Quinone-induced inhibition of urease: elucidation of its mechanisms by probing thiol groups of the enzyme

In this work we studied the reaction of four quinones, 1,4-benzoquinone (1,4-BQ), 2,5-dimethyl-1,4-benzoquinone (2,5-DM-1,4-BQ), tetrachloro-1,4-benzoquinone (TC-1,4-BQ) and 1,4-naphthoquinone (1,4-NQ) with jack bean urease in phosphate buffer, pH 7.8. The enzyme was allowed to react with different concentrations of the quinones during different incubation times in aerobic conditions. Upon incubation the samples had their residual activities assayed and their thiol content titrated. The titration carried out with use of 5,5'-di-thiobis(2-nitrobenzoic) acid was done to examine the involvement of urease thiol groups in the quinone-induced inhibition. The quinones under investigation showed two distinct patterns of behaviour, one by 1,4-BQ, 2,5-DM-1,4-BQ and TC-1,4-BQ, and the other by 1,4-NQ. The former consisted of a concentration-dependent inactivation of urease where the enzyme-inhibitor equilibrium was achieved in no longer than 10min, and of the residual activity of the enzyme being linearly correlated with the number of modified thiols in urease. We concluded that arylation of the thiols in urease by these quinones resulting in conformational changes in the enzyme molecule is responsible for the inhibition. The other pattern of behaviour observed for 1,4-NQ consisted of time- and concentration-dependent inactivation of urease with a nonlinear residual activity-modified thiols dependence. This suggests that in 1,4-NQ inhibition, in addition to the arylation of thiols, operative are other reactions, most likely oxidations of thiols provoked by 1,4-NQ-catalyzed redox cycling. In terms of the inhibitory strength, the quinones studied formed a series: 1,4-NQ approximately 2,5-DM-1,4-BQ<1,4-BQ相似文献   

Mutagenicity of thiol compounds in Escherichia coli WP2 tester strain IC203, deficient in OxyR: effects of S9 fractions from rat liver and kidney

Low doses of -cysteine (CYS), cysteinyl-glycine (CYSGLY) and reduced glutathione (GSH) activated by γ-glutamyl transpeptidase (GGT) were mutagenic in strain IC203 (oxyR), whereas higher doses were required to observe a weak mutagenicity in the oxyR⁺ strain WP2 uvrA/pKM101 (denoted IC188). This indicates that thiol mutagenesis is suppressed by OxyR-regulated antioxidant defenses and confirms its oxidative character. The mutagenesis by low doses of CYS, CYSGLY and GSH+GGT detected in IC203 was abolished by rat liver S9, through the activity of catalase, as well as by the metal chelator diethyldithiocarbamate (DETC), supporting the dependence of this mutagenesis on H₂O₂ production, probably in thiol autoxidation reactions in which transition metals are involved. Surprisingly, low DETC concentrations greatly potentiate the mutagenicity of low CYS doses. Mutagenesis by high doses of CYS and CYSGLY occurred in both IC203 and IC188 in the presence of liver S9, and was resistant to inhibition by catalase, although it was prevented by DETC. Mutagenesis by GSH activated by rat kidney S9, rich in GGT, was detected in IC203 and IC188 only at high doses since catalase and glutathione peroxidase, both present in kidney S9, might inhibit its induction by low GSH doses. In the presence of liver S9, almost deficient in GGT, GSH was not mutagenic. The mutagenicity of a high GSH dose occurring in the presence either of GGT plus liver S9 or of kidney S9 was weakly prevented by DETC.     

