Grantee Research Project Results
2000 Progress Report: Chlorotriazine Protein Binding: Biomarkers of Exposure & Susceptibility
EPA Grant Number: R828610Title: Chlorotriazine Protein Binding: Biomarkers of Exposure & Susceptibility
Investigators: Andersen, Melvin E. , Tessari, John D.
Institution: Colorado State University
EPA Project Officer: Aja, Hayley
Project Period: June 1, 2000 through May 31, 2003 (Extended to May 31, 2006)
Project Period Covered by this Report: June 1, 2000 through May 31, 2001
Project Amount: $710,617
RFA: Biomarkers for the Assessment of Exposure and Toxicity in Children (2000) RFA Text | Recipients Lists
Research Category: Environmental Justice , Children's Health , Human Health
Objective:
The main objective of this research project is to test the hypothesis that binding of chlorotriazines (Cl-TRIs) by hemoglobin and hair proteins can be used to evaluate differences in exposure and differences in individual susceptibility of children toward these Cl-TRIs. In this proposal, we will measure hemoglobin and hair binding in laboratory animals treated with triazines, predict differences in Cl-TRI pharmacokinetics for individuals of different ages, and evaluate background binding of triazines to hemoglobin and hair in limited numbers of human samples.Progress Summary:
This first year of research has covered several areas related to preparation of the laboratory and research team for the studies of atrazine binding to proteins. These areas are isolating globin for analysis of sites of atrazine binding to these proteins, measuring atrazine and similar compounds in blood and urine, assessing reactions of atrazine with blood proteins in vitro, and refining pharmacokinetic models that will eventually support biomonitoring/exposure assessment studies. A brief report on each of these areas follows:
- Separation of hemoglobin and isolation and determination of globin purity: The method that we followed was first described by Rossi Fanelli, et al. (1958). Whole blood was centrifuged and the red blood cells (RBCs) separated and washed with a phosphate-buffered saline solution. The RBCs were lysed and the cell membranes were removed. The lysate was passed through a conditioned Sephadex size exclusion-chromatography column. Hemoglobin was collected with cold acidified acetone, and globin was precipitated on ice by incubation and centrifugation. The globin was washed with cold acidified acetone and dried in a 35-40?C oven.
- Gas chromatographic (GC) determinations: We were able to develop a GC method for the analysis of triazine compounds in whole blood. This method utilizes a solid phase extraction column (C-18, 500 mg, 3.0 ml) for cleanup of the matrix prior to injection onto the GC.
- Gas chromatographic/mass selective detector (GC/MS) determinations: Basically, the extraction/cleanup method that was described above for the GC determinations was used for the GC/MS instrumentation. The GC/MS in the selected ion monitoring (SIM) mode was used. Detection limits with the GC/MS are 10 ppb for atrazine and 50 ppb for the other triazine compounds. A dynamic range of 50-1000 ppb for all the triazines easily can be attainable. It is anticipated that a lower detection limit should be attainable. A lower detection of 1.0 ppb will be attempted.
- Incubation of whole blood with 14C atrazine: The labeled atrazine work began with efficiency determination of the liquid scintillation counter. Once the efficiency was established, we determined the minimum detection limit of the instrument for 14C-labeled atrazine. The rate of atrazine binding to RBCs over time was then investigated. Two experiments were designed to determine this binding rate. The first experiment showed that the cells were being lysed even after the 1-hour incubation. There was a small amount of binding to the globin in the first experiment, but the majority of the activity was lost in the rinse step (due to excess lysing). The second experiment showed linear binding over time, with correlation coefficients >0.9. There still is a substantial amount of activity being lost in the rinse step.
