Publications by authors named "Natalie R Gassman"

50 Publications

Cytoprotective Effect of Vitamin D on Doxorubicin-Induced Cardiac Toxicity in Triple Negative Breast Cancer.

Int J Mol Sci 2021 Jul 12;22(14). Epub 2021 Jul 12.

Department of Physiology and Cell Biology, University of South Alabama College of Medicine, Mobile, AL 36688, USA.

Background: Doxorubicin (Dox) is a first-line treatment for triple negative breast cancer (TNBC), but its use may be limited by its cardiotoxicity mediated by the production of reactive oxygen species. We evaluated whether vitamin D may prevent Dox-induced cardiotoxicity in a mouse TNBC model.

Methods: Female Balb/c mice received rodent chow with vitamin D (1500 IU/kg; vehicle) or chow supplemented with additional vitamin D (total, 11,500 IU/kg). the mice were inoculated with TNBC tumors and treated with intraperitoneal Dox (6 or 10 mg/kg). Cardiac function was evaluated with transthoracic echocardiography. The cardiac tissue was evaluated with immunohistochemistry and immunoblot for levels of 4-hydroxynonenal, NAD(P)H quinone oxidoreductase (NQO1), C-MYC, and dynamin-related protein 1 (DRP1) phosphorylation.

Results: At 15 to 18 days, the mean ejection fraction, stroke volume, and fractional shortening were similar between the mice treated with vitamin D + Dox (10 mg/kg) vs. vehicle but significantly greater in mice treated with vitamin D + Dox (10 mg/kg) vs. Dox (10 mg/kg). Dox (10 mg/kg) increased the cardiac tissue levels of 4-hydroxynonenal, NQO1, C-MYC, and DRP1 phosphorylation at serine 616, but these increases were not observed with vitamin D + Dox (10 mg/kg). A decreased tumor volume was observed with Dox (10 mg/kg) and vitamin D + Dox (10 mg/kg).

Conclusions: Vitamin D supplementation decreased Dox-induced cardiotoxicity by decreasing the reactive oxygen species and mitochondrial damage, and did not decrease the anticancer efficacy of Dox against TNBC.
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http://dx.doi.org/10.3390/ijms22147439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8305038PMC
July 2021

Activated STAT3 Is a Novel Regulator of the Promoter and Selectively Increases XRCC1 Protein Levels in Triple Negative Breast Cancer.

Int J Mol Sci 2021 May 22;22(11). Epub 2021 May 22.

Department of Physiology and Cell Biology, University of South Alabama College of Medicine, 307 N University Blvd, Mobile, AL 36688, USA.

Base Excision Repair (BER) addresses base lesions and abasic sites induced by exogenous and endogenous stressors. X-ray cross complementing group 1 (XRCC1) functions as a scaffold protein in BER and single-strand break repair (SSBR), facilitating and coordinating repair through its interaction with a host of critical repair proteins. Alterations of XRCC1 protein and gene expression levels are observed in many cancers, including colorectal, ovarian, and breast cancer. While increases in the expression level of XRCC1 are reported, the transcription factors responsible for this up-regulation are not known. In this study, we identify the signal transducer and activator of transcription 3 (STAT3) as a novel regulator of through chromatin immunoprecipitation. Activation of STAT3 through phosphorylation at Y705 by cytokine (IL-6) signaling increases the expression of XRCC1 and the occupancy of STAT3 within the promoter. In triple negative breast cancer, the constitutive activation of STAT3 upregulates XRCC1 gene and protein expression levels. Increased expression of XRCC1 is associated with aggressiveness and resistance to DNA damaging chemotherapeutics. Thus, we propose that activated STAT3 regulates XRCC1 under stress and growth conditions, but constitutive activation in cancers results in dysregulation of XRCC1 and subsequently BER and SSBR.
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http://dx.doi.org/10.3390/ijms22115475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8196947PMC
May 2021

Associations between DNA Damage and PD-L1 Expression in Ovarian Cancer, a Potential Biomarker for Clinical Response.

Biology (Basel) 2021 Apr 29;10(5). Epub 2021 Apr 29.

Department of Physiology and Cell Biology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA.

Programmed death ligand-1 (PD-L1) inhibitors are currently under investigation as a potential treatment option for ovarian cancer. Although this therapy has shown promise, its efficacy is highly variable among patients. Evidence suggests that genomic instability influences the expression of PD-L1, but little is known about this relationship in ovarian cancer. To examine the relationship between PD-L1 expression and genomic instability, we measured DNA damage using Repair Assisted Damage Detection (RADD). We then correlated the presence of persistent DNA damage in the ovarian tumor with protein expression of PD-L1 using immunohistochemistry. Ovarian tumors showed a high prevalence of oxidative DNA damage. As the level of oxidative DNA damage increased, we saw a significant correlation with PD-L1 expression. The highest correlation between DNA damage and PD-L1 expression was observed for mucinous ovarian tumors (r = 0.82), but a strong correlation was also observed for high grade serous and endometrioid tumors (r = 0.67 and 0.69, respectively). These findings link genomic instability to PD-L1 protein expression in ovarian cancer and suggest that persistent DNA damage can be used as a potential biomarker for patient selection for immunotherapy treatment.
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http://dx.doi.org/10.3390/biology10050385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8146974PMC
April 2021

Chemical and Biochemical Reactivity of the Reduced Forms of Nicotinamide Riboside.

