Project 1: Modeling complex exposures and time-to-event data
Project 2: The PIN pediatric study
Project 3: Specific fatty acids alter cellular responses to environmental agents
Project 4: Genetic analysis of transcriptional regulation in liver
Project 5: Genetic susceptibility to prenatal neurotoxicants
Project 6: The role of metabolism in the modulation of toxic and cancer promoting effects of arsenic in human urinary bladder
Project 7: Human responses to UV-induced DNA damage
Project: Modeling complex exposures and time-to-event data
Investigators often go to great lengths to obtain careful, detailed measures of exposure, which may have multiple dimensions and may change over time, e.g. diet, stress, or blood pressure. In assessing the association between an individual’s exposure history and the time or rate of occurrence of a health event, it is important to reduce the dimensionality of this multivariate exposure history data in order to increase statistical power. Although replacing the multivariate exposure information with a simple summary, as is typically done in practice, can sometimes improve interpretability and statistical power, it is typically not clear how best to summarize the information at hand. In addition, reducing detailed data into naive summaries often runs counter to the study goals of obtaining the most accurate assessment of exposure possible. We are interested in developing and applying statistical methods, allowing evidence-based summaries to be constructed objectively in a manner that maximizes information about the outcomes of interest.
Project: The PIN pediatric study
Long chain fatty acids contribute to the structure and function of brain and retina during normal development, which begins in gestation and continues through early childhood. Clinical trials have indicated that supplementing infant formula with specific fatty acids may improve visual acuity and cognitive function, especially among preterm infants. Little is known, however, about whether variation in the maternal fatty acid profile during pregnancy might affect the infant’s neurodevelopment. We propose to examine the relation between maternal fatty acid levels during pregnancy and the infant’s visual and cognitive development. This study takes advantage of the Pregnancy, Infection, and Nutrition (PIN) Study and the PIN Postpartum Study. PIN collects considerable information on the women during pregnancy, including diet, stress, infection, and blood samples. PIN Postpartum will conduct a home visit with the women 3 and 12 months after delivery to assess postpartum diet, physical activity and psychosocial factors. Children born to participants of the PIN Postpartum Study will be eligible to participate in the PIN Pediatric Study. This study will add an evaluation of the child’s development to the mother’s scheduled postpartum home visit. The developmental evaluation will include a test of visual acuity using Teller Acuity Cards, assessment of mental and motor skills using the Bayley Scales of Infant Development, and assessment of early language and communication skills by the MacArthur Communicative Development Indices. This study will also analyze the maternal prenatal fatty acid profile from erythrocyte samples that were collected during pregnancy and stored by the PIN Study. We will evaluate the variability in fatty acid levels and their correlation with reported maternal diet. We will conduct a preliminary investigation of the relation between maternal fatty acid levels and child’s neurodevelopment. This preliminary pilot data will be used to apply for NIH funding of a larger study that will distinguish more subtle associations between fatty acids and neurodevelopment. In the mean time, the outcome data generated will allow an efficient investigation of the association between neurodevelopment and other maternal exposures, such as smoking, infection, and stress, which already exist among PIN data.
Project: Specific fatty acids alter cellular responses to environmental agents
Lipid rafts are specialized detergent-insoluble regions on cell plasma membrane that are entry points for viruses including influenza and HIV, and a variety of toxic proteins including bacterial toxins, prions, and plant toxins such as the potential bioterrorism agent ricin. Lipid rafts are comprised of lipids and proteins that differ from the bulk components of the plasma membrane. They contain more cholesterol and sphingomyelin as well as specific proteins, many of which are anchored by saturated fatty acids. Polyunsaturated fatty acids (PUFA) modify lipid rafts and alter their ability to regulate the entry of environmental agents, including viruses and toxins. Recently reported studies suggest that exposure to polyunsaturated fatty acids can alter the protein composition of these regions. This proposal uses influenza virus infection to model the changes in the lipid and protein composition of cell membranes and their lipid raft domains.
