TOXI 1.
Harry B Gray
Electron flow through proteins
Abstract: Biological electron transfers often occur between metal-containing cofactors that are separated by very large molecular distances. Understanding the underlying physics and chemistry of these electron transfer processes is the goal of much of the work in my laboratory. Employing laser flash-quench triggering methods, my coworkers and I have shown that 2-nm, coupling-limited Fe(II) to Ru(III) and Cu(I) to Ru(III) electron tunneling reactions in Ru-modified cytochromes and blue copper proteins occur on microsecond to nanosecond timescales. Redox equivalents can be transferred even longer distances by multistep tunneling (called hopping) through intervening tyrosines and tryptophans: notably, in our work on cytochrome P450 and azurin, we have found that long-range hole hopping through intervening tryptophans can be orders of magnitude faster than single-step tunneling. Water interactions with tryptophan radical cation intermediates play key roles in promoting these hole hopping processes.
TOXI 2.
Lawrence J Marnett
Competition between repair and oxidation of the endogenous DNA adduct, 3(2-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M1dG)
Abstract: M1dG is a major endogenous DNA adduct formed by reaction of deoxyguanosine residues with oxopropenylating agents derived from DNA and lipid oxidation. It is detectable in human genomic DNA and its levels in mitochondrial DNA are significantly higher than in genomic DNA. M1dG induces deletions as well as transitions to A and transversions to T when it is replicated in bacterial or mammalian cells. M1dG is repaired by nucleotide excision repair and appears to be recognized by proteins involved in mismatch repair. Our laboratory discovered the existence of an enzymatic activity in the nucleus that oxidizes M1dG to a 6-oxo-derivative. The kinetics of oxidation of M1dG in the genome suggest it occurs more rapidly than nucleotide excision repair. 6-Oxo-M1dG was incorporated into oligonucleotides by chemical synthesis and annealed to primers for in vitro replication. The presence of this adduct blocked DNA replication by the translesion polymerases, eta, kappa and iota to varying extents. The chemical properties of 6-oxo-M1dG and its ability to block DNA replication suggest it is more genotoxic and mutagenic than M1dG. The competition between nucleotide excision repair and genomic oxidation of M1dG may be a major determinant of mutagenic consequences of its in vivo occurrence.
TOXI 3.
Irene Chiolo
Highways for repair of heterochromatic DNA breaks
Abstract: Heterochromatin mainly comprises repeated DNA sequences that are prone to ectopic recombination. In Drosophila cells, ‘safe’ repair of heterochromatic double-strand breaks by homologous recombination relies on the relocalization of repair sites to the nuclear periphery before strand invasion. The mechanisms responsible for this movement were unknown. We discovered that relocalization occurs by directed motion along nuclear actin filaments assembled at repair sites by the Arp2/3 complex. Relocalization requires nuclear myosins associated with the heterochromatin repair complex Smc5/6 and the myosin activator Unc45, which is recruited to repair sites by Smc5/6. Similar components drive relocalization of heterochromatic repair sites in mouse cells, revealing conserved pathways. Defects in this pathway result in impaired heterochromatin repair and chromosome rearrangements. These findings identify de novo nuclear actin filaments and myosins as effectors of chromatin dynamics for heterochromatin repair and stability in multicellular eukaryotes.
TOXI 4.
John S Termini
Hyperglycemia induced DNA damage and inhibition of DNA repair: Potential mechanistic link between diabetes and increased cancer risk
Abstract: Diabetes (Type 1 and Type 2) is significantly associated with an increase in all-site cancers, yet how diabetes impacts cancer susceptibility is incompletely understood. Mechanistic hypotheses linking diabetes and cancer have invoked the mitogenic and anti-apoptotic actions of excess insulin and insulin-like growth factor 1 (IGF-1), increased adipokine secretion from fatty tissue, and steroid hormone dysregulation. However, since genomic instability plays a significant role in the initiation and promotion of cancer, we have focused on defining the mechanisms by which diabetes associated metabolic dysfunction contributes to DNA damage and reduced DNA repair. We propose that hyperglycemia-induced DNA damage and inhibition of DNA repair represent important pathological complications of diabetes which exacerbate genomic instability. We will show using tissue culture and diabetic animal models that elevated glucose significantly increases the levels of the DNA adduct N2-(1-carboxyethyl)-2’-deoxyguanosine (CEdG) and it’s RNA analog CEG, and that these adducts are significantly associated with diabetes and diabetic complications. Chronic exposure to elevated glucose also increases DNA strand breaks and inhibits nucleotide excision repair (NER), which is required for removal of CEdG from DNA. Inhibition of NER occurs at the level of gene expression due to metabolism induced destabilization of HIF1, since many genes of the NER pathway are inducible by this transcription factor. Inhibition also occurs at the level of mTORC1 regulated translation of HIF1, since DNA damage induces REDD1 (Regulated in Development and DNA Damage responses 1), which dephosphorylates Akt and attenuates mTOR signaling. Since pharmacological agents which stabilize HIF1 or increase translation by mTOR have been described, this raises the possibility of therapeutic intervention to enhanced DNA repair, limit genomic instability, and potentially reduce cancer risk associated with diabetes.
TOXI 5.
Yilun Liu
SUMO2 conjugation of PCNA facilitates chromatin remodeling to resolve transcription-replication conflicts
Abstract: During DNA synthesis, DNA replication and transcription machinery can collide, and the replication fork may temporarily dislodge RNA polymerase II (RNAPII) to resolve the transcription-replication conflict (TRC), a major source of endogenous DNA double-strand breaks (DSBs) and common fragile site (CFS) instability. However, the mechanism of TRC resolution remains unclear. I will present our recent work that reveals the mechanistic insight into how RNAPII is dislodged from the chromatin during TRC to avoid DSBs. We show that conjugation of SUMO2, but not SUMO1 or SUMO3, to the essential replication factor PCNA is induced on transcribed chromatin by the RNAPII-bound helicase RECQ5, which suppresses transcription-associated DSBs and acts as a tumor suppressor. Proteomic analysis reveals that SUMO2-PCNA enriches histone chaperones CAF1 and FACT in the replication complex via interactions with their SUMO-interacting motifs. SUMO2-PCNA enhances CAF1-dependent histone deposition, which correlates with increased histone H3.1 at CFSs and repressive histone marks in the chromatin to reduce chromatin accessibility. Hence, SUMO2-PCNA dislodges RNAPII at CFSs, and overexpressing either SUMO2-PCNA or CAF1 reduces the incidence of DSBs in TRC-prone RECQ5-deficient cells.
TOXI 6.
Lawrence J Marnett
Histone modification by bifunctional electrophiles derived from lipid and carbohydrate metabolism
Abstract: Oxidation metabolism generates a multitude of products including bifunctional electrophiles. We have developed a global strategy for identifying the protein targets of bifunctional electrophiles using alkyne-tagged electrophiles and click chemistry to attach biotin linkers to adducted proteins post-hoc. Quantifying peptide counts from digests of streptavidin-enriched protein mixtures identifies the most reactive proteins among the many that bear adducts. Bioinformatic analysis of these data sets and integration with microarray or RNA-seq data sets from treated cells enables construction of networks relating protein modification, signal transduction and transcriptional activation/inhibition. This identified key pathways of cellular response to lipid electrophiles including protein targets triggering transcriptional changes. An important class of targets of modification by bifunctional electrophiles are histones, the major structural proteins of chromatin and important sites of epigenetic regulation. We have developed a quantitative method for analysis of arginine and lysine modification (QuaRKMod), which reveals many modifications beyond acetyllysine and trimethyllysine including modifications by the bifunctional electrophiles, 4-oxo-nonenal and methylglyoxal. Application of QuaRKMod to histone samples from basal cells or cells stimulated to carry out oxidative metabolism reveals the presence of histone adducts at levels comparable to canonical adducts that regulate gene expression. Adducts from bifunctional electrophiles may link metabolism and epigenetic regulation.
TOXI 7.
Ian A Blair
Interactions of aldehydic bifunctional electrophiles and high mobility group box-1 with RAGE
Abstract: The innate immune system forms the first line of protection against infectious and non-infectious tissue injury. Cells of the innate immune system detect pathogen-associated molecular patterns. They also protect against endogenous molecules such as 4-hydroxy-2-nonenal and other α-, β-unsaturated aldehydes that are released as a result of lipid hydroperoxide decomposition during tissue injury or inflammation or the high mobility group box-1 (HMGB1) protein that is released. The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor predominantly involved in the recognition of endogenous molecules released in the context of infection, physiological stress or chronic inflammation. HMGB1 is the prototypic damage associate molecular pattern (DAMP) molecule involved in RAGE activation. It also binds avidly to platinum DNA-adducts and inhibits repair of the platinum-adducts. We have now discovered that platinum drugs secrete HMGB1 from epithelial cells and that a therapeutic concentration of cisplatin is much more potent (EC50 of 10.8 µM) than oxaliplatin at stimulating HMGB1 secretion. In contrast, carboplatin does not secrete HMGB1 from epithelial cells at therapeutic concentrations. Secretion of HMGB1 is thought to be mediated through acetylation of the two nuclear localization signals (NLSs). Cisplatin-mediated HMGB1 secretion in vivo could prime the immune system by activating RAGE. Using a novel method developed recently, which we have called stable isotope labeling of proteomes (SILAPS), no HMGB1 acetylation expected on NLS1 or NLS2 was detected. However, SILAPS revealed the HMGB1 proteoforms present in plasma after cisplatin therapy, suggesting a novel secretory mechanism. Supported by NIH Grants P42ES023720 and P30ES013508.
TOXI 8.
Vasilis Vasilou
Aldehyde dehydrogenases: From metabolic and alcohol-related diseases to cancer stem cells
Abstract: The aldehyde dehydrogenase (ALDH) superfamily contains NAD(P)+-dependent enzymes that metabolize a wide spectrum of aldehydes to their corresponding carboxylic acids. The human genome encodes nineteen ALDH isozymes. Missense mutations in ALDH genes are the molecular basis of several metabolic diseases, including Sjögren–Larsson syndrome (SLS), type II hyperprolinemia, γ-hydroxybutyric aciduria, pyridoxine-dependent seizures, De Barsy syndrome and spastic paraplegia, as well as alcohol-related diseases and late-onset Alzheimer’s disease. In addition to these identified clinical phenotypes, transgenic mouse models suggest crucial roles for ALDH isozymes in physiological processes, such as embryogenesis and development, and in protection against obesity and environmentally-induced oxidative damage. ALDHs also appear to be involved in the biology of both normal stem cells and tumor-initiating stem cells. In tumors, ALDHs influence tumor growth and resistance to chemotherapy and radiation. In summary, these observations suggest that ALDHs may play pivotal roles in physiology and pathophysiology by metabolizing endogenous and exogenous aldehydes that are either toxic or essential for life.
TOXI 9.
John D Hayes
Aldo-keto reductases and NRF2
Abstract: The aldo-keto reductase (AKR) supergene family encodes enzymes that are collectively involved in the metabolism of endogenous and exogenous carbonyl-containing compounds. Some of the AKR isoenzymes are involved in the metabolism of carcinogenic xenobiotics such as aflatoxin B1 and polycyclic aromatic hydrocarbons. As might be expected of genes involved in xenobiotic metabolism, AKR isoenzymes contribute to adaptation to environmental stress because they can be induced through the presence of cis-acting elements in their regulatory regions that recruit various transcription factors that are activated by environmental agents. One of these is NF-E2 p45-related factor 2 (NRF2, encoded by NFE2L2), a cap’n’collar basic-region leucine zipper transcription factor, which a regarded as a master regulator of redox homeostasis. NRF2 binds to antioxidant responsive element (ARE, 5′-TGACNNNGC-3′) sequences in its target genes upon exposure to oxidative stressors, including xenobiotics that contain reactive carbonyl and quinone moieties and likely serve as AKR substrates. The ability of NRF2 to induce AKR genes is not a consequence of activation of itself, but rather it entails inactivation of its repressor protein Kelch-like ECH-associated protein 1 (KEAP1), which is a E3 ubiquitin ligase substrate adaptor that enables cullin-3 to target NRF2 for proteasomal degradation, resulting from modification by inducing agents of Cys residues in KEAP1. Remarkably, although it is well known that NRF2 regulates enzymes that are intimately involved in maintaining glutathione- and thioredoxin-dependent antioxidant systems, the most inducible NRF2-target genes upon pharmacological inactivation of KEAP1 or genetic knockdown of KEAP1 are AKR1B10, AKR1C1, AKR1C2 and AKR1C3. There is therefore particular interest in how induction of these enzymes contributes to adaptation to oxidative stress. More recently, it has become apparent from work of The Cancer Genome Atlas consortium that NRF2 is constitutively active in many malignant diseases as a consequence of somatic mutations in KEAP1or in NFE2L2. Somatic mutations in KEAP1or NFE2L2 are most obvious in non-small cell lung cancer where NRF2 is constitutively activated in approximately 35% of cases. Somatic mutations in KEAP1/NFE2L2 are associated with constitutive upregulation of AKR1B10 and AKR1C family members, which raises the question of whether AKR isoenzymes contribute to the process of tumourigenesis in certain cancers.
TOXI 10.
Trevor M Penning
Role of human aldo-keto reductases (AKRs) in hormonal & chemical carcinogenesis
Abstract: Aldo-Keto Reductases (AKRs) are a gene and protein superfamily whose members are expressed in all phyla. AKRs are principally, monomeric (37 kDa), cytosolic NAD(P)(H) oxidoreductases that catalyze the reduction of aldehydes and ketones to primary and secondary alcohols for conjugation reactions and thus can be called phase I enzymes. There are more than 173 members that fall into more than 15 families with broad substrate specificity ranging from sugar and lipid aldehydes, steroid hormones, prostaglandins, chemical carcinogens to carbonyl containing drugs (visit www.upenn.edu/akr). AKRs are also stress response genes, and because they arose early in evolution, they are often induced by primordial signals e.g. osmotic stress, electrophilic stress, oxidative stress and heat shock. There are 15 human AKRs and our focus has been on the structure-function of these enzymes and their roles in steroid hormone transformation and chemical carcinogenesis. Our studies show that the repertoire of reactions catalyzed by these enzymes can be extended to steroid double-bond reduction and nitroreduction. Human AKR1C enzymes are involved in the regulation of ligands for steroid hormone receptors, a process referred to as the pre-receptor regulation of steroid hormone action. In this context the role of AKR1C3 in the formation of potent androgens in castration resistant prostate cancer and its therapeutic targeting will be discussed. Human AKR1C enzymes are also involved in the metabolic activation of NO2-polycyclic aromatic hydrocarbons (PAH). In this context their role in the metabolic activation of 3-nitrobenzanthrone and their induction by Nrf2 will be described. In the context of NO2-PAH exposure Nrf2 activation may contribute to cancer initiation revealing a hitherto unrecognized dark-side to Nrf2 signaling. [Supported by P30-ES013508 and R01-ES029294]
TOXI 11.
Jenna Fernandez
Epigenetic changes in alveolar type II lung cells of A/J mice following exposure to cigarette smoke and LPS
Abstract: 5-Methylcytosine (mC) is a stable epigenetic modification of DNA that plays a key role in controlling gene expression. Epigenetic alterations such as aberrant cytosine methylation and changes in gene expression patterns are increasingly recognized as critical events in the development of cancer. The removal of DNA methylation marks is mediated by ten eleven translocation (Tet) dioxygenases which iteratively oxidize the methyl group to give 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC). The epigenome is dynamic and responsive to external stimuli. Environmental factors such as diet, lifestyle choices, and behaviors can cause changes to the epigenome and thus effect changes in gene expression. Smoking-induced lung tumors exhibit profound epigenetic alterations, aberrant patterns of gene expression, and genetic instability. Chronic obstructive pulmonary disease (COPD) is a major risk factor for lung cancer development in smokers. Cigarette smoke contains endotoxin lipopolysaccharide (LPS), which causes COPD-like symptoms in laboratory animals. Our laboratory has shown that inhalation exposure to LPS induces early global and loci-specific epigenetic changes in Type II alveolar epithelial cells analogous to those observed in lung tumors. We hypothesize that chronic inflammation leads to changes in epigenetic marks in DNA and aberrant gene expression patterns, contributing to cancer development. In present work, we investigated the epigenetic effects of smoking and inflammation in the A/J mouse model of lung cancer. Mice were exposed to cigarette smoke or inflammatory agent LPS for 3-10 weeks. The reversibility of epigenetic changes was studied by including post-exposure groups. Tissue specific epigenetic responses were investigated by selectively sorting for Type II alveolar epithelial cells. Global levels of DNA epigenetic modifications were quantified using mass spectrometry, while loci-specific methylation and hydroxymethylation was studied by oxidized bisulfite sequencing. Gene expression levels were assessed by RNA-seq, and changes in protein abundance were studied using mass spectrometry based proteomics. Our results indicate that smoking and inflammation have a profound effect on DNA epigenetic marks and the levels of gene expression in Type II alveolar epithelial cells, which serve as precursors to pulmonary adenocarcinoma.
TOXI 12.
Immaculate Sappy | Mel Fernand Bedi
Oxidation of RNA at naturally occurring modifications
Abstract: Among nucleic acid modifications, pseudouridine (), the 5-ribosyl isomer of uridine(U) is the most abundant and is found in all domains of life and all types of RNA. There is evidence that RNA oxidation is a major player in the pathogenesis of Alzheimer’s disease (AD)and other neurodegenerative disorders. In addition, a correlation has been observed between high urinary levels of pseudouridine and oxidative stress in AD. In order to develop methods to qualitatively and quantitatively study the role that modified nucleic acids such as pseudouridine play in oxidative stress, methods are needed to facilitate the routine synthesis of oxidized pseudouridine damaged products and to analyze their degradation products. Here we present the synthesis of a radical precursor to investigate the fate of RNA damage emanating from hydrogen atom abstraction at the C5’ of pseudouridine. The precursor was photochemically converted to the C5’-radical and the resulting products were identified using LCMS and NMR.
TOXI 13.
Elizabeth R Lotsof
DNA glycosylase NEIL1 demonstrates lesion specificity from RNA editing
Abstract: Environmental toxins and toxicants can produce conditions of oxidative stress resulting in the formation of reactive oxygen and nitrogen species (RONS). The presence of RONS causes the oxidation of all four conical bases creating a diverse number of lesions including: thymine glycol (Tg), 5-hydroxycytosine (5-OHC), 5-hydroxyuracil (5-OHU), dihydrothymine (DHT), the formamidopyridines (FapyA and FapyG), guanidinohydantoin (Gh), and spiriminodihydatoin (Sp). The DNA glycosylase, NEIL1, is able to excise all of these lesions from several DNA contexts. Additionally, the pre-mRNA of NEIL1 is subject to modification by the Adenosine Deaminase Acting on RNA (ADAR1) that leads to a recoding event that converts a lysine to arginine in the lesion recognition loop of NEIL1. This leads to the presence of two isoforms of NEIL1 under different cellular conditions. Notably, the two isoforms display different enzymatic properties on Tg, where the unedited (K242) isoform showed a significantly faster rate of excision compared to edited NEIL1 (R242). We have performed detailed examinations of lesion processing by the two NEIL1 isoforms on a large number of substrates, and many interesting trends have emerged. In general, unedited NEIL1 demonstrates better excision of oxidized pyrimidines than the edited isoform. The impact of RNA editing on NEIL1 activity suggests a unique regulatory mechanism for DNA repair.
TOXI 14.
