Grantee Research Project Results
2002 Progress Report: Fetal Metabolism of Aflatoxin B1 and Susceptibility to Childhood Cancer
EPA Grant Number: R827441Title: Fetal Metabolism of Aflatoxin B1 and Susceptibility to Childhood Cancer
Investigators: Gallagher, Evan
Institution: University of Florida
EPA Project Officer: Aja, Hayley
Project Period: July 1, 1999 through June 30, 2002 (Extended to March 30, 2004)
Project Period Covered by this Report: July 1, 2001 through June 30, 2002
Project Amount: $523,123
RFA: Children's Vulnerability to Toxic Substances in the Environment (1999) RFA Text | Recipients Lists
Research Category: Children's Health , Human Health
Objective:
The specific objectives of this research project are to: (1) fully characterize and compare the expression of enzymes that catalyze aflatoxin (AFB1) oxidation and AFB1-8,9-epoxide (AFBO) detoxification in human prenatal liver and maternal placenta; (2) compare the rates of AFBO-DNA binding and DNA repair, and the frequency of AFB1-induced mutations among adult and prenatal liver tissues; and (3) evaluate the effects of chemoprotective agents on ameliorating AFB1-induced precancerous lesions in human prenatal liver slices.
Progress Summary:
Our third reporting year studies have focused on: (1) establishing a new in vitro cell model to analyze the effects of transplacental carcinogens relevant to human prenatal liver; and (2) characterizing the inducibility of key enzymes that protect against electrophilic transplacental carcinogens in cultured human liver slices. As discussed during the prior year report, progress on our studies using cultured prenatal liver slices was hampered by technical issues related to the availability of high quality human liver tissue for cell culture. Due to time delays in switching cellular models, we were granted a 1-year, no-cost extension to complete these studies (Year 4). Our new studies have entailed using human fetal liver-derived CD34+ hematopoietic progenitor cells (FLHPC) as targets for transplacental carcinogens. Injury to FLHPC is highly relevant to potential development of hematopoietic malignancies, as these cells are capable of initiating long-term hematopoiesis. We had originally proposed some limited studies using FLHPC to determine if these cells were targets for transplacental carcinogens in prenatal cell populations.
Considerable progress has been achieved in both of the aforementioned areas. Culture conditions have been largely established for FLHPC and several key experiments have been performed. Our goal was to establish culture conditions for adequate cell proliferation, while maintaining the progenitor nature of these stem cells. We have demonstrated that cells cultured in the presence of the several growth factors (GCSF, IL-3, and SCF) maintain greater than 90 percent viability over 12 days in culture. Flow cytometry analysis of cultured cells have demonstrated that these cell lines largely retain the CD34 marker characteristics, with days 7 through 12 in culture representing 17.3 percent and 2.5 percent CD34 marker characteristics respectively. CD38 was used to determine the percentage of the cell population undergoing differentiation via the myeloid lineage. Flow cytometry data indicate that after 7 days in culture, 68 percent of all cells are in the CD38 lineage. During days 10 through 12 this value decreased and plateaued at 29 percent, suggesting that between days 7 and 10 these cells are differentiating to one of the several mature myeloid cell types. CD33 was used to discern the percentage of FLHPC proceeding through the lymphoid lineage. Day 7 flow cytometry data revealed that 18.5 percent of cells are lymphoid in nature, and by day 10, this number had increased to 41.5 percent. The percentage of cells in the lymphoid lineage declines to 9 percent and 3.6 percent by day 11 and 12, respectively. CD33 identifies lymphoid cells in transition from progenitor cells and mature lymphoid cells. This data accounts for the decreased number of cells in this lineage with respect to number of days in culture. Based upon these results, we have conducted toxicant challenge experiments after 10 days of culture. At this time, cells are proliferating and undergoing differentiation into myeloid and lymphoid lineages.
