Daniel W. Nebert - US grants

The long-term objective of our research is to understand the molecular mechanisms of cell death as a consequence of harmful environmental conditions. There appear to be several distinct circumstances under which cell death takes place in multicellular organisms. For example, apoptosis (programmed cell death) is known to play an important role in embryonic development, normal cell turnover, and hormone-dependent atrophy. Environmentally adverse chemicals can lead to cytotoxicity and, ultimately, cell death. Although apoptosis and cytotoxicity appear to occur for many reasons and respond to a variety of signals, it has become increasingly evident that there exist common steps in the pathways leading to cell death. Chemicals causing oxidative damage are metabolized by "Phase I" (oxygenation) and "Phase II" (conjugate) enzymes. We propose to use the aromatic hydrocarbon-responsive [Ah] gene battery as a model system for studying the cellular response of protection against oxidative stress and cell death. In the mouse, the [Ah] battery comprises at least six genes: two Phase I genes, Cypla1 and Cypla2; and four Phase II genes, Nmo-1, Aldh- 1, Ugt-1 and Gt-1. The radiation-induced deletion homozygote c14CoS/c14CoS mouse (albino phenotype) is missing about 1 centiMorgan of chromosome 7, dies during the first 18 h post partum, and exhibits marked activation of the Nmo-1 gene, increases in Aldh-1 mRNA, and elevated Ugt-1 and Gt-1 enzyme activities. Interestingly, in the 14CoS/14CoS fetus and newborn, three growth arrest- and DNA damage-inducible (gadd) genes are also activated. We postulate that the region of mouse chromosome 7 missing in the 14CoS/14CoS mouse contains a "master switch" gene, which we have designated Nmo-1n, encoding a trans-acting factors(s) that is a negative effector of the Nmo-1 gene. Under normal conditions, Nmo-1n suppresses an unknown number of genes. In response to environmental adversity such as oxidative stress, this gene releases its negative control on the [Ah] battery Phase II genes, and perhaps the gadd genes, thereby allowing all of these genes it become expressed. This response is independent of Phase I (Cypla1 and Cypla2) gene expression. Whether this region on chromosome 7 represents a single gene, Nmo-1a, controlling both the [Ah] battery Phase II genes and the gadd genes, or whether there are two or more gene controlling the [Ah] battery Phase II genes and the gadd genes, will require further work. Our first goal is to clone, sequence and characterize by functional analysis the murine Nmo-1 and Aldh-1 genes and regulatory regions. We then propose to use this information for cloning and characterizing the murine Nmo-1n gene. Knowledge about the Nmo-1n gene and its product will surely help clarify the functional relevance of common pathways involved in protection against oxidative damage and cell death.

A new Center on Environmental Genetics is proposed by the University of Cincinnati. The focus of this Center will be to investigate the impact of genetic diversity on the response of the individual, or populations, to toxic environmental agents. The Center will include a multidisciplinary approach, from "the molecule to the human," devoted to application of molecular biology and genetics to environmental research. The fundamental strategy of the Center is to understand variation in response to toxic agents due to the underlying interindividual differences in genetic predisposition. Basic research may include everything from microbes and lower eukaryotes to mammals and human material. Center research will take advantage of the usefulness of genetic variants, i.e. genetically different subpopulations, resistant versus sensitive groups, and/or interindividual and intraspecies differences. It is clear that interindividual genetic differences can lead to dramatic dissimilarities in the response to a wide variety of environmental substances. An appreciation of these differences, and an understanding of the underlying mechanisms, are critical in the evaluation of risk of adverse health effects caused by toxic environmental agents. Individual variation often reflects allelic differences in genes encoding proteins involved in critical life functions such as receptors, drug metabolism, ion channels, multidrug resistance glycoprotein pumps, second-messenger pathways, DNA repair, and chelation of metals. The elucidation of these functions that can influence interindividual response to toxic agents, and the evaluation of their genetic diversity in the population, will be the central focus of the Center. The identification of these underlying causes of genetic differences in response to toxic agents is an important key to understanding the basic mechanisms of toxicity and provides the basis for both preventing adverse health effects and exploring opportunities for interventions in the disease process. The four Research Cores of the new Center represent existing strengths and interactions among scientists within the University of Cincinnati Medical Center that are relevant to genetics. The central focus of the Ecogenetics Research Core is the identification and characterization of genetic polymorphisms that affect the metabolism, and therefore the toxicity, of foreign chemicals. The other Research Cores--Reproductive & Developmental Toxicology, Genetic Toxicology, and Oxidative Stress Toxicology--are focused on three specific areas of toxicology of major importance. These Research Cores investigate specific toxic effects of environmental agents with an emphasis on the use of genetic techniques. The role of environmental substances in causing infertility, in utero toxicity, and birth defects is an important public health issue. The impact of environmental agents on the structure of genetic material itself, leading to mutations and cancer, is an area of vital importance. Oxidative stress is an emerging area of importance which may underlie many diverse toxic effects of environmental agents in biological systems. This new Center will be unique among NIEHS Centers and will provide. an important. resource to the environmental research community.

