Arsenic and UVR co-exposure results in unique gene expression profile identifying key co-carcinogenic mechanisms.

Changes in gene expression underlie many pathogenic endpoints including carcinogenesis. Metals, like arsenic, alter gene expression; however, the consequences of co-exposures of metals with other stressors are less understood. Although arsenic acts as a co-carcinogen by enhancing the development of UVR skin cancers, changes in gene expression in arsenic UVR co-carcinogenesis have not been investigated. We performed RNA-sequencing analysis to profile changes in gene expression distinct from arsenic or UVR exposures alone. A large number of differentially expressed genes (DEGs) were identified after arsenic exposure alone, while after UVR exposure alone fewer genes were changed. A distinct increase in the number of DEGs was identified after exposure to combined arsenic and UVR exposure that was synergistic rather than additive. In addition, a majority of these DEGs were unique from arsenic or UVR alone suggesting a distinct response to combined arsenic-UVR exposure. Globally, arsenic alone and arsenic plus UVR exposure caused a global downregulation of genes while fewer genes were upregulated. Gene Ontology analysis using the DEGs revealed cellular processes related to chromosome instability, cell cycle, cellular transformation, and signaling were targeted by combined arsenic and UVR exposure, distinct from UVR alone and arsenic alone, while others were related to epigenetic mechanisms such as the modification of histones. This result suggests the cellular functions we identified in this study may be key in understanding how arsenic enhances UVR carcinogenesis and that arsenic-enhanced gene expression changes may drive co-carcinogenesis of UVR exposure.

Arsenic is a potent co-mutagen of ultraviolet light.

Arsenic enhances the carcinogenicity of ultraviolet radiation (UVR). However, the mechanisms of arsenic-driven oncogenesis are not well understood. Here, we utilize experimental systems to investigate the carcinogenic and mutagenic properties of co-exposure to arsenic and UVR. In vitro and in vivo exposures indicate that, by itself, arsenic is not mutagenic. However, in combination with UVR, arsenic exposure has a synergistic effect leading to an accelerated mouse skin carcinogenesis and to more than 2-fold enrichment of UVR mutational burden. Notably, mutational signature ID13, previously found only in UVR-associated human skin cancers, is observed exclusively in mouse skin tumors and cell lines jointly exposed to arsenic and UVR. This signature was not observed in any model system exposed purely to arsenic or purely to UVR, making ID13, to the best of our knowledge, the first co-exposure signature to be reported using controlled experimental conditions. Analysis of existing skin cancer genomics data reveals that only a subset of cancers harbor ID13 and these exhibit an elevated UVR mutagenesis. Our results report a unique mutational signature caused by a co-exposure to two environmental carcinogens and provide comprehensive evidence that arsenic is a potent co-mutagen and co-carcinogen of UVR.

Serum autoantibodies and exploratory molecular pathways in rural miners: A pilot study.

Introduction: The Southwestern United States (SWUS) has an extensive history of coal and metal mining, including uranium (U) mining. Lung diseases, including but not limited to, lung cancer and pulmonary fibrosis, have been studied extensively in miners due to occupational, dust-related exposures. However, high-throughput autoimmune biomarkers are largely understudied in miners, despite the fact that ore miners, such as U-miners, are at an increased risk for the development of autoimmune diseases such as systemic sclerosis and systemic lupus erythematosus (SLE). Additionally, there are current gaps in knowledge regarding which signaling pathways may play a role in occupational exposure-associated autoimmunity. Methods: Most current and former miners in the SWUS live close to their previous workplaces, in remote areas, with limited access to healthcare. In this pilot study, by leveraging a mobile clinical platform for patient care and clinical outreach, we recruited 44 miners who self-identified as either U (n = 10) or non-U miners (n = 34) and received health screenings. Serum IgG and IgM autoantibodies against 128 antigens were assessed using a high-throughput molecular technique, as a preliminary health screening opportunity. Results: Even when adjusting for age as a covariate, there was a significant (p < 0.05) association between self-reported U-mining exposure and biomarkers including IgM alpha-actinin, histones H2B, and H4, myeloperoxidase (MPO) and myelin basic protein. However, adjusting for age did not result in significant associations for IgG autoantibody production in U-miners. Bioinformatic pathway analysis revealed several altered signaling pathways between IgM and IgG autoantibodies among both U and non-U miners. Conclusions: Further research is warranted regarding the mechanistic connection between U-exposure and autoantibody development, especially regarding histone-related alterations and IgM autoantibody production.

