Willi Suter - Academia.edu (original) (raw)

Papers by Willi Suter

Environmental and Molecular Mutagenesis, 2010

Appropriate follow-up actions and decisions are needed when evaluating and interpreting clear pos... more Appropriate follow-up actions and decisions are needed when evaluating and interpreting clear positive results obtained in the in vitro assays used in the initial genotoxicity screening battery (i.e., the battery of tests generally required by regulatory authorities) to assist in overall riskbased decision making concerning the potential effects of human exposure to the agent under test. Over the past few years, the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing developed a decision process flow chart to be applied in case of clear positive results in vitro. It provides for a variety of different possibilities and allows flexibility in choosing followup action(s), depending on the results obtained in the initial battery of assays and available information. The intent of the Review Subgroup was not to provide a prescriptive testing strategy, but rather to reinforce the concept of weighing the totality of the evidence. The Review Subgroup of the IVGT committee highlighted the importance of properly analyzing the existing data, and considering potential confounding factors (e.g., possible interactions with the test systems, presence of impurities, irrelevant metabolism), and chemical modes of action when analyzing and interpreting positive results in the in vitro genotoxicity assays and determining appropriate follow-up testing. The Review Subgroup also examined the characteristics, strengths, and limitations of each of the existing in vitro and in vivo genotoxicity assays to determine their usefulness in any follow-up testing.

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1997

A lambda ell positive selection was introduced (Jilkubczak et al., 1996) to assess gene mutations... more A lambda ell positive selection was introduced (Jilkubczak et al., 1996) to assess gene mutations using BigBlueGll transgen ic mice. In contrast to the color select ion method, the positive selection method increased the performance of the assay. We compared mutant frequen cy (MF) and mutation spectra of dimethylnitrosamine (DMN) between ell and lael genes. DMNinduced MF in ell (2S.8 X 10-5) was higher !han in lael (10 .7 x 10-5) as well as spontaneous MF (9.S x 10-5 in ell and 1.7 x 10-5 in lacl). The mutat ion spectra wert also different between ell and lacl genes. One base insertion at GGGGGG site was predominant in spontaneous ell mutants. One base delet ion as well as insertion at this site was also observed in DMN-induced mutants. In addition, DMN-induced mutants showed G:C to A:T trans itions in ell gene probably through d-methyl guan ine. On the other hand, deletions more than I-base were predominant among DMN-induced loci mutants as well as G:C to A:T transitions. A similar analysis is ongoing on MelQx-induced mutants.

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2004

5-(2-chloroethyl)-2'-deoxyuridine (CEDU) ... more 5-(2-chloroethyl)-2'-deoxyuridine (CEDU) is a pyrimidine nucleoside analogue formerly in development for the treatment of herpes simplex virus infections. The compound proved clearly mutagenic in the mouse spot test and exhibited weak activity in the Salmonella reverse mutation test, which led to the termination of the compound's development. In another study, CEDU, administered orally to beta-galactosidase (lacZ) transgenic mice (Muta Mouse) for five days, induced a clear increase in lacZ mutant frequencies in spleen, lung, and bone marrow. In the present follow-up study, we analyzed 32 of those lacZ mutants isolated from the bone marrow of the Muta Mouse animals of the experiments mentioned above, in order to obtain further information on the type of mutations induced by CEDU. CEDU induced a pronounced increase in A:T to G:C transitions. The distribution of A:T to G:C transitions was clearly non-random, showing a bias towards T to C substitutions in the coding DNA strand and a preference to occur in the sequence motif 5'-(G or C)-T-G-3'. Our data support the hypothesis that CEDU, after being phosphorylated, is incorporated into cellular DNA in place of thymidine, which leads to mispairing with guanosine during subsequent DNA replication. As a result, the compound is thought to exert its mutagenicity by inducing mismatches leading to T to C transitions. Our findings point towards a mode of mutagenic action of CEDU that differs fundamentally from that of other antiviral antinucleosides whose clastogenic and recombinogenic activities prevail.

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2004

5-(2-chloroethyl)-2&a... more 5-(2-chloroethyl)-2'-deoxyuridine (CEDU) had been developed for the treatment of herpes simplex infections. In the Salmonella reverse mutation test, the compound was found to be mutagenic in strains TA1535 and TA102 at very high concentrations (> or =2500 micro g/plate), both with and without S9-mix. The mutagenic potential of CEDU was further investigated in vivo and in vitro. It did not induce DNA repair in rat hepatocyte primary cultures, and was negative in the micronucleus test in V79 cells and in the comet assay in human leukocytes. In vivo, CEDU was negative in the bone marrow micronucleus test in CD1 mice. The mouse spot test provided a clearly positive result. Treatment of mice on day 9 of pregnancy with 2000 mg/kg resulted in 5.9% of the F1 animals having genetically relevant spots, whereas the corresponding vehicle control group had a spot rate of 1.9%. Since these data clearly identified CEDU as an inducer of gene mutations in vivo, this potential was further investigated in lacZ transgenic Muta Mouse. Six female animals were treated daily on five consecutive days with 2000 mg/kg/day and sacrificed, after a treatment-free sampling time, 14 days later. The data showed a clear increase in the mutant frequency in the bone marrow, the lung and in the spleen. CEDU is an exception in the group of nucleoside analogues, because it was found to be a strong gene mutagen and, in contrast to the other compounds of this group investigated so far, had no considerable clastogenic effects.