The long-term objective of our research is to understand the molecular mechanisms of cell death as a consequence of harmful environmental conditions. There appear to be several distinct circumstances under which cell death takes place in multicellular organisms. For example, apoptosis (programmed cell death) is known to play an important role in embryonic development, normal cell turnover, and hormone-dependent atrophy. Environmentally adverse chemicals can lead to cytotoxicity and, ultimately, cell death. Although apoptosis and cytotoxicity appear to occur for many reasons and respond to a variety of signals, it has become increasingly evident that there exist common steps in the pathways leading to cell death. Chemicals causing oxidative damage are metabolized by "Phase I" (oxygenation) and "Phase II" (conjugate) enzymes. We propose to use the aromatic hydrocarbon-responsive [Ah] gene battery as a model system for studying the cellular response of protection against oxidative stress and cell death. In the mouse, the [Ah] battery comprises at least six genes: two Phase I genes, Cypla1 and Cypla2; and four Phase II genes, Nmo-1, Aldh- 1, Ugt-1 and Gt-1. The radiation-induced deletion homozygote c14CoS/c14CoS mouse (albino phenotype) is missing about 1 centiMorgan of chromosome 7, dies during the first 18 h post partum, and exhibits marked activation of the Nmo-1 gene, increases in Aldh-1 mRNA, and elevated Ugt-1 and Gt-1 enzyme activities. Interestingly, in the 14CoS/14CoS fetus and newborn, three growth arrest- and DNA damage-inducible (gadd) genes are also activated. We postulate that the region of mouse chromosome 7 missing in the 14CoS/14CoS mouse contains a "master switch" gene, which we have designated Nmo-1n, encoding a trans-acting factors(s) that is a negative effector of the Nmo-1 gene. Under normal conditions, Nmo-1n suppresses an unknown number of genes. In response to environmental adversity such as oxidative stress, this gene releases its negative control on the [Ah] battery Phase II genes, and perhaps the gadd genes, thereby allowing all of these genes it become expressed. This response is independent of Phase I (Cypla1 and Cypla2) gene expression. Whether this region on chromosome 7 represents a single gene, Nmo-1a, controlling both the [Ah] battery Phase II genes and the gadd genes, or whether there are two or more gene controlling the [Ah] battery Phase II genes and the gadd genes, will require further work. Our first goal is to clone, sequence and characterize by functional analysis the murine Nmo-1 and Aldh-1 genes and regulatory regions. We then propose to use this information for cloning and characterizing the murine Nmo-1n gene. Knowledge about the Nmo-1n gene and its product will surely help clarify the functional relevance of common pathways involved in protection against oxidative damage and cell death.

The long-term goal of this project is to understand the role that the CYP1A2 (cytochrome P3450) enzyme plays in genotoxicity caused by environmental pollutants in the intact mouse. The CYP1A2 enzyme participates primarily in the oxidative metabolism of arylamines but has also been shown to metabolize polycyclic hydrocarbons. This laboratory has previously found that the murine Cypla-2 gene is constitutively expressed at high levels only in liver, and highly induced only in liver, lung and duodenum following exposure to polycyclic aromatic compounds such as those found in cigarette smoke and other combustion products. To define the involvement of the CYP1A2 enzyme in genotoxicity of environmental chemicals, we propose to develop a transgenic mouse line lacking both alleles of the Cypla-2 gene. The methods used to generate a transgenic mouse having one copy of the inactivated gene in the germ line will include knocking out the Cypla-2 gene in embryonic stem (ES) cells with an homologous recombination construct; we shall also attempt a "double-knockout" in ES cell cultures. We shall then inject the targeted ES cells into the blastocoele cavity of embryos and transfer surviving blastocysts to a foster mother by uterine implantation. If necessary, two heterozygotes will be bred in order to generate mice homozygous for the disrupted Cypla-2 gene. In the CYP1A2-deficient mouse, we have chosen to examine the genotoxicity of two environmentally important chemicals that are CYP1A2 substrates: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and aflatoxin B1 (AFB1). Pharmacokinetics, DNA adduct formation, and mutagenesis will be examined as a function of the route of administration. These two chemicals were chosen because they are environmentally relevant and the CYP1A2 enzyme plays contrasting roles in their metabolism. NNK is one of the major tobacco smoke-specific nitrosamines that require CYP1A2 for metabolic activation. AFB1, a highly carcinogenic mycotoxin contaminant found in a variety of foods, is activated by some P450 enzymes; however, CYP1A2 appears to be involved in a detoxification pathway. Thus, one would expect that, in the CYP1A2-deficient mouse, NNK-induced genotoxicity int he lung would be decreased and AFB1 genotoxicity in the liver would be enhanced. We shall measure the pharmacokinetics of these two chemicals, using commercially available radiolabeled NNK and AFB1. DNA adduct formation in several tissues will be determined by the 32P- postlabeling assay. to measure tissue-specific mutagenesis, we shall breed the CYP1A2-deficient mouse with the Stratagene Big Blue (TM) indicator mouse to establish an inbred line; mutations in the lacI transgene will be quantitated by established methodologies. The studies proposed in this application should help in evaluating the relative risk for adverse health effects in humans exposed to certain environmental pollutants. This is particularly important, since recent studies have demonstrated a trimodal distribution and >60-fold interindividual variation in the constitutive expression of CYP1A2 in human liver.

