Protective effects of transient HO-1 overexpression on susceptibility to oxygen toxicity in lung cells.
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Rat fetal lung cells (RFL-6) were transiently transfected with a full-length rat heme oxygenase (HO)-1 cDNA construct and then exposed to hyperoxia (95% O2-5% CO2) for 48 h. Total HO activity and HO-1 protein were measured as well as cell viability, lactate dehydrogenase (LDH) release, protein oxidation, lipid peroxidation, and total glutathione to measure oxidative injury. HO-1 overexpression resulted in increased total HO activity (2-fold), increased HO-1 protein (1.5-fold), and increased cell proliferation. Immunohistochemistry revealed perinuclear HO-1 localization, followed by migration to the nucleus by day 3. Decreased cell death, protein oxidation, and lipid peroxidation but increased LDH release and glutathione depletion were seen with HO-1 overexpression. Reactive iron content could not explain the apparent loss of cell membrane integrity. With the addition of tin mesoporphyrin, total HO activity was decreased and all changes in injury parameters were normalized to control values. We conclude that moderate overexpression of HO-1 is protective against oxidative injury, but we speculate that there is a beneficial threshold of HO-1 expression. (+info)
Peroxynitrite inhibits T lymphocyte activation and proliferation by promoting impairment of tyrosine phosphorylation and peroxynitrite-driven apoptotic death.
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Peroxynitrite (ONOO-) is a potent oxidizing and nitrating agent produced by the reaction of nitric oxide with superoxide. It readily nitrates phenolic compounds such as tyrosine residues in proteins, and it has been demonstrated that nitration of tyrosine residues in proteins inhibits their phosphorylation. During immune responses, tyrosine phosphorylation of key substrates by protein tyrosine kinases is the earliest of the intracellular signaling pathways following activation through the TCR complex. This work was aimed to evaluate the effects of ONOO- on lymphocyte tyrosine phosphorylation, proliferation, and survival. Additionally, we studied the generation of nitrating species in vivo and in vitro during immune activation. Our results demonstrate that ONOO-, through nitration of tyrosine residues, is able to inhibit activation-induced protein tyrosine phosphorylation in purified lymphocytes and prime them to undergo apoptotic cell death after PHA- or CD3-mediated activation but not upon phorbol ester-mediated stimulation. We also provide evidence indicating that peroxynitrite is produced during in vitro immune activation, mainly by cells of the monocyte/macrophage lineage. Furthermore, immunohistochemical studies demonstrate the in vivo generation of nitrating species in human lymph nodes undergoing mild to strong immune activation. Our results point to a physiological role for ONOO- as a down-modulator of immune responses and also as key mediator in cellular and tissue injury associated with chronic activation of the immune system. (+info)
Structural analysis of DNA-chlorophyll complexes by Fourier transform infrared difference spectroscopy.
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Porphyrins and metalloporphyrins are strong DNA binders. Some of these compounds have been used for radiation sensitization therapy of cancer and are targeted to interact with cellular DNA. This study was designed to examine the interaction of calf thymus DNA with chlorophyll a (CHL) in aqueous solution at physiological pH with CHL/DNA(phosphate) ratios (r) of 1/160, 1/80, 1/40, 1/20, 1/10, and 1/5. Fourier transform infrared (FTIR) difference spectroscopy was used to characterize the nature of DNA-pigment interactions and to establish correlations between spectral changes and the CHL binding mode, binding constant, sequence selectivity, DNA secondary structure, and structural variations of DNA-CHL complexes in aqueous solution. Spectroscopic results showed that CHL is an external DNA binder with no affinity for DNA intercalation. At low pigment concentration (r = 1/160, 1/80, and 1/40), there are two major binding sites for CHL on DNA duplex: 1) Mg-PO2 and 2) Mg-N7 (guanine) with an overall binding constant of K = 1.13 x 10(4) M-1. The pigment distributions are 60% with the backbone PO2 group and 20% with the G-C base pairs. The chlorophyll interaction is associated with a major reduction of B-DNA structure in favor of A-DNA. At high chlorophyll content (r = 1/10), helix opening occurs, with major spectral alterations of the G-C and A-T bases. At high chlorophyll concentration (1/5), pigment aggregation is observed, which does not favor CHL-DNA complexation. (+info)
Effects of vitamin C and of a cell permeable superoxide dismutase mimetic on acute lipoprotein induced endothelial dysfunction in rabbit aortic rings.