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1997

The single cell gel microelectrophoresis, or-comet-assay has become increasingly popular for meas... more The single cell gel microelectrophoresis, or-comet-assay has become increasingly popular for measuring both the magnitude and heterogeneity of damage by genotoxic agents in single cell populations. Many different techniques for quantifying comets have been reported, ranging from the simple measurement of-tail length-to more complex calculations of, for example, fraction of DNA in the comet tail, or of various moments of the DNA distribution. We have intercompared a number of alternative methods for quantitatively analyzing comet images, in order to evaluate tbe sensitivity and utility of the various approaches and their dependence on both biological and experimental variables. We conclude that the-tail moment-is one of the more versatile analytical techniques insofar as it is easily calculated, has a wide dynamic range and is relatively insensitive to experimental variables. Conversely, for specific applications, there can be advantages to the very sensitive (but quite restrictive) endpoint of tail length. Percent DNA in the tail is one of the more computationally intensive and therefore potentially subjective endpoints, but nonetheless also can be applicable to a variety of experimental questions. Supported by the National Cancer Institute of Canada.

Photochemical & Photobiological Sciences, 2008

Photosensitizing drugs increase the sensitivity of the skin and the eye toward normally harmless ... more Photosensitizing drugs increase the sensitivity of the skin and the eye toward normally harmless sunlight conditions and are known to enhance the induction of skin tumors or severe injuries to the eye. The photogenotoxicity of five common drugs (sparfloxacin, dacarbazine, chlorpromazine and 8-methoxypsoralen, promazine) was investigated in the skin as well as in the retina and cornea of Wistar rats. The compounds were administered once orally by gavage and the resulting DNA damage was analyzed in the newly developed in vivo photo comet assay. All drugs except of promazine were clearly photogenotoxic in the skin. In the cornea sparfloxacin and dacarbazine induced an increased DNA damage following irradiation. A photogenotoxic effect in the retina was observed by sparfloxacin, which is the only compound tested that absorbs wavelengths reaching the retina. The drug concentration analysis revealed that the compounds were distributed into plasma, skin and eye at concentrations, which were photogenotoxic in vitro. Additionally, histopathological analysis showed no relevant alterations or inductions of necrosis, apoptosis or inflammation in the skin or eye. In conclusion, we confirmed the photogenotoxic potential of compounds from different chemical classes in the skin. Moreover, it is the first time that photogenotoxicity has been detected in the retina and cornea in an in vivo study. Based on our results it is concluded that the photo comet assay in rat is an easy and reliable method to elucidate drug induced photogenotoxicity under conditions, which are relevant to human exposure. 23 Dacarbazine (imidazole derivative) Chemotherapeutic 250 60 24 Chlorpromazine HCl (phenothiazine derivative) Psychotic disorder 75 180

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2007

The use of flow cytometry with rat peripheral blood erythrocytes is expected to increase the sens... more The use of flow cytometry with rat peripheral blood erythrocytes is expected to increase the sensitivity of the in vivo micronucleus test and allows assessment of the genotoxic effects at doses that may be equal or close to those relevant to human exposure. However, there was a limitation to the use of rat peripheral blood erythrocytes since the spleen selectively removes micronucleated erythrocytes from circulation. In the present study, the selective analysis by flow cytometry of young MN-PCEs (micronucleated polychromatic erythrocytes or reticulocytes) by use of anti-CD71 antibodies was intended to compensate for the splenic clearance of micronucleated erythrocytes. The young polychromatic erythrocytes have on their surface a specific marker (CD71 antigen) that decreases in density during the maturation process. To investigate the usefulness of the flow cytometric micronucleus analysis combined with anti-CD71 staining of reticulocytes several compounds were tested in acute or sub-chronic treatment regimens. Furthermore, an evaluation was conducted in comparison with the standard rat bone-marrow micronucleus test with additional compounds. The results of acute studies with intraperitoneal application of ethyl methanesulfonate (EMS) (50, 100 and 200 mg/kg) and mitomycin C (MMC) (0.5, 1 and 2 mg/kg), were comparable to data published in the literature. Sub-chronic experiments were performed with cyclophosphamide (CP) (1, 2, 4 and 8 mg/(kg day)), colchicine (6, 8 mg/(kg day)) and mitomycin C (0.1 mg/(kg day)) and showed dose- and time-dependent accumulation of MN-PCEs. Parallel analysis of micronucleus induction in peripheral blood and bone marrow performed with Novartis compounds up to the highest tested dose (5 mg/kg of compound A, 200 mg/kg of compound B and 1250 mg/kg of compound C) showed concordant results. Furthermore, we performed kinetic studies of micronucleus induction in peripheral blood samples obtained at various times after a single treatment with 10 mg/kg CP and with 6 or 8 mg/kg of colchicine. Such experiments gave important supplementary information about the time course of micronucleus induction. Our data suggest that the peripheral blood flow-cytometry micronucleus test can be used for the assessment of micronucleus induction after acute and chronic exposures of rats to chemicals.

