High Content Flow Cytometry-Based Micronucleus Scoring Method is Applicable to CHO-K1 Cells
Steven Bryce,¹ Jing Shi,² Souk Phonethepswath,¹ Svetlana Avlasevich,¹ Sarojini Raja,¹ Stephen Dertinger^1
1Litron Laboratories, Rochester, NY; ²BioReliance, Rockville, MD

A flow cytometric method for scoring in vitro micronuclei (MN) in lymphoblastoid cells based on a sequential, two dye staining process has been described [Avlasevich et al., Environ. Mol. Mutagen., 47 (2006) 56-66]. The experiments reported herein were undertaken to evaluate the compatibility of the method with the attachment cell line CHO-K1. Initial work was focused on simplifying cell processing by eliminating the need for trypsin and centrifugation steps, and also by incorporating concurrent means of measuring cytotoxicity. Nine independent experiments with mitomycin C- and cyclophosphamide-treated cells verified the effectiveness of the simplified cell processing procedure for simultaneously measuring MN and cytotoxicity. Subsequently, dose-response experiments with seven prototypical genotoxicants and three non-genotoxicants indicated that the method is capable of reliably detecting MN induced by different modes of action. Further work was then directed at evaluating interlaboratory transferability. For these experiments, two laboratories used a common cell handling/analysis protocol to study each of the ten chemicals listed in Annex 3 of the Draft OECD Guidance Document 487. With the exception of benzo[a]pyrene, each site observed increased MN frequencies for the genotoxicants, whereas no significant effects were noted for the non-genotoxicants. Also noteworthy is that throughout these studies, two aneugenic signatures were evident whereby MN were larger and high frequencies of hypodiploid nuclei were induced in the cases of vinblastine, colchicine, and taxol treatments, but not with clastogens or non-genotoxicants. Taken together, these data indicate that flow cytometric scoring of MN can be accomplished in CHO-K1 cells using an efficient protocol that minimizes the number of manipulations required, and the high content assay appears capable of discriminating between aneugenic and clastogenic modes of action.

Back to Top ↑



Comparison of Micronuclei Induction in Duodenum, Colon and Bone Marrow in Sprague-Dawley Rats
Stephanie L Coffing,D.A. Dickinson, M.E. Engel, C.J. Thiffeault, T.J. Shutsky, R.A. Spellman, M.J. Schuler
Pfizer

Currently, the in vivo bone marrow micronucleus assay is one of three tests in the standard test battery to assess the genotoxic potential of a pharmaceutical candidate. In some cases, depending on results of in vitro studies, as well as the mode of action of the compound, the route of administration and/or the degree of systemic exposure, in vivo assessment of genotoxicity in the bone marrow alone may not be sufficient. Based on the potential for high gut exposures to orally administered compounds as well as the potential susceptibility of rapidly dividing cells of the intestinal tissues, we have developed a modified technique for evaluating micronuclei formation in both the duodenum and colon of rats. Adult male Sprague Dawley rats were treated once daily for 2 days with either vehicle or a positive control compound. The duodenum, colon and bone marrow were harvested, processed and analyzed for micronucleus induction. Preliminary results from studies in which micronucleus induction was compared in duodenum, colon and bone marrow, demonstrated differences in the magnitude of response between the 3 tissues. Cyclophosphamide induced micronuclei formation in both bone marrow and colon at doses of 10 and 20 milligram/kilogram but not in duodenum at any dose. Dimethylhydrazine induced micronuclei formation in duodenum at 16.5, 33, 50 and 66 milligram/kilogram and in colon at 33, 50 and 66 milligram/kilogram but not in bone marrow at any dose. Additional ongoing studies will look at the micronucleus response in the 3 tissues for Mitomycin C, Carbendazim, Vinblastine and one compound still to be determined.

