Functional Toxicogenomics: Mechanism-Centered Toxicology

Traditional toxicity testing using animal models is slow, low capacity, expensive and assesses a limited number of endpoints. Such approaches are inadequate to deal with the increasingly large number of compounds found in the environment for which there are no toxicity data. Mechanism-centered high-throughput testing represents an alternative approach to meet this pressing need but is limited by our current understanding of toxicity pathways. Functional toxicogenomics, the global study of the biological function of genes on the modulation of the toxic effect of a compound, can play an important role in identifying the essential cellular components and pathways involved in toxicity response. The combination of the identification of fundamental toxicity pathways and mechanism-centered targeted assays represents an integrated approach to advance molecular toxicology to meet the challenges of toxicity testing in the 21st century

Functional toxicology: tools to advance the future of toxicity testing

The increased presence of chemical contaminants in the environment is an undeniable concern to human health and ecosystems. Historically, by relying heavily upon costly and laborious animal-based toxicity assays, the field of toxicology has often neglected examinations of the cellular and molecular mechanisms of toxicity for the majority of compounds – information that, if available, would strengthen risk assessment analyses. Functional toxicology, where cells or organisms with gene deletions or depleted proteins are used to assess genetic requirements for chemical tolerance, can advance the field of toxicity testing by contributing data regarding chemical mechanisms of toxicity. Functional toxicology can be accomplished using available genetic tools in yeasts, other fungi and bacteria, and eukaryotes of increased complexity, including zebrafish, fruit flies, rodents, and human cell lines. Underscored is the value of using less complex systems such as yeasts to direct further studies in more complex systems such as human cell lines. Functional techniques can yield (1) novel insights into chemical toxicity; (2) pathways and mechanisms deserving of further study; and (3) candidate human toxicant susceptibility or resistance genes

A comparison between whole transcript and 3' RNA sequencing methods using Kapa and Lexogen library preparation methods.

Background3' RNA sequencing provides an alternative to whole transcript analysis. However, we do not know a priori the relative advantage of each method. Thus, a comprehensive comparison between the whole transcript and the 3' method is needed to determine their relative merits. To this end, we used two commercially available library preparation kits, the KAPA Stranded mRNA-Seq kit (traditional method) and the Lexogen QuantSeq 3' mRNA-Seq kit (3' method), to prepare libraries from mouse liver RNA. We then sequenced and analyzed the libraries to determine the advantages and disadvantages of these two approaches.ResultsWe found that the traditional whole transcript method and the 3' RNA-Seq method had similar levels of reproducibility. As expected, the whole transcript method assigned more reads to longer transcripts, while the 3' method assigned roughly equal numbers of reads to transcripts regardless of their lengths. We found that the 3' RNA-Seq method detected more short transcripts than the whole transcript method. With regard to differential expression analysis, we found that the whole transcript method detected more differentially expressed genes, regardless of the level of sequencing depth.ConclusionsThe 3' RNA-Seq method was better able to detect short transcripts, while the whole transcript RNA-Seq was able to detect more differentially expressed genes. Thus, both approaches have relative advantages and should be selected based on the goals of the experiment

Involvement of N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) in arsenic biomethylation and its role in arsenic-induced toxicity.

BackgroundIn humans, inorganic arsenic (iAs) is metabolized to methylated arsenical species in a multistep process mainly mediated by arsenic (+3 oxidation state) methyltransferase (AS3MT). Among these metabolites is monomethylarsonous acid (MMAIII), the most toxic arsenic species. A recent study in As3mt-knockout mice suggests that unidentified methyltransferases could be involved in alternative iAs methylation pathways. We found that yeast deletion mutants lacking MTQ2 were highly resistant to iAs exposure. The human ortholog of the yeast MTQ2 is N-6 adenine-specific DNA methyltransferase 1 (N6AMT1), encoding a putative methyltransferase.ObjectiveWe investigated the potential role of N6AMT1 in arsenic-induced toxicity.MethodsWe measured and compared the cytotoxicity induced by arsenicals and their metabolic profiles using inductively coupled plasma-mass spectrometry in UROtsa human urothelial cells with enhanced N6AMT1 expression and UROtsa vector control cells treated with different concentrations of either iAsIII or MMAIII.ResultsN6AMT1 was able to convert MMAIII to the less toxic dimethylarsonic acid (DMA) when overexpressed in UROtsa cells. The enhanced expression of N6AMT1 in UROtsa cells decreased cytotoxicity of both iAsIII and MMAIII. Moreover, N6AMT1 is expressed in many human tissues at variable levels, although at levels lower than those of AS3MT, supporting a potential participation in arsenic metabolism in vivo.ConclusionsConsidering that MMAIII is the most toxic arsenical, our data suggest that N6AMT1 has a significant role in determining susceptibility to arsenic toxicity and carcinogenicity because of its specific activity in methylating MMAIII to DMA and other unknown mechanisms

Short versus long silver nanowires: a comparison of in vivo pulmonary effects post instillation.

