Abstract  POLISH (Participatory Research, Organizing and Leadership Initiative for Safety and Health) will research, using a community based participatory research methodology, Asian Pacific Islander women's and girls' exposure to hazards in beauty products in Alameda County, California. POLISH will research: 1) Asian Pacific Islander teenage girls' exposures through personal use of beauty products and 2) Vietnamese women's exposures through their work in nail salons - exposures that are under-researched, particularly with this population. POLISH's long-term goal is to increase Asian Pacific Islander girls' and women's capacity to identify environmental health problems and improve the community's health status. Its specific aims are to: 1. Educate, train and mentor Asian Pacific Islander teenage girls and Vietnamese nail salons workers who will: a) become knowledgeable about women's and girls' exposures to hazardous chemicals, b) design and implement community based participatory research, c) become skilled in educating peers and community about environmental and occupational health problems and d) be mentored for careers in the sciences. 2. Increase scientific knowledge of Asian Pacific Islander low-income women's and girls' exposure to environmental hazards associated with beauty products: a) Conduct a peer consumer hazard survey and media survey, b) Test for phthalates in products used by Asian Pacific Islander teenagers c) Conduct an occupational hazard survey in nail salons and d) Test for airborne chemicals in nail salons. 3. Develop culturally relevant interventions that educate the community and achieve one to two public policy or corporate changes designed to lessen exposures to hazardous chemicals in beauty products. 4. Share research findings and intervention models with researchers, public health students, the environmental justice movement, the women's health field, and clinics.

Abstract  DESCRIPTION (provided by applicant): The overall objective of this proposal is to determine the mechanisms by which phthalate esters downregulate testosterone production in the developing fetal rat testes, resulting in antiandrogenic effects on the developing male reproductive tract. Phthalate esters are a class of environmental chemicals to which humans are ubiquitously exposed and which cause antiandrogenic effects on the developing male reproductive tract in rats. The mechanisms by which phthalate esters cause their effects remain to be determined. In preliminary studies, global changes in gene expression in the developing rat testis following in utero exposure to di(n-butyl) phthalate (DBP) were examined. A significant finding was that DBP produced a reduction in key genes in pathways associated with either steroid production or the provision of substrates for this activity. It is therefore hypothesized that phthalates decrease testosterone in the developing testes because of a coordinate disruption in cholesterol transport and steroid biosynthesis. This hypothesis will be tested by investigating the following specific aims: 1) Identify the key steps involved in cholesterol transport and testosterone biosynthesis that are targets for disruption by in utero exposure to DBP in the developing fetal rat testes, 2) Determine the consequences of DBP-induced changes in target gene and protein expression identified in Specific Aim 1 on fetal testicular cell signaling pathways, cholesterol transport, and steroidogenesis, and 3) Establish the molecular mechanism by which DBP downregulates expression of genes involved in cholesterol transport and steroid biosynthesis. Quantitative RT-PCR and protein analysis will be used to identify gene targets in the developing fetal testis following in utero exposure to DBP. The promoter regions of selected gene target will be further examined in vitro to determine the mechanism by which phthalate esters act on cholesterol transport and steroidogenesis. The proposed studies will identify critical genes and pathways associated with cholesterol transport that are targets for DBP in the male rat in utero and will aid in determining potential human risks from exposure to this class of environmental chemicals.

Abstract  More epidemiologic data are needed on the relation of background-level exposure to man-made chemicals with short half-lives to child development. The embryonic and fetal stages of development are periods of heightened susceptibility to effects of xenobiotics. For xenobiotics with short half-lives, measurement has been a challenge in the past, but new technologies now support better exposure assessment. Multiple urine specimens are frequently the medium of choice for assessing exposure to such agents. Recent data suggest: a) that exposure of pregnant women to background-levels of bisphenol A results in earlier age at menarche among female offspring, b) that background-level of exposure of pregnant women to nonpersistent pesticides can result in reduced birthweight and impaired neurodevelopment in offspring, and c) that background-level exposure to phthalates may be having adverse reproductive effects. We seek to increase our capacity to study the relation of background-level exposure to chemicals with short half-lives to child development. To achieve this goal we propose to support collection multiple urine specimens during pregnancy in a newly begun cohort, the Generation R study (described below). Freezers for long-term storage of the specimens will also be purchased. My plan is to support collection of urine from the pregnant mothers of 1,500 children in the cohort. The advantage to studying this cohort, e.g., over the Norwegian birth cohort, is that we will have multiple urine specimens, including one during the first trimester, enhancing our ability assess exposures, especially during organogenesis. Furthermore, the outcome assessment in the new cohort is more intensive and standardized than in Norway. Generation R is an ongoing prospective study of 10,000 children who will be followed from early fetal life to young adulthood, and aims to study how factors and events during pregnancy and early childhood can affect growth, development, and health in later life. All pregnant women in Rotterdam who expect to give birth between June 2002 and June 2004 are invited to participate, together with their partner. This study has been set up by the Erasmus Medical Center. The multidisciplinary characterization of the cohort, starting in early pregnancy, will produce a database containing biological, medical, genetic, psychological and community-related data which can be used to address a wide spectrum of research questions. The research questions have been subdivided as growth and physical development, cognition and behavior, illnesses and accidents, and utilization of health care resources. Our add-on to the Generation R study cohort is a small new project, but if the collaboration is successful, it could lead to some very exciting and important work.

