Anthracycline antibiotics and their synthetic analogs, an important class of anti-tumor agents, contain nucleophilic amines. Abasic (AP) sites, formed in DNA upon exposure to a variety of alkylating agents, interconvert between α and β anomers via an aldehydic intermediate susceptible to nucleophilic attack. We identified unique regiosiomeric imine conjugates between two reactive amines in pixantrone and AP sites in DNA. These were treated in the presence of sodium cyanoborohydride to form reduced conjugates suitable for structural analyses. NMR spectroscopy in the sequence 5’-d(GTTGCXCGTATG)-3’:5’-d(CACACGCGCAAC)-3’ [X = PIX-AP conjugate] revealed that both regioisomers of the PIX-AP conjugate intercalated into DNA. One of the two regioisomers was thermodynamically favored. It may hydrogen bond with the complementary cytosine. The stacking tendency of pixantrone was revealed by X-ray crystallography in the sequence 5’-d(CGCXAATTCGCG)-3’ [X = PIX-AP conjugate]. Looped-out pixantrones from neighboring duplexes engaged in a distinct stacking interaction. Anthracycline agents are frequently administered in combination chemotherapy with nitrogen mustard alkylating agents. Thus, these unique pixantrone AP-site conjugates may contribute to the cytotoxicity of this synthetic anthracycline.
In higher eukaryotic cells, mitochondria are essential subcellular organelles for energy production, cell signaling, and the biosynthesis of biomolecules. The mitochondrial DNA (mtDNA) genome is indispensable for mitochondrial function because it encodes protein subunits of the oxidative phosphorylation system and a full set of transfer and ribosomal RNAs. Mitochondrial transcription factor A (TFAM) is a major mtDNA packaging protein and a transcription factor. TFAM plays a critical role in mtDNA replication and transcription. Also, our recent research suggests a novel role of TFAM in regulating mtDNA degradation at abasic (AP) sites. AP sites are among the most abundant endogenous DNA lesions, sourced from base excision repair and spontaneous base loss. We demonstrate that the stability of AP sites is reduced dramatically upon binding to TFAM. The half-life of AP lesions within TFAM-DNA complexes is 2- to 3 orders of magnitude shorter than that in free DNA, depending on their position. The TFAM-catalyzed AP-DNA destabilization occurs with non-specific DNA and mitochondrial light-strand promoter sequence, yielding DNA single-strand breaks and DNA-TFAM cross-links. TFAM-DNA cross-links are also observed with mitochondrial extracts of human cells and in living human cells. In situ trapping of the reaction intermediates (DNA-TFAM cross-links) revealed that the reaction proceeds via Schiff base chemistry facilitated by lysine residues. Mass spectrometry analysis identified several key lysine residues in the cross-linking reaction. Collectively, our data suggest a novel role of TFAM in promoting the turnover of abasic DNA.
