Antigenotoxic and antioxidant potential of medicinal mushrooms (Immune Assist) against DNA damage induced by free radicals-an in vitro study.

Immune Assist (IA) is produced from extract of six species of medical mushrooms: Agaricus blazei - Cordyceps sinensis - Grifola frondosa - Ganoderma lucidum - Coriolus versicolor - Lentinula edodes. The genoprotective potential of IA was evaluated for the first time. Significant antigenotoxic effects were detected in human peripheral blood cells against H2O2 induced DNA damage, in the pretreatment and in the posttreatment. The most efficient concentration of IA in pretreatment was 500 µg/mL, while in posttreatment it was the concentration of 250 µg/mL. Kinetics of attenuation of H2O2 induced DNA damage in posttreatment with the optimal concentration of IA showed significant decrease in the number of damaged cells at all time periods (15-60 min), reaching the greatest reduction after 15 and 45 min. Remarkable ·OH scavenging properties and moderate reducing power, together with the modest DPPH scavenging activity, could be responsible for the great attenuation of DNA damage after 15 min of exposure to IA, while reduction of DNA damage after 45 min could be the result in additional stimulation of the cell's repair machinery. Our results suggest that IA displayed antigenotoxic and antioxidant properties. A broader investigation of its profile in biological systems is needed.

The fabrication and the use of immobilized cells as test organisms in a ferricyanide-based toxicity biosensor.

Cell immobilization is an effective method to prolong the lifetime of a microorganism and has proven feasibility in some other biosensors. Thus, we studied the use of Escherichia coli immobilized by agar, gelatin, an agar/gelatin mixture, chitosan, and polyvinyl alcohol (PVA) to screen toxicity electrochemically. The E. coli immobilized by PVA gel showed the highest apparent bioactivity and the longest storage time in pH 7.0 phosphate-buffered saline solution. Furthermore, the E. coli immobilized by different gels was applied in the toxicity determination via a reported ferricyanide-mediated electrochemical method, where 3,5-dichlorophenol (DCP) was used as a model toxin. The E. coli immobilized by PVA showed the highest sensitivity to DCP, and the corresponding value of 50% inhibition concentration was 9.62 mg L-1 . Inhibition concentrations were in the range of 6.32 to 13.75% when the E. coli immobilized by PVA was challenged by wastewater, which were comparable with values obtained with the standard luminescent bacteria method (effective inhibition were in the range 7.96-25.42% for the same samples). Given the apparent bioactivity, storage ability, and sensitivity to toxin, PVA was the best polymer to confine cells among the polymers used in the present study. Environ Toxicol Chem 2018;37:329-335. © 2017 SETAC.

Differential oxidative stress thresholds distinguishes cellular response to vascular occlusion and chemotoxicity in vivo.

BACKGROUND: A major effect of cyanide toxicity (CN) and vascular occlusion (VO) is the production of reactive oxygen species (ROS) linked with a defective energy coupling process in the mitochondria. In CN, oxygen is present but its conversion is blocked in the mitochondria (Complex V). By contrast, in VO, oxidative stress is induced via reduction of oxygen (blood) circulation to the occluded brain region. We hypothesize that differential oxygen concentration in both forms of ischemia affect ROS production rate in the mitochondria; thus distinguishing the cytotoxicity pattern for CN and VO. METHOD: Male, adult Wistar rats (N = 30) were separated into three groups. A set of n = 12 animals were treated with orally administered potassium ferricyanide. Global vascular occlusion (GVO) was induced in a second set of animals (n = 12) using neck cuffs to occlude the common carotid arteries and brachiocephalic vein. The control group (n = 6) received normal saline for the total duration of the treatment (10 days). Glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA) and acid phosphatase (ACP) levels were assayed in tissue homogenate using colorimetric assay techniques while Cathepsin D (CAD+) was demonstrated through antigen retrieval immunohistochemistry. Data were analyzed in One Way ANOVA with Tukey's post-hoc test. Significance was set p < 0.05. RESULTS/DISCUSSION: Cyanide treatment and VO caused a significant rise in SOD; attributed to ROS formation in both forms of ischemia. However, an increase in GSH levels demonstrates mitochondria-ROS production in cyanide treatment while no significant change in GSH was observed in VO versus the control (reduced mitochondria-ROS production). In addition, CN recorded a significant increase in GSH when compared with the control and VO (p < 0.001). The level of ROS was also proportional to the degree of lipid peroxidation (GPx) and autophagic cell response (ACP/CAD+). Ultimately, the lipid peroxidation/ROS effect was more significant in CN than VO. CONCLUSION: Although CN and VO induced oxidative stress through ROS production, our findings suggest a difference in the threshold of ROS production and cytotoxicity for both forms of ischemia. However, this threshold is dependent on the availability of oxygen to fuel mitochondria-ROS production in oxidative stress. Ultimately, the difference in oxygen availability in vivo determined the significance of lipid peroxidation, calcium-shift and autophagic cell response associated with the ischemia. CN treatment generated more ROS and was associated with prominent cellular changes when compared with VO.

