Neuropathological Examination of Mice Chronically Exposed to Secondhand Smoke.

INTRODUCTION: Around 21.6-35% of military personnel are smokers, while 12.26% of them have been regularly exposed to second-hand smoke (SHS). Second-hand smoke is considered an important risk factor for neurological diseases because it can induce oxidative stress, DNA damage, and disrupt DNA repair pathways. MATERIAL AND METHODS: The brain of air (sham) or SHS exposed mice was cryoperserved, sectioned, and placed on a glass slide before immunoprobing them with antibodies to observe for oxidative DNA damage (8-oxoG), oxidative DNA repair (8-oxoguanine DNA glycosylase 1, Ogg1; apurinic/apyrimidinic endonuclease, Ape1), and inflammatory (glial fibrillary acidic protein) proteins. RESULTS: Nissl staining of the prefrontal cortex (PFCTX) revealed the presence of dark, shrunken cells, hippocampal thinning, and the presence of activated astrocytes in SHS exposed mice. 8-oxoG staining was also more prominent in the PFCTX and hippocampus (HIPP) of SHS exposed mice. Ogg1 staining was reduced in the PFCTX and CA3 hippocampal neurons of SHS exposed mice, whereas it was more prominent in CA1 and CA4 hippocampal neurons. In contrast, Ape1 staining was more prominent in the PFCTX and the HIPP of SHS exposed mice. CONCLUSIONS: These studies demonstrate that oxidative DNA damage (8-oxoG) was elevated and oxidative DNA repair (Ape1 and Ogg1) was altered in the brain of SHS exposed mice. In addition, activated astrocytes (i.e., glial fibrillary acidic protein) were also observed in the brain of SHS exposed mice. Therefore, SHS induces both oxidative DNA damage and repair as well as inflammation as possible underlying mechanism(s) of the cognitive decline and metabolic changes that were observed in chronically exposed mice. A better understanding of how chronic exposure to SHS induces cognitive dysfunction among military personnel could help improve the combat readiness of U.S. soldiers as well as reduce the financial burden on the DOD and veterans' families.

Effects of Chronic Secondhand Smoke (SHS) Exposure on Cognitive Performance and Metabolic Pathways in the Hippocampus of Wild-Type and Human Tau Mice.

BACKGROUND: Exposure to secondhand smoke (SHS) is a risk factor for developing sporadic forms of sporadic dementia. A human tau (htau) mouse model is available that exhibits age-dependent tau dysregulation, neurofibrillary tangles, neuronal loss, neuroinflammation, and oxidative stress starting at an early age (3-4 months) and in which tau dysregulation and neuronal loss correlate with synaptic dysfunction and cognitive decline. OBJECTIVE: The goal of this study was to assess the effects of chronic SHS exposure (10 months' exposure to ∼30 mg/m3) on behavioral and cognitive function, metabolism, and neuropathology in mice. METHODS: Wild-type (WT) and htau female and male mice were exposed to SHS (90% side stream, 10% main stream) using the SCIREQ® inExpose™ system or air control for 168 min per day, for 312 d, 7 d per week. The exposures continued during the days of behavioral and cognitive testing. In addition to behavioral and cognitive performance and neuropathology, the lungs of mice were examined for pathology and alterations in gene expression. RESULTS: Mice exposed to chronic SHS exposure showed the following genotype-dependent responses: a) lower body weights in WT, but not htau, mice; b) less spontaneous alternation in WT, but not htau, mice in the Y maze; c) faster swim speeds of WT, but not htau, mice in the water maze; d) lower activity levels of WT and htau mice in the open field; e) lower expression of brain PHF1, TTCM1, IGF1ß, and HSP90 protein levels in WT male, but not female, mice; and f) more profound effects on hippocampal metabolic pathways in WT male than female mice and more profound effects in WT than htau mice. DISCUSSION: The brain of WT mice, in particular WT male mice, might be especially susceptible to the effects of chronic SHS exposure. In WT males, independent pathways involving ascorbate, flavin adenine dinucleotide, or palmitoleic acid might contribute to the hippocampal injury following chronic SHS exposure. https://doi.org/10.1289/EHP8428.

Protocol for High-Throughput Screening of Neural Cell or Brain Tissue Protein Using a Dot-Blot Technique with Near-Infrared Imaging.

Dot blotting allows for the rapid screening of a larger number of samples and/or targets than more traditional methods, such as a western blot or in-tissue-based methods. We have developed a dot-blot assay specifically for use with a LiCor Odyssey CLx imager, which allows for sensitive detection of proteins in the infrared range. Here, we provide a detailed protocol for the preparation of brain tissue and neural cell culture lysates for analysis of protein targets by dot blotting.