Back to Top ↑



Tetramethoxystilbene (TMS), An Inhibitor of CYP1B1, Delays Benzo[a]pyrene (BP) Metabolism and Does Not Protect MCF-7 Cells Against BP-DNA Adduct Formation
Tracey L. Einem. Miriam C. Poirier, Rao L. Divi
LCBG, NCI, NIH

Cytochrome P450 1B1 (CYP1B1) is highly expressed in steroid-responsive tissues and tumors, and is involved in estrogen metabolism. CYP1B1 and CYP1A1 activate xenobiotics, including polycyclic aromatic hydrocarbons (PAHs), such as BP, to form DNA adducts. We hypothesized that TMS, a selective CYP1B1 inhibitor, might reduce BP-DNA adduct levels, as measured by r7,t8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE)-DNA chemiluminescence immunoassay (CIA). During 96 hr of exposure, we measured BPDE-DNA adducts, and CYP1A1 and 1B1 expression changes in MCF-7 breast tumor cells exposed to 1 µM BP, with or without 1 µM or 4 µM TMS, at 2-12 hr intervals. Maximum BPDE-DNA adduct levels for BP alone, BP + 1 µM TMS, and BP + 4 µM TMS were 1572, 1658, and 1718 adducts/108 nucleotides, respectively, and were observed at 16, 24, and 36 hr, respectively. Therefore, TMS induced a right shift (delay) in BPDE-DNA adduct formation. Area under the curve (AUC4-96 hr) values of BPDE-DNA adducts for BP alone, BP + 1 µM TMS, and BP + 4 µM TMS were 76769, 94317, and 91948 adducts/108 nucleotides, respectively, with higher values (p<0.05) found in the TMS-exposed groups. Fold-increase values for maximum CYP1A1 expression induction were 758, 1713, and 2995 for BP alone, BP + 1 µM TMS, and BP + 4 µM TMS, respectively, and were observed at 16, 24, and 36 hr, respectively. Fold-increase values for maximum CYP1B1 expression induction were 50, 60 and 83 for BP alone, BP + 1 µM TMS, and BP + 4 µM TMS, respectively, with peaks at 16, 24 and 36 hr, respectively. Similar to the changes in BPDE-DNA adduct levels, the addition of TMS to BP-exposed cells caused both a right shift (delay) in induction of CYP1A1 and 1B1 expression, and significantly higher expression AUC4-96 hr values, compared to BP alone. When measured at a single point, 24 hr, BP-exposed cells, with 1 or 4 µM TMS added, had decreased CYP1A1 and 1B1 enzyme activities, as measured by ethoxyresorufin deethylase (EROD). However, complete EROD time kinetics are still in progress. Overall, in MCF-7 cells, TMS caused a late induction of CYP1A1 and 1B1 expression, and similarly delayed kinetics of BPDE-DNA adduct formation, although the overall DNA adduct burden was increased. The data suggest that TMS may not offer protection from PAH-DNA damage, since it acts only to delay PAH metabolism.

Back to Top ↑



Assessment Of In Vitro Micronucleus Assay Testing Protocols Proposed In OECD Guideline 487
Maria E Engel, C. Thiffeault, D.A. Dickinson, J.R. Cheung, R.W. Boyes, J. Sherman, M.J. Schuler
Pfizer Global Research and Development

The in vitro micronucleus assay in Chinese hamster ovary (CHO) cells has been used at PGRD since 1997 as a screening tool for test articles that have aneugenic or clastogenic potential. The CHO micronucleus assay identifies the test articles that have a high probability for positive responses in the GLP in vitro cytogenetics assay in human lymphocytes. This screening strategy has led to a significant reduction in genetic toxicology attrition. However, due to inherent differences between the two test systems (cell type and test conditions), this strategy requires multiple tests, and often times an exploratory cytogenetics follow-up assay before proceeding to the required regulatory clastogenicity testing. Switching to an in vitro micronucleus assay as the regulatory-required test could help to reduce resources required to address findings in mammalian clastogenicity testing. Prior to the 2007 Environmental Mutagen Society (EMS) meeting, the Organisation for Economic Co-operation and Development (OECD) held an expert working group meeting where significant progress was made towards a final version of the guidelines for the conduct of the in vitro mammalian micronucleus assay. In support of the proposed changes to the protocols regarding cytotoxicity and treatment, studies were performed using the treatment conditions recommended in the draft guideline TG487 for Chinese hamster ovary cells to test the ten validation compounds outlined in this document. These studies were conducted with and without the cytokinesis blocker, cytochalasin-B (CYB), for four treatment conditions.