Judy Zhu
Sequencing for 8-oxo-7,8-dihydroguanine in a mammalian genome before and after oxidative stress
Abstract: During oxidative stress, reactive oxygen species (ROS) can cause oxidation of DNA resulting in mutagenic products; one such product is 8-oxo-7,8-dihydroguanine (OG) that can cause GT transversion mutations. Inadequate repair of OG in a cell can impact cellular function and lead to disease. While OG in the genome has been considered mutagenic, recently, OG has been found to play a regulatory role. To better understand the role of OG, sequencing a genome for the modification is imperative. The location of OG in a genome, whether randomly distributed or in specific regions will provide much needed data to understand the mutagenic versus regulatory role of this modification. Herein, methods for sequencing OG are described that provide various insights to OG genomic distributions. One of the methods harnesses the lability of OG toward further oxidation for chemical ligation to biotin followed by streptavidin purification to enrich a fragmented genome for those fragments that contain OG. The biotin-ligated OG yields a characteristic mutation signature during library preparation that identifies the location of OG during sequencing. Conceptually, this is analogous to bisulfite sequencing for 5-methylcytosine. The various OG sequencing methods will be compared in order to study inflammation-derived oxidative stress on a mammalian cell line resulting in the writing of OG in the genome. The distribution of OG and how it changes as a function of oxidative stress will provide insights necessary to reveal the mutagenic versus regulatory aspects of OG.
TOXI 15.
Prabha Ranasinghe
Embryonic exposure to 2, 2′, 3, 5′, 6 polychlorinated biphenyls (PCB-95) alters GABAgenic and antioxidant transcriptome in zebrafish
Abstract: Developmental exposure to 2, 2’, 3, 5’, 6 polychlorinated biphenyls (PCB-95) influences neurodevelopmental disorders in an organism. Most invoked mechanism of PCB-95 induced neurotoxicity is through targeting and modifying ryanodine receptors (RyRs) and thereby misbalancing Ca2+ homeostasis in a cell. However, there are likely several other mechanisms that can modulate PCB oriented neurological dysfunctions. We propose that GABAgenic neurotransmitter profile as one plausible target. During development, GABA acts as an excitatory neurotrophic factor that contributes to cellular processes such as cell proliferation and migration, axonal growth, and synaptogenesis. So far little is known about the effects of embryonic PCB-95exposure and GABAgenic system. Also, we suggest that neurotoxic effects can be characterized by increasing damage of brain cells, leading to cell death with PCB-95 exposure. To characterize the effects, zebrafish embryos with intact chorions were exposed to different concentrations of PCB-95 (0.25, 0.5, 0.75 and 1ppm) with two controls (E2 solution and the 0.1 % DMSO). Gross morphological alterations, tissue up-take, neurotransmitter levels were analyzed. Different GABA related genes such as GABA (A) receptors, GABA transporters (Gat-1, Gat-3 and V-gat) and glutamic acid decarboxylase (GAD-2) were analyzed along with genes encoding antioxidant proteins such as Nrf2, Cu/Zn-superoxide dismutase (Cu/Zn-SOD), manganese superoxide dismutase (Mn-SOD), catalase (CAT), and glutathione peroxidase (GPx). Results suggested that the embryonic exposure to PCB-95 significantly induced alterations in morphology, neurotransmitter levels and the gene expressions. These findings demonstrate, that embryonic PCB-95 exposure alters GABA expressions and induces neuronal cell death which may change neuronal cell pattering and migration; thus, resulting in neurodevelopment disorders.
TOXI 16.
Jessica R Murray
Nrf2 signaling increases bioactivation of the mutagenic air pollutant 3-nitrobenzanthrone
Abstract: 3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen detected in diesel exhaust particulate and ambient air pollution. It requires metabolic activation via nitroreduction to promote DNA adduct formation and tumorigenesis. NAD(P)H:quinone oxidoreductase 1 (NQO1) has been implicated as the major nitroreductase responsible for 3-NBA activation. We investigated the roles of human aldo-keto reductases (AKR1C1-1C3) in 3-NBA reduction and found that catalytic efficiencies (kcat/KM) for AKR1C1, AKR1C3, and NQO1 were equivalent. We also determined that AKR1C1-1C3 and NQO1 contribute equally to the nitroreduction of 3-NBA in lung epithelial cell lines (A549 and HBEC3-KT) and combined they represent at least 50% of the intracellular nitroreductase activity towards 3-NBA. These enzymes are induced by Nrf2 signaling which raises the question whether Nrf2 activation as a chemopreventive strategy may exacerbate 3-NBA toxification. To evaluate the role of Nrf2 signaling on nitroarene activation, we tested the effects of Nrf2 inducers (e.g. sulforaphane, synthetic triterpenoids) in human bronchial epithelial cells (HBEC3-KT). Since A549 cells have constitutively active Nrf2 signaling due to a KEAP1 mutation, we examined the effect of heterozygous (+/-) and homozygous (-/-) Nrf2 knockout (KO) by CRISPR-Cas9 gene editing. Upregulation of AKR1C1-3 and NQO1 by Nrf2 inducers and downregulation by CRISPR-Cas9 KO was confirmed and quantified by qPCR, immunoblots, and enzyme activity assays. We observed 40-60% increases in 3-NBA bioactivation due to Nrf2 inducers in HBEC3-KT cells and a reduction of 3-NBA activation in the A549 Nrf2 heterozygous and homozygous KO cell lines (53% to 82% reduction, respectively). Enhanced 3-NBA metabolic activation due to Nrf2 activity may lead to an increase in DNA adduct burden which would promote mutagenesis. Interestingly, we did not find significant protection against cytotoxicity due to Nrf2 activators which questions whether Nrf2 induction has any protective effect during 3-NBA exposures. Nrf2 signaling is considered protective against cancer initiation despite the well-recognized dark side of Nrf2 in cancer promotion and progression. Given these data, it may be appropriate to explore whether Nrf2 activation plays a role in cancer initiation in certain exposure contexts (i.e. diesel exhaust).
TOXI 17.
Kurt Housh
Interstrand cross-links at strand breaks derived from abasic sites in duplex DNA
Abstract: Abasic (Ap) sites are one of the most common lesions in DNA. In biological systems, Ap sites can undergo either spontaneous or amine-catalyzed strand cleavage to generate an electrophilic alpha, beta unsaturated aldehyde (alkenal) at the 3’-terminus of the strand break. We will present evidence that spermine-mediated strand cleavage at an Ap site in duplex DNA can result in the formation an interstrand cross-link adjacent to the strand break via reaction of the 3′-alkenal unit with aguanine residue on the opposing strand. This chemistry may be biologically significant because interstrand cross-links can be cytotoxic, mutagenic, or induce genomic instability.
TOXI 18.
Jiabin Wu
Replicative bypass and mutagenic properties of alkylphosphotriester lesions in Escherichia coli
Abstract: Replicative bypass and mutagenic properties of alkylphosphotriester lesions in Escherichia coli Jiabin Wu1, Jun Yuan1 and Yinsheng Wang1,2 1Environmental Toxicology Graduate Program, 2Department of Chemistry, University of California, Riverside, CA92521 Endogenous metabolism, environmental exposure, and treatment with some cancer chemotherapeutic agents can give rise to DNA alkylation, which can occur on the phosphate backbone, forming alkylphosphotriester (PTE) lesions. Among the alkylated DNA lesions, PTEs were previously shown to display the longest half-life in cells, suggesting that they could be used as biomarkers for monitoring the long-term exposure to alkylating reagents and they are resistant to DNA repair. Recent research also showed that tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), could be activated to form alkyl-PTE lesions in rat tissues. Our previous study revealed the influence of alkyl group size and stereochemistry of the phosphotriester on the replication and repair of alkyl-PTE lesions. It, however, remains elusive about the impact of flanking base sequences around PTE site on cellular DNA replication. In the present study, we synthesized oligodeoxyribonucleotides (ODNs) containing a site-specifically inserted methyl-PTE lesion, with different flanking base combinations (AT, CT, GT, TA, TC and TG). We also characterized the identities and purities of these ODNs by using LC-MS/MS analysis. Moreover, we assessed how these lesions inhibit DNA replication and induce mutations in Escherichia coli cells. We found that the three XT Sp-methylphosphotriesters were not strong impediments to DNA replication and replicative bypass of these lesions were mutagenic. Similar as what we observed previously for the Sp-T(Me)T phosphotriester, mutagenic bypass of these lesions requires Ada protein. On the other hand, the three Rp-methylphosphotriesters at XT site and all TX methylphosphotriesters moderately blocked DNA replication in E. coli, with no mutagenic products being detected.
TOXI 19.
Arnold Groehler
Development of an ultra performance liquid chromatography-mass spectrometry (UPLC-MS) assay for the quantification of cisplatin-Induced DNA intra- and interstrand cross-links
Abstract: Cisplatin is a platinum-based chemotherapy drug used in the treatment of testicular, ovarian, cervical, lung, and breast cancer. Despite its clinical utility, development of resistance and toxic side effects remain big challenges associated with cisplatin therapy. Potential mechanisms of resistance include changes in drug import and efflux, cisplatin metabolism/activation, and repair rates of cisplatin-induced DNA damage. All three resistance mechanisms influence the abundance of DNA cross-links, including 1,2- and 1,3-intrastrand and interstrand cross-links. Therefore, we hypothesize that a patient’s response to cisplatin can be accurately predicted by measuring the formation and repair of these cisplatin DNA cross-links in tumors. To address this goal, we have developed an isotope dilution UPLC-MS assay to quantify both cisplatin-induced intra- and interstrand crosslinks. The DNA digestion and monomer enrichment methodology was first optimized using synthetic DNA oligos and plasmids containing specific cross-links, followed by validating the assay in cisplatin-treated cell lines. We are currently investigating the formation and repair kinetics of each lesion in cell deficient in nucleotide excision repair (addressing intrastrand crosslinks) and ICL-repair (addressing interstrand crosslinks) to investigate synergism between repair pathways in counteracting cisplatin therapy. Successful application of this assay in cells is expected to be useful in clinical trials and practice to predict the outcome of cisplatin therapy.
TOXI 20.
Morwena Jane Solivio
High-resolution/accurate-mass DNA adductomics to identify adducts formed by the hypoxia-activated alkylating agent, CP-506 and its metabolites
Abstract: Many chemotherapeutic drugs exert their cytotoxic activity through the formation of mono- or bifunctional DNA modifications (adducts), which can interfere with replication, an over-active process in rapidly dividing cancer cells. Hypoxia is a distinct characteristic of solid tumors, resulting from an imbalance between the cellular oxygen consumption rate and the tumor-oxygen supply. To selectively target tumor cells, the pro-drug CP-506 was designed to convert into strong alkylating agents under hypoxic environments. The goal is to demonstrate proof of concept, verifying CP-506 selectivity to hypoxic tumor cells. The initial work focused on adduct discovery, involving exposure of purified calf thymus DNA with CP-506 or its metabolites, followed by enzymatic digestion of the DNA into its nucleosides, adduct enrichment, and identification using high-resolution/accurate-mass (HR/AM) LC-MS3 targeted and untargeted adductomic analyses. This strategy is based on data-dependent MS2 fragmentation of the most abundant ions in the full scan. The neutral loss of the deoxyribose moiety or any of the four DNA bases in the MS2 spectra triggers MS3 fragmentation, revealing adduct identity. Validation of the identified adducts was conducted using internal standards using similarly exposed15N-labeled DNA. A targeted adductomics approach was then developed for detection of previously identified adducts in CP-506-treated cancer cells, under normoxic and hypoxic conditions to ascertain specificity. Cell exposure to CP-506 resulted to formation of various adducts with the pro-drug’s active metabolites including cross-links, only on hypoxic cancer cells. We are currently looking into off-target specimens by conducting analyses on blood samples from treated animal models. Additionally, further efficacy and specificity analyses are being conducted on tumor biopsies obtained from animal models treated with CP-506. Future work will be focused on exploring these adducts’ potential as biomarkers of therapeutic potency, to be used to select the ideal target population for a precision, patient-oriented, and personalized chemotherapeutic approach.
TOXI 21.
Yupeng Li
Identification of a new N’-nitrosonornicotine (NNN)-specific DNA adduct N6-((5-(3-pyridyl)tetrahydrofuran-2-yl)-2′-deoxyadenosine in rat liver
Abstract: N’-Nitrosonornicotine (1, NNN) has been evaluated extensively as a strong carcinogen in multiple animal models and is classified, together with the related compound 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), as a Group 1 human carcinogen by IARC. Upon metabolic activation by cytochrome P450s, NNN is oxidized to reactive electrophiles, such as a diazonium ion from 2′-hydroxylation or a diazonium ion and an oxonium ion (2) from 5′-hydroxylation, which alkylate DNA to form phosphate adducts and base adducts. A previous study from our laboratory quantified the formation of an NNN-specific base adduct, 2-(2-(3-pyridyl)-N-pyrrolidinyl)-2′-deoxyinosine (py-py-dI), in rat tissues after NaBH3CN reduction, which proceeded through the diazonium ion pathway of 5′-hydroxylation. However, this adduct was not detected in the human lung tissue. To further investigate new NNN-specific DNA adducts that have potential as biomarkers, a comprehensive study was performed using a highly sensitive and selective LC-NSI-HRMS/MS method. After confirming the existence of previously observed DNA base adducts formed by NNN in vitro and in vivo, a new adduct N6-((5-(3-pyridyl)tetrahydrofuran-2-yl)-2′-deoxyadenosine (3, Py-THF-dAdo) was detected for the first time in vivo at relatively high level when compared with py-py-dI. Its structure was further confirmed by comparison with a synthetic standard. To the best of our knowledge, this is the first DNA adduct identified in rat liver tissues that forms directly through the oxonium ion pathway of NNN metabolism. The relative stability and high abundance of Py-THF-dAdo suggest it could be a promising activation biomarker of NNN. Mass spectrometric quantitation of Py-THF-dAdo by stable isotope dilution in the rat tissues is currently in progress.
TOXI 22.
Mark Fielden
Strategies for early safety assessment of potential drug candidates at Amgen and lessons learned
Abstract: Drug discovery requires an integrated assessment and optimization of multiple parameters, including pharmacodynamics, pharmacokinetics and toxicological profiles, in order to identify a clinical candidate that is predicted to achieve the desired clinical efficacy and safety profile. At Amgen, safety assessment of novel targets and drugs starts early in order to fail fast and avoid spending resources on targets or molecules that have a low probability of success. At the early stages of target identification, a theoretical assessment of the putative liabilities related to modulating the target and closely related off-targets is conducted. This can help prioritize targets and guide early experimental strategies to assess putative liabilities that may limit development. Following high-throughput screening, in vitro predictive safety assays are used in conjunction with potency and ADME properties to help prioritize promising lead scaffolds for further optimization. As drug-like properties improve during optimization, in vitro and in vivo safety properties are further characterized to identify safety issues that will guide subsequent screening strategies at later stages of lead optimization. The suite of predictive safety assays and in vivo toxicology studies that are used to guide clinical candidate selection are intended to characterize and predict human safety, identify maximum tolerated doses and establish suitable safety margins to guide decision making and designs of future FIH-enabling GLP toxicology studies. Understanding the clinical study design, patient population and competitive landscape can help inform an efficient screening strategy to balance speed to the clinic with appropriate risk taking to avoid safety-based attrition at later stages of development. Different strategies will be presented to illustrate how the in vitro and in vivo toxicological data can be integrated into an efficient testing paradigm to rapidly screen out undesired compounds, and to speed up the identification of clinical candidates.
TOXI 23.
Joseph R Piccotti
Early safety assessment strategies for drug discovery at Celgene and lessons learned
Abstract: The overarching goal of safety assessment during drug discovery is to provide an understanding of a drug candidate’s liabilities prior to its advancement into Good Laboratory Practice (GLP) toxicology studies and clinical evaluation. This in turn is meant to shift attrition of compounds early in discovery and thus reduce failures at later stages of development due to safety issues. The tools for early safety evaluation have evolved over the past decades and include in silico approaches, such as integrated target safety assessment (TSA), and predictive and mechanistic in vitro and in vivo studies. A TSA is a white paper assessment of safety liabilities and includes proposed plans to better identify and understand risks during exploratory toxicology testing and beyond. Routine in vitro studies typically include assessment of general cytotoxicity, hERG liability, genotoxicity and effects on kinases and secondary pharmacology targets. More complex assays including 3D and organoid systems also are considered, although the utility of these approaches in predictive toxicity testing remains to be determined. In vivo exploratory toxicity studies are conducted in both rodent and non-rodent species. These studies are normally robust in the parameters evaluated, but are limited by the number of animals used; thus, restricting their use to hazard identification. More routinely now, pharmacodynamic endpoints are included in exploratory toxicology studies to demonstrate species relevance, help establish a therapeutic index and/or test putative clinical biomarkers. In this talk, an overview of early safety assessment at Celgene will be discussed, along with some case studies and insights gained from our testing strategy.
TOXI 24.
Russell Naven
Strategies towards the design of safer compounds at Takeda and lessons learned
Abstract: The early and costly attrition of candidate drugs for safety reasons, coupled with the rapid ascent and attractiveness of biologics and new modalities, elevates the importance of proactively engineering safety into the design of small molecules. At Takeda, the Chemical Toxicology Group is not only tasked with providing project teams with an assessment of toxicological risk for the chemical space within which they reside, but to influence the direction of drug design so that safety liabilities are mitigated prior to compound nomination. This is made possible through the application of an evidence-based safety screening strategy that has been validated using compound test sets that are mechanistically relevant to the toxicological endpoint being modelled and which are applicable to modern chemical space. It has been learnt that the traditional validation datasets of new assays, which often contain toxicologically potent drugs such as doxorubicin, cisplatin and sunitinb, can be useful in that they provide information on the clinical and human relevance of an assay. Often, however, they do not provide sufficient evidence, on their own, to warrant the inclusion of an assay in a predictive, risk assessment paradigm. Utility and ‘added value’ can be shown by demonstrating that an assay can differentiate the toxicological potencies of structurally-similar compounds from the same project; thus reflecting the situation that is often encountered in early drug discovery. Such robust validation studies provide the valuable evidence that helps drive the ‘Safety By Design’ concept and the development of cost-effective, risk assessment strategies. The current early safety screening strategy at Takeda will be discussed along with the validation of the component assays and its application to two recent small molecule projects.
TOXI 25.
Kevin James Coe
Reactive metabolism strategy applied to drug safety assessment: Focus on risk prevention
Abstract: Drug safety assessment, particularly as it pertains to liver, the organ responsible for the metabolism and elimination of drugs, involves a comprehensive analysis of multiple end-points to identify potential risk factors in the development of drug candidates. Given an established association between the formation of reactive metabolites and liver injury, one commonly employed safety end-point in Pharma is the detection of reactive metabolites formed from in vitro incubations using trapping agents such as glutathione (GSH). Although experimental approaches are established, a continued effort has been made to increase the sensitivity of such assays through further optimizing incubation conditions, exploring new in vitro metabolic systems (e.g. long-term culture models), and capitalizing on advances in mass spectrometry. These efforts are certainly of benefit for low turnover molecules, where appreciable levels may not be detected from conventional in vitro conditions but represent a relevant metabolic pathway in vivo. It remains to be seen if improvements in sensitivity would aid prediction or rather increase the potential for false positives, highlighting the need to further identify translational parameters both from clinical predictions (e.g. dose and exposure) and additional orthogonal safety end-points to contextualize findings. A primary focus has been to better clarify the mechanisms involved in the formation of reactive metabolites to guide programs at an early stage to shape optimization efforts. This is benefitted from increased capabilities of mass spectrometry instrumentation to provide rich spectral information to support localization endeavors. Case studies will be featured to illustrate the challenges in addition to success stories to abrogate reactivity, where resolution of adducts as a means of risk prevention obviates the need for further risk mitigation steps.
TOXI 26.
