Grantee Research Project Results
2005 Progress Report: Biomarkers of Human Exposure to Pesticides Utilizing a New PBPK/PD Model and Kinetic Data on Pesticide Metabolism in Humans
EPA Grant Number: R830683Title: Biomarkers of Human Exposure to Pesticides Utilizing a New PBPK/PD Model and Kinetic Data on Pesticide Metabolism in Humans
Investigators: Olson, James , Knaak, James B. , Kostyniak, Paul J.
Institution: The State University of New York at Buffalo
EPA Project Officer: Hahn, Intaek
Project Period: December 18, 2002 through December 17, 2005
Project Period Covered by this Report: December 18, 2004 through December 17, 2005
Project Amount: $747,704
RFA: Issues in Human Health Risk Assessment (2001) RFA Text | Recipients Lists
Research Category: Human Health
Objective:
Currently, organophosphorous pesticides (OPs) are the most commonly used pesticides in the world. OPs act as irreversible acetylcholinesterase inhibitors once bioactivated from the thiophosphate to the reactive oxon form. The primary objective of this research project is to obtain kinetic parameters (Vmax, Km values) for the metabolism of model pesticides (parathion and chlorpyrifos) in the livers from humans of various ages. Age- and gender-specific kinetic parameters (Vmax, Km) for selected pesticides will be used in a multiroute, multichemical, physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) model (i.e., Exposure Related Dose Estimating Model [ERDM]; Blancato, et al., 2000; Knaak, et al., 2004) to estimate the sensitivity of individuals within each age group based on biomarkers of susceptibility (i.e., paraoxonase genotype and CYP2B6 phenotypes), exposure (i.e., urinary metabolites), and effects (i.e., blood acetylcholinesterase [AChE]; AChE/butyrylcholinesterase inhibition). Results also will be examined and validated using available data from human monitoring studies.
Although this work focuses on organophosphates as model compounds, the differences in the ontogeny of enzyme activity can have wide applicability to other drugs and chemicals. Specific aims are to: (1) characterize the levels of specific cytochrome P450s, paraoxonase activity, and genotype of liver specimens from humans of five age groups (i.e., 0-2, 3-10, 11-20, 21-40, and > 40 years); (2) measure the kinetics (Km, Vmax) for the metabolism of parathion and chlorpyrifos in hepatic microsomal fractions from humans of five age groups; (3) use selected experimental exposure levels (i.e., 0.5 to 5.0 mg/kg of body weight) and age- and gender-specific Vmax and Km values for parathion and chlorpyrifos in the ERDEM to determine the sensitivity of individuals within each age group to these pesticides based on biomarkers of susceptibility, exposure, and effects; and (4) use human environmental exposure levels in the ERDEM to estimate biomarkers of exposure and effects. Finally, the ERDEM will be validated by comparing model biomarker estimates to monitored values (i.e., values from human monitoring studies).
Progress Summary:
Progress Summary/Accomplishments: Preliminary studies initially focused on measuring the kinetics (Km, Vmax) for the metabolism of parathion in characterized human liver microsomal specimens and recombinant human cytochrome P450 (CYPs). A sensitive and specific high performance liquid chromatography method was developed to quantify the products of biotransformation. Figure 1 illustrates the kinetic analysis of paraoxon formation (activation) from parathion (1-100 µM) in a characterized pool of human liver microsomes. The table within Figure 1 summarizes the kinetic parameters for the formation of paraoxon (activation) and p-nitrophenol (detoxification), because the kinetic values for both products are needed in the PBPK/PD model. The kinetics of parathion metabolism also were assessed in six specimens of characterized human liver microsomes from males and females, ages 19 to 54. The Km for the formation of paraoxon and p-nitrophenol ranged from 8.7 to 39.1 and from 15.2 to 55.9 µM, respectively, whereas the Vmax values ranged from 589 to 1,495 and from 588 to 1,232 pmol/min/mg, respectively. These results support the need to include a range of kinetic parameters to reflect the inherent interindividual variability in biotransformation. The Vmax for parathion to paraoxon formation from six characterized human liver microsomal specimens correlates with the formation of 6-beta hydroxytestosterone from testosterone, a marker for CYP3A4 activity (r2 = 0.96). Studies with recombinant human CYPs have established that CYP1A2, 2B6, 2C9, 2C19, 3A4, and 3A5 contribute to the metabolism of parathion to paraoxon, whereas these CYPs and CYP3A7 contribute to p-nitrophenol formation (see Tables 1 and 2). These results also indicate that CYP1A2, CYP2B6, and CYP2C19 with lower Kms, may contribute substantially to parathion metabolism at low-level human exposures.Studies with human CYPs also have established kinetic parameters for the CYP-specific metabolism of chlorpyrifos (see Tables 3 and 4). Studies with recombinant human CYPs have established that CYP1A2, 2B6, 2C19, 3A4, and 3A5 contribute to the metabolism of chlorpyrifos to chloroxon and the metabolism of chlorpyrifos to trichloro-2-pyridinol. As with parathion, the results also indicate that CYP1A2, CYP2B6, and CYP2C19, with lower Kms, may contribute substantially to chlorpyrifos metabolism at low-level human exposures.