Bladder cancer (BC) is the most lethal cancer malignancy of the urinary system. Chronic infections, genetic susceptibilities, environmental and occupational exposure are BC risk factors. Smoking is the major risk factor for BC, with up to 50% of the cases attributed to it. However, the principal chemicals in tobacco smoke and their mechanisms of action leading to BC remain unknown. Among the 70 carcinogens produced by tobacco combustion, only 4-aminobiphenyl (4-ABP) and 2-naphthylamine (2-NA) are cited as associated with BC risk. This association is based on high occupational exposures of textile dye and rubber factory workers to these chemicals. However, 4-ABP and 2-NA occur at several ng per cigarette, levels which may not be sufficient to induce BC. In contrast, other pro-carcinogens including the heterocyclic aromatic amine (HAA) 2-amino-9H-pyrido[4,3-b]indole, and aldehydes including acrolein, occur at 100-fold or higher levels in tobacco smoke. Employing the human RT4 bladder epithelial cell line, we characterized the cytotoxicity and genotoxicity of tobacco smoke condensate (TSC). TSC of Marlboro and 1R6F reference cigarettes induced a dose- and time-dependent cytotoxicity. The TSC was separated by liquid-liquid extraction into a neutral fraction containing polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs, and hydrophobic aldehydes, and a basic fraction containing aromatic amines, HAAs, and N-nitroso compounds. The cytotoxicity ofTSC is mainly attributed to the neutral fraction. TSC and the neutral fraction induced oxidative stress marked by reactive oxygen species (ROS), decreased glutathione:oxidized glutathione (GSH:GSSG) ratio, and induction of AP-sites. The presence of ROS scavengers GSH and N-acetyl-cysteine, led to cytoprotective effects implicating oxidative stress as a mechanism of action. Targeted and untargeted metabolomics-based approaches were employed to characterize glutathione-conjugates formed by TSC, neutral, and basic fractions. Over 50 GSH conjugates were detected, with the most formed by chemicals inTSC and neutral fraction or induced endogenous electrophiles. The major GSH-conjugates are formed with acrolein, crotonaldehyde, and isomeric p-benzoquinone, and o-benzoquinone. We are currently investigating the DNA adducts formed by chemicals in the neutral and basic fractions of TSC. As the next step of this work, we will assess the role of hepatic metabolism to modulate TSC-induced cytotoxicity and DNA damage in RT4 cells.
A mechanism that cells tolerate DNA damage is with translesion DNA synthesis (TLS) polymerases that bypass DNA damage. Multiple TLS polymerases exist with different fidelities and DNA damage tolerance capabilities. Since, TLS polymerases have low fidelity, their access to the replication fork must be regulated to minimize mutations. The current paradigm is that a combination of kinetic partitioning and protein-protein interactions are used to regulate TLS polymerase activity. One difficulty in elucidating the multiple roles of these polymerases is that it is impossible to identify which polymerase is active in a specific situation. We designed and synthesized a novel nucleotide analog N2-benzyl-2′-deoxyguanosine (EBndG) that is highly selective toward DNA pol kappa (POLK), a member of the Y family of TLS polymerases. POLK can bypass bulky lesions in the minor groove such as N2-(7,8,9,10-tetrahydro-7,8,9-trihydroxybenzo[a]pyren-10-yl]-2’-deoxyguanosine (N2-BP-dG). To interrogate the identity of proteins surrounding the replication fork when POLK is actively replicating DNA, we performed an extensive study using modified aniPOND (accelerated native isolation of proteins on nascent DNA) experiments. Two different human cell lines were treated with various concentrations of (R/S)-trans-benzo[a]pyrene diol epoxide (BPDE) for 1 h to allow N2-BP-dG damage to form. Subsequently 5-ethynyl-2'-deoxyuridine (EdU) or EBndG was added and incubated for various time points. The DNA was isolated, sheared, conjugated to biotin via the Click Reaction, and the DNA isolated by streptavidin precipitation. The proteins bound to the DNA were analyzed by mass spectrometry. Ongoing data analysis identifies approximately 250 common proteins earlier identified in EdU pull downs including proteins involved in DNA replication, repair, or damage like BRD, ORC, MCM, RAD50, TOP, PCNA, RPA as well as chromatin associated factors like CHAF. POLK was identified to be associated with both EdU and EBndG pull downs. Several unique proteins associated with EBndG were identified, suggesting novel roles for POLK activity in the cell. Concurrently, we are using confocal microscopy to analyze the localization of EBndG, endogenous POLK and to validate the potential candidate proteins. These data will provide first insight into POLK’s core interactome, chromatin surrounding the POLK active sites, insight into POLK’s regulation and its novel cellular roles.