Back to Top ↑



Genotoxicity in Cells Exposed to Nitric Oxide and Reactive Oxygen Species in a Novel Co-culture System
Min Young Kim, Laura J. Trudel, Gerald N. Wogan
Bioengineering Dept., Massachusetts Institute of Technology

Dysregulated production of nitric oxide (NO^•) and reactive oxygen species (ROS) by inflammatory cells in vivo may contribute to mutagenesis and carcinogenesis. Here we compare cytotoxicity and mutagenicity induced by NO^• and ROS in TK6 and AS52 cells, delivered by two methods: a well-characterized delivery system; and a novel co-culture system. NO^• delivered at a steady state concentration of 0.6 µM for 2-24 hrs caused dose-dependent cytotoxicity in TK6 cells. A steady state concentration of 7 µM for 3-8 h was required to cause comparable effects in AS52 cells. A cumulative dose of 540 µM min induced mutation fractions (MF) in TK6 at HPRT and TK1 locus of 7.7 x 10^-6 and 24.8 x 10^-6, respectively (2.7 and 3.7 fold above background). The MF induced in the AS52 gpt gene by 1260 µM min was 132 x 10^-6, 10.2-fold above background. NO^• and ROS generated by co-culture with activated macrophages induced MFs at TK1 of 13.9 x 10^-6 and HPRT of 8.8 x 10^-6, 1.9- and 2.8-fold higher than controls in TK6 suspension cells. Addition of NMA, a competitive NOS inhibitor, abrogated this cytotoxicity and genotoxicity. In contrast, in adherent cells AS52 co-culture for 48h, MF increased to 170 x 10^-6 a 9.4-fold increase. Addition of Tiron and/or uric acid, scavengers of O2^• ¯ and ONOO¯ respectively, plus NMA decreased cytotoxicity 93% and suppressed mutagenesis 97%. These results indicate that cell type and proximity to generator cells are determinants of responses induced by ROS and NO^•

Back to Top ↑



Formaldehyde Induces p53-dependent Apoptosis and Cell Cycle Changes in Human Lung H460 Cells
Haley Menard, Mindy Reynolds, Anatoly Zhitkovich
Brown University

Formaldehyde is a widely used carcinogenic chemical known to cause DNA-protein crosslinks. The mechanisms by which these lesions cause cell death and other toxic responses are currently unknown. In this work, we examined the mechanisms of formaldehyde-induced cell death and cell cycle perturbations. Lung epithelial H460 cells were selected as a cellular model because they display normal genotoxic responses, and formaldehyde exposure occurs most commonly by route of inhalation. We found that formaldehyde was a potent inducer of apoptosis as detected by the appearance of subdiploid cells and caspase-mediated PARP cleavage. Formaldehyde-treated cells also showed a strong induction of the p53 transcriptional factor associated with its phosphorylation at Ser-15. In addition, we observed an upregulation of p21, a downstream target of p53 that is known to play a major role in cell cycle arrest. To test the role of p53-dependent pathway directly, we constructed H460 cells with stable knockdown of p53 by shRNA and examined their genotoxic responses to formaldehyde. Control H460 cells displayed a significant cell cycle arrest that was much less pronounced in p53 knockdown cells. Clonogenic experiments detected an increased survival of p53 knockdown cells when compared to p53-proficient cells. We also found that formaldehyde-induced apoptosis was p53-dependent. In conclusion, our findings provide evidence that a p53-dependent signaling pathway is highly responsive to the presence of DNA-protein crosslinks, and plays a key role in formaldehyde-induced cell cycle changes and other toxic responses.

Back to Top ↑



ACB-PCR Measurement of p53 Codon 271 CGT to CAT Mutation in the Nasal Mucosa of rats Exposed to Formaldehyde
Fanxue Meng,¹ E Bermudez,² ME Andersen,² HJ Clewell, III,² BL Parsons^1
1National Center for Toxicological Research, FDA; ²The Hamner Institutes for Health Sciences

