Tingfen Zhang

Enhanced toxicity and ROS generation by doxorubicin in primary cultures of cardiomyocytes from neonatal metallothionein-I/II null mice

The clinical use of doxorubicin (Dox), a potent anticancer drug, is limited by its concurrent dose-dependent cardiotoxicity. We previously found that metallothionein-I/II (MT-I/II) null mice are more vulnerable to Dox-induced cardiomyopathy, but it is unknown whether depletion of MT would sensitize cardiomyocytes to Dox toxicity in vitro since the protective effect of MT still remains controversial. In the present study, a primary culture system of cardiomyocytes from neonatal MT-I/II null (MT(-/-)) and corresponding wild type (MT(+/+)) mice was established to unequivocally determine the effect of MT deficiency on Dox-induced toxicity. MT concentrations in the MT(-/-) cardiomyocytes were about 2.5-fold lower than those in MT(+/+) cardiomyocytes. MT(-/-) cardiomyocytes were more sensitive to Dox-induced cytotoxicity than MT(+/+) cardiomyocytes as measured by morphological alterations, lactate dehydrogenase leakage, cell viability, and apoptosis. Dox time- and concentration-dependently increased reactive oxygen species (ROS) formation in MT(+/+) cardiomyocytes, and this effect was exaggerated in MT(-/-) cardiomyocytes. Antioxidant N-acetylcysteine (NAC) and glutathione (GSH) significantly rescued MT(+/+) but not MT(-/-)cardiomyocytes from Dox-induced cell death and ROS generation. These findings suggest that basal MT provide protection against Dox-induced toxicity in cardiomyocytes, particularly highlight the important role of MT as a cellular antioxidant on scavenging ROS.

Abundance of four sulfur mustard-DNA adducts ex vivo and in vivo revealed by simultaneous quantification in stable isotope dilution-ultrahigh performance liquid chromatography-tandem mass spectrometry.

Sulfur mustard (SM) is a highly reactive alkylating vesicant and causes blisters upon contact with skin, eyes, and respiratory organs. It covalently links with DNAs by forming four mono- or cross-link adducts. In this article, the reference standards of SM-DNA adducts and deuterated analogues were first synthesized with simplified procedures containing only one or two steps and using less toxic chemical 2-(2-chloroethylthio)ethanol or nontoxic chemical thiodiglycol as starting materials. A sensitive and high-throughput simultaneous quantification method of N(7)-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (N(7)-HETEG), O(6)-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (O(6)-HETEG), N(3)-[2-[(2-hydroxyethyl)thio]-ethyl]adenine (N(3)-HETEA), and bis[2-(guanin-7-yl)ethyl]sulfide (Bis-G) in the Sprague-Dawley rat derma samples was developed by stable isotope dilution-ultrahigh performance liquid chromatography-tandem mass spectrometry (ID-UPLC-MS/MS) with the aim of revealing the real metabolic behaviors of four adducts. The method was validated, the limit of detection (S/N ratio greater than 10) was 0.01, 0.002, 0.04, and 0.11 fmol on column for N(7)-HETEG, O(6)-HETEG, Bis-G, and N(3)-HETEA, respectively, and the lower limit of quantification (S/N ratio greater than 20) was 0.04, 0.01, 0.12, and 0.33 fmol on column for N(7)-HETEG, O(6)-HETEG, Bis-G, and N(3)-HETEA, respectively. The accuracy of this method was determined to be 76% to 129% (n = 3), and both the interday (n = 6) and intraday (n = 7) precisions were less than 10%. The method was further applied for the quantifications of four adducts in the derma of adult male Sprague-Dawley rats exposed to SM ex vivo and in vivo, and all adducts had time- and dose-effect relationships. To the best of our knowledge, this is the first time that the real presented status of four DNA adducts was simultaneously revealed by the MS-based method, in which Bis-G showed much higher abundance than the result previously reported and N(3)-HETEA showed much less. It should be noted that since the interstrand cross-linked adduct is believed to stall DNA replication and finally induce a double-strand break, the higher abundance of Bis-G is a great indication of a more serious DNA lesion by SM alkylation.

