In Utero Exposure to Environmental Tobacco Smoke Increases Neuroinflammation in Offspring

The embryonic stage is the most vulnerable period for congenital abnormalities. Due to its prolonged developmental course, the central nervous system (CNS) is susceptible to numerous genetic, epigenetic, and environmental influences. During embryo implantation, the CNS is more vulnerable to external influences such as environmental tobacco smoke (ETS), increasing the risk for delayed fetal growth, sudden infant death syndrome, and immune system abnormalities. This study aimed to evaluate the effects of in utero exposure to ETS on neuroinflammation in the offspring of pregnant mice challenged or not with lipopolysaccharide (LPS). After the confirmation of mating by the presence of the vaginal plug until offspring birth, pregnant C57BL/6 mice were exposed to either 3R4F cigarettes smoke (Kentucky University) or compressed air, twice a day (1h each), for 21 days. Enhanced glial cell and mixed cell cultures were prepared from 3-day-old mouse pups. After cell maturation, both cells were stimulated with LPS or saline. To inhibit microglia activation, minocycline was added to the mixed cell culture media 24 h before LPS challenge. To verify the influence of in utero exposure to ETS on the development of neuroinflammatory events in adulthood, a different set of 8-week-old animals was submitted to the Autoimmune Experimental Encephalomyelitis (EAE) model. The results indicate that cells from LPS-challenged pups exposed to ETS in utero presented high levels of proinflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNFα) and decreased cell viability. Such a proinflammatory environment could modulate fetal programming by an increase in microglia and astrocytes miRNA155. This scenario may lead to the more severe EAE observed in pups exposed to ETS in utero.

Altered Functional Mitochondrial Protein Levels in Plasma Neuron-Derived Extracellular Vesicles of Patients With Gadolinium Deposition

The retention of the heavy metal, gadolinium, after a Gadolinium-Based Contrast Agent-assisted MRI may lead to a symptom cluster termed Gadolinium Deposition Disease. Little is known of the disorder’s underlying pathophysiology, but a recent study reported abnormally elevated serum levels of pro-inflammatory cytokines compared to normal controls. As a calcium channel blocker in cellular plasma and mitochondrial membranes, gadolinium also interferes with mitochondrial function. We applied to sera from nine Gadolinium Deposition Disease and two Gadolinium Storage Condition patients newly developed methods allowing isolation of plasma neuron-derived extracellular vesicles that contain reproducibly quantifiable levels of mitochondrial proteins of all major classes. Patients’ levels of five mitochondrial functional proteins were statistically significantly lower and of two significantly higher than the levels in normal controls. The patterns of differences between study patients and controls for mitochondrial dynamics and mitochondrial proteins encompassing neuronal energy generation, metabolic regulation, ion fluxes, and survival differed from those seen for patients with first episode psychosis and those with Major Depressive Disorder compared to their controls. These findings suggest that mitochondrial dysfunction due to retained gadolinium may play a role in causing Gadolinium Deposition Disease. Larger samples of both GDD and GSC patients are needed to allow not only testing the repeatability of our findings, but also investigation of relationships of specific mitochondrial protein deficiencies or excesses and concurrent cytokine, genetic, or other factors to GDD’s neurological and cognitive symptoms. Studies of neuronal mitochondrial proteins as diagnostic markers or indicators of treatment effectiveness are also warranted.

DMSO Concentrations up to 1% are Safe to be Used in the Zebrafish Embryo Developmental Toxicity Assay

Dimethyl sulfoxide (DMSO) is a popular solvent for developmental toxicity testing of chemicals and pharmaceuticals in zebrafish embryos. In general, it is recommended to keep the final DMSO concentration as low as possible for zebrafish embryos, preferably not exceeding 100 μL/L (0.01%). However, higher concentrations of DMSO are often required to dissolve compounds in an aqueous medium. The aim of this study was to determine the highest concentration of DMSO that can be safely used in our standardized Zebrafish Embryo Developmental Toxicity Assay (ZEDTA). In the first part of this study, zebrafish embryos were exposed to different concentrations (0–2%) of DMSO. No increase in lethality or malformations was observed when using DMSO concentrations up to 1%. In a follow-up experiment, we assessed whether compounds that cause no developmental toxicity in the ZEDTA remain negative when dissolved in 1% DMSO, as false positive results due to physiological disturbances by DMSO should be avoided. To this end, zebrafish embryos were exposed to ascorbic acid and hydrochlorothiazide dissolved in 1% DMSO. Negative control groups were also included. No significant increase in malformations or lethality was observed in any of the groups. In conclusion, DMSO concentrations up to 1% can be safely used to dissolve compounds in the ZEDTA.

