Assessment of biological activity and UPLC–MS based chromatographic profiling of ethanolic extract of Ochradenus arabicus

Natural products from wild and medicinal plants, either in the form of crude extracts or pure compounds provide unlimited opportunities for new drug leads owing to the unmatched availability of chemical diversity. In the present study, the cytotoxic potential of crude ethanolic extract of Ochradenus arabicus was analyzed by MTT cell viability assay in MCF-7 adenocarcinoma breast cancer cells. We further investigated its effect against oxidative stress induced by anticancer drug doxorubicin. In addition, Ultra Performance Liquid Chromatography–Mass Spectrometry (UPLC–MS) based chromatographic profiling of crude extract of O. arabicus was performed. The MTT assay data showed that the extract is moderately toxic to the MCF-7 cells. However, its treatment alone does not induce oxidative stress while doxorubicin increases the level of oxidative stress in MCF-7 cells. Whereas, simultaneous treatment of plant extract and doxorubicin significantly (p < 0.05) decreased the level of intracellular reactive oxygen species (ROS) and lipid peroxidation while an increase in the reduced glutathione and superoxide dismutase activity was observed in time and dose dependent manner. Hence, our finding confirmed cytotoxic and antioxidant potential of crude extract of O. arabicus in MCF-7 cells. However, further investigations on O. arabicus as a potential chemotherapeutic agent are needed. The analysis of bioactive compounds present in the plant extracts involving the applications of common phytochemical screening assays such as chromatographic techniques is discussed.     

Toxicity of Fe2O3 nanoparticles on human blood lymphocytes

Magnetic nanoparticles (NPs) are used to a large extent in the targeted delivery of therapeutic agents. In this study, we aimed to investigate the possible toxicity of Fe₂O ₃ NPs on human cells, including blood lymphocytes. We isolated blood lymphocytes from healthy humans using Ficoll polysaccharide and subsequently by gradient centrifugation. Then, the toxicity parameters, including cell viability, reactive oxygen species (ROS) formation, lipid peroxidation, cellular glutathione (GSH) level, mitochondrial and lysosomal damage, were measured in blood lymphocytes after exposure to Fe ₂O ₃ NPs. Our results indicated that Fe ₂O ₃ NPs significantly (dependent on concentration) reduced the cell viability, and the IC ₅₀ was determined to be 1 mM. With increasing concentrations, we found that Fe ₂O ₃ NPs–induced cell toxicity was associated with a significant increase in intracellular ROS and loss of mitochondrial membrane potential and lysosomal membrane leakiness. Consequently, these NPs at different concentrations affect GSH level and cause oxidative stress in human lymphocytes.     

Thioredoxin-1 suppresses lung injury and apoptosis induced by diesel exhaust particles (DEP) by scavenging reactive oxygen species and by inhibiting DEP-induced downregulation of Akt

Diesel exhaust particles (DEP) are reactive oxygen species (ROS)-inducing toxic agents that damage lungs. Thioredoxin-1 (Trx-1) is a thiol protein with antioxidant and redox-regulating effects. In this study, we demonstrate that Trx-1 scavenges ROS generated by DEP and attenuates the lung injury. Intratracheal instillation of DEP resulted in the generation of more hydroxyl radicals in control mice than in human Trx-1 (hTrx-1)-transgenic mice as measured by noninvasive L-band in vivo electron spin resonance. DEP caused acute lung damage with massive infiltration of inflammatory cells in control mice, but much less damage in hTrx-1-transgenic mice. The hTrx-1 transgene protected the mice against DEP toxicity. To investigate further the molecular mechanism of the protective role of Trx-1 against DEP-induced lung injury, we used hTrx-1-transfected L-929 cells and recombinant hTrx-1 (rhTrx-1)-pretreated A-549 cells. DEP-induced ROS generation was suppressed by hTrx-1 transfection or pretreatment with rhTrx-1. Endogenous Trx-1 expression was induced by DEP in control cells. The downregulation of Akt phosphorylation by DEP resulted in apoptosis, which was prevented by Trx-1. Moreover, an Akt inhibitor canceled this protective effect of Trx-1. Collectively, the results suggest that Trx-1 exerts antioxidant effects in vivo and in vitro and that this plays a role in protection against DEP-induced lung damage by regulating Akt-mediated antiapoptotic signaling.     

