Lisa Bain - US grants

Pesticides continue to enter the environment through human activities and pose a health risk by their presence in the food we eat, runoff into water, and persistence in soils and sediments. To determine how much of a problem pesticides pose to humans and other organisms, their accumulation and the mechanisms of elimination from cells must be ascertained, as the concentration within a cell directly effects its toxicity. The mechanisms by which pesticides are eliminated from cells and ultimately the body is not very well understood. Several membrane-bound ATP-binding cassette (ABC) transporters have been implicated in mediating their efflux, but the evidence is extremely limited. Two ABC transporters termed the human multidrug resistance-associated protein 1 (MRP1) and the human multidrug resistance-associated protein 3 (MRP3) that mediate the elimination of glucuronide, glutathione, and sulfate conjugates of physiological compounds and exogenous ligands, are probably involved in elimination of pesticides from cells. We propose to investigate nineteen pesticides that are either currently used, or have been used extensively in the past and are routinely detected as contaminants in many areas and matrices. These pesticides can be eliminated from the organism as conjugates, and therofore are likely to require MRP1 and/or MRP4 to mediate their efflux. We propose that transport and elimination of persticides by MRP1 and MRP3 results in detoxification, which protects the organism from their deleterious effects, but may alter the normal transport of physiological compounds. We will examine the actual transport of pesticides by MPR1 and MPR3, determine whether this elimination alters toxicity to cells that overexpress these proteins, and ascertain whether transport of pesticides competitively inhibits and alters the normal elimination of important physiological compounds.

[unreadable] DESCRIPTION (provided by applicant): The multidrug resistance-associated protein 1 (MRP1/ABCC1) is a member of the ATP-binding cassette (ABC) protein family. Member of this family of transporters actively transport predominantly glucuronide, glutathione, and sulfate conjugated compounds out of cells, Mice lacking the MRP1 protein (termed FVB/mrpl-/- mice) have been produced. While the animals are viable and fertile, MRP1 clearly plays a role in protecting the mice from the toxicity of a variety of compounds, including anticancer drugs such as etoposide. In preliminary studies, we have shown that MRP1 also protects the testicular tubules from methoxychlor toxicity. During the course of that study, we realized that activity of several cytochrome P450 proteins was significantly lowered in control wild type mice compared to control FVB/mrpl-/- mice. Therefore, we would like to further examine the differences in gene expression in mice lacking the multidrug resistance-associated protein 1 (MRPI/ABCC1) compared to wild-type mice. The hypothesis to be tested is that the loss of this transporter, while not lethal, results in the up- and down-regulation of a variety of genes to compensate for its loss, presumably in a tissue-specific manner. Using this information, we can better understand coordinate regulation between transporters and other genes that regulate them, or genes that are in detoxification and elimination pathways. This information will ultimately be invaluable for using these mice to determine drug disposition and the potency or specificity of anticancer drugs. [unreadable] [unreadable] [unreadable]

A grant has been awarded to the University of Texas at El Paso, under the direction of Dr. Lisa Bain, for support of instrumentation to determine altered gene expression in a variety of organisms in response to different ecological and physiological variables. Understanding what genes are changed helps to better characterize effects on organismal functions such as reproduction, growth, and development, and how these alterations will ultimately affect the organism, population, or community as a whole. The equipment requested includes an automated microarrayer and an array reader/scanner, plus the analysis software. While commercially available microarrays for human or mouse genes exist, these types of arrays are not available in alternative model species, including many microorganisms, fish, and snakes, as well as genes for targeted applications, such as neurobiology. Therefore, a microarray spotter and reader are necessary for the custom construction and analysis of these types of microarrays.

Uses of the proposed equipment would include training in bioinformatics courses and research in ecology, microbiology, physiology, endocrinology, and neurobiology. For example, studies attempting to discern the mechanisms of osmoregulation and adaptation to other environmental pressures in two estuarine fish species, using both custom-made arrays and in collaboration with the Genipol project in Europe. Rattlesnake venom and other toxins are being investigated to how understand how snake venom toxin composition may reflect evolutionary species distribution. Other investigators are collaborating on the genome project for the protist Giardia, examining how a different microorganism, Bacillus subtilis, controls sporulation, and what genes confer chlorine resistance to mycobacteria and enterohemoragic E. coli O157:H7. In terms of endocrinology and neurobiology, several investigators are examining gene expression changes in mammary glands following exposure estradiol and estrogen receptor antagonists. In addition, several of the co-investigators are developing statistical methodology for identifying genes that best discriminate the different groups among individual, as well as developing a curriculum in bioinformatics with more hands-on emphasis on microarray methods in post-genomic analysis.

The University of Texas at El Paso is a minority-serving institution for approximately 18,500 students (including 3,500 graduate students), of which 75% are of Hispanic descent. Because of the high percentage of minority students and its location on the US-Mexico border, UTEP is a national leader in the education and training of Hispanic scientists. Support of this grant will be used to train a number of post-doctoral associates, graduate students, and undergraduate students on research using the instrumentation. Hand-on training will occur specifically in our Post-Genomic Analysis class, with the students conducting a complete, hands-on array experiment from start to finish, while two other classes will make use of already scanned arrays, performing statistical analysis and interpretation of data sets. With access to this state-of-the-art instrumentation, UTEP will produce well-trained students capable of serving the science needs of the region and the nation.