Cardioprotection against doxorubicin by metallothionein Is associated with preservation of mitochondrial biogenesis involving PGC-1α pathway.

Metallothionein (MT) has been shown to inhibit cardiac oxidative stress and protect against the cardiotoxicity induced by doxorubicin (DOX), a potent and widely used chemotherapeutic agent. However, the mechanism of MT׳s protective action against DOX still remains obscure. Mitochondrial biogenesis impairment has been implicated to play an important role in the etiology and progression of DOX-induced cardiotoxicity. Increasing evidence indicates an intimate link between MT-mediated cardioprotection and mitochondrial biogenesis. This study was aimed to explore the possible contribution of mitochondrial biogenesis in MT׳s cardioprotective action against DOX. Adult male MT-I/II-null (MT(-/-)) and wild-type (MT(+/+)) mice were given a single dose of DOX intraperitoneally. Our results revealed that MT deficiency significantly sensitized mice to DOX-induced cardiac dysfunction, ultrastructural alterations, and mortality. DOX disrupted cardiac mitochondrial biogenesis indicated by mitochondrial DNA copy number and decreased mitochondrial number, and these effects were greater in MT(-/-) mice. Basal MT effectively protected against DOX-induced inhibition on the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a key regulator of mitochondrial biogenesis, and its downstream factors including mitochondrial transcription factor A. Moreover, MT was found to preserve the protein expression of manganese superoxide dismutase, a transcriptional target of PGC-1α. in vitro study showed that MT absence augmented DOX-induced increase of mitochondrial superoxide production in primary cultured cardiomyocytes. These findings suggest that MT׳s cardioprotection against DOX is mediated, at least in part, by preservation of mitochondrial biogenesis involving PGC-1α pathway.

A PGC-1α-Mediated Transcriptional Network Maintains Mitochondrial Redox and Bioenergetic Homeostasis against Doxorubicin-Induced Toxicity in Human Cardiomyocytes: Implementation of TT21C.

Chemical toxicity testing is fast moving in a direction that relies increasingly on cell-basedin vitroassays anchored on toxicity pathways according to the toxicity testing in the 21st century vision. Identifying points of departure (POD) via these assays and revealing their mechanistic underpinnings via computational modeling of the relevant pathways are critical and challenging steps. Here we used doxorubicin (DOX) as a prototype chemical to study mitochondrial toxicity in human AC16 cells. Mitochondrial toxicity has been linked to cardiovascular risk of DOX, which has limited its clinical use as an antitumor drug. Ourin vitrostudy revealed a well-defined POD concentration of DOX below which adaptive induction of proliferator-activated receptor-γ coactivator-1α (PGC-1α) -mediated mitochondrial genes, including NRF-1, MnSOD, UCP2, and COX1, concurred with negligible changes in mitochondrial superoxide and cytotoxicity. At higher DOX concentrations adversity became significant with elevated superoxide and suppressed ATP levels. A computational model was formulated to simulate the PGC-1α-mediated transcriptional network comprising multiple negative feedback loops that underlie redox and bioenergetics homeostasis in the mitochondrion. The model recapitulated the transition phase from adaptive to adverse responses, supporting the notion that saturated induction of PGC-1α-mediated gene network underpins POD. The model further predicts (follow-up experiments verified) that silencing PGC-1α compromises the adaptive function of the transcriptional network, leading to disruption of mitochondria and cytotoxicity at lower DOX concentrations. In summary, our study demonstrates that combining pathway-focusedin vitroassays and computational simulation of relevant biochemical network is synergistic for understanding dose-response behaviors in the low-dose region and identifying POD.