Acute Toxicity of Intravenously Administered Titanium Dioxide Nanoparticles in Mice

Background

With a wide range of applications, titanium dioxide (TiO₂) nanoparticles (NPs) are manufactured worldwide in large quantities. Recently, in the field of nanomedicine, intravenous injection of TiO₂ nanoparticulate carriers directly into the bloodstream has raised public concerns on their toxicity to humans.

Methods

In this study, mice were injected intravenously with a single dose of TiO₂ NPs at varying dose levels (0, 140, 300, 645, or 1387 mg/kg). Animal mortality, blood biochemistry, hematology, genotoxicity and histopathology were investigated 14 days after treatment.

Results

Death of mice in the highest dose (1387 mg/kg) group was observed at day two after TiO₂ NPs injection. At day 7, acute toxicity symptoms, such as decreased physical activity and decreased intake of food and water, were observed in the highest dose group. Hematological analysis and the micronucleus test showed no significant acute hematological or genetic toxicity except an increase in the white blood cell (WBC) count among mice 645 mg/kg dose group. However, the spleen of the mice showed significantly higher tissue weight/body weight (BW) coefficients, and lower liver and kidney coefficients in the TiO₂ NPs treated mice compared to control. The biochemical parameters and histological tissue sections indicated that TiO₂ NPs treatment could induce different degrees of damage in the brain, lung, spleen, liver and kidneys. However, no pathological effects were observed in the heart in TiO₂ NPs treated mice.

Conclusions

Intravenous injection of TiO₂ NPs at high doses in mice could cause acute toxicity effects in the brain, lung, spleen, liver, and kidney. No significant hematological or genetic toxicity was observed.     

Thunbergia laurifolia leaf extract partially recovers lead-induced renotoxicity through modulating the cell signaling pathways

This research investigated the reno-protective effect of Thunbergia laurifolia Linn. (TL) in a lead-induced toxicity test through the modulation of cell signaling pathways. The study carried out to evaluate the effect of TL leaf extracts in Swiss Albino mice exposed to lead acetate (PbAc). Prior to in vivo study, a probable kidney-protective effect of the plant leaf extract was presumed through an activity-specific (PASS) molecular docking analysis. In animal model study, albino mice were divided in seven groups and co-treated with PbAc and TL (100, 200 mg/kgBW) or vitamin E (100 mg/kgBW) for 38 days, whereas the untreated control, TL control, and vehicle control groups received sodium acetate, PbAc, sodium acetate plus mineral oil, respectively. At the end of treatment, blood and kidney tissue were collected for investigating Pb concentration, estimating biochemical profile, evaluating oxidative stress and inflammatory parameters. The histopathological change of kidney along with apoptosis was assessed from kidney sections using H & E staining and TUNEL assay. Pb-exposed mice were found to be increased concentration of Pb in the blood and kidney sample, which further led to increased MDA levels in the plasma, blood, and tissue. Followed by kidney damage, increased expression of TNF-α, iNOS, and COX-2 in kidney tissues were noticed, which were related to elevated TNF-α in the systemic circulation of Pb-treated mice. Co-treatment with TL or vitamin E significantly reduced altered structure and apoptosis of kidney tissues. Downregulation of inflammatory markers especially TNF-α, iNOS, and COX-2 with simultaneous improvement of renal function through reduced plasma BUN and creatinine levels demonstrate that TL act as a potential dietary supplement to detoxify Pb in kidney showing an antioxidant and anti-inflammatory effect.     

