SOS-LUX- and LAC-FLUORO-TEST for the quantification of genotoxic and/or cytotoxic effects of heavy metal salts

The constantly increasing industrial, agricultural and domestic activities with their growing risk of contaminating fresh water and ground water by discharged compounds lead to an increasing concern focused on the quality of water. Because of their simplicity and sensitivity, bacterial tests play an important role in the detection and screening of genotoxins or cytotoxins in water.One of those bacterial tests is the SOS-LUX- and LAC-FLUORO-TEST, which is a combination of two bioassays, that simultaneously measures the genotoxicity (SOS-LUX-TEST) and the cytotoxicity (LAC-FLUORO-TEST) of substances and mixtures of substances.The SOS-LUX-TEST is based on genetically modified Salmonella typhimurium TA1535 bacteria, which have been transformed with the plasmid pPLS-1 carrying the promoterless lux operon of Photobacterium leiognathi as reporter element under the control of a DNA damage-dependent SOS promoter from ColD as sensing element. This system reacts in a dose-dependent manner to agents which induce DNA damages inside these bacterial cells with the production of bioluminescence that can easily be measured.The analogous LAC-FLUORO-TEST has been developed for the detection of cellular responses to cytotoxins. It is based on the constitutive expression of green fluorescent protein (GFP) mediated by the bacterial protein expression vector pGFPuv (Clontech, Palo Alto, USA). In response to cytotoxic agents, this system reacts with a dose-dependent reduction of GFP-fluorescence.A panel of recombinant S. typhimurium strains carrying either the SOS-LUX plasmid or the fluorescence-mediating lac-GFPuv plasmid was used to determine in parallel on one microplate the genotoxic and the cytotoxic potential of heavy metal salts like K₂Cr₂O₇, CrCl₃, ZnSO₄, CuSO₄, NiSO₄, KH₂AsO₄ and As₂O₃ at the same time. Light and fluorescence emission of untreated and chemical-treated cells were measured in a microplate luminometer-fluorometer-photometer combination and the luminescence induction as well as the fluorescence reduction were used to determine the genotoxic and/or cytotoxic potential of the heavy metal salts.     

Utilizing Paper-Based Devices for Antimicrobial-Resistant Bacteria Detection

Antimicrobial resistance (AMR), the ability of a bacterial species to resist the action of an antimicrobial drug, has been on the rise due to the widespread use of antimicrobial agents. Per the World Health Organization, AMR has an estimated annual cost of USD 34 billion in the US and is predicted to be the number one cause of death worldwide by 2050. One way AMR bacteria can spread, and by which individuals can contract AMR infections, is through contaminated water. Monitoring AMR bacteria in the environment currently requires that samples be transported to a central laboratory for slow and labor intensive tests. We have developed an inexpensive assay using paper-based analytical devices (PADs) that can test for the presence of β-lactamase-mediated resistance. To demonstrate viability, the PAD was used to detect β-lactam resistance in wastewater and sewage and identified resistance in individual bacterial species isolated from environmental water sources.     

Utilizing Paper‐Based Devices for Antimicrobial‐Resistant Bacteria Detection

Antimicrobial resistance (AMR), the ability of a bacterial species to resist the action of an antimicrobial drug, has been on the rise due to the widespread use of antimicrobial agents. Per the World Health Organization, AMR has an estimated annual cost of USD 34 billion in the US and is predicted to be the number one cause of death worldwide by 2050. One way AMR bacteria can spread, and by which individuals can contract AMR infections, is through contaminated water. Monitoring AMR bacteria in the environment currently requires that samples be transported to a central laboratory for slow and labor intensive tests. We have developed an inexpensive assay using paper‐based analytical devices (PADs) that can test for the presence of β‐lactamase‐mediated resistance. To demonstrate viability, the PAD was used to detect β‐lactam resistance in wastewater and sewage and identified resistance in individual bacterial species isolated from environmental water sources.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric detection of bacterial biomarker proteins isolated from contaminated water, lettuce and cotton cloth