Arsenic and cancer: Evidence and mechanisms.

Arsenic is a potent carcinogen and poses a significant health concern worldwide. Exposure occurs through ingestion of drinking water and contaminated foods and through inhalation due to pollution. Epidemiological evidence shows arsenic induces cancers of the skin, lung, liver, and bladder among other tissues. While studies in animal and cell culture models support arsenic as a carcinogen, the mechanisms of arsenic carcinogenesis are not fully understood. Arsenic carcinogenesis is a complex process due its ability to be metabolized and because of the many cellular pathways it targets in the cell. Arsenic metabolism and the multiple forms of arsenic play distinct roles in its toxicity and contribute differently to carcinogenic endpoints, and thus must be considered. Arsenic generates reactive oxygen species increasing oxidative stress and damaging DNA and other macromolecules. Concurrently, arsenic inhibits DNA repair, modifies epigenetic regulation of gene expression, and targets protein function due its ability to replace zinc in select proteins. While these mechanisms contribute to arsenic carcinogenesis, there remain significant gaps in understanding the complex nature of arsenic cancers. In the future improving models available for arsenic cancer research and the use of arsenic induced human tumors will bridge some of these gaps in understanding arsenic driven cancers.

Arsenic is a potent co-mutagen of ultraviolet light.

Environmental co-exposures are widespread and are major contributors to carcinogenic mechanisms. Two well-established environmental agents causing skin cancer are ultraviolet radiation (UVR) and arsenic. Arsenic is a known co-carcinogen that enhances UVR's carcinogenicity. However, the mechanisms of arsenic co-carcinogenesis are not well understood. In this study, we utilized primary human keratinocytes and a hairless mouse model to investigate the carcinogenic and mutagenic properties of co-exposure to arsenic and UVR. In vitro and in vivo exposures revealed that, on its own, arsenic is neither mutagenic nor carcinogenic. However, in combination with UVR, arsenic exposure has a synergistic effect leading to an accelerated mouse skin carcinogenesis as well as to more than 2-fold enrichment of UVR mutational burden. Notably, mutational signature ID13, previously found only in UVR-associated human skin cancers, was observed exclusively in mouse skin tumors and cell lines jointly exposed to arsenic and UVR. This signature was not observed in any model system exposed purely to arsenic or purely to UVR, making ID13 the first co-exposure signature to be reported using controlled experimental conditions. Analysis of existing genomics data from basal cell carcinomas and melanomas revealed that only a subset of human skin cancers harbor ID13 and, consistent with our experimental observations, these cancers exhibited an elevated UVR mutagenesis. Our results provide the first report of a unique mutational signature caused by a co-exposure to two environmental carcinogens and the first comprehensive evidence that arsenic is a potent co-mutagen and co-carcinogen of UVR. Importantly, our findings suggest that a large proportion of human skin cancers are not formed purely due to UVR exposure but rather due to a co-exposure of UVR and other co-mutagens such as arsenic.

Particulate arsenic trioxide induces higher DNA damage and reactive oxygen species than soluble arsenite in lung epithelial cells.

Environmental arsenic exposure is associated with lung cancer. Arsenic is the first substance known to cause lung cancer by two distinct routes, ingestion and inhalation, in the forms of soluble arsenite and particulate arsenic trioxide, respectively. In comparison to significant progresses in research on mechanisms for lung carcinogenesis of arsenic ingestion, inhalation arsenic exposure route in particulate form and its lung carcinogenic mechanisms are relatively under-investigated. Fundamentally, it remains unclear whether particulate arsenic exposure is in a dissolved form and whether particulate exposure yields higher damage. Utilizing dynamic laser scattering, particulate arsenic trioxide exposure in cellular system was confirmed to be in particulate form instead of dissolved form. Using immunofluorescence, particulate arsenic trioxide was demonstrated to generate dramatically higher oxidative DNA damage and strand break, as well as significantly higher superoxide, in lung epithelial cell lines such as BEAS-2B, HSAEC1-KT, and SAE, comparing to soluble arsenite exposure at similar or lower concentration. This study demonstrated that particulate arsenic trioxide exposure yields higher damage in lung epithelial cells, and indicated that inhalation route of particulate arsenic exposure plays an important role in lung carcinogenesis.

