Tetrachloro-1,4-benzoquinone induces apoptosis of mouse embryonic stem cells.

Pentachlorophenol (PCP) is a widespread, persistent environmental contaminant, and it is enzymatically activated to form a reactive metabolite, tetrachloro-1,4-benzoquinone (TCBQ). To our knowledge, there is no information about TCBQ toxicity on embryonic stem cells. Here, we demonstrated that TCBQ induced significantly apoptosis of mouse embryonic stem cells in a concentration-dependent manner. We also showed that TCBQ elevated genomic 5-hydroxymethylcytosine (5hmC) by affecting ten-eleven translocation (Tet) dioxygenases in mouse embryonic stem cells. We further investigated whether Tet dioxygenases were implicated in TCBQ-induced apoptosis. By depleting all three dioxygenases (Tet1-3), we found that Tet dioxygenases slightly inhibited both early and late apoptosis induced by TCBQ at a low concentration (30µmol/L). Meanwhile, treated by TCBQ at higher concentrations (40 and 50µmol/L), the total percentage of apoptotic cells was not affected by Tet dioxygenases. However, Tet dioxygenases tended to arrest mouse ES cells to be at early apoptotic stage and to reduce the cells to enter later apoptotic stage. These results indicate that Tet dioxygenases play a role in shaping TCBQ-induced apoptosis in mouse embryonic stem cells. Our study provides new insights into the toxicology of PCP and its reactive metabolite TCBQ.

Detection and mechanistic investigation of halogenated benzoquinone induced DNA damage by photoelectrochemical DNA sensor.

Halogenated phenols are widely used as biocides and are considered to be possibly carcinogenic to humans. In this report, a previously developed photoelectrochemical DNA sensor was employed to investigate DNA damage induced by tetra-halogenated quinones, the in vivo metabolites of halogenated phenols. The sensor surface was composed of a double-stranded DNA film assembled on a SnO(2) semiconductor electrode. A DNA intercalator, Ru(bpy)(2)(dppz)(2+), was allowed to bind to the DNA film and produce photocurrent upon light irradiation. After the DNA film was exposed to 300 microM tetrafluoro-1,4-benzoquinone (TFBQ), the photocurrent dropped by 20%. In a mixture of 300 microM TFBQ and 2 mM H(2)O(2), the signal dropped by 40%. The signal reduction indicates less binding of Ru(bpy)(2)(dppz)(2+) due to structural damage of ds-DNA in the film. Similar results were obtained with tetra-1,4-chlorobenzoquinone (TCBQ), although the signal was not reduced as much as TFBQ. Fluorescence measurement showed that TFBQ/H(2)O(2) generated more hydroxyl radicals than TCBQ/H(2)O(2). Gel electrophoresis proved that the two benzoquinones produced DNA strand breaks together with H(2)O(2), but not by themselves. Using the photoelectrochemical sensor, it was also found that TCBQ covalently bound with DNA did not produce additional oxidative damage in the presence of H(2)O(2). The combined photoelectrochemistry, gel electrophoresis, and fluorescence data revealed distinctive differences between TFBQ and TCBQ in terms of DNA adduct formation and hydroxyl radical generation.

Potential mechanism for pentachlorophenol-induced carcinogenicity: a novel mechanism for metal-independent production of hydroxyl radicals.

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 H(2)O(2) 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 H(2)O(2) was identified to be trichloro-hydroxy-1,4-benzoquinone (TrCBQ-OH), and H(2)O(2) 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 H(2)O(2) is not through a semiquinone-dependent organic Fenton reaction but rather through the following novel mechanism: a nucleophilic attack of H(2)O(2) 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.

An unusual double radical homolysis mechanism for the unexpected activation of the aldoxime nerve-agent antidotes by polyhalogenated quinoid carcinogens under normal physiological conditions.

