TCHQ-induced, ROS-mediated, prolonged p-ERK expression in splenocytes.

<p>(A) Freshly isolated splenocytes were treated with various concentrations of PCP or TCHQ for 2 hr. The total protein lysates were extracted to analyze the expression of p-ERK and ERK by western blot. β-actin was served as a loading control. N: normal group; D: DMSO control group. The protein expression for the indicated periods of time is presented as (B). (C) The splenocytes were pretreated with or without 5 mM NAC for 1 hr. After washing with ice-cold PBS, the cells were treated with 100 µM PCP or 25 µM TCHQ for 15 min or 2 hr. the Total protein lysates were extracted to analyze the expression of p-ERK and ERK by western blot. ERK served as a loading control. (D) The cells were pretreated with U0126 (10 µM) for 30 min followed by treatment of 25 µM TCHQ for 2 hr. Total cell lysates were prepared and immunoblotted using antibodies to cleaved caspase-3, PARP, p-ERK, ERK and β-actin. β-actin was served as a loading control. Statistical analysis was performed using a two-tailed Student’s <i>t</i>-test. *, p<0.05.</p

PCP and TCHQ inhibit cell viability in a dose- and time-dependent manner in splenocytes.

<p>Freshly isolated mouse splenocytes were treated with various concentrations of PCP or TCHQ dissolved in DMSO for the time periods indicated. The cell viability was measured using the MTT assay. Values are the mean ± SD from six replicates of three independent experiments. Statistical analysis was performed using the two-tailed Student’s <i>t</i>-test. *, p<0.05 versus DMSO control group at the time period indicated.</p

NAC prevents TCHQ-inhibited effects on cleaved caspase-3 and PARP and caspase-3 activity in splenocytes.

<p>(A) Splenocytes were pretreated with or without 5 mM NAC for 1 hr. After washing in ice-cold PBS, the cells were treated with 100 µM PCP or 25 µM TCHQ for 15 min or 2 hr, respectively. Total protein lysates were analyzed for the expression of cleaved caspase-3 by western blot. ERK expression served as a loading control. (C) Splenocytes pretreated for 1 hr with (lower panel) or without (upper panel) NAC, were treated with 25 µM TCHQ for 2 hr. Nuclear and cytosolic extracts were prepared and subjected to western blot analysis. (B) Capase-3 activity was detected using CaspACETM Assay System and was measured by Fluoroskan Ascent FL microplate fluorometer. Statistical analysis was performed using a two-tailed Student’s <i>t</i>-test. *, p<0.05.</p

The probable cell death pathways mediated by high doses of TCHQ in splenocytes.

<p>The probable cell death pathways mediated by high doses of TCHQ in splenocytes.</p

TCHQ induces massive intracellular ROS production and persistent attenuation of membrane potential in splenocytes.

<p>(A) Splenocytes were treated with a range of PCP or TCHQ for 2 hr. DCFDA (30 µM) was added to the cell suspensions for intracellular ROS detection and 30 nM DiOC₆ was added for membrane potential analysis 30 min prior to the end of the exposures. After treatment, the cells were harvested and their fluorescence was analyzed using the FACScan and analyzed using WinMDI software. The data are shown as MFI. ROS production and the loss of membrane potential in splenocytes treated with PCP or TCHQ for the indicated periods of time were presented as (B) and (C), respectively. MFI: Mean Fluorescence Intensity. Statistical analysis was performed using the two-tailed Student’s <i>t</i>-test. *^{, #, +}, p<0.05 versus DMSO control group in treatment at the time period indicated. (D) NAC was added as an antioxidant. Splenocytes with or without 5 mM NAC pretreatment for 1 hr were collected and washed with ice-cold PBS, followed by 25 µM TCHQ treatment for 30 min. ROS, membrane potential and cell death were measured as described above. Finally (E), cell viability was analyzed using the MTT assay. Statistical analysis was performed using the two-tailed Student’s <i>t</i>-test. *, p<0.05.</p

High dose treatment with TCHQ results in inhibition of apoptosis in splenocytes.