Tat-Fused Recombinant Human SAG Prevents Dopaminergic Neurodegeneration in a MPTP-Induced Parkinson’s Disease Model

Excessive reactive oxygen species (ROS) generated from abnormal cellular process lead to various human diseases such as inflammation, ischemia, and Parkinson’s disease (PD). Sensitive to apoptosis gene (SAG), a RING-FINGER protein, has anti-apoptotic activity and anti-oxidant activity. In this study, we investigate whether Tat-SAG, fused with a Tat domain, could protect SH-SY5Y neuroblastoma cells against 1-methyl-4-phenylpyridinium (MPP⁺) and dopaminergic (DA) neurons in the substantia nigra (SN) against 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) toxicity. Western blot and immunohistochemical analysis showed that, unlike SAG, Tat-SAG transduced efficiently into SH-SY5Y cells and into the brain, respectively. Tat-SAG remarkably suppressed ROS generation, DNA damage, and the progression of apoptosis, caused by MPP⁺ in SH-SY5Y cells. Also, immunohistochemical data using a tyrosine hydroxylase antibody and cresyl violet staining demonstrated that Tat-SAG obviously protected DA neurons in the SN against MPTP toxicity in a PD mouse model. Tat-SAG-treated mice showed significant enhanced motor activities, compared to SAG- or Tat-treated mice. Therefore, our results suggest that Tat-SAG has potential as a therapeutic agent against ROS-related diseases such as PD.     

Neurotoxicity mediated by oxidative stress caused by titanium dioxide nanoparticles in human neuroblastoma (SH-SY5Y) cells

BackgroundTitanium is widely used in biomedicine. Due to biotribocorrosion, titanium dioxide (TiO₂) nanoparticles (NPs) can be released from the titanium implant surface, enter the systemic circulation, and migrate to various organs and tissues including the brain. A previous study showed that 5 nm TiO₂ NPs reached the highest concentration in the brain. Even though TiO₂ NPs are believed to possess low toxicity, little is known about their neurotoxic effects. The aim of the study was to evaluate in vitro the effects of 5 nm TiO₂ NPs on a human neuroblastoma (SH-SY5Y) cell line.MethodsCell cultures were divided into non-exposed and exposed to TiO₂ NPs for 24 h. The following were evaluated: reactive oxygen species (ROS) generation, apoptosis, cellular antioxidant response, endoplasmic reticulum stress and autophagy.ResultsExposure to TiO₂ NPs induced ROS generation in a dose dependent manner, with values reaching up to 10 fold those of controls (p < 0.001). Nrf2 nuclear localization and autophagy, also increased in a dose dependent manner. Apoptosis increased by 4- to 10-fold compared to the control group, depending on the dose employed.ConclusionsOur results show that TiO₂ NPs cause ROS increase, induction of ER stress, Nrf2 cytoplasmic translocation to the nucleus and apoptosis. Thus, neuroblastoma cell response to TiO₂ NPs may be associated with an imbalance of the oxidative metabolism where endoplasmic reticulum-mediated signal pathway seems to be the main neurotoxic mechanism.     

紫茎泽兰叶片水浸液对蚕豆的化感效应

外来入侵植物可以通过淋溶、自然挥发、根系分泌和植株凋落物分解等途径向周围环境释放化感物质,抑制伴生植物的生长、发育。该研究以不同浓度紫茎泽兰(Eupatorium adenophorum)叶片水浸液处理蚕豆(Vicia faba)种子,研究紫茎泽兰叶片水浸液对蚕豆根尖细胞微核、染色体畸变、细胞凋亡、蚕豆幼苗叶片叶绿素和N含量、光合生理特性、生物量的影响。结果表明:(1)紫茎泽兰叶片水浸液处理显著抑制蚕豆根尖的伸长和细胞的有丝分裂,并诱导蚕豆根尖细胞染色体畸变和细胞微核的产生,有丝分裂指数随着叶片水浸液浓度增加而减小,根尖细胞微核率随叶片水浸液浓度增加而增大,高浓度叶片水浸液处理对蚕豆根尖细胞的凋亡及坏死有明显影响。(2)紫茎泽兰叶片水浸液处理引起蚕豆幼苗叶片的叶绿素和N含量显著降低,并导致蚕豆幼苗最大净光合速率和生物量的显著下降。总之,紫茎泽兰叶片水浸液可能引起蚕豆根尖的氧化损伤和抑制根尖的伸长,且叶片水浸液的抑制作用呈现一定的剂量效应。紫茎泽兰叶片水浸液对蚕豆根尖的损伤和抑制作用可能影响了植株对氮素的吸收,进而对蚕豆幼苗光合生理表现以及生物量积累产生显著负面效应。     