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Low density lipoprotein (LDL) inhibits endothelium-dependent relaxation. The mechanism is uncertain, but increased production of superoxide anion O2- with inactivation of endothelium-derived NO and formation of toxic free radical species have been implicated. We investigated effects of the cell permeable superoxide dismutase mimetic manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP), the free radical scavenger vitamin C and arginine (which may reduce O2- formation) on acute LDL-induced endothelial dysfunction in rabbit aortic rings, using LDL prepared by ultracentrifugation of plasma from healthy men and aortic rings from New Zealand white rabbits. LDL (150 microg protein ml(-1) for 20 min) markedly inhibited relaxation of aortic rings (in Krebs' solution at 37 degrees C and pre-constricted to 80% maximum tension with noradrenaline) to acetylcholine 82+/-10% (mean percentage difference between sum of relaxations after each concentration of acetylcholine in the presence and absence of LDL, +/-s.e.mean, n=26, P<0.001) but not to the endothelium-independent agonist nitroprusside. MnTMPyP (10 microM) reduced inhibitory effects of LDL from 124+/-27 to 56+/-17% (n=6, P<0.05). Vitamin C (1 mM) reduced inhibitory effects of LDL from 59+/-8 to 22+/-5% (n=6, P<0.05). Inhibitory effects of LDL were similar in the absence or presence of arginine (84+/-12 vs 79+/-16%, n=14, P=0.55). Effects of L-arginine (10 mM) did not differ significantly from those of D-arginine (10 mM). Acute (20 min) exposure of aortic rings to LDL impairs endothelium-dependent relaxation which can be partially restored by MnTMPyP and vitamin C. This is consistent with LDL causing increased O2- generation. (+info)
Contribution of endogenous carbon monoxide to regulation of diameter in resistance vessels.
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Endogenous carbon monoxide was proposed to subserve vasodepressor functions. If so, inhibition of heme oxygenase may be expected to promote vascular contraction. This hypothesis was examined in large and small arteries and in isolated first-order gracilis muscle arterioles of rat. The heme oxygenase inhibitors chromium mesoporphyrin (CrMP) and cobalt protoporphyrin (0.175-102 micromol/l) decreased the diameter of pressurized (80 mmHg) gracilis muscle arterioles, whereas magnesium protoporphyrin, a weak heme oxygenase inhibitor, did not. CrMP also elicited development of isometric tension in the muscular branch of the femoral artery but not in the aorta or femoral artery. Arteriolar constrictor responses to CrMP varied in relation to the intravascular pressure, were blunted in preparations exposed to exogenous carbon monoxide (100 micromol/l), and were unaffected by an endothelin receptor antagonist. Importantly, CrMP amplified the constrictor response to increases of pressure in gracilis arterioles. Accordingly, the constrictor effect of heme oxygenase inhibitors is attributable to magnification of myogenic tone due to withdrawal of a vasodilatory mechanism mediated by endogenous carbon monoxide. The study suggests that the vascular carbon monoxide system plays a role in the regulation of basal tone in resistance vessels. (+info)
A phase I single-dose trial of gadolinium texaphyrin (Gd-Tex), a tumor selective radiation sensitizer detectable by magnetic resonance imaging.
