George Bailey - US grants

The overall aim is to enhance our understanding of the mechanisms of anti-carcinogenesis by synthetic and naturally occurring compounds, and their possible impact on human cancer. This becomes especially important in view of the results from our laboratory and others that some putative food-borne inhibitors in the human diet actually enhance tumor response experimentally. Much of the work will use a component of brassica vegetables, indole-3-carbinol (I3C), as a model for blocking agents which can also promote. We will use the rainbow trout, which has proven responsive to a number of carcinogens and inhibitors, and is especially valuable for inhibitor studies for three reasons: a) Mechanism studies in this non-mammalian vertebrate broadens our data base for extrapolation to man; b) Its economies and sensitivity permit a great range of tumor studies for investigating fundamental concepts of anti-carcinogenesis at modest cost. Many of the proposed studies would be economically unrealistic, or unfeasible for rare metabolites, in traditional rodent models. c) Use of alternative vertebrates where sound and feasible reduces use of mammalian test animals. Specific aims are: 1) Conduct tumor studies using aflatoxin B1 (AFB1) and two selected inhibitors, to rigorously establish the relationships between applied carcinogen dose, inhibitors, to rigorously establish the relationships between applied carcinogen dose, inhibitor dose, peak target tissue DNA adduct formation, and final tumor response in trout. 2) Investigate and quantitate changes in responsiveness to tumor inhibitors with increasing age, and the role of altered carcinogen pharmacokinetics, metabolism, and DNA adduct formation in this process. 3) Extend previous investigations into the detailed mechanisms of 13C inhibition of AFB1 carcinogenesis. Synthesize radiolabeled 13C and investigate its metabolism in vivo and in isolated cells. Where possible test metabolite(s) for ability to alter carcinogen metabolism and inhibit DNA adduct formation. Determine I3C effects on specific trout hepatic and intestinal phase III detoxifying activites. 4) Investigate mechanisms of promotion by 13C and quantitatively assess its potencies for promotion versus inhibition. Conduct structure/function tumor studies for promotion or inhibition by 13C analogues. Determine 13C effects on target cell proliferation, ornithine decarboxylase activity, intercellular communication, and/or DNA damage. 5) Expand the data base of inhibitor/carcinogen testing in the trout model, each with specific rationale under test, using protocols which detect promotion or inhibition. Initiate mechanism studies where inhibition or promotion occur.

Aflatoxin B1 (AFB1) is a potent toxin, mutagen, and one of only 30 substances described as a human carcinogen. The long term objective is to understand the mechanisms of carcinogenicity of this and related compounds so that rational approaches can be made toward elimination of their effects, if not their exposure, on human and animal health. The rainbow trout model has proven a useful comparative species with extremely high sensitivities which permit tumor studies of small quantities of scarce aflatoxins. The studies in this proposal will not only improve our understanding of aflatoxin toxicity, but also will help illucidate mechanisms underlying the high sensitivity of this species, and thus more clearly define the limits of its usefulness for studies related to human and environmental health. This proposal aims to describe the carcinogenicity of AFB1 and its major metabolites in trout (AFL, AFM1, AFL-M1) in terms of their cellular metabolism, detoxication, DNA adduct formation and persistence, and molecular details of epoxide-DNA interaction. In specific aim 1, chemical and enzymatic syntheses will be used to produce tritiated and untritiated aflatoxins from available precursors. These 4 aflatoxins will be chemically activated with chloroperoxybenzoic acid to their 8,9-epoxides, reacted with DNA, and their major adducts described by HPLC behavior, UV spectroscopy and mass spectral analysis. These standards will be used to determine the type of adducts formed by each aflatoxin biologically. Each aflatoxin will be incubated in isolated hepatocytes, and its relative rates of production of DNA adducts, specific metabolites, and conjugates determined. The persistence of DNA adducts from each aflatoxin will be examined in vivo and in vitro. Each aflatoxin will be reacted with a 32p-end labeled sequenced 260bp DNA fragment, cleaved where adducts form, and the nucleotide specificity and neighboring sequence effects compared on DNA sequencing gels. The effects of varying aflatoxin-epoxide structure on intercalation and reactivity with DNA will be investigated. In specific aim 2 the relative carcinogenicities and covalent binding indices of all 4 aflatoxins will be determined by embryo exposure. Very large sample sizes will provide precise tumor and DNA-binding dose response curves for AFB1 and AFL, by embryo exposure and juvenile feeding protocols. These studies will provide a comprehensive molecular description of the metabolism and initiation characteristics of AFB1 and its metabolites in trout.