We have recently shown that the pyridinium aldoximes, best-known as therapeutic antidotes for chemical warfare nerve-agents, could markedly detoxify the carcinogenic tetrachloro-1,4-benzoquinone (TCBQ) via an unusual double Beckmann fragmentation mechanism. However, it is still not clear why pralidoxime (2-PAM) cannot provide full protection against TCBQ-induced biological damages even when 2-PAM was in excess. Here we show, unexpectedly, that TCBQ can also activate pralidoxime to generate a reactive iminyl radical intermediate in two-consecutive steps, which was detected and unequivocally characterized by the complementary application of ESR spin-trapping, HPLC/MS and nitrogen-15 isotope-labeling studies. The same iminyl radical was observed when TCBQ was substituted by other halogenated quinones. The end product of iminyl radical was isolated and identified as its corresponding reactive and toxic aldehyde. Based on these data, we proposed that the reaction of 2-PAM and TCBQ might be through the following two competing pathways: a nucleophilic attack of 2-PAM on TCBQ forms an unstable transient intermediate, which can decompose not only heterolytically to form 2-CMP via double Beckmann fragmentation, but also homolytically leading to the formation of a reactive iminyl radical in double-steps, which then via H abstraction and further hydrolyzation to form its corresponding more toxic aldehyde. Analogous radical homolysis mechanism was observed with other halogenated quinones and pyridinium aldoximes. This study represents the first detection and identification of reactive iminyl radical intermediates produced under normal physiological conditions, which provides direct experimental evidence to explain only the partial protection by 2-PAM against TCBQ-induced biological damages, and also the potential side-toxic effects induced by 2-PAM and other pyridinium aldoxime nerve-agent antidotes.

The acute exposure of tetrachloro-p-benzoquinone (a.k.a. chloranil) triggers inflammation and neurological dysfunction via Toll-like receptor 4 signaling: The protective role of melatonin preconditioning.

This study is aimed to investigate the inflammation and neurological dysfunction induced by tetrachloro-p-benzoquinone (TCBQ) through Toll-like receptor 4 (TLR4) signaling. We also investigated the protective role of melatonin as an antioxidant and anti-inflammatory agent. In vitro model was established by rat pheochromocytoma PC12 cells, meanwhile, TLR4 wild-type (C57BL/6) and knockout mice (C57BL/10ScNJ TLR4-/-) were used as in vivo model. In vitro study showed TCBQ exposure enhanced the expression of TLR4, myeloid differentiation factor 88 (MyD88) at both transcriptional and post-transcriptional levels. By contrast, melatonin decreased TLR4 and MyD88 expressions. Moreover, our result indicated that melatonin disrupted the formation of TLR4/MyD88/MD2/CD14 complex. In addition, melatonin terminated TCBQ-mediated phosphorylation of c-Jun N-terminal kinase (JNK), p38, and extracellular regulated protein kinase (ERK) signaling and hampered its downstream pro-inflammatory cytokine releases. In vivo result also indicated TLR4 deficiency partially protected against TCBQ-induced morphological and neuropathological changes in mice brain, suggested the role of TLR4. In conclusion, melatonin modulates TCBQ-mediated inflammatory genes through TLR4/MyD88-dependent signaling pathway. Our current study, to the best of our knowledge, is the first time show melatonin not only disrupt the binding of TLR4 and MyD88, but also restricted the formation of TLR4/MD2/CD14 complex, suggesting that melatonin supplementary may represent a valuable therapeutic strategy for inflammatory neurological dysfunction.

Tetrachlorobenzoquinone induces acute liver injury, up-regulates HO-1 and NQO1 expression in mice model: the protective role of chlorogenic acid.

Tetrachlorobenzoquinone (TCBQ) is an active metabolite of pentachlorophenol (PCP). Although PCP has been investigated extensively, there are only a few reports describing the toxicity effect of TCBQ, and no report regarding TCBQ-induced liver injury in vivo. In the current study, we aimed to examine the acute hepatic toxicity of TCBQ in the mice model. Chlorogenic acid (CGA) exhibits promising antioxidant activity in the past studies, thus, the second aim of this study was to evaluate the protective effect of CGA on TCBQ-induced liver injury. Our results indicated TCBQ-intoxication caused marked liver cell necrosis and inflammation but not apoptosis, and this damage was alleviated by CGA treatment. Meantime, TCBQ-intoxication enhanced serum ALT, AST activities, TBIL content, hepatic oxidative stress and lipid peroxidation, decreased GSH content and inhibited the activities of antioxidant enzymes. Western blot and immunohistochemical analysis showed that TCBQ marked up-regulated HO-1 and NQO1 expression. On the other hand, pretreatment of CGA reduced TCBQ-induced liver damage remarkably. Taking together, these results revealed that TCBQ has strong hepatic toxic effect, and at least a part of this effect is initiated by free radical and relieved with CGA administration.