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2005

Sulfonic acid esters are considered as potentially alkylating agents that may exert genotoxic eff... more Sulfonic acid esters are considered as potentially alkylating agents that may exert genotoxic effects in bacterial and mammalian cell systems. One possible source of human exposure stems from drug synthesis when the salt-forming agents methanesulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid are used together with alcoholic solvents such as methanol, ethanol and propanol. In this study computer-assisted structural considerations and in vitro approaches (Ames mutagenicity test using Salmonella typhimurium strains TA98 and TA100, and the micronucleus test using L5178Y mouse lymphoma cells) were used to assess the genotoxic properties of 19 sulfonic esters. While all esters may be principally active as genotoxicants based on the presence of the sulfonate moiety, the statistical correlative multiple computer automated structure evaluation (MCASE) system (MC4PC version 1.0) using the Ames mutagenicity A2I module (version 1.54), rank-ordered the activity of the benzenesulfonic acid esters in the Ames test negligible due to an inactivating modulator and a deactivating fragment, whereas the methane-and toluenesulfonic acid esters were predicted to be positive in this test. In the Ames test, with the exception of the p-toluenesulfonic acid ethyl and iso-butyl esters, all compounds came out positive in Salmonella strain TA100. Methanesulfonic iso-propyl, sec-butyl and benzenesulfonic acid iso-propyl ester also showed mutagenic potential in strain TA98. In general, differences between results seen in Ames tests performed with or without metabolic activation were rather small. In L5178Y mouse lymphoma cells, benzenesulfonic acid n-and iso-butyl ester and p-toluenesulfonic acid iso-butyl ester did not increase the number of cells containing micronuclei. The other esters were positive in this micronucleus test; however, methanesulfonic acid iso-butyl ester was found to be only weakly positive at excessively cytotoxic concentrations. These

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2003

The alkaline Comet assay is becoming a useful tool for early genotoxicity testing of new pharmace... more The alkaline Comet assay is becoming a useful tool for early genotoxicity testing of new pharmaceutical drug candidates. The aim of this study was to elucidate the predictive value of Comet assay results for the outcome of the chromosome aberration (CA) test. For this purpose, a validation exercise with 13 drug candidates was carried out utilizing V79 Chinese hamster cells and human lymphocytes. The study demonstrates that results of the Comet assay and the chromosome aberration test show a high degree of agreement, irrespective of the cell type used. In the Comet assay, seven compounds were positive and six were negative, while in the CA test, six were positive and seven were negative. The only discrepancy was found with one compound that was positive in the Comet assay with V79 cells, negative in the Comet assay with human lymphocytes and clearly negative in the CA test with human lymphocytes. For the selection of concentrations for testing in the Comet assay, cytotoxicity by means of cell count after incubation or viability by means of Trypan-blue dye exclusion (TBDE) were used. The results show that either parameter led to analysis of a concentration range in the Comet assay similar to that chosen in the CA test, in which cell count (when using V79 cells) or mitotic index (in case of lymphocytes) were used. However, since cell count after incubation of cells is much more labour-intensive, viability was preferred as the parameter to assess cytotoxicity and for selecting concentrations for analysis in the Comet assay. The data presented in this study may contribute the regulatory acceptance of the Comet assay, e.g. for mechanistic studies.

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2001

We use the comet assay as part of our genotoxicity screening battery for newly synthesized drug c... more We use the comet assay as part of our genotoxicity screening battery for newly synthesized drug candidates. A dataset of more than 250 tests carried out with 75 drug candidates of various chemical classes was analyzed to elucidate the influence of cytotoxicity and compound precipitation on DNA migration in the comet assay. Using a V79 Chinese hamster cell line, 38 of the compounds were negative and 37 were positive in the comet assay. The reproducibility of test results between repeat experiments was 85%. Data on 72 tests with a negative call in which the compounds were tested up to highly cytotoxic concentrations demonstrated that cytotoxicity, as determined by Trypan blue dye exclusion and occurrence of cells with completely fragmented chromatin, did not lead to false positive test results. The majority (64.2%) of compounds with a positive call induced elevated DNA migration in the absence of excessive cytotoxicity. Compound precipitation was observed in 84 tests. In 88.1% of these cases, the test result at the precipitating concentration did not differ from that found at the highest soluble concentration. Half of the remaining 11.9% of contrary results (most of them weak effects) were not reproducible in the respective repeat experiment, indicating no or only a negligible influence of precipitation on test results. The data indicate that using V79 cells, the comet assay specifically detects genotoxic effects and is not confounded by cytotoxicity or compound precipitation under the conditions used.

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2002

AMP397 is a novel antiepileptic agent and the first competitive AMPA antagonist with high recepto... more AMP397 is a novel antiepileptic agent and the first competitive AMPA antagonist with high receptor affinity, good in vivo potency, and oral activity. AMP397 has a structural alert (aromatic nitro group) and was mutagenic in Salmonella typhimurium strains TA97a, TA98 and TA100 without S9, but negative in the nitroreductase-deficient strains TA98NR and TA100NR. The amino derivative of AMP397 was negative in wild-type strains TA98 and TA100. AMP397 was negative in a mouse lymphoma tk assay, which included a 24 h treatment without S9. A weak micronucleus induction in vitro was found at the highest concentrations tested in V79 cells with S9. AMP397 was negative in the following in vivo studies, which included the maximum tolerated doses of 320 mg/kg in mice and 2000 mg/kg in rats: Muta TM Mouse assay in colon and liver (5 × 320 mg/kg) at three sampling times (3, 7 and 31 days after the last administration); DNA binding study in the liver of mice and rats after a single treatment with [ 14 C]-AMP397; comet assay (1 × 2000 mg/kg) in jejunum and liver of rats, sampling times 3 and 24 h after administration; micronucleus test (2 × 320 mg/kg) in the bone marrow of mice, sampling 24 h after the second administration. Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo. In particular, no genotoxic metabolite is formed in mammalian cells, and, if formed by intestinal bacteria, is unable to exert any genotoxic activity in the adjacent intestinal tissue. These data were considered to provide sufficient safety to initiate clinical development of the compound.