Abstract  Immunoglobulin (1g) variable regions are tumor associated antigens of B cell lymphomas and meyelomas. Vaccination with intact Ig has induced tumor specified immunity, but this approach has produced variable results and has led to the outgrowth of tumor variants. We hypothesize that the variable results seen may be due to the elicitation of a limited immune response to single or very restricted set of immunodominant epitopes when whole Ig is used as the vaccine. Our goal is therefor to define discrete VH peptides representing subdominant or cryptic epitopes from both the hyper variable and conserved framework regions to design a vaccination strategy that could provoke a broader response. In addition a vaccine that provokes protective immunity against epitopes within more conserved regions including framework regions of VH could be utilized for multiple patients. Our strategy is to identify peptides from he entire VH region with predicted binding affinities for H-2Kd, and test them for their ability to generate MHC-restricted CTL. Mice are immunized with dendritic cells pulsed with synthetic peptides, with dendritic cells transfected to express the selected peptides, or with dendritic cells transfected with the entire VH region. Our rational is that by using dendritic cells and focusing upon selected peptides it will be possible to generate CTL responses to areas of the VH that would otherwise be ignored due to the suppressive effects of more dominate epitopes. To test our hypothesis, antibody forming clones derived from the immune response of BALB/c mice to dextran and to the hapten phthalate have been immortalized as hybridomas and associated VH region peptides will be used to assess the specificity of the T cell repertoire and the hybridoma cells used as tumor models. The strengths of these two models are that (a) each hybidoma represents a dominant clonotype in the primary or secondary response, and (b) the Vh regions of these hybidomas have been sequenced and MHC binding peptides from these regions have been identified. While we expect to see tumor specific MHC-restricted CTL responses to both germ line and somatically mutated peptides an attempt being made to enhance the responses by linking peptides to heat shock proteins, by the local release of cytokines from biodegradable micro spheres and by treatment of dendritic cells with CpG-containing oligodeoxynucleotides. The efficacy of vaccination is evaluated in vitro and also tested in vivo by assessing the ability of immunized mice to withstand a tumor challenge or to prevent the outgrowth of tumor in remission. Finally, the effect of vaccination upon the normal B-cell repertoire expression is assessed.

Abstract  Peroxisome proliferators elicit marked hepatic proliferation and hepatocellular carcinoma when administered to rodents. At least seventy chemicals have been identified as peroxisome proliferators including the industrial phthalate ester plasticizer di-(2-ethylhexyl)phthalate (DEHP), chlorphenoxyacetic acid herbicides, halogenated hydrocarbon solvents and some anti-hyperlipidemic drugs. Human exposure to the peroxisome proliferating agents and rodent carcinogen DEHP is well documented as it is used extensively in the manufacture of polyvinylchloride plastics to render these materials more flexible. Biological effects of peroxisome proliferators appear to be mediated via an interaction with a specific intracellular receptor protein, peroxisome proliferator-activated receptor alpha (PPARalpha), a member of the steroid/thyroid hormone receptor superfamily of ligand-dependent transcription factors. Preliminary data have been obtained suggesting that some peroxisome proliferating agents bind directly to and induce conformational changes within recombinant mouse PPARalpha (mPPARalpha). Toward the goal of a molecular description of peroxisome proliferator action, the object of this project is to test the following hypothesis: (1) the industrial phthalate ester plasticizer di-(2-ethylhexyl)phthalate and its primary metabolite mono-(2- ethylhexyl)phthalate interact directly with mRRAPalpha; (2) ligand binding by mPPARalpha is a two-step process involving ligand recognition and a subsequent ligand binding-induced receptor conformational change resulting in the formation of a unique protein interaction interface within the ligand binding domain of the receptor; (3) heterodimerization with retinoid X receptor alpha (RXRalpha) alters the conformation and function of the mPPARalpha ligand binding domain; (4) binding of mPPARalpha to peroxisome proliferator response elements requires prior dimerization with RXRalpha; and (5) ligand-induced mPPARalpha conformational changes promotes interaction of the receptor with distinct cellular proteins that serve to couple the receptor to the basic transcriptional machinery. The long-term goal of this project is to provide a complete understanding of the role of mPPARalpha-mediated transcriptional activation in peroxisome proliferation and hepatocellular carcinoma. Because of the potential for wide-spread human exposure, a better understanding of the mechanism(s) underlying the biological effects of peroxisome proliferators will be of critical importance in determining the hazard that these compounds pose to public health.

