Description: Air pollution has been clearly linked to respiratory and cardiovascular disease. In addition air pollutants may contribute to other diseases such as cancer. The exact pollutants and mechanisms of disease induction are not clearly defined. This pilot study will explore the effects of air pollution on DNA methylation and epigenetics. The Children's Health Study (CHS) is the most comprehensive study on the long-term effects of air pollution on the respiratory health of children. This study, which began in 1993, has 6000 children from 12 communities in California with variable levels of air pollution. Previous studies have shown negative effects on lung development dependent on the level of air pollution. We will quantitatively assess the DNA methylation of buccal mucosa from subjects in areas of high pollution and compare them to subjects from areas of low pollution. Specifically we will assess DNA methylation of the p16 gene and LINE-1 repetitive element. We have shown previously that DNA methylation changes in these loci were dependent upon benzene exposure. In addition we will compare these DNA methylation assessments to other factors obtained in this study such as diet, level of activity, gender, and lung function.

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Mitochondrion-Driven Apoptosis in Dopaminergic Neurons by Environmental Toxins

Description: Parkinson's disease (PD) is a progressive neurodegenerative disorder that is characterized by the loss of dopaminergic neurons in substantia nigra pars compacta (SNpc) region of the mid- brain, at least in part, due to mitochondrion-mediated apoptosis. Large scale epidemiologic studies unraveled a strong correlation between paraquat exposure and increased risk to develop PD. Paraquat had been shown to cross the bloodbrain- barrier and enter dopaminergic neurons, and mitochondria were found to be the initial sub-cellular component exhibiting structural damage. Therefore, understanding the toxicological effects of paraquat in dopaminergic neurons, especially towards mitochondrial functions, becomes critical in developing preventive/therapeutic strategies against environmental toxin-related PD. Metabolism of paraquat generates superoxide anion (O2·-) and nitric oxide (·NO) directly or indirectly. Superoxide can react with nitric oxide at a diffusion-controlled rate to form peroxynitrite [O2·-+· NO -> ONOO-], a potent oxidant. In addition, hydrogen peroxide (H2O2) can be generated from O2 ·- dis-mutation catalyzed by superoxide dismutase (Cu,Zn-SOD). S-nitrosoglutathione (GSNO), a potent S-nitrosating agent, can be formed by the reaction between GSH and reactive nitrogen species. All of the above impose a strong oxidative/nitrosative stress on mitochondria and could alter important mitochondrial protein functions (e.g. ATP synthesis; permeability transition) by posttranslational modifications of critical amino acid residues. Therefore, we propose to carry out a comprehensive and quantitative investigation into the generation of different reactive oxygen- (ROS) and nitrogen- (RNS) species in PC-12, a well established dopaminergic cell model, following paraquat treatment. The outcome, necrosis and/or apoptosis, will be identified and correlated with the kinetics of the productions of ROS and RNS. Post-translational modifications of mitochondrial proteins will be detected and correlated with the paraquatinduced oxidative/nitrosative stress and all functional impairment. Various regimens of anti-oxidative agents will be used to establish a link between paraquat-induced oxidative/nitrosative stress and (i) mitochondrial proteins post-translational modifications and (ii) cell death.

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Description: Studies on the health effects of air pollution suggest that lung injury may result from inhalation of airborne nanoparticles. Interactions of nanoparticles with alveolar epithelium may alter the passive and active transport of ions and solutes, causing epithelial barrier dysfunction. Trafficking of airborne nanoparticles across the lung following inhalation may also contribute to end-organ disease (e.g., coronary atherogenesis). Because ambient particles are undefined in terms of their constituent chemical and physical properties, it is difficult to link the specific harmful agent(s) contained in such ambient particulates to cardiopulmonary complications. Thus, it is important to study nanoparticles of defined physicochemical characteristics as a surrogate to particulate matter in air pollution. In the present study, the effects of positively-charged and negatively-charged polystyrene nanoparticles of different sizes (ultrafine, 20 - 100 nm and fine, 500 - 1000 nm) on alveolar barrier properties will be determined. More specifically, the passive and active transport of ions (Na+, Cl-, and/or K+) across rat alveolar epithelial cell monolayers (RAECM) will be measured in the presence and absence of polystyrene nanoparticles of varying surface charge and size. In addition, we will determine whether or not nanoparticles traffic across RAECM and the dependence of such trafficking on their physicochemical characteristics. It is our intention that these results will be used for a larger-scale investigation which focuses on more precise mechanisms mediating nanoparticle lung injury and transcytosis/translocation across alveolar epithelium.

