Bernard Weiss - US grants

Disulfiram (Antabuse) is an important component of many alcoholism treatment programs. One of the reasons it is not used more widely is its neurotoxicity. Chronic administration of disulfiram may produce both peripheral and central nervous system impairment ranging from sensory and motor deficits to cognitive difficulties. Among the mechanisms proposed to account for these neurotoxic reactions is the ability of disulfiram to chelate metals. Disulfiram belongs to a chemical class known as dithiocarbamates whose members are also used in agriculture, industry, and cosmetics. In medicine, they may be used specifically as chelating agents to reduce toxic levels of certain metals. Considerable data indicate that dithiocarbamate-metal complexes, however, penetrate more easily into the brain, probably because of their lipophilic properties, and may greatly enhance metal levels in brain tissue at the same time that they may reduce kidney concentrations, for example. The functional consequences of this action remain to be determined. This proposal outlines the preliminary steps of a project whose aim is to do so. One step is to measure the brain levels of methylmercury (a recognized neurotoxicant) and cadmium (at best, a weak neurotoxicant) in rats also treated with disulfiram. This part of the project is basically a dose-ranging study. Another step is to devise a suitable method for assessing the kind of neurotoxic responses that might arise with such treatment. The technique proposed relies on schedule-controlled behavior coupled with a response, wheel-running, that also reflects the capacity for coordinated movement. The eventual outcome of this research will help clarify the contribution of metal toxicity to disulfiram toxicity, and enlarge our understanding about the joint risk of disulfiram treatment and toxic metal exposure.

Dithiocarbamates are a class of chemical compounds enjoying wide use in industry, agriculture, and medicine. Most of them are potent chelating agents. As chelators, however, they may also promote elevated brain levels of metals at the same time that they enhance excretion from other organ systems. Lead, copper, nickel, methylmercury, cadmium, and thallium are among the metals for which such effects have been shown. Whether these elevated brain levels might also induce neurotoxicity, and under what circumstances, needs to be clarified. The proposed research represents an effort to combine chemical and behavioral measures to explore these issues. Rats will be exposed to lead, nickel, and thallium in drinking water and treated twice weekly with diethyldithiocarbamate (DDC). Chemical measures will be used to ascertain metal levels in various tissues, whether the metal is bound to DDC in the tissue or is in another form, and to determine a variety of biochemical effects. Behavioral measures, derived from a modified running wheel apparatus, will be used trace the appearance of neurotoxicity during the different treatment regimens.

An extensive literature indicates that the fetal brain is exquisitely sensitive to methylmercury. These data come from a multiplicity of species, including humans. Despite the extent of the current literature, however, two difficult and important questions remain unanswered. First, what are the lifetime consequences of prenatal exposure? Might they remain latent until the brain is stripped of compensatory capacity by the processes of aging? Second, what might be the consequences of lifetime exposure to relatively low levels of methylmercury? Could minute increments of damage cumulate to accelerate the processes of aging in the brain? These questions will be addressed in mice in an experimental format designed to follow the course of potential neurotoxicity through the lifespan. The mice will be exposed prenatally alone or both prenatally and postnatally. Specified groups will be observed at the age of 3, 9, 15, and 21 months for six-month periods, during which behavioral measures of motor function and cognitive performance will be secured. At the end of the observation period, brains will be examined by quantitative morphological methods to determine the structural consequences of the various exposure scenarios, and histo- chemical methods will be used for mercury localization. Both organic and inorganic content will also be assayed. These data will contribute data essential for a full risk assessment of dietary methylmercury sources, because current human data suggest adverse effects of exposure at surprisingly low levels.