Possible evidence for contribution of arbuscular mycorrhizal fungi (AMF) in phytoremediation of iron-cyanide (Fe-CN) complexes.

Arbuscular mycorrhizal fungi (AMF) are integral functioning parts of plant root systems and are widely recognized for enhancing contaminants uptake and metabolism on severely disturbed sites. However, the patterns of their influence on the phytoremediation of iron-cyanide (Fe-CN) complexes are unknown. Fe-CN complexes are of great common interest, as iron is one of the most abundant element in soil and water. Effect of ryegrass (Lolium perenne L.) roots inoculation, using mycorrhizal fungi (Rhizophagus irregularis and a mixture of R. irregularis, Funneliformis mosseae, Rhizophagus aggregatus, and Claroideoglomus etunicatum), on iron-cyanide sorption was studied. Results indicated significantly higher colonization of R. irregularis than the mixture of AMF species on ryegrass roots. Series of batch experiments using potassium hexacyanoferrate (II) solutions, in varying concentrations revealed significantly higher reduction of total CN and free CN content in the mycorrhizal roots, indicating greater cyanide decrease in the treatment inoculated with R. irregularis. Our study is a first indication of the possible positive contribution of AM fungi on the phytoremediation of iron-cyanide complexes.

Fast and sensitive optical toxicity bioassay based on dual wavelength analysis of bacterial ferricyanide reduction kinetics.

Global urban and industrial growth, with the associated environmental contamination, is promoting the development of rapid and inexpensive general toxicity methods. Current microbial methodologies for general toxicity determination rely on either bioluminescent bacteria and specific medium solution (i.e. Microtox(®)) or low sensitivity and diffusion limited protocols (i.e. amperometric microbial respirometry). In this work, fast and sensitive optical toxicity bioassay based on dual wavelength analysis of bacterial ferricyanide reduction kinetics is presented, using Escherichia coli as a bacterial model. Ferricyanide reduction kinetic analysis (variation of ferricyanide absorption with time), much more sensitive than single absorbance measurements, allowed for direct and fast toxicity determination without pre-incubation steps (assay time=10 min) and minimizing biomass interference. Dual wavelength analysis at 405 (ferricyanide and biomass) and 550 nm (biomass), allowed for ferricyanide monitoring without interference of biomass scattering. On the other hand, refractive index (RI) matching with saccharose reduced bacterial light scattering around 50%, expanding the analytical linear range in the determination of absorbent molecules. With this method, different toxicants such as metals and organic compounds were analyzed with good sensitivities. Half maximal effective concentrations (EC50) obtained after 10 min bioassay, 2.9, 1.0, 0.7 and 18.3 mg L(-1) for copper, zinc, acetic acid and 2-phenylethanol respectively, were in agreement with previously reported values for longer bioassays (around 60 min). This method represents a promising alternative for fast and sensitive water toxicity monitoring, opening the possibility of quick in situ analysis.

Development of a simple method for biotoxicity measurement using ultramicroelectrode array under non-deaerated condition.

In this paper, a mediated method by using ferricyanide under non-deaerated condition for biotoxicity measurement was proposed. Ultramicroelectrode array (UMEA) was employed for effectively amplify the electrochemical signal from the total limiting currents to distinguish a little change in toxicity. Five species of microorganisms including two bacilli (Escherichia coli and Enterobacter cloacae), two pseudomonas (Pseudomonas fluorescens and Pseucomonas putida) and one fungus (Trichosporon cutaneum) were employed. 3,5-dichlorophenol (DCP) was taken as the reference toxicant. The IC50 values we obtained were similar with the values obtained using in the deaerated method. E. coli was used as model test microorganism. The final concentration of ferricyanide is 45 mM, E. coli OD600 8 and 1 h incubation were taken in optimum conditions in this study. Four heavy metal ions (Cr(6+), Hg(2+), Cd(2+) and Bi(3+)) were examined under the optimum conditions. Comparison with the results reported previously has confirmed that this method provided a simple and rapid alternative to toxicity screening of chemicals, especially advantageous for in situ monitoring of water system.

Effect of humic substances on toxicity of inorganic oxidizer bioluminescent monitoring.

