Oligoribonucleotides containing 8-oxo-7,8-dihydroguanosine and 8-oxo-7,8-dihydro-2''-O-methylguanosine: synthesis and base pairing properties

OBJECTIVE: To test whether a rigid or a flexible ankle orthosis affects postural sway in single-limb stance as quantified by stabilometry. DESIGN: Crossover trial. SETTING: University laboratory. PARTICIPANTS: Twenty-two athletes with functional ankle instability (consecutive sample of patients with recurrent ankle sprains but without mechanical instability) and 22 healthy athletes (control group of volunteers matched to age, height, weight, physical activity). INTERVENTIONS: Stabilometry in single-limb stance on a force platform. Participants were tested on each leg with and without a rigid or a flexible ankle orthosis. The order of test conditions was randomized. MAIN OUTCOME MEASURES: Sway velocities, sway pattern, and sway area as calculated from center of pressure movements. The two groups were compared by Mann-Whitney test, and the different orthoses within each group were compared by Wilcoxon test, paired samples (type I error 5%, Bonferroni adjustment). RESULTS: In athletes with functional ankle instability, both a rigid and a flexible ankle orthosis significantly reduced mediolateral sway velocity. A flexible ankle orthosis also changed sway pattern significantly, by reducing the percentage of linear movements of less than 5 degrees per .01 sec. CONCLUSIONS: In athletes with functional ankle instability, ankle orthoses reduce mediolateral sway velocity, possibly because of improved mediolateral proprioception.     

Quinolone antibiotic photodynamic production of 8-oxo-7, 8-dihydro-2'-deoxyguanosine in cultured liver epithelial cells

To study the basis for the phototoxicity of quinolones, a class of synthetic antibacterials, the photodynamic ability to mediate 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) formation in cultured cells was measured for lomefloxacin (LMX), which is strongly associated with clinical phototoxicity in humans, and ciprofloxacin (CFX), which has few reports of phototoxicity. Adult rat liver (ARL-18) cells were exposed to the quinolones in the presence of UVA and DNA was extracted and analyzed by HPLC with electrochemical detection. Low levels of 8-oxo-dG were found in the DNA of nonirradiated ARL-18 cells and this was increased up to 6-fold in the presence of either LMX (50-400 microM) or up to 3.6-fold in the presence of CFX (50-400 microM) and UVA (20 J/cm2) when compared to the UVA control. Comparing separate experiments with LMX and CFX, LMX produced greater levels of 8-oxo-dG either after dark exposure or after UVA exposure at 20 J/cm2. Also, LMX and CFX were both shown to photodegrade in the presence of UVA, and it was determined that UVA photoinstability alone does not reflect phototoxic potential. These data suggest that the photodynamic potential of LMX and CFX to produce 8-oxo-dG may relate to their human clinical phototoxicity profile. We suggest that the observed clinical phototoxicity is mediated through a UVA photodynamic effect on the quinolone to form reactive oxygen species in the presence of molecular oxygen. The findings indicate that 8-oxo-dG formation can serve as a marker for the potential phototoxicity of new quinolones.     

Photosensitized formation of 8-hydroxy-2'-deoxyguanosine and DNA strand breakage by a cationic meso-substituted porphyrin

Cationic porphyrins, known to have a high affinity for DNA, are useful tools with which to probe a variety of interactions with DNA. In this study we have examined both DNA strand scission and oxidative DNA base damage, measured by 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation, using a photoactivated cis-dicationic porphyrin. The data demonstrated a dose-dependent formation for each type of DNA damage. Inhibition of strand scission and 8-OHdG formation with the singlet oxygen scavenger 1,3-diphenylisobenzofuran and with MgCl2 and no apparent effect by D2O suggests that a singlet oxygen mechanism generated in close proximity to the DNA may be responsible for the damage. However, a nearly complete inhibition of 8-hydroxy-2'-deoxyguanosine formation in 75% D2O and the substantial enhancement of 8-hydroxy-2'-deoxyguanosine formation in a helium atmosphere by photoactivated porphyrin rules out singlet oxygen as a primary mechanism for this process. These data indicate that distinct mechanisms lead to 8-OHdG formation and strand scission activity.     