Bacterial growth response to photoactive quinones.

Quinones are known producers of reactive oxygen species (ROS) that may be toxic in natural aquatic environments. In this study, the effects of parent quinones and their photodegradation products on bacterial growth were determined, and photochemical ROS formation rates were measured. Using (3)H-leucine incorporation to measure growth of the bacterium Pseudomonas aeruginosa and natural seawater bacterioplankton, growth inhibition was observed when samples were exposed to dichlone, chloranil and sodium anthraquinone-2-sulfonate (AQ2S). For seawater, compared with other quinones tested, dichlone showed the greatest toxicity in the dark, and AQ2S toxicity was greatest during simultaneous exposure to sunlight. Photodegraded chloranil and dichlone showed decreased toxicity compared with nonirradiated samples. For P. aeruginosa, AQ2S and its photodegradation products showed the greatest toxicity during simultaneous exposure to sunlight. Chloranil photodegradation products showed reduced toxicity compared with the parent compound during simultaneous exposure to sunlight. Dichlone was the only compound to show any toxicity to P. aeruginosa in the dark, and its photodegradation products were more toxic than the parent compound. Based on the results of dark and light controlled experiments measuring bacterial growth and estimated ROS production rates, ROS alone does not account for relative differences in toxicity between these quinones.

Nucleobase-dependent reactivity of a quinone metabolite of pentachlorophenol.

Pentachlorophenol (PCP) is a possible human carcinogen detected widely in the environment. A quinone metabolite of PCP, tetrachloro-1,4-benzoquinone (Cl4BQ), is a reactive electrophile with the capacity to damage DNA by forming bulky covalent DNA adducts. These quinone adducts may contribute to chlorophenol carcinogenesis, but their structures, occurrence, and biological consequences are not known. Previous studies have indicated that several DNA adducts are formed in vivo in rats exposed to Cl4BQ, but these adducts were not identified structurally. In the present study, we have elucidated the structure of new agent-specific DNA adducts resulting from the reaction of dGuo, dCyd, and Thd with Cl4BQ. These have been characterized chemically by liquid chromatography-electrospray ionization mass spectrometry, HPLC, UV, and NMR analysis. Two dGuo adducts and one dCyd adduct resulting from the reaction of double-stranded DNA with Cl4BQ have been identified. The results indicate that, in the structural context of DNA, Cl4BQ reacts most readily with dGuo compared to the other DNA bases and that the mode of Cl4BQ reactivity is dependent on the base structure; i.e., multiple types of adducts are formed. Finally, DNA adducts consistent with Cl4BQ reactions are observed when DNA or dGuo is treated with PCP and a peroxidase-based bioactivating system.

Sublethal effects of tetrachloro-1,2-benzoquinone--a component in bleachery effluents from pulp mills--on vertebral quality and physiological parameters in fourhorn sculpin.

The effects of tetrachloro-1,2-benzoquinone (TCQ), a component in bleached kraft mill effluents (BKME), on vertebral and physiological parameters were investigated in juvenile fourhorn sculpin, Myoxocephalus quadricornis L. After about 4.5 months of exposure to 0.1 and 0.5 mg TCQ/liter in 7% salinity brackish water, the fish demonstrated vertebral deformities, aberrant mechanical properties of the vertebrae, effects on white and red blood cell counts, enhanced activity of delta-aminolevulinic acid dehydratase in erythrocytes, and increased levels of ascorbic acid in the liver. The effects are discussed in relation to those previously observed in fish exposed to complex BKME in the field and the laboratory.

Inhibition of gamma-glutamyl transpeptidase potentiates the nephrotoxicity of glutathione-conjugated chlorohydroquinones.

