The phosphoproteome is a first responder in tiered cellular adaptation to chemical stress followed by proteomics and transcriptomics alteration.

Next-generation risk assessment (NGRA) for environmental chemicals involves a weight of evidence (WoE) framework integrating a suite of new approach methodologies (NAMs) based on points of departure (PoD) obtained from in vitro assays. Among existing NAMs, the omic-based technologies are of particular importance based on the premise that any apical endpoint change indicative of impaired health must be underpinned by some alterations at the omics level, such as transcriptome, proteome, metabolome, epigenome and genome. Transcriptomic assay plays a leading role in providing relatively conservative PoDs compared with apical endpoints. However, it is unclear whether and how parameters measured with other omics techniques predict the cellular response to chemical perturbations, especially at exposure levels below the transcriptomically defined PoD. Multi-omics coverage may provide additional sensitive or confirmative biomarkers to complement and reduce the uncertainty in safety decisions made using targeted and transcriptomics assays. In the present study, we conducted multi-omics studies of transcriptomics, proteomics and phosphoproteomics on two prototype compounds, coumarin and 2,4-dichlorophenoxyacetic acid (2,4-D), with multiple chemical concentrations and time points, to understand the sensitivity of the three omics techniques in response to chemically-induced changes in HepG2. We demonstrated that, phosphoproteomics alterations occur not only earlier in time, but also more sensitive to lower concentrations than proteomics and transcriptomics when the HepG2 cells were exposed to various chemical treatments. The phosphoproteomics changes appear to approach maximum when the transcriptomics alterations begin to initiate. Therefore, it is proximal to the very early effects induced by chemical exposure. We concluded that phosphoproteomics can be utilized to provide a more complete coverage of chemical-induced cellular alteration and supplement transcriptomics-based health safety decision making.

T-2 toxin induces cardiotoxicity by activating ferroptosis and inhibiting heme oxygenase-1.

T-2 toxin, a natural secondary sesquiterpenoid metabolite produced by numerous strains of Fusarium fungi, is prevalent in both contaminated food and the environment. T-2 toxin is known to be highly toxic to the cardiovascular system, but the precise mechanisms that lead to T-2 toxin-induced cardiotoxicity are not yet fully understood. Recent findings indicate that ferroptosis is a pivotal factor in cardiovascular damage and exhibits a strong correlation with the detrimental impacts of T-2 toxin. The present study was designed to examine the involvement of ferroptosis in T-2 toxin-induced cardiac injury. Male mice and human cardiomyocytes were subjected to T-2 toxin for 24 h to induce acute cardiotoxicity for in vivo and in vitro studies, respectively. Our results demonstrated that T-2 toxin increased reactive oxygen species production, malondialdehyde, and decreased glutathione/oxidized glutathione and adenosine triphosphate levels. Furthermore, T-2 toxin was observed to activate ferroptosis, as evidenced by an increase in iron (Fe2+) concentration and upregulation of prostaglandin endoperoxide synthase 2, downregulation of glutathione peroxidase 4 and ferritin heavy chain 1, as well as ferroptotic morphological alterations. Inhibition of ferroptosis by Liproxstatin-1 reversed T-2 toxin-induced cardiac injury. Additionally, the downregulation of heme oxgenase-1 (HO-1) expression by T-2 toxin exacerbates ferroptosis and oxidative damage, which can be further aggravated by HO-1 inhibition with Sn-protoporphyrin. These findings provide novel insights into the mechanism of T-2 toxin-induced cardiotoxicity and suggest that targeting ferroptosis and HO-1 may represent a promising cardioprotective strategy against T-2 toxin.

Using transcriptomics, proteomics and phosphoproteomics as new approach methodology (NAM) to define biological responses for chemical safety assessment.

Omic-based technologies are of particular interest and importance for hazard identification and health risk characterization of chemicals. Their application in the new approach methodologies (NAMs) anchored on cellular toxicity pathways is based on the premise that any apical health endpoint change must be underpinned by some alterations at the omic levels. In the present study we examined the cellular responses to two chemicals, caffeine and coumarin, by generating and integrating multi-omic data from multi-dose and multi-time point transcriptomic, proteomic and phosphoproteomic experiments. We showed that the methodology presented here was able to capture the complete chain of events from the first chemical-induced changes at the phosphoproteome level, to changes in gene expression, and lastly to changes in protein abundance, each with vastly different points of departure (PODs). In HepG2 cells we found that the metabolism of lipids and general cellular stress response to be the dominant biological processes in response to caffeine and coumarin exposure, respectively. The phosphoproteomic changes were detected early in time, at very low doses and provided a fast, adaptive cellular response to chemical exposure with 7-37-fold lower points of departure comparing to the transcriptomics. Changes in protein abundance were found much less frequently than transcriptomic changes. While challenges remain, our study provides strong and novel evidence supporting the notion that these three omic technologies can be used in an integrated manner to facilitate a more complete understanding of pathway perturbations and POD determinations for risk assessment of chemical exposures.