The current study deals with the effect of humic substances (HS) on toxicity of solutions of a model inorganic oxidizer, potassium ferricyanide. Chemical reactions responsible for toxicity changes are under consideration. The bioluminescent system of coupled enzymatic reactions catalyzed by bacterial luciferase and oxidoreductase was used as a bioassay. General and oxidative toxicity of ferricyanide solutions were evaluated. Ability of HS to decrease or increase general and oxidative toxicity of the solutions was revealed. Two types of chemical processes are supposed to be responsible for detoxification by HS: ferricyanide-HS complex formation and acceleration of endogenous redox reactions in the bioluminescent assay system. Decrease of oxidative toxicity of ferricyanide solution was observed under incubation with HS at all concentrations of HS used. Conditions for general toxicity decrease were prior incubation of ferricyanide with HS and low HS concentrations (< 10⁻4g/L). Acceleration of NADH auto-oxidation under higher HS concentrations was supposed to result in a toxicity increase.

A sensitive, rapid ferricyanide-mediated toxicity bioassay developed using Escherichia coli.

A need for rapid toxicity techniques has seen recent research into developing new microbiological assays and characterising their toxicity responses using a range of substances. A microbiological bioassay that determines changes in ferricyanide-mediated respiration for toxicity measurement (FM-TOX) shows particular promise. The development and optimisation of an improved FM-TOX method, incorporating novel features, is described using Escherichia coli as the biocatalyst. Omission of an exogenous carbon source, used in previously described FM-TOX assays, substantially improves the assay sensitivity. In addition, the development of a two-step procedure (toxicant exposure followed by determination of microbial respiratory activity) was found to enhance the inhibition of E. coli by 3,5-dichlorophenol and four other toxicants, compared to single-step procedures. Other assay parameters, such as the ferricyanide concentration, exposure times and optical density of the biocatalyst were also optimised, sometimes based on practical aspects. Toxicity tests were carried out using the adopted technique on both organic and inorganic toxicants, with classic sigmoid-shaped dose-response curves observed, as well as some non-standard responses. IC(50) data is presented for a number of common toxicants. The optimised assay provides a good foundation for further toxicity testing using E. coli, as well as the potential for expanding the technique to utilise other bacteria with complementary toxicity responses, thereby allowing use of the assay in a range of applications.

An evaluation of the in vitro cytotoxicities of 50 chemicals by using an electrical current exclusion method versus the neutral red uptake and MTT assays.

According to the 2001 National Institutes of Health guidance document on using in vitro data to estimate in vivo starting doses for acute toxicity, the performance of the electrical current exclusion method (ECE) was studied for its suitability as an in vitro cytotoxicity test. In a comparative study, two established in vitro assays based on the quantification of metabolic processes necessary for cell proliferation or organelle integrity (the MTT/WST-8 [WST-8] assay and the neutral red uptake [NRU] assay), and two cytoplasm membrane integrity assays (the trypan blue exclusion [TB] and ECE methods), were performed. IC50 values were evaluated for 50 chemicals ranging from low to high toxicity, 46 of which are listed in Halles Registry of Cytotoxicity (RC). A high correlation was found between the IC50 values obtained in this study and the IC50 data published in the RC. The assay sensitivity was highest for the ECE method, and decreased from the WST-8 assay to the NRU assay to the TB assay. The consistent results of the ECE method are based on technical standardisation, high counting rate, and the ability to combine cell viability and cell volume analysis for detection of the first signs of cell necrosis and damage of the cytoplasmic membrane caused by cytotoxic agents.

Further investigations regarding the toxicity of sodium nitroprusside.

Sodium nitroprusside is a valuable vasodilator, but its use has been curtailed because of numerous reports that, in the presence of blood, nitroprusside decomposes with release of toxic cyanide. We have examined the release of cyanide in terms of the known chemistry of nitroprusside and suggest that photochemical decomposition of nitroprusside and (or) its metabolism in vivo invalidates the analytical procedure used by previous workers. We also present evidence for the stability of nitroprusside in blood, based on 13C nuclear magnetic resonance studies.

Circulatory effects of haemorrhage during sodium nitroprusside induced hypotension. A study in the rat.

The haemodynamic changes induced by acute moderate blood loss were investigated in rats during normotensive halothane anaesthesia and during sodium-nitroprusside-induced hypotensive anaesthesia, respectively. Following haemorrhage in the normotensive group, mean arterial blood pressure, heart rate and left cardiac work decreased. Cardiac output was reduced non-significantly. Blood flow was redistributed to favour cerebral, coronary, renal and hepatic circulation, mainly at the expense of blood flow to the carcass. Following haemorrhage in the hypotensive group, cardiac output increased significantly. Mean arterial pressure, heart rate and left cardiac work were unchanged. Absolute values for cerebral, coronary, renal and hepatic blood flow were maintained or even increased, while blood flow to the carcass was unchanged.

