Electron Paramagnetic Resonance Spin Trapping Research Articles

Evaluation of oxidative stability of vegetable oils enriched with herb extracts by EPR spectroscopy

AbstractVegetable oils are important constituents of a healthy diet. Still, unsaturated fatty acids present in vegetable oils are susceptible to oxidation, which leads to undesirable changes in sensory, chemical and nutritional properties of oils. To prevent this problem, antioxidants are applied with herbs and spices being one of the most important sources of natural antioxidants. Electron paramagnetic resonance spectroscopy (EPR) can be used to detect free radicals, which are the short-lived intermediates of lipid oxidation, and to monitor changes in oxidation susceptibility. In this study, the ESR spin trapping technique was used as a potential method for the evaluation of the resistance to free radical formation in rapeseed and sunflower oils enriched with herb extracts. The antioxidant effect of herb extracts on vegetable oils was also investigated by measuring their ability to scavenge DPPH free radical using EPR spectroscopy. The herb extracts generally improved the radical scavenging properties of sunflower and rapeseed oils but their influence on the onset of rapid lipid oxidation as measured by spin-trapping EPR depended on the type of oil and on the extract concentration.

Evaluation of scavenging rate constants of DOPA and tyrosine enantiomers against multiple reactive oxygen species and methyl radical as measured with ESR trapping method.

The scavenging rates of DOPA (dl- and l-3-(3,4-dihydroxyphenyl)alanine) and Tyr (tyrosine (dl- and l-3-(4-hydroxyphenyl)alanine)) against five reactive oxygen species (ROS) and methyl radical were measured with the use of electron spin resonance (ESR) spin-trapping method and the scavenging rate constants of DOPA and Tyr were determined. The scavenging rate constants for multiple active species increased in the order of O2(-)<RO<(1)O2<H3C<HO for Tyr and RO≈O2(-)<(1)O2≈H3C<HO for DOPA, and the differences in the radical scavenging abilities for l-enantiomers and dl-mixtures of DOPA and Tyr were shown. Further, based on the redox potentials, we have suggested that the primary chemical process of antioxidant reactions with O2(-) and (1)O2 can be characterized with the electron transfer of antioxidants (DOPA and Tyr).

(Metallo)porphyrins as potent phototoxic anti-cancer agents after irradiation with red light.

Novel photoactive (metallo)porphyrins were synthesised and characterised. When irradiated with light at a wavelength greater than 600 nm, these porphyrins act as photosensitisers and show high cytotoxicity towards two different human cancer cell lines with IC50 values down to 0.4 μM. A paramagnetic copper(II) porphyrin is the first photosensitiser to display excellent phototoxicity, explained by the electron paramagnetic resonance (EPR) spin trapping of hydroxy radicals and experimentally confirmed by the discovery of elevated levels of reactive oxygen species (ROS) inside A2780 cells after irradiation with red light. This finding indicates that paramagnetic compounds should be considered for photodynamic therapy (PDT). Furthermore, an additive effect of cisplatin and a zinc porphyrin, both at subtherapeutic concentrations of 0.22 μM, was observed.

FD&C Yellow No. 5 (tartrazine) degradation via reactive oxygen species triggered by TiO2 and Au/TiO2 nanoparticles exposed to simulated sunlight.

When exposed to light, TiO2 nanoparticles (NPs) become photoactivated and create electron/hole pairs as well as reactive oxygen species (ROS). We examined the ROS production and degradation of a widely used azo dye, FD&C Yellow No. 5 (tartrazine), triggered by photoactivated TiO2 NPs. Degradation was found to follow pseudo-first order reaction kinetics where the rate constant increased with TiO2 NP concentration. Depositing Au on the surface of TiO2 largely enhanced electron transfer and ROS generation, which consequently accelerated dye degradation. Alkaline conditions promoted ROS generation and dye degradation. Results from electron spin resonance spin-trap spectroscopy suggested that at pH 7.4, both hydroxyl radical (•OH) and singlet oxygen ((1)O2) were responsible for dye discoloration, whereas at pH 5, the consumption of (1)O2 became dominant. Implications for dye degradation in foods and other consumer products that contain both TiO2 and FD&C Yellow No. 5 as ingredients are discussed.