Proliferating Stem Cells are Acutely Affected by DNA Damage Induced by Sulfur Mustard.

Sulfur mustard (SM), a chemical warfare agent, can form adducts with DNA, RNA, and proteins. Reactions with DNA lead to the formation of both DNA monoadducts and interstrand cross-links, resulting in DNA damage, and is an important component of SM toxicity. Our previous in vivo studies indicated that dividing cells such as hematopoietic stem cells and intestinal villi stem cells seemed to have increased sensitivity to SM. Therefore, to compare the sensitivity of somatic and stem cells to SM and to investigate the mechanism of SM cytotoxicity, we isolated human foreskin fibroblasts, reprogrammed them into pluripotent stem cells, and then compared the DNA damage repair pathways involved upon SM treatment. Our results indicated that proliferating stem cells were more sensitive to SM-induced DNA damage, and the damage mainly comprised single-stranded breaks. Furthermore, the pathways involved in DNA repair in stem cells and somatic cells were different.

Quantitative phosphoproteomics reveal cellular responses from caffeine, coumarin and quercetin in treated HepG2 cells.

Protein phosphorylation is the most common type of post-translational modification where serine, threonine or tyrosine are reversibly bound to the phosphate group of ATP in a reaction catalyzed by protein kinases. Phosphorylation plays an important role in regulation of cell homeostasis, including but not limited to signal perception and transduction, gene expression and function of proteins. Protein phosphorylation happens on a fast time scale and represents an energy-efficient way for the cell to adapt to exposure to chemical stressors. To understand the cascade of cellular signaling induced by exposure to chemicals, we have exposed HepG2 cells to three chemicals with different modes of action, namely, caffeine, coumarin, and quercetin in a concentration and time response manner. Significantly upregulated and downregulated phosphosites were screened to analyze the activation/deactivation of signaling pathways by protein kinases. In total, 69, 44 and 12 signaling pathways were found enriched in caffeine, coumarin and quercetin treated cells, respectively, of which 9 pathways were co-enriched with 11 jointly responded kinases. Among identified co-responded kinases, CDK1, MAPK1 and MAPK3 play important roles in cell cycle and insulin signaling pathways. Quantitative phosphoproteomics can sensitively distinguish the effects of different chemicals on cells, allowing the assessment of chemical safety through changes in substrates and metabolic pathways at the cellular level, which is important for the development of non-animal approaches for chemical safety assessment.

Synergistic effects of Rapamycin and Fluorouracil to treat a gastric tumor in a PTEN conditional deletion mouse model.

The tumor suppressor gene phosphatase and tensin homolog (PTEN) in PI3K/Akt/mTOR pathway is essential in inhibiting tumor growth and metastasis. However, whether the mutation of PTEN gene could induce tumorigenesis and impact the treatment of gastric cancer is still unclear. The purpose of the study was to investigate the combined treatment of gastric tumorigenesis using Rapamycin and Fluorouracil (5-Fu) through interfering with the Akt/mTOR pathway in a mouse model with PTEN conditional deletion. Three groups of mice were exposed for 5 days to Rapamycin and 5-Fu separately and together. The gene expression of the Akt/mTOR pathway, the protein expression of caspase-3 and p-Akt, p-S6K and p-4EBP1, and the pathological changes in stomachs were analyzed. Our study demonstrates that the conditional PTEN deletion in the cells of glandular stomach induces hyperplastic gastric tumors in mice. The combined Rapamycin administration with 5-Fu resulted in better outcomes than their separate administration for the treatment of gastric cancer by inhibiting the mTOR signal pathway. Our study indicates that Rapamycin has a synergistic interaction with chemotherapeutic 5-Fu, and demonstrates a potential therapeutic combination treatment on glandular stomach tumor with PTEN functional absence or aberrantly activated Akt/mTOR pathway. It provides important insights into the inhibition of the Akt/mTOR pathway in gastric cancer clinical therapy.