Journal of Pharmacological and Toxicological Methods, 2007

Journal of Investigative Dermatology, 2009

The induction and subsequent repair of photochemically induced DNA damage by sparfloxacin was ass... more 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 À1 of sparfloxacin and irradiated 3 hours later with 7 J cm À2 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.

Heart Rhythm, 2012

BACKGROUND The ventricular components (QRS and QT) on the electrocardiogram (ECG) depend on the p... more BACKGROUND The ventricular components (QRS and QT) on the electrocardiogram (ECG) depend on the properties of ventricular action potentials that can be modulated by drugs via specific ion channels. However, the correlation of ECG ventricular waveforms with underlying ion actions is not well established and has been extensively debated. OBJECTIVE To conduct a blinded in vitro assessment of the ionic mechanisms for drug-induced ECG changes. METHODS AND RESULTS Fourteen cardiac and noncardiac drugs with known effects on cardiac ion channels were selected by the study sponsor, and were tested in the rabbit left ventricular wedge preparation with recording of the ECG and contractility. The investigators who performed the experiments and analyzed the data were blinded to names, concentrations, and molecular weights of the drugs. The compounds were prepared by the sponsor and sent to the investigators as 56 stock solutions. The effects of I Kr , I Ks , I Ca,L , I Na blocker, and I KATP opener on QRS, QT, and T p-e , were evaluated. Disclosure of the names and concentrations after completion of the study revealed that there were highly correlated ECG changes with underlying ionic mechanisms and proarrhythmic potential of drugs that, respectively, target I Kr , I Ks , I Ca,L , I Na , and I KATP. Among ECG parameters, T p-e was more useful in differentiating drugs' actions. CONCLUSIONS Specific electrophysiological action and the consequent proarrhythmic potential of a drug can be accurately determined by analysis of drug-induced changes in ECG in the rabbit left ventricular wedge preparation. Change in T p-e provides the most relevant information.

European Journal of Pharmacology, 2004

Several antimalarial drugs are known to produce a QT interval prolongation via a blockade of the ... more Several antimalarial drugs are known to produce a QT interval prolongation via a blockade of the rapidly activating delayed rectifier K + current (I Kr), encoded by the human-ether-ago go related gene (hERG). We investigated the influence of lumefantrine and its major metabolite desbutyl-lumefantrine, as well as halofantrine, chloroquine, and mefloquine, on wild type hERG K + channels in stably transfected human embryonic kidney cells (HEK293) using the whole cell patch-clamp technique. All of the tested antimalarial drugs inhibited the hERG K + channels in a concentration-and time-dependent manner. Only halofantrine blocked hERG tail currents voltage-dependently. The ranking of the half-maximal inhibitory concentrations (IC 50) of the antimalarials was: halofantrine (0.04 AM) < chloroquine (2.5 AM) < mefloquine (2.6 AM) < desbutyl-lumefantrine (5.5 AM) < lumefantrine (8.1 AM). Lumefantrine and desbutyl-lumefantrine showed a slower inhibition of I Kr than the other tested antimalarials. In conclusion, lumefantrine and desbutyl-lumefantrine inhibited significantly the hERG tail current with a higher IC 50-value than mefloquine, chloroquine and halofantrine. This, together with the calculated cardiac safety indices, suggests that lumefantrine and desbutyl-lumefantrine have a weaker proarrhythmic potential than their comparator compounds.

Environmental and Molecular Mutagenesis, 2012

Most in vitro mammalian genotoxicity assays show a low specificity (high rate of irrelevant posit... more Most in vitro mammalian genotoxicity assays show a low specificity (high rate of irrelevant positive results), and therefore, lead to an increase in followup 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.

Environmental and Molecular Mutagenesis, 2009

Non-DNA binding genotoxins (e.g., aneugens), unlike DNA-binding genotoxins, are theoretically exp... more 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.

Environmental and Molecular Mutagenesis, 1998

Measurement of the frequency of micronuclei induced in cells by ionizing radiation or by chemical... more Measurement of the frequency of micronuclei induced in cells by ionizing radiation or by chemical treatment is widely used to analyze cytogenetic damage. The microscopic scoring of micronuclei is a tedious and time-consuming procedure. Therefore, attempts have been made to automate micronuclei scoring by means of image analysis or flow cytometry. A new procedure for the flow cytometric analysis of chemically induced micronuclei in V79 Chinese hamster cells has been established in our laboratory. Debris was separated from micronuclei by means of a new gating procedure using area and width fluorescence of the stained suspension of micronuclei and nuclei. In order to test the sensitivity and specificity of this improved method of flow cytometric analysis, five well-known mutagenic compounds were tested. With the new technique, the frequency of micronuclei measured and analyzed corresponded well with results obtained by conventional microscopy. In addition, a large series of negative compounds, and weak, middle, and strong micronuclei inducers, were tested in order to establish criteria for discrimination between genotoxic and nongenotoxic compounds by flow cytometry. This new procedure for flow cytometric detection of micronuclei represents a quick, reliable, and relatively simple method for in vitro micronucleus testing.