Auxin-induced ethylene production as related to auxin metabolism in leaf discs of tobacco and sugar beet

Exogenously supplied indole-3-acetic acid (IAA) stimulated ethylene production in tobacco (Nicotiana glauca) leaf discs but not in those of sugar beet (Beta vulgaris L.). The stimulatory effect of IAA in tobacco was relatively small during the first 24 hours of incubation but became greater during the next 24 hours. It was found that leaf discs of these two species metabolized [1-¹⁴C]IAA quite differently. The rate of decarboxylation in sugar beet discs was much higher than in tobacco. The latter contained much less free IAA but a markedly higher level of IAA conjugates. The major conjugate in the sugar beet extracts was indole-3-acetylaspartic acid, whereas tobacco extracts contained mainly three polar IAA conjugates which were not found in the sugar beet extracts. The accumulation of the unidentified conjugates corresponded with the rise of ethylene production in the tobacco leaf discs. Reapplication of all the extracted IAA conjugates resulted in a great stimulation of ethylene production by tobacco leaf discs which was accompanied by decarboxylation of the IAA conjugates. The results suggest that in tobacco IAA-treated leaf discs the IAA conjugates could stimulate ethylene production by a slow release of free IAA. The inability of the exogenously supplied IAA to stimulate ethylene production in the sugar beet leaf discs was not due to a deficiency of free IAA within the tissue but rather to the lack of responsiveness of this tissue to IAA, probably because of an autoinhibitory mechanism existing in the sugar beet leaf discs.     

Comparative Cardiac Toxicity of Anthracyclines In Vitro and In Vivo in the Mouse

Purpose

The antineoplastic efficacy of anthracyclines is limited by their cardiac toxicity. In this study, we evaluated the toxicity of doxorubicin, non-pegylated liposomal-delivered doxorubicin, and epirubicin in HL-1 adult cardiomyocytes in culture as well as in the mouse in vivo.

Methods

The cardiomyocytes were incubated with the three anthracyclines (1 µM) to assess reactive oxygen generation, DNA damage and apoptotic cell death. CF-1 mice (10/group) received doxorubicin, epirubicin or non-pegylated liposomal-doxorubicin (10 mg/kg) and cardiac function was monitored by Doppler echocardiography to measure left ventricular ejection fraction (LVEF), heart rate (HR) and cardiac output (CO) both prior to and 10 days after drug treatment.

Results

In HL-1 cells, non-pegylated liposomal-doxorubicin generated significantly less reactive oxygen species (ROS), as well as less DNA damage and apoptosis activation when compared with doxorubicin and epirubicin. Cultured breast tumor cells showed similar sensitivity to the three anthracyclines. In the healthy mouse, non-pegylated liposomal doxorubicin showed a minimal and non-significant decrease in LVEF with no change in HR or CO, compared to doxorubicin and epirubicin.

Conclusion

This study provides evidence for reduced cardiac toxicity of non-pegylated-liposomal doxorubicin characterized by attenuation of ROS generation, DNA damage and apoptosis in comparison to epirubicin and doxorubicin.     

Zearalenone induces ROS‐mediated mitochondrial damage in porcine IPEC‐J2 cells

In this study, the mitochondrial damage effect and mechanism of zearalenone (ZEA) in swine small intestine IPEC‐J2 cells in vitro were comprehensively characterized. The analyses revealed that ZEA at high doses (8 and 7 μg/mL) can significantly increase P < 0.05 the malondialdehyde levels and decrease antioxidant enzymes activities after 48 h of exposure. Meanwhile, the reactive oxygen species (ROS) accumulation increased in high dose ZEA‐treated groups after 2 h treatment, but decreased due to the ROS‐induced mitochondrial damage and the caused cell apoptosis after 48 h of high does ZEA treatment. Moreover, the decreasing of mitochondrial membrane potential (MMP; ΔΨ) in high dose ZEA exposure was observed in line with the increasing ROS production in mitochondria. Results suggest that ZEA exposure can induce mitochondrial damage by reducing antioxidant enzyme activities, accumulation of ROS, and decreasing MMP. The mitochondrial damage had a dramatic concentration–effects relationship with ZEA.     

