Molecular mechanism of the metal-independent production of hydroxyl radicals by thiourea dioxide and H2O2.

It is well-known that highly reactive hydroxyl radicals (HO•) can be produced by the classic Fenton system and our recently discovered haloquinone/H2O2 system, but rarely from thiol-derivatives. Here, we found, unexpectedly, that HO• can be generated from H2O2 and thiourea dioxide (TUO2), a widely used and environmentally friendly bleaching agent. A carbon-centered radical and sulfite were detected and identified as the transient intermediates, and urea and sulfate as the final products, with the complementary application of electron spin-trapping, oxygen-18 isotope labeling coupled with HPLC/MS analysis. Density functional theory calculations were conducted to further elucidate the detailed pathways for HO• production. Taken together, we proposed that the molecular mechanism for HO• generation by TUO2/H2O2: TUO2 tautomerizes from sulfinic acid into ketone isomer (TUO2-K) through proton transfer, then a nucleophilic addition of H2O2 on the S atom of TUO2-K, forming a S-hydroperoxide intermediate TUO2-OOH, which dissociates homolytically to produce HO•. Our findings represent the first experimental and computational study on an unprecedented new molecular mechanism of HO• production from simple thiol-derived sulfinic acids, which may have broad chemical, environmental, and biomedical significance for future research on the application of the well-known bleaching agent and its analogs.

Unusual enantioselective cytoplasm-to-nucleus translocation and photosensitization of the chiral Ru(II) cationic complex via simple ion-pairing with lipophilic weak acid counter-anions.

Targeted and enantioselective delivery of chiral diagnostic-probes and therapeutics into specific compartments inside cells is of utmost importance in the improvement of disease detection and treatment. The classical DNA 'light-switch' ruthenium(II)-polypyridyl complex, [Ru(DIP)2(dppz)]Cl2 (DIP = 4,7-diphenyl-1,10-phenanthroline, dppz = dipyridophenazine) has been shown to be accumulated only in the cytoplasm and membrane, but excluded from its intended nuclear DNA target. In this study, the cationic [Ru(DIP)2(dppz)]2+ is found to be redirected into live-cell nucleus in the presence of lipophilic 3,5-dichlorophenolate or flufenamate counter-anions via ion-pairing mechanism, while maintaining its original DNA recognition characteristics. Interestingly and unexpectedly, further studies show that only the Δ-enantiomer is selectively translocated into nucleus while the Λ-enantiomer remains trapped in cytoplasm, which is found to be mainly due to their differential enantioselective binding affinities with cytoplasmic proteins and nuclear DNA. More importantly, only the nucleus-relocalized Δ-enantiomer can induce obvious DNA damage and cell apoptosis upon prolonged visible-light irradiation. Thus, the use of Δ-enantiomer can significantly reduce the dosage needed for maximal treatment effect. This represents the first report of enantioselective targeting and photosensitization of classical Ru(II) complex via simple ion-pairing with suitable weak acid counter-anions, which opens new opportunities for more effective enantioselective cancer treatment.

Unprecedented enantio-selective live-cell mitochondrial DNA super-resolution imaging and photo-sensitizing by the chiral ruthenium polypyridyl DNA "light-switch".

Mitochondrial DNA (mtDNA) is known to play a critical role in cellular functions. However, the fluorescent probe enantio-selectively targeting live-cell mtDNA is rare. We recently found that the well-known DNA 'light-switch' [Ru(phen)2dppz]Cl2 can image nuclear DNA in live-cells with chlorophenolic counter-anions via forming lipophilic ion-pairing complex. Interestingly, after washing with fresh-medium, [Ru(phen)2dppz]Cl2 was found to re-localize from nucleus to mitochondria via ABC transporter proteins. Intriguingly, the two enantiomers of [Ru(phen)2dppz]Cl2 were found to bind enantio-selectively with mtDNA in live-cells not only by super-resolution optical microscopy techniques (SIM, STED), but also by biochemical methods (mitochondrial membrane staining with Tomo20-dronpa). Using [Ru(phen)2dppz]Cl2 as the new mtDNA probe, we further found that each mitochondrion containing 1-8 mtDNA molecules are distributed throughout the entire mitochondrial matrix, and there are more nucleoids near nucleus. More interestingly, we found enantio-selective apoptotic cell death was induced by the two enantiomers by prolonged visible light irradiation, and in-situ self-monitoring apoptosis process can be achieved by using the unique 'photo-triggered nuclear translocation' property of the Ru complex. This is the first report on enantio-selective targeting and super-resolution imaging of live-cell mtDNA by a chiral Ru complex via formation and dissociation of ion-pairing complex with suitable counter-anions.

