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Poster #1
The Use of 1-Methyl-2 Pyrrolidinone and N,N-Dimethylacetamide as Vehicles for Ames Assays


Poster #2
Evaluation of Oxazepam in the MicroAmes Reverse Mutation Assay

Poster #3
Development of a High Capacity In Vitro Micronucleus Screening System Based on Flow Cytometric Analysis


Poster #4
A QSAR Model to Predict the Ability of Chemicals to Induce Micronuclei In Vitro


Poster #5
Evaluation of the highest concentrations used in the in vitro chromosome aberrations assay


Poster #6
Aneugenic and clastogenic signatures in TK6 cells accomplished with a multiplexed flow cytometry-based micronuclei and mitotic cell scoring assay


Poster #7
Response Criteria Reduces Impact of Scoring Variability in the In Vitro Micronucleus Assay


Poster #8
Comparison of three closely related human lyphoblastoid cell lines with different p53 functionality in the in vitro micronucleus assay


Poster #9
3D EpiDermTM Reconstructed Human Skin Micronucleus Assay: A GLP Validation


Poster #10
Qualification of a 96-Well High Throughput In Vitro Micronucleus Assay in CHO Cells using Flow Cytometry


Poster #11
Investigation of the Dose Response Curve for Isopropyl Methanesulfonate Using the In Vivo Pig-a Mutation Assay


Poster #12
Efficient monitoring of in vivo Pig-a gene mutation and chromosomal damage: Results with 11 compounds


Poster #13
Accumulation of DNA damage in MutaTMMouse Lung Epithelial Cells after repeat exposure to EMS and ENU


Poster#14
Comparison of Comet, Micronucleus, and Pig-a Mutation Endpoints in a 28-day Oral Repeat Dose Study in Rats with MNU


Poster #15
Impact of Regenerative Anemia in Male Sprague-Dawley Rats on the Pig-A Mutagenesis Assay


Poster #16
Microfluidic Electrochemical and Electro-optical Systems for Reactive Metabolite Screening


Poster #17
An evaluation of the potential genotoxicity of the impurity 14-hydroxycodeinone


Poster #18
The Syrian Hamster Embryo (SHE) Cell Transformation Assay: Results with Three Dietary Ingredients - Menadione, Curcumin and Quercetin Hydrate


Poster #19
The in-vitro comet assay using human TK6 cells: Qualification of a high throughput 96-well screening format


Poster #20
Evaluation of Genotoxicity of Estragole and Safrole by In Vivo Comet Assay


Poster #21
Re-examination of the sensitivity and power of the in vivo bone marrow micronucleus (MN) test related to background level, and numbers of animals and cells scored


Poster #22
Genotoxicity and chemoprevention in bone marrow from DNA repair deficient and p53 haploinsufficient [Xpa(-/-)p53(+/-)] mice fed Benzo[a]pyrene (BP), with or without Chlorophyllin (CHL), for 28 days


Poster #23
Flow Cytometry-based Hematotoxicity Matrix Readily Integrates with Studies of the Effects of Ionizing Radiation or Chemical Exposure in Multiple Species


Poster #24
Investigating the Activation of the Apoptotic Pathway by a Novel Alzheimer Drug Candidate


Poster #25
Transcription Factor Activation as a Measure for Determining a Compound’s Genotoxic Mechanism


Poster #26
Interference with the UVC-induced DNA damage response by PKC activating tumor promoters in TK6 cells


Poster #27
Characterization of DNA reactive and non-DNA reactive pharmaceutical drug candidates by gene expression profiling


Poster #28
AZT-exposed Cells Show Tubulin Malfunction Related to Aberrant Stathmin 1 Expression

 

 

 

Biological Relevance and Health Concerns of Genotoxicity

October 24-25, 2012
John M. Clayton Hall Conference Center
University of Delaware
Newark, DE

Poster Abstracts

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Poster #1
The Use of 1-Methyl-2 Pyrrolidinone and N,N-Dimethylacetamide as Vehicles for Ames Assays
Emily W Dakoulas, VO Wagner, RM Hines, BM Taylor, N Moses
BioReliance by SAFC
Data published in the 1980’s supported the use of 1-methyl-2-pyrrolidinone (NMP) as a vehicle in the Ames assay; however, these studies did not use the full complement of tester strains required by OECD 471. N,N-Dimethylacetamide (DMA) was requested by a Sponsor as a vehicle for an Ames assay; however, no data were readily available to support its use. Testing NMP and DMA in the battery of strains as recommended by the guidelines allows for use of these vehicles in GLP Ames assays. Exploratory studies were conducted in our laboratory with these vehicles, using the plate incorporation method and tester strains TA98, TA100, TA1535, TA1537 and WP2 uvrA. Each strain was tested both with S9 activation (10%) and without S9 activation using various aliquots of NMP or DMA. In addition, the standard positive controls were exposed in both the presence and absence of both vehicles.  No toxicity was exhibited with NMP up to 100 microliters per plate or with DMA up to 50 microliters per plate with any of the tester strains in either the presence or absence of S9 activation. The vehicle control values were also consistent with the historical vehicle control values up to the indicated aliquot for each vehicle. In addition, the positive control values in the presence of NMP or DMA up to the indicated aliquots were consistent with the concurrent positive control values in the absence of either vehicle as well as with the historical positive control values. Therefore, NMP at aliquots up to 100 microliters or DMA at aliquots up to 50 microliters are compatible solvents for use in an Ames study via the plate incorporation method with the tested strains.

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Poster #2
Evaluation of Oxazepam in the MicroAmes Reverse Mutation Assay
Christopher S Farabaugh, Melissa M. Wells, MIchael J. Schlosser
WIL Research
The miniaturized Ames test using an agar format in 24-well plates has been used at WIL Research for 8 years to identify the genotoxic potential of drug candidates. This screen was first developed at Monsanto and used successfully at several major pharmaceutical companies for predicting the regulatory Ames test during early drug development when compound supply is minimal. In addition, this format is invaluable for assessing genotoxic potential of low-yield isolated impurities. WIL Research has adapted this screen for use with five ICH compliant tester strains.  During initial validation testing of 20 compounds in the microAmes assay, specificity was greater than 92% and sensitivity was greater than 97% for individual tester strains.  Overall, the assay was 100% predictive for mutagenicity. 

One of the compounds tested at WIL Research in the microAmes assays was oxazepam, a sedative-hypnotic and anti-anxiety agent.  Oxazepam is negative in the full plate Ames assay, produces inconclusive results for in vitro clastogenicity, and is a known in vivo mutagen and carcinogen.  The microAmes assay with oxazepam produced results that did not replicate those observed in the full plate Ames assay.  While the full plate Ames assay produced negative results with and without metabolic activation, responses that met the criteria for a positive response in the microAmes assay were observed with metabolic activation in Salmonella tester strains TA97a (2-fold increase over the mean vehicle control response), TA98 (22-fold), TA100 (2.5-fold), and TA1535 (38-fold).  Negative results were observed in tester strain WP2uvrA(PKM101) with and without metabolic activation, and in all Salmonella tester strains without metabolic activation.  Additional trials are ongoing to further investigate this response.