Abstract  Peroxisome proliferators currently include a broad spectrum of synthetic and naturally occurring compounds, such as certain hypolipidemic drugs (e.g. clofibrate), leukotriene antagonists, phthalate ester plasticizers, herbicides, solvents, and the naturally occurring steroid, dehydroepiandrosterone. Despite their structural diversity, peroxisome proliferators induce qualitatively predictable immediate and delayed pleiotropic responses in rats and mice: the immediate responses consist of hepatomegaly, proliferation of peroxisomes in hepatocytes, and the induction of several hepatic enzymes, particularly those responsible for lipid metabolism, and the delayed responses include the development of hepatocellular carcinomas. Peroxisome proliferators, irrespective of their structural diversity, are found to be nonmutagenic (nongenotoxic) in that they do not directly damage DNA, thereby leading to out hypothesis that the development of liver tumors is attributable to sustained induction of peroxisome proliferation and ensuing metabolic perturbations resulting from a receptor-mediated mechanism. These agents activate peroxisome proliferator-activated receptor alpha (PPARalpha), a nuclear receptor, and sustained induction of PPARalpha-mediated peroxisome proliferation and other metabolic alterations are considered the basis for the development of liver tumors. This raises some intriguing questions about cell/tissue specificity and possible differences in species sensitivity of this response and its presumed implications. A major focus of our ongoing research is to generate fundamental information, which can provide insights into the molecular complexity of the unique pleiotropic responses, so that meaningful extrapolations can be made regarding species sensitivity and cell specificity of gene activation. We propose the following specific aims: 1) investigate the functional implications of the disruption of genes of peroxisomal beta-oxidation system and the relationship of beta-oxidation to PPARalpha-ligand metabolism; 2) generate "humanized" mouse models of PPARalpha expression to evaluate peroxisome proliferator response in an attempt to understand the basis for species differences in response; 3) explore the role of H2O2- generating peroxisomal oxidases in carcinogenesis using the in vitro/in vivo molecular approaches and delineate the mechanism by which H2O2 initiates the cascade of events leading to neoplastic transformation; and 4) generate molecular portraits of liver with peroxisome proliferation induced by synthetic peroxisome proliferators, or occurring spontaneously in fatty acyl-CoA oxidase null mice to identify and characterize novel genes during the process of carcinogenesis.

Abstract  DESCRIPTION (Adapted from the APPLICANT'S ABSTRACT): The broad, long-term goals of the proposed research are to understand the mechanism(s) by which peroxisome proliferating agents (PPAs) interfere with signaling cross-talk between metabolic pathways and nuclear hormone receptors producing perturbations in reproduction and development. PPAs are a structurally-diverse group of chemicals including herbicides, solvents, and plasticizers, that are known fatty acid, retinoid and endocrine disrupters. These metabolic pathways interact in the regulation of gene expression for reproduction and development. Hepatic peroxisome proliferation by PPAs has species-specificity (rats are sensitive, humans are not) but little is known about the mechanisms of PPAs endocrine disruptive effects. PPA species-specificity has recently been demonstrated in fish allowing investigation of responsive and non-responsive models. Fish exposed to PPAs can bioaccumulate these xenobiotics, and serve as biomarkers of exposure and effect. Furthermore, because human consumption of fish and fish products has increased in the last decade, it is imperative that we understand the potential for adverse effects by these contaminants. Test organisms will be a commercially-important food species, the catfish (sensitive; Haasch, 1996), and a model species, the Japanese medaka (non-sensitive; Scarano et al,. 1994). The proposed research will test the hypothesis that PPAs are endocrine disrupters producing perturbations in reproduction and development. PPAs to be investigated are: clofibrate, as a model PPA, and haloxyfop enthoxyethyl, di-n-butyl-ortho-phthalate (DBoP) and tetrachloroethylene (PCE) as environmental contaminant PPAs. Specific Aims include, 1) determining if PPA-sensitivity is ruired for endocrine disruption and if PAPAS from different chemical classes cause endocrine disruption by similar mechanisms, 2) determination of specific mechanisms of endocrine disruption by examining PPA combinations, and 3) determining if PPA-mediated endocrine disruption can be correlated with reproductive or developmental problems and if PPA exposure produces metabolic alterations in offspring. The proposed investigation will provide insight into cell signaling cross-talk between metabolic pathways and nuclear hormone receptors, endocrine disruption, and the developmental and reproductive effects of PAPAS.

