Evaluation of Human Performance Aiding Live Synthetically Engineered Bacteria in a Gut-on-a-Chip.

Synbiotics are a new class of live therapeutics employing engineered genetic circuits. The rapid adoption of genetic editing tools has catalyzed the expansion of possible synbiotics, exceeding traditional testing paradigms in terms of both throughput and model complexity. Herein, we present a simplistic gut-chip model using common Caco2 and HT-29 cell lines to establish a dynamic human screening platform for a cortisol sensing tryptamine producing synbiotic for cognitive performance sustainment. The synbiotic, SYN, was engineered from the common probiotic E. coli Nissle 1917 strain. It had the ability to sense cortisol at physiological concentrations, resulting in the activation of a genetic circuit that produces tryptophan decarboxylase and converts bioavailable tryptophan to tryptamine. SYN was successfully cultivated within the gut-chip showing log-phase growth comparable to the wild-type strain. Tryptophan metabolism occurred quickly in the gut compartment when exposed to 5 µM cortisol, resulting in the complete conversion of bioavailable tryptophan into tryptamine. The flux of tryptophan and tryptamine from the gut to the vascular compartment of the chip was delayed by 12 h, as indicated by the detectable tryptamine in the vascular compartment. The gut-chip provided a stable environment to characterize the sensitivity of the cortisol sensor and dynamic range by altering cortisol and tryptophan dosimetry. Collectively, the human gut-chip provided human relevant apparent permeability to assess tryptophan and tryptamine metabolism, production, and transport, enabled host analyses of cellular viability and pro-inflammatory cytokine secretion, and succeeded in providing an efficacy test of a novel synbiotic. Organ-on-a-chip technology holds promise in aiding traditional therapeutic pipelines to more rapidly down select high potential compounds that reduce the failure rate and accelerate the opportunity for clinical intervention.

Characterization of an engineered live bacterial therapeutic for the treatment of phenylketonuria in a human gut-on-a-chip.

Engineered bacteria (synthetic biotics) represent a new class of therapeutics that leverage the tools of synthetic biology. Translational testing strategies are required to predict synthetic biotic function in the human body. Gut-on-a-chip microfluidics technology presents an opportunity to characterize strain function within a simulated human gastrointestinal tract. Here, we apply a human gut-chip model and a synthetic biotic designed for the treatment of phenylketonuria to demonstrate dose-dependent production of a strain-specific biomarker, to describe human tissue responses to the engineered strain, and to show reduced blood phenylalanine accumulation after administration of the engineered strain. Lastly, we show how in vitro gut-chip models can be used to construct mechanistic models of strain activity and recapitulate the behavior of the engineered strain in a non-human primate model. These data demonstrate that gut-chip models, together with mechanistic models, provide a framework to predict the function of candidate strains in vivo.

Perchlorate and radioiodide kinetics across life stages in the human: using PBPK models to predict dosimetry and thyroid inhibition and sensitive subpopulations based on developmental stage.

Perchlorate (ClO4(-)) is a drinking-water contaminant, known to disrupt thyroid hormone homeostasis in rats. This effect has only been seen in humans at high doses, yet the potential for long term effects from developmental endocrine disruption emphasizes the need for improved understanding of perchlorate's effect during the perinatal period. Physiologically based pharmacokinetic/dynamic (PBPK/PD) models for ClO4(-) and its effect on thyroid iodide uptake were constructed for human gestation and lactation data. Chemical specific parameters were estimated from life-stage and species-specific relationships established in previously published models for various life-stages in the rat and nonpregnant adult human. With the appropriate physiological descriptions, these kinetic models successfully simulate radioiodide data culled from the literature for gestation and lactation, as well as ClO4(-) data from populations exposed to contaminated drinking water. These models provide a framework for extrapolating from chemical exposure in laboratory animals to human response, and support a more quantitative understanding of life-stage-specific susceptibility to ClO4(-). The pregnant and lactating woman, fetus, and nursing infant were predicted to have higher blood ClO4(-) concentrations and greater thyroid iodide uptake inhibition at a given drinking-water concentration than either the nonpregnant adult or the older child. The fetus is predicted to receive the greatest dose (per kilogram body weight) due to several factors, including placental sodium-iodide symporter (NIS) activity and reduced maternal urinary clearance of ClO4(-). The predicted extent of iodide inhibition in the most sensitive population (fetus) is not significant (approximately 1%) at the U.S. Environmental Protection Agency reference dose (0.0007 mg/kg-d).

