4-hydroxy-2-nonenal and Hemolysis

Posttranslational modifications (PTMs) have been reported in hemoglobin (Hb) treated with ROS/RNS in cell-free experiments. However, little is known about oxidative PTMs of Hb occurring within the erythrocytes. The aim of this study is to characterize the patterns of Hb PTMs in erythrocytes under oxidative stress. Using mass spectrometry, we investigated specifically methionine/tryptophan oxidation, tyrosine nitration, and the modification via 4-hydroxynonenal (HNE), a product of lipid-peroxidation, on Hb. We demonstrated that the treatment with H(2)O(2)/nitrite induced higher levels of Hb oxidation/nitration in purified Hb preparations than in unpurified hemolysates and erythrocytes, indicating that ROS/RNS are primarily removed by antioxidative mechanisms. We further studied Hb from erythrocytes exposed to γ-irradiation. An irradiation of 30-100 Gy triggered a remarkable increase of intracellular ROS. However, 30 Gy did not induce apparent changes in Hb oxidation/nitration and hemolysis, while Hb oxidation/nitration and hemolysis were significantly enhanced by 100 Gy, suggesting that Hb oxidation/nitration are the consequence of overwhelmed antioxidative mechanisms after oxidative attack and reflect the severity of the oxidative damage of erythrocytes. Although irradiation was known to induce lipid-peroxidation, we could not detect HNE-Hb adducts in irradiated erythrocytes. Analyzing PTM patterns suggests Hb nitration as a more suitable indicator of the oxidative damage of erythrocytes.

Topics: Aldehydes; Amino Acid Sequence; Cells, Cultured; Dose-Response Relationship, Radiation; Erythrocyte Membrane; Erythrocytes; Gamma Rays; Hemoglobins; Hemolysis; Humans; Hydrogen Peroxide; Methionine; Molecular Sequence Data; Nitrites; Oxidation-Reduction; Oxidative Stress; Protein Processing, Post-Translational; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tryptophan; Tyrosine

Methylamine dichloramine (CH(3)NCl(2)) produced by neutrophils may promote colon tumors and colitis via architectural and oxidative changes in crypts, which are secretory granulae composed of goblet cells located in the colorectal mucosal layer. We investigated whether CH(3)NCl(2), in comparison with the other reactive oxygen species (ROS) such as H(2)O(2) and HOCl, derived from primed neutrophils in inflammatory sites in the large intestine, is a biogenic factor for the induction of colorectal disease in mice.. Male ICR-strain mice were administered each oxidant (0.5-0.7 micromol/mouse) by enema under anesthesia. The colorectal tissues were evaluated by histopathological and immunohistochemical analyses. Hemolysis and hemoglobin oxidation by the methylamine chloramines and HOCl were examined by adding them (50-400 microM) to a sheep erythrocyte suspension (1x10(8) cells/ml) and its lysate at pH 7 and 37 degrees C.. CH(3)NCl(2) oxidized erythrocyte hemoglobin more effectively than HOCl, indicating it has high cell permeability and selective oxidation ability. CH(3)NCl(2) mainly induced atrophy of crypts at 6 h after administration, while the other ROS tested did not. Furthermore, 4-hydroxy-2-nonenal (4-HNE) showed positive immunostains throughout the mucosal layer, including around the basal regions of atrophied crypts, only with CH(3)NCl(2), while positive immunostains were observed for 3-nitrotyrosine (3-NT) in the atrophied crypts and their surrounding lamina propria in the mucosal layer.. The results suggest that CH(3)NCl(2)derived from primed neutrophils may play the most important role in promoting the development of colon tumor formation and colitis by oxidative stress through its high degree of cell permeability.

Topics: Aldehydes; Animals; Chloramines; Colon; Colonic Neoplasms; Hemoglobins; Hemolysis; Hydrogen Peroxide; Hypochlorous Acid; Immunohistochemistry; Inflammatory Bowel Diseases; Intestinal Mucosa; Male; Mice; Mice, Inbred ICR; Neutrophil Activation; Oxidation-Reduction; Sheep; Tyrosine