Kinetic Evaluation of Spin Trapping Rate Constants of New CYPMPO-type Spin Traps for Superoxide and Other Free Radicals

Abstract CYPMPO (5-(2,2-dimethyl-1,3-propoxyl cyclophosphoryl)-5-methyl-1-pyrroline N-oxide) is a phosphorus containing spin trap that has been useful in aqueous media. Using a kinetic ESR spin trapping technique, we evaluated free radical trapping rate constants of two newly synthesized CYPMPO-type spin traps, including 2,2-dibutyl-substituted CYPMPO (DBCYP) and 5-phenyl-substituted CYPMPO (PhCYP). We investigated the spin trapping of five free radical species, i. e., superoxide, hydroxyl radical, alkoxyl radical, alkyl peroxyl radical and methyl radical. Other than superoxide, spin trapping rate constants of DBCYP and PhCYP are comparable to CYPMPO, ranging from 43.9% to 101% of CYPMPO's corresponding rate constants. However, PhCYP's spin-trapping rate constant against superoxide was 44% higher than that of CYPMPO. The results indicate that DBCYP and PhCYP may be the spin traps as useful as CYPMPO either in aqueous or non-aqueous medium.

Curious Case of Bactericidal Action of ZnO

ZnO nanoparticles (NPs) are well known for their bactericidal properties. Various mechanisms are proposed for their bactericidal activity. An ambiguity still prevails to know which mechanism or property is mainly influencing the bactericidal activity of ZnO NPs. The antibacterial properties of ZnO NPs were investigated against both Gram-positive and Gram-negative bacteria. Different ZnO samples with different degrees of surface oxygen vacancies were prepared from ZnO2. The surface oxygen vacancy and thereby reactive oxygen species (ROS) production in aqueous ZnO solution are quantified by photoluminescence (PL) and electron paramagnetic resonance (EPR) spin trapping experiments, respectively. Systematic experiments have been performed to validate a precise antibacterial mechanism of ZnO particle.

Alleviation effect of arbutin on oxidative stress generated through tyrosinase reaction with L-tyrosine and L-DOPA.

BackgroundHydroxyl radical that has the highest reactivity among reactive oxygen species (ROS) is generated through l-tyrosine-tyrosinase reaction. Thus, the melanogenesis might induce oxidative stress in the skin. Arbutin (p-hydroxyphenyl-β-d-glucopyranoside), a well-known tyrosinase inhibitor has been widely used for the purpose of skin whitening. The aim of the present study was to examine if arbutin could suppress the hydroxyl radical generation via tyrosinase reaction with its substrates, l-tyrosine and l-DOPA.ResultsThe hydroxyl radical, which was determined by an electron spin resonance-spin trapping technique, was generated by the addition of not only l-tyrosine but l-DOPA to tyrosinase in a concentration dependent manner. Arbutin could inhibit the hydroxyl radical generation in the both reactions.ConclusionIt is presumed that arbutin could alleviate oxidative stress derived from the melanogenic pathway in the skin in addition to its function as a whitening agent in cosmetics.

Synthesis of a Fluorescence Labeled Spin Trap and Its ESR Study

Free radicals play a key role in physiological and pathological processes including aging and cancer development. To understand the biological role of free radicals,it is of critical importance to develop highly sensitive techniques for the detection and quantitation of free radicals being produced. Among the numbers of techniques used for the detection of free radicals,electron spin resonance( ESR)-spin trapping technique,owing to its relative higher specificity,is believed to be an indispensable method for the detection of free radicals in biological systems. However,the ESR-spin trapping technique was limited becauseit has the relative low sensitivity and it's hard to achieve real-time detection of free radicals in living cells. Considering that the fluorescence probes for labeling and tracking subcellular structures in living cells are easily available,a new spin trap conjugated with fluorescence moiety( DanPPN) was synthesized and characterized. The ESR study showed that the DanPPN could be used for the detection of superoxide anion,oxygen and carbon-centered radicals. Moreover,it was observed that the fluorescence intensity of DanPPN was decreased with the increase of the concentration of AIBN. The results suggested that the new spin trap may have a potential use for the detection of free radicals in biological systems.

Measurement and characterization of superoxide generation from xanthine dehydrogenase: a redox-regulated pathway of radical generation in ischemic tissues.

