Bacterial ribonuclease: Mutagenic effect in microbial test-systems

Pure enzyme samples of ribonuclease from Bacillus intermedius 7P (known commercially as 'binase') were investigated for genotoxicity in four microbial tests: the Ames plate incorporation method, Ara(R)-assay; the prophage induction test; and the DNA-repair test. The weak mutagenic effect of binase at high concentrations (0.1 mg/plate, 1 mg/plate) was established by induction of forward Ara(R)-mutations and histidine-reverse mutations (both frameshift mutations and base pair substitution). Metabolic activation with rat or chicken liver, human placenta or plant (from tulip bulbs) microsomal fractions in vitro was seen to abolish the binase mutagenicity. Bacillus intermedius 7P ribonuclease appears to possess DNA damaging activity in uvrA- and polA- mutants, but not in the recA-deficient Escherichia coil strain, and exhibits an induction of recA-dependent mutagenesis detected by the 8-fold increase of the prophage-induction level in lysogenic Bacillus subtilis culture and by the 5-fold increase of this level in the Streptomyces lavendulae 3 lysogenic strain. The importance of the roles of both of enzyme catalytic activity and native structure is emphasized. A proposed mechanism for exogenous ribonuclease action is discussed. Bacillus intermedius 7P ribonuclease probably does not act as a direct genotoxic agent interacting with DNA, but could provoke nucleotide imbalance through its catalytic action on membrane-associated RNAs, which results in alteration of DNA replication and, as a consequence, in recA-dependent mutagenesis

SOS-inducing ability of native and mutant microbial ribonucleases

The results of genotoxicity testing of microbial ribonucleases from Bacillus species with different catalytic activity obtained by site-directed mutagenesis in SOS chromotest are reported. At the concentrations 0.1-1 mg/ml, the induction factor for wild-type bacillar binase, barnase and mutant Arg58Lys binase with 100% activity was found to be significantly higher than 1.5 (1.8-2.8). Mutant RNases having decreased catalytic activity (binases with replacements Lys26Ala, Arg61Gln, His101Glu) or through natural inhibitor barstar inactivated wild-type RNase exhibited no SOS-inducing potency. The ability of native bacillar RNases and mutant enzymes possessing high catalytic activity comparable with the activity of wild-type RNase to cause the SOS response indicates that genotoxicity is mediated through the probable cleavage of cellular RNA. The possible mechanisms of mutagenesis induced by catalytically active RNases are discussed

Genotoxicity study of a new tetraalkylammonium derivative of 6-methyluracil (agent No. 547)

Agent No. 547 (1,3-bis[x-(diethyl-ortho-nitrobenzylammonio)-pentyl]-6-methyluracil dibromide), a newly synthesized inhibitor of mammalian-specific acetyltcholinesterase (EC 3.1.1.7) was investigated for genotoxicity using the DNA-repair test, Ames test and in vivo micronucleus test with mouse peripheral blood erythrocytes. Agent No. 547 did not cause significant changes in growth of repair-deficient Escherichia coli tester strains. The compound was non-mutagenic in Salmonella typhimurium strains TA98 and TA100 with and without rat microsomal activation mixture. However, we observed a marked increase in number of His+ revertants for both tester strains in preincubation assays. The results obtained in the micronucleus test indicate that agent No. 547 possesses significant clastogenic activity. At the high dose tested (0.5 mg/kg), the compound induced a seven-fold increase in the number of micronuclei over the spontaneous background 48 h after treatment. The results suggest that further work should be promoted to identify the metabolic pathways involved in genotoxicity of agent No. 547 in mammalian cells and to evaluate the real risk of its exposure

A hydrophobic segment of some cytotoxic ribonucleases

The exact mechanism by which cytotoxic ribonucleases reach the cytosol of tumor cells remains unclear. The interaction of ribonucleases with a lipid bilayer is involved in the translocation of ribonucleases across the endosomal membrane. Here, we aimed to study the hydropathy character of toxic antitumor ribonucleases (bovine seminal ribonuclease and binase) and two non-toxic ribonucleases (bovine pancreatic ribonuclease and human pancreatic ribonuclease) by sliding-window hydrophobicity analysis. Comparative hydropathy plot analysis of the non-toxic pancreatic ribonucleases and their toxic variants was also performed. The data obtained indicate that some cytotoxic ribonucleases have a hydrophobic segment, which is sterically available for the hydrophobic interaction with a tumor cell membrane and endosomal membrane. After dissociation, subunits of dimeric ribonucleases are probably capable of thermodynamically favorable interaction with the interfacial region of a lipid bilayer. Remarkably the hydrophobic segment is not identified in the amino acid sequences of non-toxic ribonucleases. The paper describes the hydrophobic properties of toxic RNases that are essential for both the model of a lipid-protein interaction and the cytotoxicity mechanism unraveling. © 2013 Elsevier Ltd

