A potential mechanism underlying the increased susceptibility of individuals with a polymorphism in NAD(P)H:quinone oxidoreductase 1 (NQO1) to benzene toxicity.
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NAD(P)H:quinone oxidoreductase 1 (NQO1) is a two-electron reductase that detoxifies quinones derived from the oxidation of phenolic metabolites of benzene. A polymorphism in NQO1, a C609T substitution, has been identified, and individuals homozygous for this change (T/T) have no detectable NQO1. Exposed workers with a T/T genotype have an increased risk of benzene hematotoxicity. This finding suggests NQO1 is protective against benzene toxicity, which is difficult to reconcile with the lack of detectable NQO1 in human bone marrow. The human promyeloblastic cell line, KG-1a, was used to investigate the ability of the benzene metabolite hydroquinone (HQ) to induce NQO1. A concentration-dependent induction of NQO1 protein and activity was observed in KG-1a cells cultured with HQ. Multiple detoxification systems, including NQO1 and glutathione protect against benzene metabolite-induced toxicity. Indeed, exposure to a noncytotoxic concentration of HQ induced both NQO1 and soluble thiols and protected against HQ-induced apoptosis. NQO1 protein and activity increased in wild-type human bone marrow cells (C/C) exposed to HQ, whereas no NQO1 was induced by HQ in bone marrow cells with the T/T genotype. Intermediate induction of NQO1 by HQ was observed in heterozygous bone marrow cells (C/T). NQO1 also was induced by HQ in wild-type (C/C) human bone marrow CD34(+) progenitor cells. Our data suggest that failure to induce functional NQO1 may contribute to the increased risk of benzene poisoning in individuals homozygous for the NQO1 C609T substitution (T/T). (+info)
The functions of the flavin contact residues, alphaArg249 and betaTyr16, in human electron transfer flavoprotein.
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Arg249 in the large (alpha) subunit of human electron transfer flavoprotein (ETF) heterodimer is absolutely conserved throughout the ETF superfamily. The guanidinium group of alphaArg249 is within van der Waals contact distance and lies perpendicular to the xylene subnucleus of the flavin ring, near the region proposed to be involved in electron transfer with medium chain acyl-CoA dehydrogenase. The backbone amide hydrogen of alphaArg249 is within hydrogen bonding distance of the carbonyl oxygen at the flavin C(2). alphaArg249 may modulate the potentials of the two flavin redox couples by hydrogen bonding the carbonyl oxygen at C(2) and by providing delocalized positive charge to neutralize the anionic semiquinone and anionic hydroquinone of the flavin. The potentials of the oxidized/semiquinone and semiquinone/hydroquinone couples decrease in an alphaR249K mutant ETF generated by site directed mutagenesis and expression in Escherichia coli, without major alterations of the flavin environment as judged by spectral criteria. The steady state turnover of medium chain acyl-CoA dehydrogenase and glutaryl-CoA dehydrogenase decrease greater than 90% as a result of the alphaR249Ks mutation. In contrast, the steady state turnover of short chain acyl-CoA dehydrogenase was decreased about 38% when alphaR249K ETF was the electron acceptor. Stopped flow absorbance measurements of the oxidation of reduced medium chain acyl-CoA dehydrogenase/octenoyl-CoA product complex by wild type human ETF at 3 degrees C are biphasic (t(1/2)=12 ms and 122 ms). The rate of oxidation of this reduced binary complex of the dehydrogenase by the alphaR249K mutant ETF is extremely slow and could not be reasonably estimated. alphaAsp253 is proposed to function with alphaArg249 in the electron transfer pathway from medium chain acyl-CoA dehydrogenase to ETF. The steady state kinetic constants of the dehydrogenase were not altered when ETF containing an alphaD253A mutant was the substrate. However, t(1/2) of the rapid phase of oxidation of the reduced medium chain acyl-CoA dehydrogenase/octenoyl-CoA charge transfer complex almost doubled. betaTyr16 lies on a loop near the C(8) methyl group, and is also near the proposed site for interflavin electron transfer with medium chain acyl-CoA dehydrogenase. The tyrosine residue makes van der Waals contact with the C(8) methyl group of the flavin in human ETF and Paracoccus denitrificans ETF (as betaTyr13) and lies at a 30 degrees C angle with the plane of the flavin. Human betaTyr16 was substituted with leucine and alanine residues to investigate the role of this residue in the modulation of the flavin redox potentials and in electron transfer to ETF. In betaY16L ETF, the potentials of the flavin were slightly reduced, and steady state kinetic constants were modestly altered. Substitution of an alanine residue for betaTyr16 yields an ETF with potentials very similar to the wild type but with steady state kinetic properties similar to betaY16L ETF. It is unlikely that the beta methyl group of the alanine residue interacts with the flavin C(8) methyl. Neither substitution of betaTyr16 had a large effect on the fast phase of ETF reduction by medium chain acyl-CoA dehydrogenase. (+info)
Quinol-glutathione conjugate-induced mutation spectra in the supF gene replicated in human AD293 cells and bacterial MBL50 cells.