This study will concentrate on the effects of oxygen-derived free radicals. These oxidants are generated not only by lonizing radiation but also by normal aerobic metabolism in almost all cells. They have been implicated as causes of mutations, cancer, aging and of tissue damage associated with inflammation. This proposal will focus on a specific multigene system in Escherichia coli that is induced by compounds that generate superoxide. This global response includes the genes for the DNA repair enzyme endonuclease IV, for a superoxide dismutase, and for glucose-6- phosphate dehydrogenase, all of which are positively regulated at a transcriptional level by soxR and soxS, newly discovered genes that have been cloned, mapped and sequenced in this laboratory. The soxRS region will be transcriptionally mapped and tested for exogenous and endogenous regulation. Existing mutations will be sequenced and additional specific ones produced if needed. The proteins will be purified and their effect will tested in vitro on the transcription of one of the target genes, that for endonuclease IV (nfo). In the likely event that one or both or the proteins bind on the nfo promoter region, then the contact points will be identified via footprinting, in vitro modification of the DNA, sequencing of promoter-constitutive mutations, and a survey of target sites in other genes. Physical studies of the purified proteins will center around possible proteins-proteins and protein-nucleic acid interactions, conformational changes accompanying activation, and the function of four close cysteines in soxR. Posttranscriptional control will be explored by connecting the nfo (endonuclease IV) target gene to foreign promoters and possible feedback loops will be examined by measuring nfo transcription and translation in cells with different copy numbers of the gene. If the entire system cannot be reconstructed in vitro, then additional genes in this pathway will be sought through mutation, employing gene fusions as indicators. //

Cancer, mutation, and aging are common consequences of the failed enzymatic repair of DNA lesions. DNA damage is produced not only by environmental agents such as radiation but also by endogenous toxicants present in the normal cell. Almost every type of DNA repair enzyme (e.g., endonucleases for apurinic sites, mismatch repair enzymes, photolyases, ligases, glycosylases, methyl transferases, recombinases) has first been discovered and/or extensively characterized in bacteria, then found to be universal, and finally studied in mammalian cells. This proposal is for the study of a unique bacterial Dnase that can recognize lesions in irradiated DNA. Endonuclease V of Escherichia coli in a Dnase of wide-ranging specificity. Among its preferred substrates are DNAs that have been heavily irradiated by UV, treated with OsO4 (which produces some lesions similar to those of ionizing radiation), partially depurinated, alkali-treated, uracil- substituted, or treated with the carcinogen 7-bromobenz[alpha]anthracene. Although its ability to recognize a variety of DNA lesions suggests that it should be an extraordinarily useful and therefore ubiquitous DNA repair enzyme, its study has been severely limited by its paucity and by the unavailability of enzyme-deficient mutants. The objectives of this proposal are to clone its gene, to produce and characterize mutants, and to purify and characterize the enzyme from overproducers. The gene will be cloned via reverse genetics. To accomplish this, purified endonuclease V protein will first be prepared and then partially sequenced. Oligonucleotide probes will be generated to match the possible coding sequences, and the probes will be used to detect the cloned gene in an ordered genomic library. The cloned gene will be mutated and transferred to the chromosome. In order to detect the importance of the enzyme in various pathways of DNA repair, the mutants will be tested for their sensitivities to the lethal and mutagenic effects of DNA-damaging agents. The gene will be also subcloned in expression vectors for overproduction, purification, and subsequent study of the enzyme. A goal of the enzyme characterization will be to discover a common feature in its substrates or cleavage sites, thereby revealing its ultimate specificity an possibly enabling its subsequent use as a reagent for detecting certain types of lesions or structural features in DNA.