ACS Chem Biol 2021 04 30;16(4):604-614. Epub 2021 Mar 30.

All life forms require nicotinamide adenine dinucleotide, NAD, and its reduced form NADH. They are redox partners in hundreds of cellular enzymatic reactions. Changes in the intracellular levels of total NAD (NAD + NADH) and the (NAD/NADH) ratio can cause cellular dysfunction. When not present in protein complexes, NADH and its phosphorylated form NADPH degrade through intricate mechanisms. Replenishment of a declining total NAD pool can be achieved with biosynthetic precursors that include one of the reduced forms of nicotinamide riboside (NR), NRH. NRH, like NADH and NADPH, is prone to degradation via oxidation, hydration, and isomerization and, as such, is an excellent model compound to rationalize the nonenzymatic metabolism of NAD(P)H in a biological context. Here, we report on the stability of NRH and its propensity to isomerize and irreversibly degrade. We also report the preparation of two of its naturally occurring isomers, their chemical stability, their reactivity toward NRH-processing enzymes, and their cell-specific cytotoxicity. Furthermore, we identify a mechanism by which NRH degradation causes covalent peptide modifications, a process that could expose a novel type of NADH-protein modifications and correlate NADH accumulation with "protein aging." This work highlights the current limitations in detecting NADH's endogenous catabolites and in establishing the capacity for inducing cellular dysfunction.
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http://dx.doi.org/10.1021/acschembio.0c00757DOI Listing
April 2021

The Biochemical Pathways of Nicotinamide-Derived Pyridones.

Int J Mol Sci 2021 Jan 24;22(3). Epub 2021 Jan 24.

Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA.

As catabolites of nicotinamide possess physiological relevance, pyridones are often included in metabolomics measurements and associated with pathological outcomes in acute kidney injury (AKI). Pyridones are oxidation products of nicotinamide, its methylated form, and its ribosylated form. While they are viewed as markers of over-oxidation, they are often wrongly reported or mislabeled. To address this, we provide a comprehensive characterization of these catabolites of vitamin B3, justify their nomenclature, and differentiate between the biochemical pathways that lead to their generation. Furthermore, we identify an enzymatic and a chemical process that accounts for the formation of the ribosylated form of these pyridones, known to be cytotoxic. Finally, we demonstrate that the ribosylated form of one of the pyridones, the 4-pyridone-3-carboxamide riboside (4PYR), causes HepG3 cells to die by autophagy; a process that occurs at concentrations that are comparable to physiological concentrations of this species in the plasma in AKI patients.
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http://dx.doi.org/10.3390/ijms22031145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866226PMC
January 2021

Exogenous exposure to dihydroxyacetone mimics high fructose induced oxidative stress and mitochondrial dysfunction.

Environ Mol Mutagen 2021 03 6;62(3):185-202. Epub 2021 Feb 6.

Department of Physiology and Cell Biology, University of South Alabama, College of Medicine, Mobile, Alabama.

Dihydroxyacetone (DHA) is a three-carbon sugar that is the active ingredient in sunless tanning products and a by-product of electronic cigarette (e-cigarette) combustion. Increased use of sunless tanning products and e-cigarettes has elevated exposures to DHA through inhalation and absorption. Studies have confirmed that DHA is rapidly absorbed into cells and can enter into metabolic pathways following phosphorylation to dihydroxyacetone phosphate (DHAP), a product of fructose metabolism. Recent reports have suggested metabolic imbalance and cellular stress results from DHA exposures. However, the impact of elevated exposure to DHA on human health is currently under-investigated. We propose that exogenous exposures to DHA increase DHAP levels in cells and mimic fructose exposures to produce oxidative stress, mitochondrial dysfunction, and gene and protein expression changes. Here, we review cell line and animal model exposures to fructose to highlight similarities in the effects produced by exogenous exposures to DHA. Given the long-term health consequences of fructose exposure, this review emphasizes the pressing need to further examine DHA exposures from sunless tanning products and e-cigarettes.
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http://dx.doi.org/10.1002/em.22425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954877PMC
March 2021

Dihydronicotinamide riboside promotes cell-specific cytotoxicity by tipping the balance between metabolic regulation and oxidative stress.

PLoS One 2020 9;15(11):e0242174. Epub 2020 Nov 9.

Department of Physiology and Cell Biology, University of South Alabama College of Medicine, Mobile, AL, United States of America.