Arsenic impairs the lineage commitment of hematopoietic progenitor cells through the attenuation of GATA-2 DNA binding activity.

Arsenic exposure produces significant hematotoxicity in vitro and in vivo. Our previous work shows that arsenic (in the form of arsenite, AsIII) interacts with the zinc finger domains of GATA-1, inhibiting the function of this critical transcription factor, and resulting in the suppression of erythropoiesis. In addition to GATA-1, GATA-2 also plays a key role in the regulation of hematopoiesis. GATA-1 and GATA-2 have similar zinc finger domains (C4-type) that are structurally favorable for AsIII interactions. Taking this into consideration, we hypothesized that early stages of hematopoietic differentiation that are dependent on the function of GATA-2 may also be disrupted by AsIII exposure. We found that in vitro AsIII exposures disrupt the erythromegakaryocytic lineage commitment and differentiation of erythropoietin-stimulated primary mouse bone marrow hematopoietic progenitor cells (HPCs), producing an aberrant accumulation of cells in early stages of hematopoiesis and subsequent reduction of committed erythro-megakaryocyte progenitor cells. Arsenic significantly accumulated in the GATA-2 protein, causing the loss of zinc, and disruption of GATA-2 function, as measured by chromatin immunoprecipitation and the expression of GATA-2 responsive genes. Our results show that the attenuation of GATA-2 function is an important mechanism contributing to the aberrant lineage commitment and differentiation of early HPCs. Collectively, findings from the present study suggest that the AsIII-induced disruption of erythro-megakaryopoiesis may contribute to the onset and/or exacerbation of hematological disorders, such as anemia.

Arsenite exposure inhibits the erythroid differentiation of human hematopoietic progenitor CD34+ cells and causes decreased levels of hemoglobin.

Arsenic exposure poses numerous threats to human health. Our previous work in mice has shown that arsenic causes anemia by inhibiting erythropoiesis. However, the impacts of arsenic exposure on human erythropoiesis remain largely unclear. We report here that low-dose arsenic exposure inhibits the erythroid differentiation of human hematopoietic progenitor cells (HPCs). The impacts of arsenic (in the form of arsenite; As3+) on red blood cell (RBC) development was evaluated using a long-term culture of normal human bone marrow CD34+-HPCs stimulated in vitro to undergo erythropoiesis. Over the time course studied, we analyzed the expression of the cell surface antigens CD34, CD71 and CD235a, which are markers commonly used to monitor the progression of HPCs through the stages of erythropoiesis. Simultaneously, we measured hemoglobin content, which is an important criterion used clinically for diagnosing anemia. As compared to control, low-dose As3+ exposure (100 nM and 500 nM) inhibited the expansion of CD34+-HPCs over the time course investigated; decreased the number of committed erythroid progenitors (BFU-E and CFU-E) and erythroblast differentiation in the subsequent stages; and caused a reduction of hemoglobin content. These findings demonstrate that low-dose arsenic exposure impairs human erythropoiesis, likely by combined effects on various stages of RBC formation.

Arsenic co-carcinogenesis: Inhibition of DNA repair and interaction with zinc finger proteins.

Arsenic is widely present in the environment and is associated with various population health risks including cancers. Arsenic exposure at environmentally relevant levels enhances the mutagenic effect of other carcinogens such as ultraviolet radiation. Investigation on the molecular mechanisms could inform the prevention and intervention strategies of arsenic carcinogenesis and co-carcinogenesis. Arsenic inhibition of DNA repair has been demonstrated to be an important mechanism, and certain DNA repair proteins have been identified to be extremely sensitive to arsenic exposure. This review will summarize the recent advances in understanding the mechanisms of arsenic carcinogenesis and co-carcinogenesis, including DNA damage induction and ROS generation, particularly how arsenic inhibits DNA repair through an integrated molecular mechanism which includes its interactions with sensitive zinc finger DNA repair proteins.

Uranium directly interacts with the DNA repair protein poly (ADP-ribose) polymerase 1.