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Hydroquinone is a nephrocarcinogen in rats but generally tests negative in standard mutagenicity assays. However, 2,3,5-tris-(glutathion-S-yl)hydroquinone, a potent nephrotoxic metabolite of hydroquinone, and 2-bromo-bis-(glutathion-S-yl)hydroquinone, another cytotoxic quinol-glutathione (GSH) conjugate, cause extensive single strand breaks in DNA in a manner that is dependent on the formation of reactive oxygen species. We, therefore, investigated whether quinol-GSH conjugates have the potential to behave as genotoxicants. The shuttle vector pSP189, containing the supF gene, was treated with 2,3,5-tris-(glutathion-S-yl)hydroquinone and replicated in both human AD293 cells and Escherichia coli MBL50 cells. The mutation frequency increased 4.6- and 2.6-fold in human AD293 and bacterial MBL50 cells, respectively. Base substitutions were the major type of mutations, and they occurred predominantly at G:C sites in both cell types. A high frequency of deletions (30%), including < 10- and > 10-bp deletions, were observed in AD293-replicated plasmids. The most common types of mutations in AD293 cells were G:C to A:T transitions (33.8%) and G:C to T:A (29.4%) and G:C to C:G (19.1%) transversions. In MBL50 cells, the major mutations were G:C to T:A (33.8%) and G:C to C:G (31.3%) transversions and G:C to A:T transitions (27.5%). The mutation spectra were similar to those reported for *OH-induced mutations, suggesting that *OH generated from polyphenolic-GSH conjugates not only plays a role in cytotoxicity but also provides a basis for their mutagenicity and carcinogenicity. (+info)
Pathways for proton release during ubihydroquinone oxidation by the bc(1) complex.
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Quinol oxidation by the bc(1) complex of Rhodobacter sphaeroides occurs from an enzyme-substrate complex formed between quinol bound at the Q(o) site and the iron-sulfur protein (ISP) docked at an interface on cytochrome b. From the structure of the stigmatellin-containing mitochondrial complex, we suggest that hydrogen bonds to the two quinol hydroxyl groups, from Glu-272 of cytochrome b and His-161 of the ISP, help to stabilize the enzyme-substrate complex and aid proton release. Reduction of the oxidized ISP involves H transfer from quinol. Release of the proton occurs when the acceptor chain reoxidizes the reduced ISP, after domain movement to an interface on cytochrome c(1). Effects of mutations to the ISP that change the redox potential and/or the pK on the oxidized form support this mechanism. Structures for the complex in the presence of inhibitors show two different orientations of Glu-272. In stigmatellin-containing crystals, the side chain points into the site, to hydrogen bond with a ring hydroxyl, while His-161 hydrogen bonds to the carbonyl group. In the native structure, or crystals containing myxothiazol or beta-methoxyacrylate-type inhibitors, the Glu-272 side chain is rotated to point out of the site, to the surface of an external aqueous channel. Effects of mutation at this residue suggest that this group is involved in ligation of stigmatellin and quinol, but not quinone, and that the carboxylate function is essential for rapid turnover. H(+) transfer from semiquinone to the carboxylate side chain and rotation to the position found in the myxothiazol structure provide a pathway for release of the second proton. (+info)
Elevation of the cytosolic free [Ca2+] is indispensable for the transduction of the Nod factor signal in alfalfa.