Combined Developmental Toxicity of the Pesticides Difenoconazole and Dimethomorph on Embryonic Zebrafish.

Difenoconazole (DIF) and dimethomorph (DIM) are widely used pesticides frequently detected together in environmental samples, so the deleterious effects of combined exposure warrant detailed examination. In this study, the individual and combined effects of DIM and DIF on conventional developmental parameters (hatching rate, deformity rate, lethality) and gene expression were measured in embryonic zebrafish. Both DIF and DIM interfered with normal zebrafish embryo development, and the most sensitive toxicity index for both was 96 h post-fertilization (hpf) deformity rate (BMDL10 values of 0.30 and 1.10 mg/L, respectively). The combination of DIF and DIM had mainly synergistic deleterious effects on 96 hpf deformity and mortality rates. Transcriptome analysis showed that these compounds markedly downregulated expression of mcm family genes, cdk1, and cdc20, thereby potentially disrupting DNA replication and cell cycle progression. Enhanced surveillance for this pesticide combination is recommended as simultaneous environmental exposure may be substantially more harmful than exposure to either compound alone.

Individual and synergistic toxic effects of carbendazim and chlorpyrifos on zebrafish embryonic development.

The fungicide carbendazim and the insecticide chlorpyrifos are frequently used together to protect various fruit and vegetable crops in China. At high doses, carbendazim is a known carcinogen while chlorpyrifos has neurotoxic potential, but the combined toxicity of these two compounds has not been systematically investigated. In this study, we examined the separate and combined effects of these compounds on zebrafish embryonic development. The LC50 values for carbendazim and chlorpyrifos at 96 h post-fertilization (hpf) were 0.89 mg/L and 3.83 mg/L, respectively. Carbendazim dose-dependently increased the spontaneous tail-wagging frequency of 24 hpf embryos, the hatching rate of 48 hpf embryos, and the mortality and deformity rate of 96 hpf embryos, while chlorpyrifos increased the heart rate of 48 hpf embryos as well as the mortality and deformity rate of 96 hpf embryos. Mixed exposure at an equipotent concentration ratio (Mix1) and at the ratio of maximum residue limits for typical fruits (apples) (Mix2) revealed significant synergistic effects on lethality at 96 hpf within the 0%-90% effect levels range. In contrast, there was an antagonistic effect of the equipotent concentration ratio on lethality in the 90%-100% concentration range, while the ratio at the maximum residue limits still showed a synergistic effect. Mix1 exhibited antagonism on hatching rate in the 0%-35% range and synergy in the 40%-100% range, while Mix2 had a synergistic effect on hatching rate in the 0%-35% range, an additive effect at 40%, and an antagonistic effect at >40%. Both mixtures had a synergistic effect on deformity rate over all concentration ranges. Carbendazim and chlorpyrifos demonstrate synergistic developmental toxicity, indicating that health and environmental risk assessments should be conducted for various combinations of these agents.

Next generation risk assessment (NGRA): Bridging in vitro points-of-departure to human safety assessment using physiologically-based kinetic (PBK) modelling - A case study of doxorubicin with dose metrics considerations.

Using the chemical doxorubicin (DOX), the objective of the present study was to evaluate the impact of dose metrics selection in the new approach method of integrating physiologically-based kinetic (PBK) modelling and relevant human cell-based assays to inform a priori the point of departure for human health risk. We reviewed the literature on the clinical consequences of DOX treatment to identify dosing scenarios with no or mild cardiotoxicity observed. Key concentrations of DOX that induced cardiomyocyte toxicity in vitro were derived from studies of our own and others. A human population-based PBK model of DOX was developed and verified against pharmacokinetic data. The model was then used to predict plasma and extracellular and intracellular heart concentrations of DOX under selected clinical settings and compared with in vitro outcomes, based on several dose metrics: Cmax (maximum concentration) or AUC (area under concentration-time curve) in free or total form of DOX. We found when using in vitro assays to predict cardiotoxicity for DOX, AUC is a better indicator. Our study illustrates that when appropriate dose metrics are used, it is possible to combine PBK modelling with in vitro-derived toxicity information to define margins of safety and predict low-risk human exposure levels.