Exposure to certain man-made and natural environmental agents poses a significant threat to human health. In order to enable us to establish rational policies to deal with this health issue, we need to expand our knowledge about the mechanisms by which toxic and carcinogenic environmental agents compromise human health. Toxicity and cancer caused by the nongenotoxic agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) and the ubiquitous environmental combustion product benzo[a]pyrene (BaP) appear to be mediated by way of the aromatic hydrocarbon receptor (AHR). Previous work from this and other laboratories has shown that (a) there exist inbred mouse strains having genes for high- and low-affinity AHRs;(b) mice with the high-affinity-AHR phenotype metabolize numerous environmental chemicals, to form reactive intermediates, 15-20 times faster than mice with the low-affinity-AHR phenotype; (c) DNA adduct formation, mutagenesis, oncogene activation, and certain types of cancer occur more frequently in mice with the fast- metabolism phenotype; and (d) approximately one-tenth of the human population has a fast-metabolism phenotype, and in this group the risk of certain types of cancer among smokers appears to be several-fold greater than that in the slow-metabolism group. The laboratory animal data for high vs low-affinity-AHR differences in toxicity and cancer are very convincing, whereas the human AHR-related differences in toxicity and cancer remain equivocal, largely due to the ethical difficulties in carrying out definitive experiments in humans. Here we propose to: (1) determine the nucleotide difference(s) in the human AHR gene responsible for human high and low-affinity-AHR phenotype among members in one 3- generation family; (2) make heterozygous and homozygous disruptions of the Ahr gene in murine embryonic stem (ES) cells; (3) replace the murine disrupted gene with the human low-affinity (AHR) gene in one cell line, and in another cell line with the human high-affinity (AHRA) gene; and (4) generate the two human transgenic mouse lines. These animals will be helpful in examining the role of the human high vs low-affinity AHR in toxicity and cancer-- on a common mouse background where every other gene is identical. This project goes substantially beyond what we and others have already done in this field. These mice will be invaluable for defining the precise role of the human Ah receptor in innumerable studies of toxicity and cancer caused by TCDD, BaP and other environmental chemicals. These mice should become important, valid dose- response models needed for a rational approach to human risk assessment.

A new Center on Environmental Genetics is proposed by the University of Cincinnati. The focus of this Center will be to investigate the impact of genetic diversity on the response of the individual, or populations, to toxic environmental agents. The Center will include a multidisciplinary approach, from "the molecule to the human," devoted to application of molecular biology and genetics to environmental research. The fundamental strategy of the Center is to understand variation in response to toxic agents due to the underlying interindividual differences in genetic predisposition. Basic research may include everything from microbes and lower eukaryotes to mammals and human material. Center research will take advantage of the usefulness of genetic variants, i.e. genetically different subpopulations, resistant versus sensitive groups, and/or interindividual and intraspecies differences. It is clear that interindividual genetic differences can lead to dramatic dissimilarities in the response to a wide variety of environmental substances. An appreciation of these differences, and an understanding of the underlying mechanisms, are critical in the evaluation of risk of adverse health effects caused by toxic environmental agents. Individual variation often reflects allelic differences in genes encoding proteins involved in critical life functions such as receptors, drug metabolism, ion channels, multidrug resistance glycoprotein pumps, second-messenger pathways, DNA repair, and chelation of metals. The elucidation of these functions that can influence interindividual response to toxic agents, and the evaluation of their genetic diversity in the population, will be the central focus of the Center. The identification of these underlying causes of genetic differences in response to toxic agents is an important key to understanding the basic mechanisms of toxicity and provides the basis for both preventing adverse health effects and exploring opportunities for interventions in the disease process. The four Research Cores of the new Center represent existing strengths and interactions among scientists within the University of Cincinnati Medical Center that are relevant to genetics. The central focus of the Ecogenetics Research Core is the identification and characterization of genetic polymorphisms that affect the metabolism, and therefore the toxicity, of foreign chemicals. The other Research Cores--Reproductive & Developmental Toxicology, Genetic Toxicology, and Oxidative Stress Toxicology--are focused on three specific areas of toxicology of major importance. These Research Cores investigate specific toxic effects of environmental agents with an emphasis on the use of genetic techniques. The role of environmental substances in causing infertility, in utero toxicity, and birth defects is an important public health issue. The impact of environmental agents on the structure of genetic material itself, leading to mutations and cancer, is an area of vital importance. Oxidative stress is an emerging area of importance which may underlie many diverse toxic effects of environmental agents in biological systems. This new Center will be unique among NIEHS Centers and will provide. an important. resource to the environmental research community.

This is a Shannon Award providing partial support for the research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon Award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. The long-range goal of this research project is to develop a biological monitor of aquatic environmental pollution. Specifically, we propose to produce lines of transgenic fish in which DNA response elements that respond to a wide variety of environmental pollutants are able to activate an easily assayable reporter gene. We have chosen the response elements of the CYP1A1 (cytochrome P1/450), NMO1 (NAD[P]H:menadione oxidoreductase; DT diaphorase; quinone reductase; azo dye reductase) and MT (metallothionein) genes. In the mammal, the CYP1A1 gene is known to be controlled by aromatic hydrocarbon response elements (AhREs) that respond to a wide variety of polycyclic hydrocarbons and halogenated planar molecules such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) and polychlorinated biphenyls. The mammalian NMO1 gene is known to respond to quinones and a wide variety of other oxidants (potent electrophiles) via an electrophile response element (EpRE). The mammalian (as well as trout) MT gene is known to be regulated by a metal response element (MRE) and respond to heavy metals such as mercury, copper, nickel, cadmium and zinc. For the 3 years of this grant application, we propose to: [1] determine the ability of AhRE, EpRE or MRE sequences from well- characterized mouse and trout polycyclic hydrocarbon-, oxidant-, and metal-responsive gene regulatory regions to modulate transcription of the luciferase (luc) gene in zebrafish cell cultures in response to these pollutants; and [2] generate transgenic zebrafish in which the luc gene is expressed, in order to define the limits of luminescence detection in living adult albino zebrafish. The final product of this project will be a sentinel for biological monitoring of environmental pollution capable of differentiating chemical classes within a complex contaminant mixture with an easily assayable reporter gene. This will provide an alternative model which uses a lower vertebrate for the accurate assessment of environmental hazards to human health.

