Translating nanoparticle dosimetry from conventional in vitro systems to occupational inhalation exposures.

As encouraged by Toxicity Testing in the 21st Century, researchers increasingly apply high-throughput in vitro approaches to identify and characterize nanoparticle hazards, including conventional aqueous cell culture systems to assess respiratory hazards. Translating nanoparticle dose from conventional toxicity testing systems to relevant human exposures remains a major challenge for assessing occupational risk of nanoparticle exposures. Here, we explored existing computational tools and data available to translate nanoparticle dose metrics from cellular test systems to inhalation exposures of silver nanoparticles in humans. We used the Multiple-Path Particle Dosimetry (MPPD) Model to predict nanoparticle deposition of humans exposed to 20 and 110 nm silver nanoparticles at 0.9 µg/m3 over an 8 h period, the proposed National Institute of Occupational Safety and Health (NIOSH) recommended exposure limit (REL). MPPD predicts 8.1 and 3.7 µg of silver deposited in an 8 h period for 20 and 110 nm nanoparticles, respectively, with 20 nm particles displaying nearly 11-fold higher total surface area deposited. Peak deposited nanoparticle concentrations occurred more proximal in the pulmonary tract compared to mass deposition patterns (generation 4 vs. generations 20-21, respectively) due to regional differences in lung lining fluid volumes. Assuming 0.4% nanoparticle dissolution by mass measured in previous studies predicted peak concentrations of silver ions in cells of 1.06 and 0.89 µg/mL for 20 and 110 nm particles, respectively. Both predicted concentrations are below the measured toxic threshold of 1.7 µg/mL of silver ions in cells from in vitro assessments. Assuming 4% dissolution by mass predicted 10-fold higher silver concentrations in tissues, peaking at 10.6 and 8.9 µg/mL, for 20 and 110 nm nanoparticles respectively, exceeding the observed in vitro toxic threshold and highlighting the importance and sensitivity of dissolution rates. Overall, this approach offers a framework for extrapolating nanotoxicity results from in vitro cell culture systems to human exposures. Aligning appropriate dose metrics from in vitro and in vivo hazard characterizations and human pulmonary doses from occupational exposures are critical components for successful nanoparticle risk assessment and worker protection providing guidance for designing future in vitro studies aimed at relevant human exposures.

Plasma Protein Turnover Rates in Rats Using Stable Isotope Labeling, Global Proteomics, and Activity-Based Protein Profiling.

Protein turnover is important for general health on cellular and organism scales providing a strategy to replace old, damaged, or dysfunctional proteins. Protein turnover also informs of biomarker kinetics, as a better understanding of synthesis and degradation of proteins increases the clinical utility of biomarkers. Here, turnover rates of plasma proteins in rats were measured in vivo using a pulse-chase stable isotope labeling experiment. During the pulse, rats (n = 5) were fed 13C6-labeled lysine ("heavy") feed for 23 days to label proteins. During the chase, feed was changed to an unlabeled equivalent feed ("light"), and blood was repeatedly sampled from rats over 10 time points for 28 days. Plasma samples were digested with trypsin and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant was used to identify peptides and proteins and quantify heavy/light lysine ratios. A system of ordinary differential equations was used to calculate protein turnover rates. Using this approach, 273 proteins were identified, and turnover rates were quantified for 157 plasma proteins with half-lives ranging 0.3-103 days. For the ∼70 most abundant proteins, variability in turnover rates among rats was low (median coefficient of variation: 0.09). Activity-based protein profiling was applied to pooled plasma samples to enrich serine hydrolases using a fluorophosphonate (FP2) activity-based probe. This enrichment resulted in turnover rates for an additional 17 proteins. This study is the first to measure global plasma protein turnover rates in rats in vivo, measure variability of protein turnover rates in any animal model, and utilize activity-based protein profiling for enhancing turnover measurements of targeted, low-abundant proteins, such as those commonly used as biomarkers. Measured protein turnover rates will be important for understanding of the role of protein turnover in cellular and organism health as well as increasing the utility of protein biomarkers through better understanding of processes governing biomarker kinetics.

Predicting Transport of 3,5,6-Trichloro-2-Pyridinol Into Saliva Using a Combination Experimental and Computational Approach.