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Poster #3
Development of a High Capacity In Vitro Micronucleus Screening System Based on Flow Cytometric Analysis
Jeffrey C Bemis1, Steven Bryce1, Kim Luu2, Zhaoping Liu2, Joe Zock2, Bonnie Goodwin3
1Litron Laboratories
2Intellicyt Corporation
3NIH Chemical Genomics Center

Abstract not available..

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Poster #4
A QSAR Model to Predict the Ability of Chemicals to Induce Micronuclei In Vitro
R. Daniel Benz1, Barbara L. Minnier2, Kevin P. Cross3
1US FDA / CDER / OTR, Silver Spring, MD
2GlobalNet Services, Inc., Rockville, MD
3Leadscope, Inc., Columbus, OH

The final wording of the ICH Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use S2(R1), was accepted by its Expert Working Group on 9 November, 2011. In this guidance, the in vitro micronucleus assay was deemed, for the first time, to be an acceptable part of the ICH genetic toxicology test battery. We report here the results of our effort to construct a quantitative structure-activity (QSAR) relationship model to predict the results of in vitro micronucleus testing with QSAR software. For this exercise, training set data were obtained from Corvi et al., CCRIS, and other published/public sources. Of the 358 chemical substances in the training set, 67% were determined to be positive for in vitro micronucleus induction in laboratory testing, and 33% were found to be negative. In order to have the largest possible number of chemicals in the training set, results were included with no distinction made among the various cell types used for the testing. The initial model was made using software developed by Leadscope, Inc. The cross-validation performance for that model is: 68% specificity; 90% sensitivity; 85% +predictivity; 76% -predictivity; and 82% concordance. External validation is being performed using in vitro micronucleus data for chemicals reported in the Lhasa Vitic Nexus database that are not in the model's training set.

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Poster #5
Evaluation of the highest concentrations used in the in vitro chromosome aberrations assay
Lauren Brookmire1, James J. Chen2, Dan D. Levy1
1FDA Center for Food Safety and Applied Nutrition
2FDA National Center for Toxicologic Research

There is controversy over the highest concentration to which an article should be tested in in vitro mammalian cell assays of genetic toxicity. Until recently, most guidelines specified the use of concentrations of up to 10 mM or 5,000 ug/ml (whichever is lower) when not limited by the toxicity of the test article to the cells used for the test. Several recent publications have called for lowering those limits. We examined concentration/response curves for in vitro chromosome aberrations assays. Data was extracted from two published databases to evaluate the lowest dose at which a positive response was reported (the lowest "effective" concentration or LEC). Concentration/response curves were simulated using Monte Carlo procedures on log normal distributions of the data. These simulations were used to predict the loss in assay sensitivity that would be incurred by arbitrarily lowering the highest concentration to which the assay is conducted when not limited by cytotoxicity. These simulations resulted in Gaussian distributions without evidence of a significant shift to positive results at high concentrations as might be expected if high concentrations were causing undue cytotoxicity not related to genetic toxicity. The simulations suggest that lowering the current high concentration limit from 10 mM would dramatically impact the sensitivity of the assay. In contrast lowering the high concentration limit using the ug/ml scale, the most commonly applied scale in regulatory submissions, would not have a similar impact on assay sensitivity until the limit concentration was lowered to more than half of the current 5000 ug/ml limit. This analysis suggests that the current limits of 10 mM and 5000 ug/ml are not equivalent to one another and challenges the assumption that lowering the 10 mM limit will not decrease assay sensitivity.
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Poster #6
Aneugenic and clastogenic signatures in TK6 cells accomplished with a multiplexed flow cytometry-based micronuclei and mitotic cell scoring assay
Steven Bryce, Svetlana Avlasevich, Jessica Weber, Esmiranda Beha, Stephen Dertinger
Litron Laboratories
This laboratory has previously described a flow cytometric method for scoring micronuclei in cultured mammalian cells (commercially known as In Vitro MicroFlow®). Other reports indicate that the proportion of metaphase cells represents a simple endpoint that can effectively discriminate between clastogenic and aneugenic modes of action. We therefore set out to combine the flow cyotmetric micronucleus scoring method with a technique for enumerating metaphase cells and evaluate the multiplexed assay's ability to differentiate clastogenic and aneugenic responses. In order to accomplish metaphase scoring, the fluorescent reagent anti-histone H3 (pS28) antibody (anti-H3-P) was incorporated into the flow cytometric micronucleus assay procedure. TK6 cells were treated in a continuous exposure design with each of ten reference clastogens and seven reference aneugens. At the time of harvest (24 to 27 hrs after treatments were initiated), cells were processed according to In Vitro MicroFlow® kit instructions, with the exception that anti-H3-P was included in the processing steps. Data acquisition occurred for 4 minutes per sample, which provided approximately 2,000 metaphase cells per replicate. Each of the genotoxicants was observed to cause MN frequencies to increase and relative survival values to decrease in a concentration-dependant manner. Whereas the proportion of metaphase cells to total cells (as well as the proportion of metaphase cells to G2/M cells) were decreased by each of the ten reference clastogens, they were elevated by each of the seven aneugens. These data indicate that automated and multiplexed micronuclei and metaphase cell scoring of TK6 cells provides strong aneugenic versus clastogenic mode of action signatures that effectively discriminate between these classes of genotoxic agents.
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Poster #7
Response Criteria Reduces Impact of Scoring Variability in the In Vitro Micronucleus Assay
Michael L Homiski, Zhanna Sobol, Jennifer Munzner, Krista Dobo
Pfizer Inc., Groton, CT
The In Vitro Mammalian Cell Micronucleus Test (MNvit) has historically been reliant upon manual microscopic evaluation to generate study results. The OECD guideline (TG487) for the MNvit states that statistical analyses may be used to support study outcomes that are otherwise based primarily on biological relevance. Here we examined variability of the test system to measure its impact on study outcome. Three chemicals (5-fluorouracil, etoposide and noscapine) were tested, with dimethyl sulfoxide (DMSO) and vinblastine sulfate included as solvent and positive controls, respectively. Three independent assays using TK6 cells were conducted over consecutive weeks. To measure repeatability between tests we calculated and compared the cytotoxicities of all cultures based on relative population doubling. To measure reproducibility of the micronucleus response, we randomized scoring such that each dose of each compound was analyzed by 12 qualified scorers. We selected 4 concentrations per chemical for analysis, including doses with high, mid and low levels of cytotoxicity. Study outcomes were derived by assessing the biological relevance (negative control reference range) in addition to a blend of statistical analyses including the Fisher's Exact and Cochran-Armitage Trend Tests. We calculated the CV (coefficient of variation) among 12 scorers for each replicate concentration (N=24) to measure scorer to scorer variation. Results showed high repeatability of induced cytotoxicities among tests and 100% reproducibility of test outcomes despite observing some CVs as high as 52.7%. In summary, the robust test response criteria utilized herein ensure that variability inherent to the test system does not impact interpretation of assay response.
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Poster #8
Comparison of three closely related human lyphoblastoid cell lines with different p53 functionality in the in vitro micronucleus assay
Abby Myhre, Adam Faranda, Kyle Glover, E. Maria Donner
Drug Safety Research and Development, Pfizer Inc.
With the increasing use of the in vitro mammalian cell micronucleus assay, there has been more focus on relevant cell line selection. One common argument is that data generated with cell lines of human origin, that have retained intact p53 repair functions, are more predictive for assessment of human hazard and risk than data from historically and still commonly used rodent cell lines. Since the majority of these rodent cell lines (CHO-K1, V79, L5178Y, and CHL/IU) have mutated non-functional p53 proteins, and the human TK6 lymphoblast cell line is p53 competent, we wanted to investigate the effect(s) of p53 status on the outcome of the OECD TG 478 in vitro micronucleus assay. The purpose of this experiment was to determine if the p53 status of a cell line has a critical effect on the conclusion about a chemical's genotoxicity. Three closely related cell lines (TK6, WIL2-NS, and NH32) were evaluated using several of the OECD TG478 reference chemicals. All three cell lines are derived from the parent lymphoblastoid cell line (WIL-2) but have different p53 functionality. TK6 cells are p53 competent, producing a functional p53 protein; the WIL2-NS cells produces a non-functional mutant p53 protein similar to the rodent cell lines; and the NH32 cells are null for a p53 protein. The reference chemicals were tested with each of the three different cell lines and analyzed by flow cytometry using the In Vitro MicroFlow Micronucleus assay kit (Litron Laboratories, Rochester, NY). The results obtained from both the TK6 and WIL2-NS cell lines were remarkably similar. All positive compounds gave the appropriate >3-fold positive responses. For many of the compounds the p53 competent TK6 cell line actually yielded a slightly higher response then the mutant WIL2-NS cells. Regardless of p53 functionality cytotoxicity levels remained consistent across cell lines. When comparing the non-genotoxic chemicals, however, the WIL2-NS cells produced a false positive in response to treatment with Di(2-ethylhexyl)phthalate while the TK6 cells remained below the threshold for a positive response. All other non-genotoxic chemicals resulted in clear negative results as expected. Data from the NH32 cell line will be compared to the results for the TK6 and WIL2-NS cell lines.
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Poster #9
3D EpiDermTM Reconstructed Human Skin Micronucleus Assay: A GLP Validation
Shambhu Roy1, Anna Szkudlinska1, Keyan Wang1, Jing Shi1, Scott Hickman1, Wannie Madraymootoo1, Marilyn Aardema1,2
1BioReliance by SAFC, Rockville, MD, USA
2Marilyn Aardema Consulting, LLC Fairfield OH USA

