In vivo rat hemoglobin thiyl free radical formation following phenylhydrazine administration

The reaction of oxyhemoglobin with phenylhydrazine has received considerable attention for many decades. The basis for this interest stems from the ability of phenylhydrazine and hydrazine-based drugs to induce hemolytic anemia. Considerable evidence obtained from in vitro ESR experiments implicates free radicals in the events leading to red blood cell hemolysis. However, until this report, no corroborating ESR evidence for in vivo free radical formation has been presented. We have successfully employed ESR to detect the formation of a radical adduct in the blood of rats which received an intragastric dose of phenylhydrazine followed by an intraperitoneal injection of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). An immobilized radical adduct was detected by ESR when phenylhydrazine was administered in a dosage comparable to that prescribed for currently employed hydrazine-based drugs. We were also able to detect this immobilized DMPO adduct when hydrazine was employed in place of phenylhydrazine in the rat studies. The results of a series of experiments led us to ascribe this DMPO radical adduct to the trapping of a hemoglobin-derived thiyl free radical.     

Aniline-, phenylhydroxylamine-, nitrosobenzene-, and nitrobenzene-induced hemoglobin thiyl free radical formation in vivo and in vitro

We have employed the ESR spin trapping technique in vivo to detect the formation of the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)/hemoglobin thiyl free radical adduct in the blood of rats following administration of either aniline, phenylhydroxylamine, nitrosobenzene, or nitrobenzene. This DMPO adduct was a six-line, strongly immobilized, radical adduct. Using rat red blood cells, both phenylhydroxylamine and nitrosobenzene were able to induce the formation of the DMPO/glutathiyl free radical adduct and the same DMPO/hemoglobin thiyl free radical adduct was detected in in vivo samples. In experiments using purified rat oxyhemoglobin, a four-line, weakly immobilized, DMPO/hemoglobin thiyl free radical adduct was detected, in addition to the six-line strongly immobilized adduct. When this study was repeated using human red blood cells, we detected only the DMPO/glutathiyl free radical adduct and, when purified human oxyhemoglobin was employed, only the four-line, weakly immobilized, DMPO/hemoglobin thiyl radical adduct could be detected. In a study using reduced glutathione, we found that phenylhydronitroxide free radicals were reduced by glutathione and that glutathione was concomitantly oxidized to its thiyl free radical. We propose that the species responsible for the oxidation of the thiols to yield the thiyl free radicals in vivo and in vitro was the phenylhydronitroxide radical produced from the reaction of phenylhydroxylamine with oxyhemoglobin.     

In vivo formation of a free radical metabolite of ethanol

Free radical metabolism of ethanol has been suggested as a factor in its hepatotoxicity. Although evidence of lipid radical formation due to ethanol treatment in vivo has been reported, free radicals from ethanol itself have not been detected in living animals. However, by applying the EPR spectroscopy technique of spin trapping to the study of ethanol-treated alcohol dehydrogenase-deficient deermice (Peromyscus maniculatus), we have detected the alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN)/alpha-hydroxyethyl radical adduct in bile from animals administered [1-13C]ethanol and the spin trap POBN. Hyperfine coupling constants were aN = 15.48, a beta H = 2.02, and a beta 13C = 4.61 G. In addition, an ethanol-dependent but 13C-invariant radical adduct, presumably lipid derived, was detected. Hyperfine coupling constants were aN = 15.38 and a beta H = 2.5 G. This report demonstrates, for the first time, the in vivo formation of the alpha-hydroxyethyl free radical metabolite of ethanol.     

Role of free radical scavengers on phenylhydrazine induced hemolysis in rat

The mechanism of the phenylhydrazine induced oxidative hemolysis was studied on the point of role of the free radical scavengers in rats. Phenylhydrazine resulted in the degradation of hemoglobin and the lipid peroxidation of the erythrocyte membrane. Otherwise, the elevation of coenzyme Q9, endogenous CoQ in rats, levels in plasma was observed against the phenylhydrazine induced oxidative stress. Supplementation of coenzyme Q10, exogenous CoQ in rats, inhibited the phenylhydrazine induced hemolysis according to the suppression of both the degradation of hemoglobin and the lipid peroxidation of the erythrocyte membrane. These results suggest that free radical scavengers such as coenzyme Q9 and coenzyme Q10 have important roles on the phenylhydrazine induced hemolysis in rats.     