A combination experimental and computational approach was developed to predict chemical transport into saliva. A serous-acinar chemical transport assay was established to measure chemical transport with nonphysiological (standard cell culture medium) and physiological (using surrogate plasma and saliva medium) conditions using 3,5,6-trichloro-2-pyridinol (TCPy) a metabolite of the pesticide chlorpyrifos. High levels of TCPy protein binding were observed in cell culture medium and rat plasma resulting in different TCPy transport behaviors in the 2 experimental conditions. In the nonphysiological transport experiment, TCPy reached equilibrium at equivalent concentrations in apical and basolateral chambers. At higher TCPy doses, increased unbound TCPy was observed, and TCPy concentrations in apical and basolateral chambers reached equilibrium faster than lower doses, suggesting only unbound TCPy is able to cross the cellular monolayer. In the physiological experiment, TCPy transport was slower than nonphysiological conditions, and equilibrium was achieved at different concentrations in apical and basolateral chambers at a comparable ratio (0.034) to what was previously measured in rats dosed with TCPy (saliva:blood ratio: 0.049). A cellular transport computational model was developed based on TCPy protein binding kinetics and simulated all transport experiments reasonably well using different permeability coefficients for the 2 experimental conditions (1.14 vs 0.4 cm/h for nonphysiological and physiological experiments, respectively). The computational model was integrated into a physiologically based pharmacokinetic model and accurately predicted TCPy concentrations in saliva of rats dosed with TCPy. Overall, this study demonstrates an approach to predict chemical transport in saliva, potentially increasing the utility of salivary biomonitoring in the future.

A human life-stage physiologically based pharmacokinetic and pharmacodynamic model for chlorpyrifos: development and validation.

Sensitivity to some chemicals in animals and humans are known to vary with age. Age-related changes in sensitivity to chlorpyrifos have been reported in animal models. A life-stage physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model was developed to predict disposition of chlorpyrifos and its metabolites, chlorpyrifos-oxon (the ultimate toxicant) and 3,5,6-trichloro-2-pyridinol (TCPy), as well as B-esterase inhibition by chlorpyrifos-oxon in humans. In this model, previously measured age-dependent metabolism of chlorpyrifos and chlorpyrifos-oxon were integrated into age-related descriptions of human anatomy and physiology. The life-stage PBPK/PD model was calibrated and tested against controlled adult human exposure studies. Simulations suggest age-dependent pharmacokinetics and response may exist. At oral doses ⩾0.6mg/kg of chlorpyrifos (100- to 1000-fold higher than environmental exposure levels), 6months old children are predicted to have higher levels of chlorpyrifos-oxon in blood and higher levels of red blood cell cholinesterase inhibition compared to adults from equivalent doses. At lower doses more relevant to environmental exposures, simulations predict that adults will have slightly higher levels of chlorpyrifos-oxon in blood and greater cholinesterase inhibition. This model provides a computational framework for age-comparative simulations that can be utilized to predict chlorpyrifos disposition and biological response over various postnatal life stages.

Pharmacokinetics and pharmacodynamics of chlorpyrifos and 3,5,6-trichloro-2-pyridinol in rat saliva after chlorpyrifos administration.

Sensors have been developed for noninvasive biomonitoring of the organophosphate pesticide chlorpyrifos (CPF), and previous studies have suggested consistent partitioning of 3,5,6-trichloro-2-pyridinol (TCPy), a metabolite of CPF, into saliva after exposure to TCPy. The objective of this study was to quantitatively evaluate in vivo pharmacokinetics and pharmacodynamics of CPF and TCPy in saliva after CPF administration. Rats were coadministered CPF (0.5-5mg/kg) and pilocarpine (~13 mg/kg) iv. Saliva and blood were collected, and levels of CPF, TCPy, and cholinesterase (ChE) activity were quantified. Experimental results suggest that CPF is rapidly metabolized after iv administration. Formation of TCPy from administered CPF at the low dose (0.5 mg/kg) was slower than from higher CPF doses, potentially due to differences in plasma protein binding to CPF. CPF was measured in saliva only at the first time point sampled (0-15 min), indicating low partitioning and rapid metabolism. After formation, TCPy pharmacokinetics were very similar in blood and saliva. Saliva/blood TCPy concentration ratios were not affected by TCPy concentration in blood, saliva flow rate, or salivary pH and were consistent with previous studies. ChE activity in plasma demonstrated a dose-dependent decrease, and ChE activity in saliva was extremely variable and demonstrated no dose relationship. A physiologically based pharmacokinetic and pharmacodynamic model for CPF was modified and predicted the data reasonably well. It is envisioned that a combination of biomonitoring compounds like TCPy in saliva coupled with computational modeling will form an approach to measure pesticide exposure to susceptible human populations such as agricultural workers.