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of bacterial proteins were obtained from water, lettuce and cloth samples contaminated with Shigella flexneri, Escherichia coli, and Aeromonas hydrophila. Spectra were obtained using proteins directly isolated from water (or water used for rinsing samples) without culturing the bacteria. For S. flexneri and E. coli, two marker ions for specific proteins associated with a virulence-related property (acid resistance) were easily detected. For A. hydrophila, ions from two specifically selected marker proteins, as well as ions from the larger group of proteins isolated from pure cultures, all matched spectra from a contaminated water sample, providing strong evidence that A. hydrophila was the bacterial contaminant. Rinse water from contaminated lettuce and cloth samples showed the same marker ions as the contaminated water samples.     

Genotoxicity of Water Extract from Bark-Removed Rhus verniciflua Stokes

Rhus verniciflua Stokes (RVS) has been traditionally used as an herbal remedy to support the digestive functions in traditional Korean medicine. Additionally, the pharmacological effects of RVS, including antioxidative, antimicrobial and anticancer activities, have been well-reported. The genotoxicity of RVS, however, is elusive; thus, we evaluated the genotoxicity of RVS without bark (RVX) for safe application as a resource of functional food or a medical drug. To evaluate the genotoxicity of RVX, we used a bacterial reverse mutation test, chromosomal aberration test and comet assay, according to the “Organization for Economic Co-operation and Development” (OECD) guidelines. Briefly, for the reverse mutation test, samples (5000, 1667, 556, 185, 62 and 0 μg/plate of RVX or the positive control) were treated with a precultured strain (TA98, TA100, TA1535, TA1537 or WP2µvrA) with or without the S9 mix, in which RVX partially induced a reverse mutation in four bacterial strains. From the chromosomal aberration test and comet assay, the RVX samples (556, 185, 62, 20 and 0 μg/mL of RVX or the positive control) were treated in a Chinese hamster ovary cell line (CHO-K1 cells) in the conditions of the S9 mix absent or S9 mix present and in Chang liver cells and C2C12 myoblasts, respectively. No chromosomal aberrations in CHO-K1 or DNA damage in Chang liver cells and C2C12 myoblasts was observed. In conclusion, our results suggest the non-genotoxicity of RVX, which would be helpful as a reference for the safe application of bark-removed Rhus verniciflua Stokes as functional raw materials in the food, cosmetics or pharmaceutical fields.     

Genotoxicity of 2-alkylcyclobutanones, markers for an irradiation treatment in fat-containing food—Part I: cyto- and genotoxic potential of 2-tetradecylcyclobutanone

Previous experiments had indicated a slight genotoxic potential both in rat and in human colon cells of a sample of 2-dodecylcyclobutanone, a compound formed by irradiation of food containing palmitic acid in its triglycerides. Up to date, there is no evidence that 2-alkylcyclobutanones occur in non-irradiated foodstuffs, consequently it is prudent to test several members of the class of 2-alkylcyclobutanones which are produced by treatment of fat-containing food with ionising radiation. In this work, 2-tetradecylcyclobutanone (derived from stearic acid) has been tested for its cytotoxic and genotoxic potential. Human colon tumor cell lines, i.e. HT 29 and HT 29 clone 19A, were employed as models for in vitro experiments for cytotoxicity and genotoxicity tests. Cytotoxicity was measured by tetrazolium salt reduction assays (MTT and WST-1) and genotoxicity by determining DNA damage using the Comet Assay. Neither cytotoxic nor genotoxic effects were induced by 2-TCB in HT 29 or HT 29 cl 19A cells at an incubation time of 30 min at 37°C, not even at the highest concentration (400 μM) tested. After prolonged incubation times (1–2 days) at higher concentrations (>50 μM) cytotoxicity did, however, appear. Studies on other 2-alkylcyclobutanones are in progress.     

