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Director: Frederick A. Beland, Ph.D. Executive SummaryIntroductionThe Division of Biochemical Toxicology conducts fundamental and applied research specifically designed to define the biological mechanisms of action underlying the toxicity of products either regulated by or of interest to the Food and Drug Administration (FDA). This research centers on assessing the toxicities and carcinogenic risk associated with specific chemicals and gene-nutrient interactions, and the introduction of new techniques to assess toxicities and carcinogenic risk. The risk assessment research is firmly rooted in mechanistic studies focused on the understanding of toxicological endpoints, an approach that allows greater confidence in the subsequent carcinogenic risk assessments. Research within the Division capitalizes on scientific knowledge in the areas of biochemistry, organic chemistry, cellular and molecular biology, immunology, nutritional biochemistry, and pharmacology. It is supported by sound technical skills, the availability of state-of-the-art equipment, and internal and external collaborations and funding. FY 2003 AccomplishmentsA major emphasis within the Division is to conduct research on compounds nominated by the FDA for evaluation by the National Institute of Environmental Health Sciences, National Toxicology Program (NIEHS/NTP). This focus reflects the fact that the NCTR has superb animal facilities supported by a multi-disciplinary staff of scientists with strong mechanistic research experience; as such, the Center has the capability to conduct subchronic and chronic toxicological assessments in a rigorous manner to address the FDA’s needs. While acknowledging the limitations of animal bioassays, these studies currently serve as the benchmark by which toxicological assessments are made by federal agencies, including the FDA. In addition to providing basic information on toxicological endpoints, such as cancer, these experiments form the basis for mechanistic studies to ascertain if the response detected in the experimental model is pertinent to humans. During FY 2003, in response to an NTP nomination by the Center for Veterinary Medicine (CVM), division investigators completed an NTP study to determine carcinogenicity of malachite green, a therapeutic agent used in aquaculture. These studies indicated that leucomalachite green, a metabolite of malachite green, is a mouse liver carcinogen. Division investigators had previously elucidated the mechanism by which riddelliine, a pyrrolizidine alkaloid found in herbal teas, is activated to a genotoxic carcinogen. During the current year, they expanded their studies to include monocrotaline, retrorsine, heliotrine, lasiocarpine, and clivorine, and further demonstrated that the dietary supplements comfrey and coltsfoot contain pyrrolizidine alkaloids that can be activated to genotoxins. At the request of the Center for Food Safety and Applied Nutrition (CFSAN), experiments were initiated on acrylamide, a carcinogen found in fried foods. These investigations have emphasized dose-response relationships and the development of biomarkers for assessing exposure. An area of particular concern to the FDA, in particular CFSAN, is the potential toxicity of cosmetic ingredients due to their interaction with light. To address this concern, the NCTR, in collaboration with the NIEHS/NTP, constructed a phototoxicity facility that is located within the Division. Studies at the NCTR Center for Phototoxicity have focused on the co-carcinogenic effects of simulated solar light and topically-applied α- and β-hydroxy acids, Aloe vera, and retinyl palmitate. The aims of other experiments have been to quantify the chemicals used in tattoo inks, investigate the photostability and safety of pigments used for tattooing, including permanent make-up, and develop a transgenic mouse model to investigate the mechanisms for the induction of cutaneous malignant melanoma and cutaneous ocular melanoma. As part of the NTP effort, the division investigators studied a series of endocrine-active compounds, including genistein, ethinyl estradiol, nonylphenol, and vinclozolin. These studies have involved both short term and long term assessments that are unique in terms of the number of endpoints evaluated and the inclusion of chronic toxicity assessments using varied exposure windows. Anti-retroviral drugs are being used to prevent the mother-to-child transmission of human immunodeficiency virus type 1, the virus responsible for acquired immunodeficiency syndrome. While effective in preventing viral transmission, the long-term consequences of perinatal exposure to these drugs are presently unknown. Division investigators have conducted a series of investigations to examine the genotoxic consequences of the anti-retroviral reverse transcriptase inhibitors zidovudine, lamivudine, stavudine, didanosine, and zalcitabine in neonatal mice. During FY2003, these studies were expanded to assess the effects of perinatal exposure of zidovudine and lamivudine in combination with the non‑nucleoside reverse transcriptase inhibitor nevirapine and the protease inhibitor nelfinavir. Tamoxifen is an adjunct chemotherapeutic agent for the treatment of breast cancer and a chemoprotective agent for breast cancer prevention. Despite being beneficial in regard to breast cancer, tamoxifen is known to increase the risk of endometrial cancer in women. Division investigators have conducted experiments to elucidate the mechanisms for tumor induction, with emphasis on characterizing the DNA adducts formed from this drug. As part of this effort, mass spectral methods were developed with sufficient sensitivity to detect and quantify tamoxifen DNA adducts and these methods were applied to endometrial and breast samples obtained from women receiving the drug. A strong emphasis within the Division has been in the area of nutritional folic acid deficiency and tumor progression. As part of this program, Division investigators evaluated the progressive changes in global DNA hypomethylation and promoter region hypermethylation in target and non-target tissues for carcinogenesis. In FY 2004 the Division's NTP efforts will include completion of final reports on the carcinogenicity of α- and β-hydroxy acids. Final reports for the multigeneration studies on genistein, nonylphenol, and ethinyl estradiol will also be prepared. Photocarcinogenicity and mechanistic studies will continue on topically-applied Aloe vera and retinyl palmitate, and the toxicity of Aloe vera will be assessed following oral exposure. Studies will continue to assess the effects of perinatal exposure of zidovudine and lamivudine in combination with nevirapine and nelfinavir. Experiments will continue on acrylamide, including assessing its carcinogenicity following perinatal and chronic exposures. Investigators associated with the NCTR Center for Phototoxicity will continue to study the interaction of light with tattoo pigments and develop transgenic mouse models for photocarcinogenesis, with emphasis on the induction of cutaneous and ocular melanoma. They will also initiate studies of furcoumarins found in lemon and lime oil and Padimate O, a component of suntan preparations. Results from the studies with endocrine-active compounds have indicated that soy-containing diets may protect against adrenal and renal toxicities. Gene and protein expression will be used to elucidate the mechanisms behind the effect upon adrenal gland. In addition, experiments will be initiated to investigate the protective effects of soy-containing diets against the renal toxicities of nonylphenol and di(2-ethylhexyl)phathalate. Experiments with tamoxifen will be expanded to investigate the role of estrogen receptors in the formation of tamoxifen-DNA adducts. The investigations with pyrrolizidine alkaloids will be expanded to investigate if these compounds are present in a variety of dietary supplements. Finally, studies will be initiated to determine if global and locus-specific DNA hypomethylation could be a common mechanism in genotoxic and non-genotoxic hepatocarcinogenesis. Research ProjectsPI: Beland, Frederick Title: Effect of Ethanol on the Tumorigenicity of Urethane
