Assessment of ethanol exposure from hand sanitizer use and potential for developmental toxicity in nursing infants.

Ingestion of ethanol during pregnancy is known to have detrimental effects on the fetus. Although the potential developmental effects of maternal ethanol intake during lactation are less well characterized, public health guidelines recommend avoidance of alcohol or, if alcohol is consumed, to allow for 1-2 h to pass before nursing. A proposal to classify ethanol as potentially harmful to breast-fed children warrants an investigation of the potential adverse neurodevelopmental effects of low-dose ethanol exposure during lactation. There currently are no studies that have examined neurodevelopmental outcomes from lactational exposure to ethanol from the use of topical products that contain ethanol, such as alcohol-based hand sanitizers (ABHS). Furthermore, the epidemiological literature of lactational ethanol exposures from maternal alcohol consumption is limited in design, provides equivocal evidence of neurological effects in infants, and is insufficient to characterize a dose-response relationship for developmental effects. Toxicological studies that observed neurodevelopmental effects in pups from ethanol via lactation did so at exceedingly high doses that also caused maternal toxicity. In this investigation, blood ethanol concentrations (BECs) of breastfeeding women following typical-to-intense ABHS use were computationally predicted and compared to health benchmarks to quantify the risk for developmental outcomes. Margins of 2.2 to 1000 exist between BECs associated with ABHS use compared to BECs associated with neurotoxicity adverse effect levels in the toxicology literature or oral ethanol intake per public health guidelines. Neurodevelopmental effects are not likely to occur in infants due to ABHS use by breastfeeding women, even when ABHSs are used at intense frequencies.

Bioenergetic Analyses of In Vitro and In Vivo Samples to Guide Toxicological Endpoints.

Toxicology is a broad field that requires the translation of biochemical responses to xenobiotic exposures into useable information to ensure the safety of the public. Modern techniques are improving rapidly, both quantitatively and qualitatively, to provide the tools necessary to expand available toxicological datasets and refine our ability to translate that data into relevant information via bioinformatics. These new techniques can, and do, impact many of the current critical roles in toxicology, including the environmental, forensic, preclinical/clinical, and regulatory realms. One area of rapid expansion is our understanding of bioenergetics, or the study of the transformation of energy in living organisms, and new mathematical approaches are needed to interpret these large datasets. As bioenergetics are intimately involved in the regulation of how and when a cell responds to xenobiotics, monitoring these changes (i.e., metabolic fluctuations) in cells/tissues post-exposure provides an approach to define the temporal scale of pharmacodynamic responses, which can be used to guide additional toxicological techniques (e.g., "omics"). This chapter will summarize important in vitro assays and in vivo imaging techniques to take real-time measurements. Using this information, our laboratory has utilized bioenergetics to identify significant time points of pharmacodynamic relevance as well as forecast the cell's eventual fate.

Salivary cytokines as a biomarker of social stress in a mock rescue mission.

Using salivary inflammatory markers as a noninvasive biomonitoring technique within natural social contexts has become increasingly important to link social and biological responses. Many studies have associated circulating cytokines to distinct aspects of physical activity and social/emotional behavior; however, they have not been linked to success and failure in a naturalistic setting for military personnel performing tasks. In this study, salivary cytokines were studied in a group of fifteen Air Force Reserve Officers' Training Corps (ROTC; 14 males, 1 female) subjects performing three mock hostage rescue missions, designed to prompt responses associated with baseline, success, and failure. Each subject completed the tasks of the mission individually and again in randomly assigned teams. Participants were outfitted via direct skin contact with comfortable external Zephyr™ sensors to monitor heart rate, breathing rate, and activity while completing each task. Saliva samples were collected before and after the completion of each mission, and cytokine levels were quantified using enzyme-labelled immunoassay (ELISA) beads. These biomarkers were used to describe the body's immune response to success and failure when performing a mock rescue mission individually and in a team. All measured cytokine levels increased following failed missions performed individually, compared to cytokine levels associated with successful missions. When completing the tasks as a team, there were no significant differences in cytokine response between success and failure; however, being in a team stimulated an increased pre-mission cytokine response, suggesting the concept of teamwork and performing with peers for the first time had a more significant impact than the notion of failing. Additionally, none of the cytokines tested for individual missions correlated to physical activity markers (heart rate, breathing rate, activity) measured during performance. These results indicate a potentially new noninvasive method of determining social stress levels under taxing conditions.

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.

