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PubMed1993

Demonstration of hydroxyl radical and its role in hydrogen peroxide-induced myocardial injury: hydroxyl radical dependent and independent mechanisms.

We investigated the mechanism of hydrogen peroxide (H2O2) action on myocardial injury in relation to hydroxyl radical (.OH) formation. Isolated rat hearts were perfused with a concentration of H2O2 (300 microM) known to produce cardiac injury. Perfusion of H2O2 for 15 min caused severe myocardial dysfunction, morphological damage, ATP depletion, and lipid peroxidation. Hydrogen peroxide concentration in the coronary effluent was reduced approximately 40% reflecting a myocardial H2O2 consumption of 12.7 +/- 0.9 mumol/15 min/g wet tissue (n = 12). One of the .OH-generated derivatives, 2,3-dihydroxybenzoic acid (2,3-DHBA), formed from reaction with salicylic acid, was detected in the coronary effluent by high-performance liquid chromatography at 23.16 +/- 4.05 nmol/15 min/g wet tissue. Catalase (200 U/ml, n = 6) added to the perfusate attenuated all parameters of myocardial injury by eliminating H2O2 from the perfusate, and thus .OH was not detected in the effluent. Deferoxamine (5 mM, n = 7) added to the perfusate reduced morphological damage and lipid peroxidation, but not dysfunction or ATP depletion. Deferoxamine significantly reduced .OH production; 2,3-DHBA was 5.22 +/- 3.56 nmol/15 min/g wet tissue. The present study provides evidence that .OH is produced in the H2O2-perfused heart. The adverse H2O2-mediated myocardial outcomes documented in this study appear to arise from both .OH-dependent and .OH-independent mechanisms.

Adenosine Triphosphate↗
PubMed2003

Cloning and characterization of a glucosyltransferase that reacts on 7-hydroxyl group of flavonol and 3-hydroxyl group of coumarin from tobacco cells.

In higher plants, secondary metabolites are often converted to their glycoconjugates by glycosyltransferases (GTases). We cloned a cDNA encoding GTase (NtGT2) from tobacco (Nicotiana tabacum L.). The recombinant enzyme expressed in Escherichia coli (rNTGT2) showed glucosylation activity against several kinds of phenolic compounds, particularly the 7-hydroxyl group of flavonoids and 3-hydroxycoumarin. The K(m) values of kaempferol and 3-hydroxycoumarin with rNTGT2 are 6.5 microM and 23.6 microM, respectively. The deduced amino acid sequence of NTGT2 shows 60-70% identity to that of anthocyanin 5-O-glucosyltransferase (A5GT); rNTGT2 did not show activity against the anthocyanins tested. NtGT2 gene expression was induced by treating tobacco cells with plant hormones such as salicylic acid. We consider that NtGT2 gene might have evolved from the same ancestral gene as the A5GT genes to the stress-inducible GTases that react on several phenolic compounds.

Amino Acid Sequence↗
PubMed2001

Bupivacaine hydrochloride induces muscle fiber necrosis and hydroxyl radical formation-dimethyl sulphoxide reduces hydroxyl radical formation.

We induced acute skeletal muscle necrosis in rats using bupivacaine hydrochloride and found that both 2,5- and 2,3-dihydroxybenzoic acid significantly increased in skeletal muscle. A single administration of dimethyl sulphoxide, a free radical scavenger, significantly lowered concentrations of 2,5- and 2,3-dihydroxybenzoic acid. These results suggest that dimethyl sulphoxide is an effective hydroxyl radical scavenger and may be useful in the treatment of myopathy.

Anesthetics, Local↗
PubMed1989

Neutrophil degranulation inhibits potential hydroxyl-radical formation. Relative impact of myeloperoxidase and lactoferrin release on hydroxyl-radical production by iron-supplemented neutrophils assessed by spin-trapping techniques.