Hydroxyl radical formation following methamphetamine administration to rats.

Administration of neurotoxic doses of methamphetamine (8 mg/kg, intraperitoneally x 4 times, at 2 hr intervals) caused a significant decrease in dopamine and 3,4-dihydroxyphenylacetic acid and an increase in 3-methoxytyramine levels in the striatum along with a decrease in serotonin and 5-hydroxyindoleacetic acid levels in the striatum and hippocampus. In addition, the methamphetamine treatment caused an increase in rat rectal temperature. Intraventricular injection of salicylate 105 min. after the last injection of methamphetamine produced an increase in 2,3- and 2,5-dihydroxybenzoic acid in the striatum and hippocampus. Moreover, the ratio of 2,3-dihydroxybenzoic acid to salicylate was significantly increased in the striatum, but not in the hippocampus. These results indicate that the hydroxyl radical may play an important role in methamphetamine-induced neurotoxicity in rat striatum and that its formation may be the result of methamphetamine-induced release of dopamine.     

In vivo radical trapping and biliary secretion of radical adducts of carbon tetrachloride-derived free radical metabolites

Products of the well documented reductive metabolism of CCl4 to .CCl3 have been examined by free radical trapping and ESR in vivo. We have found the phenyl-N-t-butylnitrone (PBN) radical adduct of .CCl3 in the bile of rats treated with the radical trap and 13CCl4. Hypoxia or pretreatment with phenobarbital has been reported to enhance the hepatotoxicity of CCl4 in vivo; these treatments also produced an increase in the biliary concentration of the PBN/.CCl3 radical adduct and in the .CCl3-derived PBN/.CO(-)2 radical adduct as well. A 13C-invariant spectrum detected upon PBN administration, with or without carbon tetrachloride, may be due to a lipid-derived species. ESR analysis of bile from animals treated with free radical traps and xenobiotics may prove useful in monitoring hepatic free radical-adduct formation in vivo.     

Role of hydroxyl radical formation in neurotoxicity as revealed by in vivo free radical trapping

Reactive oxygen species have been implicated in dopaminergic toxicity caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and iron. Although MPTP produces a parkinsonian syndrome after its conversion to 1-methyl-4-phenylpyridine (MPP(+)) by type B monoamine oxidase (MAO-B) in the brain, the etiology of this disease remains obscure. MPP(+) is a highly potent dopaminbergic-releasing agents and dopamine (DA) autoxidation catalyzed by iron and oxidative stress may be involved in the pathogenesis of Parkinson's disease. Neuromelanine synthesis from DA produce highly reactive free radicals. Although the controversy possible neurotoxin and/or neuroprotective roles of nitric oxide (NO) was discussed, NO contributes to oxidative injury to brain neurons in vivo. An environmental estrogen-like chemical also related to MPP(+)-induced *OH generation. This review describes actual mechanism of the free radicals formation by dialysis studies of in vivo free radical trapping in the pathogenesis of neurodegenerative disorders, including in the Parkinson's disease, Alzheimer disease and traumatic brain injuries.     

Effects of chronic ethanol administration on free radical defence in rat myocardium.

Cellular protection against free radical reactions was measured in myocardium from ethanol-fed rats using ethanol administration in drinking water as a model of moderate alcohol intoxication. The activities of Cu,Zn-superoxide dismutase (SOD) and glutathione-S-transferase were higher in ethanol-fed rats than in controls, whereas Mn-SOD, catalase and glutathione peroxidase activities were not altered by ethanol treatment. Myocardial zinc was higher and selenium concentration lower in ethanol-fed rats than in controls. Ethanol consumption, which failed to modify the myocardial vitamin E level, did not result in increased lipid peroxidation, but decreased cytosolic and membraneous protein thiols.     