Development of a new E. coli strain to detect oxidative mutation and its application to the fungicide o-phenylphenol and its metabolites

Oxidative mutation is mainly induced by reactive oxygen species (ROS), such as the superoxide anion radical (O(2)(-)) and hydrogen peroxide (H(2)O(2)). However, in Escherichia coli (E. coli), ROS are eliminated by enzymes such as superoxide dismutase and catalase, which are coded by sodAB and katEG genes. In this study, to detect mutagens that induce oxidative mutation, a mutant (WP2katEGsodAB) with katEG and sodAB deleted was constructed by gene manipulation of E. coli WP2. H(2)O(2) and menadione sodium bisulfite generated mutation in WP2katEGsodAB but not in WP2. o-Phenylphenol (OPP) and its metabolites (phenylhydroquinone (PHQ) and phenyl-1,4-benzoquinone (PBQ)), which had been shown to be negative in the Ames test but reported to be carcinogenic, induced mutation in WP2katEGsodAB but not in WP2. These results suggest that the new assay may be useful for the detection of oxidative mutagens.     

Genotoxicity of stannous chloride in yeast and bacteria

Stannous chloride was found genotoxic in microbial test systems of the yeast Saccharomyces cerevisiae, in one strain of Salmonella typhimurium and in the Mutoxitest of Escherichia coli. Five isogenic haploid yeast strains differing only in a particular repair-deficiency had the following ranking in Sn2+ -sensitivity: rad52delta>rad6delta>rad2delta>rad4delta>RAD, indicating a higher relevance of recombinogenic repair mechanisms than nucleotide excision in repair of Sn2+ -induced DNA damage. Sn2+ -treated cells formed aggregates that lead to gross overestimation of toxicity when not undone before diluting and plating. Reliable inactivation assays at exposure doses of 25-75 mM SnCl2 were achieved by de-clumping with either EDTA- or phosphate buffer. Sn2+ -induced reversion of the yeast his1-798, his1-208 and lys1-1 mutant alleles, in diploid and haploid cells, respectively, and putative frameshift mutagenesis (reversion of the hom3-10 allele) was observed. In diploid yeast, SnCl2 induced intra-genic mitotic recombination while inter-genic (reciprocal) recombination was very weak and not significant. Yeast cells of exponentially growing cultures were killed to about the same extend at 0.1% of SnCl2 than respective cells in stationary phase, suggesting a major involvement of physiological parameters of post-diauxic shift oxidative stress resistance in enhanced Sn2+ -tolerance. Superoxide dismutases, but not catalase, protected against SnCl2-induced reactive oxygen species as sod1delta had a three-fold higher sensitivity than the WT while the sod2delta mutant was only slightly more sensitive but conferred significant sensitivity increase in a sod1delta sod2delta double mutant. In the Salmonella reversion assay, SnCl2 did not induce mutations in strains TA97, TA98 or TA100, while a positive response was seen in strain TA102. SnCl2 induced a two-fold increase in mutation in the Mutoxitest strain IC203 (uvrA oxyR), but was less mutagenic in strain IC188 (uvrA). We propose that the mutagenicity of SnCl2 in yeast and bacteria occurs via error-prone repair of DNA damage that is produced by reactive oxygen species.     

Genotoxicity of aminohydroxynaphthoquinones in bacteria, yeast, and Chinese hamster lung fibroblast cells

