DNA Damage In Macrophages Research Articles

Effect of sulfite-treated daylily (Hemerocallis fulva L.) flower on the production of nitric oxide and DNA damage in macrophages

The inhibitory effect of fresh or dried daylilies extract on the generation of nitric oxide, and the effects of sulfur dioxide in dried daylily on nitric oxide production and DNA damage in Raw 264.7 macrophages were examined. The extracts from sulfite-treated and untreated dried daylily flowers exhibited stronger scavenging effects on nitric oxide production by sodium nitro-prusside (SNP) than that of fresh flower. Fresh and untreated dried daylily flowers exhibited strong inhibitory effect on nitric oxide induced by lipopolysaccharide (LPS) in macrophages, but the sulfite-treated dried daylily did not. The scavenging effect of sulfite on nitric oxide production by SNP was dose-dependent, and sulfite also exhibited low inhibitory effect on nitric oxide induced by LPS in macrophages. Sodium hydrogen sulfite also slightly induced DNA damage in macrophages. Although sulfur dioxide in dried flowers is volatilized during heating, its residual adverse effects should still be concerned.

BRCA-1 depletion impairs pro-inflammatory polarization and activation of RAW 264.7 macrophages in a NF-κB-dependent mechanism.

BRCA-1 is a nuclear protein involved in DNA repair, transcriptional regulation, and cell cycle control. Its involvement in other cellular processes has been described. Here, we aimed to investigate the role of BRCA-1 in macrophages M(LPS), M(IL-4), and tumor cell-induced differentiation. We used siRNAs to knockdown BRCA-1 in RAW 264.7 macrophages exposed to LPS, IL-4, and C6 glioma cells conditioned medium (CMC6), and evaluated macrophage differentiation markers and functional phagocytic activity as well as DNA damage and cell survival in the presence and absence of BRCA-1. LPS and CMC6, but not by IL-4, increased DNA damage in macrophages, and this effect was more pronounced in BRCA-1-depleted cells, including M(IL-4). BRCA-1 depletion impaired expression of pro-inflammatory cytokines, TNF-α and IL-6, and reduced the phagocytic activity of macrophages in response to LPS. In CMC6-induced differentiation, BRCA-1 knockdown inhibited TNF-α and IL-6 expression which was accompanied by upregulation of the anti-inflammatory markers IL-10 and TGF-β and reduced phagocytosis. In contrast, M(IL-4) phenotype was not affected by BRCA-1 status. Molecular docking predicted that the conserved BRCA-1 domain BRCT can interact with the p65 subunit of NF-κB. Immunofluorescence assays showed that BRCA-1 and p65 co-localize in the nucleus of LPS-treated macrophages and reporter gene assay showed that depletion of BRCA-1 decreased LPS and CMC6-induced NF-κB transactivation. IL-4 had no effect upon NF-κB. Taken together, our findings suggest a role of BRCA-1 in macrophage differentiation and phagocytosis induced by LPS and tumor cells secretoma, but not IL-4, in a mechanism associated with inhibition of NF-κB.

Unrepaired DNA damage in macrophages causes elevation of particulate matter- induced airway inflammatory response

The inflammatory cascade can be initiated with the recognition of damaged DNA. Macrophages play an essential role in particulate matter (PM)-induced airway inflammation. In this study, we aim to explore the PM induced DNA damage response of macrophages and its function in airway inflammation. The DNA damage response and inflammatory response were assessed using bone marrow–derived macrophages following PM treatment and mouse model instilled intratracheally with PM. We found that PM induced significant DNA damage both in vitro and in vivo and simultaneously triggered a rapid DNA damage response, represented by nuclear RPA, 53BP1 and γH2AX foci formation. Genetic ablation or chemical inhibition of the DNA damage response sensor amplified the production of cytokines including Cxcl1, Cxcl2 and Ifn-γ after PM stimulation in bone marrow–derived macrophages. Similar to that seen in vitro, mice with myeloid-specific deletion of RAD50 showed higher levels of airway inflammation in response to the PM challenge, suggesting a protective role of DNA damage sensor during inflammation. These data demonstrate that PM exposure induces DNA damage and activation of DNA damage response sensor MRN complex in macrophages. Disruption of MRN complex lead to persistent, unrepaired DNA damage that causes elevated inflammatory response.

Differences between co-cultures and monocultures in testing the toxicity of particulate matter derived from log wood and pellet combustion.

