Grantee Research Project Results
Final 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 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 was 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 studies in this project addressed four specific aims: (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, which 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.
Summary/Accomplishments (Outputs/Outcomes):
Dr. Andersen continued to support this research while at CIIT-Centers for Health Research (CIIT-CHR) in Research Triangle Park, North Carolina. Dr. Andersen stayed involved in the research and visited Colorado State University (CSU) and met with researchers from time to time, although the subcontract between CSU and CIIT-CHR was completed as of May 31, 2003. Dr. Andersen continued to serve as coadvisor for one student working closely with this research. He also continued his support of the research effort as an adjunct professor, and continued to direct the efforts to develop PBPK models to atrazine and related chlorotriazines.
In Year 1 of the project, we were concerned with isolating globin for analysis of sites of atrazine binding to theses proteins, measuring atrazine and similar compounds in blood and urine, assessing reactions of atrazine with blood proteins in vitro, and refining pharmacokinetic models that would support biomonitoring/exposure assessment studies. We isolated/separated hemoglobin and determined globin purity, using the method of Fanelli, et al. (1958). We determined the purity of the isolated globin using polyacrylamide gel electrophoresis. A high pressure liquid chromatographic method also was used to analyze the globin.
We developed a GC method and a GCMS method for the analysis of triazine compounds in whole blood.
We developed a mechanistic PBPK model to describe the time course concentrations of atrazine and its metabolites in plasma, red blood cells, and various tissues.
In Year 2, we expanded our research into several main areas. We continued to look into the isolation of globin for analysis of sites of atrazine binding, developed analytical procedures for measuring atrazine and its metabolites in blood and urine, started to understand the time-course by which triazines were extracted from plasma/red blood cells, and continued our efforts in assessing reactions of atrazine with blood proteins in vitro. We expanded our PBPK model, with blood, body, and brain compartments, to estimate total plasma chlorotriazine.
In Year 3, our research centered on two main focal areas: (1) adduct determination studies using radioactivity, and (2) enzyme kinetic studies. For the adduct determination studies, we refined our techniques to prepare whole blood for liquid scintillation counting and performed efficiency tests to confirm that combination of solubilizing agent, bleaching agent, and various blood matrices do not affect the efficiency of the radioactive counter. We incubated whole blood from Sprague-Dawley (SD) rats with 30 ppm 14C atrazine or 14C diaminochlorotriazine (DACT) and analyzed the amount of radioactivity recovered in the hemoglobin. 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 not significance in the amount of radioactivity recovered in the lysate with time. 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. Based on the radioactivity studies, we calculated that the binding rate for DACT to Sprague-Dawley (SD) rat globin was 5.004 x 10-4 L/mmol-hr. We calculated that the binding rate for atrazine to 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 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 1000 molecules of globin), and that atrazine did not bind at all to human blood. Maximum binding occurred with 24 hours.
In Year 3, we also used cold studies in the identification of adduct formation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Results indicated a peak near the beta chain of globin. This peak represented additions of 104 Da in the atrazine treatment and 108 Da in the DACT treatment. These results were the strongest evidence to date that an adduct is forming in globin. We began to collaborate with Dr. T. Snow (Dupont Haskell-Stein Laboratory) in the investigation of using a tryptic peptide digestion procedure to determine and identify what specific globin chain the adduct is being formed.
During this time we developed an analytical method for measuring atrazine and its metabolites in brain samples.
Our research started to be centered on the following two main focal areas:
Adduct Determination Studies
Hemoglobin/DACT Incubations (In Vitro). Incubated rat hemoglobin for 48 hours at 37°C with 30, 60, and 90 ppm DACT and 90 ppm atrazine (n = 3 each treatment). Samples were analyzed by MALDI-TOFMS. 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 no binding was detected as we saw in the radioactivity studies at the same concentrations.
Globin Purification. Polyacrylamide gel electrophoresis was used to verify the purity of the globin obtained from 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-TOFMS. Results showed additions in approximately 100 MW to the β-chain of globin. The 24 hours showed a small addition peak, 48 hours a good sized peak, 72 hours a very large peak, 10 days a good peak, and 1 month a small peak; at 2 months the peak was gone. Addition of β-chain at about 104-107 Da. We postulated that if the nitrogens were deprotonated that would bring the addition 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, and 72 hours; 10 days; 1 month; and 2 months. Samples were analyzed using MALDI-TOFMS. Results indicated 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 the 2-month time point.
