Naihao Lu

Effects of glutathione, Trolox and desferrioxamine on hemoglobin-induced protein oxidative damage: Anti-oxidant or pro-oxidant?

Evidence to support the role of heme proteins as major inducers of oxidative damage is increasingly present. Antioxidants have been widely used to ameliorate oxidative damage in vivo and in vitro, where the mechanism of this therapeutic action was usually dependent on their anti-oxidant effects. In this study, we chose three classic antioxidants, i.e. glutathione (GSH, an important intracellular antioxidant), 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid (Trolox, a phenolic antioxidant without chelating effect) and desferrioxamine (DFO, a good iron chelator), to study their efficiencies on hemoglobin-induced protein oxidative damage. It was found that all of these antioxidants had the capacities to act as free radical scavengers and reducing agents to remove cytotoxic ferryl hemoglobin, demonstrating apparent anti-oxidant activities. However, the effects on hemoglobin-H(2)O(2)-induced protein oxidation depended on the categories and concentrations of antioxidants. GSH efficiently inhibited protein (bovine serum albumin or rat heart homogenate) carbonyl formation in a dose-dependent manner. In contrast to their protective effects at high concentrations, both Trolox and DFO could significantly aggravate protein oxidation at low concentrations. The pro-oxidant effects of Trolox and DFO on hemoglobin-mediated oxidative damage were probably related to their abilities in producing additional free radicals, such as superoxide (O·(2)(-)) and hydroxyl radical (·OH). The dual effects on hemoglobin redox reactions may provide new insights into the physiological implications of Trolox and DFO with cellular heme proteins.

Nitrite attenuated peroxynitrite and hypochlorite generation in activated neutrophils

Oxidative stress is usually considered as an important factor to the pathogenesis of various diseases. Peroxynitrite (ONOO(-)) and hypochlorite (OCl(-)) are formed in immune cells as a part of the innate host defense system, but excessive reactive oxygen species generation can cause progressive inflammation and tissue damage. It has been proven that through mediating nitric oxide (NO) homeostasis, inorganic nitrite (NO2(-)) shows organ-protective effects on oxidative stress and inflammation. However, the effects of NO2(-) on the function of immune cells were still not clear. The potential role of NO2(-) in modulating ONOO(-) and OCl(-) generation in neutrophil cells was investigated in this study. As an immune cell activator, lipopolysaccharide (LPS) increased both ONOO(-) and OCl(-) production in neutrophils, which was significantly attenuated by NO2(-). NO2(-) reduced superoxide (O2(·-)) generation via a NO-dependent mechanism and increased NO formation in activated neutrophils, suggesting a crucial role of O2(·-) in NO2(-)-mediated reduction of ONOO(-). Moreover, the reduced effect of NO2(-) on OCl(-) production was attributed to that NO2(-) reduced H2O2 production in activated neutrophils without influencing the release of myeloperoxidase (MPO), thus limiting OCl(-) production by MPO/H2O2 system. Therefore, NO2(-) attenuates ONOO(-) and OCl(-) formation in activated neutrophils, opening a new direction to modulate the inflammatory response.

Binding of human IgG to single-walled carbon nanotubes accelerated myeloperoxidase-mediated degradation in activated neutrophils.

The binding of protein to carboxylated single-walled carbon nanotubes (SWCNTs) was believed to play an important role in the biological effects of nanotubes. However, the effects of protein-SWCNTs interactions on the oxidative degradation of nanotubes were not stressed enough. Here, we investigated the binding of human immunoglobulin G (IgG) to SWCNTs, and found that the preferred binding site was located in the Fc region of IgG. The hydrophobic and electrostatic interactions might be the crucial factors in stabilizing the binding of SWCNTs with IgG. Through the competitive binding of IgG and myeloperoxidase (MPO) to nanotube surfaces, the binding of IgG could impair MPO-induced SWCNTs biodegradation in vitro. However, both SWCNTs and IgG-SWCNTs were significantly degraded in zymosan-stimulated neutrophils, and the degradation degree was more for IgG-SWCNTs. These results suggest that the binding of IgG may be an important determinant for MPO-mediated SWCNTs biodegradation in activated human inflammatory cells.

NADPH oxidase-dependent degradation of single-walled carbon nanotubes in macrophages

Previous studies have shown that carboxylated single-walled carbon nanotubes (SWCNTs) could be oxidatively biodegraded by neutrophil myeloperoxidase (MPO) and peroxynitrite (ONOO−). However, the biodegradation mechanism of nanotubes in macrophages has not been explored enough. Here, we showed that both MPO and ONOO− could effectively oxidize SWCNTs to generate shorter and oxidative nanotubes in vitro. SWCNTs were significantly degraded in zymosan-stimulated macrophages, and the degradation mechanism was dependent on MPO and ONOO−-driven oxidative pathways of activated macrophages, where NADPH oxidase was found to be a major determinant of the biodegradation process. Moreover, the functionalization of IgG to SWCNTs could stimulate MPO release and ONOO− formation in macrophages, thereby creating the conditions favorable for degradation of nanotubes and subsequently contributing to the higher degradation degree of IgG-coated SWCNTs. Therefore, our discovery of NADPH oxidase-dependent SWCNTs degradation in activated macrophages will open new opportunities for the regulation of SWCNTs fate in vivo.

