Antigenotoxic potential of the fermentation broth produced by Paenibacillus polymyxa RNC-D in vitro

2021 ◽  
Author(s):  
Jaqueline Bianchi ◽  
Rafael Cavicchioli ◽  
Lauro T Kubota ◽  
Emanuel Carrilho ◽  
Cristina P de Sousa ◽  
...  
Keyword(s):  
Comet Assay ◽  
Micronucleus Test ◽  
Fermentation Broth ◽  
Paenibacillus Polymyxa ◽  
Dna Lesions ◽  
Methyl Methanesulfonate ◽  
Oxygen Species ◽  
Tnf Α ◽  
Antigenotoxic Effect

Aim: Evaluate the chemopreventive potential of the extract from P. polymyxa RNC-D. Methods: Concentrations of P. polymyxa RNC-D extract were tested in HepG2/C3A cells to assess their genotoxic (comet assay), mutagenic (micronucleus test) and antigenotoxic potential (comet assay) in vitro. Results: 400 and 40 μg/ml concentrations induced DNA lesions, whereas the 4 μg/ml induced a desmutagenic effect. Complementary tests indicated that the extract minimized the formation of reactive oxygen species induced by methyl methanesulfonate and normalized the loss of membrane potential. The quantification of cytokines indicated that TNF-α was immunostimulated by the extract. However, when administered in conjunction with the methyl methanesulfonate, the extract blocked the TNF-α release. Conclusion: The fermentation broth from P. polymyxa RNC-D showed an antigenotoxic effect, and thus the potential to be used as chemopreventive compound.

scholarly journals Validation of the in vitro comet assay for DNA cross-links and altered bases detection

2021 ◽  
Author(s):  
Damián Muruzabal ◽  
Julen Sanz-Serrano ◽  
Sylvie Sauvaigo ◽  
Bertrand Treillard ◽  
Ann-Karin Olsen ◽  
...  
Keyword(s):  
Comet Assay ◽  
Human Health Risk ◽  
High Sensitivity ◽  
Dna Lesions ◽  
Dna Glycosylase ◽  
Vitro Assay ◽  
Cytotoxic Compound ◽  
Endonuclease Iii ◽  
Cross Links

AbstractMechanistic toxicology is gaining weight for human health risk assessment. Different mechanistic assays are available, such as the comet assay, which detects DNA damage at the level of individual cells. However, the conventional alkaline version only detects strand breaks and alkali-labile sites. We have validated two modifications of the in vitro assay to generate mechanistic information: (1) use of DNA-repair enzymes (i.e., formamidopyrimidine DNA glycosylase, endonuclease III, human 8-oxoguanine DNA glycosylase I and human alkyladenine DNA glycosylase) for detection of oxidized and alkylated bases as well as (2) a modification for detecting cross-links. Seven genotoxicants with different mechanisms of action (potassium bromate, methyl methanesulfonate, ethyl methanesulfonate, hydrogen peroxide, cisplatin, mitomycin C, and benzo[a]pyrene diol epoxide), as well as a non-genotoxic compound (dimethyl sulfoxide) and a cytotoxic compound (Triton X-100) were tested on TK-6 cells. We were able to detect with high sensitivity and clearly differentiate oxidizing, alkylating and cross-linking agents. These modifications of the comet assay significantly increase its sensitivity and its specificity towards DNA lesions, providing mechanistic information regarding the type of damage.

DNA damage induced by coal dust, fly and bottom ash from coal combustion evaluated using the micronucleus test and comet assay in vitro

2017 ◽  
Vol 324 ◽  
pp. 781-788 ◽  
Author(s):  
Cristina Araujo Matzenbacher ◽  
Ana Letícia Hilario Garcia ◽  
Marcela Silva dos Santos ◽  
Caroline Cardoso Nicolau ◽  
Suziane Premoli ◽  
...  
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Coal Combustion ◽  
Micronucleus Test ◽  
Coal Dust ◽  
Bottom Ash

scholarly journals Salvianolic Acid A Protects Against Oxidative Stress and Apoptosis Induced by Intestinal Ischemia-Reperfusion Injury Through Activation of Nrf2/HO-1 Pathways

2018 ◽  
Vol 49 (6) ◽  
pp. 2320-2332 ◽  
Author(s):  
Guo Zu ◽  
Tingting Zhou ◽  
Ningwei Che ◽  
Xiangwen Zhang
Keyword(s):  
Reactive Oxygen Species ◽  
Glutathione Peroxidase ◽  
Ischemia Reperfusion ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Salvianolic Acid ◽  
Salvianolic Acid A ◽  
Tnf Α

