Free Radicals Abroad | June 2018

Ben-Zhan (Benny) Zhu, Ph.D.
State Key Laboratory for Environmental Chemistry and Eco-Toxicology
Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences

The Research Center for Eco-Environmental Sciences (RCEES), Chinese Academy of Sciences (CAS), founded in 1973, is one of China's leading multidisciplinary research institutions with high international reputation dedicated to the basic research in broad fields of eco-environmental sciences, and to the key developments of the innovative high-technology aiming to the imperative national needs and important strategic targets.

Our research group on Free Radical Chemical Biology and Mechanism of Synergism is is located at the State Key Laboratory for Environmental Chemistry and Eco-toxicology, RCEES-CAS, in Beijing. We have been working on the classic Fe/Cu-catalyzed inorganic Fenton reaction, the novel metal-free haloquinone-mediated organic Fenton-like reaction, radical/rearrangement reactions under physiological conditions; ^•OH-dependent chemiluminescence generation by haloaromatic pollutants; new free radical pathway for DNA damage by aromatic amine carcinogens; and delivering enantioselective DNA imaging/photosensitizing Ru(II) cationic complex into live-cell nucleus via “Yin-Yang Ion-Pairing” with halophenolate counter-anions.

The Classic Inorganic Metal-catalyzed Fenton Reaction

As we all know, the hydroxyl radical (^•OH) is an extremely reactive oxidant, important in chemistry, biology, medicine, and atmospheric and environmental science. In biology, ^•OH is recognized as the most reactive and harmful of the so-called reactive oxygen species (ROS), which can cause DNA, protein, and lipid oxidation, leading to cancer, arthritis, and Parkinson’s disease. In environmental and atmospheric chemistry, ^•OH is also important because of its ability to oxidize and destroy organic pollutants efficiently and has been referred to as the atmosphere’s “detergent”. One of the most widely accepted mechanisms for ^•OH production is through the transition metal-catalyzed Fenton reaction. It had become a dogma when I pursued my PhD degree at Hebrew University in Jerusalem in the early 1990s that if ^•OH was produced, then redox-active transition metal ions (especially Fe or Cu), should be involved. In other words, transition metal ions were essential for ^•OH production. Indeed, we and several other research groups have found that intracellular iron played a critical role in H₂O₂–induced DNA damage in model cell cultural systems.

A Short Story about Discovering the Novel Metal-free Organic Fenton-like Reaction

However, during my study on the molecular mechanism of the genotoxicity of one of the most widely used biocides, pentachlorophenol (PCP), we found that ^•OH can be produced by its quinoid metabolites and H₂O₂, which is independent of transition metal ions!

As we know, the genotoxicity of PCP has been attributed to its two major quinoid metabolites: tetrachlorohydroquinone (TCHQ) and tetrachloro-1,4-benzoquinone (TCBQ). It was shown that TCHQ can induce DNA SSBs, which has been previously attributed to its ability to form ^•OH through the classic Fe-catalyzed Fenton reaction. This notion was based on the fact that TCHQ-induced DNA damage was completely prevented by the classic iron chelator DFO (deferoxamine). However, together with DTPA as another iron-chelator, we found that the protection by DFO against TCHQ-induced DNA damage was not due to its binding of iron, but rather due to its scavenging of the reactive tetrachlorosemiquinone radical (TCSQ^•) with the concurrent formation of less reactive DFO-nitroxide radicals (DFO^•).

The above findings suggest that iron was not involved in TCHQ-induced DNA damage, i.e., TCHQ-induced DNA damage may not be due to the iron-mediated ^•OH production via the classic Fenton reaction! Then, the question became what was the underlying molecular mechanism for PCP metabolites-mediated ^•OH production? To test whether ^•OH can be produced by PCP metabolites, we first employed the well-known salicylate hydroxylation method, and then the more specific secondary radical ESR spin trapping method, and foundthat ^•OH can be produced by PCP metabolites/H₂O₂metal-independently.

Further series studies showed that TCBQ, but not its corresponding semiquinone radical TCSQ^•, is essential for ^•OH production. The major reaction product for TCBQ/H₂O₂ was identified as trichloro-hydroxy-1,4-benzoquinone, and H₂O₂was found to be the source/origin of the O-atom inserted into the reaction product and ^•OH. Together we proposed that ^•OH production by TCBQ/H₂O₂ is not via a semiquinone-mediated organic Fenton reaction but rather through a novel nucleophilic substitution coupled with homolytic decomposition mechanism. These findings represent a novel mechanism of ^•OH formation not requiring the redox-active transition metal ions and may partly explain the potential carcinogenicity of the widely used biocides such as PCP and other polyhalogenated aromatic carcinogens. Then we extended our study from H₂O₂to organic hydroperoxides (ROOH) such as t-BuOOH and the more physiologically relevant 13-HPODE. We found that halogenated quinones can also facilitate ROOH decomposition to produce reactive alkoxyl, lipid alkyl radicals, the unprecedented carbon-centered quinone ketoxy radicals, and genotoxic 4-hydroxy-2-nonenal.

Lighting-up Haloaromatic Carcinogens by “Yin-Yang Fenton Reactions” Synergistically

Most chemiluminescence (CL) reactions usually generate only one-step CL, which is rarely dependent on the highly reactive and biologically/environmentally important ^•OH. As mentioned above, we found that ^•OH could be produced by H₂O₂with TCBQ and other haloquinones. Interestingly, it was observed by a Japanese research group that, together with riboflavin, CL could be produced by H₂O₂ and bromoquinones from acorn worm, a luminous marine organism. However, neither the underlying molecular mechanism nor its possible correlation with ^•OH is clear. We found that an unprecedented two-step CL can be produced by TCBQ/H₂O₂, which was well-correlated to and directly dependent on its two-step ^•OH production. We proposed that ^•OH-dependent formation of quinone-dioxetane and electronically excited carbonyl species might be responsible for this unusual two-step CL production. This represented the first report of a previously undefined two-step CL-producing system that is dependent on intrinsically formed ^•OH. More interestingly, we found that CL production by TCBQ/H₂O₂ could be further enhanced significantly by extra ^•OH produced by adding the classic Fenton reagent Fe(II)EDTA. Before this finding, we have considered the two ^•OH-producing system, TCBQ/H₂O₂ and Fe(II)EDTA/H₂O₂, as two independent, often two opposing Yin-Yang Fenton reactions, but the CL studies unify them together!

The ubiquitous distribution coupled with their carcinogenicity has raised public concerns on the potential risks to both human health and the ecosystem posed by PCP and other halogenated aromatics (XAr). Advanced oxidation processes (AOPs) have been increasingly favored as an "environmentally-green" technology for the remediation of such recalcitrant and highly toxic XAr. We found that AOPs-mediated degradation of the priority pollutant PCP and all other XAr produces an intrinsic CL that directly depends on ^•OH generation. Based on these findings, we developed a rapid, sensitive, simple, and effective CL method to quantify trace amounts of XAr and monitor their real-time degradation kinetics.