Willi Suter

Selective block of IKs plays a significant role in MAP triangulation induced by IKr block in isolated rabbit heart

INTRODUCTION The role of IKr (rapidly-activating delayed rectifier K(+) current) block in triangulation of monophasic action potentials (MAP) and in development of torsade de pointes (TdP) arrhythmia is known. Combined IKr and IKs (slowly-activating delayed rectifier K(+) current) block has been demonstrated to promote TdP. The aim of this study was to describe a possible implication of IKs block in MAP triangulation. METHODS Four contact electrodes were placed on the epicardium of the left ventricle of Langendorff-perfused rabbit hearts to record monophasic action potentials (MAP), with an IKr blocker d,l-sotalol (3 to 100 microM, n=6) or a non-selective IKr blocker, quinidine (1 to 30 microM, n=6). Their effects were assessed with or without a specific IKs blocker chromanol 293B (20 microM, n=6), on MAP duration at 30, 60 and 90% of repolarization (APD30, 60 and 90, respectively) and MAP triangulation (APD90-APD30) at 1 and 0.2 Hz. RESULTS D,L-sotalol increased significantly APD90 and triangulation with reverse use-dependency for concentrations > or =10 microM. Quinidine markedly prolonged APD90 and triangulation with reverse use-dependency at concentrations > or =3 microM. Chromanol 293B alone had no effects on APD, but when combined with D,L-sotalol or quinidine (i) increased APD prolonging effects, (ii) lowered values of pro-arrhythmic concentrations, (iii) increased incidence and length of D,L-sotalol- or quinidine-induced Early Afterdepolarizations (EADs) and TdP. All these events were primarily due to an important slowing of final repolarization, i.e. a marked increased triangulation. CONCLUSION IKs, even of low amplitude in rabbits, plays a key role in ventricular repolarization. IKs is involved in prolonged MAP duration mainly by triangulation and subsequent increased drug arrhythmogenicity. Therefore drug affinity for IKs must be evaluated with IKr studies as part of preclinical drug cardiac safety assessment.

A Flow Cytometry Based In Vitro Micronucleus Assay in TK6 Cells—Validation Using Early Stage Pharmaceutical Development Compounds

The micronucleus test (MNT) is a well established test for detecting clastogenic and aneugenic compounds. Despite the assays advantages, the MNT may produce false positive and false negative results in some conditions. This fact may be related to the underestimation of apoptosis or necrosis, the p53 status of the cell system or the cytotoxicity assay, and the top dose selection. The purpose of our studies was to contribute to the validation efforts of the flow cytometry based MNT. To identify the most reliable cytotoxicity assay for the top dose selection five parameters for relative survival were tested: relative cell count, relative population doubling, trypan blue supravital staining, relative ratio of scored nuclei to latex beads, and ethidium monoazide staining. For all compounds the least sensitive method was the relative cell count and the most reliable was the nuclei/beads ratio. The comparative evaluation of micronuclei induction in TK6 cells, analyzed with microscopy and flow cytometry, was performed with reference compounds and internal Novartis early development compounds with positive, weak positive, equivocal, and negative genotoxic effects. Our data document a good correlation between the MNT results obtained by flow cytometry and by microscopy. The results confirm that the method may be applied for routine testing in the pharmaceutical industry for the tested group of compounds, including compounds which require metabolic activation. However, further validation and miniaturization may be required. Environ. Mol. Mutagen., 2011.

Flow cytometry peripheral blood micronucleus test in vivo: Determination of potential thresholds for aneuploidy induced by spindle poisons

Non‐DNA binding genotoxins (e.g., aneugens), unlike DNA‐binding genotoxins, are theoretically expected to show thresholded concentration‐effect response curves. This is a major issue in genetic toxicology testing because the identification of thresholds in vivo can provide a safety margin for exposure to a particular compound. In the current study we measured micronucleus induction by flow cytometry to determine the dose‐response curves for tubulin interacting agents, a specific class of aneugens. All experiments with aneugens, which include colchicine, vinblastine, vincristine, as well as the clastogen cyclophosphamide (CP) were performed in mice to avoid the splenic elimination of micronucleated reticulocytes, which has been described in rats. Flow cytometry analysis revealed a non‐linear dose‐dependent increase in micronuclei frequencies for all tested aneugens, and a linear dose response curve for the clastogen, CP. To determine whether micronucleus induction at higher doses was due to chromosome loss (aneuploidy) or chromosome breakage (clastogenicity), flow sorting of the micronucleated reticulocytes and fluorescent in situ hybridization (FISH) with a mouse pan centromeric probe were performed for vinblastine, vincristine, and colchicine. Statistical evaluation of the flow cytometry and FISH data was performed to determine the threshold levels for chromosome loss in vivo. The threshold concentrations for vinblastine, vincristine, and colchicine were found at 0.35, 0.017, and 0.49 mg kg−1, respectively. Environ. Mol. Mutagen., 2010.

