In vitro micronucleus assay with Chinese hamster V79 cells — results of a collaborative study with in situ exposure to 26 chemical substances

Hude, Wilhelm von der; Kalweit, S.; Engelhardt, G.; McKiernan, Sinead; Kasper, P.; Slacik-Erben, Renate; Miltenburger, H.G.; Honarvar, Naveed; Fahrig, Rudolf; Görlitz, Bernd; Albertini, Silvio; Kirchner, Stephan; Utesch, Dietmar; Pötter-Locher, Franziska; Stopper, Helga; Madle, Stephan

doi:10.1016/s1383-5718(00)00045-0

Cited by 85 publications

(29 citation statements)

References 38 publications

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“…In addition to L5178Y cells, CHO [24][25][26], V79 [27], HepG2 [28], TK6 [29], and other cell lines have various advantageous characteristics. For instance, preliminary work with the attachment cell lines CHO-K1 and HepG2 indicate that the EMA dye and subsequent lysis solutions described herein can be directly applied to cells that are affixed to growth vessels, thereby liberating nuclei and MN without the need for trypsinization.…”

Section: Compatibility With Other Cell Linesmentioning

confidence: 99%

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

Bryce

Avlasevich

Bemis

et al. 2008

Mutation Research/Genetic Toxicology and Environmental Mutagene

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An international, multi-lab trial was conducted to evaluate a flow cytometry-based method for scoring micronuclei in mouse lymphoma L5178Y cells [Avlasevich et al., Environ. Molec. Mutagen. 47 (2006) [56][57][58][59][60][61][62][63][64][65][66]. A reference laboratory investigated the potential of six chemicals to induce micronuclei-the genotoxicants mitomycin C, etoposide, and vinblastine, and the non-genotoxicants sucrose, staurosporine, or dexamethasone. The latter two non-genotoxicants were selected as extreme challenges to the assay because of their potent apoptogenic activity. Three collaborating laboratories were supplied with prototype In Vitro MicroFlow ™ kits, and each was assigned one genotoxicant and one non-genotoxicant. Cells were treated continuously for 24 hrs over a range of concentrations up to 5 mg/ml, or overtly cytotoxic concentrations. Micronuclei were scored via standard microscopy and flow cytometry. In addition to enumerating micronucleus frequencies, a cytotoxicity measurement that is simultaneously acquired with the flow cytometric micronucleus scoring procedure was evaluated (Flow-NBR). With this method, latex particles served as counting beads, and facilitated relative survival measurements that exclude the presence of dead/dying cells. For comparison purposes, additional cytotoxicity endpoints were measured, including several that are based on cell number, and others that reflect compromised membrane integrity, including dye permeability and/or phospholipid distribution. Key findings for this set of compounds include the following: (1) significant discrepancies in top concentration selection were found when cytotoxicity measurements were based on different methods, with the Flow-NBR approach tending to be the most sensitive, (2) both microscopy-and flow cytometry-based scoring methods detected concentrationdependent micronucleus formation for the three genotoxic agents studied, with good agreement between the reference laboratory and the collaborating laboratories, and (3) whereas flow cytometric analyses showed no significant increases for the non-genotoxicants when top concentration selection was based on Flow-NBR, significantly elevated micronucleus frequencies were observed for concentrations that were chosen based on less-sensitive cytotoxicity assays. Collectively, these results indicate that rapid assessment of genotoxicity can be accomplished with a relatively simple Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

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Section: Compatibility With Other Cell Linesmentioning

confidence: 99%

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

Bryce

Avlasevich

Bemis

et al. 2008

Mutation Research/Genetic Toxicology and Environmental Mutagene

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“…Compounds were dissolved in DMSO and tested over the concentration range of 0.25 to 250 g/ml. The in vitro micronucleus test (49) was performed with and without S9 activation over the concentration range of 0.5 to 280 g/ml, using linearly growing V79-4 Chinese hamster lung fibroblasts (ATCC strain CCL-93) (1% DMSO in each assay well).…”

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confidence: 99%

In Vitro and In Vivo Properties of Dihydrophthalazine Antifolates, a Novel Family of Antibacterial Drugs

