Tobacco-Specific Nitrosamines (NNAL, NNN, NAT, and NAB) Exposures in the US Population Assessment of Tobacco and Health (PATH) Study Wave 1 (2013-2014).

INTRODUCTION: The tobacco-specific nitrosamines (TSNAs) are an important group of carcinogens found in tobacco and tobacco smoke. To describe and characterize the levels of TSNAs in the Population Assessment of Tobacco and Health (PATH) Study Wave 1 (2013-2014), we present four biomarkers of TSNA exposure: N'-nitrosonornicotine, N'-nitrosoanabasine, N'-nitrosoanatabine, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) which is the primary urinary metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. METHODS: We measured total TSNAs in 11 522 adults who provided urine using automated solid-phase extraction coupled to isotope dilution liquid chromatography-tandem mass spectrometry. After exclusions in this current analysis, we selected 11 004 NNAL results, 10 753 N'-nitrosonornicotine results, 10 919 N'-nitrosoanatabine results, and 10 996 N'-nitrosoanabasine results for data analysis. Geometric means and correlations were calculated using SAS and SUDAAN. RESULTS: TSNA concentrations were associated with choice of tobacco product and frequency of use. Among established, every day, exclusive tobacco product users, the geometric mean urinary NNAL concentration was highest for smokeless tobacco users (993.3; 95% confidence interval [CI: 839.2, 1147.3] ng/g creatinine), followed by all types of combustible tobacco product users (285.4; 95% CI: [267.9, 303.0] ng/g creatinine), poly tobacco users (278.6; 95% CI: [254.9, 302.2] ng/g creatinine), and e-cigarette product users (6.3; 95% CI: [4.7, 7.9] ng/g creatinine). TSNA concentrations were higher in every day users than in intermittent users for all the tobacco product groups. Among single product users, exposure to TSNAs differed by sex, age, race/ethnicity, and education. Urinary TSNAs and nicotine metabolite biomarkers were also highly correlated. CONCLUSIONS: We have provided PATH Study estimates of TSNA exposure among US adult users of a variety of tobacco products. These data can inform future tobacco product and human exposure evaluations and related regulatory activities.

Epigenetic Vulnerability of Insulator CTCF Motifs at Parkinson's Disease-Associated Genes in Response to Neurotoxicant Rotenone.

CCCTC-binding factor (CTCF) is a regulatory protein that binds DNA to control spatial organization and transcription. The sequence-specific binding of CTCF is variable and is impacted by nearby epigenetic patterns. It has been demonstrated that non-coding genetic variants cluster with CTCF sites in topological associating domains and thus can affect CTCF activity on gene expression. Therefore, environmental factors that alter epigenetic patterns at CTCF binding sites may dictate the interaction of non-coding genetic variants with regulatory proteins. To test this mechanism, we treated human cell line HEK293 with rotenone for 24 h and characterized its effect on global epigenetic patterns specifically at regulatory regions of Parkinson's disease (PD) risk loci. We used RNA sequencing to examine changes in global transcription and identified over 2000 differentially expressed genes (DEGs, >1.5-fold change, FDR < 0.05). Among these DEGs, 13 were identified as PD-associated genes according to Genome-wide association studies meta-data. We focused on eight genes that have non-coding risk variants and a prominent CTCF binding site. We analyzed methylation of a total of 165 CGs surrounding CTCF binding sites and detected differential methylation (|>1%|, q < 0.05) in 45 CGs at 7 PD-associated genes. Of these 45 CGs, 47% were hypomethylated and 53% were hypermethylated. Interestingly, 5 out of the 7 genes had correlated gene upregulation with CG hypermethylation at CTCF and gene downregulation with CG hypomethylation at CTCF. We also investigated active H3K27ac surrounding the same CTCF binding sites within these seven genes. We observed a significant increase in H3K27ac in four genes (FDR < 0.05). Three genes (PARK2, GPRIN3, FER) showed increased CTCF binding in response to rotenone. Our data indicate that rotenone alters regulatory regions of PD-associated genes through changes in epigenetic patterns, and these changes impact high-order chromatin organization to increase the influence of non-coding variants on genome integrity and cellular survival.

