ToxSci Advance Access originally published online on May 15, 2007
Toxicological Sciences 2007 98(2):348-365; doi:10.1093/toxsci/kfm119
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An Age-Dependent Physiologically Based Pharmacokinetic/Pharmacodynamic Model for the Organophosphorus Insecticide Chlorpyrifos in the Preweanling Rat
Battelle Pacific Northwest Division, Center for Biological Monitoring and Modeling, Richland, Washington 99352
1 To whom correspondence should be addressed at Battelle Pacific Northwest Division, Center for Biological Monitoring and Modeling, 902 Battelle Boulevard, Richland, WA 99352. Fax: (509) 376-9064. E-mail: charles.timchalk{at}pnl.gov.
Received January 5, 2007; accepted May 8, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Juvenile rats are more susceptible than adults to the acute toxicity of organophosphorus insecticides like chlorpyrifos (CPF). Age- and dose-dependent differences in metabolism may be responsible. Of importance are CYP450 activation and detoxification of CPF to chlorpyrifos-oxon (CPF-oxon) and trichloropyridinol (TCP), as well as B-esterase (B-est) and PON-1 (A-esterase) detoxification of CPF-oxon to TCP. In the current study, a physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) model incorporating age-dependent changes in CYP450, PON-1, and tissue B-est levels for rats was developed. In this model, age was used as a dependent function to estimate body weight which was then used to allometrically scale both metabolism and tissue cholinesterase (ChE) levels. In addition, age-dependent changes in brain, liver, and fat volumes and brain blood flow were obtained from the literature and used in the simulations. Model simulations suggest that preweanling rats are particularly sensitive to CPF toxicity, with levels of CPF-oxon in blood and brain disproportionately increasing, relative to the response in adult rats. This age-dependent nonlinear increase in CPF-oxon concentration may potentially result from both the depletion of nontarget B-est and a lower PON-1 metabolic capacity in younger animals. The PBPK/PD model behaves consistently with the general understanding of CPF toxicity, pharmacokinetics, and tissue ChE inhibition in neonatal and adult rats. Hence, this model represents an important starting point for developing a computational model to assess the neurotoxic potential of environmentally relevant organophosphate exposures in infants and children.
Key Words: chlorpyrifos; PBPK/PD; preweanling rat; age-dependent sensitivity.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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There is currently a significant concern and focus over the potential increased sensitivity of infants and children to the toxic effects of chemicals. The importance of this issue is highlighted by the National Research Council's report on Pesticides in the Diets of Infants and Children (National Academy of Sciences, 1993
) and the establishment of the Food Quality Protection Act (FQPA, 1996). Children are not small adults, but rather a unique subpopulation that may have altered vulnerability to chemical insult. Age-dependent changes in a child's physiology (i.e., body size, blood flow, organ functions) and metabolic capacity (i.e., phase I and II metabolism) may significantly impact their response to a chemical insult, resulting in either beneficial or detrimental effects (Alcorn and McNamara, 2002; Ginsberg et al., 2004; Johnson, 2003; Makri et al., 2004; Miller et al., 1997). Clear age-dependent variability in the capacity to detoxify environmental chemicals has been established in both animals and humans. However, the current risk assessment paradigms may not adequately consider the implications of these differences on the risk to infants and children.
Numerous studies have demonstrated that juvenile animals are more susceptible to the acute effects of organophosphorus (OP) insecticides than adults (Benke and Murphy, 1975; Brodeur and DuBois, 1963; Gaines and Linder, 1986; Harbison, 1975; Moser and Padilla, 1998; Pope and Liu, 1997; Pope et al., 1991). The primary toxicological effect of OP insecticides is associated with the inhibition of acetylcholinesterase (AChE) in both central and peripheral nerve tissues (Murphy, 1986; Sultatos, 1994). The greater neonatal sensitivity has primarily been attributed to the lack of complete metabolic competence during development (Benke and Murphy, 1975). In this regard age-dependent sensitivity may be associated with maturational differences in CYP450 activation/detoxification, detoxification by PON-1 and B-esterase [B-est; AChE, butyrylcholinesterase (BuChE) and carboxylesterase (CaE)] enzyme activity (Atterberry et al., 1997; Li et al., 1997; Mortensen et al., 1996, 1998). These findings in animals are in agreement with observations in which newborn and young humans have lower metabolic capacity for CYP450 and PON-1 activity compared to adults (Augustinsson and Barr, 1963; Johnson, 2003; Mueller et al., 1983).
The application of physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) modeling offers a unique opportunity to integrate age-dependent changes in metabolic activation and detoxification pathways into a comprehensive model that is capable of quantifying dosimetry and response across all ages (for review see Corley et al., 2003). In this context, PBPK models are being extended to the modeling of chemical exposure in developing/juvenile animals and in children (Byczkowski et al., 1994; Clewell et al., 2002, 2003, 2004; Fisher et al., 1990; Price et al., 2003; Sundberg et al., 1998).
