Mechanism of the Formation of Electronically Excited Species by Oxidative Metabolic Processes: Role of Reactive Oxygen Species

It is well known that biological systems, such as microorganisms, plants, and animals, including human beings, form spontaneous electronically excited species through oxidative metabolic processes. Though the mechanism responsible for the formation of electronically excited species is still not clearly understood, several lines of evidence suggest that reactive oxygen species (ROS) are involved in the formation of electronically excited species. This review attempts to describe the role of ROS in the formation of electronically excited species during oxidative metabolic processes. Briefly, the oxidation of biomolecules, such as lipids, proteins, and nucleic acids by ROS initiates a cascade of reactions that leads to the formation of triplet excited carbonyls formed by the decomposition of cyclic (1,2-dioxetane) and linear (tetroxide) high-energy intermediates. When chromophores are in proximity to triplet excited carbonyls, the triplet-singlet and triplet-triplet energy transfers from triplet excited carbonyls to chromophores result in the formation of singlet and triplet excited chromophores, respectively. Alternatively, when molecular oxygen is present, the triplet-singlet energy transfer from triplet excited carbonyls to molecular oxygen initiates the formation of singlet oxygen. Understanding the mechanism of the formation of electronically excited species allows us to use electronically excited species as a marker for oxidative metabolic processes in cells

Effect of the General Anaesthetic Ketamine on Electrical and Ca²⁺ Signal Propagation in <i>Arabidopsis thaliana</i>

The systemic electrical signal propagation in plants (i.e., from leaf to leaf) is dependent on GLUTAMATE RECEPTOR-LIKE proteins (GLRs). The GLR receptors are the homologous proteins to the animal ionotropic glutamate receptors (iGluRs) which are ligand-gated non-selective cation channels that mediate neurotransmission in the animal’s nervous system. In this study, we investigated the effect of the general anaesthetic ketamine, a well-known non-competitive channel blocker of human iGluRs, on systemic electrical signal propagation in Arabidopsis thaliana. We monitored the electrical signal propagation, intracellular calcium level [Ca2+]cyt and expression of jasmonate (JA)-responsive genes in response to heat wounding. Although ketamine affected the shape and the parameters of the electrical signals (amplitude and half-time, t1/2) mainly in systemic leaves, it was not able to block a systemic response. Increased [Ca2+]cyt and the expression of jasmonate-responsive genes were detected in local as well as in systemic leaves in response to heat wounding in ketamine-treated plants. This is in contrast with the effect of the volatile general anaesthetic diethyl ether which completely blocked the systemic response. This low potency of ketamine in plants is probably caused by the fact that the critical amino acid residues needed for ketamine binding in human iGluRs are not conserved in plants’ GLRs

