An assessment of serum-dependent impacts on intracellular accumulation and genomic response of per- and polyfluoroalkyl substances in a placental trophoblast model.

Per- and polyfluoroalkyl substances (PFAS), a class of environmental contaminants, have been detected in human placenta and cord blood. The mechanisms driving PFAS-induced effects on the placenta and adverse pregnancy outcomes are not well understood. This study investigated the impact of perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), and a replacement PFAS known as hexafluoropropylene oxide dimer acid (HFPO-DA, tradename GenX) on placental trophoblasts in vitro. Several key factors were addressed. First, PFAS levels in cell culture reagents at baseline were quantified. Second, the role of supplemental media serum in intracellular accumulation of PFAS in a human trophoblast (JEG3) cell line was established. Finally, the impact of PFAS on the expression of 96 genes involved in proper placental function in JEG3 cells was evaluated. The results revealed that serum-free media (SFM) contained no detectable PFAS. In contrast, fetal bovine serum-supplemented media (SSM) contained PFNA, PFUdA, PFTrDA, and 6:2 FTS, but these PFAS were not detected internally in cells. Intracellular accumulation following 24 hr treatments was significantly higher when cultured in SFM compared to SSM for PFOS and PFOA, but not HFPO-DA. Treatment with PFAS was associated with gene expression changes (n = 32) in pathways vital to placental function, including viability, syncytialization, inflammation, transport, and invasion/mesenchymal transition. Among the most robust PFAS-associated changes were those observed in the known apoptosis-related genes, BAD and BAX. These results suggest a complex relationship between PFAS, in vitro culture conditions, and altered expression of key genes necessary for proper placentation.

Streamlined assessment of membrane permeability and its application to membrane engineering of Escherichia coli for octanoic acid tolerance.

The economic viability of bio-production processes is often limited by damage to the microbial cell membrane and thus there is a demand for strategies to increase the robustness of the cell membrane. Damage to the microbial membrane is also a common mode of action by antibiotics. Membrane-impermeable DNA-binding dyes are often used to assess membrane integrity in conjunction with flow cytometry. We demonstrate that in situ assessment of the membrane permeability of E. coli to SYTOX Green is consistent with flow cytometry, with the benefit of lower experimental intensity, lower cost, and no need for a priori selection of sampling times. This method is demonstrated by the characterization of four membrane engineering strategies (deletion of aas, deletion of cfa, increased expression of cfa, and deletion of bhsA) for their effect on octanoic acid tolerance, with the finding that deletion of bhsA increased tolerance and substantially decreased membrane leakage.

Formation of PFAAs in fish through biotransformation: A PBPK approach.

A physiologically-based pharmacokinetic (PBPK) model for perfluorinated alkyl acids (PFAAs) in rainbow trout has been updated to include formation of perfluorooctanoic acid (PFOA) from the biotransformation of 8:2 fluorotelomer carboxylic acid (8:2 FTCA). The updated model is dynamic and simulates both uptake and depuration phases. Two empirical studies are used to parameterize and test the model. In the first case, parameters related to fecal elimination and protein binding were optimized. In the second case, parameters were sourced either from literature or from optimized values based on the first study to test model performance. Optimization of parameters resulted in a decrease in the difference between experimental data and simulation results by 57 and 23 percent for the first and the second case, respectively, compared to the original case. Sensitivity analysis was performed to identify important parameters, and uncertainty in model prediction propagated by these parameters was assessed using Monte Carlo analysis. For each case, 80 and 89 percent, respectively, of median predicted values were within the limits of experimental error when comparing simulated and experimental data. This is the first toxicokinetic model that incorporates biotransformation of PFAA precursors and simultaneously predicts the distribution of the precursor and metabolite in different tissues. The model is mechanistic, and could be applied to simulate a variety of scenarios by using the organism-specific physiological properties compiled here with other chemical-specific parameters (e.g. protein interactions).

Indirect vs direct assessment of gastric emptying: A randomized crossover trial comparing C-isotope breath analysis and MRI.