(Ethyl Carbamate) in B6C3F1 Mice Objective(s): Determine the effect of ethanol on the tumorigenicity of urethane (ethyl carbamate) in B6C3F1 mice. Status: Completed on 5/19/2003 Title: Perinatal Carcinogenicity of Drug Combinations used to
Prevent Mother-to-Child Transmission of HIV Objective(s): Determine the carcinogenicity, genotoxicity and metabolism of antiretroviral drug combinations administered to mice transplacentally, perinatally, or neonatally. Status: Started/Ongoing Title: Genotoxicity and Carcinogenicity of Acrylamide and its
Metabolite, Glycidamide, in Rodents - Two-Year Chronic Carcinogenicity Study
Objective(s): Compare the carcinogenicity of acrylamide and its metabolite glycidamide in B6C3F1 mice and F344 rats treated chronically for two years. Status: Project Under Review Title: DNA Adducts of Tamoxifen Objective(s): The nonsteroidal antiestrogen tamoxifen, which is currently being used in clinical trials as a chemoprotective agent against breast cancer, has been associated with the induction of certain malignancies. In order to determine if tamoxifen is acting through a genotoxic mechanism, this project will characterize DNA adducts from suspected tamoxifen metabolites, and develop methods for their detection and quantitation. Additional objectives include assessing the mutagenicity of tamoxifen and its metabolites in relation to DNA adduct formation in tissues of the Big Blue rat, determining the extent adduct formation in women treated with tamoxifen, and investigating the extent of DNA adduct formation from tamoxifen analogues including toremifene and GW5638. Status: Started/Ongoing Title: Genotoxicity ad Carcinogenicity of Acrylamide And Its
Metabolite, Glycidamide, in Rodents: Neonatal Mouse Bioassay Objective(s): Compare the extent of DNA adduct formation, induction of micronuclei, and carcinogenicity of acrylamide and its metabolite glycidamide in B6C3F1 mice treated neonatally. Status: Project Under Review Title: Salmonella Mutagenicity Testing for Regulatory Needs Objective(s): Use the Ames Salmonella mutagenicity test system to determine the mutagenicity of compounds of regulatory interest to the Agency. Status: Started/Ongoing Title: In Vivo DNA Adduct Standards Objective(s): Develop methods to detect, characterize, and quantify DNA adducts in vivo. Status: Started/Ongoing PI: Boudreau, Mary Title: Effects of Aloe Vera Components on Cell Proliferation
and DNA Adduct Formation in SKH-1 Mice Following Simulated Solar Light Exposure
Objective(s): Determine the dose-response and acute kinetics of topical exposure to Aloe vera plant components on the structure of SKH-1 mouse skin in the absence of simulated solar light exposure; Determine the effects of topical exposure of Aloe vera plant components on the amount of simulated solar light required to induce skin edema in the SKH-1 mouse; Determine the subchronic effects of repeated co-exposure to Aloe vera plant components and simulated solar light on skin cell edema, proliferation, and DNA damage in the SKH-1 mouse; Determine the tumor-promoting activities of Aloe vera plant components following simulated solar light tumor initiation; and Determine the influence of Aloe vera components on simulated solar light-induced tumor formations in mice. Title: Bioassays in the F-344 Rat and the B6C3F1 Mouse
Administered Aloe Vera Plant constituents in the Drinking Water Objective(s): The use of Aloe vera is not limited to over-the-counter dermal therapeutics and cosmetics. Aloe vera is also taken internally. Aloe vera for internal consumption is also widely used as a prophylaxis and treatment for a variety of unrelated systemic conditions. In view of the complexities inherent in aloe pharmacology and the inconsistencies reported in literature, the objective of these studies is to conduct bioassays in rats and mice using standardized preparations of Aloe vera to explore the limits of safety for the Aloe vera leaf constituents present in commercial products. Status: Started/Ongoing PI: Chou, Ming Title: A Collaborative Research Proposal to Assess Cancer Risk
Posed by Intermittent Exposure to Aflatoxin B1 in Rats Objective(s): Test the hypothesis that a chemically-induced tumor incidence is a function of the accumulated lifetime exposure, and is predictable from the average daily dose for various dosing regimens, such as continuous and intermittent dosing; and Study correlations between the chemically-induced tumor incidence and various biomarkers of the initiation and the promotion stage of carcinogenesis for continuous and intermittent dosing. Status: Started/Ongoing Title: Effects of Dietary Restriction on the Post-Initiation
Stages in Aflatoxin B1-Induced Carcinogenesis on Male F-344 Rats Fed
Methyl-Deficient Diets Objective(s): Study the interactions of dietary restriction (DR) and methyl deficiency (MD) on the alterations of hepatic oxidative DNA damages, DNA methylation, cell proliferation, oncogene and tumor suppressor gene mutation, preneoplastic foci formation and tumor incidence during the post-initiation stages of AFB1-induced carcinogenesis in male F344 rats. The results of these studies will: 1) test the hypothesis that DR may be an antagonist to the promotional effect of MD in the AFB1-induced carcinogenesis; and 2) evaluate the correlations between the effects of DR and MD on the formation of AFB1-induced preneoplastic foci and tumors and various biomarkers during the post-initiation stages of carcinogenesis. Status: Started/Ongoing Title: A Study of Genotoxic Mechanisms of Carcinogenic
Pyrrolizidine Alkaloids and Pyrrolizidine Alkaloid N-Oxides Objective(s): Characterize the structures of the eight DHP-derived DNA adducts; Study metabolism of retronecine-based pyrrolizidine alkaloids, heliotridine-based pyrrolizidine alkaloids, otonecine-based pyrrolizidine alkaloids, and pyrrolizidine alkaloid N-oxides by liver microsomes of F344 rats, B6C3F1 mice, and humans of both sexes, and compare metabolism profiles; Study the DNA adduct formation in vitro (from liver microsomal metabolism of the pyrrolizidine alkaloids described above in the presence of calf thymus DNA) and in vivo, and determine whether or not the same set of DHP-derived DNA adducts is formed in all cases; Determine whether or not the levels of DHP-derived DNA adducts from different types of necine-based pyrrolizidine alkaloids formed in target tissues (liver) are significantly higher than those in non-target tissues; Determine whether or not pyrrolizidine alkaloid N-oxides can be metabolized by rat and mouse liver microsomes to the parent pyrrolizidine alkaloids and whether or not DHP-derived DNA adducts are formed in significant amounts both in vivo and in vitro; Determine whether or not some dietary supplements sold in the United States contain genotoxic pyrrolizidine alkaloids; Determine the effect of liver carboxyesterases on DHP-derived DNA adduct formation from rat and human liver microsomal metabolism in the presence of calf thymus DNA; Determine the effect of liver carboxyesterase inhibitors on DHP-derived DNA adduct formation from rat and human liver microsomal metabolism in the presence of calf thymus DNA; and Determine the effect of Chinese herbs, such as liquorice, and their active components, such as glycyrrhizin and glycyrrhetinic acid, on inhibition of DHP-derived DNA adduct formation in vivo and in vitro. Status: Started/Ongoing PI: Culp, Sandra Title: Two-Year Bioassay in Mice Administered Malachite Green
or Leucomalachite Green in the Diet Objective(s): Determine the risk associated with exposure to malachite green or leucomalachite green. Status: Started/Ongoing Title: Two-year Bioassay in Rats Administered Malachite Green
or Leucomalachite Green in the Diet Objective(s): Determine the risk associated with exposure to malachite green or leucomalachite green and establish if the demethylated derivatives of malachite or leucomalachite green found in tissues of treated rodents lead to the reactive species that bind to DNA. Status: Started/Ongoing PI: Delclos, Kenneth Title: Range Finding Study for the Evaluation of the Toxicity