Nicotinamide adenine dinucleotide (NAD+), the essential cofactor derived from vitamin B3, is both a coenzyme in redox enzymatic processes and substrate in non-redox events; processes that are intimately implicated in all essential bioenergetics. A decrease in intracellular NAD+ levels is known to cause multiple metabolic complications and age-related disorders. One NAD+ precursor is dihydronicotinamide riboside (NRH), which increases NAD+ levels more potently in both cultured cells and mice than current supplementation strategies with nicotinamide riboside (NR), nicotinamide mononucleotide (NMN) or vitamin B3 (nicotinamide and niacin). However, the consequences of extreme boosts in NAD+ levels are not fully understood. Here, we demonstrate the cell-specific effects of acute NRH exposure in mammalian cells. Hepatocellular carcinoma (HepG3) cells show dose-dependent cytotoxicity when supplemented with 100-1000 μM NRH. Cytotoxicity was not observed in human embryonic kidney (HEK293T) cells over the same dose range of NRH. PUMA and BAX mediate the cell-specific cytotoxicity of NRH in HepG3. When supplementing HepG3 with 100 μM NRH, a significant increase in ROS was observed concurrent with changes in the NAD(P)H and GSH/GSSG pools. NRH altered mitochondrial membrane potential, increased mitochondrial superoxide formation, and induced mitochondrial DNA damage in those cells. NRH also caused metabolic dysregulation, altering mitochondrial respiration. Altogether, we demonstrated the detrimental consequences of an extreme boost of the total NAD (NAD+ + NADH) pool through NRH supplementation in HepG3. The cell-specific effects are likely mediated through the different metabolic fate of NRH in these cells, which warrants further study in other systemic models.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0242174PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652347PMC
January 2021

Transcriptional dysregulation of base excision repair proteins in breast cancer.

DNA Repair (Amst) 2020 09 7;93:102922. Epub 2020 Jul 7.

Department of Physiology and Cell Biology, University of South Alabama College of Medicine, Mobile, AL, USA; Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA.

Base excision repair (BER) addresses the numerous base lesions and strand breaks induced by exogenous and endogenous stressors daily. The complexity and importance of BER requires careful regulation of basal levels of these proteins and inducible responses following DNA damage. Several reports have noted the dysregulation of BER proteins and defects in BER capacity in cancer. Modulated gene and protein expression of several BER proteins, including APE1, PARP1, POL β, and XRCC1, have been observed in breast cancer. Overexpression of these factors has been associated with chemoresistance and cancer aggressiveness, but the regulatory mechanisms that drive overexpression have not been defined. Here, we review the known transcriptional regulators of these key BER proteins and examine potential mechanisms that may drive overexpression in breast cancer.
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http://dx.doi.org/10.1016/j.dnarep.2020.102922DOI Listing
September 2020

Exploiting DNA repair defects in triple negative breast cancer to improve cell killing.

Ther Adv Med Oncol 2020 18;12:1758835920958354. Epub 2020 Sep 18.

Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, AL 36607, USA.

Background: The lack of molecular targets for triple negative breast cancer (TNBC) has limited treatment options and reduced survivorship. Identifying new molecular targets may help improve patient survival and decrease recurrence and metastasis. As DNA repair defects are prevalent in breast cancer, we evaluated the expression and repair capacities of DNA repair proteins in preclinical models.

Methods: DNA repair capacity was analyzed in four TNBC cell lines, MDA-MB-157 (MDA-157), MDA-MB-231 (MDA-231), MDA-MB-468 (MDA-468), and HCC1806, using fluorescence multiplex host cell reactivation (FM-HCR) assays. Expression of DNA repair genes was analyzed with RNA-seq, and protein expression was evaluated with immunoblot. Responses to the combination of DNA damage response inhibitors and primary chemotherapy drugs doxorubicin or carboplatin were evaluated in the cell lines.

Results: Defects in base excision and nucleotide excision repair were observed in preclinical TNBC models. Gene expression analysis showed a limited correlation between these defects. Loss in protein expression was a better indicator of these DNA repair defects. Over-expression of PARP1, XRCC1, RPA, DDB1, and ERCC1 was observed in TNBC preclinical models, and likely contributed to altered sensitivity to chemotherapy and DNA damage response (DDR) inhibitors. Improved cell killing was achieved when primary therapy was combined with DDR inhibitors for ATM, ATR, or CHK1.

Conclusion: Base excision and nucleotide excision repair pathways may offer new molecular targets for TNBC. The functional status of DNA repair pathways should be considered when evaluating new therapies and may improve the targeting for primary and combination therapies with DDR inhibitors.
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http://dx.doi.org/10.1177/1758835920958354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502856PMC
September 2020

A cancer amidst us: the plexiform lesion in pulmonary arterial hypertension.

Am J Physiol Lung Cell Mol Physiol 2020 06 19;318(6):L1142-L1144. Epub 2020 Mar 19.

Department of Physiology and Cell Biology, University of South Alabama, Mobile, Alabama.

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http://dx.doi.org/10.1152/ajplung.00092.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347268PMC
June 2020

DNA damage measurements within tissue samples with Repair Assisted Damage Detection (RADD).

Curr Res Biotechnol 2019 Nov 15;1:78-86. Epub 2019 Nov 15.

University of South Alabama College of Medicine, Mobile, AL 36688 USA.

