The anti-cancer drug candidate CBL0137 induced necroptosis via forming left-handed Z-DNA and its binding protein ZBP1 in liver cells.

CBL0137, a promising small molecular anti-cancer drug candidate, has been found to effectively induce apoptosis via activating p53 and suppressing nuclear factor-kappa B (NF-κB). However, it is still not clear whether CBL0137 can induce necroptosis in liver cancer; and if so, what is the underlying molecular mechanism. Here we found that CBL0137 could significantly induce left-handed double helix structure Z-DNA formation in HepG2 cells as shown by Z-DNA specific antibody assay, which was further confirmed by observing the expression of Z-DNA binding protein 1 (ZBP1) and adenosine deaminase acting on RNA 1 (ADAR1). Interestingly, we found that caspase inhibition significantly promoted CBL0137-induced necroptosis, which was further supported with the increase of the late apoptosis and necrosis assessed by the flow cytometry. Furthermore, we found that CBL0137 can also induce the expression of the three necroptosis-related proteins: receptor interacting serine/threonine kinase 1 (RIPK1), receptor interacting serine/threonine kinase 3 (RIPK3), and mixed lineage kinase domain-like (MLKL). Taken together, it was assumed that CBL0137-indued necroptosis in liver cells was due to induction of Z-DNA and ZBP1, which activated RIPK1/RIPK3/MLKL pathway. This represents the first report on the induction of the Z-DNA-mediated necroptosis by CBL0137 in the liver cancer cells, which should provide new perspectives for CBL0137 treatment of liver cancer.

Structure-Activity Relationship Investigation on Reaction Mechanism between Chlorinated Quinoid Carcinogens and Clinically-Used Aldoxime Nerve-Agent Antidote under Physiological Condition.

Pyridinium aldoximes are best-known therapeutic antidotes used for clinical treatment of poisonings by organophosphorus nerve-agents and pesticides. Recently, we found that pralidoxime (2-PAM, a currently clinically used nerve-agent antidote) could also detoxify tetrachloro-1,4-benzoquinone (TCBQ), which is a carcinogenic quinoid metabolite of the widely used wood preservative pentachlorophenol under normal physiological conditions, via an unusually mild and facile Beckmann fragmentation mechanism accompanied by radical homolysis. However, it is not clear whether the less-chlorinated benzoquinones (CnBQs, n ≤ 3) act similarly; if so, what is the structure-activity relationship? In this study, we found that (1) The stability of reaction intermediates produced by different CnBQs and 2-PAM was dependent not only on the position but also the degree of Cl-substitution on CnBQs, which can be divided into TCBQ- and DCBQ (dichloro-1,4-benzoquinone)-subgroup; (2) The pKa value of hydroxlated quinones (Cn-1BQ-OHs, the hydrolysis products of CnBQs), determined the stability of corresponding intermediates, that is, the decomposition rate of the intermediates depended on the acidity of Cn-1BQ-OHs; (3) The pKa value of the corresponding Cn-1BQ-OHs could also determine the reaction ratio of Beckmann fragmentation to radical homolysis in CnBQs/2-PAM. These new findings on the structure-activity relationship of the halogenated quinoid carcinogens detoxified by pyridinium aldoxime therapeutic agents via Beckmann fragmentation and radical homolysis reaction may have broad implications on future biomedical and environmental research.

Different modes of synergistic toxicities between metam/copper (II) and metam/zinc (II) in HepG2 cells: apoptosis vs. necrosis.

Both metam sodium and copper/zinc-containing compounds are widely used as fungicides. They therefore may co-occur in the biosphere. Despite certain studies of individual toxicity for either metam or copper (II)/zinc (II), their synergistic toxicity has not been examined. In this paper, a remarkable synergistic toxicity was observed in HepG2 cells when metam and copper (II)/zinc (II) at non-toxic and sub-toxic levels were combined. Unexpectedly, cell death modes between metam/copper (II) and metam/zinc (II) were different: For metam/copper (II), apoptosis was evident from morphological characteristics including cytoplasm-chromatin condensation, phosphatidylserine (PS) exposure, SubG0 /G1 DNA fragmentation, mitochondrial membrane potential decrease, pro/anti-apoptotic protein activation, and cytochrome c release; for metam/zinc (II), necrosis was evident from organelle swelling and uncontrolled collapse. To our knowledge, this work first not only demonstrates the synergistic toxicities of metam and both copper (II)/zinc (II), but also verifies the different modes of apoptosis/necrosis between metam/copper (II) and metam/zinc (II). © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1964-1973, 2016.