Hydroxyl radical (.OH) formation by neutrophils in vitro requires exogenous iron. Two recent studies [Britigan, Rosen, Thompson, Chai & Cohen (1986) J. Biol. Chem. 261, 17026-17032; Winterbourn (1987) J. Clin. Invest. 78, 545-550] both reported that neutrophil degranulation could potentially inhibit the formation of .OH, but differed in their conclusions as to the responsible factor, myeloperoxidase (MPO) or lactoferrin (LF). By using a previously developed spin-trapping system which allows specific on-line detection of superoxide anion (O2-) and .OH production, the impact of MPO and LF release on neutrophil .OH production was compared. When iron-diethylenetriaminepenta-acetic acid-supplemented neutrophils were stimulated with phorbol myristate acetate or opsonized zymosan, .OH formation occurred, but terminated prematurely in spite of continued O2- generation. Inhibition of MPO by azide increased the magnitude, but not the duration, of .OH formation. No azide effect was noted when MPO-deficient neutrophils were used. Anti-LF antibody increased both the magnitude and duration of .OH generation. Pretreatment of neutrophils with cytochalasin B to prevent phagosome formation did not alter the relative impact of azide or anti-LF on neutrophil .OH production. An effect of azide or anti-LF on spin-trapped-adduct stability was eliminated as a confounding factor. These data indicate that neutrophils possess two mechanisms for limiting .OH production. Implications for neutrophil-derived oxidant damage are discussed.

Antibodies↗
PubMed1993

Direct detection of endogenous hydroxyl radical production in cultured adult cardiomyocytes during anoxia and reoxygenation. Is the hydroxyl radical really the most damaging radical species?

Isolated adult rat cardiac myocytes were subjected to anoxia and substrate deprivation for 15, 30, 60, 90, and 120 minutes and reoxygenation for 120 seconds. The supernatant and cell extract were analyzed for hydroxyl radicals (.OH) with high-performance liquid chromatography using salicylate as a trapping agent. The production of intracellular H2O2 as a possible precursor of .OH was also documented using the fluorescent probe dichlorofluorescein diacetate. The release of the cytosolic enzyme lactate dehydrogenase (LDH) and malondialdehyde (MDA) formation were used as cell injury markers. Trypan blue and horseradish peroxidase stains were used as markers for altered membrane permeability. Maximum formation of .OH was observed in myocytes subjected to 15 minutes of anoxia/reoxygenation (2.83 +/- 0.27 nmol/mg protein), at which time no injury was observed at light and ultramicroscopic levels. On the other hand, there was no correlation between the amount of .OH production and different parameters of cell injury in myocytes subjected to anoxia/reoxygenation longer than 15 minutes. Myocytes developed extensive blebbing, loss of cell membrane permeability, and ultrastructural damage. The enzyme leakage was minimal at 15 minutes (0.094 +/- 0.021 units/mg protein) and increased fivefold after 120 minutes (0.428 +/- 0.069 units/mg protein). Similarly, MDA increased from 0.78 +/- 0.14 nmol/mg protein at 15 minutes to 1.65 +/- 0.35 nmol/mg protein at 120 minutes. Incubation with 1 mM deferoxamine reduced the .OH production at all anoxic intervals, most significantly at 15 minutes, but did not decrease LDH and MDA release or provide ultrastructural preservation. However, preincubation with 2.5 microM diphenylphenylenediamine markedly reduced both LDH and MDA release and offered prominent ultrastructural protection. These results suggest that 1) myocytes were able to generate .OH endogenously; 2) maximum .OH was produced at 15 minutes after anoxic reoxygenation without compromising cell viability; 3) prolongation of the anoxic period exacerbated cell damage without parallel increase in .OH generation; 4) there was no significant production of .OH after 15 minutes of anoxia/reoxygenation with or without treatment of deferoxamine, suggesting that prolonged anoxia/reoxygenation does not induce additional .OH formation and thus mediate cell injury; and 5) it is likely that the damage to myocytes in this system was still mediated by free radicals other than .OH, as indicated by the protection by diphenylphenylenediamine against the cellular injury.

Animals↗
PubMedDate not supplied

Genetic polymorphism of cytochrome P-450-dependent phencyclidine hydroxylation in mice. Comparison of phencyclidine hydroxylation in humans.