Compounds 2,4,6-trinitrotoluene and 2,4-diamino-6-nitrotoluene: The absence of recA-dependent mutagenesis?

The genotoxicity of 2,4,6-trinitrotoluene (2,4,6-TNT) and its amino derivative, 2,4-diamino-6-nitrotoluene (2,4-DA-6-NT), was studied using the Escherichia coli tester strain PQ37 in the SOS chromotest. The compound 2,4,6-TNT, without metabolic activation, virtually failed to induce an SOS effect in cells of the tester bacteria. Consequently, mutagenic activity of 2,4,6-TNT, which was shown earlier in the Ames test, does not depend on SOS mutagenesis. It was demonstrated that metabolic activation with the microsomal S9 human placenta fraction results in a threefold increase in the induction factor of the SOS effect caused by 2,4,6-TNT. The absence of the SOS-inducing activity of 2,4-DA-6-NT, regardless of the presence of a microsomal activating mixture, is shown. Thus, 2,4-DA-6-NT does not belong to metabolites of 2,4,6-TNT, responsible for the genotoxicity of this compound

Cytotoxic and genotoxic effects of β-(triphenylphosphonio)ethyl carboxylate and of N,N′-bis(dihexylphosphinoylmethyl)-1,4- diaminocyclohexane

Background: Several organophosphorous compounds (OPs) are now being tested therapeutically. Cholinesterase inhibition, which in large doses makes these agents effective pesticides, may also be useful in other doses for treating dementia. Metrifonate, for example, has been used to treat schistosomiasis and is undergoing trials for the treatment of primary degenerative dementia. Material/Methods: Here we report the characterization of newly synthesized OPs from the group of phosphobetaines [β-(triphenylphosphonio)ethyl carboxylate, PB] and of alpha-aminophosphoryl compounds [N,N′- bis(dihexylphosphinoylmethyl)-1,4-diaminocyclohexane, AP] according to their toxic and genotoxic properties determined in prokaryotic and eukaryotic test systems. Results: The absence of toxicity towards Gram-negative bacteria and of genotoxicity in Ames mutagenicity assay and in SOS-chromotest did not exclude the cytotoxic effect of PB towards NIH3T3 mouse fibroblasts, which supports the notion of an extremely diverse interspecies response to OPs. In contrast, AP demonstrated toxic properties detected by antibacterial effect as well as by the inhibition of the proliferation and respiration of fibroblasts. The enzymatic transformation of the compound is necessary to reveal the genotoxic properties of AP. The role of mammalian microsomal enzymes and of bacterial C-P lyase in the formation of AP genotoxic metabolites is under discussion. Conclusions: Neither toxicity nor genotoxicity of PB was found in bacterial tests. Cytotoxic and mutagenic effects of AP were detected. The data contribute to the investigation of the biological activity of novel organophosphates which could be useful for the future development of OP-based therapeutics

The Effect of high hydrostatic pressure on the viability and mutagenesis of Salmonella typhimurium

© 2017, Pleiades Publishing, Ltd. This work is devoted to the study of the influence of high hydrostatic pressure (HHP) on the viability and level of mutagenesis of Salmonella typhimurium. It was established that the viability of bacteria significantly decreases under hydrostatic pressure of 200 MPa or higher. In addition, the viability index of the bacteria is six orders of magnitude lower with respect to the number of colony-forming units (CFUs) compared to the data of the flow cytofluorometry analysis. This is probably due to the transition of some part of the bacterial population to a viable but nonculturable state (VBNC). HHP of 50 MPa caused a 1.9-fold increase in the number of His + revertants of the S. typhimurium strain TA98, which indicates the potential of the induction of gene mutations under these conditions. The mechanisms to reduce the viability and genetic changes in bacterial cells under HHP conditions are discussed