Epimedium protects against dyszoospermia in mice with Pex3 knockout by exerting antioxidant effects and regulating the expression level of P16

Oxidative stress (OS) is one of the primary factors leading to male infertility. Oral administration of antioxidants has thus far been found to significantly improve the quality of human sperm. Therefore, antioxidant treatment has become the consensus among international experts on male infertility. In this study, peroxisomal biogenesis factor 3 (Pex3)-knockout (KO, −/−) mice were used as a model to compare the efficacy of three types of traditional Chinese medicine (TCM) granules (Epimedium [YYH], Cuscuta [TSZ], and Rhodiola [HJT]) for male reproductive function rescue. YYH was revealed to be the best and exerted a rescue effect on Pex3−/− mice with spermatogenesis defects. In addition, YYH prominently reduced ROS levels in the testes, inhibited DNA oxidative damage in spermatogenic cells, promoted the proliferation of spermatogenic cells, and inhibited apoptosis in Pex3−/− male mice. Furthermore, the mechanism by which YYH ameliorated dyszoospermia was confirmed via the establishment of cyclin-dependent kinase inhibitor 2 A (P16Ink4a)-KO mice. Specifically, Pex3−/− mice produced elevated amounts of ROS, which damaged germ cell DNA and further activated the signaling pathway of the cell senescence regulatory protein P16-CDK6, resulting in cell cycle arrest and eventually contributing to spermatogenesis dysfunction. YYH supplementation partially corrected the associated phenotype in gene KO mice by affecting P16 expression levels, thus improving the reproductive outcome to a certain extent.Subject terms: Senescence, Embryology, Infertility, Experimental models of disease, Translational research     

Effect of ethylene on [C]indole-3-acetic Acid metabolism in leaf tissues of woody plants

The effect of ethylene on [¹⁴C]indole-3-acetic acid (IAA) metabolism was investigated in defoliation sensitive leaf tissues of citrus (Citrus sinensis) and resistant leaf tissues of eucalyptus (Eucalyptus camaldulensis). IAA metabolites were fractionated into 80% ethanol-soluble, H₂O-soluble, NaOH-soluble, and insoluble components. In citrus, pretreatment with 25 microliters per liter ethylene for 24 hours significantly increased the amount of ethanol- and H₂O-extractable conjugates during the first hour of incubation in [¹⁴C]IAA and increased 3- to 4-fold the formation of NaOH-extractable conjugates during the entire 6-hour incubation period. However, induction of the IAA-aspartate conjugation system was inhibited by ethylene. In eucalyptus, ethylene pretreatment only slightly stimulated the formation of IAA metabolites. Increased formation of ethanol-extractable conjugates in ethylene-pretreated eucalyptus tissues was observed only after 6 hours of incubation. Chromatographic analysis indicated that the ethanol and H₂O extracts of both species contained various low molecular weight conjugates, whereas in citrus leaf tissues high molecular weight conjugates accounted for most of the greater radioactivity detected in the NaOH extracts as a result of ethylene-pretreatment. It is suggested that ethylene may reduce the level of endogenous IAA in citrus leaf tissues by stimulating IAA conjugation.     