Detection in Orchards of Predominant Azole-Resistant Candida tropicalis Genotype Causing Human Candidemia, Taiwan.

Fluconazole-resistant clade 4 Candida tropicalis causing candidemia in humans has been detected in tropical/subtropical areas, including those in China, Singapore, and Australia. We analyzed 704 individual yeasts isolated from fruits, soil, water, and farmers at 80 orchards in Taiwan. The most common pathogenic yeast species among 251 isolates recovered from farmers were Candida albicans (14.7%) and C. parapsilosis (11.6%). In contrast, C. tropicalis (13.0%), C. palmioleophila (6.6%), and Pichia kudriavzevii (6.0%) were prevalent among 453 environmental isolates. Approximately 18.6% (11/59) of C. tropicalis from the environment were resistant to fluconazole, and 81.8% (9/11) of those belonged to the clade 4 genotype. C. tropicalis susceptibility to fluconazole correlated with susceptibilities to the agricultural azole fungicides, difenoconazole, tebuconazole, and triadimenol. Tandem gene duplications of mutated ERG11 contributed to azole resistance. Agriculture environments are a reservoir for azole-resistant C. tropicalis; discontinuing agricultural use of azoles might reduce emergence of azole-resistant Candida spp. strains in humans.

Molecular mechanism for the unusual enhancement of the second-step chemiluminescence production from the carcinogenic tetrabromohydroquinone and H2O2.

We have found recently that two-step intrinsic hydroxyl radical (·OH)-dependent chemiluminescence (CL) could be produced by carcinogenic tetrahaloquinone and H2O2. However, the first-step CL was too fast to clearly detect the stepwise generation of ·OH and CL, and to distinguish the exact dividing point between the first-step and second-step CL. Here we found that, extremely clear two-step intrinsic CL could be produced by the relative slow reaction of tetrabromohydroquinone (TBHQ) with H2O2, which was directly dependent on the two-step ·OH generation. Interestingly, the second-step, but not the first-step CL production of TBHQ/H2O2 (CRET donor) was markedly enhanced by fluorescein (a typical xanthene dye, CRET acceptor) through a unique chemiluminescence resonance energy transfer (CRET) process. The novel CRET system of TBHQ/H2O2/fluorescein was successfully applied for the sensitive detection of TBHQ with the detection limit as low as 2.5 µmol/L. These findings will help to develop more sensitive and highly efficient CL or CRET systems and specific CL sensor to detect the carcinogenic haloquinones, which may have broad environmental applications.

Mechanistic Study on Oxidative DNA Damage and Modifications by Haloquinoid Carcinogenic Intermediates and Disinfection Byproducts.

Haloquinones (XQs) are a group of carcinogenic intermediates of the haloaromatic environmental pollutants and newly identified chlorination disinfection byproducts (DBPs) in drinking water. The highly reactive hydroxyl radicals/alkoxyl radicals and quinone enoxy/ketoxy radicals were found to arise in XQs and H2O2 or organic hydroperoxides system, independent of transition-metal ions. However, it was not clear whether these haloquinoid carcinogens and hydroperoxides can cause oxidative DNA damage and modifications, and if so, what are the underlying molecular mechanisms. We found that 8-oxodeoxyguanosine (8-oxodG), DNA strand breaks, and three methyl oxidation products could arise when DNA was treated with tetrachloro-1,4-benzoquinone and H2O2 via a metal-independent and intercalation-enhanced oxidation mechanism. Similar effects were observed with other XQs, which are generally more efficient than the typical Fenton system. We further extended our studies from isolated DNA to genomic DNA in living cells. We also found that potent oxidation of DNA to the more mutagenic imidazolone dIz could be induced by XQs and organic hydroperoxides such as t-butylhydroperoxide or the physiologically relevant hydroperoxide 13S-hydroperoxy-9Z,11E-octadecadienoic acid via an unprecedented quinone-enoxy radical-mediated mechanism. These findings should provide new perspectives to explain the potential genotoxicity, mutagenesis, and carcinogenicity for the ubiquitous haloquinoid carcinogenic intermediates and DBPs.

