Physiologically Based Pharmacokinetic Modeling of Salivary Concentrations for Noninvasive Biomonitoring of 2,4-Dichlorophenoxyacetic Acid (2,4-D).

Saliva has become a favorable sample matrix for biomonitoring due to its noninvasive attributes and overall flexibility in collection. To ensure measured salivary concentrations reflect the exposure, a solid understanding of the salivary transport mechanism and relationships between salivary concentrations and other monitored matrices (ie, blood, urine) is needed. Salivary transport of a commonly applied herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), was observed in vitro and in vivo and a physiologically based pharmacokinetic (PBPK) model was developed to translate observations from the cell culture model to those in animal models and further evaluate 2,4-D kinetics in humans. Although apparent differences in experimental in vitro and in vivo saliva:plasma ratios (0.034 and 0.0079) were observed, simulations with the PBPK model demonstrated dynamic time and dose-dependent saliva:plasma ratios, elucidating key mechanisms affecting salivary transport. The model suggested that 2,4-D exhibited diffusion-limited transport to saliva and was additionally impacted by protein binding saturation and permeability across the salivary gland. Consideration of sampling times post-exposure and potential saturation of transport mechanisms are then critical aspects for interpreting salivary 2,4-D biomonitoring observations. This work utilized PBPK modeling in in vitro to in vivo translation to explore benefits and limitations of salivary analysis for occupational biomonitoring.

Evaluation of non-invasive biomonitoring of 2,4-Dichlorophenoxyacetic acid (2,4-D) in saliva.

The objective of this study was to evaluate the potential for non-invasive biomonitoring of 2,4-Dichlorophenoxyacetic acid (2,4-D) in saliva. Using an in vitro rat salivary gland epithelial cell (SGEC) system, a collection of experiments investigating chemical protein binding, temporal and directional transport, as well as competitive transport with para-aminohippuric acid (PAH), a substrate for renal organic anion transporters, was conducted to identify cellular transport parameters required to computationally model salivary transport of 2,4-D. Additionally, a physiological protein gradient was implemented to mimic physiologically relevant concentrations of protein in rat plasma and saliva, and under these conditions the transfer of 2,4-D was markedly slower, driven by increased protein binding (i.e. reduced free 2,4-D species available to cross salivary barrier). The rate of transfer was directly proportional to the amount of unbound 2,4-D and demonstrated no indication of active transport. An in vivo assessment of 2,4-D exposure in rats revealed non-linear protein binding in plasma, indicating saturated protein binding and increased levels of unbound 2,4-D species at higher doses. A strong correlation between 2,4-D concentrations in saliva and unbound 2,4-D in plasma was observed (Pearson correlation coefficient = 0.95). Saliva:plasma 2,4-D ratios measured in vivo (0.0079) were consistent within the linear protein binding range and expected 2,4-D levels from occupational exposures but were significantly different than ratios measured in vitro (physiological conditions) (0.034), possibly due to 2,4-D concentrations in saliva not being at equilibrium with 2,4-D concentrations in blood, as well as physiological features absent in in vitro settings (e.g. blood flow). We demonstrated that 2,4-D is consistently transported into saliva using both in vitro and in vivo models, making 2,4-D a potential candidate for human non-invasive salivary biomonitoring. Further work is needed to understand whether current sensor limits of detection are sufficient to measure occupationally relevant exposures.

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.

Non-invasive saliva human biomonitoring: development of an in vitro platform.

Direct measurements of exposure represent the most accurate assessment of a subject's true exposure. The clearance of many drugs and chemicals, including pesticides such as chlorpyrifos (CPF), can be detected non-invasively in saliva. Here we have developed a serous-acinar transwell model system as an in vitro screening platform to prioritize chemicals for non-invasive biomonitoring through salivary clearance mechanisms. Rat primary serous-acinar cells express both α-amylase and aquaporin-5 proteins and develop significant tight junctions at postconfluence - a feature necessary for chemical transport studies in vitro. CPF exhibited bidirectional passage across the serous-acinar barrier that was disproportional to the passage of a cell impermeable chemical (lucifer yellow), consistent with a hypothesized passive diffusion process. CPF was metabolized to trichlorpyridinol (TCPy) by serous-acinar cells, and TCPy also displayed bidirectional diffusion in the transwell assay. This model system should prove useful as an in vitro screening platform to support the non-invasive monitoring of toxicons and pharmacons in human saliva and provide guidance for development of advanced in vitro screening platforms utilizing primary human salivary gland epithelial cells.