Tetrabromobisphenol A induces neuronal cytotoxicity by inhibiting PINK1-Parkin-mediated mitophagy via upregulating ATF3 expression.

Tetrabromobisphenol A (TBBPA), as a widely used brominated flame retardant, has been implicated as a potential neurotoxicant. However, the mechanism of TBBPA-induced neurotoxicity has not been fully elucidated yet. In this study, using mouse hippocampal neuron cell HT22 as the in vitro model, the neuronal cytotoxicity of TBBPA and the mechanism by focusing on mitophagy have been studied. We found that neuronal cytotoxic effects were indeed induced by TBBPA exposure at concentrations of >20 µM for 24 h, including decreased cell viability (to 92.38 % at 20 µM; 18.25 % at 80 µM), enhanced ROS (enhanced 53.26 % at IC50 of 60 µM, compared with that in the control group) and mitochondrial ROS (mtROS) levels (enhanced 24.12 % at 60 µM), reduced mitochondrial membrane potential (MMP) (decreased 33.60 % at 60 µM). As a protective mechanism in cells, autophagy was initiated; however, mitophagy was inhibited, where PINK1 (PINK1-Parkin activation is critical in the depolarized MMP-induced mitophagy) expression was found to be repressed and decreased, further leading to the failure of Parkin recruitment to the damaged mitochondria. Mitophagy activator, nicotinamide mononucleotide (ß-NMN) that activates the PINK1-Parkin pathway, could alleviate TBBPA-induced mitophagy deficiency and further reduce the neuronal cytotoxicity, demonstrating that TBBPA-induced PINK1-Parkin-mediated mitophagy deficiency contributed to the neuronal cytotoxicity. Furthermore, we found TBBPA caused the upregulation of Atf3 (activating transcription factor 3) gene transcription and expression levels, alongside reduced Pink1 levels; whereas enhanced Pink1 transcript levels were observed after ATF3 depletion even under TBBPA treatment, demonstrating TBBPA-induced overexpression of ATF3 should be responsible for the reduced PINK1 expression. Therefore, for the first time, here we demonstrate that TBBPA can inhibit PINK1-Parkin-mediated mitophagy via upregulating ATF3 expression, which further contributes to its neuronal cytotoxicity. This study should be able to improve our understanding of the mechanism of TBBPA-induced neuronal cytotoxicity.

Unusual enantioselective cytoplasm-to-nucleus translocation and photosensitization of the chiral Ru(II) cationic complex via simple ion-pairing with lipophilic weak acid counter-anions.

Targeted and enantioselective delivery of chiral diagnostic-probes and therapeutics into specific compartments inside cells is of utmost importance in the improvement of disease detection and treatment. The classical DNA 'light-switch' ruthenium(II)-polypyridyl complex, [Ru(DIP)2(dppz)]Cl2 (DIP = 4,7-diphenyl-1,10-phenanthroline, dppz = dipyridophenazine) has been shown to be accumulated only in the cytoplasm and membrane, but excluded from its intended nuclear DNA target. In this study, the cationic [Ru(DIP)2(dppz)]2+ is found to be redirected into live-cell nucleus in the presence of lipophilic 3,5-dichlorophenolate or flufenamate counter-anions via ion-pairing mechanism, while maintaining its original DNA recognition characteristics. Interestingly and unexpectedly, further studies show that only the Δ-enantiomer is selectively translocated into nucleus while the Λ-enantiomer remains trapped in cytoplasm, which is found to be mainly due to their differential enantioselective binding affinities with cytoplasmic proteins and nuclear DNA. More importantly, only the nucleus-relocalized Δ-enantiomer can induce obvious DNA damage and cell apoptosis upon prolonged visible-light irradiation. Thus, the use of Δ-enantiomer can significantly reduce the dosage needed for maximal treatment effect. This represents the first report of enantioselective targeting and photosensitization of classical Ru(II) complex via simple ion-pairing with suitable weak acid counter-anions, which opens new opportunities for more effective enantioselective cancer treatment.

A fluorescent sensor recognized by the FA1 site for highly sensitive detection of HSA.