Environmental and Molecular Mutagenesis, 2010

Appropriate follow-up actions and decisions are needed when evaluating and interpreting clear pos... more Appropriate follow-up actions and decisions are needed when evaluating and interpreting clear positive results obtained in the in vitro assays used in the initial genotoxicity screening battery (i.e., the battery of tests generally required by regulatory authorities) to assist in overall riskbased decision making concerning the potential effects of human exposure to the agent under test. Over the past few years, the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing developed a decision process flow chart to be applied in case of clear positive results in vitro. It provides for a variety of different possibilities and allows flexibility in choosing followup action(s), depending on the results obtained in the initial battery of assays and available information. The intent of the Review Subgroup was not to provide a prescriptive testing strategy, but rather to reinforce the concept of weighing the totality of the evidence. The Review Subgroup of the IVGT committee highlighted the importance of properly analyzing the existing data, and considering potential confounding factors (e.g., possible interactions with the test systems, presence of impurities, irrelevant metabolism), and chemical modes of action when analyzing and interpreting positive results in the in vitro genotoxicity assays and determining appropriate follow-up testing. The Review Subgroup also examined the characteristics, strengths, and limitations of each of the existing in vitro and in vivo genotoxicity assays to determine their usefulness in any follow-up testing.

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1997

A lambda ell positive selection was introduced (Jilkubczak et al., 1996) to assess gene mutations... more A lambda ell positive selection was introduced (Jilkubczak et al., 1996) to assess gene mutations using BigBlueGll transgen ic mice. In contrast to the color select ion method, the positive selection method increased the performance of the assay. We compared mutant frequen cy (MF) and mutation spectra of dimethylnitrosamine (DMN) between ell and lael genes. DMNinduced MF in ell (2S.8 X 10-5) was higher !han in lael (10 .7 x 10-5) as well as spontaneous MF (9.S x 10-5 in ell and 1.7 x 10-5 in lacl). The mutat ion spectra wert also different between ell and lacl genes. One base insertion at GGGGGG site was predominant in spontaneous ell mutants. One base delet ion as well as insertion at this site was also observed in DMN-induced mutants. In addition, DMN-induced mutants showed G:C to A:T trans itions in ell gene probably through d-methyl guan ine. On the other hand, deletions more than I-base were predominant among DMN-induced loci mutants as well as G:C to A:T transitions. A similar analysis is ongoing on MelQx-induced mutants.

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2004

5-(2-chloroethyl)-2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-deoxyuridine (CEDU) ... more 5-(2-chloroethyl)-2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-deoxyuridine (CEDU) is a pyrimidine nucleoside analogue formerly in development for the treatment of herpes simplex virus infections. The compound proved clearly mutagenic in the mouse spot test and exhibited weak activity in the Salmonella reverse mutation test, which led to the termination of the compound&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s development. In another study, CEDU, administered orally to beta-galactosidase (lacZ) transgenic mice (Muta Mouse) for five days, induced a clear increase in lacZ mutant frequencies in spleen, lung, and bone marrow. In the present follow-up study, we analyzed 32 of those lacZ mutants isolated from the bone marrow of the Muta Mouse animals of the experiments mentioned above, in order to obtain further information on the type of mutations induced by CEDU. CEDU induced a pronounced increase in A:T to G:C transitions. The distribution of A:T to G:C transitions was clearly non-random, showing a bias towards T to C substitutions in the coding DNA strand and a preference to occur in the sequence motif 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-(G or C)-T-G-3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;. Our data support the hypothesis that CEDU, after being phosphorylated, is incorporated into cellular DNA in place of thymidine, which leads to mispairing with guanosine during subsequent DNA replication. As a result, the compound is thought to exert its mutagenicity by inducing mismatches leading to T to C transitions. Our findings point towards a mode of mutagenic action of CEDU that differs fundamentally from that of other antiviral antinucleosides whose clastogenic and recombinogenic activities prevail.

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2004

5-(2-chloroethyl)-2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;a... more 5-(2-chloroethyl)-2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-deoxyuridine (CEDU) had been developed for the treatment of herpes simplex infections. In the Salmonella reverse mutation test, the compound was found to be mutagenic in strains TA1535 and TA102 at very high concentrations (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; or =2500 micro g/plate), both with and without S9-mix. The mutagenic potential of CEDU was further investigated in vivo and in vitro. It did not induce DNA repair in rat hepatocyte primary cultures, and was negative in the micronucleus test in V79 cells and in the comet assay in human leukocytes. In vivo, CEDU was negative in the bone marrow micronucleus test in CD1 mice. The mouse spot test provided a clearly positive result. Treatment of mice on day 9 of pregnancy with 2000 mg/kg resulted in 5.9% of the F1 animals having genetically relevant spots, whereas the corresponding vehicle control group had a spot rate of 1.9%. Since these data clearly identified CEDU as an inducer of gene mutations in vivo, this potential was further investigated in lacZ transgenic Muta Mouse. Six female animals were treated daily on five consecutive days with 2000 mg/kg/day and sacrificed, after a treatment-free sampling time, 14 days later. The data showed a clear increase in the mutant frequency in the bone marrow, the lung and in the spleen. CEDU is an exception in the group of nucleoside analogues, because it was found to be a strong gene mutagen and, in contrast to the other compounds of this group investigated so far, had no considerable clastogenic effects.