Rat 7,8-dihydro-8-oxoguanine DNA glycosylase: substrate specificity, kinetics and cleavagemechanism at an apurinic site

Reactive oxygen species produce different lesions in DNA. Among them, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major oxidative products implicated in mutagenesis. This lesion is removed from damaged DNA by base excision repair, and genes coding for 8-oxoG-DNA glycosylases have been isolated from bacteria, yeast and human cells. We have isolated and characterized the cDNA encoding the rat 8-oxoG-DNA glycosylase (rOGG1). Expression of the cDNA in the fgp mutY Escherichia coli double mutant allowed the purification of the untagged rOGG1 protein. It excises 8-oxoG from DNA with a strong preference for duplex DNA containing 8-oxoG:C base pairs. rOGG1 also acts on formamidopyrimidine (FaPy) residues, and the K m values on 8-oxoG and FaPy residues are 18.8 and 9.7 nM, respectively. When acting on an oligonucleotide containing an 8-oxoG residue, rOGG1 shows a beta-lyase activity that nicks DNA 3' to the lesion. However, rOGG1 acts on a substrate containing an apurinic site by a beta-delta elimination reaction and proceeds through a Schiff base intermediate. Expression of rOGG1 in E.coli fpg mutY suppresses its spontaneous mutator phenotype.     

(E)-2-hexenal-induced DNA damage and formation of cyclic 1,N2-(1,3-propano)-2'-deoxyguanosine adducts in mammalian cells

(E)-2-Hexenal (hexenal), a natural flavor compound, acts as directly genotoxic agent and forms cyclic 1,N2-propano adducts with deoxyguanosine. Formation of this adduct in isolated DNA and in cells was studied with a modified 32P-postlabeling procedure including HPLC separation, nuclease P1 enrichment, two-dimensional TLC of adducted nucleotide bisphosphates on PEI-cellulose, and quantification of adduct spots by liquid scintillation counting. Adduct formation with the more reactive crotonaldehyde was included for comparison. Synthesized adducted dG-3'-phosphates served as external standards for identification and quantification. In calf thymus DNA, hexenal (0.2 mM) shows a time dependent formation of adducts, yielding 1.55 pmol/mumol of DNA at 5 h incubation. With crotonaldehyde (0.2 mM) the adduct rate was about 10-fold higher. Hexenal also generated 1,N2-propano-dG adducts in the human lymphoblastoid Namalva cell line (0.2 mM, 1 h, 86 fmol/mumol of DNA) and in primary rat colon mucosa cells (0.4 mM, 30 min, 50 fmol/mumol of DNA). In primary colon mucosa cells from rats and humans, hexenal and crotonaldehyde (0.4 mM, 30 min) induced DNA damage, detected by single cell microgel electrophoresis (comet assay). In primary rat gastric mucosa cells, hexenal was only weakly active, inducing detectable DNA damage in 20% of cells at 0.8 mM concentration. In contrast, primary mucosa cells from rat esophagus were as sensitive as colon cells. After single oral application of hexenal to rats (up to 320 mg/kg body wt) DNA damage was not detectable in gastrointestinal mucosa. Analysis of hexenal in selected flavored foods revealed concentrations up to 14 ppm (0.14 mM) that are comparable to its natural occurrence in some fruits and vegetables (up to 30 ppm). Thus, the concentration range selected for the toxicological studies described here clearly is relevant: Hexenal, at concentrations found in food, exerts genotoxic effects in cells from rat and human gastrointestinal tract.     

