Grantee Research Project Results
Final Report: Biomarkers for Air Pollutants: Development of Hemoglobin Adduct Methodology for Assessment of Exposure to Butadienes and Polycyclic Aromatic Hydrocarbons, SEER project of SIP: Experimental Program To Stimulate Competitive Research (EPSCoR) From The Commonwealth Of Kentucky
EPA Grant Number: R829419E02Title: Biomarkers for Air Pollutants: Development of Hemoglobin Adduct Methodology for Assessment of Exposure to Butadienes and Polycyclic Aromatic Hydrocarbons, SEER project of SIP: Experimental Program To Stimulate Competitive Research (EPSCoR) From The Commonwealth Of Kentucky
Investigators: Hurst, Harrell E. , Myers, Steven R.
Institution: University of Louisville
EPA Project Officer: Chung, Serena
Project Period: October 1, 2002 through September 30, 2004 (Extended to September 30, 2005)
Project Amount: $219,287
RFA: EPSCoR (Experimental Program to Stimulate Competitive Research) (2000) RFA Text | Recipients Lists
Research Category: EPSCoR (The Experimental Program to Stimulate Competitive Research)
Objective:
This is a Science and Engineering Environmental Research (SEER) project of the Strategic Implementation Plan (SIP). The objective of this research project was to develop methodology that will measure systemic exposures to chloroprene (2-chloro-1,3-butadiene) and selected polycyclic aromatic hydrocarbons (PAHs), specifically fluoranthene and benzo(a)pyrene. The methods involved detection and measurement of covalent adducts to the abundant blood protein hemoglobin (Hb) as biomarkers of exposure. The postulated adducts are formed by electrophilic epoxide metabolites of these compounds. Analysis involved synthesis of derivatives of chloroprene epoxide adducts through Edman cleavage of globin N-terminal valine adducts, or hydrolysis of PAH adducts bound at other more labile sites. Quantification of adducts derived from chloroprene was accomplished by selected ion monitoring gas chromatography/mass spectrometry (SIM-GC/MS) using stable isotope internal standards. Adducts from PAH exposure are analyzed after acid hydrolysis of labile PAH-Hb carboxylate adducts.
Summary/Accomplishments (Outputs/Outcomes):
Component 1: Development of Methods To Detect Hemoglobin (Hb) Adducts of Chloroprene—Harrell E. Hurst, Ph.D.
During the final year of this grant, research focused on refinement of working assays for measurement of Hb adducts. Selected ion monitoring GC/MS methods have been developed for assay of Hb adducts formed from (1-chloroethenyl) oxirane, also known as chloroprene epoxide or CEO. The adduct detection methods rely on sequential Edman cleavage and trimethylsilylation reactions. Limits of detection and quantification of analytical methods of CEO-valine adducts are of the order of 1 and 10 picomoles of adduct per mg globin. These limits are expected to be inadequate to use this assay for chloroprene human exposure assessment at parts-per-billion levels in ambient air; however, significant exposure over days to levels in the parts-per-million range should be detectable. This research provided insight into the mechanism of detoxification of the toxicologically significant metabolite of chloroprene, CEO. Reactions of CEO with N-terminal valines in mouse red cell Hb have been characterized kinetically, and these studies indicate a half-life of formation in vitro of about 6 hours. Additionally, a method was developed to resolve chromatographically the R- and S-enantiomers of CEO. The methods were used to explore a glutathione-dependent mechanism of degradation of CEO, an active metabolite of chloroprene. From the latter work, enantiomeric selectivity in the destruction of chloroprene epoxide was discovered using a red blood cell incubation system in vitro. These studies noted that the S-enantiomer of CEO was destroyed very quickly in vitro, with a half-life of about 0.09 hours, whereas the R-enantiomer half-life was about 2 hours. This marked difference is abolished if the red cells are pretreated with diethyl maleate (DEM), which depletes the cellular glutathione. This discovery led to formulation of the hypothesis that the enantiomeric selectivity results from glutathione S-transferase mediated degradation of the S-enantiomer of CEO. When these kinetic studies were paired with analyses of adducts in the cells, levels of adducts increased 25-fold in cells treated with DEM + S-CEO over those treated with S-CEO alone. A similar study using the R-enantiomer of CEO indicated higher levels of adducts and less that 2-fold increase in N-terminal adducts with DEM pretreatment. Taken together, it is evident that detoxification of the S-CEO occurs rapidly by glutathione conjugation. The R-enantiomer is more persistent, however, and less susceptible to detoxification via glutathione dependent mechanisms.
Component 2: Development of Hemoglobin Adduct Methodology for Assessment of Exposure Polycyclic Aromatic Hydrocarbons—Steven R. Myers, Ph.D.
The binding of selected PAH epoxides to Hb was examined to determine the overall extent of binding and to investigate the kinetics of binding of the PAH epoxide to Hb. These experiments were carried out using C57BL/6 male mouse Hb in vitro. Another set of experiments replicated the in vitro studies after giving the parent PAH compounds via intraperitoneal injection in C57BL/6 male mice. These relied on the innate mouse oxidation mechanisms to form the reactive epoxides. The assay involved hydrolytic cleavage of adducts followed by HPLC. The limit of sensitivity fluorometric detection of the released PAH tetrols is greater than 10 pmols PAH epoxide/mg protein. Following both in vitro and in vivo studies, it was noted that epoxide derivatives of PAHs form covalent adducts with Hb. These adduct levels are formed in relationship to the overall carcinogenicity of the parent compound. Similar kinetics of adduct formation were found in both mouse and human red cells suggesting that the mouse model developed could serve as a model for human adduct formation and detection.
Journal Articles:
No journal articles submitted with this report: View all 14 publications for this projectSupplemental Keywords:
hemoglobin adducts, chloroprene, polycyclic aromatic hydrocarbons, PAH, SIM-GC/MS,, RFA, Health, Scientific Discipline, Geographic Area, Ecosystem Protection/Environmental Exposure & Risk, Sustainable Industry/Business, cleaner production/pollution prevention, Environmental Chemistry, Health Risk Assessment, amphibians, State, Risk Assessments, Biochemistry, EPA Region, pollution prevention research, biomarkers, population decline, amphibian decline, clean technology, hazardous emissions, Region 3, animal model, exposure, alternative materials, air pollution, PAH, butadiene, human exposure, amphibian bioindicator, causal mechanisms, air emissions, pollution prevention, Kentucky (KY), biomarker based exposure inference, biomarker, biomarker measurements, exposure assessmentRelevant Websites:
http://www.murraystate.edu/qacd/cos/hbs/epaepscor_grant.html 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.