Structure-Activity Relationship Investigation on Reaction Mechanism between Chlorinated Quinoid Carcinogens and Clinically-Used Aldoxime Nerve-Agent Antidote under Physiological Condition.

Pyridinium aldoximes are best-known therapeutic antidotes used for clinical treatment of poisonings by organophosphorus nerve-agents and pesticides. Recently, we found that pralidoxime (2-PAM, a currently clinically used nerve-agent antidote) could also detoxify tetrachloro-1,4-benzoquinone (TCBQ), which is a carcinogenic quinoid metabolite of the widely used wood preservative pentachlorophenol under normal physiological conditions, via an unusually mild and facile Beckmann fragmentation mechanism accompanied by radical homolysis. However, it is not clear whether the less-chlorinated benzoquinones (CnBQs, n ≤ 3) act similarly; if so, what is the structure-activity relationship? In this study, we found that (1) The stability of reaction intermediates produced by different CnBQs and 2-PAM was dependent not only on the position but also the degree of Cl-substitution on CnBQs, which can be divided into TCBQ- and DCBQ (dichloro-1,4-benzoquinone)-subgroup; (2) The pKa value of hydroxlated quinones (Cn-1BQ-OHs, the hydrolysis products of CnBQs), determined the stability of corresponding intermediates, that is, the decomposition rate of the intermediates depended on the acidity of Cn-1BQ-OHs; (3) The pKa value of the corresponding Cn-1BQ-OHs could also determine the reaction ratio of Beckmann fragmentation to radical homolysis in CnBQs/2-PAM. These new findings on the structure-activity relationship of the halogenated quinoid carcinogens detoxified by pyridinium aldoxime therapeutic agents via Beckmann fragmentation and radical homolysis reaction may have broad implications on future biomedical and environmental research.

Sulfur-centered hemi-bond radicals as active intermediates in S-DNA phosphorothioate oxidation.

Phosphorothioate (PS) modifications naturally appear in bacteria and archaea genome and are widely used as antisense strategy in gene therapy. But the chemical effects of PS introduction as a redox active site into DNA (S-DNA) is still poorly understood. Herein, we perform time-resolved spectroscopy to examine the underlying mechanisms and dynamics of the PS oxidation by potent radicals in free model, in dinucleotide, and in S-oligomer. The crucial sulphur-centered hemi-bonded intermediates -P-S∴S-P- were observed and found to play critical roles leading to the stable adducts of -P-S-S-P-, which are backbone DNA lesion products. Moreover, the oxidation of the PS moiety in dinucleotides d[GPSG], d[APSA], d[GPSA], d[APSG] and in S-oligomers was monitored in real-time, showing that PS oxidation can compete with adenine but not with guanine. Significantly, hole transfer process from A+• to PS and concomitant -P-S∴S-P- formation was observed, demonstrating the base-to-backbone hole transfer unique to S-DNA, which is different from the normally adopted backbone-to-base hole transfer in native DNA. These findings reveal the distinct backbone lesion pathway brought by the PS modification and also imply an alternative -P-S∴S-P-/-P-S-S-P- pathway accounting for the interesting protective role of PS as an oxidation sacrifice in bacterial genome.

Targeted live-cell nuclear delivery of the DNA 'light-switching' Ru(II) complex via ion-pairing with chlorophenolate counter-anions: the critical role of binding stability and lipophilicity of the ion-pairing complexes.

