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Dysregulation of DNA repair is a hallmark of cancer, though few cancer-specific mechanisms that drive the overexpression of DNA repair proteins are known. We previously identified STAT3 as a novel transcriptional regulator of X-ray cross-complementing group 1 (XRCC1), an essential scaffold protein in base excision repair in triple-negative breast cancers. We also identified an inducible response to IL-6 and epidermal growth factor stimulation in the non-tumorigenic embryonic kidney cell line HEK293T. As IL-6 and EGF signaling are growth and inflammatory-inducible responses, we examined if glucose challenge can increase STAT3 activation, promoting adaptive changes in XRCC1 expression in different cell types. Acute high glucose exposure promoted XRCC1 expression through STAT3 activation, increasing the repair of methyl methanesulfonate-induced DNA damage in HEK293T cells and the osteosarcoma cell line U2OS. Sustained exposure to high glucose promoted the overexpression of XRCC1, which can be reversed upon glucose restriction and down-regulation of STAT3 activation. Thus, we have identified a novel link between XRCC1 expression and STAT3 activation following exogenous exposures, which could play a critical role in dictating a cancer cell's response to DNA-damaging agents. [ABSTRACT FROM AUTHOR]

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 in vitro and in vivo models. These interactions clearly influence cellular responses and impact endpoint measures and need to be considered when evaluating BPA exposures and their health effects. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Fluorescence-based methods offer new opportunities for assessing DNA adducts. • Fluorescent-tagging of DNA repair proteins allow in vivo damage measurements. • Labeled dNTPs inserted at damage sites by repair enzymes also allow damage detection. • Specific DNA adducts are labeled using covalent binding to chemical modifiers. Detection and characterization of DNA damage is essential for evaluating genotoxicity, monitoring DNA repair, developing biomarkers for exposures, and evaluating the efficacy of chemotherapies. These diverse applications for DNA damage measurements have spurred the continual development and refinement of methodologies for detecting, characterizing, and quantifying DNA damage from isolated DNA and in cells and tissues. Current damage detection methods cover a wide range of techniques from radiolabeling to mass spectrometry, and use of these techniques varies widely based on expense, expertise, and knowledge of adduct formation. More generalizable, easy-to-use methods for detecting and quantifying DNA damage are needed, and there has been an emergence of fluorescence-based methodologies to address this need. Developments in these fluorescence-based strategies are reviewed here. [ABSTRACT FROM AUTHOR]

BACKGROUND: Exposure to bisphenol A (BPA) has been reported to alter global gene expression, induce epigenetic modifications, and interfere with complex regulatory networks of cells. In addition to these reprogramming events, we have demonstrated that BPA exposure generates reactive oxygen species and promotes cellular survival when co-exposed with the oxidizing agent potassium bromate (KBrO3). OBJECTIVES: We determined the cellular microenvironment changes induced by co-exposure of BPA and KBrO3 versus either agent alone. METHODS: Ku70-deficient cells were exposed to 150 µM BPA, 20 mM KBrO3, or co-exposed to both agents. Four and 24 hr post-damage initiation by KBrO3, with BPA-only samples timed to coincide with these designated time points, we performed whole-genome microarray analysis and evaluated chromatin structure, DNA lesion load, glutathione content, and intracellular pH. RESULTS: We found that 4 hr post-damage initiation, BPA exposure and co-exposure transiently condensed chromatin compared with untreated and KBrO3-only treated cells; the transcription of DNA repair proteins was also reduced. At this time point, BPA exposure and co-exposure also reduced the change in intracellular pH observed after treatment with KBrO3 alone. Twenty-four hours post-damage initiation, BPA-exposed cells showed less condensed chromatin than cells treated with KBrO3 alone; the intracellular pH of the co-exposed cells was significantly reduced compared with untreated and KBrO3-treated cells; and significant up-regulation of DNA repair proteins was observed after co-exposure. CONCLUSION: These results support the induction of an adaptive response by BPA co-exposure that alters the microcellular environment and modulates DNA repair. Further work is required to determine whether BPA induces similar DNA lesions in vivo at environmentally relevant doses; however, in the Ku70-deficient mouse embryonic fibroblasts, exposure to a high dose of BPA was associated with changes in the cellular microenvironment that may promote survival. [ABSTRACT FROM AUTHOR]