There are few studies on the biological activity of aminohydroxy derivates of 1,4-naphthoquinone (1,4-NQ) on prokaryotic and eukaryotic cells. We determined the mutagenic activity of 5-amino-8-hydroxy-1,4-naphthoquinone (ANQ) and 5-amino-2,8-dihydroxy-1,4-naphthoquinone (ANQ-OH) as compared to the unsubstituted 1,4-NQ in Salmonella/microsome assay. Potential mutagenic and recombinogenic effects and cytotoxicity were analyzed in haploid and diploid cultures of the yeast Saccharomyces cerevisiae. In Salmonella/microsome assay, 1,4-NQ was not mutagenic, whereas aminohydroxynaphthoquinones were weakly mutagenic in TA98 and TA102 strains. In haploid yeast in stationary growth phase (STAT), mutagenic response was only observed for the hom3 locus at the highest dose. In diploid yeast, aminohydroxynaphthoquinones did not induce any recombinogenic events, but 1,4-NQ was shown to be a recombinogenic agent. These results suggest that aminohydroxynaphthoquinones are weak mutagenic agents only in prokaryotic cells. The cytotoxicity of 1,4-NQ in yeast stationary cells was more significant in diploid cells as compared to that observed in haploid cells. However, ANQ and ANQOH were slightly cytotoxic in all treatments. Genotoxicity of these naphthoquinone compounds was also determined in V79 Chinese hamster lung fibroblast cells using standard Comet, as well as modified Comet assay with the bacterial enzymes formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (ENDOIII). Both 1,4-NQ and ANQ induced pronounced DNA damage in the standard Comet assay. The genotoxic effect of ANQ-OH was observed only at the highest dose. In presence of metabolic activation all substances showed genotoxic effects on V79 cells. Post-treatment of V79 cells with ENDOIII and FPG proteins did not have a significant effect on ANQ-OH-induced oxidative DNA damage as compared to standard alkaline Comet assay. However, all naphthoquinones were genotoxic in V79 cells in the presence of metabolic activation and post-treatment with enzymes, indicating that all compounds induced oxidative DNA damage in V79 cells. Our data suggest that aminohydroxynaphthoquinone pro-oxidant activity, together with their capability of DNA intercalation, have an important role in mutagenic and genotoxic activities.     

Genotoxicity of aminohydroxynaphthoquinones in bacteria, yeast, and Chinese hamster lung fibroblast cells

There are few studies on the biological activity of aminohydroxy derivates of 1,4-naphthoquinone (1,4-NQ) on prokaryotic and eukaryotic cells. We determined the mutagenic activity of 5-amino-8-hydroxy-1,4-naphthoquinone (ANQ) and 5-amino-2,8-dihydroxy-1,4-naphthoquinone (ANQ-OH) as compared to the unsubstituted 1,4-NQ in Salmonella/microsome assay. Potential mutagenic and recombinogenic effects and cytotoxicity were analyzed in haploid and diploid cultures of the yeast Saccharomyces cerevisiae. In Salmonella/microsome assay, 1,4-NQ was not mutagenic, whereas aminohydroxynaphthoquinones were weakly mutagenic in TA98 and TA102 strains. In haploid yeast in stationary growth phase (STAT), mutagenic response was only observed for the hom3 locus at the highest dose. In diploid yeast, aminohydroxynaphthoquinones did not induce any recombinogenic events, but 1,4-NQ was shown to be a recombinogenic agent. These results suggest that aminohydroxynaphthoquinones are weak mutagenic agents only in prokaryotic cells. The cytotoxicity of 1,4-NQ in yeast stationary cells was more significant in diploid cells as compared to that observed in haploid cells. However, ANQ and ANQOH were slightly cytotoxic in all treatments. Genotoxicity of these naphthoquinone compounds was also determined in V79 Chinese hamster lung fibroblast cells using standard Comet, as well as modified Comet assay with the bacterial enzymes formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (ENDOIII). Both 1,4-NQ and ANQ induced pronounced DNA damage in the standard Comet assay. The genotoxic effect of ANQ-OH was observed only at the highest dose. In presence of metabolic activation all substances showed genotoxic effects on V79 cells. Post-treatment of V79 cells with ENDOIII and FPG proteins did not have a significant effect on ANQ-OH-induced oxidative DNA damage as compared to standard alkaline Comet assay. However, all naphthoquinones were genotoxic in V79 cells in the presence of metabolic activation and post-treatment with enzymes, indicating that all compounds induced oxidative DNA damage in V79 cells. Our data suggest that aminohydroxynaphthoquinone pro-oxidant activity, together with their capability of DNA intercalation, have an important role in mutagenic and genotoxic activities.     

