UVB-dependent generation of reactive oxygen species by catalase and IgG under UVB light: Inhibition by antioxidants and anti-inflammatory drugs.

Catalase, which can decompose H2O2, has recently been found to generate unspecified reactive oxygen species (ROS) as a result of ultraviolet B (UVB) irradiation. Many proteins, hemes, and iron compounds were first tested to determine that this ROS generation was unique to catalase and immunoglobulin G (IgG). An increase in absorbance at 502 nm due to 2',7'-dichlorofluorescein, the oxidized product of 2',7'-dichlorodihydrofluorecein diacetate as a result of UVB (310 nm) irradiation, was measured in order to estimate this ROS generation. Catalase and IgG generated a pronounced amount of ROS when irradiated with UVB. Another heme protein, cytochrome c, and heat-inactivated catalase had no such effect. ROS generation by catalase was at least 5 times more potent than that reported for IgG with UVB and without antigens. This catalasemediated ROS generation was largely temperaturedependent in the range of 25 to 42°C. As IgG is considered an evolutionally important bactericidal component, the same was considered true for this enzyme. Next, inhibitory effects of various drugs, including antioxidants and anti-inflammatory drugs, on catalase-mediated and UVB-induced ROS generation were examined. Many of the drugs, including catalase inhibitors, had inhibitory effects with different potencies. Melanin was found to be the most effective inhibitor of this ROS generation (IC(30), 0.2 µg/mL), followed by Indigo Carmine and rutin. Also inhibiting this ROS generation were ascorbic acid, α-tocopherol, indomethacin, coenzyme Q10, ß-carotene, uric acid, piroxicam, diclofenac, and glutathione, in that order of potency. Various ROS were apparently generated by catalase under UVB, creating a cycle or chain which was thought due to the biphasic effects of some drugs such as 3-aminotriazole or sodium azide. Excess ROS generation induces inflammation. Catalase might serve dual roles, removing H2O2 and generating various ROS depending on the H2O2 concentration and other factors.

Effect of nilvadipine in weak acidic medium by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay.

A new medium (pH 5.6 reached by addition of 0.1 mol/l acetate buffer, 37 degrees C, 60 min) was established for detecting anti-free radical compounds in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. DPPH bleaching activities of typical antioxidants appeared generally weaker at pH 8.0 (the measured ethanol solution pH by the classical DPPH assay method) than at pH 5.6. Nilvadipine (CAS 75530-68-6), a lipophilic calcium antagonist, enhanced the bleaching much more at pH 5.6 than at pH 8.0. Nifedipine (CAS 21829-25-4) and amiodipine (amiodipine besilate, CAS 88150-42-9) showed some effect, but the other five calcium antagonists failed to bleach DPPH at any pH tested (pH 4.4-8.0). Probucol and beta-carotene (standard antioxidant) showed nearly the same bleaching activity as nilvadipine. Captopril, glutathione and bilirubin were weaker anti-free radical compounds at pH 5.6. No intermediating participation of superoxide radicals and hydroxyl radicals were observed in the DPPH assay, both at pH 8.0 and 5.6. Thus, nilvadipine was shown to be an efficient free-radical scavenger only at around pH 5.6, a weak acidic condition which may occur as a result of inflammation and/or ischemia-reperfusion.

Immunosuppressants enhance superoxide radical/nitric oxide-dependent dexamethasone suppression of ischemic paw edema in mice.

A possible new common action of immunosuppressants, besides suppression of the genes for cytokines like interleukin-2, was investigated in in vivo models. Dexamethasone (0.1 mg/kg, s.c.) failed to suppress ischemic paw edema in mice 1 h after its injection, but maximal suppression was achieved at 3 h (20%) whereafter the suppression decreased at 6 and 18 h (11% and 10%). Pretreatment with oral FK506 (chemical name is recently donated as tacrolimus, 0.1 mg/kg) resulted in 38%, 52%, 23% and 17% suppression at 1, 3, 6 and 18 h, respectively. Cyclosporin A (1 mg/kg), rapamycin (0.1 mg/kg) and deoxyspergualin (1 mg/kg) showed a similar pattern of suppressions after dexamethasone. Transforming growth factor-beta1 (TGF-beta1, 0.3 microg/kg, i.p.) maintained the suppression elicited by an immunosuppressant (42-58%) at 6 h after dexamethasone, whereas transforming growth factor-beta1 and/or an immunosuppressant were not suppressive. Suppression, irrespective of the agent that elicited it, was blocked by nitric oxide (NO) synthase inhibitor, anti-oxidant enzymes and cycloheximide. Endogenous nitric oxide or oxyradicals are essential for the action of dexamethasone in vivo. The four immunosuppressants bound to specific heat-hock proteins (hsp) in the glucocorticoid receptor complex and might enhance the synthesis of anti-inflammatory protein(s).