To use the data in Tables 3 and 4 to establish estimates of the hepatic metabolism of parathion and chlorpyrifos in humans, it is necessary to have the content or concentration of specific CYPs in human liver microsomal protein. Table 5 summarizes human hepatic microsomal CYP content as a function of age from published studies (Tateishi, et al., 1997; Wrighton, et al.,1990; Koukouritaki, et al., 2004; Stevens, et al., 2003).
Using the equation below, it then is possible to estimate the age-dependent hepatic metabolism of parathion and chlorpyrifos based on CYP-specific kinetic constants and the age-dependent specific CYP content in human liver microsomal protein.
Figure 1. Paraoxon Formation From Parathion in Pooled Sample of Human Liver Microsomes
Table 1. Kinetic Constants for the Metabolism of Parathion to Paraoxon
CYP |
Km |
Vmax pmol/min/pmol P450 |
Vmax pmol/hr/mg microsome |
Vmax pmol/hr/g liver |
Vmax umol/hr/kg |
Vmax/Km |
1A2 |
1.608 |
6.2835 |
16,965.45 |
508,963.5 |
13.7420145 |
8.546028918 |
2B6 |
0.363 |
3.3007 |
7,723.638 |
231,709.14 |
6.25614678 |
17.23456413 |
2C9 |
10.12 |
1.302 |
7,499.52 |
224,985.6 |
6.0746112 |
0.600258024 |
2C19 |
0.477 |
3.382 |
3,855.48 |
115,664.4 |
3.1229388 |
6.547041509 |
3A4 |
48.07 |
12.07 |
78,213.6 |
2,346,408 |
63.353016 |
1.317932515 |
3A5 |
14.29 |
0.802 |
48.12 |
1,443.6 |
0.0389772 |
0.002727586 |
3A7 |
ND |
DN |
Table 2. Kinetic Constants for the Metabolism of Parathion to P-Nitrophenol
CYP |
Km |
Vmax pmol/min/pmol P450 |
Vmax pmol/hr/mg microsome |
Vmax pmol/hr/g liver |
Vmax |
Vmax/Km |
1A2 |
2.17 |
5.832 |
15,746.4 |
472,392 |
12.754584 |
5.877688479 |
2B6 |
0.45 |
0.82588 |
1,932.5592 |
57,976.776 |
1.565372952 |
3.47860656 |
2C9 |
13.97 |
0.952 |
5,483.52 |
164,505.6 |
4.4416512 |
0.317942105 |
2C19 |
0.92 |
1.745 |
1,989.3 |
59,679 |
1.611333 |
1.751448913 |
3A4 |
35.3 |
16.9 |
109,512 |
3,285,360 |
88.70472 |
2.512881586 |
3A5 |
19.69 |
0.787 |
47.22 |
1,416.6 |
0.0382482 |
0.001942519 |
3A7 |
47.8 |
0.8358 |
501.48 |
15,044.4 |
0.4061988 |
0.008497883 |
Table 3. Kinetic Constants for the Metabolism of Chlorpyrifos to Chloroxon
CYP |
Km |
Vmax pmol/min/pmol P450 |
Vmax pmol/hr/mg microsome |
Vmax pmol/hr/g liver |
Vmax umol/hr/kg |
Vmax/Km |
1A2 |
0.71 |
2.83724 |
7,660.548 |
229,816.44 |
6.20504388 |
8.739498423 |
|
|
|
|
|
|
|
2B6 |
0.32 |
9.668 |
22,623.12 |
678,693.6 |
18.3247272 |
57.2647725 |
|
|
|
|
|
|
|
2C19 |
1.406 |
3.673 |
4,187.22 |
125,616.6 |
3.3916482 |
2.412267568 |
|
|
|
|
|
|
|
3A4 |
20.85 |
9.328 |
60,445.44 |
1,813,363.2 |
48.9608064 |
2.348240115 |
|
|
|
|
|
|
|
3A5 |
11.739 |
2.482 |
148.92 |
4,467.6 |
0.1206252 |
0.010275594 |
Table 4. Kinetic Constants for the Metabolism of Chlorpyrifos to Trichloro-2-pyridinol
CYP |
Km |
Vmax pmol/min/pmol P450 |
Vmax pmol/hr/mg microsome |
Vmax pmol/hr/g liver |
Vmax umol/hr/kg |
Vmax/Km |
1A2 |
3.09 |
0.7283 |
1966.41 |
58992.3 |
1.5927921 |
0.515967638 |
|
|
|
|
|
|
|
2B6 |
0.8426 |
1.403 |
3283.02 |
98490.6 |
2.6592462 |
3.156000712 |
|
|
|
|
|
|
|
2C19 |
1.2032 |
15.013 |
17114.82 |
513444.6 |
13.8630042 |
11.52177876 |
|
|
|
|
|
|
|
3A4 |
19.19 |
7.878 |
51049.44 |
1531483.2 |
41.3500464 |
2.154770526 |
|
|
|
|
|
|
|
3A5 |
5.457 |
1.738 |
104.28 |
3128.4 |
0.0844668 |
0.015478615 |
Table 5. Human Hepatic Cytochrome P-450 Content as a Function of Age (pmol/mg microsomal protein)
|
Age Group |
CYP1A2 |
CYP2B6 |
CYP2C9 |
CYP2C19 |