Abstract  DESCRIPTION: (Adapted from the Investigator's Abstract)Adverse trends in male reproductive health may result, at least in part, from exposure to environmental chemicals during development. The overall objective of this proposal is to characterize the impact of di-(2-ethylhexyl) phthalate (DEHP), the most widely used plasticizer, on neonatal development of the testis and to identify mechanisms underlying the observed toxicant-induced injury. The investigator's preliminary findings, along with published data, suggest the DEHP reduces Sertoli cell/gonocyte numbers in neonatal rats, damages the testis permanently and impairs fertility of the adult. Based on pilot data, they hypothesize that exposure to low levels of DEHP in neonates reduces Sertoli cell and gonocyte population size [1] by inhibiting proliferation and inducing apoptosis through perturbation of cell cycle machinery and altered expression of immediate-early response genes and [2] by disrupting critical Sertoli cell-gonocyte interactions. The specific aims to be addressed are: [1] to identify the precise effects of low levels of phthalates on neonatal development of Sertoli cells and gonocytes in vivo and in vitro. [2] To evaluate the effects of phthalates on cell cycle machinery and determine how these effects impact on development of the Sertoli cell population. [3] To investigate the potential involvement of immediate-early response genes in phthalate-induced impairment of Sertoli cell development. [4] To determine whether phthalates impair Sertoli cell and/or gonocyte development by interfering with normal NCAM-and/or c-kit-based cell-cell interactions. Male rats 3-15 days old will be treated orally with DEHP and its impact on Sertoli cell/gonocyte maturation at the cellular and molecular level determined by evaluating proliferation, apoptosis and, for gonocytes, directed migration. An in vitro model system, neonatal Sertoli cell-gonocyte co-cultures, will also be used to explore mechanism(s) underlying these effects. The overall goal of this research is to increase our understanding of basic mechanisms important for testicular development and of how environmental toxicants interfere with these mechanisms and injure developing testes. In the long term, the findings will improve the investigator's ability to safeguard developing human males from the risks of exposure to the these toxicants.

Abstract  Human exposure to environmental pollutants has been proposes as a risk factor for endocrine disruption and the development of cancers of the breast and reproductive tract. Environmental pollutants are thought to exert effects on the endocrine system in part by influences the activities of nuclear hormone receptors that regulate diver aspects of growth, development and homeostasis. Some of the most common environmental pollutants found at Superfund Sites, including polychlorinated biphenyls (PCBs) and phthalates, can bind to members of the nuclear receptor superfamily and regulate their activities. These chemicals therefore have the potential for disrupting normal programs of endocrine regulation, resulting in disease. The major goals of this multi disciplinary research program are to use recombinant DNA approaches to assess the impact of exposure to Superfund chemicals on cellular and molecular events that affect the regulation of gene expression. In this project, we propose to take advantage of recent developments that affect the regulation of gene expression. In this project, we propose to take advantage of recent developments in the molecular biology of nuclear receptors to develop novel methods to assess effects of environmental pollutants on specific endocrine signaling pathways in transgenic mice. These methodologies, which can be tailored to allow evaluation of effects of environmental pollutants on human receptors in an in vivo setting, are potentially applicable to all members of the nuclear receptor super family. These methods will initially be developed to assess effects of Superfund site chemicals on estrogen and peroxisome proliferator activated receptor function. Estrogen receptors represent one of the most intensively studied targets of endocrine disruptors, allowing validation of these new methodologies by comparison to existing assay systems. The peroxisome proliferator activated receptors are much less well understood, but represent important emerging targets of endocrine disrupting compounds. The specific aims of this proposal are to: 1) Develop transgenic reporter systems that allow quantitative assessment of the transcriptional activities of estrogen and peroxisome endogenous hormones and identify physiologic target genes; and 3) Assess effects of endocrine disrupters found at Superfund Sites on the activities of estrogen and peroxisome- proliferator-activated receptors in vivo and their effects on the expression of downstream target genes.

Abstract  DESCRIPTION: (Adapted from the Investigator's Abstract) : Phthalate compounds are ubiquitous environmental contaminants and because they are endocrine disruptors with weak estrogenic properties, there is concern about their toxic effects on male reproductive organs. Phthalates are also peroxisome proliferators which activate a receptor called the peroxisome proliferator-activated receptor (PPAR). The goal of this proposal is to identify the underlying molecular and cellular mechanisms by which phthalates and peroxisome proliferators disrupt testicular function. The hypothesis to be tested is that exposure to phthalates or other peroxisome proliferators cause PPAR activation which interferes with the retinoid signaling pathway that is vital for embryonic and postnatal testis development. A heterodimer of retinoic acid receptor alpha (RARalpha) and retinoid X receptor (RXR) is essential for normal testicular function. RXR also forms a heterodimer with PPAR which is required for its transcriptional activity, thus RXR is a common factor for retinoic acid and peroxisome proliferator action. Specific aims are to determine the signaling mechanisms by which phthalates and other peroxisome proliferators activate PPAR and show that PPAR may interfere with normal retinoid signaling in the testis. The dose dependent and developmental -specific changes in the expression of PPAR, retinoid receptors, and target genes involved in lipid, retinoid, or steroid metabolism, will be examined in testes from embryos and postnatal rats. and testes from PPARalpha knockout mice treated with phthalates and other peroxisome proliferators. The PI will also examine the effects of peroxisome proliferators on transcriptional activation of PPAR and retinoid receptors in Sertoli cells to establish whether there is a potential competition for RXR. Over expression of RARalpha or PPARalpha will be achieved to ascertain whether PPARalpha and RARalpha compete for endogenous RXR. The results will demonstrate that exposure to phthalates and other peroxisome proliferators leads to alteration in the activation of PPAR and RAR mediated by increased activities of protein kinase C and MAP kinase, and decreased activities of FSH and CAMP, and this activation will lead to altered expression of target genes in testes of embryonic and postnatal rats. Completion of this research will elucidate the mechanism by which phthalates and other peroxisome proliferators alter testicular function and contribute to our understanding of the health risks from these compounds to humans. Understanding the molecular signaling mechanism may facilitate development of a method to screen for other chemicals that have similar properties as phthalates on the male reproductive system.