Microscopy and micro-irradiation imaging techniques have significantly advanced our knowledge of DNA damage tolerance and the assembly of DNA repair proteins at the sites of damage. While these tools have been extensively applied to the study of nucleotide excision repair and double-strand break repair, their application to the repair of oxidatively-induced base lesions and single-strand breaks is just beginning to yield new insights. This review will focus on examining micro-irradiation techniques reported to create base lesions and single-strand breaks; these lesions are considered to be primarily addressed by proteins involved in the base excision repair (BER) pathway. By examining conditions for generating these DNA lesions and reviewing information on the assembly and dissociation of repair complexes at the induced lesion sites, we hope to promote further investigations into BER and to stimulate further development and enhancement of these techniques for the study of BER. [ABSTRACT FROM AUTHOR]

Bisphenol A (BPA) is a biologically active industrial chemical used in production of consumer products. BPA has become a target of intense public scrutiny following concerns about its association with human diseases such as obesity, diabetes, reproductive disorders, and cancer. Recent studies link BPA with the generation of reactive oxygen species, and base excision repair (BER) is responsible for removing oxidatively induced DNA lesions. Yet, the relationship between BPA and BER has yet to be examined. Further, the ubiquitous nature of BPA allows continuous exposure of the human genome concurrent with the normal endogenous and exogenous insults to the genome, and this co-exposure may impact the DNA damage response and repair. To determine the effect of BPA exposure on base excision repair of oxidatively induced DNA damage, cells compromised in double-strand break repair were treated with BPA alone or co-exposed with either potassium bromate (KBrO3) or laser irradiation as oxidative damaging agents. In experiments with KBrO3, co-treatment with BPA partially reversed the KBrO3-induced cytotoxicity observed in these cells, and this was coincident with an increase in guanine base lesions in genomic DNA. The improvement in cell survival and the increase in oxidatively induced DNA base lesions were reminiscent of previous results with alkyl adenine DNA glycosylase-deficient cells, suggesting that BPA may prevent initiation of repair of oxidized base lesions. With laser irradiation-induced DNA damage, treatment with BPA suppressed DNA repair as revealed by several indicators. These results are consistent with the hypothesis that BPA can induce a suppression of oxidized base lesion DNA repair by the base excision repair pathway. [ABSTRACT FROM AUTHOR]

Base excision repair (BER) is a primary mechanism for repair of base lesions in DNA such as those formed by exposure to the DNA methylating agent methyl methanesulfonate (MMS). Both DNA polymerase β (pol β)- and XRCC1-deficient mouse fibroblasts are hypersensitive to MMS. This is linked to a repair deficiency as measured by accumulation of strand breaks and poly(ADP-ribose) (PAR). The interaction between pol β and XRCC1 is important for recruitment of pol β to sites of DNA damage. Endogenous DNA damage can substitute for MMS-induced damage such that BER deficiency as a result of either pol β- or XRCC1-deletion is associated with sensitivity to PARP inhibitors. Pol β shRNA was used to knock down pol β in Xrcc1 +/+ and Xrcc1 −/− mouse fibroblasts. We determined whether pol β-mediated cellular resistance to MMS and PARP inhibitors resulted entirely from coordination with XRCC1 within the same BER sub-pathway. We find evidence for pol β-dependent cell survival independent of XRCC1 expression for both types of agents. The results suggest a role for pol β-dependent, XRCC1-independent repair. PAR immunofluorescence data are consistent with the hypothesis of a decrease in repair in both pol β knock down cell variants. [ABSTRACT FROM AUTHOR]