Inactivity and fatty liver disease

Rác Marek, Skladaný Ľubomír, Szulc Adam, Mesárošová Zuzana, Szántová Mária. Inactivity and fatty liver disease. Journal of Education Health and Sport. 2016;6(13):200-210. eISSN 2391-8306. DOI http://dx.doi.org/10.5281/zenodo.248873 http://ojs.ukw.edu.pl/index.php/johs/article/view/4173 The journal has had 7 points in Ministry of Science and Higher Education parametric evaluation. Part B item 754 (09.12.2016). 754 Journal of Education, Health and Sport eISSN 2391-8306 7 © The Author (s) 2016; This article is published with open access at Licensee Open Journal Systems of Kazimierz Wielki University in Bydgoszcz, Poland Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. This is an open access article licensed under the terms of the Creative Commons Attribution Non Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. This is an open access article licensed under the terms of the Creative Commons Attribution Non Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. The authors declare that there is no conflict of interests regarding the publication of this paper. Received: 05.12.2016. Revised 20.12.2016. Accepted: 31.12.2016. Inactivity and fatty liver disease Marek Rác1,5, Ľubomír Skladaný3, Adam Szulc4, Zuzana Mesárošová3, Mária Szántová2 1 Department of Internal Medicine, Teaching Hospital, Nitra, Slovakia 2 3rd Department of Internal Medicine, University Hospital Bratislava, Faculty of Medicine, Comenius University Bratislava, Slovakia 3 2nd Department of Internal Medicine, Hepatology, Gastroenterology and Liver transplantation unit - HEGITO, F.D. Roosevelt University Hospital, Banská Bystrica, Slovakia 4 Institute of Physical Education, Kazimierz Wielki University, Bydgoszcz, Poland 5 St. Elizabeth University of Health and Social Work, Bratislava, Slovakia Corresponding author: Ľubomír Skladaný, [email protected] Abstract Physical activity represents a key element in the prevention and management of many chronic diseases. On other hand physical inactivity is a primary cause of obesity, metabolic syndrome and nonalcoholic liver disease. A higher body weight is associated with an increased incidence of a number of conditions, including diabetes mellitus, cardiovascular disease and nonalcoholic fatty liver disease. Obesity is associated with a increased risk of all-cause mortality. Hepatic consequence of sedentary lifestyle is nonalcoholic fatty liver disease (NAFLD), which is now in Western countries the most common cause of chronic liver disease. NAFLD primary affects hepatic structure and function. NAFLD cause morbidity and mortality from liver cirrhosis, liver failure and hepatocellular carcinoma. The majority of deaths among NAFLD patients are attributable to cardiovascular disease (CVD) and cancer. NAFLD is strongly associated with the clinical features of insulin resistance and is the hepatic component of metabolic syndrome

Oxidative damage of U937 human leukemic cells caused by hydroxyl radical results in singlet oxygen formation.

The exposure of human cells to oxidative stress leads to the oxidation of biomolecules such as lipids, proteins and nuclei acids. In this study, the oxidation of lipids, proteins and DNA was studied after the addition of hydrogen peroxide and Fenton reagent to cell suspension containing human leukemic monocyte lymphoma cell line U937. EPR spin-trapping data showed that the addition of hydrogen peroxide to the cell suspension formed hydroxyl radical via Fenton reaction mediated by endogenous metals. The malondialdehyde HPLC analysis showed no lipid peroxidation after the addition of hydrogen peroxide, whereas the Fenton reagent caused significant lipid peroxidation. The formation of protein carbonyls monitored by dot blot immunoassay and the DNA fragmentation measured by comet assay occurred after the addition of both hydrogen peroxide and Fenton reagent. Oxidative damage of biomolecules leads to the formation of singlet oxygen as conformed by EPR spin-trapping spectroscopy and the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. It is proposed here that singlet oxygen is formed by the decomposition of high-energy intermediates such as dioxetane or tetroxide formed by oxidative damage of biomolecules

Luminescence imaging of leaf damage induced by lipid peroxidation products and its modulation by β‐cyclocitral

International audienceLipid peroxidation is a primary event associated with oxidative stress in plants. This phenomenon secondarily generates bioactive and/or toxic compounds such as reactive carbonyl species (RCS), phytoprostanes, and phytofurans, as confirmed here in Arabidopsis plants exposed to photo‐oxidative stress conditions. We analyzed the effects of exogenous applications of secondary lipid oxidation products on Arabidopsis plants by luminescence techniques. Oxidative damage to attached leaves was measured by autoluminescence imaging, using a highly sensitive CCD camera, and the activity of the detoxification pathway, dependent on the transcription regulator SCARECROW‐LIKE 14 (SCL14), was monitored with a bioluminescent line expressing the firefly LUCIFERASE (LUC) gene under the control of the ALKENAL REDUCTASE (AER) gene promoter. We identified 4‐hydroxynonenal (HNE), and to a lesser extent 4‐hydroxyhexenal (HHE), as highly reactive compounds that are harmful to leaves and can trigger AER gene expression, contrary to other RCS (pentenal, hexenal) and to isoprostanoids. Although the levels of HNE and other RCS were enhanced in the SCL14‐deficient mutant (scl14), exogenously applied HNE was similarly damaging to this mutant, its wild‐type parent and a SCL14‐overexpressing transgenic line (OE:SCL14). However, strongly boosting the SCL14 detoxification pathway and AER expression by a pre‐treatment of OE:SCL14 with the signaling apocarotenoid β‐cyclocitral canceled the damaging effects of HNE. Conversely, in the scl14 mutant, the effects of β‐cyclocitral and HNE were additive, leading to enhanced leaf damage. These results indicate that the cellular detoxification pathway induced by the low‐toxicity β‐cyclocitral targets highly toxic compounds produced during lipid peroxidation, reminiscent of a safener‐type mode of action