BACKGROUND: Indirect methods to assess gastric emptying (GE), such as 13 C breath tests (BT), are commonly used. However, BT usually use a sampling time of 4+ hours. The current study aims to assess the validity of BT for four liquid meals differing in physicochemical properties. To this aim, we compared them to MRI GE-measurements. METHODS: Fifteen healthy males (age 22.6 ± 2.4 years, BMI 22.6 ± 1.8 kg/m2 ) participated in a randomized 2 × 2 crossover experiment. Test foods were liquid meals, which were either thin/thick and 100/500 kcal, labeled with 100 mg of 13 C-octanoate. GE was measured with MRI and assessed by 13 C recovery from breath. Participants were scanned every 10 minutes and at six time points breath samples were collected up to t = 90 minutes. Two curves were fitted to the data to estimate emptying halftime (t50 Ghoos and t50 Bluck ). T50 times were ranked per participant and compared between methods. KEY RESULTS: On average, MRI and BT showed similar t50 rankings for the four liquid meals. In comparison to MRI, t50 Ghoos overestimated, while t50 Bluck underestimated GE time. Moreover, more viscous foods were overestimated. In most participants individual t50 time rankings differed significantly between methods. CONCLUSIONS & INFERENCES: BT can assess relative emptying differences on group level and collecting breath data for 90 minutes constitutes a lower burden for participants and the research facility. However, BT has severe shortcomings compared to MRI for individual GE assessment. Notably, food matrix effects should be considered when interpreting the results of BT.

Application of the OECD 301F respirometric test for the biodegradability assessment of various potential endocrine disrupting chemicals.

The biodegradability of several potential endocrine disrupting compounds, namely 4-n-nonylphenol (4-n-NP), nonylphenol monoethoxylate (NP1EO), nonylphenol diethoxylate (NP2EO), bisphenol A (BPA), triclosan (TCS), di-(2-ethylhexyl)-phthalate (DEHP), perfluorooctanoate (PFOA) and perfluorononanoate (PFNA) was evaluated in this study, using OECD method 301F (manometric respirometry test) and activated sludge as inoculum. According to the results, 4-n-NP and BPA meet the strict definition of ready biodegradability and they are not expected to be persistent during the activated sludge process. Partial biodegradation was observed for DEHP (58.7+/-5.7%, n=3), TCS (52.1+/-8.5%, n=3) and NP1EO (25.9+/-8.1%, n=3), indicating their possible biodegradation in wastewater treatment systems, while no biodegradation was observed for NP2EO, PFOA and PFNA. Experiments in the co-presence of a readily biodegradable compound showed the absence of co-metabolic phenomena during 4-n-NP, BPA and TCS biodegradation. Using first order kinetics to describe biodegradation of the target compounds, half-lives of 4.3+/-0.6, 1.3+/-0.2, 1.8+/-0.5, 6.9+/-2.6 days were calculated for 4-n-NP, BPA, TCS and DEHP, respectively. Toxicity tests using marine bacterium Vibrio fischeri showed that biodegradation of 4-n-NP, NP1EO, BPA and TCS is a simultaneous detoxification process, while possible abiotic or biotic transformations of NP2EO, DEHP, PFOA and PFNA during respirometric test resulted to significant increase of their toxicities.

Exposure assessment and risk characterization for perfluorooctanoate in selected consumer articles.

An exposure assessment and risk characterization was conducted to better understand the potential human health significance of trace levels of perfluorooctanoate (PFO) detected in certain consumer articles. PFO is the anion of perfluorooctanoic acid (PFOA). Concentrations of PFO in the consumer articles were determined from extraction tests and product formulation information. Potential exposures during consumer use of the articles were quantified based on an assessment of behavior patterns and regulatory guidance. Health benchmarks were developed and then compared to the exposure estimates to yield margins of exposure (MOEs). A simple one-compartment model was also developed to estimate contributions of potential consumer exposures to PFO concentrations in serum. While there are considerable uncertainties in this assessment, it indicates that exposures to PFO during consumer use of the articles evaluated in this study are not expected to cause adverse human health effects in infants, children, adolescents, adult residents, or professionals nor result in quantifiable levels of PFO in human serum.

