2004 Pilot Project Summaries             

El Mustapha Bahassi PhD
Department of Cell Biology, Neurobiology and Anatomy

Regulation of BRCA2 Function by ChK2 in response to Irradiation

Breast cancer is the most common malignancy among women worldwide, and is a leading cause of cancer-related deaths. A minority of breast cancer cases is attributable to inherited mutations in rare high-penetrance breast cancer susceptibility genes such as BRCA1 and BRCA2, but the majority appears to be due to multiple low-penetrance genes. Several such susceptibility alleles have been previously suggested, but among them the Chk2 low-penetrance alleles show the most convincing statistical significance. We have established the first known connection between BRCA2 and Chk2. We show that Chk2 phosphorylates BRCA2 on a threonine residue (T3246) located in the C-terminus region of the protein coded by exon 27. Deletion of exon 27 in mice leads to hypersensitivity to DNA crosslinks, chromosome instability, and reduced life span. Frameshift mutations deleting a part of the C-terminus of BRCA2 have been reported in hereditary breast cancer patients. The importance of the C-terminal region of BRCA2 comes from its key role in homologous recombination via its interaction with Rad51. We showed that in a mouse cell line with targeted mutations in Brca2 (Brca2lex1/lex2), which deletes exon 27, DNA repair proteins failed to localize to nuclear foci of DNA damage in response to genotoxic stress. Similarly, a GFP-BRCA2 exon 27 peptide, carrying a T3246A substitution failed to localize to DNA damage-induced foci after stress, whereas the wild type peptide did. Our results indicate that the DNA damage pathway involving the highly conserved Chk2 protein regulates BRCA2 by direct phosphorylation, which controls its localization after DNA damage.


 
William Hardie, MD
Division of Pulmonary Biology, Children's Hospital

Mechanisms of Protection from Nickel-induced Lung Injury by Transforming Growth Factor α Expression

Acute lung injury has a diverse etiology, a poor prognosis (30-60% mortality), and can lead to severe sequelae (pulmonary fibrosis). Numerous precipitating factors have been identified, yet questions remain about the pathophysiological mechanisms controlling this complex condition and their relationship to therapeutic strategies, which are mainly supportive measures. We propose that key events in acute lung injury intersect with select growth-factor mediated pathways, and that knowledge of these pathways will provide insights into the mechanisms of this condition and may redirect or strengthen current clinical approaches. Transforming growth factor-α (TGFa) exclusively binds to the epidermal growth factor receptor (EGFR). Activation of the EGFR leads to phosphorylation of several intracellular signaling pathways that activate gene expression controlling several cellular processes critical to lung function including cellular proliferation and migration, cell survival and extracellular matrix and repair. We have previously generated transgenic mice that constitutively express TGFa in the lung epithelium and survive aerosolized nickel (NiSO4)-induced acute lung injury. Recently, we generated transgenic mice that have lung-specific TGFa expression controlled by a conditional promoter. Using this regulatable TGFa transgenic mouse model our project will determine mechanisms of TGFa protection by testing the hypothesis that expression of TGFa prior to lung injury induces modulators that confer survival by reducing induction of proinflammatory cytokines and reducing neutrophil influx. Our project will test this hypothesis by first determining the timing of TGFa induction conferring survival, and second, by using microarray of gene expression, we will identify other candidate modulators and pathways that may regulate proinflammatory cytokines and neutrophil influx.