Gemifloxacin mesylate (GFM) stability evaluation applying a validated bioassay method and in vitro cytotoxic study

The validation of a microbiological assay applying the cylinder-plate method to determine the quinolone gemifloxacin mesylate (GFM) content is described. Using a strain of Staphylococcus epidermidis ATCC 12228 as the test organism, the GFM content in tablets at concentrations ranging from 0.5 to 4.5 μg mL⁻¹ could be determined. A standard curve was obtained by plotting three values derived from the diameters of the growth inhibition zone. A prospective validation showed that the method developed is linear (r = 0.9966), precise (repeatability and intermediate precision), accurate (100.63%), specific and robust. GFM solutions (from the drug product) exposed to direct UVA radiation (352 nm), alkaline hydrolysis, acid hydrolysis, thermal stress, hydrogen peroxide causing oxidation, and a synthetic impurity were used to evaluate the specificity of the bioassay. The bioassay and the previously validated high performance liquid chromatographic (HPLC) method were compared using Student's t test, which indicated that there was no statistically significant difference between these two validated methods. These studies demonstrate the validity of the proposed bioassay, which allows reliable quantification of GFM in tablets and can be used as a useful alternative methodology for GFM analysis in stability studies and routine quality control. The GFM reference standard (RS), photodegraded GFM RS, and synthetic impurity samples were also studied in order to determine the preliminary in vitro cytotoxicity to peripheral blood mononuclear cells. The results indicated that the GFM RS and photodegraded GFM RS were potentially more cytotoxic than the synthetic impurity under the conditions of analysis applied.     

Chemometric analysis of groundwater quality data of alluvial aquifer of Gangetic plain, North India

Water quality data set from the alluvial region in the Gangetic plain in northern India, which is known for high fluoride levels in soil and groundwater, has been analysed by chemometric techniques, such as principal component analysis (PCA), discriminant analysis (DA) and partial least squares (PLS) in order to investigate the compositional differences between surface and groundwater samples, spatial variations in groundwater composition and influence of natural and anthropogenic factors. Trilinear plots of major ions showed that the groundwater in this region is mainly of Na/K-bicarbonate type. PCA performed on complete data matrix yielded six significant PCs explaining 65% of the data variance. Although, PCA rendered considerable data reduction, it could not clearly group and distinguish the sample types (dug well, hand-pump and surface water). However, a visible differentiation between the water samples pertaining to two watersheds (Khar and Loni) was obtained. DA identified six discriminating variables between surface and groundwater and also between different types of samples (dug well, hand pump and surface water). Distinct grouping of the surface and groundwater samples was achieved using the PLS technique. It further showed that the groundwater samples are dominated by variables having origin both in natural and anthropogenic sources in the region, whereas, variables of industrial origin dominate the surface water samples. It also suggested that the groundwater sources are contaminated with various industrial contaminants in the region.     

GC–MS Determination of Antioxidants in Ground Water Contaminated with JP-8

Accidental spills and leaks of kerosene-based fuel require the differentiation of the exact fuel type between kerosene and JP-8. The detection of the antioxidants, 2,6-Di-tert-butylphenol (DTBP) and 2,4-dimethyl-6-tert-butylphenol (DMTBP) in ground water can be an important clue to distinguish between the two. We have developed a method to determine trace phenolic antioxidants in ground water without derivatization by a gas chromatography–mass spectrometry, and then applied it to distinguish JP-8 from kerosene. 25 ground water samples were collected from 25 monitoring wells in an area contaminated with kerosene-based fuel. The antioxidants from ground water were extracted with methylene chloride. Solid phase extraction (SPE) was compared to liquid extraction (LLE), but LLE was selected due to poorer reproducibility and recovery of SPE. Extraction of the compounds from ground water gave recoveries of about 90% and a detection limit of 0.02 μg L^?1. The method was used to analyze groundwater samples contaminated with fuel. DMTBP was detected in concentrations of 0.05–4.65 μg L^?1 in 12 of the samples. Since DMTBP is the only antioxidant used in JP-8 in Korea, this suggests that the fuel in the contaminated samples is JP-8.     