Zinc Oxide Nanoparticles Induce Mitochondrial Biogenesis Impairment and Cardiac Dysfunction in Human iPSC-Derived Cardiomyocytes.

BACKGROUND: Zinc oxide nanoparticles (ZnO NPs) are one of the most widely used nanomaterials in a variety of fields such as industrial, pharmaceutical, and household applications. Increasing evidence suggests that ZnO NPs could elicit unignorable harmful effect to the cardiovascular system, but the potential deleterious effects to human cardiomyocytes remain to be elucidated. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been increasingly used as a promising in vitro model of cardiomyocyte in various fields such as drug cardiac safety evaluation. Herein, the present study was designed to elucidate the cardiac adverse effects of ZnO NPs and explore the possible underlying mechanism using hiPSC-CMs. METHODS: ZnO NPs were characterized by transmission electron microscopy and dynamic light scattering. The cytotoxicity induced by ZnO NPs in hiPSC-CMs was evaluated by determination of cell viability and lactate dehydrogenase release. Cellular reactive oxygen species (ROS) and mitochondrial membrane potential were measured by high-content analysis (HCA). Mitochondrial biogenesis was assayed by detection of mtDNA copy number and PGC-1α pathway. Moreover, microelectrode array techniques were used to investigate cardiac electrophysiological alterations. RESULTS: We demonstrated that ZnO NPs concentration- and time-dependently elicited cytotoxicity in hiPSC-CMs. The results from HCA revealed that ZnO NPs exposure at low-cytotoxic concentrations significantly promoted ROS generation and induced mitochondrial dysfunction. We further demonstrated that ZnO NPs could impair mitochondrial biogenesis and inhibit PGC-1α pathway. In addition, ZnO NPs at insignificantly cytotoxic concentrations were found to trigger cardiac electrophysiological alterations as evidenced by decreases of beat rate and spike amplitude. CONCLUSION: Our findings unveiled the potential harmful effects of ZnO NPs to human cardiomyocytes that involve mitochondrial biogenesis and the PGC-1α pathway that could affect cardiac electrophysiological function.

Integration of in vitro data from three dimensionally cultured HepaRG cells and physiologically based pharmacokinetic modeling for assessment of acetaminophen hepatotoxicity.

Selection of appropriate fit-for-purpose in vitro and in silico models is critical for non-animal safety assessment of chemical-induced hepatoxicity. The present study evaluated the feasibility of integrating in vitro data from three-dimensionally (3D)-cultured HepaRG cells and physiologically based pharmacokinetic (PBPK) modeling to predict chemical-induced liver toxicity. A 3D organoid culture system was established using an ultralow attachment method. HepaRG cells cultured in a two-dimensional (2D) monolayer and under 3D conditions were exposed to acetaminophen (APAP) at concentrations of 0.16-20 mM. The results showed that the viability of both 3D- and 2D cultured cells was significantly decreased by APAP in a concentration-dependent manner. Furthermore, 3D cultures were more sensitive to APAP-induced mitochondrial damage than 2D cultures were, based on measurements of mitochondrial superoxide accumulation and mitochondrial membrane potential loss. PBPK simulations using nominal in vitro concentrations showed that the APAP concentration eliciting mitochondrial damage was closer to the predicted peak liver concentration in humans in 3D cultures than it was in 2D cultures. In summary, our results suggest that combining in vitro data from 3D HepaRG cultures and PBPK modeling provides a promising tool for assessment of liver injury.

Integrating in vitro testing and physiologically-based pharmacokinetic (PBPK) modelling for chemical liver toxicity assessment-A case study of troglitazone.

In vitro to in vivo extrapolation (IVIVE) for next-generation risk assessment (NGRA) of chemicals requires computational modeling and faces unique challenges. Using mitochondria-related toxicity data of troglitazone (TGZ), a prototype drug known for liver toxicity, from HepaRG, HepG2, HC-04, and primary human hepatocytes, we explored inherent uncertainties in IVIVE, including cell models, cellular response endpoints, and dose metrics. A human population physiologically-based pharmacokinetic (PBPK) model for TGZ was developed to predict in vivo doses from in vitro point-of-departure (POD) concentrations. Compared to the 200-800 mg/d dose range of TGZ where liver injury was observed clinically, the predicted POD doses for the mean and top one percentile of the PBPK population were 28-372 and 15-178 mg/d respectively based on Cmax dosimetry, and 185-2552 and 83-1010 mg/d respectively based on AUC. In conclusion, although with many uncertainties, integrating in vitro assays and PBPK modeling is promising in informing liver toxicity-inducing TGZ doses.