The impact of cytokine responses in the intra- and extracellular signaling network of a traumatic injury.

Investigations of cellular responses involved in injury and repair processes have generated valuable information contributing to the advancement of wound healing and treatments. Intra- and extracellular regulators of healing mechanisms, such as cytokines, signaling proteins, and growth factors, have been described to possess significant roles in facilitating optimal recovery. This study explored a collection of 30 spatiotemporal responses comprised of cytokines (IL-1α, IL-1ß, IL-2, IL-6, TNF-α, MIP-1α), intracellular proteins (Akt, c-Jun, CREB, ERK1/2, JNK, MEK1, p38, p53, p90RSK), phosphorylated proteins (p-Akt, p-c-Jun, p-CREB, p-ERK1/2, p-GSK-3α/ß, p-HSP27, p-IκBα, p-JNK, p-MEK1, p-p38, p-p70S6K, p-p90RSK, p-STAT2, p-STAT3), and a protease (Caspase-3), measured in skeletal muscle tissue following a traumatic injury (rodent Gustilo IIIB fracture). To optimize the analysis of context-specific data sets, a network centrality parameter approach was used to assess the impact of each response in relation to all other measured responses. This approach identified proteins that were substantially amplified and potentially central in the wound healing network by evaluation of their corresponding centrality parameter, radiality. Network analysis allowed us to distinguish the progression of healing that occurred at certain time points and regions of injury. Notably, new tissue formation was proposed to occur by 168 h post-injury in severely injured tissue, while tissue 1-cm away from the site of injury that experienced relatively minor injury appeared to exhibit signs of new tissue formation as early as 24 h post-injury. In particular, hallmarks of inflammation, cytokines IL-1ß, IL-6, and IL-2, appear to have a pronounced impact at earlier time points (0-24 h post-injury), while intracellular proteins involved in cell proliferation, differentiation, or proteolysis (c-Jun, CREB, JNK, p38, p-c-Jun; p-MEK1, p-p38, p-STAT3) are more significant at later times (24-168 h). Overall, this study demonstrates the feasibility of a network analysis approach to extract significant information and also offers a spatiotemporal visualization of the intra- and extracellular signaling responses that regulate healing mechanisms.

In vitro cytotoxicity assessment of a West Virginia chemical spill mixture involving 4-methylcyclohexanemethanol and propylene glycol phenyl ether.

Thousands of gallons of industrial chemicals, crude 4-methylcyclohexanemethanol (MCHM) and propylene glycol phenyl ether (PPh), leaked from industrial tanks into the Elk River in Charleston, West Virginia, USA, on January 9, 2014. A considerable number of people were reported to exhibit symptoms of chemical exposure and an estimated 300,000 residents were advised not to use or drink tap water. At the time of the spill, the existing toxicological data of the chemicals were limited for a full evaluation of the health risks, resulting in concern among those in the impacted regions. In this preliminary study, we assessed cell viability and plasma membrane degradation following a 24-h exposure to varying concentrations (0-1000 µM) of the two compounds, alone and in combination. Evaluation of different cell lines, HEK-293 (kidney), HepG2 (liver), H9c2 (heart), and GT1-7 (brain), provided insight regarding altered cellular responses in varying organ systems. Single exposure to MCHM or PPh did not affect cell viability, except at doses much higher than the estimated exposure levels. Certain co-exposures significantly reduced metabolic activity and increased plasma membrane degradation in GT1-7, HepG2, and H9c2 cells. These findings highlight the importance of examining co-exposures to fully understand the potential toxic effects.

Spatiotemporal phosphoprotein distribution and associated cytokine response of a traumatic injury.

Molecular mechanisms of wound healing have been extensively characterized, providing a better understanding of the processes involved in wound repair and offering advances in treatment methods. Both spatial and temporal investigations of injury biomarkers have helped to pinpoint significant time points and locations during the recovery process, which may be vital in managing the injury and making the appropriate diagnosis. This study addresses spatial and temporal differences of phosphoproteins found in skeletal muscle tissue following a traumatic femur fracture, which were further compared to co-localized cytokine responses. In particular, several proteins (Akt, ERK, c-Jun, CREB, JNK, MEK1, and p38) and post-translational phosphorylations (p-Akt, p-c-Jun, p-CREB, p-ERK1/2, p-MEK1, p-p38, p-GSK3α/ß, p-HSP27, p-p70S6K, and p-STAT3) associated with inflammation, new tissue formation, and remodeling were found to exhibit significant spatial and temporal differences in response to the traumatic injury. Quadratic discriminant analysis of all measured responses, including cytokine concentrations from previously published findings, was used to classify temporal and spatial observations at high predictive rates, further confirming that distinct spatiotemporal distributions for total protein, phosphorylation signaling, and cytokine (IL-1α, IL-1ß, IL2, IL6, TNF-α, and MIP-1α) responses exist. Finally, phosphoprotein measurements were found to be significantly correlated to cytokine concentrations, suggesting coordinated intracellular and extracellular activity during crucial periods of repair. This study represents a first attempt to monitor and assess integrated changes in extracellular and intracellular signaling in response to a traumatic injury in muscle tissues, which may provide a framework for future research to improve both our understanding of wounds and their treatment options.