Bioremediation of PCB-contaminated sediments and evaluation of their pre- and post-treatment ecotoxicity

The paper deals with the application of two perspective and promising bioremediation approaches, bioaugmentation and biostimulation, applied to sediment contaminated with polychlo-rinated biphenyls (PCBs) with the aim to enhance biodegradation of PCBs. Sediments were sampled from Strá?sky canal, an industrial waste canal that flows from a former PCB-factory through the Laborec river into the Zemplínska ?írava water reservoir, all located in the eastern part of Slovakia. Bioaugmentation of sediments was performed in microcosms using two bacterial isolates with PCB-degradation ability obtained from the contaminated sediment: Achromobacter xylosoxidans, Stenotrophomonas maltophilia. Biostimulation was performed using an addition of cut plants containing terpenes, known as PCB-inducers (ivy leaves and pine needles). Ecotoxicity of the contaminated sediments was evaluated pre- and post-treatment using biotests of the standard aquatic plant Lemna minor, standard contact test using Lactuca sativa var. capitata, and the bioluminescent bacteria Vibrio fischeri. Biostimulation treatment using ivy leaves revealed higher degradation of detected PCB congeners than that achieved by the addition of pine needles, but moderately higher post-treatment toxicity of the sediment to the bioindicator Lemna minor.     

Antimicrobial Activity of Water‐Soluble Triazole Phenazine Clickamers against E. coli

The antimicrobial potency of phenazine derivatives is attenuated by their inherently hydrophobic nature, complicating their use as antibiotic drugs. We have analyzed the cytotoxicity and mode of action of water‐soluble bis‐triazolyl phenazines against E. coli and a human epithelial (HaCat) cell line. We observed complete inhibition of bacterial growth over concentration ranges that do not affect the viability of human epithelial cells. Confocal fluorescence microscopy revealed a high degree of interaction between the phenazine compounds and E. coli, as well as evidence of membrane damage in phenazine‐treated E. coli. Additional data suggests that the potency of these particular water‐soluble phenazine compounds does not result from the production of reactive oxygen species, but rather from cytotoxic interference with metabolic electron‐transfer cascades.     

The development of a screening molecularly imprinted polymer optosensor for detecting xylenes in water samples

We describe a rapid, sensitive, fluorescent screening test for xylenes in water samples that avoids more costly time-consuming methods. The screening test is based on a molecularly imprinted polymer and it runs without the need for any pre-concentration step, thus rendering it suitable for routine use in water-quality-control laboratories. The test recognizes contaminated samples rapidly (50 s) and inexpensively with a cut-off level of 10 μg mL^{− 1}, which is the value that the International Organization has laid down in its assessment of the water quality for human consumption. The reliability of the screening test was 23% false positives and 0% false negatives in 30 samples. The applicability was confirmed by analyzing mineral, tap and river water samples.     

Arsenic speciation and other parameters of surface and ground water samples of Jamshoro,Pakistan

This study evaluated and interpreted complex data sets of water samples collected from different sampling origins of ground water (hand pump and tube well) and surface water (municipal, river and canal). The aim was to provide information concerning the apportionment of pollution sources to obtain better information about water quality and possible distribution of As with respect to its speciation. The As (III) formed complex with ammonium pyrrolidinedithiocarbamate (APDC) and extracted by surfactant-rich phases in the non-ionic surfactant Triton X-114, while total iAs in water samples was adsorbed on titanium dioxide (TiO₂) and determined by electrothermal atomic absorption spectrometry. The accuracy of the proposed methodologies was confirmed by standard addition method. The recoveries of As (III) and total inorganic arsenic (iAs) were found to be >98%. The results revealed that the ground water of the area under study was more contaminated as compared to surface water samples. The mean concentration of As (III) and As (V) in the surface water samples was found to be 15.8 and 6.00?µg?L^?1, respectively, whereas, in the case of ground water samples, the contents of As (III) and As (V) ranged from 6.20 to 51.0 and 6.40 to 53.0?µg?L^?1, respectively. Principal component analysis performed on a combined (tube well and hand pump) samples data set extracted two significant factors explaining more than 60% of total variance, which suggested that the contamination sources might be natural or anthropogenic.     

