Kedderis G.L.,1803 Jones Ferry Road |
Shepard K.G.,Integrated Laboratory Systems, Inc. |
Recio L.,Integrated Laboratory Systems, Inc.
Chemico-Biological Interactions | Year: 2014
Chronic inhalation exposure to high concentrations of naphthalene produced nasal tumors in rats and lung tumors in female mice. Naphthalene bioactivation is required for target organ toxicity and cytotoxicity in target organs may be involved in tumor development. The present studies characterized the dose-response relationships for naphthalene-induced glutathione (GSH) depletion, effects on cellular ATP, and cytotoxicity in cells from both target (lung, nasal epithelium) and non-target (liver) organs in vitro using cells from F-344 rats, B6C3F1 mice and humans. The cells were incubated with various concentrations of naphthalene in sealed glass flasks for 3 h, then placed in monolayer culture in fresh media for 24 h to examine the repair or progression of damage. Naphthalene was a low potency cytotoxicant in vitro, with 500 μM frequently observed as a no-observed adverse effect concentration or lowest observed adverse effect concentration. Naphthalene exposure produced dose-dependent decreases in cellular GSH, ATP and viability in rat, mouse and human hepatocytes at concentrations >500 μM. Human nasal respiratory epithelial cells exhibited greater naphthalene cytotoxicity than rat or mouse nasal respiratory epithelial cell preparations. Rat nasal respiratory epithelial cell preparations metabolized naphthalene through pathways leading to the preferential formation of 1,2-naphthoquinone GSH conjugates rather than 1,4-naphthoquinone GSH conjugates observed in rat hepatocytes or mouse nasal respiratory epithelial cells, consistent with the suggestion that this bioactivation pathway may be involved in rat nasal tumor development. Naphthalene exposures of ≥500 μM decreased cellular GSH and ATP in rat, mouse and human lung cell preparations. The variability of the responses of the human lung cell preparations was consistent with the known variability of CYP activities in human lung tissue. The results of these studies can be used as the basis for future studies of the mechanisms involved in naphthalene-induced cytotoxicity and the relevance of the bioactivation pathways for human exposure to naphthalene. © 2013 Elsevier B.V. All rights reserved.
Recio L.,Integrated Laboratory Systems, Inc. |
Shepard K.G.,Integrated Laboratory Systems, Inc. |
Hernandez L.G.,National Health Research Institute
Toxicological Sciences | Year: 2012
The dose-response relationship for the induction of micronuclei (MN) and the impact of glutathione (GSH) detoxication on naphthalene-induced cytotoxicity and genotoxicity were investigated in human TK6 cells. TK6 cells were exposed to 10 concentrations ranging from 0.0625 to 30μM naphthalene in the presence of β-naphthoflavone- and phenobarbital (βNP/PB)-induced rat liver S9 with a nicotinamide adenine dinucleotide phosphate-generating system. Three approaches were used to identify a no-observed-effect level (NOEL) for naphthalene-induced genotoxicity: (1) laboratory criteria of ≥ twofold increase over the concurrent solvent controls (NOEL = 10μM), (2) ANOVA with Bonferroni correction (NOEL = 2.5μM), and (3) the benchmark dose approach (BMCL 10 = 3.35μM). The NOEL and point of departure micronucleus frequency for naphthalene-induced MN are between the tested naphthalene concentrations of 2.5-10.0μM in this experimental system. Supplementation of the exposure system with physiological relevant concentrations of 5mM GSH eliminated naphthalene-induced cytotoxicity and genotoxicity; no increased cytotoxicity or genotoxicity was observed at concentrations of up to 500μM naphthalene in the presence of GSH compared with 2.5-10.0μM in the absence of GSH. Naphthalene bioactivation by βNP/PB-induced rat liver S9 exhibits a nonlinear dose-response for the induction of MN in TK6 cells with a NOEL of 2.5-10μM that in the presence of GSH is shifted upward greater than 50- to 200-fold. These data demonstrate a nonlinear dose-response for naphthalene-induced genotoxicity that is eliminated by GSH, and both observations should be considered when assessing human risk from naphthalene exposures. © The Author 2012. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
Yin Z.,U.S. National Institutes of Health |
Menendez D.,U.S. National Institutes of Health |
Resnick M.A.,U.S. National Institutes of Health |
French J.E.,U.S. National Institutes of Health |
And 2 more authors.