CYP3A4 |
CYP3A5 |
CYP3A7 |
Gentest |
|
45 |
39 |
96 |
19 |
49 |
1 |
|
Hines |
0-2yrs |
|
|
14.191 |
7.395 |
7.662 |
3.388 |
56.355 |
n = |
|
|
118 |
118 |
49 |
114 |
49 |
|
2-10yrs |
|
|
17.158 |
11.006 |
16.425 |
4.633 |
2.69 |
|
n = |
|
|
28 |
28 |
14 |
27 |
14 |
|
10-20yrs |
|
|
16.905 |
15.771 |
11.798 |
0.752 |
2.088 |
|
n = |
|
|
20 |
20 |
4 |
18 |
3 |
|
Wrighton |
0-18yrs |
|
|
|
|
92.4 |
35.4 |
CYP3A4/7 |
n = |
|
|
|
|
13 |
5 |
|
|
18+ yrs |
|
|
|
|
86 |
8 |
CYP3A4/7 |
|
n = |
|
|
|
|
7 |
2 |
|
|
Tateishi |
0-1yrs |
3.27 |
2.65 |
79.78 |
|
173.31 |
|
|
n = |
10 |
10 |
10 |
|
10 |
|
|
|
1+ yrs |
24.93 |
19.36 |
74.39 |
|
239.40 |
|
|
|
n = |
10 |
10 |
10 |
|
10 |
|
|
By using published data on the content of the above CYPs, the metabolic range for total Vmax for the conversion of parathion to paraoxon in infants and adults is 1.62 to 28.0 and 34.9 to 121 mmol/hr/kg body weight respectively. In the conversion of chlorpyrifos to chlorpyrifos oxon, the total Vmax range is 1.86 to 24.0 and 40.3 to 156 mmol/hr/kg body weight for infants and adults respectively. Therefore, our best estimates at this time suggest that the rate of CYP-dependent activation of these OPs to the toxic oxon metabolite is less active in infants. The PBPK/PD and ERDEM models, however, will require additional inputs, such as CYP-specific Kms, paraoxonase (PON1) activity, genotype analysis, real-world exposure levels, and other refinements to obtain better estimates of relative risk.
Future Activities:
At this time our laboratory is collecting additional kinetic data on metabolism and serum protein binding and data on specific CYP content in human liver. Current studies also are investigating the statistical relationships of how age, gender, and CYP levels, affect the content of a specific CYP in human liver microsomal protein. Future analyses will use mean, median, and 95 percent confidence intervals to express the concentration of specific CYPs in human liver microsomal protein. Recently, Lang, et al. (2001) conducted the first systematic investigation of genetic polymorphism in the CYP2B6 gene on chromosome 19. A total of nine novel point mutations were identified. Following analysis of 215 Caucasians, only 48 (22.3%) were shown to be wildtype with respect to the newly identified mutations. Thus, in terms of frequency of mutant alleles, CYP2B6 appears to be one of the most polymorphic human P450s. By analyzing a large number of human liver samples, significantly reduced CYP2B6 protein expression and S-mephenytoin N-demethylase activity were found in carriers of the C1459T (R487C, exon 9 Arg487Cys) mutation (alleles *5 and *7) (Lang, et al., 2001). These data demonstrate that the extensive interindividual variability of CYP2B6 expression and function is caused not only by regulatory phenomena, but also by a common genetic polymorphism. Because CYP2B6 is one of the most active enzymes that activates parathion and chlorpyrifos to the active axon metabolite, future studies will assess the genotype of CYP2B6 in human liver specimens to assess the role of genetic variability in the metabolic activation of these model Ops more effectively. Refined estimates of the age-specific hepatic CYP content and the CYP-specific data on the kinetics of biotransformation then will be used in the PBPK/PD and ERDEM models to improve risk assessment for these and other prototypical pesticides.