9304092 Weiss The soxRS (superoxide response) regulon of Escherichia coli an adaptation to oxidative stress. It includes the genes for a superoxide dismutase, endonuclease IV (a DNA repair enzyme), and glucose 6-phosphate dehydrogenase. Induction occurs in two stages. SoxR is first altered by an oxidative signal to become a transcriptional activator of SoxS. The overexpressed SoxS then activates the target genes of the regulon. The first question to be answered is whether or not the two-stage mechanism provides a temporally programmed response; i.e., are there genes other than soxS that are turned on directly by the activated SoxR and that are therefore expressed before those genes targeted by SoxS? To answer this question, 2-D gel protein patterns are being examined in regulon-constitutive (soxR) mutants that have a soxS deletion. The major part of this research deals with purification of SoxS and SoxR proteins from overproducing strains and a study of their interactions with their DNA binding sites and with RNA polymerase. Because the Sox S protein is a member of the large AraC family, so many analogs of which are being separately studied, investigations is limited to examining only its unique properties; thus, its specific recognition sequence will be delineated via DNA footprinting and mutational analysis of the for promoter region. SoxR, which is activated directly and/or indirectly by superoxide, will be studied more extensively. An attempt will be made to reconstruct SoxR-mediated soxS induction in vitro an to use it to identify the possible effector (s). The physical traits of the native SoxR protein will be compared with both mutationally activated and chemically activated forms to provide an insight into the mechanism of induction. The physical traits to be examined will include evidence of phosphorylation or proteolysis and changes in metal content, DNA footprint, titratable thiols, absorption spectrum, fluorescence, and multimer formation. %%% Because bacterial regulons are controlled in diverse ways, they provide many useful models for the coordinate regulation of dispersed genes in all organisms. Ultimate goals of the studies on the soxRS regulon are (a) the understanding of an additional system of multigene regulation and (b) the elaboration of the mechanism whereby organisms that grow in the presence of oxygen can deal with toxic by products of oxidative growth. ***

embryo /fetus toxicology; neurotoxins; developmental neurobiology; cocaine; methanol; methylmercury; laboratory rat; laboratory mouse;

In one study, we are analyzing a bacterial DNA repair enzyme and its gene in order to understand mechanisms of repair of damaged genes. We are testing the hypothesis that the enzyme recognizes single-stranded or distorted regions of DNA next to normal ones. In a second study, we are examining the pathway of activation of a protein that senses superoxide, a mutagen, and regulates a bacterial response to it. We are testing the hypothesis that the pathway recognizes a depletion of reducing compounds that leads to oxidation of the regulatory protein and a change in the conformation of the DNA to which it is bound.

DESCRIPTION: (Adapted from the Investigator's Abstract) Metallic mercury, a neurotoxicant usually viewed as an occupational hazard, is a more general environmental hazard as well. Although we know a great deal about its effects in adults, our knowledge of its impact on the developing brain is fragmentary. This information gap is disquieting. (1) Many women in the work force are exposed to mercury vapor. (2) Many, perhaps most, women have dental amalgam fillings. These emit mercury vapor. Maternal tissue and fetal levels are correlated with the number of fillings. (3) Mercury is a more common contaminant of indoor environments than is generally recognized. Because of its physical properties it can lurk undetected and continue to evaporate. (4) Young children are considerably more vulnerable to mercury toxicity than adults; the syndrome of Pink Disease is a childhood affliction. (5) Organic mercury compounds are potent disrupters of brain development; some authorities believe that its conversion to the inorganic form in brain cells is the toxic mechanism responsible. (6) The experimental literature devoted to neurobehavioral effects consists of a handful of studies. To enhance our ability to evaluate the risks of metallic mercury to the developing brain, we intend to expose both pregnant rats and neonates to mercury vapor. Pregnant rats will be exposed to air, 30 ug/cubic meter, 100 ug/cubic meter, the OSHA permissible level, and 300 ug/cubic meter. Neonatal rats will be exposed to air, 10, 30, and 100 ug/cubic meter. Exposure will last two hours. The different levels are based on reports indicating widely different sensitivities between prenatal and neonatal exposures. Dam and pup blood levels will be monitored, and tissue assays, especially of brain, will be conducted at selected times. To determine the functional consequences of developmental exposure, three behavioral endpoints will be assayed: (1) The acoustic startle response, which will provide measures of habituation, sensory dysfunction, and the total neuromuscular response pattern to sound. (2) Spontaneous locomotor activity in the rats, a common index of neurotoxicity. (3) Acquisition of schedule-controlled operant behavior. Histochemical techniques will be deployed to map localization of brain mercury deposits.