A response to stress within the human body involves the C11-hydroxylation of 11-deoxycorticosterone (DOC) to corticosterone by the enzyme cytochrome P450 CYP11B2 (Figure 1). The inhibition of this pathway is a target for stress management techniques, and there are many postulated inhibitors of the enzyme. The phytochemical sutherlandioside B (SU1) of Sutherlandia frutescens lowers corticosterone and acts as a natural inhibitor (Sergent, 2009). Additional naturally occurring inhibitors of CYP11B2 include ethyl caffeate and labiatenic acid which are sourced from Bidens pilosa and Rosmarinus officinalis (Menard et al., 2014). Our research explores the mechanistic qualities of the enzymatic hydroxylation of deoxycorticosterone via CYP11B2 to further understand possible inhibition mechanisms.
An initial study was performed on a small model system containing the iron/parent porphyrin complex, the apical ligand methyl sulfide and a hydrocarbon propane. This study provided a foundation for electronic structure and spin distributions with chemical accuracy. The system was computed at the quantum mechanical level. Results suggest a stepwise radical mechanism that initializes with hydrogen abstraction and subsequent hydroxyl group migration.
A more realistic model system was developed from the crystal structure of CYP11B2 Cytochrome P450 complexed to deoxycorticosterone (PDB 4DVQ). We employed multilevel methods (ONIOM) for accurate computation of the system. The active site contained the iron-porphyrin complex with a cysteine ligand and the steroid deoxycorticosterone, which were treated with density functional theory. Critical amino acid residues were carefully selected to obtain a region surrounding the active site and were described using semi-empirical methods. The propane-oxidation model provided the foundation for the ONIOM study, which establishes how thermochemical parameters are affected by the enzymatic environment.
Figure 1. Hydroxylation of 11-deoxycorticosterone to corticosterone by cytochrome P450 CYP11B2.
The opioid crisis continues to grow resulting in a large number of fatal and nonfatal overdoses each year in the United States. Increasing use of fentanyl and fentanyl related analogues poses a significant risk of fatal overdose due to their high potency and unpredictable pharmacokinetics. Fentanyl is a synthetic opioid primarily prescribed for pain management and anesthesia procedures but is also produced and used illegally often mixed with other drugs. As part of the South Carolina (SC) statewide response to the opioid crisis, the SC Public Health Laboratory (PHL) began a pilot study to test specimens for fentanyl and fentanyl analogs from suspected opioid-related emergency department visits from across the state. The SC public Health laboratory analyzed over 500 residual urine samples for the detection of fentanyl analogs. Of these, about 20% samples were tested positive for fentanyl or fentanyl analogues. The samples were analyzed quantitatively on a High Performance Liquid Chromatography Tandem Mass Spectrometer (LC-MS/MS) and qualitatively on a High Performance Liquid Chromatography Quadrupole Time of Flight Mass Spectrometer (LC-QTOF). Herein, we will provide a comparison of both techniques for the analysis of fentanyl and fentanyl related analogues. The LC-MS/MS provides better sensitivity and lower detection limits, whereas the LC-QTOF can scan for more fentanyl analogs; allowing the laboratory to identify novel fentanyl analogs that are being used within affected communities. These novel fentanyl analogs can be added to the growing list of fentanyl analogs to help other public health laboratories and stakeholders recognize a new threat on the market. Specimens from opioid-related emergency department visits from hospitals around the state are resulting in an expansion of biosurveillance of fentanyl analog usage in a broader segment of the SC population and this data will help to provide fidelity on the opioid epidemic within South Carolina.