Synovial fluid degradation induced by free radicals. In vitro action of several free radical scavengers and anti-inflammatory drugs

The action of several free radical scavengers and anti-inflammatory drugs to decrease the viscosity of the synovial fluid induced by the reaction between hypoxanthine and xanthine oxidase is evaluated. It confirms the significance of the hydroxyl and Superoxide radicals in the synovial fluid degradation and it shows that, with 1,3 diphenilisobenzofuran, singlet oxygen is generated in the xanthine oxidase reaction and suggests that it plays a role in the synovial fluid deterioration. The nonsteroidal anti-inflammatory drugs (Indometacine > Niflumic Acid > Acetaminophen > Imidazole > Mefenamic Acid > Phenylbutazone) as well as the steroidal ones (Prednisone > Dexamethasone > Triamcinolone Acetonide > Hydrocortisone) at 80 μM show the capacity to inhibit the synovial fluid degradation in the indicated order. Acetylsalicylic Acid is inactive. Paradoxically, Acetaminophen and Imidazole are more active for this system than as anti-inflammatory agents. To conclude, the results presented in this paper indicate that the capacity of anti-inflammatory dugs to inhibit the synovial fluid degradation is due to their ability to absorb or destroy free radicals.     

Inhibitory activity of kinetin on free radical formation of activated platelets in vitro and on thrombus formation in vivo

Kinetin has been shown to have anti-aging effects on several different systems, including plants and human cells. Recently, we demonstrated that kinetin markedly inhibited platelet aggregation in washed human platelets. In the present study, an electron spin resonance (ESR) method was used to further evaluate the scavenging activity of kinetin on the free radicals formed. Kinetin (70 and 150 microM) concentration dependently reduced the ESR signal intensity of hydroxyl radicals in collagen (1 microg/ml)-activated platelets. Furthermore, kinetin was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice when administered intravenously at doses of 4 and 6 mg/kg. In addition, intravenous injection of kinetin (4 and 6 mg/kg) significantly prolonged the bleeding time by approximately 1.9- and 2.1-fold as compared with normal saline in severed mesenteric arteries of rats. A continuous infusion of kinetin (0.6 mg/kg/min) for 10 min also significantly increased the bleeding time by about 2.3-fold, and the bleeding time returned to baseline within 120 min after cessation of kinetin infusion. Platelet thrombi formation was induced by irradiation of mesenteric venules with filtered light in mice pretreated intravenously with fluorescein sodium. When kinetin was administered at 13 and 14 mg/kg in mice pretreated with fluorescein sodium (5 mg/kg), the occlusion time was significantly prolonged. In conclusion, these results suggest that kinetin has effective free radical-scavenging activity in vitro and antithrombotic activity in vivo. Treatment with kinetin may lower the risk of thromboembolic-related disorders. Therefore, kinetin may be a potential therapeutic agent for arterial thrombosis, but its toxicity must be further assessed.     

Effects of nicotine on hydroxyl free radical formation in vitro and on MPTP-induced neurotoxicity in vivo

Parkinson’s disease (PD) is one of the most frequent disorders of the basal ganglia. From epidemiological studies there is a controversial discussion on the question whether tobacco smoking is correlated with a decreased incidence of PD. The present study aimed to elucidate the role of nicotine and its potential neuroprotective effects in a rodent model of PD. These effects may be related to an altered hydroxyl radical formation; this possibility was studied in vitro. Nicotine and α-phenyl-N-tert-butyl nitrone (PBN) were examined in a cell-free in vitro Fenton system (Fe³⁺/EDTA + H₂O₂) for their radical scavenging properties using the salicylate trapping method. Salicylic acid (0.5 mM) was incubated in the presence and absence of nicotine or PBN and the main products of the reaction of hydroxyl radicals with salicylic acid, namely 2,3- and 2,5-dihydroxybenzoic acid, were immediately determined using HPLC in combination with electrochemical detection. Nicotine and PBN were both able to significantly reduce hydroxyl radical levels at concentrations of 1, 2.5 and 5 mM. Interestingly, at 5 mM nicotine was able to reduce hydroxyl radical levels significantly more than the radical scavenger PBN (5 mM). To investigate the in vivo effects of nicotine, male C57BL/6 mice were used in the MPTP mouse model of PD. Nicotine (0.1 or 0.4 mg/kg s.c.) was administered twice daily for a period of 14 days. On day 8 a single injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 30 mg/kg s.c.) was given as well as an enhanced protocol of nicotine treatment (0.1 or 0.4 mg/kg s.c., 30 min before MPTP and 30, 90, 210, 330, 450, 570 min after MPTP) for a total of seven injections of nicotine. High dosage nicotine treatment significantly increased the MPTP-induced loss of body weight and resulted in a significantly decreased striatal dopamine content and an increased dopamine turnover in comparison with the MPTP-treated controls at day 15. However, the lower dosage of nicotine did not significantly alleviate the MPTP-induced effects, although some parameters showed a slight tendency in this direction. These results demonstrate that in vitro nicotine has radical scavenging properties which might suggest neuroprotective effects. In vivo experiments with nicotine, however, showed that a low dosage of nicotine did not alleviate the MPTP-induced dopamine depletion, but a large dosage even enhanced it. Received: 20 March 1998 / Accepted: 23 June 1998     