Human serum albumin (HSA), as the most abundant protein in blood plasma, plays a crucial role in many physiological processes. The abnormal HSA level in serum or in urine is often associated with various diseases. Therefore, to achieve highly sensitive and selective quantification of HSA is of great importance for disease diagnosis and preventive medicine. Herein, an HSA-selective light-up fluorescent sensor, DCM-ML, was successfully developed for quantitative detection of HSA. DCM-ML exhibited good (photo-) stability and strong fluorescence enhancement around 630 nm in the presence of HSA in complex samples containing numerous biological analytes. Upon addition of HSA into DCM-ML containing solution, a good linear relationship (R2 > 0.99) between the fluorescence intensity of DCM-ML and HSA concentration from 0 to 0.08 mg/mL was obtained with the detection limit of 0.25 µg/mL. The sensing mechanism of the sensor towards HSA was demonstrated to be via recognition in the fatty acid site 1 (FA1), instead of the most reported binding sites (Sudlow I and II) in HSA, for the first time, by both the displacement experiments and molecular docking simulation. Thus, DCM-ML can also be assumed as a potential FA1 site-binding marker for examining drugs binding to the FA1 site in HSA. At last, the utilization of sensor DCM-ML for quantification and validation of HSA in urine samples and cell culture medium was effectively demonstrated. Therefore, the development of DCM-ML should find great application potentials in the fields of analytical chemistry and clinical medicine as a highly sensitive HSA sensor.

Pb induced mitochondrial fission of fibroblast cells via ATM activation.

Previous study showed that lead (Pb) could induce ATM-dependent mitophagy. However, whether Pb has any impact on mitochondrial fusion and fission, the upstream events of mitophagy, and how ATM connects to these processes remain unclear. In this study, we found that Pb can disrupt mitochondrial network morphology as indicated by increased percentage of shortened mitochondria and by decreased mitochondrial footprints. Correspondingly, the expression of fission protein Drp1 and its association with mitochondrial marker Hsp60 were significantly increased, while those of fusion proteins Mfn2 and Opa1 and their co-localization with Hsp60 were drastically attenuated. Notably, the expression of p-Drp1 (Ser616) and its translocation to mitochondria were dramatically elevated. Moreover, a small amount of ATM could be detected in the cytoplasm around mitochondria in response to Pb, and the co-localization of p-ATM (Ser1981) with Drp1 and p-Drp1 (Ser616) was obviously increased while its co-localization with Mfn2 and Opa1 was dramatically decreased. Furthermore, siRNA silencing of ATM evidently promoted greater fission in response to Pb stress, indicating that ATM is involved in mitochondrial fragmentation. Our results suggest that cytoplasmic ATM is an important regulator of Pb-induced mitochondrial fission.

Light-Driven Cascade Mitochondria-to-Nucleus Photosensitization in Cancer Cell Ablation.

Nuclei and mitochondria are the only cellular organelles containing genes, which are specific targets for efficient cancer therapy. So far, several photosensitizers have been reported for mitochondria targeting, and another few have been reported for nuclei targeting. However, none have been reported for photosensitization in both mitochondria and nucleus, especially in cascade mode, which can significantly reduce the photosensitizers needed for maximal treatment effect. Herein, a light-driven, mitochondria-to-nucleus cascade dual organelle cancer cell ablation strategy is reported. A functionalized iridium complex, named BT-Ir, is designed as a photosensitizer, which targets mitochondria first for photosensitization and subsequently is translocated to a cell nucleus for continuous photodynamic cancer cell ablation. This strategy opens new opportunities for efficient photodynamic therapy.

Highly Photostable Fluorescent Tracker with pH-Insensitivity for Long-Term Imaging of Lysosomal Dynamics in Live Cells.