[unreadable] DESCRIPTION (provided by applicant): Arsenic is an enormous public health problem as it is a contaminant of drinking water in many parts of the world. A number of recent epidemiological studies have correlated arsenic exposure with adverse developmental outcomes such as stillbirths, spontaneous abortions, neonatal mortality, low birth weight, and delays in the use of musculature. Studies in rodents also demonstrate that offspring of pregnant dams exposed to arsenic have increases in preterm death, low birth weight, and changes in locomotor activity. Using a model fish species termed mummichogs, our laboratory has shown developmental abnormalities in offspring exposed to 230ppb arsenic during gametogenesis, which correlated with an upregulation of genes involved the musculature, such as myosin light chain 2, tropomyosin, and parvalbumin. We have also demonstrated that exposure of C2C12 myocyte cells to 20nM arsenic resulted in a delay in myoblast differentiation that correlated to a reduction in myogenin expression and a reduction in methyltransferase expression. Thus, we propose to investigate the mechanisms underlying the increase in developmental abnormalities and changes in myogenesis after exposure to arsenic using the C2C12 cell line. Our hypothesis is that inappropriate expression of transcription factors involved in skeletal muscle development, coupled with altered gene methylation, is a mechanism partly responsible for altered myogenesis and the developmental abnormalities in arsenic-exposed offspring. We will test this hypothesis by exposing C2C12 myoblast cells to increasing concentrations of arsenic to investigate the time and dose-dependent changes in differentiation, multinucleation, muscle-specific genes, and myogenic transcription factor expression by real-time PCR and immunoblotting. To investigate the mechanisms responsible for the lack of differentiation, DNA methylation, methyltransferase activity and expression, and methylation precursors will be examined. Finally, methylation inducers and inhibitors, and transfection of methyltransferase genes will be used to determine whether the myogenic phenotype and myogenic protein expression can be altered. Our laboratory is well positioned to carry out the proposed studies because we have experience in examining the effects of arsenic on development, and on differential gene and protein expression. We have previously been examining phenotypic and gene expression changes after arsenic exposure in a model fish species, and now wish to further our in vivo findings by using a cell model to examine arsenic's mechanisms of action as it relates to altered development and altered myogenesis. PUBLIC HEALTH RELEVANCE: The ultimate benefit of this work is to assess the mechanisms of how environmentally realistic arsenic exposure impacts development, and help to examine whether the drinking water standard for arsenic is protective of human health. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Arsenic is a contaminant of drinking water that affects millions of people throughout the world. Exposure during embryonic development is associated with low birth weight, altered locomotor activity, and reduced neuronal development, likely because of reductions in the number or localization of skeletal myocytes and neurons. Indeed, our data indicate that arsenite exposure to stem cell-derived embryoid bodies reduces the expression of MyoD, Myf5, myogenin, and NeuroD, neurogenin-1, and neurogenin-2, all transcription factors needed to convert stem cells into skeletal myocytes and sensory neurons, respectively. Both of these cell types share a common lineage in the neural plate border specifier cells, which express transcription factors involved in myogenesis, such as Pax3, Pax7, Dlx5, Msx1, and Msx2, and are responsible for secreting factors that lead to the formation of the neural crest specifier cells, which produce sensory neurons. In embryonic bodies exposed to arsenite, Msx2 expression is reduced and Pax3 spatial localization within the embryoid body is altered. These data suggest that the number and spatial localization of neural plate border specifier cells may play a critical role in the developmental toxicity of arsenic. The goal of this application is to determine whether arsenic alters the number and/or localization of neural plate border specifier cells and to determine the specific inductive or repressive signaling pathway targeted by arsenic during the formation of the neural plate, neural crest, and the mesoderm. In the first aim, we will examine changes in the localization and number of the neural plate border specifier cells following exposure to arsenic or its methylated metabolites. These results will enable us to both determine if the specifier cells are altered, and also determine whether neural crest cell production is inhibited. The goal of the second aim is to determine the inductive and repressive signaling pathways that are targeted by arsenic. Specifically, we will use Wnt1, Wnt3, Wnt3a, Wnt5a, noggin, Bmp2, and Bmp4 to differentiate the embryonic stem cells and investigate whether arsenic abrogates sensory neuron and skeletal myotube formation. Our long-term objective is to understand why arsenic-exposed populations are at increased risk for defects in skeletal muscle and neuronal development, and how this leads to functional changes such as low birth weight and altered neuronal function. We can then use this generated data to investigate the specific mechanisms by which arsenic alters the development of neural crest cells and paraxial mesoderm.