Identification of cobalt protoporphyrin IX formation in vivo following cobalt administration to rats

An electron paramagnetic resonance method for the identification and quantitation of cobalt protoporphyrin IX has been developed to provide definitive evidence for the formation of the cobalt chelate of protoporphyrin IX in vivo. Both authentic and enzymatically generated cobalt protoporphyrin exhibited a characteristic primary resonance at g = 2.32 at 100° Kelvin (K) in the reduced state. At low concentrations, signal averaging was found to improve considerably the signal to noise ratio and thereby allow for the measurement of low concentrations of cobalt protoporphyrin. It was possible to determine concentrations of cobalt protoporphyrin IX as low as 0.5 μM in biologic preparations, and the signal height of the e.p.r. resonance was linear with cobalt protoporphyrin concentrations up to at least 8.0 μM. Following the administration of cobaltous chloride to rats, cobalt protoporphyrin IX was demonstrated in livers at times when alterations in heme biosynthesis are known to occur. Cobalt protoporphyrin IX at the level of 4.8 nmoles/g liver was detected 120 min after the administration of cobaltous chloride at a dose of 60 mg/kg of body weight. This finding supports the concept that cobalt protoporphyrin IX is rapidly produced in vivo after the administration of cobaltous chloride and may be responsible for the observed inhibition of hepatic heme biosynthesis.     

In vivo increase in hypothalamic cyclic AMP following 5-hydroxytryptophan administration in rats

Summary The administration of 5-hydroxytryptophan (5-HTP, 100 mg/kg, i.p.) consistently increased hypothalamic cyclic AMP levels in rats treated 10 days earlier with the serotonin neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), to produce 5-HT receptor supersensitivity. However 5-HTP (100 mg/kg), failed to cause an increase in hypothalamic cyclic AMP in rats not pretreated with 5,7-DHT. The 5-HTP-induced increase in cyclic AMP was blocked by the decarboxylase inhibitor, benserazide (RO 44602, 800 mg/kg) and by the 5-HT antagonist metergoline (5 mg/kg). Other treatments that caused a significant elevation of hypothalamic cyclic AMP included: (a) l-Tryptophan plus the monoamine oxidase inhibitor, tranylcypromine, and (b) the serotonin agonist, 1-(m-trifluromethylphenyl)-1-piperazine.The 5-HT antagonist, methysergide, blocked the serotonin receptor mediated behavioral syndrome, but failed to prevent the increase in hypothalamic cyclic AMP. Moreover, the 5-HT agonist, 5-methoxy-N, N-dimethyltryptamine, (5-Me-DMT), induced a strong behavioral syndrome but failed to significantly increase hypothalamic cyclic AMP. These findings suggest that activation of 5-HT receptors some-where in the brain causes an increase in hypothalamic cyclic AMP, but further studies will be needed to determine whether this is a direct result of activation of the 5-HT receptors in the hypothalamus.     

In vivo effects of meloxicam,celecoxib, and ibuprofen on free radical metabolism in human erythrocytes

One of the major groups of chemical mediators involved in the inflammatory response is the prostaglandins, which are synthesized from arachidonic acid by the enzyme cyclooxygenase. The aim of this study is to compare the in vivo effects of celecoxib, meloxicam, and ibuprofen on the activities of catalase (CAT), glutathione peroxidase (GSHPx), superoxide dismutase (SOD) as well as malondialdehyde (MDA), and antioxidant potential levels (AOP) in human erythrocytes. Patients diagnosed as osteoarthritis were included in the study. Patients were treated with Celecoxib (200 mg/d) (n = 12), Meloxicam (15 mg/d) (n = 12), and Ibuprufen (1200 mg/d) (n = 9) for 21 days. SOD, CAT, GSHPx activities, MDA, and AOP levels were investigated in human erythrocyte haemolysates. SOD activity and AOP levels were significantly decreased in all NSAID groups when we compared the values before and after 21 days of celecoxib, meloxicam, ibuprofen treatment. There were no significant difference in CAT, GSHPx activities, and MDA levels before and after treatment in each group. Decreased SOD activities are thought to be related with the increased superoxide anion. Decreased AOP levels may indicate impairment in the total antioxidant defence system. These NSAIDs have similar effects on free radical metabolism on human erythrocytes; despite some difference in action mechanisms.     

