure. We assessed the role of arsenic biomethylation in oxidative DNA damage (ODD) using a recently developed immuno-spin trapping method. Immuno-spin trapping was used to measure ODD after chronic exposure of cultured TRL1215 vs RWPE-1 cells, or of methylation-competent UROtsa/F35 vs methylation-deficient UROtsa human urothelial cells, to sodium arsenite. Secreted matrix metalloproteinase (MMP)-2 and -9 activity, as analyzed by zymography, cellular invasiveness by using a transwell assay, and colony formation by using soft agar assay were compared in cells exposed to arsenite with and without selenite, an arsenic biomethylation inhibitor, to assess the role of ODD in the transition to an in vitro cancer phenotype. Exposure of methylation-competent TRL1215 cells to up to 1.0 microM sodium arsenite was followed by a substantial increase in ODD at 5-18 weeks (eg, at 16 weeks with 1.0 microM arsenite, 1138% of control, 95% confidence interval [CI] = 797% to 1481%), whereas exposure of methylation-deficient RWPE-1 cells to up to 5.0 microM arsenite did not increase ODD for a 30-week period. Inhibition of arsenic biomethylation with sodium selenite abolished arsenic-induced ODD and invasiveness, colony formation, and MMP-2 and -9 hypersecretion in TRL1215 cells. Arsenic induced ODD in methylation-competent UROtsa/F35 cells (eg, at 16 weeks, with 1.0 microM arsenite 225% of control, 95% CI = 188% to 262%) but not in arsenic methylation-deficient UROtsa cells, and ODD levels corresponded to the levels of increased invasiveness, colony formation, and hypersecretion of active MMP-2 and -9 seen after transformation to an in vitro cancer phenotype. Arsenic biomethylation appears to be obligatory for arsenic-induced ODD and appears linked in some cells with the accelerated transition to an in vitro cancer phenotype.

Increased chicken-derived fat and fructose consumption in the human diet is paralleled by an increasing prevalence of obesity and metabolic syndrome (MS). Herein, we aimed at developing and characterizing a mouse model of diet-induced obesity (DIO) resembling most of the key features of the human MS. To accomplish this, we fed male C57BL/6J mice for 4, 8, 12, and 16 weeks with either a low-fat diet (LFD) or a high-chicken-fat diet (HFD) and tap water with or without 10% fructose (F). This experimental design resulted in the following four experimental groups: LFD, LFD + F, HFD, and HFD + F. Over the feeding period, and on a weekly basis, the HFD + F group had more caloric intake and gained more weight than the other experimental groups. Compared to the other groups, and at the end of the feeding period, the HFD + F group had a higher adipogenic index, total cholesterol, low-density lipoprotein cholesterol, fasting basal glycemia, insulin resistance, hypertension, and atherogenic index and showed steatohepatitis and systemic oxidative stress/inflammation. A mouse model of DIO that will allow us to study the effect of MS in different organs and systems has been developed and characterized.

Myeloperoxidase (MPO) released by activated neutrophils can initiate and promote carcinogenesis. MPO produces hypochlorous acid (HOCl) that oxidizes the genomic DNA in inflammatory cells as well as in surrounding epithelial cells. DNA-centered radicals are early intermediates formed during DNA oxidation. Once formed, DNA-centered radicals decay by mechanisms that are not completely understood, producing a number of oxidation products that are studied as markers of DNA oxidation. In this study we employed the 5,5-dimethyl-1-pyrroline N-oxide-based immuno-spin trapping technique to investigate the MPO-triggered formation of DNA-centered radicals in inflammatory and epithelial cells and to test whether resveratrol blocks HOCl-induced DNA-centered radical formation in these cells. We found that HOCl added exogenously or generated intracellularly by MPO that has been taken up by the cell or by MPO newly synthesized produces DNA-centered radicals inside cells. We also found that resveratrol passed across cell membranes and scavenged HOCl before it reacted with the genomic DNA, thus blocking DNA-centered radical formation. Taken together our results indicate that the formation of DNA-centered radicals by intracellular MPO may be a useful point of therapeutic intervention in inflammation-induced carcinogenesis.

We have investigated the role played by GSH efflux in apoptosis of human HaCaT keratinocytes induced by UVA irradiation. UVA irradiation of HaCaT cells caused a rapid rise in GSH efflux across the intact cell membrane, followed by an increase in apoptosis. GSH efflux was stimulated by glucose and was reduced by the addition of exogenous GSH and intracellular GSH depletion by buthionine sulfoximine, suggesting that GSH transport is active and is influenced by the GSH concentration gradient across the cell membrane. Verapamil and cyclosporin A, blockers of the multidrug resistance-associated protein, decreased UVA-induced GSH efflux. GSH efflux occurred within 2 h of UVA irradiation, suggesting that the stimulation of GSH efflux is due to an increase in the activity of pre-existing multidrug resistance-associated protein transporter carrier. Although inhibition of GSH efflux did not affect caspase activation and DNA fragmentation, it delayed the gradual increase in plasma membrane permeability and reduced phosphatidylserine translocation in HaCaT cells. It is therefore likely that upon UVA irradiation, GSH efflux increased the intracellular oxidative stress without intervention of reactive oxygen species, thus resulting in more phosphatidylserine externalization and membrane rearrangement. These provide targets for macrophage recognition and phagocytosis and thus minimize the potential to invoke inflammation or neoplastic transformation.