In vitro primary human lymphocyte flow cytometry based micronucleus assay: simultaneous assessment of cell proliferation, apoptosis and MN frequency

In order to minimise the number of positive in vitro cytogenetic results which are not confirmed in rodent carcinogenicity tests, biological systems that are p53 and DNA repair proficient should be recommended. Moreover, an appropriate cytotoxicity parameter for top dose selection should be considered. Recent International Conference on Harmonisation draft S2 and Organisation for Economic Co-operation and Development (OECD) 487 guideline accepted the in vitro micronucleus test (MNT) as a valid alternative method for in vitro chromosome aberration test within the in vitro cytogenetic test battery. Since mitosis is a prerequisite for expression of the micronuclei, it is compulsory to demonstrate that cell division occurred, and if possible, to identify the cells that completed mitosis. The OECD guideline recommends the use of a cytokinesis block for the assessment of proliferation in primary T-lymphocytes. The work presented in this manuscript was initiated to develop a novel flow cytometry-based primary human lymphocyte MNT method. This new assay is based on a three-step staining procedure: carboxyfluorescein succinimidyl ester as a proliferation marker, ethidium monoazide for chromatin of necrotic and late apoptotic cells discrimination and 4,6-diaminodino-2-phenylindole as a DNA marker. The proof of principle of the method was performed using genotoxic and non-genotoxic compounds: methyl methanesulfonate, mitomycin C, vinblastine sulphate, cyclophosphamide, sodium chloride and dexamethasone. It has been shown that the new flow cytometry-based primary human lymphocyte MNT method is at least equally reliable method as the standard Cytochalasin B MNT. However, further validation of the assay using a wide selection of compounds with a variety of mechanisms of action is required, before it can be used for regulatory purposes. Moreover, a miniaturisation of the technology may provide an additional advantage for early drug development.

Repair of Sparfloxacin-Induced Photochemical DNA Damage In Vivo

The induction and subsequent repair of photochemically induced DNA damage by sparfloxacin was assessed in different tissues of juvenile Wistar rats. The animals were treated once orally with 500 mg kg(-1) of sparfloxacin and irradiated 3 hours later with 7 J cm(-2) UVA. Induction and repair of DNA damage was studied in the skin, retina and cornea using the alkaline comet assay. After a tissue-specific increase in the initial DNA damage (higher in the cornea than in skin and retina), an exponential decrease was found in the skin and retina, whereas in cornea a further increase of the DNA damage after 1 hour followed by an exponential decrease was observed. The half-lives for DNA repair were approximately 3 hours for skin and retina and 1 hour for cornea. After a recovery time of 6 hours, the majority of the induced DNA damage detectable with the comet assay had been removed. In conclusion, the data indicate that (1) photochemically induced DNA damage by sparfloxacin is efficiently removed in skin, retina and cornea, (2) repair of these DNA lesions follows an exponential decrease, (3) the induction and repair of sparfloxacin-mediated photochemical DNA damage might be tissue specific.

Miniaturized flow cytometry-based in vitro primary human lymphocyte micronucleus assay—validation study

Most in vitro mammalian genotoxicity assays show a low specificity (high rate of irrelevant positive results), and therefore, lead to an increase in follow‐up in vivo genotoxicity testing. One of the sources of the high rate of in vitro irrelevant positive results that find no confirmation in in vivo studies may be the characteristics of the test system used. It has been shown that cells that are p53 deficient or carry an alteration in DNA repair genes may be more prone to produce high rate of false/irrelevant positive results. Primary human lymphocytes (HuLy) are considered to show a higher specificity in predicting the in vivo genotoxic potential of a tested compound. We recently developed a flow cytometry‐based primary human T‐lymphocyte micronucleus test (MNT) and showed that the technology is promising and reliable in detecting genotoxic compounds. The purpose of the present work was to develop and validate a miniaturized format of the assay. For validation purposes of the flow cytometry HuLy MNT a wide selection of compounds with different mechanisms of genotoxicity was used. The evaluation covered 30 compounds: 19 commercially available genotoxicants and nongenotoxicants and 11 early pharmaceutical development compounds. Being faster and less tedious than the microscopic analysis, the miniaturized flow cytometry‐based methodology showed very promising results. Conveniently, cell division is verified within the same sample as the MN frequency. Moreover analysis of hypodiploid events may provide an indication for a mode of action, i.e. clastogenic versus aneugenic mechanism, for further follow‐up testing. Mol. Mutagen. 2012.

Mutation Research/Genetic Toxicology and Environmental Mutagenesis

In the present study, the cyto-genotoxic effects of 12 CSCs prepared from a diverse set of cigarettes on human B lymphoblastoid cells were compared using five in vitro assays. The cells were exposed to CSCs at doses of 2.5, 5.0, 7.5, 10.0, and 12.5 × 10