Fructose-1,6-diphosphate enhanced oxyradicals and nitric oxide-dependent suppressions by dexamethasone of ischemic and histamine paw edema of mice.

Dexamethasone (Dex, 0.1 mg/kg, s.c.) suppressions of ischemic paw edema in mice at 1, 3, 6, 8, 18 hr were; 2, 22, 12, 11, 7%. Dex suppression in fructose-1,6-diphosphate (FDP, 100 mg/kg, i.p.)-treated mice were; 5, 49, 51, 42, 33%. Suppressions by this dose of FDP alone were less than 10% during 0-18 hr. ED30 at 6 hr of Dex +/- FDP was: 80 versus 500 mg/kg in ischemic-, and 5 versus 30 mg/kg in histamine edema. Endogenous oxyradicals or NO and protein synthesis were essential for suppressions. FDP may not change glucocorticoid receptor (GR) conformation, but increase ATP-dependent GR recycling efflux from nucleus. FDP is possible to supply this ATP. Clinical trial of FDP with low dose of Dex seems advantageous.

Cromoglycate and nedocromil enhanced the reactive oxygen species-dependent suppressions with, but not without, dexamethasone in ischaemic and histamine paw oedema of mice.

Anti-inflammatory actions of two anti-allergic drugs, alone or with dexamethasone (Dex) were examined in two models, because inflammation is claimed to be important for allergic events, especially for asthma. Cromoglycate and nedocromil were tested in ischaemic- and histamineinduced paw oedema models of mice. These antiallergic drugs (1-100 mg/kg, i.p.) failed to suppress these oedemata, but enhanced the suppressions by a low dose of dexamethasone (0.1 mg/kg, s.c.) at 3-8 h after Dex injection. The mode of effects by anti-allergic drugs resembled that of a natural antioxidant (alpha-tocopherol, beta-carotene etc.), and was different from that of an immunosuppressant like FK506. The enhancing potencies of the two anti-allergic drugs were similar at 6 h after Dex in both oedemata, and were diminished by superoxide dismutase (SOD) or catalase (i.p.). Cycloheximide completely abolished suppressions. Nedocromil, but not cromoglycate, inhibits inflammatory events. Therefore, there are common unknown actions by which the two anti-allergics enhance suppression by Dex. A possible mechanism of this action was supposed to enhance the superoxide and/or hydrogen peroxide-dependent glucocorticoid receptor (GR) signalling in the target cells.

Natural antioxidants enhance and prolong the oxyradical/no-related suppression by dexamethasone of ischemic and histamine paw edema in mice.

Dexamethasone (0.1 mg/kg, s.c.) suppressed 2, 22, 12, 6, and 4% of ischemic paw edema of mice at 1, 3, 6, 18, and 40 h. Various antioxidants when given with 0.1 mg/kg Dex enhanced and prolonged the suppression. For example, 30 mg/kg of tannic acid induced suppression of 6, 52, 59, 42 and 27%, whereas no increase of Dex suppression at 1 h was observed with any of the antioxidants tested. The prolonging effect of antioxidants may be due to the change of glucocorticoid (GC) receptor signaling under new redox state, because oxyradicals modified the suppression, whereas nitric oxide (NO) showed no effect on Dex suppression enhanced by antioxidants. ED30 (mg/kg, i.p.) at 6 h after Dex was: morin (0.02) > alpha-tocopherol (0.03) > tannic acid (0.12) > rutin (0.2) = bilirubin (0.2) > beta-carotene (0.3) >> quercetin (6) > melatonin (8) > ascorbic acid (12), whereas none of the antioxidants alone afforded more than 10% suppression even at 30 mg/kg. Similar Dex plus antioxidant actions were observed in histamine paw edema. Clinical trials of nontoxic natural antioxidants might be successful when coinjected with low dose of GC which shows very little side effects.