Formaldehyde is classified as a Group I human carcinogen with respect to the induction of nasopharyngeal cancers. Formaldehyde exposures are both occupational and environmental because formaldehyde is used in the manufacture of building materials, household products and industrial chemicals, is found in vehicle emissions, and is released indoors from building materials. To accurately assess the risk of formaldehyde exposures, sensitive and relevant biomarker data are needed to describe the formaldehyde mode of action (MOA) at different exposure levels. Mutation in the p53 tumor suppressor gene has been implicated in the mechanism of formaldehyde-induced rat nasal tumor development because abnormal p53 protein accumulated in rat nasal mucosa following formaldehyde exposure and p53 mutations were detected in several squamous cell carcinomas (SCC) generated in a two-year bioassay. The goals of this study were to: 1) determine the spontaneous level of p53 mutation in rat nasal mucosa, 2) to describe the dose-dependence on the induction of p53 mutation following 13 weeks of exposure to formaldehyde, and 3) to correlate the induction of p53 mutation with other endpoints (i.e., induction of cell proliferation and changes in gene expression). An allele-specific competitive blocker-PCR (ACB-PCR) was developed to quantify p53 codon 271 CGT to CAT mutation in nasal mucosa of rats exposed to formaldehyde. Male Fisher 344 rats (6-7 weeks old, 5 rats/group) were exposed to formaldehyde via whole-body inhalation at target concentrations of 0.7, 2.0, 6.0, 10 and 15.0 ppm (0.86, 2.46, 7.38, 12.3, 18.5 mg/m3) for 6 h/day, 5 days/week for 13 weeks. Formaldehyde concentrations of 6 ppm and above are carcinogenic in a two-year bioassay. DNA was isolated from sites of nasal mucosa where the incidence of SCC was greatest and p53 codon 271 CGT to CAT mutant fraction (MF) was determined by three replicate ACB-PCR measurements. Only 8/30 samples had measurable levels of p53 CGT to CAT MFs (>10^-5). The geometric mean MFs for the treatment groups were: 8.64 X 10^-6, 0 ppm; 2.89 x 10-6, 0.7 ppm; 2.71 x 10^-6, 2 ppm; 8.49 x 10^-6, 6 ppm; and 2.67 x 10^-6, 10 ppm; 1.21 x 10-5, 15 ppm. Further, the results indicated a large range of p53 mutation exist in the nasal mucosa of control rats (from below 10^-5 to 1.85 X 10^-4). Thirteen weeks of exposure to formaldehyde did not cause a significant induction of p53 codon 271 CGT to CAT mutation at the doses tested, even though some of these doses have been shown to cause cytotoxicity, changes in gene expression, and to stimulate cell proliferation. This abstract does not necessarily reflect the views or policies of the US FDA.

Back to Top ↑



γ-H2AX Formation in Three-Dimensional Human Tissue Models Following In Vitro Exposure to Sulfur Mustard
Adele L Miller, Offie Clark, Eric Nealley, Kenneth Leiter, William Smith
United States Army Medical Research Inst of Chemical Defense

Sulfur mustard (2-2'-dichlorodiethyl sulfide, SM) is a cytotoxic chemical warfare agent known for its vesicating properties. SM exposure results in DNA damage, eventually leading to cell and tissue death. To elucidate the genotoxic effects of SM, a commercially available, multicellular skin tissue construct, EpiDerm (MatTek Corp.), was used to observe the presence of γ-H2AX foci. γ-H2AX is a phosphorylated derivative of the H2AX histone and is tightly bound to double strand DNA breaks sites thus, it is an indicator of genotoxic injury. EpiDerm constructs were exposed to 0, 50, 100 and 300 μM concentrations of SM topically. Following exposure, tissues underwent immunohistochemistry staining using a mouse anti-γ-H2AX antibody with propidium iodine (PI) as a nuclear stain. Our results show that SM exposure results in the formation of γ-H2AX foci in EpiDerm tissue constructs. In the future, γ-H2AX studies will be investigated in additional models representing the bronchial epithelium and cornea. Together, these constructs correspond to the principal target sites for in vivo toxicity in response to HD exposure.

Back to Top ↑



Studies on Mycotoxins in Human Food Commodities from Cameroon
Patrick B. Njobeh,¹ MF Dutton,1 SH Koch,² SD Stoev,³ AA Chuturgoon^4
1FEHRG, University of Johannesburg, South Africa; ²PPRI- Agricultural Research Council of South Africa; ³Faculty of Veterinary Medicine, Trakia University, Bulgaria; ⁴University of KwaZulu-Natal, Durban, South Africa