Cancer Research | Year: 2012
The ubiquitin interaction motif-containing protein RAP80 was recently found to play a key role in DNA damage response (DDR) signaling by facilitating the translocation of several DDR mediators, including BRCA1, to ionizing irradiation (IR)-induced foci. In this study, we examine the effect of the loss of RAP80 on genomic stability and the susceptibility to cancer development in RAP80 null (RAP80-/-) mice. RAP80-/- mice are viable and did not exhibit any apparent developmental defects. Mouse embryonic fibroblasts (MEF) derived from RAP80-/- mice underwent premature senescence compared with wild-type (WT) MEFs, were more sensitive to IR, and exhibited a higher level of spontaneous and IR-induced genomic instability. RAP80-/- thymocytes were more sensitive to IR-induced cell death than WT thymocytes. RAP80-/- mice were more susceptible to spontaneous lymphoma development and the development of 7,12-dimethylbenz(a)anthracene-induced mammary gland tumors. Moreover, the loss of RAP80 accelerated tumor formation in both p53-/- and p53+/- mice. Our data indicate that RAP80-deficiency promotes genomic instability and causes an increase in cancer risk consistent with the concept that RAP80 exhibits a tumor suppressor function. ©2012 AACR.
Waters M.D.,Integrated Laboratory Systems, Inc. |
Jackson M.,Integrated Laboratory Systems, Inc. |
Lea I.,Integrated Laboratory Systems, Inc.
Mutation Research - Reviews in Mutation Research | Year: 2010
The results of predictive toxicogenomics investigations over the past 6 years reviewed in this report have shed new light on the potential of molecular expression analysis to more properly classify both genotoxic and nongenotoxic carcinogens and to predict the carcinogenicity of untested chemicals. Predictive toxicogenomics uses global molecular expression data resulting from genomic perturbation (e.g., transcription or gene expression profiles) to predict a toxicological outcome, such as carcinogenicity. The classification of carcinogens has become an essential and highly debatable component of cancer risk assessment largely because of the default assumptions that drive regulatory decision-making regarding the presumed linearity of the dose-response curve for genotoxic carcinogens. Nongenotoxic mechanisms of carcinogenesis complicate the well-established relationship between genotoxicity and carcinogenicity and challenge the interpretation of the results of rodent carcinogenicity studies in terms of their relevance to humans. Although the number of presumed nongenotoxic rodent carcinogens has dramatically increased over the past two decades, the fact remains that more than 90% of the known human carcinogens are detected in conventional short-term tests for genotoxicity and induce tumors at multiple sites in rodents. In toxicogenomics studies, a strong DNA damage response at the gene expression level suggests direct DNA modification whereas increased expression of genes involved in cell cycle progression is more characteristic of the indirect-acting agents such as those that induce oxidative stress. Metabolism genes are prominently represented among gene expression profiles that discriminate nongenotoxic modes of action (e.g., cytotoxicity and regenerative proliferation, xenobiotic receptor agonists, peroxisome proliferator-activated receptors, or hormonal-mediated processes). The evidence accumulated to date suggests that gene expression profiles reflect underlying modes or mechanisms of action, such that they will be useful in the prediction of chemical carcinogenicity, especially in conjunction with conventional short-term tests for gene mutation, chromosomal aberration and aneuploidy. © 2010 Elsevier B.V.