Immunosuppressants and TGF-beta 1 accelerated and prolonged the nitric oxide/oxyradicals-dependent suppression by dexamethasone in paw edema of mice.

Dexamethasone (Dex, 0.3 mg/kg, s.c.) did not suppress histamine and ischemic paw edema of mice up to 1 hr. However, given TGF-beta 1 (0.3 microgram/kg, i.p.), Dex suppression appeared early as 30 min (36% and 42%). When Dex (0.1 mg/kg, s.c.) was injected 6 hr before the assay, Dex alone, TGF-beta 1 +/- Dex, FK506 (10 mg/kg, oral) +/- Dex, cyclosporin (CsA, 30 mg/kg, oral) +/- Dex, rapamycin (Rapa, 10 mg/kg, i.p.) +/- Dex, deoxyspergualin (DSP, 10 mg/kg, i.p.) +/- Dex, did not suppress the edemata (less than 11%). Nevertheless, if Dex and TGF-beta 1 were dosed together with one of these immunosuppressants, suppressions of histamine and ischemic edema were 53%, 45% (FK506), 45%, 49% (CsA), 44%, 48% (Rapa) and 39%, 51% (DSP), respectively. Glucocorticoid (GC) receptor (GR) complex contains heat shock proteins such as hsp56 (or CsA-binding protein: CyP-40), hsp70 and hsp90. FK506, Rapa and TGF-beta 1 receptor I (TR-I) bind FK-binding protein-12 (FKBP-12). FK506 and Rapa bind also hsp56. CsA binds CyP-40. DSP binds hsp70 and/or hsp90. These bindings might change or stabilize the conformation of GR complex resulting in edema suppressions. Nitric oxide synthase (NOS) inhibitors, superoxide dismutase (SOD), catalase, mannitol and cycloheximide, reversed the edema suppressions by TGF-beta1 +/- immunosuppressant at 30 min and 6 hr after Dex. Endogenous NO, O2- and/or .OH seemed to be essential for edema suppressions. Our demonstration in vivo may offer a theoretical support for clinicians to adopt combination therapy of immunosuppressant(s) and GC.

Nitric oxide- and hydrogen peroxide-mediated gene expression by glucocorticoids and FK506 in histamine paw edema of mice.

An immunosuppressant FK506 binds with a component (hsp 56) of glucocorticoid receptor (GR) complex. Dexamethasone (Dex) never suppressed histamine paw edema of mice before 1 hr after its dosing as new protein(s) synthesis is required. However, FK506 (0.01-10 mg/kg, oral) 1.5 hr before 0.1 mg/kg Dex (s.c.), suppressed edema at 30 min. This suppression and that at 3 hr, were abolished by nitric oxide (NO) synthesis inhibitors (1-300 mg/kg). Nitroprusside (NO donor), catalase and molybdate (GR complex stabilizing protease inhibitor) enhanced the suppression. FK506, not cyclosporin A, was demonstrated for the first time in vivo to enhance GR and a hypothesis is proposed that FK506 might enhance GR and AP-1 signalings in a system reciprocally controlled by NO and H2O2.

Nitric oxide and superoxide radical are involved in both initiation and development of adjuvant arthritis in rats.

Nitric oxide synthase(NOS) inhibitor,N omega-nitro-L-arginine methyl ester (L-NAME, 10-300 mg/kg) and L-NG-monomethyl-arginine (L-NMMA, 30-300 mg/kg) suppressed the swellings of adjuvant-injected paw of rats (25-54%) at day 2 and 8 when dosed intraperitoneally and orally for 4 days from day -1 to day 2 after adjuvant. L-NAME (30-300 mg/kg) also suppressed the edema of the non adjuvant-injected paws (15-42%) at day 28. Local injection of this inhibitor (2 and 10 mg/kg) was without effect. L-arginine (1 g/kg, i.p.), impaired the suppression by L-NAME. Bovine blood Cu, Zn-superoxide dismutase (SOD, 3 mg/kg, i.p.: 28% suppression) and L-NAME (30 mg/kg i.p.: 36% suppression) showed additive effect (52%) in adjuvant-injected paws at day 8 when co-injected. As the effect of 30 mg/kg L-NAME corresponded nearly to that of 10 mg/kg VoltarenR, this NOS inhibitor would be worth considering as an anti-inflammatory agent. Sodium nitroprusside (NO-donor) and methylene blue (guanylate cyclase inhibitor) had no effect. L-NAME was also suppressive when dosed after adjuvant inoculation and NO is involved in the development and maintenance of swelling.