Atorvastatin induces mitochondrial dysfunction and cell apoptosis in HepG2 cells via inhibition of the Nrf2 pathway.

Atorvastatin (ATO) is a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor widely used to treat hypercholesterolemia. However, clinical application is limited by potential hepatotoxicity. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a master regulator of cellular antioxidants, and oxidative stress is implicated in statin-induced liver injury. This study investigated mechanisms of ATO-induced hepatotoxicity and potential mitigation by Nrf2 signaling. ATO reduced Nrf2 and antioxidant enzyme superoxide dismutase-2 (SOD2) expression in human hepatocarcinoma HepG2 cells. ATO also induced concentration-dependent HepG2 cell toxicity, reactive oxygen species (ROS) accumulation, and mitochondrial dysfunction as evidenced by decreased mitochondrial membrane potential (MMP) and cellular adenosine triphosphate (ATP). Further, ATO induced mitochondria-dependent apoptosis as indicated by increased Bax/Bcl-2 ratio, cleaved caspase-3, mitochondrial cytochrome c release and Annexin V-fluorescein isothiocyanate/propidium iodide staining. Tert-butylhydroquinone enhanced Nrf2 and SOD2 expression, and partially reversed ATO-induced cytotoxicity, ROS accumulation, MMP reduction, ATP depletion and mitochondria-dependent apoptosis. In conclusion, the present study demonstrates that ATO induces mitochondrial dysfunction and cell apoptosis in HepG2 cells, at least in part, via inhibition of the Nrf2 pathway. Nrf2 pathway activation is a potential prevention for ATO-induced liver injury.

Therapeutic Effects of Human Umbilical Cord Mesenchymal Stromal Cells in Sprague-Dawley Rats with Percutaneous Exposure to Sulfur Mustard.

Sulfur mustard (SM) exposure, whose symptoms are similar to radiation exposure, can lead to acute injury. Because mesenchymal stromal cells (MSCs) have been used to experimentally and clinically treat acute radiation syndrome, in this study, MSCs were intravenously injected into rats after percutaneous SM exposure. Then, we examined sternum and spleen samples by histopathological and immunohistochemical methods to observe pathological changes. Furthermore, blood samples were taken to test the white blood cell (WBC) count, blood platelet count (BPC), red blood cell count, and the levels of cytokines in the serum. The number of bone marrow karyocytes and the WBC in the MSC + SM group were higher than those in the SM group, and the levels of granulocyte colony-stimulating factor, granulocyte-macrophage colony stimulating factor, monocyte chemoattractant protein-1, interleukin (IL)-1α, IL-5, and interferon-γ in the MSC + SM group remained high at different time points after SM exposure. In addition, the BPC, the level of erythropoietin and the relative weight of the spleen in the MSC + SM group were significantly higher than those in the SM group. Meanwhile, spleens in the MSC + SM group were more hyperplastic and hematopoietic, and had fewer apoptotic cells than in the SM group. Furthermore, rat body weight and locomotion ability in the MSC + SM group were higher than in the SM group. This evidence supports the potential ability of MSCs in immunoregulation and functional improvements to the hemopoietic microenvironment. Intravenous injection of MSCs exerted significant therapeutic effects in rats with percutaneous exposure to SM.

Prolonged exposure to carbon nanoparticles induced methylome remodeling and gene expression in zebrafish heart.

Growing black carbon (BC) emission has become one of the major urgent environmental issues facing human beings. Usually, BC or BC-containing carbon nanoparticles (CNPs) were recognized as non-directly toxic components of atmospheric particulate matter. However, epidemiology studies have provided much evidence of the associations of exposure of particulate-containing carbon particles with cardiovascular diseases. There are still no related studies to support the epidemiological conclusions. Hence, in this article we exposed adult zebrafish to CNPs for 60 days, and then explored the heart location and potential adverse effects on cardiac tissues of these nanosized carbon particles. Our results first showed direct visualization of cardiac endothelial uptake and heart deposition of CNPs in zebrafish. In addition, CNPs caused significant ultrastructural alterations in myocardial tissue and induced the expression of inflammatory cytokines in a dose-dependent manner, resulting in sub-endocardial inflammation and cell apoptosis. Moreover, our data demonstrated the perturbations caused by CNPs on DNA methylation, suggesting that DNA methylome remodeling might play a critical role in CNP-induced cardiotoxicity in zebrafish heart. Therefore, this study not only proved a laboratory link between CNP exposure and cardiotoxicity in vivo, but also indicated a possible toxicity mechanism involved.