In vivo biodistribution of prion- and GM1-targeted polymersomes following intravenous administration in mice

Due to the aging of the population, the incidence of neurodegenerative diseases, such as Parkinson's and Alzheimer's, is expected to grow and, hence, the demand for adequate treatment modalities. However, the delivery of medicines into the brain for the treatment of brain-related diseases is hampered by the presence of a tight layer of endothelial cells that forms the blood-brain barrier (BBB). Furthermore, most conventional drugs lack stability and/or bioavailability. These obstacles can be overcome by the application of nanocarriers, in which the therapeutic entity has been incorporated, provided that they are effectively targeted to the brain endothelial cell layer. Drug nanocarriers decorated with targeting ligands that bind BBB receptors may accumulate efficiently at/in brain microvascular endothelium and hence represent a promising tool for brain drug delivery. Following the accumulation of drug nanocarriers at the brain vasculature, the drug needs to be transported across the brain endothelial cells into the brain. Transport across brain endothelial cells can occur via passive diffusion, transport proteins, and the vesicular transport pathways of receptor-mediated and adsorptive-mediated transcytosis. When a small lipophilic drug is released from its carrier at the brain vasculature, it may enter the brain via passive diffusion. On the other hand, the passage of intact nanocarriers, which is necessary for the delivery of larger and more hydrophilic drugs into brain, may occur via active transport by means of transcytosis. In previous work we identified GM1 ganglioside and prion protein as potential transcytotic receptors at the BBB. GM1 is a glycosphingolipid that is ubiquitously present on the endothelial surface and capable of acting as the transcytotic receptor for cholera toxin B. Likewise, prion protein has been shown to have transcytotic capacity at brain endothelial cells. Here we determine the transcytotic potential of polymersome nanocarriers functionalized with GM1- and prion-targeting peptides (G23, P50 and P9), that were identified by phage display, in an in vitro BBB model. In addition, the biodistribution of polymersomes functionalized with either the prion-targeting peptide P50 or the GM1-targeting peptide G23 is determined following intravenous injection in mice. We show that the prion-targeting peptides do not induce efficient transcytosis of polymersomes across the BBB in vitro nor induce accumulation of polymersomes in the brain in vivo. In contrast, the G23 peptide is shown to have transcytotic capacity in brain endothelial cells in vitro, as well as a brain-targeting potential in vivo, as reflected by the accumulation of G23-polymersomes in the brain in vivo at a level comparable to that of RI7217-polymersomes, which are targeted toward the transferrin receptor. Thus the G23 peptide seems to serve both of the requirements that are needed for efficient brain drug delivery of nanocarriers. An unexpected finding was the efficient accumulation of G23-polymersomes in lung. In conclusion, because of its combined brain-targeting and transcytotic capacity, the G23 peptide could be useful in the development of targeted nanocarriers for drug delivery into the brain, but appears especially attractive for specific drug delivery to the lung.     

Non-invasive measurement of in vivo free radical reactions]

It was proven that in vivo free radicals roles in various physiological phenomena and many diseases such as inflammation, ischemic disease, digestive problems and neurodegeneration, thus making measurements of free radicals very important for determining the mechanisms of these diseases. Spin trapping method using ESR, salicylic acid methods using high performance liquid chromatography and chemiluminescent methods are reported for ex vivo and in situ measurements. And, among these techniques, in vivo ESR is the most powerful for performing non-invasive measurements in animals. In in vivo ESR, exogenous nitroxyl radicals are administered to animal and then by measuring the signal change, the radical reaction can be indirectly determined. The signal decay rate is enhanced by oxidative stress, and this enhanced signal decay is suppressed by giving radical scavengers, etc. By analyzing the enhancement of this attenuation in detail, it is possible to specify the type of radical or the location at which the free radicals are produced. Such non-invasive techniques to measure free radicals like the present in vivo ESR technique should provide considerable information for elucidating the mechanisms of diseases.