Grantee Research Project Results
2003 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, 2003 through May 31, 2004
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 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 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) 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:
During this time period, there were no significant issues associated with conducting the proposed research or the quality control/quality assurance associated with the work. In addition, there were no changes in the scope or objectives.
Dr. Melvin Andersen continues to support this research while he is at CIIT-Centers for Health Research (CHR) in Research Triangle Park, NC. Dr. Andersen still is involved in the research and visited Colorado State University (CSU) and met with researchers in August 2004, although the subcontract between CSU and CIIT-CHR was completed as of May 31, 2003. Dr. Andersen continues to serve as coadvisor to Ms. Tami McMullin, a Ph.D. student working on developing the PBPK models to support our biomarker research efforts with atrazine. Dr. Andersen served as coadvisor to Ms. Pilar Prentiss (Tillberg), an M.S. student working on determining hemoglobin adduct formation. Ms. Prentiss completed her degree requirements in the summer of 2004.
Dr. Andersen will continue his support of the research effort as an adjunct professor and will continue to direct the efforts to develop PBPK models to atrazine and related chlorotriazines. A poster session will be presented at the U.S. Environmental Protection Agency Human Health Symposium–A Science To Achieve Results Progress Review Workshop in Philadelphia, PA, on October 28-29, 2004.
Research
Year 4 of the project has centered on two main focal areas.
Adduct Determination Studies
Hemoglobin/Diaminochlorotriazine (DACT) Incubations (in vitro). We incubated rat hemoglobin for 48 hours at 37ºC, with 30, 60, and 90 ppm DACT and 90 ppm atrazine (n = 3 each treatment). Samples were analyzed by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. The 30 ppm was comparable to the radioactivity studies, and the 90 ppm represented peak plasma concentrations of DACT in rat blood after treatment with 300 mg/kg atrazine. Results indicated that no binding was detected in the radioactivity studies at the same concentrations.
Globin Purification. Polyacrylamide gel electrophoresis was used to verify the purity of the globin obtained from the isolation method used. It also was used to compare the purity of globin isolated by the ethyl acetate method to that of the commonly employed acid acetone method. We could not resolve the σ-chains and β-chains, although there was a clear band at approximately 16,000 Da. No other bands were detected. A decision was made not to use the acid acetone method for the isolation of globin.
Atrazine Dose (in vivo) Study 1. Sprague Dawley rats were dosed with atrazine at 0, 10, 30, 100, and 300 ppm for 0, 24, 48, and 72 hours, 10 days, 1 month, and 2 months. We analyzed samples using MALDI-TOF MS. The results showed additions in approximately 100 MW to the β-chain of globin. A small addition peak was shown at 24 hours, a good-sized peak was shown at 48 hours, a very large peak was shown at 72 hours, a good peak was shown at 10 days, a small peak was shown at 1 month, and at 2 months the peak was gone. β-chain was added at about 104-107 Da. We postulated that if the nitrogens were deprotonated, the addition would be brought down to approximately 105-107 Da.
Atrazine Dose (in vivo) Study 2. Sprague Dawley rats were dosed with 0, 10, 30, 100, and 300 mg/kg atrazine for 3 days (n = 3) for each treatment (n = 2 controls). Blood samples were taken at the following time points: 0, 24, 48, 72 hours, 10 days, 1 month, and 2 months. Samples were analyzed using MALDI-TOF MS. The results indicated that the approximate 106 MW addition to the β-chain appeared after 24 hours in the 100 and 300 mg/kg dose groups. The addition peak gets larger after 24 hours, with a maximum at 72 hours. The peak is still present after 10 days, gets smaller after 1 month, and virtually disappears at 2 months.
Treatment of rats with both atrazine and DACT caused the formation of an additional peak that appears to be a hemoglobin adduct. As with the in vitro study, the MALDI-TOF MS analysis was not able to resolve each individual globin chain. The peaks at m/z 15152.4 and m/z 15195.4 in the control sample represent the α-chains. The four individual β-chains were not resolved and appeared as a large peak with a shoulder at m/z 15847.1. The m/z 16059.1 peak is an artifact of the acids used as the MALDI-TOF matrix. Peak resolution has an error of +/- 10 Da.
Atrazine treatment elicited a peak that was 103.9 Da heavier than the tallest peak of the combined β-chains, with 21.4 percent of the β-chains modified. DACT treatment formed a peak that was 107.7 Da heavier than the combined β-chain peak, with 36.6 percent of the β-globin modified. Determination of the percent of modified globin was achieved by dividing the peak height of modified β-globin (the adduct) by the total peak height of β-globin (modified plus unmodified). Peak height was normalized to the largest peak, which was the taller peak of unmodified ß-globin chains.
Qualitative analysis showed the peak height of the adduct to increase with respect to dose and time. Analysis of variance confirmed that there was a significant effect of dose (p < 0.0001), time (p < 0.0001), and the interaction between dose and time (p < 0.0001). The largest amount of adduct was yielded 24 hours after the third dose at 300 mg/kg/day. No adduct was detected at the dosing level of 10 mg/kg/day. For treatments of 30, 100, and 300 mg/kg/day, adduct still was present in all samples 30 days after the first dose. By 60 days post dose, most to all of the modified globin was no longer detectable. The half-life of the adduct was found to average 12 days, and the elimination rate (Kel) was 0.06 day-1.
Enzyme Kinetic Studies
We have continued our efforts in assessing the in vitro determination of atrazine metabolite formation rates using isolated primary rat hepatocytes and incorporated 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.
