Two-Coordinate Dinuclear Donor-Gold(I)-Acceptor Complexes Exhibiting Multiple Excitation Wavelength Dependent Phosphorescence.

Two-coordinate Au(I) complexes with a donor-metal-acceptor (D-M-A) structure have shown rich luminescent properties. However, charge-neutral dinuclear donor-metal-acceptor type Au(I) complexes featuring aurophilic interactions have been seldom explored. Herein, we describe the structures and photoluminescence properties of two dinuclear Au(I) complexes, namely DiAu-Ph and DiAu-Me. Single crystal X-ray structural analysis of DiAu-Ph reveals a short intramolecular Au-Au distance of 3.224 Å. In dilute solution and doped films, excitation wavelength dependent multiple phosphorescence phenomena were observed for these dinuclear complexes. Theoretical calculations reveal that the aurophilic interaction causes increased contribution of the Au d orbital to the highest occupied molecular orbitals. Thus, the gap between singlet and triplet excited states (ΔEST) is enlarged, which disables the thermally activated delayed fluorescence (TADF). Moreover, the large energy separation (0.45-0.52 eV) and the different orbital configurations between the various excited states result in an inefficient internal conversion, accounting for their multiple phosphorescence properties.

Development of a reporter gene-based assay for the bioactivity determination of rhLH pharmaceutical products.

To establish an in vitro biological activity detection method for luteinizing hormone (LH), the hLHCGR-CREB-HEK293 cell line was constructed to stably express human luteinizing hormone/chorionic gonadotropin receptor (hLHCGR). After optimization, the rhLH starting working concentration was 800 mIU/mL with 4-fold serial dilutions, 10 concentrations and an incubation time of 5 h. The method was confirmed to be highly specific, with good accuracy, precision and linearity, meeting the needs of process research and release testing, and can be used as a routine detection method for LH biological activity. With the increasing demand for research and development of rhLH biologically similar drugs, establishing a stable and simple activity assay method to evaluate the biological activity of rhLH can provide technical support for quality control of rhLH products and powerful tools for comparability research of similar products.

Two-Coordinate Gold(I) Complex with Rotational Freedom: A Platform Integrating Thermally Activated Delayed Fluorescence, Polymorphism, and Linearly Polarized Luminescence.

Two-coordinate coinage metal complexes have been exploited for various applications. Herein, a new donor-metal-acceptor (D-M-A) complex PZI-Au-TOT, using bulky pyrazine-fused N-heterocyclic carbene (PZI) and trioxytriphenylamine (TOT) ligands, was synthesized. PZI-Au-TOT displays decent thermally activated delayed fluorescence (TADF) with a quantum yield of 93% in doped film. The crystals of PZI-Au-TOT show simultaneous TADF, polymorphism, and linearly polarized luminescence (LPL). The polymorph-dependent emission properties with widely varied peaks from 560 to 655 nm are attributed to different packing modes in terms of isolated monomers, discrete π-π stacked dimers or dimer PLUS. Two well-defined microcrystals of PZI-Au-TOT exhibit linearly polarized thermally activated delayed fluorescence with a degree of polarization up to 0.64. This work demonstrates that the molecular rotational flexibility of D-M-A type complexes endows an integration of multiple functions into one complex through manipulation of supramolecular aggregation. This type of complexes is expected to serve as a versatile platform for the fabrication of crystal materialsfor advanced photonic applications.

Gold Coordination-Accelerated Multi-Resonance TADF Emission for Efficient Solution-Processible Ultrapure Deep-Blue OLEDs.

Multi-resonance (MR) type emitters have emerged as highly promising candidates for high-resolution organic light-emitting diodes (OLEDs). However, thermally activated delayed fluorescence (TADF) emissions with simultaneous short excited state lifetimes and ultrapure blue color (a CIEy close to 0.046 and an emission peak > 440 nm) have rarely been obtained for MR emitters. Herein, we report a design of dual gold-coordinated MR molecules to achieve efficient and short-lived ultrapure blue TADF emission. The dinuclear Au(I) complex, namely iPrAuBN, shows a narrowband deep-blue emission with a peak maximum of 448 nm and a full width at half maximum (FWHM) of 29 nm in doped film. The coordination with two Au atoms significantly shortens the delayed fluorescence lifetime to 7.8 µs in comparing with 60.6 µs for the organic analogue. Solution-processed OLED doped with iPrAuBN demonstrates an ultrapure blue electroluminescence with a peak maximum of 442 nm, a FWHM of 19 nm, Commission International de l'Éclairage (CIE) coordinates of (0.154, 0.036), and a maximum external quantum efficiency of 14.8%.