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Gadolinium Texaphyrin (Gd-Tex) is a radiation sensitizer with a novel mechanism of action that sensitizes both oxic and hypoxic cells, localizes selectively in tumors, and is detectable by magnetic resonance imaging (MRI). This Phase I single-dose trial of Gd-Tex administered concurrently with radiation therapy was carried out to determine the maximally tolerated dose (MTD), dose-limiting toxicities, pharmacokinetics, and biolocalization of Gd-Tex as determined by MRI. Adults with incurable cancers of any histology requiring radiation therapy were eligible. A single i.v. dose of Gd-Tex was followed at least 2 h later by radiation therapy. The Gd-Tex dose was escalated in cohorts of 3 to 5 patients. Thirty-eight patients (median age, 58 years; range, 35-77 years) with incurable cancers of the lung (26), cervix (3), or other solid tumors (9) received a total of 41 single administrations of Gd-Tex. The Gd-Tex dose was escalated from 0.6 to 29.6 mg/kg. Irradiated sites included the thorax, brain, pelvis, bone, soft tissue, and sites of nodal metastases. The MTD was 22.3 mg/kg, determined by reversible acute tubular necrosis as the dose-limiting toxicities. Gd-Tex selectively accumulated in primary and metastatic tumors as demonstrated by MRI. No increase in radiation toxicity to normal tissues was seen. The median half-life of Gd-Tex after single-dose administration is 7.4 h. This study demonstrates that Gd-Tex is well tolerated in doses below the MTD, and that there is selective biolocalization in tumors. The maximum recommended dose for single administrations is 16.7 mg/kg. (+info)
Peroxynitrite induces haem oxygenase-1 in vascular endothelial cells: a link to apoptosis.
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Peroxynitrite (ONOO-) is a potent oxidizing agent generated by the interaction of nitric oxide (NO) and the superoxide anion. In physiological solution, ONOO- rapidly decomposes to a hydroxyl radical, one of the most reactive free radicals, and nitrogen dioxide, another species able to cause oxidative damage. In the present study we investigated the effect of ONOO- on the expression of haem oxygenase-1 (HO-1), an inducible protein that is highly up-regulated by oxidative stress. Exposure of bovine aortic endothelial cells to ONOO- (250-1000 microM) produced a concentration-dependent increase in haem oxygenase activity and HO-1 protein expression. This effect was completely abolished by the ONOO- scavengers uric acid and N-acetylcysteine, and partly attenuated by 1,3-dimethyl-2-thiourea, a scavenger of hydroxyl radicals. ONOO- also produced a concentration-dependent increase in apoptosis and cytotoxicity, which were considerably decreased by uric acid and N-acetylcysteine. A 70% decrease in apoptosis was observed when cells were exposed to ONOO- in the presence of 10 microM tin protoporphyrin IX (SnPPIX), an inhibitor of haem oxygenase activity. When SnPPIX was added 5 min after ONOO-, apoptosis decreased by only 40%, which suggests that an interaction between ONOO- and the protoporphyrin occurs in our system. Increased haem oxygenase activity by pretreatment of cells with haemin resulted in elevated bilirubin production and was associated with a substantial decrease (35%) in ONOO--mediated apoptosis. These results indicate the ability of ONOO- to modulate the expression of the stress protein HO-1 and suggest that the haem oxygenase pathway contributes to protection against the cytotoxic action of ONOO-. (+info)
PO2 measurements in the rat intestinal microcirculation.
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Microvascular partial oxygen pressure (PO2) data measured with the quenched phosphorescence of palladium-porphyrin (Pd-porphyrin) with the use of optical fibers have provided new insight into the behavior of the microvascular oxygenation in models of shock. However, the actual microcirculatory compartment measured by this fiber technique has not yet been demonstrated. The purpose of this study was to investigate whether the PO2 of the intestines, as measured using a fiber phosphorometer, reflects the microvascular compartment. To this end, a new intravital phosphorometer with an improved sensitivity to high-PO2 levels (up to 180 mmHg) was developed and validated. With this setup, PO2 values were measured at different inspired oxygen fractions (15, 25, and 50% oxygen) in first-order arterioles, capillaries, and venules of the ileum of rats. Simultaneously, the PO2 was measured with an optical fiber attached to another phosphorometer. PO2 measurements with the fiber phosphorometer show excellent correlation with the PO2 in the capillaries and first-order venule vessels (r2 = 0.94, slope 0.99). We therefore conclude that values measured with the fiber phosphorometer correlate with the capillary and venular PO2. (+info)