Background/Aims: Ischemia-reperfusion (I/R) adversely affects the intestinal mucosa. The major mechanisms of I/R are the generation of reactive oxygen species (ROS) and apoptosis. Salvianolic acid A (SalA) is suggested to be an effective antioxidative and antiapoptotic agent in numerous pathological injuries. The present study investigated the protective role of SalA in I/R of the intestine. Methods: Adult male Sprague-Dawley rats were subjected to intestinal I/R injury in vivo. In vitro experiments were performed in IEC-6 cells subjected to hypoxia/ reoxygenation (H/R) stimulation to simulate intestinal I/R. TNF-α, IL-1β, and IL-6 levels were measured using enzyme-linked immunosorbent assay. Malondialdehyde and myeloperoxidase and glutathione peroxidase levels were measured using biochemical analysis. Apoptosis was measured by terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling staining or flow cytometry in vivo and in vitro. The level of reactive oxygen species (ROS) was measured by dichlorodihydrofluorescin diacetate (DCFH-DA) staining. Western blotting was performed to determine the expression of heme oxygenase-1 (HO-1), Nrf2 and proteins associated with apoptosis. The mRNA expressions of Nrf2 and HO-1 were detected by quantitative real-time polymerase chain reaction in vivo and in vitro. Results: Malondialdehyde level and myeloperoxidase and glutathione peroxidase, TNF-α, IL-1β, and IL-6 levels group in intestinal tissue decreased significantly in the SalA pretreatment groups compared to the I/R group. SalA markedly abolished intestinal injury compared to the I/R group. SalA significantly attenuated apoptosis and increased Nrf2/HO-1 expression in vivo and in vitro. However, Nrf2 siRNA treatment partially abrogated the above mentioned effects of SalA in H/R-induced ROS and apoptosis in IEC-6 cells. Conclusion: The present study demonstrated that SalA ameliorated oxidation, inhibited the release of pro-inflammatory cytokines and alleviated apoptosis in I/R-induced injury and that these protective effects may partially occur via regulation of the Nrf2/ HO-1 pathways.

scholarly journals Neutrophil azurophilic granule exocytosis is primed by TNF-α and partially regulated by NADPH oxidase

2016 ◽  
Vol 22 (8) ◽  
pp. 635-646 ◽  
Author(s):  
Renee M Potera ◽  
Melissa J Jensen ◽  
Brieanna M Hilkin ◽  
Gina K South ◽  
Jessica S Hook ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Tissue Damage ◽  
Chronic Inflammatory Disease ◽  
Host Tissue ◽  
Ros Generation ◽  
Oxygen Species ◽  
Granule Exocytosis ◽  
Azurophilic Granule ◽  
Tnf Α

Neutrophil (polymorphonuclear leukocyte) activation with release of granule contents plays an important role in the pathogenesis of acute lung injury, prompting clinical trials of inhibitors of neutrophil elastase. Despite mounting evidence for neutrophil-mediated host tissue damage in a variety of disease processes, mechanisms regulating azurophilic granule exocytosis at the plasma membrane, and thus release of elastase and other proteases, are poorly characterized. We hypothesized that azurophilic granule exocytosis would be enhanced under priming conditions similar to those seen during acute inflammatory events and during chronic inflammatory disease, and selected the cytokine TNF-α to model this in vitro. Neutrophils stimulated with TNF-α alone elicited intracellular reactive oxygen species (ROS) generation and mobilization of secretory vesicles, specific, and gelatinase granules. p38 and ERK1/2 MAPK were involved in these components of priming. TNF-α priming alone did not mobilize azurophilic granules to the cell surface, but did markedly increase elastase release into the extracellular space in response to secondary stimulation with N-formyl-Met-Leu-Phe (fMLF). Priming of fMLF-stimulated elastase release was further augmented in the absence of NADPH oxidase-derived ROS. Our findings provide a mechanism for host tissue damage during neutrophil-mediated inflammation and suggest a novel anti-inflammatory role for the NADPH oxidase.

Comparison of in vitro and in situ genotoxicity in the Danube River by means of the comet assay and the micronucleus test

2010 ◽  
Vol 700 (1-2) ◽  
pp. 11-17 ◽  
Author(s):  
Melanie Boettcher ◽  
Stefanie Grund ◽  
Steffen Keiter ◽  
Thomas Kosmehl ◽  
Georg Reifferscheid ◽  
...  
Keyword(s):  
Comet Assay ◽  
Micronucleus Test ◽  
Danube River ◽  
The Danube River

Nicotine derived genotoxic effects in human primary parotid gland cells as assessed in vitro by comet assay, cytokinesis-block micronucleus test and chromosome aberrations test

