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Student & Post-Doctoral Scholar Symposium: Student & Post-Doctoral Scholar Symposium
08:00am - 10:00am USA / Canada - Eastern - August 23, 2021 | Room: B213 - B214
Erin Prestwich, Organizer, University of Toledo; Ujjal Sarkar, Organizer, MIT; Ujjal Sarkar, Presider, MIT; Erin Prestwich, Presider, University of Toledo
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Hybrid
Division/Committee: [TOXI] Division of Chemical Toxicology
Monday
Ribonucleotide containing DNA facilitates the identification of cross-linked amino acid residues in DNA protein cross links
08:00am - 08:15am USA / Canada - Eastern - August 23, 2021 | Room: B213 - B214
Jin Tang, Presenter; Wenxin Zhao; Linlin Zhao, University of California, Riverside
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Hybrid
DNA-protein cross-links (DPC) are important DNA modifications, resulting from endogenous and exogenous factors, such as reactive oxygen species, chemotherapeutics, ionizing radiation, and DNA repair. Generally, DPC are extremely toxic to the cell; however, occasionally they form as beneficial intermediates during DNA repair. DPC have broad applications in mapping DNA-protein interactions, isolating interacting complexes, and providing structural insights into macromolecular structures when used in combination with mass spectrometry (i.e., cross-linking mass spectrometry). Cross-linking mass spectrometry enables the identification of cross-linked amino acid residues but often limited by the sample preparation, ionization, and fragmentation of DPC. For example, enzymatic digestion of the nucleic acid components of DPC can be incomplete due to steric hindrance. Also, the labile chemical linkages in the nucleotide component can lead to ion suppression or insufficient peptide fragmentation, resulting in low confidence in identification of peptides and cross-linking sites. Here, we designed a ribonucleotide-containing DNA with the size-based enrichment workflow to circumvent the challenges in mass spectrometric analysis of DPC. The alkali-labile ribonucleotides in DNA facilitates the formation of defined nucleic acid structures in DPC and eliminates the need for enzymatic digestion. As a proof of concept, we applied the method to human mitochondrial transcription factor A (TFAM)-DNA cross-links and obtained high-quality fragmentation for TFAM peptides and identified the cross-linking sites. We envision the ribonucleotide-containing DNA can find its applications in mass spectrometry analysis and synthesis of structurally defined nucleic acid-protein cross-links.
Monday
Experimental study of Lumbricus teresstris to arsenic bioaccumulation
08:15am - 08:30am USA / Canada - Eastern - August 23, 2021 | Room: B213 - B214
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Hybrid
Arsenic (As) is widely spread in soil and is poisonous to plants, animals and humans. Arsenic contamination from both anthropogenic and natural origins is one of the biggest problems around the globe. The present research was aimed to find the bioaccumulation of arsenic in earthworms. To examine the bioaccumulation, Lumbricus terrestris were exposed to arsenic-contaminated soil having doses of 210, 270 and 330mg arsenic per 3kg of soil. Two control experiments and six treatment experiments with three replicates were carried out for one month. The accumulation of metals in digested samples were analysed by using atomic absorption spectrophotometer. The size of earthworms, morphological characteristics, and weight were examined and contrasted with different earthworms species along with initially recorded parameters. The biomass (length and weight) of the earthworms was increased in the control group and decreased in the treated groups. Dark colour patterns were observed in the skin that shows the accumulation of metals. Cocoon production reduced in the treated groups. All the parameters used in this study showed arsenic toxicity in earthworms. No change was seen in the behaviour of earthworms after the trial. No mortality occurred during the said period. Treatments were examined through Analysis of Variance and T-test. It was inferred that earthworms' bio-accumulative ability was directly proportional to the concentration of arsenic in the soil where they lived1.
Monday
Removal of common alterations in genomic DNA for accurate damage sequencing
08:30am - 08:45am USA / Canada - Eastern - August 23, 2021 | Room: B213 - B214
Nikolai Püllen, Presenter, ETHZ; Hailey Gahlon; Shana J. Sturla, ETH Zurich
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Hybrid
The structural integrity of DNA is constantly altered by exposure to metabolic byproducts, chemicals or radiation. Persisting DNA damage can result in mutations and genome instability. To better understand biological influences of DNA damage formation, new DNA damage sequencing methods are rapidly emerging. One common strategy for single-nucleotide resolution damage sequencing relies on having a reactive 3′-hydroxyl group at the damage position of interest. Such targeted 3′-hydroxyl ends are generated through enzymes to specifically excise damage products and produce interim apurinic sites and single strand breaks. However, such generic damage sites are by nature abundant in genomic DNA, making the possibility to uncover low abundance damage patterns without an effective background removal strategy impossible. Therefore, we used a ligation-mediated fluorescent probe-based damage quantitation approach to develop a strategy to minimize background damage in genomic DNA. Free 3′-hydroxyl groups in DNA were labeled by enzymatic incorporation of an alkyne-modified nucleobase and subsequently reacted with a fluorescent azide by click chemistry. By this approach, we could accurately detect enzyme-induced single strand breaks in oligonucleotide DNA in concentrations as low as 20 pM. We similarly processed genomic DNA and could detect elevated levels of 3′-hydroxyl groups on the basis of increased fluorescence intensity between enzymatically treated vs. native DNA. With an aim to remove pre-existing damage, we used an optimized enzyme mixture capable of repairing apurinic sites, gaps and single strand breaks in genomic DNA samples and validated the strategy by observing decreases in fluorescence signal compared to native genomic DNA. In conclusion, we developed an effective and general method for the quantitation and removal of adventitious common DNA damage products. Furthermore, the fluorescence-based ligation strategy used to optimize the enzymatic clean-up procedure could have wide use for developing sample preparation protocols for reliable DNA damage sequencing analysis.
Monday
Influence of food on the in vivo bioavailability of DDT and its metabolites in soils
08:45am - 09:00am USA / Canada - Eastern - August 23, 2021 | Room: B213 - B214
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Hybrid
Incidental soil ingestion is considered an important route of exposure to hydrophobic organic contaminants (HOCs), such as dichlorodiphenyltrichloroethane (DDT). Contaminant ingestion often occurs during food consumption, however, knowledge on the influence of food components on DDT bioavailability remains limited. In this study, the relative bioavailability (RBA) of soil DDTr (i.e., DDT and metabolites) was determined using an in vivo mouse model, in the presence of eight kinds of food including rice, egg, pork, pear, soybean, bread, spinach, and milk powder. The values of DDTr-RBA ranged from 19.8 ± 10.9 to 114 ± 25.1%. DDTr-RBA was positively correlated with fat (r=0.71), and negatively correlated with fiber content (r=0.63) in foods. A mechanistic study showed that fat enhanced micellarization and promoted the formation of chylomicron, which facilitated the dissolution and transport of DDTr in the intestinal tract. DDTr-RBA decreased with an increase in dietary fiber, possibly attributed to the role of fiber in the destruction of micelle formation. Bioaccessibility of DDTr was determined using a physiologically based in vitro method. The addition of lipase significantly improved the ability of the method to predict DDTr-RBA, indicating that the “fasted state” in vitro method required optimization for food scenarios. To the best of our knowledge, this is the first study to explore the mechanistic influence of food on DDTr-RBA and provide important knowledge on dietary approaches for reducing exposure to HOCs.

