Shuangqing Peng

Toxic effects of silica nanoparticles on zebrafish embryos and larvae.

Silica nanoparticles (SiNPs) have been widely used in biomedical and biotechnological applications. Environmental exposure to nanomaterials is inevitable as they become part of our daily life. Therefore, it is necessary to investigate the possible toxic effects of SiNPs exposure. In this study, zebrafish embryos were treated with SiNPs (25, 50, 100, 200 µg/mL) during 4–96 hours post fertilization (hpf). Mortality, hatching rate, malformation and whole-embryo cellular death were detected. We also measured the larval behavior to analyze whether SiNPs had adverse effects on larvae locomotor activity. The results showed that as the exposure dosages increasing, the hatching rate of zebrafish embryos was decreased while the mortality and cell death were increased. Exposure to SiNPs caused embryonic malformations, including pericardial edema, yolk sac edema, tail and head malformation. The larval behavior testing showed that the total swimming distance was decreased in a dose-dependent manner. The lower dose (25 and 50 µg/mL SiNPs) produced substantial hyperactivity while the higher doses (100 and 200 µg/mL SiNPs) elicited remarkably hypoactivity in dark periods. In summary, our data indicated that SiNPs caused embryonic developmental toxicity, resulted in persistent effects on larval behavior.

Silica nanoparticles enhance autophagic activity, disturb endothelial cell homeostasis and impair angiogenesis

BackgroundGiven that the effects of ultrafine fractions (<0.1 μm) on ischemic heart diseases (IHD) and other cardiovascular diseases are gaining attention, this study is aimed to explore the influence of silica nanoparticles (SiNPs)-induced autophagy on endothelial cell homeostasis and angiogenesis.Methods and resultsUltrastructural changes of autophagy were observed in both vascular endothelial cells and pericytes in the heart of ICR mice by TEM. Autophagic activity and impaired angiogenesis were further confirmed by the immunohistochemistry staining of LC3 and VEGFR2. In addition, the immunohistochemistry results showed that SiNPs had an inhibitory effect on ICAM-1 and VCAM-1, but no obvious effect on E-selectin in vivo. The disruption of F-actin cytoskeleton occurred as an initial event in SiNPs-treated endothelial cells. The depolarized mitochondria, autophagic vacuole accumulation, LC3-I/LC3-II conversion, and the down-regulation of cellular adhesion molecule expression were all involved in the disruption of endothelial cell homeostasis in vitro. Western blot analysis indicated that the VEGFR2/PI3K/Akt/mTOR and VEGFR2/MAPK/Erk1/2/mTOR signaling pathway was involved in the cardiovascular toxicity triggered by SiNPs. Moreover, there was a crosstalk between the VEGFR2-mediated autophagy signaling and angiogenesis signaling pathways.ConclusionsIn summary, the results demonstrate that SiNPs induce autophagic activity in endothelial cells and pericytes, subsequently disturb the endothelial cell homeostasis and impair angiogenesis. The VEGFR2-mediated autophagy pathway may play a critical role in maintaining endothelium and vascular homeostasis. Our findings may provide experimental evidence and explanation for cardiovascular diseases triggered by nano-sized particles.

Repeated PM2.5 exposure inhibits BEAS-2B cell P53 expression through ROS-Akt-DNMT3B pathway-mediated promoter hypermethylation

Long-term exposure to fine particulate matter (PM2.5) has been reported to be closely associated with the increased lung cancer risk in populations, but the mechanisms underlying PM-associated carcinogenesis are not yet clear. Previous studies have indicated that aberrant epigenetic alterations, such as genome-wide DNA hypomethylation and gene-specific DNA hypermethylation contribute to lung carcinogenesis. And silence or mutation of P53 tumor suppressor gene is the most prevalent oncogenic driver in lung cancer development. To explore the effects of PM2.5 on global and P53 promoter methylation changes and the mechanisms involved, we exposed human bronchial epithelial cells (BEAS-2B) to low concentrations of PM2.5 for 10 days. Our results indicated that PM2.5-induced global DNA hypomethylation was accompanied by reduced DNMT1 expression. PM2.5 also induced hypermethylation of P53 promoter and inhibited its expression by increasing DNMT3B protein level. Furthermore, ROS-induced activation of Akt was involved in PM2.5-induced increase in DNMT3B. In conclusion, our results strongly suggest that repeated exposure to PM2.5 induces epigenetic silencing of P53 through ROS-Akt-DNMT3B pathway-mediated promoter hypermethylation, which not only provides a possible explanation for PM-induced lung cancer, but also may help to identify specific interventions to prevent PM-induced lung carcinogenesis.

T-2 toxin induces developmental toxicity and apoptosis in zebrafish embryos

T-2 toxin is one of the most important trichothecene mycotoxins occurring in various agriculture products. The developmental toxicity of T-2 toxin and the exact mechanism of action at early life stages are not understood precisely. Zebrafish embryos were exposed to different concentrations of the toxin at 4-6 hours post fertilization (hpf) stage of development, and were observed for different developmental toxic effects at 24, 48, 72, and 144 hpf. Exposure to 0.20 μmol/L or higher concentrations of T-2 toxin significantly increased the mortality and malformation rate such as tail deformities, cardiovascular defects and behavioral changes in early developmental stages of zebrafish. T-2 toxin exposure resulted in significant increases in reactive oxygen species (ROS) production and cell apoptosis, mainly in the tail areas, as revealed by Acridine Orange staining at 24 hpf. In addition, T-2 toxin-induced severe tail deformities could be attenuated by co-exposure to reduced glutathione (GSH). T-2 toxin and GSH co-exposure induced a significant decrease of ROS production in the embryos. The overall results demonstrate that T-2 toxin is able to produce oxidative stress and induce apoptosis, which are involved in the developmental toxicity of T-2 toxin in zebrafish embryos.