Synthesis and In Vitro Biological Evaluation of Psoralen‐Linked Fullerenes

Photodynamic therapy (PDT) is a widely used medicinal treatment for the cancer therapy that utilizes the combination of a photosensitizer (PS) and light irradiation. In this study, we synthesized two novel C₆₀ fullerene derivatives, compounds 1 and 2 , with a psoralen moiety that can covalently bind to DNA molecules via cross‐linking to pyrimidine under photoirradiation. Along with several fullerene derivatives, the biological properties of several novel compounds have been evaluated. Compounds 1 and 2 , which have been shown to induce the production of hydroxyl radicals using several ROS detecting reagents, induced DNA strand breaks with relatively weak activities in the in vitro detection system using a supercoiled plasmid. However, the psoralen‐bound fullerene with carboxyl groups ( 2 ) only showed genotoxicity in the genotoxicity assay system of the umu test. Compound 2 was also seen to have cytotoxic activities in several cancer cell lines at higher doses compared to water‐soluble fullerenes.     

Evaluation of cytogenotoxic effects of cold aqueous extract from Achyrocline satureioides by Allium cepa L test

Achyrocline satureioides ("marcela del campo") is native to America. Numerous investigations have reported several bioactive properties such as anti-inflammatory, hepatoprotective, immunomodulatory, antimicrobial and antiviral. Nowadays, few medicinal plants have been scientifically evaluated to test its safety, efficacy and potential benefits, despite the great public interest in these herbs. The aim of this work was to evaluate the cytotoxic and genotoxic activities of cold aqueous extract obtained from A. satureioides using Allium cepa L test. The results demonstrated the absence of genotoxicity of the extract. Only higher concentrations induced cytotoxicity but interestingly this effect was reversible and was not associated with mutagenicity. The contribution of this research provides assurance of safety in the application of Achyrocline satureioides in treatment of microbial diseases and other pathologies helping to define selective toxicity.     

Multi-Hollow Surface Dielectric Barrier Discharge for Bacterial Biofilm Decontamination

The plasma-activated gas is capable of decontaminating surfaces of different materials in remote distances. The effect of plasma-activated water vapor on Staphylococcus epidermidis, methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli biofilm contamination was investigated on the polypropylene nonwoven textile surface. The robust and technically simple multi-hollow surface dielectric barrier discharge was used as a low-temperature atmospheric plasma source to activate the water-based medium. The germicidal efficiency of short and long-time exposure to plasma-activated water vapor was evaluated by standard microbiological cultivation and fluorescence analysis using a fluorescence multiwell plate reader. The test was repeated in different distances of the contaminated polypropylene nonwoven sample from the surface of the plasma source. The detection of reactive species in plasma-activated gas flow and condensed activated vapor, and thermal and electrical properties of the used plasma source, were measured. The bacterial biofilm decontamination efficiency increased with the exposure time and the plasma source power input. The log reduction of viable biofilm units decreased with the increasing distance from the dielectric surface.     

Influence of Agronomic Practice on Total Phenols,Carotenoids, Chlorophylls Content,and Biological Activities in Dry Herbs Water Macerates

Oregano (Origanum vulgare L.) and thyme (Thymus vulgaris L.) have long been known for their organoleptic properties. Both plants are widely used in cuisine worldwide in fresh and dried form and as a pharmaceutical raw material. The study aimed to assess if the type of cultivation influenced chosen chemical parameters (total polyphenols by Folin-Ciocalteu method; carotenoids and chlorophyll content by Lichtenthaler method), antimicrobial activity (with chosen reference microbial strains) and shaped cytotoxicity (with L929 mouse fibroblasts cell line) in water macerates of dry oregano and thyme. Polyphenols content and antimicrobial activity were higher in water macerates obtained from conventional cultivation (independently from herb species), unlike the pigments in a higher amount in macerates from organic herbs cultivation. Among all tested macerates stronger antimicrobial properties (effective in inhibiting the growth of Pseudomonas aeruginosa, Bacillus cereus and Salmonella enteritidis) and higher cytotoxicity (abilities to diminish the growth of L929 fibroblasts cytotoxicity) characterized the conventionally cultivated thyme macerate.     