Systemic and carotid haemodynamics and plasma renin activity during deliberate hypotension in dogs: a comparison of sodium nitroprusside with nicergoline.

This study was designed to compare at the same level of hypotension (-30%) nitroprusside (SNP) and nicergoline (NIC) effects on systemic haemodynamics and carotid haemodynamics (pulsed Doppler) and on plasma renin activity (PRA) in 20 anaesthetized dogs before and at the 20th minute of hypotension. In SNP group (n = 9) cardiac output (CO) and heart rate (HR) increased. Stroke volume (SV), pulmonary wedge pressure (PWP), central venous pressure (CVP) and systemic vascular resistance (SVR) decreased. Common carotid diameter (D) increased and blood flow velocity (V) decreased with constant common carotid blood flow (CCBF). CCBF/CO ratio was unchanged. PRA levels increased. In NIC group (n = II), HR, CO, SV were unchanged and PWP, CVP, SVR decreased. D, V and CCBF were unchanged. CCBF/CO ratio increased and PRA was unchanged. NIC provoked mild hypotension without reflex sympathetic activation unlike SNP. Only SNP dilated large arteries. The autoregulation of CCBF is maintained with both drugs but CCBF/CO ratio is increased only with NIC.

Ferricyanide can replace pyruvate to stimulate growth and attachment of serum restricted human melanoma cells.

Addition of potassium ferricyanide to RPMI 1640 medium can stimulate cell attachment and replication, in a closely correlated fashion, of a human melanoma line when serum is a limiting growth factor. Ferricyanide is more effective than pyruvate on a molar basis but toxic effects at concentrations greater than 0.03mM limit its full potential. Since ferricyanide cannot itself provide nutrients for the cell and is extracellular but may be involved in transmembrane electron flow, it is suggested that its mechanism of action may be to provide energy for cell surface processes concerned with attachment and thus secondarily for replication.

Paradoxical bradycardia following the administration of sodium nitroprusside in the rat is abolished by indomethacin.

Severe bradycardia and hypotension associated with the administration of organic nitrates or nitrites has been observed in patients with ischaemic heart disease and hypertension. Experiments were performed on rats to elucidate the mechanism by which these vasodilators may effect their anomalous bradycardia-hypotensive effect. In sodium pentobarbital (50 mg.kg-1, ip) anaesthetised rats of the Wistar strain, hypotension and a paradoxical bradycardia were observed following the intra-arterial injection of 10 microgram.kg-1 sodium nitroprusside or 1 mg.kg-1 arachidonic acid. Bilateral vagotomy abolished the bradycardia to both sodium nitroprusside and arachidonic acid but did not eliminate their hypotensive effects. Indomethacin (5 mg.kg-1) abolished the blood pressure and heart rate responses to arachidonic acid. However, indomethacin abolished only the bradycardia to sodium nitroprusside without reducing its effect on blood pressure. These data suggest that in the rat sodium nitroprusside can stimulate prostaglandin biosynthesis which then initiates a reflex bradycardia.

The antidote effect of thiosulphate and hydroxocobalamin in formation of nitroprusside intoxication of rabbits.

Sodium nitroprusside (SNP) and hydroxocobalamin (HC) - in molar ratios of 1:4, 1:5, and 1:8, respectively - were infused simultaneously during 4 h into two veins of separate ears of conscious rabbits. Controls received HC only. Sodium thiosulphate (ST) was infused with SNP at molar ratios of 1:4, 1:5, and 1:10. The observation period was 48 h. With the lowest dose of HC (1:4), SNP produced a severe metabolic acidosis; three of ten animals died during the infusion, an additional six within 24 h. When the 1:5 ratio was administered, the acidosis was less marked, but still three of seven animals succumbed within 24 h. The highest dose (1:8) prevented acidosis, however three of eight animals died. All doses of HC caused histological changes in the liver, the myocardium, and the kidney, independently if given alone or with SNP. In contrast to this, ST had a complete antidote effect, if administered in a 1:5 ratio; no acidosis was demonstrable and death did not occur. In neither dosage ST could prevent histological changes in the liver, but the kidney and the heart were not affected. In contrast to HC ST alone did not cause histological alterations. Consequently, ST is the preferable antidote and is superior to HC for preventing or treating intoxications with SNP.