Inhibition of Myeloperoxidase- and Neutrophil-Mediated Hypochlorous Acid Formation in Vitro and Endothelial Cell Injury by (−)-Epigallocatechin Gallate

Myeloperoxidase (MPO) plays important roles in various diseases through its unique chlorinating activity to catalyze excess hypochlorous acid (HOCl) formation. Epidemiological studies indicate an inverse correlation between plant polyphenol consumption and the incidence of cardiovascular diseases. Here we showed that (-)-epigallocatechin gallate (EGCG), the main flavonoid present in green tea, dose-dependently inhibited MPO-mediated HOCl formation in vitro (chlorinating activities of MPO: 50.2 ± 5.7% for 20 μM EGCG versus 100 ± 5.6% for control, P < 0.01). UV-vis spectral and docking studies indicated that EGCG bound to the active site (heme) of MPO and resulted in the accumulation of compound II, which was unable to produce HOCl. This flavonoid also effectively inhibited HOCl generation in activated neutrophils (HOCl formation: 65.0 ± 5.6% for 20 μM EGCG versus 100 ± 6.2% for control, P < 0.01) without influencing MPO and Nox2 release and superoxide formation, suggesting that EGCG specifically inhibited MPO but not NADPH oxidase activity in activated neutrophils. Moreover, EGCG inhibited MPO (or neutrophil)-mediated HOCl formation in human umbilical vein endothelial cells (HUVEC) culture and accordingly protected HUVEC from MPO (or neutrophil)-induced injury (P < 0.05, all cases), although it did not induce cytotoxicity to HUVEC (P > 0.05, all cases). Our results indicate that dietary EGCG is an effective and specific inhibitor of MPO activity and may participate in the regulation of immune responses at inflammatory sites.

Effects of serum albumin on the degradation and cytotoxicity of single-walled carbon nanotubes

Neutrophil myeloperoxidase (MPO) and peroxynitrite (ONOO-) can oxidatively biodegrade carboxylated single-walled carbon nanotubes (SWCNTs). The protein-SWCNTs interactions will play an important role in the degradation and cytotoxicity of nanotubes. Here, we investigated the binding of bovine serum albumin (BSA, a common and well-characterized model blood serum protein) to SWCNTs, and found that the hydrophobic and electrostatic interactions might be crucial factors in stabilizing the binding of SWCNTs with BSA. The binding of BSA could impair SWCNTs biodegradation in vitro through the competitive adsorption to nanotube. Both SWCNTs and BSA-SWCNTs were significantly degraded in zymosan-stimulated macrophages, and the degradation degree was more for BSA-SWCNTs. The mechanism for SWCNTs degradation in activated macrophages was further investigated to demonstrate the dominant participation of MPO and ONOO--driven pathways. Moreover, binding of BSA to SWCNTs reduced cytotoxicity and degraded nanotubes induced less cytotoxicity than non-degraded nanotubes. The binding of BSA may be an important determinant for the biodegradation and cytotoxicity of SWCNTs in inflammatory cells, and therefore, provide a new route to mitigate the potential toxicity of nanotubes in future biomedical applications.

Inhibition of myeloperoxidase-mediated oxidative damage by nitrite in SH-SY5Y cells: Relevance to neuroprotection in neurodegenerative diseases.

Myeloperoxidase (MPO) and MPO-catalyzed hypochlorous acid (HOCl) is elevated in many neurodegenerative diseases, and lead to severe tissue injuries. Nitrite (NO2(-)) is a widespread inorganic molecule that has recently been proposed as a direct NO donor to exert antioxidant properties in vivo and vitro. Since NO2(-) and MPO (and/or HOCl) were important mediators in brain function and disease, we investigated the effects of NO2(-) on MPO-mediated damage to human neuroblastoma SH-SY5Y cells. Here, we showed that exposure of SH-SY5Y cells to MPO (or HOCl) resulted in a significant loss in viability, ATP and glutathione levels, and treatment of neuronal cells with NO2(-) substantially attenuated MPO (or HOCl)-dependent cellular toxicity. The protective effects of NO2(-) on MPO (or HOCl)-induced cytotoxicity were because that (1) NO2(-) at high concentrations competed effectively with Cl(-) for MPO, thus limiting OCl(-) production by the enzyme; (2) HOCl was removed by reacting with NO2(-), forming less damaging compound; (3) NO2(-) significantly inhibited MPO-mediated inactivation of brain protein (enolase) and protein oxidation. Therefore, NO2(-) could show novel protective effects in some neurodegenerative diseases by preventing MPO-mediated oxidative damage.