People living in southwest part of United States are exposed to uranium (U) through drinking water, air, and soil. U is radioactive, but independent of this radioactivity also has important toxicological considerations as an environmental metal. At environmentally relevant concentrations, U is both mutagenic and carcinogenic. Emerging evidence shows that U inhibits DNA repair activity, but how U interacts with DNA repair proteins is still largely unknown. Herein, we report that U directly interacts with the DNA repair protein, Protein Poly (ADP-ribose) Polymerase 1 (PARP-1) through direct binding with the zinc finger motif, resulting in zinc release from zinc finger and DNA binding activity loss of the protein. At the peptide level, instead of direct competition with zinc ion in the zinc finger motif, U does not show thermodynamic advantages over zinc. Furthermore, zinc pre-occupied PARP-1 zinc finger is insensitive to U treatment, but U bound to PARP-1 zinc finger can be partially replaced by zinc. These results provide mechanistic basis on molecular level to U inhibition of DNA repair.

Inhibition of red blood cell development by arsenic-induced disruption of GATA-1.

Anemia is a hematological disorder that adversely affects the health of millions of people worldwide. Although many variables influence the development and exacerbation of anemia, one major contributing factor is the impairment of erythropoiesis. Normal erythropoiesis is highly regulated by the zinc finger transcription factor GATA-1. Disruption of the zinc finger motifs in GATA-1, such as produced by germline mutations, compromises the function of this critical transcription factor and causes dyserythropoietic anemia. Herein, we utilize a combination of in vitro and in vivo studies to provide evidence that arsenic, a widespread environmental toxicant, inhibits erythropoiesis likely through replacing zinc within the zinc fingers of the critical transcription factor GATA-1. We found that arsenic interacts with the N- and C-terminal zinc finger motifs of GATA-1, causing zinc loss and inhibition of DNA and protein binding activities, leading to dyserythropoiesis and an imbalance of hematopoietic differentiation. For the first time, we show that exposures to a prevalent environmental contaminant compromises the function of a key regulatory factor in erythropoiesis, producing effects functionally similar to inherited GATA-1 mutations. These findings highlight a novel molecular mechanism by which arsenic exposure may cause anemia and provide critical insights into potential prevention and intervention for arsenic-related anemias.

Serum-borne factors alter cerebrovascular endothelial microRNA expression following particulate matter exposure near an abandoned uranium mine on the Navajo Nation.

BACKGROUND: Commercial uranium mining on the Navajo Nation has subjected communities on tribal lands in the Southwestern United States to exposures from residual environmental contamination. Vascular health effects from these ongoing exposures are an active area of study. There is an association between residential mine-site proximity and circulating biomarkers in residents, however, the contribution of mine-site derived wind-blown dusts on vascular and other health outcomes is unknown. To assess neurovascular effects of mine-site derived dusts, we exposed mice using a novel exposure paradigm, the AirCARE1 mobile inhalation laboratory, located 2 km from an abandoned uranium mine, Claim 28 in Blue Gap Tachee, AZ. Mice were exposed to filtered air (FA) (n = 6) or concentrated ambient particulate matter (CAPs) (n = 5) for 2 wks for 4 h per day. RESULTS: To assess miRNA differential expression in cultured mouse cerebrovascular cells following particulate matter (PM) exposure (average: 96.6 ± 60.4 µg/m3 for all 4 h exposures), the serum cumulative inflammatory potential (SCIP) assay was employed. MiRNA sequencing was then performed in cultured mouse cerebrovascular endothelial cells (mCECs) to evaluate transcriptional changes. Results indicated 27 highly differentially expressed (p < 0.01) murine miRNAs, as measured in the SCIP assay. Gene ontology (GO) pathway analysis revealed notable alterations in GO enrichment related to the cytoplasm, protein binding and the cytosol, while significant KEGG pathways involved pathways in cancer, axon guidance and Wnt signaling. Expression of these 27 identified, differentially expressed murine miRNAs were then evaluated in the serum. Nine of these miRNAs (~ 30%) were significantly altered in the serum and 8 of those miRNAs demonstrated the same directional change (either upregulation or downregulation) as cellular miRNAs, as measured in the SCIP assay. Significantly upregulated miRNAs in the CAPs exposure group included miRNAs in the let-7a family. Overexpression of mmu-let-7a via transfection experiments, suggested that this miRNA may mediate mCEC barrier integrity following dust exposure. CONCLUSIONS: Our data suggest that mCEC miRNAs as measured in the SCIP assay show similarity to serum-borne miRNAs, as approximately 30% of highly differentially expressed cellular miRNAs in the SCIP assay were also found in the serum. While translocation of miRNAs via exosomes or an alternative mechanism is certainly possible, other yet-to-be-identified factors in the serum may be responsible for significant miRNA differential expression in endothelium following inhaled exposures. Additionally, the most highly upregulated murine miRNAs in the CAPs exposure group were in the let-7a family. These miRNAs play a prominent role in cell growth and differentiation and based on our transfection experiments, mmu-let-7a may contribute to cerebrovascular mCEC alterations following inhaled dust exposure.