Flutamide Induces Hepatic Cell Death and Mitochondrial Dysfunction via Inhibition of Nrf2-Mediated Heme Oxygenase-1.

Flutamide is a widely used nonsteroidal antiandrogen for prostate cancer therapy, but its clinical application is restricted by the concurrent liver injury. Increasing evidence suggests that flutamide-induced liver injury is associated with oxidative stress, though the precise mechanism is poorly understood. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master transcription factor regulating endogenous antioxidants including heme oxygenase-1 (HO-1). This study was designed to delineate the role of Nrf2/HO-1 in flutamide-induced hepatic cell injury. Our results showed that flutamide concentration dependently induced cytotoxicity, hydrogen peroxide accumulation, and mitochondrial dysfunction as indicated by mitochondrial membrane potential loss and ATP depletion. The protein expression of Nrf2 and HO-1 was induced by flutamide at 12.5 µM but was downregulated by higher concentrations of flutamide. Silencing either Nrf2 or HO-1 was found to aggravate flutamide-induced hydrogen peroxide accumulation and mitochondrial dysfunction as well as inhibition of the Nrf2 pathway. Moreover, preinduction of HO-1 by Copp significantly attenuated flutamide-induced oxidative stress and mitochondrial dysfunction, while inhibition of HO-1 by Snpp aggravated these deleterious effects. These findings suggest that flutamide-induced hepatic cell death and mitochondrial dysfunction is assoicated with inhibition of Nrf2-mediated HO-1. Pharmacologic intervention of Nrf2/HO-1 may provide a promising therapeutic approach in flutamide-induced liver injury.

Developing a robust LC-MS/MS method to quantify Zn-DTPA, a zinc chelate in human plasma and urine.

Quantitation of Zn-DTPA (zinc diethylenetriamene pentaacetate, a metal chelate) in complex biological matrix is extremely challenging on account of its special physiochemical properties. This study aimed to develop a robust and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of Zn-DTPA in human plasma and urine. The purified samples were separated on Proteonavi (250 × 4.6 mm, 5 µm; Shiseido, Ginza, Tokyo, Japan) and a C18 guard column. The mobile phase consisted of methanol-2 mm ammonium formate (pH 6.3)-ammonia solution (50:50:0.015, v/v/v), flow rate 0.45 mL/min. The linear concentration ranges of the calibration curves for Zn-DTPA were 1-100 µg/mL in plasma and 10-2000 µg/mL in urine. The intra- and inter-day precisions for quality control (QC) samples were from 1.8 to 14.6% for Zn-DTPA and the accuracies for QC samples were from -4.8 to 8.2%. This method was fully validated and successfully applied to the quantitation of Zn-DTPA in plasma and urine samples of a healthy male volunteer after intravenous infusion administration of Zn-DTPA. The result showed that the concentration of Zn-DTPA in urine was about 20 times that in plasma, and Zn-DTPA was completely (94.7%) excreted through urine in human.

Doxorubicin-induced mitophagy and mitochondrial damage is associated with dysregulation of the PINK1/parkin pathway.

The usefulness of doxorubicin (DOX), a potent anticancer agent, is limited by its cardiotoxicity. Mitochondria play a central role in DOX-induced cardiotoxicity though the precise mechanisms are still obscure. Increasing evidence indicates that excessive activation of mitophagy and mitochondrial dysfunction are key causal events leading to DOX-induced cardiac injury. The PINK1/parkin pathway has emerged as a critical pathway in regulation of mitophagy as well as mitochondrial function. The present study was aimed to investigate the role of PINK1/parkin pathway in DOX-induced mitochondrial damage and cardiotoxicity. Our results showed that DOX concentration-dependently induced cytotoxicity and mitochondrial toxic effects including mitochondrial superoxide accumulation, decreased mitochondrial membrane potential and mitochondrial DNA copy number, as well as mitochondrial ultrastructural alterations. DOX induced mitophagy as evidenced by increases of the markers of autophagosomes, LC3, Beclin 1, reduction of p62, and co-localization of LC3 in mitochondria. DOX activated PINK1/parkin pathway and promoted translocation of PINK1/parkin to mitochondria. Meanwhile, DOX inhibited the expression of PGC-1α and its downstream targets nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM), and reduced the expression of mitochondrial proteins. Inhibition of mitophagy by mdivi-1 was found to attenuate activation of the PINK1/parkin pathway by DOX and preserve mitochondrial biogenesis, consequently mitigating DOX-induced mitochondrial superoxide overproduction and mitochondrial dysfunction. Moreover, scavenging mitochondrial superoxide by Mito-tempo was also found to effectively attenuate activation of the PINK1/parkin pathway and rescue the cells from DOX-induced adverse effects. Taken together, these findings suggest that DOX-induced mitophagy and mitochondrial damage in cardiomyocytes are mediated, at least in part, by dysregulation of the PINK1/parkin pathway.