Description: Ten to twenty percent of all couples attempting pregnancy are infertile. Male factor infertility accounts for 40-50% of these cases for which the etiology is often idiopathic. Possible etiologies include genetic predisposition and environmental exposures toxic to the reproductive system. A unifying theory, postulating both genetic and environmental factors is that a number of men with idiopathic infertility may have a genetic component which makes them more susceptible to reproductive toxicants; or alternatively, select toxicants may induce heritable changes in the exposed man. One possible mechanism for this proposed synergism is that chemicals induce epigenetic changes during spermatogenesis. Recent animal data suggest that exposure to specific reproductive toxicants induces epigenetic changes in spermatozoa via alterations in DNA methylation. Preliminary clinical data suggest an association between abnormal genomic imprinting and hypospermatogenesis and a possible correlation between pregnancy outcomes in the IVF/ICSI setting and DNA methylation changes in the spermatozoa selected for ova injection. We propose that changes in methylation patterns in the male germ line provide an opportunity for the development of sensitive biomarkers of disrupted epigenetic programming and assessment of fertility potential. The mature spermatozoan is an ideal candidate to serve as a biomarker of epigenetic abnormalities. Sperm are readily collected in large numbers and methylation of DNA is the reaction of epigenetic programming for which tissue-specific and developmental patterns are most extensively described and laboratory assays are most developed. Thus, the immediate goal of this proposal is to develop and validate methodology to assess epigenetic changes during spermatogenesis and to investigate the relationship between epigenetic changes in the male germ line and fertility potential. A longer term objective is to understand mechanisms whereby environmental exposure may compromise fertility in the male. We hypothesize that spermatozoa can serve as biomarkers of epigenetic events induced by environmental exposures. We will begin investigating this hypothesis by 1) establishing a sensitive assay for DNA-M in human spermatozoa, 2) validating the assay by assessing DNA-M in the spermatozoa of a crosssection of men of reproductive age with and without infertility, and 3) correlating the results to the standard semen analysis and pregnancy outcomes.

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Description: We propose to develop new methods for studying traffic related exposures and their relationship to oxidant stress. Urinary isoprostanes, specifically 8-iso-prostaglandin-F2-alpha (F2-IsoP), are promising new markers for the oxidative stress associated with atherosclerotic heart disease, asthma, obesity and other conditions. However, little is known about the levels or determinants of variability of F2-IsoP in children, preliminary data which are necessary to design larger population based epidemiological studies. Urinary markers of exposure to traffic related polyaromatic hydrocarbons (PAH's) have not been examined in the context of recent studies of heavy school commuting exposures in Los Angeles that may account for a major proportion of exposure to ambient oxidant pollutants, nor have methods for modeling these exposures been developed. The goals of this study are: (1) to assess the components of variability in urinary F2-IsoP due to handling procedures in the field and temporal physiologic variability in children; (2) to model on-road and residential pollutant exposures to a sample of children representing the extremes of school commuting time in Los Angeles; and 3) to measure urinary PAH metabolites and examine the association with modeled exposure. The within- and between-child variability in urinary F2-IsoP will be determined in a convenience sample of 20 children who will provide first-morning urine on two occasions separated by two weeks. The samples from ten of these children also will be split in the field (with parental help) to assess the contribution of handling to variability. Methods for assigning traffic volume on road segments derived from a Mapquest TM algorithm of likely route from home to school for each child will be developed and calibrated to parent reported route of commuting. Other on-road travel patterns and times will be collected by diary report of the parent. Exposure to traffic may also be further refined using temporally resolved personal GPS logged measurements during the study period, for which support has been requested from another funding source. The methods developed in the convenience sample will then be refined in a sample of 20 children from classrooms of 4th and 5th graders in a school with children coming from different parts of Los Angeles, who will have a large gradient both in commuting time and regional ambient pollutant exposure. In this population we will assess willingness of families to participate and provide urine; we will calibrate the modeled on-road traffic volumes to additional information on time and location of activities; and we will estimate the range and distribution of F2-IsoP and of urinary PAH metabolites. We will conduct preliminary analyses examining the associations of urinary PAH metabolites, F2-IsoP, and modeled on-road exposure (adjusted for traffic-modeled exposure at homes and regional air pollutants at homes). These analyses will enable us to estimate required sample sizes for subsequent studies to examine associations with respiratory health. This pilot study will develop new tools for exposure assessment in epidemiologic studies, for evaluation of potentially important and little-studied on-road exposures, and for the assessment of oxidative stress.

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