A mouse model of P-450 phencyclidine 3-cyclohydroxylase (P-450 PCP 3-cyclohydroxylase) genetic polymorphism is described. Up to a 3-fold difference was observed in the constitutive liver microsomal activity of P-450 PCP 3-cyclohydroxylase between "slow" (A/J and DBA/2J) and "rapid" (C57BL/6J and BALB/CJ) phencyclidine (PCP) metabolizers. The segregation of slow and rapid hydroxylator phenotypes between 17 recombinant inbred mouse strains derived from A/J and C57BL/2J mice suggests control of the activity by a single gene located on the X-chromosome or, less likely, on chromosome 17. A liver deficiency of P-450 PCP 3-cyclohydroxylase was observed in the New Zealand rabbit and Wistar rat, as well as in some human subjects. Any relationship of P-450 PCP 3-cyclohydroxylase polymorphism to other well characterized P-450 polymorphisms in mice (aryl hydrocarbon hydroxylase and coumarin hydroxylase) was excluded on the basis of differences in inducibility and activity distribution among the inbred mouse strains. Lack of relationship to the P-450 debrisoquine hydroxylase was confirmed by direct comparison of both activities in the same mouse and human liver microsomes. The pharmacological consequence of the observed polymorphism in mice appears to be that the rapid PCP metabolizers are more resistant to the effects of PCP compared to the slow metabolizers as based upon its ED50 and duration of action in A/J and C57BL/6J mice. The relevance of this data to humans remains to be determined, but clearly the latter show marked differences in PCP 3-cyclohydroxylase activity, which separate into low, intermediate, and high groups.

Animals↗
PubMed1993

[Interrelationship of microsomal and mitochondrial systems for hydroxylating steroids from cattle adrenal cortex: effect of highly-purified components of the mitochondrial monoxygenase system on hydroxylation of steroids by microsomes].

The possibility of functioning of microsomal steroid hydroxylases in the adrenal cortex in the presence of the electron-transporting proteins in mitochondrial monooxygenase systems has been studied. It was found that adrenodoxin reductase stimulates both the 21- and 17 alpha-steroid hydroxylation activities in microsomes of the adrenal cortex with the same efficiency as does NADPH-dependent cytochrome P-450 reductase. Using cytochrome b5 and its specific antibody, an attempt has been made to investigate possible mechanisms of the stimulating effect of adrenodoxin reductase.

Adrenal Glands↗
PubMed1982

Specific inhibition of hnRNA synthesis by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Requirement of a free 3'-hydroxyl group, but not 2'- or 5'-hydroxyls.

Five structural analogues of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), all with modified sugar moieties, have been examined for their inhibitory activities on RNA transcription in salivary glands of Chironomus tentans. The well-known ability of the parent DRB at 65 microM concentration to selectively inhibit hnRNA/mRNA synthesis by approx. 90% was essentially abolished on methylation of the 3'-OH; but, at an overdose the analogue suppressed labeling of all RNA classes examined (hnRNA/mRNA, rRNA, 4-5 S RNA) by 70-80%. By contrast, the 2'-O-methyl derivative of DRB was almost as effective as DRB itself in blocking transcription of hnRNA/mRNA genes. Blocking of both the 2' and 3' hydroxyls (2',3'-O-isopropylidene-DRB) completely abolished inhibitory activity, irrespective of the concentration employed. The 5'-deoxy-5'-chloro derivative of DRB was only slightly less effective than the parent DRB. An unusual aspect of the activities of 2'-O-methyl-DRB and 5'-deoxy-5'-chloro-DRB was their ability to stimulate synthesis of 4-5 S RNA by 25-45%. Also investigated was the influence of the various analogues on the rate of formation of [3H]UTP from [3H]uridine used as an RNA precursor. The rate of such formation of [3H]UTP was suppressed 2-6-fold by treatment with 2'-O-methyl or 3'-O-methyl-DRB, but was unaffected by 5'-deoxy-5'-chloro-DRB or 5,6-dichloro-1-alpha-D-arabinofuranosylbenzimidazole. The overall data point to the importance of a free 3'-OH in the ribose moiety of DRB for selective inhibitory activity. The alpha-D-arabinofuranosyl analogue, although less selective in inhibition of RNA transcription, still exhibits about 50% of the activity of DRB.