Antinociceptive evaluation of 14 beta-(bromoacetamido)-7,8-dihydro- N(cyclopropylmethyl)-normorphinone in mice

This study evaluated the supraspinal opioid effects of 14 beta-(bromoacetamido)-7,8-dihydro-N(cyclopropylmethyl)-normorphinone+ ++ (N-CPM-H2BAMO) in the mouse acetic acid-induced writhing and tail-flick assays. In the writhing test, N-CPM-H2BAMO produced a time- and dose-dependent antinociception after i.c.v. administration, with a 50% antinociceptive response being obtained with 0.28 (0.19-0.39) nmol when given 10 min before testing. The antinociceptive effect of N-CPM-H2BAMO was antagonized in a dose-dependent manner by the kappa-selective opioid receptor antagonist, nor-binaltorphimine. In the mouse tail-flick assay, N-CPM-H2BAMO failed to produce any antinociception after i.c.v. administration. N-CPM-H2BAMO produced a dose-dependent antagonism of morphine-induced antinociception but not antinociception induced by the delta-opioid receptor agonist [D-Pen2,D-Pen5]enkephalin. Nor-binaltorphimine (0.3 nmol) at dose that completely antagonized N-CPM-H2BAMO-induced antinociception in the writhing assay did not prevent the antagonistic effect of N-CPM-H2BAMO on morphine-induced antinociception. Therefore, these data indicate that N-CPM-H2BAMO produces antinociception by acting at supraspinal kappa-opioid receptors in the writhing assay, and also acts as a mu-opioid receptor antagonist.     

Mg(2+)-mediated binding of 6-substituted quinolones to DNA: relevance to biological activity

The interaction of a number of novel 6-substituted quinolone derivatives with DNA in the presence/absence of magnesium ions has been investigated by fluorometric techniques. The drug-single-stranded nucleic acid interaction is invariantly mediated by the metal ion. In all cases optimal complex formation is found at physiological Mg2+ concentration. From titrations at different [Mg2+] the binding constant for the ternary drug-DNA-Mg2+ complex (KT) has been evaluated. Interestingly, a good relationship is found between KT and gyrase poisoning activity of the test quinolones (IC50), which confirms that DNA-affinity of the quinolone, modulated by Mg2+, plays an important role in poisoning the cleavable gyrase-DNA complex and, consequently, in eliciting antibacterial activity in this family of drugs. The results obtained with different 6-substituted compounds supports the idea that position 6 of the drug, besides playing a pharmacokinetic role, is involved in recognition of the enzyme pocket. Our data do not support a mechanism of action based upon quinolone intercalation into B-DNA.     

Synthesis and pharmacological activities of ethyl 5-cyano-1,6-dihydro-6-oxo-2-(2,3,4-pyridyl)-3-pyridinecarboxylates and derivatives

The synthesis of ethyl esters of 5-cyano-1,6-dihydro-6-oxo-3-pyridinecarboxylic acids carrying as 2-substituent the 2-,3- or 4-pyridyl group is described. By alkaline hydrolysis followed by acidification, these esters gave the corresponding carboxylic acids, which were decarboxylated to 1,2-dihydro-2-oxo-6-(2,3,4-pyridyl)-3-pyridinecarbonitriles. As milrinone analogues, the above compounds were tested on contractile activity and frequency rate of spontaneously beating atria from reserpine-treated guinea-pigs. Ethyl 5-cyano-1,6-dihydro-6-oxo-2-(2-pyridyl)-3-pyridinecarboxylate showed an appreciable positive inotropic activity, although inferior to that of milrinone; moreover, some other compounds bearing the above 2-substitution pattern showed interesting antiinflammatory, analgesic and hypotensive activity.     

Singlet oxygen involvement in ultraviolet (254 nm) radiation-induced formation of 8-hydroxy-deoxyguanosine in DNA

In the present article, we report that ultraviolet (UV 254 nm) radiation substantially induced the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in purified DNA. The formation of 8-OHdG, a hallmarker of oxidative DNA damage, increased linearly up to 25 kJ/m2 and was dependent on the presence of oxygen in the solution. Deoxygenation by nitrogen significantly reduced the yield of 8-OHdG by UV radiation, whereas oxygenation with 100% oxygen substantially enhanced the yield. The hydroxyl radical (HO.) scavenger dimethysulfoxide (DMSO) dramatically quenched the formation of 8-OHdG by the ionizing radiation and Fenton reaction, but enhanced the formation of UV-induced 8-OHdG. Further studies showed that DMSO and mannitol, two predominant HO. scavengers, enhanced the levels of UV-induced 8-OHdG in a dose-dependent fashion, suggesting that UV-induced 8-OHdG is independent of the generation of HO.. The use of deuterium oxide (D2O), which prolongs the half life of singlet oxygen (1O2), substantially enhanced the yield of 8-OHdG by UV radiation, but not that by Fenton reaction. In contrast, sodium azide, a more and less specific 1O2 quencher, substantially reduced the levels of 8-OHdG by both UV radiation and Fenton reaction, indicating that sodium azide lacks the quenching specificity of 1O2 and HO.. It is proposed that UV induced 8-OHdG proceeds through a singlet oxygen involvement mechanism, rather than the generation of hydroxyl radicals.     