of Genistein Administered in the Feed to CD (Sprague-Dawley) Rats Determine the doses of genistein to be used in a multigeneration bioassay for establishing the effects of this naturally occurring isoflavone on development of reproductive organs, reproduction, cancer of the reproductive organs, and neurological and immunological function. Status: Completed on 1/10/2003 Title: Range Finding Study for the Evaluation of the Toxicity
of Nonylphenol Administered in the Feed to CD (Sprague-Dawley) Rats Objective(s): Determine the doses of nonylphenol for use in a multigeneration bioassay for assessing the effects of this compound on the development of the reproductive tract, reproduction, and neurological and immunological function. Status: Completed on 10/23/2003 Title: Range Finding Study for the Evaluation of the Toxicity
of Vinclozolin Administered in the Feed to CD (Sprague-Dawley) Rats Objective(s): Determine the doses of vinclozolin for use in a multigeneration bioassay for assessing the effects of this compound on the development of the reproductive tract, reproduction, and neurological and immunological function. Status: Started/Ongoing Title: Range Finding Study for the Evaluation of the Effects of
Ethinyl Estradiol Administered in the Feed to CD (Sprague-Dawley) Rats During
Development Objective(s): Determine the doses of ethinyl estradiol (EE2) for use in a multigeneration bioassay for assessing the effects of this compound on the development of the reproductive tract, reproduction, and neurological and immunological function. Status: Started/Ongoing Title: A Comparison of Weight Gain and Fertility in CD Rats Fed
a Standard Diet (NIH-31) or a Soy- and Alfalfa-free, Casein-containing Diet
(NIH-31C) Objective(s): Evaluate effects of NIH-31C on fertility by comparing pregnancy rates and litter size and weight in CD rats treated according to the treatment regimen to be used in the F0 generation of the multigeneration. Status: Started/Ongoing Title: Genistein: Evaluation of Reproductive Effects Over
Multiple Generations and the Chronic Effects of Exposure during Various Life
Stages Objective(s): Determine the effects of genistein, a naturally occurring isoflavone, on reproduction and on the development of reproductive and other selected hormone-sensitive organs when administered to CD rats over multiple generations; Determine if subtle effects observed in the dose range finding study are magnified through multiple generations; Evaluate the reversibility of any observed effects; Evaluate the chronic toxicity of genistein, particularly potential induction of cancer of the reproductive organs, following exposures that will include various life stages (in utero through early adulthood, in utero and continuous for two years after birth, in utero and lactational only, and postweaning only); and Assess plasma and tissue genistein levels and evaluate additional endpoints modulated by genistein. These additional endpoints may be mechanistically associated with compound-induced alterations observed in the dose range finding studies that are also anticipated in animals from the multigeneration studies. Status: Started/Ongoing Title: Para-Nonylphenol: Evaluation of Reproductive Effects
over Multiple Generations Objective(s): Determine the effects of p-nonylphenol, an intermediate in the production of surfactants and other industrial products, on reproduction and on the development of reproductive and selected other hormone-sensitive organs when administered to CD rats over multiple generations; Determine if subtle effects observed in the dose range finding study are magnified through multiple generations; Evaluate the reversibility of any observed effects; and Assess the effect of nonylphenol on aromatase activity in the central nervous system in male rats at birth and on testosterone production and steroid receptor expression at puberty and at PND 140. Status: Started/Ongoing Title: Ethinyl Estradiol: Evaluation of Reproductive Effects
over Multiple Generations and the Chronic Effects of Exposure during Various
Life Stages Objective(s): Evaluate the effects of ethinyl estradiol, a potent synthetic estrogen widely used in prescription drugs, on reproduction and on the development of reproductive and selected other hormone-sensitive organs when administered to CD rats in the diet over multiple generations; Determine if subtle effects observed in the dose range finding study are magnified through multiple generations; Evaluate the reversibility of any observed effects; Evaluate the chronic toxicity of ethinyl estradiol, particularly the potential induction of cancer of the reproductive organs, following exposures that will include various life stages; and Assess pharamacokinetics of ethinylestradiol (F0 generation only) by measuring serum levels using high pressure liquid chromatography and atmospheric pressure chemical ionization tandem mass spectrometry following an oral gavage dose. Status: Started/Ongoing Title: Sexual Dimorphism in the Inflammatory Response to
Biomaterials Objective(s): Determine if a sex difference in the in vitro response of human monocytes and mouse peritoneal macrophages to various biomaterials can be demonstrated. Based on existing literature, we hypothesize that there will be a significant sex difference in the synthesis and release of inflammatory mediators that could influence the biocompatibility of the material. Status: Completed on 10/27/2003 Title: The Effects of Dietary Genistein on the Growth of
Chemically-Induced Mammary Tumors in Ovariectomized and Intact Rats Objective(s): Determine whether or not, in the absence of endogenous ovarian estrogens, dietary genistein can promote or suppress the growth of neoplastic mammary tissue at various stages of the carcinogenic process; and Evaluate the expression and activity of CYP1B1 in NCTR CD (Sprague-Dawley) female rats fed a soy- and alfalfa-free diet (5K96) relative to the same rats fed NIH-31 chow and to commercially available Sprague-Dawley (SD) rats. Based on the previously observed sensitivity of NCTR CD rats fed 5K96 diet to DMBA-induced adrenal toxicity, we hypothesize that either the 5K96 diet up-regulates CYP1B1 activity in the rat adrenal or that the NCTR CD (SD) rat contains higher CYP1B1 activity than SD rats from common commercial suppliers (Charles River and Harlan). Status: Started/Ongoing Title: Effects of Endocrine-active Agents on Bone Objective(s): Determine if the administration of the endocrine-active agents genistein and ethinyl estradiol (EE2) will alter bone growth and remodeling and if the direction and extent of the effect depends on the window of exposure to the compounds. Status: Started/Ongoing Title: Protective Effects of Soy-Containing Diets Against the
Renal Toxicity of P-nonylphenol (NP) and Di(2-ethylhexyl)phthalate (DEHP) Objective(s): Determine that the cystic kidney disease previously shown to be induced by nonylphenol in developing NCTR CD rats fed a soy-free diet is decreased in incidence and/or severity in rats fed soy-containing diets and to determine if the degree of protection is related to the amount of soy in the diet; Evaluate the renal toxicity of dietary DEHP in developing rats maintained on a soy-free diet; Evaluate potential early markers of renal cystogenesis in nonylphenol - and DEHP-treated rats and their modulation by soy-containing diets; Evaluate the roles of modulation of antioxidant defenses and cyclooxygenase activities in the protective effect of soy against nonylphenol - and, if demonstrated, DEHP-induced renal toxicity; and If renal toxicity of DEHP and a protective effect of soy against that toxicity are demonstrated, the effect of diet on the hepatic and testicular toxicity of DEHP will be assessed by measurement of the peroxisomal enzyme palmitoyl coA-oxidase in liver and histopathological evaluation of the tests. Status: Project Under Review Title: Optimization of Procedures for 1) Laser Capture
Microdissection of Rat Kidney for Gene and Protein Expression Studies; and 2)
Measurement of Renal Cyclooxygenases, Antioxidant Enzymes and Isoprostanes Objective(s): Determine optimal parameters for laser capture microdissection to collect distinct renal cell populations for analysis of mRNA and proteins; Optimize conditions for the measurement of cox-1, cox-2, glutathione peroxidase, superoxide dismutase and quinone reductase; and Evaluate the feasibility of utilizing commercial ELISA kits for the determinations of prostaglandin and isoprostane levels in renal cortex and medulla. Status: Started/Ongoing PI: Doerge, Daniel Title: ADDEND: Evaluation of Lactational Transfer of Genistein