Peroxynitrite contributes to arsenic-induced PARP-1 inhibition through ROS/RNS generation.

Arsenic, in the trivalent form (AsIII), is a human co-carcinogen reported to enhance mutagenesis effects of other carcinogens such as UV radiation by inhibiting DNA repair. The zinc finger DNA repair protein Poly (ADP-ribose) polymerase 1 (PARP-1) is a sensitive target of AsIII and both reactive oxygen and nitrogen species (ROS/RNS) generated by AsIII contribute to PARP-1 inhibition. However, the mechanisms of ROS/RNS-mediated PARP inhibition and how AsIII-generated ROS/RNS may be interconnected are still unclear. In this study, we found AsIII exposure of normal human keratinocyte (HEKn) cells generated peroxynitrite through superoxide and nitric oxide production in an AsIII concentration dependent manner. Peroxynitrite inhibited PARP-1 activity and caused zinc loss from PARP-1 protein while scavenging peroxynitrite was protective of the impacts on PARP-1. We identified peroxynitrite was responsible for S-nitrosation on cysteine residues resulting in PARP-1 zinc finger conformational changes. Taken together, the evidence indicates AsIII generates peroxynitrite through superoxide and nitric oxide production, induces S-nitrosation on PARP-1, leading to zinc loss and activity inhibition of PARP-1, thus enhancing DNA damage caused by UV radiation. These findings highlight a role for peroxynitrite as a key molecule of ROS/RNS mediated DNA repair inhibition by AsIII which should inform the development of prevention and intervention strategies against AsIII co-carcinogenesis.

The 10th conference on metal toxicity and carcinogenesis: Overview and recent advances.

Environmental and occupational metal exposures promote numerous diseases including cardiovascular, pulmonary and neurological disorders as well as various types of cancers. Research in metal toxicity and carcinogenesis focuses on addressing major health concerns associated with metal exposure. In October 2018, the 10th Conference on Metal Toxicity and Carcinogenesis held in Albuquerque, New Mexico, assembled scientists from across the United States to discuss current status and future directions in this unique and important field of research. Here, we summarize the on-going research and recent advances presented at this conference and provide insights on future progression and challenges of the field.

Oral bisphosphonates and incidence of cancers in patients with osteoporosis: a systematic review and meta-analysis.

PURPOSE: Several previous studies have shown that oral bisphosphonates (BPs) are associated with the incidence of 13 specific cancers, including lung cancer, esophageal cancer, gastric cancer, and colorectal cancer (CRC). However, the findings are heterogeneous. METHODS AND RESULTS: Relevant studies published in databases such as PubMed, Embase database, and Cochrane library were systematically retrieved from inception to August 25th, 2018, regardless of language, by two investigators independently. Afterwards, the maximum adjusted hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) were extracted from the retrieved studies. Finally, 13 cohort studies involving 1,510,763 participants were enrolled into this meta-analysis. No significant relationship was found between oral BPs and the risk of all-cause cancer in osteoporosis (OP) patients among the entire population (HR 0.97, 95% CI 0.80-1.18; I2 92.5%). Besides, oral BPs could remarkably reduce the incidence of breast cancer (HR 0.79, 95% CI 0.68-0.92; I2 54%) and endometrial cancer (HR 0.79, 95% CI 0.64-0.96; I2 0%) in postmenopausal OP females. In addition, oral BPs were also found to evidently reduce the incidence of upper gastrointestinal cancer in OP patients among the entire population (HR 0.73, 95% CI 0.54-0.98; I2 36.1%). However, oral BPs may lead to increased risk of liver cancer in mixed genders (HR 1.69, 95% CI 1.03-2.77; I2 30.7%). CONCLUSIONS: Taken together, oral BPs do not increase the risk of incidence of all-cause cancer; instead, they can reduce the incidence of breast, endometrial, and upper gastrointestinal cancers among the postmenopausal OP females. Our analysis stratified by gender suggests that oral BPs may increase the incidence of liver cancer in mixed genders, while no significant association was observed in females. Careful analysis of post-marketing data should be conducted to address the clinical relevance of our results on the putative association of oral BP use and liver cancer suggested by our meta-analysis.