Effect of Bacillus pumilus ribonuclease on the paramagnetic centers of microbial cells

The potential clinical application of Bacillus pumilus cytotoxic ribonuclease (binase) for selectively inducing the death of tumor cells makes it imperative to investigate its effect on the normal human microflora. Flow cytometry was used to determine that binase concentration causing the apoptosis of cancer cells had no effect of the viability of Escherichia coli K12. The changes in the paramagnetic centers of E. coli K12 cells in the presence of nontoxic binase concentrations revealed by EPR spectroscopy included higher EPR signals from iron-containing proteins (including those from the Fe-S clusters) and of the Mn(II) hyperfine structure. The TMTH spin probe (N-(1-hydroxy-2,2,6,6-tetramethylpiperidine-4-il)-2-methylpropanamide hydrochloride) was used to reveal a twofold increase in the levels of reactive oxygen species (ROS) in the cells, which induced oxidative stress in the enzyme-treated bacteria. Inductively coupled plasma mass spectrometry revealed elevated contents of alkaline (Li, Na, K), alkali earth (Mg, Ca), transition (Cr, Mn, Fe, Cu, Zn), and post-transition metals (Bi, Pb) in the cells. Elevated levels of Cu and Zn (which impair the activity of the respiratory chain enzymes) and of Mn, which is known as a superoxide dismutase cofactor, confirmed development of the oxidative stress in bacteria. © 2013 Pleiades Publishing, Ltd

A Comparative Evaluation of Antimicrobial Effect of Thymus capitatus Ethanolic Extract on the Different Respiratory Tract Infections Isolates

© 2017, Springer Science+Business Media, LLC. Natural components, particularly those derived from medicinal plants, provide a promising source of new antimicrobial agents for the treatment of respiratory infections. In this work, we studied the antimicrobial potency of Thymus capitatus ethanolic extract against nine microorganisms isolated from clinical material obtained from patients admitted to hospitals in Egypt and Germany with infections of the respiratory tract (bacterial sinusitis, laryngitis, pharyngitis and bronchitis) using the disc diffusion assay and minimum inhibitory concentration (MIC) values. All isolates obtained from the Giessen University Clinic (Germany) exhibit susceptibility of different extent to the plant extract tested. The growth inhibition zone diameters for nine strains were in the range of 13–15.5 mm at a concentration of 5 mg/disc. The extract produced large inhibition zones of 14–17 mm diameters for seven microbial strains obtained in Egypt although it failed to inhibit the growth of isolates of Staphylococcus epidermidis and Enterococcus faecalis. In fact, most of the respiratory tract infections isolates from Egypt were generally more susceptible to the ethanolic extract of Thymus capitatus at lower MIC values than those obtained from Giessen University Clinic MIC values. The results of this study suggest that further delineation of bioactive phytomolecules of Thymus capitatus ethanolic extract will improve their potential application for the treatment of respiratory tract diseases

Antioxidant and Antimutagenic Potential of Extracts of Some Agavaceae Family Plants

© 2016, Springer Science+Business Media New York.The application of natural antimutagens and antioxidants, particularly those derived from higher plants has been seen as a promising approach to the protection of human health. In this work, we studied methanolic extracts from Sansevieria cylindrica, Sansevieria trifasciata, and Polianthes tuberosa plants focusing on their antioxidative and antimutagenic capacities based on the following parameters: inhibitory activity on lipid peroxidation, suppressing ability on direct-acting mutagen sodium azide-induced mutagenesis in Salmonella typhimurium cells. A clear dose-dependent decrease in lipid peroxidation was observed with all the extracts tested. Extracts from leaves of P. tuberosa and rhizomes of S. cylindrica and S. trifasciata (1 mg/mL) displayed the highest antioxidant effect. At the same time, extracts from rhizomes of S. cylindrica and S. trifasciata significantly reduced the sodium azide-induced mutations. The highest antimutagenic activity (76 %) in the S. typhimurium TA100 strain was obtained for the S. cylindrica rhizomes extract (1 mg/plate). We propose that the observed protective effects of plant extracts tested may correspond to a synergic participation of several secondary metabolites and mainly to polyphenolic compounds