Pharmacokinetics and pharmacodynamics of chlorpyrifos in adult male Long-Evans rats following repeated subcutaneous exposure to chlorpyrifos.

Chlorpyrifos (CPF) is a commonly used organophosphorus pesticide. Several pharmacokinetic and pharmacodynamic studies have been conducted in rats in which CPF was administered as a single bolus dose. However, there is limited data regarding the pharmacokinetics and pharmacodynamics following daily exposure. Since occupational exposures often consist of repeated, daily exposures, there is a need to evaluate the pharmacokinetics and pharmacodynamics of CPF under exposure conditions which more accurately reflect real world human exposures. In this study, the pharmacokinetics and pharmacodynamics of CPF were assessed in male Long-Evans rats exposed daily to CPF (0, 3 or 10mg/kg/day, s.c. in peanut oil) over a 10 day study period. Throughout the study, multiple pharmacokinetic (urinary TCPy levels and tissue CPF and metabolite levels) and pharmacodynamic (blood and brain AChE activity) determinants were measured. Average blood AChE activity on day 10 was 54% and 33% of baseline among animals in the 3 and 10mg/kg/day CPF treatment groups, respectively, while average brain AChE activity was 67% and 28% of baseline. Comparable dose-response relationships between brain AChE inhibition and blood AChE inhibition, suggests that blood AChE activity is a valid biomarker of brain AChE activity. The pharmacokinetic and pharmacodynamic measures collected in this study were also used to optimize a rat physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) model for multiple s.c. exposures to CPF based on a previously published rat PBPK/PD model for CPF following a single bolus injection. This optimized model will be useful for determining pharmacokinetic and pharmacodynamic responses over a wide range of doses and durations of exposure, which will improve extrapolation of results between rats and humans.

In vitro age-dependent enzymatic metabolism of chlorpyrifos and chlorpyrifos-oxon in human hepatic microsomes and chlorpyrifos-oxon in plasma.

Age-dependent chlorpyrifos (CPF) metabolism was quantified by in vitro product formation in human hepatic microsomes (ages 13 days to 75 years) and plasma (ages 3 days to 43 years) with gas chromatography-mass spectrometry. Hepatic CPF cytochrome P450 desulfuration [CPF to chlorpyrifos-oxon (CPF-oxon)] and dearylation (CPF to 3,5,6-trichloro-2-pyridinol) V(max) values were 0.35 ± 0.21 and 0.73 ± 0.38 nmol · min(-1) · mg microsomal protein (-1) (mean ± S.D.), respectively. The mean (±S.D.) hepatic CPF-oxon hydrolysis (chlorpyrifos-oxonase [CPFOase]) V(max) was 78 ± 44 nmol · min(-1) · mg microsomal protein (-1). None of these hepatic measures demonstrated age-dependent relationships on a per microsomal protein basis using linear regression models. Ratios of CPF bioactivation to detoxification (CPF desulfuration to dearylation) V(max) values were consistent across ages. CPFOase in plasma demonstrated age-dependent increases on a volume of plasma basis, as did total plasma protein levels. Mean (±S.D.) CPF-oxon hydrolysis V(max) values for children <6 months of age and adults (≥16 years) were 1900 ± 660 and 6800 ± 1600 nmol · min(-1) · ml(-1), respectively, and at environmental exposure levels, this high- capacity enzyme is likely to be sufficient even in infants. Plasma samples were phenotyped for paraoxonase status, and frequencies were 0.5, 0.4, and 0.1 for QQ, QR, and RR phenotypes, respectively. These results will be integrated into a physiologically based pharmacokinetic and pharmacodynamic model for CPF and, once integrated, will be useful for assessing biological response to CPF exposures across life stages.