Abstract  In exciting new studies, we showed that phthalic acid esters and lipid lowering drugs (e.g., diethylhexylphthalate (DEHP) and WY-14,643) activate phagocytosis by the resident hepatic macrophages, Kupffer cells. Further, increased cell proliferation was blocked in vivo by both an antibody to TNFalpha and methyl palmitate, an inactivator of Kupffer cells. Based on this new information, we hypothesize that phthalates activate Kupffer cells to produce mitogenic cytokines that stimulate cell proliferation and cause tumors. To test this hypothesis, three questions will be posed. First, are Kupffer cells responsible for increased cell proliferation due to plasticizers? Kupffer cells will be treated with DEHP and the lipid lowering drug WY-14,643 in vitro or isolated from rats treated in vivo and cultured. Conditioned media from Kupffer cells will be added to cultured hepatocytes and cell proliferation will be assessed. Similar experiments will be performed with human macrophages and hepatocytes since whether or not these chemicals cause cancer in humans remains controversial. Nimodipine, methyl palmitate and dietary glycine will be used to prevent or minimize activation of Kupffer cells. We expect these experiments to demonstrate conclusively that TNFalpha produced by Kupffer cells is responsible for enhanced cell proliferation caused by plasticizers. Second, experiments will be performed to understand how phthalates activate Kupffer cells to produce mitogens. We propose that phthalates will enter Kupffer cells membranes based on their lipophilicity, increase intracellular calcium, and activate PKC by altering second messenger systems. Accordingly, uptake of radiolabelled drugs of different lipid solubility will be compared. Further, the effect of phthalate treatment in vivo on intracellular calcium and PKC will be measured in isolated Kupffer cells. To determine if PKC activates NADPH oxidase and increases NF/kappa/B-mediated TNFalpha production, superoxide and NF/kappa/B will be monitored and correlated with changes in TNFalpha mRNA. Third, can phthalate-stimulated increases in cell proliferation and tumors be prevented in vivo by modulation of Kupffer cells? Dietary glycine, which inhibits Kupffer cell activation, will be used to prevent cell proliferation, changes in apoptosis, and the formation of preneoplastic foci and tumors, which is the "gold-standard." By pursuing these new hypotheses, we will fill important gaps in our knowledge regarding mechanisms. Another important impact of this work will be to provide governmental regulators with mechanistic information which will allow them to shift emphasis away from the fact that these chemicals induce peroxisomes and focus on cell proliferation.

Abstract  DESCRIPTION: Dr. Jirtle's group has recently demonstrated that the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2r) gene is an important tumor suppressor in human and rat hepatocellular carcinoma (HCC). The proposed studies will focus on mouse model systems. In Aim 1, HCCs from two carcinogen models will be analyzed to demonstrate that M6P/IGF2r gene lesions are present. Aim 2 studies will evaluate the effect of M6P/IGF2r gene replacement in an HCC line that lacks a functional gene. In Aim 3, a M6P/IGF2r transgenic that overexposes the gene will be evaluated for its sensitivity to liver carcinogenesis by two agents, diethylnitrosamine (DEN) and di[2-ethyl-hexyl]phthalate (DEHP). Finally, for Aim 4 studies, a transgenic mouse will be constructed with inducible knock-out of the M6P/IGF2r gene for evaluation in the carcinogenesis models.

Abstract  The purpose of this contract is to procure detailed chemical disposition data from a number of studies of selected environmental contaminants or model compounds per year. Such studies are designed to provide both applied knowledge of the fate of chemicals in the intact animal in support of toxicity tests conducted by the National Toxicology Program and basic knowledge of mechanisms of chemical toxicity. These data will help to predict more accurately the correct doses for compounds to be studied in animal bioassays. Developing fundamental information on chemical structure-activity relationships will permit a more accurate prediction of the fate and toxicity of chemicals that have similar structural or physical properties. Most of these studies are performed in intact laboratory rats and/or mice although some studies may require the use of human and rodent liver slice incubations in vitro for more accurate extrapolation of results from rodents to humans and for mechanistic studies. Disposition and metabolism studies are being conducted on the industrial intermediate cyclohexene oxide. Methyl styryl ketone is being studied to provide data to determine the appropriate route of exposure for the subchronic and chronic toxicity tests. Four peroxisome proliferators, dibutyl phthalate, Wyeth 14,643, 2,4-dichlorophenoxyacetic acid, and gemfibrozil, are being studied in rat, mouse and human liver and kidney slices. These data will provide data for risk assessments from human exposure to these chemicals. The comparative biotransformation in rodent and human hepatocytes for eugenol, isoeugenol, methyleugenol and myristicin are being compared to the known carcinogen safrole for risk comparison purposes. Additional studies on this class study include unscheduled DNA synthesis, DNA binding and analysis of adducts, and toxicity to human and rodent hepatocytes.