We will use a knockout mouse with aberrant lipid composition and cultured cells exposed to different fatty acids in order to delineate alterations in lipid raft function that may affect the entry of environmental toxins. We develop methods for phospholipid fatty acid analysis. These methods will be used to analyze the fatty acid composition of phospholipids from a variety of samples: influenza virus that have been isolated from GPAT KO and WT mice, lung tissue and macrophages from these mice, PUFA-treated cells infected with influenza virus, and virus itself after replication in cells exposed to different fatty acids that alter viral replication. Proteins present in lipid rafts will be identified before and after the lipid modifications, as will the viral coat proteins hemaglutinin and neuraminidase. We will also analyze macrophage eicosanoids from KO and WT mice. These studies will delineate the changes that occur in lipid rafts that restrict entry of environmental agents and toxins into cells.
Project: Genetic analysis of transcriptional regulation in liver
Exposure to environmental agents is inevitable and all individuals encounter various chemicals, doses and times of exposure throughout the lifetime. The end-effects usually vary greatly across a population of similarly exposed individuals. Genetic variation in commonly used animal models, such as mice, is as complex as in man, but the availability of genetically controlled “inbred” animal populations provides a significant benefit impossible to achieve in humans. These genetic tools become even more powerful as the complete haplotype maps of many commonly used mouse inbred strains become available. This application aims to combine microarray-based assays of mRNA level with gene mapping methods to detect polymorphic loci that co-regulate extensive molecular networks in mouse liver. Our specific experimental approach is to analyze the variation in basal gene expression among genetically controlled mice by using a panel of BXD recombinant inbred (RI) strains derived from C57BL/6J and DBA/2J. Steady-state mRNA levels will be measured and used as quantitative traits to map potential susceptibility and resistance genes and, more importantly, networks of gene interactions related to liver injury susceptibility. The latter will be achieved by combining the data from this project with an already established web interface containing genotypes, phenotypic data and already collected gene expression measurements from other organs (e.g., brain) of BXD mice. This will allow exploration of networks of correlated molecular and phenotypic traits, and to uncover shared upstream modulation of expression in several diverse tissue types targeting genes known to be potential targets for toxicants. Collectively, this project will serve as a springboard to future studies on the genetic dissection of novel genotype-phenotype correlations associated with in vivo responses to toxic agents that affect the liver. This project will allow us to collect pilot data necessary to infer genetic causes for phenotypic variation across RI strains. Furthermore, this approach will serve as an example of how genetics, animal models, genomics and traditional mechanistic toxicology can be combined to bring new discoveries and technical advances into the mainstream of environmental science.
Project: Genetic susceptibility to prenatal neurotoxicants
The aim of this project is to investigate gene-environment interactions in the etiology of developmental disabilities, like autism, using a mouse model for Smith-Lemli-Opitz Syndrome (SLOS). This project tests the general hypothesis that individuals with genetic predisposition to low cholesterol due to mutations in delta 7-dehydrocholesterol reductase (Dhcr7) may be at increased risk for abnormal brain development following prenatal exposure to neurotoxicants that target neurotransmitter systems. This hypothesis is based on: 1) evidence for vulnerability of developing GABA, serotonin (5-HT) and catecholamine neurons following exposure to organochlorine pesticides (e.g., dieldrin) that act as potent GABAA receptor antagonists; and 2) evidence for aberrant development of 5-HT neurons in mice with a targeted mutation in Dhcr7. The Specific Aims test the hypothesis that Dhcr7 mice will be more vulnerable to prenatal exposure to dieldrin or the GABAA receptor antagonist, bicuculline (positive control) than their wildtype counterparts. Pregnant mice will be exposed to different doses of pesticide or bicuculline from gestational day (GD) 11-16. In Specific Aim 1, dose-dependent treatment effects will be evaluated in adult offspring using a behavioral test battery to screen effective doses of prenatal treatments and determine long-term effects of prenatal exposure on social and perseverative behaviors. In Specific Aim 2, prenatal treatments will be limited to doses of pesticide or bicuculline that produce deficits in social behavior or promote perseverative responses. Affymetrix microarrays will be used to profile gene expression in brains of neonatal and adult offspring. Gene expression data will be validated using Real -Time PCR. Future studies will analyze proteins encoded by significantly altered genes using Western blotting or radiolabel immunocytochemistry on brain sections. Taken together, these studies should provide important insights into roles played by gene-environment interactions in the etiology of developmental disabilities, like autism, that involve deficits in social behavior and perseverative responses, and give clues to possible underlying cellular and molecular mechanisms.
