RESUMO
Balancing surface functionalization and low immune recognition of nanomedicines is a major challenge. Opsonization with the third component of the complement protein (C3) plays a major role in immune cell recognition of nanomedicines. We used dextran-coated superparamagnetic iron oxide nanoworms (SPIO NWs) to study the effect of surface functionalization on C3 opsonization in mouse serum and subsequent macrophage/leukocyte recognition in vitro as well as on intravenous injection into mice. Previously, we found that in mouse serum, SPIO NWs became opsonized with C3 via complement lectin pathway. Crosslinking the dextran shell with epichlorohydrin significantly decreased C3 opsonization and uptake by mouse peritoneal macrophages. Crosslinked nanoworms (NWs) further functionalized with polyethylene glycol (PEG) or with PEG-antibody (Ab) (~160 IgG molecules/particle) did not show an increase in C3 opsonization and peritoneal macrophage uptake in vitro. Following tail vein injection into mice, plain crosslinked NWs and PEGylated crosslinked NWs showed very low C3 opsonization and mouse leukocyte uptake. However, Ab-decorated crosslinked NWs showed significant C3 opsonization and high level of complement-dependent uptake by leukocytes in mice. Decreasing the number of conjugated Abs to 46 IgG molecules/particle significantly reduced C3 opsonization and leukocyte uptake. Using fresh mouse lepirudin plasma rather than serum showed better correlation with C3 opsonization in vivo. The reason for this difference could be related to the known instability of complement classical pathway in mouse sera. Our data illustrate that fine-tuning in nanoparticle surface functionalization with Abs is required to avoid excessive complement activation and complement-mediated immune uptake in mice, and raise issues with in vitro immunological assays of nanomedicines intended to mimic in vivo conditions.
RESUMO
Notwithstanding rapid advances of nanotechnology in diagnostic imaging and drug delivery, the engineered nanocarriers still exhibit substantial lack of hemocompatibility. Thus, when injected systemically, nanoparticles are avidly recognized by blood leukocytes and platelets, but the mechanisms of immune recognition are not well understood and strategies to mitigate these phenomena remain underexplored. Using superparamagnetic dextran iron oxide (SPIO) nanoworms (NWs) we demonstrate an efficient and predominantly complement-dependent uptake by mouse lymphocytes, neutrophils and monocytes from normal and tumor bearing mice in vitro. Following intravenous injection into wild type mice, blood leukocytes as well as platelets became magnetically labeled, while the labeling was decreased by 95% in complement C3-deficient mice. Using blood cells from healthy and cancer patient donors, we demonstrated that neutrophils, monocytes, lymphocytes and eosinophils took up SPIO NWs, and the uptake was prevented by EDTA (a general complement inhibitor) and by antiproperdin antibody (an inhibitor of the alternative pathway of the complement system). Cross-linking and hydrogelation of SPIO NWs surface by epichlorohydrin decreased C3 opsonization in mouse serum, and consequently reduced the uptake by mouse leukocytes by more than 70% in vivo. Remarkably, the cross-linked particles did not show a decrease in C3 opsonization in human serum, but showed a significant decrease (over 60%) of the uptake by human leukocytes. The residual uptake of cross-linked nanoparticles was completely blocked by EDTA. These findings demonstrate species differences in complement-mediated nanoparticle recognition and uptake by leukocytes, and further show that human hemocompatibility could be improved by inhibitors of complement alternative pathway and by nanoparticle surface coating. These results provide important insights into the mechanisms of hemocompatibility of nanomedicines.
Assuntos
Complemento C3/metabolismo , Dextranos/metabolismo , Leucócitos/metabolismo , Nanopartículas/química , Proteínas Opsonizantes/metabolismo , Animais , Plaquetas/metabolismo , Ativação do Complemento , Reagentes de Ligações Cruzadas/química , Dextranos/química , Endocitose , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Nanopartículas de Magnetita , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Propriedades de Superfície , Distribuição TecidualRESUMO
Our recent studies in SKH-1 hairless mice have demonstrated that topical exposure to nitrogen mustard (NM), an analog of sulfur mustard (SM), triggers the inflammatory response, microvesication and apoptotic cell death. Here, we sought to identify the mechanism/s involved in these NM-induced injury responses. Results obtained show that NM exposure of SKH-1 hairless mouse skin caused H2A.X and p53 phosphorylation and increased p53 accumulation, indicating DNA damage. In addition, NM also induced the activation of MAPKs/ERK1/2, JNK1/2 and p38 as well as that of Akt together with the activation of transcription factor AP1. Also, NM exposure induced robust expression of pro-inflammatory mediators namely cyclooxygenase 2 and inducible nitric oxide synthase and cytokine tumor necrosis factor alpha, and increased the levels of proteolytic mediator matrix metalloproteinase 9. NM exposure of skin also increased lipid peroxidation, 5,5-dimethyl-2-(8-octanoic acid)-1-pyrroline N-oxide protein adduct formation, protein and DNA oxidation indicating an elevated oxidative stress. We also found NM-induced increase in the homologous recombinant repair pathway, suggesting its involvement in the repair of NM-induced DNA damage. Collectively, these results indicate that NM induces oxidative stress, mainly a bi-phasic response in DNA damage and activation of MAPK and Akt pathways, which activate transcription factor AP1 and induce the expression of inflammatory and proteolytic mediators, contributing to the skin injury response by NM. In conclusion, this study for the first time links NM-induced mechanistic changes with our earlier reported murine skin injury lesions with NM, which could be valuable to identify potential therapeutic targets and rescue agents.