Theoretical studies on thermally activated delayed fluorescence of "carbene-metal-amide" Cu and Au complexes: geometric structures, excitation characters, and mechanisms.

"Carbene-metal(I)-amide" (CMA) complexes have garnered significant attention due to their remarkable properties and potential TADF applications in organic electronics. However, the atomistic working mechanism is still elusive. Herein, we chose two CMA complexes, i.e., cyclic (alkyl)(amino) carbene-copper[gold](I)-carbazole (CAAC-Cu[Au]-Cz), and employed both DFT and TD-DFT methods, in combination with radiative and nonradiative rate calculations, to investigate geometric and electronic structures of these two complexes in the ground and excited states, including orbital compositions, electronic transitions, absorption and emission spectra, and the luminescence mechanism. It is found that the coplanar or perpendicular conformations are coexistent in the ground state (S0), the lowest excited singlet state (S1), and the triplet state (T1). Both the coplanar and perpendicular S1 and T1 states have similar ligand-to-ligand charge transfer (LLCT) character between CAAC and Cz, and some charge-transfer character between metal atoms and ligands, which is beneficial to minimize the singlet-triplet energy gaps (ΔEST) and increase the spin-orbit coupling (SOC). An interesting three-state (S0, S1, T1) model involving two regions (coplanar and perpendicular) is proposed to rationalize the experimental TADF phenomena in the CMA complexes. In addition to the coplanar ones, the perpendicular S1 and T1 states also play a role in promoting the repopulation of the coplanar S1 exciton, which is a primary source for the delayed fluorescence.

Regulation of Multiple Resonance Delayed Fluorescence via Through-Space Charge Transfer Excited State towards High-Efficiency and Stable Narrowband Electroluminescence.

B- and N-embedded multiple resonance (MR) type thermally activated delayed fluorescence (TADF) emitters usually suffer from slow reverse intersystem crossing (RISC) process and aggregation-caused emission quenching. Here, we report the design of a sandwich structure by placing the B-N MR core between two electron-donating moieties, inducing through-space charge transfer (TSCT) states. The proper adjusting of the energy levels brings about a 10-fold higher RISC rate in comparison with the parent B-N molecule. In the meantime, a high photoluminescence quantum yield of 91 % and a good color purity were maintained. Organic light-emitting diodes based on the new MR emitter achieved a maximum external quantum efficiency of 31.7 % and small roll-offs at high brightness. High device efficiencies were also obtained for a wide range of doping concentrations of up to 20 wt % thanks to the steric shielding of the B-N core. A good operational stability with LT95 of 85.2 h has also been revealed. The dual steric and electronic effects resulting from the introduction of a TSCT state offer an effective molecular design to address the critical challenges of MR-TADF emitters.

Thermally Activated Delayed Fluorescence of a Dinuclear Platinum(II) Compound: Mechanism and Roles of an Upper Triplet State.

A dinuclear Pt(II) compound was reported to exhibit thermally activated delayed fluorescence (TADF); however, the luminescence mechanism remains elusive. To reveal relevant excited-state properties and luminescence mechanism of this Pt(II) compound, both density function theory (DFT) and time-dependent DFT (TD-DFT) calculations were carried out in this work. In terms of the results, the S1 and T2 states show mixed intraligand charge transfer (ILCT)/metal-to-ligand CT (MLCT) characters while the T1 state exhibits mixed ILCT/ligand-to-metal CT (LMCT) characters. Mechanistically, a four-state (S0 , S1 , T1 , and T2 ) model is proposed to rationalize the TADF behavior. The reverse intersystem crossing (rISC) process from the initial T1 to final S1 states involves two up-conversion channels (direct T1 →S1 and T2 -mediated T1 →T2 →S1 pathways) and both play crucial roles in TADF. At 300 K, these two channels are much faster than the T1 phosphorescence emission enabling TADF. However, at 80 K, these rISC rates are reduced by several orders of magnitude and become very small, which blocks the TADF emission; instead, only the phosphorescence is observed. These findings rationalize the experimental observation and could provide useful guidance to rational design of organometallic materials with superior TADF performances.