in CD (Sprague-Dawley) Rats Objective(s): Measure transfer of genistein from the lactating dam to nursing pups by measuring genistein in serum and milk using high pressure liquid chromatography (LC) and electrospray (ES) tandem mass spectrometry (LC-ES/MS/MS) following consumption of genistein-fortified soy-free basal diet. Status: Started/Ongoing Title: ADDEND: Evaluation of Serum Nonylphenol in CD (Sprague-Dawley)
Rats Exposed to Dietary Nonylphenol Objective(s): Assess serum and tissue nonylphenol levels (F2 generation only) by high pressure liquid chromatography and atmospheric pressure chemical ionization mass spectrometry (LC-APCI/MS). Status: Started/Ongoing Title: Genotoxicity, Mutagenicity and Exposure Biomarkers of
Acrylamide and Its Metabolite, Glycidamide, in Rodents Objective(s): Synthesize chemically and characterize spectroscopically the major glycidamide-DNA adducts; Develop and validate LC-ES/MS/MS assays to quantify the major glycidamide-DNA adducts; Determine glycidamide-DNA adduct levels in rodent tissues following short-term exposures of rodents to acrylamide and to glycidamide; Determine toxicokinetics and compare bioavailability of acrylamide and glycidamide following exposure by intravenous, oral gavage, and dietary administration; Correlate the levels and kinetics of glycidamide-DNA adduct in target tissues and circulating lymphocytes with acrylamide- and glycidamide-hemoglobin adducts in rodent exposure studies for future use in monitoring human exposure through occupation, smoking, and the diet; and Determine in vivo mutagenesis of acrylamide and glycidamide using transgenic mice (Big Blue). Status: Started/Ongoing Title: Toxic Hazards from Anti-Thyroid Chemicals Objective(s): Determine inhibition mechanisms for environmental goitrogens using purified thyroid peroxidase and lactoperoxidase; Determine the mechanism for covalent binding suicide substrates to purified peroxidases using electrospray-mass spectrometry to analyze intact adducted proteins and/or proteolytic fragments; Determine mechanism of goitrogen uptake into isolated thyroid cells in primary culture and subsequent inhibition of iodination/coupling reactions involved in thyroid hormone synthesis; Determine the structure-activity relationship for uptake of goitrogens into the thyroid and inhibition of thyroid hormone synthesis rats; Correlate in vivo thyroid hormone status endpoints from the NTP endocrine disruption rat bioassay with the in vitro activity of rat microsomal thyroid peroxidase to better define the anti-thyroid mechanism for genistein; Determine whether administration of genistein to rats results in an increase in the amounts of circulating auto-antibodies to TPO; and Develop analytical methodology to quantify genistein/daidzein and the respective sulfate and glucuronide conjugates in blood and mammary tissue. Status: Completed on 10/29/2003 Title: Development of Methods for Analysis and Confirmation of
β-Agonists Objective(s): Develop determinative and confirmatory procedures using Liquid Chromatography-Atmospheric Pressure Chemical Ionization Mass Spectrometry (LC-APCI/MS) for multiresidue screening β-agonists in livestock tissues; Develop synthetic procedures to produce authentic β-agonist standards for use in regulatory screening. These methods will provide the flexibility to adapt to the potential for “designer drug” modifications by clandestine laboratories; and Explore the use of packed column supercritical fluid chromatography (SFC) coupled to APCI/MS as a more efficient technique for chromatographic separation in the screening of large numbers of β-agonists in livestock tissues. Status: Started/Ongoing Title: ADDEND: Development of Methods for Analysis and
Confirmation of β-Agonists Objective(s): Generate incurred ractopamine residues in livestock species that may be targets of off-label ractopamine use. Tissues and excreta from dosed animals will be used to validate immunochemical and confirmatory assays of ractopamine. The USDA will provide tissues from an incurred residue feeding study and NCTR will expand our multiresidue LC/MS method to include an additional compound, ractopamine, that was recently approved for commercial use by CVM. Status: Started/Ongoing Title: Measurement of Oxidative DNA Damage in Normal and
Hepatitis C-Infected Human Liver Objective(s): Develop simple synthetic methods to produce stable labeled analogs of 8-oxo-dG, etheno-dA, etheno-dC, and M1-dG; Develop an automated on-line sample preparation method to maximize detection sensitivity for 8-oxo-dG, etheno-dA, etheno-dC, and M1-dG, in a single sample analysis, using liquid chromatography and tandem mass spectrometry; Apply methodology to the analysis of hepatic DNA from humans and animals; and Determine feasibility for application to clinical trials of therapeutic agents and toxicity/carcinogenicity testing in experimental animals. Status: Completed on 10/27/2003 Title: Effect of Soy-Containing Diets on Ammonium Perchlorate-Induced
Thyroid Toxicity in Sprague-Dawley Rats Objective(s): Determine the effect of dietary soy and genistein, the principal soy isoflavone, on the dose-response characteristics for perchlorate-induced thyroid toxicity in male Sprague-Dawley rats. Status: Started/Ongoing Title: Human Studies of Isoflavone Safety and Efficacy Objective(s): Bioanalytical analysis of soy isoflavones (and metabolites) in support of clinical trials at the University of Miami and Wayne State University. Status: Started/Ongoing PI: Fu, Peter Title: Effect of Topically Applied Skin Creams Containing
Retinyl Palmitate on the Photocarcinogenicity of Simulated Solar Light in SKH-1
Mice Objective(s): Study the effects of topically applied skin cream containing retinyl palmitate on the photocarcinogenicity of simulated solar light in SKH-1 mice. Status: Started/Ongoing Title: The Evaluation of Selected Benzodiazepine and
Antihistamine Drugs in the Neonatal Mouse Tumorigenicity Bioassay and in
Transgenic Human Lymphoblastoid Cells Objective(s): Determine if the neonatal mouse bioassay can be employed to evaluate the tumorigenic potential of therapeutic drugs; Examine concurrently as positive controls the genotoxic carcinogens: 4-aminobiphenyl, benzo(a)pyrene, 6-nitrochrysene, and aflatoxin B1; Study the metabolism and DNA adduct formation of benzodiazepine and antihistamine drugs by mouse and human liver microsomes to determine which if any cytochrome P450 is responsible for metabolic activation in mice and humans; Transgenic human lymphoblastoid cell lines expressing appropriate CYP isozymes will also be employed to study the mutations and DNA binding of the subject drugs; Determine whether or not the B6C3F11 neonatal mouse tumorigenicity bioassay is sensitive to chemical carcinogens that exert their tumorigenic activity by a secondary mechanism; Assess the carcinogenicity of methylphenidate hydrochloride (ritalin), 4-hydroxy-2-nonenal, malondialdehyde, crotonaldehyde, and acrolein; Assess the carcinogenicity of phenolphthalein and Tacrin; Determine the tumorigenicity, mutagenicity, and DNA adduct formation of anti-HIV nucleosides in the B6C3F1 neonatal mouse bioassay; and Assess the tumorigenicity and estimate DNA adduct formation of a set of known or suspected human carcinogens in the B6C3F1neonatal mouse. Status: Started/Ongoing Title: A Study of the Secondary Mechanisms of Carcinogenesis:
Lipid Peroxidiation and Endogenous DNA Adduct Formation from Chloral Hydrate,