Visualizing and tracking lysosomal dynamic changes is crucially important in the fields of physiology and pathology. Most currently used pH-dependent small-molecule lysotrackers and sensors usually fail to visualize and track the changes due to (1) their leakage from lysosomes when the lysosomal pH increases and (2) their low photostability. Therefore, it is of significant interest to develop lysosomal probes for visualizing and tracking lysosomal dynamics independent of pH fluctuations and with high photostability. Herein, we found that the popular dicyanomethylene-4H-pyran (DCM) derivative DCM-NH2 can selectively target and label lysosomes with bright red fluorescence regardless of pH changes. The fluorescence enhancement in lysosomes has probably resulted from their microenvironment of polarity and viscosity. Compared with the commonly used commercial lysosomal molecular probes (LysoTracker Deep Red (LTDR) and LysoTracker Red DND-99), DCM-NH2 was demonstrated to exhibit a much stronger tolerance in lysosomes against various treatments and microenvironmental changes, and lysosomal membrane permeability could not cause DCM-NH2 to lose imaging of their targets as well. Moreover, DCM-NH2 exhibited a superior anti-photobleaching ability and low (photo-) cytotoxicity, which, along with pH-insensitivity, ensured its capability of long-term visualizing and tracking lysosomal dynamics. Lysosomal dynamic events such as the kiss-and-run process, fusion-fission, and mitophagy were successfully recorded with DCM-NH2. Our study thus confirms that DCM-NH2 is highly competitive for lysosomal imaging by overcoming the limitations of the commercial LysoTrackers and highlights the unexplored application of DCM-NH2 in bioimaging.

Targeted live-cell nuclear delivery of the DNA 'light-switching' Ru(II) complex via ion-pairing with chlorophenolate counter-anions: the critical role of binding stability and lipophilicity of the ion-pairing complexes.

We have found recently that nuclear uptake of the cell-impermeable DNA light-switching Ru(II)-polypyridyl cationic complexes such as [Ru(bpy)2(dppz)]Cl2 was remarkably enhanced by pentachlorophenol (PCP), by forming ion-pairing complexes via a passive diffusion mechanism. However, it is not clear whether the enhanced nuclear uptake of [Ru(bpy)2(dppz)]2+ is only limited to PCP, or it is a general phenomenon for other highly chlorinated phenols (HCPs); and if so, what are the major physicochemical factors in determining nuclear uptake? Here, we found that the nuclear uptake of [Ru(bpy)2(dppz)]2+ can also be facilitated by other two groups of HCPs including three tetrachlorophenol (TeCP) and six trichlorophenol (TCP) isomers. Interestingly and unexpectedly, 2,3,4,5-TeCP was found to be the most effective one for nuclear delivery of [Ru(bpy)2(dppz)]2+, which is even better than the most-highly chlorinated PCP, and much better than its two other TeCP isomers. Further studies showed that the nuclear uptake of [Ru(bpy)2(dppz)]2+ was positively correlated with the binding stability, but to our surprise, inversely correlated with the lipophilicity of the ion-pairing complexes formed between [Ru(bpy)2(dppz)]Cl2 and HCPs. These findings should provide new perspectives for future investigations on using ion-pairing as an effective method for delivering other bio-active metal complexes into their intended cellular targets.

What Are the Major Physicochemical Factors in Determining the Preferential Nuclear Uptake of the DNA "Light-Switching" Ru(II)-Polypyridyl Complex in Live Cells via Ion-Pairing with Chlorophenolate Counter-Anions?

Delivering potential theranostic metal complexes into preferential cellular targets is becoming of increasing interest. Here we report that nuclear uptake of a cell-impermeable DNA "light-switching" Ru(II)-polypyridyl complex can be significantly facilitated by chlorophenolate counter-anions, which was found, unexpectedly, to be correlated positively with the binding stability but inversely with the lipophilicity of the formed ion pairs.

The Critical Role of X Chromosome-Linked Inhibitor of Apoptosis (XIAP) in Differential Synergism Induced by Pentachlorophenol and Copper-1,10-Phenanthroline Complex in Normal and Cancer Liver Cells.