 
Gary E. Shull, PhD
Department of Molecular Genetics

Effects of perturbations in calcium homeostasis on tumorigenesis

This laboratory has generated an Atp2a2 heterozygous mouse model, in which one allele of the Atp2a2 gene, encoding the principal, ubiquitously expressed calcium-sequestering pumps (SERCA2a/b), has been disrupted. These mutant mice spontaneously develop squamous cell carcinomas upon aging with a penetrance of nearly 100%. Thapsigargin, a known inhibitor of SERCA2 activity, is known to be a strong tumor-promoter. The affected tissues include the oral cavity, esophagus, forestomach, genitalia and skin, all with keratinized, squamous epithelia. PCR analysis of genomic DNA from neoplastic cells, isolated by laser-capture microscopy from tumor-sections, has shown that the tumors result not from the loss of heterozygozity but due to the presence of a single, functional copy of the Atp2a2 gene. The objective of this project is to determine the effects of SERCA2 haploinsufficiency on responses to specific environmental carcinogens using the multistage mouse-skin carcinogenesis model. The studies included in this project are significant for two reasons: (1) They would indicate whether individuals with perturbed calcium homeostasis constitute an “outlier population” with increased susceptibility to environmental mutagens. This is highly relevant to an aging population: calcium handling becomes increasingly dysfunctional with age and the incidence of cancer rises exponentially with age. Currently, the relationship between aging and cancer is often viewed simply in terms of the kinetics of the several mutations needed to transform a cell. (2) They would address the interplay between somatic mutations, environmental mutagens and cancer; research into the mechanistic details underlying cancer often ignores the role of environmental agents in carcinogenesis.


 
Howard Shertzer, PhD
Department of Environmental Health

CYP1 Gene Expression and Lung Cancer


 
Dorothy Supp, PhD;
Department of Surgery
Mutations in the Melanocortin 1 Receptor Gene Increase the Risk for Skin Cancer by Disrupting DNA Repair Pathways

 
Craig Tomlinson, PhD;
Department of Environmental Health

Gene Expression Profiles to Predict Disease


 
Nancy Steinberg Warren, MS;
Genetic Counseling Program, UC and Children’s Hospital

Changing the Face of the Genetic Counseling Profession

Genetics is fast becoming important in all areas of clinical medicine and public health. Genomic research has identified genetic factors in the etiology of heart disease, diabetes, cancer, and psychiatric disorders and advances in genetics hold tremendous hope for better disease management. Along with this hope, however, comes concern about whether all people will have access to genetic services that are provided in a culturally appropriate manner. It is documented in many health professions that minority providers are more likely to serve minority populations and that underserved patients are more likely to seek out care from providers who share similar cultural and community values. Given the diverse, and constantly changing, population demographics, it is critical to train genetic counselors from diverse backgrounds to best meet the needs of the changing US population. The current minority membership of the National Society of Genetic Counselors is less than 5%. Our primary goal is to establish a Midwest Consortium of Genetic Counseling Programs to develop a three-year regional plan to promote collaborative minority recruitment efforts for genetic counseling training, and to publish a white paper for stakeholders describing the process and outcomes. The Consortium will conduct an intensive two-day Retreat, conduct activities that to increase the number of minority candidates who are interested in and prepared for admission to a genetic counseling graduate program, and improve understanding of and address the unique barriers to minority recruitment into the profession.

Update: An invited retreat focusing on minority recruitment, entitled: "Changing the Face of the Genetic Counseling Profession" was held on August 2-3, at the Hueston Woods Resort & Conference Center. View the brochure and agenda or click here to see pictures from the event.


 
Jianhua Zhang, PhD;
Department of Cell Biology, Neurobiology and Anatomy

An endonuclease in breast cancer development

Breast cancer is a devastating disease. One in eight women is at risk in North America. Deficient apoptosis is generally believed to enable normal cells to gain survival advantages and renders cancer cells resistant to the cytotoxic effects of chemotherapeutic agents. In cooperation with increased cell proliferation, decreased apoptosis results in predisposition to tumorigenesis and resistance to therapy. How apoptosis is regulated during normal mammary gland involution and dysregulated in breast cancer development is unclear.

In collaboration with Dr. Nelson Horseman, we found that deficiencies of a unique endonuclease cofactor, DNA fragmentation factor 45 (DFF45), result in a profound phenotype of deficient apoptosis in mammary gland involution. Based on this observation and our general understanding of mammary gland physiology and mechanisms of apoptosis regulation, we hypothesize that DFF activity is necessary and sufficient for mammary gland apoptosis and is important for prevention of mammary gland tumorigenesis. To address the issue of whether indeed a reduction of apoptosis by DFF loss-of-function predisposes to breast cancer, we will take advantage of our already generated DFF45-/- mouse model that is defective of DFF function and mammary gland involution to examine the involvement of DFF in tumorigenesis in response to carcinogen and oncogene overexpression. To explore therapeutic potential of DFF therapy, we will examine whether DFF gain-of-function is sufficient to suppress mammary gland tumorigenesis in xenografts. This study may provide a new understanding of the development and progression of mammary gland tumorigenesis, and help design new strategies for treatment for human breast cancer.