Monday
Fishing for damage: Recognition and repair of OG:A mispairs by MutY
09:00am - 09:15am USA / Canada - Eastern - August 23, 2021 | Room: B213 - B214
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Hybrid
Oxidation of guanine (G) to 8-oxo-guanine (OG) by reactive oxygen species can result in G:C to T:A transversion mutations in the genome due to the ability of OG to mispair with adenine (A). The DNA glycosylase MutY is responsible for recognition of the OG:A mispair and removal of the undamaged, but contextually miscoding A. The role of this enzyme in maintaining genomic integrity is highlighted by the cancer association of functionally compromised variants of the human MutY homolog (MUTYH); a syndrome known as MUTYH-Associated Polyposis (MAP). Elucidating the additional features of OG detection and A removal by MutY can provide a deeper understanding into why some MAP variants have compromised functions and how the enzyme can locate and process the OG:A mispairs. The crystal structure of Geobacillus stearothermophilus (Gs) MutY in complex with DNA containing a transition state analog (TSAC-OG:1N) identifies a hydrogen bond network that connects the C-terminal (CTD) OG recognition domain to the N-terminal (NTD) catalytic domain of the enzyme. In this study, we replaced OG with G to discover how MutY detects OG:A mispairs and obtained a new crystal structure of Gs MutY in complex with DNA containing G:1N (TSAC-G:1N). This structure highlights a serine residue (S308) in OG versus G specificity. Interestingly, S308 is part of a CTD loop comprising residues F307, S308 and H309, also known as FSH loop. The significance of Ser308 and its neighboring residues was further explored by mutational analysis to unravel the importance of this region for OG specific recognition in cells and in vitro. In fact, the FSH loop was found to be critical for function and OG specific recognition. These results suggest that FSH loop can be targeted as a druggable allosteric site for drug discovery.
Monday
Post-translational modifications (PTMs) regulate enzyme structure and function to expand the functional proteome; many of these PTMs are a direct result of cellular metabolism, serving as regulatory mechanisms. Specifically, modifications by the glycolytic by-product methylglyoxal (MGO) have been implicated in diabetic nephropathy and non-alcoholic fatty liver disease (NAFLD). Furthermore, decreased expression of glyoxalase I (GLO1), the major enzyme to detoxify MGO, has also been associated with metabolic disorders. Herein, we demonstrate the generation and characterization of Glo1 knockout mice (KO). When fed a high-fat, high sucrose (HFHS) diet, wild-type mice show significant hepatic lipid accumulation, liver injury, and serum alanine aminotransferase activity. These effects are blunted in KO mice. Furthermore, wild-type mice show significant glucose intolerance following this feeding regimen, an effect that is blocked in KO mice. Using QuARKMod to quantify the global Arg and Lys PTM landscape, we show that MGO-derived PTMs are significantly elevated in HFHS fed KO mice, while HFHS fed WT mice do not exhibit these changes. Our data support the growing notion that low levels of MGO may play a role in hormesis, finely tuning metabolism to restore homeostasis.
<b>Figure 1. Glucose tolerance is restored in KO mice </b>(a) The glyoxalase cycle. (b) Western blotting of WT and KO mice. (c) Body weight was measured once per week. (d) Change in body weight over the 16-week study in each cohort.

Figure 1. Glucose tolerance is restored in KO mice (a) The glyoxalase cycle. (b) Western blotting of WT and KO mice. (c) Body weight was measured once per week. (d) Change in body weight over the 16-week study in each cohort.

<b>Figure 2. MGO modifications and Lys acetylation are elevated in HFHS-fed KO mice. </b>(a) MGO-generated Lys and Arg PTMs. (b) Hepatic MGO levels. (c-e) MGO-derived liver PTMs are significantly elevated in HFHS-fed KO mice compared to HFHS-fed WT mice. (f) acLys is significantly elevated in HFHS-fed KO mice.

Figure 2. MGO modifications and Lys acetylation are elevated in HFHS-fed KO mice. (a) MGO-generated Lys and Arg PTMs. (b) Hepatic MGO levels. (c-e) MGO-derived liver PTMs are significantly elevated in HFHS-fed KO mice compared to HFHS-fed WT mice. (f) acLys is significantly elevated in HFHS-fed KO mice.


Monday
Analysis of post-transcriptional modifications in tRNAs associated with hyperglycemia in diabetes associated cells
09:30am - 09:45am USA / Canada - Eastern - August 23, 2021 | Room: B213 - B214
Taylor Dodson, Presenter; Chao Liu; Samuel Senyo; Erin Prestwich, University of Toledo
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Hybrid
Diabetes has many comorbidities including cardiovascular, renal, and hepatic diseases. Diabetic metabolic abnormalities can cause an overproduction of reactive oxygen species (ROS) in blood vessels and the myocardium, activating inflammatory pathways involved in diabetic cardiomyopathy. Additionally, ROS and insulin resistance have been reported to affect other tissues. Prolonged exposure to glucose (hyperglycemia) may produce increased intracellular levels of ROS which can cause sustained epigenetic changes driving the expression of proinflammatory genes. Thus, epigenomic profiles may give insight to the pathogenesis of diabetes and related diseases. Indeed, recent reports suggest that translation can be affected by the presence of certain tRNA modifications which can lead to decreases in insulin secretion. We developed a method to study tRNA post-transcriptional modifications in mammalian cells. Cultures of cardiac cell lines and other cells known to be affected in diabetes were maintained in either high glucose or low glucose conditions to mimic hyperglycemic or control conditions. Novel methods for nucleic acid isolation and analysis were developed and subsequently utilized on tRNA from these different cell types. After digestion to nucleosides, the tRNA was analyzed via liquid chromatography tandem mass spectrometry (LC-MS/MS) utilizing isotope dilution mass spectrometry using 13C labeled modified RNA nucleosides isolated from Pseudomonas aeruginosa. Preliminary data suggests that specific tRNA modifications are decreased or elevated in cells grown in excess glucose. Interestingly, some of these trends differ with cell type. We are continuing these studies to further investigate the effects of hyperglycemia to the epitranscriptome and explore the downstream biochemical consequences.
Monday
Uncovering the landscape of phosphorothioates in the human gut microbiome
09:45am - 10:00am USA / Canada - Eastern - August 23, 2021 | Room: B213 - B214
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Hybrid
There are now dozens of enzymatically-installed DNA modifications – the epigenome – in all forms of life, with the greatest diversity occurring in prokaryotes and bacteriophage. While bacterial DNA modifications are best known from restriction-modification systems, they are now known to regulate gene expression. Phosphorothioation (PT) of DNA, in which a sulfur atom replaces a non-bridging oxygen in the sugar-phosphate backbone, is the only known naturally occurring DNA backbone modification and is inserted by coordinated action of 4-5 enzymes encoded by the ssp and dnd families of modification and restriction genes. Using chromatography-coupled mass spectrometry (LC-MS), we recently found that 5-10% of the bacteria in the human gut microbiome possess PTs. Here we describe efforts to define the landscape of PT-possessing gut organisms using a combination of LC-MS, new next-generation sequencing (NGS) technologies, and metagenomic analysis. Nuclease digestion and LC-MS analysis of fecal DNA isolated from 11 healthy human donors revealed 8 of the 16 possible dinucleotide contexts, with each donor possessing an average of 2-3 PT dinucleotide species and wide variation among donors. A year-long time course in one donor revealed striking changes in PT levels over time. To identify organisms possessing PTs, fecal DNA was treated with iodine to convert PTs to strand-breaks and the breaks mapped by poly-T tailing NGS and bacteria identified by metagenomic analysis referenced to sequenced genomes of >9000 bacterial isolates from the gut microbiome. Ten PT sequence motifs from Bacteroidales, Clostridiales, and Enterobacterales were identified in the sequencing analysis of 3 donors. Although more studies are needed to define the determinants of the PT-containing gut microbiome, the redox-active and nucleophilic PT sulfur is known to affect bacterial fitness in the face of oxidative stress. Future studies will address the effects of inflammatory bowel disease on PT epigenetics in the human gut microbiome.
TOXI at 25: Chemical Toxicology Division Anniversary Symposium:
10:30am - 12:30pm USA / Canada - Eastern - August 23, 2021 | Room: B213 - B214
Sarah Shuck, Organizer, City of Hope; Michael Trakselis, Organizer, Baylor University; Michael Trakselis, Presider, Baylor University; Sarah Shuck, Presider, City of Hope
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Hybrid
Division/Committee: [TOXI] Division of Chemical Toxicology
Monday
Introductory Remarks
10:30am - 10:35am USA / Canada - Eastern - August 23, 2021 | Room: B213 - B214
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Hybrid