The breast cancer 1 (BRCA1) protein is a tumor suppressor playing roles in DNA repair and cell cycle regulation. Studies of DNA repair functions of BRCA1 have focused on double-strand break (DSB) repair pathways and have recently included base excision repair (BER). However, the function of BRCA1 in BER is not well defined. Here, we examined a BRCA1 role in BER, first in relation to alkylating agent (MMS) treatment of cells and the BER enzyme DNA polymerase β (pol β). MMS treatment of BRCA1 negative human ovarian and chicken DT40 cells revealed hypersensitivity, and the combined gene deletion of BRCA1 and pol β in DT40 cells was consistent with these factors acting in the same repair pathway, possibly BER. Using cell extracts and purified proteins, BRCA1 and pol β were found to interact in immunoprecipitation assays, yet in vivo and in vitro assays for a BER role of BRCA1 were negative. An alternate approach with the human cells of immunofluorescence imaging and laser-induced DNA damage revealed negligible BRCA1 recruitment during the first 60 s after irradiation, the period typical of recruitment of pol β and other BER factors. Instead, 15 min after irradiation, BRCA1 recruitment was strong and there was γ-H2AX co-localization, consistent with DSBs and repair. The rapid recruitment of pol β was similar in BRCA1 positive and negative cells. However, a fraction of pol β initially recruited remained associated with damage sites much longer in BRCA1 positive than negative cells. Interestingly, pol β expression was required for BRCA1 recruitment, suggesting a partnership between these repair factors in DSB repair. [ABSTRACT FROM AUTHOR]

Regulation of poly(ADP-ribose) (PAR) synthesis and turnover is critical to determining cell fate after genotoxic stress. Hyperactivation of PAR synthesis by poly(ADP-ribose) polymerase-1 (PARP-1) occurs when cells deficient in DNA repair are exposed to genotoxic agents; however, the function of this hyperactivation has not been adequately explained. Here, we examine PAR synthesis in mouse fibroblasts deficient in the base excision repair enzyme DNA polymerase b (pol b). The extent and duration of PARP-1 activation was measured after exposure to either the DNA alkylating agent, methyl methanesulfonate (MMS), or to low energy laser-induced DNA damage. There was strong DNA damage-induced hyperactivation of PARP-1 in pol b nullcells, but not in wild-type cells. In the case of MMS treatment, PAR synthesis did not lead to cell death in the pol b null cells, but instead resulted in increased PARylation of the nonhomologous end-joining (NHEJ) protein Ku70 and increased association of Ku70 with PARP-1. Inhibition of the NHEJ factor DNA-PK, under conditions of MMS-induced PARP-1 hyperactivation, enhanced necrotic cell death. These data suggest that PARP-1 hyperactivation is a protective mechanism triggering the classical-NHEJ DNA repair pathway when the primary alkylated base damage repair pathway is compromised. [ABSTRACT FROM AUTHOR]

In mammalian cells, the nucleosome-binding protein HMGN1 (high mobility group N1) affects the structure and function of chromatin and plays a role in repair of damaged DNA. HMGN1 affects the interaction of DNA repair factors with chromatin and their access to damaged DNA; however, not all of the repair factors affected have been identified. Here, we report that HMGN1 affects the self-poly(ADP-ribosyl)ation (i.e., PARylation) of poly(ADP-ribose) polymerase-1 (PARP-1), a multifunctional and abundant nuclear enzyme known to recognize DNA lesions and promote chromatin remodeling, DNA repair, and other nucleic acid transactions. The catalytic activity of PARP-1 is activated by DNA with a strand break, and this results in self-PARylation and PARylation of other chromatin proteins. Using cells obtained from Hmgn1-/- and Hmgn1-/- littermate mice, we find that in untreated cells, loss of HMGN1 protein reduces PARP-1 self-PARylation. A similar result was obtained after MMS treatment of these cells. In imaging experiments after low energy laser-induced DNA damage, less PARylation at lesion sites was observed in Hmgn1-/- than in Hmgn1+/+ cells. The HMGN1 regulation of PARP-1 activity could be mediated by direct protein-protein interaction as HMGN1 and PARP-1 were found to interact in binding assays. Purified HMGN1 was able to stimulate self-PARylation of purified PARP-1, and in experiments with cell extracts, self-PARylation was greater in Hmgn1+/+ than in Hmgn1-/- extract. The results suggest a regulatory role for HMGN1 in PARP-1 activation. [ABSTRACT FROM AUTHOR]