The enzyme xanthine oxidoreductase (XOR) is an important source of oxygen free radicals and related postischemic injury. Xanthine dehydrogenase (XDH), the major form of XOR in tissues, can be converted to xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. The conversion of XDH to XO has been assumed to be required for radical generation and tissue injury. It is also possible that XDH could generate significant quantities of superoxide, •O2–, for cellular signaling or injury; however, this possibility and its potential ramifications have not been previously considered. To unambiguously determine if XDH can be a significant source of •O2–, experiments were performed to measure and characterize •O2– generation using XDH from chicken liver that is locked in the dehydrogenase conformation. Electron paramagnetic resonance spin trapping experiments with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide demonstrated that XDH in the presence of xanthine produces significant amounts of •O2–. NAD+ and NADH inhibited the generation of •O2– from XDH in a dose-dependent manner, with NAD+ exhibiting stronger inhibition than NADH at low physiological concentrations. Decreased amounts of NAD+ and NADH, which occur during and following tissue ischemia, enhanced the generation of •O2– from XDH in the presence of xanthine. It was observed that XDH-mediated oxygen radical generation markedly depressed Ca2+-ATPase activity of isolated sarcoplasmic reticulum vesicles from cardiac muscle, and this was modulated by NAD+ and NADH. Thus, XDH can be an important redox-regulated source of •O2– generation in ischemic tissue, and conversion to XO is not required to activate radical formation and subsequent tissue injury.

Synergistic interaction between wavelength of light and concentration of H2O2 in bactericidal activity of photolysis of H2O2

The present study aimed to evaluate the interaction between wavelength of light in the range of ultra violet A-visible and concentration of H2O2 in the reaction of photolysis of H2O2 from the point of view of hydroxyl radical (·OH) generation and the bactericidal activity. Light emitting diodes (LEDs) emitting the light at wavelengths of 365, 385, 400 and 465 nm were used at an irradiance of 1000 mW/cm(2). H2O2 was used at the final concentrations of 0, 250, 500, and 1000 mM. Quantitative analysis of ·OH generated by the LED irradiation of H2O2 were performed using an electron spin resonance-spin trapping technique. In a bactericidal assay, a bacterial suspension of Staphylococcus aureus prepared in sterile physiological saline was irradiated with the LEDs. The bactericidal activity of each test condition was evaluated by viable counts. When H2O2 was irradiated with the LEDs, ·OH was generated and bacteria were killed dependently on the concentration of H2O2 and the wavelength of LED. The two-way analysis of variance revealed that the wavelength, the H2O2 concentration and their interaction significantly affected the yield of ·OH and the bactericidal activity of the photolysis of H2O2. Therefore, it is suggested that bactericidal activity of photolysis of H2O2 could be enhanced by controlling the wavelength and the concentration of H2O2, which may contributes to shortening the treatment time and/or to reducing the concentration of H2O2.

2D Porous graphitic C3N4 nanosheets/Ag3PO4 nanocomposites for enhanced visible-light photocatalytic degradation of 4-chlorophenol

A novel visible-light-activated photocatalyst consisting of porous graphitic C3N4 nanosheets and Ag3PO4 nanoparticles were synthesized through a tunable in situ deposition method. The morphology and microstructure of the C3N4/Ag3PO4 nanocomposites were carefully characterized by scanning electron microscope, TEM, X-ray diffraction (XRD), energy-dispersive X-ray analysis (EDX), X-ray photoelectron spectra, and so on. The porous C3N4/Ag3PO4 nanocomposites were featured by the typical platelet-like morphology, with an average crystallite size of about 39 nm. The coexistence of porous graphitic C3N4 nanosheets and Ag3PO4 nanoparticles is confirmed via the XRD and EDX results. Owing to its narrower band gap of Ag3PO4, the nanocomposites displayed a significant redshift of UV–Vis spectral absorption edge together with expanded light absorption in comparison with g-C3N4 nanosheets. The photocatalytic activity of the prepared C3N4/Ag3PO4 nanocomposites is demonstrated by the photodegradation of 4-chlorophenol (4-CP). The C3N4/Ag3PO4 nanocomposites displayed higher photocatalytic activity than pure C3N4 or Ag3PO4, which could be attributed to the interfacial effect of the nanocomposites in inhibiting the unfavorable recombination of photogenerated electrons and holes. Electron spin resonance spin-trap study implied that the generation of hydroxyl radicals plays the key role in the photodegradation of 4-CP by the C3N4/Ag3PO4 nanocomposites under visible light irradiation.

