The hydroxyl radical (•OH) has been considered to be one of the most reactive oxygen species produced in biological systems. It has been shown that •OH can cause DNA, protein, and lipid oxidation. One of the most widely accepted mechanisms for •OH production is through the transition metal-catalyzed Fenton reaction. Pentachlorophenol (PCP) was one of the most widely used biocides, primarily for wood preservation. PCP is now ubiquitously present in our environment and even found in people who are not occupationally exposed to it. PCP has been listed as a priority pollutant by the U.S. Environmental Protection Agency (EPA) and classified as a group 2B environmental carcinogen by the International Association for Research on Cancer (IARC). The genotoxicity of PCP has been attributed to its two major quinoid metabolites: tetrachlorohydroquinone and tetrachloro-1,4-benzoquinone (TCBQ). Although the redox cycling of PCP quinoid metabolites to generate reactive oxygen species is believed to play an important role, the exact molecular mechanism underlying PCP genotoxicity is not clear. Using the salicylate hydroxylation assay and electron spin resonance (ESR) secondary spin-trapping methods, we found that •OH can be produced by TCBQ and H2O2 independent of transition metal ions. Further studies showed that TCBQ, but not its corresponding semiquinone radical, the tetrachlorosemiquinone radical (TCSQ•), is essential for •OH production. The major reaction product between TCBQ and H2O2 was identified to be trichloro-hydroxy-1,4-benzoquinone (TrCBQ-OH), and H2O2 was found to be the source and origin of the oxygen atom inserted into this reaction product. On the basis of these data, we propose that •OH production by TCBQ and H2O2 is not through a semiquinone-dependent organic Fenton reaction but rather through the following novel mechanism: a nucleophilic attack of H2O2 to TCBQ, leading to the formation of an unstable trichloro-hydroperoxyl-1,4-benzoquinone (TrCBQ-OOH) intermediate, which decomposes homolytically to produce •OH. These findings represent a novel mechanism of •OH formation not requiring the involvement of redox-active transition metal ions and may partly explain the potential carcinogenicity of the widely used biocides such as PCP and other polyhalogenated aromatic compounds.

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We have shown recently that halogenated quinones could enhance the decomposition of hydroperoxides and formation of alkoxyl/ hydroxyl radicals through a metal-independent mechanism. However, neither the proposed quinone enoxy radical intermediate, nor the major reaction products were unambiguously identified. In the present study, one of the major reaction products between 2,5-dichloro-1,4-benzoquinone (DCBQ) and t-butylhydroperoxide (t-BuOOH) was isolated and purified by semipreparative HPLC, and identified as 2-hydroxy-3-t-butoxy-5-chloro-1,4-benzoquinone [CBQ(OH)-O-t-Bu], which is the rearranged isomer of the postulated quinone-peroxide reaction intermediate. The formation of CBQ(OH)-O-t-Bu was found to be inhibited by the spin trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and concurrently, a new DMPO adduct with 1-chlorine isotope peak clusters at m/z 268 was observed. Further electron spin resonance (ESR) spintrapping, 1 H-NMR and HPLC/Fourier transform ion cyclotron resonance (FTICR) mass spectrometric studies with oxygen-17-labeled and unlabeled hydrogen peroxide strongly suggest that the radical trapped by DMPO is a carbon-centered quinone ketoxy radical, which is the spin isomer of the proposed oxygen-centered quinone enoxy radical. Analogous results were observed when DCBQ was substituted by other halogenated quinones. This study represents the first detection and identification of an unusual carbon-centered quinone ketoxy radical, which provides direct experimental evidence to further support and expand our previously proposed mechanism for metal-independent decomposition of hydroperoxides by halogenated quinones.ESR spin-trapping ͉ spin isomerization ͉ carbon-centered quinone ketoxy radical ͉ oxygen-centered quinone enoxy radical ͉ keto-enol tautomerization W e have shown recently that alkoxyl radicals can be produced by organic hydroperoxides and halogenated quinones independent of transition metal ions, and a reaction mechanism was proposed (1): A nucleophilic reaction may take place between 2,5-dichloro-1,4-benzoquinone (DCBQ) and t-butylhydroperoxide (t-BuOOH), forming a quinone-peroxide reaction intermediate 2-chloro-5-t-butylperoxyl-1,4-benzoquinone (CBQ-OO-t-Bu), which can decompose homolytically to produce t-butoxyl radical (t-BuO • ) and 2-chloro-5-hydroxy-1,4-benzoquinone enoxy radical (CBQ-O • ). CBQ-O• then disproportionate to form the ionic form of 2-chloro-5-hydroxy-1,4-benzoquinone (CBQ-OH) (see scheme 1 in ref. 1). We also found that hydroxyl radicals could be produced metal-independently by hydrogen peroxide and halogenated quinones (2-4). However, neither the major reaction products, nor the proposed quinone-peroxide reaction intermediate CBQ-OO-t-Bu and quinone enoxy radical CBQ-O • were unambiguously identified in the previous studies.Therefore, in the present study, we addressed the following questions: (i) was it possible to isolate and purify the proposed quinone-peroxide reaction intermediate; (ii) could CBQ-OH or any other major reaction products be isolated, purifi...