Human mismatch repair proteins MSH2-MSH6 play an essential role in maintaining genetic stability and preventing disease. While protein functions have been extensively studied, the substantial amino-terminal region (NTR*) of MSH6 that is unique to eukaryotic proteins, has mostly evaded functional characterization. We demonstrate that a cluster of three nuclear localization signals (NLS) in the NTR direct nuclear import. Individual NLSs are capable of partially directing cytoplasmic protein into the nucleus; however only cooperative effects between all three NLSs efficiently transport MSH6 into the nucleus. In striking contrast to yeast and previous assumptions on required heterodimerization, human MSH6 does not determine localization of its heterodimeric partner, MSH2. A cancer-derived mutation localized between two of the three NLS significantly decreases nuclear localization of MSH6, suggesting altered protein localization can contribute to carcinogenesis. These results clarify the pending speculations on the functional role of the NTR in human MSH6 and identify a novel, cooperative nuclear localization signal. [ABSTRACT FROM AUTHOR]

The lack of efficient identification and isolation methods for specific molecular binders has fundamentally limited drug discovery. Here, we have developed a method to select peptide nucleic acid (PNA) encoded molecules with specific functional properties from combinatorially generated libraries. This method consists of three essential stages: (1) creation of a Lab-on-Bead™ library, a one-bead, one-sequence library that, in turn, displays a library of candidate molecules, (2) fluorescence microscopy-aided identification of single target-bound beads and the extraction - wet or dry - of these beads and their attached candidate molecules by a micropipette manipulator, and (3) identification of the target-binding candidate molecules via amplification and sequencing. This novel integration of techniques harnesses the sensitivity of DNA detection methods and the multiplexed and miniaturized nature of molecule screening to efficiently select and identify target-binding molecules from large nucleic acid encoded chemical libraries. Beyond its potential to accelerate assays currently used for the discovery of new drug candidates, its simple bead-based design allows for easy screening over a variety of prepared surfaces that can extend this technique's application to the discovery of diagnostic reagents and disease markers. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: Harnessing the new bioremediation and biotechnology applications offered by the dissimilatory metal-reducing bacteria, Shewanella oneidensis MR-1, requires a clear understanding of its transcription machinery, a pivotal component in maintaining vitality and in responding to various conditions, including starvation and environmental stress. Here, we have reconstituted the S. oneidensis RNA polymerase (RNAP) core in vivo by generating a co-overexpression construct that produces a long polycistronic mRNA encoding all of the core subunits (α, β, β′, and ω) and verified that this reconstituted core is capable of forming fully functional holoenzymes with the S. oneidensis σ factors σ70, σ38, σ32, and σ24. Further, to demonstrate the applications for this reconstituted core, we report the application of single-molecule fluorescence resonance energy transfer (smFRET) assays to monitor the mechanisms of transcription by the S. oneidensis σ70-RNAP holoenyzme. These results show that the reconstituted transcription machinery from S. oneidensis, like its Escherichia coli counterpart, “scrunches” the DNA into its active center during initial transcription, and that as the holoenzyme transitions into elongation, the release of σ70 is non-obligatory. [Copyright &y& Elsevier]