BackgroundIn vitro studies with monocultures of human alveolar cells shed deeper knowledge on the cellular mechanisms by which particulate matter (PM) causes toxicity, but cannot account for mitigating or aggravating effects of cell-cell interactions on PM toxicity.MethodsWe assessed inflammation, oxidative stress as well as cytotoxic and genotoxic effects induced by PM from the combustion of different types of wood logs and softwood pellets in three cell culture setups: two monocultures of either human macrophage-like cells or human alveolar epithelial cells, and a co-culture of these two cell lines. The adverse effects of the PM samples were compared between these setups.ResultsWe detected clear differences in the endpoints between the mono- and co-cultures. Inflammatory responses were more diverse in the macrophage monoculture and the co-culture compared to the epithelial cells where only an increase of IL-8 was detected. The production of reactive oxygen species was the highest in epithelial cells and macrophages seemed to have protective effects against oxidative stress from the PM samples. With no metabolically active cells at the highest doses, the cytotoxic effects of the PM samples from the wood log combustion were far more pronounced in the macrophages and the co-culture than in the epithelial cells. All samples caused DNA damage in macrophages, whereas only beech and spruce log combustion samples caused DNA damage in epithelial cells. The organic content of the samples was mainly associated with cytotoxicity and DNA damage, while the metal content of the samples correlated with the induction of inflammatory responses.ConclusionsAll of the tested PM samples induce adverse effects and the chemical composition of the samples determines which pathway of toxicity is induced. In vitro testing of the toxicity of combustion-derived PM in monocultures of one cell line, however, is inadequate to account for all the possible pathways of toxicity.

The Role of Innate Immune Dysfunction in Inherited Bone Marrow Failure

The 16 Fanconi anemia (FA) genes encode proteins that facilitate cellular responses to DNA damage. A nuclear complex of eight of these proteins facilitates the monoubiquitination of FANCD2 and FANCI and permits the proper orchestration of DNA crosslink repair. Loss of any one of these “core complex” proteins disrupts repair. While the resulting genetic instability phenotype is broadly shared across tissues and cell types, the clinical phenotype involves a more narrow set of organ systems. Bone marrow failure (BMF) is nearly universal and progresses at a currently unpredictable pace in individual patients. The relative risk for clonal evolution to myelodysplasia (MDS) and acute myeloid leukemia (AML) is also high. Exposure to both exogenous (in human and knockout mouse models) and endogenous (knockout mouse) crosslinking agents rapidly results in fatal aplastic anemia, so there is a general tendency for those in the field to attribute FA BMF simply to DNA damage caused by endogenous crosslinking agents such as aldehydes. However, currently there exists no formal biochemical evidence that stem cells suffering from exposure to endogenous aldehydes fail to survive solely as a consequence of DNA damage inflicted upon them by aldehydes per se. Moreover, FA proteins are known to interact with mitochondrial and cytoplasmic proteins that modulate responses to inflammation and oxidative stress. We and others have tested and confirmed the hypothesis that inflammatory cytokines contribute to hematopoietic suppression in FA. For example, FANCC and FANCA progenitor cells are hypersensitive to the suppressive effects of IFNγ, Mip1α, TNF, TGFβ, and IL-1. Moreover, FANCC- and FANCA-deficient macrophages exposed to specific toll-like receptor agonists (TLR7/8) overproduce precisely the same cytokines to which FA HSC are intolerant. Critically important in vivo studies have shown that repeated activation of the inflammatory response in FA-deficient mice using lps, TNF, or antigen/adjuvant exposure results in stem cell exhaustion, a phenomenon that can be reliably modeled in vitro with cultured multipotential hematopoietic stem and progenitor (Kit+Sca+Lin- [LSK]) cells. Two potential explanations for FA stem cell exhaustion have been proposed. The first is that the inflammatory process induces “replicative stress,” followed by replication fork collapse, DNA damage and stem cell death. The second is that FA proteins are multifunctional and that the core complex proteins have evolved to independently: (1) promote self-replication and cytokine resistance of hematopoietic stem and progenitor cells; and (2) restrain production of inflammatory cytokines by macrophages. Experimental evidence reconciling both views will be reviewed including recent studies demonstrating that the TLR response in FA macrophages is dependent upon p38 MAPK activation, enhanced by endogenous aldehydes, and independent of macrophage DNA damage. These models have informed our concepts of clonal evolution in FA as well and have demonstrated that stem cell exhaustion is a selective force that favors evolution of neoplastic clones. Pang’s group, for example, has shown that when FA LSK cells are exposed to both survival factors and TNF ex-vivo, TNF-resistant preleukemic stem cells can be selected.1When transplanted into sub-lethally radiated mice, these selected TNF-resistant cells lead to the evolution of overt AML. These findings indicate that a somatic mutation or epigenetic change in a single stem cell that results in cytokine resistance has a high likelihood of selective expansion. Consequently, interdicting the hyperactive innate immune response may not only enhance stem cell survival in FA, but might forestall clonal evolution as well. Reference: Li J, Sejas DP, Zhang X et al. TNF-alpha induces leukemic clonal evolution ex vivo in Fanconi anemia group C murine stem cells. J.Clin.Invest. 2007;117:3283-3295. Disclosures No relevant conflicts of interest to declare.