Forecasting cell death dose-response from early signal transduction responses in vitro.

The rapid pharmacodynamic response of cells to toxic xenobiotics is primarily coordinated by signal transduction networks, which follow a simple framework: the phosphorylation/dephosphorylation cycle mediated by kinases and phosphatases. However, the time course from initial pharmacodynamic response(s) to cell death following exposure can have a vast range. Viewing this time lag between early signaling events and the ultimate cellular response as an opportunity, we hypothesize that monitoring the phosphorylation of proteins related to cell death and survival pathways at key, early time points may be used to forecast a cell's eventual fate, provided that we can measure and accurately interpret the protein responses. In this paper, we focused on a three-phased approach to forecast cell death after exposure: (1) determine time points relevant to important signaling events (protein phosphorylation) by using estimations of adenosine triphosphate production to reflect the relationship between mitochondrial-driven energy metabolism and kinase response, (2) experimentally determine phosphorylation values for proteins related to cell death and/or survival pathways at these significant time points, and (3) use cluster analysis to predict the dose-response relationship between cellular exposure to a xenobiotic and plasma membrane degradation at 24 h post-exposure. To test this approach, we exposed HepG2 cells to two disparate treatments: a GSK-3ß inhibitor and a MEK inhibitor. After using our three-phased approach, we were able to accurately forecast the 24 h HepG2 plasma membrane degradation dose-response from protein phosphorylation values as early as 20 min post-MEK inhibitor exposure and 40 min post-GSK-3ß exposure.

An approach to investigate intracellular protein network responses.

Modern toxicological evaluations have evolved to consider toxicity as a perturbation of biological pathways or networks. As such, toxicity testing approaches are shifting from common end point evaluations to pathway based approaches, where the degree of perturbation of select biological pathways is monitored. These new approaches are greatly increasing the data available to toxicologists, but methods of analyses to determine the inter-relationships between potentially affected pathways are needed to fully understand the consequences of exposure. An approach to construct dose-response curves that use graph theory to describe network perturbations among three disparate mitogen-activated protein kinase (MAPK) pathways is presented. Mitochondrial stress was induced in human hepatocytes (HepG2) by exposing the cells to increasing doses of the complex I inhibitor, deguelin. The relative phosphorylation responses of proteins involved in the regulation of the stress response were measured. Graph theory was applied to the phosphorylation data to obtain parameters describing the network perturbations at each individual dose tested. The graph theory results depicted the dynamic nature of the relationship between p38, JNK, and ERK1/2 under conditions of mitochondrial stress and revealed shifts in the relationships between these MAPK pathways at low doses. The inter-relationship, or crosstalk, among these 3 traditionally linear MAPK cascades was further probed by coexposing cells to deguelin plus SB202190 (JNK and p38 inhibitor) or deguelin plus SB202474 (JNK inhibitor). The cells exposed to deguelin plus SB202474 resulted in significantly decreased viability, which could be visualized and attributed to the decrease of ERK1/2 network centrality. The approach presented here allows for the construction and visualization of dose-response curves that describe network perturbations induced by chemical stress, which provides an informative and sensitive means of assessing toxicological effects on biological systems.

Catalytic azide reduction in biological environments.

In the quest for the identification of catalytic transformations to be used in chemical biology and medicinal chemistry, we identified iron(III) meso-tetraarylporphines as efficient catalysts for the reduction of aromatic azides to their amines. The reaction uses thiols as reducing agents and tolerates water, air, and other biological components. A caged fluorophore was employed to demonstrate that the reduction can be performed even in living mammalian cells. However, in vivo experiments in nematodes (Caenorhabditis elegans) and zebrafish (Danio rerio) revealed a limitation to this method: the metabolic reduction of aromatic azides.