Grantee Research Project Results
2001 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, 2001 through May 31, 2002
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 overall objective of this research project is to test the hypothesis that the binding of chlorotriazines by hemoglobin and hair proteins can be used to evaluate differences in exposure and in individual sensitivity toward chlorotriazines. The specific objectives of this research project are to: (1) further refine gas chromatography/mass spectroscopy (GC/MS) methods to assess the reactivity of chlorotriazines and metabolites with thiol-containing amino acid residues in hemoglobin; (2) determine whether hair binding of sulfhydryl reactive triazines can be used as noninvasive measures of exposure to these triazines; (3) develop physiologically based pharmacokinetic (PBPK) models for juvenile and adult ages utilizing blood protein and hair protein binding. Binding will be used to assess tissue exposure to total chlorotriazines in relation to ambient exposure; and (4) develop these PBPK models with protein binding measurements to recreate exposure characteristics in laboratory animals and in a limited set of human blood and hair samples.
Progress Summary:
During this time period, there were no significant issues associated with conducting the proposed research or the quality assurance/quality control (QA/QC) associated with the work. There were no changes in the scope or objectives. However, Dr. Andersen has accepted a position as Director, Division of Biomathematics and Physical Sciences, at Chemical Industry Institute of Toxicology (CIIT) Center for Health Research (CIIT-CHR) in Research Triangle Park, NC. Due to this change, Dr. Tessari will become the Principal Investigator and the primary contact at Colorado State University (CSU).
Our research in the second year of the project has expanded into several main areas. We continue to look into the isolation of globin for analysis of sites of atrazine binding, we have developed an analytical method for measuring atrazine and metabolites in blood and urine, we are beginning to understand the time-course by which triazines are extracted from plasma/red blood cells (RBCs), and we are continuing our efforts in assessing reactions of atrazine with blood proteins in vitro. We have developed a PBPK model, with blood, body, and brain compartments, to estimate total plasma chlorotriazine. This model will help support our biomonitoring/exposure assessment studies. We also have started to investigate the question of which triazines are extracted from plasma into hair in amounts sufficient to detect and quantify in localized sections along hair fibers.
Globin Isolation/Hemoglobin Binding. We have successfully isolated globin from the whole blood of rodents dosed with atrazine and diaminochlorotriazine (DACT). Portions of this isolate have been sent to Dr. Heiko Kafferlein (working with Dr. Andersen) at CIIT-CHR for analysis using high performance liquid chromatography (HPLC)/mass spectrometry (MS) for purity. We also have been working on refining our radiometric techniques to determine the in vitro binding of radioactive atrazine and DACT to hemoglobin in whole blood samples.
Analytical Method. During the past year, we have developed an analytical method for the analysis of atrazine and its chlorinated metabolites from plasma. This method was presented to the Society of Toxicology in March 2002. This is a very important method, as analytical methods for major metabolites in blood have not yet been reported.
Time-Course Studies. Several time-course studies were undertaken during this time period. We investigated the time rate for parent atrazine to form metabolites, after a single-oral gavage dose. Parent atrazine was eliminated within the 72-hour time point with the major metabolite DACT peaking at 8 hours after dosing. We also investigated the time-course of the major metabolite DACT. The peak DACT concentrations appeared at 4 hours after dosing. We also investigated time-course elimination of the two other atrazine metabolites desisopropylatrazine, and desethylatrazine. This test is still in progress, and the results will be tabulated at completion.
PBPK Model Development. A manuscript that describes the modeling work has been submitted for publication to the Journal of Toxicology and Environmental Health. The title of this manuscript is "Pharmacokinetic modeling with disposition and time-course studies with 14C-atrazine."
Hair. We have started to define objectives for our hair experiments, and after reviewing the relevant literature, we are starting to plan a course of action for determining the extent of binding of chlorotriazine to cysteine residues in hair proteins. We are attempting to answer the following questions: Which triazines are excreted from plasma/RBCs into hair in amounts sufficient to detect and quantify in localized sections along hair fibers? Quantitatively, how do the various triazines distribute between keratin and melanin in the hair matrix? How stable is trazine binding? How variable is the hair partitioning rate between triazine compounds? What are the stoichiometric and temporal relationships between triazine plasma concentrations and the binding of triazines to keratin and melanin? We have investigated techniques by which we can pretreat and extract hair.
Future Activities:
Future activities include:
(1) Confirming the present PBPK model by measuring plasma protein and Hb binding kinetics in vitro.
(2) Completing time-course experiments for atrazine, desethylatrazine, deisopropylatrazine and 2-chloro-4,6-diamino-1,3,5-triazine DACT.
(3) Investigating the pretreatment and digestion/extraction of hair. Most reviews in the literature are for the determinations of amphetamines. These methods will have to be refined and further developed to achieve our goals for the triazines.
(4) Continuing work with Drs. Andersen and Heiko Kafferlein (Postdoctoral Fellow at CIIT-CHR), to whom we have sent isolated globin for liquid chromatography (LC)/MS analysis and identification.
(5) Continuing construction and refining of PBPK models that will link exposure and circulate triazine levels to produce a comprehensive model of triazine binding to hemoglobin/hair proteins.
(6) Continuing enhancement of current risk assessment procedures for the triazine compounds.
Journal Articles on this Report : 4 Displayed | Download in RIS Format
Other project views: | All 28 publications | 7 publications in selected types | All 7 journal articles |
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Barton HA, Deisinger PJ, English JC, Gearhart JM, Faber WD, Tyler TR, Banton MI, Teeguarden J, Andersen ME. Family approach for estimating reference concentrations/doses for series of related organic chemicals. Toxicological Sciences 2000;54(1):251-261. |
R828610 (2001) R828610 (Final) |
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Brzezicki JM, Andersen ME, Cranmer BK, Tessari JD. Quantitative identification of atrazine and its chlorinated metabolites in plasma. Journal of Analytical Toxicology 2003;27(8):569-573. |
R828610 (2001) R828610 (2002) R828610 (Final) |
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McMullin TS, Brzezicki J, Cranmer B, Tessari J, Andersen M. Pharmacokinetic modeling of disposition and time-course studies with [14C] atrazine. Journal of Toxicology and Environmental Health-Part A 2003;66(10):941-964. |
R828610 (2001) R828610 (2002) R828610 (Final) |
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Pinnella KD, Cranmer BK, Tessari JD, Cosma GN, Veeramachaneni DNR. Gas chromatographic determination of catecholestrogens following isolation by solid-phase extraction. Journal of Chromatography B: Biomedical Sciences and Applications 2001;758(2):145-152. |
R828610 (2001) R828610 (Final) |
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Supplemental Keywords:
exposure, risk assessment, health effects, children, susceptibility, chemicals, atrazine, chlorotriazine, hemoglobin, hair proteins, adducts, physiologically based pharmacokinetic, PBPK, biomarkers., 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
- 2000 Progress Report
- Original Abstract
7 journal articles for this project