Genomic instability has been proposed as a predictive biomarker for immunotherapy in ovarian cancer. We tested a method for measuring DNA damage, a direct measure of genomic instability, in ovarian tumors and its ability to predict immunotherapy response to Vigil (gemogenovatucel-T). Eighty-two formalin-fixed paraffin-embedded tumors from the VITAL trial (NCT02346747) underwent DNA damage assessment using Repair Assisted Damage Detection (RADD). VITAL tested maintenance Vigil therapy vs. placebo for stage IIIB-IV newly diagnosed ovarian cancer in clinical complete response. DNA lesion levels determined by RADD were scored and assessed against patient survival outcomes, expression of CD39, and gene expression signatures. A graduated distribution of RADD scores occurred across all 82 ovarian samples. RADD scores were able to predict HR status (p < 0.001). RADD demonstrated a significant Pearson's correlation with suggested Vigil biomarker CD39 (r = 0.473; p < 0.001), specifically within HRP tumors (r = 0.57; p = 0.002). High RADD scores correlated with worse recurrent free survival (RFS) in the placebo arm of the trial (7.9 vs. 14.7 months, high vs. low; p = 0.066). High RADD scores were also predictive of significant RFS over 39.4 months with Vigil compared to placebo (25.1 vs. 11.7 months, p = 0.005) and improved, but not significantly, OS with 38.8 vs. 31.8 months. RADD revealed DNA repair proficiency without mutation signatures or expression profiling. High DNA damage levels show improved survival for Vigil maintenance therapies and are correlated with immune evasion proteins. The persistence of DNA lesions in the genomic DNA offers a new biomarker for immunotherapy patient stratification. • DNA damage levels within tumors are measured using RADD. • RADD damage scores detect differences between homologous recombination proficient and deficient tumors. • High DNA damage levels correlate with improved response to the immunotherapy Vigil. • High DNA damage levels also correlate with expression changes in immune evasion signatures. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Inhalation exposures to dihydroxyacetone (DHA) occur through spray tanning and e-cigarette aerosols. Several studies in skin models have demonstrated that millimolar doses of DHA are cytotoxic, yet the genotoxicity was unclear. We examined the genotoxicity of DHA in cell models relevant to inhalation exposures. Human bronchial epithelial cells BEAS-2B, lung carcinoma cells A549, cardiomyocyte Ac16, and hepatocellular carcinoma HepG3 were exposed to DHA, and low millimolar doses of DHA were cytotoxic. IC90 DHA doses induced cell cycle arrest in all cells except the Ac16. We examined DHA's genotoxicity using strand break markers, DNA adduct detection by Repair Assisted Damage Detection (RADD), metaphase spreads, and a forward mutation assay for mutagenesis. Similar to results for skin, DHA did not induce significant levels of strand breaks. However, RADD revealed DNA adducts were induced 24 h after DHA exposure, with BEAS-2B and Ac16 showing oxidative lesions and A549 and HepG3 showing crosslink-type lesions. Yet, only low levels of reactive oxygen species or advanced glycation end products were detected after DHA exposure. Metaphase spreads revealed significant increases in chromosomal aberrations in the BEAS-2B and HepG3 with corresponding changes in ploidy. Finally, we confirmed the mutagenesis observed using the supF reporter plasmid. DHA increased the mutation frequency, consistent with methylmethane sulfonate, a mutagen and clastogen. These data demonstrate DHA is a clastogen, inducing cell-specific genotoxicity and chromosomal instability. The specific genotoxicity measured in the BEAS-2B in this study suggests that inhalation exposures pose health risks to vapers, requiring further investigation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We have characterized the nuclear localization signal (NLS) of XRCC1 structurally using X-ray crystallography and functionally using fluorescence imaging. Crystallography and binding studies confirm the bipartite nature of the XRCC1 NLS interaction with Importin α (Impα) in which the major and minor binding motifs are separated by >20 residues, and resolve previous inconsistent determinations. Binding studies of peptides corresponding to the bipartite NLS, as well as its major and minor binding motifs, to both wild-type and mutated forms of Impα reveal pronounced cooperative binding behavior that is generated by the proximity effect of the tethered major and minor motifs of the NLS. The cooperativity stems from the increased local concentration of the second motif near its cognate binding site that is a consequence of the stepwise binding behavior of the bipartite NLS. We predict that the stepwise dissociation of the NLS from Impα facilitates unloading by providing a partially complexed intermediate that is available for competitive binding by Nup50 or the Importin β binding domain. This behavior provides a basis for meeting the intrinsically conflicting high affinity and high flux requirements of an efficient nuclear transport system. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: Heat shock factor 1 (HSF1) is a transcription factor crucial for cellular stress responses. HSF1 activates heat shock proteins (HSPs) in response to proteotoxic stress, aiding in protein folding and maintaining proteostasis. HSF1 is often overexpressed in various cancer cells, fueling malignancy and indicating a poor prognosis. The mechanisms behind HSF1-induced tumorigenesis are complex and cancer type-dependent. Targeting HSF1 presents a novel cancer treatment strategy. Heat shock factor 1 (HSF1) is a transcription factor crucial for regulating heat shock response (HSR), one of the significant cellular protective mechanisms. When cells are exposed to proteotoxic stress, HSF1 induces the expression of heat shock proteins (HSPs) to act as chaperones, correcting the protein-folding process and maintaining proteostasis. In addition to its role in HSR, HSF1 is overexpressed in multiple cancer cells, where its activation promotes malignancy and leads to poor prognosis. The mechanisms of HSF1-induced tumorigenesis are complex and involve diverse signaling pathways, dependent on cancer type. With its important roles in tumorigenesis and tumor progression, targeting HSF1 offers a novel cancer treatment strategy. In this article, we examine the basic function of HSF1 and its regulatory mechanisms, focus on the mechanisms involved in HSF1′s roles in different cancer types, and examine current HSF1 inhibitors as novel therapeutics to treat cancers. [ABSTRACT FROM AUTHOR]

