Endothelial cell activation in vascular disease mediated by hydrogen peroxide in vitro

yesThe development of cardiovascular disease (CVD) is the main cause of death among chronic kidney disease (CKD) patients (1). Endothelial injury and dysfunction are critical steps in atherosclerosis, a major CVD (2). Increased production of reactive oxygen species (ROS) has been associated with the pathogenesis of cardiovascular diseases such as atherosclerosis, hypertension and heart failure (3). However, hydrogen peroxide (H2O2) modulates endothelial cell function by intricate mechanisms. Ambient production of O2.− and subsequently H2O2 at low levels, maintained via basal activity of pre-assembled endothelial NAD (P) H oxidases (4). Endothelial cells play an important regulatory role in the circulation as a physical barrier and as a source of a variety of regulatory substances. Dysfunction of the vascular endothelium is thus leading to atherosclerosis which is characterised by overexpression of adhesion molecule expression, comprising vascular cell adhesion molecule 1(VCAM1). This adhesion molecule has been found to be up-regulation in human atherosclerotic lesions. The aim of this study is to evaluate the effect of H2O2 on the endothelial cells adhesion molecules expression. Primary cultures of Human Umbilical Vascular Endothelial Cells (HUVECs) will be maintained in endothelial growth medium supplemented with penicillin-streptomycin and supplement mix of fetal calf serum in a 37C humidified incubator in an atmosphere of 5% v/v CO2. HUVECs will be treated with in the presence and absences of 50 μM of H 2O2 for 2, 6, 12 and 24 h. Intracellular superoxide anion production in HUVECs will be detected by using p-Nitro Blue Tetrazolium (NBT) assay to demonstrate whether H2O2 induce the generation of superoxide anions intracellularly in HUVECs. The formation of blue formazan will be measured spectrophotometrically at 570 nm. Total RNA will be extracted from non-treated and treated cells and RNA quantity and quality will be checked by OD260/280 measurements. VCAM-1 mRNA expression will be assessed using RT-PCR. Our results show that H2O2 could potentially significantly induce EC activation through increased mRNA expression of ICAM-1 adhesion molecules in cultured HUVECs. Treatment with N-acetyl cysteine (NAC) (bulk/nano form) could significantly attenuate the effect of H2O2 administration on adhesion molecule protein expression. This strongly suggests the role of ROS in the endothelial cell damage sustained. Future work is to find reliable methods to test endothelial function. Non-invasive studies such as brachial ultrasound testing are also needed to determine its predictive value as a potential predictor for cardiovascular disease

In vitro studies on genotoxicity and gene expression in spermatogenic cells: mechanisms and assay development

Spermatogenesis is a complex process of male germ cell development from diploid spermatogonia to haploid fertile spermatozoa. Apoptosis plays a vital role in limiting cell numbers and eliminating defective germ cells. This requires novel gene products, and precise and well-coordinated programmes of gene expression. It is therefore possible that a disruption of transcription factor function would significantly impact germ cell development. The present work was undertaken to use Staput separation followed by culture of purified germ cells of rodent testis since mammalian spermatogenesis cannot yet be recreated in vitro. Specificity of separation was assessed using immunocytochemistry to identify spermatogonia, spermatocytes and spermatids. The genotoxins H2O2, doxorubicin, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, 6-mercaptopurine, 5-bromodeoxyuridine, methyl methanesulphonate and ethyl methanesulphonate were investigated. Cells were cultured and treated with different concentrations for each agent. DNA damage and apoptosis were measured by Comet and TUNEL assay respectively. Up-regulation of expression of the transcription factors Tbpl1, FHL5 and Gtf2a1l that are important post-meiotically, were examined using RT- PCR and qPCR. Protein production was evaluated using Western blotting. Tbpl1, FHL5 and Gtf2a1l were cloned in-frame into the inducible expression vector pET/100-TOPO. The recombinant clones were induced and successful expression of the proteins in E. coli was confirmed by SDS-PAGE and Western blotting. The recombinant clones obtained were used to demonstrate genotoxin induced impairment of gene expression. Thus, Staput-isolated rodent testicular germ cells seem to be a suitable model to study genotoxicity in vitro yielding result comparable to those reported in vivo. Furthermore, the work shows that genotoxins can impair gene expression

In vitro responses to known in vivo genotoxic agents in mouse germ cells

YesGenotoxic compounds have induced DNA damage in male germ cells and have been associated with adverse clinical outcomes including enhanced risks for maternal, paternal and offspring health. DNA strand breaks represent a great threat to the genomic integrity of germ cells. Such integrity is essential to maintain spermatogenesis and prevent reproduction failure. The Comet assay results revealed that the incubation of isolated germ cells with n-ethyl-n-nitrosourea (ENU), 6-mercaptopurine (6-MP) and methyl methanesulphonate (MMS) led to increase in length of Olive tail moment and % tail DNA when compared with the untreated control cells and these effects were concentration-dependent. All compounds were significantly genotoxic in cultured germ cells. Exposure of isolated germ cells to ENU produced the highest concentration-related increase in both DNA damage and gene expression changes in spermatogonia. Spermatocytes were most sensitive to 6-MP, with DNA damage and gene expression changes while spermatids were particularly susceptible to MMS. Real-time PCR results showed that the mRNA level expression of p53 increased and bcl-2 decreased significantly with the increasing ENU, 6-MP and MMS concentrations in spermatogonia, spermatocytes and spermatids respectively for 24 hr. Both are gene targets for DNA damage response and apoptosis. These observations may help explain the cell alterations caused by ENU, 6-MP and MMS in spermatogonia, spermatocytes and spermatids. Taken together, ENU, 6-MP and MMS induced DNA damage and decreased apoptosis associated gene expression in the germ cells in vitro.Libyan Governmen