Michael Gill
Strategies for early safety assessment of potential drug candidates at Bristol-Myers Squibb: Preclinical evaluation of hepatobiliary toxicity
Abstract: Drug discovery requires an integrated assessment and optimization of multiple parameters, including pharmacodynamics, pharmacokinetics and toxicological profiles, in order to identify a clinical candidate that is predicted to achieve the desired clinical efficacy and safety profile. At Amgen, safety assessment of novel targets and drugs starts early in order to fail fast and avoid spending resources on targets or molecules that have a low probability of success. At the early stages of target identification, a theoretical assessment of the putative liabilities related to modulating the target and closely related off-targets is conducted. This can help prioritize targets and guide early experimental strategies to assess putative liabilities that may limit development. Following high-throughput screening, in vitro predictive safety assays are used in conjunction with potency and ADME properties to help prioritize promising lead scaffolds for further optimization. As drug-like properties improve during optimization, in vitro and in vivo safety properties are further characterized to identify safety issues that will guide subsequent screening strategies at later stages of lead optimization. The suite of predictive safety assays and in vivo toxicology studies that are used to guide clinical candidate selection are intended to characterize and predict human safety, identify maximum tolerated doses and establish suitable safety margins to guide decision making and designs of future FIH-enabling GLP toxicology studies. Understanding the clinical study design, patient population and competitive landscape can help inform an efficient screening strategy to balance speed to the clinic with appropriate risk taking to avoid safety-based attrition at later stages of development. Different strategies will be presented to illustrate how the in vitro and in vivo toxicological data can be integrated into an efficient testing paradigm to rapidly screen out undesired compounds, and to speed up the identification of clinical candidates.
TOXI 27.
Kaushik Mitra
Integrated platform, utilizing transcriptomic profiling and metabolite identification studies, to derisk drug bioactivation-mediated liver injury
Abstract: Drug-induced liver injury (DILI) is a major reason for attrition during development, denied commercialization, withdrawal from the marketplace and restricted prescribing indications of new pharmaceuticals. Metabolic bioactivation is thought to contribute to a significant number of liver-associated adverse drug reactions in humans that fail to be detected in standard preclinical animal studies. By measuring transcriptional pathways activated by electrophilic metabolites of a well-curated list of commercial drugs and internal compounds, we have developed in vivo and in vitro hepatic gene signatures to discern drugs with bioactivation-mediated hepatotoxic potential. Case examples, utilizing complementary tools such as metabolite identification-guided structural modifications, describe the application of this approach in lead optimization. When used in conjunction with exposure estimates, this approach improves prediction of clinical liver liabilities, and combination with assays of other potential DILI mechanisms such as inhibition of bile salt export protein (BSEP) or mitochondrial toxicity may further improve sensitivity. When used together with strategies that can identify liver toxicity liabilities resulting from other established mechanisms of DILI, such as inhibition of bile salt export protein (BSEP) or mitochondrial toxicity, overall prediction sensitivity is expected to rise. Additional qualification and refinement of these experimental models are ongoing, with growing confidence that they continue to add important new information for guiding an improved weight-of-evidence approach that facilitates lead optimization and candidate selection.
TOXI 28.
Antony J Williams
Delivering web-based access to data and algorithms to support computational toxicology: US EPA CompTox Chemicals Dashboard
Abstract: The US-EPA National Center for Computational Toxicity (NCCT) has been generating data and building software applications and web-based chemistry databases for over a decade. During this period the center has analyzed thousands of chemicals in hundreds of bioassays, has researched high-throughput physicochemical property measurements and investigated approaches for high throughput toxicokinetics. NCCT continues to expand the battery of assays and number of chemicals under examination and is now investigating the application of transcriptomics. In parallel to these experimental efforts, and to support our efforts to develop new approaches to prioritize chemicals based on potential human health risks, we aggregate and curate data streams of various types to support prediction models. Over the past few years some of the data have been delivered through prototype web-based “dashboards” for public consumption. The latest of these web applications, the CompTox Chemicals Dashboard, is an integrated access point to obtain information associated with 875,000 chemical substances and providing experimental and predicted data of various types. This includes physicochemical and fate and transport data, bioactivity data, exposure data and integrated literature searches. Real-time predictions and generalized read-across are possible and advanced search capabilities are available to support EPA-related projects including mass spectrometry non-targeted analysis. This presentation will provide an overview of the CompTox Chemicals Dashboard and the its role in delivering access to the outputs of NCCT. This abstract does not necessarily represent the views or policies of the U.S. Environmental Protection Agency.
DARPA’s microphysiological systems (MPS) program
In 2012 DARPA challenged the bioengineering community to create an interconnected network of 10 human microphysiological systems that could revolutionize the way drugs are screened and tested. Flash forward 7 years and the MPS program has successfully drawn to a close with two separate research groups, one led by Dr. Linda Griffith at MIT and the other by Dr. Don Ingber at the Harvard Wyss Institute, accomplishing this audacious goal. This presentation will describe all of the research associated with the MPS program including the development of multiple human organs-on-a-chip as well as the modeling and scaling of those systems into an interconnected network mimicking the human state. Validation experiments will be presented where MPS were exposed to a number of well characterized drugs and characterized through a number of physiologically-relevant measurements. Additional experiments will also be discussed including pathogen exposure to lung chips, microbiome development in gut chips and an interconnected 3-way MPS to model the metabolic flow at the brain-gut axis.
TOXI 30.
Sheila S David
Dare to repair: From DNA chemistry to cancer
Abstract: The damaged base 8-oxoguanine (OG) is particularly sinister due to its mimicry of thymine that evades detection during replication and results in incorrect insertion of A to form OG:A mismatches. The MutY glycosylase is a unique [4Fe-4S]2+-cofactor containing enzyme that prevents mutations by excising adenine from OG:A mismatches to initiate base excision repair (BER). MutY has captured the spotlight due to a direct correlation between inherited defects in the human MutY homologue (MUTYH) and colorectal cancer, referred to as MUTYH-associated polyposis (MAP). Our research laboratory played a key role in the discovery of MAP by providing functional analysis of the two most common variants in MUTYH revealing a hampered ability to recognize OG. We have also used a combination of synthesis of modified substrates, enzymology and X-ray crystallography to reveal features associated with damage recognition and adenine excision by MutY and MUTYH. Notably, using modified substrates and modified enzymes in in vitro and cellular assays we have revealed the key features of OG:A base pairs that MUTYH uses to “find” substrate and ignore structurally similar T:A base pairs, and these insights help illuminate the most deleterious types of MAP variants. We have also synthesized several azaribose transition state analog containing DNA duplexes and determined crystal structures that provide insight into the adenine excision mechanism catalyzed by MutY. Together with measurements of product stereochemistry, we have proposed a revised mechanism for MutY that involves two nucleophilic displacement steps akin to the mechanisms accepted for “retaining” O-glycosidases. This new-for-MutY yet familiar mechanism may also be operative in related BER glycosylases and provides a critical framework for analysis of MAP variants. Our structural and mechanistic insights have also revealed new ways to selectively target MUTYH and other BER glycosylases. Notably, along this line, we have prepared a “clickable” TS mimic that provides a unique strategy to prepare high affinity specific inhibitors of MUTYH, and related glycosylases.
TOXI 31.
Irina Stepanov
E-cigarette aerosols for in vitro and in vivo toxicology studies: Key considerations
Abstract: Toxicology research on electronic cigarettes (e-cigarettes) has been mounting over the past decade, propelled by their increasing popularity and the discord in the public health community regarding the potential health impact of their use. Most of the early studies have focused on chemical characterization of aerosols and the resulting exposures in humans, demonstrating significant reductions in many tobacco-related chemical toxicants and carcinogens compared to cigarette smoke levels. Such studies also revealed the important role of e-cigarette device characteristics and e-liquid composition in the potential toxicological profile of e-cigarette aerosol. Accounting for these factors is critical, but not sufficient in designing adequate in vitro and in vivo toxicology studies with e-cigarettes. Other important factors are the choice of puffing parameters and regimens, the aerosol collection technique and handling conditions, and the exposure method (direct, extracts, or solutions). Variations in these and other relevant factors can lead to changes in the aerosol particle size distribution, inconsistencies in nicotine delivery, losses of important volatile toxicants, and other changes to important physico-chemical characteristics of the final material delivered to cells or laboratory animals. As the result, some seemingly trivial modifications to experimental design can have dramatic impact on the outcomes of such studies and lead to discrepancies in findings across research groups. This presentation will discuss key considerations related to the generation of e-cigarette aerosol for in vitro and in vivo toxicology studies and will provide recommendations for future research.
TOXI 32.
Robert Sobol
Developing DNA repair pathway specific genotoxic signatures
Abstract: DNA repair pathways maintain the integrity of the genome, prevent the onset of mutations and genetic change that leads to cancer, disease and aging phenotypes and play a significant role in the cellular and organismal response to environmental exposures. As such, identifying a defect in DNA repair capacity or in the cellular response to endogenous or environmental stresses is a precision prevention strategy that may help identify those at high risk from environmental genotoxins. However, functional defects require a direct analysis of basal genome integrity and a direct measure of the cellular capacity to repair the genome in response to exogenous genotoxins. To clarify DNA repair pathway-specific DNA damage and repair fingerprints, we have begun a program to iteratively define (i) the cellular response induced by a spectrum of DNA damaging agents, (ii) the impact on the cellular response by DNA damage response modifiers and (iii) the change in signature in human cells with defined deletions (knockouts) in genes across all DNA repair pathways. The DNA Repair Pathway (DRP) signature or fingerprint includes classical genotoxic analyses including cell survival, apoptosis and cell cycle effects. Further, both basal and induced DNA damage levels as well as genomic DNA repair kinetics are quantified using a 96-well CometChip platform. In this initial cohort analysis, we describe the DRP-fingerprint of base excision repair (BER) deficiency, employing cells with gene deletions in base damage repair (OGG1) as well as key components of BER including XRCC1, Pol and PARP1. Finally, we will present an analysis of 40 environmental compounds to begin to define those genotoxins that trigger BER-specific DNA damage. Our overall goal is to further classify genotoxins based on DNA repair pathway involvement using our DRP-fingerprint analysis platform.
Gary Patti
Annotating the small-molecule exposome by subtraction
When using liquid chromatography/mass spectometry to perform metabolomics, it is routine to detect thousands of signals from most biological specimens. At this time, however, very few of these signals can be biochemically annotated with a specific structure. Using a test sample, we wished to determine how many signals could not be identified because they resulted from artifacts and/or contaminants. The test sample was uniquely labeled with isotopes using the credentialing approach. Strikingly, in our test dataset, we found that more than half of the total signals detected with our standard metabolomics platform corresponded to artifacts and contaminants. Comparable analyses on other samples (including serum) provided similar results. The implications of these data for profiling chemical exposures will be discussed.
TOXI 34.
Peter W Villalta
E.coli-produced genotoxin colibactin: DNA adduct identification using untargeted adductomics and in vivo detection using high resolution mass spectrometry
Abstract: Strains of Escherichia coli present in the human gut produce colibactin, a small molecule genotoxin implicated in colorectal cancer pathogenesis. In spite of significant efforts over the past decade, the chemical structure of colibactin and molecular mechanism by which it exerts its genotoxic effects remain unknown. Here, we report on the use of our untargeted DNA adductomic methodology to discover covalent DNA modifications present in human cell lines treated with colibactin-producing E. coli. Two putative DNA adducts, with the same [M+H]+ m/z of 540.1765, were found only in the cells treated with pks+ E. coli. Both peaks triggered MS3 fragmentation events upon observation of the neutral loss of adenine (135.0545 amu) in the MS2 fragmentation spectra, and confirmed to be associated with pks through cell infection assays using individual stable isotope labeled L-amino acids known to be used by the pks NRPS-PKS assembly line and integrated into pks-associated metabolites. The accurate mass measurement of m/z 540.1765 [M+H]+ (calculated, 540.1772) yielded a molecular formula of C23H25N9O5S with 16 degrees of unsaturation. MS2 fragmentation of both adducts identified the same major fragment ions indicating these compounds were likely stereoisomeric. Using this information and knowledge regarding the chemical nature of colibactin, potential structures were postulated and confirmed through synthesis of adduct standards and confirmation of identical mass spectral and chromatographic behaviour, including co-injection of the synthetic standard and colibactin-adduct containing E.coli +pks sample. Finally, the adducts were detected using targeted high resolution LC-MS2 analysis in DNA extracted from the colonic epithelial cells of Germ-free wild-type C57BL/6J mice which had been incolated with pks+ E. coli for 2 weeks, and were not found in the same mice inculated with pks- E. coli. Additional experiments are underway to identify of crosslinking adducts, thought to be central to the genotoxicity of colibactin.
TOXI 35.
Jiri Zavadil
Integrative experimental modelling reveals novel genome-scale mutation fingerprints of carcinogens
Abstract: The International Agency for Research on Cancer (IARC) aims to identify preventable cancer causes; to this end, the ongoing research programs employ innovative experimental models and computational approaches to investigate previously unrecognized mutagenic effects of human carcinogens. Such effects of candidate substances are studied in clonally expanded cell lines and by massively parallel sequencing conducted at the genome-scale. Highly complementary in vitro adductomics experiments as well as sequencing of rodent tumors associated with the corresponding chemical exposures further elucidate the underlying mutagenic processes. The experimental results are matched with the sequencing data from human pan-cancer genome repositories, to identify relevant exposure fingerprints in humans. Several novel mutational signatures associated with dietary compounds, mycotoxins, therapeutic agents and industrial chemicals will be presented, alongside mechanistic insights into the possible causes of particular cancer types. Selection of the studied compounds reflects the priorities set for carcinogen evaluation by IARC, yet other sources, such as the results from independent adductomics and metabolomics studies, can also inform the design of these integrative analyses. Overall, this powerful approach has a considerable potential for improved understanding of cancer causes and for cancer prevention efforts aimed at modifiable exposures.
TOXI 36.
Silvia Balbo
High resolution mass spectrometry-based approaches for the investigation of chemical carcinogenesis
Abstract: Characterizing chemical exposures and their health effects in humans, remains a significant challenge. This is due in part to the difficulties related to capturing the complexity of exposures and their interaction with biological systems, and to the fact that markers that can help disentangle this complexity are often at trace levels. Improved tools providing greater analytical sensitivity, combined with more comprehensive screening, are needed to move this field of research forward. Our laboratory is focusing on developing state-of-the-art high-resolution mass spectrometry-based methods to characterize the exposome and determine how it may interact with cellular targets which play key roles in carcinogenesis. We have recently developed a neutral loss screening and relative quantitation strategy to characterize reactive carbonyls in biological fluids and a metabolic profiling method for the tobacco-specific carcinogen NNK. In parallel, we are working on establishing a DNA adductomic approach based on a data dependent-constant neutral loss-MS3 (DDA-CNL/MS3) methodology as a comprehensive screening method to characterize all covalent DNA modifications (DNA adducts), induced by various exposures. These methods are based on the use of high-field orbital trap instrumentation (Thermo Scientific Orbitrap Fusion) which is very well suited for the analysis of challenging low-abundance, high-complexity samples. A number of examples will be presented in which the integrated MS-based methods we are developing are aimed at generating new comprehensive tools for the characterization of exposures in humans and for the elucidation of their effects on DNA.
TOXI 37.
Brian Berridge
Embracing translation in toxicology: National toxicology program strategy
The National Toxicology Program (NTP) has a 40-year history of success and public health impact studying some of the most concerning agents in our environment. A lot has changed over that 40 years including the number and diversity of agents we’re exposed to, expectations of our stakeholders and technology. Over a decade ago, our discipline aspired to significantly increase the efficiency with which we could provide insights into putative hazards of novel environmental agents to support our efforts to mitigate those hazards but also to enable industry to develop safe and useful products. We’ve struggled to achieve that in a meaningful and translational way. The NTP is embracing that challenge and invigorating our focus on enhancing our ability to meaningfully translate mechanistic bioactivity to human outcomes. This presentation will provide insights into our strategy, evolution of our portfolio, a translational toxicology pipeline of capabilities and prioritized areas of ‘health effect innovation’.
TOXI 38.
Niladri Bhowmik
Combined in vitro assays for eye irritation assessment
Abstract: Two in vitro primary eye irritation studies, designed as an alternative approach to the in vivo rabbit eye test, were submitted to support the registration of a cleaner and sanitizer (“Product M”) for use on food- and non-food- contact surfaces. The first study was performed using Epiocular™ Human Cell Construct Assay. This assay is based on the enzymatic conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dye after topical application of the test material as a measure of cell viability. The ET50 (time needed to reduce cell viability by 50%) value for “Product M” was < 2 minutes, which classifies it as an EPA Category I eye irritant. Since the Epiocular assay cannot distinguish between Category I or II, a second confirmatory study was conducted using the Bovine Corneal Opacity and Permeability (BCOP) assay which measures changes in opacity and fluorescein dye permeability in isolated bovine corneas. The test resulted in a corrected mean opacity value of 90.3 and a corrected mean optical permeability (OD490 value) score of 0.051, which gave rise to an in vitro irritancy score (IVIS) of 91.1 and resulted in the classification of “Product M” as a Category I eye irritant. Results obtained from the two in vitro studies are consistent with a previous in vivo study with rabbits which showed that 4% lactic acid, similar to the concentration of lactic acid as an active ingredient in “Product M”, induced similar Category I irritancy. Observations from the present investigation showed that these two in vitro studies, in combination, provide a promising alternative approach in identifying potential eye irritancy of chemicals.
TOXI 39.
Byeong Hwa Yun
Kinetics of formation and removal of DNA adducts in mice exposed to tobacco and meat carcinogens by UPLC-mass spectrometry
Abstract: Exposure to aromatic amines via smoking or the environment is a significant risk factor for bladder cancer (BC) and renal cancer. Epidemiological studies suggest that genotoxic heterocyclic aromatic amines (HAAs) found in well-done cooked meats also may contribute to BC. 4-Aminobiphenyl (4-ABP) is a tobacco smoke carcinogen that forms DNA adducts in human bladder. However, the identities of other chemicals that contribute to the etiology of BC are mainly unknown. We examined the ability of 4-ABP, 2-naphthylamine (2-NA), and 2-amino-9H-pyrido[2,3-b]indole (AC), carcinogens present in tobacco smoke; 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, carcinogens formed in cooked meats, and 8-methoxy-6-nitrophenanthro[3,4-d]-1,3-dioxole-5-carboxylic acid (AA-I), a potent urothelial carcinogen present in herbal remedies, to form DNA adducts in bladder, kidney and liver tissues, using a male mouse model. Carcinogens mixed in the drinking water (100 ppm) were administered to mice for four consecutive days. The levels of DNA adducts in bladder, liver, and kidney were monitored by mass spectrometry after one and 33 days post-dosing. 4-ABP produced the highest adduct levels in bladder and liver among all carcinogens tested. On day 33, >50% of the adducts persisted in the bladder, and 90% were present in liver and kidney at day 33. AA-I formed the highest levels of DNA adducts in the kidney. The AA-I adduct level in the bladder was 50-fold lower than 4-ABP. AA-I adducts persisted in renal tissue, but 90% were removed from the bladder by day 33. The AC-DNA adduct formed in the bladder occurred at 50-fold lower levels than for 4-ABP, and more than 95% of the adducts were removed by day 33. DNA adducts of the other carcinogens formed in bladder, kidney, and liver at 200- 5000-fold lower levels than 4-ABP adducts and were efficiently removed from all three organs by day 33. Our data demonstrate that 4-ABP and AA-I are potent DNA-damaging carcinogens in the mouse urinary system, and their adducts persist in these tissues over time. Our goal is to develop a noninvasive method to detect DNA-adducts of bladder carcinogens in humans using exfoliated urinary cells. The male mouse model is a promising approach to develop this screening technology.
TOXI 40.