Mutation spectra of chemical mutagens determined by Lac+ reversion assay with Escherichia coli WP3101P-WP3106P tester strains

We previously reported the development of mutation-specific Escherichia coli B tester strains WP3101 to WP3106 from strain WP2uvrA. In this study we constructed their pKM101-containing derivatives WP3101P to WP3106P, and further isolated their rfa derivatives WP4101-WP4106 and WP4101P-WP4106P. The six kinds of F' plasmids (lacI-, lacZ-, proAB+), each of which carries a different lacZ allele, contained in the above strains were originally derived from E. coli K-12 strains CC101-CC106. All the tester strains show Lac- and Trp- phenotype. Assays for transitions and transversions are based upon Lac+ reversion of a specific mutation located within the lacZ gene on an F' plasmid. The trpE65(ochre) allele in the same strains enables them to be used for Trp+ reversion assays as well. In the present paper, we evaluated the sensitivity, specificity, and usefulness of the newly developed tester strains. Strains WP3101P-WP3106P were highly sensitive to determine mutational profile of heterocyclic amines with S9 mix-mediated metabolic activation and most of the oxidative mutagens and free radical generators tested. Every type of base-pair substitutions induced by 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) or 5-diazouracil were detected in strains WP3101P-WP3106P, while A:T-->C:G and G:C-->A:T mutations induced by MeIQ, and A:T-->C:G, G:C-->A:T, and G:C-->C:G by 5-diazouracil were not detected in pKM101-free tester strains. In pKM101-carrying strains, cumene hydroperoxide induced all types of base substitutions, while formaldehyde preferentially induced G:C-->T:A transversions. Phenazine methosulfate induced predominantly G:C-->A:T transitions and G:C-->T:A transversions, while H2O2 induced predominantly G:C-->T:A and A:T-->T:A transversions. Introduction of the rfa mutation considerably enhanced sensitivity to bulky mutagens such as polycyclic aromatic compounds. All six possible base substitutions induced by 9, 10-dimethyl-1,2-benzanthracene (DMBA) were detected in tester strains WP4101P-WP4106P. In conclusion, our tester strains WP3101P-WP3106P and WP4101P-WP4106P permitted rapid and simple detection of specific mutations induced by variety of mutagens.     

Disparity in the induction of glutathione depletion, ROS formation, poly(ADP-ribose) polymerase-1 activation, and apoptosis by quinonoid derivatives of naphthalene in human cultured cells

The purpose of this study is to examine the differences in the induction of cytotoxic effects and poly(ADP-ribose) polymerase-1 activation in human MCF-7 breast cancer cells by quinonoid derivatives of naphthalene, including 1,2-naphthalenediol (NCAT), 1,4-naphthalenediol (NHQ), 1,2-naphthoquinone (1,2-NQ), and 1,4-naphthoquinone (1,4-NQ). Results from the cytotoxic response analyses in cells indicated that all naphthalene quinonoids induced cell death in MCF-7 cells at concentrations ranging from 0.1 to 100microM where NHQ and 1,4-NQ were more efficient than NCAT and 1,2-NQ in the induction of cell death. Results from Western blot analyses confirmed that treatment of cells with NCAT and NHQ resulted in up-regulation of p53 protein expression and a significant shift in bax/bcl2 ratio, suggesting the induction of p53-dependent apoptosis in MCF-7 cells. Additionally, we observed that all naphthalene quinonoids induced increases in reactive oxygen species (ROS) formation and glutathione (GSH) depletion in MCF-7 cells. The induction of ROS formation and GSH depletion in cells by naphthalene quinonoids decreases in the rank order 1,4-NQ>NHQ>1,2-NQ approximately equal to NCAT. Further investigation indicated that least-squares estimates of the overall rates of elimination (k(e)) of naphthalene quinonoids in MCF-7 cells decreased in the rank order 1,4-NQ>1,2-NQ>NHQ>NCAT. Values of k(e) were estimated to be between 0.280h(-1)(T(1/2)=151min) and 13.8h(-1)(T(1/2)=3.05min). These results provide evidence that the para-isomeric form of naphthalene quinonoids tend to induce acute production of ROS and alterations in intracellular redox status in cells, leading to the subsequent cell death. Further, all naphthalene quinonoids induced decreases in intracellular NAD(P)H and NAD(+) in MCF-7 cells at non-cytotoxic concentrations. The reduction of intracellular NAD(P)H in cells exposed to NCAT and 1,2-NQ was blocked by two types of poly(ADP-ribose) polymerase (PARP) inhibitors whereas PARP inhibitors did not prevent the reduction of NAD(P)H in cells exposed to NHQ and 1,4-NQ. Further investigation confirmed that increases in the number of DNA single-strand breaks were detected in MCF-7 cells exposed to NCAT and 1,2-NQ as measured by the single-cell gel electrophoresis (Comet) assay whereas NHQ and 1,4-NQ did not induce increases in the number of single-strand breaks in MCF-7 cells. Overall, results from our investigation suggest that while NHQ and 1,4-NQ are more efficient in the induction of cell death, NCAT and 1,2-NQ are prone to induce depletion of NAD(P)H and NAD(+) mediated by PARP-1 activation through formation of DNA single-strand breaks in human cultured cells.     