The 3D reconstructed human skin micronucleus assay (RSMN) in EpiDermTM is a promising new in vitro assay for the assessment of genotoxicity. Analysis of micronuclei in a 3D primary skin model offers a more biologically relevant in vitro approach to assess genotoxicity of many types of dermal exposures including drugs, chemicals and cosmetics, compared to standard in vitro genotoxicity assays. EpiDermTM provides a functional stratum corneum that takes into account permeability, appears to have normal dermal metabolic capability, normal DNA repair and cell-cycle control. The RSMN assay provides a new animal alternative for following up chemicals that are positive in current in vitro genotoxicity assays and is especially useful for cosmetics that can no longer be tested in in vivo assays according to the 7th Amendment to the EU Cosmetics Directive. To meet the increasing interest in this assay, we have developed a GLP RSMN assay. Results for model chemicals with various modes of action including crosslinker (mitomycin C), aneugen (vinblastine sulphate), clastogen (methylmethane sulfonate) show dose-dependent increases of MN and demonstrated good reproducibility and comparability to previously published results. The CREST staining technique was used for identification of aneugenic versus clastogenic mechanisms. Studies are ongoing with genotoxins and nongenotoxins with various modes of action. Results to date demonstrate the transferability of this novel assay into a robust GLP setting.

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Poster #10
Qualification of a 96-Well High Throughput In Vitro Micronucleus Assay in CHO Cells using Flow Cytometry
Leon F. Stankowski, Jr.1, Timothy E. Lawlor1, Marilyn Aardema1,2
1BioReliance by SAFC
2Marilyn Aardema Consulting, LLC

A 96-well high throughput flow cytometric in vitro micronucleus assay in CHO cells was qualified using MicroFlowTM kits (Litron Laboratories) and ten reference compounds identified in OECD TG 487. All compounds were evaluated in ten independent experiments, in duplicate cultures and at ten concentrations, using a 4-hour treatment with S9 and a 24-hour treatment without S9, to assess inter- and intra-experimental variability, as well as sensitivity and specificity. In these ten trials, the frequencies of micronucleated cells in the pooled vehicle and untreated controls was 1.35 ± 0.49 and 1.66 ± 0.53, while the % hypodiploid cells were 0.30 ± 0.15 and 0.68 ± 0.29 (average ± 1 SD; n = 862 or 861; with or without S9, respectively). Using an empirical analysis of this data set, it was possible to reduce the criteria for a positive response for micronuclei and hypodiploidy to 2- and 6-fold concurrent vehicle control values, respectively, thereby increasing sensitivity without any loss of specificity. Mitomycin C and cytosine arabinoside were reproducibly positive without S9, as were benzo(a)pyrene and cyclophosphamide with S9; all four compounds produced a clastogenic signature. Colchicine and vinblastine were positive with and/or without S9, and both produced a significant increase in micronuclei and hypodiploid cells, indicative of an aneugenic mechanism of action. In contrast, di(2-ethylhexyl)phthalate, nalidixic acid, pyrene and sodium chloride were reproducibly negative in all trials and at all dose levels with and without S9. Additional analyses of these data are ongoing as related to modification of evaluation criteria and threshold effects.

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Poster #11
Investigation of the Dose Response Curve for Isopropyl Methanesulfonate Using the In Vivo Pig-a Mutation Assay
Joel I. Ackerman, Michelle Kenyon, Stephanie Coffing, Thomas Shutsky, Krista Dobo
Pfizer Inc., Groton, CT
It is generally accepted that DNA reactive compounds exhibit linear dose responses. However, it has recently been demonstrated that at least some mutagens exhibit a sub-linear dose response. Evidence of a sub-linear dose response could be beneficial in the management of pharmaceutical genotoxic impurities if it allowed for daily exposures above the default control level for mutagens of 1.5 µg/day, the Threshold of Toxicological Concern. The aim of this work is to investigate the dose response for a pharmaceutically relevant alkyl ester, isopropyl methanesulfonate (IPMS). The induction of mutations in the peripheral blood of Wistar rats dosed with IPMS either acutely (single doses) or sub-chronically over 28 days was examined using the in vivo Pig-a mutation assay. With both acute and chronic administration of IPMS, mutations accumulated over the course of the study. The dose response curve appeared to be linear in nature at acute doses of 3.5 to 56 mg/kg, while with chronic dosing only total cumulative doses of 14 to 56 mg/kg appeared linear based on an approximately two-fold-increase in mutant frequency with a doubling in dose. Additionally, a comparison of the mutant frequency from acute and 28-day dosing regimens indicated a less than additive dose-response relationships, such that a single dose resulted in a much higher mutant frequency than the same dose fractionated over 28 days. The data suggests a sub-linear dose-response for IPMS.