An investigation on the metabolite profile of various stored food commodities from Cameroon was conducted. A total of 82 samples were analyzed for various mycotoxins of known genotoxic and carcinogenic properties to include fumonisin B1 (FB1), zearalenone (ZEA), deoxynivalenol (DON), aflatoxins (AF) and ochratoxin A (OTA) by HPLC. An unknown metabolite of Penicillium polonicum previously found in our laboratory to be cytotoxic to human lymphocytes was also screened and its atomic mass determined by TLC and LC-MS, respectively. HPLC results showed that of these samples, 54% contained FB1, 77% had ZEA, 76% had DON, 77% had AF and 4% were with OTA, while co-occurrence frequency of more than one mycotoxin in the same sample was 84%. Significantly much higher FB1 contents in maize averaging 3415 ppb were recorded, while the overall range was 1.9 to 24,225 ppb. Zearalenone mean levels were much higher in peanuts (87.2 ppb) and maize (57.4 ppb) than for beans (43.3 pp b), rice (33.7 ppb) and soybean (2.5 ppb). DON contents were low ranging from 2.4 to 273 ppb. For AF, an average content was found to be 2.3 ppb with peanut and maize as principal substrate. Lowest recorded incidence was recorded for OTA with mean level of 4.5 ppb observed. For the unknown metabolite, LC-MS data indicate the actual [M+H]+ value of 679.4425, thus giving a mass of 678.4347 that does not equate to that of any known mycotoxin. Of the 82 samples analyzed, 45% contained this unknown metabolite. Although a large proportion of samples had fairly low levels of individual mycotoxins, persistent occurrence of more than one mycotoxin in the same matrix indicates that such samples are likely to be of public health hazard. There is need for the structural elucidation of this unknown cytotoxic metabolite since it may possibly be genotoxic.

Back to Top ↑



Mouse Mutation Assay Based on the Pig-a Gene
Souk Phonethepswath. Steven Bryce, Jeffrey Bemis, Stephen Dertinger
Litron Laboratories, Rochester, NY

Glycosylphosphatidylinositol (GPI) anchors attach specific proteins to the cell surface of hematopoietic cells. Of the genes required to form GPI anchors, only Pig-a is located on the X-chromosome. Prior work with rats suggests that the GPI anchor deficient phenotype is a reliable indicator of Pig-a mutation [Bryce et al., Environ. Mol. Mutagen., 49 (2008) 256-264]. The current report extends this line of investigation by describing simplified blood handling procedures, and by testing the assay principle in a second species, Mus musculus. With this method, erythrocytes are isolated, incubated with anti-CD24-PE, and stained with SYTO 13. Flow cytometric analyses quantify GPI anchor-deficient erythrocytes and reticulocytes. For these experiments, CD-1 mice were treated with mutagenic chemicals three times over the course of one week (DMBA at 75 mg/kg/day or ENU at 40 mg/kg/day). Two weeks after the final treatment, DMBA-treated mice were found to exhibit markedly elevated frequencies of GPI anchor deficient erythrocytes and reticulocytes. For the ENU experiment, blood specimens were collected at weekly intervals over a five week period. Whereas the frequencies of mutant reticulocytes were significantly elevated one week after the last administration, the erythrocyte population was unchanged until the second week. Thereafter, both populations exhibited persistently elevated frequencies for the duration of the experiment (mean frequency at termination = 310 x 10^-6 and 523 x 10^-6 for erythrocyte and reticulocyte populations, respectively). These data provide evidence that Pig-a mutation does not convey an appreciable positive or negative cell survival advantage to affected erythroid progenitors, although they do suggest that affected erythrocytes have a reduced lifespan in circulation. Collectively, accumulated data support the hypothesis that flow cytometric enumeration of GPI anchor deficient erythrocytes and/or reticulocytes represents an effective in vivo mutation assay that is applicable across species of toxicological interest.

Back to Top ↑



Genotoxicity Assessments Using Combined Comet and Micronucleus Assays: In Vitro and In Vivo Studies
Leslie Recio,¹ C Hobbs-Riter,¹ C Swartz,¹ K Shepard,¹ C Baldetti,¹ J Winters,¹ W Caspary,² K Witt^2
1Genetic Toxicology Division, ILS, RTP, NC 27709; ²National Toxicology Program, NIEHS, RTP, NC 27709