Benzodiazepines, Antihistamines, and Other Chemicals Objective(s): The specific aims outlined below are critical for the development of methodologies to study secondary mechanisms of carcinogenesis, including lipid peroxidation and endogenous DNA adduct formation, for determination of the mechanisms by which chemicals, such as FDA-regulated drugs including benzodiazepines and antihistamines, may induce cancer, and for the continued development of the neonatal mouse bioassay as a regulatory alternative tumorigenicity bioassay: Develop analytical methodologies for analysis of lipid peroxidation products and endogenous DNA adducts; Determine whether or not the drugs of FDA interest, including benzodiazepines and antihistamines studied in E687901, and other chemicals induce lipid peroxidation and endogenous DNA adduct formation in vitro; Determine the inhibitory effect of lipid- and water-soluble antioxidants on drug-induced lipid peroxidation and endogenous DNA adduct formation in vitro; Determine whether or not the malondialdehyde-modified MG-1 DNA adduct and/or other endogenous DNA adducts can be used as biomarkers of lipid peroxidation; and Determine the mutagenicity of the benzodiazepine and antihistamine drugs in Salmonella typhimurium TA 104 and determine whether or not mutagenicity in Salmonella typhimurium TA104 can be used as a biomarker of lipid peroxidation induced by chemicals that generate free radicals upon metabolism. Status: Started/Ongoing PI: Howard, Paul Title: The Role of Fumonisin B1 in Fusarium sp. Tumorigenicity
in Rats Objective(s): Determine the effect of fumonisin B1 on signal transduction pathways in cultured human esophageal epithelial tissues; Determine if DNA damage occurs in vivo in F344 rats when fed in the diet cultures of Fusarium graminearum, Fusarium subglutinans, Fusarium moniliforme or a combination of the three fungi, using 32P-postlabeling technique; Determine the pharmacokinetics of fumonisin B1 in B6C3F1 mice and F344 rats under conditions similar to those used in the chronic bioassay, and in non-human primates; and Use the Rhesus monkey as a model to determine if fumonisin B1 crosses the placenta. Status: Started/Ongoing Title: Comparative Toxicity of Fumonisin Derivatives in Female
B6C3F1 Mice Objective(s): The primary objective of the study is to compare the toxicity of several fumonisin derivatives in female B6C3F1mice. Status: Started/Ongoing Title: The Effects of Chemoexfoliation using α- and β-hydroxy
Acids on Cell Proliferation and DNA Adduct Formation in SKH-1 Mice Exposed to
Simulated Solar Light Objective(s): The NIEHS/FDA Phototoxicity Center is designed to address the effects of compounds on the induction of skin cancer in mice using light sources that are relevant to humans. Input into the design of the facility has been obtained from experts in phototoxicity and photocarcinogenicity. These experts will continue to provide critical advice on the design of the experimental protocols. As a result, a facility will be developed that will meet the rigors of scientific scrutiny, and will generate data for human health risks from the effects of compounds on light-induced skin cancer. The facility is also designed for expansion to allow simultaneous examination of the toxicity or cocarcinogenicity of compounds in the presence of either simulated sunlight or fluorescent UVB light. The mechanistic studies in this proposal will provide the data necessary to design and interpret properly the future α-hydroxy acid and simulated solar light cocarcinogenicity studies. Status: Started/Ongoing Title: Effect of Topically Applied Skin Creams Containing
Glycolic and Salicylic Acid on the Photocarcinogenicity of Simulated Solar Light
in SKH-1 Mice Objective(s): Determine if the application of creams containing α- and β-hydroxy acids to the skin of male and female SKH-1 hairless mice alters the tumor incidence induced by simulated solar light in the mouse skin. Status: Started/Ongoing Title: The Use of DNA Microarray Technology to Quantify the
Effects of 8-methoxypsoralen (8-MOP) and UVA Light Treatment on SKH-1 Mouse Skin
Objective(s): Determine the effects of PUVA treatment on gene expression in the skin of SKH-1 mice. Success of this project will lead to a more extensive protocol in collaboration with NIEHS. Status: Started/Ongoing Title: DNA Adduct Formation by Nicotine Metabolites Objective(s): Determine the structural identity of the nicotine delta 1',2'- and delta 1',5'-iminiumion DNA adducts, and modify existing 32P-post labeling techniques to detect the adduct; and Quantify the presence of these adducts in vitro and in vivo in mice. Status: Started/Ongoing Title: Purification of Ceramide Synthase Objective(s): Isolate rat ceramide synthase; Identify the gene coding for rat ceramide synthase; Develop antibodies to rat ceramide synthase; and Use the antibodies to study tissue specific expression of ceramide synthase. Status: Started/Ongoing Title: Methodology for Safety Testing of Pigments Used for
Tattooing, Including Permanent Make-up Objective(s): Determine the chemicals in tattoo pigments and their metabolism in vitro; Develop methodology for tattooing SKH-1 hairless mice in a quantitative and reproducible manner; Determine the extent of inflammation induced by the implanted pigment, and determine the time of recovery following tattooing; Determine the acute toxicity of several tattoo inks and permanent make-up inks in SKH-1 hairless mice in the presence and absence of simulated solar light; Determine if tattoo pigments are photococarcinogenic in the SKH-1 hairless mouse using simulated solar light; and Determine if visual or infrared frequency imaging technology can be used for the detection of skin tumors in SKH-1 mice. Status: Started/Ongoing Title: Development of a Research Plan and Research Protocol for
Furocoumarins in Lemon and Lime Oil Objective(s): Conduct a literature search and summarize the occurrence, use, pharmacokinetics, toxicity, mutagenicity, and carcinogenicity of lemon and lime oil furocoumarins; and Develop a draft research protocol for submission to the NCTR Protocol Review and IAG Toxicology Study Selection and Review Committee. Status: Started/Ongoing Title: Historical Database of Skin Tumor Formation in SKH-1
Mice Objective(s): Enter into the NCTR MultiGen system the historical data (Argus Research Laboratories) of the tumor incidence in SKH-1 mice treated with simulated solar light (SSL) (no test compounds). Argus Research Laboratories will provide the weekly individual animal observation records for as many studies as deemed reasonable by Argus Research Laboratories. NCTR will be responsible for entering the data into the NCTR MultiGen database; NCTR will generate tumor incidence reports summarizing the occurrence of tumors in the animal groups from the animal tumor data that were obtained from Argus Research Laboratories. The data will then be analyzed for various parameters pertinent to these types of studies (e.g. time-to-first-tumor, mean time- to-first-tumor, tumors/mouse). The data, summary data, and statistical analyses will be shared with Argus Research Laboratories; NCTR will share with Argus Research Laboratories the incidence data on the occurrence of tumors in control SKH-1 mice treated with SSL at NCTR. NCTR will also share the development of any statistical methods for analyzing the tumor incidence data with SKH-1 mice; and Both parties agree to share information concerning future development of animal rack and caging systems; devices for holding the SSL lamps; control of SSL lamps; devices for housing fluorescent light fixtures; devices, software, or protocol for monitoring the irradiance from SSL or fluorescent lights; and database systems for collecting animal information. Status: Started/Ongoing PI: James Gaylor, Sandra Title: Nutritional Modulation of Apoptosis and Chemosensitivity:
A Novel Anticancer Strategy Objective(s): In Nitroso methylurea (NMU)-initiated mammary epithelial cells, to determine whether nutritional manipulation of the cell cycle combined with low-dose chemotherapy will permanently eliminate p53-independent and p53-dependent preneoplastic and neoplastic cells; and Determine the mechanisms of cell death induced by nutritional manipulation and low-dose chemotherapy by examining molecular endpoints associated with p53-dependent and independent pathways of apoptosis. Status: Started/Ongoing Title: Genes, Micronutrients, and Homeobox-Related