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1997

The single cell gel microelectrophoresis, or-comet-assay has become increasingly popular for meas... more The single cell gel microelectrophoresis, or-comet-assay has become increasingly popular for measuring both the magnitude and heterogeneity of damage by genotoxic agents in single cell populations. Many different techniques for quantifying comets have been reported, ranging from the simple measurement of-tail length-to more complex calculations of, for example, fraction of DNA in the comet tail, or of various moments of the DNA distribution. We have intercompared a number of alternative methods for quantitatively analyzing comet images, in order to evaluate tbe sensitivity and utility of the various approaches and their dependence on both biological and experimental variables. We conclude that the-tail moment-is one of the more versatile analytical techniques insofar as it is easily calculated, has a wide dynamic range and is relatively insensitive to experimental variables. Conversely, for specific applications, there can be advantages to the very sensitive (but quite restrictive) endpoint of tail length. Percent DNA in the tail is one of the more computationally intensive and therefore potentially subjective endpoints, but nonetheless also can be applicable to a variety of experimental questions. Supported by the National Cancer Institute of Canada.

Photochemical & Photobiological Sciences, 2008

Photosensitizing drugs increase the sensitivity of the skin and the eye toward normally harmless ... more Photosensitizing drugs increase the sensitivity of the skin and the eye toward normally harmless sunlight conditions and are known to enhance the induction of skin tumors or severe injuries to the eye. The photogenotoxicity of five common drugs (sparfloxacin, dacarbazine, chlorpromazine and 8-methoxypsoralen, promazine) was investigated in the skin as well as in the retina and cornea of Wistar rats. The compounds were administered once orally by gavage and the resulting DNA damage was analyzed in the newly developed in vivo photo comet assay. All drugs except of promazine were clearly photogenotoxic in the skin. In the cornea sparfloxacin and dacarbazine induced an increased DNA damage following irradiation. A photogenotoxic effect in the retina was observed by sparfloxacin, which is the only compound tested that absorbs wavelengths reaching the retina. The drug concentration analysis revealed that the compounds were distributed into plasma, skin and eye at concentrations, which were photogenotoxic in vitro. Additionally, histopathological analysis showed no relevant alterations or inductions of necrosis, apoptosis or inflammation in the skin or eye. In conclusion, we confirmed the photogenotoxic potential of compounds from different chemical classes in the skin. Moreover, it is the first time that photogenotoxicity has been detected in the retina and cornea in an in vivo study. Based on our results it is concluded that the photo comet assay in rat is an easy and reliable method to elucidate drug induced photogenotoxicity under conditions, which are relevant to human exposure. 23 Dacarbazine (imidazole derivative) Chemotherapeutic 250 60 24 Chlorpromazine HCl (phenothiazine derivative) Psychotic disorder 75 180

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2007

The use of flow cytometry with rat peripheral blood erythrocytes is expected to increase the sens... more The use of flow cytometry with rat peripheral blood erythrocytes is expected to increase the sensitivity of the in vivo micronucleus test and allows assessment of the genotoxic effects at doses that may be equal or close to those relevant to human exposure. However, there was a limitation to the use of rat peripheral blood erythrocytes since the spleen selectively removes micronucleated erythrocytes from circulation. In the present study, the selective analysis by flow cytometry of young MN-PCEs (micronucleated polychromatic erythrocytes or reticulocytes) by use of anti-CD71 antibodies was intended to compensate for the splenic clearance of micronucleated erythrocytes. The young polychromatic erythrocytes have on their surface a specific marker (CD71 antigen) that decreases in density during the maturation process. To investigate the usefulness of the flow cytometric micronucleus analysis combined with anti-CD71 staining of reticulocytes several compounds were tested in acute or sub-chronic treatment regimens. Furthermore, an evaluation was conducted in comparison with the standard rat bone-marrow micronucleus test with additional compounds. The results of acute studies with intraperitoneal application of ethyl methanesulfonate (EMS) (50, 100 and 200 mg/kg) and mitomycin C (MMC) (0.5, 1 and 2 mg/kg), were comparable to data published in the literature. Sub-chronic experiments were performed with cyclophosphamide (CP) (1, 2, 4 and 8 mg/(kg day)), colchicine (6, 8 mg/(kg day)) and mitomycin C (0.1 mg/(kg day)) and showed dose- and time-dependent accumulation of MN-PCEs. Parallel analysis of micronucleus induction in peripheral blood and bone marrow performed with Novartis compounds up to the highest tested dose (5 mg/kg of compound A, 200 mg/kg of compound B and 1250 mg/kg of compound C) showed concordant results. Furthermore, we performed kinetic studies of micronucleus induction in peripheral blood samples obtained at various times after a single treatment with 10 mg/kg CP and with 6 or 8 mg/kg of colchicine. Such experiments gave important supplementary information about the time course of micronucleus induction. Our data suggest that the peripheral blood flow-cytometry micronucleus test can be used for the assessment of micronucleus induction after acute and chronic exposures of rats to chemicals.