The long-range goal of this research project is to study the relationship between oxidative stress and the [Ah] gene battery. It is becoming increasingly clear that we "age" because our genes undergo more and more damage by reactive oxygen metabolites (ROMs) as a function of time. When genes are damaged, several escape mechanisms"--including programmed cell death (apoptosis)--occur with increasing frequency. During the past 5 years, an oxidative stress signal transduction pathway has been defined, comprising more than 15 steps and initiated by ROMs by way of physical agents (ionizing and UV irradiation) as well as the metabolism of both endogenous and foreign chemicals. This laboratory has taken a genetic approach to study the role of oxidative stress in gene regulation and cell death. The 14CoS/14CoS mouse contains a 3,800-kb deletion on chromosome 7 and dies during the first 24 h post partum. We found that this mouse exhibits a constitutive oxidative stress response, in which expression of the NAD(P)H:menadione oxidoreductase (Nmol) and other [Ah] Phase II genes is increased. Recent work in other laboratories has shown that homozygous disruption of the fumarylacetoacetate hydrolase (Fah) gene--located in the 3,800-kb deleted region--completely mimics the 14CoS/14CoS mouse, due to ROMs generated by blockade of the tyrosine degradation pathway. Now that we understand more about the 14CoS/14CoS mouse, we can investigate directly the role of oxidative stress in aging through construction of transgenic mouse lines having defects in the control of redox homeostasis. In the next 5 years, we therefore propose to: [1] develop a conventional, as well as an inducible, knockout transgenic mouse having a homozygous disruption in the Fah gene, which will allow us to study ROM pharmacokinetics and cell type- and organ-specific responses of aging secondary to endogenous ROMs; [2] characterize in cells in culture, as well as in the intact animal, the mechanism(s) by which the intracellular levels of reduced glutathione (GSH) are regulated; and [3] develop a conventional, as well as an inducible, knockout transgenic mouse having a homozygous disruption in the gamma glutamylcysteine synthase (Gcs) gene. The GCS enzyme controls GSH production and thus affects at least two critical, distinct steps in the less than 15-step oxidative stress pathway. These studies will greatly enhance our understanding of the cellular responses, and consequences of the role of endogenous ROM-mediated oxidative stress, during the aging process.

DESCRIPTION (from Applicant's Abstract): The long-term goal of this project is to understand the role that SYP1A1 (cytochrome P1-450) plays in toxicity caused by environmental pollutants. Cyp1a1 is a member of the dioxin-inducible [ah] battery, the genes of which are up-regulated by the ah receptor (AHR). The CYP1A1 enzyme oxgenates halogenated and polycyclic aromatic hydrocarbons, e.g. polychlorniated biphenyls (PCBs) and benzo[a]pyrene(BaP). AHR ligands include dioxin (which is metabolised extremely slowly) and PCBs and BaP (which are metabolised more rapidly). The [Ah] battery plays a major role in toxicity of the skin, bone marrow, liver, eye, ovary, and immune system--as well as carcinogenesis. In the mouse the dosage and route of BaP administration are important determinants in target organ toxicity. Genetic differences in BaP toxicity depends upon the high-affinity C57BL/6-type (Ahr(b) allele) or the low-affinity DBA/2-type(Ahr(d) allele) of Ah receptor. Expression of mouse CYP1A1 mRNA is constitutively low absent, but is highly inducible in virtually every tissue and cell type in the body--following exposure to polycyclic aromatic chemicals. Toxicity can occur by either metabolism-dependent or receptor-dependent (metabolism- independent) mechanism. This laboratory has recently collaborated in making the Ahr(-/-) knockout mouse line. To investigate the mechanisms of CYP1A1 metabolism-mediated, vs. AHR-mediated toxicity caused by environmental chemicals, we therefore propose to: [1] develop a conventional, as well as an inducible, Cypla(-/-) knockout transgenic mouse line; [2] develop a (global, rather than tissue-specific) Cyp1a1(u/u) (ultra-expression) transgenic mouse line, and then generate, by breeding, the combined mouse lines Cyp1a1(-/-)Ahr(-/-), Cyp1a1 (-/-)Ahrd/d), Cyp1a1(-/-)Ahr(b/b), Cyp1a1(u/u)Ahr(-/-), Cyp1a1(u/u)Ahr(d/d), and Cyp1a1(u/u)Ahr(b/b). [3] study bone marrow toxicity and skin inflammation, following treatment of these mouse lines with oral BaP and with topical 7,12-dimethylbenzo[a]anthracene(DMBA), respectively, to determine which forms of environmental toxicity are dependent on CYP1A1 metabolism, and which forms of toxicity are dependent on the Ah receptor. These studies will greatly enhance our understanding of CYP1A1 metabolism-dependent, compared with AHR-dependent, toxicity caused by environmental pollutants. Because of conservation between human and mouse, and human polymorphisms in the CYP1A1 and AHR genes are known to exist, studies in these intact mice should help elucidate the mechanisms surrounding genetic differences in susceptibility to toxicity caused by substrates of CYP1A1, as well as ligands of the AHR.

This Core will focus on the role of genetic susceptibility in toxic responses to environmental agents. Its stated long-range goal is, "to identify and characterize genetic polymorphisms concerning differences in response to environmental agents - responses include tumorigenesis as well as acute and chronic toxicity." There are six specific aims stated for this Research Core: 1) to elucidate mechanisms of gene expression and regulation through genetic variants; 2) to screen populations and three- generation families for differences in toxic responses; 3) to determine associations between a genetic locus and a phenotypic trait; 4) to understand the molecular basis of a polymorphism; 5) to study the interaction of genes and agents during embryogenesis; and 6) to evaluate the impact of each polymorphism on human health.