Radical Scavenging of White Tea and Its Flavonoid Constituents by Electron Paramagnetic Resonance (EPR) Spectroscopy

White tea (WT) presents high levels of catechins, which are known to reduce oxidative stress. WT is the least processed tea, unfermented and prepared only from very young tea leaves. The subject of this paper is the use of the spin trap method and electron paramagnetic resonance (EPR) spectroscopy as the analytical tool to measure, for the first time, the radical scavenging activity of WT and its major catechin components, epicatechin (EC), epicatechin-3-gallate (ECG), epigallocatechin (EGC), and epigallocatechin-3-gallate (EGCG), against the methoxy radical, using ferulic acid as antioxidant pattern. The antioxidant activity has been measured by the decrease of the intensity of the spectral bands of the adduct DMPO-OCH3 in the EPR with the amount of antioxidant in the reactive mixture. Tea leaves and buds were extracted with waterless methanol. It has been proved that tea compounds with more antiradical activity against methoxy radical are those with the gallate group, EGCG and ECG.

Photomanipulation of vasodilation with a blue-light-controllable nitric oxide releaser.

Spatiotemporally controllable nitric oxide (NO)-releasers allow us to analyze the physiological effects of NO, a gaseous mediator that modulates many biological signaling networks, and are also candidate chemotherapeutic agents. We designed and synthesized a blue-light-controllable NO releaser, named NOBL-1, which bears an N-nitrosoaminophenol moiety for NO release tethered to a BODIPY dye moiety for harvesting blue light. Photoinduced electron transfer from N-nitrosoaniline to the antenna moiety upon irradiation with relatively noncytotoxic blue light (470-500 nm) should result in NO release with formation of a stable quinone moiety. NO release from NOBL-1 was confirmed by ESR spin trapping and fluorescence detection. Spatially controlled NO release in cells was observed with DAR-4M AM, a fluorogenic NO probe. We also demonstrated temporally controlled vasodilation of rat aorta ex vivo by blue-light-induced NO release from NOBL-1. This compound should be useful for precise examination of the functions of NO with excellent spatiotemporal control.

Radical Model of Arsenic(III) Toxicity: Theoretical and EPR Spin Trapping Studies

Arsenic is one of the most environmentally significant pollutants and a great global health concern. Although a growing body of evidence suggests that reactive oxygen species (ROS) mediate the mechanism of arsenic toxicity, the exact mechanism remains elusive. In this study, we examine the capacity of trivalent arsenic species arsenous acid (iAs(III)), monomethylarsonous acid (MMA(III)), and dimethylarsinous acid (DMA(III)) to generate ROS through a theoretical analysis of their structures, redox properties, and their reactivities to various ROS using a density functional theory (DFT) approach at the B3LYP/6-31+G**//B3LYP/6-31G* level of theory and by employing electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap. Results show that the oxidized forms (As(IV)) are structurally more stable compared to the reduced forms (As(II)) that impart elongated As-O bonds leading to the formation of As(III) and hydroxide anion. Enthalpies of one-electron reduction and oxidation indicate that increasing the degree of methylation makes it harder for As(III) to be reduced but easier to be oxidized. The order of increasing favorability for arsenical activation by ROS is O2 < O2(•-) < HO(•), and the oxidation of DMA(III) to DMA(V) is highly exoergic in multiple redox pathways with concomitant generation of radicals. This is followed by MMA(III) and by iAs(III) being the least favorable. Spin trapping studies showed a higher propensity for methylated arsenicals to generate radicals than iAs(III) upon treatment with H2O2. However, in the presence of Fe(II,III), all showed radical generation where MMA(III) gave predominantly C-centered adducts, while acidified iAs (III) and DMA(III) gave primarily HO-adducts, and their formation was affected in the presence of SOD suggesting a As(III)-OO/OOH radical intermediate. Therefore, our results suggest a basis for the increased redox activity of methylated arsenicals that can be applied to the observed trends in arsenic methylation and toxicity in biological systems.