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Poster #12
Efficient monitoring of in vivo Pig-a gene mutation and chromosomal damage: Results with 11 compounds
Stephen Dertinger1, Souk Phonethepswath1, Svetlana Avlasevich1, Dorothea Torous1, Jared Mereness1, Steven Bryce1, Jeffrey Bemis1, Sara Bell1, Pamella Weller1, James MacGregor2
1Litron Laboratories, Rochester, NY
2Toxicology Consulting Services, Arnold, MD

The ability to effectively monitor gene mutation and micronucleated reticulocyte (MN-RET) frequency in short-term and repeated dosing schedules was investigated using the recently developed flow cytometric Pig-a mutation assay and the established flow cytometric micronucleus method. Eight genotoxicants (chlorambucil, melphalan, thiotepa, cyclophosphamide, azathioprine, 2-acetylaminofluorene, hydroxyurea and methyl methanesulfonate) and three non DNA-reactive compounds (o-anthranilic acid, sulfisoxazole and sodium chloride) were studied in male Sprague-Dawley rats. Groups of animals were treated via oral gavage for 3 or 28 consecutive days with several dose levels of each chemical up to maximum tolerated doses. Blood samples were collected at several time points up to Day 45, and were analyzed for Pig-a mutation with a dual-labeling method that facilitates mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. An immunomagnetic separation technique was used to increase the efficiency of scoring mutant cells. Blood samples collected on Day 4 for both study designs, and also Day 29 for the 28-day study, were evaluated for MN-RET frequency. The three non-genotoxicants did not induce Pig-a or MN-RET responses. All genotoxicants except hydroxyurea increased the frequency of mutant reticulocytes and erythrocytes. Significant increases in MN-RET frequency were observed for each of the genotoxicants at both time points. Whereas the highest Pig-a responses tended to occur in the 28-day studies, the highest induction of MN-RET was observed in the 3-day studies. There was no clear relationship between the maximal Pig-a response of a given chemical and its corresponding maximal MN-RET response, despite the fact that both endpoints were determined in the same cell lineage. Taken together with previously published results, these data demonstrate the value of integrating Pig-a and micronucleus endpoints into in vivo toxicology studies, thereby providing information about mutagenesis and chromosomal damage in the same animals from which toxicity, toxicokinetics, and metabolism data are obtained.
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Poster #13
Accumulation of DNA damage in MutaTMMouse Lung Epithelial Cells after repeat exposure to EMS and ENU
William C. Gunther, Zhanna Sobol, Elizabeth E. Rubitski, Michelle O. Kenyon, Donna A. Dickinson, Joel Ackerman, Donna M. Peckham, Maik Schuler and Krista L. Dobo
Pfizer Inc., Groton, CT
The dose-response relationship of a genotoxin provides important information for risk assessment. The aim of this study was to determine if an in vitro mutation assay can be used to establish a dose-response curve and detect cumulative DNA damage after repeat exposure to the model genotoxins EMS and ENU.

Epithelial cells derived from MutaTMMouse lung tissue contain the lacZ positive selection system for measuring mutations [P.A. White et al, Env and Mol Mut 2003]. These cells were exposed to EMS and ENU to obtain acute dose-response curves for mutation and micronucleus induction. A concentration was chosen from the NOEL portion of the acute dose response curve for each compound and cells were repeatedly exposed for a total of 8 treatments over a two week period. Both EMS and ENU led to a cumulative increase in mutations but not micronuclei. These initial data indicate that the MutaTMMouse lung tissue cell system may be useful for measuring cumulative DNA damage and studying dose-response relationships prior to expensive in vivo studies.

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Poster #14
Comparison of Comet, Micronucleus, and Pig-a Mutation Endpoints in a 28-day Oral Repeat Dose Study in Rats with MNU
Daniel J. Roberts, Steven Nicotra, Marie Mack, Carol Gleason, Laura L. Custer
Bristol-Myers Squibb
This laboratory has previously reported Pig-a, micronucleus, and comet assay results after treatment of male Sprague Dawley rats with N-Methyl-N-Nitrosourea (MNU) daily, for 28-days, as part of the international "Stage III" interlaboratory Pig-a evaluation trials (Lynch et al., 2011, EMM 52(9):699-710). A higher throughput version of the assay using magnetic column enrichment of mutant cells was conducted to compare sensitivities and assay power between the two methodologies. The same treatment schedule and animal strain was used, with doses of 0.9 to 5.0 mg/kg/day MNU. Peripheral blood was sampled from the tail vein on Days -4, 15, and 29 for assessment of Pig-a mutation (ie, CD59NEG phenotype) in young reticulocytes (RET) and total erythrocytes. On Days 4 and 29 of the study, blood was processed for micronuclei (MN-RET) measurements via flow cytometry, and approximately 24-hours after the last dose, DNA damage was evaluated in the liver using the alkaline Comet assay. Statistical significance was observed on Day 29 for every dose of MNU, in each endpoint evaluated. The MN-RET endpoint and the higher-throughput version of the Pig-a assay demonstrated similar sensitivity in detecting biologically meaningful increases in genetic damage. The Comet assay had the highest sensitivity when considering fold-response at the lowest dose group, but demonstrated only small differences in response between low and high doses. Threshold doses for mutagenicity were lower than those identified previously using the original "Stage III" protocol, which can be attributed to reduction of counting error from scoring many more cells.

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Poster #15
Impact of Regenerative Anemia in Male Sprague-Dawley Rats on the Pig-A Mutagenesis Assay
Paul A Sonders, Joel Murray, Bruce LeRoy
Abbott Laboratories
Red blood cells (RBCs) and reticulocytes (RETs) from normal animals exhibit a low background level of spontaneous mutations in the Pig-A gene, which is an X-linked membrane glycoprotein important in paroxysmal nocturnal hemoglobinuria. However, it is not known if the proportion of RBCs and RETs harboring Pig-A mutations is increased in regenerative anemia. The in vivo rat Pig-A mutation assay identifies mutant RBCs or RETs lacking the CD-59 surface marker, presumably due to a mutation in the Pig-A gene. A study was conducted to quantify the number of RBCs and RETs with Pig-A mutations in anemic rats compared to background mutation level from non-anemic control rats. Pig-A mutation rates were assayed using flow cytometry. Results from this study provided key data to indicate whether a false-positive Pig-A mutagenicity signal would occur when erythropoiesis is stimulated in rats recovering from an anemic episode.

This study shows that there was no increase of mutations in anemic rats in either RBCs or RETs .