PBPK model for radioactive iodide and perchlorate kinetics and perchlorate-induced inhibition of iodide uptake in humans.

Detection of perchlorate (ClO4-) in several drinking water sources across the U.S. has lead to public concern over health effects from chronic low-level exposures. Perchlorate inhibits thyroid iodide (I-) uptake at the sodium (Na+)-iodide (I-) symporter (NIS), thereby disrupting the initial stage of thyroid hormone synthesis. A physiologically based pharmacokinetic (PBPK) model was developed to describe the kinetics and distribution of both radioactive I- and cold ClO4- in healthy adult humans and simulates the subsequent inhibition of thyroid uptake of radioactive I- by ClO4-. The model successfully predicts the measured levels of serum and urinary ClO4- from drinking water exposures, ranging from 0.007 to 12 mg ClO4-/kg/day, as well as the subsequent inhibition of thyroid 131I- uptake. Thyroid iodine, as well as total, free, and protein-bound radioactive I- in serum from various tracer studies, are also successfully simulated. This model's parameters, in conjunction with corresponding model parameters established for the male, gestational, and lactating rat, can be used to estimate parameters in a pregnant or lactating human, that have not been or cannot be easily measured to extrapolate dose metrics and correlate observed effects in perchlorate toxicity studies to other human life stages. For example, by applying the adult male rat:adult human ratios of model parameters to those parameters established for the gestational and lactating rat, we can derive a reasonable estimate of corresponding parameters for a gestating or lactating human female. Although thyroid hormones and their regulatory feedback are not incorporated in the model structure, the model's successful prediction of free and bound radioactive I- and perchlorate's interaction with free radioactive I- provide a basis for extending the structure to address the complex hypothalamic-pituitary-thyroid feedback system. In this paper, bound radioactive I- refers to I- incorporated into thyroid hormones or iodinated proteins, which may or may not be bound to plasma proteins.

PBPK predictions of perchlorate distribution and its effect on thyroid uptake of radioiodide in the male rat.

Due to perchlorate's (ClO4-) ability to competitively inhibit thyroid iodide (I-) uptake through the sodium-iodide symporter (NIS), potential human health risks exist from chronic exposure via drinking water. Such risks may include hypothyroidism, goiter, and mental retardation (if exposure occurs during critical periods in neurodevelopment). To aid in predicting perchlorate's effect on normal I- kinetics, we developed a physiologically-based pharmacokinetic (PBPK) model for the adult male rat. The model structure describes simultaneous kinetics for both anions together with their interaction at the NIS, in particular, the inhibition of I- uptake by ClO4-. Subcompartments and Michaelis-Menten (M-M) kinetics were used to describe active uptake of both anions in the thyroid, stomach, and skin. Separate compartments for kidney, liver, plasma, and fat were described by passive diffusion. The model successfully predicts both 36ClO4- and 125I- kinetics after iv doses of 3.3 mg/kg and 33 mg/kg, respectively, as well as inhibition of thyroid 125I- uptake by ClO4- after iv doses of ClO4- (0.01 to 3.0 mg/kg). The model also predicts serum and thyroid ClO4- concentrations from 14-day drinking water exposures (0.01 to 30.0 mg ClO4-/kg/day) and compensation of perchlorate-induced inhibition of radioiodide uptake due to upregulation of the thyroid. The model can be used to extrapolate dose metrics and correlate observed effects in perchlorate toxicity studies to other species and life stages, such as rat gestation (Clewell et al., 2003). Because the model successfully predicts perchlorate's interaction with iodide, it provides a sound basis for future incorporation of the complex hypothalamic-pituitary-thyroid feedback system.

Predicting neonatal perchlorate dose and inhibition of iodide uptake in the rat during lactation using physiologically-based pharmacokinetic modeling.