Exposures to genotoxic carcinogens and reactive species result in strand breaks and a spectrum of covalent modifications to DNA that can induce mutations and contribute to the initiation and progression of cancer. Measurements of DNA damage within tissue or tumor samples can serve as a biomarker for exposures or assess changes in DNA repair capacity relevant in cancer development and treatment. Numerous methods to characterize DNA damage exist. However, these methods are primarily applicable to isolated DNA or cultured cells, often require a substantial amount of material, and may be limited to the detection and quantification of only a handful of DNA adducts. Here, we used the Repair Assisted Damage Detection (RADD) assay to detect and excise DNA adducts using a cocktail of DNA repair enzymes, then the damage site within the genome are tagged for detection using a modified nucleotide. We previously demonstrated the RADD assay can detect lesions within isolated DNA and fixed cells, and now RADD can be used to detect DNA adducts and DNA strand breaks in formalin-fixed paraffin-embedded (FFPE) tissue samples. We verified the ability of the RADD assay to detect DNA damage in tissue by exogenously inducing DNA damage with X-rays and restriction enzymes. We also showed that RADD can be multiplexed with antibodies to detect cell cycle markers or other proteins of interest. Finally, we showed that RADD can detect DNA damage within clinically relevant ovarian tumor samples. RADD is a flexible and easy-to-use assay that allows relative damage levels to be determined within FFPE samples and allows the heterogeneity of DNA adducts and strand breaks within clinically relevant samples to be measured.
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http://dx.doi.org/10.1016/j.crbiot.2019.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8118132PMC
November 2019

Defective base excision repair in the response to DNA damaging agents in triple negative breast cancer.

PLoS One 2019 9;14(10):e0223725. Epub 2019 Oct 9.

Mitchell Cancer Institute, University of South Alabama, Mobile, AL, United States of America.

DNA repair defects have been increasingly focused on as therapeutic targets. In hormone-positive breast cancer, XRCC1-deficient tumors have been identified and proposed as targets for combination therapies that damage DNA and inhibit DNA repair pathways. XRCC1 is a scaffold protein that functions in base excision repair (BER) by mediating essential interactions between DNA glycosylases, AP endonuclease, poly(ADP-ribose) polymerase 1, DNA polymerase β (POL β), and DNA ligases. Loss of XRCC1 confers BER defects and hypersensitivity to DNA damaging agents. BER defects have not been evaluated in triple negative breast cancers (TNBC), for which new therapeutic targets and therapies are needed. To evaluate the potential of XRCC1 as an indicator of BER defects in TNBC, we examined XRCC1 expression in the TCGA database and its expression and localization in TNBC cell lines. The TCGA database revealed high XRCC1 expression in TNBC tumors and TNBC cell lines show variable, but mostly high expression of XRCC1. XRCC1 localized outside of the nucleus in some TNBC cell lines, altering their ability to repair base lesions and single-strand breaks. Subcellular localization of POL β also varied and did not correlate with XRCC1 localization. Basal levels of DNA damage correlated with observed changes in XRCC1 expression, localization, and measure repair capacity. The results confirmed that XRCC1 expression changes indicate DNA repair capacity changes but emphasize that basal DNA damage levels along with protein localization are better indicators of DNA repair defects. Given the observed over-expression of XRCC1 in TNBC preclinical models and tumors, XRCC1 expression levels should be assessed when evaluating treatment responses of TNBC preclinical model cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0223725PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785058PMC
March 2020

Simultaneous detection of multiple DNA damage types by multi-colour fluorescent labelling.

Chem Commun (Camb) 2019 Sep;55(76):11414-11417

Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University, Tel Aviv 6997801, Israel.

Herein we present an assay allowing concurrent detection of oxidative DNA damage and photoproducts. We apply DNA repair enzymes specific for each lesion type to incorporate spectrally distinct fluorescent nucleotides, enabling simultaneous quantification of the lesions on individual DNA molecules. We follow the repair of both damage types in skin cells exposed to artificial sunlight.
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http://dx.doi.org/10.1039/c9cc05198hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783632PMC
September 2019

Dihydroxyacetone Exposure Alters NAD(P)H and Induces Mitochondrial Stress and Autophagy in HEK293T Cells.

Chem Res Toxicol 2019 08 2;32(8):1722-1731. Epub 2019 Aug 2.

Department of Physiology and Cell Biology , University of South Alabama College of Medicine , Mobile , Alabama 36688 , United States.

Dihydroxyacetone phosphate (DHAP) is the endogenous byproduct of fructose metabolism. Excess DHAP in cells can induce advanced glycation end products and oxidative stress. Dihydroxyacetone (DHA) is the triose precursor to DHAP. DHA is used as the active ingredient in sunless tanning products, including aerosolized spray tans, and is formed by the combustion of solvents found in electronic cigarettes. Human exposure to DHA has been increasing as the popularity of sunless tanning products and electronic cigarettes has grown. Topically applied DHA is absorbed through the viable layers of the skin and into the bloodstream. Exogenous exposure to DHA is cytotoxic in immortalized keratinocytes and melanoma cells with cell cycle arrest induced within 24 h and cell death occurring by apoptosis at consumer-relevant concentrations of DHA within 72 h. Less is known about systemic exposures to DHA that occur following absorption through skin, and now through inhalation of the aerosolized DHA used in spray tanning. In the present study, HEK293T cells were exposed to consumer-relevant concentrations of DHA to examine the cytotoxicity of systemic exposures. HEK293T cells were sensitive to consumer-relevant doses of DHA with an IC value of 2.4 ± 0.3 mM. However, cell cycle arrest did not begin until 48 h after DHA exposure. DHA-exposed cells showed altered metabolic activity with decreased mitochondrial function and decreased lactate and ATP production observed within 24 h of exposure. Autofluorescent imaging and NAD sensors also revealed an imbalance in the redox cofactors NAD/NADH within 24 h of exposure. Cell death occurred by autophagy indicated by increases in LC3B and SIRT1. Despite DHA's ability to be converted to DHAP and integrated into metabolic pathways, the metabolic dysfunction and starvation responses observed in the HEK293T cells indicate that DHA does not readily contribute to the energetic pool in these cells.
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http://dx.doi.org/10.1021/acs.chemrestox.9b00230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6701868PMC
August 2019

Bisphenol A co-exposure effects: a key factor in understanding BPA's complex mechanism and health outcomes.