Differential sensitivities of cellular XPA and PARP-1 to arsenite inhibition and zinc rescue.

Arsenite directly binds to the zinc finger domains of the DNA repair protein poly (ADP ribose) polymerase (PARP)-1, and inhibits PARP-1 activity in the base excision repair (BER) pathway. PARP inhibition by arsenite enhances ultraviolet radiation (UVR)-induced DNA damage in keratinocytes, and the increase in DNA damage is reduced by zinc supplementation. However, little is known about the effects of arsenite and zinc on the zinc finger nucleotide excision repair (NER) protein xeroderma pigmentosum group A (XPA). In this study, we investigated the difference in response to arsenite exposure between XPA and PARP-1, and the differential effectiveness of zinc supplementation in restoring protein DNA binding and DNA damage repair. Arsenite targeted both XPA and PARP-1 in human keratinocytes, resulting in zinc loss from each protein and a pronounced decrease in XPA and PARP-1 binding to chromatin as demonstrated by Chip-on-Western assays. Zinc effectively restored DNA binding of PARP-1 and XPA to chromatin when zinc concentrations were equal to those of arsenite. In contrast, zinc was more effective in rescuing arsenite-augmented direct UVR-induced DNA damage than oxidative DNA damage. Taken together, our findings indicate that arsenite interferes with PARP-1 and XPA binding to chromatin, and that zinc supplementation fully restores DNA binding activity to both proteins in the cellular context. Interestingly, rescue of arsenite-inhibited DNA damage repair by supplemental zinc was more sensitive for DNA damage repaired by the XPA-associated NER pathway than for the PARP-1-dependent BER pathway. This study expands our understanding of arsenite's role in DNA repair inhibition and co-carcinogenesis.

S-nitrosation on zinc finger motif of PARP-1 as a mechanism of DNA repair inhibition by arsenite.

Arsenic, a widely distributed carcinogen, is known to significantly amplify the impact of other carcinogens through inhibition of DNA repair. Our recent work suggests that reactive oxygen/nitrogen species (ROS/RNS) induced by arsenite (AsIII) play an important role in the inhibition of the DNA repair protein Poly(ADP-ribose) polymerase 1 (PARP-1). AsIII-induced ROS lead to oxidation of cysteine residues within the PARP-1 zinc finger DNA binding domain. However, the mechanism underlying RNS-mediated PARP inhibition by arsenic remains unknown. In this work, we demonstrate that AsIII treatment of normal human keratinocyte (HEKn) cells induced S-nitrosation on cysteine residues of PARP-1 protein, in a similar manner to a nitric oxide donor. S-nitrosation of PARP-1 could be reduced by 1400W (inducible nitric oxide synthase inhibitor) or c-PTIO (a nitric oxide scavenger). Furthermore, AsIII treatment of HEKn cells leads to zinc loss and inhibition of PARP-1 enzymatic activity. AsIII and 1400W/c-PTIO co-treatment demonstrate that these effects occur in an iNOS- and NO-dependent manner. Importantly, we confirmed S-nitrosation on the zinc finger DNA binding domain of PARP-1 protein. Taken together, AsIII induces S-nitrosation on PARP-1 zinc finger DNA binding domain by generating NO through iNOS activation, leading to zinc loss and inhibition of PARP-1 activity, thereby increasing retention of damaged DNA. These findings identify S-nitrosation as an important component of the molecular mechanism underlying AsIII inhibition of DNA repair, which may benefit the development of preventive and intervention strategies against AsIII co-carcinogenesis.

Nicotinamide Phosphoribosyltransferase Upregulation by Phenylephrine Reduces Radiation Injury in Submandibular Gland.