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Poster #16
Microfluidic Electrochemical and Electro-optical Systems for Reactive Metabolite Screening
Dhanuka P Wasalathanthri1, James F Rusling1,2,3
1Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States
2National University of Ireland at Galway, Ireland
3Department of Cell Biology, University of Connecticut Health Center, Farmington, Connecticut 06032, United States

Despite the utility of sophisticated array of toxicity assessment tools for the prediction of adverse reactions by new molecular entities (NME), toxicity issues become apparent in some NME after reaching human clinical trials, and a few even after marketing, enlightening the need of a simple, versatile, reliable and high-throughput detection platform for better drug development pipeline.(1,2,3,4) Covalent DNA nucleobase adducts formed by NME or their metabolites are important biomarkers of genetic toxicity, which can be utilized for successful prediction of drug toxicity.(5) Our research efforts on developing molecular-based chemical and drug toxicity screening novel technologies are based on 20-50 nm thick layer by layer (LbL) films that combine metabolic enzymes with DNA on carbon electrode arrays housed in to microfluidic devices, where metabolites produced by the enzymes react with DNA nucleo-bases and the rate of DNA damage is measured as a voltammetric or electrochemiluminescent (ECL) response using a specific ruthenium based redox polymer [Ru(bpy)2(PVP)10](ClO4) {(PVP = poly(4-vinylpyridine)} – (Ru(II)-PVP).(6,7,8) The validation of these devices with a common set of compounds demonstrated excellent correlation with rodent genotoxicity metric TD50 holding considerable promise as a molecular chemistry complement to toxicity bioassays.

Reference(s):
1. Paul, S.M.; Mytelka, D. S.; Dunwiddie, C. T.; Persinger, C. C.; Munos, B. H.; Lindborg, S. R.; Schacht, A. L. Nature Rev. Drug Discov. 2010, 9, 203-215.
2. Stokstad, E. Science 2009, 325, 294-295.
3. Kramer, J. A.; Sagartz, J. E.; Morris, D. L. Nature Rev. Drug Discov. 2007, 6, 636-649.
4. Rusling, J. F.; Hvastkovs, E. G.; Schenkman, J. B. Curr. Opin. Drug Discovery Dev. 2007, 10, 67–73.
5. Park, B. K.; Boobis, A.; Clarke, S.; Goldring, C. E. P.; David Jones, D.; Kenna, J. G.; Lambert, C.; Laverty, H. G.; Naisbitt, D. J.; Nelson, S.; Nicoll-Griffith, D. A.; Obach, R. S.; Routledge, P.; Smith, D. A.; Tweedie, D. J.; Vermeulen, N.; Williams, D. P.; Wilson, I. D. and Baillie, T. A. Nature Rev. Drug Discov. 2011, 10, 292-307.
6. Wasalathanthri, D. P.; Mani, V.; Tang, C. K.; Rusling, J. F. Anal. Chem. 2011, 83, 9499–9506.
7. Wasalathanthri, D. P.; Faria, R. C.; Spundana, M.; Joshi, A. A.; Schenkman, J. B.; Rusling, J. F. Anal. Chem. 2012, Submitted.
8. Wasalathanthri, D. P.; Spundana, M.; Bist, I.; Tang, C. K.; Faria, R. C.; Rusling, J. F. 2012, Paper in preparation.

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Poster #17
An evaluation of the potential genotoxicity of the impurity 14-hydroxycodeinone
Patricia O'Neill, Zoryana Cammerer and Sandy Weiner
Janssen Pharmaceutical Companies of Johnson & Johnson
The chemical 14-hydroxycodeinone (14-HC) is a potential impurity found in several drug substances intended for the treatment of pain. 14-HC contains an alpha,beta-unsaturated ketone structural alert. Assuming that any impurity with a structural alert is potentially genotoxic (DNA reactive), this alert requires controlling the substance to the default threshold of toxicological concern (TTC) levels, consequently leading to restrictive specification levels. Purified 14-hydroxycodeinone was tested in a battery of genetic toxicology assays. Specifically, neat 14 hydroxycodeinone was tested for point mutations (mutagenicity) in an Ames Assay and chromosome rearrangements (clastogenicity) in a chromosome aberration assay using primary human peripheral blood lymphocytes. The Ames test was negative demonstrating an absence of mutagenic potential for purified 14-hydroxycodeinone. Clastogenicity was however observed in the in vitro chromosomal aberration assay. As a follow-up to the in vitro chromosomal aberration finding, an in vivo bone marrow micronucleus study was conducted in ICR mice to evaluate the potential for in vivo clastogenicity. The in vivo micronucleus study was clearly negative for clastogenicity at doses up to 320 mg/kg/bw (the MTD). An in vivo mouse comet assay analyzing both glandular stomach and liver was conducted to evaluate the potential for in vivo clastogenicity/DNA damage. This assay confirmed negative result observed in mouse bone marrow assay. The overall result of testing indicates that 14-hydroxycodeinone is not a genotoxic agent. Therefore 14-hydroxycodeinone has been qualified as a non genotoxic impurity per the appropriate guidelines (ICH Q3A & ICH Q3B) designed for qualifying the safety risks of impurities.

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Poster #18
The Syrian Hamster Embryo (SHE) Cell Transformation Assay: Results with Three Dietary Ingredients - Menadione, Curcumin and Quercetin Hydrate
Kamala Pant1, Shannon W. Bruce1, Jamie E. Sly1, Michelle Klug-Laforce1, Andrew D. Scott2, Marilyn J. Aardema1
1BioReliance by SAFC, Rockville, MD, USA
2Safety and Environmental Assurance Centre, Unilever Colworth Science Park, Bedfordshire, MK441LQ, UK