Amanitins↗
PubMed1992

Ferric-ion-photosensitized damage to DNA by hydroxyl and non-hydroxyl radical mechanisms.

Iron(III) and UVA (320-400 nm) light strongly diminished the transforming activity of Haemophilus influenzae DNA in the presence of oxygen. Iron(III) alone in the absence of light had no measurable effect on the transforming activity. The chelating agent ethylenediaminetetraacetic acid (EDTA) conferred virtually complete protection, but hydroxyl radical scavengers (mannitol, methanol, ethanol, isopropanol and dimethyl sulfoxide) inhibited only a small fraction of the inactivation. Treatment of plasmid DNA (pBR322) with iron(III) results in the conversion of the covalently closed circular form of the plasmid to open circles and ultimately to the linear form. Concomitant with the alteration in the conformation of the plasmid, the ability to transform Escherichia coli was reduced. In model systems, iron(III) photoreacted with the DNA backbone causing nicking and double-strand breakage. The results are consistent with a mechanism involving a preliminary complexation of iron(III) by DNA followed by the generation of reactive free radicals other than .OH. We suggest that bound iron, or other UV-absorbing transition metal complexes, may be chromophores capable of causing DNA damage in the long-wave near-UV region.

DNA Damage↗
PubMed2003

Epigenetic toxicity of hydroxylated biphenyls and hydroxylated polychlorinated biphenyls on normal rat liver epithelial cells.

2-Biphenylol, 3-biphenylol, 2,2'-biphenyldiol, 3,3'-biphenyldiol, 3-chloro-2-biphenylol, and 4,4'-dichloro-3-biphenylol were evaluated using the scrape-loading/dye transfer (SL/ DT) technique to determine in vitro gap junctional intercellular communication (GJIC) in a normal rat liver epithelial cell line as a measure of the epigenetic toxicity. Cytotoxicity was determined using the neutral red uptake assay. A dose range of 0-300 microM was examined. Only 3,3'-biphenyldiol and 4,4'-dichloro-3-biphenylol induced cytotoxicity within the tested dose ranges. Noncytotoxic doses were selected for evaluation of epigenetic toxicity. 4,4'-Dichloro-3-biphenylol was most inhibitory to GJIC at the lowest dose. The cytotoxicity and GJIC inhibitory effects observed for 4,4'-dichloro-3-biphenylol might be, although not exclusively, a consequence of the lipophilic nature of this chemical. 3-Chloro-2-biphenylol was least inhibitory to GJIC. 3-Chloro-2-biphenylol was less inhibitory to GJIC than 2-biphenylol because of the presence of the chlorine functional group, which appears to attenuate the toxic effect of the ortho-hydroxyl group. Although cells were capable of complete recovery of GJIC after removal of each of the chemicals, only with 2,2'-biphenyldiol and 4,4,'-dichloro-3-biphenylol did the cells demonstrate partial recovery without removal of the chemical. The more noncoplanar conformation of 2,2'-biphenyldiol and 2-biphenylol might explain their more inhibitory behavior in comparison to 3,3'-biphenyldiol and 3-biphenylol, respectively.

Animals↗
PubMed2003

Short synthesis of tert-butyl-hydroxylated 3,5-di-tert-butyl-4-hydroxybenzaldehyde: Synthesis of tert-butyl-hydroxylated S-2474.

We have developed a very short synthesis of tert-butyl-hydroxylated di-tert-butyl-4-hydroxybenzaldehyde in which the HBr-DMSO system is used as an effective oxidant (overall yield of 45% for the entire four-step process from 2-tert-butyl-p-cresol). We also accomplished the synthesis of a major metabolite of the antiarthritic drug candidate S-2474.