 

2003 Pilot Project Summaries
Michael T Borchers, PhDDepartment of Environmental Health
Michael.Borchers@uc.edu

CD8+ Cells in Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a progressive disease of the airways. Smoking is the single major cause of COPD but the disease also results from or becomes exacerbated by occupational and environmental exposures. COPD affects > 10 million adults and is the fourth leading cause of death in the United States. Only 10-20 % of smokers develop COPD suggesting the influence of genetic heterogeneity in the pathogenesis of this disease. COPD is marked by the presence of increased macrophages, neutrophils and CD8+ T cells around the airways. A role for T cells in the pathogenesis of COPD is suggested from associations of increased CD8+ T cells in the lung of smokers that develop COPD. Acrolein (CH2=CH-CHO) is a respiratory tract irritant that reproduces the lesions associated with COPD in experimental animals. Macrophage likely play a role in the pathogenesis of COPD through the release of proteases capable of breaking down connective tissue and stimulating mucus secretion. However, the roles of CD8+ T cells in the development of COPD are unknown. One hypothesis is that CD8+ T cells represent a protective function of the immune system that maintains the integrity of the airways following injury but, when accumulation persists, the effector functions of these cells (e.g. macrophage recruitment/activation) increase lung pathology. This proposal seeks to identify the temporal relationships between CD8+ T cell and macrophage accumulation in the lung as they coincide with the development of COPD and examine the CD8+ T cell dependence in COPD pathologies.


 

Iain Cartwright, PhD
Department of Molecular Genetics
Email: Iain.Cartwright@uc.edu

Expression Profiling of Arsenic Susceptibility Variants

In several parts of the world arsenic contamination of the drinking water supply is having severe pathological consequences. Investigations in this laboratory of close to 60 geographically distinct strains of Drosophila melanogaster have led to the characterization of strains that show significant variability in their susceptibility to the toxic effects of sodium arsenite. Detailed genetic analysis of those strains that lie at the extremes of the susceptibility distribution has enabled us to define a small region of the X chromosome that contains one or more loci that are currently being tested in multiple ways for their specific contributions to this arsenite susceptibility phenotype. We plan to capitalize on this information in two ways. First, we will investigate, by microarray-based expression profiling, differences in gene expression between the most sensitive and most resistant Drosophila strains, both in the presence and the absence of sodium arsenite. Second, an RNA interference assay is currently being used to evaluate the effects of loss of function of candidate susceptibility genes on survival of tissue culture cells in arsenite-containing media. We will subject cells that show dramatically enhanced arsenite susceptibility upon specific mRNA ablation to expression profiling both before and after such mRNA ablation. By these combined approaches we aim to provide a comprehensive picture of relative gene expression patterns in both cells and organisms that differ genetically in respect of their sensitivity to arsenite, and thereby provide a data intensive platform on which to further investigate the cellular response to this form of environmental stress.

 

Ranjan Deka, PhD
Department of Environmental Health
Email: Ranjan.Deka@uc.edu

Genetics of Type 2 Diabetes Related Phenotypes in Adolescents: Development of a Resource