In vitro assessment of cytotoxicity of spent fluid catalytic cracking refinery catalysts on cell lines and identification of critical toxic metals.

The Purpose of the present study was to quantify the responses of ten cell lines (HeLa, HepG2, HEK293, MDA-MB-231, A498, A549, A357, 3 T3, BALB-C3 T3, and NIH-3 T3) to spent fluid catalytic cracking catalysts (SFCCCs) from different petroleum refineries, and relate these responses to metal concentrations of SFCCC leachates (SFCCCLs). Cytotoxicity of SFCCCs were significantly different depending on cell lines. A357 and 3 T3 cell were the most sensitive, and A498 and HeLa cells were the least sensitive. HEK293 cells showed the least fluctuation in toxic response to different SFCCCLs among all cells. Cytotoxic IC50 values of SFCCCs to 7 kinds of cells were the most correlated with vanadium (V) concentration in SFCCCLs. V is the most critical toxic factor of SFCCC. Glutathione synthesis was induced in HepG2 cells exposed to higher concentrations of SFCCCLs. SFCCCLs with low concentration of V can induce the decrease of GSH/GSSG ratio in HepG2 cells, suggesting that high concentration of V inhibits the detoxification of glutathione.

Bisphenol A at a low concentration boosts mouse spermatogonial cell proliferation by inducing the G protein-coupled receptor 30 expression.

Bisphenol A (BPA) is one of the most prevalent chemicals in daily-use materials, therefore, human exposure to BPA is ubiquitous. We found that low concentrations of BPA stimulate the spermatogonial GC-1 cells proliferation by G protein-coupled receptor 30 (GPR30)-mediated epidermal growth factor receptor (EGFR)-extracellular regulated kinase (ERK)-c-Fos pathway. However, through the same pathway GPR30 expression has been shown to be induced by EGF, an EGFR ligand. Thus, we want to know if low concentrations of BPA are able to induce the GPR30 expression and the possible mechanism(s) in GC-1 cells. By transient transfection with expression plasmids, 10(-9)M BPA significantly transactivates the Gpr30-5'-flanking region through activating the GPR30, cGMP-dependent protein kinase (PKG), estrogen receptor-α (ER-α), and EFGR-ERK pathways. Furthermore, an activator protein-1 (AP-1) site located within this region is found to be responsible for the transactivation of BPA. Expectedly, through the same pathways, BPA significantly induces the gene and protein expression of GPR30. c-Fos is further observed to be strongly recruited to the AP-1 site in a chromatin immunoprecipitation assay and its dysfunction on the AP-1 site markedly suppresses the expression of GPR30, p-ERK1/2, p-Ser118-ER-α and cell proliferation by BPA. Our results demonstrate that a low-concentration BPA induces GPR30 expression through the GPR30-EFGR-ERK-c-Fos, ER-α, and PKG pathways, presumably boosting the cells proliferation via a regulatory loop. The present study provides a novel insight into the potential role of GPR30 in the initiation and progression of male germ cell cancer induced by environmentally relevant BPA.

Ofloxacin induces apoptosis via ß1 integrin-EGFR-Rac1-Nox2 pathway in microencapsulated chondrocytes.