Perchlorate (ClO4-), a contaminant in drinking water, competitively inhibits active uptake of iodide (I-) into various tissues, including mammary tissue. During postnatal development, inhibition of I- uptake in the mammary gland and neonatal thyroid and the active concentration ClO4- in milk indicate a potentially increased susceptibility of neonates to endocrine disruption. A physiologically based pharmacokinetic (PBPK) model was developed to reproduce measured ClO4- distribution in the lactating and neonatal rat and predict resulting effects on I- kinetics from competitive inhibition at the sodium iodide symporter (NIS). Kinetic I- and ClO4- behavior in tissues with NIS (thyroid, stomach, mammary gland, and skin) was simulated with multiple subcompartments, Michaelis-Menten (M-M) kinetics and competitive inhibition. Physiological and kinetic parameters were obtained from literature and experiment. Systemic clearance and M-M parameters were estimated by fitting simulations to tissue and serum data. The model successfully describes maternal and neonatal thyroid, stomach, skin, and plasma, as well as maternal mammary gland and milk data after ClO4- exposure (from 0.01 to 10 mg/kg-day ClO4-) and acute radioiodide (2.1 to 33,000 ng/kg I-) dosing. The model also predicts I- uptake inhibition in the maternal thyroid, mammary gland, and milk. Model simulations predict a significant transfer of ClO4- through milk after maternal exposure; approximately 50% to 6% of the daily maternal dose at doses ranging from 0.01 to 10.0 mg ClO4-/kg-day, respectively. Comparison of predicted dosimetrics across life-stages in the rat indicates that neonatal thyroid I- uptake inhibition is similar to the adult and approximately tenfold less than the fetus.

Predicting fetal perchlorate dose and inhibition of iodide kinetics during gestation: a physiologically-based pharmacokinetic analysis of perchlorate and iodide kinetics in the rat.

Perchlorate (ClO4-) disrupts endocrine homeostasis by competitively inhibiting the transport of iodide (I-) into the thyroid. The potential for health effects from human exposure to ClO4- in drinking water is not known, but experimental animal studies are suggestive of developmental effects from ClO4- induced iodide deficiency during gestation. Normal hormone-dependent development relies, in part, on synthesis of hormones in the fetal thyroid from maternally supplied iodide. Although ClO4- crosses the placenta, the extent of inhibition in the fetal thyroid is unknown. A physiologically-based pharmacokinetic (PBPK) model was developed to simulate ClO4- exposure and the resulting effect on iodide kinetics in rat gestation. Similar to concurrent model development for the adult male rat, this model includes compartments for thyroid, stomach, skin, kidney, liver, and plasma in both mother and fetus, with additional compartments for the maternal mammary gland, fat, and placenta. Tissues with active uptake are described with multiple compartments and Michaelis-Menten (M-M) kinetics. Physiological and kinetic parameters were obtained from literature and experiment. Systemic clearance, placental-fetal transport, and M-M uptake parameters were estimated by fitting model simulations to experimental data. The PBPK model is able to reproduce maternal and fetal iodide data over five orders of magnitude (0.36 to 33,000 ng/kg 131I-), ClO4- distribution over three orders of magnitude (0.01 to 10 mg/kg-day ClO4-) and inhibition of maternal thyroid and total fetal I- uptake. The model suggests a significant fetal ClO4- dose in late gestation (up to 82% of maternal dose). A comparison of model-predicted internal dosimetrics in the adult male, pregnant, and fetal rat indicates that the fetal thyroid is more sensitive to inhibition than that of the adult.

Evidence for competitive inhibition of iodide uptake by perchlorate and translocation of perchlorate into the thyroid.

Various published data sets that investigate the potential effect of exogenous perchlorate (ClO4-) on the uptake of iodide in the thyroid and subsequent changes in thyroid hormone levels are available. In order to best use the data towards the prediction of human health effects resulting from ClO4- exposure, the available literature data must be integrated into a self-consistent, coherent, and parsimonious quantitative model based on the most likely mode of action of perchlorate effect on thyroid function. We submit that the simplest mode of action for ClO4- in the thyroid that remains consistent with all available data involves competitive inhibition of iodide transport into the thyroid follicle, transport of perchlorate into the thyroid follicle against a concentration gradient, further transport into the thyroid lumen (where it may again interfere with iodide transport), and, finally, passive diffusion back into the blood. We believe this description of perchlorate's kinetic behavior should serve as the foundation for predictive physiologically based pharmacokinetic (PBPK) models and as a working hypothesis for further experimental exploration.