P106 Photo-manipulation of vasodilation with a novel photo-controllable nitric oxide releaser based on N-nitrosoaminophenol

Nitric oxide (NO) is synthesized in the human body and plays main roles in some physiological event, such as vasodilation, neurotransmission, and immune response. Due to its instability in physiological conditions, NO releasers, which can store and release NO, had been utilized for the research, so far. Among them, photo-controllable NO releasers are expected as precisely controllable chemical tools or agents for photodynamic therapy. Although many photo-controllable NO releasers had been reported, there are few report of visible-light controllable NO releaser which could photo-control physiological events ex vivo. We designed and synthesized a novel visible light controllable NO releaser, named NOBL-1, composed of N-nitrosoaminophenol as an NO releasing moiety and BODIPY as a visible light harvesting moiety. NO release from NOBL-1 induced by blue light was confirmed by means of ESR spin trapping method with iron-dithiocarbamate complex. Furthermore, it was confirmed that the NO release was under control in temporal and spatial precision in vitro and in cell by using a fluorogenic NO probe, DAR-4M. Finally, we achieved vasodilation control of rat aorta slice with NOBL-1 and the triggering light in highly temporal manner. NOBL-1 is expected to be a useful chemical tool for NO research, and it may also have potential for phototherapy.

The cytotoxicity of the anticancer drug elesclomol is due to oxidative stress indirectly mediated through its complex with Cu(II)

Elesclomol is an anticancer drug that is currently undergoing clinical trials. Elesclomol forms a strong 1:1 complex with Cu(II) and may exert its anticancer activity through the induction of oxidative stress and/or its ability to transport copper into the cell. A UV–vis spectrophotometric titration showed that Cu(I) also formed a 1:1 complex with elesclomol. Ascorbic acid, but not glutathione or NADH, potently reduced the Cu(II)-elesclomol complex to produce hydrogen peroxide. Even though hydrogen peroxide mediated reoxidation of the copper(I) produced by ascorbic acid reduction has the potential to lead to hydroxyl radical formation, electron paramagnetic resonance spin trapping experiments, either with or without added hydrogen peroxide, showed that the ascorbic acid-reduced Cu(II)-elesclomol complex could not directly generate damaging hydroxyl radicals. Both Cu(II)-elesclomol and elesclomol potently oxidized dichlorofluorescin in K562 cells. The highly specific copper chelators tetrathiomolybdate and triethylenetetramine were found to greatly reduce the cytotoxicity of both elesclomol and Cu(II)-elesclomol complex towards erythroleukemic K562 cells, consistent with a role for copper in the cytotoxicity of elesclomol. The superoxide dismutating activity of Cu(II)-elesclomol was much lower than that of Cu(II). Depletion of glutathione levels in K562 cells by treatment with buthionine sulfoximine sensitized cells to both elesclomol and Cu(II)-elesclomol. In conclusion, these results showed that elesclomol indirectly inhibited cancer cell growth through Cu(II)-mediated oxidative stress.

Metal and metal-free photocatalysts: mechanistic approach and application as photoinitiators of photopolymerization.

In the present paper, the photoredox catalysis is presented as a unique approach in the field of photoinitiators of polymerization. The principal photocatalysts already reported as well as the typical oxidation and reduction agents used in both reductive or oxidative cycles are gathered. The chemical mechanisms associated with various systems are also given. As compared to classical iridium-based photocatalysts which are mainly active upon blue light irradiation, a new photocatalyst Ir(piq)2(tmd) (also known as bis(1-phenylisoquinolinato-N,C2’)iridium(2,2,6,6-tetramethyl-3,5-heptanedionate) is also proposed as an example of green light photocatalyst (toward the long wavelength irradiation). The chemical mechanisms associated with Ir(piq)2(tmd) are investigated by ESR spin-trapping, laser flash photolysis, steady state photolysis, cyclic voltammetry and luminescence experiments.

Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps

Reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide (H2O2), have a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of their production. For measuring ROS production in cells, the ESR spin trapping technique using cyclic nitrones distinguishes itself from other methods by its specificity for superoxide and hydroxyl radical. However, several drawbacks, such as the low spin trapping rate and the spontaneous and cell-enhanced decomposition of the spin adducts to ESR-silent products, limit the application of this method to biological systems. Recently, new cyclic nitrones bearing a triphenylphosphonium (Mito-DIPPMPO) or a permethylated β-cyclodextrin moiety (CD-DIPPMPO) have been synthesized and their spin adducts demonstrated increased stability in buffer. In this study, a comparison of the spin trapping efficiency of these new compounds with commonly used cyclic nitrone spin traps, i.e., 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and analogs BMPO, DEPMPO, and DIPPMPO, was performed on RAW 264.7 macrophages stimulated with phorbol 12-myristate 13-acetate. Our results show that Mito-DIPPMPO and CD-DIPPMPO enable a higher detection of superoxide adduct, with a low (if any) amount of hydroxyl adduct. CD-DIPPMPO, especially, appears to be a superior spin trap for extracellular superoxide detection in living macrophages, allowing measurement of superoxide production in unstimulated cells for the first time. The main rationale put forward for this extreme sensitivity is that the extracellular localization of the spin trap prevents the reduction of the spin adducts by ascorbic acid and glutathione within cells.