A PBPK/PD model (see Fig. 1A) has been previously developed for the phosphorothionate insecticide chlorpyrifos (CPF) ([O,O'-diethyl (O-3,5,6-trichloro-2-pyridyl) phosphorothionate]) (Timchalk et al., 2002). CPF does not directly inhibit AChE, but must first undergo CYP450 mediated oxidative desulfation, to form chlorpyrifos-oxon (CPF-oxon) as is illustrated in Figure 1B (Chambers and Chambers, 1989; Murphy, 1986; Sultatos, 1994). A balance between the extent of metabolic activation and detoxification determines individual susceptibility to the toxicity of this compound and is most likely associated with individual and age-dependent sensitivity (Ma and Chambers, 1994). The working hypothesis is that a decrement in CYP450 and/or esterase (PON-1 and B-est)–mediated detoxification alters the balance between activation and detoxification and correlates with increased sensitivity of young animals and the significant variability in human sensitivity to OP insecticides. To address this issue, the previously published CPF PBPK/PD model (Timchalk et al., 2002) was modified by incorporating age-dependent scaling to adjust physiology, organ volumes, and blood flows, metabolism rates, B-est tissue levels, and bimolecular inhibition rates for CPF-oxon and AChE as a function of age. The model was then used to predict tissue dosimetry, and pharmacodynamic (PD) response (i.e., esterase inhibition) in preweanling and adult rats exposed to CPF. The ultimate goal is to apply this model to evaluate the risk associated with environmental exposure of infants and children to OP insecticides.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Model structure.
A diagram of the PBPK/PD model structure is illustrated in Figure 1A. The CPF model was developed to describe the time-course of CPF, CPF-oxon, and TCP, as well as the inhibition of target esterases by the oxon in adult rats and humans, and was originally based on the model developed for diisopropylfluorophosphate by Gearhart et al. (1990)
. New research has made it possible to update a number of key model parameters including those associated with ChE inhibition, metabolism, and tissue partition for CPF and CPF-oxon (Kousba et al., 2004, 2007; Lowe et al., 2006; Timchalk et al., 2002). Briefly, in the model CYP450 mediated activation of CPF to CPF-oxon and detoxification to TCP occurs only in the liver and is described as a Michaelis–Menten process. PON-1 mediated metabolism of CPF-oxon occurs in the liver and blood and is likewise described as a Michaelis–Menten process. Interaction of CPF-oxon with the B-esterases (B-est) AChE, BuChE, and CaE is modeled as a second-order process occurring in the liver, blood, diaphragm, and brain. The B-est enzyme levels (µmol) in blood (plasma and red blood cells[RBC]), brain, liver, and diaphragm were calculated based on the enzyme turnover rates and enzyme activities reported by Maxwell et al. (1987), and these were based on a balance between basal degradation and enzyme synthesis. In the model scaling equations were developed to address age-dependent changes in CYP450, PON-1, and B-est activity in the rat.
Model parameters.
To develop the model, physiological constants, partition coefficients, and biochemical constants were obtained from the literature, through experimentation, or via optimization of model output using computer simulation. The PBPK/PD model code for simultaneous solution of both algebraic and differential equations was originally developed in SIMUSOLV (The Dow Chemical Co., Midland, MI), and adapted to acslXtreme (Aegis Technology, Huntsville, AL) for sensitivity analysis. The model parameter estimates that were used to describe the adult CPF pharmacokinetic and PD responses are presented in Tables 1 and 3; for a more detailed description of the sources and rational for parameter estimates see Timchalk et al. (2002). In addition, the model code, parameter estimates, and data files used as part of this PBPK/PD model have been posted with Toxicological Sciences as Supplemental Data (for model code, experimental data, and model parameters see supplemental data files 7, 8, and 9 for additional details).
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Parameter scaling.
To simulate the kinetics of CPF dosimetry and ChE inhibition in neonatal rats, the PBPK/PD model was modified to scale metabolism and ChE activity as a function of age, allometric scaling equations, and parameter estimates are presented in Tables 2 and 4. A polynomial equation was fit to the age-dependent body weight data for Sprague–Dawley rats (up to 75 days of age) using published values from male neonates (Carr et al., 2001
), and from the historical body weight ranges obtained from the animal supplier (Charles River Laboratory, Raleigh, NC). A similar scaling approach was used to fit age-dependent changes in liver and brain volumes using compiled data (Gentry et al., 2004). As illustrated in Figure 2, the polynomial equations accurately reflected body weight, liver, and brain volume as a function of age in the Sprague–Dawley rat, up to 
60–75 days of age. Body weights were then used to allometrically scale both metabolism rates and tissue esterase levels, whereas liver and brain volume changes were calculated and used directly in the model (see supplemental data files 1, 2, and 3 for additional details).
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To allometrically scale CPF and CPF-oxon metabolism in the preweanling rat, in vitro data on age-dependent CYP450 and PON-1 metabolism were utilized (Atterberry et al., 1997
). In the adult rat and human PBPK/PD model (Timchalk et al., 2002) in vitro metabolic rates were scaled across species as a function of body weight utilizing a standard allometric approach (Bwt0.74), which is consistent with the commonly used methods for adults (e.g., Krishnan and Andersen, 1994). However, applying this scaling factor to preweanling rats resulted in a substantial overestimate of the in vivo metabolism, based on model simulations (data not shown). As previously reported by Atterberry et al. (1997) the age-dependent increase in CYP450 metabolic activity paralleled the increase in the amount of hepatic CYP450 enzyme. Hence, the age-dependent preweanling CYP450 as well as PON-1 activity was based upon the experimentally measured in vivo results (see Atterberry et al., 1997), utilizing a modified allometric scaling equation, and the overall fit to the experimental results are presented in Figure 3A. The modification to the allometric equation is illustrated in Equation 1 and the individual equations (Vmax(1–4)) with their specific parameter estimates are presented in Table 2. As is illustrated in Equation 1, the age-dependent Vmax for CYP450 or PON-1 activity was determined for each age by adjusting the VmaxC by a scaling factor (VmSC) and the exponent for the body weight adjustment (Bwtn). Based on this approach allometric scaling was utilized to determine Vmax(1–4) for both the CYP450 and PON-1 metabolism and was used to scale metabolism in preweanling through adult rats (see supplemental data file 6 for more details).