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by insulin resistance, dysfunction of the pancreatic beta cells and hyperglycemia. Generally considered to be a disease of the adults, there has been an alarming increase in its incidence in children and adolescents recently. It has been demonstrated that T2DM has both genetic and environmental components. While a number of studies have been undertaken to elucidate the natural history, epidemiology and pathophysiology of T2DM in children and adolescents, to our knowledge, no major study has yet been initiated to understand the genetics of intermediate phenotypes associated with development of T2DM. Our goal is to develop a major study in this direction focusing on the genetics of intermediate phenotypes that progress to T2DM in adolescents. In this pilot project we will develop a resource and preliminary data for this proposed study. We will build this resource based on an ongoing study ‘The landmarks in the progression to type 2 diabetes study’ (NIH R01 DK59183; PI: LM Dolan). Through this funded project, over 2500 blood samples have been collected from children in the Princeton School district, grades 5 through 12 and archived in Dr. Deka’s laboratory. Our aims are to extract DNA from these blood samples and type genetic polymorphisms in two genes, calpain 10 and adiponectin, putative candidate genes for T2DM and obesity. These DNA samples and the preliminary data on the two candidate genes will form the base for the proposed major study on genetics of T2DM in adolescents.


 
Kathleen Dixon, PhD
Department of Environmental Health
Email: Kathleen.Dixon@uc.Edu

DNA Damage-Induce Phosphorylation and interactions
between BLM and RPA

The purpose of this proposal is to examine the role of phosphorylation of two DNA replication/repair proteins (BLM and RPA) in protein/protein interaction and protein function. Both proteins are phosphorylated in response to certain genotoxic agents and both are required for maintenance for genomic stability. BLM is defective in the genetic instability syndrome, Bloom’s syndrome (BS)[1]; RPA phosphorylation is defective in the human genetic instability syndrome, ataxia telangiectasia (AT) [2]. The specific aims of this proposal are as follows:

Aim 1: To characterize DNA damage-specific phosphorylation of BLM and RPA

Aim 2: To determine the effect of phosphorylation on BLM/RPA interaction

Aim 3: To determine the effect of phosphorylation on the DNA unwinding activity.

The long term goal of this proposed research is to understand the role of DNA damage-induced phosphorylation of BLM and RPA in DNA replication and DNA double strand break repair. Ultimately these studies may provide insights into the relationship between these particular genes (BLM and ATM) and environmental susceptibility; in addition, they may serve as a paradigm for the role of protein phosphorylation in the regulation of DNA replication and DNA repair following environmental insult.


 

Mary Beth Genter, PhD
Department of Environmental Health
Email: MaryBeth.Genter@uc.edu

Mechanism and Gender Specificity of Naphtalene Carcinogenicity

Naphthalene (NP) is the major chemical component of both whole and filtered cigarette smoke and is a rodent respiratory tract carcinogen. These data cloud the assessment and perception of human health risks associated with NP, as NP-based consumer products have been widely available for many years. Cigarette smoke is a complex mixture of direct and indirect acting carcinogens, irritants, and particles, but the precise contribution of NP to tobacco smoke-associated pulmonary disease is unclear. Several studies suggest that human females are more susceptible to components of tobacco smoke than males, and female mice are susceptible to NP-induced lung cancer, whereas male mice are resistant. These observations suggest that gender-specific metabolic pathways and/or hormone-mediated pathways may contribute to susceptibility to NP-induced carcinogenesis. While it is known that several cytochromes P450 contribute to the bioactivation of NP, and several Phase II enzymes aid in detoxification, differences in their levels in male vs. female lungs are unknown. We will gain an understanding of NP-induced respiratory tract carcinogenesis by testing the hypothesis that direct comparison of the metabolic responses of male and female mouse lungs to NP will reveal genes and proteins critical in NP carcinogenesis. We will do this by determining the ratio of cyp2f2 to microsomal epoxide hydrolase in female vs. male mouse lung; comparing gene expression in control and NP-treated female and male mouse lung; and determining whether one or more target tissue-generated NP metabolites is capable of estrogen receptor activation.