Highly Efficient Au(I) Alkynyl Emitters: Thermally Activated Delayed Fluorescence and Solution-Processed OLEDs.

Two-coordinate donor-metal-acceptor type coinage metal complexes displaying efficient thermally activated delayed fluorescence (TADF) have been unveiled to be highly appealing candidates as emitters for organic light-emitting diodes (OLEDs). Herein a series of green to yellow TADF gold(I) complexes with alkynyl ligands has been developed for the first time. The complexes exhibit high photoluminescence quantum yields (PLQYs) of up to 0.76 in doped films (5 wt % in PMMA) at room temperature. The modifications of alkynyl ligands with electron-donating amino groups together with the use of electron-deficient carbene ligands induce ligand-to-ligand charge transfer excited states that give rise to TADF emission. Spectroscopic and density functional theory (DFT) calculations reveal the roles of electron-donating capability of the alkynyl ligand in tuning the excited-state properties. Solution-processed organic light-emitting diodes (OLEDs) using the present complexes as emitters achieve maximum external quantum efficiency (EQE) of up to 20 %.

Thermally Activated Delayed Fluorescence Mechanism of a Bicyclic "Carbene-Metal-Amide" Copper Compound: DFT/MRCI Studies and Roles of Excited-State Structure Relaxation.

Herein we investigated the luminescence mechanism of one "carbene-metal-amide" copper compound with thermally activated delayed fluorescence (TADF) using density functional theory (DFT)/multireference configuration interaction, DFT, and time-dependent DFT methods with the polarizable continuum model. The experimentally observed low-energy absorption and emission peaks are assigned to the S1 state, which exhibits clear interligand and partial ligand-to-metal charge-transfer character. Moreover, it was found that a three-state (S0, S1, and T1) model is sufficient to describe the TADF mechanism, and the T2 state should play a negligible role. The calculated S1-T1 energy gap of 0.10 eV and proper spin-orbit couplings facilitate the reverse intersystem crossing (rISC) from T1 to S1. At 298 K, the rISC rate of T1 → S1 (∼106 s-1) is more than 3 orders of magnitude larger than the T1 phosphorescence rate (∼103 s-1), thereby enabling TADF. However, it disappears at 77 K because of a very slow rISC rate (∼101 s-1). The calculated TADF rate, lifetime, and quantum yield agree very well with the experimental data. Methodologically, the present work shows that only considering excited-state information at the Franck-Condon point is insufficient for certain emitting systems and including excited-state structure relaxation is important.

Highly Efficient Thermally Activated Delayed Fluorescence from Pyrazine-Fused Carbene Au(I) Emitters.

Metal-based thermally activated delayed fluorescence (TADF) is conceived to inherit the advantages of both phosphorescent metal complexes and purely organic TADF compounds for high-performance electroluminescence. Herein a panel of new TADF Au(I) emitters has been designed and synthesized by using carbazole and pyrazine-fused nitrogen-heterocyclic carbene (NHC) as the donor and acceptor ligands, respectively. Single-crystal X-ray structures show linear molecular shape and coplanar arrangement of the donor and acceptor with small dihedral angles of <6.5°. The coplanar orientation and appropriate separation of the HOMO and LUMO in this type of molecules favour the formation of charge-transfer excited state with appreciable oscillator strength. Together with a minor but essential heavy atom effect of Au ion, the complexes in doped films exhibit highly efficient (Φ∼0.9) and short-lived (<1 µs) green emissions via TADF. Computational studies on this class of emitters have been performed to decipher the key reverse intersystem crossing (RISC) pathway. In addition to a small energy splitting between the lowest singlet and triplet excited states (ΔEST ), the spin-orbit coupling (SOC) effect is found to be larger at a specific torsion angle between the donor and acceptor planes which favours the RISC process the most. This work provides an alternative molecular design to TADF Au(I) carbene emitters for OLED application.