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2005

Sulfonic acid esters are considered as potentially alkylating agents that may exert genotoxic eff... more Sulfonic acid esters are considered as potentially alkylating agents that may exert genotoxic effects in bacterial and mammalian cell systems. One possible source of human exposure stems from drug synthesis when the salt-forming agents methanesulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid are used together with alcoholic solvents such as methanol, ethanol and propanol. In this study computer-assisted structural considerations and in vitro approaches (Ames mutagenicity test using Salmonella typhimurium strains TA98 and TA100, and the micronucleus test using L5178Y mouse lymphoma cells) were used to assess the genotoxic properties of 19 sulfonic esters. While all esters may be principally active as genotoxicants based on the presence of the sulfonate moiety, the statistical correlative multiple computer automated structure evaluation (MCASE) system (MC4PC version 1.0) using the Ames mutagenicity A2I module (version 1.54), rank-ordered the activity of the benzenesulfonic acid esters in the Ames test negligible due to an inactivating modulator and a deactivating fragment, whereas the methane-and toluenesulfonic acid esters were predicted to be positive in this test. In the Ames test, with the exception of the p-toluenesulfonic acid ethyl and iso-butyl esters, all compounds came out positive in Salmonella strain TA100. Methanesulfonic iso-propyl, sec-butyl and benzenesulfonic acid iso-propyl ester also showed mutagenic potential in strain TA98. In general, differences between results seen in Ames tests performed with or without metabolic activation were rather small. In L5178Y mouse lymphoma cells, benzenesulfonic acid n-and iso-butyl ester and p-toluenesulfonic acid iso-butyl ester did not increase the number of cells containing micronuclei. The other esters were positive in this micronucleus test; however, methanesulfonic acid iso-butyl ester was found to be only weakly positive at excessively cytotoxic concentrations. These

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2003

The alkaline Comet assay is becoming a useful tool for early genotoxicity testing of new pharmace... more The alkaline Comet assay is becoming a useful tool for early genotoxicity testing of new pharmaceutical drug candidates. The aim of this study was to elucidate the predictive value of Comet assay results for the outcome of the chromosome aberration (CA) test. For this purpose, a validation exercise with 13 drug candidates was carried out utilizing V79 Chinese hamster cells and human lymphocytes. The study demonstrates that results of the Comet assay and the chromosome aberration test show a high degree of agreement, irrespective of the cell type used. In the Comet assay, seven compounds were positive and six were negative, while in the CA test, six were positive and seven were negative. The only discrepancy was found with one compound that was positive in the Comet assay with V79 cells, negative in the Comet assay with human lymphocytes and clearly negative in the CA test with human lymphocytes. For the selection of concentrations for testing in the Comet assay, cytotoxicity by means of cell count after incubation or viability by means of Trypan-blue dye exclusion (TBDE) were used. The results show that either parameter led to analysis of a concentration range in the Comet assay similar to that chosen in the CA test, in which cell count (when using V79 cells) or mitotic index (in case of lymphocytes) were used. However, since cell count after incubation of cells is much more labour-intensive, viability was preferred as the parameter to assess cytotoxicity and for selecting concentrations for analysis in the Comet assay. The data presented in this study may contribute the regulatory acceptance of the Comet assay, e.g. for mechanistic studies.

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2001

We use the comet assay as part of our genotoxicity screening battery for newly synthesized drug c... more We use the comet assay as part of our genotoxicity screening battery for newly synthesized drug candidates. A dataset of more than 250 tests carried out with 75 drug candidates of various chemical classes was analyzed to elucidate the influence of cytotoxicity and compound precipitation on DNA migration in the comet assay. Using a V79 Chinese hamster cell line, 38 of the compounds were negative and 37 were positive in the comet assay. The reproducibility of test results between repeat experiments was 85%. Data on 72 tests with a negative call in which the compounds were tested up to highly cytotoxic concentrations demonstrated that cytotoxicity, as determined by Trypan blue dye exclusion and occurrence of cells with completely fragmented chromatin, did not lead to false positive test results. The majority (64.2%) of compounds with a positive call induced elevated DNA migration in the absence of excessive cytotoxicity. Compound precipitation was observed in 84 tests. In 88.1% of these cases, the test result at the precipitating concentration did not differ from that found at the highest soluble concentration. Half of the remaining 11.9% of contrary results (most of them weak effects) were not reproducible in the respective repeat experiment, indicating no or only a negligible influence of precipitation on test results. The data indicate that using V79 cells, the comet assay specifically detects genotoxic effects and is not confounded by cytotoxicity or compound precipitation under the conditions used.

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2002

AMP397 is a novel antiepileptic agent and the first competitive AMPA antagonist with high recepto... more AMP397 is a novel antiepileptic agent and the first competitive AMPA antagonist with high receptor affinity, good in vivo potency, and oral activity. AMP397 has a structural alert (aromatic nitro group) and was mutagenic in Salmonella typhimurium strains TA97a, TA98 and TA100 without S9, but negative in the nitroreductase-deficient strains TA98NR and TA100NR. The amino derivative of AMP397 was negative in wild-type strains TA98 and TA100. AMP397 was negative in a mouse lymphoma tk assay, which included a 24 h treatment without S9. A weak micronucleus induction in vitro was found at the highest concentrations tested in V79 cells with S9. AMP397 was negative in the following in vivo studies, which included the maximum tolerated doses of 320 mg/kg in mice and 2000 mg/kg in rats: Muta TM Mouse assay in colon and liver (5 × 320 mg/kg) at three sampling times (3, 7 and 31 days after the last administration); DNA binding study in the liver of mice and rats after a single treatment with [ 14 C]-AMP397; comet assay (1 × 2000 mg/kg) in jejunum and liver of rats, sampling times 3 and 24 h after administration; micronucleus test (2 × 320 mg/kg) in the bone marrow of mice, sampling 24 h after the second administration. Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo. In particular, no genotoxic metabolite is formed in mammalian cells, and, if formed by intestinal bacteria, is unable to exert any genotoxic activity in the adjacent intestinal tissue. These data were considered to provide sufficient safety to initiate clinical development of the compound.