DNA damage of macrophages induced by metal nanoparticulates using an air–liquid interface exposure model

DNA damage of macrophages induced by metal nanoparticulates using an air–liquid interface exposure model

DNA damage of macrophages at an air-tissue interface induced by metal nanoparticles

An air-tissue interface model was used to assess nanoparticulate-induced DNA damage to airway macrophages. Human Mono Mac 6 cells and rat alveolar macrophages were cultured on a collagen membrane and the deposition of metal nanoparticles in nitrogen enhanced using electrostatic charge. Cells were exposed to nanoparticles of iron, gold, silver for up to 10 min, then cultured in medium for 24 hours. Damage to DNA was assessed using the Comet assay. Nanoparticle dose delivered to cells varied with metal. Significant DNA damage to macrophages was induced by all three metal nanoparticles. Transmission electron microscopy showed deposition of discrete nanoparticles of gold and silver, but not iron. We conclude that an air-tissue model is a useful method for modelling DNA damage to airway cells from inhalation of metal nanoparticles.

Internalization of Libby Amphibole Asbestos and Induction of Oxidative Stress in Murine Macrophages

The community members of Libby, MT, have experienced significant asbestos exposure and developed numerous asbestos-related diseases including fibrosis and lung cancer due to an asbestos-contaminated vermiculite mine near the community. The form of asbestos in the contaminated vermiculite has been characterized in the amphibole family of fibers. However, the pathogenic effects of these fibers have not been previously characterized. The purpose of this study is to determine the cellular consequences of Libby amphibole exposure in macrophages compared to another well-characterized amphibole fiber; crocidolite asbestos. Our results indicate that Libby asbestos fibers are internalized by macrophages and localize to the cytoplasm and cytoplasmic vacuoles similar to crocidolite fibers. Libby asbestos fiber internalization generates a significant increase in intracellular reactive oxygen species (ROS) as determined by dichlorofluorescein diacetate and dihydroethidine fluorescence indicating that the superoxide anion is the major contributing ROS generated by Libby asbestos. Elevated superoxide levels in macrophages exposed to Libby asbestos coincide with a significant suppression of total superoxide dismutase activity. Both Libby and crocidolite asbestos generate oxidative stress in exposed macrophages by decreasing intracellular glutathione levels. Interestingly crocidolite asbestos, but not Libby asbestos, induces significant DNA damage in macrophages. This study provides evidence that the difference in the level of DNA damage observed between Libby and crocidolite asbestos may be a combined consequence of the distinct chemical compositions of each fiber as well as the activation of separate cellular pathways during asbestos exposure.

Chemically induced DNA damage in isolated rabbit lung cells

By use of an isolation procedure including centrifugal elutriation and density gradient centrifugation, relatively pure fractions of Clara cells and type II cells were obtained from rabbit lungs. These cells and alveolar macrophages isolated by lavage were exposed to methyl methanesulfonate (MMS), 1,2-dibromo-3-chloropropane (DBCP), 1-nitropyrene (1-NP), 2-nitrofluorene (2-NF), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosonornicotine (NNN), N-nitrosoheptamethyleneimine (NHMI) or phorbol 12-myristate 13-acetate (TPA). DNA damage measured as alkali-labile sites and/or single-strand breaks was then determined in the different lung cells by an automated alkaline elution system. The direct-acting compound MMS showed similar DNA-damaging effect in Clara cells, type II cells and alveolar macrophages. The nematocide DBCP, activated by both P450- and glutathione S-transferase(s)-dependent pathways, caused considerably less DNA damage in macrophages than in Clara or type II cells. Similar differences between the lung cells in induction of DNA damage as observed with DBCP were demonstrated after exposure to the activation-dependent nitrosamines NNK and NHMI and the tumor promoter TPA. The other test substances (1-NP, 2-NF, NNN) did not caused by marked DNA damage measured by the alkaline elution technique. These findings are in agreement with the known metabolic capacity of these cell types, indicating that Clara and type II cells are possible primary targets for lung toxic/carcinogenic compounds.

Oxidative stress induced by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) is one of the most potent toxins and tumor promoters known to man. It is prototypical of many halogenated polycyclic hydrocarbons that occur as environmental contaminants. Pathologic lesions produced by these compounds are mediated by an intracellular receptor protein called the TCDD (Ah) receptor which functions as a rans-acting effector of gene expression. However, the ultimate posttranslational pathways and mechanisms involved in the expressions of the toxic manifestations of TCDD have received little attention and remain unclear, yet constitute an important segment in our understanding of the overall mechanism of action of TCDD. Recent studies have demonstrated that an oxidative stress occurs in various tissues ot TCDD-treated animals. Evidence indicating production of an oxidative stress by TCDD in rodents is reviewed and includes: enhanced in vitro and in vivo hepatic and extrapatic lipid peroxidation: increased hepatic and macrophage DNA damage; increased urinary excretion of malondialdehyde; decreased hepatic membrane fluidity; increased production of superoxide anion by peritoneal macrophage; and decreased glutathione, nonprotein sulfhydryl, and NADPH contents in liver. The potential roleof reactive oxygen species in tumor promotion by TCDD is discussed. Possible sources and mechanisms of production of reactive oxygen species in response to TCDD are considered in light of current information. Evidence demonstrating the involvement of iron in TCDD-induced formation of reactive oxygen species and DNA damage is reviewed. Oxidative damage may contribute to many of the toxic responses produced by TCDD and its bioisosteres, and may be common to most of the tissue-damaging effects.