Quinolones (QNs)-induced arthropathy is an important toxic side-effect in immature animals leading to the restriction of their therapeutic use in pediatrics. Ofloxacin, a typical QN, was found to induce the chondrocytes apoptosis in the early phase (12-48 h) of arthropathy in our previous study. However, the exact mechanism(s) is unclear. Microencapsulated juvenile rabbit joint chondrocytes, a three-dimensional culture system, is utilized to perform the present study. Ofloxacin, at a therapeutically relevant concentration (10 µg/ml), disturbs the interaction between ß1 integrin and activated intracellular signaling proteins at 12 h, which is inhibited when supplementing Mg(2+). Intracellular reactive oxygen species (ROS) significantly increases in a time-dependent manner after exposure to ofloxacin for 12-48 h. Furthermore, ofloxacin markedly enhances the level of activated Rac1 and epidermal growth factor receptor (EGFR) phosphorylation, and its inhibition in turn reduces the ROS production, apoptosis and Rac1 activation. Silencing Nox2, Rac1 or supplementing Mg(2+) inhibits ROS accumulation, apoptosis occurrence and EGFR phosphorylation induced by ofloxacin. However, depletion of Nox2, Rac1 and inhibition of EGFR do not affect ofloxacin-mediated loss of interaction between ß1 integrin and activated intracellular signaling proteins. In addition, ofloxacin also induces Vav2 phosphorylation, which is markedly suppressed after inactivating EGFR or supplementing Mg(2+). These results suggest that ofloxacin causes Nox2-mediated intracellular ROS production by disrupting the ß1 integrin function and then activating the EGFR-Vav2-Rac1 pathway, finally resulting in apoptosis within 12-48 h exposure. The present study provides a novel insight regarding the potential role of Nox-driven ROS in QNs-induced arthropathy.

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.

An unprecedented free radical mechanism for the formation of DNA adducts by the carcinogenic N-sulfonated metabolite of aristolochic acids.

The carcinogenicity of aristolochic acids (AAs) has been attributed mainly to the formation of stable DNA-aristolactam (DNA-AL) adducts by its reactive N-sulfonated metabolite N-sulfonatooxyaristolactam (N-OSO3--AL). The most accepted mechanism for such DNA-AL adduct formation is via the postulated but never unequivocally-confirmed aristolactam nitrenium ion. Here we found that both sulfate radical and two ALI-derived radicals (N-centered and C-centered spin isomers) were produced by N-OSO3--ALI, which were detected and unequivocally identified by complementary applications of ESR spin-trapping, HPLC-MS coupled with deuterium-exchange methods. Both the formation of the three radical species and DNA-ALI adducts can be significantly inhibited (up to 90%) by several well-known antioxidants, typical radical scavengers, and spin-trapping agents. Taken together, we propose that N-OSO3--ALI decomposes mainly via a new N-O bond homolysis rather than the previously proposed heterolysis pathway, yielding reactive sulfate and ALI-derived radicals, which are together and in concert responsible for forming DNA-ALI adducts. This study presents strong and direct evidence for the production of free radical intermediates during N-OSO3--ALI decomposition, providing an unprecedented free radical perspective and conceptual breakthrough, which can better explain and understand the molecular mechanism for the formation of DNA-AA adducts, the carcinogenicity of AAs and their potential prevention.

Molecular mechanism of the unusual biphasic effects of the natural compound hinokitiol on iron-induced cellular DNA damage.

Hinokitiol is a natural monoterpene compound found in the heartwood of cupressaceous plants that have anticancer and anti-inflammatory properties. However, few studies have focused on its effect on iron-mediated cellular DNA damage. Here we show that hinokitiol exhibited unusual biphasic effects on iron-induced DNA damage in a molar ratio (hinokitiol/iron) dependent manner in HeLa cells. Under low ratios (<3:1), hinokitiol markedly enhanced DNA damage induced by Fe(II) or Fe(II)-H2O2; However, when the ratios increased over 3:1, the DNA damage was progressively inhibited. We found that the total cytoplasmic and nuclear iron concentration increased as the ratios of hinokitiol/iron increased. However, the cellular level of labile iron pool (LIP) only increased at ratios lower than 3, and the ROS generation is consistent with LIP change. Hinokitiol was found to interact with iron to form lipophilic hinokitiol-iron complexes with different stoichiometry and redox-activity by complementary applications of various analytical methods. Taken together, we propose that the enhancement of iron-induced cellular DNA damage by hinokitiol at low ratios (<3:1) was due to formation of lipophilic and redox-active iron complexes which facilitated cellular iron uptake and •OH production, while the inhibition at ratios higher than 3 was due to formation of redox-inactive iron complexes. These new findings will help us to design more effective drugs for the prevention and treatment of a series of iron-related diseases via regulating the two critical physicochemical factors (lipophilicity and redox activity of iron complexes) by simple natural compounds with iron-chelating properties.

Mechanistic investigation of the differential synergistic neurotoxicity between pesticide metam sodium and copper or zinc.