Malformations Objective(s): A more specific understanding of the genetic and environmental factors that contribute to the etiology of birth defects is a necessary prerequisite for the design of effective preventive strategies to reduce infant and maternal risk. This project has the potential to significantly advance current knowledge of specific etiological factors involved in maternal and infant risk, to aid in the design of nutritional intervention strategies, and to provide a basis for future mechanistic studies of human malformation. NCTR's main objective is investigation of maternal risk factor for neural tube defects and congenital heart defects. Status: Started/Ongoing PI: Pogribna, Marta Title: Folic Acid Metabolism in Children with Down Syndrome Objective(s): Determine whether supplementation with the nutrients folic acid and betaine will increase plasma levels of methionine, S-adenosylmethionine (SAM) and S-adenosylhomocysteine SAH, which have shown to be low in children with Down Syndrome (DS). The experiments will focus on the biochemical lesions in one-carbon metabolism stemming from trisomy 21 gene overdose and the potential to normalize metabolic imbalance with targeted nutritional intervention. With better understanding of the metabolic and molecular aberrations of CBS gene overdose in DS, the potential to ameliorate or prevent these progressive disease processes with nutritional intervention could become a reality. In the proposed study, baseline levels of homocysteine, methionine, cystathionine, cysteine, glutathione, cysteinyl-glycine, SAM, SAH, and adenosine in plasma of DS children will be determined at baseline and after 3 months supplementation with folic acid and betaine. This will define the metabolic abnormalities in one-carbon metabolism caused by the presence of an extra copy of chromosome 21 and is an important first step in determining whether there is a potential for nutritional intervention to correct the metabolic imbalance. The long-term goal for this study is to determine whether nutritional intervention in children with DS at 2-10 years of age will have a positive effect on their growth, immunologic function, and cognitive development. Adults with DS have already reached a plateau of growth and development and therefore the likelihood that nutritional intervention will affect their growth and development is minimal. Status: Started/Ongoing PI: Pogribny, Igor Title: Mechanisms and Consequences of DNA Damage and
Methylation Dysregulation during Rat Hepatocarcinogenesis Objective(s): Confirm that the presence of uracil and abasic sites in preneoplastic DNA from folate/methyl deficient rats creates nonproductive high affinity binding sites for the DNA methyltransferase that compromise normal DNA methylation at the replication fork resulting in genome-wide hypomethylation; Determine a) whether the double-stranded loss of cytosine methylation is maintained in folate/methyl deficient rats after nutritional repletion of methyl donors or whether the original methylation pattern and chromatin structure can be reestablished; and b) whether the increase in dnmt1 expression is stimulated by global loss of methyl groups and whether dmnt1 expression is decreased by methyl repletion; Determine the temporal relationship between the appearance of DNA lesions and site-specific methylation within the CpG island of the p16 promoter region in p16 gene expression with alterations in local chromatin structure and DNA methyltransferase mRNA levels and activity; and Use microarray slides printed with the rat cDNA library in collaboration with Dr. James Fuscoe, of the Center for Functional Genomics, as a tool to screen for methylation-related down-regulation of candidate genes in hepatic preneoplastic foci, preneoplastic nodules, and tumor tissue from folate/methyl deficient rats. Status: Started/Ongoing PI: Roberts, Dean Title: Antigenic Biomarkers of Estrogen Catechol Metabolism for
Use in Epidemiological Studies Objective(s): Prepare immunogenic conjugates for immunization of rabbits and antigenic conjugates for the characterization of antisera and for affinity purification of antibodies; Develop IA/LC/MS methods to detect the antigenic biomarkers in urine and/or serum; and Initiate studies to validate the use of the antibody reagents and IA/LC/MS methods developed in aim 1 and 2 using human urine and serum samples collected in an ongoing study of reproductive events, carcinogen metabolism, and interindividual variability. Status: Completed on 12/13/2002 Title: Padimate O: Development of a Research Plan and Research
Protocol Objective(s): Conduct a literature search and summarize the occurrence, use, stability, vehicle effects, pharmacokinetics and toxicity of padimate O; and Develop a draft research protocol for submission to the NCTR Protocol Review and IAG Toxicology Study Selection and Review Committee. Status: Started/Ongoing PI: Tolleson, William Title: Molecular Basis of Tumor Promotion and Increased Somatic
Growth in Yellow Avy/a Mice: Mitogenic Effects of Agouti Protein In Vitro Objective(s): Determine whether or not the agouti protein stimulates mitogenesis in vitro. Status: Started/Ongoing Title: The Role of Human Metabolism in Endocrine Disruption Objective(s): Humans may be exposed to compounds in the diet or in the environment that disrupt endogenous endocrine responses in various tissues. We propose to utilize cell biological approaches to determine the role of human cytochromes P-450, UDP-glucuronosyltransferases, and sulfotransferases in the antiestrogens. The relative abilities of the various human enzyme systems expressed by individual cell lines to alter the extent of green fluorescent protein synthesis will indicate those human enzyme activities that activate or deactivate endocrine disrupting agents. Status: Started/Ongoing Title: Photoinduction of Cutaneous Malignant Melanoma in TP-ras/ink4A
(+/-) Transgenic Mice Objective(s): Characterize photochemical DNA damage in the skin of TP-ras/ink-4a mice exposed to UVA+UVB radiation; Determine whether cutaneous malignant melanoma can be induced in neonatal TP-ras (+) ink4a (+/-) transgenic mice using UVA+UVB radiation; Identify photochemically-induced mutations within the ink4a/p16/CDKN2A and p53 foci in tumor tissues; and Determine whether UVA+UVB exposure at an early age creates a greater risk for developing cutaneous melanoma in TP-ras (+)ink4a(+/-) mice compared with chronic UVA+UVB exposure of older animals. Status: Started/Ongoing PI: Wolff, George Title: Caloric Restriction and Gene Expression in Agouti Mice
Objective(s): The total amount of fat and calories we consume in our diet is highly correlated with the occurrence of cancer in North America and other highly developed nations. The studies proposed will help us learn how calories modify the development of cancer in mice and the mechanism underlying cancer development in humans. Status: Started/Ongoing Title: Tumor Promotion and Neurochemical Changes in Mice During
Chronic Feeding of the Antidepressant Fluoxetine Objective(s): Determine if chronic feeding of fluoxetine (Prozac) results in promotion of mouse mammary carcinomas; and Determine if chronic feeding of fluoxetine: a) produces changes in the concentrations of serotonin and its metabolite, 5-hydroxyindoleacetic acid, in different regions of the mouse brain; and b) induces changes in serotonergic receptor and uptake sites in different regions of the mouse brain. Status: Started/Ongoing Title: Cellular and Molecular Responses to Chronic Iron