Comparison of the carbon 13-labeled octanoic acid breath test and ultrasonography for assessment of gastric emptying of a semisolid meal in dogs.

OBJECTIVE: To compare the rate of gastric emptying of a semisolid meal by use of the carbon 13-labeled octanoic acid breath test (13C-OBT) and gastric emptying ultrasonography (GEU) in dogs. ANIMALS: 10 healthy dogs. PROCEDURE: Food was withheld from dogs for 12 hours before ingestion of a test meal (bread, egg, and skimmed milk) containing 13C-octanoic acid. The gastric antrum was visualized by use of a 6.5-MHz microconvex transducer, and the area of the ellipse defined by the craniocaudal and ventrodorsal diameters of the stomach was measured. Samples of expired air and antral images were obtained 30 minutes before ingestion of the test meal and then every 15 minutes for 4 hours and every 30 minutes for a further 2 hours. The half-dose recovery time with the 13C-OBT (t1/2[BT]) and the gastric half emtying time with GEU (t50%[GEU]) was calculated. RESULTS: Mean +/- SD values for the t1/2(BT) and t50%(GEU) were 3.44 +/- 0.48 hours and 1.89 +/- 0.78 hours, respectively. A significant correlation was detected between the t1/2(BT) and t50%(GEU), although there was a large (1.55 hours) mean difference between these indices. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that there was a correlation between the rate of solid-phase gastric emptying assessed by use of GEU and the 13C-OBT in dogs. Gastric emptying ultrasonography may be a useful, noninvasive method for assessment of the rate of solid-phase gastric emptying in dogs.

Assessment of the rate of solid-phase gastric emptying in ponies by means of the 13C-octanoic acid breath test: a preliminary study.

The aim of this study was to assess the feasibility of applying the 13C-octanoic acid breath test for assessment of gastric emptying in ponies by investigating the pattern of 13C enrichment in breath following the administration of a test meal +/- 13C-octanoic acid. After a 14 h fast, the ponies received either no meal (Test I) or a standardised test meal labelled with 0 mg (Test II), 125 mg (Test III), 250 mg (Test IV) or 500 mg (Test V) 13C-octanoic acid. For each test (I-V), exhaled breath samples were collected in duplicate at 1 h and immediately before ingestion of the test meal and at frequent intervals thereafter for 12 h. Breath samples were analysed by continuous flow isotope ratio mass spectrometry. Three indices of breath 13C-enrichment were computed; half dose recovery time (t1/2), gastric emptying coefficient (GEC) and time to peak breath 13C-enrichment t(max). For Tests I and II, the ratio of 13CO2:12CO2 remained stable for the duration of the sampling period. For Tests III, IV and V, an increase in the ratio of 13CO2:12CO2 was detected. The test was reproducible within individuals, and intersubject variation was low. Further validation studies of this noninvasive technique are justified.

Use of the 13C-octanoic acid breath test for assessment of solid-phase gastric emptying in dogs.

OBJECTIVE: To assess the 13C-octanoic acid breath test for determining gastric emptying in dogs. ANIMALS: 6 healthy adult dogs. PROCEDURE: Food was withheld for 12 hours before each test. Expired air was collected 30 minutes and immediately before each test and at frequent intervals thereafter for 6 hours. Concentration of 13CO2 in expired air was determined by use of continuous-flow isotope-ratio mass spectrometry. Basal concentration of 13CO2 was measured in dogs that were not fed a test meal. Effects of the standard unlabeled test meal on basal concentration of 13CO2 were then assessed. The optimum dose of substrate was determined by measuring 13CO2 concentration after ingestion of the standard test meal containing 50 or 100 mg of 13C-octanoic acid, whereas effect of energy density of the test meal on gastric emptying was determined after ingestion of the standard or high-energy labeled test meal. Gastric emptying coefficient (GEC), time to peak 13CO2 concentration (tmax), and half-dose recovery time (t(1/2)) were calculated. RESULTS: Basal concentration of 13CO2 in expired air was not significantly affected by ingestion of the unlabeled test meal. However, 13CO2 concentration significantly increased in a dose-dependent manner after ingestion of the labeled meal. Gastric emptying coefficient, and were significantly different between dogs fed the standard and high-energy test meals, indicating that ingestion of a high-energy meal delays gastric emptying. CONCLUSIONS AND CLINICAL RELEVANCE: The 13C-octanoic acid breath test may be a useful noninvasive and nonradioactive method for assessment of gastric emptying in dogs.