As reported previously, we further modified/developed the analytical method for the determination of atrazine and major metabolites in a primary rat hepatocyte matrix. This method uses GC/MS/selection ion monitoring (SIM) and derivatization using tetrabutyl ammonium hydroxide and methyl iodide. Recoveries of the triazines extracted from hepatocytes at time 0 still remain a question (low recoveries). The method still is validated to 15 ng/ml (ppb). We still are investigating 50 percent recoveries at time 0.
Our results still indicate that all three alkylated triazines are metabolized to DACT. High concentrations of atrazine in the medium, however, inhibit oxidation of mono-dealkylated traizines to DACT, indicative of competitive interactions among these compounds.
We have developed a PBPK model (as previously reported) with blood, body, and brain compartments to estimate total plasma chlorotriazine. This model currently is being modified, and improvement will help support our biomonitoring/exposure assessment studies. An example of this model is shown below:
Figure 1. PBPK Model to Estimate Total Plasma Chorotriazine
Upon reviewing the time course results, it was evident that the concentration of substrates and products changed over time and that there were characteristics of inhibition occurring in these incubations. Therefore, basic Michaelis-Menten enzyme kinetics would not adequately produce Vmax and Km for this study. A more complex kinetic analysis, building upon the Michaelis-Menten equation, was necessary to generate kinetic values. A kinetic analysis was completed to address changes in parent compound and chlorinated metabolites over time and multisubstrate inhibition. A kinetic analysis using Berkeley-Madonna allowed us to evaluate quantitatively the changes in substrate and product concentration over time while accounting for competitive inhibition. To accomplish this, three models were generated and used to determine kinetic constants. Future studies include experiments to obtain kinetic parameters from desisopropyl-atrazine and desethyl-atrazine metabolism to DACT.
Significance of Findings
The results of these studies provide a strong basis for the objectives put forth by this research project. Atrazine exposure can and does lead to the formation of hemoglobin adducts in rats, and these adducts meet several requirements for a biomarker of exposure. First, they are sensitive and reflect internal dose, as shown by the significant association between dose level and percent of modified globin. Second, they provide a measure of toxic effect because the formation of this adduct may be positively correlated with dose-dependent luteinizing hormone surge suppression in Sprague Dawley rats dosed with atrazine at the same levels. Finally, the adducts appear to be chemically stable, and to some extent they reflect the individual susceptibility of the Sprague Dawley rat strain to the endocrine-disrupting toxic effects of atrazine. This research project is showing that atrazine-induced hemoglobin conjugates behave differently between humans and rats and between in vivo versus in vitro treatment in rats. These discoveries raise questions as to what mechanisms are involved in order for adducts to form, as well as exactly what the reactive metabolite is. It will be important to determine the exact mechanism of adduction because this study indicates that conjugate formation relies on metabolic activation. One of the long-term objectives of this research project is to use hemoglobin biomarkers as a measure of tissue exposure to active compounds. They also could be used to determine individual susceptibility because those that have the capacity to produce the reactive intermediate may be more vulnerable to atrazine toxicity. Understanding the reaction of proteins with atrazine would bring us closer to that objective and help us understand the exact mode of action by which atrazine produces its toxic effects.
Our work will be instrumental in improving our understanding of the 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 risk. 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 assess accurately 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 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 calculations of expected triazine binding in various populations. Study design criteria for biomonitoring in children and workers can be established, at least partially, on the basis of these calculations.
Future Activities:
We will continue to enhance current risk assessment procedures for the triazine compounds and continue to construct and refine PBPK models that will link exposure and circulating triazine levels to produce a comprehensive model of triazine binding to hemoglobin/hair proteins.
In addition, we will: (1) separate and isolate the alpha and beta subunits of Sprague Dawley rat globin and human globin, attempting to use gel electrophoresis to achieve this separation; (2) continue our investigation into the use of tryptic peptide digestion for the subunits of the two globin chains; (3) initiate in vitro experiments using human hepatocytes; (4) continue our studies to incubate whole human blood with atrazine and DACT and analyze the globin for binding as described previously; (5) confirm the present PBPK model by measuring plasma, hemoglobin, and brain binding kinetics in vitro; and (6) continue to investigate the pretreatment and digestion/extraction of hair.
Journal Articles on this Report : 1 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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McMullin TS, Andersen ME, Nagahara A, Lund TD, Pak T, Handa RJ, Hanneman WH. Evidence that atrazine and diaminochlorotriazine inhibit the estrogen/progesterone induced surge of luteinizing hormone in female Sprague-Dawley rats without changing estrogen receptor action. Toxicological Sciences 2004;79(2):278-286. |
R828610 (2003) R828610 (Final) |
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Supplemental Keywords:
exposure, risk assessment, health effects, susceptibility, chemicals, atrazine, diaminochlorotriazine, DACT, chlorotriazine, hemoglobin, hair proteins, adducts, pharmacokinetic biomarkers, physiologically based pharmacokinetic, PBPK, PBPK models, blood protein binding, hair protein binding, triazines, human blood samples, incubated rat hemoglobin, peak plasma concentrations, polyacrylamide gel electrophoresis, Sprague Dawley rats, ethyl acetate method, acid acetone method, individual globin chain, rat hepatocytes, total plasma chlorotriazine,, 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
- 2002 Progress Report
- 2001 Progress Report
- 2000 Progress Report
- Original Abstract
7 journal articles for this project