DESCRIPTION (provided by applicant): Arsenic is found in drinking water and rice, resulting in exposure to millions of people throughout the world. Exposure to arsenic during embryogenesis is associated with low birth weight, altered locomotor activity, and reduced weight gain, likely because of reductions in the number of skeletal muscle progenitor cells. Indeed, our data indicate that arsenite exposure to stem cell-derived embryoid bodies reduces the expression of MyoD, Myf5, and myogenin, all transcription factors needed to differentiate stem cells into skeletal myocytes. We have also shown that arsenic impedes the ability of myocytes to differentiate into mature myotubes. Muscle progenitor cells, or satellite cells, arise during embryogenesis and are needed for new fiber formation later in life. We have been using the killifish as a model species to examine changes in muscle development following embryonic arsenic exposure. Our preliminary data indicates that arsenite exposure only during embryogenesis reduces weight gain in offspring even 4 months later. This reduced weight gain appears to be due to reductions in the total muscle fiber numbers, as embryonically-exposed fish have 15-20% less fibers than control fish at 4 months of age, even when accounting for difference in size. Thus, the goal of this application is to assess the mechanism by which arsenic reduces muscle fiber number by determining the number, location, and function of satellite or muscle stem cells. We will examine these processes from the juvenile period and into adulthood, and during both normal development and during recovery after an injury. In the first aim, we will determine whether exposure to low arsenite concentrations during embryogenesis reduces the number and/or localization of the satellite cells as the organism develops into an adult. These results will enable us to determine if the satellite cells are targeted, and also determine whether muscle mass is inhibited. The goal of the second aim is to determine whether organisms exposed embryonically to arsenic can recover from a muscle injury later in life, which will indicate whether arsenic exposure during embryogenesis has latent effects that only become apparent in adulthood. Our long-term objective is to understand why arsenic-exposed populations are at increased risk for defects in muscle development, and how this leads to functional changes such as reduced weight gain. It will also help to determine whether a standard for arsenic levels in food should be set - one that will be protective of embryonic health.

7. Project Summary/Abstract Arsenic is a contaminant found in drinking water and food stuffs, resulting in the exposure of millions of people to concentrations above the current US EPA and WHO drinking water standard. Despite its prevalence, the mechanisms by which it impacts development are still not well understood. Evidence from epidemiological and rodent studies indicates that embryonic and fetal exposure to arsenic is associated with altered muscle function, reduced weight gain, and neurobehavioral effects such as reduced IQ, impaired spatial memory, and changes in behavior. We have using mouse embryonic stem (ES) cells to examine changes in differentiation following a short-term arsenic exposure, and found that arsenic inhibits their differentiation into skeletal myotubes and neurons by reducing the expression of lineage-specific transcription factors, including MyoD, myogenin, NeuroD, and neurogenin. Our lab has recently created a series of ES cell lines that have been chronically exposed to 0.1?M or 7.5pppb arsenic, a concentration below the drinking water standard. Our preliminary data shows that cells ES exposed to arsenic for >16 weeks have lost their ability to differentiate into adult cell types. Thus, the goal of this application in to assess the time course, the persistence of the phenotype, the mechanisms behind this loss of differentiation ability, and whether a similar loss of differentiation happens to neural stem cell in vivo. In the first and second aims, we determine the time course of this phenotype, assess whether the loss of differentiation persists following a recovery period, and assess the mechanisms responsible for aberrant differentiation, including changes in apoptosis, proliferation, apoptosis, and epigenetic modifications. In the third aim, we will ascertain whether neuronal stem cell proliferation, migration, and differentiation is also impacted during an in vivo embryonic arsenic exposure by investigating whether certain cell types are more sensitive to particular exposure time points. These studies will further our understanding by which arsenic exposure during development causes impaired repair after an injury, reduced memory development, sensory impairment, and motor changes. More importantly, our research will determine whether exposed populations can ever ?recover? from a prenatal arsenic exposure

7. Project Summary/Abstract Arsenic is a contaminant found in drinking water and food, resulting in the exposure of millions of people to concentrations above the current US EPA and WHO drinking water standard. Evidence from epidemiological studies has demonstrated that in utero, childhood, and adult exposure to arsenic is associated with reduced birth weight and weight gain, although the mechanisms responsible are not well understood. In addition, even though ingestion is the main route of arsenic absorption, almost nothing is known about the effects of arsenic on the small intestine itself. Our lab has conducted pilot studies exposing intestinal organoids to arsenic, and found that exposure reduces stem cell differentiation and inhibits production of specific adult cell types. Thus, the goal of this application in to assess the time course, the persistence of the phenotype, the mechanisms behind this loss of differentiation, and whether a similar loss of differentiation happens to the intestinal epithelium in vivo. In the first aim, we determine the dose-response, assess whether arsenic and its mono- and di-methylated metabolites are equivalently toxic, and assess the mechanisms responsible for aberrant differentiation, including changes in apoptosis, proliferation, and cell signaling. In the second and third aim, we will ascertain whether intestinal niche function, stem cell differentiation, and function is impaired during an in vivo arsenic exposure. We will also study the role of mesenchymal cells in maintaining the niche. These studies will further our understanding of how arsenic alters cellular differentiation and reduces growth. More importantly, our research will determine whether exposed populations can ever ?recover? from an arsenic exposure