DESCRIPTION: The applicants propose a workshop on Environmental Contaminants and Developmental Disabilities as part of the Rochester Conference Series on Environmental Toxicity. The conference is to be held June 1977 in Rochester, N.Y. The two-and-a-half-day conference is designed to familiarize participants with concepts of exposure, incidence, prevalence, etiology, severity and pathophysiology of developmental disabilities. The organizers seek to unite the disparate disciplines of research and clinical practice and to identify potentially fruitful areas for future research and collaboration. There will be approximately 50 participants. Participation in the conference is contingent upon invitation by the steering committee and potential invitees include clinicians, regulators from federal agencies and university researchers.

DESCRIPTION: (Adapted from the Investigator's Abstract) Endocrine disruptors are a class of environmentally prevalent compounds that include the dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the polychlorinated biphenyls, and some isoflavonoid phytoestrogens. Their potential health hazards, until recently, were dominated by concerns over carcinogenicity following exposure of adults to high doses. Developmental toxicity now seems to be achieving a leading role and may even displace cancer as the basis for assessing the human risks posed by these compounds. One source of this shifting concern is data describing impaired sexual development in rats exposed prenatally to surprisingly low doses of TCDD. TCDD and related endocrine disrupters appear to alter the perinatal gonadal hormone milieu. Such activity triggers a cascade of disturbances in brain development and neuroendocrine function. Although such disturbances might be seen most readily in reproductive studies, a constellation of much more subtle consequences is conceivable. These outcomes might include altered gender-specific cognition. For example, female rats tend to emit different patterns of responding than male rats on schedule-controlled operant behavior assays. The current proposal is guided by the hypothesis that prenatal TCDD modifies the usual pattern of differences between male and female rats. Tests of the hypothesis will be based on three situations in which gender-specific learning, memory, and spatial cognition are assessed. One assessment situation will examine a sexually-motivated behavior in exposed females, a second situation will examine sexual motivation in exposed males, and the third situation will examine nonsexual cognitive function in both males and females. In order to determine sensitive critical periods of prenatal development, the behavioral endpoints will be examined across groups of offspring that have been exposed under one of three different prenatal schedules. All of these measures will be examined in subjects exposed to a new, low dose range of TCDD that approaches background levels in the environment. The coupling of several functional measures is adduced as a model of risk assessment.

With a single known exception (a virus), the DNA, or genetic material, of every organism contains thymine rather than its simpler analog, uracil. This is true even though uracil was used long before thymine in evolution and even though, in the test tube, uracil-containing DNA can be replicated and encodes genetic traits. The reason for the preference of thymine over uracil is a major unanswered question in biology. A major long term goal of this project is to produce a thymineless bacterial chromosome. A multiple mutant in a common gastrointestinal bacterium (Escherichia coli) has been already been constructed that can replace 30% of its chromosomal thymine with uracil. By gradually forcing this bacterium to use increasing amounts of an alternative DNA precursor, deoxyuridine, from the medium, it may be possible to drive the evolution of a thymineless chromosome. The bacterium used in this project has been widely used for the discovery of universal metabolic pathways. It has multiple ways to make thymine compounds, to keep uracil out of DNA, and to remove it once it is incorporated. Some pathways that are still unknown will be identified by mutation. By successive mutation, it may be possible to "reverse" evolution and produce a bacterium that no longer excludes uracil from the chromosome. Whether or not the goal of a completely thymineless chromosome is reached, the results should provide valuable information about alternate pathways of thymine synthesis, about what genes and enzymes keep uracil out of DNA, and about differences in the biological properties of uracil-containing versus thymine-containing DNA.