Epidemiological studies suggest neurological disorders have been associated with the co-exposure to certain pesticides and transition metals. The present study aims to investigate whether co-exposure to the widely-used pesticide metam sodium and copper (Cu2+) or zinc ion (Zn2+) is able to cause synergistic neurotoxicity in neural PC12 cells and its possible mechanism(s). We found that both metam/Cu2+ and metam/Zn2+ synergistically induced apoptosis, intracellular Cu2+/Zn2+ uptake, reactive oxygen species (ROS) accumulation, double-strand DNA breakage, mitochondrial membrane potential decrease, and nerve function disorder. In addition, metam/Cu2+ was shown to release cytochrome c and apoptosis-inducing factor (AIF) from mitochondria to cytoplasm and nucleus, respectively, and activate the caspase 9, 8, 3, 7. However, metam/Zn2+ induced caspase 7 activation and AIF translocation and mildly activated cytochrome c/caspase 9/caspase 3 pathway. Furthermore, metam/Cu2+ activated caspase 3/7 by the p38 pathway, whereas metam/Zn2+ did so via both the p38 and JNK pathways. These results demonstrated that metam/Cu2+ or metam/Zn2+ co-exposure cause synergistic neurotoxicity via different mechanisms, indicating a potential risk to human health when they environmentally co-exist.

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.

Caffeic Acid Phenyl Ester (CAPE) Protects against Iron-Mediated Cellular DNA Damage through Its Strong Iron-Binding Ability and High Lipophilicity.

Caffeic acid phenethyl ester (CAPE) and its structurally-related caffeic acid (CA), ferulic acid (FA) and ethyl ferulate (EF) are constituents of honeybee propolis that have important pharmacological activities. This study found that CAPE-but not CA, FA, and EF-could effectively prevent cellular DNA damage induced by overloaded iron through decreasing the labile iron pool (LIP) levels in HeLa cells. Interestingly, CAPE was found to be more effective than CA in protecting against plasmid DNA damage induced by Fe(II)-H2O2 or Fe(III)-citrate-ascorbate-H2O2 via the inhibition of hydroxyl radical (•OH) production. We further provided more direct and unequivocal experimental evidences for the formation of inactive CAPE/CA-iron complexes. CAPE was found to have a stronger iron-binding ability and a much higher lipophilicity than CA. Taken together, we propose that the esterification of the carboxylic moiety with phenethyl significantly enhanced the iron-binding ability and lipophilicity of CAPE, which is also responsible for its potent protection against iron-mediated cellular DNA damage. A study on the iron coordination mechanism of such natural polyphenol antioxidants will help to design more effective antioxidants for the treatment and prevention of diseases caused by metal-induced oxidative stress, as well as help to understand the structure-activity relationships of these compounds.

The critical role of superoxide anion radicals on delaying tetrachlorohydroquinone autooxidation by penicillamine.

We have recently found that penicillamine, a classic copper-chelating thiol-drug for Wilson's disease, can delay tetrachlorohydroquinone (TCHQ) autooxidation via a previously unrecognized redox-activity. However, its underlying molecular mechanism remains not fully understood. In this study, we found, interestingly and unexpectedly, that superoxide dismutase (SOD) can significantly shorten the delay of TCHQ autooxidation by penicillamine, but not by ascorbate; SOD can also markedly increase the yields of the oxidized form of penicillamine. Similar effects were observed with a recently-developed specific and sensitive superoxide anion radical (O2•-) probe CT-02H, which was also employed to successfully measure O2•- generated from both TCHQ and TCHQ/penicillamine systems for the first time. More importantly, addition of extra O2•- (KO2/18-crown-6) can further prolong the delaying effects by penicillamine and slow down penicillamine consumption. Taken together, an unexpected critical role of O2•- in TCHQ/penicillamine interaction was proposed: O2•- may regenerate penicillamine, thereby continuously reducing TCSQ•- to TCHQ and finally delaying TCHQ autooxidation; In contrast, if O2•- were eliminated, which can not only markedly change the reaction equilibrium, accelerate the rate of interaction, and ultimately shorten the delay of TCHQ autooxidation by penicillamine, but can also accelerate penicillamine oxidation to form its corresponding disulfide solely via redox reaction without any minor nucleophilic reaction. These findings not only further support our previously-proposed redox mechanism for the protection against TCHQ-induced cytotoxicity by penicillamine, but also reveal a new mode of action for O2•- in the inhibition of haloquinoids-induced toxicity by thiol antioxidants.