Despite well-established links between exposure to ultraviolet radiation (UVR) and skin cancer, UVR-based tanning behaviors persist among college students. Understanding tanning motivations, perceptions, barriers, and demographic characteristics of this population is critical to modifying these behaviors, but is limited by variability in study design, sample size, and outcomes measured in the current literature. To help clarify the tanning behaviors of this population and provide a concise reference for future studies, this review examines existing reports to determine the comparability of tanning behaviors across multiple U.S. college populations. A systematic review of the literature was performed in July 2016 to identify studies investigating tanning behaviors among U.S. college students. Twenty-three studies met inclusion criteria. High rates of indoor tanning (IT) and outdoor tanning (OT) were found among college students. Key motivators included appearance, emotion, health perceptions, and the influence of parents, peers, and the media. Misconceptions regarding skin protection, low rates of sun protective behaviors, and tanning dependence were barriers against safe UVR exposure. Understudied demographic factors may account for variance in observed tanning behaviors, emphasizing the need for standardization efforts to consistently identify trends associated with geographical region, age, year in college, and sex. The findings presented in this review reaffirm that college students are at high risk for tanning-associated skin cancer, emphasizing the critical need for effective, targeted interventions. Improved interventions will reduce the burden of skin cancer within this group, ultimately contributing to longer, healthier lives. [ABSTRACT FROM AUTHOR]

Despite well-established links between exposure to ultraviolet radiation (UVR) and skin cancer, UVR-based tanning behaviors persist among college students. Understanding tanning motivations, perceptions, barriers, and demographic characteristics of this population is critical to modifying these behaviors, but is limited by variability in study design, sample size, and outcomes measured in the current literature. To help clarify the tanning behaviors of this population and provide a concise reference for future studies, this review examines existing reports to determine the comparability of tanning behaviors across multiple U.S. college populations. A systematic review of the literature was performed in July 2016 to identify studies investigating tanning behaviors among U.S. college students. Twenty-three studies met inclusion criteria. High rates of indoor tanning (IT) and outdoor tanning (OT) were found among college students. Key motivators included appearance, emotion, health perceptions, and the influence of parents, peers, and the media. Misconceptions regarding skin protection, low rates of sun protective behaviors, and tanning dependence were barriers against safe UVR exposure. Understudied demographic factors may account for variance in observed tanning behaviors, emphasizing the need for standardization efforts to consistently identify trends associated with geographical region, age, year in college, and sex. The findings presented in this review reaffirm that college students are at high risk for tanning-associated skin cancer, emphasizing the critical need for effective, targeted interventions. Improved interventions will reduce the burden of skin cancer within this group, ultimately contributing to longer, healthier lives. [ABSTRACT FROM AUTHOR]

Highlights: [•] C12–C20 oxidized and reduced forms of XRCC1 were verified in mouse fibroblasts. [•] Pol β binding affinity to the XRCC1 NTD decreased with the reduced C12A mutant. [•] Folding properties of full-length C12A and wild-type XRCC1 were indistinguishable. [•] MMS-induced cellular PAR level was highly elevated in C12A-expressing cells. [•] PARP inhibitor sensitization to MMS was compromised in C12A-expressing cells. [ABSTRACT FROM AUTHOR]