Abstract  DESCRIPTION: Dr. Jirtle's group has recently demonstrated that the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2r) gene is an important tumor suppressor in human and rat hepatocellular carcinoma (HCC). The proposed studies will focus on mouse model systems. In Aim 1, HCCs from two carcinogen models will be analyzed to demonstrate that M6P/IGF2r gene lesions are present. Aim 2 studies will evaluate the effect of M6P/IGF2r gene replacement in an HCC line that lacks a functional gene. In Aim 3, a M6P/IGF2r transgenic that overexposes the gene will be evaluated for its sensitivity to liver carcinogenesis by two agents, diethylnitrosamine (DEN) and di[2-ethyl-hexyl]phthalate (DEHP). Finally, for Aim 4 studies, a transgenic mouse will be constructed with inducible knock-out of the M6P/IGF2r gene for evaluation in the carcinogenesis models.

Abstract  DESCRIPTION (Adapted from the Investigator's Abstract) Understanding the mechanism of nongenotoxic liver carcinogens including peroxisome proliferators is important due to the wide range of sensitivity of different species. One mechanism for controlling the expression of genes is DNA methylation, i.e., specific changes in 5-methylcytosine (5-MEC) content of DNA. The principal investigator (PI) proposes that the effect of peroxisome proliferators on methylation of genes would be useful in understanding their mechanism and as biomarkers for their species sensitivity. There are four aims to test this hypothesis. Aim 1 will determine in rat liver, a sensitive species, whether dibutyl phthalate, gemfibrozil, Wy-14,643, and 2,4-D decrease methylation of DNA, IGF2 and c-myc while increasing methylation of connexin 32. By evaluating genes that are expected to be hypomethylated and a gene that should be hypermethylated, we hope to demonstrate specificity for the effect of peroxisome proliferators on DNA methylation. Aim 2 will determine whether the alterations in methylation found in rat liver also occurs in mouse liver, another sensitive species, and to a lesser degree in a less sensitive species, Syrian hamsters. Aim 3 will determine dose-response relationships. Aim 4 will determine whether hypomethylation of IGF2 and c-myc are associated with an increased expression of their mRNA and protein and whether hypermethylation of the connexin 32 gene is associated with decreased expression. The species sensitivity of the effect of the four peroxisome proliferators on the methylation of DNA and genes and on the expression of their mRNA will be compared to known species sensitivity of their carcinogenic activity, enhancement of cell proliferation, induction of peroxisomes, and other biological and molecular activity. A correlation between the species sensitivity of the effect of peroxisome proliferators on DNA (gene) methylation and the expression of their mRNA and/or protein would indicate usefulness as biomarkers of exposure to these chemicals. Furthermore, a correlation with carcinogenic activity would indicate the involvement of the gene(s) in a nongenotoxic mechanism for peroxisome proliferators.

Abstract  DESCRIPTION (Adapted from the APPLICANT'S ABSTRACT): The broad, long-term goals of the proposed research are to understand the mechanism(s) by which peroxisome proliferating agents (PPAs) interfere with signaling cross-talk between metabolic pathways and nuclear hormone receptors producing perturbations in reproduction and development. PPAs are a structurally-diverse group of chemicals including herbicides, solvents, and plasticizers, that are known fatty acid, retinoid and endocrine disrupters. These metabolic pathways interact in the regulation of gene expression for reproduction and development. Hepatic peroxisome proliferation by PPAs has species-specificity (rats are sensitive, humans are not) but little is known about the mechanisms of PPAs endocrine disruptive effects. PPA species-specificity has recently been demonstrated in fish allowing investigation of responsive and non-responsive models. Fish exposed to PPAs can bioaccumulate these xenobiotics, and serve as biomarkers of exposure and effect. Furthermore, because human consumption of fish and fish products has increased in the last decade, it is imperative that we understand the potential for adverse effects by these contaminants. Test organisms will be a commercially-important food species, the catfish (sensitive; Haasch, 1996), and a model species, the Japanese medaka (non-sensitive; Scarano et al,. 1994). The proposed research will test the hypothesis that PPAs are endocrine disrupters producing perturbations in reproduction and development. PPAs to be investigated are: clofibrate, as a model PPA, and haloxyfop enthoxyethyl, di-n-butyl-ortho-phthalate (DBoP) and tetrachloroethylene (PCE) as environmental contaminant PPAs. Specific Aims include, 1) determining if PPA-sensitivity is ruired for endocrine disruption and if PAPAS from different chemical classes cause endocrine disruption by similar mechanisms, 2) determination of specific mechanisms of endocrine disruption by examining PPA combinations, and 3) determining if PPA-mediated endocrine disruption can be correlated with reproductive or developmental problems and if PPA exposure produces metabolic alterations in offspring. The proposed investigation will provide insight into cell signaling cross-talk between metabolic pathways and nuclear hormone receptors, endocrine disruption, and the developmental and reproductive effects of PAPAS.