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B-esterase (B-est) scaling.
The amount of tissue ChE (AChE and BuChE) and CaE were also allometrically scaled based upon age-dependent changes in published preweanling tissue enzyme levels (Atterberry et al., 1997
; Carr et al., 2001; Mortensen et al., 1998, 1998; Tang et al., 1999), and the enzyme activity (B-estC(1–12); µmol/h/kg) for each tissue was calculated (see Table 4) based on the enzyme turnover rates and activities reported by Maxwell et al. (1987). For plasma AChE the enzyme activity (Maxwell et al., 1987) was increased from 523 to 5230 µmol/h/kg in order to reasonably simulate the time-course of ChE inhibition (see Table 4) at all ages (see supplemental data files 4 and 5 for additional details). For the liver and diaphragm compartments where age-dependent enzyme activity was not available the default scaling was arbitrarily based on the plasma. The basal levels of RBC AChE were calculated based on the ratio of AChE activity in brain and RBC as previously described (Timchalk et al., 2002). These estimates of tissue ChE and CaE levels were used in the PBPK/PD model to establish steady-state enzyme levels (µmol) and to enable calculation of the relative extent of enzyme inhibition as a function of CPF-oxon concentration in the various tissue compartments. These calculated changes in the plasma B-est (CaE, AChE and BuChE) levels (µmol) as a function of age in the rat are presented in Figure 3B. Similar analyses were conducted for the brain, diaphragm, and liver (data not shown). The enzyme degradation (Kd) rates for AChE, BuChE, and CaE were initially based upon the first-order loss of rat brain AChE as described by Gearhart et al. (1990). In general, BuChE activity is similar to AChE and as a first approximation the synthesis and loss rates for BuChE were set the same as for AChE. Whereas for CaE, reasonable parameter estimates for synthesis and loss rates were not available so the rates were optimized with the PD model to fit CaE inhibition data from Chanda et al. (1997; Timchalk et al., 2002). For example, the equations describing AChE inhibition in the brain are
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Those PBPK/PD model parameters that were modified as a function of age or changed relative to the adult model (Timchalk et al., 2002) are presented in Table 4. Since there was no information on the extent of oral CPF absorption in preweanling pups, it was set at 100% for all ages. The percentage of body fat in the pups was based on an extrapolation of age-dependent changes in fat reported for the Sprague–Dawley rat (Schoeffner et al., 1999). The fractional binding of CPF and CPF-oxon with blood proteins was both set at 95% based upon recently reported results (Lowe et al., 2006), in both preweanling and adult rats which is slightly changed from the 97% to 98% binding used in the original adult model (Timchalk et al., 2002). The brain AChE bimolecular inhibition rate constant (Ki) was measured in vitro at each postnatal age and was shown to decrease with age (Kousba et al., 2007). The experimentally determined Ki was applied to the brain, plasma, liver, and diaphragm compartments. However, the Ki for the RBC AChE was initially based upon the parameter estimate in the previous PBPK/PD model (Timchalk et al., 2002), and was proportionally scaled based on the measured age-dependent changes reported by Kousba et al. (2007). The Ki's for BuChE and CaE inhibition were not determined experimentally, so these parameter estimates were also based on the previously published model (Timchalk et al., 2002).
Sensitivity analysis.
A sensitivity analysis was conducted using the subroutines within acslXtreme to identify the importance of selected parameters relative to their impact on brain AChE inhibition response. This analysis primarily focused on those model parameters that substantially changed as a function of age. The sensitivity of the model to these parameters was assessed for simulations at postnatal days (PND)-5, PND-17 (data not shown), and in adult rats following a single oral gavage dose of 5 mg/kg of body weight. The 5 mg/kg dose was selected since model simulations (see "PND-5 vs. Adult Oxon Area Under Concentration Curve (AUC)") suggest that the brain CPF-oxon concentration was disproportionately increased in PND-5 relative to adult rats at this dose level. The analysis measured a change in model output (brain AChE) corresponding to a 1% change in the parameter of interest (e.g., Vmax) when all other parameters were held fixed. Sensitivity parameters were qualitatively categorized as having low, medium, or high uncertainty based on the following criteria: low, experimental data obtained in the rat in our laboratory or from the published literature; medium, scaled value from a different species or optimized based on multiple data sets; high, parameters optimized from limited data or lacking published or historical values. These criteria are similar to those utilized by Teeguarden et al. (2005) for ranking uncertainty of model parameter estimates (see supplemental data file 10 for additional detail).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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In order to adequately address age-dependent changes in both the pharmacokinetics of CPF and the resulting PD (i.e., ChE) response, a number of metabolic and physiological parameters were scaled as a function of age. The initial evaluation focused on the capability of the updated model to adequately simulate blood CPF and CPF-oxon dosimetry in the adult rat and the results from these comparisons are presented in Figure 4. In general the model adequately simulated the available CPF blood concentrations at doses ranging from 1 to 50 mg/kg (data from Timchalk et al., 2002
). In addition, although CPF-oxon was only detectable in a very limited number of blood samples (peak concentrations at 10 and 50 mg/kg) the model also provided a reasonable simulation of these data. Since CPF-oxon is responsible for the observed ChE inhibition, the good fits for blood and brain ChE inhibition (see Figs. 7 and 9) in the adult rat also suggest that the model reasonably predicts CPF-oxon blood and tissue concentrations. These model simulations include a number of updated model parameters (Kousba et al., 2004, 2007; Lowe et al., 2006; Poet et al., 2003) and show a slightly improved fit to the experimental data relative to the previously published model (Timchalk et al., 2002).