Monday
Sixteen years of catching DNA polymerases in the act of mistakes: What have we learned about DNA adducts?
10:35am - 10:53am USA / Canada - Eastern - August 23, 2021 | Room: B213 - B214
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Hybrid
Our laboratories published LC-MS methods for analysis of oligonucleotide primer extension products in 2005, along with our first X-ray crystal structure of the translesion DNA polymerase Sulfolobus solfataricus Dpo4 copying past the adduct 1,N2-etheno (ε) G. Subsequently we obtained crystal structures of Dpo4 mispairs with N2,3-εG, O6-MeG, O6-BzG, 8-oxoG, N2,N2-Me2G, and N2-Me naphthalene. These structures revealed a number of aspects of Dpo4, along with a study of kinetics with tryptophan mutants and proton-deuterium exchange work. We have solved a number of structures of human translesion DNA polymerases with DNA adducts, including: (a) pol k and 8-oxoG; (b) pol ι with O6-MeG and N2,3-εG; and (c) pol η with 8-oxoG, O6-MeG, 1,N6-εA, 1,N2-εG, an abasic (AP) site, and a ribonucleotide. Some interesting findings included the similarity of AP sites and 1,N6-εA in driving the insertion of purines and frameshifts. Although pol η has steric gate residues, it has activity with ribo sugars and is also able to copy ribonucleotide templates, i.e. reverse transcriptase activity. Current interests also involve DNA-protein crosslinks, although crystal structures are not yet in hand.
Monday
Impact of structurally distinct DNA lesions on the unwinding of double-stranded DNA by helicases
10:53am - 11:11am USA / Canada - Eastern - August 23, 2021 | Room: B213 - B214
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Hybrid
In human cells, environmental contaminants like polycyclic aromatic hydrocarbons (PAH) or aromatic amines (AA), are metabolized to highly reactive intermediates that bind chemically to nucleotides to generate DNA lesions. If these lesions are unrepaired, error-prone translesion bypass can give rise to mutations in critical tumor suppressor genes and oncogenes that can play a role in cancer etiology and cancer initiation. DNA helicases play important roles in the removal of genotoxic DNA lesions by the Nucleotide Excision Repair (NER) mechanism that employs a two-stage system for recognizing and excising DNA lesions. The first is the DNA damage-sensing factor XPC-RAD23B XPC), and the second is a helicase-driven unwinding mechanism that distinguishes between true chemical DNA damage and aberrant but nontoxic DNA lesions. While significant progress has been made in elucidating the structural features of DNA lesions that affect the XPC recognition step, much less is known about the impact of structurally different NER substrates on unwinding rates. This presentation will highlight recent work in our laboratory focused on the unwinding of selected DNA lesions by prokaryotic and human polymerases embedded in the NER factor TFIIH, as well as structural insights based on modeling and molecular dynamic simulation methods.
Monday
The role of pyridyloxobutyl DNA adducts in the carcinogenic properties of tobacco specific nitrosamines
11:11am - 11:29am USA / Canada - Eastern - August 23, 2021 | Room: B213 - B214
Lisa Peterson, Presenter, University of Minnesota Cancer Center: Masonic Cancer Center
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Hybrid
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N¢-nitrosonornicotine (NNN) are two tobacco smoke nitrosamines. NNK induces lung tumors and NNN causes esophageal tumors in laboratory animals. Both are listed as human carcinogens by the International Agency for Research on Cancer. NNK and NNN require hydroxylation adjacent to the nitrosamine group to exert their genotoxic effects. They both generate a pyridyloxobutylating agent that generates a variety of adducts including 7-[4-3-(pyridyl)-4-oxobut-1-yl]-guanine (7-pobG), O6-[4-3-(pyridyl)-4-oxobut-1-yl]-guanine (O6-pobG), O2-[4-3-(pyridyl)-4-oxobut-1-yl]thymidine (O2-pobT), and O2-[4-3-(pyridyl)-4-oxobut-1-yl]-cytosine (O2-pobC). Mechanistic studies in cell lines and laboratory animals have provided important clues as to how these adducts contribute to the carcinogenic properties of NNK and NNN. This talk will review the highlights from 25 years of research on this subject.
Monday
Intermission
11:29am - 11:34am USA / Canada - Eastern - August 23, 2021 | Room: B213 - B214
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Hybrid