Crit Rev Toxicol 2019 05 1;49(5):371-386. Epub 2019 Jul 1.

Department of Oncologic Sciences, University of South Alabama Mitchell Cancer Institute, Mobile, AL, USA.

Bisphenol A (BPA) is an environmental endocrine disrupting chemical widely used in the production of consumer products, such as polycarbonate plastics, epoxies, and thermal receipt paper. Human exposure to BPA is ubiquitous due to its high-volume production and use. BPA exposure has been associated with obesity, diabetes, reproductive disorders, and cancer. Yet, the molecular mechanisms or modes of action underlying these disease outcomes are poorly understood due to the pleiotropic effects induced by BPA. A further confounding factor in understanding BPA's impact on human health is that co-exposure of BPA with endogenous and exogenous agents occurs during the course of daily life. Studies investigating BPA exposure effects and their relationship to adverse health outcomes often ignore interactions between BPA and other chemicals present in the environment. This review examines BPA co-exposure studies to highlight potentially unexplored mechanisms of action and their possible associations with the adverse health effects attributed to BPA. Importantly, both adverse and beneficial co-exposure effects are observed between BPA and natural chemicals or environmental stressors in and models. These interactions clearly influence cellular responses and impact endpoint measures and need to be considered when evaluating BPA exposures and their health effects.
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http://dx.doi.org/10.1080/10408444.2019.1621263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823117PMC
May 2019

Targets for repair: detecting and quantifying DNA damage with fluorescence-based methodologies.

Curr Opin Biotechnol 2019 02 13;55:30-35. Epub 2018 Aug 13.

University of South Alabama Mitchell Cancer Institute, 1660 Springhill Ave, Mobile, AL 36607, USA.

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http://dx.doi.org/10.1016/j.copbio.2018.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372338PMC
February 2019

Variations in nuclear localization strategies among pol X family enzymes.

Traffic 2018 Jun 22. Epub 2018 Jun 22.

National Institute of Environmental Health Sciences, Genome Integrity and Structural Biology Laboratory, National Institutes of Health, Research Triangle Park, North Carolina.

Despite the essential roles of pol X family enzymes in DNA repair, information about the structural basis of their nuclear import is limited. Recent studies revealed the unexpected presence of a functional nuclear localization signal (NLS) in DNA polymerase β, indicating the importance of active nuclear targeting, even for enzymes likely to leak into and out of the nucleus. The current studies further explore the active nuclear transport of these enzymes by identifying and structurally characterizing the functional NLS sequences in the three remaining human pol X enzymes: terminal deoxynucleotidyl transferase (TdT), DNA polymerase mu (pol μ) and DNA polymerase lambda (pol λ). NLS identifications are based on Importin α (Impα) binding affinity determined by fluorescence polarization of fluorescein-labeled NLS peptides, X-ray crystallographic analysis of the Impα∆IBB•NLS complexes and fluorescence-based subcellular localization studies. All three polymerases use NLS sequences located near their N-terminus; TdT and pol μ utilize monopartite NLS sequences, while pol λ utilizes a bipartite sequence, unique among the pol X family members. The pol μ NLS has relatively weak measured affinity for Impα, due in part to its proximity to the N-terminus that limits non-specific interactions of flanking residues preceding the NLS. However, this effect is partially mitigated by an N-terminal sequence unsupportive of Met1 removal by methionine aminopeptidase, leading to a 3-fold increase in affinity when the N-terminal methionine is present. Nuclear targeting is unique to each pol X family enzyme with variations dependent on the structure and unique functional role of each polymerase.
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http://dx.doi.org/10.1111/tra.12600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684861PMC
June 2018

Analysis of single, cisplatin-induced DNA bends by atomic force microscopy and simulations.

J Mol Recognit 2018 10 3;31(10):e2731. Epub 2018 Jun 3.

Department of Physics, Wake Forest University, Winston-Salem, NC, USA.

Bent DNA, or DNA that is locally more flexible, is a recognition motif for many DNA binding proteins. These DNA conformational properties can thus influence many cellular processes, such as replication, transcription, and DNA repair. The importance of these DNA conformational properties is juxtaposed to the experimental difficulty to accurately determine small bends, locally more flexible DNA, or a combination of both (bends with increased flexibility). In essence, many current bulk methods use average quantities, such as the average end-to-end distance, to extract DNA conformational properties; they cannot access the additional information that is contained in the end-to-end distance distributions. We developed a method that exploits this additional information to determine DNA conformational parameters. The method is based on matching end-to-end distance distributions obtained experimentally by atomic force microscopy imaging to distributions obtained from simulations. We applied this method to investigate cisplatin GG biadducts. We found that cisplatin induces a bend angle of 36° and softens the DNA locally around the bend.
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http://dx.doi.org/10.1002/jmr.2731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168373PMC
October 2018

Camptothecin Efficacy to Poison Top1 Is Altered by Bisphenol A in Mouse Embryonic Fibroblasts.