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 XRCC1 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 XRCC1 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Exogenous exposures to the triose sugar dihydroxyacetone (DHA) occur from sunless tanning products and electronic cigarette aerosol. Once inhaled or absorbed, DHA enters cells, is converted to dihydroxyacetone phosphate (DHAP), and incorporated into several metabolic pathways. Cytotoxic effects of DHA vary across the cell types depending on the metabolic needs of the cells, and differences in the generation of reactive oxygen species (ROS), cell cycle arrest, and mitochondrial dysfunction have been reported. We have shown that cytotoxic doses of DHA induced metabolic imbalances in glycolysis and oxidative phosphorylation in liver and kidney cell models. Here, we examine the dose-dependent effects of DHA on the rat cardiomyocyte cell line, H9c2. Cells begin to experience cytotoxic effects at low millimolar doses, but an increase in cell survival was observed at 2 mM DHA. We confirmed that 2 mM DHA increased cell survival compared to the low cytotoxic 1 mM dose and investigated the metabolic differences between these two low DHA doses. Exposure to 1 mM DHA showed changes in the cell's fuel utilization, mitochondrial reactive oxygen species (ROS), and transient changes in the glycolysis and mitochondrial energetics, which normalized 24 h after exposure. The 2 mM dose induced robust changes in mitochondrial flux through acetyl CoA and elevated expression of fatty acid synthase. Distinct from the 1 mM dose, the 2 mM exposure increased mitochondrial ROS and NAD(P)H levels, and sustained changes in LDHA/LDHB and acetyl CoA-associated enzymes were observed. Although the cells were exposed to low cytotoxic (1 mM) and non-cytotoxic (2 mM) acute doses of DHA, significant changes in mitochondrial metabolic pathways occurred. Further, the proliferation increase at the acute 2 mM DHA dose suggests a metabolic adaption occurred with sustained consequences in survival and proliferation. With increased exogenous exposure to DHA through e-cigarette aerosol, this work suggests cell metabolic changes induced by acute or potentially chronic exposures could impact cell function and survival. [Display omitted] • Non-cytotoxic doses of DHA induce dose-dependent changes in mitochondrial metabolic pathways. • Increased cell proliferation occurs in H9c2 at 2 mM DHA. • Cytotoxic doses of DHA induce mitophagy and suppression of mitochondrial metabolic pathways. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 H 2 O 2 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. [ABSTRACT FROM AUTHOR]

The multi-domain protein XRCC1 is without catalytic activity, but can interact with a number of known repair proteins. The interaction between the N-terminal domain (NTD) of XRCC1 and DNA polymerase β (pol β) is critical for recruitment of pol β to sites of DNA damage and repair. Crystallographic and NMR approaches have identified oxidized and reduced forms of the XRCC1 NTD, and the corresponding forms of XRCC1 have been identified in cultured mouse fibroblast cells. Both forms of NTD interact with pol β, but the interaction is much stronger with the oxidized form. The potential for formation of the C12-C20 oxidized conformation can be removed by alanine substitution at C12 (C12A) leading to stabilized reduced XRCC1 with a lower pol β binding affinity. Here, we compare cells expressing C12A XRCC1 (XRE8) with those expressing wild-type XRCC1 (XC5). Reduced C12A XRCC1 is detected at sites of micro-irradiation DNA damage, but provides slower recruitment of pol β. Expression of reduced XRCC1 does not affect sensitivity to MMS or H 2 O 2. In contrast, further oxidative stress imposed by glutathione depletion results in increased sensitization of reduced XRCC1-expressing cells to H 2 O 2 compared with wild-type XRCC1-expressing cells. There is no indication of enhanced H 2 O 2 -generated free radicals or DNA strand breaks in XRE8 cells. However, elevated cellular PAR is found following H 2 O 2 exposure, suggesting BER deficiency of H 2 O 2 -induced damage in the C12A expressing cells. [ABSTRACT FROM AUTHOR]