Monday
Recent studies on phenanthrene and acrolein biomarkers in cigarette smokers and non-smokers
11:34am - 11:52am USA / Canada - Eastern - August 23, 2021 | Room: B213 - B214
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Hybrid
Phenanthrene (Phe) is a polycyclic aromatic hydrocarbon (PAH) with a bay region but unlike many of its higher homologues it is non-carcinogenic, and its metabolites are mainly excreted in urine. We have used urinary Phe metabolites as biomarkers of uptake plus metabolic activation of PAH. This is possible because Phe forms bay region diol epoxides structurally similar to those of the highly carcinogenic benzo[a]pyrene (BaP) but unlike BaP diol epoxides, Phe diol epoxides are non-carcinogenic. Phe diol epoxides are hydrolyzed in vivo and excreted in urine as Phe tetraols (PheT), biomarkers of exposure plus metabolic activation of PAH, with concentrations in human urine far greater than those of BaP tetraols. We developed a strategy in which deuterated Phe ([D10]Phe) was administered to humans and excretion of [D10]PheT and Phe phenols ([D9]PhOH) were monitored. In a clinical study of 170 cigarette smokers and 57 non-smokers, we demonstrated that the [D10]Phe bioactivation ratio was 2.3-4.8-fold higher (P<0.01) in smokers than non-smokers showing for the first time in humans that induction of aryl hydrocarbon hydroxylase activity by smoking results in increased metabolic activation of PAH. Acrolein is a highly toxic and mildly carcinogenic constituent of cigarette smoke. We have used its urinary metabolites and tissue DNA adducts as biomarkers of acrolein exposure and possibly cancer risk. In one recent study, we developed a high resolution mass spectrometric method for determination of acrolein DNA adducts and etheno-DNA adducts in oral cells of cigarette smokers and non-smokers. Levels of these adducts in cells obtained by oral rinse were more than 20 times higher in smokers than non-smokers demonstrating a large effect of smoking on DNA damage in the oral cavity. Collectively these studies provide some important new insights on mechanisms of carcinogenesis in cigarette smokers.
Monday
Chemical toxicology of human gut microbiota
11:52am - 12:10pm USA / Canada - Eastern - August 23, 2021 | Room: B213 - B214
Shana J. Sturla, Presenter, ETH Zurich
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Hybrid
Metabolic transformations have a profound impact on susceptibility to the effects of drugs and chemicals. For this reason, a core focus in Chemical Toxicology involves delineating how enzymatic processes in various types of human tissue and fluids control the fate and disposition of chemicals in the human body. Yet, various chemicals and drugs reach the human colon in either unchanged or metabolized forms where they can undergo a completely new slate of toxicologically relevant reactions catalyzed by bacterial enzymes. Moreover, the complex ecological dynamics of human gut microbial communities appear to be a basis of inter-individual variations in responses to drugs and chemical exposures and susceptibility to disease. Understanding the toxicological relevance of the gut microbiome in chemical disposition and toxicity requires insight on the chemical transformation capacities of the human gut microbiome, the uptake potential of microbial metabolites, and the influence of microbial metabolites on the host. The presentation will highlight emerging examples and experimental approaches integrating these aspects of chemistry, functional genomics, and toxicology. The findings suggest that gut microbial transformation have a strong potential to reduce toxicological impacts and these approaches can help to evaluate the fate and impacts of microbial metabolites produced in the human gut.
Monday
Adventures of a chemist in toxicology: DNA adducts, epigenetic deregulation, and proteins trapped on DNA strands
12:10pm - 12:28pm USA / Canada - Eastern - August 23, 2021 | Room: B213 - B214
Dr Natalia Tretyakova, Presenter, University of Minnesota
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Hybrid
In all living systems, genetic information is encoded by a simple chemical code of four major nucleotide bases (A, T, G, and C). However, up to 2% of the nucleobases in our genome are not in their canonical form, but are instead structurally altered. Some of these bases are intentionally modified by cellular enzymes to regulate specific genes; other bases are inadvertently modified as a result of exposure to reactive agents present in our environment to form DNA adducts. For example, cytosine and adenine bases of DNA can be enzymatically methylated and demethylated to regulate gene expression in cells and tissues; this process known as “epigenetics” is essential for normal development, immune response, and DNA repair. On the other hand, unintentional nucleobase modifications (DNA adducts) that occur as a result of environmental exposures such UV light and dietary/environmental chemicals can cause errors during DNA replication, leading to heritable mutations and the development of cancer. Specifically, proteins can become trapped on DNA strands to generate bulky DNA-protein crosslinks. My laboratory investigates both naturally occurring DNA modifications and toxic/mutagenic DNA adducts. In this presentation, I will describe our efforts to identify novel DNA modifications, to elucidate their biological impacts, and to develop ultra-sensitive methodologies for their detection in humans.
Thinking Outside the Well: Novel Assays in Drug Discovery & Development:
02:00pm - 03:50pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 49
Donna Huryn, Organizer, University of Pittsburgh; Michael Walters, Organizer, The University of Minnesota; Donna Huryn, Presider, University of Pittsburgh; Michael Walters, Presider, The University of Minnesota
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Virtual
Division/Committee: [TOXI] Division of Chemical Toxicology
Monday
Introductory Remarks
02:00pm - 02:05pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 49
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Virtual

Monday
Scaling up: Zebrafish behaviors as scalable phenotypes for small molecule screening
02:05pm - 02:25pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 49
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Virtual
Phenotypic screening is a powerful approach for discovering the therapeutic and toxic effects of small molecules. Although cellular phenotypes are frequently used for screening, they are relatively limited in their utility for modeling nervous system function. We have therefore focused on zebrafish behaviors as readouts for phenotypic screening, including such diverse behaviors as threat responses, light-evoked responses, opioid self-administration, and response to social stimuli. Sociality is broadly conserved across the animal kingdom, facilitating cooperation, reproduction, and protection from predation. In humans, social dysfunction is a hallmark of several neurodevelopmental disorders, including autism spectrum disorder. To systematically identify environmental factors that influence development of sociality, we established an automated screening system named Fishbook to search for chemicals that disrupt development of social behavior in zebrafish (Danio rerio). A screen of 1120 drugs revealed that the α isoform of topoisomerase II (Top2a) is required for zebrafish social behavior development. In mice, prenatal exposure to the Top2 inhibitor ICRF-193 caused lasting behavioral deficits related to core symptoms of autism without affecting other major behavioral categories. Top2a depletion in zebrafish selectively altered expression level and epigenetic status of autism related genes, and a custom upstream analysis pipeline unveiled Ezh2 and H3K27me3 as the key mediators of this effect. Together, these findings identify Top2a as a key component of an evolutionarily conserved molecular mechanism that regulates the development of social behavior.
Monday
Zebrafish assays to drive medicinal chemistry: Challenges and opportunities
02:25pm - 02:45pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 49
Donna Huryn, Presenter, University of Pittsburgh
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Virtual
Assays in whole organisms like zebrafish present great opportunities to generate multi-factorial data, including biological efficacy, toxicity, and drug-like properties. However, these assays also provide some challenges when trying to interpret structure-activity relationships (SAR) during medicinal chemistry optimization efforts. While medicinal chemists can use a number of validated metrics and in vitro assays to predict permeability through mammalian cells, there is little information on the properties of molecules that permeate zebrafish. Therefore, during medicinal chemistry optimization programs, when relying on data from zebrafish assays, it can be difficult to differentiate compounds that are inactive from compounds that are not permeable. This talk will highlight a study that suggested guidelines to predict zebrafish absorption, and application of these metrics to a medicinal chemistry optimization program
Monday
Go with the flow and reduce animal testing: Application of microphysiological system (MPS) technology using liver-on-chips to genotoxicity testing
02:45pm - 03:05pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 49
Dr Thalita Boldrin Zanoni, Presenter, Charles River Laboratories; Ammer Khawam; Karen DiPerna; Annie Hamel
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Virtual
Divergence across animal species metabolism is a frequent cause of unexpected toxic effects that contribute to drug development failure. Since great part of genotoxicants require metabolic activation, the current pre-clinical genetic toxicology test battery evaluates this by using in vitro assays with an exogenous rat metabolic system (S9), and with in vivo experiments using rodents. The objective of this study was to create a metabolically human competent Liver-on-chip (LOC) with improved species-and organ specific metabolism that would allows for communication between two compartments and that could be applied to genotoxiciy testing. For this, two LOC , one comprised with primary human hepatocytes (PHH-LOC) an another with HepaRG cells (HepaRG-LOC) were tested and co-culture with human lymphoblastoid (TK6 cells). Both systems allowed the performance of multiple endpoints analysis i.e. the comet assay (% tail DNA intensity) in liver cells and the micronucleus (MN) assay in TK6 cells. Both LOC (extracellular matrix, endothelial cells, PHH or HepaRG and matrigel) were maintained under CN BIO® microfluids flow technology, with TK6 cells cultured on top in transwells. Both compartments were treated for 0, 24 and 45 h with either negative control, and with diferent concentrations of the direct mutagens methanesulfonate (MMS 2.5, 5 and 10 µg/mL) and ethyl metanesulfonate (EMS 5, 10 and 25 µg/mL ) or with indirect mutagens benzo[a]pyrene (BaP at 10 and 20 µg/mL) and cyclophosphamide (CP 50, 100 and 150 µg/mL). Three independent experiments were performed and the liver and TK6 cells were collected. Hepatocytes functions were evaluated by quantification of urea and albumin in the culture media. The results suggested that hepatocyte functions were well maintained in the system. Furthermore, low levels of DNA damage (% tail DNA intensity) in the negative control and a dose related statistically significant increase in all tested positive controls were observed when compared to the negative control, except for CP (data compatible with in vivo results). Furthermore, increase in the % MN after treatment with CP, MMS, EMS and BaP were observed when compared to the negative control. In conclusion, both LOC showed metabolizing properties and the capability of adressing two genotoxic adverse outcomes within one system, i.e. induction of chromosomal damage or damage to the mitotic apparatus (micronucleus test) and DNA strand breakage (comet assay).
Monday
Complex (not complicated) in vitro scale-able models for phenotypic drug screening
03:05pm - 03:25pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 49
Banupriya Sridharan, Presenter, GlaxoSmithKline; Jason Ekert
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Virtual
Traditional HTS that utilize lab-adapted cell lines in high density cell plates do not sufficiently recapitulate the complex biological environment and thus do not translate well to in vivo and clinical outcomes. Complex models on well-plates such as iPSC-derived cell types, patient organoids, and multicellular systems are increasingly adopted as they are more predictive, disease relevant, and will eventually reduce the drug attrition rates when employed early in the drug discovery & development process. As an exemplar of scale-able human-relevant complex model for screening, we systematically developed a triculture system of non-small cell lung cancer (NSCLC) incorporating cancer associated cells such as lung fibroblasts and bronchiolar epithelial cells, and, components of the extracellular matrix (ECM), in the context of drug combination screen with Poly(ADP-ribose) polymerase (PARP) inhibitors. Tumor cell cytotoxicity was assessed via inherent fluorescent expression by high content analysis (HCA) across drug resistant and sensitive cell lines yielded excellent Z’ ( >0.5) and S:B ( >30) that can support a long-term (7 day) combinatorial drug screening approach thus highlighting the impact of testing in a more complex but translationally relevant system.
Monday
Drug screening using 3D Biofabricated tissues
03:25pm - 03:45pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 49
Marc Ferrer, Presenter
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Virtual
A wide range of in vitro 3D organotypic cellular models are being developed for scientific research and preclinical drug efficacy and safety testing. The hope is that these in vitro complex 3D cellular models will closely mimic human physiology and pathology and predict clinical responses more accurately than the cellular assays currently used in drug discovery and development. The integration of these 3D organotypic cellular models into the drug discovery and development pipeline requires rigorous scientific validation, including cellular, morphological, and functional characterization; benchmarking of clinical biomarkers; and operationalization as robust and reproducible screening platforms. It is critical to establish the degree of physiological complexity that is needed in each 3D organotypic model to accurately reproduce native-like homeostasis and disease phenotypes, as well as clinical pharmacological responses. Choosing which 3D organotypic model to use at each stage of the drug discovery and development pipeline will be driven by a fit-per-purpose approach, based on the specific disease pathomechanism to model and screening throughput needed. This presentation will illustrate the work being done by the 3D Tissue Bioprinting Laboratory at the NIH Center for Advancing Translational Sciences (NCATS) to develop and operationalize the use of 3D organotypic models as physiologically relevant assay platforms for safety and efficacy testing in drug discovery and development pipelines.
Monday
Concluding Remarks
03:45pm - 03:50pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 49
Division: [TOXI] Division of Chemical Toxicology
Session Type: Oral - Virtual