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Pharmacokinetics of CPF and TCP
The experimentally determined time-course of CPF and TCP in blood of preweanling rats (PND-5, -12, and -17) following oral gavage administration of CPF at doses of 1 or 10 mg/kg body weight (Timchalk et al., 2006
), and model simulations (solid lines) of these experimental data are presented in Figure 5. Both the data and model simulations suggest that CPF is rapidly absorbed and metabolized since peak blood levels of both CPF and the major metabolite TCP were observed between 3- and 6-h postdosing. Within all age groups a comparison of the measured maximum blood concentration (Cmax) and the PBPK model predictions suggests that the kinetics of both CPF and TCP are linear over the dose range (1–10 mg/kg). The experimentally determined Cmax for CPF is comparable across preweanling ages; whereas, the model predicts a consistently lower Cmax at both doses and ages, and also suggests a decreasing trend with age. Of interest to note is that the model underpredicts the CPF blood concentration, particularly in PND-12 and -17 rats and simulates a faster blood elimination relative to the experimental data. With regard to the blood TCP kinetics, in PND-5 and -12 rats the blood time-course and Cmax (observed and predicted) for TCP are very comparable, and both the experimental data and model predictions increase 3-fold at both dose levels in the PND-17 rats.
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Domoradski et al. (2004) compared the pharmacokinetics of CPF and TCP in PND-5 rats following exposure by different routes (oral vs. subcutaneous), formulations (corn oil vs. milk vs. dimethylsulfoxide), and rates (single bolus vs. fractionated) of administration. The time-course of CPF and TCP in blood following a single oral bolus 1 mg CPF/kg body weight dose in corn oil or as three fractionated doses of 0.33 mg/kg each in PND-5 rats is presented in Figure 6. In this experiment, a substantial number of blood concentration time-points for both CPF and TCP were determined. Peak blood concentrations of CPF and TCP were attained at 3- to 6-h postdosing (Fig. 6A). Compared to the results seen in Figures 5A and 5D the Cmax for CPF was slightly higher (0.14 vs. 0.07 µmol/l) while the blood TCP concentration was lower (1.61 vs. 3.85 µmol/l); however, the overall pharmacokinetic profile was very comparable and the PBPK/PD model reasonably simulated the CPF and TCP blood time-course. Likewise the model reasonably simulated the CPF and TCP blood time-course following the split dose exposure experiment (Fig. 6B). As anticipated, the Cmax for CPF and TCP following the fractionated CPF doses were lower (2.8–7.4 fold) than following the single bolus administration and the PBPK/PD model reasonably simulated this response.
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PD of ChE Inhibition
The PBPK/PD model (Timchalk et al., 2002
) developed to describe the CPF-oxon stoichiometric inhibition of both target (i.e., AChE) and nontarget B-est (i.e., CaE and BuChE) was adapted to incorporate age-dependent parameter estimates. Parameter estimates and scaling equations for the PD model are presented in Tables 3 and 4. In the current model, the tissue B-est enzyme levels were scaled as a function of age (see Fig. 3B), and the measured Ki for AChE in PND-5, -12, and -17 rats of 950, 500, and 220 (µM h)–1, respectively, were applied (Kousba et al., 2007). The Ki for RBC AChE in the adult model was set at 100 (µM h)–1, while in the current model the RBC AChE Ki for PND-5 and -12 rats was proportionally adjusted to 431 and 227 (µM h)–1, respectively, based on changes reported by Kousba et al. (2007) with brain AChE. The CaE and BuChE Ki values and the reactivation (Kr) and aging (Ka) rates for the B-est were based on the previously published model (Timchalk et al., 2002).
The time-courses of plasma ChE, RBC AChE, and brain AChE inhibition were previously determined (Timchalk et al., 2002, 2006) in the preweanling (PND-5 to PND-17) and adult (plasma and brain only) rats through 24-h postdosing following single oral gavage administration of CPF at doses of 1 or 10 mg/kg and along with the PBPK/PD model predictions (solid lines) are presented in Figures 7–9. In these experiments, CPF produced a dose- and age-dependent (i.e., younger more sensitive) inhibition of tissue B-est activity.
