Stem cell-derived systems in toxicology assessment.

Industrial sectors perform toxicological assessments of their potential products to ensure human safety and to fulfill regulatory requirements. These assessments often involve animal testing, but ethical, cost, and time concerns, together with a ban on it in specific sectors, make appropriate in vitro systems indispensable in toxicology. In this study, we summarize the outcome of an EPAA (European Partnership of Alternatives to Animal Testing)-organized workshop on the use of stem cell-derived (SCD) systems in toxicology, with a focus on industrial applications.

Advancing the 3Rs in regulatory toxicology - Carcinogenicity testing: Scope for harmonisation and advancing the 3Rs in regulated sectors of the European Union.

Different government agencies operating in the European Union regulate different types of chemical products but all require testing for carcinogenicity to support applications for product marketing and commercialisation. A conference was held in Brussels in 2013 where representatives of the pharmaceutical, animal health, chemical and plant protection industries, together with representatives of regulatory agencies, universities and other stakeholders, met under the auspices of The European Partnership for Alternative Approaches to Animal Testing (EPAA) to discuss the varying requirements for carcinogenicity testing, and how these studies might be refined to improve hazard evaluation and risk assessment while implementing principles of the 3Rs (replacement, refinement and reduction in animal studies). While there are some similarities, the regulatory approaches in pharmaceutical, animal health, chemical and plant protection sectors have varying degrees of flexibility in requirements for carcinogenicity testing, to an extent reflecting concerns over the magnitude and duration of human exposure, either directly as in therapeutic exposure to pharmaceuticals, or indirectly through the ingestion of residues of veterinary drugs or plant protection chemicals.

Report from the EPAA workshop: in vitro ADME in safety testing used by EPAA industry sectors.

There are now numerous in vitro and in silico ADME alternatives to in vivo assays but how do different industries incorporate them into their decision tree approaches for risk assessment, bearing in mind that the chemicals tested are intended for widely varying purposes? The extent of the use of animal tests is mainly driven by regulations or by the lack of a suitable in vitro model. Therefore, what considerations are needed for alternative models and how can they be improved so that they can be used as part of the risk assessment process? To address these issues, the European Partnership for Alternative Approaches to Animal Testing (EPAA) working group on prioritization, promotion and implementation of the 3Rs research held a workshop in November, 2008 in Duesseldorf, Germany. Participants included different industry sectors such as pharmaceuticals, cosmetics, industrial- and agro-chemicals.

Efomycine M, a new specific inhibitor of selectin, impairs leukocyte adhesion and alleviates cutaneous inflammation.

Specific interference with molecular mechanisms guiding tissue localization of leukocytes may be of great utility for selective immunosuppressive therapies. We have discovered and characterized efomycines, a new family of selective small-molecule inhibitors of selectin functions. Members of this family significantly inhibited leukocyte adhesion in vitro.

Antiarrhythmic effects of increasing the daily intake of magnesium and potassium in patients with frequent ventricular arrhythmias. Magnesium in Cardiac Arrhythmias (MAGICA) Investigators.

Objectives: This study sought to assess potential antiarrhythmic effects of an increase in the daily oral intake of magnesium and potassium in patients with frequent ventricular arrhythmias.

Background: Magnesium and potassium contribute essentially to the electrical stability of the heart. Despite experimental and clinical evidence for the antiarrhythmic properties of the two minerals, controlled data in patients with stable ventricular arrhythmias are lacking.

In vitro evaluation of BAY Y3118, a new full-spectrum fluoroquinolone.

BAY Y3118, 1-cyclopropyl-7-(2,8-diazabicyclo[4.3.0]non-8-yl)-6-fluoro-8- chloro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid hydrochloride, is a new fluoroquinolone with antibacterial activity against an expanded spectrum of species including Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae, Streptococcus pyogenes, and also anaerobes such as Bacteriodes fragilis and Clostridium perfringens.

Modulation of leukotriene metabolism from human polymorphonuclear granulocytes by bifonazole.

The influence of bifonazole and other azoles on the leukotriene-metabolism of human neutrophil granulocytes (PMN) was investigated. The cells were stimulated with the Ca-ionophore A23187 over 15 minutes in the presence of the azoles. Subsequently the supernatants were analyzed for their leukotriene content by reversed-phase HPLC.

Application of high-performance liquid chromatography of some antibiotics in clinical microbiology.

During recent years high-performance liquid chromatography has become an excellent tool for the determination of antibiotics in biological fluids. Compared with biological assays, the major benefits of this method are specificity and rapidity. In particular, the determination of biologically inactive metabolites emphasizes that this technique plays an outstanding role for the analysis of antibiotics.

Concentration of azlocillin in human chondral tissue.

The concentration of azlocillin was determined using high performance liquid chromatography in serum and chondral tissue after intravenous infusion of azlocillin (75 mg/kg body weight). In serum the levels of ten patients (2 to 27 years of age, body weight 12 to 69 kg) decreased from 478 (30 min post infusion) to 120 micrograms/ml (120 min). In contrast, the concentrations in chondral tissue ranged between 24 and 35 mg/g tissue at the corresponding times.

Degradation of acylaminopenicillins with regard to their pH dependency.

Determination of antibiotic concentration is performed in many biological fluids and tissues which all have different pH values. Therefore, we investigated the in vitro stability of three acylaminopenicillins (azlocillin, mezlocillin and piperacillin) in borate buffer by the HLPC technique with regard to pH dependency. HPLC allows the detection of all three substances together with their metabolites, penicilloate and penilloate, within 15 min.

Modulation of granulocyte functions by bacterial exotoxin and endotoxins.