chemical carcinogenesis;

The focus of this proposal is the application and continued development of the rainbow trout model as an alternative vertebrate species for investigating genotoxicity, especially carcinogenesis and tumor modulation, by environmental agents an factors. The aim is to exploit and develop aspect of the trout model, such as nanogram carcinogen sensitivity, which make it a valuable additional system for bioassay of carcinogens and modulating agents, and an important non-mammalian species for comparative mechanism studies. The proposed projects will provide detailed mechanistic information which should allow results to be related clearly to mammalian carcinogenesis. The six projects which address this common theme are summarized as follows: 1) Basic model improvement (submicogram bioassay of initiators, anti-carcinogens, promoters; creation of totally homozygous cloned lines of trout; tumor data base expansion); 2) Characterization of xenobiotic metabolizing enzymes and their developmental regulation in trout; 3) Characterization on non-P450 co-oxidative mechanisms for procarcinogen activation and carcinogenesis in trout; 4) Investigation of temperature effects on xenobiotic pharmacodynamics, metabolism, and carcinogenesis in this poikilothermic model; 5) Exploitation of the trout embryo microinjection model to explore quantitative relationships among related aflatoxins in relative carcinogenicities, overall DNA binding and persistence, chemistry of adducts formed, and sequence-related preferences for site-specific oncogene adduction and transformation; 6) Investigation of the effects of chromatin structure on gene target site-specific aflatoxin susceptibility, using homogeneous nucleosomes in vitro and c-ras genes in vivo.

chemical carcinogenesis;

biomedical equipment purchase;

saltwater environment; fresh water environment; chemical carcinogenesis; trout /salmon;

The aim of this Center is to facilitate investigator, graduate student, and postdoctoral research activities that develop and apply unique features of trout and other fish models to problems of biomedical concern. Our focus is on carcinogenesis, immunotoxicity, neurotoxicity, and their modulation by dietary and environmental factors. Center Investigators bring together multidisciplinary expertise in tumor histopathology, statistics, genetics, enzymology, protein chemistry, cell biology, neurobiology, immunology, pharmacology, biophysics, and molecular biology. Though emphasis will remain on basic mechanistic research, our findings have had clinical application at the national level and environmental application at the regional and local levels.

This proposal investigates mechanisms of genotoxicity in rainbow trout as a central theme, with a primary emphasis on the genetics, molecular genetics, and biochemistry of carcinogenesis and tumor promotion. Many attributes justify use of this aquatic model in environmental health research. Trout reduce dependence on mammals for cancer research, provide comparative mechanism information, show high sensitivity (nanogram tumor response), have such low cost that many thousands of animals per tumor study are reasonable, have zero and near-zero background incidence, and are readily manipulated to produce clones or polyploids. An emphasis on mechanisms will broaden our comparative base for extrapolating cancer studies from surrogates to humans, while also increasing understanding of tumor development in feral fish species. The four interactive projects within this theme are: 1. Investigate the molecular basis for tumor initiation and development in trout, including: mechanisms that determine the incidence, site-specific generation and selective propagation of Ki-ras p21 allelomorphs; the influence of carcinogen dose, proliferative regeneration, DNA adduction, and repair capacity on the incidence of Ki-ras activation, down to 10-3 tumor risk (ED0.1% tumor study); and the influence of triploid as a selective environment for oncogenesis involving dominant p53 point mutations. 2. Examine the genetic control of variation in tumor response in outbred, clonal, and triploid populations of trout. Utilize ploidy manipulation to assess tumor suppressor gene involvement in trout carcinogenesis, and establish the use of RFLP markers and known suppressor gene probes to investigate deletional events in diploid and triploid populations of isogenic trout. Initiate development of a genetic map of DNA markers for rainbow trout. 3. Establish the metabolic capacity of early trout life stages for procarcinogen bioactivation and alkylation repair. Clone, sequence, and establish the expression of three new trout cytochrome P450 genes. Establish the time- and tissue-dependent expression of these and two additional trout P450s, their inductive response to selected environmental agents, and their potential to bioactivate selected procarcinogens. Establish the levels, developmental expression, and tissue distribution of 06- alkyltransferase. 4. For clinical application, establish the mechanisms through which the naturally occurring compound indole-3-carbinol, a putative "anticarcinogen" currently proposed for human intervention trials, also behaves as a tumor promoter. Investigate if the promotional mechanism is mediated in trout and mice through Ah receptor agonism. Rigorously quantify its dose-responsive promotional potency and the potential presence of a threshold dose for promotion, in order to assess promotional risk vs. chemopreventive benefit.