Genotoxicity assessment by using combined test protocols for the micronucleus and Comet assays in vitro and in vivo enables the evaluation of these two endpoints in the same cell cultures or animals, saving resources and limiting animal use. ILS has established a limited-compound-requiring (? 50 mg) medium-throughput in vitro assay that enables the determination of DNA damage by the Comet assay and micronucleus (MN) induction from the same exposed cell population. ATP levels and the neutral diffusion assay were used as measures of cytotoxicity for the in vitro Comet assay and cell growth or binucleated cell index was used as a measure of cytotoxicity for the in vitro MN assay. After establishing appropriate dose levels based on cytotoxicity, cells are exposed to the test compound for 4 hr (± S9), after which an aliquot of cells is removed for Comet assay analysis. The remaining cells are allowed to grow for an additional 20-24 hrs post-exposure to collect cells for MN determination. This in vitro multiplex assay was used to examine the cytotoxicity and genotoxicity of model compounds and cigarette smoke condensate (CSC) in mouse lymphoma and human TK6 cells. For these studies, ATP levels and cell growth showed similar dose-response for cytotoxicity while the neutral diffusion assay did not show any correlation to cytotoxicity levels based on either ATP or cell growth. These data indicate that ATP level-based but not neutral diffusion-based cytotoxicity assessments are useful to assess cytotoxicity-based dose-setting for the Comet assay. All CSC samples showed genotoxicity with either the Comet or MN assay with the Comet assay exhibiting a broader range of responses among the CSC samples. Although the in vivo MN assay has proven an effective measure of a chemical's potential for genotoxicity in a single tissue (bone marrow), combining the MN assay with the Comet assay permits genotoxicity assessments in additional tissues such as liver, stomach, and colon. We initially evaluated a combined test protocol using the in vivo micronucleus and Comet assay with four model genotoxic compounds: acrylamide, ethyl methanesulfonate, cyclophosphamide, and vincristine. Test compounds were administered over 3 to 4 consecutive days to male B6C3F1 mice or F344 rats, and mice and rats were killed 4 hours after the last administration. Micronucleus frequencies in peripheral blood erythrocytes were determined using flow cytometry, and DNA damage was measured using the Comet assay in blood leukocytes, liver, and either stomach or duodenum. For the Comet assay, 3 measures were examined: % tail DNA, tail length, and Olive tail moment (OTM). For the 4 model genotoxic compounds, positive results in the Comet assay were observed for acrylamide, ethyl methanesulfonate, and cyclophosphamide in multiple tissues while vincristine was negative in all tissues. We have now expanded our testing to include 11 structurally diverse chemicals. Among these, 3 were positive in both assays, 2 were negative in the MN assay but positive for DNA damage in blood leukocytes, and the rest were negative in both assays. None induced MN-RET without showing evidence of DNA damage in at least one tissue. Further studies with additional test compounds are ongoing in mice and Wistar-Han rats to more thoroughly evaluate the usefulness of data obtained through this combined testing protocol. Results of these studies demonstrate that the MN and Comet assays can be used effectively in combined test protocols, providing useful genotoxicity assessments.

Back to Top ↑



Haplotype Analysis of the Full XPC Genomic Sequence Reveals a Cluster of Variants Associated with Sensitivity to the Genotoxic Effects of Tobacco Smoke
Catherine M Rondelli,¹ Jeffery K Wickliffe,² Randa A El-Zein,³ Carol Etzel,³ Sherif Z Abdel-Rahman^1,2
1Department of Neuroscience and Cell Biology, UTMB; ²Department of Preventive Medicine and Community Health,UTMB; ³Department of Epidemiology, MD Anderson Cancer Center

The Xeroderma pigmentosum complementation group C protein, encoded by the XPC gene, plays a key role in the nucleotide excision repair process. XPC is highly polymorphic, but only a few single nucleotide polymorphisms (SNPs) have been studied as modifiers of cancer risk. To date, the phenotypic effects of these SNPs have not been characterized, nor has their impact on DNA damage-response and DNA repair capacity been determined. In this study, we constructed a comprehensive haplotype map encompassing the common SNPs in the XPC gene, and evaluated their effect on DNA damage associated with smoking, using chromosome aberrations (CA) as a biomarker. We hypothesized that if certain haplotypes have phenotypic effects, there would be a correlation between these haplotypes and CA in smokers. Our results indicate that out of 92 SNPs identified, 35 had a minor allele frequency ?0.05. A haplotype-tagging (ht) approach was used to identify 12 htSNPs representing these 35 SNPs. We used these htSNPs to genotype a population of smokers matched to non-smokers (n=123), and to construct corresponding haplotypes. Out of 48 haplotypes we identified 20 that existed with a frequency ?0.05. Phylogenetic analysis revealed that these 20 haplotypes segregate into 5 distinct phylogenic groups of haplotypes (PGHs A to E). When we evaluated the relationship between these PGHs and CA, we observed that smokers with PGH-E had double the mean CA frequency (mean CA/100 cells ±SEM=1.25±0.257) compared to smokers with other haplotypes (0.568±0.137). We also observed significant interactions between smoking and PGH-B (P=0.042). Among those negative for PGH-B, CA was 1.35 times higher in smokers compared to non-smokers; whereas among those who were positive for PGH-B, CA was 41% less in smokers compared to non-smokers. Given the strong association between CA and cancer, our data suggest that XPC haplotypes could significantly affect the risk of smoking-related cancers.