Comparative analysis of 8-oxo-2' -deoxyguanosine in DNA by 32P- and 33P-postlabeling and electrochemical detection

8-Oxo-2'-deoxyguanosine (8-oxo-dG) is emerging as a useful marker for oxidative DNA damage. Reported basal levels determined by 32P-postlabeling (PPL) method were 10-fold or more higher than those obtained with HPLC/electrochemical detection (ECD). This discrepancy was investigated. In commercial calf thymus DNA, levels of 4 +/- 1 and 64 +/- 14 8-oxo-dG per 10(6) 2'-deoxynucleosides (dN) were measured by the standard HPLC/ECD and PPL methods, respectively. DNA digestion by micrococcal nuclease/spleen phosphodiesterase and nuclease P1 (as used in the standard PPL method), followed by ECD analysis resulted in a level of 8 +/- 3. In calf thymus DNA spiked with chemically synthesized 8-oxo-dGp to give an increment of 9 8-oxo-dG/10(6) dN, the added standard produced a significant increase with HPLC/ECD but not PPL. After spiking the DNA with 90 8-oxo-dG/10(6) dN, the added 8-oxo-dGp was detectable also with PPL, with a labeling efficiency of 65%. In order to investigate the role of ionizing radiation from 32P for the higher 8-oxo-dG levels in PPL, incubation times and amounts of radioactivity in the phosphorylation reaction with commercial dGp were increased, and external irradiation of commercial dG with 32P was investigated. All modifications resulted in higher values of 8-oxo-dG measured, but the effect was not large enough to fully explain the discrepancy between PPL and HPLC/ECD. Using [gamma-33P]ATP instead of [gamma-32P]ATP or adding [33P]phosphate to a 32P-PPL assay resulted in even higher levels of 8-oxo-dG measured. The increase in 8-oxo-dG levels during the PPL workup is attributed to the presence and oxidation of unmodified dGp in the reaction mixture. For a determination of true basal levels, the PPL method will have to be modified, including the removal of dGp prior to the phosphorylation reaction.     

Synthesis and biochemical study of N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-(alpha,beta-imido)triphosphate (BuPdGMPNHPP): a non-substrate inhibitor of B family DNA polymerases

BuPdGMPNHPP was synthesized and assayed as a non-incorporable inhibitor of B family DNA polymerases. The derivative was synthesized by preparation of the imidophosphorane of BuPdG followed by reaction with orthophosphate using the imidazolide method. BuPdGMPNHPP inhibited human DNA polymerase alpha and T4 DNA polymerase 10 and 3.5-times more potently than BuPdGTP, respectively, and was not a substrate for either enzyme. BuPdGMPNHPP acts as an active site affinity probe that could find use in co-crystallization trials of B family DNA polymerases.     

Elevated 8-hydroxy-2'-deoxyguanosine levels in lung DNA of A/J mice and F344 rats treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and inhibition by dietary 1,4-phenylenebis(methylene)selenocyanate