The rainbow trout is a useful lower vertebrate model for comparative issues in human environmental carcinogenesis and an appropriate surrogate to investigate the molecular basis for tumor development by feral fish populations in contaminated waterways. However, the genetic events accompanying cancer development in aquatic species are not well understood. The focus of this proposal is to characterize the molecular and cellular basis for Ki-Ras proto-oncogene activation in trout neoplasia, and its interactions with carcinogen dose, DNA adduct levels, cell proliferation and target organ toxicity down to 0.1% tumor incidence, an order of magnitude lower than any previous animal studies. We also propose a limited examination of p53 suppressor gene dominant mutation during tumor development in polyploid animals, studies not feasible with mammalian models. Specific Aims are: 1. Establish the fundamental parameters that govern the frequency, and types, of mutant Ki-ras alleles among various trout tumor phenotypes. Examine the hypothesis that the balance of ras and non-ras tumorigenesis pathways is altered by carcinogen doses (see Aim 3) or tumor promoters that induce regenerative proliferations. 2. Examine the hypothesis that selective differences in dysfunctional fitness of mutant ras p21 proteins during clonal expansion and progression, not solely site-specific rates of carcinogen-DNA adduction and mutagenesis during initiation, determine the eventual spectrum of Ki-ras mutant alleles detected in end-stage tumors. 3. Establish molecular dosimetry relationships between Ki-ras activation and carcinogen dose, DNA adduction, cell proliferation and tumor response in two ED0.1% tumor studies - By appropriate mechanistic studies explore four hypotheses: 1) that any non-linearity is explained by dose-dependent toxicity, DNA adduction, or persistence; 2) that Ki- ras oncogenesis decreases with increasing carcinogen-induced toxicity and proliferative regeneration; 3) that DMBA tumorigenesis in vivo may be driven primarily by unstable adducts from one electron oxidation, rather than stable adducts; 4) that DEN dosimetry and Ki-ras mutant allele spectrum is dependent on host excision repair proficiency, not solely alkyltransferase repair. 4. Examine the hypothesis that increased gene dosage for suppressor genes accounts for the greatly increased resistance to development of most tumor phenotypes in triploid trout. Specifically, we hypothesize that those few tumors that do develop in triploids have developed in an environment selective for dominantly transforming p53 point mutations, and thus will show a much greater prevalence of mutant p53 than will tumor from diploid siblings.

The aim of this Center is to facilitate investigator, graduate student, and postdoctoral research activities that develop and apply unique features of trout and other fish models to problems of biomedical concern. Our focus is on carcinogenesis, immunotoxicity, neurotoxicity, and their modulation by dietary and environmental factors. Center Investigators bring together multidisciplinary expertise in tumor histopathology, statistics, genetics, enzymology, protein chemistry, cell biology, neurobiology, immunology, pharmacology, biophysics, and molecular biology. Though emphasis will remain on basic mechanistic research, our findings have had clinical application at the national level and environmental application at the regional and local levels.