We have found recently that nuclear uptake of the cell-impermeable DNA light-switching Ru(II)-polypyridyl cationic complexes such as [Ru(bpy)2(dppz)]Cl2 was remarkably enhanced by pentachlorophenol (PCP), by forming ion-pairing complexes via a passive diffusion mechanism. However, it is not clear whether the enhanced nuclear uptake of [Ru(bpy)2(dppz)]2+ is only limited to PCP, or it is a general phenomenon for other highly chlorinated phenols (HCPs); and if so, what are the major physicochemical factors in determining nuclear uptake? Here, we found that the nuclear uptake of [Ru(bpy)2(dppz)]2+ can also be facilitated by other two groups of HCPs including three tetrachlorophenol (TeCP) and six trichlorophenol (TCP) isomers. Interestingly and unexpectedly, 2,3,4,5-TeCP was found to be the most effective one for nuclear delivery of [Ru(bpy)2(dppz)]2+, which is even better than the most-highly chlorinated PCP, and much better than its two other TeCP isomers. Further studies showed that the nuclear uptake of [Ru(bpy)2(dppz)]2+ was positively correlated with the binding stability, but to our surprise, inversely correlated with the lipophilicity of the ion-pairing complexes formed between [Ru(bpy)2(dppz)]Cl2 and HCPs. These findings should provide new perspectives for future investigations on using ion-pairing as an effective method for delivering other bio-active metal complexes into their intended cellular targets.

Detecting and Quantifying Polyhaloaromatic Environmental Pollutants by Chemiluminescence-Based Analytical Method.

Polyhaloaromatic compounds (XAr) are ubiquitous and recalcitrant in the environment. They are potentially carcinogenic to organisms and may induce serious risks to the ecosystem, raising increasing public concern. Therefore, it is important to detect and quantify these ubiquitous XAr in the environment, and to monitor their degradation kinetics during the treatment of these recalcitrant pollutants. We have previously found that unprecedented intrinsic chemiluminescence (CL) can be produced by a haloquinones/H2O2 system, a newly-found ●OH-generating system different from the classic Fenton system. Recently, we found that the degradation of priority pollutant pentachlorophenol by the classic Fe(II)-Fenton system could produce intrinsic CL, which was mainly dependent on the generation of chloroquinone intermediates. Analogous effects were observed for all nineteen chlorophenols, other halophenols and several classes of XAr, and a novel, rapid and sensitive CL-based analytical method was developed to detect these XAr and monitor their degradation kinetics. Interestingly, for those XAr with halohydroxyl quinoid structure, a Co(II)-mediated Fenton-like system could induce a stronger CL emission and higher degradation, probably due to site-specific generation of highly-effective ●OH. These findings may have broad chemical and environmental implications for future studies, which would be helpful for developing new analytical methods and technologies to investigate those ubiquitous XAr.

Unusual Two-Step Claisen-type Rearrangement Reaction under Physiological Conditions.

N-aryl hydroxamic acids, which are best known for their metal-chelating properties in chemical and biomedical research, have been found to markedly detoxify carcinogenic halogenated quinones. However, the exact chemical mechanism underlying such detoxication remains unclear. Here, we show that a very fast reaction took place between N-phenylbenzohydroxamic acid (N-PhBHA) and 2,5-dichloro-1,4-benzoquinone (DCBQ), forming an unexpected new carbon-carbon bonding phenyl-quinone product with high yield. In contrast, no reaction was observed with O-benzoyl N-PhBHA. Analogous results were observed for other N-aryl hydroxamic acids and halogenated quinones, which have an ortho-hydrogen adjacent to the reaction site (DCBQ-type). Interestingly, no free radical intermediates could be detected by both ESR spin-trapping and radical-scavenging methods during the reaction process. Taken together, we proposed that nucleophilic substitution followed by an unusual two-step Claisen-type rearrangement reaction was responsible for the formation of a new C-C bonding compound and the detoxication reaction. This represents the first report of an unusually mild and facile two-step Claisen-type rearrangement, which could take place under normal physiological conditions.

First Direct and Unequivocal Electron Spin Resonance Spin-Trapping Evidence for pH-Dependent Production of Hydroxyl Radicals from Sulfate Radicals.

Recently, the sulfate radical (SO4•-) has been found to exhibit broad application prospects in various research fields such as chemical, biomedical, and environmental sciences. It has been suggested that SO4•- could be transformed into a more reactive hydroxyl radical (•OH); however, no direct and unequivocal experimental evidence has been reported yet. In this study, using an electron spin resonance (ESR) secondary radical spin-trapping method coupled with the classic spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and the typical •OH-scavenging agent dimethyl sulfoxide (DMSO), we found that •OH can be produced from three SO4•--generating systems from weakly acidic (pH = 5.5) to alkaline conditions (optimal at pH = 13.0), while SO4•- is the predominant radical species at pH < 5.5. A comparative study with three typical •OH-generating systems strongly supports the above conclusion. This is the first direct and unequivocal ESR spin-trapping evidence for •OH formation from SO4•- over a wide pH range, which is of great significance to understand and study the mechanism of many SO4•--related reactions and processes. This study also provides an effective and direct method for unequivocally distinguishing •OH from SO4•-.