Histamine paw edema of mice was increased and became H2-antagonist sensitive by co-injection of nitric oxide forming agents, but serotonin paw edema was decreased.

Nitric oxide (NO) surprisingly caused the opposite effect on histamine and serotonin edema. The local injection of acidified nitrite (0.3-30 micrograms/paw which correspond 10 micrograms-1 mg/kg) increased histamine edema of mice up to 45 +/- 4% and suppressed serotonin edema to 90 +/- 3%. Other NO-generators (nitroprusside sodium and hydroxylamine) showed similar effects. These results were in accordance with our previous data on endogenous NO. Methylene blue (MB, 30 ng/paw which corresponds to 1 microgram/kg) suppressed histamine edema (62 +/- 3%) and increased serotonin edema (43 +/- 3%) in normal mice, being reversed by acidified nitrite. This suggests the involvement of guanosine 3',5'-cyclic monophosphate (cGMP) formation for the action of NO. Histamine edema became sensitive to H2-antagonist, cimetidine, by co-injection of 30 micrograms/paw (which corresponds to 1 mg/kg) acidified nitrite (ED50 = 30 micrograms/kg versus >> 1 mg/kg). NO seemed to modify the histamine receptor(s) or tautomeric form of histamine. NO, O2- and other oxyradicals might finely control the vascular permeability together with inflammatory mediators.

Superoxide dismutases and anti-oxidants protected mice from no-reflow and necrotic damage induced by ischemia.

A simple method in mice was established to screen anti-ischemic compounds. Thirteen times binding of rubber ring (1 x 1 mm, d = 42 mm) for 4.5 hrs, swelled the paws of 60% mice applied and 14 times binding swelled only of 5% mice. Critically reversible limit lay between these conditions. "All or none" rule dominated the paw swelling perhaps due to different endogenous anti-oxidants' levels of individual mice. Determination of paw reversibility at 90 min of recirculation, was proved to be suitable. Swollen paws at this time returned normal and the paws with no-reflow dropped out by muscle necrosis after several days. Intravenous (i.v.) bovine Cu, Zn-SOD and bacterial Mn-SOD (3-10 x 10(4) U/kg) or liposomal Cu, Zn-SOD (0.3-3 x 10(4) U/kg) were protective (35-50%) by 14 times binding. Allopurinol (10-100 mg/kg) and D-mannitol (3-30 mg/kg) was effective (25-55%). Catalase (i.v., up to 10(5) U/kg) showed little protection, but local injection of 100 U/kg resulted in 50% protection. Glutathione (30 mg/kg) was suppressive only by local injection suggesting the importance of administration route. Desferal, heparin and nitric oxide synthesis inhibitor showed some protection, but indomethacin, mepyramine, ascorbate, vitamin E and dexamethasone were without effect. Excess dosing of all anti-oxidants tested, dramatically decreased their effects demanding caution for therapeutic trials.

Opposite effect of superoxide dismutase, L-arginine analogues, methylene blue and desferal: suppression of histamine-induced and stimulation of serotonin-induced paw edema in mice.

