Grantee Research Project Results
2002 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, 2002 through May 31, 2003
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 binding of chlorotriazines by hemoglobin (Hb) 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 spectrometry (GC/MS) methods to assess the reactivity of chlorotriazines and metabolites with thiol-containing amino acid residues in Hb; (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) use 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:
Year 3 of the project has centered on two main focal areas: adduct determination studies and enzyme kinetic studies. Our progress in each of these two areas is summarized below.
Adduct Determination Studies
Radioactivity Studies
• We have refined our techniques to prepare whole blood for liquid scintillation counting, so that light/color quenching would not interfere with the instrumental analysis.
• We have performed efficiency tests to confirm that combining a solubilizing agent (Soluene-350), a bleaching agent (H2O2), and various blood matrices (plasma, washes in 0.9 percent NaCl, whole red blood cells, and Hb) do not affect the efficiency of the radioactive counter.
• We have incubated whole blood from Sprague-Dawley rats with 30 ppm 14C-Atrazine or 14C-diaminochlorotriazine (DACT) for 0, 24, and 48 hours, and analyzed the amount of radioactivity recovered in the Hb. For the DACT treatment, we found that the fraction of radioactivity recovered in the lysate was significant in relation to time. For atrazine, we found that there was no significance in the amount of radioactivity recovered in the lysate with time. Therefore, we decided to isolate, clean, and analyze the globin.
• We found that both compounds bind to rat globin in a significant time-dependent manner. These results indicate that both atrazine and DACT form protein adducts with globin; however, the amount of binding for DACT was very low (estimated to be about 3 molecules of DACT per 1,000 globin molecules), and was even lower for atrazine (about 0.5 molecules of atrazine per 1,000 globin molecules).
• Based on the radioactivity studies, we calculated that the binding rate for DACT to Sprague-Dawley rat globin was 5.004 x 10-4 L/mmol-hr. We calculated that the binding rate for atrazine to Sprague Dawley (SD) rat globin was 1.499 x 10-4 L/mmol-hr.
• We incubated whole blood from humans with 30 ppm 14C-atrazine or 14C-DACT for 0, 24, and 48 hours, and analyzed the amount of radioactivity recovered in the globin. For the DACT treatment, we found that the fraction of radioactivity recovered in the globin was even lower in humans than in rats (0.6 molecules of DACT per 1,000 molecules globin), and that atrazine did not bind at all to human blood. Maximum binding occurred within 24 hours.
Cold Studies
• We have investigated our globin isolation procedures. We wanted to confirm that we were isolating a pure globin. We tested different isolation techniques using ethyl acetate and acetone. We also used the Sephadex column chromatography procedure. We used polyacrylamide gel electrophoresis to check for globin isolation and purity. The ethyl acetate method without purification showed a clean band at 16,000 daltons; therefore, we will use this technique for future studies.
• We are investigating the identification of adduct formation (cold DACT) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We have investigated adduct formation using rat red blood cells (RBCs), whole blood, and powdered Hb.
• We have incubated whole blood from Sprague-Dawley rats with 30 ppm cold DACT for 24 hours, and have analyzed samples using MALDI-TOF MS. If adduct formed, they were below the detection limit of this technology.
• Using powdered lyophilized rat Hb (Sigma H-3883), we incubated 10 mg with 1,000 ppm DACT. Samples were analyzed using MALDI-TOF MS. The analyses showed the and globin chains are present, but also showed excessive noise and no indication of a 145 MW addition, or 110 MW if the Cl was removed from the molecule. We may be introducing some confounding factors by using lypholized powered rat Hb.
• We analyzed samples dosed with atrazine and DACT (aliquots of the samples that we sent to Heiko Kafferlein) using MALDI-TOF MS. Results indicated a peak near the b chain in both treatments. This peak represents additions of 104 daltons, atrazine treatment, and 108 daltons in DACT treatment. This is the strongest evidence yet that an adduct is formed.
• We have completed an in vitro study incubating Sprague-Dawley rat whole blood with cold DACT (0, 30, 60, and 90 ppm) and cold atrazine (90 ppm). These samples currently are being analyzed using MALDI-TOF GC/MS.
• We currently are finishing up on an in vivo dosing study using cannulated rats. Eighteen rats have been dosed with 0, 10, 30, 100, and 300 mg/kg atrazine and DACT. Blood is being drawn at 0, 24, 48, and 72 hours. Globin is being isolated and will be analyzed using MALDI-TOF GC/MS.
• In collaboration with Dr. T. Snow (DuPont Haskell-Stein Laboratory), we are investigating the tryptic peptide digestion procedure to determine and identify what specific globin chain is being formed by the adduct.
• We will continue to determine whether binding of sulfhydryl reactive triazines to hair proteins could be used as noninvasive measures of exposure to these triazines. Hair from the in vivo Hb-binding study will be used for analysis. We are continuing to develop/modify PBPK models for juveniles and adults that utilize blood protein and hair adduct levels to assess tissue exposure to total chlorotriazines in relation to ambient exposure. We will use 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.
• We have developed an analytical method for measuring atrazine and metabolites in brain samples. Brain samples from rats are homogenized and ground with sand, water, and phosphoric acid. The blended homogenate is loaded onto a serum protein electrophoresis (SPE) column. The column is rinsed with acetone and then eluted with 5 percent NH4OH in acetone. Samples are derivatized for 30 minutes at room temperature with tetrabutyl ammonium hydroxide (TBAOH) in methanol and methyl iodide. The methylated derivatives of atrazine and metabolites are analyzed using GC/MS with selective ion monitoring (SIM). The method was validated between 0.04 and 0.24 µg/mL for atrazine, and 2-12 µg/mL for DACT.