The cell-impermeable Ru(II) polypyridyl complex as a potent intracellular photosensitizer under visible light irradiation via ion-pairing with suitable lipophilic counter-anions.

Developing the cell-impermeable Ru(II) polypyridyl cationic complexes as effective photosensitizers (PS) which have high cellular uptake and photo-toxicity, but low dark toxicity, is quite challenging. Here we found that the highly reactive singlet oxygen (1O2) can be generated by the irradiation of a typical Ru(II) polypyridyl complex Ru(II)tris(tetramethylphenanthroline) ([Ru(TMP)3]2+) under visible light irradiation by ESR with TEMPO (2,2,6,6-tetramethyl-4-piperidone-N-oxyl) as 1O2 probe. Effective cellular and nuclear delivery of cationic [Ru(TMP)3]2+ was achieved through our recently developed ion-pairing method, and 2,3,4,5-tetrachlorophenol (2,3,4,5-TeCP) was found to be the most effective among all chlorophenols tested. The accelerated cellular, especially nuclear uptake of [Ru(TMP)3]2+ results in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and DNA strand breaks, caspase 3/7 activation and cell apoptosis in HeLa cells upon light irradiation. More importantly, compared with other traditional photosensitizers, [Ru(TMP)3]2+ showed significant photo-toxicity but low dark toxicity. Similar effects were observed when 2,3,4,5-TeCP was substituted by the currently clinically used anti-inflammatory drug flufenamic acid. This represents the first report that the cell-impermeable Ru(II) polypyridyl complex ion-paired with suitable lipophilic counter-anions functions as potent intracellular photosensitizer under visible light irradiation mainly via a 1O2-mediated mechanism. These findings should provide new perspectives for future investigations on other metal complexes with similar characteristics as promising photosensitizers for potential photodynamic therapy.

Unexpected reversible and controllable nuclear uptake and efflux of the DNA "light-switching" Ru(ii)-polypyridyl complex in living cells via ion-pairing with chlorophenolate counter-anions.

An in-depth understanding of the mechanisms of cellular uptake and efflux would facilitate the design of metal complexes with not only better functionality and targeted theranostic efficiency, but also with controlled toxicity. Here we find, unexpectedly, that the DNA "light-switching" Ru(ii)-polypyridyl complex [Ru(phen)2(dppz)]2+ already delivered to the nucleus via ion-pairing with chlorophenolate counter-anions can gradually efflux to the cytoplasm when the cells were washed and incubated with fresh culture-medium. Interestingly, [Ru(phen)2(dppz)]2+ effluxed to the cytoplasm can be redirected back to the nucleus when the chlorophenolate counter-anions were added again. The efflux of nuclear [Ru(phen)2(dppz)]2+ was found to be mediated mainly via ATP-binding cassette (ABC) transporter proteins. Analogous reversible, but enantio-selective nuclear uptake and efflux were observed with the two pure chiral forms of [Ru(phen)2(dppz)]Cl2. This represents the first report of reversible and controllable nuclear uptake and efflux of a DNA "light-switching" Ru(ii)-complex in living-cells via ion-pairing, which should provide novel insights for future research on using ion-pairing as an effective approach to control the cellular uptake and redistribution of other potential theranostic metal complexes.

Mechanism of synergistic DNA damage induced by caffeic acid phenethyl ester (CAPE) and Cu(II): Competitive binding between CAPE and DNA with Cu(II)/Cu(I).

Caffeic acid phenethyl ester (CAPE) is an active polyphenol of propolis from honeybee hives, and exhibits antioxidant and interesting pharmacological activities. However, in this study, we found that in the presence of Cu(II), CAPE exhibited pro-oxidative rather than antioxidant effect: synergistic DNA damage was induced by the combination of CAPE and Cu(II) together as measured by strand breakage in plasmid DNA and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, which is dependent on the molar ratio of CAPE:Cu(II). Production of Cu(I) and H2O2 from the redox reaction between CAPE and Cu(II), and subsequent OH formation was found to be responsible for the synergistic DNA damage. DNA sequencing investigations provided more direct evidence that CAPE/Cu(II) caused preferential cleavage at guanine, thymine and cytosine residues. Interestingly, we found there are competitive binding between CAPE and DNA with Cu(II)/Cu(I), which changed the redox activity of Cu(II)/Cu(I), via complementary applications of different analytical methods. The observed DNA damage was mainly attributed to the formation of DNA-Cu(II)/Cu(I) complexes, which is still redox active and initiated the redox reaction near the binding site between copper and DNA. Based on these data, we proposed that the synergistic DNA damage induced by CAPE/Cu(II) might be due to the competitive binding between CAPE and DNA with Cu, and site-specific production of OH near the binding site of copper with DNA. Our findings may have broad biological implications for future research on the pro-oxidative effects of phenolic compounds in the presence of transition metals.