Journal of Pharmacological and Toxicological Methods, 2007

Journal of Investigative Dermatology, 2009

The induction and subsequent repair of photochemically induced DNA damage by sparfloxacin was ass... more 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 À1 of sparfloxacin and irradiated 3 hours later with 7 J cm À2 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.

Heart Rhythm, 2012

BACKGROUND The ventricular components (QRS and QT) on the electrocardiogram (ECG) depend on the p... more BACKGROUND The ventricular components (QRS and QT) on the electrocardiogram (ECG) depend on the properties of ventricular action potentials that can be modulated by drugs via specific ion channels. However, the correlation of ECG ventricular waveforms with underlying ion actions is not well established and has been extensively debated. OBJECTIVE To conduct a blinded in vitro assessment of the ionic mechanisms for drug-induced ECG changes. METHODS AND RESULTS Fourteen cardiac and noncardiac drugs with known effects on cardiac ion channels were selected by the study sponsor, and were tested in the rabbit left ventricular wedge preparation with recording of the ECG and contractility. The investigators who performed the experiments and analyzed the data were blinded to names, concentrations, and molecular weights of the drugs. The compounds were prepared by the sponsor and sent to the investigators as 56 stock solutions. The effects of I Kr , I Ks , I Ca,L , I Na blocker, and I KATP opener on QRS, QT, and T p-e , were evaluated. Disclosure of the names and concentrations after completion of the study revealed that there were highly correlated ECG changes with underlying ionic mechanisms and proarrhythmic potential of drugs that, respectively, target I Kr , I Ks , I Ca,L , I Na , and I KATP. Among ECG parameters, T p-e was more useful in differentiating drugs' actions. CONCLUSIONS Specific electrophysiological action and the consequent proarrhythmic potential of a drug can be accurately determined by analysis of drug-induced changes in ECG in the rabbit left ventricular wedge preparation. Change in T p-e provides the most relevant information.

European Journal of Pharmacology, 2004

Several antimalarial drugs are known to produce a QT interval prolongation via a blockade of the ... more Several antimalarial drugs are known to produce a QT interval prolongation via a blockade of the rapidly activating delayed rectifier K + current (I Kr), encoded by the human-ether-ago go related gene (hERG). We investigated the influence of lumefantrine and its major metabolite desbutyl-lumefantrine, as well as halofantrine, chloroquine, and mefloquine, on wild type hERG K + channels in stably transfected human embryonic kidney cells (HEK293) using the whole cell patch-clamp technique. All of the tested antimalarial drugs inhibited the hERG K + channels in a concentration-and time-dependent manner. Only halofantrine blocked hERG tail currents voltage-dependently. The ranking of the half-maximal inhibitory concentrations (IC 50) of the antimalarials was: halofantrine (0.04 AM) < chloroquine (2.5 AM) < mefloquine (2.6 AM) < desbutyl-lumefantrine (5.5 AM) < lumefantrine (8.1 AM). Lumefantrine and desbutyl-lumefantrine showed a slower inhibition of I Kr than the other tested antimalarials. In conclusion, lumefantrine and desbutyl-lumefantrine inhibited significantly the hERG tail current with a higher IC 50-value than mefloquine, chloroquine and halofantrine. This, together with the calculated cardiac safety indices, suggests that lumefantrine and desbutyl-lumefantrine have a weaker proarrhythmic potential than their comparator compounds.

Environmental and Molecular Mutagenesis, 2012

Most in vitro mammalian genotoxicity assays show a low specificity (high rate of irrelevant posit... more Most in vitro mammalian genotoxicity assays show a low specificity (high rate of irrelevant positive results), and therefore, lead to an increase in followup 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.

Environmental and Molecular Mutagenesis, 2009

Non-DNA binding genotoxins (e.g., aneugens), unlike DNA-binding genotoxins, are theoretically exp... more 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.

Environmental and Molecular Mutagenesis, 1998

Measurement of the frequency of micronuclei induced in cells by ionizing radiation or by chemical... more Measurement of the frequency of micronuclei induced in cells by ionizing radiation or by chemical treatment is widely used to analyze cytogenetic damage. The microscopic scoring of micronuclei is a tedious and time-consuming procedure. Therefore, attempts have been made to automate micronuclei scoring by means of image analysis or flow cytometry. A new procedure for the flow cytometric analysis of chemically induced micronuclei in V79 Chinese hamster cells has been established in our laboratory. Debris was separated from micronuclei by means of a new gating procedure using area and width fluorescence of the stained suspension of micronuclei and nuclei. In order to test the sensitivity and specificity of this improved method of flow cytometric analysis, five well-known mutagenic compounds were tested. With the new technique, the frequency of micronuclei measured and analyzed corresponded well with results obtained by conventional microscopy. In addition, a large series of negative compounds, and weak, middle, and strong micronuclei inducers, were tested in order to establish criteria for discrimination between genotoxic and nongenotoxic compounds by flow cytometry. This new procedure for flow cytometric detection of micronuclei represents a quick, reliable, and relatively simple method for in vitro micronucleus testing.