Electric field controlled uphill electron migration along α-helical oligopeptides.

A systematic study on applied electric field effects (Eapp) on electron transfer along the peptides is very important for the regulation of electron transfer behaviors so as to realize the functions of proteins. In this work, we computationally investigated the uphill migration behaviors of excess electrons along the peptide chains under Eapp using the density functional theory method. We examined the electronic property changes of the model α-helical oligopeptides, the dynamics behavior of an excess electron along the peptide chains under Eapp opposite to the internal dipole field of peptides. We found that Eapp of different intensities can effectively modulate the electron-binding abilities, Frontier molecular orbital (FMO) energies and distributions, dipole moments and other corresponding properties with different degrees. The electron-binding abilities of α-helical oligopeptides revealed by vertical electron affinity and FMO energies decrease in weak Eapp and then increase greatly in high Eapp, while the dipole moments change mildly in weak Eapp and increase significantly until a threshold and then become gentle in high Eapp. Analysis of FMO and electron distributions indicates that an excess electron can migrate uphill from the N-terminus to the C-terminus of the α-helical peptides in an irregular jump mode as Eapp linearly increases. Another interesting finding is that α-helical peptides with diverse chain lengths have different sensitivities to Eapp. The longer the peptide is, the more obvious the effects of Eapp are. Additionally, compared to the Eapp effect on linear oligopeptides, we summarized the systematic rule about the Eapp effect on excess electron migration uphill along the peptide chains. Clearly, this work not only enriches the information of the Eapp effect on electronic properties and electron transfers in the helical peptides, but also provides a new perspective for modulating electron migration behaviors in protein electronics engineering.

Dynamic relaying properties of a ß-turn peptide in long-range electron transfer.

The relay stations play a significant role in long-range charge hopping transfer in proteins. Although studies have clarified that many more protein structural motifs can function as relays in charge hopping transfers by acting as intermediate charge carriers, the relaying properties are still poorly understood. In this work, taking a ß-turn oligopeptide as an example, we report a dynamic character of a relay with tunable relaying properties using the density functional theory calculations. Our main finding is that a ß-turn peptide can serve as an effective electron relay in facilitating long-range electron migration and its relay properties is vibration-tunable. The vibration-induced structural transient distortions remarkably affect the lowest occupied molecular orbital (LUMO) energy, vertical electron affinity and electron-binding mode of the ß-turn oligopeptide and the singly occupied molecular orbital (SOMO) energy of the corresponding electron adduct and thus the relaying properties. Different vibration modes lead to different structural distortions and thus have different effects on the relaying properties and ability of the ß-turn peptide. For the relaying properties, there approximately is a linear negative correlation of electron affinity with the LUMO energy of the ß-turn or the SOMO energy of its electron adduct. Besides, such relaying properties also vary in the vibration evolution process, and the electron-binding modes may be tunable. As an important addition to the known static charge relaying properties occurring in various protein structural motifs, this work reports the dynamic electron-relaying characteristics of a ß-turn oligopeptide with variable relaying properties governed by molecular vibrations which can be applied to different proteins in mediating long-range charge transfers. Clearly, this work reveals molecular vibration effects on the electron relaying properties of protein structural motifs and provides new insights into the dynamics of long-range charge transfers in proteins. © 2018 Wiley Periodicals, Inc.

Nonlinear Migration Dynamics of Excess Electrons along Linear Oligopeptides Controlled by an Applied Electric Field.