DESCRIPTION: (Adapted from the Investigator's Abstract) The long-term goal of this project is the elucidation of cytochrome P450 CYP1A2's role in toxicity and cancer caused by environmentally hazardous chemicals. CYP1A2 substrates include environmentally important heterocyclic amines, nitrosamines, aflatoxins, nitrated polycyclic aromatic hydrocarbons, and arylamines such as 4-aminobiphenyl (ABP). The mouse CYP1A2 gene shows high basal activity only in the liver; is inducible in liver, lung, and GI tract following exposure to inducers such as dioxin and chemicals found in cigarette smoke; and is not expressed in blood cells, kidney or urinary bladder. The mouse CYP1A2 and human CYP1A2 are known to exhibit large differences (6- to 10-fold) in activity/substrate specificity. Acutely, ABP causes hemoglobin (Hb) adducts, methemoglobinemia, and liver toxicity. Chronically, ABP causes bladder ABP-DNA adducts and tumor formation as the consequence of hepatic CYP1A2 and N-acetyltransferase (NAT2) levels, as well as urinary pH. Our hypothesis is that ABP toxicity and cancer will be correlated with hepatic CYP1A2 and low NAT2 activities. To define the role of CYP1A2 in toxicity and cancer, we have generated during the first 4.25 years of this grant a viable, fertile CYP1A2(-/-) genotype with other relevant genotypes: the Stratagene Big Blue indicator and marked differences in Ah receptor affinity [Ahr(b1/b1), Ahr(d/d) or Ahr(-/-)] and rapid vs slow N-acetylation [Nat(b/b), Nat(a/a)]. We are now ready to use these mice to answer questions relevant to human environmental diseases. Comparing Cyp1a2(-/-) with Cyp1a2(+/+) wild-type and the above genotypes, we predict the Cyp1a2(+/+), Nat(a/a) mouse will show more ABP acute toxicity and more DNA adducts/mutations/tumors in the bladder, and dioxin-treated Ahr(b1/b1) will show even higher toxicity/cancer. We also believe that human-mouse differences in ABP metabolism by CYP1A2 are very important. Thus, for the next funding period we will: (1) measure acute toxicity (red cell; liver), ABP-DNA adducts, mutagenesis and tumor genesis (liver, bladder, and kidney)--comparing Cyp1a2(-/-) vs (+/+), and Ahr(b1/b1) vs (d/d) or (-/-), and Nat(b/b) vs (a/a)--following topical ABP treatment, as low vs high urine pH, and with or without dioxin pretreatment; (2) generate an hCYP1A2 mouse line by targeted replacement of the mouse Cyp1a2 gene with the human CYP1A2 gene as a single copy under 5' flanking regulatory controls of the mouse Cyp1A2 gene; and (3) examine in the "humanized" hCYP1A2 mouse line the parameters as listed in Specific Aim 1. The studies proposed in the applications should help in evaluating the relative risk for adverse health effects in humans exposed to a known human bladder carcinogen that is present in cigarette smoke. This is particularly important, since studies have shown >60-fold interindividual variation in the basal levels of human liver CYP1A2, >30-fold variation in AHR affinity, and slow acetylators comprise about 50 percent of the U.S. population.

The long-term goal of this project is to identify and characterize the gene(s) responsible for interindividual differences in response to cadmium (Cd++). Cadmium is a widespread environmental pollutant and is classifed as an IARC "Category I" human carcinogen. Cadmium can also cause severe renal toxicity and cardiovascular disease. Genetic differences in susceptibility to cadmium toxicity have been suggested in humans, whereas in inbred mice there are unequivocal genetic data. Resistance to cadmium-induced testicular damage was reported in 1973 to be associated with a single recessive gene, named Cdm, found on mouse chromosome (Chr) 3. With the help of recent advances in the Mouse Genome Project and studying semiquantitative histological parameters, we have now corroborated the original 1973 data and, using polymorphic microsatellite markers, have refined the chromosomal location of the Cdm gene from more than 24 centiMorgans (cM) to 0.64 cM (estimated 40-80 genes). We have used recombinant inbred lines generated from C57BL/6J (B6, resistant) and DBA/2J (D2, sensitive) inbred mice to determine that the Cdm gene maps between microsatellite markers D3Mit110 and D3Mit255. There is strong evidence that Cd++ exerts its toxic effects by causing cell type-specific oxidative stress which can result in the covalent modification of cellular macromolecules or disruption of the cell cycle-leading to enhanced cell division, apoptosis or growth arrest. We will locate and identify the Cdm gene, and our hypothesis is that the Cdm gene encodes a protein in specific cell types that plays an important role in cadmium-induced disruption of cellular redox homeostasis. Thus, we propose to [1] narrow the Cdm-gene-containing region on Chr 3, by identifying single nucleotide polymorphisms (SNPs) between B6 and D2 mice, and then determining the genotype of these new markers in the BXD14/Ty mouse ; [2] sequence portions of the Cdm-gene- containing region and identify a BAC clone that will confer testicular sensitivity to Cd++ on a resistant B6 mouse background; and [3] identify the Cdm gene from the genomic sequences produced in Spec.Aims number 1 and number 2. Although toxicity to numerous heavy metals is well known, molecular mechanisms have yet to be uncovered - in humans or laboratory mammals. These studies will enhance our understanding of heavy metal toxicity by identifying and characterizing, for the first time, a major gene responsible for susceptibility to cadmium-induced toxicity.