Metabolic activation of 1-naphthol and phenol by a simple superoxide-generating system and human leukocytes

Phenol and 1-naphthol, products of benzene and naphthalene biotransformation, are metabolized during O2- generation by xanthine oxidase/hypoxanthine and phorbol myristate acetate (PMA)-stimulated human neutrophils. The addition of 1-naphthol to xanthine oxidase/hypoxanthine incubations resulted in the formation of 1,4-naphthoquinone (1,4-NQ) whereas phenol addition yielded only small quantities of hydroquinone, catechol and a unidentified reducible product but not 1,4-benzoquinone. This formation of 1,4-NQ was dependent upon hypoxanthine, xanthine oxidase, and 1-naphthol and was inhibited by the addition of superoxide dismutase (SOD) demonstrating that the conversion was O2-mediated. During O2- generation by PMA-stimulated neutrophils, the addition of phenol interfered with luminol-dependent chemiluminescence and resulted in covalent binding of phenol to protein. Protein binding was 80% inhibited by the addition of azide or catalase to the incubations indicating that bioactivation was peroxidase-mediated. In contrast, the addition of 1-naphthol to PMA-stimulated neutrophils interfered with superoxide-dependent cytochrome c reduction as well as luminol-dependent chemiluminescence and also resulted in protein binding. Protein binding was only partially inhibited by azide or catalase. The addition of SOD in combination with catalase resulted in a significantly greater inhibition of binding when compared to that of catalase alone. The results of these experiments indicate that phenol and 1-naphthol are converted to reactive metabolites during superoxide generating conditions but by different mechanisms. The formation of reactive metabolites from phenol was almost exclusively peroxidase-mediated whereas the bioactivation of 1-naphthol could occur by two different mechanisms, a peroxidase-dependent and a direct superoxide-dependent mechanism.     

Molecular insights into human monoamine oxidase (MAO) inhibition by 1,4-naphthoquinone: evidences for menadione (vitamin K3) acting as a competitive and reversible inhibitor of MAO