Migration of an excess electron along linear oligopeptides governed by the external electric field (Eex ) which is against the inner dipole electric field is theoretically investigated, including the effects of Eex on the structural and electronic properties of electron migration. Two structural properties including electron-binding ability and the dipole moment of linear oligopeptides are sensitive to the Eex values and can be largely modulated by Eex due to the competition of Eex and the inner electric field and electron transfer caused by Eex . In the case of low Eex values, two structural properties decrease slightly, while for high Eex values, the electron-binding ability continually increases strongly, with dipole moments firstly increasing significantly and then increasing more slowly at higher Eex . Additionally, linear oligopeptides of different chain lengths influence the modulation extent of Eex and the longer the chain length is, the more sensitive modulation of Eex is. In addition, electronic properties represented by electron spin densities and singly occupied molecular orbital distributions vary with Eex intensities, leading to an unusual electron migration behavior. As Eex increases, an excess electron transfers from the N-terminus to the C-terminus and jumps over a neighboring dipole unit of two termini to other units, respectively, instead of transferring by means of a one-by-one dipole unit hopping mechanism. These findings not only promote a deeper understanding of the connection between Eex and structural and electronic properties of electron transfer behavior in peptides, but also provide a new insight into the modulation of electron migration along the oligopeptides.

Activating Transcription Factor 4 (ATF4)-ATF3-C/EBP Homologous Protein (CHOP) Cascade Shows an Essential Role in the ER Stress-Induced Sensitization of Tetrachlorobenzoquinone-Challenged PC12 Cells to ROS-Mediated Apoptosis via Death Receptor 5 (DR5) Signaling.

Tetrachlorobenzoquinone (TCBQ) is a downstream metabolite of pentachlorophenol (PCP). Previously, we demonstrated that TCBQ caused cytotoxicity due to mitochondrial-related apoptosis. Here, we confirmed the upregulation of death receptor 5 (DR5) followed by the construction of the death-inducing signaling complex (DISC). We also detected the activation of the caspase cascade, which was correlated with TCBQ-induced apoptotic cell death in PC12 cells. The upregulation of DR5 included transcriptional activation and de novo protein synthesis in response to TCBQ. We also identified the endoplasmic reticulum (ER) as a new target for the TCBQ challenge in PC12 cells. The protein kinase R-like ER kinase/eukaryotic translation initiation factor 2α (PERK/eIF2α)-mediated activating transcription factor 4 (ATF4)-ATF3-C/EBP homologous protein (CHOP) signaling pathway contributed to the process of TCBQ-induced ER stress. Blocking ATF4, ATF3, or CHOP signaling by gene silencing technology resulted in decreased cell apoptosis after exposure to TCBQ. Finally, NAC ameliorated TCBQ-induced apoptosis and ER stress, which illustrated that TCBQ-induced apoptosis is somehow ROS-dependent. In summary, this study provided important mechanistic insight into how TCBQ utilizes ER stress-related signaling to exhibit pro-apoptotic activity in PC12 cells.

Unpredicted Downregulation of RAD51 Suggests Genome Instability Induced by Tetrachlorobenzoquinone.

We previously demonstrated that halogenated quinone induces DNA double strand breaks (DSBs) in a ROS-dependent manner, which coordinates with downstream repair cascade including nonhomologous end joining, base excision repair, and nucleotide excision repair. However, these error-prone processes may cause the potential risk of genome instability, and current has no information on how faithful repair route, such as homologous recombination (HR), was affected. RAD51 is a key protein in the HR pathway of DSBs repair. Here, we found that tetrachlorobenzoquinone (TCBQ) causes a time-dependent reverse U-shape biphasic trend of RAD51 expression. An increase in the early stage and a following decrease of RAD51 expression were found in both 12.5 and 25 µM TCBQ groups, wherein higher concentration faced a faster response. The upregulated RAD51 in the early phase suggested the attempting to repair TCBQ-induced DNA damage; however, the downregulation of RAD51 in the late phase implicated that the rescue probably be abandoned with severe DNA damage. This phenomenon is a general toxic manner of TCBQ regardless of cell type. Surprisingly, TCBQ showed minimum effect on RAD51 mRNA (or protein) synthesis as well as RAD51 degradation. Specific inhibition of RAD51 by siRNA amplified TCBQ-induced DNA damage and cytotoxicity, while cells with enhanced RAD51 expression resisted TCBQ-induced toxicity. The modulation of RAD51 is correlated with p53 level, which suggests p53 has a role in TCBQ-induced RAD51 clearance. Together, our data suggested that TCBQ increases genome instability and cell death through a unique mechanism of inducing DNA damage and inhibiting DNA repair.