Toxicity Assessment of Silica Coated Iron Oxide Nanoparticles and Biocompatibility Improvement by Surface Engineering

We have studied in vitro toxicity of iron oxide nanoparticles (NPs) coated with a thin silica shell (Fe₃O₄/SiO₂ NPs) on A549 and HeLa cells. We compared bare and surface passivated Fe₃O₄/SiO₂ NPs to evaluate the effects of the coating on the particle stability and toxicity. NPs cytotoxicity was investigated by cell viability, membrane integrity, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) assays, and their genotoxicity by comet assay. Our results show that NPs surface passivation reduces the oxidative stress and alteration of iron homeostasis and, consequently, the overall toxicity, despite bare and passivated NPs show similar cell internalization efficiency. We found that the higher toxicity of bare NPs is due to their stronger in-situ degradation, with larger intracellular release of iron ions, as compared to surface passivated NPs. Our results indicate that surface engineering of Fe₃O₄/SiO₂ NPs plays a key role in improving particles stability in biological environments reducing both cytotoxic and genotoxic effects.     

Functional Characterisation of Anticancer Activity in the Aqueous Extract of Helicteres angustifolia L. Roots

Helicteres angustifolia L. is a shrub that forms a common ingredient of several cancer treatment recipes in traditional medicine system both in China and Laos. In order to investigate molecular mechanisms of its anticancer activity, we prepared aqueous extract of Helicteres angustifolia L. Roots (AQHAR) and performed several in vitro assays using human normal fibroblasts (TIG-3) and osteosarcoma (U2OS). We found that AQHAR caused growth arrest/apoptosis of U2OS cells in a dose-dependent manner. It showed no cytotoxicity to TIG-3 cells at doses up to 50 μg/ml. Biochemical, imaging and cell cycle analyses revealed that it induces ROS signaling and DNA damage response selectively in cancer cells. The latter showed upregulation of p53, p21 and downregulation of Cyclin B1 and phospho-Rb. Furthermore, AQHAR-induced apoptosis was mediated by increase in pro-apoptotic proteins including cleaved PARP, caspases and Bax. Anti-apoptotic protein Bcl-2 showed decrease in AQHAR-treated U2OS cells. In vivo xenograft tumor assays in nude mice revealed dose-dependent suppression of tumor growth and lung metastasis with no toxicity to the animals suggesting that AQHAR could be a potent and safe natural drug for cancer treatment.     

Phloroglucinol (1,3,5-trihydroxybenzene) protects against ionizing radiation-induced cell damage through inhibition of oxidative stress in vitro and in vivo

Exposure of cells to γ-rays induces the production of reactive oxygen species (ROS) that play a main role in ionizing radiation damage. We have investigated the radioprotective effect of phloroglucinol (1,3,5-trihydroxybenzene), phlorotannin compound isolated from Ecklonia cava, against γ-ray radiation-induced oxidative damage in vitro and in vivo. Phloroglucinol significantly decreased the level of radiation-induced intracellular ROS and damage to cellular components such as the lipid, DNA and protein. Phloroglucinol enhanced cell viability that decreased after exposure to γ-rays and reduced radiation-induced apoptosis via inhibition of mitochondria mediated caspases pathway. Phloroglucinol reduced radiation-induced loss of the mitochondrial membrane action potential, reduced the levels of the active forms of caspase 9 and 3 and elevated the expression of bcl-2. Furthermore, the anti-apoptotic effect of phloroglucinol was exerted via inhibition of mitogen-activated protein kinase kinase-4 (MKK4/SEK1), c-Jun NH₂-terminal kinase (JNK) and activator protein-1 (AP-1) cascades induced by radiation exposure. Phloroglucinol restored the level of reduced glutathione (GSH) and protein expression of a catalytically active subunit of glutamate-cysteine ligase (GCL), which is a rate-limiting enzyme in GSH biosynthesis. In in vivo study, phloroglucinol administration in mice provided substantial protection against death and oxidative damage following whole-body irradiation. We examined survival with exposure to various radiation doses using the intestinal crypt assay and determined a dose reduction factor (DRF) of 1.24. Based on our findings, phloroglucinol may be possibly useful as a radioprotective compound.     