General Posters:
11:00am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Virtual Room
Erin Prestwich, Organizer, University of Toledo; Ujjal Sarkar, Organizer, MIT; Grover Miller, Organizer, Dept of Biochem and Mol Biol
Division: [TOXI] Division of Chemical Toxicology
Session Type: Poster - Virtual
Division/Committee: [TOXI] Division of Chemical Toxicology

This poster session can be accessed by going the the TOXI Divisions website: http://www.acschemtox.org/

Tuesday
Amyotrophic lateral sclerosis (ALS): What do we really know about the role of the environment?
11:00am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Virtual Room
Melanie Newell, Presenter; Sangeet Adhikari, Arizona State University; Rolf Halden
Division: [TOXI] Division of Chemical Toxicology
Session Type: Poster - Virtual
Even as incidence and prevalence for Amyotrophic lateral sclerosis (ALS) are increasing in the United States, the motor neuron disease will continue to be considered an orphan disease for many generations. People 65 years and older residing in the United States can expect to live another 10-20 years on average, but an ALS diagnosis currently cuts short the period of time remaining for 50% of patients to 5 years or less. We conducted a systematic literature review using PRISMA methodology to survey hypothesis-driven, cohort, case-controlled, population-based studies that explored the role of environmental factors. A low article-to-review ratio of approximately 4 articles for every review paper suggests a need to evaluate how the field assesses environmental factors for association or causal relationships to ALS. We applied the Bradford Hill criteria to reclassify environmental factors for causality or association with current findings in the literature. Results ranked the cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA), formaldehyde, selenium, and heavy metals, specifically mercury, manganese, and zinc and copper among the most likely to be causal. Significant gaps in research for even the highest-ranked environmental factors include a lack of metabolic pathway specificity and human cohort studies. A further survey into model research and studies with a geospatial focus indicates gaps in our ability to align etiology pathways with epidemiology between animals and humans, as well as across geographic regions. Reported incidence rates predict a high number of cases in at-risk groups (ages 65-80) relative to population size in Scotland (incidence rate in units of cases per 100,000 people per year of 3.9 per 100,000 person-years predicts 282 incidences), France (incidence rate of 3.2 per 100,000 person-years predicts 304 incidences), and Japan (incidence rate of 0.69 per 100,000 person-years predicts 165 incidences) if extended across the country. Meanwhile, very little is known of incidence rates outside of Europe and the Western Pacific. If future research begins to focus resources toward the highly ranked environmental toxins, energy may be subsequently spent associating these triggers to changes in the genome or epigenome.
Tuesday
Docking study on the position of nitro groups affecting the mutagenicity of nitroarenes
11:00am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Virtual Room
Division: [TOXI] Division of Chemical Toxicology
Session Type: Poster - Virtual
Nitro-polycyclic aromatic hydrocarbons (NO2PAHs) generated and released in combustion systems are environmental pollutants with strong mutagenic and carcinogenic properties. The mutagenic nitro derivative of benzo(a)pyrene (BaP) includes 1- and 3-nitroform, 1,6- and 3,6-dinitroisomers. Compared to the mononitro form, the dinitro form is a more potent mutagen. The mutagenicity of mono- and dinitro-BaP with a nitro group at position 3 is much more potent than that with the nitro group at position 1. The nitro group of NO2PAHs is reductively metabolized to hydroxylamine (HONPAHs) and then acetylated by acetyltransferase (AcONPAHs) to form nitronium ions (+NPAHs), which bind mainly to the guanine 8 positions of DNA. Since the nitro group is reductively activated, the nitro group's reductive property is useful in evaluating mutagenicity. However, the difference in mutagenicity between the 1-nitro and 3-nitro compounds cannot be explained by the reducing property. We hypothesized that the difference in the position of the nitro group would significantly affect the DNA-binding mode of the reductively activated product. Therefore, we explored the stable conformation of AcONPAHs for each nitro compound by docking calculations targeting the DNA base-pair layers. We optimized the docking pose structure in which the distance (dis) between the nitrogen of AcONPAHs and the 8-position of guanine is 5 Å and obtained the distance of the structure with the minimum binding free energy. As a result, mono- and dinitro-BaPs with a nitro group at the 3-position rather than the 1-position are more likely to form an adduct with DNA due to the closer distance between the reductively activated form and the guanine 8-position. For both 1- and 3-nitro-BaP with a cyano group at 6-position, the reductively activated form of the 3-nitro compound, which has strong mutagenic activity, was closer to the guanine 8 positions. These results indicate that the strength of mutagenicity of NO2PAH is greatly influenced not only by the reduction properties of the nitro group but also by the distance between the metabolic activator and DNA.
Tuesday
C-Methylated fisetins with strong antioxidative activities
11:00am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Virtual Room
Division: [TOXI] Division of Chemical Toxicology
Session Type: Poster - Virtual
Although the beneficial effects of fisetin, a natural antioxidant found in strawberries, on prevention and treatment of oxidative stress-related diseases have become broadly recognized, the radical scavenging activities of fisetin required for direct neutralization of free radicals generated during disease progression are not that strong. In this study, fisetin derivatives with electron-donating methyl substituents at the ortho positions relative to the catechol hydroxyl groups were synthesized, and their radical scavenging activities were evaluated. The radical scavenging activity of fisetin increased with the introduction of a methyl group, and especially 5’-methyl fisetin showed the most potent activity. Molecular orbital calculations revealed that the planarity of the entire molecule of 5’-methyl fisetin was maintained, while the B ring of 2’-methyl and 2’,5’-dimethyl fisetin was twisted relative to the AC ring due to the steric hindrance of the 2'-methyl group. The dissociation energy (BDE) and ionization potential (IP) of the O-H bond of 5’-methyl fisetin, which has a strong radical-scavenging effect, were both the lowest among these compounds. The radical scavenging activity of these methyl derivatives showed a good negative correlation with IP, indicating that the radical scavenging reaction proceeds by a one-electron reduction reaction.
In conclusion, introducing a methyl group into the ortho-position of the catechol structure of fisetin showed potent radical scavenging activity. The strongest radical scavenging activity of 5’-methyl fisetin is attributed to its coplanar structure, which is effective in the delocalizing radical cation formed by the one-electron transfer reaction. Due to the potent radical scavenging activity resulting from the inherent planar structure, 5’-methyl fisetin would exhibit not only the variety of biological activities of fisetin but also potent antioxidant activity. The simple derivatization by methyl substituents makes it possible to improve the radical scavenging activity without causing a destructive change in the overall structure that may be significantly associated with its inherent biological activities. This strategy is likely to be of value in medicinal chemistry applications.