<p>(A) Freshly isolated mouse splenocytes were treated with indicated concentrations of PCP or TCHQ for 2 hr. Total protein lysates were analyzed for the expression of cleaved caspase-3 and PARP by western blot. β-actin was served as a loading control. N: normal group; D: DMSO control group. The expression of the target proteins at the indicated time points are shown in (B). Capase-3 activity was detected by a fluorogenic assay, CaspACETM Assay System (Promega, WI, USA), and Caspase-3 released free fluoro-chrome 7-amino-4-methyl coumarin (AMC) was measured at 360 nm excitation/460 nm emission in a Fluoroskan Ascent FL microplate fluorometer (Thermo Scientific, USA). Statistical analysis was performed using a two-tailed Student’s <i>t</i>-test. *, p<0.05. (D) Cell death analysis was performed using Annexin V and PI double-staining. The treated splenocytes were stained for detection of PS with Annexin V-FITC and for DNA content with PI. The necrotic cells were characterized by loss of plasma membrane integrity (annexin V<b>+/</b>PI<b>+</b>). The fluorescence was measured using a FACScan and analyzed by WinMDI software. The statistical analysis was performed using a two-tailed Student’s <i>t</i>-test. *, p<0.05 versus DMSO control group at the time period indicated. (E) Transmission electron microscopy (JEOL JEM-1200EX, Japan) was used to examine cell morphology. Scale bar: 0.5 µm.</p

TCHQ treatment in higher doses inhibits sub-diploid DNA content and DNA ladder in splenocytes.

<p>(A, B) After treatment with PCP or TCHQ for 0, 5 min, 15 min, 30 min, 1 hr, 2 hr or 6 hr, the splenocytes were collected and stained with PI. The cell cycle distribution was analyzed using the FACScan (Becton Dickinson, San Jose, CA, USA) and analyzed using WinMDI software (Becton Dickson, San Jose, CA, USA). Cells with sub-diploid DNA content (Sub-G0/G1 phase) were defined as apoptotic. Values are the mean ± SD from at least three separate experiments. The statistical analysis was performed using the two-tailed Student’s <i>t</i>-test. *, p<0.05 versus DMSO control group at the time period indicated. (C) To further confirm these effects, analysis of DNA fragmentation in splenocytes was performed. Following PCP or TCHQ treatment for 2 hr, the cells were lysed and the DNA was extracted and electrophoresed on a 2% agarose gel. The gel was stained with EtBr and photographed. M: marker; N: normal splenocytes group; D: DMSO control group.</p

Intrinsic Chemiluminescence Generation during Advanced Oxidation of Persistent Halogenated Aromatic Carcinogens

The ubiquitous distribution coupled with their carcinogenicity has raised public concerns on the potential risks to both human health and the ecosystem posed by the halogenated aromatic compounds (XAr). Recently, advanced oxidation processes (AOPs) have been increasingly favored as an “environmentally-green” technology for the remediation of such recalcitrant and highly toxic XAr. Here, we show that AOPs-mediated degradation of the priority pollutant pentachlorophenol and all other XAr produces an intrinsic chemiluminescence that directly depends on the generation of the extremely reactive hydroxyl radicals. We propose that the hydroxyl radical-dependent formation of quinoid intermediates and electronically excited carbonyl species is responsible for this unusual chemiluminescence production. A rapid, sensitive, simple, and effective chemiluminescence method was developed to quantify trace amounts of XAr and monitor their real-time degradation kinetics. These findings may have broad biological and environmental implications for future research on this important class of halogenated persistent organic pollutants

The Unexpected and Exceptionally Facile Chemical Modification of the Phenolic Hydroxyl Group of Tyrosine by Polyhalogenated Quinones under Physiological Conditions

The phenolic hydroxyl group of tyrosine residue plays a crucial role in the structure and function of many proteins. However, little study has been reported about its modification by chemical agents under physiological conditions. In this study, we found, unexpectedly, that the phenolic hydroxyl group of tyrosine can be rapidly and efficiently modified by tetrafluoro-1,4-benzoquinone and other polyhalogenated quinones, which are the major genotoxic and carcinogenic quinoid metabolites of polyhalogenated aromatic compounds. The modification was found to be mainly due to the formation of a variety of fluoroquinone–<i>O</i>-tyrosine conjugates and their hydroxylated derivatives via nucleophilic substitution pathway. Analogous modifications were observed for tyrosine-containing peptides. Further studies showed that the blockade of the reactive phenolic hydroxyl group of tyrosine in the substrate peptide, even by very low concentration of tetrafluoro-1,4-benzoquinone, can prevent the kinase catalyzed tyrosine phosphorylation. This is the first report showing the exceptionally facile chemical modification of the phenolic hydroxyl group of tyrosine by polyhalogenated quinones under normal physiological conditions, which may have potential biological and toxicological implications

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 <i>O</i>-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