Analysis of the medians of comet, head, and tail parameters in the control, the H₂O₂-treated and the Fenton reagent-treated U937 cells.

<p>Data are presented as mean values and standard deviations. The mean value represents the average value from at least three measurements.</p><p>Analysis of the medians of comet, head, and tail parameters in the control, the H₂O₂-treated and the Fenton reagent-treated U937 cells.</p

Detection of lipid peroxidation product malondialdehyde by HPLC analysis.

<p>The chromatogram of DNPH-MDA complex in U937 cells (A) and DNPH-MDA standard (B). In A, chromatogram of DNPH-MDA complex was measured in the control (trace a), the H₂O₂-treated (trace b) and the Fenton reagent-treated (trace c) U937 cells. The U937 cells were treated with 5 mM H₂O₂ (b) and Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) (c) for 30 min. After the treatment, lipids were separated from proteins and DNPH was added to lipids. In B, the chromatogram of DNPH-MDA standard shows the retention time of 3 min 50 s. The insert shows the dependence of average peak area on the concentration of DNPH-MDA standard. Based on the calibration curve, the concentrations of DNPH-MDA complex determined from calibration curve were as following: 0.029±0.003 nmol ml⁻¹ (control), 0.030±0.003 (H₂O₂) and 0.09±0.02 nmol ml⁻¹ (Fenton reagent). The coefficient of determination R² was determined as 0.9997. Data are presented as mean values and standard deviations. The mean value represents the average value from at least three measurements.</p

Analysis of DNA strand breaks by Comet assay.

<p>Comet assay of the control (A), the H₂O₂-treated (B) and the Fenton reagent-treated (C) U937 cells. The U937 cells were treated with 5 mM H₂O₂ (B) and Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) (C) for 30 min. After the treatment, U937 cells were stained by SYBR Green.</p

Detection of singlet oxygen by EPR spin-trapping spectroscopy.

<p>TEMPONE EPR spectra were measured in the control (A and B), the H₂O₂-treated (C and D) and the Fenton reagent-treated (E and F) U937 cells in the presence of 100 mM TEMPD. U937 cells were treated with no addition (A), 5 mM H₂O₂ (C) and Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) (E) for a period indicated in Fig. In C and E, top traces show the simulation of TEMPONE EPR signal using hyperfine coupling constants <i>a</i>^N = 16 G. Chemical source of ¹O₂ (10 mM molybdic acid + 10 mM H₂O₂ measured right after the preparation) was used as a positive control (C,E). Bar graphs represent the hight of the middle peak of TEMPONE EPR signal in the control (B), the H₂O₂-treated (D) and the Fenton reagent-treated (F) U937 cells. Experimental EPR conditions were as follows: microwave power, 10 mW; modulation amplitude, 1 G; modulation frequency, 100 kHz; sweep width, 100 G; scan rate, 1.62 G s-1, gain 500. Bars represent 4000 (A) and 8000 (C and E) relative units. Data are presented as mean values and standard deviations. The mean value represent the average value from at least three measurements.</p

Determination of the U937 cell viability.

<p>The cell viability was determined 30 min after the addition of 5 mM H₂O₂ or Fenton reagent to the U937 cells. The results are normalized to control U937 cells. The data are presented as the mean and standard deviation of at least 3 measurements.</p