Grantee Research Project Results
2000 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, 1999 through June 30, 2000
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 long-term objective of this project is to determine the role of developmental age and interindividual variation on the ability of fetal liver tissues to detoxify dietary transplacental carcinogens. We are using the potent dietary transplacental carcinogen aflatoxin B1 (AFB1) as a model. Our goal is to identify risk factors for fetal cell injury that are based upon biotransformation enzyme expression and may be linked to the development of cancers in childhood.Progress Summary:
Substantial progress has been made in two areas. In the first study, we have been characterizing interindividual and ontogenic variation in the expression of AFB1 metabolizing enzymes among individuals, as such variation may contribute to individual and developmental differences in the susceptibility to AFB1- induced cancers. In this study, we have examined the interindividual variability in AFB1 metabolizing enzymes in a panel of 10 prenatal and 10 adult liver tissues using quantitative Western blotting. We observed interindividual variation (> three-fold) in the expression of enzymes that bioactivate AFB1 to the potent DNA-binding intermediate, aflatoxin B1-8,9-exo-epoxide (AFBO), including adult CYP1A2 and 3A4, and fetal CYP3A7. The expression of lipoxygenase, another AFB1 bioactivating enzyme, was 9-fold higher in prenatal liver tissues than in adult liver tissues. hGSTM1-1, a glutathione S-transferase enzyme reported to be protective against AFBO-DNA injury, was detected in 30 percent of the adult livers, as well as 40 percent of the prenatal livers examined. The levels of hGSTM1-1 expression were higher in the adult hGSTM1-1-positive tissues compared to hGSTM1-1-positive prenatal tissues. The expression of other enzymes that may be important in protecting against AFB1, including AFB1 reductase and microsomal epoxide hydrolase, did not markedly vary (< three-fold) among donors. Thus, this study demonstrated important ontogenic and individual differences in the expression of AFB1 metabolizing enzymes. The relationship among the expression of AFB1 metabolizing enzymes and in vitro AFB1-DNA and AFB1-protein adduct formation are currently being analyzed to determine if interindividual differences in enzyme expression are correlated to DNA injury.A second study is underway that is designed to evaluate and compare AFB1-induced alterations in stress gene expression patterns in human adult and prenatal liver slices. Precision-cut liver slices were prepared from adult and second trimester prenatal liver and incubated in the presence of 0.5 µM AFB1 for 18 hour. Control and AFB1-exposed slices were harvested for preparation and labeling of poly(A)+ RNA, which was used to probe a commercial nylon stress gene microarray comprising 234 cDNA clones involved in cellular defense against chemical toxicity. A comparison of the control adult and prenatal liver slices revealed that a number of genes involved in chemical biotransformation, excision repair, and oxidative defense, were expressed at markedly lower levels in prenatal liver compared to adult liver. These data are supportive of our first study showing differences in AFB1 activating and detoxification enzymes among the two age classes. Prenatal liver slices exposed to AFB1 increased the expression of human glutathione S-transferases pi-1 (hGSTP1) and omega-1 (hGSTO1), as well as DNA excision repair ERCC1 and heat shock protein 27 (HSP27). Collectively, our data suggest that a number of protective pathways, including GST, HSP and DNA excision repair may be increased in prenatal liver on exposure to AFB1. The results of this preliminary study also indicate that DNA microarray technology may be used in conjunction with cultured liver slices to examine ontogenic differences in chemical-mediated expression of relevant human genes. This approach has potential applications for evaluating human risk to transplacental exposure to dietary carcinogens.
Future Activities:
The major objectives for the next reporting period will be to: (1) complete studies using the stress gene array and cultured liver slices to determine developmental differences in the pattern of stress gene induction by AFB1, (2) complete the comparative in vitro DNA binding experiments using archived liver tissues, and (3) initiate a comparative in vitro study with archived placental tissues to determine interindividual variation in expression of AFB1 metabolizing genes and to in vitro AFB1-DNA binding. By the end of this reporting period we hope to better understand if there are substantial developmental differences in susceptibility to AFB1-induced cell injury in vitro that could form the basis for an increased sensitivity to developing cancer during childhood from transplacental AFB1 exposure.Journal Articles on this Report : 1 Displayed | Download in RIS Format
| Other project views: | All 18 publications | 5 publications in selected types | All 5 journal articles |
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Gallagher EP, Sheehy KM. Effects of phenytoin on glutathione status and oxidative stress biomarker gene mRNA levels in cultured precision human liver slices. Toxicological Sciences 2001;59(1):118-126. |
R827441 (2000) 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, decision making, southeast, EPA Region 4., RFA, Scientific Discipline, Health, Genetics, Health Risk Assessment, Epidemiology, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Biochemistry, Children's Health, genetic susceptability, Molecular Biology/Genetics, sensitive populations, aflatoxin B1, heterocyclic amines, adolescents, carcinogenesis, childhood cancer, fetal metabolism, cytochrome P450, detoxification enzymes, exposure, DNA reactive metabolites, dietary procarcinogens, children, cancer risks, human exposure, susceptibility, children's vulnerablity, assessment of exposure, genetic risk factors, biotransformation, epidemeology, environmentally caused disease, bioactivated environmental toxicants, transplacental exposure to mutagenic agents, aflotoxin, biomedical research, genetic susceptibility, toxicsRelevant Websites:
http://www.ufbi.ufl.edu/physdept/gallagher.htmProgress 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.