Antioxidants↗
PubMed1984

Reactivity of hydroxyl and hydroxyl-like radicals discriminated by release of thiobarbituric acid-reactive material from deoxy sugars, nucleosides and benzoate.

Hydroxyl radicals (OH.) can be formed in aqueous solution by a superoxide (O2.-)-generating system in the presence of a ferric salt or in a reaction independent of O2.- by the direct addition of a ferrous salt. OH. damage was detected in the present work by the release of thiobarbituric acid-reactive material from deoxy sugars, nucleosides and benzoate. The carbohydrates deoxyribose, deoxygalactose and deoxyglucose were substantially degraded by the iron(II) salt and the iron(III) salt in the presence of an O2.- -generating system, whereas deoxyinosine, deoxyadenosine and benzoate were not. Addition of EDTA to the reaction systems producing radicals greatly enhanced damage to deoxyribose, deoxyinosine, deoxyadenosine and benzoate, but decreased damage to deoxygalactose and deoxyglucose. Further, OH. scavengers were effective inhibitors only when EDTA was present. Inhibition by catalase and desferrioxamine confirmed that H2O2 and iron salts were essential for these reactions. The results suggest that, in the absence of EDTA, iron ions bind to the carbohydrate detector molecules and bring about a site-specific reaction on the molecule. This reaction is poorly inhibited by most OH. scavengers, but is strongly inhibited by scavengers such as mannitol, glucose and thiourea, which can themselves bind iron ions, albeit weakly. In the presence of EDTA, however, iron is removed from these binding sites to produce OH. in 'free' solution. These can be readily intercepted by the addition of OH. scavengers.

Benzoates↗
PubMed1987

Superoxide-dependent and ascorbate-dependent formation of hydroxyl radicals from hydrogen peroxide in the presence of iron. Are lactoferrin and transferrin promoters of hydroxyl-radical generation?

Apo-lactoferrin and apo-transferrin protect against iron-ion-dependent hydroxyl-radical (.OH) generation from H2O2 in the presence of superoxide radicals or ascorbic acid at pH 7.4, whether the necessary iron is added as ionic iron or as ferritin. Iron-loaded transferrin and lactoferrin [2 mol of Fe(III)/mol] show no protective ability, but do not themselves accelerate .OH production unless chelating agents are present in the reaction mixture, especially if the proteins are incorrectly loaded with iron. At acidic pH values, the protective ability of the apoproteins is diminished, and the fully iron-loaded proteins can release some iron in a form able to accelerate .OH generation. The physiological significance of these observations is discussed.

Apoproteins↗
PubMed2004

High-level ab initio studies of the electronic excited states of the hydroxyl radical and water-hydroxyl complex.

The lowest-energy electronic transitions in the hydroxyl radical and the hydrogen bound complex H(2)O.HO have been studied using ab initio methods. We have used the complete active-space self-consistent field and multireference configuration interaction (MRCI) methods to calculate vertical excitation energies and oscillator strengths. At the MRCI level the lowest-lying (2)Sigma(+)<--(2)Pi electronic transition is redshifted by about 2500 cm(-1) upon formation of the H(2)O.HO complex. We propose that this transition could be used to identify the complex in the gas phase, which in turn could be used to examine the role of H(2)O.HO in atmospheric reactions.

Journal Article↗
PubMed2000

Steady-state plasma levels of nortriptyline and its hydroxylated metabolites in Japanese patients: impact of CYP2D6 genotype on the hydroxylation of nortriptyline.