Manganese-induced Parkinsonism in mice is reduced using a novel contaminated water sediment exposure model.

Heavy metals enter the aquatic environment and accumulate within water sediments, but these metal-sediment interactions remain to be explored within toxicity studies. We developed an exposure model in mice that encapsulates the aquatic microenvironment of metals before exposure. Male and female C57/BL6 mice were exposed via their drinking water to manganese contaminated sediment (Sed_Mn) or to manganese without sediment interaction (Mn) for six weeks. Sediment interaction did not alter weekly manganese ingestion from water in males or females. We analyzed motor impairment, a common feature in manganese-induced Parkinsonism, using the beam traversal, cylinder, and accelerating rotarod tests. Sed_Mn mice performed better overall compared to Mn mice and males were more sensitive to manganese than females in both Sed_Mn and Mn treatment groups. Our study indicates that metal-sediment interactions may alter metal toxicity in mammals and introduces a new exposure model to test the toxicity of metal contaminants of drinking water.

The conserved DNMT1-dependent methylation regions in human cells are vulnerable to neurotoxicant rotenone exposure.

BACKGROUND: Allele-specific DNA methylation (ASM) describes genomic loci that maintain CpG methylation at only one inherited allele rather than having coordinated methylation across both alleles. The most prominent of these regions are germline ASMs (gASMs) that control the expression of imprinted genes in a parent of origin-dependent manner and are associated with disease. However, our recent report reveals numerous ASMs at non-imprinted genes. These non-germline ASMs are dependent on DNA methyltransferase 1 (DNMT1) and strikingly show the feature of random, switchable monoallelic methylation patterns in the mouse genome. The significance of these ASMs to human health has not been explored. Due to their shared allelicity with gASMs, herein, we propose that non-traditional ASMs are sensitive to exposures in association with human disease. RESULTS: We first explore their conservancy in the human genome. Our data show that our putative non-germline ASMs were in conserved regions of the human genome and located adjacent to genes vital for neuronal development and maturation. We next tested the hypothesized vulnerability of these regions by exposing human embryonic kidney cell HEK293 with the neurotoxicant rotenone for 24 h. Indeed,14 genes adjacent to our identified regions were differentially expressed from RNA-sequencing. We analyzed the base-resolution methylation patterns of the predicted non-germline ASMs at two neurological genes, HCN2 and NEFM, with potential to increase the risk of neurodegeneration. Both regions were significantly hypomethylated in response to rotenone. CONCLUSIONS: Our data indicate that non-germline ASMs seem conserved between mouse and human genomes, overlap important regulatory factor binding motifs, and regulate the expression of genes vital to neuronal function. These results support the notion that ASMs are sensitive to environmental factors such as rotenone and may alter the risk of neurological disease later in life by disrupting neuronal development.

In vitro and in silico Models to Study Mosquito-Borne Flavivirus Neuropathogenesis, Prevention, and Treatment.

Mosquito-borne flaviviruses can cause disease in the nervous system, resulting in a significant burden of morbidity and mortality. Disease models are necessary to understand neuropathogenesis and identify potential therapeutics and vaccines. Non-human primates have been used extensively but present major challenges. Advances have also been made toward the development of humanized mouse models, but these models still do not fully represent human pathophysiology. Recent developments in stem cell technology and cell culture techniques have allowed the development of more physiologically relevant human cell-based models. In silico modeling has also allowed researchers to identify and predict transmission patterns and discover potential vaccine and therapeutic candidates. This review summarizes the research on in vitro and in silico models used to study three mosquito-borne flaviviruses that cause neurological disease in humans: West Nile, Dengue, and Zika. We also propose a roadmap for 21st century research on mosquito-borne flavivirus neuropathogenesis, prevention, and treatment.

Toxicity, recovery, and resilience in a 3D dopaminergic neuronal in vitro model exposed to rotenone.