The Syrian hamster embryo (SHE) cell transformation assay (CTA) is an in vitro test for potential carcinogenicity that has been approved for development into an OECD guideline (EURL-ECVAM recommendation, In-vitro Carcinogenicity Testing 2012). In the present study, three chemicals were tested in the SHE CTA, menadione (Vitamin K3, CAS number: 58-27-5), curcumin (CAS number: 458-37-7) and quercetin hydrate (CAS number: 849061-97-8). Menadione is a synthetic chemical compound sometimes used as a nutritional supplement because of its vitamin K activity. Curcumin and quercetin hydrate are dietary flavonoids; quercetin is found in citrus fruit, buckwheat and onions and curcumin is part of the Indian spice turmeric. Both exhibit antioxidant effects at low dose and pro-oxidant effects at higher concentrations. Accordingly, these materials show adverse effects in some in vitro genotoxicity tests that do not manifest in vivo. The purpose of this study was to investigate the predictivity of the SHE CTA as a means to inform consumer safety cancer risk assessment. The SHE CTA was performed using a seven-day exposure regimen following the ECVAM (European Center for the Validation of Alternative methods) recommended protocol. Concentrations for the SHE CTA were selected based on initial dose range finding data. The relative plating efficiency for menadione (2.5 µg/ml), curcumin (2.5 µg/ml) and quercetin (5.0 µg/ml) at the highest concentration scored was 41.3%, 49.3% and 43.6%, respectively. None of these test article concentrations induced a statistically significant (p < 0.05) increase in the number of morphologically transformed colonies in comparison to the vehicle control. Thus, these three dietary ingredients do not cause cell transformation in the SHE CTA under the conditions described.
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Poster #19
The in-vitro comet assay using human TK6 cells: Qualification of a high throughput 96-well screening format
Kamala Pant, Shannon W. Bruce, David Albert, Sandra Springer, Yong Xu, Timothy Lawlor, Marilyn Aardema
BioReliance by SAFC, Rockville, MD, USA
There is increased interest in the field of genetic toxicology to use p53 competent human cells in in vitro assays instead of transformed rodent cell lines that are associated with misleading positive results. To this end, we have qualified a 96 well screening Comet assay in human p53 proficient TK6 cells. Following the protocol used in the JaCVAM (Japanese Center for Validation of Alternative Methods) Comet validation studies (version 6.2), six reference compounds were tested: ethyl methanesulfonate, cycloheximide, triton-X, 9-aminoacridine, 2-amino anthracene and methyl methanesulfonate. Four independent experiments were conducted using duplicate cultures, and seven to ten test article concentrations, using a 4-hour treatment with and without exogenous metabolic activation to assess inter- and intra-experimental variability, as well as sensitivity and specificity. The number of clouds (hedgehogs) on the slides was used as the toxicity measure. The median % tail DNA was used as the parameter to evaluate the DNA damage. Reproducible positive results were obtained with ethyl methanesulfonate, 9-aminoacridine, 2-amino anthracene and methyl methanesulfonate as expected and reproducible negative results were obtained with cycloheximide and triton-X. This assay works well for screening a large number of compounds and is in use by BioReliance for the EPA ToxCast screening program.
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Poster #20
Evaluation of Genotoxicity of Estragole and Safrole by In Vivo Comet Assay
Wei Ding1, Dan Levy2, Michelle E. Bishop1, Mason G. Pearce1, Gene A. White3, Lascelles E. Lyn-Cook1, Mugimane G. Manjanatha1
1FDA Division of Genetic and Molecular Toxicology
2FDA Center for Food Safety and Applied Nutrition
3FDAToxicologic Pathology Associates

Estragole and safrole are members of alkenylbenzenes that are naturally-occuring constituents of various herbs and spices. Estragole is used as a flavoring and fragrance agent in foods, perfumes, soaps and detergents. Safrole was the first of the class of alkenylbenzenes discovered to induce liver malignancies in experimental animals. More recently estragole was shown to induce malignancy in liver of F344 male rats when dosed at 600 mg/kg bw for 3 months in a NTP study. While the compounds both form DNA adducts, neither is positive in the Ames test. Safrole is positive in the mouse lymphoma test whereas estragole is negative in the in vivo micronucleus assay. To further understand their modes of action for cancer, we evaluated the genotoxicity of estragole and safrole using the in vivo Comet assay. hOGG1- and EndoIII-modified in vivo Comet assays were performed to measure oxidative DNA damages. Groups of 5 male F344 rats were treated with corn oil (vehicle control) or with 300, 600, and 1,000 mg/kg bw estragole and 125, 250 and 450 mg/kg bw safrole at 0, 24, and 45 hr. Treated and control rats were sacrificed at 48 hr and several tissues including liver were processed for measuring comets. As measured using the alkaline Comet assay, estragole treatment resulted in a near-linear increase in DNA strand breaks in the liver with significant (p≤0.05) increases detected in rats treated with 600 and 1000 mg/kg bw estragole. There also were near-linear dose-dependent increases for EndoIII-sensitive DNA damage and hOGG1-sensitive DNA damage, both with significant (p≤0.05) increases detected in rats treated with 300, 600 and 1000 mg/kg bw estragole. Safrole treatment resulted in a near-linear increase in DNA strand breaks in the liver with significant (p≤0.05) increases detected in rats treated with 450 mg/kg safrole. Near-linear dose responses for EndoIII-sensitive DNA damage and hOGG1-sensitive DNA damage were also detected in safrole-treated rat livers, both with significant (p≤0.05) increases detected in all dose groups. These data show that estragole and safrole damage liver DNA, both via direct strand breakage and oxidative damage at doses which cause liver malignancy. This suggests that both DNA strand breaks and oxidative DNA damage may be causative factors in the hepatocarcinogenicity of estragole and safrole.
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Poster #21
Re-examination of the sensitivity and power of the in vivo bone marrow micronucleus (MN) test related to background level, and numbers of animals and cells scored
Sheila M. Galloway, Daniel J. Holder, Michael J. Armstrong
Merck Research Laboratories, West Point, PA
The ICH S2(R1) guideline recommends ensuring that sufficient cells/animals are scored to provide adequate sensitivity taking into account an individual laboratory's background level of micronucleated cells. It states: "…Rat blood can be used for micronucleus analysis provided methods are used to ensure analysis of the newly formed reticulocytes (Hayashi et al., 2007; MacGregor et al., 2006), and the sample size is sufficiently large to provide appropriate statistical sensitivity given the lower micronucleus levels in rat blood than in bone marrow (Kissling et al., 2007). Whichever method is chosen, bone marrow or blood, automated or manual analysis, each laboratory should determine the appropriate minimum sample size to ensure that scoring error is maintained below the level of animal-to-animal variation." While adequate sensitivity is not defined, it is inferred from the publications cited above, and from IWGT reports on the in vivo MN assay, that typically 2 – 3 fold increases in cells with MN should be detectable with high confidence. Kissling et al carried out power calculations using various background levels of MN, scores from 5 animals per group and various numbers of cells scored per animal; they estimated "true" MN levels and animal to animal variability from flow cytometric studies that provide large cell samples. With tests integrated into toxicology studies there are options to score cells from more than 5 animals (e.g., 10 per dose level) and 2 sexes, so we examined the power of various study designs given our typical background level of MN in rat bone marrow scored 'manually', of 0.18 percent MN-PCE. We also assessed the study design for a single administration and two sampling times, with mice (historical background level of 0.14 percent MN PCE). We assessed the relative merits of scoring more than 2000 cells per animal, or more animals per group. We have traditionally used a larger number of animals for vehicle controls (10, cf 5 per treatment group) to enhance the power. With the background levels in our lab for rats, this results in very high power (>90 percent) for detecting a 2.5 fold increase when scoring 2000 PCE per animal, even if only one sex or time point is used. The results of the other analyses will be presented.
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Poster #22
Genotoxicity and chemoprevention in bone marrow from DNA repair deficient and p53 haploinsufficient [Xpa(-/-)p53(+/-)] mice fed Benzo[a]pyrene (BP), with or without Chlorophyllin (CHL), for 28 days
Ariadna M Marrogi1, Michael O. On'gele1, Kaarthik John1, M. Margaret Pratt1, Igor P. Pogribny2, Miriam C. Poirier1, Ofelia A. Olivero1
1Carcinogen-DNA Interactions Section, LCBG, CCR, National Cancer Institute, NIH
2Division of Biochemical Toxicology, National Center for Tox