Monoamine oxidase (MAO) catalyzes the oxidative deamination of biogenic and exogenous amines and its inhibitors have therapeutic value for several conditions including affective disorders, stroke, neurodegenerative diseases and aging. The discovery of 2,3,6-trimethyl-1,4-naphthoquinone (TMN) as a nonselective and reversible inhibitor of MAO, has suggested 1,4-naphthoquinone (1,4-NQ) as a potential scaffold for designing new MAO inhibitors. Combining molecular modeling tools and biochemical assays we evaluate the kinetic and molecular details of the inhibition of human MAO by 1,4-NQ, comparing it with TMN and menadione. Menadione (2-methyl-1,4-naphthoquinone) is a multitarget drug that acts as a precursor of vitamin K and an inducer of mitochondrial permeability transition. Herein we show that MAO-B was inhibited competitively by 1,4-NQ (K_i = 1.4 μM) whereas MAO-A was inhibited by non-competitive mechanism (K_i = 7.7 μM). Contrasting with TMN and 1,4-NQ, menadione exhibited a 60-fold selectivity for MAO-B (K_i = 0.4 μM) in comparison with MAO-A (K_i = 26 μM), which makes it as selective as rasagiline. Fluorescence and molecular modeling data indicated that these inhibitors interact with the flavin moiety at the active site of the enzyme. Additionally, docking studies suggest the phenyl side groups of Tyr407 and Tyr444 (for MAO-A) or Tyr398 and Tyr435 (for MAO-B) play an important role in the interaction of the enzyme with 1,4-NQ scaffold through forces of dispersion as verified for menadione, TMN and 1,4-NQ. Taken together, our findings reveal the molecular details of MAO inhibition by 1,4-NQ scaffold and show for the first time that menadione acts as a competitive and reversible inhibitor of human MAO.     

Characterization of metabolic activation of pentachlorophenol to quinones and semiquinones in rodent liver

Pentachlorophenol (PCP), a widely used biocide, induces liver tumors in mice but not in rats. Metabolic activation of PCP to chlorinated quinones and semiquinones in liver cytosol from Sprague-Dawley rats and B6C3F1 mice was investigated in vitro (1) with microsomes in the presence of either beta-nicotinamide adenine dinucleotide phosphate (NADPH) or cumene hydroperoxide (CHP), (2) with CHP in the absence of microsomes, and (3) with horseradish peroxidase (HRP) and H2O2. Mono-S- and multi-S-substituted adducts of tetrachloro-1,4-benzoquinone (Cl4-1,4-BQ) and Cl4-1,2-BQ and their corresponding semiquinones [i.e. tetrachloro-1,4-benzosemiquinone (Cl4-1,4-SQ) and tetrachloro-1,2-benzosemiquinone (Cl4-1,2-SQ)] were measured by gas chromatography-mass spectrometry (GC-MS). Qualitatively, the metabolites of PCP were the same in both rats and mice for all activation systems. Induction of PCP metabolism by either 3MC or PB-treated microsomes was observed in NADPH- but not in CHP-supported systems. In rats, the amount of induction was comparable with either 3MC or PB. 3MC was a stronger inducer than PB in mice and also induced a greater amount of metabolism than in rats. This suggests that induction of specific P450 isozymes may play a role in the toxicity of PCP to mice. Both HRP/H2O2 and CHP led to production of the full spectrum of chlorinated quinones and semiquinones, confirming the direct oxidation of PCP. CHP (with or without microsomes) converted PCP into much greater quantities of quinones and semiquinones than did microsomal P450/NADPH or HRP/H2O2 in both species. This implies that, under conditions of oxidative stress, endogenous lipid hydroperoxides may increase PCP metabolism sufficiently to enhance the toxicity and carcinogenicity of PCP.     

A 50 Hz, 14 mT magnetic field is not mutagenic or co-mutagenic in bacterial mutation assays

We used bacterial mutation assays to assess the mutagenic and co-mutagenic effects of power frequency magnetic fields (MF). For the former, we exposed four strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and two strains of Escherichia coli (WP2 uvrA, WP2 uvrA/pKM101) to 50Hz, 14mT circularly polarized MF for 48h. All results were negative. For the latter, we treated S. typhimurium (TA98, TA100) and E. coli (WP2 uvrA, WP2 uvrA/pKM101) cells with eight model mutagens (N-ethyl-N'-nitro-N-nitrosoguanidine, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, 4-nitroquinoline-N-oxide, 2-aminoanthracene, N(4)-aminocytidine, t-butyl hydroperoxide, cumen hydroperoxide, and acridine orange) with and without the MF. The MF induced no significant, reproducible enhancement of mutagenicity. We also investigated the effect of MF on mutagenicity and co-mutagenicity of fluorescent light (ca. 900lx for 30min) with and without acridine orange on the most sensitive tester strain, E. coli WP2 uvrA/pKM101. Again, we observed no significant difference between the mutation rates induced with and without MF. Thus, a 50Hz, 14mT circularly polarized MF had no detectable mutagenic or co-mutagenic potential in bacterial tester strains under our experimental conditions. Nevertheless, some evidence supporting a mutagenic effect for power frequency MFs does exist; we discuss the potential mechanisms of such an effect in light of the present study and studies done by others.     