Assuntos
Dano ao DNA/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Mecloretamina/toxicidade , Estresse Oxidativo , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Substâncias para a Guerra Química/toxicidade , Regulação Enzimológica da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Peroxidação de Lipídeos , Masculino , Camundongos , Camundongos Pelados , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Oxirredução , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Peptídeos/genética , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Pele/efeitos dos fármacos , Pele/metabolismo , Absorção Cutânea , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Currently, there is no effective antidote to prevent skin injuries by sulfur mustard (SM) and nitrogen mustard (NM), which are vesicating agents with potential relevance to chemical warfare, terrorist attacks, or industrial/laboratory accidents. Our earlier report has demonstrated the therapeutic efficacy of silibinin, a natural flavanone, in reversing monofunctional alkylating SM analog 2-chloroethyl ethyl sulfide-induced toxic effects in mouse skin. To translate this effect to a bifunctional alkylating vesicant, herein, efficacy studies were carried out with NM. Topical application of silibinin (1 or 2mg) 30 min after NM exposure on the dorsal skin of male SKH-1 hairless mice significantly decreased NM-induced toxic lesions at 24, 72 or 120 h post-exposure. Specifically, silibinin treatment resulted in dose-dependent reduction of NM-induced increase in epidermal thickness, dead and denuded epidermis, parakeratosis and microvesication. Higher silibinin dose also caused a 79% and 51%reversal in NM-induced increases in myeloperoxidase activity and COX-2 levels, respectively. Furthermore, silibinin completely prevented NM-induced H2A.X phosphorylation, indicating reversal of DNA damage which could be an oxidative DNA damage as evidenced by high levels of 8-oxodG in NM-exposed mouse skin that was significantly reversed by silibinin. Together, these findings suggest that attenuation of NM-induced skin injury by silibinin is due to its effects on the pathways associated with DNA damage, inflammation, vesication and oxidative stress. In conclusion, results presented here support the optimization of silibinin as an effective treatment of skin injury by vesicants.
Assuntos
Antídotos/farmacologia , Irritantes/toxicidade , Mecloretamina/toxicidade , Silimarina/farmacologia , Pele/efeitos dos fármacos , 8-Hidroxi-2'-Desoxiguanosina , Administração Cutânea , Animais , Antídotos/administração & dosagem , Apoptose/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Citoproteção , Dano ao DNA , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Relação Dose-Resposta a Droga , Histonas/metabolismo , Masculino , Camundongos Pelados , Necrose , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/metabolismo , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Silibina , Silimarina/administração & dosagem , Pele/metabolismo , Pele/patologia , Fatores de TempoRESUMO
Vesicating agents sulfur mustard (SM) and nitrogen mustard (NM) are reported to be easily absorbed by skin upon exposure causing severe cutaneous injury and blistering. Our studies show that topical exposure of NM (3.2mg) onto SKH-1 hairless mouse skin, not only caused skin injury, but also led to significant body weight loss and 40-80% mortality (120 h post-exposure), suggesting its systemic effects. Accordingly, further studies herein show that NM exposure initiated an increase in circulating white blood cells by 24h (neutrophils, eosinophils and basophils) and thereafter a decrease (neutrophils, lymphocytes and monocytes). NM exposure also reduced both white and red pulp areas of the spleen. In the small intestine, NM exposure caused loss of membrane integrity of the surface epithelium, abnormal structure of glands and degeneration of villi. NM exposure also resulted in the dilation of glomerular capillaries of kidneys, and an increase in blood urea nitrogen/creatinine ratio. Our results here with NM are consistent with earlier reports that exposure to higher SM levels can cause damage to the hematopoietic system, and kidney, spleen and gastrointestinal tract toxicity. These outcomes will add to our understanding of the toxic effects of topical vesicant exposure, which might be helpful towards developing effective countermeasures against injuries from acute topical exposures.