Quinones Derived from Polychlorinated Biphenyls Induce ROS-Dependent Autophagy by Evoking an Autophagic Flux and Inhibition of mTOR/p70S6k.

Autophagy is a "self-eating" destructive process that eliminates damaged organelles to maintain cellular homeostasis. Polychlorinated biphenyls (PCBs) are one of the most infamous industrial pollutants, which are ubiquitous in nature. In the present study, we found that an active, quinone-type PCB metabolite (PCB29-pQ) treatment causes an autophagic response through mTOR/p70S6k inhibition in HepG2 and MDA-MB-231 cells. Furthermore, our data suggested that PCB29-pQ enhances autophagosome formation through autophagic vacuole (AV) biogenesis, which evokes autophagic flux and induces AV-lysosome colocalization. The inhibition of autophagy enhanced PCB29-pQ-caused cytotoxicity, suggesting that autophagy serves as pro-survival machinery that plays a protective role in the early stage of PCB29-pQ-induced insult. However, higher concentration of PCB29-pQ exposure (>5 µM) caused autophagic cell death, which implied a shift from "pro-survival" to "pro-death" upon autophagic signaling. N-Acetylcysteine suppressed PCB29-pQ-induced autophagy and cytotoxicity, suggesting that ROS plays an important role in the regulation of PCB29-pQ-induced autophagy. Because autophagy shows significant implications in various human diseases and conditions, our current study provides a new mechanism for PCB-associated toxicity.

Tetrachlorobenzoquinone Stimulates NLRP3 Inflammasome-Mediated Post-Translational Activation and Secretion of IL-1ß in the HUVEC Endothelial Cell Line.

Our previous studies suggested that tetrachlorobenzoquinone (TCBQ) elicits pro-inflammatory activities; however, the mechanism of its toxicity toward vascular endothelial cell has not been characterized. Although TCBQ has been shown to stimulate interleukin-1 beta (IL-1ß) expression, it is unknown whether TCBQ regulates post-translational IL-1ß activation. Using human umbilical vein endothelial cells, we discovered that TCBQ not only promotes the expression of NOD-like receptor family, pyrin domain-containing protein 3 (NLRP3) components [composed of NLRP3, adaptor molecule apoptosis-associated speck like protein containing a caspase activation and recruitment domain (ASC), and pro-caspase 1] but also participates in priming the NLRP3 inflammasome. Activation of the NLRP3 inflammasome results in the maturation and release of IL-1ß. Further experiments showed that K(+) efflux, reactive oxygen species (ROS) production, and mitochondrial DNA damage may be involved in NLRP3 inflammasome activation mediated by TCBQ. Moreover, TCBQ downregulates the ubiquitination of NLRP3, further facilitating the activation of the NLRP3 inflammasome. These results suggest that the NLRP3/IL-1ß signaling pathway plays an important role in TCBQ-induced endothelial cell pro-inflammatory responses, which may point to potential therapeutic approaches against TCBQ-mediated toxicity.

Polychlorinated biphenyl quinone induces oxidative DNA damage and repair responses: The activations of NHEJ, BER and NER via ATM-p53 signaling axis.

Our previous studies demonstrated that polychlorinated biphenyl (PCB) quinone induced oxidative DNA damage in HepG2 cells. To promote genomic integrity, DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair and apoptosis. PCB quinone-induced cell cycle arrest and apoptosis have been documented, however, whether PCB quinone insult induce DNA repair signaling is still unknown. In this study, we identified the activation of DDR and corresponding signaling events in HepG2 cells upon the exposure to a synthetic PCB quinone, PCB29-pQ. Our data illustrated that PCB29-pQ induces the phosphorylation of p53, which was mediated by ataxia telangiectasia mutated (ATM) protein kinase. The observed phosphorylated histone H2AX (γ-H2AX) foci and the elevation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) indicated that DDR was stimulated by PCB29-pQ treatment. Additionally, we found PCB29-pQ activates non-homologous end joining (NHEJ), base excision repair (BER) and nucleotide excision repair (NER) signalings. However, these repair pathways are not error-free processes and aberrant repair of DNA damage may cause the potential risk of carcinogenesis and mutagenesis.