Overload in Animal Models Project Number: E0691201 Objective(s): Determine the health effects of chronic iron overload in mice and rats; Determine neurochemical changes after chronic iron overload in mice and rats; and Develop an animal model for identifying the cellular and molecular mechanisms underlying the hepatic and pancreatic effects of chronic iron overload which are characteristic of the human disease idiopathic hemochromatosis and possible neurochemical mechanisms which associate effects of iron with neurological disorders, e.g., Parkinson and Alzheimer diseases. Status: Started/Ongoing PublicationsCarmel, R. and James Gaylor, S.J., Alcohol abuse: an important cause of severe hyperhomocysteinemia, Nutrition Reviews. Accepted: 5/1/2002 (E0712801) Chang, H., Huffer, D.M., Chiarelli, M.P., Blankenship, L., Culp, S. and Cho, B.P., Characterization of DNA Adducts Derived from syn-Benzo[ghi]fluoranthene-3,4-Dihydrodiol-5,5a-Epoxide and Comparative DNA Binding Studies with Structurally-Related anti-Diolepoxides of Benzo[ghi]fluoranthene and Benzo[c]phenanthrene, Chemical Research in Toxicology, 15:198-208. Accepted: 12/11/2001 (S00179) Chen, T., Gamboa da Costa, G., Marques, M.M., Shelton, S.D., Beland, F.A. and Manjanatha, M., Mutations induced by alpha-hydroxytamoxifen in the lacI and cII genes of Big Blue transgenic rats, Carcinogenesis, 23:1751-1757. Accepted: 7/8/2002 (E0701101) Christopher, S.A., Melnyk, S.B., James Gaylor, S.J. and Kruger, W.D., S-Adenosylhomocystein, but not homocystein, is toxic to yeast lacking cystathionine B-synthase, Molecular Genetic and Metabolism, 75:335-343. Accepted: 2/15/2002 (E0706501) Churchwell, M.I., Beland, F.A. and Doerge, D.R., Quantification of multiple DNA adducts formed through oxidative stress using liquid chromatography and electrospray tandem mass spectrometry, Chemical Research in Toxicology. Accepted: 5/20/2002 (E0706401) Churchwell, M.I., Holder, C.L., Little, D., Preece, S.W., Smith, D.J. and Doerge, D.R., LC-ES/MS/MS Analysis of incurred ractopamine residues in livestock tissues, Rapid Communications in Mass Spectrometry, 16:1261-1265. Accepted: 5/7/2002 (E0694501) Couch, L.H. and Howard, P., Quantification of glycolic acid in cosmetic products using reversed-phase high performance liquid chromatography, International J. Cosmetic Science. Accepted: 10/4/2001 (E0213101) Culp, S., Beland, F.A., Heflich, R.H., Benson, R.W., Blankenship, L., Webb, P., Mellick, P.W., Trotter, R.W., Shelton, S.D., Greenlees, K. and Manjanatha, M., Mutagenicity and Carcinogenicity in Relation to DNA Adduct Formation in Rats Fed Leucomalachite Green, Mutation Research. Accepted: 2/8/2002 (E0212821) Dalu, A.N., Blaydes, B.J., Bryant, C.W., Latendresse, J.R., Weis, C.C. and Delclos, K.B., Estrogen receptor expression in the prostate of rats treated with dietary genistein, Journal of Chromatography, 777:225-236. Accepted: 5/16/2002 (E0213211) Delclos, K.B., Germolec, D., Newbold, R. and Weis, C.C., Genistein Modulates Splenic Natural Killer Cell Activity, Antibody-Forming Cell Response, and Phenotypic Marker Expression in F0 and F1 generations of Sprague Dawley Rats, Toxicological Sciences, 181:219-227. Accepted: 3/12/2002 (E0212214) Delclos, K.B., Evaluation of phytoestrogen safety and toxicity in rodent models that include developmental exposure, Phytoestrogen and Health. Accepted: 3/20/2002 (E0212201) Divi, R.L., Beland, F.A., Fu, P.P., Von Tungeln, L.S., Schoket, B., Rothman, N., Sinha, R. and Poirier, M.C., Highly-sensitive chemiluminescence immunoassay for benzo[a]pyrene-DNA adducts: validation by comparison with other methods, and use in human biomonitoring, Carcinogenesis. Accepted: 8/27/2002 (S00198) Doerge, D.R. and Chang, H.C., Inactivation of thyroid peroxidase by soy isoflavones, in vitro and in vivo, Journal of Chromatography. Accepted: 10/25/2001 (E0213201) Doerge, D.R., Churchwell, M.I. and Beland, F.A., Analysis of DNA adducts from chemical carcinogens and lipid peroxidation using liquid chromatography and electrospray mass spectrometry, Env. Carcin. Ecotoxicol. Rev., C20:1-20. Accepted: 12/28/2001 (E0706401) Doerge, D.R., Luecke, R. and Young, J.F., Metabolism and disposition of genistein, the principal soy isoflavone, Phytoestrogens and Health. Accepted: 3/1/2002 (E0713201) Doerge, D.R., Twaddle, N.C., Banks, E.P., Jefferson, W.N. and Newbold, R., Pharmacokinetic analysis in serum of genistein administered subcutaneously to neonatal mice, Cancer Letters, 184:21-27. Accepted: 4/2/2002 (E0213201) Doerge, D.R., Twaddle, N.C., Churchwell, M.I., Chang, C., Newbold, R. and Delclos, K.B., Mass spectrometric determination of p-nonylphenol metabolism and disposition following oral administration to Sprague-Dawley rats, Reproductive Toxicology, 16:45-56. Accepted: 10/13/2001 (E0213501) Dong, S., Fu, P.P., Hwang, H. and Yu, H., Effect of histidine on light-induced DNA single strand cleavage by selected polycyclic aromatic hydrocarbons, Polycyclic Aromat. Cpd., 22:451-458. Accepted: 2/7/2002 (E0657300) Dong, S., Fu, P.P., Hwang, H. and Yu, H., UVA light-induced DNA cleavage by isomeric methylbenz[a]anthracenes, Chem. Res. Toxicol., 15:400-407. Accepted: 1/8/2002 (E0657300) Dong, S., Hwang, H., Stewart, G., Fu, P.P. and Yu, H., Effect of Organic Solvents and Biologically Relevant Ions on the light-induced DNA cleavage by PAHs, Intl. J. Mol. Sci., Accepted: 9/3/2002 (E0657300) Drachtman, R.A., Cole, P.D., Golden, C.B., James Gaylor, S.J., Melnyk, S.B., Aisner, J. and Kamen, B.A., Dextromethorphan is effective in the treatment of subacute methotrexate neurotoxicity, Cancer. Accepted: 1/11/2002 (E0706501) Fang, G., Chang, C., Wu, Y., Fu, P.P. and Chang, S., Concentration of atmospheric particulates during a dust storm period in central Taichung, Taiwan, Sci. Total Environ., 287:141-145. Accepted: 6/7/2002 (E0657300) Fang, G., Chang, C., Wu, Y., Fu, P.P., Chang, S., Yang, I. And Yang, C., Characteristic study of particulate and metal elements at traffic sampling site in central Taiwan, Intl. J. Environ. Pollut. Sci., 18:64-75. Accepted: 7/6/2002 (E0657300) Fang, G., Chang, C., Wu, Y., Fu, P.P., Chen, C. and Chang, S., The study of water-soluble ions in ambient air suspended particles in Taichung, Taiwan, Aerosol Air Qual. Sci. Accepted: 6/26/2002 (E0657300) Fang, G., Chang, C., Wu, Y., Fu, P.P., Yang, C., Chen, C. and Chang, S., Ambient suspended particulate matters and related chemical species study in central Taichung, Taiwan, during 1998-2001, Atm. Environ. Sci. Accepted: 3/2/2002 (E0657300) Fang, J., Beland, F.A., Doerge, D.R., Wiener, D., Guillemette, C., Marques, M.M. and Lazarus, P., Characterization of benzo[a]pyrene-7,8-dihydrodiol glucuronidation by human tissue microsomes and over-expressed UDP-glucuronosyltransferase enzymes, Cancer Research, 62:1978-1986. Accepted: 1/31/2002 (S00198) Feng, Z., Hu, W., Rom, W.N., Beland, F.A. and Tang, M., N-Hydroxy-4-aminobiphenyl-DNA Binding in Human p53 Gene: Sequence Peference and the Effect of C5 Cytosine Methylation, Biochemistry, 41:6414-6421. Accepted: 6/24/2002 (S00198) Ferguson, S.A., Flynn, K.M., Delclos, K.B., Newbold, R. and Gough, B.J., Effects of lifelong dietary exposure to genistein or nonylphenol on amphetamine-stimulated striatal dopamine release in male and female rats, Neurotoxicology and Teratology, 24:37-45. Accepted: 10/26/2001 (E0213213) Finnell, R.H., Spiegelstein, O., Wlodarczyk, B., Triplett, A., Pogribny, I.P., Melnyk, S.B. and James Gaylor, S.J., DNA methylation in FOLBP1 knockout mice supplemented with folinic acid during gestation, J. Nutrition, in press. Accepted: 10/1/2001 (E0706501) Fu, P.P., Von Tungeln, L.S., Xia, Q., Zhan, D. and Heflich, R.H., Effect of Nitro Orientation on Ras-Protooncogene Mutation in Liver Tumors from 7-Nitrodibenz[A,H]Anthracene-Treated Mice, Polycyclic Aromatic Compounds, 22:853-859. Accepted: 12/22/2001 (E0687901) Fu, P.P., Xia, Q. and Chou, M.W., Genotoxic Pyrrolizidine Alkaloids – Mechanisms Leading to DNA Adduct Formation and Tumorigenicity, Int'l J. Mol. Sci. Accepted: 7/30/2002 (E0710401) Fu, P.P., Yang, Y., Xia, Q., Chou, M.W., Cui, Y. and Lin, G., Pyrrolizidine alkaloids-tumorigenic components in Chinese herbal medicines and dietary supplements, J. Food Drug Analys. Accepted: 8/28/2002 (E0710401) Ge, Y., Jensen, T., James Gaylor, S.J., Weinstein, H.J., Becton, D.L., Massey, G.V., Ravindranath, Y., Matherly, L.H. and Taub, J.W., High frequency of the 844ins68 cystathionine-B-synthase gene variant in Down syndrome children with acute myeloid leukemia, Leukenua, 16. Accepted: 6/15/2002 (E0708501) Gorlewska, K., Teitel, C.H., Lay, J.O., Roberts, D.W. and Kadlubar, F.F., The role of lactoperoxidase-catalyzed activation of aromatic and heterocyclic amines in relation to breast carcinogenesis, Chemical Research in Toxicology, in press. Accepted: 7/11/2002 Gorlewska, K., Teitel, C.H., Roberts, D.W. and Kadlubar, F.F., Lactoperoxidase-catalyzed activation of aromatic and heterocyclic amines and its potential role in breast carcinogenesis, Chem Res in Tox. Accepted: 7/12/2002 (E0697801) Halsted, C., Villanueva, J., Devlin, A.M., Parkkila, S., Niemela, O., Garrow, T., Wallock, L.M., Shigenaga, M.K., Melnyk, S.B. and James Gaylor, S.J., Folate