[unreadable] DESCRIPTION (provided by applicant): In our ES08147 grant, the goals are to: [1] study the toxicity of benzo[a]pyrene (BaP) vs. dioxin in various, tissues (bone marrow, liver, developing embryo) of the Cyp1a1(-/-) knockout and Cyp1a1(+/+) wild-type mouse as a function of dose of the environmental pollutant and route of administration, and [2] create mouse lines having exclusively mitochondrial CYP1A 1 (mt1A1) vs. exclusively microsomal CYP1A1 (mc1A1) to study such toxicity further. Preliminary data from Specific Aim #1 studies, however, have convinced us that we should supplement our ES08147 research with double and triple knockouts of the Cyp1genes as soon as possible. Previous results with cell culture or in vitro studies differ from that in the intact animal. For example, we found that Cyp1a1(-/-) mice are strikingly sensitized to oral BaP-induced immunotoxicity. Also, Cyp1a1(-/-) showed only modest protection against BaP-induced liver damage, compared with Cyp1a1(+/+) mice, whereas BaP-DNA adducts were unexpectedly >4-fold higher in Cyp1a1(-/-) mice. It would appear that the biological functions of the dioxin-inducible CYP1 enzymes are more redundant than we had imagined, with overlapping substrate specificities of all three enzymes, plus unique tissue- and cell type-specific locations of the three enzymes, thereby making it extremely difficult to assess the role of CYP1A1 in environmental toxicity using only the Cyp1a1(-/-) mouse. Thus, the function of CYP1A1 in environmental toxicity is best studied with and without the presence of CYP1A2 and/or CYP1B1. Conventional and Cre-inducible knockout Cyp1 mice provide the systems to tease apart the contribution of each CYP1 enzyme that contributes to cell type-specific toxicity. Therefore, concomitantly with the ES08147 grant, we believe it is imperative that we also: [1] generate conventional and inducible Cyp1a1/1a2(-/-) and Cyp1a1/1bl(-/-) double-knockout lines and the Cyp1a1/1a2/1b1(-/-) triple-knockout mouse lines; and [2] assess BaP- vs dioxin-induced toxicity of the marrow, liver and developing embryo in these double- and triple-knockout lines. Our original aim to generate the mt1A1 and mc1A1 mice and then study BaP vs dioxin toxicity in these lines remains unchanged.