Potent Oxidation of DNA by Haloquinoid Disinfection Byproducts to the More Mutagenic Imidazolone dIz via an Unprecedented Haloquinone-Enoxy Radical-Mediated Mechanism.

Halogenated quinones are a class of carcinogenic intermediates and newly identified chlorination disinfection byproducts in drinking water. We found recently that halogenated quinones could enhance the decomposition of hydroperoxides independent of transition-metal ions and formation of the novel quinone enoxy/ketoxy radicals. Here, we show that the major oxidation product was 2-amino-5-[(2-deoxy-ß-d-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (dIz) when the nucleoside 2'-deoxyguanosine (dG) was treated with tetrachloro-1,4-benzoquinone (TCBQ) and t-butyl hydroperoxide (t-BuOOH). The formation of dIz was markedly inhibited by typical radical spin-trapping agents. Interestingly and unexpectedly, we found that the generated quinone enoxy radical played a critical role in dIz formation. Using [15N5]-8-oxodG, dIz was found to be produced either directly from dG or through the transient formation of 8-oxodG. Based on these data, we proposed that the production of dIz might be through an unusual haloquinone-enoxy radical-mediated mechanism. Analogous results were observed in the oxidation of ctDNA by TCBQ/t-BuOOH and when t-BuOOH was substituted by the endogenously generated physiologically relevant hydroperoxide 13S-hydroperoxy-9Z,11E-octadecadienoic acid. This is the first report that halogenated quinoid carcinogens and hydroperoxides can induce potent oxidation of dG to the more mutagenic product dIz via an unprecedented quinone-enoxy radical-mediated mechanism, which may partly explain their potential carcinogenicity.

Mechanism of unprecedented hydroxyl radical production and site-specific oxidative DNA damage by photoactivation of the classic arylhydroxamic acid carcinogens.

The carcinogenicity of N-hydroxy-2-acetamidofluorene (N-OHAAF), the major genotoxic metabolite of the classic model aromatic amine (AA) carcinogen 2-acetylaminofluorene, has been attributed mainly to the formation of DNA adducts via arylnitrenium upon enzymatic activation. Here, we show, unexpectedly, that exposure of N-OHAAF to UV or sunlight irradiation can not only induce the formation of the well-known covalent DNA adducts, but, more interestingly, simultaneous generation of oxidative DNA damage was also observed as measured by the formation of DNA single-/double-strand breaks (SSBs/DSBs) and 8-oxo-2'-deoxyguanosine (8-oxodG), which were partly inhibited by the typical hydroxyl radical (•OH) scavengers. Electron spin resonance spin-trapping and fluorescent studies unequivocally confirmed that the highly reactive •OH was generated from photolysis of N-OHAAF. Further DNA sequencing investigations suggest that photoactivation of N-OHAAF caused preferential cleavage at guanine, thymine and cytosine sites. More importantly, the formation of 8-oxodG and DSBs were also observed when fibroblast Balb/c-3T3 cells were co-exposed to N-OHAAF/UV irradiation as measured by double immunofluorescence staining. Taken together, we propose that both •OH and amidyl radicals can be readily produced via N-OH homolysis in N-OHAAF by photoirradiation, which can induce both oxidative and covalent DNA damage. This represents the first report of •OH production and site-specific DNA damage via photoactivation of the genotoxic hydroxamic acid intermediate, which provides a new free radical perspective to better understand the molecular mechanism for the carcinogenicity of AAs.

Reactive Nitrogen Species Are Also Involved in the Transformation of Micropollutants by the UV/Monochloramine Process.