Chem Res Toxicol 2018 06 5;31(6):510-519. Epub 2018 Jun 5.

Department of Oncologic Sciences , University of South Alabama Mitchell Cancer Institute , 1660 Spring Hill Avenue , Mobile , Alabama 36604 , United States.

Bisphenol A (BPA) is used heavily in the production of polycarbonate plastics, thermal receipt paper, and epoxies. Ubiquitous exposure to BPA has been linked to obesity, diabetes, and breast and reproductive system cancers. Resistance to chemotherapeutic agents has also been shown in cancer cell models. Here, we investigated BPA's ability to confer resistance to camptothecin (CPT) in mouse embryonic fibroblasts (MEFs). MEFs are sensitive to CPT; however, co-exposure of BPA with CPT improved cell survival. Co-exposure significantly reduced Top1-DNA adducts, decreasing chromosomal aberrations and DNA strand break formation. This decrease occurs despite BPA treatment increasing the protein levels of Top1. By examining chromatin structure after BPA exposure, we determined that widespread compaction and loss of nuclear volume occurs. Therefore, BPA reduced CPT activity by reducing the accessibility of DNA to Top1, inhibiting DNA adduct formation, the generation of toxic DNA strand breaks, and improving cell survival.
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http://dx.doi.org/10.1021/acs.chemrestox.8b00050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374779PMC
June 2018

Broad spectrum detection of DNA damage by Repair Assisted Damage Detection (RADD).

DNA Repair (Amst) 2018 Jun - Jul;66-67:42-49. Epub 2018 Apr 27.

Department of Oncologic Sciences, University of South Alabama Mitchell Cancer Institute, Mobile, AL, 36604, USA. Electronic address:

Environmental exposures, reactive by-products of cellular metabolism, and spontaneous deamination events result in a spectrum of DNA adducts that if un-repaired threaten genomic integrity by inducing mutations, increasing instability, and contributing to the initiation and progression of cancer. Assessment of DNA adducts in cells and tissues is critical for genotoxic and carcinogenic evaluation of chemical exposure and may provide insight into the etiology of cancer. Numerous methods to characterize the formation of DNA adducts and their retention for risk assessment have been developed. However, there are still significant drawbacks to the implementation and wide-spread use of these methods, because they often require a substantial amount of biological sample, highly specialized expertise and equipment, and depending on technique, may be limited to the detection and quantification of only a handful of DNA adducts at a time. There is a pressing need for high throughput, easy to implement assays that can assess a broad spectrum of DNA lesions, allowing for faster evaluation of chemical exposures and assessment of the retention of adducts in biological samples. Here, we describe a new methodology, Repair Assisted Damage Detection (RADD), which utilizes a DNA damage processing repair enzyme cocktail to detect and modify sites of DNA damage for a subsequent gap filling reaction that labels the DNA damage sites. This ability to detect and label a broad spectrum of DNA lesions within cells, offers a novel and easy to use tool for assessing levels of DNA damage in cells that have been exposed to environmental agents or have natural variations in DNA repair capacity.
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http://dx.doi.org/10.1016/j.dnarep.2018.04.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6375075PMC
October 2018

A truly safer alternative? Sunless tanning products and the unknown.

Prev Med 2018 07 5;112:45-46. Epub 2018 Apr 5.

University of South Alabama, Mitchell Cancer Institute, United States. Electronic address:

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http://dx.doi.org/10.1016/j.ypmed.2018.04.007DOI Listing
July 2018

Intentional tanning behaviors among undergraduates on the United States' Gulf Coast.

BMC Public Health 2018 04 3;18(1):441. Epub 2018 Apr 3.

Department of Surgery, University of South Alabama College of Medicine, 2451 Fillingim Street, Mobile, AL, 36617, USA.

Background: Rates of melanoma have dramatically increased among adolescents and young adults in recent years, particularly among young women. Exposure to ultraviolet radiation from intentional tanning practices is likely a major contributor to this epidemic. Southern and coastal regions have higher melanoma mortality rates among non-Hispanic whites in other parts of the U.S., yet little is known about tanning practices of adolescents and young adults in these regions. This study determines the prevalence and methods of intentional tanning utilized by an undergraduate population located on the United States' Gulf Coast.

Methods: Undergraduate students enrolled at a university on the Gulf Coast completed an online survey from March-April 2016, self-reporting their engagement, knowledge, and attitudes regarding outdoor tanning (OT), indoor tanning (IT) and spray tanning (ST). Univariate and multivariate analyses were performed to identify factors associated with tanning behaviors.

Results: 2668 undergraduates completed the survey. Of these, 64.9% reported OT tanning, 50.7% reported ever IT, and 21.2% reported ever ST.