DESCRIPTION (provided by applicant): Public anxieties about mercury exposure have been aroused by reports about the hazards of fish consumption, dental amalgams, and preservatives in vaccines. Although scientific support for such concerns may be tenuous, it is also true that sizable gaps remain in our understanding of mercury neurotoxicity, especially with respect to combined exposures to different species, a commonplace condition. The two forms that present the greatest health risks are methylmercury, found in fish and marine mammals, and metallic mercury vapor, found in industry, cosmetics, and dental amalgams. Mechanistic analyses and limited experimental data indicate that co-exposure to the two forms may enhance the neurotoxicity of either alone, an especially worrisome outcome for the developing brain. Because joint exposure is such a common occurrence, with assays in pregnant women indicating that the inorganic form accounts for more than 25% of mercury in blood, this project is designed to more clearly define the associated risks to brain development. Pregnant rats will be exposed concurrently to mercury vapor by inhalation and to methylmercury in drinking water. Neurobehavioral measures in the offspring will include schedule-controlled operant behavior to assess cognitive behavior, motivation, and memory; hindlimb splay as an assay for motor function; and locomotor activity, a common assay for neurobehavioral status. Mercury concentrations in offspring brains will also be measured. The research will proceed in stages. (1) Exposure levels will produce mild to moderate degrees of neurotoxicity and reliable dose-effect functions for each form by itself. (2 and 3) Here we determine if a specified concentration of vapor changes the dose-effect function for methylmercury and if a specified dose of methylmercury alters the concentration-effect function of mercury vapor. These results will be used to calculate, by Benchmark Dose modeling, vapor and methyl exposures inducing a 10% and 25% change in the neurotoxic indices. (4) The combined effects of these levels will be determined. The acquisition of these data offers a firm foundation for determining the risks to neurobehavioral development of concurrent exposures to both mercury species.