Diethylstilbestrol induces oxidative DNA damage, resulting in apoptosis of spermatogonial stem cells in vitro

YesThe spermatogonial stem cells (SSCs) are the only germline stem cells in adults that are responsible for the transmission of genetic information from mammals to the next generation. SSCs play a very important role in the maintenance of progression of spermatogenesis and help provide an understanding of the reproductive biology of future gametes and a strategy for diagnosis and treatment of infertility and male reproductive toxicity. Androgens/oestrogens are very important for the suitable maintenance of male germ cells. There is also evidence confirming the damaging effects of oestrogen-like compounds on male reproductive health. We investigated the effects in vitro, of diethylstilbestrol (DES) on mouse spermatogonial stem cells separated using Staput unit-gravity velocity sedimentation, evaluating any DNA damage using the Comet assay and apoptotic cells in the TUNEL assay. Immunocytochemistry assays showed that the purity of isolated mouse spermatogonial cells was 90%, and the viability of these isolated cells was over 96%. Intracellular superoxide anion production (O2−) in SSCs was detected using p-Nitro Blue Tetrazolium (NBT) assay. The viability of cells after DES treatment was examined in the CCK8 (cell counting kit-8) cytotoxicity assay. The results showed that DES-induced DNA damage causes an increase in intracellular superoxide anions which are reduced by the flavonoid, quercetin. Investigating the molecular mechanisms and biology of SSCs provides a better understanding of spermatogonial stem cell regulation in the testis

Silver nanoparticle-mediated cellular responses in isolated primary Sertoli cells in vitro

YesThe present study explored the mechanism of cytotoxic and genotoxic effects of AgNPs on a primary culture of mouse Sertoli cells in vitro. To understand the possible molecular mechanisms of testicular lesions following exposure to AgNPs, isolated Sertoli cells were exposed to 5, 10, or 15 μg/ml. DNA damage in the Comet assay and apoptosis in the TUNEL assay were evaluated. The mRNA expression of p53 and bcl-2 genes and their proteins involved in apoptosis was also investigated. The antioxidant status of treated Sertoli cells was determined by measuring superoxide dismutase (SOD-1), catalase (CAT), and glutathione peroxidase (GPX-1) and superoxide dismutase (SOD-1) using quantitative polymerase chain reaction (qPCR)qPCR. The superoxide anions were detected using the nitroblue tetrazolium (NBT) reduction assay. Results indicated that AgNP exposure causes increased oxidative stress levels. The activation of p53, repression of bcl-2 and reduction of endogenous antioxidant enzymes were also involved in these mechanistic pathways, leading to reduced cell numbers and cell detachment.The Sponsorship of the Libyan Government of a PhD studentship to Khaled Haba

Germ Cell Responses to Doxorubicin Exposure in Vitro

YesAnthracyclines such as doxorubicin (Dox), widely used to treat various types of tumours, may result in induced testicular toxicity and oxidative stress. The present investigation was designed to determine whether exposure of isolated and purified mouse germ cells to Dox induces DNA damage in the form of strand breaks (presumably) resulting in apoptosis and to investigate the relative sensitivity of specific cell types. DNA damage was assessed using the Comet assay and the presence of apoptosis was determined by TUNEL assay. Isolated mouse germ cells were treated with different concentrations (0.05, 0.5 and 1 mM, respectively) of Dox, and fixed 1 h after treatment. The incidences of both DNA damage shown by single cell gel-electrophoresis and of apoptosis increased significantly in each specific cell type in a concentration-dependent manner. The DNA damage and apoptosis incidences gradually increased with concentration from 0.05 to 1 mM with Dox. Our results indicate that apoptosis plays a vital role in the induction of germ cell phase-specific toxicity caused by Dox with pre-meiotically and meiotically dividing spermatogonia and spermatocytes respectively as highly susceptible target cells.Higher Education Funding Council for England (HEFCE

Alterations in intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in human endothelial cells