Cytotoxic and pro-apoptotic effects of Abrus precatorius L. on human metastatic breast cancer cell line, MDA-MB-231

Abrus precatorius is highly regarded as a universal panacea in the herbal medicine with diverse pharmacological activity spectra. This experimental study on the mechanism of the anticancer activity of A. precatorius leaf extracts, may offer new evidence for A. precatorius in the treatment of breast cancer in clinical practice. Cell death was determined by using MTT assay. Further analyses were carried out by doing DNA laddering, PARP cleavage, FACS, semi-quantitative RT-PCR and detection of cellular reactive oxygen species (ROS) by DCFDA assay. A. precatorius showed very striking inhibition on MDA-MB-231 cells. MTT assay showed more than 75 % inhibition of the cells and treated cells indicated visible laddering pattern with thick compact band. PARP cleavage produced 89 kDa cleavage product which was associated with apoptosis. Flow cytometer exhibited a sub-G0/G1 peak as an indicative of apoptosis. mRNA expression level of apoptosis-related genes p21 and p53 was markedly increased in cells treated with the extract as compared to control. The up-regulation of p21 and p53 may be the molecular mechanisms by which A. precatorius extract which induces apoptosis. An increase in the concentration of A. precatorius extract does not generate ROS, instead it reduces ROS formation in MDA-MB-231 cells, as evident from the shift in fluorescence below untreated control. This is the first report showing that A. precatorius leaf extract exhibits a growth inhibitory effect by induction of apoptosis in MDA-MB-231 cells. Our results contribute towards validation of the A. precatorius extract as a potentially effective chemopreventive or therapeutic agent against breast cancer.     

DNA Damage Responses and Oxidative Stress in Dyskeratosis Congenita

Dyskeratosis congenita (DC) is an inherited multisystem disorder of premature aging, cancer predisposition, and bone marrow failure caused by selective exhaustion of highly proliferative cell pools. DC patients also have a poor tolerance to chemo/radiotherapy and bone marrow transplantation. Although critically shortened telomeres and defective telomere maintenance contribute to DC pathology, other mechanisms likely exist. We investigate the link between telomere dysfunction and oxidative and DNA damage response pathways and assess the effects of antioxidants. In vitro studies employed T lymphocytes from DC subjects with a hTERC mutation and age-matched controls. Cells were treated with cytotoxic agents, including Paclitaxel, Etoposide, or ionizing radiation. Apoptosis and reactive oxygen species (ROS) were assessed by flow cytometry, and Western blotting was used to measure expression of DNA damage response (DDR) proteins, including total p53, p53S15, and p21^WAF. N-acetyl-cysteine (NAC), an antioxidant, was used to modulate cell growth and ROS. In stimulated culture, DC lymphocytes displayed a stressed phenotype, characterized by elevated levels of ROS, DDR and apoptotic markers as well as a proliferative defect that was more pronounced after exposure to cytotoxic agents. NAC partially ameliorated the growth disadvantage of DC cells and decreased radiation-induced apoptosis and oxidative stress. These findings suggest that oxidative stress may play a role in the pathogenesis of DC and that pharmacologic intervention to correct this pro-oxidant imbalance may prove useful in the clinical setting, potentially alleviating untoward toxicities associated with current cytotoxic treatments.     

Exogenous nitric oxide improves antioxidative capacity and reduces auxin degradation in roots of Medicago truncatula seedlings under cadmium stress

The effects of nitric oxide (NO) on cadmium toxicity in Medicago truncatula seedlings were studied by investigating root growth and uptake of antioxidants, IAA and ions. Exposure to cadmium reduced root growth and NO accumulation, and increased the production of reactive oxygen species (ROS) in roots. Supplementation with NO improved root growth and reduced ROS accumulation in roots. The NO-scavenger cPTIO, the nitrate reductase (NR) inhibitor tungstate, and the NO synthase (NOS) inhibitor L-NAME all inhibited the accumulation of NO in roots and reversed the effects of NO in promoting the root growth and accumulation of proline and glutathione. Application of NO reduced auxin degradation by inhibiting the activity of IAA oxidase. Exogenous NO also enhanced the uptake of K⁺ and Ca²⁺. These results suggest that NO improves cadmium tolerance in plants by reducing oxidative damage, maintaining the auxin equilibrium and enhancing ion absorption.