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.

Protective Effects of Metallothionein on Isoniazid and Rifampicin-Induced Hepatotoxicity in Mice

Isoniazid (INH) and Rifampicin (RFP) are widely used in the world for the treatment of tuberculosis, but the hepatotoxicity is a major concern during clinical therapy. Previous studies showed that these drugs induced oxidative stress in liver, and several antioxidants abated this effect. Metallothionein (MT), a member of cysteine-rich protein, has been proposed as a potent antioxidant. This study attempts to determine whether endogenous expression of MT protects against INH and RFP-induced hepatic oxidative stress in mice. Wild type (MT+/+) and MT-null (MT−/−) mice were treated intragastrically with INH (150 mg/kg), RFP (300 mg/kg), or the combination (150 mg/kg INH +300 mg/kg RFP) for 21 days. The results showed that MT−/− mice were more sensitive than MT+/+ mice to INH and RFP-induced hepatic injuries as evidenced by hepatic histopathological alterations, increased serum AST levels and liver index, and hepatic oxidative stress as evidenced by the increase of MDA production and the change of liver antioxidant status. Furthermore, INH increased the protein expression of hepatic CYP2E1 and INH/RFP (alone or in combination) decreased the expression of hepatic CYP1A2. These findings clearly demonstrate that basal MT provides protection against INH and RFP-induced toxicity in hepatocytes. The CYP2E1 and CYP1A2 were involved in the pathogenesis of INH and RFP-induced hepatotoxicity.

Combined exposure to nano-silica and lead induced potentiation of oxidative stress and DNA damage in human lung epithelial cells.

Growing evidence has confirmed that exposure to ambient particulate matters (PM) is associated with increased morbidity and mortality of cardiovascular and pulmonary diseases. Ambient PM is a complex mixture of particles and air pollutants. Harmful effects of PM are specifically associated with ultrafine particles (UFPs) that can adsorb high concentrations of toxic air pollutants and are easily inhaled into the lungs. However, combined effects of UFPs and air pollutants on human health remain unclear. In the present study, we elucidated the combined toxicity of silica nanoparticles (nano-SiO2), a typical UFP, and lead acetate (Pb), a typical air pollutant. Lung adenocarcinoma A549 cells were exposed to nano-SiO2 and Pb alone or their combination, and their combined toxicity was investigated by focusing on cellular oxidative stress and DNA damage. Factorial analyses were performed to determine the potential interactions between nano-SiO2 and Pb. Our results showed that exposure of A549 cells to a modest cytotoxic concentration of Pb alone induced oxidative stress, as evidenced by elevated reactive oxygen species generation and lipid peroxidation, and reduced glutathione content and superoxide dismutase and glutathione peroxidase activities. In addition, exposure of A549 cells to Pb alone induced DNA damage, as evaluated by alkaline comet assay. Exposure of A549 cells to non-cytotoxic concentration of nano-SiO2 did not induce cellular oxidative stress and DNA damage. However, exposure to the combination of nano-SiO2 and Pb potentiated oxidative stress and DNA damage in A549 cells. Factorial analyses indicated that the potentiation of combined toxicity of nano-SiO2 and Pb was induced by additive or synergistic interactions.

Distribution of DNA Adducts and Corresponding Tissue Damage of Sprague–Dawley Rats with Percutaneous Exposure to Sulfur Mustard

Sulfur mustard (SM) is a highly reactive alkylation vesicant and cytotoxic agent that has been recognized as an animal and human carcinogen. Although the exact mechanism of toxicology is vague, DNA alkylation seems to be responsible for the triggering of apoptosis. In this study, after male adult Sprague-Dawley rats were cutaneous exposed to a low concentration of SM at parts-per-million levels, their lungs, livers, pancreases, spleens, marrow, and brains were collected at 11 different time points and analyzed. N7-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (N7-HETEG), N3-[2-[(2-hydroxyethyl)thio]-ethyl]adenine (N3-HETEA), and bis[2-(guanin-7-yl)ethyl]sulfide (Bis-G) as the biomarkers for DNA damage were measured in the vital tissues by isotope dilution ultraperformance liquid chromatography tandem mass spectrometry (ID-UPLC-MS/MS). At the same time, general variations and pathological changes were monitored and detected to evaluate the tissue damage. Time- and dose-dependent data showed that SM had strong permeability and reactivity and that three SM-DNA adducts were detected in all investigated tissues only after 10 min after exposure. Obvious dose-dependency was observed except in the brain and pancreas. Most times to peak (tmax) of all three adducts were less than 3 h, while half-lifetimes (t1/2) were less than 24 h. We also suggested that the lipophilic SM can easily pass through the blood-brain barrier and can be stored in the fatty organs. To the best of our knowledge, the abundant adducts in marrow were found and reported for the first time. The surveillance of N7-HETEG in vivo, which was the most abundant adduct, may be the most efficient indicator to validate SM exposure even without any symptoms. Bis-G can be regarded as a biomarker of effect, which is directly related to the extent of damage. The most abundant Bis-G was found in the most sensitive tissues, marrow, spleen, and lung, which is in good accordance with histopathologic results. General variations and pathological changes were evaluated as well. After cutaneous exposure to SM, the body weights of rats heavily decreased in the first 4 days and were inversely proportional to the applied doses, and then recovered at the last experimental day except for those of the rats at the highest dosing level, in which the relative weights of rat spleens were obviously lost. Moreover, we found remarkable histological changes of the lung and skin, such as encephalemia, at the very beginning of the sampling procedure, and plentiful mononuclear cells in marrow appeared 6 h after exposure. The micronucleus test of marrow cells showed that the micronucleus rate had a positively dose-dependent effect.

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.