The purpose of this study was to examine mouse bone marrow cells (n=1000) for genotoxic end points, including micronuclei (MN, formed when all or part of a chromosome is lost during mitosis), nucleoplasmic bridges (NPBs, formed when chromosomes fail to separate during anaphase), and nuclear buds (NBUDs, possibly derived from lagging chromosomes at anaphase). Tumor-susceptible mice, deficient in nucleotide excision repair (NER) and haploinsufficient for the tumor suppressor p53, designated [Xpa(-/-)p53(+/-) ] mice, were fed 0 or 100ppm BP, with or without 0.3% CHL, for 28 days, and the groups examined included: unexposed, BP-exposed, CHL alone, and BP+CHL-exposed. Bone marrow was obtained at euthanasia from 4-6 mice per group (males and females combined), and fixed and stained with DAPI to visualize nuclei. Genotoxic end points (MN, NBUDs and NPBs) were examined with BP alone, as well as with BP plus the chemopreventive agent CHL, hypothesizing that CHL would lower genotoxicity. For percentage of cells with MN, values were low in the control and CHL alone groups, and 30-50% higher in the BP and BP+CHL groups. Comparison of the BP and BP+CHL groups showed no suggestion that CHL reduced BP-induced MN in the males; however, the females showed an apparent protection by CHL when BP+CHL MN were compared to the BP group. In addition, for percentage of cells with NBUDs and with NPBs there was no indication that co-incubation with CHL resulted in lower levels of BP-induced genotoxicity. The DNA adduct data obtained in esophagus, liver, and lung from these same animals (K. John et al., Carcinogenesis 2012, Epublished), also showed that CHL did not consistently lower BP-DNA adduct levels. Whereas this study is still in progress, currently we must conclude that MN were reduced in the presence of CHL, while NBUDs and NPBs were not reduced. Although the long-term study design used here further suggests that chronic CHL use is not likely to protect human tissues from polycyclic aromatic hydrocarbon exposures, more experiments are needed to understand the apparent difference in the protective effect in males and females.

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Poster #23
Flow Cytometry-based Hematotoxicity Matrix Readily Integrates with Studies of the Effects of Ionizing Radiation or Chemical Exposure in Multiple Species
Jared A. Mereness1, Jeffrey C. Bemis1, Yuhchyau Chen2, Steven M. Bryce1, Souk Phonethepswath1, Pamela C. Weller1, Eric Hernady2, Jackie Williams2, J Finkelstein2, S Dertinger1
1Litron Laboratories
2University of Rochester Dept. of Radiation Oncology

The ability to rapidly and efficiently generate data on the condition of peripheral blood cell populations has obvious value in studies designed to investigate the impact of agents like ionizing radiation or chemicals. A HemAtotoxicity Response Matrix, or HARM assay, was devised that involves the addition of a minimal volume of whole blood to a solution containing a fluorescent nucleic acid dye, mitochondrial membrane potential dye and counting beads. Following a short incubation, the sample is analyzed by flow cytometry and cell populations are identified based on light scatter characteristics and nucleic acid content. Initial assessment of the method involved exposure of rats to a wide range of radiation doses and demonstrated time- and dose-dependent alterations in several important peripheral blood cell populations including lymphocytes, neutrophils, reticulocytes and platelets. As examples of the utility of the HARM assay, data from two study designs will be discussed. Mice were injected with 100 µCi 137Cs as a model of internal, systemic exposure to radiation. Absolute lymphocyte counts were reduced as early as 6 hours and up to 4 weeks post exposure, however no effects were observed in the erythroid population. Integration of the HARM assay into the standard 28-day repeat dose rat study used in pharmaceutical and industrial product safety assessment was investigated. Exposure of rats to the antineoplastic agents chlorambucil, cyclophosphamide, melphalan, or thiotepa for 28 consecutive days elicited changes in lymphoid and erythroid populations that were dose and compound specific. The initial method that employed a single-tube approach has since been successfully translated into both 96 and 384-well plate formats. Utilizing autosampler technologies, these miniaturized methods provided data equivalent to those obtained by the single- tube format while decreasing the analysis time from nearly 7 minutes to around 30 seconds per sample. The ability of the HARM assay to readily assess a multitude of hematological endpoints that display varying responses to different exposures highlights the utility of this assay for situations involving industrial hygiene and safety assessments as well as environmental biomonitoring.
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Poster #24
Investigating the Activation of the Apoptotic Pathway by a Novel Alzheimer Drug Candidate
Jennifer R. Cheung, Elizabeth Rubitski, Richard Spellman, Maria Engel, Mike Schuler, Krista Dobo, Pamela Heard
Pfizer Inc, Groton, CT
Apoptosis can be a confounding factor in the evaluation of micronuclei. Determining whether the micronuclei are a byproduct of the apoptosis itself or caused by a true genotoxin is important for risk managing the potential drug product as it moves through the pharmaceutical pipeline. Novel therapeutic (compound X) developed for the treatment of Alzheimer's was evaluated in a an in vitro micronucleus (IVMN) assay in TK6 human lymphoblastoid cells, chromosome aberration assay, a flow cytometry Annexin V-FITC test, and via Western Blot to support clinical investigations. In the 27-hour continuous (-S9) treatment with compound X, small but statistically significant increases in micronuclei were observed when tested up to 249µM. However, analysis was confounded by a high frequency (~50%) of apparently apoptotic cells, which can lead to misleading positive findings in this assay [1]. To investigate whether apoptosis was responsible for the increases in micronuclei rather than clastogenicity, the percentage of apoptotic cells in treated cultures was evaluated by flow cytometry, using Annexin V-FITC and propidium iodide staining. Increases in early apoptosis were not observed following 12-hour treatment (+/-S9). However, consistent with the micronucleus assay results, dose-related increases in apoptosis were observed in the 27 hour treatment. The lack of clastogenic potential of the compound was confirmed by a negative result in an in vitro assessment for structural chromosome aberrations in the same test system. The Western Blot was utilized to confirm the presence of proteins within the apoptotic pathway by probing for Cleaved Caspase 3 and Caspase 9 which were both detected in the high dose 24 hour treatment samples. Furthermore, a negative result in an (IVMN) assay in TK6 cells treated with cytoclasin B in which there was no increase in micronuclei confirms the absence of genotoxicity by compound X. Considering the nature of the in vitro results, the overall weight of evidence indicates that the weak increases in micronuclei were the consequence of apoptosis induction and not caused by a genotoxic event. Therefore compound X was not considered to pose a genotoxic hazard and progression to clinical experimentation was recommended.
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Poster #25
Transcription Factor Activation as a Measure for Determining a Compound's Genotoxic Mechanism
Maria E. Engel, Jennifer Cheung, Krista Dobo, Maik Schuler, Pamela Heard
Pfizer Inc, Groton, CT
Identification of safe and effective compounds remains a challenge in drug discovery. Toxicity is also one of the major reasons for approved drugs to be withdrawn from the market. Thus, there is a clear need for the development of predictive, high throughput, in vitro screening strategies for earlier detection of compound toxicities and elucidation of mechanism of action. Cells respond to exogenous stressors such as chemical toxicants and xenobiotics by inducing specific cellular stress response pathways. Induction of these signaling transduction pathways commonly triggers the activation of transcriptional factors that can act protectively by reestablishing cellular homeostatis. In the case of the DNA damage response, highly conserved checkpoint pathways are involved and become activated upon genotoxic stress. Here we describe the development and validation of a panel of reporter gene cell lines representing cellular mechanisms of compound toxicities frequently observed with genotoxic stress. The cell lines express stress inducible reporter constructs in form of firefly luciferase under the control of a pathway specific transcriptional response element. Reporter assays for a number of signal transduction pathways representative of various cell stress responses (such as Oxidative stress, Apoptosis and Cell Cycle Progression) were established. A set of known toxicants were screened against the panel of reporters in order to produce a baseline "toxicity signature" against which future developmental compounds might be compared. The ultimate goal is development of a reporter assay panel to understand mechanism of toxicity that will be predictive and useful in gaining further insight into the mechanism involved in genotoxic stress.
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Poster #26
Interference with the UVC-induced DNA damage response by PKC activating tumor promoters in TK6 cells
Kyle P. Glover, Lauren Markell, E. Maria Donner, Xing Han
DuPont Haskell
Non-genotoxic carcinogens do not directly cause fixed mutations but interfere with many cellular processes during the promotion stage of cancer development.  The purpose of this study was to investigate the effect of tumor promoting compounds on the DNA damage response (DDR), a critical cellular pathway for preventing oncogenesis.   12-O-tetradecanoyl-phorbol-13-acetate (TPA) has been widely used as a model compound to induce cellular changes associated with tumor promotion and belongs to a class of tumor promoting compounds that activate protein kinase C (PKC).  In this study, TK6 cells were treated with TPA, or other PKC activating tumor promoters, for 72 hours which altered growth morphology (increased aggregation and attachment) and slowed the growth rate without significantly affecting viability prior to UVC induced DNA damage.  TPA treated cells were then exposed to 10 J/m2 UVC and analyzed for apoptosis (annexin V/PI) and DNA repair (gammaH2AX) by flow cytometry.  At 24 or 48 hours post UVC exposure, TPA treated cells (0.1 - 1 nM) displayed a dose dependent synergistic increase in apoptosis compared to the UVC exposed vehicle control.  This synergistic effect was mimicked by other PKC activating tumor promoters such as other phorbol esters, mezerein, sapintoxin D or indolactam V.  Phosphorylation of histone H2AX (gammaH2AX) at serine 139 occurs following UVC induced DNA damage and is an established marker for double strand breaks and DNA repair complexes.  Maximum gammaH2AX positive cells were observed 2 hours post UVC exposure (56%).   At 8 and 24 hours post exposure, the percent of gammaH2AX positive cells was reduced to 23% and 6% respectively.  TPA treated cells had a similar percentage gammaH2AX positive cells at 2 hours (50%) but levels were significantly higher than the control at 8 hours (36%) and 24 hours (22%) post exposure, indicating inhibition of DNA repair processes.  DNA damage inducible genes with different biological functions such as p53 feedback control (MDM2), cell cycle arrest (p21), DNA repair (DDB2, GADD45a, XPC) and apoptosis (PUMA, NOXA, BAX, BCL-2) were analyzed at 8 and 24 hours post UVC exposure.  TPA caused a significant decrease in MDM2 expression and a significant increase in p21, GADD45a and NOXA expression compared to DMSO treated cells 8 hours post UVC exposure.  These differences indicate that TPA has a unique gene expression profile following exposure to DNA damage.  Overall, these results show how tumor promoting chemicals can alter critical cellular processes required for preventing neoplastic transformation.  Future work will further elucidate the interacting molecular pathways responsible for the altered DDR.
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Poster #27
Characterization of DNA reactive and non-DNA reactive pharmaceutical drug candidates by gene expression profiling
Pamela L Heard1, Jiri Aubrecht1, Donna Dickinson1, Ahmed Enayetallah1, Albert Fornace2, Peter Henstock1, Petra Koza-Taylor1, Henghong Li2, Yizheng Li1, Maik Schuler1
1Pfizer Inc., Groton, CT
2Georgetown University