Paraoxonase (PON1) is a key enzyme that detoxifies paraoxon and chlorpyrifos oxon. PON1 is produced in the liver and carried in the blood bound to APO-A1 in HDL. Current studies are measuring PON1 activity in human liver microsomes and will attempt to investigate age-dependent differences in PON1 activity and content in human liver specimens. There are currently two polymorphisms identified within the coding region of PON1 (L55M and Q192R) that have been shown to alter the functional activity of this key detoxification enzyme. Studies currently are assessing PON1 activity and functional polymorphism to assess both age and genetic variability of PON1 activity. PON1 specific parameter then will be used in the PBPK/PD and ERDEM models to improve risk assessment for these and other prototypical pesticides.
Finally, a major goal over the next year will be to publish the results of our findings in peer-reviewed journals.
References:
Blancato JN, Knaak JB, Power F, Cary CC. Use of PBPK models for assessing absorbed dose and ChE inhibition from aggregate exposure of infants and children to organophosphorus insecticides. Presented at the 10th Annual Conference of the International Society of Exposure Analysis, Asilomar Conference Center, Monterey, CA, October 24-27, 2000 (abstract 3F-09o).
Knaak JB, Dary CC, Power F, Thompson CB, et al. Physiochemical and biological data for the development of predictive organophosphorus pesticide QSARs and PBPK/PD models for human risk assessment. Critical Reviews in Toxicology 2004;34(2):143-207.
Koukouritaki SB, Manro JR, et al. Developmental expression of human hepatic CYP2C9 and CYP2C19. Journal of Pharmacology & Experimental Therapeutics 2004;308(3):965-974.
Lang T, Klein K, Fischer J, Nüssler AK, Neuhaus P, Hofmann U, Eichelbaum M, Schwab M, Zanger UM. Extensive genetic polymorphism in the human CYP2B6 gene with impact on expression and function in human liver. Pharmacogenetics 2001;11(5):399-415.
Stevens JC, Hines RN, et al. Developmental expression of the major human hepatic CYP3A enzymes. Journal of Pharmacology and Experimental Therapeutics 2003;307(2):573-582.
Tateishi T, Nakura H, et al. A comparison of hepatic cytochrome P450 protein expression between infancy and postinfancy. Life Sciences 1997;61(26):2567-2574.
Wrighton SA, Brian WR, et al. Studies on the expression and metabolic capabilities of human liver cytochrome P450IIIA5 (HLp3). Molecular Pharmacology 1990;38(2):207-213.
Journal Articles on this Report : 4 Displayed | Download in RIS Format
| Other project views: | All 8 publications | 4 publications in selected types | All 4 journal articles |
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Ellison C, Smith J, Lein P, Olson J. Pharmacokinetics and pharmacodynamics of chlorpyrifos in adult male Long-Evans rats following repeated subcutaneous exposure to chlorpyrifos. TOXICOLOGY 2011;287(1-3):137-144. |
R830683 (2005) |
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Ellison C, Tian Y, Knaak J, Kostyniak P, Olson J. Human hepatic cytochrome P450-specific metabolism of the organophosphorus pesticides methyl parathion and diazinon. DRUG METABOLISM AND DISPOSITION 2012;40(1):1-5. |
R830683 (2005) |
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Foxenberg RJ, McGarrigle BP, Knaak JB, Kostyniak PJ, Olson JR. Human hepatic cytochrome P450-specific metabolism of parathion and chlorpyrifos. Drug Metabolism and Disposition 2007;35(2):189-193. |
R830683 (2005) |
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Foxenberg RJ, Ellison CA, Knaak JB, Ma CX, Olson JR. Cytochrome P450-specific human PBPK/PD models for the organophosphorus pesticides: chlorpyrifos and parathion. Toxicology 2011;285(1-2):57-66. |
R830683 (2005) |
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Supplemental Keywords:
pesticides, biomarkers, parathion, chlorpyrifos, organophosphates, PBPK/PD model, paraoxon, chlorpyrifos oxon, pesticide metabolism,, Health, RFA, Scientific Discipline, Toxics, Susceptibility/Sensitive Population/Genetic Susceptibility, Health Risk Assessment, pesticides, Risk Assessments, genetic susceptability, Biochemistry, Toxicology, environmental hazard exposures, sensitive populations, metabolism, PBPK modeling, kinetic studies, human exposure, insecticides, pharmacokinetic models, children, pharmokinetic models, P450 gene expression, biomarker, human health risk, biomarkersProgress 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.