Plasma ChE Inhibition
For both doses, the maximum plasma ChE inhibition (MaxI) expressed as the percentage of control activity and PBPK/PD model simulations increased as a function of age such that the extent of ChE inhibition in preweanling rats followed the pattern PND-5 > PND-12 > PND-17 (see Figs. 7A–C). Maximum inhibition at the low dose was achieved by 3-h postdosing, and 6 h at the high dose; recovery of enzyme activity was marginal through 24 h. As noted in the Methods section, to more reasonably fit the overall plasma ChE response at all ages the basal plasma AChE activity reported by Maxwell et al. (1987) was increased by a factor of 10. With this modification the PBPK/PD model consistently simulated the maximum ChE inhibition at 6-h postdosing, and model simulation of enzyme recovery through 24-h postdosing was consistent with the experimental data. For adult rats administered the same doses (see Fig. 7D) the maximum plasma ChE inhibition was comparable to the response seen in PND-12 and -17 rats. Overall, these results indicate that the PD model provided a reasonable simulation of the age- and dose-dependent inhibition of plasma ChE activity in the neonatal and adult rats.
RBC AChE Inhibition
As illustrated in Figure 8, the experimental result time-course of RBC AChE also demonstrated an age- and dose-dependency in preweanling rats with the extent of inhibition following the pattern PND-5 > PND-12 > PND-17 at both doses (Timchalk et al., 2006). The PBPK/PD model (solid lines) accurately simulated both the dose- and age-dependent RBC AChE inhibition through 24-h postdosing. Although the timing to maximal inhibition of RBC AChE (3- to 6-h postdosing) was very comparable to the response seen with plasma ChE, consistent with previous observations in rats (Timchalk et al., 2002) the magnitude of the RBC AChE inhibition as reflected by both the experimental data and model simulations was less than for the plasma ChE at all ages.
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Brain AChE Inhibition
The time-course for brain AChE inhibition (Timchalk et al., 2002
, 2006) and model simulations are presented in Figure 9 and are similar to the responses seen with plasma ChE and RBC AChE. The experimentally determined time-course of brain AChE inhibition demonstrated both an age- and dose-dependency in the preweanling rats (Timchalk et al., 2006), and at all dose levels and ages the PBPK/PD model reasonably simulated the dynamics of brain AChE inhibition (Fig. 9). At a dose of 1 mg/kg only the PND-5 rats exhibited any brain AChE inhibition (MaxI, 78% of control); whereas, no inhibition was noted in PND-12 and -17 rats as compared with the controls. In contrast, a dose of 10 mg/kg resulted in substantial inhibition of brain AChE at all preweanling ages with the experimentally determined MaxI at PND-5, -12, and -17 being 16%, 20%, and 41% of control, respectively. In adult rats administered 1 or 10 mg CPF/kg of body weight, measured brain AChE activity (MaxI) was 103% and 85% of control, respectively (see Fig. 8D). These results suggest that the adult rat brain AChE is fairly refractory to the CPF-oxon inhibition at a dose as high as 10 mg/kg; whereas, at all ages in the preweanling rats the enzyme activity was more substantially inhibited. This age- and dose-dependent response as reflected by both the experimental data and model simulations is consistent with the greater sensitivity of juvenile rats to the acute high-dose effects of CPF relative to adults (Moser and Padilla, 1998; Pope and Liu, 1997; Pope et al., 1991).
Model Simulation of ChE Recovery
Since the previous study (Timchalk et al., 2006) only evaluated CPF dosimetry and ChE inhibition through 24-h postdosing, the full recovery of ChE activity was not observable. To evaluate the capability of the model to simulate recovery of ChE activity the model simulation was compared against several different published data sets. Won et al. (2001) evaluated the extent of brain (frontal cortex) AChE inhibition through 96-h postdosing following oral gavage administration of CPF at doses corresponding to the LD10 and 50% of the LD10 in neonatal (PND-7: 15 and 7.5 mg/kg), juvenile (PND-21: 47 and 23.5 mg/kg), and adult (PND-90: 136 and 60 mg/kg) Sprague–Dawley rats (see Fig. 10). The low and high doses were anticipated to produce comparable responses across ages, and the authors reported that the brain AChE inhibition ranged from 40% to 60% (50% LD10) and 80% to 90% (LD10), respectively (Won et al., 2001). As illustrated in Figure 10, the PBPK/PD model simulations (solid lines) did result in comparable brain AChE inhibition for all ages, consistent with the experimental design (50% and 100% of LD10). However, in PND-7 rats the model overpredicted the observed inhibition following the low dose (7.5 mg/kg), and in adults the model-predicted brain AChE recovery was slightly faster than the observed response at 24- and 96-h postdosing for both dose levels (68 and 136 mg/kg). Nonetheless, the model simulations are reasonably consistent with the observed experimental data and experimental design.
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Moser and Padilla (1998)
likewise evaluated the toxicity of CPF in male and female PND-17 Long Evans rats that were orally administered CPF by gavage at a maximum tolerated dose of 15 mg/kg. In this study the time-course of brain and RBC AChE activity were determined through 14 days (336 h) postexposure. The results of the model simulations for both brain and RBC AChE are presented in Figure 11. Moser and Padilla (1998) reported average body weights for PND-17 male and female Long Evans rats as 34 and 33 g, respectively; which is consistent with the 33g predicted using the polynomial equation developed for Sprague–Dawley rats (see Table 2, Fig. 2A). In brains analyzed from both genders, the AChE time-course was very comparable and the maximum measured AChE inhibition was 11% of control (6-h postdosing) which recovered to 80% by 162-h postdosing. Model simulations of these data resulted in a maximum inhibition of 27% of control which was nearly fully recovered (95%) by 160 h. Although the model predicted less inhibition than was experimentally observed, the simulations accurately reflected the enzyme recovery rate. With regard to RBC AChE, enzyme activity was significantly inhibited to 3–5% of control activity for both genders by 6-h postdosing, which was very consistent with the model's maximum prediction of 8% of control activity. However, the overall RBC AChE recovery rate appeared to be slightly faster in the females than in males, with the model predicting near maximal recovery by 160-h postdosing, which was consistent with the female data set. It is of interest to note that an improved fit to the experimental data (dashed line) could be obtained by allowing the model to optimize the fit to the experimental data by increasing the administered dose of CPF. By fitting the maximum inhibition, particularly for the brain AChE, the model provided a better fit of the overall brain AChE recovery profile through 350-h postdosing. However, the same approach for RBC AChE did not provide any substantial improvement.