Metallothioneins attenuate paraquat-induced acute lung injury in mice through the mechanisms of anti-oxidation and anti-apoptosis

Paraquat (PQ) is a widely used herbicide, and lung is the primary target of PQ poisoning. Metallothionein (MT) is a potent antioxidant and free radical scavenger, and has been shown to play a protective role in lung injury induced by different stressors. This study was undertaken to evaluate the protective potential of MT against PQ-induced acute lung injury using MT-I/II null (MT(-/-)) mice. Wild-type (MT(+/+)) mice and MT(-/-) mice were given one intragastric administration of 50mg/kg PQ for 24h, and it was revealed that MT(-/-) mice were more susceptible to PQ-induced acute lung injury than MT(+/+) mice evidenced by the following findings. As compared with MT(+/+) mice, MT(-/-) mice presented more severe histopathological lesions in the lung, higher pulmonary malondialdehyde content, and more reduced pulmonary antioxidative enzymes activities. PQ also induced more apoptosis in pneumocytes from MT(-/-) mice, and the expressions of apoptosis-related proteins Bax, Bcl-2, cleaved-caspase-3, and the ratio of Bax/Bcl-2 were all more significantly increased in PQ-treated MT(-/-) mice. Our results clearly demonstrate that endogenous MT can attenuate PQ-induced acute lung injury, possibly through the mechanisms of anti-oxidation and anti-apoptosis.

Erythropoietin activates SIRT1 to protect human cardiomyocytes against doxorubicin-induced mitochondrial dysfunction and toxicity

The hormone erythropoietin (EPO) has been demonstrated to protect against chemotherapy drug doxorubicin (DOX)-induced cardiotoxicity, but the underlying mechanism remains obscure. We hypothesized that silent mating type information regulation 2 homolog 1 (SIRT1), an NAD+-dependent protein deacetylase that activates peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), plays a crucial role in regulating mitochondrial function and mediating the beneficial effect of EPO. Our study in human cardiomyocyte AC16 cells showed that DOX-induced cytotoxicity and mitochondrial dysfunction, as manifested by decreased mitochondrial DNA (mtDNA) copy number, mitochondrial membrane potential, and increased mitochondrial superoxide accumulation, can be mitigated by EPO pretreatment. EPO was found to upregulate SIRT1 activity and protein expression to reverse DOX-induced acetylation of PGC-1α and suppression of a suite of PGC-1α-activated genes involved in mitochondrial function and biogenesis, such as nuclear respiratory factor-1 (NRF1), mitochondrial transcription factor A (TFAM), citrate synthase (CS), superoxide dismutase 2 (SOD2), cytochrome c oxidase IV (COXIV), and voltage-dependent anion channel (VDAC). Silencing of SIRT1 via small RNA interference sensitized AC16 cells to DOX-induced cytotoxicity and reduction in mtDNA copy number. Although with SIRT1 silenced, EPO could reverse to some extent DOX-induced mitochondrial superoxide accumulation, loss of mitochondrial membrane potential and ATP depletion, it failed to normalize protein expression of PGC-1α and its downstream genes. Taken together, our results indicated that EPO may activate SIRT1 to enhance mitochondrial function and protect against DOX-induced cardiotoxicity.

Non-cytotoxic concentrations of acetaminophen induced mitochondrial biogenesis and antioxidant response in HepG2 cells

Mitochondrial dysfunction has been implicated in acute, severe liver injury caused by overdose of acetaminophen (APAP). However, whether mitochondrial biogenesis is involved is unclear. Here we demonstrated that mitochondrial biogenesis, as indicated by the amounts of mitochondrial DNA and proteins, increased significantly in HepG2 cells exposed to low, non-cytotoxic concentrations of APAP. This heightened response was accompanied by upregulated expression of PGC-1α, NRF-1 and TFAM, which are key transcriptional regulators of mitochondrial biogenesis. Additionally, antioxidants including glutathione, MnSOD, HO-1, NQO1, and Nrf2 were also significantly upregulated. In contrast, for HepG2 cells exposed to high, cytotoxic concentration of APAP, mitochondrial biogenesis was inhibited and the expression of its regulatory proteins and antioxidants were concentration-dependently downregulated. In summary, our study indicated that mitochondrial biogenesis, along with antioxidant induction, may be an important cellular adaptive mechanism counteracting APAP-induced toxicity and overwhelming this cytoprotective capacity could result in liver injury.