The authors investigated the impact of the CYP2D6 genotype on steady-state concentrations of nortriptyline (NT) and its metabolites, trans-10-hydroxynortriptyline (EHNT) and cis-10-hydroxynortriptyline in a Japanese population of psychiatric patients. Forty-one patients (20 men and 21 women) were orally administered nortriptyline hydrochloride. The allele frequencies of the CYP2D6*5 and CYP2D6*10 were 4.9% and 34.1%, respectively. Significant differences in NT concentrations corrected for dose and weight were observed between the subjects with no mutated alleles and those with one mutated allele (mean +/- SD for no mutated alleles vs. one mutated allele: 70.3 +/- 25.4 vs. 98.4 +/- 36.6 ng/mL x mg(-1) x kg(-1); t = 2.54, dcf = 33, p < 0.05) and between the subjects with no mutated alleles and two mutated alleles (no mutated alleles vs. two mutated alleles: 70.3 +/- 25.4 vs. 147 +/- 31.1 ng/mL x mg(-1) x kg(-1); t = 5.87, df = 19, p < 0.0001). Also, a significant difference in the NT/EHNT ratio, which is representative of the hydroxylation ratio of NT, was observed between the subjects with no mutated alleles and those with two mutated alleles (no mutated alleles vs. two mutated alleles: 0.82 +/- 0.30 vs. 2.71 +/- 0.84; t = 7.86, df = 19, p < 0.0001). Multiple regression analysis showed that the number of mutated alleles of CYP2D6, which was the only significant factor, accounted for 41% and 48% of the variability in log(NT corrected for dose and weight) and log(NT/EHNT), respectively.

Adjustment Disorders↗
PubMed1993

Cytochrome P4502A-mediated coumarin 7-hydroxylation and testosterone hydroxylation in mouse and rat lung.

Pulmonary coumarin 7-hydroxylase, testosterone hydroxylase and other P450-mediated activities were compared in the mouse and rat. Coumarin 7-hydroxylase activity was 20 pmol/mg/min. in mouse and 4 pmol/mg/min. in rat lung microsomes. Liver values were 180 (mouse) and 1 (rat) pmol/mg/min. Km values of rat and mouse lung coumarin 7-hydroxylase were about 1 microM whereas the rat liver Km value was > 100 microM. Phenobarbital and 3-methylcholanthrene did not affect rat lung (or liver) coumarin 7-hydroxylase activity. Anti-Cyp2a-5 antibody effectively inhibited mouse and rat lung coumarin 7-hydroxylase and testosterone 15 alpha-hydroxylations but failed to block these activities in the rat liver. In immunoblot analysis anti-Cyp2a-5 antibody recognized the 50-kDa Cyp2a-4/5 protein in mouse lung microsomes. A P450 protein co-migrating with Cyp2a-5 was also detected in rat lung microsomes. Cyp2a-5 cDNA probe hybridized with a 1.8-kb mRNA species in rat lung RNA fraction. The hybridization signal was not increased by 3-methylcholanthrene or phenobarbital. These data suggest that the mouse lung expresses Cyp2a-5 which differs from the liver enzyme only in its regulation and that the rat lung contains a P450 isoform(s) belonging to the 2A subfamily which may be orthologous with the mouse Cyp2a-4/5 catalyzing coumarin 7-hydroxylase and testosterone 15 alpha-hydroxylase activities. The recently reported rat lung CYP2A3 (Kimura et al.) gene product is a candidate for the observed coumarin 7-hydroxylase activity in the rat lung.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1978

Hydroxyl radical production by free and DNA-bound aminoquinone antibiotics and its role in DNA degradation. Electron spin resonance detection of hydroxyl radicals by spin trapping.

The reduced antitumor antibiotic mitomycin C in aqueous solution exposed to air gives a 36-line electron spin resonance spectrum of the semiquinone identified by computer simulation. Incubation of this radical with the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) gives the PBN.OH nitroxide radical identified by independent generation. This nitroxide radical is also formed from similar treatment of a DNA to which mitomycin C is covalently attached. Incubation of the semiquinone from mitomycin C, mitomycin B, or streptonigrin (SN) with catalase or with superoxide dismutase inhibits the generation of OH, implying the intermediacy of H2O2 and O2 in its formation. The formation of the spin-trapped nitroxide radical is similarly inhibited by EDTA, suggesting the intermediacy of trace metal ions in the generation of hydroxyl radicals from SN. The results are consistent with the generation by the aminoquinone antibiotics in vivo of OH. already implicated in the degradation of DNA.

Catalase↗