 
Gurjit Khurana Hershey, MD, PhD
Division of Asthma and Allergy; CCHMC
Email: Gurjit.Hershey@cchmc.org

Gene-Environment Interactions in Asthma Outcome

The pathogenesis of asthma is complex, depending on genetic and environmental variables. As the pool of candidate genes for asthma continues to grow, a necessary next step is to understand how environmental and genetic factors work together to contribute to asthma, especially in early childhood when asthma often begins. The central hypothesis of this proposal is that genetic and environmental factors are critical to the natural history of asthma in children. To test this hypothesis we will focus on 3 specific aims. In Aim #1, we will determine whether immune-related polymorphisms are associated with asthma in children and elucidate genotype:phenotype relationships. We will recruit children with asthma from the Allergy/Immunology clinics at Children’s Hospital Medical Center and then genotype them for snps implicated in asthma. We will examine the effect of each gene individually as well as together with other genes as complex haplotypes. In Aim #2, we will relate genotype to disease outcome. The Children's Health Survey for Asthma (CHSA) will be used to quantify disease related quality of life for comparison across genotypes. In Aim #3, we will relate the effect of mold and dust mite allergen levels to asthma severity and outcome in sensitive subjects. We will determine if and how indoor allergen levels correlate with asthma symptoms and quality of life in sensitive and non-sensitive subjects. Furthermore, we will relate this to the genetic data to determine if a given genotype is associated with a certain response to the environmental load of mold or dust mite.

 

Dan Nebert, MD
Department of Environmental Health
Email: Dan.Nebert@uc.edu

Association between the AHR Genotype and Risk of Head- and Neck Cancer among Smokers

Cytochromes P450 1A1, 1A2 and 1B1 (CYP1A1, CYP1A2, CYP1B1) are responsible for both the detoxication and metabolic activation of innumerable polycyclic hydrocarbons (PCHCs) and arylamines present in cigarette smoke. The dioxin- and PCHC-inducible CYP1A1/1A2/1B1 genes are up-regulated by the aromatic hydrocarbon receptor (AHR), which binds these inducing chemicals and activates transcription. Metabolic activation of many of these and other environmental chemicals is associated with toxicity and cancer. Only 7-10% of cigarette smokers develop cancer of the lung or head-and-neck, strongly suggesting a gradient from “highly sensitive” to “highly resistant” patients with regard to cigarette smoking-induced cancers. Numerous laboratory animal studies have shown that striking genetic differences in the CYP1A1/1A2/1B1 enzyme levels and in AHR affinity are directly correlated with cancer risk caused by PCHCs and arylamines. A systematic study to search for allelotypes and haplotypes of the human CYP1A2/1A2/1B1 and AHR genes has not yet been done but is now possible, due to available data in the Celera database. The CYP1A1 (6.31 kb) and CYP1A2 (7.8 kb) genes are oriented head-to-head, and 24,455 bp apart, on Chr 15q22-24. The CYP1B1 gene spans 8.5 kb on Chr 2p21. The AHR gene spans 47.2 kb on Chr 7p15. Hypothesis: specific allelotypes and haplotypes of the CYP1A1/1A2/1B1 and AHR genes are associated with increased risk of head-and-neck cancer in cigarette smokers. We propose to select 100 “most sensitive” and 100 “most resistant” head-and-neck cancer patients from Professor Gluckman’s patient population (cohort of more than 4,200) and perform SNP-typing of these four genes.


 

Yoli Sanchez, PhD
Department of Molecular Genetics
Email: sanchey@email.uc.edu

The Role of Sfp1 in the response to DNA damage

Genomic integrity is safeguarded in part by checkpoints, which are biochemical pathways that provide cells with a mechanism to detect DNA damage and respond by arresting the cell cycle to allow DNA repair. The checkpoint response also involves the transcriptional induction of genes whose products are involved in DNA metabolism, DNA repair and cell cycle arrest.

Chk1 is a conserved checkpoint kinase that is differentially phosphorylated in response to different types of DNA damage lesions. In the past 5 years the picture of how the kinases signal cell cycle arrest in response to different lesions has begun to emerge. However, little is known regarding the mechanism by which signals from different lesions are integrated by the transcriptional regulatory network. We have found that the transcription factor Sfp1, like Chk1, is phosphorylated in response to ionizing radiation and oxidative stress. This suggests that Sfp1 function is regulated by the response to oxidative damage to DNA. Like Chk1, the transcription factor Sfp1 is differentially modified in response to different genotoxic agents. Therefore, Sfp1 represents a transcriptional target for specific DNA lesions. Using biochemical and genomic approaches in conjunction with the genetically amenable budding yeast model system we will dissect the regulatory switches controlling transcriptional response (or transcriptional repression) to specific lesions caused by cellular and environmental agents. A goal of this proposal is to determine the role that checkpoint signaling plays in the regulation of the transcriptional response to oxidative and radiation-induced DNA damage.