Jiehong Guo
Mass spectrometric quantitation of apurinic/apyrimidinic sites in tissues of rats treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N’-nitrosonornicotine
Abstract: The tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N’-nitrosonornicotine (NNN) are considered carcinogenic to humans by the International Agency for Research on Cancer. Metabolic activation of NNK and NNN results in formation of reactive electrophiles that modify DNA to produce a variety of products including methyl, 4-(3-pyridyl)-4-oxobutyl (POB)-, and 4-(3-pyridyl)-4-hydroxybutyl (PHB)- adducts that have been previously characterized. Among these are adducts such as 7-POB-deoxyguanosine and O2-POB-deoxycytidine that can result in apurinic/apyrimidinic (AP) sites by facile hydrolysis of the base-deoxyribonucleoside bond. The purpose of this study was to quantitatively examine the AP sites induced by NNN and NNK in different tissues of rats using a novel validated mass spectrometric method. Incubation of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc), a model compound for methyl hydroxylation of NNK, with rat liver homogenate, produced apurinic sites quantified by derivatization with O-(pyridin-3-ylmethyl)hydroxylamine (PMOA) and analysis by LC-ESI-MS/MS. The amount found in the NNKOAc incubations was 4.54 ± 0.41 mol/107 mol nucleotides (n=4), significantly greater (p<0.001) than in controls (2.00 ± 0.16 mol/107 mol nucleotides, n=4) lacking NNKOAc. No significant difference (p>0.05) was found in the levels of AP sites in the livers of rats treated with 5 ppm NNK in their drinking water for 10 weeks or 70 weeks, compared to control untreated rats in a carcinogenicity study. Further investigation of AP site formation in different tissues of NNK and NNN exposed rats is in progress. This is the first study to examine AP site formation by carcinogenic tobacco-specific nitrosamines.
TOXI 41.
Brent V Powell
Mutagenicity of the C8-adenine adduct derived from the environmental carcinogen 6-nitrochrysene in Escherichia coli and human cells
Abstract: 6-Nitrochrysene (6-NC) is a potent mutagen in bacteria. It is an exceptionally potent carcinogen in newborn mice, and it induces mammary carcinoma in rats. The genotoxicity of 6-NC is derived from its ability to generate DNA adducts such as N-(2′-deoxyadenosine-8-yl)-6-aminochrysene, N-(dA-8-yl)-6-AC which was shown to be repaired inefficiently by the nucleotide excision repair system in humans. To investigate its replication properties, we have synthesized N-(dA-8-yl)-6-AC adducted nucleoside using palladium cross-coupling chemistry. The adducted nucleoside was converted to its corresponding protected 3’-phosphoramidite, which was used to synthesize site-specific 12 mer and 15-mer oligonucleotides. To investigate the mutagenicity and translesion synthesis (TLS) efficiency of N-(dA-8-yl)-6-AC, the modified oligonucleotides were inserted into a plasmid to construct site-specifically modified vectors using recombinant DNA technology. These vectors were replicated in E. Coli and human embryonic kidney 293T (HEK293T) cells. Preliminary results in E. Coli showed that viability of the lesion is ~66% relative to control, and the major type of mutation induced by N-(dA-8-yl)-6-AC is A G (2%) transitions and AT transversions at the base 5′ to the lesion site as well as other semi targeted mutations (3%). In HEK293T cells, N-(dA-8-yl)-6-AC is a strong replication block, but significant TLS also occurred. Mutational analyses in HEK293T cells are in progress.
TOXI 42.
Hong Mu
Rad4/XPC initial binding discriminates repair-resistant lesions from efficiently-repaired ones in global genomic nucleotide excision repair
Abstract: Nucleotide excision repair (NER) recognizes a wide variety of DNA lesions derived from environmental factors. However, the NER efficiencies for structurally different DNA lesions can vary by several orders of magnitude, from efficiently repaired to repair-resistant. These repair-resistant lesions are the most hazardous, as they can survive to replication and cause mutations that initiate cancer. However, the origins of the differences in NER efficiency are elusive. The goal of our work is to develop computational strategies to identify repair-resistant lesions, toward the objective of predicting these computationally. We utilize as benchmarks a library of lesions derived from polycyclic aromatic chemicals and UV light; their structures and relative NER efficiencies, from resistant to well-repaired, are known. The xeroderma pigmentosum C protein complex (XPC) is a key factor in lesion recognition for global genomic-NER. Its productive binding to lesion-containing DNA duplexes is required for the assembly of subsequent factors to achieve successful NER. Previous experimental and computational studies have suggested that XPC can employ different pathways for structurally different lesions. Furthermore, lesion-dependent differences are manifested upon initial binding. We hypothesize that impeded initial binding can lead to repair resistance by preventing achievement of the productive complex. We have carried out molecular dynamics simulations for Rad4/yeast XPC binding to 12 different lesions from our library; these include various lesions produced by UV light or derived from benzo[a]pyrene, dibenzo[a,l]pyrene, and the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. We have identified promising structural descriptors of the initial binding states that show correlation with the relative NER efficiencies of the lesions. While efficiently repaired lesions manifest significant duplex unwinding near the lesion site, extensive binding of the Rad4 BHD2 hairpin with the minor groove around the lesion site, and conformational capture of a lesion partner base, the repair-resistant lesions fail to display these binding characteristics. The lesion structural origins that underlie their differing initial binding characteristics with Rad4 are characterized, and offer insight on their differing NER efficiencies.
TOXI 43.
Guannan Zhang
Elucidating the roles of estrogens in the development and prognosis of malignant pleural mesothelioma
Abstract: Asbestos is a carcinogen that causes mesothelioma rare cancer that arises from the mesothelial lining of the pleura. Malignant pleural mesothelioma is an aggressive tumor that is resistant to conventional treatment including chemotherapy, surgery or radiation. Epidemiological observations show that for non-occupational exposure the incidence of mesothelioma is higher in women than men. However, women diagnosed with malignant pleural mesothelioma respond better to the treatment and have a better prognosis than men. This prompted us to assess the role of estrogen and estrogen receptors in determining the risk and prognosis of this cancer. Our hypothesis is that mesothelioma cells express estrogen receptors and they can generate their own ligands for these receptors. As a positive control, MCF-7 cells were shown to express ER at the transcript and protein level, proliferate in response to 17β-estradiol and proliferation was blocked with fulvestrant an ERα antagonist. qPCR showed that three different malignant mesothelioma cell lines (MSTO-211H, REN, and IST) expressed ERα at very low levels when compared with MCF-7 cells and this was supported by immunoblot analysis. However, treatment of MSTO-211H cells with 17β-estradiol induced growth proliferation that was not blocked by fulvestrant. These data suggest that 17β-estradiol exerts survival and proliferative effects in malignant mesothelioma cells through a pathway that is independent of ERα. Using an antagonist for GPR30 preliminary studies indicate that the growth proliferation observed with 17&beta-estradiol is blocked in MSTO-211H cells. We now aim to measure the expression of ERα, ERβ and GPR30 in malignant mesothelioma cells with IP-based LC-MS/MS proteomics and their clinical and biological significance.
TOXI 44.
Kurt Housh
Facile synthesis of DNA duplexes containing a chemically-defined, derivatizable, covalent cross-link
Abstract: Methods for chemical synthesis DNA duplexes containing site-specific, chemically-defined interstrand cross-links have potential applications in biophysics, biochemistry, structural biology, and materials science. We will describe the facile installation of an interstrand DNA cross-link that contains a derivatizable ethylamine “handle”. Our approach employs reductive amination reactions to cross-link two Ap sites on opposing strands of a DNA duplex with a triamine linker. We further showed that these cross-linked duplexes can be derivatized with various activated esters to install fluorophores, biotin tags, or photo affinity labels.
TOXI 45.
Michael Malfatti
Iterative approach for the development of a CNS-active oxime reactivator for nerve agent exposure
Abstract: Organophosphorus-based (OP) nerve agents represent one of the most toxic, man-made chemicals ever assembled, and within the chemical warfare agent (CWA) arena, they are certainly the most feared due to their lethal efficiency and ease of large-scale production. Given the rapid toxicity observed after nerve agent exposure, a fast acting, efficacious therapeutic is needed to counteract these effects immediately after exposure. The current standard of care for nerve agent poisoning relies on the use of small molecule-based oximes that can efficiently restore acetylcholinesterase (AChE) activity. Despite their efficacy in reactivating AChE, the action of drugs like Pralidoxime(2-PAM) is strictly limited to the peripheral nervous system (PNS) and thus, provides no protection to the central nervous system (CNS). This lack of action in the CNS stems from the ionic nature of the drugs that prevents them from crossing the blood brain barrier (BBB) and thus access any nerve agent-inhibited AChE therein. The current challenge lies in developing neutral (hydrophobic) oximes that not only cross the BBB but are efficacious enough to reactivate inhibited AChE in both the PNS and CNS. The work described herein uses an iterative cycle involving parallel efforts in synthetic chemistry, computation, in vitroand in vivocapabilities. This cyclical approach seeks to balance the often competing aims of achieving BBB permeability and reactivation activity simultaneously. Using this approach, we have developed and characterized a new candidate oxime molecule LLNL-02 with superior BBB penetration, significant reactivation capacity and minimal toxicity in less than three years’ time.
TOXI 46.
Melissa Lizette Liriano
Probing the thermostability and DNA loading efficiency of beta sliding clamp variants with perturbed dimer interfaces
Abstract: In Escherichia coli, the beta sliding clamp is an essential subunit of the replicative polymerase DNA polymerase III (Pol III). In the presence of the beta sliding clamp, the efficiency and processivity of DNA replication is significantly enhanced, with a rate of >50 kb added versus 10 nucleotides in the absence of the clamp. The beta sliding clamp is a homodimeric ring structure, with monomers arranged in a head-to-tail orientation yielding two identical interfaces. Loading of the beta sliding clamp onto DNA requires opening of the clamp ring, which is facilitated by a second multiprotein subunit of the replisome called the clamp loader, or the gamma complex. The gamma clamp loader complex is composed of three proteins: delta, delta prime, and gamma. While a partially opened beta is presumably required for beta loading onto DNA by the gamma complex, the exact clamp opening mechanism remains unknown. One model proposes that the delta subunit opens the beta clamp, in a wrench-like action, dissociating Domain I in one monomer from Domain III of the other monomer. We previously showed that the beta sliding clamp is highly dynamic in Domain I, transiently sampling conformational states in solution. We have made mutations at the dimer interface and have probed the stability of each variant and assessed their DNA loading efficiencies. We have also designed and purified a beta clamp variant that has one interface fused with an 18-amino acid long linker. This linked beta variant is homodimeric but each monomer has a distinct DNA sequence, allowing us to make mutations in only one monomer. The linked beta variant supports many beta functions; thus, we can gain more insight into how restricting interactions to one interface of the beta clamp affects its thermostability, interactions with the gamma complex, and DNA loading efficiency.
TOXI 47.
Pamela Cebrowski
Application of a read-across approach to address data gaps in human health screening level risk assessments and the chemicals management plan
Abstract: The Chemicals Management Plan (CMP) is a multi-phase, Government of Canada initiative jointly managed between Health Canada (HC) and Environment and Climate Change Canada (ECCC) dedicated to protecting human health and the environment from chemical risks. Some substances that required assessment as part of the CMP had limited substance-specific health effects data available, which presented a challenge in assessing the potential risks to human health. One way to address data gaps is through the use of a ‘read-across’ approach. Read-across is the methodology where by toxicity test results for one or more chemical(s) are used to predict the critical health effects or endpoints for another chemical that is considered to be similar based upon scientific justification. In prinicple, read-across can be used to predict physicochemical properties, (eco)toxicity, and persistence and bioaccumulation potential of chemicals, and require a rationale for chemical similarity. Similarity may be based on a common functional group(s), chemical class(es), carbon chain length range(s), and/or reactivity in chemical or biological processes. In the past, read-across has been conducted by HC and ECCC on an ad hoc basis by following guidance developed by the OECD in 2014, and by using internal expertise and judgement. Recent application of the read-across approach to address data gaps in human health screening level risk assessments of existing substances in CMP will be discussed.
TOXI 48.
Katherine Hurley
Endogenous microbial generation of acrolein: Strategies to assess the effects of intestinal acrolein formation
Abstract: Human exposure to acrolein results from its presence in car exhaust and frying oil vapors, due to lipid peroxidation and polyamine catabolism, and it has recently emerged as a product of glycerol metabolism by human gut microbes. Human gut bacteria that possess glycerol dehydratase (GDH) convert glycerol to 3-HPA, which is spontaneously degraded to acrolein in the lumen of the intestines. Acrolein reacts with DNA forming potentially mutagenic guanine adducts (Acr-dG) and with amino acids leading to possible loss of protein function and premature proteolysis. Beneficially, dietary heterocyclic amines react with acrolein to form glycerol conjugates that block the mutagenic potential of the amines. While acrolein toxicity is well characterized, there is little understanding of the chemical-biomolecular interactions and their biological consequences resulting from intestinal acrolein formation. We assess the effects of acrolein on the intestinal epithelium in vitro by 1) quantifying Acr-dG adducts and 2) measuring levels of proteins known to be affected by acrolein exposure. We quantified Acr-dG adducts utilizing capLC-ESI-MS and observed a dose-dependent increase of Acr-dG adducts in colon epithelial cells exposed to acrolein. Furthermore, there was a 2-fold decrease in the levels of XPA, a key protein in the nucleotide excision repair pathway known to repair Acr-dG adducts, and a dose-dependent increase in the levels of vimentin, an intermediate filament protein that maintains cellular integrity. These findings support the potential for gut microbial acrolein to react with biomolecular targets in the colon epithelium in vivo. In initial in vivo studies in a mouse model colonized with GDH-competent microbes, we detected heterocyclic amine-glycerol conjugate formation in the feces, which was not present in animals lacking GDH-producing microbes. These bioanalytical tools will allow us to connect the chemical-biomolecular alterations of acrolein to adverse or protective biological consequences in future gnotobiotic studies.
TOXI 49.
Rui Qi
Effect of sequence context on 1, N6-ethenoadenine repair by the AlkB family DNA repair enzymes
Abstract: DNA adducts are constantly generated from exogenous and endogenous processes, causing genome instability and eventually lead to cancer and other genetic diseases. To avoid the damages from DNA lesions, organisms have evolved an array of DNA repair strategies to protect cells from the adverse effects. One of the repair proteins, the adaptive response enzyme AlkB in E.coli, has been reported to repair various adducts including m1A, m3C and eA by directly reversing those damaged nucleotides to the normal ones. The repair efficiency may vary depending on the sequence context, especially the neighboring bases at both pre- and post-lesion sites. In this project, we are aiming to test the in-vitro and in-cell repair efficiencies of AlkB and its human homologs ABH2 and ABH3 by applying enzymatic reactions and replicative bypass and mutagenicity assays. The information obtained from this research may provide explanation for the mutational spectra of etheno adducts and the interconnection with repair enzymes.
TOXI 50.
James Starr
Is food type important for in vitro post ingestion bioaccessibility models of polychlorinated biphenyls sorbed to soil
Abstract: Soils are sorbents for many regulated organic compounds and children consume relatively large amounts of soil. To better the estimate the risk from this exposure pathway, requires an understanding of the behavior of sorbed chemicals following soil ingestion. We examined the role of co-ingested foods in determining the post-ingestion bioaccessibility (mobilization) of 18 polychlorinated biphenyls (PCBs) sorbed to 10 characterized soils. The bioaccessibility test system (DIN 19738, 2004) was an in vitro, 3-compartment, digestive tract containing salts, protein, and bile. Each soil was fortified with PCBs, then, digestive fluids appropriate to each compartment, were added sequentially. Next, digestive fluid and soil were seperated and PCB concentrations in both were measured. Bioaccessibility was defined as percent of each PCB in the fluid. The “complete” assay was then reduced to assess contributions of individual endogenous digestive fluid constituents (water, salts, pancreatin, bile, and mucin), and representative foods: protein (bovine serum albumin (BSA)), sugar (glucose), and fat (oleic acid). Finally, increasing concentrations of BSA, glucose, and oleic acid were added (individually) to “complete” test systems. In a subset of the samples, solid phase microextraction (SPME) measured freely dissolved PCBs. Across all treatments, percent soil carbon was the primary bioaccessibility determinant, accounting for 87% of the explained variation. All sink materials (protein, bile, and fat), effectively increased PCB bioaccessibility and reduced freely dissolved PCB concentrations. This suggests competitive removal of desorbed PCBs. Without sink material, intra-PCB mobilization differences were observed as freely dissolved PCB concentrations correlated (negative, p < 0.05) with their respective log Kow’s, When added to the complete digestive fluid, increasing oleic acid mass increased PCB bioaccessibility (p < 0.05), while adding more BSA or glucose did not (P > 0.05). This indicates that fat intake may be the sole consideration when modeling dietary contributions to bioaccessibility of soil sorbed PCBs.
TOXI 51.
Spandana Naldiga
Translesion synthesis past DNA-peptide crosslinks is dependent on local DNA sequence context
Abstract: Tandem lesions, comprised of two adjacent damaged nucleotides, make up a family of DNA damages formed by nucleobase radicals frequently generated by ionizing radiation. One such tandem lesion contains a thymine glycol (Tg) flanked on its 5’-side by a 2-deoxyribonolactone (L*). To study replication properties of this tandem lesion, we prepared three modified plasmid constructs containing L*Tg and single lesions Tg and L*, which were replicated in HEK 293T cells. After incubation for 2 days, progeny DNA was collected, purified, and used to transform E. coli DH10B cells, and the transformants were analyzed by oligonucleotide hybridization followed by DNA sequencing. While viability of Tg containing constructs was ~95% in this 5’-CTTgG-3’ sequence, suggesting that in this sequence context Tg is not a strong block of DNA replication in human cells, the L* lesion reduced the viability to <20%. Viability was further decreased in the tandem lesion 5’-CL*TgG-3’ to ~6%. Analyses of the progeny from Tg showed that nearly 97% replication involved dA incorporation opposite Tg with 1.6% other nucleotide incorporations and 1.6% targeted single-base deletions. In contrast, the tandem L*Tgconstruct showed 19% one-base deletions. In the full-length products from L*Tg, ~80% and 88% dA incorporations occurred opposite L* and Tg, respectively. Therefore, Tg is nearly 4-times more mutagenic when it is 3’ to L*. To determine the roles of translesion synthesis (TLS) DNA polymerases,we examined the replication of these constructs in HEK 293T cells either with knockout in human DNA polymerase η (hPol η) expression, or with knockdown of hPol ζ or hPol ι. For the tandem lesion, the rate of one base deletions increased from 19% in the HEK 293T cells to 26% and 34%, respectively, in the hPol ζ and hPol ι knockdown cell lines; additionally, ~3% two-base deletions occurred in these pol knockdown cells. In contrast, neither the viability nor the deletion frequency was altered in hPol η knockout cells. This suggests that the TLS polymerases ζ and but not hpol η play a critical role in generating full-length products from replication of this tandem lesion. In the full-length products,dA incorporation also was lowered from 81% to 71% and 63%, respectively, in the hPol ζ and hPol ι deficient cells, suggesting that these polymerases are involved in dA incorporations opposite both L* and Tg. The biological implication of these results will be discussed. [Supported by NIEHS grant ES027558.]
TOXI 52.
Jun Yuan
Replication studies of chemically stable analogs of N7-methylguanine and N3-methyladenine in Escherichia coli cells
Abstract: N7-methyl-2’-deoxyguanosine (m7dG) and N3-methy-2’-deoxyladenosine (m3dA) can be generated in DNA from SN2–type methylation reactions. Owing to the presence of a positive charge located at the methylated nitrogen atom, these modified nucleosides can undergo spontaneous N-glycosidic bond cleavage, which leads to depurination and formation of abasic site. It remains unclear how these two chemically unstable lesions perturb DNA replication and how they are repaired in cells. Here, we applied both transition-state destabilization and carbon replacement strategies with the use of 2′-fluro-m7dG, 7-deaza-m7dG, 2′-fluro-m3dA, 3-deaza-m3dA as stable analogues. Oligodeoxyribonucleotides (ODNs) containing a site-specifically inserted analogue have been synthesized and characterized. Those ODNs will be incorporated into single-stranded M13 plasmids and introduced into E. colicells to investigate their biological consequences regarding how they impede DNA replication and induce mutations. We will also define the identities of translesion synthesis DNA polymerases involved in their replicative bypass and the DNA glycosylases involved in their repair.