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) is an effective chemopreventive agent against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung adenoma in female A/J mice. While p-XSC can effectively inhibit NNK-induced DNA methylation in female A/J mice and in male F344 rats, its effect on NNK-induced oxidative DNA damage had not been determined. Thus, the effect of p-XSC on the levels of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in lung DNA from A/J mice and F344 rats treated with NNK was examined. Mice were given NNK by gavage (0.5 mg/mouse in 0.2 ml corn oil, three times per week for 3 weeks) or by a single i.p. injection (2 mg/mouse in 0.1 ml saline) while maintained on a control diet (AIN-76A) or control diet containing p-XSC at 10 or 15 p.p.m. (as Se) starting 1 week before NNK administration and continuing until termination. Mice were killed 2 h after the last NNK gavage in the multiple administration protocol or 2 h after the single i.p. injection. Treatment with NNK by gavage significantly elevated the levels of 8-OH-dG in lung DNA of A/J mice from 0.7 +/- 0.1 to 1.6 +/- 0.2 adducts/10(5) 2'-deoxyguanosine (dG) (P < 0.001), while dietary p-XSC (at 10 p.p.m. Se) prevented significant elevation of the levels of this lesion caused by NNK, keeping them at 0.9 +/- 0.1 adducts/10(5) dG (P < 0.003). Injection of NNK in saline also significantly increased the levels of 8-OH-dG in lung DNA of A/J mice from 1.2 +/- 0.6 to 3.6 +/- 0.8/10(5) dG adducts (P < 0.01), while dietary p-XSC (at 15 p.p.m. Se) kept these levels at 1.9 +/- 0.5 adducts/10(5) dG (P < 0.03). Rats were given a single i.p. injection of NNK (100 mg/kg body wt) in saline while being maintained on control diet (AIN-76A) or control diet containing p-XSC (15 p.p.m. as Se) starting 1 week before NNK administration and continuing until termination. The rats were killed 2 h after injection. Treatment with NNK using this protocol significantly elevated the levels of 8-OH-dG in lung DNA of F344 rats from 2.6 +/- 0.5 to 3.5 +/- 0.5 adducts/10(5) dG (P < 0.03), while dietary p-XSC (at 15 p.p.m. Se) kept the levels of this lesion at 2.2 +/- 0.6 adducts/10(5) dG (P < 0.01). Our findings suggest that the chemopreventive efficacy of p-XSC against NNK-induced lung tumorigenesis in A/J mice and F344 rats may be due in part to inhibition of oxidative DNA damage.     

N2,7-bis(1-hydroxy-2-oxopropyl)-2'-deoxyguanosine: identical noncyclic adducts with 1,3-dichloropropene epoxides and methylglyoxal

cis- and trans-1,3-dichloropropene epoxides (1,3-D-epoxides) are proposed to be the penultimate or ultimate genotoxic metabolites of the major soil fumigant nematicide 1,3-dichloropropene. The 1, 3-D-epoxide isomers and the potential aldehydes from their degradation readily form adducts with 2'-deoxyguanosine (dGuo) but not with 2'-deoxyadenosine or 2'-deoxycytidine. The reaction of dGuo with the 1,3-D-epoxides (1:20 molar equiv) in phosphate buffer at pH 7.4 for 24 h at 37 degreesC results in complete conversion to four adducts that can be separated by HPLC with the same UV spectra and electrospray (ES)/MS molecular ion and fragmentation patterns. These adducts contain no chlorine and are identical to those obtained more rapidly with methylglyoxal in place of the 1,3-D-epoxides. The four isomeric methylglyoxal adducts with dGuo were proposed originally by others to be the cyclic adducts 1,N2-(1, 2-dihydroxy-2-methyl)ethano-dGuo, but they are reassigned here as the four diastereomers of the noncyclic bis adducts N2, 7-bis(1-hydroxy-2-oxopropyl)-dGuo. The assignments are based on HPLC/UV and HPLC/ES/MS experiments and 1H NMR spectral analysis of the first of the four adducts eluted with HPLC. Acid-catalyzed depurination converts the four dGuo derivatives to the two corresponding isomers of N2,7-bis(1-hydroxy-2-oxopropyl)guanine, assigned by ES/MS and 1H and 13C NMR. Although identical adducts are formed from dGuo with the 1,3-D-epoxides or methylglyoxal, the latter alpha,beta-dicarbonyl compound is not an intermediate in the reaction; instead, the 1,3-D-epoxides hydrolyze to 3-chloro-2-hydroxypropanal which adds to dGuo at N2 and N7. The adducts dehydrochlorinate, in a rate-limiting reaction, thereby giving the same end products obtained on direct reaction with methylglyoxal. Thus, 3-chloro-2-hydroxypropanal (not the 1, 3-D-epoxides or methylglyoxal) is the derivatizing agent for dGuo and therefore probably the mutagenic agent on 1,3-D bioactivation. On the basis of the dGuo model studied here, the DNA adducts of 1, 3-D and its epoxides may be the same as those with methylglyoxal [Vaca, C. E., Fang, J.-L., Conradi, M., and Hou, S.-M. (1994) Carcinogenesis 15, 1887-1894].     