2002 Pilot Project Summaries


Zalfa Abdel-Malek, PhD.
Dermatology

Email: abdelmza@email.uc.edu

Loss-of-function mutations in the melanocortin 1 receptor gene sensitize melanocytes to the photodamaging effects of UV radiation

The synthesis of eumelanin in human melanocytes (hMC) is mainly regulated by the melanocortin 1 receptor (MCIR), the receptor for the melanocortins alpha-melanocyte stimulating hormone (alpha-MSH) and ACTH. Population studies have shown that specific mutations in the human MCIR gene are associated with red hair phenotype, poor tanning ability, and increased risk for melanoma and nonmelanoma skin cancer. We have found that hMC that naturally express these mutations fail to respond to alpha-MSH and are hypersensitive to the cytotoxic effect of ultraviolet radiation (UVR). Further, we found that alpha-MSH reduces the UV-induced apoptosis and promotes the survival of hMC. The survival of hMC is only beneficial if accompanied with genetic stability. The main goal of this pilot project is to investigate the hypothesis that loss-of-function mutations in the human MC1R gene sensitize hMC to the photodamaging effects of ultraviolet radiation (UVR), thus increasing the risk for skin cancer.


 

Scott M Belcher, Ph.D.
Pharmacology and Cell Biophysics
Email: Scott.Belcher@uc.edu

Proteomic analysis of differential estrogen sensitivity in developing neurons

The Specific Aim of this proposal is to define changes in expressed proteins that may contribute to differential sensitivity of developing and mature cerebellar neurons to estrogenic compounds. This aim is built around our laboratory's observation that transient activation of mitogen activated protein kinase (MAPK) by 0.01nM 17b-estradiol (E2) differentially influences viability of immature and mature cerebellar neurons. We hypothesize that specific changes in protein expression and phosphorylation underlay the differential E2-sensitivity of these two neuronal populations. The proposed pilot studies employ two-dimensional (2D) gel electrophoresis to identify differences in expression levels and phosphorylation-state of the expressed proteins (proteome) of primary cultures of E2-sensitive and that of insensitive mature cerebellar neurons. The identity of differentially expressed proteins will be determined by MALDI-mass spectrometric (MS) peptide mass fingerprinting analysis by the CEG's protein analysis core. These studies will likely identify important proteomic differences that contribute to neuronal sensitivity to endogenous and environmental estrogens during neuronal development.



Nira Ben-Jonathan, Ph.D.
Cell Biology, Neuorobiology and Anatomy
Email: Nira.Ben-jonathan@uc.edu

Xenoestrogens and differential pituitary gene expression profiles in estrogen-sensitive and estrogen-insensitive rat strains

Xenoestrogens are chemicals with diverse structures that mimic or interfere with the action of endogenous estrogens. Bisphenol A (BPA), a monomer of polycarbonate plastics, is abundant in the environment, binds to the estrogen receptor (ER) and act as a partial estrogen agonist or as an antagonist. We previously reported that BPA induced lactotroph proliferation, increased pituitary prolactin (PRL) secretion, altered the expression of ER-alpha, ER-beta and c-fos in the pituitary and downregulated tyrosine hydroxylase (TH) in the hypothalamus. Given the multiple interacting pathways and the cascade of signaling molecules that are activated by estrogens and xenoestrogens, the best approach for comparing their overall effect is by using the gene array technique. This technique can simultaneously determine the relative expression of multiple hormonally-responsive genes rather than one or two genes at a time. Our overall objective is to use two strains of rats that differ in their estrogen-sensitivity and compare the effects of BPA and estradiol (E2) on global pituitary gene activation. The DNA gene profiling is a new direction undertaken by our laboratory to better understand the spectrum of biological effects of xenoestrogens with the ultimate goal of generating sufficient preliminary results to serve as a foundation for extramural funding.