Tuesday
Withania somnifera metabolites withanone, withaferin A, and their GSH conjugates are potential irreversible inhibitors of the SARS-CoV-2 protease Mpro
11:00am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Virtual Room
Division: [TOXI] Division of Chemical Toxicology
Session Type: Poster - Virtual
The current COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has created an unprecedented global crisis since December 2019. Because of emerging new strains, virulence, and morbidity of SARS-CoV-2, there is an urgent need for an effective cure. Although vaccines have been approved, there are concerns regarding the time frame of their effectiveness and against emerging new strains. SARS-CoV-2 being an RNA virus is prone to accrue mutations. With the extensive spread of SARS-CoV-2 infection and the selection pressure coming from vaccines and currently used drugs, the evolution of new virulent strains is inevitable. Moreover, there are numerous coronaviruses in nature that have the potential to mutate and jump species to infect humans. Hence, a broad-spectrum antiviral effective against coronaviruses, in general, is necessary. New drug candidates including those designed using AI, mini-, and decoy-proteins are being pursued although it might take years to reach the clinics. Repurposing existing drugs is a quicker approach, however, considering they are typically designed and developed for specific diseases, success depends on a rare “chance” factor. Herbal medicines, extracts, and their active ingredients, which are used for years, provide a reasonably safe alternative. Withania somnifera (Ashwagandha), is a medicinal plant used for thousands of years in India against various diseases. Withanone (win) and withaferin A (wifA) are two metabolites of Withania somnifera having epoxide and Michael acceptor functional groups. MPro is a ubiquitous coronavirus protein with no human homolog which renders it a lucrative target for antiviral drug development. Although a plethora of potential reversible and irreversible inhibitors (including aldehydes, Michael acceptors, and epoxides) of Mpro has been identified, none has been approved. Herein, using experimental and docking studies, we show that not only win and wifA can for adducts with cysteine and glutathione (GSH) but can also act as covalent inhibitors of Mpro. Our studies also show that the GSH adducts of win and wifA has the potential to inhibit Mpro. We propose to win, wifA and Withania somnifera extract as preventative and therapeutic interventions for COVID-19.
Tuesday
Monitoring the formation and repair kinetics of cisplatin-induced DNA-DNA cross-links by comprehensive ultra-performance liquid chromatograph-selective ion monitoring (UPLC-SIM)
11:00am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Virtual Room
Dr. Arnold Scott Groehler IV, Presenter, IBS Center for Genomic Integrity; Anuar Makhmut; Nhat Mai Dao; Orlando Schärer
Division: [TOXI] Division of Chemical Toxicology
Session Type: Poster - Virtual
Cisplatin is a platinum-based chemotherapy drug used in the treatment of testicular, ovarian, cervical, lung, and breast cancer. The anti-tumor activity of cisplatin is attributed to the formation of DNA 1,2- and 1,3-intrastrand and interstrand cross-links. Despite its clinical utility, the development of resistance and toxic side effects remain big challenges associated with cisplatin therapy. Potential mechanisms of resistance include changes in drug influx and efflux, cisplatin metabolism/activation, and repair rates of cisplatin-induced DNA damage. All three resistance mechanisms influence the abundance and distribution of DNA-DNA cross-links. Therefore, we hypothesize that the biological response to cisplatin can be accurately predicted by measuring the formation and repair of these cisplatin DNA cross-links in cells or tumors.

To address this goal, we have developed an isotope dilution UPLC-SIM assay to quantify cisplatin-induced 1,2-GG, 1,2-AG, 1,3-GCG, and 1,3-GTG intrastrand and interstrand crosslinks. The DNA digestion and monomer enrichment methodology for each analyte were optimized using synthetic DNA oligonucleotides containing specific cross-links and platinated calf thymus DNA. We then monitored the formation and repair of cross-links over time in nucleotide excision repair (NER)-deficient cell lines and isogenic control cell lines and are extending these studies to additional cell lines with mutations in DNA repair genes. Successful application of this assay in cells is expected to be useful in clinical practice to predict the outcome of cisplatin therapy.