Inhibition of klenow fragment (exo-) catalyzed DNA polymerization by (5R)-5,6-dihydro-5-hydroxythymidine and structural analogue 5,6-dihydro-5-methylthymidine

Oligonucleotides containing 5R-5,6-dihydro-5-hydroxythymidine (5R-3) and structural analogue 5,6-dihydro-5-methylthymidine (9) at defined sites were chemically synthesized via a method that obviates the use of NH4OH. Oligonucleotides prepared by this method were used to examine the effects of 5R-3 and 9 on the fidelity of Klenow (exo-) in vitro. The presence of lesions 5R-3 and 9 in DNA templates was shown to inhibit polymerization of primers hybridized to these templates. Inhibition was observed for both translesional synthesis and extension one nucleotide past the lesion, with the latter being more pronounced. The fidelity of Klenow (exo-) was reduced only slightly when utilizing substrates containing either dihyropyrimidine nucleotide. These results provide the first experimental verification of computational studies carried out on the effects of 3 on DNA templates, and are consistent with a structural model in which the C5-methyl group of 5R-3 adopts a pseudoaxial orientation resulting in a disruption in base stacking.     

Synthesis of 7,8-(methylenedioxy)-1-phenyl-3,5-dihydro-4H-2, 3-benzodiazepin-4-ones as novel and potent noncompetitive AMPA receptor antagonists

A group of 7,8-(methylenedioxy)-1-phenyl-3,5-dihydro-4H-2, 3-benzodiazepin-4-ones was synthesized and assayed for antagonism of rat brain alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors expressed in Xenopus oocytes. The benzodiazepinones inhibited AMPA-activated membrane current responses in a manner consistent with noncompetitive, allosteric inhibition of the receptor-channel complex. The most potent compound in the series was 1-(4-aminophenyl)-7,8-(methylenedioxy)-3,5-dihydro-4H-2, 3-benzodiazepin-4-one (6), which had an IC50 of 2.7 microM. For comparison, the reference compound GYKI 52466 (2) had an IC50 of 6.9 microM. Compound 6 also had potent anticonvulsant activity in a mouse maximum electroshock-induced seizure (MES) assay: the ED50 was 2.8 mg/kg iv, whereas the ED50 for GYKI 52466 was 4.6 mg/kg iv. In contrast to a previous report, the 7,8-dimethoxy analogue of 6 was a low-potency AMPA antagonist (IC50 >100 microM) and weak anticonvulsant (ED50 >10 mg/kg iv). The benzodiazepinones described herein are potent noncompetitive AMPA receptor antagonists that could have therapeutic potential as anticonvulsants and neuroprotectants.     

Brain mitochondria catalyze the oxidation of 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxyli c acid (DHBT-1) to intermediates that irreversibly inhibit complex I and scavenge glutathione: potential relevance to the pathogenesis of Parkinson's disease