 
Jonathan Bernstein, MD
Internal Medicine
Email: Jonathan.Bernstein@UC.Edu

Genetics of nonallergic vasomotor rhinits

Idiopathic environmental intolerance (IEI) represents a complex gene-environment interaction, about which little is known. We propose to use a genomics approach to begin to dissect this complex disease. A subset of IEI is vasomotor rhinitis (VMR), which can be further divided into allergic and nonallergic types. There is experimental evidence suggesting that nonallergic VMR has a strong olfactory/gustatory component. The major histocompatibility region (MHC) on human Chr 6 is known to be involved in numerous allergic and immune responses. Our hypothesis is: differences in the genotype [SNPs in olfactory receptor (OR) genes] can be associated with the phenotype (nonallergic VMR), whereas the genotype of the MHC region, known to be associated with allergic/immune conditions, will not be correlated with the nonallergic VMR phenotype. In the 1 year of funding, we therefore will: (a) select 25 "most severely affected/unequivocal cases of non allergic VMR" patients who are documented to have no atopy and who have both a mother and father available for testing; (b)] use the candidate-gene-region approach, examining DNA from these patients and their parents for polymorphisms in the two largest OR gene clusters, located on Chr 11, plus the MHC region on Chr 6 (as control); and (c) search in these family studies for genotype-phenotype associations, using the transmission disequilibrium test (TDT)-in which the "other chromosome" serves as the control. Finding a significant genotype-phenotype association for explaining this complex environmental /genetic disease would not only be exciting and publishable, but would also provide important preliminary data for submitting a more substantial RO 1 proposal to request NIH funding. This approach might lead to important insight into a complex disease about which almost nothing is known.

 

Bandana Chakraborty, Ph.D.
Environmental Health
Email: Bandana.Chakraborty@uc.edu

Effects of lead exposure and genes on postural balance

Researchers at the Department of Environmental Health and their collaborators have had a long-term interest in studying the effects of chronic early life lead exposure on children's health, leurobiological, and cognitive impairments. A key observation from the data from the Cincinnati Lead Program Project (CLPP), a study initiated in late 1970s in the inner Cincinnati neighborhoods to examine the effects of chronic lead exposure on health, behavior, and growth of children, is that the ability to maintain upright postural balance in six-year old children is significantly impaired with an increase in their blood lead levels, independent of the socio-economic, racial and environmental factors. An independent study showed that postural balance might be influenced by genetic factors, such as the genotypes at a glutathione S-transferase (GST) gene locus (Bhattacharya et al., unpublished data). Evidences are also accumulating, suggesting that one or more genes, acting singly or in interaction with each other, may modify the toxicokinetics of lead absorption. Polymorphisms at the d-Aminolevuline Acid Dehydratase (ALAD), Vitamin D Receptor (VDR), and members of the GST-gene cluster (e.g., GSTM -locus genotypes) are examples of such genetic modifiers of lead exposure induced leurobiological consequences in human. Although associations with lead exposure are yet to be proven, polymorphisms at dopamine receptor (DRD2, DRD3, and DRD4), dopamine transporter, Monoamine oxidase-A and B (MAO-A, MAO-B), CYP1D6, and Nat-2 loci are also implicated in leurocognitive disabilities. From such data, we hypothesize that certain specific genotypes, singly, or acting in combination with each other, may differentially affect the neurobiological impairment in children induced by lead exposure. From the cohort recruited in the Cincinnati Lead program Project (CLPP), approximately 160 children (for whom informed consent for genetic analyses of blood are available and who participated in earlier postural balance measurement studies) will be typed for genotype determinations (through DNA malysis) at 10 candidate genes. Pilot data on these genotypes will be analyzed to seek for evidence of gene-gene and gene-lead exposure interaction effects on upright postural imbalance (an indicator of leuromotor impairement), an end-point earlier shown to be at least partially lead exposure induced.