Interaction of cytochrome P-450 with a hydroperoxide derived from butylated hydroxytoluene. Mechanism of isomerization

Attack of O2 on the phenoxy radical derived from butylated hydroxytoluene resulted in the formation of 2,6-di-t-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BOOH). This hydroperoxide was rapidly consumed when incubated with rat liver microsomes in the absence of NADPH. The destruction of BOOH was accompanied by formation of the corresponding alcohol (BOH) and a derivative of the alcohol (B(OH)2) in which a t-butyl methyl group was hydroxylated. This diol was produced also when BOH was incubated with microsomes and NADPH, but at a slower rate. Mass spectral analyses of B(OH)2 formed from substrates labeled with either 2H or 18O, showed that oxygen was transferred from the peroxy group to a t-butyl group (via the heme iron of P-450) without migration of the intermediate alcohol from the enzyme active site. The results support a mechanism involving heterolytic O-O bond cleavage during isomerization of the hydroperoxide to B(OH)2. The chiral diol was produced from BOOH nonstereoselectively, but the NADPH/O2-supported hydroxylation of BOH resulted in the formation of a 20% excess of one enantiomer of B(OH)2. Analyses of products formed from the interaction of cumene hydroperoxide with cytochrome P-450 showed that this substrate undergoes rearrangement also; 2-phenyl-1,2-propanediol was produced, together with cumyl alcohol and acetophenone. These results indicate that isomerization competes with other pathways of hydroperoxide destruction by cytochrome P-450.     

Assessment of Escherichia coli B with enhanced permeability to fluorochromes for flow cytometric assays of bacterial cell function

BACKGROUND: Flow cytometry has become a choice methodology for microbiological research. However, functional cytometric assays in live bacteria are still limited. This is due, in part, to the cell wall impairing penetration of vital dyes in bacteria, thus imposing permeabilization procedures. These manipulations may affect cell physiology, provoke cell aggregation or lysis, and they are time-consuming. Escherichia coli B strains have been used for mutagenic assays because of an altered lipopolysaccharide that provokes increased membrane permeability. We assessed the use of these strains as possible alternatives for flow cytometric assays to avoid the permeabilization steps. METHODS: Suspensions of E. coli K-12 (strain AB1157) and E. coli B (strain WP2 uvrA/pKM101, denoted as strain IC188) were stained with several fluorochromes, including fluorescein isothiocyanate, propidium iodide, Nile Red, bis-(1,3-dibutylbarbituric acid) trimethine oxonol, hydroethidine, and dihydro-dichlorofluorescein diacetate, under basal conditions and following permeabilization, impairment of membrane potential, inhibition of dye efflux pump, and oxidative stress. Fluorescent staining of both strains was compared by epifluorescence microscopy and flow cytometry. RESULTS: The E. coli B strain IC188 exhibited more efficient staining with vital fluorochromes than the E. coli K-12 strain AB1157 and maintained a similar membrane potential. In addition, IC188 showed higher sensitivity than AB1157 to reveal oxidative stress when challenged with prooxidants. CONCLUSIONS: E. coli B strains may be useful for biochemical and toxicological studies based on flow cytometry and fluorescence microscopy.     

Interaction of camel lens zeta-crystallin with quinones: portrait of a substrate by fluorescence spectroscopy.