ESR study of spin trapping in Fenton media in the presence of taxifolin

The effect of taxifolin on the production of hydroxyl and C-centered radicals via the Fenton reaction in the presence of methanol and ethanol was investigated using spin trapping ESR. The results showed that with the increase of taxifolin concentration the production of hydroxyl radicals increased and the generation of C-centered radicals decreased. The FDMPO/taxifolin radical adduct (AN=13.33G, AF=2.8G) was observed in Fenton reaction with methanol in the presence of 1–10mM of taxifolin. Experimental results were supplemented with DFT calculations. All geometry optimizations for taxifolin, its radical and the corresponding spin adducts were performed at the DFT-B3LYP/6-31G(d,p) level using the PCM model to consider solvent effect. The optimized geometry of taxifolin in the B3LYP/6-31G(d,p) approach is consistent with its crystal structure. Based on the calculated Gibbs energy values the order of increasing favorability of the radical reaction with spin traps is: OH>CH2OH>CH(CH3)OH>taxifolin radical. The calculated values of the HOMO–LUMO gap for FDMPO/taxifolin and 4-POBN/taxifolin radical adducts are 4.43eV and 3.67eV, respectively, indicating high reactivity of those adducts. ESR hyperfine splittings were also calculated for FDMPO/taxifolin spin adduct at the B3LYP/6-31++G//EPR-II level of theory (6-31++G: AN=13.37G, AF=2.9G, and EPR-II: AN=9.3G, AF=3.0G).

Inhibition of the phosphoinositide 3-kinase pathway decreases innate resistance to lipopolysaccharide toxicity in TLR4 deficient mice

BackgroundUpon lipopolysaccharide (LPS) stimulation, activation of both the Toll-like receptor 4 (TLR4) and phosphoinositide 3-kinase (PI3K) pathways serves to balance proinflammatory and anti-inflammatory responses. Although the antagonist to TLR4 represents an emerging promising target for the treatment of sepsis; however, the role of the PI3K pathway under TLR4-null conditions is not well understood. This goal of this study was to investigate the effect of inhibition of PI3K on innate resistance to LPS toxicity in a murine model.ResultsThe overall survival of the cohorts receiving intraperitoneal injections of 100, 500, or 1000 μg LPS from Escherichia coli serotype 026:B6 after 7 d was 100%, 10%, and 10%, respectively. In contrast, no mortality was noted after 500-μg LPS injection in Tlr4-/- mice. When the PI3K inhibitor LY294002 was injected (1 mg/25 g body weight) 1 h prior to the administration of LPS, the overall survival of the Tlr4-/- mice was 30%. In the Tlr4-/- mice, the LPS injection induced no NF-κB activation but an increased Akt phosphorylation in the lung and liver, when compared to that of the C57BL/6 mice. Injection of 500 μg LPS led to a significant induction in O2- detected by electron paramagnetic resonance (EPR) spin trapping spectroscopy in the lung and liver at 3 and 6 h in C57BL/6 but not Tlr4-/- mice. Addition of LY294002 only significantly increased the O2- level in the lung and liver of the Tlr4-/- mice but not in the C57BL/6 mice following 500-μg LPS injection. In addition, the serum IL-1β and IL-2 levels were more elevated in C57BL/6 mice than in Tlr4-/- mice. Notably, IL-1β and IL-2 were significantly increased in Tlr4-/- mice but not in the C57BL/6 mice when the PI3K pathway was inhibited by LY294002 prior to LPS injection.ConclusionsIn this study, we demonstrate that innate resistance to LPS toxicity in Tlr4-/- mice is impaired by inhibition of the PI3K pathway, with a corresponding increase in mortality and production of tissue O2- and inflammatory cytokines.