BackgroundSilver nanowires (Ag NWs) are increasingly being used to produce touchscreens for smart phones and computers. When applied in a thin film over a plastic substrate, Ag NWs create a transparent, highly-conductive network of fibers enabling the touch interface between consumers and their electronics. Large-scale application methods utilize techniques whereby Ag NW suspensions are deposited onto substrates via droplets. Aerosolized droplets increase risk of occupational Ag NW exposure. Currently, there are few published studies on Ag NW exposure-related health effects. Concerns have risen about the potential for greater toxicity from exposure to high-aspect ratio nanomaterials compared to their non-fibrous counterparts. This study examines whether Ag NWs of varying lengths affect biological responses and silver distribution within the lungs at different time-points.MethodsTwo different sizes of Ag NWs (2 μm [S-Ag NWs] and 20 μm [L-Ag NWs]) were tested. Male, Sprague-Dawley rats were intratracheally instilled with Ag NWs (0, 0.1, 0.5, or 1.0 mg/kg). Broncho-alveolar lavage fluid (BALF) and lung tissues were obtained at 1, 7, and 21 days post exposure for analysis of BAL total cells, cell differentials, and total protein as well as tissue pathology and silver distribution.Results and conclusionsThe two highest doses produced significant increases in BAL endpoints. At Day 1, Ag NWs increased total cells, inflammatory polymorphonuclear cells (PMNs), and total protein. PMNs persisted for both Ag NW types at Day 7, though not significantly so, and by Day 21, PMNs appeared in line with sham control values. Striking histopathological features associated with Ag NWs included 1) a strong influx of eosinophils at Days 1 and 7; and 2) formation of Langhans and foreign body giant cells at Days 7 and 21. Epithelial sloughing in the terminal bronchioles (TB) and cellular exudate in alveolar regions were also common. By Day 21, Ag NWs were primarily enclosed in granulomas or surrounded by numerous macrophages in the TB-alveolar duct junction. These findings suggest short and long Ag NWs produce pulmonary toxicity; thus, further research into exposure-related health effects and possible exposure scenarios are necessary to ensure human safety as Ag NW demand increases

Probing the Mechanochemistry of Metal-Organic Frameworks with Low-Frequency Vibrational Spectroscopy

The identification and characterization of low-frequency vibrational motions of metal-organic frameworks (MOFs) allows for a better understanding of their mechanical and structural response upon perturbation by external stimuli such as temperature, pressure, and adsorption. Here, we describe the combination of an experimental temperature- and pressure-dependent terahertz spectroscopy system with quantum mechanical simulations to measure and assign specific low-frequency vibrational modes that directly drive the mechanochemical properties of this important class of porous materials. More specifically, those intense spectral features in the terahertz region of the vibrational spectrum of ZIF-8 are identified, which are directly connected to its mechanochemical response. In particular, the mechanical compressibility of pristine ZIF-8 is found to follow a peculiar non-linear trend upon pressure: its bulk modulus initially increases up to 0.1 GPa and decreases at higher pressures, which is simultaneously reflected in the terahertz vibrational spectra. This work highlights the interplay between structural, vibrational, and mechanochemical phenomena, all of which are key to the effective exploitation of MOFs. The importance of terahertz vibrational motions on the function of MOFs is demonstrated, and a method presented for their measurement and interpretation, which can be applied widely to any supramolecular material

A P-type ATPase importer that discriminates between essential and toxic transition metals

Transition metals, although being essential cofactors in many physiological processes, are toxic at elevated concentrations. Among the membrane-embedded transport proteins that maintain appropriate intracellular levels of transition metals are ATP-driven pumps belonging to the P-type ATPase superfamily. These metal transporters may be differentiated according to their substrate specificities, where the majority of pumps can extrude either silver and copper or zinc, cadmium, and lead. In the present report, we have established the substrate specificities of nine previously uncharacterized prokaryotic transition-metal P-type ATPases. We find that all of the newly identified exporters indeed fall into one of the two above-mentioned categories. In addition to these exporters, one importer, Pseudomonas aeruginosa Q9I147, was also identified. This protein, designated HmtA (heavy metal transporter A), exhibited a different substrate recognition profile from the exporters. In vivo metal susceptibility assays, intracellular metal measurements, and transport experiments all suggest that HmtA mediates the uptake of copper and zinc but not of silver, mercury, or cadmium. The substrate selectivity of this importer ensures the high-affinity uptake of essential metals, while avoiding intracellular contamination by their toxic counterparts

A Systems Biology Approach Reveals a Calcium-Dependent Mechanism for Basal Toxicity in Daphnia magna.

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Environmental Science & Technology, copyright © American Chemical Society after peer review and technical editing by the publisher.The expanding diversity and ever increasing amounts of man-made chemicals discharged to the environment pose largely unknown hazards to ecosystem and human health. The concept of adverse outcome pathways (AOPs) emerged as a comprehensive framework for risk assessment. However, the limited mechanistic information available for most chemicals and a lack of biological pathway annotation in many species represent significant challenges to effective implementation of this approach. Here, a systems level, multistep modeling strategy demonstrates how to integrate information on chemical structure with mechanistic insight from genomic studies, and phenotypic effects to define a putative adverse outcome pathway. Results indicated that transcriptional changes indicative of intracellular calcium mobilization were significantly overrepresented in Daphnia magna (DM) exposed to sublethal doses of presumed narcotic chemicals with log Kow ≥ 1.8. Treatment of DM with a calcium ATPase pump inhibitor substantially recapitulated the common transcriptional changes. We hypothesize that calcium mobilization is a potential key molecular initiating event in DM basal (narcosis) toxicity. Heart beat rate analysis and metabolome analysis indicated sublethal effects consistent with perturbations of calcium preceding overt acute toxicity. Together, the results indicate that altered calcium homeostasis may be a key early event in basal toxicity or narcosis induced by lipophilic compounds