On establishing culture conditions, several toxicant challenge experiments have been conducted. Exposure to physiologically relevant levels of aflatoxin B1 did not result in loss of viability or in the formation of AFB-DNA or AFB-protein adduct formation in FLHPC. Western blotting experiments revealed a potential mechanism for the lack of sensitivity to AFB1. It appears that the FLHPC do not appreciably express the cytochrome P450 isoenzymes (CYP1A2 and CYP 3A4) that are primarily responsible for activation of aflatoxin B1 to the toxic DNA-binding derivative aflatoxin B1-8,9-epoxide. In contrast, further dose response studies revealed that FLHPC lose viability when cultured in the presence of 4-hydroxynonenal (HNE), a model mutagenic and cytotoxic aldehyde produced upon oxidative injury. The levels of 4-HNE that caused injury were relevant to those encountered under cellular oxidative stress. Furthermore, exposure to 4-HNE resulted in the formation of one or more protein adducts which are currently being identified by 2-D gel electrophoresis and MALDI-TOF analysis. Experiments performed using stem cell preparations from different donors indicate that interindividual differences may exist with respect to sensitivity to 4-HNE injury. Collectively, our results to date strongly suggest that human liver cells are not disproportionally sensitive to injury to AFB1 as originally hypothesized. However, metabolic byproducts of transplacental compounds that cause oxidative injury (e.g., 4-HNE production from maternal alcohol exposure) may be more toxicologically relevant than AFB1 with regards to FLHPC injury.
Future Activities:
Our major focus for the next reporting period will be to use the hematopoietic precursor cell line model to complete studies of the effects of low dietary relevant levels of transplacental toxicant exposure on DNA injury and gene expression. These studies will involve one or more cDNA microarray experiments. We will complete our chemoprotection studies using adult liver slices to determine if genes that protect against dietary carcinogens are readily inducible in human liver. However, because chemoprotection studies that were originally proposed may be of limited value in our new model cell line (e.g. it is highly unlikely that the physiological levels of a dietary chemoprotectant available to FLHPC via maternal transfer would be sufficient to modulate protective genes in FLHPC), we will also include studies to determine if low level exposure to relevant transplacental toxicants may affect FLHPC differentiation. This also will entail toxicant studies at earlier stages of FLHPC differentiation to establish any critical windows of susceptibility to chemical exposure. Ultimately, establishing the effects of transplacental carcinogen exposure to FLHPC with respect to cell differentiation and gene expression will have important ramifications in the etiology of blood-borne disorders that may occur during development.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 18 publications | 5 publications in selected types | All 5 journal articles |
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Doi AM, Patterson PE, Gallagher EP. Variability in aflatoxin B1-macromolecular binding and relationship to biotransformation enzyme expression in human prenatal and adult liver. Toxicology and Applied Pharmacology 2002;181(1):48-59. |
R827441 (2002) R827441 (Final) |
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Gardner JL, Doi AM, Pham RT, Huisden CM, Gallagher EP. Ontogenic differences in human liver 4-hydroxynonenal detoxification are associated with in vitro injury to fetal hematopoietic stem cells. Toxicology and Applied Pharmacology 2003;191(2):95-106. |
R827441 (2002) R827441 (Final) |
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Supplemental Keywords:
risk, risk assessment, effects, health effects, human health, metabolism, vulnerability, sensitive populations, carcinogen, teratogen, mutagen, cellular, population, enzymes, infants, children, age, diet, genetic predisposition, susceptibility, chemicals, toxics, decisionmaking, southeast, EPA Region 1, EPA Region 2, EPA Region 3, EPA Region 4, EPA Region 5, EPA Region 6, EPA Region 7, EPA Region 8, EPA Region 9, EPA Region 10., RFA, Scientific Discipline, Health, Genetics, Health Risk Assessment, Epidemiology, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Biochemistry, Children's Health, Molecular Biology/Genetics, genetic susceptability, aflatoxin B1, sensitive populations, heterocyclic amines, childhood cancer, cytochrome P450, fetal metabolism, detoxification enzymes, adolescents, carcinogenesis, DNA reactive metabolites, dietary procarcinogens, exposure, children, genetic risk factors, assessment of exposure, children's vulnerablity, susceptibility, human exposure, cancer risks, biotransformation, epidemeology, environmentally caused disease, bioactivated environmental toxicants, transplacental exposure to mutagenic agents, biomedical research, genetic susceptibility, aflotoxin, environmental hazard exposuresRelevant Websites:
http://www.ufbi.ufl.edu/physdept/gallagher.htm Exit
http://www.floridatox.org/ Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.