Interaction of camel lens zeta-crystallin, an NADPH:quinone oxidoreductase, with several quinone derivatives was examined by fluorescence spectroscopy and activity measurements. Fluorescence of zeta-crystallin was quenched to different levels by the different quinones:juglone (5-OH, 1,4 naphthoquinone), 1,4 naphthoquinone (1,4-NQ), and 1,2 naphthoquinone (1,2-NQ) considerably quenched the fluorescence of zeta-crystallin, where as the commonly used substrate, 9,10-phenanthrenequinone (PQ) did not induce significant quenching. Activity measurements showed only PQ served as a substrate for camel lens zeta-crystallin, while juglone, 1,4-NQ, and 1,2-NQ were inhibitors. Thus quinones that interacted with zeta-crystallin directly inhibited the enzyme, whereas the substrate had very low affinity for the enzyme in the absence of NADPH. Another substrate, dichlorophenol indophenol (DCIP), conformed to the same pattern; DCIP did not quench the fluorescence of the enzyme significantly, but served as a substrate. This pattern is consistent with an ordered mechanism of catalysis with quinone being the second substrate. All three naphthoquinones were uncompetitive inhibitors with respect to NADPH and noncompetitive with respect to PQ. These kinetics are similar to those exhibited by cysteine- and/or lysine-modifying agents. Juglone, 1,4-NQ, and 1,2-NQ interacted with and quenched the fluorescence of camel lens alpha-crystallin, but to lesser extent than that of zeta-crystallin.     

Reaction products of 1-naphthol with reactive oxygen species prevent NADPH oxidase activation in activated human neutrophils, but leave phagocytosis intact

Activation of human neutrophils with opsonized particles in the presence of a nontoxic dose of 1-naphthol resulted in inhibition of superoxide anion production but not of the phagocytotic activity of the cells. In this study we have investigated the mechanism of action of 1-naphthol. The inhibition is not at the level of cellular activation since the FMLP-induced rise of intracellular free calcium was unaffected. Our results show that the (metabolic) activation of 1-naphthol to 1,4-naphthoquinone by reaction with H2O2 from the oxidative burst is a necessary event for the inhibition to occur. The study provides evidence that by its reactivity with essential thiol groups 1,4-naphthoquinone (1,4-NQ) prevents the assembly of a functional NADPH-oxidase in the neutrophil membrane.     

A novel and potent biological antioxidant, Kinobeon A, from cell culture of safflower

Kinobeon A was originally isolated from cultured cells of safflower (Carthamus tinctorius L., Compositae). It had never previously been directly isolated from safflower or other plants, animals or microorganisms. In this report, we demonstrate the anti-oxidative effects of kinobeon A and compare the results with those two known natural antioxidants, lignan (nordihydroguaiaretic acid) and quercetin. The NADPH-induced microsomal lipid peroxidation system was employed to assess anti-oxidative effects of kinobeon A. Addition of kinobeon A to the system significantly decreased the formation of thiobarbituric acid reactive substances (TBARS) in a dose-dependent manner with effects similar to those of lignan and quercetin. Formation of TBARS was completely inhibited at 10 microM of kinobeon A. Employing the xanthine/xanthine oxidase/nitroblue tetrazolium system and the KO2/XTT system, the superoxide anion scavenging activity of kinobeon A was greater than that of lignan or quercetin. IC50 values calculated for kinobeon A in these two systems were 1 microM and 0.8 microM, respectively. Kinobeon A exerted cytoprotective effects following oxidative treatments with hydrogen peroxide, cumene hydroperoxide, menadione and xanthine oxidase (XOD). Addition of kinobeon A to the systems markedly enhanced survival ratios of Madin-Darby bovine kidney cells, while their survival significantly decreased with the oxidative treatment alone. Kinobeon A exhibited stronger effect on the cell viability than lignan or quercetin when menadion or XOD were used as inducing reagents of oxidative stress. The present study demonstrates for the first time that kinobeon A prevents oxidative stresses and could be a useful cytoprotective reagent.