Endogenous nitric oxide (NO, endothelium-derived relaxing factor) was stimulatory for histamine- and suppressive for serotonin-induced paw edema of mice. This action was mediated by guanosine 3',5'-cyclic monophosphate production. Local injection of superoxide dismutase (SOD), catalase, NG-monomethyl-L-arginine (L-NMMA), methylene blue and Desferal (iron chelator) mixed with mediator suppressed histamine-induced edema at doses between 0.1 and 100 micrograms/kg and showed no or little stimulatory effect at higher doses. L-arginine reversed the effect of L-NMMA. Serotonin edema was enhanced by a high dose of these drugs. Their effect became suppressive as the histamine ratio was increased in edema induced by a histamine-serotonin mixture. This suggested that serotonin-induced vascular permeability decreased with a greater production of either O2- or NO. Cimetidine (H2-antagonist) was not effective in histamine edema of normal mice, but became suppressive (ED50 = 70 micrograms/kg) when 10 mg/kg L-NMMA was coinjected. SOD and methylene blue also rendered this edema sensitive to cimetidine. A simultaneous decrease in sensitivity to mepyramine (H1-antagonist) indicated that NO and oxyradicals kept H1-receptors activated. L-NMMA had no effect on bradykinin edema, but suppressed thrombin-, acetylcholine-, platelet-activating factor-, substance P- and FeCl3-induced paw edemata. Nitroprusside (NO donator) suppressed serotonin edema. N-Acetylcysteine and cytochrome c, but not ascorbate and hydroxyl radical scavengers suppressed histamine edema.

Inhibition of carrageenan-induced paw edema by superoxide dismutase that binds to heparan sulfates on vascular endothelial cells.

The effect of heparin-binding superoxide dismutase (HB-SOD), a fusion gene product consisting of human Cu/Zn-SOD and a C-terminal basic domain with high affinity for heparin-like proteoglycans, was examined on carrageenan-induced paw edema in mice and rats. When injected intravenously to mice just before carrageenan, HB-SOD suppressed significantly paw edema. ED30 of HB-SOD (1000 units/kg) was markedly lower than that of SOD (bovine free Cu/Zn-SOD, 7000 units/kg). When HB-SOD was administered with heparin (500-2000 units/kg), edema was suppressed more markedly than by HB-SOD alone. In contrast, the suppressive action of SOD was decreased by heparin. HB-SOD also suppressed carrageenan paw edema in rats with an ED30 of 2500 units/kg which was also obtained by SOD. Heparin prolonged significantly the duration of HB-SOD suppression of edema. The inhibitory effect of HB-SOD alone disappeared within 5 hr of injection, while more than 80% of the effect remained at this time when HB-SOD has been injected with 1000 units/kg of heparin. Heparin failed to enhance the anti-inflammatory effect of SOD under any of the conditions tested and heparin alone showed no suppression up to 5 hr after injection. HB-SOD might permit studies on pathophysiological events in and around vascular endothelial cells where reactive oxygen species play critical roles.

Inhibition by nilvadipine of ischemic and carrageenan paw edema as well as of superoxide radical production from neutrophils and xanthine oxidase.

1. Nilvadipine (FK 235, FR 34235) suppressed ischemia (20 min)-reflow (20 min)-induced paw edema of mice (ED30:0.4 mg/kg i.v. and 2 mg/kg p.o.). Other calcium entry blockers of dihydropyridine-type also suppressed the edema, but 30-fold higher doses were required. 2. Oral dosing of nilvadipine suppressed carrageenan-induced paw edema (ED30:15 mg/kg in rats and 20 mg/kg in mice) at a potency corresponding to that of an anti-inflammatory drug, ibuprofen. Nifedipine, nicardipine and nimodipine resulted in a suppression of 30% only with 100 mg/kg oral dosing in rats. Nitrendipine, diltiazem and verapamil were without effect. 3. Nilvadipine inhibited superoxide radical (O-2production from xanthine oxidase (XOD) both with lactate dehydrogenase + NADH method and cytochrome c method (IC50:90 and 100 micrograms/ml, respectively). Nifedipine and nicardipine showed some inhibition, but the other calcium entry blockers failed to inhibit significantly even at 320 micrograms/ml. As uric acid formation was not reduced by the tested drugs, the inhibitory action might be due to their O-2scavenging effects. 4. Superoxide production of neutrophils from casein-induced peritoneal fluid in rats was most strongly inhibited by nilvadipine when the cells were stimulated by a calcium ionophore, A23187 (IC50:4 micrograms/ml). Inhibition by this drug when stimulated by f-methonyl-leucyl-phenylalanine and phorbol myristate acetate was less effective (IC50:20 and 30 micrograms/ml, respectively). Nifedipine and nicardipine inhibited neutrophil O-2production at higher concentrations (30-200 micrograms/ml) with all stimulants. Inhibitory actions by other drugs were weak. 5. Triggering of atherosclerosis depends largely on the oxidative stress on blood vessels after recently established concept.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of carrageenan paw oedema in rats and mice by heparin-induced EC-SODs.