- Atrazine physiologically based pharmacokinetic (PBPK) modeling: We have developed a mechanistic PBPK model to describe the time course concentrations of atrazine and its metabolites in plasma, RBCs, and various tissues. Atrazine is metabolized to chlorinated metabolites via P450 and nonchlorinated, conjugated metabolites via glutathione (GSH). The PBPK model tracks the metabolites as two separate pools: Cl-TRIs and GSH conjugates. All kinetic data available to develop the model measured total atrazine equivalents, not individual metabolites. Using data sets on urinary metabolites and amounts over time, this PBPK model allows assessment of tissue and plasma exposures to Cl-TRIs separate from total parent compound exposure.
Previous radiolabeled studies for RBC reactivity with Cl-TRIs (Hamboeck, et al., 1981) indicate binding of Cl-TRIs to hemoglobin. Therefore, we initially incorporated this binding into our PBPK model. Based on multiple dose data of total radioactivity accumulated in the RBCs (Thede, et al., 1986), we estimated a second-order rate constant of 0.007 L/hr/mmol for hemoglobin binding to Cl-TRIs. For both single and multiple dose data, we observed that the model with only RBC binding under estimated plasma levels at longer time points after parent compound and GSH conjugates had been removed from the plasma. It appears that bound components are present at these longer times. By adding a term for plasma protein binding to Cl-TRIs, the new PBPK model simultaneously describes urine and plasma radioactivity more accurately. Although there are no experimental data describing plasma protein binding of Cl-TRIs, our PBPK modeling results strongly indicate the general ability of Cl-TRIs to covalently react with other proteins in blood plasma. We currently are performing 14C atrazine-binding studies with whole blood and with plasma proteins to confirm this hypothesis.
Future Activities:
We will continue experiments with radiolabeled forms of atrazine and atrazine metabolites to measure rates of binding of the Cl-TRIs with hemoglobin. These incubations will be performed using RBCs from rats and packed outdated RBCs from humans. We will continue to incubate RBCs with labeled reactant for various times and at various reactant concentrations. The relationship between bound radioactivity and duration/concentration will be used to estimate second-order rate constants for each reactant. The results from the experimental studies to determine the rate constant for RBC binding will be compared with the constants inferred from the PBPK model. We also are performing studies to assess the extent of plasma protein binding to atrazine. Once we confirm these binding results, we will be submitting a paper on the PBPK model for publication.We will determine whether binding of sulfhydryl reactive triazines to hair proteins could be used as a noninvasive measure of exposure to these triazines. The existing atrazine PBPK model will be refined by addition of a hair compartment, including hair adduct formation. After comparing the predicted model results with actual data from in vivo studies, the PBPK model will be refined to relate ambient exposure to tissue exposure as stated in the grant proposal.
We will further refine GC/MS techniques for measuring the reactivity of Cl-TRIs and metabolites with thio-containing amino acid residues in hemoglobin. We will continue to answer the questions?can existing GC/MS methods for hemoglobin-cysteine adducts be easily adapted to assess atrazine and atrazine metabolite reactions with hemoglobin? Are protein-triazine bonds sufficiently stable to integrate exposures over the entire life of the rat blood cell? If existing analytical methods are not sufficient, we will attempt to develop new techniques of measurement.
Journal Articles:
No journal articles submitted with this report: View all 28 publications for this projectSupplemental Keywords:
children, biomarkers of exposure, susceptibility., RFA, Scientific Discipline, Health, Toxics, Environmental Chemistry, Health Risk Assessment, pesticides, Susceptibility/Sensitive Population/Genetic Susceptibility, Biochemistry, Children's Health, genetic susceptability, Biology, health effects, pesticide exposure, metabolites, hemaglobin binding, tissue reactivity, endocrine disruptors, Human Health Risk Assessment, chlorotriazine protein binding, susceptibility, harmful environmental agents, pharmacokinetc model, triazine herbicides, atrazine, biological markers, growth & development, chlorotriazine, protein bindingRelevant Websites:
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.
Project Research Results
- Final Report
- 2004 Progress Report
- 2003 Progress Report
- 2002 Progress Report
- 2001 Progress Report
- Original Abstract
7 journal articles for this project