deficiency disturbs hepatic methionine metabolism and promotes liver injury in the ethanol fed micropig, PNAS, In Press. Accepted: 6/1/2002 (E0706501) Hobbs, C.A. and James Gaylor, S.J., Preferential transmission of the MTHFR 677T allele to infants with Down syndrome: implications for a survival advantage, Am. J. Med. Genet., 2002:In Press. Accepted: 2/1/2002 (E0701601) Howard, P., Couch, L.H., Patton, R.E., Eppley, R., Doerge, D.R., Churchwell, M.I., Marques, M.M. and Okerberg, C.V., Comparison of the toxicity of seven fumonisin derivatives in a 28-day feeding study with female B6C3F1 mice, Toxicology and Applied Pharmacology. Accepted: 9/30/2002 (E0212401) James Gaylor, S.J., Hobbs, C.A. and Hine, R., Folate deficiency and the molecular determinants of chromosomal instability: possible link to Down syndrome and meiotic nondisjunction, Folate and Human Development, Chp 3:43-70. Accepted: 1/1/2002 (E0701601) James Gaylor, S.J., Melnyk, S.B., Pogribna, M.V., Pogribny, I.P. and Caudill, M.A., Elevation in S-adenosylhomocysteine and DNA hypomethylation: potential epigenetic mechanism for homocystein-related pathology, Journal of Nutrition, In Press. Accepted: 11/1/2001 (E0706501) Jernigan, S.L., Melchior, W.B., Jenkins, R.E., Rowland, K.L., Roberts, D.W., Howard, P. and Tolleson, W.H., Characterization of a POROStm fumonisin B1 affinity column for isolating ceramide synthase from rat liver, Journal of the Arkansas Academy of Science, 55:75-81. Accepted: 10/16/2001 (E0705901) Ju, Y.H., Doerge, D.R., Allred, K.F., Allred, C.D. and Helferich, W.G., Interaction of genistein with tamoxifen on growth of estrogen-dependent human breast cancer (MCF-7) cells implanted on ovariectomized athymic mice, Cancer Research, 62:2474-7. Accepted: 2/15/2002 (E0713201) Laurenzana, E.M., Balasubramanian, G., Weis, C.C., Blaydes, B.J., Newbold, R. and Delclos, K.B., Effect of Nonylphenol on Serum Testosterone Levels and Testicular Steroidogenic Enzyme Activity in Neonatal and Pubertal Rats, Chemico-Biological Interactions, 139:23-41. Accepted: 10/10/2001 (E0213501) Marques, M.M., Gamboa da Costa, G., Blankenship, L., Culp, S. and Beland, F.A., The effect of deuterium and fluorine substitution upon the mutagenicity of N-hydroxy-2,6-dimethylaniline, Mutation Research. Accepted: 4/16/2002 (S00198) Moody, J.D., Doerge, D.R., Freeman, J.P. and Cerniglia, C.E., Degradation of biphenyl by Mycobacterium sp. PYR-1, Applied Microbiology and Biotechnology, 58:364-369. Accepted: 10/19/2001 (E0690101) Moody, J.D., Freeman, J.P., Fu, P.P. and Cerniglia, C.E., Biotransformation of mirtazapine by Cunninghamella elegans, Drug Metabolism and Disposition. Accepted: 7/21/2002 (E0690101) Parsons, B.L., Culp, S., Manjanatha, M. and Heflich, R.H., Occurrence of H-ras codon 61 CAA to AAA mutation during mouse liver tumor progression, Carcinogenesis, 23:943-948. Accepted: 1/23/2002 (E0704101) Pogribny, I.P. and James Gaylor, S.J., De novo methylation of the p16ink4A gene in early preneoplastic liver and tumors induced by folate/methyl deficiency in rats, Cancer Letters, in press. Accepted: 1/1/2002 (E0687501) Pogribny, I.P. and James Gaylor, S.J., Methylation of the p53 promoter region is associated with reduction of gene expression in human primary hepatocellular hepatoma, Cancer Letter, 176:169-74. Accepted: 12/1/2001 (E0687501) Ritter, C.L., Culp, S., Freeman, J.P., Marques, M.M., Beland, F.A. and Malejka-Giganti, D., DNA Adducts from Nitroreduction of 2,7-Dinitrofluorene, a Mammary Gland Carcinogen, Catalyzed by Rat Liver or Mammary Gland Cytosol, Chem. Res. Tox., 15:536-544. Accepted: 2/15/2002 (S00198) Sams, R.L., Couch, L.H., Miller, B.J., Okerberg, C.V., Warbritton, A.R., Wamer, W., Beer, J. and Howard, P., The effects of alpha- and Beta-Hydroxy acids on the edemal response induced in female SKH-1 mice by simulated solar light, Toxicology and Applied Pharmacology. Accepted: 7/12/2002 (E0213101) Sheehan, D.M. and Doerge, D.R., Goitrogenic activity of soy isoflavones, Environmental Health Perspectives, 110(53):349-353. Accepted: 1/7/2002 (E0213201) Sibani, S., Melnyk, S.B., Pogribny, I.P., Wang, W., Hiou-Tim, F., Deng, L., Trasler, J., James Gaylor, S.J. and Rozen, R., Studies of methionine cycle intermediates (SAM, SAH), DNA methylation and impact of folate deficiency on tumor numbers in Min mice, Carcinogenesis, 23:61-65. Accepted: 10/1/2001 (E0706501) Tolleson, W.H., Doerge, D.R., Churchwell, M.I., Marques, M.M. and Roberts, D.W., Metabolism of biochanin A and formononetin by human liver microsomes in vitro, Journal of Agricultural and Food Chemistry, 50(17):4783-4790. Accepted: 6/3/2002 (E0705701) Trasler, J., Deng, L., Melnyk, S.B., Pogribny, I.P., Hiou-Tim, F., Sibani, S., Oakes, C., Li, E., James Gaylor, S.J. and Rozen, R., Impact of Dnmt1 deficiency, with and without low folate diets, on tumor numbers and DNA methylation in Min mice, Carcinogenesis. Accepted: 8/15/2002 (E0712801) Twaddle, N.C., Churchwell, M.I. and Doerge, D.R., High throughput quantification of soy isoflavones in human and rodent blood using LC with electrospray-MS and MS/MS detection, Journal of Chromatography. Accepted: 10/25/2001 (E0213201) Von Tungeln, L.S., Hamilton, L.P., Dobrovolsky, V.N., Bishop, M.E., Shaddock, J.G., Heflich, R.H. and Beland, F.A., Frequency of Tk and Hprt lymphocyte mutants and bone marrow micronuclei in B6C3F1ITK+/-mice treated neonatally with zidovudine and lamivudine, Carcinogenesis. Accepted: 6/3/2002 (E0687901) Von Tungeln, L.S., Yi, P., Bucci, T.J., Lomax, L.G., Samokyszyn, V.M., Chou, M.W., Kadlubar, F.F. and Fu, P.P., Tumorigenicity of chloral hydrate, trichloroacetic acid, trichloroethanol, malondialdehyde, 4-hydroxy-2-nonenal, crotonaldehyde, and acrolein in the B6C3F1 neonatal mouse, Cancer Letters, 185:13-19. Accepted: 7/8/2002 (E0657300) Williams, L.D., Chou, M.W., Yan, J., Young, J.F., Chan, P.C. and Doerge, D.R., Toxicokinetics of riddelliine, a carcinogenic pyrrolizidine alkoloid, and metabolites in rats and mice, Toxicology and Applied Pharmacology. Accepted: 5/13/2002 (E0213301) Wu, Y., Fang, G., Fu, P.P. and Yang, C., The measurements of ambient particulates (TSP, PM2.5, PM2.5-10), chemical component concentration variation, and mutagenicity study during 1988-2001 in Central Taiwan, Environ. Carcino. & Ecotox. Revs., C20(1):45-59. Accepted: 12/18/2001 (E0657300) Wu, Y., Fang, G., Moody, J.D., Von Tungeln, L.S., Fu, P.P., Hwang, H. and Yu, H., In vitro Metabolism of Dibenzo[a,l]pyrene, 2-Chlorodibenzo[a,l]pyrene and 10-Chlorodi-benzo[a,l]pyrene – Effects of Chloro Substitution, Intl. J. Mol. Sci., In Press. Accepted: 6/28/2002 (E0657300) Yan, J., Nichols, J.A., Yang, Y., Fu, P.P. and Chou, M.W., Detection of Riddelliine-derived DNA Adducts in Blood of Rats Fed Riddelliine, Internatl J of Molecular Sciences. Accepted: 8/1/2002 (E0213301) Yi, P., Pogribny, I.P. and James Gaylor, S.J., Multiplex PCR for simultaneous detection of 677 C-T and 1298 A-C polymorphisms in methylenetetrahydrofolate reductase gene, Cancer Letters, 181:209-213. Accepted: 1/1/2002 (E0701601) Yu, H., Dong, S., Fu, P.P. and Hwang, H., UVA Light-Induced Single Strand Cleavage by Hydroxybenzo[A]Pyrenes, Polycyclic Aromatic Cpd, 22:861-870. Accepted: 12/22/2001 (E0657300) Zeng, K., Hwang, H., Fu, P.P. and Yu, H., Identification of 1-Hydroxypyrene Photoproducts and Study of the Effect of Humic Substances on its Photolysis, Polycyclic Aromatic Compounds, 22:459-467. Accepted: 1/1/2002 (E0657300) Concept PapersPI: Delclos, KennethTitle: Concept - Protective Effects of Soy-containing Diets Against Renal
Toxicity: Implications for Animal Toxicity Assessments Objective(s): Examine the effects of diet and timing of exposure on renal toxicity induced by nonylphenol, di(2-ethylhexyl)phthalate (DEHP), and mono(2-ethylhexyl)phalate (MEHP). Title: Optimization of Procedures for 1) laser capture microdissection of
rat kidney for gene and protein expression studies and 2) measurement of renal
cyclooxygenases, antioxidant enzymes and isoprostanes Objective(s):
PI: Roberts, DeanTitle: Concept - Two-Dimensional micro-LC Proteomics using Stable-isotope
Affinity Tags for Differential Display of Toxicity-induced Biomarkers Objective(s):
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