The main goal of this undergraduate research project was to develop analytical, chemistry-based methodologies for studying (a) the interaction of silver nanoparticles (AgNPs), a well-known antibacterial agent, with human red blood cells (RBCs), and (b) their potential cytotoxicity mechanism. To achieve this, inductively coupled plasma optical emission spectroscopy (ICP-OES) and graphite furnace atomic absorption spectroscopy (GFAAS) in conjunction with U.S. Environmental Protection Agency (EPA) quality control guidelines were employed for (a) the quantification of the total amount of Ag interacting with RBCs (5 % hematocrit, -12.5 mV) and (b) the toxic effects on the Na+/K+ ATPase after RBCs exposure to 150 µg.mL-1 of AgNPs (5 % glucose) for 60 min. Negatively charged, citrate-capped AgNPs (-15 mV) of an average diameter of ~10 nm served as a nano-model due to their smaller size, wide use, and biocompatibility (trisodium citrate is heavily used as a food preservative and excipient pharmaceutical agent). It was found that approximately half of the administered AgNPs (~48 ± 5 %) interacted with RBCs, inhibited Rb+ influx (~50 %) and reduced the cellular K+ ion concentration (~15 %) of RBCs. Rb+ was utilized as a congener for Na+. These results are indicative of membrane damage and activation of a potentially highly active ion channel in the RBC membrane due to AgNPs exposure. Thus, citrate capped AgNPs may remain concentrated in the blood circulatory system and cause RBC toxicity despite their negative charge and biocompatible capping agent.
As a result of catastrophic nuclear accidents such as Fukushima and Chernobyl, the proliferation of actinides in the environment has become a major concern. Actinides, such as isotopes of uranium and thorium, can spread through the environment both naturally and because of anthropogenic activities, such as mining and smelting. Distributed actinides can be taken in by humans via ingestion from sources such as contaminated drinking water, or by inhalation, whether oral or nasal. Once ingested, actinides either exhibit chemical and/or radiological toxicity, e.g., 238U and 232Th mainly exhibit chemical toxicity. Potential non-radiological adverse effects include long-term accumulation in the skeleton and arrests in bone growth. A major area of concern is the teeth, since they are one of the first points of contact of actinides entering the body, and consequences of contact include delays in tooth eruption and development. The major mineral component of both bone and teeth is hydroxyapatite (HAP,) and this present work studies and models the influence of pH, contact time, initial metal concentration, and buffer solution on the uptake and removal of a long-lived actinide (238U) onto synthetic HAP. pH had negligible effects on the uptake of U, and the kinetics of uptake were extremely fast, as 98% of U was uptaken in one second of exposure. The uptake followed pseudo-second order kinetics and a Freundlich isotherm model. A 0.2 M sodium carbonate solution removed all the U from HAP after one hour. Another series of in vitro tests were performed with real teeth as targets. We found that for a 50 mg/L U in PBS solution adjusted to physiological pH, ~35% of the uranyl was uptaken by the tooth after one hour, following pseudo-first order kinetics. Among several washing solutions tested, a commercially available carbonate as well as a commercially available fluoride solution, enabled removal of all the uranyl taken up by the teeth. Finally, characterization of the interaction between U and the tooth surface was performed using FT-IR spectroscopy. Similar studies were also conducted using 232Th, and will be reported later.
Multiple regulatory agencies including the National Institute for Occupational Safety and Health (NIOSH), and the U.S. Environmental Protection Agency (EPA), have outlined the need to improve the risk assessment and control associated with the heavy use of nanomaterial-based consumer products. The Nanotechnology Product Database indicates that silver nanoparticles (AgNPs) are the main component of ~ 70% of the reported biomedical products. This work addresses this knowledge gap by combining CytoViva and Raman hyperspectral imaging to study the uptake, distribution, and toxicity of silver nanoparticles in human red blood cells. To achieve this, RBCs in 5% glucose solution were incubated at 37 °C, for 60 min, with 150 µg mL-1 of negatively charged, spherical AgNPs of an average diameter of ~ 20 nm, in the 10-35 nm size range. These citrate-capped AgNPs were selected as a nano-model due to their wide use, biocompatible capping agent, low cost, and simple fabrication. The physicochemical properties of AgNPs were characterized following the U.S. EPA recommendations after size-selecting, concentrating, and purifying them by tangential flow filtration (TFF) for improved homogeneity and reproducibility. CytoViva images showed that 48 ± 5 % of the total administered Ag was absorbed by cells and ~ 70% of the interacting AgNPs were present at the cell membrane. The deconvolution of the corresponding CytoViva hyperspectral data indicated changes in the content of RBC membrane spectrin (520 nm) and phospholipid (521 and 591 nm) components as a result of this interaction when compared to the negative controls. These toxicity effects of AgNPs were further confirmed through Raman hyperspectral imaging and a lipid peroxidation assay (two-fold increase in malondialdehyde). In addition, the spectral changes observed for the characteristic Raman vibrational modes of hemoglobin demonstrated the transformation of oxyhemoglobin (1226 cm-1) to deoxyhemoglobin (1214 cm-1) after AgNP exposure. Overall, the results of this study suggest that citrate-capped AgNPs may remain concentrated in the blood circulatory system despite their negative charge and biocompatible capping agent, and thereby raise major concerns with respect to the biomedical applications of AgNPs in humans.