We have proposed that a very early step in the pathogenesis of idiopathic Parkinson's disease is the elevated translocation of L-cysteine into neuromelanin-pigmented dopaminergic neurons in the substantia nigra. This influx of L-cysteine was proposed to divert the normal neuromelanin pathway by scavenging dopamine-o-quinone, formed by autoxidation of cytoplasmic dopamine, to give initially 5-S-cysteinyldopamine, which is further oxidized to 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1). In a recent report, it was demonstrated that DHBT-1 evokes inhibition of complex I respiration when incubated with intact rat brain mitochondria and a time-dependent irreversible inhibition of NADH-coenzyme Q1 (CoQ1) reductase when incubated with mitochondrial membranes. In this study, it is established that the time dependence of NADH-CoQ1 reductase inhibition reflects the oxidation of DHBT-1, catalyzed by an unknown constituent of the inner mitochondrial membrane, to an o-quinone imine intermediate that rearranges to 7-(2-aminoethyl)-5-hydroxy-1,4-benzothiazine-3-carboxylic acid (BT-1) and decarboxylates to 7-(2-aminoethyl)-5-hydroxy-1,4-benzothiazine (BT-2), which are further catalytically oxidized to o-quinone imine intermediates. The electrophilic o-quinone imine intermediates formed in these mitochondria-catalyzed oxidations of DHBT-1, BT-1, and BT-2 are proposed to bind covalently to key sulfhydryl residues at the complex I site, thus evoking irreversible inhibition of NADH-CoQ1 reductase. Evidence for this mechanism derives from the fact that greater than equimolar concentrations of glutathione completely block inhibition of NADH-CoQ1 reductase by DHBT-1, BT-1, and BT-2 by scavenging their electrophilic o-quinone imine metabolites to form glutathionyl conjugates. The results of this investigation may provide insights into the irreversible loss of glutathione and decreased mitochondrial complex I activity, which are both anatomically specific to the substantia nigra and exclusive to Parkinson's disease.     

Induction of 8-hydroxydeoxyguanosine in isolated DNA and HeLa cells exposed to fecapentaene-12: evidence for the involvement of prostaglandin H synthase and iron

The genotoxic/mutagenic mechanism(s) of action of fecapentaene-12 (fec-12) is complex but there is evidence to suggest that the generation of active oxygen species (AOS) may be involved. This has been assessed by measuring the formation of 8-hydroxydeoxyguanosine (8-OHdG) in isolated DNA and HeLa cells exposed in vitro to fec-12. The possibility that fec-12 may form AOS via peroxidative 'activation' by prostaglandin H synthase (PHS) has been investigated by measuring 8-OHdG in HeLa cells exposed to fec-12 in the absence or presence of PHS inhibitors. The role of iron as a catalyst in this pathway has also been investigated. A 4-fold increase in the level of 8-OHdG in isolated DNA was seen after exposure to fec-12 (1 mM) alone. This increase was enhanced synergistically by ferrous iron. Fec-12 exposure of HeLa cells at 50 and 100 microM induced 2- and 3-fold increases (P < 0.001) respectively in the level of 8-OHdG in cellular DNA. No increase was seen at 10 microM fec-12. The PHS inhibitors indomethacin and acetylsalicylate blocked the formation of 8-OHdG induced by fec-12 (50 microM) but did not inhibit the formation of 8-OHdG in these cells after exposure to H2O2 and Fe2+. Addition of the iron chelating agent o-phenanthroline to cells prior to fec-12 exposure blocked the increase in 8-OHdG induced by fec-12 (50 microM). Addition of the radical scavenging agent DMSO (10%) to cells prior to fec-12 exposure reduced the level of 8-OHdG to within 10% of control. Specific inhibition of fec-12 induced 8-OHdG formation in HeLa cells by PHS inhibitors suggests that this enzyme may be involved in 'activating' fec-12 to form AOS in cells. Inhibition of fec-12 induced 8-OHdG formation in cells by o-phenanthroline suggests a role for intracellular iron as a catalyst in this process.     

Evidence for the formation of a novel cyclopentenone isoprostane, 15-A2t-isoprostane (8-iso-prostaglandin A2) in vivo

A2/J2-Isoprostanes (IsoPs) are prostaglandin (PG) A2/J2-like compounds that are produced in vivo as dehydration products of D2/E2-IsoPs. One A2-IsoP that should be formed is 15-A2t-IsoP (8-iso-PGA2). Analogous to cyclopentenone PGs, 15-A2t-IsoP readily undergoes nucleophilic addition to various biomolecules suggesting the compound is capable of exerting potent bioactivity. However, proof that it is definitively formed in vivo is lacking. Evidence is now presented that 15-A2t-IsoP, in fact, is generated in vivo by demonstrating that an endogenous A2-IsoP with a retention time on capillary GC identical with that 15-A2t-IsoP co-chromatographs through four high resolving HPLC purification procedures with authentic radiolabeled 15-A2t-IsoP.