Back to Top ↑



Coupling Cytotoxic Biomarkers with Genotoxicity Tests
Jing Shi, Sandra Springer, Shannon Bruce, Jamie Sly, Maria Scherer, Mark Cecil, Kamala Pant, Patricia Escobar
Genetic Toxicology Department, BioReliance Corporation

In in vitro genotoxicity tests, the maximum concentrations of test compounds are determined by cytotoxicity, unless limited by solubility in solvents or culture medium. Cytotoxicity, which measures the degree of cell damage or death due to a chemical, varies in different test systems depending on the method to estimate it and the levels of toxicity that must be achieved. According to ICH S2A, in cytogenetic assays, the acceptable level of toxicity is defined as a 'significant reduction (>50%)' in cell number or culture confluency whereas mammalian cell mutation tests require relative total growth or cloning efficiency (CE) to be reduced by 80-90%. Conventional cytotoxicity assessment methods, including trypan blue exclusion assay and mitotic index, often underestimate cytotoxicity, revealing the need for developing reliable cytotoxic biomarkers to provide high sensitivity and certain indications of cytotoxic mechanisms in genotoxicity tests. Here, we introduced the ATP determination assay and Caspase-3 assay in combination with the in vitro Comet assay to test a number of cytotoxins, genotoxins and apoptosis inducers in in vitro mammalian cell culture systems. The ATP determination assay quantifies the ATP amount in metabolically active (live) cells, thus correlates well with cell number and viability. The Caspase-3 assay measures the activation of the Caspase-3 protease, which indicates the onset of apoptosis. The use of these cytotoxic biomarkers greatly aids in determination of maximum concentration of test articles in genotoxicity tests and provides a powerful tool to understand the mode of action (MOA) for the tested chemicals.

Back to Top ↑



Mouse Lymphoma Assay Data Mining Can Provide Insight Into the Various Measures of Cytotoxicity
Tracie L Verkler, Dr. Martha Moore
National Center for Toxicological Research

As routinely conducted, the MLA requires counting cells in the negative controls and treated cultures prior to treatment and again approximately 24 and 48 hrs following the initiation of treatment. In addition, the mutant selection phase provides a plating efficiency (PE) for each culture. There is currently a lively debate concerning the best way to assess cytotoxicity for the in vitro micronucleus assay. Various methods including relative cell number and population doubling are currently used. Much of the discussion revolves around trying to determine the fate of cells affected by the chemical treatment. By mining our MLA database, we see cytotoxicity patterns from chemical treatment. With the available data, we can calculate relative (to the negative control) cell number, population fold increase and population doubling time. These can be calculated at 24 and 48 hrs. The PE of cultures (plated approximately 48 hrs following the initiation of treatment) is also available. In addition the "normal" cytotoxicity measure for the MLA--the relative total growth (RTG)--is available. This cytotoxicity measure accounts for relative cell growth in the expression phase and relative PE in the cloning phase of the assay. When all of these various measures are compiled for a specific chemical, patterns of cytotoxicity emerge. (1) Some chemicals cause most of their cytotoxicity during the first 24 hours and then grow and plate almost as well as the negative control. (2) Some chemicals have a delayed cytotoxicity. Their cell growth and PE indicate lingering effects that continue to affect the cells. (3) Some chemicals show a pattern intermediate to patterns 1 and 2. Our assessment shows that cell counts taken only approximately 24 hrs after the initiation of treatment are unable to reveal the cytotoxicity pattern caused by the test chemical. Furthermore, this analysis provides a means to compare the various cytotoxicity measures that can be applied to MLA data. This analysis reveals that the various measures will give different "cytotoxicity" values and because dose selection is based upon covering the appropriate cytotoxicity range, the various measures will result in chemicals being tested over different dose ranges. Because of the various growth patterns after treatment, it is not possible to readily convert some of the cytotoxicity measures to other measures.

Back to Top ↑