Screening and characterizing the quality markers of Selaginella tamariscina (P. Beauv.) Spring using metabonomics and molecular networking

Quality control of traditional Chinese medicine (TCM) should be linked with the authentication and efficacy of TCM. Selaginella tamariscina is a frequently used traditional Chinese herbal medicine. However, its quality control is still difficult due to its multiple adulterants. We established quality markers (Q-markers) of S. tamariscina by using metabolomics, molecular networking and network pharmacology to improve the authenticity study and quality control of S. tamariscina. In this study, ultra high performance liquid chromatography-mass spectrum (UHPLC-MS) coupled with multivariate statistical analyses was applied to distinguish between S. tamariscina samples and their confusing adulterants. Principal component analysis, hierarchical clustering analysis (PCA), hierarchical clustering analysis (HCA) and partial least squares discriminant analysis (PLS-DA) were employed to screen the distinguishing markers from S. tamariscina samples and their adulterants. The top-2 distinguishing markers were isolated from S. tamariscina and identified by molecular networking together with nuclear magnetic resonance spectroscopy (NMR). Network pharmacology predicted the bioactivity and cytotoxicity of the top-2 distinguishing markers. The top-2 distinguishing markers were adopted as Q-markers of S. tamariscina for content determination. Based on the results of ultra performance liquid chromatography-quardrupole-time of flight mass spectrometry (UHPLC-QTOF-MS) metabolomics, we revealed that selaginellins could only be detected in S. tamariscina samples and contributed greatly to discriminating S. tamariscina samples from their confused species. The top-2 distinguishing markers were isolated and purified from S. tamariscina extract. Then, they were further identified as selaginellin and selaginellin A by molecular networking and NMR. Network pharmacology predicted the antitumor activity of selaginellin and selaginellin A, while the cytotoxicity assay verified their bioactivity. In conclusion, selaginellin and selaginellin A were selected as Q-markers for the determination and quality evaluation of S. tamariscina based on ultra performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-QQQ-MS). The ranges of the concentrations of selaginellin and selaginellin A were 41.57–44.89 μg/g and 15.09–16.75 μg/g, respectively. This study provides a novel strategy combining Ultra performance liquid chromatography mass spectrometry based (UHPLC-MS-based) metabolomics with molecular networking for rapid species identification of S. tamariscina and discovery of the Q-markers of TCM.     

Paper-based chromatic toxicity bioassay by analysis of bacterial ferricyanide reduction

Water quality assessment requires a continuous and strict analysis of samples to guarantee compliance with established standards. Nowadays, the increasing number of pollutants and their synergistic effects lead to the development general toxicity bioassays capable to analyse water pollution as a whole. Current general toxicity methods, e.g. Microtox^®, rely on long operation protocols, the use of complex and expensive instrumentation and sample pre-treatment, which should be transported to the laboratory for analysis. These requirements delay sample analysis and hence, the response to avoid an environmental catastrophe. In an attempt to solve it, a fast (15 min) and low-cost toxicity bioassay based on the chromatic changes associated to bacterial ferricyanide reduction is here presented. E. coli cells (used as model bacteria) were stably trapped on low-cost paper matrices (cellulose-based paper discs, PDs) and remained viable for long times (1 month at −20 °C). Apart from bacterial carrier, paper matrices also acted as a fluidic element, allowing fluid management without the need of external pumps. Bioassay evaluation was performed using copper as model toxic agent. Chromatic changes associated to bacterial ferricyanide reduction were determined by three different transduction methods, i.e. (i) optical reflectometry (as reference method), (ii) image analysis and (iii) visual inspection. In all cases, bioassay results (in terms of half maximal effective concentrations, EC₅₀) were in agreement with already reported data, confirming the good performance of the bioassay. The validation of the bioassay was performed by analysis of real samples from natural sources, which were analysed and compared with a reference method (i.e. Microtox). Obtained results showed agreement for about 70% of toxic samples and 80% of non-toxic samples, which may validate the use of this simple and quick protocol in the determination of general toxicity. The minimum instrumentation requirements and the simplicity of the bioassay open the possibility of in-situ water toxicity assessment with a fast and low-cost protocol.