[unreadable] DESCRIPTION (provided by applicant): Cytochromes P450 1B1, 1A1 & 1A2 (CYP1B1, CYP1A1, and CYP1A2) are responsible for both detoxifying and metabolically activating innumerable polycyclic aromatic hydrocarbons (PAHs), nitrosamines, and A/-heterocyclics present in combustion processes including cigarette smoke. The PAH- inducible CYP1B1/1A1/1A2 genes are up-regulated by the aromatic hydrocarbon receptor (AHR). Many in vitro, cell culture and animal studies have shown that high CYP1 enzyme levels and AHR high-affinity are correlated with increased genotoxicity caused by PAHs; recent studies with knockout mice, however, show that, whereas high CYP1B1 activity causes metabolic activation, CYP1A1 and CYP1A2 are far more important in detoxication than metabolic activation. Data in humans have been inconclusive, perhaps because many are assuming that all three CYP1 enzymes cause only higher cancer risk. We have a large cohort of head-and-neck squamous-cell carcinoma (HNSCC) patients with a history of one to 40 cigarette pack-years ("highly sensitive," HS) and heavy smokers with >80 pack-years having no cancer ("highly resistant," HR)?strongly suggesting a genetic component. These two extremes will be studied, using the extreme discordant phenotype (EDP) approach. A systematic search (SNP-discovery followed by SNP- typing) for haplotypes of the human,CYP1B1 and AHR genes is now possible; we have completed such a study of the CYP1A1_1A2 locus (39.6 kb) and discovered 85 SNPs, 49 of which were not yet in any database. Our hypothesis is: Specific haplotypes involving one or more of these four genes leading to high CYP1B1 and low CYP1A1/1A2 activities are associated with increased risk of HNSCC cancer in smokers. In the 3 years of this proposed project, we therefore will: [a] carry out whatever SNP-discovery and SNP-typing that still needs to be done, from six major geographically-isolated subgroups; we will collect blood and prepare DMA from 200 HS HNSCC patients and 200 HR non-cancer heavy smokers in our cohort; and [b] examine the association between haplotypes and risk of HNSCC by performing SNP-typing of the CYP1B1, CYP1A1, CYP1A2 and AHR genes in the 200 HS versus the 200 HR individuals. Establishing important phenotype-genotype associations between HNSCC and these four genes would provide the first unequivocal data that humans are similar to laboratory animals regarding cancer and one or more of these genes. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this project is to identify and characterize the gene(s) responsible for interindividual differences in response to cadmium (Cd). Environmental Cd is absorbed by the small intestine and lung, is bound to metallothionein and glutathione in hepatocytes, and is deposited in the kidney. The kidney is the major target organ for human and lab animal Cd exposure. During the current funding period, we identified the Slc39a8 gene as the Cdm locus responsible for Cd-induced testicular necrosis. Slc39a8 encodes the ZIPS transporter protein, which we show is a high-affinity apically-oriented rogue Cd/HCO3- cotransporter. ZIPS expression in culture increases Cd influx and sensitizes (>30-fold) the cells to Cd. ZIPS is expressed in a cell-type-specific manner and is highest in alveolar and tubular epithelial cells in the lung and kidney, respect- tively. A BAC-transgenic (BTZIP8) mouse line containing 2 + 1 additional copies of the Slc39a8 gene shows a gene-dose-dependent increase in ZIPS mRNA in all tissues tested, and is acutely sensitive to Cd-induced kidney dysfunction. Slc39a8 is one of 14 members of a solute-carrier (SLC) family of metal transporters; Slc39a14 shares the most recent ancestry with Slc39a8; ZIP14 exists as two peptides, ZIP14A and ZIP14B, as a consequence of alternate exon splicing. The ZIP14s are expressed highest in small intestine and liver and have transport and toxicologic properties similar to ZIPS. Our hypothesis is that the ZIPS and ZIP14 transporters are the principal mediators of Cd-induced renal dysfunction. For the next funding period, we propose to: [1] characterize the transport and physiological properties of ZIPS, ZIP14A and ZIP14B in vitro; [2] study the physiological role of ZIPS in Cd-induced renal dysfunction using our BTZIP8 overexpresser and Slc39a8(-/-) knockout mouse models; and [3] develop and characterize similar mouse models for Slc39a14 These studies will improve our understanding of heavy-metal toxicity and may lead to uncovering new targets that might be useful for preventive strategies as well as therapeutic intervention in heavy-metal diseases. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Polycyclic aromatic hydrocarbons (PAHs), nitrosamines, and N-heterocyclics are present in combustion products, e.g. grilled foods & cigarette smoke. Cytochromes P450 1A1 & 1B1 (CYP1A1, CYP1B1) are responsible for the metabolism of numerous PAHs, the prototype of which is benzo[a]pyrene (BaP). CYP1A2 is responsible for metabolizing nitrosamines and N-heterocyclics, but also PAHs (in particular, BaP) to a lesser extent. Using Cyp1a1(-/-) and Cyp1b1(-/-) knockout mice, we have shown that CYP1A1 is more important in detoxication than metabolic activation, whereas CYP1B1 causes metabolic activation of BaP to unwanted reactive intermediates. In other words, CYP1A1 is more good than bad in the intact mouse ingesting BaP, and CYP1B1 is more bad than good in the intact mouse administered PAHs by various routes. The importance of mesenteric lymphatics vs. the portal system (mesenteric blood vessels, liver, bile) is not known for oral BaP. This lab now has seven-all three single, all three double, and the one triple-Cyp1 knockout mouse lines. Our hypothesis is: lymph BaP uptake and CYP1B1 in distal tissues (e.g. immune cells, spleen, and bone marrow) are the principal causes of oral BaP toxicity, whereas inducible CYP1A1 in liver and intestine is the principal cause of BaP detoxication. In this proposed project, we therefore will: [a] identify and determine the amounts of metabolites vs. unchanged parent BaP in mesenteric lymph, portal vein blood, liver, and bile in wild-type and all seven Cyp1 knockout mouse lines, and the role and mechanism of chylomicrons in delivering BaP to target organs; [b] generate liver- and intestinal epithelium-specific Cyp1a1 conditional knockout lines; [c] replace the Cyp1b1 gene (in the genome) with the Cyp1a1 gene, and vice versa; and [d] repeat our BaP pharmacokinetics studies (see [a]) in these four newly generated mouse lines. Understanding the tissue- specific roles for each of the three CYP1 enzymes in the intact mouse receiving oral BaP will provide us with a greater understanding of BaP detoxification vs. metabolic activation. We expect this knowledge will provide a blueprint for understanding the mechanisms of elimination vs. dissemination of ingested BaP and will be informative in clinical studies in which we would determine which haplotypes of these three human genes might be associated with resistance vs. sensitivity to PAH-induced toxicity and cancer. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Cytochromes P450 1B1, 1A1 & 1A2 (CYP1B1, CYP1A1, and CYP1A2) are responsible for both detoxifying and metabolically activating innumerable polycyclic aromatic hydrocarbons (PAHs), nitrosamines, and A/-heterocyclics present in combustion processes including cigarette smoke. The PAH- inducible CYP1B1/1A1/1A2 genes are up-regulated by the aromatic hydrocarbon receptor (AHR). Many in vitro, cell culture and animal studies have shown that high CYP1 enzyme levels and AHR high-affinity are correlated with increased genotoxicity caused by PAHs; recent studies with knockout mice, however, show that, whereas high CYP1B1 activity causes metabolic activation, CYP1A1 and CYP1A2 are far more important in detoxication than metabolic activation. Data in humans have been inconclusive, perhaps because many are assuming that all three CYP1 enzymes cause only higher cancer risk. We have a large cohort of head-and-neck squamous-cell carcinoma (HNSCC) patients with a history of one to 40 cigarette pack-years ("highly sensitive," HS) and heavy smokers with >80 pack-years having no cancer ("highly resistant," HR)?strongly suggesting a genetic component. These two extremes will be studied, using the extreme discordant phenotype (EDP) approach. A systematic search (SNP-discovery followed by SNP- typing) for haplotypes of the human,CYP1B1 and AHR genes is now possible; we have completed such a study of the CYP1A1_1A2 locus (39.6 kb) and discovered 85 SNPs, 49 of which were not yet in any database. Our hypothesis is: Specific haplotypes involving one or more of these four genes leading to high CYP1B1 and low CYP1A1/1A2 activities are associated with increased risk of HNSCC cancer in smokers. In the 3 years of this proposed project, we therefore will: [a] carry out whatever SNP-discovery and SNP-typing that still needs to be done, from six major geographically-isolated subgroups; we will collect blood and prepare DMA from 200 HS HNSCC patients and 200 HR non-cancer heavy smokers in our cohort; and [b] examine the association between haplotypes and risk of HNSCC by performing SNP-typing of the CYP1B1, CYP1A1, CYP1A2 and AHR genes in the 200 HS versus the 200 HR individuals. Establishing important phenotype-genotype associations between HNSCC and these four genes would provide the first unequivocal data that humans are similar to laboratory animals regarding cancer and one or more of these genes. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Environmental exposures to polychlorinated biphenyls (PCBs) are known in humans as well as lab animals to cause immunosuppression, thyroid disease, endocrine disruption, and damage to the central nervous system. Not all humans or laboratory animals respond similarly to the same dose -indicating interindividual genetic differences. In rodents to elicit these pathologies, planar PCBs must bind to, and activate, the aryl hydrocarbon receptor (AHR). Despite this overriding role for planar-PCB-mediated AHR activation in toxicity, the AHR up-regulates CYP1A2, which in liver sequesters and protects distant tissues against planar PCBs. Both the AHR and CYP1A2 are polymorphic in humans: the AHR exhibits >12-fold differences in ligand-binding affinity; liver basal CYP1A2 shows >60-fold differences in subjects having no known exposure to inducers. With regard to fetal exposure, our studies in mice demonstrate that risk of birth defects by planar TCDD depends on the high-affinity AHR and is also greatly increased in fetuses carried by dams that lack CYP1A2. PCBs represent mixtures having many dozens of different congeners; which congener is toxic, and the rates of uptake, metabolism and excretion are difficult to determine in humans, and most studies in lab animals look at a single congener. We have studied mice with the high- (Ahrb, B6) vs low- (B6.D2-Ahrd) affinity AHR, and with or without the Cyp1a2 gene. Using these mice, we hypothesize that Ahrb fetuses carried by Cyp1a2(-/-) dams will be most susceptible, and Ahrd fetuses carried by Cyp1a2(+/+) dams most resistant, to deficits in learning, memory, and other behaviors caused by planar PCBs. For the funding period, we propose to: [1] determine tissue distribution of each of eight PCB congeners (most relevant to humans) given as a mixture -comparing B6 vs B6.D2-Ahrd, and Cyp1a2(+/+) vs Cyp1a2(-/-) dams and their offspring; and [2] evaluate the in utero and lactational effects of this orally administered PCB mixture on learning, memory, and other behaviors in offspring of these treated dams, starting at postnatal day 60. These studies will define the impact of a fetal basis for adult disease. The Ahr and Cyp1a2 genotypes in these mice represent the extremes for variability of these two genes in the human population. There exist genetic differences in mouse (and human) populations, which represent a gradient of at-risk individuals. Project Narrative: Polychlorinated biphenyls (PCBs) are widespread persistent organic pollutants linked to numerous human health problems, including learning and memory deficits in children of exposed mothers. The Ahr and Cyp1a2 genotypes in our mouse models represent the extremes for variability in these two genes in the human population, and both genes likely play a role in susceptibility following PCB exposure. These studies will define the fetal basis for adult disease and help to identify individuals at greatest risk of PCB-induced neurotoxicity. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant) The goal of the Gene-Environment Interactions Training Program (GEITP) is to train pre-doctoral and post-doctoral students who will be versed in ways that both environment exposure and genetics diversity interact to alter the onset of disease. Achieving this objective requires an interdisciplinary team approach integrating an understanding of genetic diversity, epigenetic alterations, high-throughput genomics, biostatistics, biomarkers, and exposure assessment approaches. The collaborative efforts of research faculty, clinicians, postdoctoral and pre-doctoral trainees are needed in order to meet this objective. A mentorship team approach, combining the expertise of well-regarded scientists in the areas of exposure assessment, genetic variability, biomarkers of disease, and individual/tailored medicine will be used to educate trainees in multiple areas of gene-environment interactions. Pre-doctoral training will include required coursework in addition to the student's matriculated Ph.D. program, laboratory rotations with the team of mentors, and hands-on work in several areas of exposure assessment, high-throughput genetic variability measures, and biomarkers or exposure and/or disease. Postdoctoral training will include programs in laboratory and personnel management, pilot grant applications, and an intensive year long grant writing workshop to prepare them for independent research. All trainees will be required to attend "Technologies in genomics, exposure, a biomarkers" workshop, which will be created as part of this program, and present research results at an annual GEITP Student Symposium. This program will include a dedicated governance structure to assure that appropriate trainees are recruited, education goals are met, and the aims of this grant are achieved.