TOXI 53.
James McIsaac
Probing interactions between E. coli DNA polymerase III and single-stranded DNA binding protein to gain insights into polymerase management
Abstract: The goal of this project is to elucidate the interactions between E. coli DNA polymerase III (pol III) and single-stranded DNA binding protein (SSB) and how these interactions contribute to polymerase management at the replication fork. Pol III is responsible for producing exact copies of the template strand during DNA replication and is composed of 10 subunits forming the holoenzyme, while the polymerase core contains three subunits. SSB has been shown to inhibit polymerization activity unless the holoenzyme is assembled, suggesting a potential role in polymerase regulation. Computational methods including model construction, protein-protein docking, inter and intra-protein residue coevolution analysis as well as multisequence alignments were employed to predict regions of interaction between pol III core and SSB. The in silico phase of this project provides a focused experimental approach leading to the construction of protein variants with changes in predicted key residues as well as truncations, which are used to probe interaction between pol III and SSB. Early work in the project has shown a clear interaction between wild type forms of these proteins as well as several alpha truncations. To probe the extent of the change in interaction between variants, experiments to identify deviation in DNA binding, substrate specificity, and fidelity are being pursued. Preliminary results bear evidence of more than one interaction point in the alpha subunit, which is in line with the in silico predictions. Ammonium sulfate precipitation serves as the initial screening method for these variants.
TOXI 54.
Guang Cheng
Quantitation of carboxyethylguanine adducts in leukocyte DNA from smokers and nonsmokers using LC-NSI-HRMS/MS
Abstract: 7-(2′-Carboxyethyl)guanine (7-CEG) and N2-(1′-carboxyethyl)guanine (N2-CEG) are carboxyethyl DNA adducts originating from different sources. 7-CEG could potentially be formed upon the metabolism of the carcinogenic nitrosamines 3-(methylnitrosamino)propionic acid (MNPA) and 1-nitroso-5,6-dihydrouracil, or by reaction of DNA with acrylic acid. N2-CEG could be formed upon exposure to methylglyoxal (MG), a mutagenic compound which is a major breakdown product of carbohydrates and is present in many foods and in cigarette smoke. We have previously analyzed human liver DNA for 7-CEG. The method was now further developed to include the quantitation of N2-CEG. A standard for N2-CEG was synthesized from dG and 1,1-dihydroxyacetone, purified and characterized by NMR and MS. The accuracy and precision of this new quantitative LC-NSI-HRMS/MS method was determined for both adducts. Investigation of potential artefactual formation of the adducts was performed. Finally, samples from 20 smokers and 20 nonsmokers were analyzed. Leukocyte DNA was isolated and hydrolyzed enzymatically, the samples were purified by cation exchange/reverse phase solid-phase extraction, and the resulting fraction containing the adducts was treated with acetyl chloride in methanol to convert the adducts to their methyl esters. Further purification and enrichment was performed using polymeric reverse phase solid-phase extraction, with the final fraction containing both adducts analyzed by LC-NSI-HRMS/MS with extracted ion chromatogram generation of the m/z 238 [M+H]+ to152.0565 [BH]+ and m/z 238 [M+H]+ to 178.0723 [M+H -COOCH3]+ transitions respectively, with a mass tolerance of 5 ppm. The analysis resulted in levels of 7-CEG (mean ± SD) of 602 ± 245 fmol/µmol dG in smokers, and 555 ± 171 fmol/µmol dG in nonsmokers, and of N2-CEG of 39.5 ± 10.6 pmol/µmol dG in smokers, and 33.8 ± 8.0 pmol/µmol dG in nonsmokers. Although a significant difference between smokers and nonsmokers was not observed, the method described here is unique in the use of high resolution mass spectrometry and establishes for the first time the ability to simultaneously quantify these two carboxyethyl adducts in leukocyte DNA.
TOXI 56.
Jun Yuan
Replication studies of purine 5′,8-cyclonucleosides in cells
Abstract: N7-methyl-2’-deoxyguanosine (m7dG) and N3-methy-2’-deoxyladenosine (m3dA) can be generated in DNA from SN2–type methylation reactions. Owing to the presence of a positive charge located at the methylated nitrogen atom, these modified nucleosides can undergo spontaneous N-glycosidic bond cleavage, which leads to depurination and formation of abasic site. It remains unclear how these two chemically unstable lesions perturb DNA replication and how they are repaired in cells. Here, we applied both transition-state destabilization and carbon replacement strategies with the use of 2′-fluro-m7dG, 7-deaza-m7dG, 2′-fluro-m3dA, 3-deaza-m3dA as stable analogues. Oligodeoxyribonucleotides (ODNs) containing a site-specifically inserted analogue have been synthesized and characterized. Those ODNs will be incorporated into single-stranded M13 plasmids and introduced into E. colicells to investigate their biological consequences regarding how they impede DNA replication and induce mutations. We will also define the identities of translesion synthesis DNA polymerases involved in their replicative bypass and the DNA glycosylases involved in their repair.
TOXI 57.
YiTzai Chen
DNA base sequence effects on 4-aminobiphenyl lesion-induced mutational spectra
Abstract: Many agents in environment result DNA damage directly or through metabolic processes. N-acetyltransferase, one of detoxification enzymes, in fact can activate 4-aminobiphenyl (ABP) to from C8-substituted dG-ABP as a major DNA lesion. The ABP lesion is a bulky lesion which has been demonstrated to provide conformational heterogeneities and to affect DNA replication and repair. In this study, we used chemical synthesis and identified the ABP adduct containing oligonucleotides. Then the Competitive Replication and Adduct Bypass (CRAB) and Restriction Endonuclease and Post-labeling (REAP) Assays were used to examine the frequency and the type of mutation of the lesions. The results show the environmental toxin causes changes of mutational spectra in a sequence dependent manner.
TOXI 58.
Ying Tan
Human translesion synthesis DNA polymerases function in transcriptional bypass and repair of N2-alkyl-2′-deoxyguanosine lesions
Abstract: DNA, the key component of genetic material, is constantly under attack by exogenous environmental factors, as well as natural cellular metabolites, resulting in an array of covalent DNA modifications. Alkylation represents a common type of DNA modification, where the size and position of alkyl group may vary depending on the chemical nature of the alkylating agents. These DNA modifications may perturb the flow of genetic information by impeding DNA replication and transcription and eliciting mutations during the processes. To maintain genome stability, cells are equipped with DNA repair and damage tolerance mechanism in all domains of life. Translesion synthesis (TLS) is one damage tolerance mechanism where specialized DNA polymerase can synthesize across the damage or extend from it when replicative DNA polymerase is blocked. While the role of TLS polymerases in replication is well documented, it’s largely unknown whether TLS polymerase would assist transcriptional bypass. Recently, growing evidence revealed the versatile roles of TLS polymerases aside from canonical TLS. For example, human DNA polymerase h has reverse transcriptase activity in cellular environment. In this study, we employed our recently developed competitive transcription and adduct bypass assay (CTAB), where non-replicative double-stranded plasmids harboring site-specifically inserted N2-alkyl-dG lesions are used, to assess their transcriptional bypass efficiency and mutation frequency in HEK293T human embryonic kidney cells. We also conducted the transcription experiments in the isogenic cells where individual translesion synthesis DNA polymerases were depleted by the CRISPR-Cas9 genome editing method. Thus far, our results showed that, N2-alkyl-dG lesions are moderately blocking to transcription, and deficiency of Pol k and Pol i, but not Pol z, further exacerbate the effect. In addition, simultaneous ablation of Pol k and Pol i resulted in diminished transcriptional bypass efficiency than depletion either polymerase alone, indicating that Pol k and Pol i function in separate pathways. Together, our results provide new insights into transcriptional perturbation of N2-alkyl-dG lesions, and the biological functions of TLS polymerases.
TOXI 59.
Li Zhang
Evaluation of tobacco smoke and diet as sources of exposure to two heterocyclic aromatic amines for the U.S. population: NHANES 2013-2014
Abstract: Heterocyclic aromatic amines (HAAs) are a group of hazardous chemicals produced during combustion of tobacco or high-temperature cooking of meats. 2-Amino-9H-pyrido[2,3-b]indole (AαC) is a major carcinogenic HAA that is found in tobacco smoke. Urinary AαC, used as a marker of AαC exposure, was measured in spot urine samples from adult participants of the 2013-2014 cycle of the National Health and Nutrition Examination Survey (NHANES; N=1,820). AαC was measured using isotope-dilution liquid chromatography-tandem mass spectrometry. Exclusive tobacco smokers were distinguished from non-users of tobacco products through a combination of self-report and serum cotinine data. Among exclusive smokers, sample-weighted median urinary AαC was 36 times higher than non-users. Sample-weighted regression models showed that urinary AαC increased significantly with increasing serum cotinine among both exclusive tobacco users and non-users with second-hand smoke exposure. Among non-users, eating beef cooked at high temperature was associated with a significant increase in urinary AαC, while consuming vegetables was associated with decreased AαC. In addition, smoking one-half pack of cigarettes per day was associated with a significant increase of 23.7 pg AαC/mL (calculated at geometric mean of AC, controlling for potential confounders). In comparison, increase in AC attributable to consuming the 99th percentile of beef cooked at high temperature was 0.99 pg AαC/mL. In conclusion, both exclusive smokers and non-users of tobacco in the general U.S. population are exposed to AαC from tobacco smoke, with additional, lesser contributions from certain dietary constituents. AC is an important biomarker that is associated with tobacco smoke exposure. (Acknowledge Center for Tobacco Products, FDA for providing funds for this project.)
TOXI 60.
Qiyuan Han
Multi-omics characterization of an inflammatory bowel disease mouse model
Abstract: Inflammatory bowel disease (IBD) is an important risk factor for colorectal cancer (CRC) development. Several studies have detected disordered methylation patterns induced by inflammatory bowel disease in mouse model, suggesting that epigenetic dysregulation may provide a key mechanistic link between IBD and CRC. In the present study, we employed a multi-omics approach to characterize epigenetic changes associated with IBD in an established model of human IBD: IL10-/- Rag2-/- mice infected with Helicobacter hepaticus. In addition to DNA methylation, we have focused on novel DNA epigenetic mark, 5-hydroxymethylcytosine (5hmC), which is generated from 5-methylcytosine (5mC) via Ten-eleven-translocation enzyme (TET) mediated oxidation. First of all, we mapped DNA methylome and hydroxymethylome in mouse proximal colon via reduced representative bisulfite sequencing (RRBS) combined with oxidized RRBS (Ox-RRBS). In addition, we employed RNA-seq to characterize the changes in gene expression patterns in same tissue. Our results showed that the inflammation in mouse proximal colon is accompanied by a global decrease of DNA hydroxymethylation. Interestingly, differentially methylated region(DMR) and differentially hydroxymethylated region(DhMR) are enriched in genes that are correlated with gastrointestinal disease, inflammatory disease and cancer. Furthermore, the corresponding transcriptome showed an initial feature of colorectal cancer. Taken together, our results suggest a potential epigenetic mechanism for the initiation of inflammation mediated colorectal cancer.
TOXI 61.
HENGJIANG LIU
Heavy metal toxicants deposition and destruction in pulmonary surfactant layer: Classical density functional prediction
Abstract: The deposition and destruction of toxicants in pulmonary surfactant layer are important processes in human health and medical care. We have introduced classical density functional theory (CDFT) to provide insight into this process. Nine typical heavy metal toxicants with different size and shape in PM2.5 were considered, and their free energy and structural information have been examined. The free energy profile indicates various valuable information. When considering single-molecule transport, there exist energy well during the transport and the toxicant molecules will trap in the surfactant layer. PbO, As2O3, and CdO are the three toxicants most easily deposited in the pulmonary alveolus, which is consistent with survey data. CuO appears to be the easiest toxicant to transport through the surfactant layer. However, for larger spherical particles, the energy well trends to transform into energy barrier and the barrier height is proportional to particles’ diameter. Moreover, the change in the interfacial tension reveals that CrO3, V2O5, and FeO are the three toxicants most decreased the interfacial tension and may be the most ruinous toxicants in PM2.5. Structural analysis indicates that the toxicants tend to pass through the surfactant layer with rotation. The configuration of the pulmonary surfactant was examined by extending our previous work to polymer systems, and it appears that both the configurational entropy and the direct interaction between the surfactant and the toxicant dominate the configuration of the pulmonary surfactant layer.
TOXI 62.
Alexander Siyasatovich Bunev
Investigation of kinase inhibitor hepatotoxicity with induced pluripotent stem cell-derived hepatocytes
Abstract: iPSCs were differentiated into hepatocytes in two-dimensional (2D) monolayers and three-dimensional (3D) hepatocyte spheroid cultures; this process required growth factors such as BMP4, bFGF, HGF, and OSM. Immunofluorescence staining and flow cytometry were performed to confirm that the mature hepatocytes expressed cytokeratin 18, anti–alpha-1 antitrypsin, and albumin. MTX toxicity was evaluated via monitoring of cell viability, alanine aminotransferase, and mitochondrial status after MTX treatment in 2D and 3D cultures.
TOXI 63.
Gurdat Premnauth
Reactive oxygen species-activated DNA damage agents to selectively targeting cancer by oxidative-cyclizing
Abstract: Targeted therapy reduces off-target inhibitor effects by releasing payloads specifically at the site of disease like tumors or cancer microenvironments. Current strategies for release include reduction, enzyme-assisted release, and other very stable linkers that require internalization. This research is focused on the design of a release strategy dependent on reactive oxygen species (ROS). These entities are common at cancer microenvironments sites at high levels and thus can lead to selective release of a prodrug. Using a delivery vehicle such as an aptamer, antibody or short targeting peptide is possible to target a particular type of cancer for the delivery, doubling the specificity features for a selective drug release at tumor site. In my design, I will use a ROS-activatable self-cyclizing molecule that will be attached to a leukemia targeting peptide and a poly ADP ribose polymerase (PARP) inhibitor, NU1025 as the cytotoxic payload. The linker carrying the drug will first be synthesized with an available alkene and then conjugated with the delivery vehicle, a cysteine terminated peptide, via thiol-ene click chemistry. In vitro and in cellulo studies will then be carried to show the release of the cytotoxic payload under oxidative stress (ROS) conditions and the increased cell cytotoxicity and selectivity to the targeted cancerous cells.
TOXI 64.
Xu Guo
Unhooking of DNA-DNA interstrand cross-links by the base excision repair glycosylase NEIL3
Abstract: Interstrand DNA-DNA cross-links (ICLs) can be generated by endogenous processes, anticancer drugs, and environmental agents. ICLs prevent strand separation and compromise the function of cellular DNA. We have characterized a group of ICLs derived from abasic (AP) sites that are common lesions in cellular DNA. Recent studies revealed that a cross-link derived from the reaction of an AP site with an adenine residue in duplex DNA can be repaired by the base excision repair glycosylase NEIL3. We will describe chemical and structural features that allow cross-linked DNA duplexes to be unhooked by this novel glycosylase-dependent mechanism.
TOXI 65.
Eun Ko | Songhee Lee
Oxidative stress and mitochondrial dysfunction in tail of zebrafish induced by persistent organic pollutant
Abstract: Effects of toxicological pollution in aquatic ecosystem have been main issues in research and human health. Persistent Organic Pollutants(POPs), a lipid soluble toxin that is not easily degraded by any chemical or biological process, is known to be associated with metabolic diseases. A key characteristic of metabolic diseases is mitochondrial dysfunction. Here, we developed a novel POPs exposing system giving POPs on zebrafish by water flow in water tank and carried out the POPs exposure on zebrafish for 3 months with low doses. As for the study, we chose a tail of zebrafish representing skeletal muscle known as one of representative organs testing metabolic abnormality for metabolic diseases. We isolated mitochondria, measured mitochondrial oxidative phosphorylation(OXPHOS) and found that POPs influenced mitochondrial function of tail.
TOXI 66.
Arnold Groehler
Studying the assembly of nucleotide excision repair complexes by biochemical and crosslinking mass spectrometry approaches
Abstract: Nucleotide excision repair (NER) is the main DNA repair pathway responsible for the removal of bulky DNA lesions induced by UV radiation, environmental mutagens, and chemotherapeutic agents. Deficiencies in NER are associated with the genetic disorder xeroderma pigmentosum (XP), which is characterized by an extreme sensitivity to ultraviolet rays from the sun. XP patients have a 2000-fold increased incidence of skin cancer due to the inability to repair UV-induced DNA lesions. Although the core NER factors and reactions have been discovered, additional studies are required to fully understand the assembly and regulation of NER complexes. Specifically, many relatively weak and dynamic interactions between NER factors and DNA that drive the assembly of the NER complex have yet to be elucidated. To address this need, we are developing cross-linking mass spectrometry-based techniques (XL-MS) in combination with biochemical and cryo-EM approaches to elucidate all transient DNA-protein and protein-protein interactions formed during the steps of NER assembly. Preliminary XL-MS experiments with purified XPC-RAD23B, which initiates global genome-NER by recognizing and binding to distorted DNA,1 has identified specific amino acid-amino acid and amino acid-nucleoside interactions between XPC-RAD23B and damaged DNA. After optimization of the XL-MS techniques and analysis, we will investigate the formation of downstream NER complex formation using purified factors. Our studies are expected to provide unprecedented insights towards understanding the mechanisms of dynamic and ordered assembly of NER factors on damaged DNA and the progression through the NER pathway. Furthermore, a more complete understanding of NER assembly is needed for understanding the carcinogenicity of environmental mutagens and could lead to improved anti-tumor treatments.
DNA probe-based screening strategy for selectivity engineering of 8-oxoguanine glycosylase
DNA glycosylase enzymes play a key role in the recognition and excision of damaged bases, protecting genomic DNA from endogenous and exogenous damaging agents. Moreover, DNA glycosylases are useful tools for evaluating the distribution of oxidative damage in genomic DNA. However, these strategies have limitations as many DNA glycosylases can recognize and remove similar adducts on different bases (for example oxidation both on adenine and guanine). We are focusing on human 8-oxoguanine DNA glycosylase (hOGG1) which recognizes and removes 8-oxoguanine (8-oxoG), 8-oxoadenine, 2, 6-diamino-4-oxo-5-formamidopyrimidine (FapyG) and methyl-fapyG. To engineer glycosylases with altered substrate scope, new analytical strategies are needed to speed the evaluation of their adduct specificity. Toward this goal, we have developed a fluorescent DNA probe based strategy for rapid profiling of glycosylase variants with altered substrate scopes. Nucelotides with oxidative damage (8-oxoG and FapyG) were synthetically incorporated into duplex DNA probes, containing fluorophores color-matched to report on the excision proficiency toward different adducts, as well as a specific quencher. When damaged bases were effectively removed by a given glycosylase, strand hydrolysis and dissociation led to separation of fluorophore and quencher. The signal intensity of the different fluorophores indicated the enzyme activity targeting 8-oxoG and fapyG. hOGG1 variants were tested and, based on signal intensity and ratio of fluorophores, glycosylase selectivities were determined. The chemical probe strategy is expected to impact the development of engineered enzymes for DNA damage mapping applications, as well as providing insight on the relationship of glycosylase structure with substrate recognition and excision.
TOXI 68.