Comparative pharmacokinetics of chlorpyrifos versus its major metabolites following oral administration in the rat.

Chlorpyrifos (CPF) is a commonly used diethylphosphorothionate organophosphorus (OP) insecticide. Diethylphosphate (DEP), diethylthiophosphate (DETP) and 3,5,6-trichloro-2-pyridinol (TCPy) are products of both in vivo metabolism and environmental degradation of CPF and are routinely measured in urine as biomarkers of exposure. Hence, urinary biomonitoring of TCPy, DEP and DETP may be reflective of an individual's contact with both the parent pesticide and exposure to these metabolites in the environment. In the current study, simultaneous dosing of 13C- or 2H-isotopically labeled CPF (13C-labeled CPF, 5 13C on the TCPy ring; or 2H-labeled CPF, diethyl-D10 (deuterium labeled) on the side chain) were exploited to directly compare the pharmacokinetics and metabolism of CPF with TCPy, and DETP. The key objective in the current study was to quantitatively evaluate the pharmacokinetics of the individual metabolites relative to their formation following a dose of CPF. Individual metabolites were co-administered (oral gavage) with the parent compound at equal molar doses (14 micromol/kg; approximately 5 mg/kg CPF). Major differences in the pharmacokinetics between CPF and metabolite doses were observed within the first 3h of exposure, due to the required metabolism of CPF to initially form TCPy and DETP. Nonetheless, once a substantial amount of CPF has been metabolized (> or =3h post-dosing) pharmacokinetics for both treatment groups and metabolites were very comparable. Urinary excretion rates for orally administered TCPy and DETP relative to 13C-CPF or (2)H-CPF derived 13C-TCPy and 2H-DETP were consistent with blood pharmacokinetics, and the urinary clearance of metabolite dosed groups were comparable with the results for the 13C- and 2H-CPF groups. Since the pharmacokinetics of the individual metabolites were not modified by co-exposure to CPF; it suggests that environmental exposure to low dose mixtures of pesticides and metabolites will not impact their pharmacokinetics.

Comparative chlorpyrifos pharmacokinetics via multiple routes of exposure and vehicles of administration in the adult rat.

Chlorpyrifos (CPF) is a commonly used organophosphorus pesticide. A number of toxicity and mechanistic studies have been conducted in animals, where CPF has been administered via a variety of different exposure routes and dosing vehicles. This study compared chlorpyrifos (CPF) pharmacokinetics using oral, intravenous (IV), and subcutaneous (SC) exposure routes and corn oil, saline/Tween 20, and dimethyl sulfoxide (DMSO) as dosing vehicles. Two groups of rats were co-administered target doses (5 mg/kg) of CPF and isotopically labeled CPF (L-CPF). One group was exposed by both oral (CPF) and IV (L-CPF) routes using saline/Tween 20 vehicle; whereas, the second group was exposed by the SC route using two vehicles, corn oil (CPF) and DMSO (L-CPF). A third group was only administered CPF by the oral route in corn oil. For all treatments, blood and urine time course samples were collected and analyzed for 3,5,6-trichloro-2-pyridinol (TCPy), and isotopically labeled 3,5,6-trichloro-2-pyridinol (L-TCPy). Peak TCPy/L-TCPy concentrations in blood (20.2 micromol/l), TCPy/L-TCPy blood AUC (94.9 micromol/lh), and percent of dose excreted in urine (100%) were all highest in rats dosed orally with CPF in saline/Tween 20 and second highest in rats dosed orally with CPF in corn oil. Peak TCPy concentrations in blood were more rapidly obtained after oral administration of CPF in saline/Tween 20 compared to all other dosing scenarios (>1.5 h). These results indicate that orally administered CPF is more extensively metabolized than systemic exposures of CPF (SC and IV), and vehicle of administration also has an effect on absorption rates. Thus, equivalent doses via different routes and/or vehicles of administration could potentially lead to different body burdens of CPF, different rates of bioactivation to CPF-oxon, and different toxic responses. Simulations using a physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model for CPF are consistent with these possibilities. These results suggest that exposure route and dosing vehicle can substantially impact target tissue dosimetry. This is of particular importance when comparing studies that use varying exposure paradigms, which are then used for extrapolation of risk to humans.