Gene expression profiling is expanding our understanding of genotoxic mechanisms involving DNA reactive or non-DNA reactive toxicants.  In this study, TK6 cells were exposed for 4 hrs to compounds of various mechanisms and then examined for gene expression changes using the Agilent Human 4x44 or 6x60 arrays.  In total, 39 treatments were included, a training set (27) and a test set (12).  Class discrimination was based on Random Forest modeling for 18k differentially expressed genes from the training set.  Results show that two compounds within the test set are DNA reactive and 10 compounds are non-DNA reactive.  To gain more mechanistic information for the test set of compounds, the gene expression profiles were interrogated using causal reasoning analysis (CRE) to generate hypotheses for mechanism of genotoxicity.  We identified a set of CRE hypotheses for further testing and validation.

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Poster #28
AZT-exposed Cells Show Tubulin Malfunction Related to Aberrant Stathmin 1 Expression
Andrea V. Rivera, V. C. Sanchez, O. A. Olivero, M. C. Poirier
Center for Cancer Research, National Cancer Institute, NIH
Previous studies from this laboratory have shown that the nucleoside reverse transcriptase inhibitor (NRTI) zidovudine (AZT) induced genomic instability along with many other indicators of genotoxicity.  In addition, we have found abnormal distribution of polymerized β-tubulin and a reduction of unpolymerized β-tubulin in normal human mammary epithelial cells (NHMECs) exposed to AZT for 24 hours. MicroRNA analysis of AZT exposed NHMECs revealed downregulation of hsa-miR-770-5p, a microRNA with multiple cellular targets including Stathmin 1. Stathmin 1 is a phosphoprotein, which depolymeries microtubules by sequestering free tubulin, and when Stathmin 1 is depleted, overexpressed or hypo-phosphorylated, improper spindle formation and chromosome misalignment occur.  Because hsa-miR-770-5p is downregulated in AZT-exposed cells, we hypothesized that upregulation of Stathmin 1 would occur as a consequence of abnormal β-tubulin distribution in these cells.  In this study we exposed NHME-hTERT transformed cells and MCF-10A cells to 100 µM AZT continuously for 1 to 3 passages.  Proteins were extracted and Total Stathmin 1 and Phosphorylated Stathmin 1 were assessed by Western Blot analysis.  We found a 3.5-fold increase in Total Stathmin 1, and a 3.5-fold increase in Phosphorylated Stathmin1 at passage 1, and a 1.3 fold increase in Phosphorylated Stathmin1 at passage 3.  Therefore, Stathmin 1 may be involved in the abnormal distribution of β-tubulin observed in AZT treated cells.  Further studies to elucidate mechanism will comprise confirmation of gene expression with RT-PCR, and Stathmin localization by immunoflourescence, to discover mechanisms involved in regulation of the protein.

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