[unreadable] DESCRIPTION (provided by applicant): This R21 proposal is based on the hypothesis that prenatal exposure to phthalate esters will alter normal brain development and associated behavioral outcomes. Phthalates are ubiquitous environmental contaminants found in cosmetics, household furnishings, medical devices, toys, and numerous other products. Human exposures are universal. Recent human data, showing a significant relationship between levels of phthalate metabolites in maternal urine during pregnancy and shortened anogenital distance in male children, provoke concerns that phthalates may pose significant threats to neurobehavioral development. In rats, exposure during gestation to certain phthalates induces malformations in the reproductive tract of male offspring as well as markers of feminization such as shortened anogenital distance. This syndrome signifies a disturbance in androgen-mediated development attributable to a decrease in fetal testicular production of testosterone. Because testosterone governs sexual differentiation of the brain, reductions in fetal output would be expected to engender demasculinization of brain anatomy and its expression in behavior. To test this hypothesis, pregnant rats will be administered selected phthalate esters (diethylhexyl phthalate, or DEHP, and dibutyl phthalate, or DBP), or a combination of the two, during gestational days 12-20, which encompass the testosterone surge in fetal males. Both male and female offspring will be evaluated behaviorally and morphologically, with an emphasis on behaviors and structures recognized as sexually dimorphic. Behavioral measures include play behavior, exploratory behavior, saccharin preference, sex partner preference, and schedule-controlled operant behavior. Morphological assessments are based on the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA). This research addresses an urgent question arising from the human data. It will enable us to use the rat as a laboratory model for human phthalate exposures during fetal development, to carefully control exposure dose and timing, and to to amplify and extend the epidemiological findings by subjecting certain of their implications to laboratory investigation. Phthalate esters are chemicals used as plasticizers in personal care items such as shampoos, medical tubing, plastic toys, food packaging, and many other products. They impair male reproductive system development in animal models, and recent studies show similar effects in humans. Because phthalates act as anti-androgens, they have the potential to interfere with human brain and behavioral development. The proposed research will examine this question in rats, and translate its implications into human relevance. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Virtually all US inhabitants carry detectable levels of bisphenol-A (BPA) in their tissues. Its estrogenic properties have been recognized for decades. Although BPA had been viewed as weakly estrogenic in the past, it is now likely that exposure to low doses of BPA can produce disruptive effects in androgen and estrogen responsive tissues. Among the potential health consequences of BPA exposure of concern, interference with the normal processes of brain development is viewed as one of the most worrisome. These concerns arise because the developing nervous system is a key target of BPA;gonadal hormone levels mold brain organization, particularly sexual differentiation, during perinatal development. Aberrant brain development is the basis of aberrant behavioral function, whose effects extend across the entire lifespan, even into the realm of neurodegenerative disease. Despite the growing volume of reports on the neurobehavioral consequences of early developmental exposure to BPA, the literature remains fragmentary and somewhat inconsistent. Among the reasons are: data too limited to allow for characterization of dose-response relationships, the tendency to focus on isolated outcome(s), and, glaringly, the absence of definitive information on humans. This proposal confronts these limitations in several ways. (1) It coordinates, to the degree possible, human and animal behavioral indices. (2) It measures exposure biomarkers in both species (in urine, blood, or both). (3) It encompasses several behavioral endpoints, including cognitive function. (4) It provides mechanistic measures. (5) It includes a range of exposure levels from low to high. The project contains three components. In the human component a cohort of children whose mothers were enrolled in a large prospective study in 2000-2005 and provided samples of blood and urine during mid-pregnancy will be contacted again. Previously urinary phthalate metabolites were measured in a subset of these samples and significant associations were discovered with male genital development. A subset of these children were then recontacted at 4-7 years and examined for prenatal phthalate exposure in relation to mother's reports of play behavior, and significant associations (described in this application) were found. The children are now 5-9 years old and mothers will be asked to provide information on their children's cognitive skills, sex-linked play behaviors, social behaviors, and physical activity. These will be examined using stored urine and blood samples, in relation to conjugated and unconjugated BPA, as well as phthalate metabolites when available. In the animal behavior component we will expose rats will be exposed perinatally to BPA and a positive control (DES) and to measure aspects of cognitive function, play behavior, and activity patterns. Conjugated and unconjugated BPA will be determined in rodent serum. In the mechanistic component brain structures and functions especially relevant to sexually dimorphic indices will be assessed. To this end, perinatal exposure to BPA will be related to the volume of two sexually dimorphic brain regions, the anterior ventral periventricular nucleus (AVPV) and the sexually dimorphic nucleus of the preoptic area (SDN-POA) and associated neural markers. The proposed research addresses a question seen by both the public and by scientific review panels as crucial to weighing the risks posed by environmental exposure to BPA. Namely, what are its effects on brain development, and are these gender-specific? By focusing on endpoints recognized as sexually dimorphic, and which arise from sexual differentiation of the brain during early development, a coordinated base of information will ensue that should yield answers to that and related questions in a more definitive manner than has not been possible until now. Data produced will be novel and may not only indicate a new and important endpoint for BPA toxicity based on environmentally relevant levels, but should promote a field that is rather new to environmental health science: use of gender-specific neurodevelopmental endpoints to identify endocrine disruption. Demonstration of differing effects for a currently used (and novel) environmental chemical on male and female developmental endpoints may become a flag for the ability of that chemical to produce endocrine disruption. Moreover, follow-up studies beyond early development may show persistent effects later in life, including potentially aberrant social behaviors, impaired learning and school performance, impaired maternal and child-rearing behaviors and, later, enhanced risks of neurodegenerative disease. These issues are not confined to discussions within the scientific community. The public is an active participant. Pregnant women are especially anxious about exposing their babies to chemicals that may alter brain development. The comprehensive, coordinated body of information this project is designed to procure will provide the basis for decisions by regulatory authorities charged with protecting public health and by women concerned about the future health of their children. PUBLIC HEALTH RELEVANCE: This project will provide unique information on how exposure to bisphenol A (BPA) before birth can alter masculinization and feminization of the brain during prenatal development. Subsequent studies, beyond infancy and childhood, may show persistent effects later in life as well, including potentially aberrant social behaviors, impaired learning and school performance, impaired maternal and child-rearing behaviors and, later, enhanced risks of neurodegenerative disease.