Enzyme Kinetic Studies
• We continue our efforts in assessing the in vitro determination of atrazine metabolite formation rates using isolated primary rat hepatocytes and incorporation of these rates into an in vitro enzyme kinetic model. We have developed a procedure to study atrazine metabolism by dosing and incubating hepatocytes in suspension and monitoring product formation and atrazine disappearance over a 90-minute period.
• We have developed an analytical method for the determination of atrazine and major metabolites in a primary rat hepatocyte matrix. This method uses GC/MS/SIM, and derivatization using TBAOH and methyl iodide. Recoveries of the triazines extracted hepatocytes ranges from 50 percent at time 0 and decreases over time because of glutathione (GSH) conjugation. The method was validated to 15 ng/mL (ppb). We are still investigating 50 percent recoveries at time 0, as an 80-100 percent recovery was expected at this time point.
• An in vitro kinetic enzyme model has been constructed (see Figure 1) and mono-dealkylated and di-dealkylated metabolite formation rates will be included in this model. This information can be incorporated into our in vivo PBPK model, where we currently are using relative metabolism rates to predict dose to tissue atrazine concentrations.
• Our results already show that all three alkylated triazines are metabolized to DACT. However, high concentrations of atrazine in the medium inhibit oxidation of mono-dealkylated triazines to DACT, which is indicative of competitive interactions among these compounds.
We are beginning to understand the time course by which triazines are extracted from plasma/RBCs.
A single oral gavage dose is administered at 90 mg atrazine/kg in a 1 percent methyl-cellulose suspension. The majority of total area under the curve (AUC) for chlorotriazines in plasma was DACT (> 95 percent total AUC) followed in order by de-isopropylatrazine (DeIp-atra), de-ethylatrazine (DeEt-atra), and atrazine. Equimolar oral dosing studies were completed with DACT, DeEt-atra, and DeIp-atra. Maximum absorption of DACT was observed 8 hours after dosing, followed by approximate first-order elimination with a plasma half-life of 12 hours and a rate of elimination equal to 0.0578. DeIp-atra reached higher initial plasma concentrations than did DeEt-atra following oral doses of each compound. With each compound, absorption appeared to be multiphasic, probably representing solubilization of the slurried dosing solutions after gavage. Because of rapid metabolic clearance from blood, the plasma AUC observed after dosing with DeEt-atra, DeIp-atra, or atrazine is dominated by DACT.
Figure 1. The in vitro PBPK Metabolite Model
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. An example of this model is shown in Figure 2.
Figure 2. The PBPK Model to Estimate Total Plasma Chlorotriazine
Significance of Findings
Our work will be instrumental in improving our understanding of risks of these herbicides to children. Its value, however, has to be measured in relation to two phases: (1) development of accurate tools to assess both exposure and potential susceptibility to triazine herbicides in children; and (2) use of these tools with specific populations of children who may be at higher risks. Currently, methods for assessing exposure in children are based on a series of assumptions regarding uptake and metabolic rate differences in children without general methods to accurately assess the validity of these assumptions. With the triazines, metabolite identification in urine and/or salivary or urinary analysis of atrazine lacks the sensitivity for use as anything other than a monitor for acute rather than high exposures. By completing the analytical methods, we will have broadly integrated biomarkers of exposure and susceptibility that can be applied to different juvenile populations. The PBPK model will permit calculation of expected triazine binding in various populations. Study design criteria for biomonitoring in children and workers can be established partially at least on the basis of these calculations.
Future Activities:
Our research group will continue to enhance current risk assessment procedures for the triazine compounds. We will continue to construct and refine PBPK models that will link exposure and circulating triazine levels to produce a comprehensive model of triazine binding to Hb/hair proteins. We will continue to investigate the globin analysis with the analysis and identification of adduct formation by MALDI-TOF. We hope that these results will confirm the results from the radioactive experiments. Additional future activities are as follows:
· We plan on continuing our collaboration with Dr. Snow, investigating the tryptic peptide digestion procedure and the identification of the globin chain adduct formation.
· We will complete the analysis of the in vitro study incubating Sprague-Dawley rat whole blood with DACT and atrazine.
· We will complete the in vivo dosing study using cannulated Sprague-Dawley rats with DACT and atrazine.
· We will continue our studies to incubate whole human blood with atrazine and DACT, and analyze the globin for binding as described previously.
· We will confirm the present PBPK model by measuring plasma, Hb, and brain binding kinetics in vitro.
· We will continue to investigate the pretreatment and digestion/extraction of hair.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 28 publications | 7 publications in selected types | All 7 journal articles |
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Type | Citation | ||
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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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Supplemental Keywords:
exposure, risk assessment, health effects, susceptibility, chemicals, atrazine, 14C-Atrazine, 14C-diaminochlorotriazine, DACT, chlorotriazine, hemoglobin, Hb, hair proteins, adducts, pharmacokinetic biomarkers, physiologically based pharmacokinetic, PBPK, PBPK models., 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
- 2001 Progress Report
- 2000 Progress Report
- Original Abstract
7 journal articles for this project