Metabolism in the gastrointestinal (GI) space has many components that highly impact the orally administered drugs. We will capture the manner that we dissect various metabolic liabilities; from human-derived luminal content, microbiome to the gut wall. With species differences in metabolic liabilities from hydrolysis, reduction to oxidation. To recapitulate the in vivo gut metabolism and explain PK variabilities, multiple ex vivo microbiome platforms and screening tools are examined. In addition, we describe tool compounds and the standardized microbiome platform that allow us to benchmark assays and scale the contribution of metabolism to drug elimination.
With the improvement of analytical technologies, the Chemical Biology field now recognizes that the overwhelming majority of small molecule metabolites that are found in any organism remain unknown. It is believed that some of the uncharacterized metabolites from this so-called and reproducible ‘dark matter’ of the metabolome could regulate complex molecular interactions that significantly influence human health and disease. To explore this question more broadly, we highlight four novel metabolic pathways that we have characterized in the well-studied gut bacterium, E. coli. These include: 1) folate/monapterin pathway-derived pteridines that upregulate IL-10 in the host and reduce disease symptoms in a colitis mouse model; 2) indole-functionalized metabolites that regulate IL-6 in the host and are structurally conserved with products of the plant innate immune system; 3) pyrazinones that regulate bacterial virulence programs and IL-8 in the host; and 4) polyketide synthase-nonribosomal peptide synthetase hybrid products that crosslink DNA and are associated with colorectal cancer initiation. Based on these findings and other novel metabolic pathways that we are currently characterizing in E. coli, we argue that much remains to be discovered even in organisms that have been studied for generations.
Despite knowledge for over 10 years that P-glycoprotein(P-gp) plays a central role in GI homeostasis, the precise molecular mechanism that controls its regulation and function remains unclear. Since the resident microbiota contribute to tolerance and homeostasis, our studies address how the functional core microbiome governs intestinal homeostasis via maintenance of P-gp by uniting with a key pathway in xenobiotic metabolism, and how loss of this control leads to disease.
- 12:00pm USA / Canada - Eastern
- August 25, 2021
| Room: Virtual Room
Division: [TOXI] Division of Chemical Toxicology
Session Type: Networking Events - Virtual
Division/Committee: [TOXI] Division of Chemical Toxicology
Curious about the publishing process in an ACS journal and the best ways to interact with editors? Come to this workshop with members of the editorial board of Chemical Research in Toxicology and meet the editors! Geared toward early career researchers and open and applicable to everyone!
- 03:00pm USA / Canada - Eastern
- August 25, 2021
| Room: Virtual Room
Division: [TOXI] Division of Chemical Toxicology
Session Type: Networking Events - Virtual
Division/Committee: [TOXI] Division of Chemical Toxicology
We are hosting a panel of individuals from the toxicology field that represent industry, academia, and government positions. Each panel member will give a brief description of their career path and how mentoring has played a role in their trajectory. We will then open the discussion so that participants may ask questions to the panel members, which can focus on how to find mentors, establish relationships with colleagues, and investigate new career paths.