Administration of either 2,5-dichloro-3-(glutathion-S-yl)-1, 4-benzoquinone (DC-[GSyl]BQ) or 2,5,6-trichloro-3-(glutathion-S-yl)-1,4-benzoquinone (TC-[GSyl]BQ) to male Sprague-Dawley rats caused dose-dependent (50-200 mumol/kg; iv) renal proximal tubular necrosis, as evidenced by elevations in blood urea nitrogen (BUN), and in the urinary excretion of lactate dehydrogenase (LDH), gamma-glutamyl transpeptidase (gamma-GT) and glucose. Renal proximal tubular necrosis was also confirmed by histological examination of kidney slices prepared from DC-(GSyl)BQ- and TC-(GSyl)BQ-treated animals. Administration of the corresponding hydroquinone conjugates (DC-[GSyl]HQ and TC-[GSyl]HQ), prepared by reducing the quinones with a threefold molar excess of ascorbic acid, resulted in a substantial increase in nephrotoxicity. Moreover, in contrast to other glutathione (GSH)-conjugated hydroquinones, the nephrotoxicity of both DC-(GSyl)HQ and TC-(GSyl)HQ was potentiated when rats were pretreated with AT-125, an irreversible inhibitor of gamma-GT. Neither the quinone-GSH nor the hydroquinone-GSH conjugates caused any effect on liver histology or serum glutamate-pyruvate transaminase levels. The results suggest that coadministration of ascorbic acid with DC-(GSyl)BQ or TC-(GSyl)BQ decreases their interactions with extrarenal nucleophiles, including plasma proteins, and thus increases the concentration of the conjugates delivered to the kidney, and hence toxicity. Furthermore the ability of AT-125 to potentiate the nephrotoxicity of DC-(GSyl)HQ and TC-(GSyl)HQ suggests that metabolism of these conjugates by gamma-GT constitutes a detoxication reaction.

Oxidative DNA lesions in V79 cells mediated by pentachlorophenol metabolites.

Incubation of the pentachlorophenol (PCP) metabolites, tetrachloro-p-benzoquinone (chloranil, TCpBQ), tetrachloro-p-hydroquinone (TCpHQ) and tetrachloro-p-benzoquinone (TCoBQ) with V79 Chinese hamster cells led to a significant enhancement of the amount of 8-hydroxydeoxyguanosine (8-OH-dG) in DNA. With PCP itself and the metabolite tetrachloro-o-hydroquinone (TCoHQ) no distinct induction of this lesion could be observed. The average yields of 8-OH-dG were about 2-2.5 times above background levels. In addition, TCpBQ and TCpHQ were able to generate DNA single-strand breaks, while PCP, TCoHQ and TCoBQ failed to induce this lesion. All incubations were performed for 1 h without exogenous metabolic activation and concentrations were 25 microM of the respective agent. It is concluded that these metabolites may contribute to the carcinogenicity of PCP observed in mice, by generating reactive oxygen species (ROS) through their redox cycling properties.

Immunomodulatory effects of tetrachlorobenzoquinone, a reactive metabolite of hexachlorobenzene.

Hexachlorobenzene (HCB) is an environmental pollutant that causes autoimmune-like effects in humans and rats. It is not completely clear whether T cells are involved and, if so, how they are stimulated after oral exposure to HCB. HCB as a rather inert chemical is not likely to bind covalently to macromolecules. The oxidative metabolite of HCB, tetrachlorobenzoquinone (TCBQ), which is in a redox equilibrium with tetrachlorohydroquinone (TCHQ), can bind to macromolecules, hence may form hapten-carrier complexes in vivo. We have assessed in the reporter antigen-popliteal lymph node assay whether HCB or TCHQ and TCBQ are able to induce a 2,4,6-trinitrophenyl (TNP) specific IgG1 response to the T cell-independent antigen TNP-Ficoll, which is indicative of neoantigen specific T cell help. To this end, these compounds and silica were injected into the footpad of Balb/c mice. Silica was included as an inert model compound, which causes autoimmune-like effects by activating macrophages. Seven days later, cell number and TNP specific antibody-secreting cells (ASC) in the popliteal lymph node (PLN) were determined. Furthermore, a secondary PLNA was performed to find out if TCHQ was capable of eliciting a memory response. Silica, TCHQ, and TCBQ, but not HCB, increased PLN cellularity and the number of IgM-producing ASC by ELISPOT. Both oxidative metabolites were able to induce the formation of germinal centers as assessed by immunohistochemistry and an IgG1 response to TNP-Ficoll. In the secondary PLNA, only mice primed with TCHQ and challenged with TCHQ together with TNP-Ficoll showed a significant increase in TNP specific IgG1 ASC. Present data show that TCHQ and TCBQ are capable of inducing neoantigen specific T cell help and that TCHQ can induce a compound specific memory response.