PURPOSE: Radiation therapy for head and neck cancer commonly leads to radiation sialadenitis. Emerging evidence has indicated that phenylephrine pretreatment reduces radiosensitivity in the salivary gland; however, the underlying cytoprotective mechanism remains unclear. Nicotinamide phosphoribosyltransferase (NAMPT) is not only a key enzyme for the nicotinamide adenine dinucleotide salvage pathway, but also a cytokine participating in cell survival, metabolism, and longevity, with a broad effect on cellular functions in physiology and pathology. However, the regulatory events of NAMPT in response to the irradiated salivary gland are unknown. METHODS AND MATERIALS: The cell viability of primary cultured submandibular gland cells was determined using the PrestoBlue assay. NAMPT expression was measured using reverse transcriptase polymerase chain reaction and Western blotting in vitro and in vivo. Silent information regulator 1 (SIRT1) and phosphorylated Akt protein levels were examined by Western blotting. The cellular locations of NAMPT and SIRT1 were detected by immunohistochemistry. NAMPT promoter activity was assessed using the luciferase reporter gene assay. RESULTS: NAMPT was mainly distributed in the cytoplasm of granular convoluted tubule cells and ductal cells in normal submandibular glands. mRNA and protein expression of NAMPT was downregulated after radiation but upregulated with phenylephrine pretreatment both in vivo and in vitro. Moreover, the protein expression of phosphorylated Akt and SIRT1 was decreased in irradiated glands, and phenylephrine pretreatment restored the expression of both. SIRT1 was mainly located in the cell nucleus and cytoplasm in the normal submandibular gland. Phenylephrine dramatically enhanced the expression of SIRT1, which was significantly reduced by radiation. Furthermore, phenylephrine induced a marked increase of NAMPT promoter activity. CONCLUSIONS: These findings reveal the regulatory mechanisms of NAMPT expression, which help to understand the mechanism of the cytoprotective role of phenylephrine on irradiated tissues.

Kinetics and thermodynamics of zinc(II) and arsenic(III) binding to XPA and PARP-1 zinc finger peptides.

Inhibition of DNA repair is an established mechanism of arsenic co-carcinogenesis, and may be perpetuated by the binding of As(III) to key zinc finger (zf) DNA repair proteins. Validated molecular targets of As(III) include the first zinc finger domain of Poly (ADP-Ribose) Polymerase 1 (PARP-1), and the zinc finger domain of Xeroderma Pigmentosum Complementation Group A (XPA). In order to gain an understanding of the thermodynamic and kinetic parameters of the interaction of As(III) with these two zinc finger motifs, a fluorescence based approach was used to investigate Zn(II) and As(III) binding to synthetic model peptides corresponding to the zf motif of XPA and first zf motif of PARP-1, referred to in this paper as XPAzf and PARP-1zf-1, respectively. While XPAzf and PARP-1zf-1 display similar relative affinities for As(III), PARP-1zf-1 shows a potential kinetic advantage over XPAzf for As(III) binding, with a rate constant for the fast phase of formation of As(III)-PARP-1zf-1 approximately 4-fold higher than for As(III)-XPAzf. However, the binding of Zn(II) with either peptide proceeds at a faster rate than As(III). Notably, XPAzf demonstrates comparable affinities for binding both metals, while PARP-1zf-1 shows a slightly higher affinity for Zn(II), suggesting that the relative concentrations of Zn(II) and As(III) in a system may significantly influence which species predominates in zinc finger occupancy. These results provide insight into the mechanisms underlying interactions between zinc finger structures and As(III), and highlight the potential utility of zinc supplementation in mitigating adverse effects of As(III) on zinc finger functions in vivo.

Selective Sensitization of Zinc Finger Protein Oxidation by Reactive Oxygen Species through Arsenic Binding.

Cysteine oxidation induced by reactive oxygen species (ROS) on redox-sensitive targets such as zinc finger proteins plays a critical role in redox signaling and subsequent biological outcomes. We found that arsenic exposure led to oxidation of certain zinc finger proteins based on arsenic interaction with zinc finger motifs. Analysis of zinc finger proteins isolated from arsenic-exposed cells and zinc finger peptides by mass spectrometry demonstrated preferential oxidation of C3H1 and C4 zinc finger configurations. C2H2 zinc finger proteins that do not bind arsenic were not oxidized by arsenic-generated ROS in the cellular environment. The findings suggest that selectivity in arsenic binding to zinc fingers with three or more cysteines defines the target proteins for oxidation by ROS. This represents a novel mechanism of selective protein oxidation and demonstrates how an environmental factor may sensitize certain target proteins for oxidation, thus altering the oxidation profile and redox regulation.