The metal-independent decomposition of organic hydroperoxides and the formation of organic alkoxyl radicals in the absence or presence of halogenated quinones were studied with electron spin resonance (ESR) and the spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO). We found that 2,5-dichloro-1,4-benzoquinone (DCBQ) markedly enhanced the decomposition of tert-butylhydroperoxide (t-BuOOH), leading to the formation of the DMPO adducts with t-butoxyl radicals (t-BuO • ) and methyl radicals ( • CH3). The formation of DMPO/t-BuO • and DMPO/ • CH3 was dosedependent with respect to both DCBQ and t-BuOOH and was not affected by iron-or copper-specific metal chelators. Comparison of the data obtained with DCBQ and t-BuOOH with those obtained in a parallel study with ferrous iron and t-BuOOH strongly suggested that t-BuO • was produced by DCBQ and t-BuOOH through a metal-independent mechanism. Other halogenated quinones were also found to enhance the decomposition of t-BuOOH and other organic hydroperoxides such as cumene hydroperoxide, leading to the formation of the respective organic alkoxyl radicals in a metal-independent manner. Based on these data, we propose a mechanism for DCBQ-mediated t-BuOOH decomposition and formation of t-BuO • : a nucleophilic attack of t-BuOOH on DCBQ, forming a chloro-t-butylperoxyl-1,4-benzoquinone intermediate, which decomposes homolytically to produce t-BuO • . This represents a mechanism of organic alkoxyl radical formation not requiring the involvement of redox-active transition metal ions.electron spin resonance spin-trapping ͉ 2,5-dichloro-1,4-benzoquinone ͉ 2,5-dichlorosemiquinone anion radical ͉ reactive intermediate ͉ metal chelators O rganic hydroperoxides (ROOH) can be formed both nonenzymatically by reaction of free radicals with polyunsaturated fatty acids and enzymatically by lipoxygenase-or cyclooxygenasecatalyzed oxidation of linoleic acid and arachidonic acid (1, 2). It has been shown that organic hydroperoxides can undergo transition metal ion-catalyzed decomposition to alkoxyl radicals (Reaction 1), which may initiate de novo lipid peroxidation or further decompose to ␣,␤-unsaturated aldehydes that can react with and damage DNA and other biological macromolecules (1, 2).where Me represents a transition metal, such as iron or copper. Using the salicylate hydroxylation assay and electron spin resonance (ESR) spin-trapping methods, we reported previously that HO • can be produced from H 2 O 2 by halogenated quinones independent of transition metal ions (3, 4). However, it is not clear whether halogenated quinones react in a similar fashion with organic hydroperoxides to produce alkoxyl radicals independent of transition metal ions.Therefore, in the present study we addressed the following questions. (i) Can halogenated quinones enhance the decomposition of organic hydroperoxides to produce alkoxyl radicals? (ii) if so, is the production of alkoxyl radicals dependent or independent of transition metal ions? (iii) And what is the underlying molecular mechanism? To c...

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Hydroxamic acids, which are best-known for their metal-chelating properties in biomedical research, have been found to effectively detoxify the carcinogenic polyhalogenated quinoid metabolites of pentachlorophenol and other persistent organic pollutants. However, the chemical mechanism underlying such detoxication is unclear. Here we show that benzohydroxamic acid (BHA) could dramatically accelerate the conversion of the highly toxic tetrachloro-1, 4-benzoquinone (p-chloranil) to the much less toxic 2, 5-dichloro-3, 6-dihydroxy-1, 4-benzoquonine (chloranilic acid), with rate accelerations of up to 150,000-fold. In contrast, no enhancing effect was observed with O-methyl BHA. The major reaction product of BHA was isolated and identified as O-phenylcarbamyl benzohydroxamate. On the basis of these data and oxygen-18 isotope-labeling studies, we proposed that suicidal nucleophilic attack coupled with an unexpected double Lossen rearrangement reaction was responsible for this remarkable acceleration of the detoxication reaction. This is the first report of an unusually mild and facile Lossen-type rearrangement, which could take place under normal physiological conditions in two consecutive steps. Our findings may have broad biological and environmental implications for future research on hydroxamic acids and polyhalogenated quinoid carcinogens, which are two important classes of compounds of major biomedical and environmental interest.

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