Ryan Dilworth
Mapping the interactions of UmuD and the beta clamp loader in E. coli
Abstract: Cells are constantly faced with DNA damage. In order to ensure survival, they require processes that allow for tolerance or repair of these sites, and one such tolerance process is translesion synthesis (TLS). TLS plays a critical role in the SOS response that occurs during DNA damage. In the Escherichia coli SOS response, UmuD is expressed due to damage-induced replication fork stalling which causes a buildup of RecA and ssDNA. Upon expression UmuD forms the homodimer UmuD2, which is a key regulator of the Y family polymerases DinB and UmuC as well as of the replicative DNA polymerase pol III. UmuD seems to function as a switch between these mutagenic repair polymerases and the non-mutagenic DNA replication pathways. The homodimer UmuD2 self-cleaves to form UmuD’2 which combines with UmuC to form DNA polymerase V. Both UmuD2 and UmuD’2 have been shown to interact with the replicative DNA polymerase of E. coli. Whereas interactions between UmuD and the processivity clamp and the DNA polymerase subunit of pol III have been well characterized, far less is known about interactions of UmuD and the processivity clamp loader complex. The clamp loader, consisting of tau, gamma, delta, and delta’ subunits, interact with other proteins within the replisome and coordinate polymerases during DNA replication. Preliminary data suggests that UmuD2 and UmuD’2 interact with clamp loader subunits and may provide additional regulation of DNA replication in response to DNA damage. This study will provide additional insights into the interactions and potential regulation of TLS by UmuD2 and UmuD’2 through these clamp loader subunits, as well as insight into the coordination of the incoming repair polymerases to the stalled fork via the clamp loader assembly.
TOXI 69.
Junzhou Wu
Amplification and sequencing of 5′-aldehyde lesions resulting from DNA oxidation
Abstract: DNA single strand breaks (SSBs) are the most abundant DNA oxidative damage resulting from reactive oxygen species (ROS). Persistent SSBs could lead to replication folk collapse and double strand break (DSB) formation upon interaction with the replisome, which may further lead to genome rearrangements, cell death and disease. There are numerous chemical forms of SSBs including 5’ aldehyde lesions, which involves several different repair enzymes. Despite advances in understanding of SSB repair, the genome-wide landscape of SSBs remains largely unknown because of the lack of methods for sequencing this damage with high specificity and resolution. Therefore, we developed a new strategy to locate the 5’-aldehyde terminus at single nucleotide resolution. The principle of the method involves labelling the 5’-aldehyde terminus with a hydroxylamine-functionalized oligonucleotide, giving rise to a biocompatible altered DNA linkage and allowing labelled sites to be amplified by the polymerase chain reaction. The specificity of labelling and bypass of other aldehyde modifications, such as abasic site and 5-formylcytosine were characterized, supporting that various sites are labelled but only those derived from 5’-aldehyde precursors could be bypassed and amplified by polymerase. The results of this work provide a new strategy for studies aiming to provide valuable knowledge on the biological and toxicological impacts of SSB in a genome scale.
TOXI 70.
Carlos Antonio Hernandez
MG-adducts as biomarkers of metabolic disease
Abstract: Atypical metabolic conditions are associated with type 1 and type 2 diabetes (T1D and T2D) and diabetic kidney disease (DKD). Current diagnostic and prognostic indicators do not present before substantial and irreparable injury has taken place. Altered metabolism generates biomarkers that may be able to predict and track disease progression. Metabolic changes are associated with increased oxidative and carbonyl stress, leading to the production of the -oxoaldehyde methylglyoxal (MG). MG reacts with DNA, RNA, and proteins, producing N2-(1-carboxyethyl)-2′-deoxyguanosine (CEdG), the RNA analog CEG, and N-(carboxyethyl)-lysine (CEL), respectively. These adducts can be quantified in urine, providing a tool to monitor disease-inducing metabolic changes. We have developed a multiplex mass spectrometric method to simultaneously measure CEdG, CEG, and CEL from a single urine sample. We applied this method to a clinical study including subjects without diabetes (N=25) and patients with T2D (N=50). Patients provided 24-hour urine collections monthly for six months, from which CEdG and CEG were measured. Patients with T2D had significantly higher levels of both CEdG and CEG, with diagnostic cutoffs defined as CEdG: 15 nmol/24 hr and CEG: 35 nmol/24 hr. Future studies aim to define the predictive and diagnostic utility of CEdG, CEG, and CEL for DKD. We have been awarded samples from the DCCT/EDIC study in which all patients have T1D. We designed a nested case-control study in which cases develop DKD (N=118) and controls (N=118) do not. We will measure analytes pre- and post-DKD diagnosis to determine diagnostic and prognostic utility. We have access to extensive clinical data from this cohort and have developed a Python program to readily retrieve and analyze patient data. We also have a comprehensive statistical and mathematical modeling plan to establish a multicomponent biomarker panel for DKD prediction.
TOXI 71.
Cindy Khuu
Investigating the metabolic and biological effects of glyoxalase 1 inhibition and knockdown in cancer cell lines
Abstract: Environmental toxins and toxicants are sources of oxidative stress in cells due to the production of reactive oxygen species (ROS), which can damage cellular components such as proteins and DNA. One of the most common products of DNA oxidation is 8-oxoguanine (OG) that becomes mispaired with adenine (A) during DNA replication, ultimately producing G:C to T:A transversion mutations. MUTYH is a base excision repair (BER) glycosylase that initiates the repair of OG:A mispair by removing the adenine base. Notably, inherited germline mutations in the MUTYH gene can alter enzyme function and lead to a condition called MUTYH-associated Polyposis (MAP), which increases lifetime risk of colorectal cancer. However, proper functioning of MUTYH under high ROS conditions could lead to genomic instability due to higher levels of MUTYH generated AP sites that may lead to single and double stranded breaks in DNA. Due to this conundrum, we wish to explore the regulatory role of ROS on the activity of MUTYH. We hypothesize that high ROS conditions regulate MUTYH activity through the oxidation of exposed Cys residues. We have also previously identified a Cys-coordinate Zn(II) site which is required for MUTYH repair activity, and we propose that oxidation of one of these Cys residues may lead to loss of Zn(II) and reduced activity. In this work, we characterize the in vitro effects of different oxidants on MUTYH activity and conduct cellular assays to identify the ROS reactive moieties in MUTYH and understand how ROS exposure affects enzymatic activity and cell survival.
TOXI 72.
Rachana Tomar
Structural insights into aflatoxin B1 (AFB1)-associated mutational spectrum
Abstract: The etiology of hepatocellular carcinoma (HCC) is correlated with chronic dietary exposures to aflatoxin B1 (AFB1). The genotoxicity of AFB1 is mainly attributed to the AFB1-FAPY DNA adduct. It induces G to T transversions associated with AFB1 mutagenesis in bacteria and mammalian cells. AFB1-FAPY adducts exist in DNA as complex mixtures of configurational and conformational isomers. The extent to which these equilibrium populations of isomers depend upon sequence could modulate biological processing of AFB1-FAPY adducts in sequence-dependent manners, leading to specific AFB1-mutagenic signatures. In different sequence contexts, DNA processing enzymes may differentially recognize the AFB1-FAPY adduct. We are examining how 5′ and 3′ neighbor bases with respect to AFB1-FAPY adducts might regulate the formation of and the repair of AFB1-FAPY-dG lesions by the base-excision repair enzyme, DNA glycosylase NEIL1. NEIL1 repairs the AFB1-FAPY adduct. However, the specific configuration or conformation of the AFB1-FAPY adduct recognized by NEIL1 remains unclear. Here, we compare frequently mutated “hot-spot” sequences (e.g. 5′-CGC-3′, 5′-GGC-3′ and 5′-CGG-3′) and under-representated “cold-spot” sequences (e.g. 5′-TGT-3′, 5′-AGT-3′ and 5′-CGA-3′) derived from AFB1-FAPY-induced mutational signatures in murine hepatocytes (John Essigmann and Lawrence Loeb labs). Using NMR and crystallography, we seek to understand how the BER-specific base flipping mechanism is achieved in the case of AFB1-FAPY damaged nucleotides, and if it is favored in specific sequences by a particular combination of configurational or conformational isomers of the AFB1-FAPY adduct.
TOXI 73.
Clementina Mesaros
Environmental polycyclic aromatic hydrocarbons and risk of lung cancer in never smokers
Abstract: Lung cancer is the leading cause of cancer-related deaths worldwide. Lung cancer in nonsmokers is the seventh leading cause of cancer deaths worldwide and the proportion of lung cancer in never smokers is expected to increase as successful smoking prevention and cessation programs are implemented. In Pennsylvania, the adjusted rates of lung cancer incidence for Philadelphia is almost double that of rural areas in PA. If smoking was the only cause of lung cancer, these incidence rates should be the same suggesting that other environmental exposures may contribute. Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental pollutants and are elevated in urban areas. PAHs, including the prototypical benzo[a]pyrene, are activated by several pathways (involving P450 and AKR enzymes) to electrophiles that can cause DNA lesions and initiate lung cancer. Studies outside US showed a positive correlation between PAH levels in non-smokers urine and lung cancer risk. Alterations in different metabolic pathways following PAH exposure in cell and animal models, suggest that dysregulation of plasma metabolomics may provide potential biomarkers of PAH effect. Metabolomics biomarkers have been identified in lung cancer and other cancer types. Identification of cancer-associated mutations in genes encoding enzymes with significant roles in cellular metabolism lead to oncometabolites (small-molecule components of normal metabolism whose accumulation causes signaling dysregulation to establish a milieu that initiates carcinogenesis) being quantified in different cancer types. In non-small cell lung cancer (NSCLC) cases, several biomarkers were able to discriminate NSCLC cases from healthy controls. Significant associations between specific plasma metabolites and the pathological type or stage of NSCLC were also observed. These findings suggest that metabolomics (by identifying oncometabolites) can serve as a potential tool to predict NSCLC disease stage and select lung cancer patients for precision treatment. We tested the hypothesis that PAHs are major modifiers of NSCLC risk in never-smokers by generating PAH-associated metabolic profiles and metabolomics profiles from plasma in never smokers with NSCLC and compare these levels in matched controls. The levels of the dysregulated metabolomic biomarkers were correlated with individual PAH-metabolites, to determine if they confer excess risk to NSCLC.
TOXI 74.
Chidiebere Amarachi Dim
Independent synthesis and fate of DNA lesions generated from oxidative damage at the C-5′ of thymidine
Abstract: Under conditions of oxidative stress, the mechanisms of the cell designed to detoxify reactive oxygen and nitrogen species are overwhelmed. These radical species can then proliferate and cause damage to DNA, RNA and other macromolecules. An initial hydrogen atom abstraction can occur, in the case of some reactive species, leading to the formation of a carbon centered radical that reacts with cellular oxygen to form a peroxyl radical. Ultimately strand breaks and abasic sites can be generated along with a variety of other reactive lesions specific to the position of the original hydrogen atom abstraction. In this study, lesions emanating from oxidation at the C-5′ of thymidine have been independently synthesized and their ability to form adducts via Michael addition with selected thiol containing biomolecules has been studied. The formation and identity of adducts were investigated using LCMS and NMR.
Chemical metabolism and toxicological relevance of microbial glycerol dehydratase from human gut microbiota
Human gut microbial communities can metabolize chemicals and alter their toxicity. Understanding the toxicological relevance of the gut microbiome in chemical disposition and toxicity requires insight on the functional transformation capacities of the human gut microbiome, uptake potential of microbial metabolites, and the influence of microbial transformation on chemical toxicity. The heterocylic amine 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a mutagen and probable human carcinogen that reaches the human colon and can be transformed to 9-hydroxyl-2,7-dimethyl-7,9,10,11-tetrahydropyrimido[2,1:2,3]imidazo[4,5-f]quinoxaline (MeIQx-M1) by the function of a microbial glycerol dehydratase enzyme. Our data suggests that this transformation is relevant also for the glucuronidated form of the compound, and that the transformation involves microbial production of acrolein. Results suggest that MeIQx and its microbial metabolite are similarly transported from the mucosal side to the serosal side of intestinal tissue. Physiologically based pharmacokinetic modeling, taking microbial biotransformation into account, suggests the requirement of high levels of intestinal acrolein to significantly alter the availability of MeIQx. Furthermore, in vitro evaluation of cytotoxicity and mutagenicity are consistent with the glycerol dehydratase-catalyzed transformation being a detoxification process. Moreover, metagenomics profiles from healthy individuals vs cancer patients suggest a protective role of the presence of this microbial gene. These findings delineate a chemical basis for how inter-individual differences in human gut microbial communities have the potential to influence toxicological impacts of dietary chemicals.
TOXI 77.
Jonathan Sczepanski
Investigating base excision repair using “designer” chromatin
Abstract: Like all genomic processes, DNA repair must take place within the structural constraints of chromatin. However, due to the lack of tools to precisely manipulate chromatin in vivo, the relationship between chromatin structure and DNA repair remains poorly defined, especially at the molecular level. To address this issue, the Sczepanski laboratory focuses on investigating DNA repair using chemically defined nucleosome arrays reconstituted in vitro (referred to as “designer” chromatin). Uncoupled from the more complex cellular environment, this powerful experimental platform enables quantitative interrogation of DNA repair in the context of specific chromatin structures.For example, we assembled nucleosome arrays containing positioned 2-deoxyuridine (dU) residues and systematically examined the efficiency of base excision repair (BER) within various chromatin environments. We show that, depending on the position of the lesion, BER can be either inhibited by as much as 20-fold or accelerated by more than 5-fold within compact chromatin (i.e. the 30 nm fiber) relative to naked DNA. Moreover, we demonstrate thatinter-nucleosome interactions mediated by the histone H4 tail domain play an important role in regulating BER within higher-order chromatin structures. More recently, we assembled novel nucleosome arrays consisting of both site-specifically damaged DNA and homogeneously acetylated histones in order to investigate, for the first time, the impact of histone acetylation on BER within higher-order chromatin structures. Details regarding these studies, as well as our efforts to expand this experimental approach into other areas of DNA repair, will be presented.
TOXI 78.
Salako N Olatunji
Medical countermeasures for the individual service member to maintain combat effectiveness and prevent or reduce injury from the battlefield
Abstract: The Medical Countermeasures programs for the Warfighter/ Service Members is very much important to the Service members/ Warfighter in the battlefield to protect their territory and its aliens. To maintain this combat effectiveness, good standings and to prevent or reduce injury from the battlefield for the Warfighter/ Services member; the respiratory, ocular, neuro and dermal pathophysiology of the service Members must be well connected to the tissues organs and the Organs-Networking system must resist any internal and external injuries, by achieving these aims, the new concepts for prophylaxes, pretreatments, and therapeutic countermeasures; development of skin protectants. Service Members/ Warfighters must be prevented from sudden cardiac arrest (SCA) and sudden cardiac death (SCD) which occur when the heart abruptly begins to beat in an abnormal or irregular rhythm (arrhythmia) before, during and after the battlefield or warfront. The new therapeutic Countermeasures develop is OXONITROGENSIC Intensive Skin protectant Ferrilatecheratine , which base series is Ferrilatepolyethyleneaminase (FPETA) develops for skin protector to protect the skin of the Service Members/ Warfighter in the battlefield against any contaminations and infectious (Cocci, Bacilli, fungi, Viral infectious )through the Skin.This skin protectant acts as a protector barrier and as an active Matrix against proliferation of destructive weapons such as Biological, Ammunitions, and Nuclear and Chemical agents in the battle field. These efforts are to define specific requirements for incorporating antimicrobial textiles (AMTs) and ferrilatePolythyeneaminase (FPETA) in Warfighter Clothing and Individual Equipment (CIE) components to reduce the incidence of these conditions, which are costing the military up to $1 billion in treatment costs per year and are compromising mission accomplishment. Four groups of health and medical issues relevant to Warfighters are discussed. These creams are composite materials consisting of a base material (RSP) and a reactive moiety. Using an established base of Ferrilatedpolyethylaminase (FPETA) and hydrated Organometallics (HOM’s) Compound .We incorporated over 15 reactive components. Classes tested include organic polymers, organic/inorganic hybrid materials, polyoxometallate (POM’s), enzymes, inorganic oxides, metal alloys and small molecules. These Materials are to be characterized under light microscopy and FTIR.
TOXI 79.
Kai Luo
Multiple deuterated phenanthrene metabolites as probes for the metabolism of carcinogenic PAHs in humans: Phenotyping, genotyping and potential application in human health risk assessment
Abstract: Individual susceptibility plays an important role in cancer development in humans exposed to chemical carcinogens, thus identification of high-risk individuals is critical in cancer prevention and therapy. Benzo[a]pyrene (BaP), an important polycyclic aromatic hydrocarbon (PAH) in tobacco smoke and the environment, is considered carcinogenic to humans by the International Agency for Research on Cancer. BaP requires metabolic activation by the diol epoxide pathway to exert its carcinogenic effect, and this process differs greatly among individuals, who can’t be identified based on exposure measurements only. In this study, we used deuterated phenanthrene ([D10]Phe), a non-carcinogenic PAH with structural features and enzymology profile similar to that of BaP, as a probe substrate. The bioactivation of Phe bythe diol epoxide pathway yields the urinary metabolites phenanthrene dihydrodiol(Phe-Diol), phenanthrene tetraol(PheT) and phenanthrene quinone (PheQ), whereas phenanthrols (HOPhe) signify detoxification. A previous prospective epidemiological study has confirmed the statistically significant relationship between urinary levels of PheT and lung cancer risk. PheQ has been associated with the generation of reactive oxygen species (ROS) and oxidative damage, while Phe-Diol is the precursor of PheT and PheQ. We administered [D10]Phe, with FDA approval, to smokers (N=137, 10 µg oral dose) and nonsmokers (N=89, 1 µg oral dose) and quantified the excretion of its urinary metabolites noted above. The smoking status of all subjects was determined by urinary cotinine. We have already shown that this metabolism phenotyping approach is practical, accurate and precise. There were great variations among subjects in conversion of [D10]Phe to [D10]Phe-1,2-diol (31-fold/497-fold in 10 µg/1 µg dose group), [D10]PheT (51-fold/84-fold) and [D9]HOPhe (33-fold/84-fold), [D8]PheQ (18-fold/47-fold). We hypothesize that individuals who have a higher ratio of bioactivation metabolites ([D10]Phe-Diol+[D10]PheT+[D8]PheQ) to detoxification metabolites ([D9]HOPhe) are at higher risk for lung cancer. Oral cell genotyping of GSTs were also determined to explore genetic causes of individual differences. The results of this study promises to expand our understanding of metabolism of carcinogenic PAH in humans, thus providing new insights for human health risk assessment and lung cancer prevention.
TOXI 80.
Feng Tang
Base-resolution analysis of thymine glycol (Tg) in DNA by polymerase-aided differential coding
Abstract: Thymine glycol (Tg) is the most common pyrimidine lesion produced in DNA by oxidation or ionizing radiation. Because of the addition of hydroxyl groups to the C5 and C6 positions of thymine, Tg becomes a strong block to DNA replication and elicits cytotoxic effects. However, the genome-wide distribution of Tg remains unknown owing to the lack of a reliable and sensitive sequencing method for this lesion. Recently, we found that two commercially available DNA polymerases can bypass the Tg lesion and display distinct properties in nucleotide incorporation opposite or near the lesion site. By comparing in parallel the primer extension products formed with these two DNA polymerases, we developed a quantitative analytical method for locating the site of Tg at single-base resolution in synthesized DNA. We are in the process of developing an enrichment method for Tg-containing DNA, which, together with the differential coding properties conferred by the aforementioned polymerases, may allow for the development of a genome-wide sequencing method for this modified nucleoside.
TOXI 81.