Inhibitory effect of human serum albumin on Cu-induced Aβ40 aggregation and toxicity

It has been suggested that the aggregation and cytotoxicity of amyloid-β (Aβ) peptide with transition-metal ions in neuronal cells is involved in the development and progression of Alzheimers disease (AD). As the most abundant protein in blood plasma and in cerebrospinal fluid, human serum albumin (HSA) can bind Aβ in vivo and subsequently inhibit Aβ fibril growth. However, the roles of albumin in Cu-induced Aβ aggregation and toxicity, and its potential biological relevance to AD therapy, were not stressed enough. Here, we showed that HSA was capable of binding Cu (I) with much higher affinity than Aβ, competitively inhibiting the interaction of Aβ and Cu ions. In the presence of biological reducing agent ascorbate, HSA inhibited Cu (II)/Cu (I)-mediated Aβ40 aggregation, reactive oxygen species production, and neurotoxicity. However, in the absence of Cu (II)/Cu (I), HSA could not effectively inhibit Aβ40 aggregation and neurotoxicity at 24 h (or less) incubation time, but decreased Aβ40 aggregation at much longer incubation (120 h). Our data suggested that through competitively decreasing Cu-Aβ interaction, HSA could effectively inhibit Cu (II)/Cu (I)-induced Aβ40 aggregation and neurotoxicity, and play important roles in regulating redox balance as well as metal homeostasis in AD prevention and therapy.

Inhibitive effects of quercetin on myeloperoxidase-dependent hypochlorous acid formation and vascular endothelial injury

Myeloperoxidase (MPO) from activated neutrophils plays important roles in multiple human inflammatory diseases by catalyzing the formation of powerful oxidant hypochlorous acid (HOCl). As a major flavonoid in the human diet, quercetin has been suggested to act as antioxidant and anti-inflammatory agent in vitro and in vivo. In this study, we showed that quercetin inhibited MPO-mediated HOCl formation (75.0 ± 6.2% for 10 μM quercetin versus 100 ± 5.2% for control group, P < 0.01) and cytotoxicity to endothelial cells in vitro, while this flavonoid was nontoxic to endothelial cell cultures ( P > 0.05, all cases). Moreover, quercetin inhibited HOCl generation by stimulated neutrophils (a rich source of MPO) and protected endothelial cells from neutrophils-induced injury. Furthermore, quercetin could inhibit HOCl-induced endothelial dysfunction such as loss of cell viability, and decrease of nitric oxide formation in endothelial cells ( P < 0.05, all cases). Consistent with these in vitro data, quercetin attenuated lipopolysaccharide-induced endothelial dysfunction and increase of MPO activity in mouse aortas, while this flavonoid could protect against HOCl-mediated endothelial dysfunction in isolated aortas ( P < 0.05). Therefore, it was proposed that quercetin attenuated endothelial injury in inflammatory vasculature via inhibition of vascular-bound MPO-mediated HOCl formation or scavenging of HOCl. These data indicate that quercetin is a nontoxic inhibitor of MPO activity and MPO/neutrophils-induced cytotoxicity in endothelial cells and may be useful for targeting MPO-dependent vascular disease and inflammation.

Effects of pharmacological ascorbate on hemoglobin-induced cancer cell proliferation

The high heme content in red meat is associated with an increased risk of developing cancer. Pharmacologic concentrations of ascorbate can specifically kill a wide range of cancer cells. In this study, the impact of ascorbate at pharmacologic concentrations on hemoglobin (Hb)-modulated human hepatoma HepG2 cell survival was investigated. It was found that HepG2 cells were proliferated by Hb (5-25μM), but killed by high pharmacologic concentrations of ascorbate (2-10mM). Although ascorbate at the low pharmacologic concentration (0.5mM) alone exhibited insignificant effect on cell viability, it effectively inhibited Hb (10μM)-induced cancer cell proliferation. The mechanism of this cytotoxicity was based on the production of extracellular H2O2 and involved transition iron. The influence of ascorbate on Hb-dependent redox reactions (i.e. the oxidative stability of Hb and its cytotoxic ferryl intermediate) was further investigated to illustrate the reaction mechanism of ascorbate toxicity, where H2O2 was generated in the reaction of ascorbate with Hb. Furthermore, circular dichroism demonstrated no significant change in the secondary structure of Hb after ascorbate addition and molecular docking revealed binding modes of ascorbate with Hb. These results demonstrated that ascorbate could possess anti-cancer activity through interfering in Hb-dependent redox reactions.