DNA adducts and strand breaks are induced by various exogenous and endogenous agents. Accumulation of DNA damage is implicated in many disease processes, including cancer, aging, and neurodegeneration. The continuous acquisition of DNA damage from exogenous and endogenous stressors coupled with defects in DNA repair pathways contribute to the accumulation of DNA damage within the genome and genomic instability. While mutational burden offers some insight into the level of DNA damage a cell may have experienced and subsequently repaired, it does not quantify DNA adducts and strand breaks. Mutational burden also infers the identity of the DNA damage. With advances in DNA adduct detection and quantification methods, there is an opportunity to identify DNA adducts driving mutagenesis and correlate with a known exposome. However, most DNA adduct detection methods require isolation or separation of the DNA and its adducts from the context of the nuclei. Mass spectrometry, comet assays, and other techniques precisely quantify lesion types but lose the nuclear context and even tissue context of the DNA damage. The growth in spatial analysis technologies offers a novel opportunity to leverage DNA damage detection with nuclear and tissue context. However, we lack a wealth of techniques capable of detecting DNA damage in situ. Here, we review the limited existing in situ DNA damage detection methods and examine their potential to offer spatial analysis of DNA adducts in tumors or other tissues. We also offer a perspective on the need for spatial analysis of DNA damage in situ and highlight Repair Assisted Damage Detection (RADD) as an in situ DNA adduct technique with the potential to integrate with spatial analysis and the challenges to be addressed. • DNA adducts are biomarkers of exogenous and endogenous exposures. • Spatial analysis should be extended to DNA adducts to understand their biological context. • Fluorescence-based methods offer new opportunities for spatial analysis of DNA adducts. • Measuring DNA adducts and their spatial context may provide new insight into changes in DNA repair. [ABSTRACT FROM AUTHOR]

Simple Summary: Prostate cancer is the most diagnosed cancer among men in the United States. African American men are diagnosed with and succumb to prostate cancer at higher rates than other demographic groups. Previously published works described the biological differences in prostate tumors that may contribute to poorer outcomes in African American men compared to European American men. This study was designed to explore the DNA lesion profiles found in prostate tissues. Using tissue microarrays, we found that prostate tumors from African American patients have more uracil and pyrimidine damage, elevated UNG levels, and reduced XRCC1 levels than European American tumors, which may indicate defects in the base excision repair pathway. In addition, these men had higher UMP and lower expression of folate cycle metabolites, suggesting that metabolic rewiring may also contribute to the dysregulation of base excision repair. African Americans (AA) are two times more likely to be diagnosed with and succumb to prostate cancer (PCa) compared to European Americans (EA). There is mounting evidence that biological differences in these tumors contribute to disparities in patient outcomes. Our goal was to examine the differences in DNA damage in AA and EA prostate tissues. Tissue microarrays with matched tumor-benign adjacent pairs from 77 AA and EA PCa patients were analyzed for abasic sites, oxidative lesions, crosslinks, and uracil content using the Repair Assisted Damage Detection (RADD) assay. Our analysis revealed that AA PCa, overall, have more DNA damage than EA PCa. Increased uracil and pyrimidine lesions occurred in AA tumors, while EA tumors had more oxidative lesions. AA PCa have higher levels of UMP and folate cycle metabolites than their EA counterparts. AA PCa showed higher levels of UNG, the uracil-specific glycosylase, than EA, despite uracil lesions being retained within the genome. AA patients also had lower levels of the base excision repair protein XRCC1. These results indicate dysfunction in the base excision repair pathway in AA tumors. Further, these findings reveal how metabolic rewiring in AA PCa drives biological disparities and identifies a targetable axis for cancer therapeutics. [ABSTRACT FROM AUTHOR]

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 D3 (1500 IU/kg; vehicle) or chow supplemented with additional vitamin D3 (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. [ABSTRACT FROM AUTHOR]

Simple Summary: This work establishes that ovarian tumors contain persistent oxidative DNA damage levels that can be measured using Repair Assisted Damage Detection (RADD). The persistent DNA damage correlates with increased protein expression of PD-L1, establishing a link between genomic instability and PD-L1 expression across ovarian tumors. DNA damage may be a potential biomarker for immunotherapy. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]