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ChE Inhibition for Single and Repeat CPF Oral Doses
Zheng et al. (2000)
evaluated the age-dependent changes in neurochemical indicators of toxicity including brain AChE over a broad range of acute and repeated dose exposures to CPF in PND-7 and -21 Sprague–Dawley rats. Beginning on PND-7, groups of rats were given either a single or 14 daily oral gavage doses, and sacrificed 4 h after the first or 14th dose, respectively. The inhibition of plasma ChE, RBC AChE, and brain AChE was evaluated and the observed and model-predicted response (single and repeat) is presented in Table 5 and the simulation of the 14 daily repeat doses is illustrated in Figure 12. As reported by Zheng et al. (2000) the observed dose-dependent plasma ChE and brain AChE inhibition was comparable for both the PND-7 and -21 animals that received either a single or repeated administration of CPF. In contrast, repeated CPF doses resulted in a greater RBC AChE inhibition (10–38%) at all dose levels relative to the single dose (Table 5, B). The PBPK/PD model accurately simulated the plasma ChE inhibition in PND-7 rats following single oral exposures ranging from 0.15 to 1.5 mg/kg. However, the lack of a dose–response for plasma ChE at doses 
4.5 mg/kg resulted in the model overpredicting the amount of inhibition at these higher doses. The model also overpredicted the plasma ChE inhibition in PND-21 rats following the repeated exposure at all dose levels (Table 5, A). However, the model reasonably simulated the RBC AChE response following the single or repeated exposures (Table 5, B). The dose-dependent brain AChE inhibition was adequately simulated at doses ranging from 0.15 to 0.75 mg/kg, but was underpredicted at doses 1.5 mg/kg following the single (PND-7) exposures. The model tended to slightly underpredict the extent of brain AChE inhibition following the repeated (PND-21) doses, but the overall trend was consistent with the experimental results. Following repeated dose administration (see Figure 12) the time-course of plasma ChE, and RBC and brain AChE inhibition suggest that the maximum inhibition in plasma and RBC was achieved within two to four doses, whereas in the brain the maximum inhibition was not achieved until six to eight doses. It is also of interest to note that the brain AChE inhibition response was marginal at 0.75 mg/kg/day or less, but was substantially inhibited following repeated dosing > 1.5 mg/kg/day.
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PND-5 versus Adult Oxon Area Under Concentration Curve
A comparison of the simulated CPF-oxon AUC ratios (PND-5 vs. adult) in both blood and brain over a broad range of CPF doses is illustrated in Figure 13. At doses ranging from 0.001 to 1 mg/kg the neonatal to adult CPF-oxon ratio [PND – 5(AUC)]/Adult(AUC) for blood and brain were
1.3; however, at CPF doses > 1 mg/kg the ratio rapidly increased in both blood and brain and approached 2 at 10 mg/kg. This suggests that age-dependent difference in brain oxon concentration may be an important contributing factor associated with the increased sensitivity of preweanling rats relative to adults particularly at the higher doses utilized in toxicology studies.
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comparing neonatal (PND-5) and adult rats PND-5(AUC)/Adult(AUC) following simulation of an acute oral exposure to a broad range of CPF doses.Sensitivity Analysis
A model sensitivity analysis was conducted to compare the relative impact of metabolism, brain AChE parameters, tissue volume changes, and blood flows on brain AChE response in PND-5 versus adult rats. A summary of these results is presented in Table 6 (acslXtreme®; see supplemental data). It was anticipated that hepatic metabolism and blood flow would most likely be important determinants of the extent of CPF-oxon formation and detoxification which could modify brain oxon dosimetry and associated AChE inhibition. In addition, the bimolecular inhibitory rate constant (Ki) for brain AChE binding/inhibition will also be an important determinant of brain AChE inhibition. For all analyses, the metabolic parameters (Km and Vmax) associated with CYP450 and PON-1 metabolism were more sensitive in PND-5 versus adult rats with regard to brain AChE inhibition. The model was more sensitive to changes in hepatic PON-1 than to blood activity, which would be consistent with the importance of the liver for overall metabolism (activation as well as detoxification). As expected, the model was very sensitive to changes in the Ki for brain AChE inhibition, especially in PND-5 animals relative to the adults. The greater sensitivity in the PND-5 rats may be associated with an approximate 4x higher Ki for CPF-oxon binding to AChE than is seen in the adult rat. Since these parameters were measured in vitro (Kousba et al., 2007
), there is confidence in utilizing these different age-dependent parameter estimates. Overall, the PND-5 preweanling rats appeared to be more sensitive to slight changes in model parameters than the adult rats, and they were particularly sensitive to changes in CYP450-related metabolism (activation and detoxification).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Previous studies suggest that juvenile rats are sensitive to the high-dose toxicity of OP insecticides, and a lack of maturation in detoxification pathways appears to be a contributing factor (Atterberry et al., 1997
; Li et al., 1997; Mortensen et al., 1996, 1998). It has been proposed that PBPK/PD models can be applied to assess this age-dependent sensitivity (Clewell et al., 2004; Ginsberg et al., 2004; Price et al., 2003; Timchalk, 2001). The current paper extends our previously developed rodent PBPK/PD model for CPF by incorporating age-dependent physiological and metabolic parameters.