Conclusions: In the largest study to date of intentional tanning behaviors of adolescents and young adults from coastal regions, we found high rates of intentional tanning behaviors. There was also significant engagement in spray tanning by this population, not previously reported for adolescents and young adults in a sample of this size. We also identified a high association between different tanning methods, indicating this population engages in multiple tanning behaviors, a phenomenon whose health consequences are not yet known.
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http://dx.doi.org/10.1186/s12889-018-5345-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5883533PMC
April 2018

XRCC1 phosphorylation affects aprataxin recruitment and DNA deadenylation activity.

DNA Repair (Amst) 2018 04 15;64:26-33. Epub 2018 Feb 15.

Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address:

Aprataxin (APTX) is a DNA-adenylate hydrolase that removes 5'-AMP blocking groups from abortive ligation repair intermediates. XRCC1, a multi-domain protein without catalytic activity, interacts with a number of known repair proteins including APTX, modulating and coordinating the various steps of DNA repair. CK2-phosphorylation of XRCC1 is thought to be crucial for its interaction with the FHA domain of APTX. In light of conflicting reports, the importance of XRCC1 phosphorylation and APTX function is not clear. In this study, a phosphorylation mutant of XRCC1 designed to eliminate APTX binding was stably expressed in Xrcc1 cells. Analysis of APTX-GFP accumulation at micro-irradiation damage confirmed that phosphorylated XRCC1 is required for APTX recruitment. APTX-mediated DNA deadenylation activity (i.e., 5'-AMP removal) was measured in extracts of cells expressing wild-type XRCC1 or the XRCC1 phosphorylation mutant, and compared with activity in APTX-deficient and APTX-complemented human cells. APTX activity was lower in extracts from Xrcc1 and XRCC1 phosphorylation mutant cells compared to the robust activity in extract from wild-type XRCC1 expressing cells. Taken together, results verify that interaction with phosphorylated XRCC1 is a requirement for significant APTX recruitment to cellular DNA damage and enzymatic activity in cell extracts.
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http://dx.doi.org/10.1016/j.dnarep.2018.02.004DOI Listing
April 2018

Significant Engagement in Tanning Behaviors by Men at a U.S. University.

J Community Health 2018 08;43(4):656-659

University of South Alabama Mitchell Cancer Institute, 1660 Springhill Avenue, Mobile, AL, 36604, USA.

Adolescent and young adult men are a potentially overlooked population with respect to risky tanning behaviors. This study sought to determine the prevalence of various modes of tanning and associated variables among young men in a university setting in the southeastern United States. Undergraduate students at a public institution in Mobile, Alabama were surveyed electronically in March 2016. Of the 818 undergraduate men surveyed, over 90% reported tanning behaviors, with 37% reporting engaging in indoor tanning. Additionally, over 25% reported engaging in two or more types of tanning concurrently. These findings indicate that early intervention efforts targeting young men are needed to reduce risky tanning behaviors and associated negative health outcomes.
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http://dx.doi.org/10.1007/s10900-017-0464-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935420PMC
August 2018

Dihydroxyacetone induces G2/M arrest and apoptotic cell death in A375P melanoma cells.

Environ Toxicol 2018 Mar 29;33(3):333-342. Epub 2017 Nov 29.

Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Ave, Mobile, Alabama, 36604-1405.

The active ingredient in sunless tanning products (STPs) is a simple sugar, dihydroxyacetone (DHA). Several studies have demonstrated that DHA is absorbed within the viable layers of skin and not fully contained within the stratum corneum. Additionally, spray tanning and other aerosolized application methods have increased the risk of internal exposure through mucous membranes and inhalation. Beyond its presence in STPs, DHA also occurs as an endogenous by-product of fructose metabolism, and an excess of DHA in cells can induce advanced glycation end (AGE) products and oxidative stress. Therefore, exogenous and endogenous exposures to DHA may be harmful to cells, and it has already been demonstrated that exogenous exposure to DHA is cytotoxic in immortalized keratinocytes. Still, little is known about the exogenous DHA exposure effects on other skin components. In this study, we explore the effects of exogenous DHA exposure in a human melanoma cell line, A375P. Melanoma cells were sensitive to DHA and displayed a transient burst of reactive oxygen species within an hour of exposure. Cell cycle arrest at G2/M was observed within 24 h of exposure, and apoptosis, monitored by the cleavage of PARP-1 and Caspase-3, was detected within 72 h of exposure to DHA. Together, these demonstrate that exogenous exposure to DHA has cytotoxic effects in our selected cell model and indicates the need to further investigate the exogenous exposure effects of DHA in other relevant exposure models.
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http://dx.doi.org/10.1002/tox.22520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809210PMC
March 2018

PARP1 changes from three-dimensional DNA damage searching to one-dimensional diffusion after auto-PARylation or in the presence of APE1.

Nucleic Acids Res 2017 Dec;45(22):12834-12847

Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15213, USA.