An Adverse Outcome Pathway for Decreased Lung Function Focusing on Mechanisms of Impaired Mucociliary Clearance Following Inhalation Exposure

Adverse outcome pathways (AOPs) help to organize available mechanistic information related to an adverse outcome into key events (KEs) spanning all organizational levels of a biological system(s). AOPs, therefore, aid in the biological understanding of a particular pathogenesis and also help with linking exposures to eventual toxic effects. In the regulatory context, knowledge of disease mechanisms can help design testing strategies using in vitro methods that can measure or predict KEs relevant to the biological effect of interest. The AOP described here evaluates the major processes known to be involved in regulating efficient mucociliary clearance (MCC) following exposures causing oxidative stress. MCC is a key aspect of the innate immune defense against airborne pathogens and inhaled chemicals and is governed by the concerted action of its functional components, the cilia and airway surface liquid (ASL). The AOP network described here consists of sequences of KEs that culminate in the modulation of ciliary beat frequency and ASL height as well as mucus viscosity and hence, impairment of MCC, which in turn leads to decreased lung function.

The Future of Neurotoxicology: A Neuroelectrophysiological Viewpoint

Neuroelectrophysiology is an old science, dating to the 18th century when electrical activity in nerves was discovered. Such discoveries have led to a variety of neurophysiological techniques, ranging from basic neuroscience to clinical applications. These clinical applications allow assessment of complex neurological functions such as (but not limited to) sensory perception (vision, hearing, somatosensory function), and muscle function. The ability to use similar techniques in both humans and animal models increases the ability to perform mechanistic research to investigate neurological problems. Good animal to human homology of many neurophysiological systems facilitates interpretation of data to provide cause-effect linkages to epidemiological findings. Mechanistic cellular research to screen for toxicity often includes gaps between cellular and whole animal/person neurophysiological changes, preventing understanding of the complete function of the nervous system. Building Adverse Outcome Pathways (AOPs) will allow us to begin to identify brain regions, timelines, neurotransmitters, etc. that may be Key Events (KE) in the Adverse Outcomes (AO). This requires an integrated strategy, from in vitro to in vivo (and hypothesis generation, testing, revision). Scientists need to determine intermediate levels of nervous system organization that are related to an AO and work both upstream and downstream using mechanistic approaches. Possibly more than any other organ, the brain will require networks of pathways/AOPs to allow sufficient predictive accuracy. Advancements in neurobiological techniques should be incorporated into these AOP-base neurotoxicological assessments, including interactions between many regions of the brain simultaneously. Coupled with advancements in optogenetic manipulation, complex functions of the nervous system (such as acquisition, attention, sensory perception, etc.) can be examined in real time. The integration of neurophysiological changes with changes in gene/protein expression can begin to provide the mechanistic underpinnings for biological changes. Establishment of linkages between changes in cellular physiology and those at the level of the AO will allow construction of biological pathways (AOPs) and allow development of higher throughput assays to test for changes to critical physiological circuits. To allow mechanistic/predictive toxicology of the nervous system to be protective of human populations, neuroelectrophysiology has a critical role in our future.

Development and Application of Nanoparticle-Nanopolymer Composite Spheres for the Study of Environmental Processes

Plastics have long been an environmental contaminant of concern as both large-scale plastic debris and as micro- and nano-plastics with demonstrated wide-scale ubiquity. Research in the past decade has focused on the potential toxicological risks posed by microplastics, as well as their unique fate and transport brought on by their colloidal nature. These efforts have been slowed by the lack of analytical techniques with sufficient sensitivity and selectivity to adequately detect and characterize these contaminants in environmental and biological matrices. To improve analytical analyses, microplastic tracers are developed with recognizable isotopic, metallic, or fluorescent signatures capable of being identified amidst a complex background. Here we describe the synthesis, characterization, and application of a novel synthetic copolymer nanoplastic based on polystyrene (PS) and poly(2-vinylpyridine) (P2VP) intercalated with gold, platinum or palladium nanoparticles that can be capped with different polymeric shells meant to mimic the intended microplastic. In this work, particles with PS and polymethylmethacrylate (PMMA) shells are used to examine the behavior of microplastic particles in estuarine sediment and coastal waters. The micro- and nanoplastic tracers, with sizes between 300 and 500 nm in diameter, were characterized using multiple physical, chemical, and colloidal analysis techniques. The metallic signatures of the tracers allow for quantification by both bulk and single-particle inductively-coupled plasma mass spectrometry (ICP-MS and spICP-MS, respectively). As a demonstration of environmental applicability, the tracers were equilibrated with sediment collected from Bellingham Bay, WA, United States to determine the degree to which microplastics bind and sink in an estuary based of grain size and organic carbon parameters. In these experiments, between 80 and 95% of particles were found to associate with the sediment, demonstrative of estuaries being a major anticipated sink for these contaminants. These materials show considerable promise in their versatility, potential for multiplexing, and utility in studying micro- and nano-plastic transport in real-world environments.