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 1.40M | Year: 2015
DESCRIPTION provided by applicant Thousands of new industrial chemicals are produced each year Knowledge about the potential carcinogenicity of these compounds is therefore critical to public health It is well established that DNA damage can lead to the mutations that cause cancer Therefore being able to screen chemicals for their potential to damage DNA offers an effective strategy for improving public health Several genotoxicity screens have been developed and are used today effectively with high throughput screening technology However there remains a critical gap in the tools that we currently have available Specifically while man types of DNA damage can be detected using existing technologies bulky DNA adducts remain undetectable It is well established that bulky lesions can be carcinogenic For example aflatoxin is known to play a major role in causing liver cancer around the world Furthermore polycyclic aromatic hydrocarbons present in our environment are known to increase the risk of cancer Using todayandapos s technologies a novel chemical that causes analogous DNA lesions would not be identified as being a potential hazard One of the key challenges lies in the fact that many types of DNA damaging compounds are benign unless they are metabolically activated which usually happens in hepatocytes Therefore use of cell types other than hepatocytes limits detection ability To overcome these limitations we propose a combination of scientific and engineering innovation to create a high throughput comet assay based technology that detects bulky DNA lesions The principle of the comet assay is that damaged DNA can be detected by its ability to migrate more readily than undamaged DNA when electrophoresed While effective for detecting strand breaks abasic sites and alkali sensitive sites bulky lesions are not detectable using the comet assay under standard conditions Here we propose to refine established approaches to develop a method for detecting DNA base adducts Furthermore we propose refining these approaches for use with hepatocytes that are able to metabolically activate potential carcinogens Finally we propose to harness our recently developed CometChip platform a microfabricated system that dramatically increases both sensitivity and throughput In Phase I of this proposal we propose to establish approaches that are effective for detecting bulky lesions in the hepatocytes and to demonstrate efficacy of high throughput screening by analyzing a small library of select compounds In Phase we will leverage this technology to screen up to compounds available through the National Toxicology Program of the NIEHS and develop a general screening platform to detect DNA damage caused by agents from a variety of chemical classes Results of the proposed studies will fill a critical gap in genotoxicity testing both nationally and internationally Resulting technologies will provide an effective platform for the NTP as well as enhancing the efficacy of genotoxicity testing in industrial settings As such it is anticipated that the proposed technology will have a significan impact on public health PUBLIC HEALTH RELEVANCE Knowing that a chemical has the potential to induce cancer is critical to public health DNA damage is known to promote toxicity and mutations that drive cancer but there is a major gap in our current assays for DNA damage due to the fact that lesions that bind DNA rather than breaking DNA are not detected We already know of examples where DNA binding chemicals cause cancer therefore it is critical to develop a screening method that ensure that agents that bind to DNA are identified before they cause cancer in people who are exposed
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 224.97K | Year: 2014
DESCRIPTION (provided by applicant): Thousands of new industrial chemicals are produced each year. Knowledge about the potential carcinogenicity of these compounds is therefore critical to public health. It is well-established that DNA damage can lead to the mutations that cause cancer. Therefore, being able to screen chemicals for their potential to damage DNA offers an effective strategy for improving public health. Several genotoxicity screens have been developed and are used today effectively with highthroughput screening technology. However, there remains a critical gap in the tools that we currently have available. Specifically, while man types of DNA damage can be detected using existing technologies, bulky DNA adducts remain undetectable. It is well-established that bulky lesions can be carcinogenic. For example, aflatoxin is known to play a major role in causing liver cancer around the world. Furthermore, polycyclic aromatic hydrocarbons present in our environment are known to increase the risk
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 149.94K | Year: 2012