Comparative developmental toxicity of a comprehensive suite of polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants that occur in complex mixtures. Several PAHs are known or suspected mutagens and/or carcinogens, but developmental toxicity data is lacking for PAHs, particularly their oxygenated and nitrated derivatives. Such data are necessary to understand and predict the toxicity of environmental mixtures. 123 PAHs were assessed for morphological and neurobehavioral effects for a range of concentrations between 0.1 and 50 µM, using a high throughput early-life stage zebrafish assay, including 33 parent, 22 nitrated, 17 oxygenated, 19 hydroxylated, 14 methylated, 16 heterocyclic, and 2 aminated PAHs. Additionally, each PAH was evaluated for AHR activation, by assessing CYP1A protein expression using whole animal immunohistochemistry (IHC). Responses to PAHs varied in a structurally dependent manner. High-molecular weight PAHs were significantly more developmentally toxic than the low-molecular weight PAHs, and CYP1A expression was detected in five distinct tissues, including vasculature, liver, skin, neuromasts and yolk.

Investigating the application of a nitroreductase-expressing transgenic zebrafish line for high-throughput toxicity testing.

Nitroreductase enzymes are responsible for the reduction of nitro functional groups to amino functional groups, and are found in a range of animal models, zebrafish (Danio rerio) excluded. Transgenic zebrafish models have been developed for tissue-specific cell ablation, which use nitroreductase to ablate specific tissues or cell types following exposure to the non-toxic pro-drug metronidazole (MTZ). When metabolized by nitroreductase, MTZ produces a potent cytotoxin, which specifically ablates the tissue in which metabolism occurs. Uses, beyond tissue-specific cell ablation, are possible for the hepatocyte-specific Tg(l-fabp:CFP-NTR)s891 zebrafish line, including investigations of the role of nitroreductase in the toxicity of nitrated compounds. The hepatic ablation characteristics of this transgenic line were explored, in order to expand its potential uses. Embryos were exposed at 48, 72, or 96 hours post fertilization (hpf) to a range of MTZ concentrations, and the ablation profiles were compared. Ablation occurred at a 10-fold lower concentration than previously reported. Embryos were exposed to a selection of other compounds, with and without MTZ, in order to investigate alternative uses for this transgenic line. Test compounds were selected based on: their ability to undergo nitroreduction, known importance of hepatic metabolism to toxicity, and known pharmaceutical hepatotoxins. Selected compounds included nitrated polycyclic aromatic hydrocarbons (nitro-PAHs), the PAHs retene and benzo[a]pyrene, and the pharmaceuticals acetaminophen and flutamide. The results suggest a range of potential roles of the liver in the toxicity of these compounds, and highlight the additional uses of this transgenic model in toxicity testing.

Formation of Developmentally Toxic Phenanthrene Metabolite Mixtures by Mycobacterium sp. ELW1.

Mycobacterium sp. ELW1 co-metabolically degraded up to 1.8 µmol of phenanthrene (PHE) in ∼48 h, and hydroxyphenanthrene (OHPHE) metabolites, including 1-hydroxyphenanthrene (1-OHPHE), 3-hydroxyphenanthrene (3-OHPHE), 4-hydroxyphenanthrene (4-OHPHE), 9-hydroxyphenanthrene (9-OHPHE), 9,10-dihydroxyphenanthrene (1,9-OHPHE), and trans-9,10-dihydroxy-9,10-dihydrophenanthrene (trans-9,10-OHPHE), were identified and quantified over time. The monooxygenase responsible for co-metabolic transformation of PHE was inhibited by 1-octyne. First-order PHE transformation rates, kPHE, and half-lives, t1/2, for PHE-exposed cells were 0.16-0.51 h-1 and 1.4-4.3 h, respectively, and the 1-octyne controls ranged from 0.015-0.10 h-1 to 7.0-47 h, respectively. While single compound standards of PHE and trans-9,10-OHPHE, the major OHPHE metabolite formed by ELW1, were not toxic to embryonic zebrafish (Danio rerio), single compound standards of minor OHPHE metabolites, 1-OHPHE, 3-OHPHE, 4-OHPHE, 9-OHPHE, and 1,9-OHPHE, were toxic, with effective concentrations (EC50's) ranging from 0.5 to 5.5 µM. The metabolite mixtures formed by ELW1, and the reconstructed standard mixtures of the identified OHPHE metabolites, elicited a toxic response in zebrafish for the same three time points. EC50s for the metabolite mixtures formed by ELW1 were lower (more toxic) than those for the reconstructed standard mixtures of the identified OHPHE metabolites. Ten unidentified hydroxy PHE metabolites were measured in the derivatized mixtures formed by ELW1 and may explain the increased toxicity of the ELW1 metabolites mixture relative to the reconstructed standard mixtures of the identified OHPHE metabolites.

Mechanistic Investigations Into the Developmental Toxicity of Nitrated and Heterocyclic PAHs.