The modulation of granulocyte functions by bacterial exotoxins (Streptolysin O, alveolysin, theta toxin) and endotoxins from salmonella and lipid A is described here. Incubation of polymorphonuclear granulocytes with thiol-activated toxins resulted in an increased leukotriene generation. Toxin-pretreated PMNs revealed an increased omega oxidation of LTB4, which may explain why toxin-stimulated cells release more LTC4 than LTB4.

Determination of aspoxicillin (TA-058) by high-performance liquid chromatography. Stability at different temperatures.

A rapid and sensitive HPLC-method has been developed for the determination of serum concentrations of aspoxicillin (TA-058), a new semisynthetic beta-lactam antibiotic. Aspoxicillin was chromatographed with a phosphate buffer/methanol (92:8 v/v) mobile phase and a C-18 reversed phase column and was detected at a wavelength of 220 nm. The stability of aspoxicillin in serum and buffer at different temperatures was studied over a time period of 3 months.

The role of leukotriene-inducing and -metabolizing enzymes in inflammation.

Leukotrienes are potent mediators of allergy and inflammation. Polymorphonuclear granulocytes which participate in acute and chronic disease processes can be activated by immunological and nonimmunological stimuli to generate leukotrienes. It appears that on activation of the cells 5-lipoxygenase is released into the supernatant and thus may exert a potent role with regard to interdependent cellular interactions.

Determination of ciprofloxacine, norfloxacine, and ofloxacine by high performance liquid chromatography.

A fast, versatile and precise method is described for the determination of quinolone derivatives in biological fluids and tissues by high performance liquid chromatography (HPLC) and fluorescence detection. Using a solvent consisting of acetonitrile/methanol/water/tetrabutylammoniumhydroxide (pH 3.0) and reversed phase material (5C18), ciprofloxacine, norfloxacine and ofloxacine were identified within 10 min, with a detection limit of 10 pg (ofloxacine 40 pg).

Determination of mezlocillin and its penicilloate and penilloate by high-performance liquid chromatography and stability of mezlocillin at different temperatures.

A method is described for the determination of mezlocillin and its metabolites penicilloic acid and penilloic acid in biological fluids by high-performance liquid chromatography. The stability of mezlocillin in serum and buffer was studied at different temperatures (4, -20, -70, and -196 degrees C) over a period of 6 months. Mezlocillin remained stable at -70 and -196 degrees C, whereas degradation was observed at -20 and 4 degrees C in serum and buffer.

Effect of thiol-activated toxins (streptolysin O, alveolysin, and theta toxin) on the generation of leukotrienes and leukotriene-inducing and -metabolizing enzymes from human polymorphonuclear granulocytes.

The generation of leukotrienes (LTC4, LTD4, LTE4, and LTB4; 12-epi-LTB4 isomer) from human granulocytes by thiol-activated toxins (streptolysin O, alveolysin from Bacillus alvei, and theta toxin from Clostridium perfringens) is described. The release occurs under noncytolytic conditions. Although LTB4 is the major component after calcium ionophore stimulation, more LTC4 as compared with LTB4 is released with the toxins.

Degradation of azlocillin in human serum.

We developed a rapid and precise high performance liquid chromatographic method (HPLC) for the determination of azlocillin and its metabolites penicilloate and penilloate in serum and tissue as well as in vivo as in vitro. The linear relationship (r greater than 0.99) of determination ranged between 0.

Functional characteristics of leukotriene C4- and D4-metabolizing enzymes (gamma-glutamyl transpeptidase, dipeptidase) within human plasma.

Several properties of the leukotriene C4- and leukotriene D4-metabolizing enzymes within human plasma were studied after fractionation of the plasma proteins using ammonium sulfate precipitation. Leukotriene D4-metabolizing enzymes were widely distributed among the fractions obtained. They showed different pH optima (pH 6.

The metabolism of leukotrienes in blood plasma studied by high-performance liquid chromatography.

The metabolism of leukotrienes (B4, C4, D4, and E4) within human plasma was studied and a simple sample preparation is presented. It was demonstrated that leukotriene E4 and leukotriene B4 were stable during incubation at 37 degrees C using the in vitro system. In contrast, leukotriene C4 was metabolized by gamma-glutamyl transpeptidase activities into leukotriene D4 which was further metabolized by dipeptidase activities of plasma into leukotriene E4.

Generation of slow-reacting substance (leukotrienes) by endotoxin and lipid A from human polymorphonuclear granulocytes.

Leukotrienes were released from human polymorphonuclear granulocytes on incubation with endotoxins and lipid A. The analysis was performed by their smooth muscle contracting properties, reversed phase high-pressure liquid chromatography and radioimmunoassay for leukotrienes C4 and D4. The active component of the lipopolysaccharides seems to be the lipid A portion.

Generation of leukotrienes from human granulocytes by alveolysin from Bacillus alvei.

We investigated the effect of alveolysin on human granulocytes. Alveolysin is an exoprotein produced by Bacillus alvei and belongs to the group of sulfhydryl-activated cytolysins. Other members of this group are streptolysin O and theta-toxin from Clostridium perfringens.

Subcellular localization of enzymes involved in leukotriene formation within human polymorphonuclear granulocytes.

The subcellular localization of enzymes involved in leukotriene formation was analysed according to biological (chemotaxis, spasmogenic properties) and analytical methods. By subcellular fractionation the major activity for 5--lipoxygenase and L-gamma-glutamyltranspeptidase coeluted with the 200,000 g precipitate while glutathione-S-transferase activity was mainly present in the 200,000 g supernatant. Our data were supported by results indicating that the 200,000 g precipitate and supernatant fractions proved to be most active in generating 5-HETE and leukotriene C4 (LTC4) respectively.