Chemical pollutants often co-occur and can interact to cause unexpected combined toxic effects. Both pentachlorophenol (PCP) and copper-1,10-phenanthroline [Cu(OP)2], used as wood preservatives, coexist in fluids and tissues of ordinary population. Our previous studies demonstrate that a combination of subtoxic PCP and Cu(OP)2 causes synergistic toxicity on Escherichia coli and hepatocarcinoma cells. However, it is not clear whether this effect also occurs in normal hepatocytes; and if so, what are the differences as compared with the hepatocarcinoma cells. We demonstrate that the combination of low-toxic PCP and Cu(OP)2 (0-1.6 µM; PCP/Cu(OP)2 molar ratio: 2:1) induces a concentration-dependent intracellular copper accumulation, apoptosis, caspase-3/9 activation, depolarization of mitochondrial membrane potential, and oxidative stress (reactive oxygen species increasing and glutathione/oxidized glutathione ratio decreasing) in both normal hepatocytes HL-7702 and hepatocarcinoma HepG2 cells. However, HepG2 cells are more susceptible to the above molecular events as compared with HL-7702 cells. Further data reveal that PCP/Cu(OP)2 markedly decreases X chromosome-linked inhibitor of apoptosis (XIAP), p-ERK-1/2, and p-JNK protein expression in HepG2, but not HL-7702. Overexpression of XIAP gene in HepG2 significantly blocks PCP/Cu(OP)2-induced cytotoxicity, caspase activity, apoptosis, ROS accumulation, and antioxidant genes expression. These results suggest that the combination of low-toxic PCP and Cu(OP)2 preferentially induce synergistic cytotoxicity in human hepatocarcinoma cells by XIAP-ROS-apoptosis pathway, compared with the normal hepatocytes. The present data not only confirm the synergistic toxicity of PCP/Cu(OP)2 combination in normal liver cells, but also suggest a possible opportunity in developing new therapeutic approaches for liver cancer by sensitizing cancer cells to chemotherapy.

Cationic Organochalcogen with Monomer/Excimer Emissions for Dual-Color Live Cell Imaging and Cell Damage Diagnosis.

Studies on the development of fluorescent organic molecules with different emission colors for imaging of organelles and their biomedical application are gaining lots of focus recently. Here, we report two cationic organochalcogens 1 and 2, both of which exhibit very weak green emission (Φ1 = 0.12%; Φ2 = 0.09%) in dilute solution as monomers, but remarkably enhanced green emission upon interaction with nucleic acids and large red-shifted emission in aggregate state by the formation of excimers at high concentration. More interestingly, the monomer emission and excimer-like emission can be used for dual color imaging of different organelles. Upon passively diffusing into cells, both probes selectively stain nucleoli with strong green emission upon 488 nm excitation, whereas upon 405 nm excitation, a completely different stain pattern by staining lysosomes (for 1) or mitochondria (for 2) with distinct red emission is observed because of the highly concentrated accumulation in these organelles. Studies on the mechanism of the accumulation in lysosomes (for 1) or mitochondria (for 2) found that the accumulations of the probes are dependent on the membrane permeabilization, which make the probes have great potential in diagnosing cell damage by sensing lysosomal or mitochondrial membrane permeabilization. The study is demonstrative, for the first time, of two cationic molecules for dual-color imaging nucleoli and lysosomes (1)/mitochondria (2) simultaneously in live cell based on monomer and excimer-like emission, respectively, and more importantly, for diagnosing cell damage.

Red fluorescent probes for real-time imaging of the cell cycle by dynamic monitoring of the nucleolus and chromosome.

Two cationic molecular rotors, 1 and 2, capable of real-time cell-cycle imaging by specifically dynamic monitoring of nucleolus and chromosome changes were developed. A further study shows that fluorescence enhancements in the nucleolus and chromosome are attributed to a combination effect of interaction with nucleic acid and high condensation of the nucleolus and chromosome.

A coumarin Schiff's base two-photon fluorescent probe for hypochlorite in living cells and zebrafish.

Selective and sensitive fluorescent probes for ClO- are desirable due to the importance of ClO- in biological processes. Here, a coumarin Schiff's base, compound 1, has been developed and successfully used as a one- and two-photon fluorescent probe for ClO- with high selectivity. This probe can recognize ClO- with obvious color change from yellow-green to colorless and green to blue fluorescence emission, which can be observed by the naked eye. The properties of low cytotoxicity and good cell permeability allow it to be used for ClO- detection in living cells and zebrafish by both one- and two-photon microscopy imaging. All these results indicate that the compound is a sensitive probe with potential for analysis of ClO- in biological samples. The mechanism by which probe 1 recognizes ClO- is possibly nucleophilic addition followed by hydrolysis.

Delivering the cell-impermeable DNA 'light-switching' Ru(ii) complexes preferentially into live-cell nucleus via an unprecedented ion-pairing method.