YesAlterations of Endothelial cells (ECs) play a critical role in different pathogenesis of many serious human diseases, and dysfunction of the vascular endothelium is an indicator for human disorders. Endothelial dysfunction is considered to be an early indicator for atherosclerosis, which is characterised by overexpression of adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Hydrogen peroxide (H2O2) released via neutrophils is an important mediator of endothelial cell function. Ambient production of superoxide anion (O2−) and subsequently H2O2 at low levels is critical for regulating endothelial cell functions and proliferation. In this study, we investigated the effects of H2O2 on the expression of adhesion molecules VCAM-1 and ICAM-1 in cultured human umbilical vein endothelial cells (HUVECs). Intracellular superoxide anion production was detected by using p-Nitro Blue Tetrazolium (NBT) assay. Our results showed that administration of 100μM of H2O2 on HUVECs for 2, 6, 12 and 24 h induced a time-dependent increase in ICAM-1 and VCAM-1 mRNA and protein expression levels with a significant increase observed from 6 h. HUVECs exposed to H2O2 exhibit increased O2−, suggesting that H2O2 induced oxidative stress may be a reasonable for atherosclerosis. This increase can be reduced by the flavonoid, N-acetyl cysteine (NAC). The modulation of endothelial cell function through this mechanism may underlie the contribution of H2O2 to the development of vascular disease

DNA damage protection by bulk and nano forms of quercetin in lymphocytes of patients with chronic obstructive pulmonary disease exposed to the food mutagen 2-amino-3-methylimidazo [4,5-f]quinolone (IQ)

YesChronic obstructive pulmonary disease (COPD) in humans, describes a group of lung conditions characterised by airflow limitation that is poorly reversible. The airflow limitation usually progresses slowly and is related to an abnormal inflammatory response of the lung to toxic particles. COPD is characterised by oxidative stress and an increased risk of lung carcinoma. The 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) is one of a number of mutagenic/carcinogenic heterocyclic amines found mainly in well-cooked meats which are thus part of the regular diet. Antioxidants are very important in order to protect the cells against oxidative damage. The aim of the present study was to assess the effects of IQ on the level of DNA damage and susceptibility to a potent mutagen in peripheral blood cells of COPD patients. DNA damage and the frequency of micronuclei (MNi) were evaluated using the Comet and micronucleus assays, respectively. Differential expressions of both mRNA and protein of the endogenous antioxidant enzyme catalase were evaluated with quantitative polymerase chain reaction (qPCR) and Western blot analysis, respectively. Furthermore, the effect of bulk and nano forms of quercetin and their combination with IQ were examined. Results of the present study clearly demonstrated that MNi frequency in the peripheral blood lymphocytes exhibited a positive correlation with the DNA damage as evident from the different Comet assay parameters. Increase of the endogenous antioxidant catalase also showed there was a stimulation of this enzyme system by IQ. Whereas, the endogenous antioxidant quercetin significantly reduced oxidative stress in COPD patients and healthy individuals.Libyan Governmen

Tackling the Covid-19 pandemic

YesSince December 2019, a new type of coronavirus called novel coronavirus (2019-nCoV, or COVID-19) was identified in Wuhan, China and on March 11, 2020, the World Health Organization (WHO) has declared the novel coronavirus (COVID-19) outbreak a global pandemic. With more than 101,797,158 confirmed cases, resulting in 3,451,354 deaths as of May 21, 2021, the world faces an unprecedented economic, social, and health impact. The clinical spectrum of COVID-19 has a wide range of manifestations, ranging from an asymptomatic state or mild respiratory symptoms to severe viral pneumonia and acute respiratory distress syndrome. Several diagnostic methods are currently available for detecting the coronavirus in clinical, research, and public health laboratories. Some tests detect the infection directly by detecting the viral RNA using real time reverse transcriptase polymerase chain reaction (RT-PCR) and other tests detect the infection indirectly by detecting the host antibodies. Additional techniques are using medical imaging diagnostic tools such as X-ray and computed tomography (CT). Various approaches have been employed in the development of COVID-19 therapies. Some of these approaches use drug repurposing (eg Remdesivir and Dexamethasone) and combinational therapy (eg Lopinavir/Ritonavir), whilst others aim to develop anti-viral vaccines (eg mRNA and antibody). Additionally, health experts integrate data sharing, provide with guidelines and advice to minimize the effects of the pandemic. These guidelines include wearing masks, avoiding direct contact with infectious people, respiratory and personal hygiene

Evaluation of the toxicity of two electron-deficient half-sandwich complexes against human lymphocytes from healthy individuals

YesElectron‐deficient half‐sandwich complexes are a class of under‐studied organometallics with demonstrated potential as metallodrug candidates. The present study investigates the effect of two 16‐electron organoruthenium complexes ([( p‐ cym)Ru(benzene‐1,2‐dithiolato)] ( 1 ) and [( p ‐cym)Ru(maleonitriledithiolate)] ( 2 )) on the cell viability of non‐immortalised human lymphocytes from healthy individuals. The genotoxic effects of 1 and 2 in lymphocytes using the Comet and cytokinesis‐block micronucleus assays is also investigated. Gene expression studies were carried out on a panel of genes involved in apoptosis and DNA damage repair response. Results show that the two 16‐electron complexes do not have significant effect on the cell viability of human lymphocytes from healthy individuals. However, an increase in DNA damage is induced by both compounds, presumably through oxidative stress production.This project was supported by the Royal Society (University Research Fellowship No. UF150295 to NPEB), the University of Bradford (RDF Award), and by the Academy of Medical Sciences/the Wellcome Trust/ the Government Department of Business, Energy and Industrial Strategy/ the British Heart Foundation Springboard Award [SBF003\1170 to NPEB]