Assessment of gastric emptying in the mouse using the [13C]-octanoic acid breath test.

1. Gastric emptying studies in small laboratory animals are hampered by the deficiency of a technique that is non-invasive and repeatable. The aim of the present study was to adapt the non-invasive [13C]-octanoic acid breath test, which has been validated in humans, to assess both liquid and solid gastric emptying in the mouse. 2. Gastric emptying rates were investigated for a liquid meal (Intralipid; Kabi Pharmacia AB, Stockholm, Sweden; n = 7) and two solid meals (egg yolk and mouse chow; n = 7) incorporating [13C]-octanoic acid. All meals were analysed for natural enrichment of [13C]. Mathematical analysis of the 13CO2 excretion rate allowed the determination of gastric emptying parameters. 3. Gastric emptying of Intralipid was more rapid than egg yolk (P < 0.0001). Gastric emptying of mouse chow could not be assessed due to intragastric separation of [13C]-octanoic acid and natural [13C] enrichment of the pellet. 4. The [13C]-octanoic acid breath test can reproducibly assess both liquid and solid gastric emptying non-invasively in the mouse. This method can now be used to assess gastric emptying in drug studies and disease studies for which there are established mouse models.

Reproducibility of the 13C-octanoic acid breath test for assessment of gastric emptying in healthy preterm infants.

BACKGROUND: The 13C-octanoic acid breath test has been used to measure gastric emptying in preterm infants, but the reproducibility of the test has not been evaluated in this population. METHODS: Fifty-six paired breath test analyses were performed on 28 healthy preterm infants 1 to 5 days apart using the same food type, volume, and energy content for each paired sample. Breath samples were taken before the feeding, at 5-minute intervals after feeding for 30 minutes, then each 15 minutes for 4 hours. Samples were analyzed using an isotope-ratio mass spectrometer, and 3C recovery was used to calculate values for gastric-emptying coefficient and gastric half-emptying time. RESULTS: There was no significant difference between test results on different days in the paired samples studied. gastric-emptying coefficients for the first and subsequent samples were 2.6+/-0.1 (mean+/-SEM) and 2.7+/-0.1, respectively, and half-emptying times were 44.5+/-3.7 minutes and 41.4+/-3.2 minutes. CONCLUSION: The 13C-octanoic acid breath test is a reliable, noninvasive, and reproducible measure of gastric emptying in preterm infants that should have wide application for use in this population.

Non-invasive assessment of intraluminal lipolysis using a 13CO2 breath test.

Techniques available for the study of lipase activity in the gut are unsatisfactory. Breath tests measuring labelled carbon dioxide (13CO2) may provide a useful means for this assessment. Six subjects with cystic fibrosis and pancreatic insufficiency and 10 controls received a test meal containing [13C] trioctanoin, and breath 13CO2 was measured using a dual inlet, dual detector isotope ratio mass spectrometer. Comparison of postprandial breath 13CO2 enrichment allowed complete separation between children with pancreatic insufficiency and controls. Administration of one capsule of pancreatic enzyme with the test meal resulted in an increase in 13CO2 production in all six patients, and four capsules produced a further increase in five of the six. Serial fat balance studies on four of the patients while receiving comparable doses of oral enzyme failed to demonstrate a progressive improvement in fat absorption. The [13C]trioctanoin breath test may prove a safe, non-invasive technique not only for the detection of pancreatic insufficiency, but also for the quantitative study of intraluminal lipolysis.