Treatment of rats with both atrazine and DACT caused the formation of an additional peak that appears to be a hemoglobin (Hb) adduct. As with the in vitro study, the MALDI-TOFMS 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 represents 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 b-chains modified. DACT treatment formed a peak that 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 β-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 300mg/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 was still present in all samples 30 days after the first dose. By 60 days postdose, 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 incorporating 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 we have reported before, we have further modified/developed the analytical method for the determination of atrazine and major metabolites in primary rat hepatocyte matrix. This method uses GC/MS/selected ion monitoring (GC/MS/SIM) 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 are still investigating 50 percent recoveries at time 0.
Our results indicate 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, 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 improved, which will help support our biomonitoring /exposure assessment studies. An example of this model is shown in Figure 1.
Figure 1. An Improved PBPK Model
On 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 on 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 quantitatively evaluate 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.
Summary/Significance of Findings
The principal atrazine metabolite DACT is thought to be responsible for the in vivo hemoglobin adduct seen in this study. The absence of atrazine adducts and adducts of desethylatrazine or desisopropylatrazine is likely caused by rapid metabolism to DACT. McMullin, et al. (2003) showed complete metabolism of atrazine to DACT within 48 hours of a single 90 mg/kg/body weight atrazine dose in SD rats. This adduct formation was confirmed using in vitro incubation of globin obtained from controlsand rats dosed with 90 ppm DACT.
The results of these studies provide a strong basis for the goals put forth by the research plan. 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 SD 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 SD rat strain to the endocrine disrupting toxic effects of atrazine. This research is showing that atrazine-induced Hb 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 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 goals of this project was to use hemoglobin biomarkers as a measure of tissue exposure to active compounds. They also could be used to determine individual susceptibility, because those who 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 goal, as well as to understanding the exact mode of action by which atrazine produces its toxic effects.
Some of the most important areas of future research should be related to reactivity of DACT with cellular constituents within the pituitary, development of kinetic parameters for metabolism of atrazine and the major metabolites in human hepatocytes/liver microsomes, and experiments that will allow the extension of the current PBPK models to lower doses where atrazine is present in solution rather than in suspension.
Conclusions
The analysis of protein adducts may provide an effective method for biomonitoring environmental exposure to chemicals such as atrazine and other pesticides. Previous data from our laboratory suggests that the commonly applied herbicide atrazine may covalently bond with hemoglobin in SD rats. Our work will be instrumental in improving our understanding of risks of these herbicides to children. Its value though 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 methods in general 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 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.
During this time period, we did an extensive literature review on the use of hair to assess exposure in humans. Accumulation of environmental chemicals in hair is a potentially attractive biomarker because hair growth patterns and sequential sampling along hair shafts can evaluate past history of exposure patterns. Among several of our planned research items were the following: we attempted to develop a long-lived in vitro skin tissue culture system that would be able to grow hair and to study the underlying biology of drug, xenobiotic, and metabolite deposition into hair. This information would subsequently be used in PBPK computer models to reconstruct administered dose, tissue concentration, or environmental exposure. As primary goals, we attempted to answer the following questions:
- Which trazines were excreted from plasma 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 variable is the hair partitioning rate between triazines?
- What are the stoichiometric and temporal relationships between triazine plasma concentrations and the binding of triazines to keratin and melanin?
Because of unanticipated personal changes and difficulties in developing skin tissue culture systems, we were not able to be successful with determining whether hair has any binding of sulfhydryl reactive triazines. We were not able to quantitatively determine how the various triazines distribute between keratin and melanin in the hair matrix. We were not able to determine the variability of the hair partitioning rate between triazines or the stoichiometric and temporal relationships between triazine plasma concentrations and the binding of triazines to keratin and melanin.
Journal Articles on this Report : 7 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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Dooley GP, Prenni JE, Prentiss PL, Cranmer BK, Andersen ME, Tessari JD. Identification of a novel hemoglobin adduct in Sprague Dawley rats exposed to atrazine. Chemical Research in Toxicology 2006;19(5):692-700. |
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. |
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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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McMullin TS, Andersen ME, Tessari JD, Cranmer B, Hanneman WH. Estimating constants for metabolism of atrazine in freshly isolated rat hepatocytes by kinetic modeling. Toxicology in Vitro 2007;21(3):492-501. |
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, susceptibility, chemicals, atrazine, DACT, chlorotriazine, hemoglobin, hair proteins, adducts, pharmacokinetic biomarkers, 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.