Abstract  IPA COPYRIGHT: ASHP The effects of the dissolution pH value of hydroxypropylmethylcellulose phthalate (pH 5), cellulose acetate phthalate (pH 6) and Eudragit (pH 7) on the intestinal absorption of enteric coated aspirin tablets were studied. No significant difference was observed between salicylate blood levels corresponding to tablets coated with either cellulose preparation, however, 2 of 6 volunteers receiving the Eudragit coated tablet did not absorb aspirin. These results show that enteric coating materials should not dissolve at a pH value higher than 6. Moreover, they confirm that disintegration tests of enteric products should be performed in simulated intestinal fluids at a pH value lower than 7.

Abstract  IPA COPYRIGHT: ASHP The relationships between in vitro dissolution rates of acetylsalicylic acid (aspirin) from enteric tablets coated with cellulose acetate phthalate and maximal blood levels of salicylate measured after administration of the tablets to human volunteers are reported.

Abstract  HEEP COPYRIGHT: BIOL ABS. RRM ABSTRACT ESCHERICHIA-COLI VIBRIO-CHOLERAE NITRO BENZENE M CRESOL PHTHALATE GROWTH MEMBRANE COMPOSITION PLASMID CARRIAGE CHESAPEAKE BAY USA

Abstract  HEEP COPYRIGHT: BIOL ABS. Models which predict the geochemical behavior of Np in nuclear waste require thermodynamic parameters and stability constants. Conditional stability constants, enthalpies %W%000004%% and entropies %W%000004%% of complexation at pH 7.5 and ionic strength 0.1 were determined for Np (V) complexes of phosphate, salicylate, phthalate and citrate. Phosphate formed a complex with log beta = 2.36 | 0.42 at 25? C, %W%000004%% = -69.9 kJ/mol and %W%000004%% = -188 J/mol-K. At pH 7.5 salicylate did not for complex with Np(V) due to the low charge density of the NpO2+ ion and incomplete ionization of the salicylate ion. Phthalate formed a complex with log beta = 3.43 | 0.33 at 25? C, %W%000004%% = 33.5 kJ/mol and %W%000004%% = 182 J/mol-K. Citrate forms a complex with log beta = 4.84 | 0.72 at 25? C, %W%000004%% = 14.0 kJ/mol and %W%000004%% = 140 J/mol-K. In all cases, only 1:1 complexes were identified.

Abstract  HEEP COPYRIGHT: BIOL ABS. RRM PHTHALATE ESTER PESTICIDE

Abstract  An industrial hygiene survey was performed in a polyvinyl-chloride (9002862) (PVC) plasticized film production facility. The concentration of chemical substances in the workplace was determined. Measures for improving working conditions were implemented. To evaluate the effectiveness of these measures, the morbidity rate with loss of work time was investigated by shop, sex, age, job, nosological illness category, and by groups of people who were ill. Release of excess heat from the equipment, insufficient ventilation, precipitation of dust, vapors and gases from chemical substances, and precipitation of toxic substance at the roller and feeding presses were found. Concentrations of PVC resin dust, lead (7439921) and its compounds, vapors of complex phthalic-acid-esters, and vinyl-chloride (75014) were elevated in the factory. Installation of a dehumidification system improved the microclimate at the work sites. Use of a pneumatic conveyor and automation of transporting PVC from the mixer to the sheet presses decreased dustiness and gas release in the factory. Local exhaust ventilation was improved which decreased concentrations of toxic substances 3 to 4 times. The highest morbidity for the production shops was 89.5 cases out of 100 workers for males and 95.0 cases for females; in the auxiliary shops, the morbidity was 74.9 for males and 84.5 for females. Over the following 2 years, after hygienic improvements in the facility, morbidity for both sexes decreased significantly and remained at the decreased rate when measured for an additional year. Increases of productivity of 20 percent for the mixer operators and 22 percent for roller press operators were seen. The author concludes that implementation and execution of hygienic, technological, and sanitary measures in a PVC film production facility improves working conditions, decreases workers' illness, and increases productivity. (Russian)