DESCRIPTION (provided by applicant): Current assays employed to assess the potential of drugs and environmental agents for their potential to cause birth defects involve the use of animal models. Not only are these models costly and time consuming, but they also have poor concordance to human data. SteminaTM Biomarker Discovery (Stemina) has developed a screening assay that utilizes human embryonic stem (hES) cells and metabolomics to study the secretome of hES cells exposed to test compounds in an all human model system to identify compounds with teratogenic potential. Integrated Laboratory Systems (ILS) and Stemina have formalized a partnership to advance devTOXTM, Stemina's stem cell-based toxicology testing platform. This SBIR will deepen and extend theunique devTOXTM platform by interrogating impact on the human genome and epigenome of hES cells from toxicant exposures. Toxicity testing screens focused on effects to the epigenome are aspect of toxicity based screening that with a few exceptions is virtually absent in high content cellular based toxicity assays. The focus of this SBIR is to: 1. conduct time course and dose-response studies directed at evaluating known and investigative epigenetic biomarkers indicative of aberrant hES cell function usingthe devTOXTM platform and 2. integrate toxicogenomics-based mRNA and miRNA biomarkers to the devTOXTM platform to assess the impact of toxicants on the genome and epigenome in three specific aims. Specific Aim 1 is to assess stability of biomarker genes and miRNAS associated with maintaining self- renewal and pluripotency of hES cells during cell culture expansion. Specific aim 2 Assess the impact of a reference set of test compounds on genomic and epigenomic response biomarkers in hES cells. Specific aim 3will test 10 blinded chemicals provided by DOW Chemical Company in DevTOXTM integrated with genomic and epigenomic response biomarkers, then compare results to Dow in-house test results from other developmental toxicology screens. We anticipate that the integration of the gene expression and epigenomic profiling with the devTOXTM will increase the predictivity of the devTOXTM platform increasing its use as a weight- of-evidence tool in predicting potential development and reproductive toxicity.PUBLIC HEALTH RELEVANCE: Stem cells have the unique ability among all of the cells of the human body of self- renewal, that is, they can remain in a primitive unspecialized state. Under the right conditions, they can give rise to specialized cells of thebody (differentiation) like th heart, liver, or pancreas. Human embryonic stem cells are being developed as a toxicity testing platform for the assessment of developmental toxicity. These cells present a unique model system to understand and assess the effects of environmental agents and new drug candidates to predict or anticipate toxicity in humans.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 224.31K | Year: 2013
PROJECT SUMARY/ABSTRACT This proposed project is in response to the need for more extensive toxicological evaluation of environmental chemicals and for predictive models to assess associated risks of chemical exposure to humans. There is also need for innovative methods for evaluating the effects of chemicals on pluripotent stem cells and the differentiation process. A novel chromatin structure bearing both repressive and activating histone modifications ( bivalent domains ) that direct gene expression is highly enriched in embryonic stem (ES) cells as compared to differentiated cells. Bivalently marked histones silence developmental genes in ES cells while keeping them primed for activation upon initiation of specific differentiation programs. The uniquecharacteristics of chromatin and poised status of bivalently marked genes in ES and induced pluripotent stem (iPS) cells may render them particularly sensitive targets for epigenetic effects resulting from chemical exposure. To expand the scope of toxicological evaluation of chemicals, an objective of this project is the development of an assay platform designed to monitor histone modifications in stem cells using a biomarker panel of developmentally-relevant genes characterized by the bivalent histone signature. Using chromatin immunoprecipitation (ChIP) and PCR arrays, promoter regions of a panel of human stem cell transcription factors, Polycomb and Trithorax target genes, and markers of cell lineage progression will be screened to identify promoters exhibiting bivalent histone marks in stem cells. A subset of these consensus genes that exhibit the greatest response to chemical inhibitors of histone modifying enzymes will be identified and validated as biomarkers for detecting an epigenetic response in stem cells. In addition, a Matrix ChIP assay platform that allows for all steps to be performed in a single 96-well plate will be established and validated. To accomplish these goals the following specific aims are proposed: 1) Employ ChIP using antibodies against specific opposing histone marks (i.e., H3K4me3, H3K27me3, and H3K9ac) and PCR arrays to: a) identify a consensus panel of bivalently marked genes relevant to stem cell differentiation in several pluripotent stem cell lines and b) use canonical chemical inhibitors of histone methylation, acetylation, and deacetylation to define a signature subset of epigenetically responsive bivalently marked genes; 2) Validate the ability of the signature biomarker panel to detect chemical-induced effects on thestem cell epigenome using a panel of reference chemicals; and 3) Establish and validate a 96-well Matrix ChIP assay platform for evaluating the effects of chemicals on epigenetic marks located at bivalently marked genes in stem cells. This project will result in a set of bivalently marked biomarker genes useful for evaluating effects of chemicals on histone modifications relevant to stem cell development and a validated 96-well plate method for measuring histone markers. The long term objective is to adaptthe assay to a higher throughput platform for rapid and efficient screening of effects of environmental toxicants on the human epigenome that could predispose an individual to disease. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: There is a need formore extensive toxicological evaluation of environmental chemicals and for predictive models to assess associated risks of chemical exposure to humans. The overall goal of this project is to develop an assay to evaluate the effects of chemicals directly on specific patterns of biochemical modification ( epigenetic marks ) of proteins located at regulatory domains of genes that control stem cell growth and maturation into different organs of the body. The long term objective is to adapt the assay to a highthroughput platform for rapid and efficient screening of effects of environmental chemicals on the human epigenome that could predispose an individual to disease, including cancer.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 299.36K | Year: 2013
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 1.49M | Year: 2015
DESCRIPTION provided by applicant This proposed project is in response to the need for more extensive toxicological evaluation of environmental chemicals and for better predictive models to assess associated risks of chemical exposure to humans There is also need for innovative methods for evaluating the effects of chemicals on pluripotent stem cells and the differentiation process Maintenance of stemness and the stem cell differentiation processes is regulated by networks of genes controlled at least in part by the stem cell epigenome Disruption of this finely tuned regulatory circuit by exposure to certain environmental agents can lead to adverse health effects Chromatin in pluripotent stem cells is characterized by several unique properties including a novel structure highly enriched in embryonic stem ES cells in which nucleosomes bear juxtaposed repressive and activating histone modifications andquot bivalent domainsandquot that direct gene expression Bivalently marked histones silence developmental genes in ES cells while keeping them andquot primedandquot for activation upon initiation of specific differentiation programs The unique characteristics of chromatin and poised status of bivalently marked genes in stem cells may render them particularly sensitive targets for epigenetic effects resulting from chemical exposure To expand the biological landscape of toxicological evaluation of chemicals the objective of this project is to develop a medium throughput assay platform to monitor pertinent histone modifications at a panel of developmentally relevant genes in human embryonic stem hES cells A comprehensive toxicogenomics evaluation of gene expression changes will be performed using a training set of chemicals representing various classes of chemicals known to affect enzymes that modulate histone acetylation and methylation including some known teratogens Correlating changes in specific active and repressive histone marks at the promoters of consensus differentially expressed genes will then be identified to define a biomarker signature indicative of an andquot epigenetic responseandquot in stem cells The signature will be validated by blind testing of a set of chemicals using a ChIP assay platform that allows for all immunoprecipitation steps to be performed in a single well plate The following specific aims are proposed to accomplish these goals conduct expression profiling and ChIP evaluation of hES cells exposed to chemicals known to influence histone acetylation methylation using a training set of compounds to construct a predictive transcriptome based signature of epigenetic impact on developmental processes establish methodology for preparing sheared chromatin from hES cells directly in the chemical exposure plate and for conducting ChIP in a well assay plate develop and implement bioassay standard procedures and quality control criteria and test the epigenetics based signature derived from the training set of chemicals with a blinded set of test chemicals This project will result in a medium throughput platform for rapid and efficient screening of effects of environmental toxicants on the human epigenome that could lead to developmental defects or predispose an individual to disease PUBLIC HEALTH RELEVANCE There is a need for more extensive toxicological evaluation of environmental chemicals and for predictive models to assess associated risks of chemical exposure to humans The overall goal of this project is to develop an assay to evaluate the effects of chemicals directly on specific patterns of biochemical modification andquot epigenetic marksandquot of proteins located at regulatory domains of genes that control stem cell growth and maturation into different organs of the body The assay platform is intended for rapid and efficient early screening of effects of environmental toxicants that could lead to developmental defects and or predispose an individual to disease or cognitive disorders