PARP1-dependent poly-ADP-ribosylation (PARylation) participates in the repair of many forms of DNA damage. Here, we used atomic force microscopy (AFM) and single molecule fluorescence microscopy to examine the interactions of PARP1 with common DNA repair intermediates. AFM volume analysis indicates that PARP1 binds to DNA at nicks, abasic (AP) sites, and ends as a monomer. Single molecule DNA tightrope assays were used to follow the real-time dynamic behavior of PARP1 in the absence and presence of AP endonuclease (APE1) on AP DNA damage arrays. These experiments revealed that PARP1 conducted damage search mostly through 3D diffusion. Co-localization of APE1 with PARP1 on DNA was found capable of inducing 1D diffusion of otherwise nonmotile PARP1, while excess APE1 also facilitated the dissociation of DNA-bound PARP1. Moreover, auto-PARylation of PARP1 allowed the protein to switch its damage search strategy by causing a 3-fold increase in linear diffusion. Finally, we demonstrated that PARP inhibitor olaparib did not significantly alter the rate of PARP1 dissociation from DNA, but instead resulted in more motility of DNA-bound PARP1 molecules.
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http://dx.doi.org/10.1093/nar/gkx1047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728402PMC
December 2017

DNA polymerase β: A missing link of the base excision repair machinery in mammalian mitochondria.

DNA Repair (Amst) 2017 12 28;60:77-88. Epub 2017 Oct 28.

Genome Integrity and Structural Biology Laboratory, National Institutes of Health, NIEHS, 111 T.W. Alexander Drive, P.O. Box 12233, Research Triangle Park, NC 27709, USA. Electronic address:

Mitochondrial genome integrity is fundamental to mammalian cell viability. Since mitochondrial DNA is constantly under attack from oxygen radicals released during ATP production, DNA repair is vital in removing oxidatively generated lesions in mitochondrial DNA, but the presence of a strong base excision repair system has not been demonstrated. Here, we addressed the presence of such a system in mammalian mitochondria involving the primary base lesion repair enzyme DNA polymerase (pol) β. Pol β was localized to mammalian mitochondria by electron microscopic-immunogold staining, immunofluorescence co-localization and biochemical experiments. Extracts from purified mitochondria exhibited base excision repair activity that was dependent on pol β. Mitochondria from pol β-deficient mouse fibroblasts had compromised DNA repair and showed elevated levels of superoxide radicals after hydrogen peroxide treatment. Mitochondria in pol β-deficient fibroblasts displayed altered morphology by electron microscopy. These results indicate that mammalian mitochondria contain an efficient base lesion repair system mediated in part by pol β and thus pol β plays a role in preserving mitochondrial genome stability.
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http://dx.doi.org/10.1016/j.dnarep.2017.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919216PMC
December 2017

XRCC1-mediated repair of strand breaks independent of PNKP binding.

DNA Repair (Amst) 2017 12 19;60:52-63. Epub 2017 Oct 19.

Genomic Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address:

Repair of DNA-protein crosslinks and oxidatively damaged DNA base lesions generates intermediates with nicks or gaps with abnormal and blocked 3'-phosphate and 5'-OH ends that prevent the activity of DNA polymerases and ligases. End cleaning in mammalian cells by Tdp1 and PNKP produces the conventional 3'-OH and 5'-phosphate DNA ends suitable for completion of repair. This repair function of PNKP is facilitated by its binding to the scaffold protein XRCC1, and phosphorylation of XRCC1 by CK2 at several consensus sites enables PNKP binding and recruitment to DNA damage. To evaluate this documented repair process, a phosphorylation mutant of XRCC1, designed to eliminate PNKP binding, was stably expressed in Xrcc1 mouse fibroblast cells. Analysis of PNKP-GFP accumulation at micro-irradiation induced damage confirmed that the XRCC1 phosphorylation mutant failed to support efficient PNKP recruitment, whereas there was rapid recruitment in cells expressing wild-type XRCC1. Recruitment of additional fluorescently-tagged repair factors PARP-1-YFP, GFF-XRCC1, PNKP-GFP and Tdp1-GFP to micro-irradiation induced damage was assessed in wild-type XRCC1-expressing cells. PARP-1-YFP recruitment was best fit to two exponentials, whereas kinetics for the other proteins were fit to a single exponential. The similar half-times of recruitment suggest that XRCC1 may be recruited with other proteins possibly as a pre-formed complex. Xrcc1 cells are hypersensitive to the DNA-protein cross-link inducing agent camptothecin (CPT) and the DNA oxidative agent HO due in part to compromised PNKP-mediated repair. However, cells expressing the PNKP interaction mutant of XRCC1 demonstrated marked reversal of CPT hypersensitivity. This reversal represents XRCC1-dependent repair in the absence of the phosphorylation-dependent PNKP recruitment and suggests either an XRCC1-independent mechanism of PNKP recruitment or a functional back-up pathway for cleaning of blocked DNA ends.
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http://dx.doi.org/10.1016/j.dnarep.2017.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696015PMC
December 2017

High prevalence of combination tanning among undergraduates: Survey at a southeastern US university.

J Am Acad Dermatol 2017 11;77(5):968-970

Division of Oncological Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama; Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama.

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http://dx.doi.org/10.1016/j.jaad.2017.05.039DOI Listing
November 2017

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells.

J Vis Exp 2017 09 5(127). Epub 2017 Sep 5.

Department of Oncologic Sciences, University of South Alabama Mitchell Cancer Institute;

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.
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http://dx.doi.org/10.3791/56265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752190PMC
September 2017
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