Tuesday
Role of Aldo-Keto reductases in the nitroreduction of 1-nitropyrene and 1,8-dinitropyrene
11:00am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Virtual Room
Dr. Anthony L Su, Presenter, University of Pennsylvania; Trevor Penning
Division: [TOXI] Division of Chemical Toxicology
Session Type: Poster - Virtual
1-Nitropyrene (1-NP) and 1,8-dinitropyrene (1,8-DNP) are nitroarenes commonly found in diesel exhaust and classified as either probable or possible human carcinogens by IARC. Significantly, both compounds require metabolic activation by nitroreduction to generate metabolites capable of forming DNA adducts. Xanthine oxidase (XO) has been identified as one enzyme implicated in the nitroreduction of 1-NP and 1,8-DNP to form DNA adducts in rat liver; however, the enzymes involved in this process in human lung remain to be elucidated. We have previously shown that human aldo-keto reductases (AKR1C1, AKR1C2 and AKR1C3) and NAD(P)H quinone oxidoreductase 1 (NQO1) catalyze the nitroreduction of another diesel exhaust nitroarene, 3-nitrobenzanthrone (3-Nitro-7H-benzo[d,e]anthracen-7-one, 3-NBA), raising the possibility that these enzymes could be involved in the nitroreduction of 1-NP and 1,8-DNP. Furthermore, studies have shown that AKR1C1, AKR1C2 and AKR1C3 are induced by the Nrf2-Keap1 pathway. In the current study, we showed that human recombinant AKR1C1 catalyzed 1-NP nitroreduction using a UV-HPLC-based discontinuous assay that measured the formation of 1-aminopyrene (1-AP), a product of 1-NP nitroreduction. By contrast, AKR1C2 and AKR1C3 catalyzed 1-NP nitroreduction to a more limited extent, while AKR1A1, AKR1B1, AKR1B10, AKR1C4, AKR1C9, AKR1D1, AKR7A2, AKR7A3, and NQO1 had negligible nitroreductase activity with 1-NP as substrate. Investigation of 1,8-DNP nitroreduction showed that AKR1C3 was capable of producing 1-amino-8-nitropyrene (1,8-ANP); however, the reaction failed to go to completion within 24 hours. Other tested enzymes minimally catalyzed 1,8-DNP nitroreduction. Interestingly, our work showed that purified XO catalyzed 1-NP and 1,8-DNP nitroreduction poorly under either aerobic or anaerobic conditions. By contrast, XO catalyzed the robust reduction of cytochrome c. Ongoing work will further elaborate the role of nitroreductases in the metabolic activation of 1-NP and 1,8-DNP in human lung cells.
Tuesday
Mechanism of DNA damage by the anticancer agent sepantronium bromide (YM155)
11:00am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Virtual Room
Division: [TOXI] Division of Chemical Toxicology
Session Type: Poster - Virtual
Sepantronium bromide (YM155) is a small molecule that selectively inhibits the expression of anti-apoptotic protein survivin. Being a nodal protein specifically expressed in transformed and cancer cells, survivin is considered a lucrative anti-cancer drug target. This prompted several phase I and II clinical trials with YM155 for the treatment of various cancers. However, based on several reports of this drug causing DNA damage in tumor cells, there have been concerns regarding its actual function. Recently, we have shown that YM155 triggers reactive oxygen species (ROS) generation through multiple mechanisms including redox cycling and mitochondrial membrane depolarization that in turn causes DNA damage and survivin suppression. We have also demonstrated an additional reductively activated, radical-mediated pathway of DNA damage by YM155 that is distinct from that of typical quinones and involves the abstraction of hydrogen atom by the drug radical. A clear-cut understanding of the mode of action of YM155 is absolutely necessary for determining its therapeutic efficacy. Therefore, we have performed further chemical analysis of the drug-induced DNA damage. End-product analysis of the YM155-damaged DNA in the presence of catalase has revealed selective abstraction of C4’-H atom. Unlike hydroxyl radical, DNA damage by YM155 in the presence of catalase does not involve C1’ and C5’-H abstraction. Taken together, these indicate that YM155-mediated DNA damage in the presence of catalase does not involve hydroxyl radical and supports our previously proposed alternate pathway.
Tuesday
DNA adducts as molecular markers of aging
11:00am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Virtual Room
Division: [TOXI] Division of Chemical Toxicology
Session Type: Poster - Virtual
DNA damage occurs continuously in cells of living organisms and can disrupt genomic integrity. While most damaged nucleotides are repaired, DNA adducts have been postulated to accumulate and possibly participated in the aging process. Most evidence of DNA adducts accumulating with age have been indirect rather than direct chemical quantification. Here we characterize tissue-specific accumulation of DNA adducts both in rat tissues and human non-failing heart tissue. The characterization and identification of age-related DNA adducts was addressed using a high-throughput stepped MRM approach scanning a wide range of DNA modifications losing m/z 116, the mass of the deoxyribose (from transition m/z 225 → 109 to transition m/z 525 → 409), using high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Most DNA adducts did not accumulate with age, but in each tissue, some specific adducts did increase with age. We found that N2-CMdG, an advanced glycation end-products resulting from the reaction between glyoxal with 2’-deoxyguanosine, increased significantly with age in rat liver and heart but not in human heart. However, we identified two novel DNA adducts that are significantly and positively correlated with age in the human non-failing heart: 1, N6 etheno-dA, which can arise from lipid peroxidation of polyunsaturated fatty acids, and a currently unidentified adduct with a m/z of 328 (“328”). Interestingly, we also found that the “328” adduct significantly increased with age in rat heart and brain. We hypothesize that the increase of 1, N6 etheno-dA and the 328 molecule with age might induce alterations in gene expression in the heart, although our approach cannot determine which cell type accumulates the DNA adduct. These experiments indicate that while most DNA adducts do not accumulate with age, there are specific molecules that do increase with age. Furthermore, we identify two molecules that increase with age in the human heart.
Tuesday
Biosynthesis of acrolein with a coupled enzyme system to emulate continuous acrolein production by gut microbiota
11:00am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Virtual Room
Division: [TOXI] Division of Chemical Toxicology
Session Type: Poster - Virtual
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 been identified to be an endogenous product of glycerol metabolism by human gut microbes. Despite the recent designation of acrolein as a probable human carcinogen, there remains limited data concerning molecular initiating and key cellular events associated with biomolecular interactions of endogenously produced acrolein, especially with respect to exposure in the gastrointestinal tract. Acrolein is a highly reactive and volatile molecule, that is not stable in buffer or cell culture media making it difficult to characterize its biological effects in vitro. To circumvent the volatile nature of acrolein and emulate the persistent production of acrolein by gut bacteria for toxicological evaluation, we established a cell culture-compatible coupled-enzyme system for the in-situ production and testing the effects of acrolein. The continuous acrolein production is executed by combining a polyamine catabolism enzyme, polyamine substrate, and catalase to scavenge hydrogen peroxide formed as a side product. We performed cell-culture compatibility studies with the coupled enzyme system and observed reductions in cell viability after exposure of colon epithelial cells to acrolein generated by the coupled enzyme system. To validate this system for controlled continuous in vitro acrolein exposure, we first characterized the formation of the most abundant Acrolein-DNA adduct acrolein-deoxyguanosine (Acr-dG) following acute exposure of colon epithelial cells to acrolein. There was a dose-dependent increase in adduct levels after 6 and 24 hours. Next, we evaluated changes in levels of gene transcripts of HMOX1 and SRXN1, which are activated by the Nrf2 pathway under conditions of oxidative stress and, also, observed a dose-dependent induction after 6 and 24 hours. We used DNA adduct formation as an internal molecular dosimeter for acrolein produced by the enzyme system, consistent with exogenous acute exposures of 50-75 µM acrolein. Therefore, the system can produce acrolein at levels that cause DNA damage when compared to acute exposure and provides a novel adaptable in vitro method to understand biological consequences of endogenous acrolein formation.
Tuesday
Withdrawn
11:00am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Virtual Room
Division: [TOXI] Division of Chemical Toxicology
Session Type: Poster - Virtual