To date, most in vitro toxicity testing has focused on acute effects of compounds at high concentrations. This testing strategy does not reflect real-life exposures, which might contribute to long-term disease outcome. We used a 3D-human dopaminergic in vitro LUHMES cell line model to determine whether effects of short-term rotenone exposure (100 nM, 24 h) are permanent or reversible. A decrease in complex I activity, ATP, mitochondrial diameter, and neurite outgrowth were observed acutely. After compound removal, complex I activity was still inhibited; however, ATP levels were increased, cells were electrically active and aggregates restored neurite outgrowth integrity and mitochondrial morphology. We identified significant transcriptomic changes after 24 h which were not present 7 days after wash-out. Our results suggest that testing short-term exposures in vitro may capture many acute effects which cells can overcome, missing adaptive processes, and long-term mechanisms. In addition, to study cellular resilience, cells were re-exposed to rotenone after wash-out and recovery period. Pre-exposed cells maintained higher metabolic activity than controls and presented a different expression pattern in genes previously shown to be altered by rotenone. NEF2L2, ATF4, and EAAC1 were downregulated upon single hit on day 14, but unchanged in pre-exposed aggregates. DAT and CASP3 were only altered after re-exposure to rotenone, while TYMS and MLF1IP were downregulated in both single-exposed and pre-exposed aggregates. In summary, our study shows that a human cell-based 3D model can be used to assess cellular adaptation, resilience, and long-term mechanisms relevant to neurodegenerative research.

Metal ion coordination in the E. coli Nudix hydrolase dihydroneopterin triphosphate pyrophosphatase: New clues into catalytic mechanism.

Dihydroneopterin triphosphate pyrophosphatase (DHNTPase), a member of the Mg2+ dependent Nudix hydrolase superfamily, is the recently-discovered enzyme that functions in the second step of the pterin branch of the folate biosynthetic pathway in E. coli. DHNTPase is of interest because inhibition of enzymes in bacterial folate biosynthetic pathways is a strategy for antibiotic development. We determined crystal structures of DHNTPase with and without activating, Mg2+-mimicking metals Co2+ and Ni2+. Four metal ions, identified by anomalous scattering, and stoichiometrically confirmed in solution by isothermal titration calorimetry, are held in place by Glu56 and Glu60 within the Nudix sequence motif, Glu117, waters, and a sulfate ion, of which the latter is further stabilized by a salt bridge with Lys7. In silico docking of the DHNTP substrate reveals a binding mode in which the pterin ring moiety is nestled in a largely hydrophobic pocket, the ß-phosphate activated for nucleophilic attack overlays with the crystallographic sulfate and is in line with an activated water molecule, and remaining phosphate groups are stabilized by all four identified metal ions. The structures and binding data provide new details regarding DHNTPase metal requirements, mechanism, and suggest a strategy for efficient inhibition.

Structural basis for misfolding in myocilin-associated glaucoma.

Olfactomedin (OLF) domain-containing proteins play roles in fundamental cellular processes and have been implicated in disorders ranging from glaucoma, cancers and inflammatory bowel disorder, to attention deficit disorder and childhood obesity. We solved crystal structures of the OLF domain of myocilin (myoc-OLF), the best studied such domain to date. Mutations in myoc-OLF are causative in the autosomal dominant inherited form of the prevalent ocular disorder glaucoma. The structures reveal a new addition to the small family of five-bladed ß-propellers. Propellers are most well known for their ability to act as hubs for protein-protein interactions, a function that seems most likely for myoc-OLF, but they can also act as enzymes. A calcium ion, sodium ion and glycerol molecule were identified within a central hydrophilic cavity that is accessible via movements of surface loop residues. By mapping familial glaucoma-associated lesions onto the myoc-OLF structure, three regions sensitive to aggregation have been identified, with direct applicability to differentiating between neutral and disease-causing non-synonymous mutations documented in the human population worldwide. Evolutionary analysis mapped onto the myoc-OLF structure reveals conserved and divergent regions for possible overlapping and distinctive functional protein-protein or protein-ligand interactions across the broader OLF domain family. While deciphering the specific normal biological functions, ligands and binding partners for OLF domains will likely continue to be a challenging long-term experimental pursuit, atomic detail structural knowledge of myoc-OLF is a valuable guide for understanding the implications of glaucoma-associated mutations and will help focus future studies of this biomedically important domain family.