 
Gary Dean, Ph.D.
Molecular Genetics, Biochemistry and Microbiology
Email: Gary.Dean@uc.edu

Worms and bacteria as environmental tools

How we teach science education in the United States is currently undergoing a thorough re-evaluation. An inquiry based science curriculum is the current working model on how to teach science effectively (National Science Standards, Project 2061) .We propose to give school children the opportunity to actively investigate a problem in biodegradation by a sound scientific process. The system involves the microbe Pseudomonas aeruginosa to detoxify chemical agents in soil. This detoxification will be measured by the ability of earthworms, Allolbophora caliginosa to survive and lignify toxins. Measurement of earthworm survival will be measured either directly by determining their biomass or indirectly by growing a known quantity of e.g. soybeans and measuring their biomass. The scientific education directions unlocked by this program for teachers include microbiology, chemical degradation, spectrophotometry, soil pH, earthworm anatomy, photosynthesis, etc. to name a very few. In addition, the number of potentially interesting and scientifically novel projects (that could yield useful data) that could arise could be equally large; these might include cataloguing the types of chemicals that Pseudomonas can treat, isolation of mutants that treat novel compounds, selection of earthworms for particular traits, etc.

 

Mary Beth Genter, PhD
Environmental Health
Email: MaryBeth.Genter@uc.edu

Genetic factors contributing to alachlor carcinogenesis

The chloracetanilide herbicides, including alachlor, are used world wide for preemergent weed control in such economically-important crops as corn, soybeans, peanuts, and rice. It was estimated that >120 million pounds of alachlor, metolachlor and acetochlor was used in the United States, with other chloracetanilides (e.g. butachlor and pretilachlor) widely used in Asian countries for rice production. These herbicides are associated with a complex, species-dependent range of neoplasm: in laboratory animals; rats treated chronically with alachlor develop tumors of the thyroid gland, stomach, and nasal cavity; brain tumors found in some rats were felt to be extensions of olfactory mucosal tumors, rather than primary tumors. Our laboratory has shown that the outbred Long-Evans rat strain develops olfactory mucosal tumors following 5 mo of daily exposure to alachlor (126 mg/kg/day in the diet), and rats have 10-20 olfactory mucosal polypoid adenomas/adenocarcinomas per rat with >6 mo of treatment with alachlor. Mice, on the other hand, are reported to develop only lung adenocarcinomas upon chronic alachlor exposure. The results of the mouse bioassay should be interpreted with caution. This study was conducted in female CD-1 mice, which tend to display a slow acetylation phenotype; however, we have data suggesting that acetylation is important in the bioactivation of alachlor in the olfactory mucosa. Therefore slow acetylators would be predicted to be resistant to alachlor-induced olfactory mucosa tumors, and we would propose that rapid acetylators, e.g. C57BL mice, would be susceptible.

 

Susan Pinney, Ph.D.
Environmental Health
Email: Susan.Pinney@uc.edu

Population representative case: Control series for an association study of Parkinson's disease

Our primary hypothesis remains that there are specific genotypes that greatly increase the risk of developing Parkinson's disease through interaction with pesticides, metals or other environmental exposures. In the US, incidence of Parkinson's Disease is expected to triple over the next 50 years as the population ages. Numerous studies of PD have suggested have suggested both genetic and environmental factors in its multifactorial etiology. A small fraction (<5%) of PD cases occur in high-risk families suggesting inheritance as an autosomal dominant trait. However, susceptibility genes for the common adult onset idiopathic form of PD have not been identified. Clusters of genes with low penetrance alleles that interact with environmental, occupational, or lifestyle risk factors may be involved in the multifactorial etiology of PD. Previous studies have not addressed whether gene-environment interactions have significant impact on disease risk additional to the separate effects of heredity and environment. The Specific Aims of this pilot project are: 1) To identify and recruit an additional 50 case and 50 control subjects for the proposed study, and obtain detailed exposure history and clinical information; 2) To obtain blood samples from these case and control subjects and use extracted DNA to examine for genetic polymorphisms of candidate genes; 3) To improve our methodology for control identification, recruitment and specimen collection.

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