The UV/monochloramine (NH2Cl) process is an emerging advanced oxidation process (AOP) in water treatment via radicals produced from the UV photolysis of NH2Cl. This study investigated the degradation of micropollutants by the UV/NH2Cl AOP, with ibuprofen (IBP) and naproxen (NPX) selected as representative micropollutants. Hydroxyl radical (HO•) and chlorine atom (Cl•) were identified in the process, and unexpectedly, we found that reactive nitrogen species (RNS) also played important roles in the transformation of micropollutants. The electron paramagnetic resonance (EPR) analysis proved the production of •NO as well as HO•. The concentrations of HO•, Cl•, and •NO in UV/NH2Cl remained constant at pH 6.0-8.6, resulting in the slightly changed UV fluence-based pseudo-first-order rate constants (k') of IBP and NPX, which were about 1.65 × 10-3 and 2.54 × 10-3 cm2/mJ, respectively. For IBP, the relative contribution of RNS to k' was 27.8% at pH 7 and 50 µM NH2Cl, which was higher than that of Cl• (6.5%) but lower than that of HO• (58.7%). For NPX, the relative contribution of RNS to k' was 13.6%, which was lower than both Cl• (23.2%) and HO• (46.9%). The concentrations of HO•, Cl•, and •NO increased with the increasing NH2Cl dosage. Water matrix components of natural organic matter (NOM) and bicarbonate can scavenge HO•, Cl•, and RNS. The presence of 5 mg/L NOM decreased the k' of IBP and NPX by 66.9 and 57.6%, respectively, while 2 mM bicarbonate decreased the k' of IBP by 57.4% but increased the k' of NPX by 10.5% due to the contribution of CO3•- to NPX degradation. Products containing nitroso-, hydroxyl-, and chlorine-groups were detected during the degradation of IBP and NPX by UV/NH2Cl, indicating the role of nitrogen oxide radical (•NO) as well as HO• and Cl•. Trichloronitromethane formation was strongly enhanced in the UV/NH2Cl-treated samples, further indicating the important roles of RNS in this process. This study first demonstrates the involvement of RNS in the transformation of micropollutants in UV/NH2Cl.

The Expensive-Tissue Hypothesis in Vertebrates: Gut Microbiota Effect, a Review.

The gut microbiota is integral to an organism’s digestive structure and has been shown to play an important role in producing substrates for gluconeogenesis and energy production, vasodilator, and gut motility. Numerous studies have demonstrated that variation in diet types is associated with the abundance and diversity of the gut microbiota, a relationship that plays a significant role in nutrient absorption and affects gut size. The Expensive-Tissue Hypothesis states (ETH) that the metabolic requirement of relatively large brains is offset by a corresponding reduction of the other tissues, such as gut size. However, how the trade-off between gut size and brain size in vertebrates is associated with the gut microbiota through metabolic requirements still remains unexplored. Here, we review research relating to and discuss the potential influence of gut microbiota on the ETH.

Unusual Double Beckmann Fragmentation Reaction under Physiological Conditions.

Pyridinium aldoximes, which are best-known as therapeutic antidotes for organophosphorus chemical warfare nerve-agents and pesticides, have been found to markedly detoxify polyhalogenated quinones, which are a class of carcinogenic intermediates and recently identified disinfection byproducts in drinking water. However, the exact chemical mechanism underlying this detoxication remains unclear. Here we demonstrate that pralidoxime can remarkably facilitate the dechlorination/hydroxylation of the highly toxic tetrachloro-1,4-benzoquinone in two-consecutive steps to generate the much less toxic 2,5-dichloro-3,6-dihydroxy-1,4-benzoquonine, with rate enhancements of up to 180 000-times. On the contrary, no accelerating effect was noticed with O-methylated pralidoxime. The major reaction product from pralidoxime was identified as its corresponding nitrile (2-cyano-1-methylpyridinium chloride). Along with oxygen-18 isotope-labeling studies, a reaction mechanism was proposed in which nucleophilic substitution coupled with an unprecedented double Beckmann fragmentation reaction was responsible for the dramatic enhancement in the detoxification process. This represents the first report of an unusually mild and facile Beckmann-type fragmentation that can occur under normal physiological conditions in two-consecutive steps. The study may have broad biomedical and environmental significance for future investigations of aldoxime therapeutic agents and carcinogenic polyhalogenated quinones.

Mechanism of Intrinsic Chemiluminescence Production from the Degradation of Persistent Chlorinated Phenols by the Fenton System: A Structure-Activity Relationship Study and the Critical Role of Quinoid and Semiquinone Radical Intermediates.