In this model, age was utilized as a dependent function to estimate body weight, and organ growth was determined as a percentage of body weight. Liver and brain volumes were based on best-fit polynomial equations (see Fig. 2 and supplemental data). This approach was comparable to the scaling approach applied to characterize physiological and anatomical changes from birth to adolescence in humans (Haddad et al., 2001). However, due to limited amounts of juvenile rat data it was not feasible to develop comprehensive age-dependent scaling algorithms as has been done for human models (Clewell et al., 2004; Price et al., 2003). The default approach for cross species scaling of metabolism is to scale the metabolic rate as the 3/4th power of body weight (Vmax = VmaxC x Bwt0.74) (Krishnan and Andersen, 1994). In the current model, alternative scaling exponents were developed (see Tables 2 and 4) for CYP450 and PON-1 metabolism and for B-est tissue levels based upon published data in the preweanling rat (Atterberry et al., 1997; Carr et al., 2000; Mortensen et al., 1996, 1998; Tang et al., 1999).
Knaak et al. (2004) reviewed the physiochemical and biological literature needed to develop OP insecticide PBPK/PD models, and noted that many of the in vitro and in vivo data needed for model development are inadequate or lacking. To this end, in vivo and in vitro pharmacokinetic/PD studies were previously conducted to provide needed experimental data to facilitate model development (Kousba et al., 2007; Timchalk et al., 2006). Based on the observed blood time-course of CPF, absorption following gavage oral administration in the preweanling rat was relatively rapid (see Figure 5; Cmax 3–6 h) and consistent with the absorption rate previously seen in the adult rat (see Figure 4A). Hence, the current model utilized the same structure and parameter estimates to determine the absorption rate as previously described (Timchalk et al., 2002). It was assumed that absorption from the gut was complete at all ages; this simplifying assumption was done in full recognition that the extent of oral absorption has been shown to be variable and dependent upon the dose formulation (Nolan et al., 1984; Timchalk et al., 2002). However, there are supporting data suggesting that absorption for a number of xenobiotics in both rodents and humans may be greater in juveniles than for adults (Ginsberg et al., 2004).
Of particular importance was the observation that even in rats as young as PND-5, the CYP450 metabolic capacity was adequate to metabolize CPF to both TCP and CPF-oxon based on the detection of TCP in blood and extensive ChE inhibition. In addition, the increase in the blood TCP concentration (3-fold) in PND-17 rats relative to the response in the younger animals, and the lower blood concentrations of CPF (1.7 to 7.5-fold) in adults (Timchalk et al., 2002) relative to preweanling rats is consistent with an increase in CYP450 metabolic capacity with age. This suggests that CPF was rapidly absorbed and metabolized, and the extent of metabolism was age-dependent.
The current model is capable of predicting B-est inhibition based upon the stoichiometric enzyme interaction with CPF-oxon. The extent and rate of B-est inhibition and recovery is dependent upon the amount of available enzyme, the bimolecular inhibition rate constant (Ki), and the exposure duration (Vale, 1998). The relative quantity of available B-est binding sites (µmol) followed the general order: CaE >> BuChE > AChE as was previously described for the adult rat (Maxwell et al., 1987; Timchalk et al., 2002). Age-related changes in regional brain distribution of AChE and the molecular forms of the enzyme have been reported (Bisso et al., 1991; Mendeguz et al., 1992; Skau and Triplett, 1998). Although age-dependent changes in Ki values have not been extensively investigated, our research group has recently reported age-dependent differences in the apparent Ki values for CPF-oxon (Kousba et al., 2007). Studies suggest that Ki differences could be related to the presence of a secondary peripheral binding site, which can impact the capability of the substrate to reach the active site of AChE (Kousba et al., 2004; Taylor and Radic, 1994). The potential for CPF-oxon to interact with secondary proteins that modify the binding affinity of the oxon with AChE is also feasible; in this regard, Murphy (1982) suggested that a lower toxicity in adult rats may be in part due to a higher binding of the oxon to noncritical tissues in the brain. Consistent with this hypothesis, Mortensen et al. (1998) showed that the brain protein concentrations increased from 7% to 12% from PND-4 to -90. Regardless of the specific underlying mechanism for the observed age-related differences in the apparent Ki parameters, the use of the measured Ki parameters (Kousba et al., 2007) for tissue AChE enables the model to reasonably simulate tissue ChE as a function of age and dose in the rat.