Carrageenan-induced paw edemata of mice and rats were suppressed by 1-4 x 10(3) U/kg intravenous injection of heparin. High doses were less suppressive, corresponding well to the increase in plasma SOD activity. This biphasic dose response curve was also observed in our ischemic paw model of mice. Increased SOD appeared as high molecular EC-SOD C (in mice) and B (in rats) as a result of its sensitivity to a copper chelator and long retention time in the blood stream, compared to the short life of cytosolic Cu, Zn-SOD. EC-SOD C (135 kDa) failed to be detected in the plasma of heparin-injected mice by way of nitroblue tetrazolium staining after PAGE electrophoresis. Instead, SOD activity was found near 270 kDa. An excess heparin-loaded subunit of this enzyme might become inactivated or might not be able to fix to a pocket where EC-SOD eliminates O2-, to protect the endothelium, Electrophoresis dissociates the excess heparin resulting in an active form of the enzyme. Paw edemata of rats were less sensitive because this species lacks the strongly heparin-binding EC-SOD C and has only the weakly heparin-binding EC-SOD B, Ischemia-induced mitochondrial swelling of the paw muscle was observed by electron microscopy and was prevented by heparin injection.

Heparin, a potent releasing agent of extracellular superoxide dismutase (EC-SOD C), suppresses ischaemic paw oedema in mice.

Heparin (2,000 U/kg, i.v.) increases the plasma superoxide dismutase (SOD) activity by 2-3 times after 5 min. followed by a gradual decrease. A high dose of heparin (4 x 10(3) and 10 x 10(3) U/kg) exhibits a lower increase in the release of SOD. Ischaemic paw oedema in mice was suppressed by various types of SOD and heparin also suppresses this oedema. The dose-dependent curve of heparin of oedema suppression corresponds well with the increased plasma level of SOD. Inducibility with heparin, slow clearance from the bloodstream and blocking of oedema suppression by the copper chelator, diethyldithiocarbamate (DDC), suggest that the oedema suppressing SOD was the extracellular (EC)-SOD C. Other anticoagulants such as citrate and EDTA had no effect. Chondroitin sulphate A and C or carrageenan exhibited weak suppression. A complex of EC-SOD C and heparin appears not to bind to the endothelium in contrast to the injected free EC-SOD C. When heparin is re-injected, more than 1 week was required to get the same degree of oedema suppression. This indicates the necessity of newly synthesized enzyme. A biological role for heparin-induced release of plasma SOD is demonstrated for the first time in this investigation.

[Possible clinical application of SOD and free radical scavengers].

Ischemia-reperfusion injury in various organs has been discussed in connection with reactive oxygen species (ROS). Xanthine oxidase (XOD) has been believed to be the source of O2- to produce this injury of dogs and rats but XOD is not detected in hearts of men, pigs or rabbits. This suggests the importance of O2- produced by leukocyte NAD(P)H oxidase. We demonstrated in 1976 that 3 injections of Cu, Zn-SOD (superoxide dismutase) (i.v.) suppressed rat carrageenan paw edema. McCord succeeded with a single injection in the same model using polyethyleneglycol (PEG-SOD) of long retention time in the blood stream. Michelson's liposomal SOD had clinical effects on Behçet's disease, Crohn's disease etc. Stylenemaleimide (SMA)-SOD (Inoue) is now under experimental trial and recombinant human SOD (r-h-SOD) is today in phase II stage. The aim is to prevent myocyte damage or for kidney transplantation. So-called SOD mimics (Cu-complex etc), antioxidants (synthetic propyl gallate or natural flavonoids or tannins) and hydroxyl radical (.OH) scavengers such as DMTU (dimethylthiourea) are considered as a prototype for clinical application. Fe-chelators also attract attention, because Fe+2 produces the most reactive ROS, .OH radical.