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.

Low concentrations of bisphenol A induce mouse spermatogonial cell proliferation by G protein-coupled receptor 30 and estrogen receptor-α.

BACKGROUND: Bisphenol A (BPA) is one of the most prevalent chemicals in daily-use materials; therefore, human exposure to BPA is ubiquitous. The estrogenicity of BPA is generally mediated by nuclear estrogen receptors (ERs). However, low concentrations of BPA stimulate seminoma cell proliferation by an uncertain mechanism that does not involve activation of ERs. OBJECTIVE: We investigated the possible promoting effects of low-concentration BPA and the possible mechanism(s) using the murine ER-ß negative spermatogonial GC-1 cell line. METHODS AND RESULTS: Using the specific signaling inhibitor, BPA at test concentrations ranging from 10-10 to 10-8 M markedly induced proliferation of GC-1 cells by activating both cGMP-dependent protein kinase (PKG) and epidermal growth factor receptor (EGFR) extracellular regulated kinase (ERK) pathways. BPA stimulated a rapid (15-min) phosphorylation of the transcription factor cAMP response element binding protein (CREB) and the cell cycle regulator retinoblastoma protein (Rb). Interestingly, ER-α phosphorylation is involved in the proliferation, whereas BPA does not directly transactivate ER-α in gene reporter assays. Using specific agonists and gene silencing, we further observed that BPA mediates the proliferation and fos gene expression of GC-1 cells by G protein-coupled receptor 30 (GPR30) and ER-α. CONCLUSIONS: Our data suggest that low concentrations of BPA activate the PKG and EGFR/ERK/c-fos pathways through a cross-talk between GPR30 and ER-α, which in turn stimulates GC-1 cell proliferation. The present study provides a novel insight regarding the potential role of GPR30 and ER-α in mediating the proliferative effects of BPA in male germ cells.

Apoptosis in microencapsulated juvenile rabbit chondrocytes induced by ofloxacin: role played by beta(1)-integrin receptor.

Quinolone(s) (QNs) is widely used in infection therapy due to its good antimicrobial characteristics. However, QNs-induced arthropathy of immature animals has led to restrictions on the therapeutic use of these antimicrobial agents. The exact mechanism(s) of QNs-induced chondrotoxicity remain unknown. In the present study, we investigated the possible mechanism of ofloxacin (one typical QNs)-induced injuries of chondrocytes. Juvenile rabbit joint chondrocytes cultured in alginate microspheres were incubated with ofloxacin at concentrations of 0, 2, 5, 10, 20, and 40 microg/ml for up to 96 h. Concentration of 10 microg/ml ofloxacin induced apoptosis of chondrocyte with visible apoptotic signs, including degradation of poly(ADP-ribose) polymerase, caspase-3 activation, and DNA ladder formation. Furthermore, extracellular signal-regulated kinase 1/2 (phospho-ERK1/2) and growth factor receptor-bound protein 2 (Grb2) were significantly reduced, and similar changes were also observed in the beta(1)-integrin receptor as assessed by immunoblotting. However, the mRNA level of beta(1)-integrin obtained from reverse transcription-polymerase chain reaction remained unchanged. Results of beta(1)-integrin immunoprecipitation have also shown that beta(1)-integrin did not interact with activated intracellular signaling proteins. In addition, ofloxacin did not induce apoptosis and decrease beta(1)-integrin expression in chondrocytes supplemented with Mg(2+), and the ofloxacin-induced apoptosis was caspase-8-dependent, inhibition of which did not affect the expression mode of phospho-ERK1/2 and beta(1)-integrin. Our results demonstrate that ofloxacin affects beta(1)-integrin receptor functions and the ERK mitogen-activated protein kinase signaling pathway, causing caspase-8-dependent apoptosis after exposure of 48 h.