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In root hairs of alfalfa (Medicago sativa), the requirement of Ca(2+) for Nod factor signaling has been investigated by means of ion-selective microelectrodes. Measured 50 to 100 microm behind the growing tip, 0.1 microM NodRm-IV(C16:2,S) increased the cytosolic free [Ca2+] by about 0.2 pCa, while the same concentration of chitotetraose, the nonactive glucosamine backbone, had no effect. We demonstrate that NodRm-IV(C16:2,S) still depolarized the plasma membrane at external Ca(2+) concentrations below cytosolic values if the free EGTA concentration remained low (+info)
Role of a mitogen-activated protein kinase pathway in the induction of phase II detoxifying enzymes by chemicals.
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Mitogen-activated protein kinase (MAPK) cascades are activated by diverse extracellular signals and participate in the regulation of an array of cellular programs. In this study, we investigated the roles of MAPKs in the induction of phase II detoxifying enzymes by chemicals. Treatment of human hepatoma (HepG2) and murine hepatoma (Hepa1c1c7) cells with tert-butylhydroquinone (tBHQ) or sulforaphane (SUL), two potent phase II enzyme inducers, stimulated the activity of extracellular signal-regulated protein kinase 2 (ERK2) but not c-Jun N-terminal kinase 1. tBHQ and SUL also activated MAPK kinase. Inhibition of MAPK kinase with its inhibitor, PD98059, abolished ERK2 activation and impaired the induction of quinone reductase, a phase II detoxifying enzyme, and antioxidant response element (ARE)-linked reporter gene by tBHQ and SUL. Overexpression of a dominant-negative mutant of ERK2 also attenuated tBHQ and SUL induction of ARE reporter gene activity. Interestingly, although expression of Ras and its mutant forms showed distinct effects on basal ARE reporter gene activity, they did not affect the activation of reporter gene by the inducers. Furthermore, a dominant-negative mutant of Ras had little effect on ERK2 activation by tBHQ and SUL, implicating a Ras-independent mechanism. Indeed, both tBHQ and SUL were able to stimulate Raf-1 kinase activity in vivo as well as in vitro. Thus, our results indicate that the induction of ARE-dependent phase II detoxifying enzymes is mediated by a MAPK pathway, which may involve direct activation of Raf-1 by the inducers. (+info)
Pro-oxidant challenge in vivo provokes the onset of NIDDM in the insulin resistant obese Zucker rat.
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We investigated the ability of an acute pro-oxidant challenge in vivo to deteriorate glycaemic control and insulin action in the obese Zucker rat, a model of insulin resistance associated with oxidant stress. In obese animals, the daily administration for 1 week of hydroquinone (HQ) in combination with L-buthionine sulphoximine (BSO), elevated fasting plasma glycaemia and insulinaemia and markedly aggravated i.v. glucose-stimulated hyperinsulinaemia without significantly affecting i.v. glucose tolerance, suggesting exacerbated insulin resistance. Intermediate effects on hyperinsulinaemia in obese animals were determined with HQ treatment alone while BSO treatment alone had no effect. In contrast, none of the pro-oxidant treatments affected age-matched, insulin sensitive, lean Zucker rats. Our data therefore demonstrate for the first time, a vulnerability to deterioration in insulin action in an established insulin resistant state following an environmental pro-oxidative insult. This may have relevance in the conversion of insulin resistance syndrome (IRS) to non-insulin dependent diabetes mellitus (NIDDM). (+info)
PcpA, which is involved in the degradation of pentachlorophenol in Sphingomonas chlorophenolica ATCC39723, is a novel type of ring-cleavage dioxygenase.
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The pentachlorophenol (PCP) mineralizing bacterium Sphingomonas chlorophenolica ATCC39723 degrades PCP via 2,6-dichlorohydroquinone (2,6-DCHQ). The pathway converting PCP to 2,6-DCHQ has been established previously; however, the pathway beyond 2,6-DCHQ is not clear, although it has been suggested that a PcpA plays a role in 2, 6-DCHQ conversion. In this study, PcpA expressed in Escherichia coli was purified to homogeneity and shown to have novel ring-cleavage dioxygenase activity in conjunction with hydroquinone derivatives, and converting 2,6-DCHQ to 2-chloromaleylacetate. (+info)