2014 ◽  
Vol 28 (5) ◽  
pp. 838-846 ◽  
Author(s):  
Christian Ginzkey ◽  
Gudrun Steussloff ◽  
Christian Koehler ◽  
Marc Burghartz ◽  
Agmal Scherzed ◽  
...  
Keyword(s):  
Comet Assay ◽  
Parotid Gland ◽  
Chromosome Aberrations ◽  
Micronucleus Test ◽  
Genotoxic Effects ◽  
Gland Cells

scholarly journals Pharmacological Disruption of Phosphorylated Eukaryotic Initiation Factor-2α/Activating Transcription Factor 4/Indian Hedgehog Protects Intervertebral Disc Degeneration via Reducing the Reactive Oxygen Species and Apoptosis of Nucleus Pulposus Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Junping Bao ◽  
Zhanyang Qian ◽  
Lei Liu ◽  
Xin Hong ◽  
Hui Che ◽  
...  
Keyword(s):  
Cell Apoptosis ◽  
Intervertebral Disc Degeneration ◽  
Initiation Factor ◽  
Oxygen Species ◽  
Cell Degeneration ◽  
Activating Transcription Factor 4 ◽  
Tnf Α ◽  
Activating Transcription Factor ◽  
Transcription Factor 4

Excessive reactive oxygen species (ROS) and apoptosis in nucleus pulposus (NP) cells accelerate the process of intervertebral disc degeneration (IDD). Here, we integrated pathological samples and in vitro and in vivo framework to investigate the impact of phosphorylation of eukaryotic initiation factor-2α (eIF2α)/activating transcription factor 4 (ATF4)/Indian hedgehog (Ihh) signaling in the IDD. From the specimen analysis of the IDD patients, we found phosphorylated eIF2α (p-eIF2α), ATF4 and Ihh protein levels were positively related while the NP tissue went degenerative. In vitro, tumor necrosis factor (TNF)-α caused the NP cell degeneration and induced a cascade of upregulation of p-eIF2α, ATF4, and Ihh. Interestingly, ATF4 could enhance Ihh expression through binding its promoter region, and silencing of ATF4 decreased Ihh and protected the NP cells from degeneration. Moreover, ISRIB inhibited the p-eIF2α, which resulted in a suppression of ATF4/Ihh, and alleviated the TNF-α-induced ROS production and apoptosis of NP cells. On the contrary, further activating p-eIF2α aggravated the NP cell degeneration, with amplification of ATF4/Ihh and a higher level of ROS and apoptosis. Additionally, applying cyclopamine (CPE) to suppress Ihh was efficient to prevent NP cell apoptosis but did not decrease the ROS level. In an instability-induced IDD model in mice, ISRIB suppressed p-eIF2α/ATF4/Ihh and prevented IDD via protecting the anti-oxidative enzymes and decreased the NP cell apoptosis. CPE prevented NP cell apoptosis but did not affect anti-oxidative enzyme expression. Taken together, p-eIF2α/ATF4/Ihh signaling involves the ROS level and apoptosis in NP cells, the pharmacological disruption of which may provide promising methods in preventing IDD.

scholarly journals Evaluation of genotoxicity and cytotoxicity of water samples from the Sinos River Basin, southern Brazil

2015 ◽  
Vol 75 (2 suppl) ◽  
pp. 68-74 ◽  
Author(s):  
E Bianchi ◽  
A Goldoni ◽  
L Trintinaglia ◽  
G Lessing ◽  
CEM Silva ◽  
...  
Keyword(s):  
Comet Assay ◽  
River Basin ◽  
Water Samples ◽  
Micronucleus Test ◽  
Vero Cells ◽  
Neutral Red ◽  
Genetic Damage ◽  
Middle Section ◽  
Genotoxic Compounds

<p>Some water bodies in the Sinos River Basin (SRB) have been suffering the effects of pollution by residential, industrial and agroindustrial wastewater. The presence of cytotoxic and genotoxic compounds could compromise the water quality and the balance of these ecosystems. In this context, the research aimed to evaluate the genotoxicity and cytotoxicity of the water at four sites along the SRB (in the cities of Santo Antônio da Patrulha, Parobé, Campo Bom and Esteio), using bioassays in fish and cell culture. Samples of surface water were collected and evaluated <italic>in vitro</italic> using the <italic>Astyanax jacuhiensis</italic> fish species (micronucleus test and comet assay) and the Vero lineage of cells (comet assay and cytotoxicity tests, neutral red - NR and tetrazolium MTT). The micronucleus test in fish showed no significant differences between the sampling sites, and neither did the comet assay and the MTT and NR tests in Vero cells. The comet assay showed an increase in genetic damage in the fish exposed to water samples collected in the middle and lower sections of the basin (Parobé, Campo Bom and Esteio) when compared to the upper section of the basin (Santo Antônio da Patrulha). The results indicate contamination by genotoxic substances starting in the middle section of the SRB.</p>

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