In the current study the model-predicted ChE inhibition is generally comparable with the experimentally determined results which are also consistent with the observed age-dependent sensitivity of juvenile rats (Atterberry et al., 1997; Benke and Murphy, 1975; Broderu and DuBois, 1963; Gaines and Linder, 1986; Harbison, 1975; Mortensen et al., 1996, 1998; Moser and Padilla, 1998). Of importance is the magnitude of the age- and dose-dependent inhibition of brain AChE. In the preweanling rats, the dose–response for brain AChE was very steep as demonstrated by the limited inhibition predicted by the model at 1 mg/kg (78–98% of control), but substantial inhibition (16–41% of control) at 10 mg/kg (see Figs. 9A–C). In adult rats we have previously noted (Timchalk et al., 2002) a similar steep dose–response for brain AChE inhibition which occurs at doses > 10 mg/kg; whereas, in the current study doses that produce substantial brain AChE inhibition in preweanling rats (10 mg/kg) resulted in minimal to no AChE inhibition (85–103% of control; Fig. 9D) in adults. This observation is consistent with the model simulations comparing the ratio of blood and brain CPF-oxon AUC of PND-5 and adult rats (see Fig. 13) that suggest a disproportionate increase in oxon levels in preweanlings relative to adults. This increase in blood and brain CPF-oxon concentration is consistent with the hypothesis that the greater sensitivity of preweanling rats is primarily due to a lack of maturation in detoxification pathways, as has been suggested by a number of other investigators (Atterberry et al., 1997; Li et al., 1997; Mortensen et al., 1996, 1998).
Although the PBPK/PD model simulates the overall age-dependent dosimetry and ChE inhibition response, not all of the experimental data are as well described by the model, which suggests some potential areas for improvement in model structure (i.e., inadequate description of the biological system) or inadequate model parameterization, and future research needs. For example, in the preweanling rats (see Fig. 5) the simulations of CPF blood concentrations generally underpredicted the experimental data and suggested a faster blood clearance than was experimentally determined. These observed dosimetry differences suggest that rates of absorption, changes in tissue distribution and protein binding characteristics, or the extent of CYP450 metabolism may not be adequately characterized. Likewise, with regard to plasma ChE, the model consistently overpredicted peak preweanling inhibition (Table 5, A; Figs. 7 and 12), even when we increased the reported plasma AChE activity by a factor of 10, yet more reasonably simulated the RBC AChE response (Table 5, B, Figs. 8 and 12). This observed difference may be related to the complexity of the plasma where three B-est enzymes (AChE, BuChE and CaE) are present and function to stoichiometrically bind with and detoxify CPF-oxon. Of the three B-est, AChE was the only enzyme where age-dependent data for the bimolecular inhibition rate constant (Ki) were determined (Kousba et al., 2007) in brain and applied to all tissue (plasma, RBC, diaphragm, and liver); whereas, in the current model the inhibition rate constants for BuChE and CaE were maintained at the adult levels. The complexity of the plasma compartment with regard to B-est activity could contribute to the observed difference in plasma ChE response relative to RBC AChE. Further refinement of the model fit may be possible by obtaining additional experimental data to define these potential age-dependent model parameters. Finally, one of the important functional limitations of the current model is that it does not take into account observed cholinergic toxicity which is clearly known to modify a range of physiological and metabolic parameters (see Lotti, 2001). Hence, some of the poorer fits to experimental data particularly at acutely toxic doses may be partially accounted for by this limitation in the current model structure.
The PBPK/PD model developed here represents one potential computational framework for development of an age-dependent human model. As noted by Ginsberg et al. (2004), current risk assessment methods utilize an across species extrapolation that is typically done between adults; however, based on similar patterns for developmental ontogeny of metabolism and clearance mechanisms the evaluation of toxicity in juvenile animals may have more direct relevance to children. Therefore, the development of age-dependent models offers great promise to better identify "early life-stage" related sensitivity (Clewell et al., 2004; Price et al., 2003). In general, metabolic functions develop over the first 2–3 weeks in rats and 2–3 months in children (Ginsberg et al., 2004). However, there is still considerable uncertainty concerning the species-specific developmental rates of pharmacokinetic systems, particularly in juvenile animals where there are limited data. There is a need to continue developing new data with animal models that can then be used to facilitate the extrapolation and development of early life-stage models in humans. Nonetheless, early life-stage models are being developed and applied to children (Clewell et al., 2004; Pelekis et al., 2001; Price et al., 2003). The development of comparable life-stage animal models holds great promise to enhance age- and species-specific dosimetry and dynamic response extrapolations and should reduce the uncertainty associated with age-specific risk assessments.
In summary, these results indicate that the age-dependent PBPK/PD model behaves consistently with the general understanding of CPF toxicity, pharmacokinetics, and tissue ChE inhibition in neonatal and adult rats. Secondly, the model suggests that neonatal rats are quantitatively more sensitive to the high-dose acute effects of CPF exposure than adult rats. Future research must entail further development and validation with the ultimate goal of developing a model that is capable of predicting biological response in infants and children.
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SUPPLEMENTARY DATA |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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FUNDING |
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Centers for Disease Control and Prevention (R01 OH003629-04, R01 OH008173-01); U.S. EPA's STAR program (R828608).
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NOTES |
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2 Current address: TargeGen Inc., 9380 Judiccial Dr., San Diego, CA 92121.
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ACKNOWLEDGMENTS |
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The contents of this paper are solely the responsibility of the authors and have not been subject to any review by CDC or EPA and therefore do not necessarily represent the official view of CDC or EPA, and no official endorsement should be inferred.
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