Haptoglobin (Hp), a conserved plasma glycoprotein, forms very stable soluble complexes with free plasma haemoglobin. Haemoglobin binding by haptoglobin is thought to be important in the rapid hepatic clearance of haemoglobin from the plasma and in the inhibition of glomerular filtration of haemoglobin. It is thought to reduce haemoglobin-induced renal damage during haemolysis. To evaluate these functions, Hp knockout (Hp-/-) mice were created. The Hp-/- mouse was generated by a standard gene replacement technique in mouse embryonic stem cells. These mice were evaluated with and without haemolysis using several parameters: mortality, haemoglobin clearance, renal tissue damage and function. Hp-/- mice were viable but had a small, significant reduction in postnatal viability. The lack of Hp did not impair clearance of free plasma haemoglobin. Induction of severe haemolysis by phenylhydrazine caused extensive haemoglobin precipitation in the renal tubular cells. However, haemoglobin precipitation in the kidney was not increased in Hp-/- mice. Nevertheless, Hp-/- mice were more susceptible to phenylhydrazine with a mortality rate of 55% in Hp-/- mice versus 18% in Hp+/+ mice. In general, phenylhydrazine-treated Hp-/- mice suffered greater tissue damage, as evidenced by the induction of a hepatic acute phase response, resulting in increased plasma alpha1-acidic glycoprotein (AGP) levels and higher plasma malonaldehyde (MDA) and 4-hydroxy-2(E)-nonenal (HNE) levels. Gross pathological analysis indicated that the kidney was the most affected tissue in phenylhydrazine-treated Hp-/- and Hp+/+ mice, and Hp-/- mice were more severely affected. They had lower mitotic indices in their kidneys, higher basal levels of renal lipid peroxidation, as evidenced by levels of malonaldehyde and 4-hydroxy-2(E)-nonenal (MDA/HNE) and elevated levels of 8-hydroxyguanine (but not other products of oxidative DNA damage). There also was increased induction of haem oxygenase-1. The more severe renal damage in Hp-/- mice was also evident in the delayed erythropoietin gene expression and poorer renal clearance of [3H]-inulin. The reduction in glomerular filtration function in Hp+/+ and Hp-/- mice could be restored to baseline by vasodilators (prazosin or diazoxide), implicating renal vasoconstriction as a major mechanism of acute renal failure during induced haemolysis. These data suggest that Hp plays a pivotal role in reducing renal oxidative damage during haemolysis.

Topics: Aldehydes; Animals; Cysteine Proteinase Inhibitors; Diazoxide; Haptoglobins; Hemoglobins; Hemolysis; Kidney; Malondialdehyde; Mice; Mice, Knockout; Nitric Oxide; Orosomucoid; Oxidative Stress; Phenotype; Prazosin; Protein Binding; Vasodilator Agents

Haptoglobin knockout (Hp-/-) mice are more sensitive to phenylhydrazine-induced hemolysis than Hp+/+ mice.. Hemolysis was induced in Hp-/- and Hp+/+ mice using phenylhydrazine. Relative renal tissue damage and function were then assessed.. Hp-/- mice had higher basal levels of renal lipid peroxidation, as evidenced by levels of malonaldehyde and 4-hydroxy-2(E)-nonenal (MDA/HNE). After the administration of phenylhydrazine, levels of 8-hydroxyguanine (but not other products of oxidative DNA damage) were significantly elevated in the renal DNA. There was also increased induction of heme oxygenase-1. The more severe renal damage in Hp-/- mice was also evident in the delayed erythropoietin gene expression and poorer renal clearance of 3H-inulin. This reduction in glomerular filtration function in Hp+/+ and Hp-/- mice could be restored to baseline by vasodilators (prazosin or diazoxide), implicating renal vasoconstriction as a major mechanism of acute renal failure during induced hemolysis. Precipitation of hemoglobin in the kidney was not increased in Hp-/- mice.. Haptoglobin appears to play an important physiological role as an antioxidant, particularly during hemolysis.

Topics: Acute-Phase Reaction; Aldehydes; Animals; Antioxidants; DNA; Erythropoietin; Gene Expression Regulation, Enzymologic; Guanine; Haptoglobins; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Hemoglobins; Hemolysis; Inulin; Kidney; Kidney Function Tests; Lipid Peroxidation; Liver; Malondialdehyde; Membrane Proteins; Mice; Mice, Knockout; Oxidative Stress; Phenylhydrazines; Tritium

In order to evaluate the pro-hemolytic action exerted by different classes of biogenic aldehydes, normal red cells obtained from human beings of both sexes were incubated at 37 degrees C under iso or hypo-osmotic conditions in the presence of hydroxyalkenals or alkanals, in a concentration compatible with those actually recovered during red cell lipid peroxidation. None of the tested aldehydes showed a direct hemolytic effect, i.e. red cell lysis in iso-osmotic conditions. Conversely, almost all assayed alkanals and hydroxyalkenals exhibited a pre-lytic damage of human erythrocytes, as detected in the red cells suspended in hypo-osmotic medium. The highest pro-hemolytic effect was displayed by hexanal, nonanal, 2-nonenal and 4-hydroxynonenal.

Topics: Aldehydes; Erythrocytes; Female; Hemolysis; Humans; Lipid Peroxidation; Male