Subchronic toxicity study of yttrium nitrate by 90-day repeated oral exposure in rats

ABSTRACT Concerns regarding the adverse effects of long‐term exposure to low levels of rare earth elements (REEs) from foods on human health have arisen in recent years. Nevertheless, no official acceptable daily intake (ADI) has yet been proposed for either total REEs or individual REE. In accordance with the Organization for Economic Co‐operation and Development (OECD) testing guideline, the present study was undertaken to evaluate the subchronic toxicity of yttrium, a representative heavy REE with higher contaminated level in foods in China, to achieve a no observed adverse effect level (NOAEL) which is a critical basis for the establishment of an ADI. Yttrium nitrate was orally administered to rats at doses of 0, 10, 30 and 90 mg/kg/day for 90 days followed by a recovery period of 4 weeks. The following toxicity indices were measured: mortality, clinical signs, daily food consumption and weekly body weight; urinalysis, hematology, blood coagulation, clinical biochemistry and histopathology at the end of administration and recovery periods. No toxicologically significant changes were found in any yttrium‐treated group as compared to the concurrent control group. Under the present experimental condition, the NOAEL in rats was thus set at 90 mg/kg for yttrium nitrate, i.e. 29.1 mg/kg for yttrium. HighlightsSubchronic toxicity study was performed in accordance with OECD TG 408.The NOAEL in rats was estimated to be 29.1 mg/kg for yttrium.Daily intake of yttrium from foods is acceptable for adults in China.

Comparison of freely-moving telemetry Chinese Miniature Experiment Pigs (CMEPs) to beagle dogs in cardiovascular safety pharmacology studies.

INTRODUCTION Telemetry beagle dogs are the most frequently used species in cardiovascular telemetry assessments. However, beagle dogs may not be always suitable for all of the tests. Recently minipigs have received increased attention for these studies. Differences between the two species regarding the response of their cardiovascular systems to environmental stimuli are unclear. This study investigates how the telemetry minipig compares to beagle dog as a test subject and also refines the experimental protocols necessary to obtain accurate data. METHODS Beagle dogs and Chinese Miniature Experiment Pigs (CMEPs) were implanted with telemetry transmitters and the influences of gavage, feeding and the circadian cycle on various cardiovascular parameters were investigated. RESULTS ECG signal quality from CMEPs was superior to that of the beagle dogs. Poor ECG signal quality, elevated HR, BP and locomotor activity associated with gavage and feeding were observed in both species. ECG signal quality, BP and locomotor activity recovered more quickly in the CMEPs than in the beagle dogs. Residual elevation of HR found in CMEPs lasted approximately 4h post-feeding, which has a profound influence on the circadian cycle. A diurnal rhythm in CMEP with a significant increase of body temperature during the dark period and a clear circadian rhythm of locomotor activity in both species were observed. DISCUSSION The present data demonstrated that gavage, feeding and circadian cycle were having an enormous influence on BP, HR and locomotor activity in both species. If drug-induced effects are expected rapidly after oral administration and feeding, CMEP seems to be a favorable choice. Also, due to the effects of feeding on HR, CMEPs should fast at least 5h before the start of recording or should not be fed during the study where the Tmax of a given compound might occur very late. It also should be taken into consideration when the test article has a potential effect on body temperature by using CMEPs. In summary, the telemetry CMEP is a valuable alternative to the beagle dog for cardiovascular telemetry studies.

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.