The Pilot Projects Program (PPP) is one of the most important components of the CEG. The primary objective of the PPP is to provide seed support for new initiatives in basic, translational, and clinical research that will shed new light on the interaction between genes and the environment in which they operate. The goal of the program is to allow investigators, either established or new to the field, to obtain significant preliminary data that can become the basis of a new training grant (K-series) or R01-type grant proposals (or their equivalent) and associated publications. The program is also viewed by CEG as an important vehicle to help junior investigators in their career development (see Section 4. Career Development Program or CDP, pages 624-634). The PPP has been enormously successful in the past and during the last funding cycle [see F &G below, and Tables D2 (pages 612-617), E1 and E2]. Success of the PPP will continue to be measured by the following matrices: a) recruitment of new investigators at all levels to this area of research, b) subsequent attainment of independence among funded junior investigators, and c) funding of additional investigator-initiated grants, and d) resulting publications with high impact in the field. Special attention is given to whether the funded projects are integrated into the collaborative and integrative nature of the CEG, their creativity in utilizing technologies offered by the facilities and services cores, and their potentials in clinical translation. An important element of the PPP is to support synergistic, innovative, high-risk/high-reward research with a multidisciplinary foundation. The funding mechanisms for FY17-21 will be closely aligned to the mission of the CEG (see Section 1, page 444) and the two-track training model of the CDP (see, Section 4, page 624). Specifically, the PPP will introduce four new award mechanisms (see Figure 1): 1) mentee-mentor partnership awards, 2) new-to-EHS investigator awards, 3) Innovator awards for established investigators to pursue a new direction or acquire/utilize a new technology, and 4) affinity-group awards that aim at building functional multidisciplinary group research. The PPP will seek to fund projects that pioneer leading-edge directions and future leaders in the forefront of research in geneenvironment interactions. Given the completion of the Human Genome Project, the PPP is especially interested in funding studies on human populations or diseases. All PPP recipients will be fully supported by three state-of-the-art, highly integrated facilities and services cores: the Integrative Technologies Support Core, the Integrative Health Services Core, and the Bioinformatics Core.