Nitrated polycyclic aromatic hydrocarbons (NPAHs) and heterocyclic PAHs (HPAHs) are recognized environmental pollutants. However, the health risks of NPAHs and HPAHs to humans and environmental systems are not well-studied. The developmental zebrafish (Danio rerio) model was used to evaluate the toxicity of a structurally diverse set of 27 NPAHs and 10 HPAHs. The individual activity of each compound towards the aryl hydrocarbon receptor (AHR), including the role of the AHR in observed toxicity, and genetic markers of oxidative stress and cardiac toxicity were evaluated. Zebrafish embryos were exposed from 6 to 120 hours post fertilization (hpf), to a broad concentration range of individual compounds, and evaluated for 22 developmental endpoints. The potential role of AHR was determined using the transgenic Tg(cyp1a:nls-egfp) reporter zebrafish line. All compounds were screened computationally through molecular docking using a previously developed AHR models of zebrafish isoforms 1A, 1B, and 2. Some compounds did not induce observable developmental toxic responses, whereas others produced statistically significant concentration-dependent toxicity. The tested compounds also exhibited a range of predicted AHR binding and cyp1a/GFP induction patterns, including cyp1a expression in the liver, vasculature, skin, and yolk, which we determined to be due to distinct isoforms of the AHR, using morpholino oligonucleotide knockdown. Furthermore, we investigated mRNA expression of oxidative and cardiac stress genes at 48 and 120 hpf, which indicated several potential mechanisms-of-action for NPAHs. Overall, we observed a range of developmental toxicities, cyp1a/GFP expression patterns, and gene expression profiles, suggestive of several potential mechanisms of action.

Optimizing multi-dimensional high throughput screening using zebrafish.

The use of zebrafish for high throughput screening (HTS) for chemical bioactivity assessments is becoming routine in the fields of drug discovery and toxicology. Here we report current recommendations from our experiences in zebrafish HTS. We compared the effects of different high throughput chemical delivery methods on nominal water concentration, chemical sorption to multi-well polystyrene plates, transcription responses, and resulting whole animal responses. We demonstrate that digital dispensing consistently yields higher data quality and reproducibility compared to standard plastic tip-based liquid handling. Additionally, we illustrate the challenges in using this sensitive model for chemical assessment when test chemicals have trace impurities. Adaptation of these better practices for zebrafish HTS should increase reproducibility across laboratories.

Quantitation and prediction of sorptive losses during toxicity testing of polycyclic aromatic hydrocarbon (PAH) and nitrated PAH (NPAH) using polystyrene 96-well plates.

Developing zebrafish are increasingly being used for rapid assessments of chemical toxicity, and these assays are frequently conducted in multi-well plastic plates. This study investigated the sorptive behavior of polycyclic aromatic hydrocarbons (PAHs) and nitrated PAHs (NPAHs) to uncoated 96-well polystyrene plates typically used for zebrafish (Danio rerio) testing. We measured the percent sorption in the presence and absence of zebrafish embryos, at two exposure concentrations, as well as using two different procedures (addition of embryos to polystyrene plates either before analyte addition, or allowing 24h of equilibrium between analyte addition and embryo addition to the polystyrene plates). Following exposure, the plates were extracted with hexane and analyzed using gas chromatography coupled with mass spectrometry (GC/MS). Allowing 24h of pre-incubation between the addition of analytes and embryos did not significantly impact the percent sorption. The percent sorption was higher for both PAHs and NPAHs at the lower exposure concentration, and sorption was lower in the presence of zebrafish embryos. A mass balance model was developed to predict the sorption to polystyrene plates, based on the PAH and NPAH mass distribution ratios between polystyrene and water. While PAH sorption was significantly correlated with subcooled liquid solubility, NPAH sorption did not correlate with any of the physical-chemical properties investigated. This indicates the need to better understand the sorptive behavior of hydrophobic analytes to plastics, and to better account for sorptive losses during toxicity testing in polystyrene plates.

Identification and Toxicological Evaluation of Unsubstituted PAHs and Novel PAH Derivatives in Pavement Sealcoat Products.

Pavement sealcoat products contain high concentrations of unsubstituted polycyclic aromatic hydrocarbons (PAHs), but the assessment of the potential toxicological impact is limited without the inclusion of PAH derivatives. This study determined the concentrations of 23 unsubstituted PAHs, 11 high molecular weight-PAHs (MW302-PAHs), and 56 PAH derivatives, including 10 methyl-PAHs (MPAHs), 10 heterocyclic-PAHs (Hetero-PAHs), 26 nitrated-PAHs (NPAHs), and 10 oxygenated-PAHs (OPAHs) in coal-tar and asphalt based sealcoat products and time point scrapes. Inclusion of MW302-PAHs resulted in an increase of 4.1-38.7% in calculated benzo[a]pyrene-carcinogenic equivalent (B[a]Peq) concentrations for the coal-tar based products. Increases in some NPAH and OPAH concentrations were measured after application, suggesting the possibility of photochemical transformation of unsubstituted PAHs. The Ames assay indicated that the asphalt based product was not mutagenic, but the coal-tar based sealcoat products were. The zebrafish developmental toxicity tests suggested that fractions where NPAHs and OPAHs eluted have the most significant adverse effects.