Assuntos
Substâncias para a Guerra Química/toxicidade , Sistema Hematopoético/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Rim/efeitos dos fármacos , Mecloretamina/toxicidade , Pele/efeitos dos fármacos , Baço/efeitos dos fármacos , Administração Cutânea , Animais , Apoptose/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Intestino Delgado/patologia , Rim/patologia , Contagem de Leucócitos , Masculino , Camundongos Pelados , Tamanho do Órgão/efeitos dos fármacos , Pele/lesões , Baço/patologia , Análise de SobrevidaRESUMO
One of the core issues of nanotechnology involves masking the foreignness of nanomaterials to enable in vivo longevity and long-term immune evasion. Dextran-coated superparamagnetic iron oxide nanoparticles are very effective magnetic resonance imaging (MRI) contrast agents, and strategies to prevent immune recognition are critical for their clinical translation. Here we prepared 20 kDa dextran-coated SPIO nanoworms (NWs) of 250 nm diameter and a high molar transverse relaxivity rate R2 (â¼400 mM(-1) s(-1)) to study the effect of cross-linking-hydrogelation with 1-chloro-2,3-epoxypropane (epichlorohydrin) on the immune evasion both in vitro and in vivo. Cross-linking was performed in the presence of different concentrations of NaOH (0.5 to 10 N) and different temperatures (23 and 37 °C). Increasing NaOH concentration and temperature significantly decrease the binding of anti-dextran antibody and dextran-binding lectin conconavalin A to the NWs. The decrease in dextran immunoreactivity correlated with the decrease in opsonization by complement component 3 (C3) and with the decrease in the binding of the lectin pathway factor MASP-2 in mouse serum, suggesting that cross-linking blocks the lectin pathway of complement. The decrease in C3 opsonization correlated with the decrease in NW uptake by murine peritoneal macrophages. Optimized NWs demonstrated up to 10 h circulation half-life in mice and minimal uptake by the liver, while maintaining the large 250 nm size in the blood. We demonstrate that immune recognition of large iron oxide nanoparticles can be efficiently blocked by chemical cross-linking-hydrogelation, which is a promising strategy to improve safety and bioinertness of MRI contrast agents.
Assuntos
Compostos Férricos/sangue , Compostos Férricos/química , Nanopartículas , Animais , Transporte Biológico , Dextranos/química , Compostos Férricos/imunologia , Compostos Férricos/toxicidade , Meia-Vida , Macrófagos/metabolismo , Masculino , Camundongos , Tamanho da Partícula , Hidróxido de Sódio/química , TemperaturaRESUMO
BACKGROUND: The complement system is a key component of innate immunity implicated in the neutralization and clearance of invading pathogens. Dextran coated superparamagnetic iron oxide (SPIO) nanoparticle is a promising magnetic resonance imaging (MRI) contrast agent. However, dextran SPIO has been associated with significant number of complement-related side effects in patients and some agents have been discontinued from clinical use (e.g., Feridex™). In order to improve the safety of these materials, the mechanisms of complement activation by dextran-coated SPIO and the differences between mice and humans need to be fully understood. METHODS: 20 kDa dextran coated SPIO nanoworms (SPIO NW) were synthesized using Molday precipitation procedure. In vitro measurements of C3 deposition on SPIO NW using sera genetically deficient for various components of the classical pathway (CP), lectin pathway (LP) or alternative pathway (AP) components were used to study mechanisms of mouse complement activation. In vitro measurements of fluid phase markers of complement activation C4d and Bb and the terminal pathway marker SC5b-C9 in normal and genetically deficient sera were used to study the mechanisms of human complement activation. Mouse data were analyzed by non-paired t-test, human data were analyzed by ANOVA followed by multiple comparisons with Student-Newman-Keuls test. RESULTS: In mouse sera, SPIO NW triggered the complement activation via the LP, whereas the AP contributes via the amplification loop. No involvement of the CP was observed. In human sera the LP together with the direct enhancement of the AP turnover was responsible for the complement activation. In two samples out of six healthy donors there was also a binding of anti-dextran antibodies and C1q, suggesting activation via the CP, but that did not affect the total level of C3 deposition on the particles. CONCLUSIONS: There were important differences and similarities in the complement activation by SPIO NW in mouse versus human sera. Understanding the mechanisms of immune recognition of nanoparticles in mouse and human systems has important preclinical and clinical implications and could help design more efficient and safe nano-formulations.