Special Issue: Novel Methods for Labeling Nanomaterials for Biological and Environmental Tracing Radiolabeling of Nanomaterials: Advantages and Challenges

Quantifying the distribution of nanomaterials in complex samples is of great significance to the toxicological research of nanomaterials as well as their clinical applications. Radiotracer technology is a powerful tool for biological and environmental tracing of nanomaterials because it has the advantages of high sensitivity and high reliability, and can be matched with some spatially resolved technologies for non-invasive, real-time detection. However, the radiolabeling operation of nanomaterials is relatively complicated, and fundamental studies on how to optimize the experimental procedures for the best radiolabeling of nanomaterials are still needed. This minireview looks back into the methods of radiolabeling of nanomaterials in previous work, and highlights the superiority of the “last-step” labeling strategy. At the same time, the problems existing in the stability test of radiolabeling and the suggestions for further improvement are also addressed.

A Low-Serum Culture System for Prolonged in Vitro Toxicology Experiments on a Macrophage System

Immunotoxicology sensu lato comprises not only toxicity toward immune cells, but also biological reactions from immune cells exposed to toxicants, reactions that may have deleterious effects at the organismal level. Within this wide frame, a specific case of interest is represented by the response of macrophages to particulate materials, with the epitome examples of asbestos and crystalline silica. For such toxicants that are both persistent and often encountered in an occupational setting, i.e. at low but repeated doses, there is a need for in vitro systems that can take into account these two parameters. Currently, most in vitro systems are used in an acute exposure mode, i.e., with a single dose and a readout made shortly if not immediately after exposure. We describe here how adequate changes of the culture methods applied to the murine macrophage cell line J774A.1 enable longer periods of culture (several days), which represents a first opportunity to address the persistence and dose-rate issues. To respond to this, the protocol uses a reduction in the concentration of the animal serum used for cell culture, as well as a switch from fetal to adult serum, which is less rich in proliferation factors. By doing so, we have considerably reduced cell proliferation, which is a problem with cell lines when they are supposed to represent slowly-dividing cells such as resident macrophages. We also succeeded in maintaining the differentiated functions of macrophages, such as phagocytosis or inflammatory responses, over the whole culture period. Furthermore, the presence of serum, even at low concentrations, provides excellent cell viability and keeps the presence of a protein corona on particulate materials, a feature that is known to strongly modulate their effects on cells and is lost in serum-free culture. Besides data showing the impact of these conditions on macrophages cell line cultures, illustrative examples are shown on silica- and cobalt-based pigments.

Using Live Imaging and FUCCI Embryonic Stem Cells to Rank DevTox Risks: Adverse Growth Effects of PFOA Compared With DEP Are 26 Times Faster, 1,000 Times More Sensitive, and 13 Times Greater in Magnitude

Fluorescent ubiquitination-based cell cycle indicator (FUCCI) embryonic stem cells (ESCs), which fluoresce green during the S-G2-M phases, generate an S-shaped curve for the accumulation of cells during normal stemness (NS) culture with leukemia-inhibitory factor (LIF). Since it was hypothesized that a culture of ESCs was heterogeneous in the cell cycle, it was expected that increased S-G2-M-phases of the cell cycle would make an S-shaped curve parallel to the accumulation curve. Unexpectedly, it was observed that the fraction of FUCCI ESCs in green decreases over time to a nadir at ∼24 h after previous feeding and then rapidly enters S-G2-M-phases after medium change. G1 delay by infrequent medium change is a mild stress, as it does not affect growth significantly when frequency is increased to 12 h. Perfluoro-octanoic acid (PFOA) and diethyl phthalate (DEP) were used as examples of members of the per- and polyfluoroalkyl substances (PFAS) and phthalate families of chemicals, respectively. Two adverse outcomes were used to compare dose- and time-dependent effects of PFOA and DEP. The first was cell accumulation assay by time-lapse confluence measurements, largely at Tfinal/T74 h. The second was by quantifying dominant toxicant stress shown by the suppression of mild stress that creates a green fed/unfed peak. In terms of speed, PFOA is 26 times faster than DEP for producing a time-dependent LOAEL dose at 100 uM (that is, 2 h for PFOA and 52 h for DEP). PFOA has 1000-fold more sensitive LOAEL doses than DEP for suppressing ESC accumulation (confluence) at day 3 and day 2. There were two means to compare the magnitude of the growth suppression of PFOA and DEP. For the suppression of the accumulation of cells measured by confluence at Tfinal/T74h, there was a 13-fold suppression at the highest dose of PFOA > the highest dose of DEP. For the suppression of entry into the cell cycle after the G1 phase by stress on day 1 and 2, there is 10-fold more suppression by PFOA than DEP. The data presented here suggest that FUCCI ESCs can assay the suppression of accumulated growth or predict the suppression of future growth by the suppression of fed/unfed green fluorescence peaks and that PFOA’s adverse effects are faster and larger and can occur at more sensitive lower doses than DEP.