We found recently that intrinsic chemiluminescence (CL) could be produced by all 19 chlorophenolic persistent organic pollutants during environmentally friendly advanced oxidation processes. However, the underlying mechanism for the structure-activity relationship (SAR, i.e., the chemical structures and the CL generation) remains unclear. In this study, we found that, for all 19 chlorophenol congeners tested, the CL increased with an increasing number of chlorine atoms in general; and for chlorophenol isomers (such as the 6 trichlorophenols), the CL decreased in the order of meta- > ortho-/para-Cl-substituents with respect to the -OH group of chlorophenols. Further studies showed that not only chlorinated quinoid intermediates but also, more interestingly, chlorinated semiquinone radicals were produced during the degradation of trichlorophenols by the Fenton reagent; and the type and yield of which were determined by the directing effects, hydrogen bonding, and steric hindrance effect of the OH- and/or Cl-substitution groups. More importantly, a good correlation was observed between the formation of these quinoid intermediates and CL generation, which could fully explain the above SAR findings. This represents the first report on the structure-activity relationship study and the critical role of quinoid and semiquinone radical intermediates, which may have broad chemical and environmental implications for future studies on remediation of other halogenated persistent organic pollutants by advanced oxidation processes.

Molecular mechanism of metal-independent decomposition of organic hydroperoxides by halogenated quinoid carcinogens and the potential biological implications.

Halogenated quinones (XQ) are a class of carcinogenic intermediates and newly identified chlorination disinfection byproducts in drinking water. Organic hydroperoxides (ROOH) can be produced both by free radical reactions and enzymatic oxidation of polyunsaturated fatty acids. ROOH have been shown to decompose to alkoxyl radicals via catalysis by transition metal ions, which may initiate lipid peroxidation or transform further to the reactive aldehydes. However, it is not clear whether XQ react with ROOH in a similar manner to generate alkoxyl radicals metal-independently. By complementary applications of ESR spin-trapping, HPLC/high resolution mass spectrometric and other analytical methods, we found that 2,5-dichloro-1,4-benzoquinone (DCBQ) could significantly enhance the decomposition of a model ROOH tert-butylhydroperoxide, resulting in the formation of t-butoxyl radicals independent of transition metals. On the basis of the above findings, we detected and identified, for the first time, an unprecedented C-centered quinone ketoxy radical. Then, we extended our study to the more physiologically relevant endogenous ROOH 13-hydroperoxy-9,11-octadecadienoic acid and found that DCBQ could also markedly enhance its decomposition to generate the reactive lipid alkyl radicals and the genotoxic 4-hydroxy-2-nonenal (HNE). Similar results were observed with other XQ. In summary, these findings demonstrated that XQ can facilitate ROOH decomposition to produce reactive alkoxyl, quinone ketoxy, lipid alkyl radicals, and genotoxic HNE via a novel metal-independent mechanism, which may explain partly their potential genotoxicity and carcinogenicity.

A combined experimental and computational investigation on the unusual molecular mechanism of the Lossen rearrangement reaction activated by carcinogenic halogenated quinones.

The classic Lossen rearrangement is a well-known reaction describing the transformation of an O-activated hydroxamic acid into the corresponding isocyanate. In this study, we found that chlorinated benzoquinones (CnBQ) serve as a new class of agents for the activation of benzohydroxamic acid (BHA), leading to Lossen rearrangement. Compared to the classic one, this new kind of CnBQ-activated Lossen rearrangement has the following unique characteristics: (1) The stability of CnBQ-activated BHA intermediates was found to depend not only on the degree but also on the position of Cl-substitution on CnBQs, which can be divided into two subgroups. (2) It is the relative energy of the anionic CnBQ-BHA intermediates that determine the rate of this CnBQ-activated rearrangement, which is the rate-limiting step, and the Cl or H ortho to the reaction site at CnBQ is crucial for the stability of the anionic intermediates. (3) A pKa-activation energy correlation was observed, which can explain why the correlation exists between the rate of the rearrangement and the acidity of the conjugate acid of the anionic leaving group, the hydroxlated quinones. These findings may have broad implications for future research on halogenated quinoid carcinogens and hydroxamate biomedical agents.