Tuesday
Withdrawn
11:00am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Virtual Room
Division: [TOXI] Division of Chemical Toxicology
Session Type: Poster - Virtual

Tuesday
Beyond the lesion: The switch back to high fidelity synthesis
11:00am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Virtual Room
Joseph Kaszubowski, Presenter; Michael Trakselis, Baylor University
Division: [TOXI] Division of Chemical Toxicology
Session Type: Poster - Virtual
Upon encountering a template-strand lesion during DNA replication, high fidelity (HiFi) polymerases (Pols) stall and are replaced by specialized translesion synthesis (TLS) Pols. Generally, TLS Pols have low fidelity but are specialized for faithful insertions opposite lesions. Therefore, extension past the lesion by a TLS Pol should be limited to maintain fidelity. While the first polymerase switch and subsequent lesion bypass have been studied in some detail, the ‘switch-back’ to the HiFi Pol has not been explored. Studies have shown that this switch-back does not always occur at the same position beyond all lesions. Further complicating this process is the occasional presence of multiple TLS Pols contributing to bypass of the same lesion. Here, we explore the kinetic process and structural context for limiting extension by a TLS Pol beyond the lesion. We will focus on this switch-back using a model system from Saccharolobus solfataricus as well as draw parallels with the human TLS system to highlight conserved polymerase behavior between multiple TLS Pols, Pol families, and species.
<b>Figure 1. </b>Translesion synthesis along a DNA template with “x” lesion. The shaded blue area marks the switch-back from TLS to a HiFi Pol.

Figure 1. Translesion synthesis along a DNA template with “x” lesion. The shaded blue area marks the switch-back from TLS to a HiFi Pol.


Tuesday
Structural modelling of MIE interactions – docking neonicotinoids into human nAChR
11:00am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Virtual Room
Karin Grillberger, Presenter, University of Vienna; Dominik Loser; Jonathan Blum; Marcel Leist; Gerhard Ecker, Presenter
Division: [TOXI] Division of Chemical Toxicology
Session Type: Poster - Virtual
The purpose of the EU-ToxRisk project is to investigate new approach methods (NAM) for toxicity risk assessment, for which a case study about a prominent group of pesticides, known as neonicotinoids, and their potential developmental neurotoxicity (DNT) hazard was implemented. This could be further used in an integrated approach for a testing and assessment (IATA) framework to develop an enhanced DNT testing battery for an OECD guidance document. The goal is to gather data and information concerning the molecular initiating event of these compounds with the human nicotinic acetylcholine receptor (nAChR) with a focus on the subtypes α7 and α4β2. These membrane proteins are classified as ligand-gated ion channels, allowing the entry of Na+ and Ca2+ into cells which can lead to subsequent signal transduction pathways and play a key role in mammalian brain development and function.
In order to achieve the goal of this study, induced fit docking (IFD) and subsequent hierarchical clustering methods were applied and illustrated that there is a common binding mode of neonicotinoids in the nAChR, which has also been reported in homologous co-crystalised structures and relevant literature. Further analysis revealed that there is a difference concerning the interactions of neonicotinoids and the more potent nicotinoids, which is mainly based on a flip of the imidazole ring thus mimicking the agonistic binding mode of nicotine. Using the IFD-method, binding energy calculations and scoring parameters, allowed to distinguish between Neonicotinoids and Nicotinoids since their generated docking scores reflect the experimentally derived values. Furthermore, according to our docking experiments different interactions in the binding pocket can be formed, depending on the electronegative pharmacophore being a nitrogen- or a cyano-functionality.

Tuesday
The DNA adductome portal: A one-stop search site for DNA adduct Information
11:00am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Virtual Room
Division: [TOXI] Division of Chemical Toxicology
Session Type: Poster - Virtual
Environmental and dietary genotoxicants, endogenous electrophiles, and ionizing radiation damage the human genome through covalent modifications of DNA, known as DNA adducts. DNA adductomics measures the totality of DNA damage resulting from chemical exposure and endogenous processes and enables a better understanding of how DNA adducts influence disease.
To advance the study of DNA adducts, we have developed a DNA adductome spectral library and database containing more than 4000 high-quality MS2 and MS3 spectra from over 200 synthetic DNA adduct standards provided by collaborators worldwide. Many of these spectra have been incorporated into the public databases of MassBank of North America (MoNA), PubChem, and mzCloud (Thermo Fisher Scientific) and include associated metadata such as the common and IUPAC names of the adducts, chemoinformatic information including structural SMILES, and the parameters for MS acquisition. The “DNA Adductome Portal” (www.z.umn.edu/DNAAdductomePortal) is being developed for our DNA adductome database including the DNA adduct spectral library. This portal will serve as a one-stop location for the research investigator to obtain comprehensive information on DNA adduct-related topics. DNA adduct precursor and fragment ions gathered from our adductome spectral library will be available as a downloadable file in multiple MS data formats. Other data will include additional information on product ion formulae to aid in the interpretation of DNA adduct mass spectra. A compilation of published literature on mechanisms of DNA adduct formation and mass spectrometry-based methods to detect and measure DNA adducts in humans will be available.

Tuesday
Analysis of interactions of human polymerase kappa and DinB polymerases with damaged DNA
11:00am - 12:30pm USA / Canada - Eastern - August 24, 2021 | Room: Virtual Room
Mansi Kinare, Presenter; Lakindu Pathira Kankanamge; Penny Beuning, Presenter; Dr. Mary Jo Ondrechen, Northeastern University
Division: [TOXI] Division of Chemical Toxicology
Session Type: Poster - Virtual
The purpose of this project is to probe interactions of Y-family DNA polymerases with damaged DNA. DNA faces a variety of damaging factors every day, such as UV rays, cigarette smoke, and other carcinogens, and the ability for cells to bypass this routine damage is essential for the continuation of life. Y-family polymerases are found in all domains of life and specialized to copy damaged DNA. Human DNA Pol Kappa and E. coli DinB are orthologs and bypass similar types of DNA damage. However, human DNA Pol Kappa is more accommodating in terms of the types of damage it can bypass and is more efficient at extending DNA from a damaged site. In order to understand the basis for these differences, we are performing a computational analysis of Pol Kappa and DinB to determine which amino acids are most important for activity and to predict the effect of specific amino acid changes on activity. First, homology models of protein variants are generated. DinB variants are analyzed with POOL (Partial Order Optimum Likelihood), developed at Northeastern, which is a machine learning method that provides a rank-ordered list of residues according to their predicted functional importance. The prediction is based on computed electrostatic properties from THEMATICS as well as ligand pocket binding information from ConCavity. This analysis is compared to a similar analysis carried out with human Pol Kappa to understand DNA damage bypass efficiency and then propose mutations in DinB and Pol Kappa to test the bypass efficiency of the variants. This project is expected to reveal the differences in DinB and Pol Kappa that contribute to their different biochemical activity and will provide new insights into how DNA polymerases accomplish bypass of DNA damage.
Tuesday
TOXI Virtual Poster Session
11:00am - 12:30pm USA / Canada - Eastern - August 24, 2021
Division: [TOXI] Division of Chemical Toxicology
Session Type: Networking Events - Virtual
Division/Committee: [TOXI] Division of Chemical Toxicology

This poster session can be accessed by going the the TOXI Divisions website: http://www.acschemtox.org/