Medjda Bellamri
Biological effects of the cooked meat carcinogen PhIP in human prostate cancer
Abstract: Prostate cancer (PC) is the most commonly diagnosed cancer in men and the second leading cause of cancer-related death in men worldwide. Consumption of well-done cooked meats is a risk factor for PC. Among the 25 heterocyclic aromatic amines (HAAs) formed in cooked meats, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most mass-abundant carcinogenic HAA. PhIP is a rodent and possible human prostate carcinogen. Employing mass spectrometry, we identified DNA adducts of PhIP, but not other prominent HAAs, including 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-9H-pyrido[2,3-b]indole (AαC), in the prostate genome of PC patients. Moreover, using LNCaP cells, a well-characterized human prostate cell line, we showed that the bioactivated metabolite of PhIP, HONH-PhIP, is cytotoxic and forms high levels of DNA adducts. However, HONH-MeIQx, HONH-IQ, and HONH- AαC were not cytotoxic and formed DNA adducts at levels 20-fold or lower than HONH-PhIP. In addition to DNA damage, PhIP induces oxidative stress and inflammation in the prostate of rodents. These features are associated with human PC. Thus, we aimed to characterize the biological mechanisms that may underlie the induction of PC by PhIP. For this purpose, LNCaP cells were treated chronically with PhIP and HONH-PhIP at levels approaching human exposure. Using MTT assay, we observed a concentration-dependent cell growth decrease associated with phenotypic changes toward a neuroendocrine cell morphology, suggestive of a senescence state. This senescence state may have been attributed to the accumulation of unrepaired DNA damage or other cellular stresses induced by HONH-PhIP. In addition to the effects on the cell cycle, chronic exposure to HONH-PhIP led to the induction of oxidative stress resulting in decreased levels of reduced glutathione with a concomitant formation of oxidized glutathione. In contrast, the treatment of LNaP cells with PhIP did not induce these biological events. These data highlight the need for more mechanistic studies to better characterize the role of PhIP in the etiology of PC.
TOXI 82.
Takafumi Takai
Evaluating the value of primary human cells for the prediction of in vivo toxicity in early drug discovery
Abstract: The in vitro cytotoxicity assay using the human hepatocellular carcinoma (HepG2) cell line is a widely-used screen for the both the detection of hepatotoxic potential and the identification of detrimental pharmacological activities associated with a candidate drug. A retrospective attrition analysis at Takeda showed that only 31% of internal candidate drugs that displayed adverse findings in an in vivo toxicity study at low doses (Total Cmax < 10uM) were cytotoxic against the HepG2 cell line. To understand the value of additional or replacement cytotoxicity assays towards the prediction of low-dose in vivo toxicity, we evaluated five types of primary human cells against 117 internal candidate compounds with in vivo toxicity data. Our results indicate that primary human hepatocytes and HUVECs are the most sensitive (hit rates 41% and 44%, respectively) when compared to HepG2 (hit rate 26%). However, these cells were less specific than HepG2 towards the prediction of compounds known to have toxic findings below the Cmax level of 10uM. This may limit their impact in early drug discovery where high specificity is valued so that compounds are not unnecessarily deprioritized. Primary cells may be more sensitive to specific pharmacological activities, as opposed to mechanisms associated with cell health, and a deeper investigation into the type of toxicity observed may be necessary to fully understand the value of these assays. Nevertheless, both primary hepatocytes and HUVECs may be useful to follow up the event of a negative response in the HepG2 assay. In this case, primary cells may be able to differentiate undesirable off-target pharmacology and metabolism profiles of lead compounds within the same chemotype. Case studies of recent projects at Takeda will be presented to support this conclusion as well as the activity profiles of these assays against the compounds in the dataset.
TOXI 83.
Isabelle Lee
Estrogenic activity of polycyclic aromatic hydrocarbon metabolites in human endometrial cells
Abstract: Polycyclic aromatic hydrocarbons (PAHs) are byproducts of incomplete combustion of organic materials, including fossil fuels, food, and tobacco. Cigarette smoking is associated with reproductive abnormalities in women, and some PAHs are uterine toxicants in rodents. Moreover, PAHs or their metabolites can activate estrogen receptors (ERs), resulting in endocrine disruption. Once in the body, PAH are metabolized by the family of CYP-450 enzymes to more water-soluble hydroxy-PAHs (OH-PAHs). Additionally, aldo-keto reductases (AKRs) convert PAH trans-dihydrodiols into PAH ortho (o)-quinones. Given the similarity between planar PAH and estrogens, we hypothesize that PAH and PAH metabolites activate ERs in estrogen target tissues e.g. endometrium. We used inducible alkaline phosphatase activity in Ishikawa cells, a human endometrial adenocarcinoma cell-line, and estrogen response element (ERE) luciferase activity as the read-out for ER activation. We tested the estrogenicity of various PAH and their metabolites, including benzo[a]pyrene (BaP), 3 PAH o-quinones (benzo [a] pyrene-7,8-dione (BPQ), benz[a]anthracene-3,4-dione and 5-methyl-chrysene-1,2-dione), and 3-OH-BaP in endometrial cells. We demonstrated that these compounds induce ER activity with EC50 values in the low micromolar range, and that this activation is inhibited by Fulvestrant, an ER antagonist. We have also shown that nanomolar concetrations of BPQ modulates cell cycle gene expression. Using high performance liquid chromatography and APCI mass spectrometry in the selected reaction monitoring mode, we find that BaP can be metabolized to the estrogenic BPQ and 3-OH-BaP in Ishikawa cells. Low nanomolar concentrations of BPQ increase Ishikawa cell proliferation to the same level observed with picomolar concentrations of 17--ethinyl estradiol. Our work indicates that planar PAH metabolites may play a role in the disruption of ER signaling in the endometrium. Additionally, differences in reporter gene response and cell proliferation were noted, raising the question of whether or not the estrogenic effects are mediated through ER.
TOXI 84.
Cindy Khuu
Investigating the role of cysteine oxidation by environmentally induced oxidative stress on the DNA glycosylase MUTYH
Abstract: Environmental toxins and toxicants are sources of oxidative stress in cells due to the production of reactive oxygen species (ROS), which can damage cellular components such as proteins and DNA. One of the most common products of DNA oxidation is 8-oxoguanine (OG) that becomes mispaired with adenine (A) during DNA replication, ultimately producing G:C to T:A transversion mutations. MUTYH is a base excision repair (BER) glycosylase that initiates the repair of OG:A mispair by removing the adenine base. Notably, inherited germline mutations in the MUTYH gene can alter enzyme function and lead to a condition called MUTYH-associated Polyposis (MAP), which increases lifetime risk of colorectal cancer. However, proper functioning of MUTYH under high ROS conditions could lead to genomic instability due to higher levels of MUTYH generated AP sites that may lead to single and double stranded breaks in DNA. Due to this conundrum, we wish to explore the regulatory role of ROS on the activity of MUTYH. We hypothesize that high ROS conditions regulate MUTYH activity through the oxidation of exposed Cys residues. We have also previously identified a Cys-coordinate Zn(II) site which is required for MUTYH repair activity, and we propose that oxidation of one of these Cys residues may lead to loss of Zn(II) and reduced activity. In this work, we characterize the in vitro effects of different oxidants on MUTYH activity and conduct cellular assays to identify the ROS reactive moieties in MUTYH and understand how ROS exposure affects enzymatic activity and cell survival.
TOXI 85.
Shawna Vreeke
Underestimation of toxin levels in the aerosol of electronic cigarettes
Abstract: Electronic cigarette use continues to increase among youth and young adults. The reliable prediction and measurement of e-cigarette aerosol toxin levels are needed to better understand their health effects. Our investigation focuses on identifying not only the precise characteristics of device components that modulate toxin levels but also to show how the choice of specific analytical method can lead to inaccuracies in aerosol component determination. By studying the heating properties of an e-cigarette, such as wicking efficiency, a mathematical model was created that enables a relatively straightforward prediction of aldehyde levels in eleven devices. In addition, we found that levels of some toxic products in e-cigarette aerosol were underestimated by the widely-used DNPH derivatization followed by HPLC analysis. When compared to the quantitative NMR method measured levels of acrolein and formaldehyde were several-fold lower. These findings will contribute to better understanding of parameters impacting e-cigarette safety and its accurate assessment.
TOXI 86.
Valeria Guidolin
DNA damage as a toxicity mechanism of NMC (LixNi1/3Mn1/3Co1/3O2) nanomaterial to bacteria Shewanella oneidensis MR-1 and Bacillus subtilis
Abstract: In the last decade, significant technological achievements have resulted in the widespread use of nanomaterials including NMC (LixNi1/3Mn1/3Co1/3O2), a nanoscale cathode material used Lithium ion batteries. The rapid increase in the use of these materials calls for a better understanding of their impact on human health and the environment. Exposure to engineered nanoparticles has been shown to induce DNA damage in various organisms. Nanoscale NMC has been found to inhibit bacterial oxygen uptake, however the specific cytotoxic mechanisms involved remain unclear. One hypothesis is that nanoscale NMC-induced DNA damage is due to the generation of covalently modified nucleobases (DNA adducts). In this study, we investigated bacterial DNA damage in two environmentally relevant bacterial strains upon NMC exposure through optimization and use of our recently developed DNA adductomic approach. This technique allows the characterization of DNA damage by screening for DNA modifications, beyond the traditionally studied 8-OHdG. Shewanella oneidensis and Bacillus subtilis were exposed to nanoscale NMC. The bacterial DNA was extracted and enzymatically digested with purification by off-line HPLC fraction collection and analysis via nanoLC/MS3 with data dependent scanning and MS3 triggering upon detection of the neutral loss of deoxyribose (-116.0474 Da). Data analysis was carried out using newly in-house developed software. Several DNA modifications were observed to be greater or only present in exposed bacteria in both strains. The mechanisms leading to DNA adducts upon exposure to nanoscale NMC are yet to be elucidated, however the chemical nature of the DNA adducts detected suggests that reactive oxygen species and increased lipid peroxidation are likely involved in the DNA damage process. These findings confirm the ability of our approach to chemically characterize DNA damage induced by nanoscale NMC in bacteria and provide insight to elucidate the responsible chemical mechanisms.
TOXI 87.
Gurdat Premnauth
Reactive oxygen species (ROS)-dependent release of an anticancer drug from a targeting peptide
Abstract: Targeted therapy reduces off-target inhibitor effects by releasing payloads specifically at the site of disease like tumors or cancer microenvironments. Current strategies for release include reduction, enzyme-assisted release, and other very stable linkers that require internalization. This research is focused on the design of a release strategy dependent on reactive oxygen species (ROS). These entities are common at cancer microenvironments sites at high levels and thus can lead to selective release of a prodrug. Using a delivery vehicle such as an aptamer, antibody or short targeting peptide is possible to target a particular type of cancer for the delivery, doubling the specificity features for a selective drug release at tumor site. In my design, I will use a ROS-activatable self-cyclizing molecule that will be attached to a leukemia targeting peptide and a poly ADP ribose polymerase (PARP) inhibitor, NU1025 as the cytotoxic payload. The linker carrying the drug will first be synthesized with an available alkene and then conjugated with the delivery vehicle, a cysteine terminated peptide, via thiol-ene click chemistry. In vitro and in cellulo studies will then be carried to show the release of the cytotoxic payload under oxidative stress (ROS) conditions and the increased cell cytotoxicity and selectivity to the targeted cancerous cells.
TOXI 88.
Spandana Naldiga
Translesion synthesis of tandem DNA lesions containing 2-deoxyribonolactone 5′ to thymine glycol in human cells
Abstract: Tandem lesions, comprised of two adjacent damaged nucleotides, make up a family of DNA damages formed by nucleobase radicals frequently generated by ionizing radiation. One such tandem lesion contains a thymine glycol (Tg) flanked on its 5’-side by a 2-deoxyribonolactone (L*). To study replication properties of this tandem lesion, we prepared three modified plasmid constructs containing L*Tg and single lesions Tg and L*, which were replicated in HEK 293T cells. After incubation for 2 days, progeny DNA was collected, purified, and used to transform E. coli DH10B cells, and the transformants were analyzed by oligonucleotide hybridization followed by DNA sequencing. While viability of Tg containing constructs was ~95% in this 5’-CTTgG-3’ sequence, suggesting that in this sequence context Tg is not a strong block of DNA replication in human cells, the L* lesion reduced the viability to <20%. Viability was further decreased in the tandem lesion 5’-CL*TgG-3’ to ~6%. Analyses of the progeny from Tg showed that nearly 97% replication involved dA incorporation opposite Tg with 1.6% other nucleotide incorporations and 1.6% targeted single-base deletions. In contrast, the tandem L*Tgconstruct showed 19% one-base deletions. In the full-length products from L*Tg, ~80% and 88% dA incorporations occurred opposite L* and Tg, respectively. Therefore, Tg is nearly 4-times more mutagenic when it is 3’ to L*. To determine the roles of translesion synthesis (TLS) DNA polymerases,we examined the replication of these constructs in HEK 293T cells either with knockout in human DNA polymerase (hPol ) expression, or with knockdown of hPol or hPol . For the tandem lesion, the rate of one base deletions increased from 19% in the HEK 293T cells to 26% and 34%, respectively, in the hPol and hPol knockdown cell lines; additionally, ~3% two-base deletions occurred in these pol knockdown cells. In contrast, neither the viability nor the deletion frequency was altered in hPol knockout cells. This suggests that the TLS polymerases and but not hpol play a critical role in generating full-length products from replication of this tandem lesion. In the full-length products,dA incorporation also was lowered from 81% to 71% and 63%, respectively, in the hPol and hPol deficient cells, suggesting that these polymerases are involved in dA incorporations opposite both L* and Tg. The biological implication of these results will be discussed. [Supported by NIEHS grant ES027558.]
TOXI 89.
Marie Bourgeois
Evaluation of postnatal Chlorpyrifos administration in rat pups
Abstract: Chlorpyrifos (CPF) is a widely used insecticide currently under EPA registration review. Several published studies have claimed neurologic effects in postnatal day 1 (PND1) rats following either intraperitoneal (ip) or subcutaneous injections of chlorpyrifos. The results suggested induction of bradycardia. However, the data used for these evaluations did not include direct measurement of heart rate. Indirect extrapolations were utilized. This study reports new data utilizing direct measurement of heart rate in PND1 rats. Low dose CPF was administered to PND1 rat pups and heart rates subsequently measured. CPF was administered in DMSO. Study groups included low dose ip injections of CPF. Body weights ranged from 7.5 to 8.5 grams. Groups of 5-6 PND1 rat pups were included in the DMSO and CPF comparisons. PND1 pups were administered 1 mg/kg body weight CPF in DMSO or 2 mg/kg body weight CPF in DMSO. Heart rates were measured at multiple timepoints using direct reading Visual Sonics. Non-invasive ultrasound imaging was used for the heart rate measurements. Saline control, DMSO control and CPF+DMSO groups were compared. CPF did not produce bardycardia at 30, 60 or 120 minutes following administration. Bradycardia was not observed in any of the PND 1 pups administered CPF.
TOXI 90.
Stanley K. Mauldin
Expression of the catalytic domain of DNA polymerase zeta from dictyostelium discoideum
Abstract: DNA polymerase zeta (Pol zeta) is a translesion DNA polymerase in which the enzyme will replicate over DNA damage at the replication fork. Pol-zeta mediated translesion synthesis is an important mechanism to prevent DNA replication fork collapse. Pol zeta is composed of two subunits REV3 and REV7. REV3 is the catalytic subunit, while REV7 is a regulatory subunit that stimulates REV3 activity. DNA pol zeta is categorized as a B family DNA polymerase in contrast to other translesion polymerases which are members of the Y-family. Dictyostelium discoideum is an excellent model system to study DNA repair. The organism is highly resistant to most known DNA damaging agents and, in many ways, mimics cancer cells that have become resistant to chemotherapeutic agents. The purpose of this study is to express in E. coli the 564 amino acid catalytic domain of the D. discoideum Pol zeta and purify the protein for structural determination. The structure of Pol zeta would then be compared to computerized molecular modelling of the D. discoideum Pol zeta. The nucleotide sequence of the D. discoideum Pol zeta catalytic domain (1692 bp) was obtained from the genomic database (DictyBase.org). Using this genomic information, primers were designed to PCR amplify the fragment. The amplified fragment was then cloned into pGEX-4T-1 expression vector using BamHI and XhoI at the 5’ and 3’ ends respectively. This expression vector will tag the Pol zeta with Glutathione-S-transferase (GST). The cloned fragment was then expressed in E. coli BL21 by induction with isopropyl-thiogalactoside (IPTG). An approximately 93 kilodalton protein was observed from several independent clones after SDS-PAGE. The presence of Pol zeta was confirmed using an antibody to the GST tag. Future work includes purifying the Pol zeta using glutathione-Sepharose chromatography and then preparing the protein for structural determination using either X-ray crystallography or cryo-EM.
TOXI 91.
Akrimah Akrimah
Synthesis and study of RNA oxidative damage product, 3′-deoxy-3′,4′-didehydrouridine-5′-aldehyde
Abstract: RNA is prone to oxidative damage due to the exposure of its single strand regions and high abundance in cells. RNA damage has been linked to the initiation of neurodegenerative diseases, such as Alzheimer’s, the disease etiology related to this oxidation is still unknown. Based on previous monomer studies that photochemically generate the 5′ uridinyl radical under aerobic conditions, it was found that a 5′ aldehyde and the base elimination product, uracil, were the major compounds identified when the reaction is performed at pH 7.0 and 3.5 in the presence of glutathione. In this study, the RNA oxidative damage product, 3′-deoxy-3′,4′-didehydrouridine-5′-aldehyde was synthesized and its reactivity towards natural nucleophiles such as glutathione under physiological conditions was analyzed by LC-MS and NMR.
TOXI 92.
Eun Ko
Unregulated Mitochondrial function exposed in Persistent organic pollutants Mixture
Abstract: Synthetic environmental toxins are widely distributed with adverse effect on human health. Persistent organic pollutants(POPs) are synthetic chemicals that are resistant to degradation through chemical, biological and photolytic process. Meanwhile, several animal models like mouse or rats are used to study the effect of toxic materials. Zebrafish, well-known vertebrate organism, has strength as novel animal model in research of disease or toxicology due to its rapid ontogeny process and easier genetic and proteinic manipulation than other animal models. Also, it is beneficial to examine several exposure-effect from its water-survival nature. Here, 5 kinds of persistent organic pollutants mixture (β-HCH, Heptachlor, Hexachlorobenzene, 4, 4’-DDT and Chlordane) are directly treated to isolated mitochondria of zebrafish’s liver to analyze its effect on mitochondrial activity. After adding POPs mixture, activity of each mitochondrial complex I, II, III and IV was measured immediately to examine the short-term exposing effect by spectrophotometric method. As a result, higher concentration of POPs treated mitochondria exhibited much more enhanced enzymatic activity than non-treated one except complex III. In the case of complex III, conversion of hexachlorobenzene contained in POPs into redox-active form of tetrachloro-1,4-benzoquinone in oxidative condition is competitively able to inhibit the Q binding site of mitochondrial complex III resulting in decreased enzymatic activity. Furthermore, competitive inhibitory action was confirmed by steady-state kinetic analysis of complex III with different concentrations of substrate and POPs mixture. Accordingly, production of ATP was decreased in POPs treated mitochondria compare to non-treated one. This study suggests that brief in-vitro effects of POPs on mitochondrial OXPHOS system and suitability of zebrafish as toxicological risk-assessment model to analyze function of mitochondria with toxins.
TOXI 93.
