1-3-dimethylthiourea and 3-nitrotyrosine

Previous studies have shown that in situ exposure to arsenic induced increased vascular leakage. However, the underlying mechanism remains unclear. Reactive nitrogen and oxygen species such as nitric oxide (NO) and hydroxyl radical (OH(-)) are known to affect vascular permeability. Therefore, the goal of our present studies is to investigate the functional impact of the generation of NO or OH(-) on arsenic-induced vascular leakage. Vascular permeability changes were evaluated by means of Evans blue (EB) assay. Rats were anesthetized and intravenously injected with EB. Permeability changes were induced in back skin by intradermal injections of sodium arsenite mixed with NOS inhibitor: N(omega)-Nitro-L-arginine methyl ester (L-NAME) or aminoguanidine (AG) and OH(-) scavenger: 1,3 Dimethyl-2 thiourea (DMTU). Experiments were also performed to determine whether DMTU mixed with L-NAME would further inhibit arsenic-induced vascular leakage as compared with attenuation effects by either DMTU or L-NAME. One hour after administration, EB accumulated in the skin was extracted and quantified. Both L-NAME (0.02, 0.1 and 0.5 micromol/site) and DMTU (0.05, 0.2 and 1.2 micromol/site) inhibited the increase in vascular leakage induced by arsenite. However, only high dose (1 micromol/site) of AG significantly attenuated arsenite-induced vascular leakage. In contrast, neither D-NAME (0.02, 0.1 and 0.5 micromol/site) nor AG (0.04 and 0.2 micromol/site) attenuated increased vascular leakage by arsenic. DMTU mixed with L-NAME caused no further inhibition of arsenic-induced vascular leakage by either DMTU or L-NAME. The techniques of India ink and immunostaining were used to demonstrate both vascular labeling and nitrotyrosine staining in tissue treated with arsenic. L-NAME apparently reduced the density of leaky vessels and the levels of peroxynitrite staining induced by arsenite. These results suggest that NO, OH(-) and peroxynitrite play a role in increased vascular permeability induced by arsenic exposure.

Topics: Animals; Arsenites; Blood Vessels; Capillary Permeability; Carbon; Dose-Response Relationship, Drug; Drug Therapy, Combination; Enzyme Inhibitors; Evans Blue; Free Radical Scavengers; Guanidines; Hydroxyl Radical; Injections, Intradermal; Injections, Intravenous; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Skin; Sodium Compounds; Thiourea; Tyrosine

Light has been shown to modulate protein nitration in rat retinas. To better understand the role of protein nitration in photoreceptor cell death induced by intense light, we examined retinal protein nitration and identified target proteins in rod outer segments (ROS).. Cyclic light-reared rats, treated or not with the antioxidant, dimethylthiourea (DMTU), were exposed to intense green light for 8 h. A subset of these rats was kept in the dark for 24 h after 8 h of light exposure. Western analysis of ROS proteins with an anti-nitrotyrosine antibody was performed to examine changes in protein nitration. 2D-immunoblots with anti-nitrotyrosine antibody followed by liquid chromatography tandem mass spectrometry was used to identify nitrated proteins in ROS. The expression levels of three nitric oxide synthase (NOS) isoforms, inducible, neuronal-, and endothelial-NOS were semi-quantified by immunoblot analysis.. Western analysis revealed that the level of ROS protein nitration increased during the dark recovery period after 8 h of light treatment in both DMTU treated and untreated rats. However, DMTU effectively reduced protein nitration in ROS during light exposure and during the subsequent dark recovery period. Using 2D-immunoblotting followed by liquid chromatography tandem mass spectrometry analysis, we identified ten ROS proteins as nitration targets. Most of these proteins were glycolytic enzymes. The level of inducible-NOS in the retina was increased by light exposure.. The effect of DMTU in reducing ROS protein nitration during and after light suggests the involvement of protein nitration during light-induced photoreceptor cell death. Nitration of glycolytic enzymes specifically may alter their activities. Increased levels of iNOS during and after intense light exposure suggest that this isoform is responsible for intense light induced protein nitration in ROS during the dark recovery period. The limited nitration seen in ROS during light exposure may reflect a quenching effect by endogenous antioxidants on the generation of reactive oxygen and nitrogen species.

Topics: Animals; Antioxidants; Blotting, Western; Chromatography, Liquid; Darkness; Eye Proteins; Light; Male; Nitrates; Nitric Oxide Synthase; Rats; Rats, Sprague-Dawley; Rod Cell Outer Segment; Tandem Mass Spectrometry; Thiourea; Tyrosine

Lead-induced hypertension has previously been shown to be closely associated with an increase in reactive oxygen species in low lead (100 ppm)-treated rats. The present study has attempted to define the specific moiety involved by noting the blood pressure (BP), reactive oxygen species (MDA-TBA), hydroxyl radical, and nitrotyrosine responses to infusion of the reactive oxygen species scavenger dimethylthiourea. Dimethylthiourea, a reputed scavenger of hydroxyl radical, normalized BP and MDA-TBA in the lead-treated rats but had no effect in normal control animals. MDA-TBA, hydroxyl radical, and nitrotyrosine, the tissue end product of peroxynitrite, were reduced to or toward normal by dimethylthiourea. The results, therefore, are consistent with the suggestion that either hydroxyl radical or peroxynitrite may be the reactive species affected by lead.

Topics: Animals; Blood Pressure; Free Radical Scavengers; Hydroxyl Radical; Hypertension; Lead; Lipid Peroxides; Male; Rats; Rats, Sprague-Dawley; Thiourea; Tyrosine

Thiourea and, more recently, dimethylthiourea, have been used as hydroxyl radical (OH.) scavengers in experiments both in vitro and in vivo. We show that both compounds can inhibit nitration of the amino acid tyrosine on addition of peroxynitrite, and also the inactivation of alpha1-antiproteinase by peroxynitrite. Hence, protective effects of (dimethyl) thiourea could be due to inhibition of peroxynitrite-dependent damage as well as to OH. scavenging, and these compounds must not be regarded as specific OH. scavengers.

Topics: alpha 1-Antitrypsin; Enzyme Inhibitors; Free Radical Scavengers; Hydroxyl Radical; Nitrates; Thiourea; Tyrosine