Exposure to environmental chemicals is considered a contributing factor to increased human susceptibility to cancer, reproductive and developmental disorders, and other diseases. The mechanism(s) of action of these chemicals is not well understood, however, most of these environmental chemicals are metabolized to epoxide intermediates via CYP2E1. We therefore hypothesized that epoxidation of these chemicals may be responsible for the induction of mutagenicity. Using acrylamide (AA) as a model chemical, studies were undertaken to investigate the metabolic and molecular basis for the induction of somatic and germ cell mutagenicity by epoxide-forming chemicals. AA is an animal carcinogen, neurotoxin, and reproductive toxin. AA is formed in baked and fried carbohydrate-rich foods as a result of processing at high temperatures. Metabolism of AA occurs via epoxidation to glycidamide (GA) or direct conjugation with glutathione. Studies were undertaken to assess the role of CYP2E1 in the epoxidation of AA to GA and the formation of DNA and hemoglobin (HGB) adducts. AA was administered to CYP2E1-null or wild-type mice at 50 mg/kg. Using LC-ES/MS/MS, AA, GA, and DNA- and HGB-adducts were measured. While the plasma levels of AA and GA were 115?14.0 and 1.7?0.31uM in CYP2E1-null mice, they were 0.84?0.80 and 33.0?6.3uM in the plasma of AA-treated wild-type mice. Administration of AA to wild-type mice caused a large increase in N7-GA-Gua and N3-GA-Ade adducts in the liver, lung, and testes. While traces of N7-GA-Gua adducts were measured in the tissues of AA-treated CYP2E1-null mice, these levels were 52-66-fold lower than in wild-type mice. Significant elevation of both AA- and GA-HGB adducts was detected in AA-treated wild-type mice. To explore the role of CYP2E1 metabolism in the germ cell mutagenicity of AA, CYP2E1-null and wild-type were treated by intraperitoneal injection with 50mg AA/kg/day for 5 consecutive days. At defined times after exposure, males were mated to untreated B6C3F1 females. Females were sacrificed in late gestation and uterine contents were examined. Dose-related increases in resorption moles (chromosomally aberrant embryos) and decreases in the numbers of pregnant females and the proportion of living fetuses were seen in females mated to AA-treated wild-type mice. No changes in any fertility parameters were seen in females mated to AA-treated CYP2E1-null mice. In a second set of experiments, we examined the role of CYP2E1 in AA-induced somatic cell damage. Female wild type and CYP2E1-null mice were administered AA (0, 25, 50 mg/kg) by intraperitoneal injection once daily for 5 consecutive days. Erythrocyte micronucleus frequencies were determined using flow cytometry and DNA damage was assessed in leukocytes, liver, and lung using the alkaline (pH>13) single cell gel electrophoresis (Comet) assay. Results were consistent with our observations in male germ cells: significant dose-related increases in micronucleated erythrocytes and DNA damage in somatic cells were induced in acrylamide-treated wild type but not in the CYP2E1-null mice. These results constitute the first unequivocal demonstration that genetic damage in somatic and germ cells of mice-treated with AA is dependent upon metabolism of the parent compound to GA by CYP2E1.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Intramural Research (Z01)
Project #
1Z01ES101589-03
Application #
7174348
Study Section
(LPC)
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2005
Total Cost
Indirect Cost
Name
U.S. National Inst of Environ Hlth Scis
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Ghanayem, Burhan I; Bai, Re; Burka, Leo T (2009) Effect of dose volume on the toxicokinetics of acrylamide and its metabolites and 2-deoxy-D-glucose. Drug Metab Dispos 37:259-63
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Hoffler, Undi; Ghanayem, Burhan I (2005) Increased bioaccumulation of urethane in CYP2E1-/- versus CYP2E1+/+ mice. Drug Metab Dispos 33:1144-50
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Hoffler, Undi; Dixon, Darlene; Peddada, Shyamal et al. (2005) Inhibition of urethane-induced genotoxicity and cell proliferation in CYP2E1-null mice. Mutat Res 572:58-72

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