The dipyridophenazine (dppz) based ruthenium polypyridyl complexes are known as molecular 'light-switches' for DNA. This property is poised to serve in diagnostic and therapeutic applications, but the poor cellular uptake restricts their use in live cells. Herein, we show that the cellular uptake, and more interestingly and surprisingly, the nuclear uptake of cell-impermeable Ru(ii)-polypyridyl cationic complexes such as [Ru(bpy)2(dppz)]2+ were remarkably enhanced by three structurally unrelated biochemical agents (pentachlorophenol, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone and tolfenamic acid), by forming lipophilic and relatively stable ion-pair complexes, via a passive diffusion mechanism. Enantioselective imaging of live-cell nuclear DNA was observed between the two chiral forms of Ru(ii) complexes. This represents the first report of an unprecedented new method for delivering the DNA 'light-switching' Ru(ii) complexes into the nucleus of living cells via ion-pairing, which could serve as a promising general live-cell delivery method for other potentially bio-medically important but cell-impermeable metal complexes.

Lethal synergism between organic and inorganic wood preservatives via formation of an unusual lipophilic ternary complex.

We have shown previously that exposing bacteria to wood preservatives pentachlorophenol (PCP) and copper-containing compounds together causes synergistic toxicity. However, it is not clear whether these findings also hold true in mammalian cells; and if so, what is the underlying molecular mechanism? Here we show that PCP and a model copper complex bis-(1,10-phenanthroline) cupric (Cu(OP)(2)), could also induce synergistic cytotoxicity in human liver cells. By the single crystal X-ray diffraction and atomic absorption spectroscopy assay, the synergism was found to be mainly due to the formation of a lipophilic ternary complex with unusual structural and composition characteristics and subsequent enhanced cellular copper uptake, which markedly promoted cellular reactive oxygen species (ROS) production, leading to apoptosis by decreasing mitochondrial membrane potential, increasing pro-apoptotic protein expression, releasing cytochrome c from mitochondria and activating caspase-3, and -9. Analogous results were observed with other polychlorinated phenols (PCPs) and Cu(OP)(2). Synergistic cytotoxicity could be induced by PCP/Cu(OP)(2) via formation of an unusual lipophilic complex in HepG2 cells. The formation of ternary complexes with similar lipophilic character could be of relevance as a general mechanism of toxicity, which should be taken into consideration especially when evaluating the toxicity of environmental pollutants found at currently-considered non- or sub-toxic concentrations.

Low concentrations of bisphenol a suppress thyroid hormone receptor transcription through a nongenomic mechanism.

Bisphenol (BPA) is one of the highest-volume chemicals produced worldwide, and human exposure to BPA is thought to be ubiquitous. Various rodent and in vitro studies have shown that thyroid hormone (TH) function can be impaired by BPA. However, it is still unknown if low concentrations of BPA can suppress the thyroid hormone receptor (TR) transcription. The present study aims to investigate the possible suppressing effects of low concentrations of BPA on TR transcription and the involved mechanism(s) in CV-1 cells derived from cercopithecus aethiops monkey kidneys. Using gene reporter assays, BPA at concentrations as low as 10(-9)M suppresses TR or steroid receptor coactivator-1(SRC-1)-enhanced TR transcription, but not reducing TR/SRC-1 interaction in mammalian two-hybrid and glutathione S-transferase pull-down studies. It has been further shown that both nuclear receptor co-repressor (N-CoR) and silencing mediator for retinoid and thyroid hormone receptors (SMRT) are recruited to the TR-ß1 by BPA in the presence of physiologic concentrations of T3 or T4. However, the overexpression of ß3 integrin or c-Src significantly reduces BPA-induced recruitment of N-CoR/SMRT to TR or suppression of TR transcription. Furthermore, BPA inhibits the T3/T4-mediated interassociation of the ß3 integrin/c-Src/MAPK/TR-ß1 pathways by the co-immunoprecipitation. These results indicate that low concentrations of BPA suppress the TR transcription by disrupting physiologic concentrations of T3/T4-mediated ß3 integrin/c-Src/MAPK/TR-ß1 pathways, followed by recruiting N-CoR/SMRT to TR-ß1, providing a novel insight regarding the TH disruption effects of low concentration BPA.