Polychlorinated biphenyl quinone induces endoplasmic reticulum stress, unfolded protein response, and calcium release.

Organisms are able to respond to environmental insult to maintain cellular homeostasis, which include the activation of a wide range of cellular adaptive responses with tightly controlled mechanisms. The endoplasmic reticulum (ER) is an organelle responsible for protein folding and calcium storage. ER stress leads to the accumulation of unfolded proteins in the ER lumen. To be against or respond to this effect, cells have a comprehensive signaling system, called unfolded protein response (UPR), to restore homeostasis and normal ER function or activate the cell death program. Therefore, it is critical to understand how environmental insult regulates the ingredients of ER stress and UPR signalings. Previously, we have demonstrated that polychlorinated biphenyl (PCB) quinone caused oxidative stress, cytotoxicity, genotoxicity, and apoptosis in HepG2 cells. Here, we investigated the role of a PCB quinone, PCB29-pQ on ER stress, UPR, and calcium release. PCB29-pQ markedly increased the hallmark genes of ER stress, namely, glucose-regulated protein 78 (GRP78), GRP94, and C/EBP homologous protein (CHOP) on both protein and mRNA levels in HepG2 cells. We also confirmed PCB29-pQ induced ER morphological defects by using transmission electron microscopy. Moreover, PCB29-pQ induced intracellular calcium accumulation and calpain activity, which were significantly inhibited by the pretreatment of BAPTA-AM (Ca(2+) chelator). These results were correlated with the outcome that PCB29-pQ induces ER stress-related apoptosis through caspase family gene 12, while salubrinal and Z-ATAD-FMK (a specific inhibitor of caspase 12) partially ameliorated this effect, respectively. N-Acetyl-l-cysteine (NAC) scavenged ROS formation and consequently alleviated PCB29-pQ-induced expression of ER stress-related genes. In conclusion, our result demonstrated for the first time that PCB quinone leads to ROS-dependent induction of ER stress, and UPR and calcium release in HepG2 cells, and the evaluation of the perturbations of ER stress, UPR, and calcium signaling provide further information on the mechanisms of PCB-induced toxicity.

Polychlorinated Biphenyl Quinone Metabolite Promotes p53-Dependent DNA Damage Checkpoint Activation, S-Phase Cycle Arrest and Extrinsic Apoptosis in Human Liver Hepatocellular Carcinoma HepG2 Cells.

Polychlorinated biphenyls (PCBs) are a group of persistent organic pollutants. The toxic behavior and mechanism of PCBs individuals and congeners have been extensively investigated. However, there is only limited information on their metabolites. Our previous studies have shown that a synthetic PCB metabolite, PCB29-pQ, causes oxidative damage with the evidence of cytotoxicity, genotoxicity, and mitochondrial-derived intrinsic apoptosis. Here, we investigate the effects of PCB29-pQ on DNA damage checkpoint activation, cell cycle arrest, and death receptor-related extrinsic apoptosis in human liver hepatocellular carcinoma HepG2 cells. Our results illustrate that PCB29-pQ increases the S-phase cell population by down-regulating cyclins A/D1/E, cyclin-dependent kinases (CDK 2/4/6), and cell division cycle 25A (CDC25A) and up-regulating p21/p27 protein expressions. PCB29-pQ also induces apoptosis via the up-regulation of Fas/FasL and the activation of caspase 8/3. Moreover, p53 plays a pivotal role in PCB29-pQ-induced cell cycle arrest and apoptosis via the activation of ATM/Chk2 and ATR/Chk1 checkpoints. Cell cycle arrest and apoptotic cell death were attenuated by the pretreatment with antioxidant N-acetyl-cysteine (NAC). Taken together, these results demonstrate that PCB29-pQ induces oxidative stress and promotes p53-dependent DNA damage checkpoint activation, S-phase cycle arrest, and extrinsic apoptosis in HepG2 cells.