A novel α-conopeptide Eu1.6 inhibits N-type (CaV2.2) calcium channels and exhibits potent analgesic activity.

We here describe a novel α-conopeptide, Eu1.6 from Conus eburneus, which exhibits strong anti-nociceptive activity by an unexpected mechanism of action. Unlike other α-conopeptides that largely target nicotinic acetylcholine receptors (nAChRs), Eu1.6 displayed only weak inhibitory activity at the α3ß4 and α7 nAChR subtypes and TTX-resistant sodium channels, and no activity at TTX-sensitive sodium channels in rat dorsal root ganglion (DRG) neurons, or opiate receptors, VR1, KCNQ1, L- and T-type calcium channels expressed in HEK293 cells. However, Eu1.6 inhibited high voltage-activated N-type calcium channel currents in isolated mouse DRG neurons which was independent of GABAB receptor activation. In HEK293 cells expressing CaV2.2 channels alone, Eu1.6 reversibly inhibited depolarization-activated Ba2+ currents in a voltage- and state-dependent manner. Inhibition of CaV2.2 by Eu1.6 was concentration-dependent (IC50 ~1 nM). Significantly, systemic administration of Eu1.6 at doses of 2.5-5.0 µg/kg exhibited potent analgesic activities in rat partial sciatic nerve injury and chronic constriction injury pain models. Furthermore, Eu1.6 had no significant side-effect on spontaneous locomotor activity, cardiac and respiratory function, and drug dependence in mice. These findings suggest α-conopeptide Eu1.6 is a potent analgesic for the treatment of neuropathic and chronic pain and opens a novel option for future analgesic drug design.

Impairment of Mitochondrial Biogenesis and Dynamics Involved in Isoniazid-Induced Apoptosis of HepG2 Cells Was Alleviated by p38 MAPK Pathway.

Isoniazid (INH), a widely used first-line antitubercular drug, has been noted to be associated with hepatotoxicity. In spite of extensive researches over many decades, the mechanism of INH-induced hepatotoxicity still remains poorly understood. Recently, mitochondrial toxicity has been emerging as a new paradigm for INH-induced hepatotoxicity. In this study, we showed that INH impaired mitochondrial biogenesis and dynamics in human hepatocarcinoma HepG2 cells. INH reduced mitochondrial membrane potential (MMP) and induced mitochondria swelling. INH also inhibited the protein expressions of three major mitochondrial biogenesis regulators, SIRT1, PGC1α and NRF1, along with increased acetylation of PGC1α. Meanwhile, INH decreased the number of mitochondria, accompanied by decreased expression of mitochondrial protein COX IV. INH caused mitochondrial fragmentation involving decreased levels of the fusion protein MFN2 as well as the fission protein DRP1. INH-reduced DRP1 expression was associated with the increase of apoptosis, suggesting the existence of pro-survival fission and its involvement in mitochondrial quality control. INH activated p38 MAPK, whereas inhibition of p38 MAPK aggravated INH-induced decreases of SIRT1, PGC1α, NRF1, COX IV and DRP1 expressions. P38 MAPK inhibition also further up-regulated the acetylation of PGC1α and exacerbated INH-induced MMP loss, mitochondrial swelling and apoptosis. Taken together, INH-activated p38 MAPK induced mitochondrial biogenesis to alleviate apoptosis through partly recovering SIRT1-PGC1α pathway activation. In the meantime, p38 MAPK activation by INH promoted protective mitochondrial fission to alleviate apoptosis by partial recovery of DRP1 expression.