Caspers

Bury

Gaucher

et al. 2009

Antimicrob Agents Chemother

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Racemic 2,4-diaminopyrimidine dihydrophthalazine derivatives BAL0030543, BAL0030544, and BAL0030545 exhibited low in vitro MICs toward small, selected panels of Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae, Moraxella catarrhalis, and Mycobacterium avium, though the compounds were less active against Haemophilus influenzae. The constellation of dihydrofolate reductases (DHFRs) present in 20 enterococci and 40 staphylococci was analyzed and correlated with the antibacterial activities of the dihydrophthalazines and trimethoprim. DHFRs encoded by dfrB, dfrA (S1 isozyme), dfrE, and folA were susceptible to the dihydrophthalazines, whereas DHFRs encoded by dfrG (S3 isozyme) and dfrF were not. Studies with the separated enantiomers of BAL0030543, BAL0030544, and BAL0030545 revealed preferential inhibition of susceptible DHFRs by the (R)-enantiomers. BAL0030543, BAL0030544, and BAL0030545 were well tolerated by mice during 5-and 10-day oral toxicity studies at doses of up to 400 mg/kg of body weight. Using a nonoptimized formulation, the dihydrophthalazines displayed acceptable oral bioavailabilities in mice, and efficacy studies with a septicemia model of mice infected with trimethoprim-resistant, methicillin-resistant Staphylococcus aureus gave 50% effective dose values in the range of 1.6 to 6.25 mg/kg.Despite sustained efforts to identify new drugs against antibiotic-resistant bacteria, treatment of infections caused by vancomycin-resistant enterococci and methicillin-resistant staphylococci continues to challenge clinicians (5). Enterococci are often associated with urinary tract infections, intra-abdominal abscesses, endocarditis, and bacteremia, whereas staphylococci are common causes of skin and skin structure infections, endocarditis, osteomyelitis, prosthetic joint infections, catheterrelated bacteremia, and nosocomial pneumonia. Methicillinresistant Staphylococcus aureus (MRSA) accounts for approximately 50% of all S. aureus infections in the United States (24). Once confined to healthcare settings, MRSA has emerged as a serious cause of community-acquired skin and skin structure infections (32), and an increased frequency of skin cultures positive for S. aureus is associated with nasal and intestinal colonization (2).Dihydrofolate reductase (DHFR; EC 1.5.1.3) is an essential enzyme in most pathogenic bacteria, and the clinical success of trimethoprim (28,33,41,43) confirms DHFR as an important chemotherapeutic target. A phenylalanine3tyrosine substitution at position 98 in the wild-type, nontransferable, chromosomally encoded DHFR renders S. aureus resistant to trimethoprim (15); a corresponding tyrosyl residue occurs in transferable plasmid-encoded wild-type staphylococcal DHFR isozymes S1, S2, and S3 (13,14,40) and in the enterococcal DHFRs encoded by dfrE and dfrF. The gene encoding DHFR S1 may also be associated with staphylococcal chromosomal cassette mec N1 (SCCmec-N1) (37).BAL0030543, BAL0030544, and BAL0030545 are novel dihydrophthalazine antifolates with potent activities a...

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“…For example, our industrial set of results was derived in a single testing facility using one cell line for each in vitro assay and standardized test response criteria when compared with a similar industrial evaluation by Miller et al [1997], in which there were four contributing laboratories with multiple cell lines and variable test response criteria utilized. In our study, we used chemical unknowns (potential pharmaceuticals) similar to Miller's industrial experience [Miller et al, 1997] but very different from other studies in which commercially available compounds were utilized [Miller et al, 1998;Matsushima et al, 1999;von der Hude et al, 2000]. In addition, because we did not include a cytotoxic cutoff in the CBMN assay and because we did not have to achieve minimal allowable levels of cytotoxicity for a valid test, we were able to take a unique look at the effect on concordance with the CA assay that resulted from seemingly high or low levels of compound-induced cytotoxicity in the CBMN assay.…”

Section: Resultsmentioning

confidence: 99%

“…The GUM working group looked at published data from 30 compounds and reported 80% (24/30) correlation between the IVMN and CA assays; they attributed 6 of 30 (20%) discordant results to known or suspected aneugens [Miller et al, 1998] that are not readily detected in the CA assay. Matsushima et al [1999] Hude et al, 2000]. Although the amount of published data for the IVMN assay was relatively small at the time, Kirkland et al [2005] reported that, despite low specificity, the IVMN assay had the highest sensitivity (up to 80.9%) among individual tests (IVMN, CA, or the mouse lymphoma assay) for detecting rodent carcinogens.…”

Section: Introductionmentioning

confidence: 99%

Concordance analysis of an in vitro micronucleus screening assay and the regulatory chromosome aberration assay using pharmaceutical drug candidates

Homiski

Muehlbauer

Dobo

et al. 2009

Environ and Mol Mutagen

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The in vitro micronucleus assay is under consideration by regulatory agencies as a suitable alternative to the in vitro chromosome aberration (CA) assay. At Pfizer, we utilized a non-Good Laboratory practices cytokinesis-block in vitro micronucleus (CBMN) assay in CHO cells as a screen to predict the regulatory outcome of the human lymphocyte CA assay, and we have retrospectively analyzed a highly select set of 112 internal drug candidates to measure concordance. Overall, our exploratory CBMN correctly classified 97 of 112 (86.6%) compounds in the CA assay. Specificity was high with 87 of 92 (94.6%) CA negative compounds correctly classified by CBMN. Sensitivity was low at 50% with 10 of 20 CA positive compounds correctly classified by CBMN; this may be attributed to the low number of CA positives in the select set. In an attempt to improve sensitivity, we increased the number of CA positives by combining our internal set with an industrial data set previously published (Miller B et al. 1997: Mutat Res 392:45-59). When combined, concordance was 86.7% (143/165), specificity was 91.2% (114/125), and sensitivity increased to 72.5% (29/40). Because cytotoxicity is considered a confounding factor of in vitro test systems, we also examined, within the Pfizer data set, the influence of cytotoxicity in the CBMN assay, and the results indicated that seemingly low (<50%) or excessively high (>70%) levels of cytotoxicity did not significantly alter predicted CA outcome. These collective analyses contribute to growing evidence that the CBMN assay is a suitable regulatory option in the standard battery of genetic toxicology tests.

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In vitro micronucleus assay with Chinese hamster V79 cells — results of a collaborative study with in situ exposure to 26 chemical substances

Cited by 85 publications

References 38 publications

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

In Vitro and In Vivo Properties of Dihydrophthalazine Antifolates, a Novel Family of Antibacterial Drugs

Concordance analysis of an in vitro micronucleus screening assay and the regulatory chromosome aberration assay using pharmaceutical drug candidates

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