Natalia Yurievna Tretyakova
Epigenetic regulation via oxidized forms of Me C and inflammation-mediated epigenetic deregulation
Abstract: While the key role of 5-methyl-dC (MeC) in epigenetic regulation is widely recognized, the corresponding contributions of its oxidized forms 5-hydroxymethyl-dC (hmC) and 5-formyl-dC (fC) are incompletely understood. In cells, hmC and fC residues are generated via oxidation of 5-methylcytosines in DNA by ten eleven translocation (TET) dioxygenases. It has been reported that 5fC bases are recognized by thymine DNA glycosylases and are removed via the base excision repair pathway, suggesting that they serve as intermediates in DNA demethylation. However, recent evidence supports a possible direct role for oxidized forms of MeC in epigenetic regulation. 5fC and hmC have been found in regulatory genomic regions and reported to modulate DNA-protein interactions, to influence DNA structure and dynamics, and to interact with unique sets of protein “readers”. We recently discovered that the aldehyde group of 5fC residues in DNA and Lys or Arg side chains of proteins form reversible Schiff base conjugates under physiological conditions. Such covalent histone-5fC conjugates could help orchestrate reversible changes in chromatin structure and allow for protein recruitment to regulatory regions, ultimately influencing the levels of gene expression. Interestingly, global levels of hmC and fC are significantly decreased in many human cancers, potentially triggering epigenetic deregulation. We discovered that in two distinct mouse models, tissue inflammation can lead to epigenetic changes that mirror epigenetic changes observed in cancer. These results are suggestive of an important function of TET and oxidized variants of MeC in epigenetic regulation and in cancer.
TOXI 94.
Yael David
Reversible histone glycation drives disease-associated changes in chromatin architecture
Abstract: Cells are continuously subjected to an array of reactive chemical species which are produced both endogenously through metabolic pathways and taken up exogenously by diet and exposure to drugs or toxins. As a result, proteins often undergo non-enzymatic covalent modifications (NECMs) by these chemically reactive/toxic species, which can alter protein structure, function, stability and binding partner affinity. NECMs accumulate over time and are linked to various diseases such as Alzheimer’s disease, cancer and diabetes. In the cellular proteome, histones have some of the longest half-lives, making them prime targets for NECMs. In addition, histones have emerged as key regulators of transcription, a function that is primarily controlled by modification to their tails. These modifications are usually installed or removed enzymatically, but recent evidence suggests that some may also occur non-enzymatically. We found that the non-enzymatic reaction between sugars and amines (termed glycation), which is the hallmark of diabetes, specifically accumulates on histones. This pathophysiological modification has global ramifications on histone enzymatic modifications, the assembly and stability of nucleosomes, and the three-dimensional topology of chromatin. Importantly, we identified a physiologic regulation mechanism for restraining this damage in the form of DJ-1, which functions as a potent histone deglycase (Zheng Q. et al, Nature Communications, In press). Collectively, our results suggest a new mechanism for cellular metabolic damage through epigenetic perturbation, with far-reaching implications in pathogenesis and potential future therapeutics.
TOXI 95.
Aaron M Fleming
Base modifications in DNA non-canonical structures regulate transcription
Abstract: Regulation of transcription is a delicate cellular process with many checkpoints along the path from DNA to protein that are modulated during oxidative and inflammatory stress. One possible modulator of gene expression occurs when stress results in oxidative modification of guanine (G) to 8-oxo-7,8-dihydroguanine (OG) in G-rich gene promoter elements. A curious feature of some oxidation prone G-rich elements is their ability to adopt non-canonical structures such as G-quadruplexes (G4s) and i-motifs in the strand opposite, or Z-DNA. Studies in our laboratory have found G oxidation to OG in certain promoter regions can regulate gene expression via a shift in the structure to a G4 non-canonical fold. The NEIL3 DNA repair gene promoter possesses a potential G4 sequence sensitive to oxidation that induces transcription. A plasmid with the potential NEIL3 BG4 sequence driving a luciferase gene was transfected into mammalian cells and found to be activated under oxidative stress conditions. Studies in cellulo with OG synthesized into the reporter plasmid, as well as in vitro experiments on model sequences support the proposal of OG providing a target for the DNA repair protein OGG1 to activate the BG4 switch. Once switched APE1 functions in the initial steps to upregulate gene expression. The findings provide molecular-level details addressing one point at which the NEIL3 gene is upregulated during oxidative stress. Examples in other potential G-rich structure-switching sequences, like Z-DNA, will provide additional data supporting OG as a regulatory epigenetic-like modification in DNA.
TOXI 96.
Dong Wang
Transcription-coupled recognition of DNA lesions and endogenous epigenetic modifications
Abstract: During transcription elongation, RNA polymerase II (Pol II) moves along the DNA template strand and can encouter a number of obstructions, including covalent DNA lesions and endogenous epigenetic DNA modifications. These DNA modifications lead distinct transcriptional outcomes including transient transcriptional pausing and prolonged transcriptional arrest. Here we report our recent structural and functional studies to understand the molecular basis of transcription-coupled recognition of DNA lesions and endogenous modifications.
TOXI 97.
Yinsheng Wang
Targeted quantitative proteomic approaches toward understanding epitranscriptomic regulations
Abstract: RNA is known to harbor more than 100 distinct types of post-transcriptional modifications, which can modulate its splicing, localization, stability and translation. It was found that methylation at the N6 position of adenosine (m6A) in mRNA is involved in the epigenetic control of gene expression. Recent transcriptome-wide mapping also revealed the presence of m1A, pseudouridine, N7-methylguanosine, and 2¢-O-methylated nucleosides in mRNA. We reason that a more complete understanding of the roles of these RNA modifications in epitranscriptomics requires a better understanding about how the reader, writer, and eraser proteins are regulated, and how these RNA modifications modulate cell signaling. In this presentation, I will discuss our recent development of a parallel-reaction monitoring (PRM)-based quantitative proteomic strategy for monitoring the levels of expression of reader, writer, and eraser proteins of RNA epitranscriptomic marks, and the application of this method for assessing how exposure to toxic chemicals modulates the expression levels of these epitranscriptomic modifiers. In addition, the application of the method for profiling how these epitranscriptomics modifiers are altered during metastatic transformation of cancer will also be described. Moreover, I will discuss our investigation about how changes in expression levels of METTL3, the catalytic component of the methyltransferase for m6A formation, and m6A demethylating enzymes, including FTO and ALKBH5 alter the expression levels of small GTPases and kinases. The method allowed for a systematic discovery of small GTPases and kinases that are under m6A-mediated epitranscriptomic regulation. Together, our quantitative proteomic approaches allowed for comprehensive assessments about how exposure to toxic chemicals alters epitranscriptomic modifiers and about the systematic discovery of small GTPases and kinases that are under m6A-mediated epitranscriptomic control.
Magic of the chemosynthetic livers which is the best porphyrin
We report advances in proprietary in vitro green chemistry-based technology, mimicking in vivo metabolism of several chemical entities used in pharmaceuticals, cosmetics, and agrochemicals. Our catalysts enable prediction of metabolism patterns with soft-spot analysis and the methodology introduces new paradigms for drug discovery and drug-drug interactions for clinical diagnostics.
TOXI 99.
Chris Vulpe
Genome-wide CRISPR screening to identify modulators of formaldehyde toxicity in erythroid cells
Metabolites are implicated in adverse drug reactions, and are the subject of intense scrutiny in drug R&D. Present-day processes involving animal studies are expensive, labor-intensive and chemically inconclusive.
TOXI 100.
Hauh-Jyun Candy Chen
Higher advanced oxidative modifications in hemoglobin of oral cancer patients as measured by nanoflow liquid chromatography tandem mass spectrometry
Humans are continually exposed to various reactive oxygen species (ROS) due to endogenous aerobic metabolism as well as from environmental pollution. Excessive production of ROS plays an important role in disease onset and progression, including cancer. Post-translational oxidative modifications of hemoglobin have been used as surrogate biomarker for monitoring the oxidative stress in vivo. In this study, Fenton reaction was used as a model to generate ROS and to react with human hemoglobin in vitro. After trypsin digestion, the sites and types of modification were identified by nanoflow liquid chromatography nanospray ionization coupled with high-resolution mass spectrometry. In addition to oxidation at certain sites of Met, His, Tyr, and Lys, conversion of His to Asp and hydroxyl-Asp was also identified. Furthermore, advanced oxidation of His to hydroxyl Asp was identified at two sites. Because elevated oxidative stress is tightly associated with oral cancer, we measured the relative extent of modification at the identified sites were quantified in hemoglobin from oral cancer patients and compared them with those from healthy subjects. Our results showed that the extents of oxidation at -His-45, -Lys-99, and -His-77 in globin were significantly higher in oral cancer patients than in healthy subjects, while the extents of conversion of histidine residues at -20, -50, and -2 to aspartate were significantly lower. Moreover, the advanced oxidation of His to hydroxyl Asp at -His-50 and -His-2 are also significantly higher in oral cancer patients than in healthy subjects (p < 0.0005). To our knowledge, this is the first report on advanced oxidation of His to hydroxyl Asp in human hemoglobin. This advanced oxidative modification in hemoglobin might serve as potential biomarker to assess oxidative stress in oral cancer patients.
TOXI 101.
Vladimir Shafirovich
Competitive binding of human DNA glycosylase hNEIL1 and DNA damage-sensing factor XPC-RAD23B to oxidatively generated guanine lesions
Our catalysts (azamacrocycles) are sterically protected and electronically activated, providing speed, stability and scalability. We predict structures of metabolites, prepare them on a large scale by oxidation, and elucidate chemical structures. Comprehensive safety evaluation enables researchers to conduct more complete in vitro metabolism studies, confirm structure and generate quantitative measures of toxicity. We define an animal-free platform that identifies a more complete set of safety-relevant drug metabolites to accelerate the pace of drug discovery and development.
TOXI 102.
Michael P Stone
Probing chemical biology of DNA damage using NMR
We have utilized NMR to delineate the chemistry and biology of complex DNA damage arising subsequent to N7-alkylation of dG by environmental mutagens and chemotherapeutic agents. One manner in which N7-dG adducts rearrange is to form N6-(2-Deoxy-D-erythro-pentofuranoysl-)-2,6-diamino-3,4-dihydro-4-oxo-5-N-alkylformamidopyrimidine (AlkylFapy-dG). Using NMR, we probed the equilibrium chemistry of the Me-Fapy-dG and the AFB1-Fapy-dG adducts in DNA. These lesions equilibrate between or anomers, and for each anomer, atropisomerism may occur around the MeFapy-dG C5-N5 bond to produce Ra and Sa atropisomers. Each atropisomer also exhibits geometrical isomerism about the MeFapy-dG formyl bond yielding E and Z conformations. The relative abundances of these species have been determined in single strand and duplex DNA. These may be differentially recognized during DNA processing, providing a mechanistic basis for the generation of sequence-specific repair and replication processing. Initially formed N7-dG adducts also depurinate to form apurinic sites (AP sites) in DNA. AP sites, once formed, epimerize via a reactive aldehyde intermediate, which may conjugate with anthracycline anti-tumor agents such as doxorubicin, or form DNA interstrand cross-links (ICL), both of which are potential cytotoxic endpoints. Progress in utilizing NMR to examine the chemistry and biology of AP sites, including an ICL involving the conjugation of AP sites with the N6-dA in the complementary DNA strand will be presented.
TOXI 103.
Rachelle Joyce Bienstock
Molecular modeling of genotoxic azo dyes, Sudan I and Sudan II, and their metabolites
Polypharmacy, involving co-administration of several drugs, is common among the elderly and chronically ill. It is a risk factor for adverse drug reactions (ADRs) and drug-drug interactions (DDIs). One plausible DDI occurs when a drug interferes with another, causing irreversible changes to formation of metabolites from one or both. Such suppression or attenuation of metabolism could cause variances in toxicity and efficacy. We report experiments to predict and confirm modulation of oxidative metabolites from several combinations of common drugs for cancer, diabetes, hypercholesterolemia and hypertension in the presence of each other. Recent papers indicate best evidence for the dimerization of some compounds in dilute aqueous solution or assorted complex formation between disparate compounds.
TOXI 104.
Neelam Vaidya
Comprehensive toxicity information of every chemical for better R&D decisions
Abstract: In recent years there has been tremendous corporate and consumer interest in moving towards the commercialization of more sustainable products. Understanding the toxicity of products and their raw materials is a crucial for chemical hazard and risk assessment. Unfortunately, there is not enough trustworthy and comprehensive toxicity data that can help in avoiding toxic chemicals, understanding the risks involved in exposure to complex mixture of chemicals within laboratories, to consumers, and through toxic waste generated. Scientists in academia and industry that are responsible for product development processes usually not trained or well-equipped to make objective assessments of chemicals. There is, therefore, an urgent need for solutions that offer trusted, comprehensive, easily obtained and understood environmental, safety and health hazard information. This presentation will highlight a self-learning software platform that provides comprehensive chemical and toxicological information of over 90 million chemicals and a real-time hazard evaluation for new drug-targets or proprietary chemicals. It will describe how careful and constant data-curation and utilization of sophisticated read-across and deep-learning-neural-networks technologies can be used to achieve high accuracy of prediction not achieved in the past. Use of this platform will enable R&D scientists to make better decisions during product design and development.
TOXI 105.
Peter Byrley
Emission of respirable particles from fused deposition modeling 3D printers
Abstract: Fused deposition modeling (FDM) 3D printers, the most popular type of 3D printer among home hobbyists, have been shown to release volatile organic chemicals (VOCs) and billions of airborne particles per minute, indicating the potential for inhalation exposure and consequent health risks among 3D printer users including children. Publications on FDM 3D printer emissions, however, contain large heterogeneity of testing methods and analytical procedures making it difficult to reach overall conclusions for particle characteristics or particle number emission rates. We conducted a meta-analysis of published particle emission data collected during FDM 3D printer operation using two popular feed stocks: acrylonitrile butadiene styrene (ABS) or poly-lactic acid (PLA), expressed as either particle count diameters (PCDs) (nanometers), particle number concentrations (PNCs) (particles/cm3), or particle number emission rates (PNERs) (particles/min). The results showed a majority of particles were released as ultrafine in size (i.e., <100 nm), a size range capable of penetrating to deep lung tissues. A linear mixed model fit to mean PNCs for the studies indicated an association with nozzle temperature and filament material. Overall means of PNC data were found to be 300,980 particles/cm3 for ABS and 65,482 particles/cm3 for PLA, suggesting increased exposure to ultrafine particles from the use of ABS. Finally, the PNER calculation method varied widely across studies, especially regarding losses, and directly impacted the PNERs reported. It is recommended that standard emissions testing protocols be used for FDM 3D printers with particle influxes and losses more uniformly calculated. The potential health implications of inhalation of respirable particles from 3D printers will depend on accurate measurements of particle emission rates, as well as other factors such as particle composition, frequency of use, measures to mitigate exposures, and susceptibility of the exposed individuals, including children. This abstract does not reflect EPA policy.
TOXI 106.
Gunnar Boysen
Glutamine drives glutathione synthesis and contributes to radiation sensitivity of A549 and H460 lung cancer cell lines
Abstract: Increased glutamine uptake is known to drive cancer cell proliferation, making tumor cells glutamine-dependent. Studying lymph node aspirates containing malignant lung tumor cells showed a strong correlation between glutamine consumption and glutathione (GSH) excretion. Subsequent validation in A549 and H460 lung tumor cell lines provided additional evidence that glutamine drives GSH synthesis and excretion at molar amounts. Surprisingly, using stable isotope labeled glutamine as tracer metabolite, demonstrated that the glutamate group in GSH is directly derived from glutamine, linking glutamine utilization to GSH syntheses. To understand the possible mechanistic link between glutamine consumption and GSH excretion we studied GSH metabolism in more detail. Inhibition of glutaminase with BPTES and CB-839, two known inhibitors, essentially abolished GSH synthesis and excretion in lung tumor cells. Inhibition of glutaminase markedly radiosensitizes the lung tumor cell lines, suggesting an important role of glutamine-derived GSH in determining radiation sensitivity. In subsequent mouse xenografts, short term CB-839 treatment reduced serum GSH by >50% and increases response to radiation therapy of H460 derived tumors by 30%, suggesting a clinical relevant mechanism. GSH is the most abundant and most widely studied endogenous antioxidant and concentrations in tumor tissue have been reported to be as high a 10 mM. Extracellular GSH is metabolized by -glutamyl-transferase (GGT) to various -glutamyl-amino acid derivates. Further analysis of our clinical specimens and cell culture media and cell samples showed presence of various -glutamyl-amino acids. To elucidate why lung tumor cells excrete large amounts of GSH and make -glutamyl-amino acids we incubated none-transformed NIH3T3 and small airway epithelial cells (SAEC) with GSH and -glutamyl-glutamine. -Glutamyl-glutamine induced transformations in both cell lines, while GSH induced transformations only in GGT positive SAEC. Together these results provide evidence that lung tumors utilize glutamine to promote GSH synthesis to increase defense against radiation injury and excrete GSH to recruit neighboring cells producing a heterogeneous tumor phenotype.
TOXI 107.
Lisa A Peterson
Carbon dioxide enhances the pulmonary tumorigenic activity of the tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)
Abstract: Tobacco is a complex mixture of chemicals, some of which are toxic and/or carcinogenic. Animal studies designed to assess risk of tobacco smoke exposure have predominantly focused on either single chemical exposures or the more complex mixtures of tobacco smoke or its fractions. There are fewer studies investigating the interactions between specific tobacco smoke chemicals. Previous studies from our lab investigated the ability of inhaled formaldehyde or acetaldehyde to modify the pulmonary carcinogenic properties of NNK in A/J mice. These aldehydes significantly increased the number of lung adenomas with dysplasia and adenomas with progression without a significant enhancement of total lung adenomas. More recently, we tested the ability of a 3 h nose-only carbon dioxide (0, 5, 10 or 15%) co-exposure to influence lung adenoma formation by NNK (0, 2.5 or 7.5 µmol, i.p. in saline). Tobacco smoke contains 12.5% carbon dioxide and literature reports indicated that carbon dioxide inhalation can induce inflammation in the mouse lungs. While carbon dioxide alone did not increase lung adenomas in A/J mice, the co-exposure to carbon dioxide more than doubled the number of lung adenomas induced by 2.5 µmol NNK, with the maximal effect observed with 10% carbon dioxide (0%: 1.8 ± 1.9; 5%: 3.9 ± 2.8; 10%: 7.1 ± 3.5; 15%; 5.5 ± 2.9 lung adenomas/mouse). The co-exposure effect was less with 7.5 µmol NNK (0%: 11 ± 6.2; 5%: 13 ± 7.9; 10%: 18 ± 6.0; 15%; 12 ± 5.3 lung adenomas/mouse). These data indicate that carbon dioxide had a significant additive and synergistic effect on NNK-derived pulmonary adenomas (p value = 1.0 x 10-14). The mechanism of this interaction is under investigation. This study supports the hypothesis that the chemicals in tobacco smoke interact to form a more potent lung carcinogen [funded by CA-184987].
TOXI 108.
Michael J Calandra
Oxidative decarboxylation of 2-oxoacids by hydroperoxides can be used to lower peroxide values in citrus oils
Abstract: Many terpenes may autoxidize under certain conditions to form terpene hydroperoxides, which are reported to be sensitizers that may cause allergic contact dermatitis. The fragrance industry is currently required to monitor terpene hydroperoxide levels in many raw materials by iodometric titration (aka; the Peroxide Value, or POV test), and to reject lots that exceed a specification limit. We have found that compounds containing the 2–oxoacid moiety (the “pyruvic acid” moiety) react readily with organic hydroperoxides via an oxidative decarboxylation mechanism. The reaction products include an alcohol corresponding to the reduced hydroperoxide, carbon dioxide, and a carboxylic acid that is one carbon shorter than the starting 2–oxoacid. Because the hydroperoxide is irreversibly consumed by this reaction, the POV of a 2–oxoacid treated sample is effectively lowered. It follows that the skin sensitizing potential of the treated sample should also be lowered as a result of the hydroperoxide removal.