Project: The role of metabolism in the modulation of toxic and cancer promoting effects of arsenic in human urinary bladder
Cancer of the urinary bladder is one of the most common forms of cancer associated with chronic exposure to inorganic arsenic (iAs) from drinking water. In humans the metabolism of iAs yields methylarsenic (MAs) and dimethylarsenic (DMAs) metabolites that contain arsenic in +3 or +5 oxidation state. The key enzyme in this pathway, AsIII-methyltransferase (Cyt19), is expressed in liver, but not in normal human urothelial (UROtsa) cells. Recent studies have shown that methylated trivalent arsenicals, methylarsonous acid (MAsIII) and dimethylarsinous acid (DMAsIII), are more potent than iAs as cytotoxins, genotoxins, and enzyme inhibitors. We have shown that in human bladder cells, MAsIII and DMAsIII are considerably more potent than iAsIII in activating ERK-mediated AP-1 DNA binding, a mechanism that is known to regulate cell proliferation and transcription of genes involved in bladder carcinogenesis (e.g., TGF-a). Notably, preliminary experiments have shown that iAsIII and methylated trivalent arsenicals do induce TGF-a expression by UROtsa cells. Thus the rate of the production and the yields of MAsIII and DMAsIII may be a critical step in cancer promoting effects of iAs. Recombinant wild-type (w/t) and mutated forms of rat and human Cyt19 are now available in our laboratory. The rates and patterns of in vitro methylation reactions catalyzed by these proteins depend strongly on the extent and character of mutation. Using a retroviral vector encoding w/t and mutated forms of Cyt19, we plan to established a genetically modified UROtsa cell lines with modified methylation capacities. We plan to use this cell line to study the role of enzymatic methylation in the modulation of toxic and cancer promoting effects of iAs in human bladder cells.
Project: Human responses to UV-induced DNA damage
This pilot project seeks to acquire preliminary data in support of a competitive application for a program project grant on “Human responses to UV-induced DNA damage.” Four independent researchers and a junior investigator with expertise in biochemistry, proteomics, molecular biology and radiobiology will pursue interdisciplinary research projects focused on the responses of human cells to UV-induced DNA damage. Project investigators independently have established strong records of productivity and accomplishment in the fields of DNA repair and cell cycle checkpoints. The joint effort by these investigators will be directed toward developing in vitro systems that are amenable to biochemical manipulations and responsive to DNA damage. The primary goal will be to demonstrate that these model systems could support mechanistic studies on the primary components of DNA damage checkpoints, mainly damage sensors, mediators, signal transducers, and effectors of specific responses. The focus of this pilot project will be on the intra-S phase checkpoint and the signal transduction reactions that underlie the inhibition of replicon initiation. Synergistic approaches fostered by this pilot project will lead to a new program of study to investigate the multiple interactions among cell cycle checkpoint and DNA repair proteins in human keratinocytes and fibroblasts damaged by UV. Solar UV radiation is a ubiquitous environmental carcinogen accounting for over a million new cases of skin cancer each year. Information gained by study of human responses to UV will facilitate understanding of responses to other environmental toxins that damage DNA and induce cancer at internal sites.
Funded by NIEHS Grant # P30 ES010126