Abstract  Research published on the toxicological and environmental effects of phthalic-acid esters since 1978 was reviewed. Human studies were discussed. These have focused on the release and breakdown of di(2-ethylhexyl)phthalate (117817) (DEHP) and mono(ethylhexyl)phthalate (4376209) (MEHP) from polyvinyl-chloride materials used for blood storage or hemodialysis. The studies have shown that DEHP can be enzymatically converted into MEHP in stored plasma. The concentrations of DEHP and MEHP in stored blood samples are sufficient to cause toxicity in human cell lines. Autopsies have revealed that hemodialysis and transfusion patients may have increased tissue concentrations of phthalate esters. The significance of this finding is unknown since the frequency of detectable tissue phthalate concentrations in the general population is not known. Except for one Italian study using a small cohort, epidemiological studies on phthalate plasticizer workers have not found an increase in neurological symptoms or chromosome aberrations. The Italian study found a high prevalence of motor and sensorimotor polyneuropathy; however, the significance of the findings is limited because of the small number of cases. Studies in laboratory animals were discussed. These have shown that DEHP and other esters induce increases in liver weight and drug metabolizing enzymes in rats. DEHP also causes significant decreases in plasma testosterone in male rats. DEHP and MEHP have induced teratogenic effects; however, the doses used caused significant maternal toxicity. MEHP and DEHP have shown mutagenic activity in bacterial systems. Carcinogenesis bioassays using butylbenzyl-phthalate (85687) (BBP), diallyl-phthalate (131179), and DEHP conducted by the National Cancer Institute are in progress. Studies on mammalian metabolism of DEHP and butylglycolbutyl-phthalate (85701) were considered. Studies in fish and wildlife were discussed. BBP at concentrations of 0.5 to 5.0 milligrams per liter has been found to be toxic to algae, invertebrates, and fish. BBP seems to have a greater potential for bioconcentration than DEHP. Studies on the environmental fate and monitoring of phthalic-acid esters were discussed. These have shown that although there are many microorganisms in soil and water that can degrade phthalate esters, they do not apparently biodegrade rapidly.

Abstract  Asthma caused by acid anhydrides was studied immunologically. Using sera from seven patients with asthma caused by inhalation of tetrachlorophthalic-anhydride (117088) (TCPA) and from patients with asthma caused by phthalic-anhydride (85449) (PA), trimellitic-anhydride (552307) (TMA), and maleic-anhydride (108316), the specificity of immunoglobulin-E (IgE) for epitopes formed by conjugation of anhydride with albumin was studied. In patients sensitized to TCPA and TMA, anhydride/human serum albumin (HSA) conjugate was a more effective inhibitor of radioallergosorbent test (RAST) than either the sodium salt of the anhydride or an anhydride/bovine serum albumin conjugate. In patients sensitized to PA, free hapten was as effective an inhibitor as PA conjugate. HSA conjugates of anhydrides to which individuals were not sensitized were weaker inhibitors of RAST than the sensitizing acid anhydride/HSA conjugate. Although there were no additional cases of TCPA induced asthma, specific IgE antibody binding to TCPA/HSA conjugate was present in 24 of 300 factory workers exposed to TCPA. The intensity of exposure and cigarette smoking were major factors associated with the presence of specific IgE to TCPA/HSA. Eighty three percent of subjects with specific IgE were current cigarette smokers and 48 percent of those without specific IgE were current cigarette smokers. The percent with specific IgE was 16.1 for atopic smokers and for nonatopic nonsmokers it was zero. During a 5 year followup with avoidance of exposure, specific IgE to TCPA/HSA conjugate fell exponentially in six patients, with a half life of one year. At the end of the observation period, all six patients complained of episodic respiratory symptoms. The authors suggest that persistence of airway hyperresponsiveness in asthmatic patients after avoidance of exposure may indicate a permanent hyperresponsiveness caused by the asthma inducing exposure.

Abstract  Pathogenic mechanisms involved in occupational asthma were reviewed and its characteristics summarized. Occupational asthma is a generalized, usually reversible, airway obstruction caused by inhalation of substances that a worker handles or are present at the worksite. Predisposing factors include atopy, continuous or intermittent exposure to agents such as diisocyanates and Western-red-cedar dust, and chronic bronchitis. Atopic workers have a greater risk of sensitization and developing asthma than nonatopics. Mechanisms proposed to explain the occurrence of occupational asthma were discussed. These include immunologic responses, airway smooth muscle hyperresponsiveness, receptor mediated responses to intracellular nucleotides, changes in the deposition and clearance of particles from the lungs, airway inflammation, and airway epithelial damage. Induced increases in airway responsiveness associated with late, but not early, asthmatic responses are considered to be very important in the pathogenesis of occupational asthma. Exposure to sensitizing agents probably causes the nonspecific hyperresponsiveness seen in asthma patients rather than the hyperresponsiveness being a predisposing factor. Agents that can cause occupational asthma were discussed. These include substances of animal origin including immunoglobulin-E mediated responses to proteolytic agents, animal hair, dander, and proteins excreted in the feces and urine, plant substances such as grain, fungi and mite infected grain, and exposure to low molecular weight chemicals such as diisocyanates, phthalic-anhydride (85449), and plicatic-acid (16462650) in Western-red-cedar dust. Reactive airways dysfunction syndrome (RADS) was discussed. RADS is a clinical entity in which inhalation of an irritant substance causes a constellation of signs and symptoms that resembles asthma. The symptoms include cough, dyspnea, and wheeze. The pathogenesis of RADS is thought to involve a nonimmunologic inflammatory mechanism.