Assuntos
Ativação do Complemento/efeitos dos fármacos , Meios de Contraste/farmacologia , Dextranos/farmacologia , Adulto , Animais , Biomarcadores/sangue , Via Alternativa do Complemento/efeitos dos fármacos , Via Clássica do Complemento/efeitos dos fármacos , Lectina de Ligação a Manose da Via do Complemento/efeitos dos fármacos , Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/metabolismo , Humanos , Nanopartículas de Magnetita , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade da Espécie , Propriedades de SuperfícieRESUMO
Our previous studies and other published reports on the chemical warfare agent sulfur mustard (SM) and its analog 2-chloroethyl ethyl sulfide (CEES) have indicated a role of oxidative stress in skin injuries caused by these vesicating agents. We examined the effects of the catalytic antioxidant AEOL 10150 in the attenuation of CEES-induced toxicity using our established skin injury models (skin epidermal cells and SKH-1 hairless mice) to validate the role of oxidative stress in the pathophysiology of mustard vesicating agents. Treatment of mouse epidermal JB6 and human HaCaT cells with AEOL 10150 (50µM) 1h post-CEES exposure resulted in significant (p < 0.05) reversal of CEES-induced decreases in both cell viability and DNA synthesis. Similarly, AEOL 10150 treatment 1h after CEES exposure attenuated CEES-induced DNA damage in these cells. Similar AEOL 10150 treatments also caused significant (p < 0.05) reversal of CEES-induced decreases in cell viability in normal human epidermal keratinocytes. Cytoplasmic and mitochondrial reactive oxygen species measurements showed that AEOL 10150 treatment drastically ameliorated the CEES-induced oxidative stress in both JB6 and HaCaT cells. Based on AEOL 10150 pharmacokinetic studies in SKH-1 mouse skin, mice were treated with a topical formulation plus subcutaneous injection (5mg/kg) of AEOL 10150 1h after CEES (4mg/mouse) exposure and every 4h thereafter for 12h. This AEOL 10150 treatment regimen resulted in over 50% (p < 0.05) reversal of CEES-induced skin bi-fold and epidermal thickness, myeloperoxidase activity, and DNA oxidation in mouse skin. Results from this study demonstrate the potential therapeutic efficacy of AEOL 10150 against CEES-mediated cutaneous lesions, supporting AEOL 10150 as a medical countermeasure against SM-induced skin injuries.
Assuntos
Antídotos/farmacologia , Antioxidantes/farmacologia , Substâncias para a Guerra Química/toxicidade , Metaloporfirinas/farmacologia , Gás de Mostarda/análogos & derivados , Pele/efeitos dos fármacos , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Humanos , Camundongos , Camundongos Pelados , Gás de Mostarda/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Pele/patologiaRESUMO
Nitrogen mustard (NM), a structural analog of chemical warfare agent sulfur mustard (SM), forms adducts and crosslinks with DNA, RNA and proteins. Here we studied the mechanism of NM-induced skin toxicity in response to double strand breaks (DSBs) resulting in cell cycle arrest to facilitate DNA repair, as a model for developing countermeasures against vesicant-induced skin injuries. NM exposure of mouse epidermal JB6 cells decreased cell growth and caused S-phase arrest. Consistent with these biological outcomes, NM exposure also increased comet tail extent moment and the levels of DNA DSB repair molecules phospho H2A.X Ser139 and p53 Ser15 indicating NM-induced DNA DSBs. Since DNA DSB repair occurs via non homologous end joining pathway (NHEJ) or homologous recombination repair (HRR) pathways, next we studied these two pathways and noted their activation as defined by an increase in phospho- and total DNA-PK levels, and the formation of Rad51 foci, respectively. To further analyze the role of these pathways in the cellular response to NM-induced cytotoxicity, NHEJ and HRR were inhibited by DNA-PK inhibitor NU7026 and Rad51 inhibitor BO2, respectively. Inhibition of NHEJ did not sensitize cells to NM-induced decrease in cell growth and cell cycle arrest. However, inhibition of the HRR pathway caused a significant increase in cell death, and prolonged G2M arrest following NM exposure. Together, our findings, indicating that HRR is the key pathway involved in the repair of NM-induced DNA DSBs, could be useful in developing new therapeutic strategies against vesicant-induced skin injury.
Assuntos
Substâncias para a Guerra Química/toxicidade , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Queratinócitos/metabolismo , Mecloretamina/toxicidade , Pele/metabolismo , Animais , Compostos de Boro/farmacologia , Linhagem Celular , Cromonas/farmacologia , Reparo do DNA/genética , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Histonas/genética , Histonas/metabolismo , Queratinócitos/patologia , Camundongos , Morfolinas/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Rad51 Recombinase/antagonistas & inibidores , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Pele/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The pathologic mechanisms of skin injuries, following the acute inflammatory response induced by vesicating agents sulfur mustard (SM) and nitrogen mustard (NM) exposure, are poorly understood. Neutrophils which accumulate at the site of injury, abundantly express myeloperoxidase (MPO), a heme protein that is implicated in oxidant-related antimicrobial and cytotoxic responses. Our previous studies have shown that exposure to SM analog 2-chloroethyl ethyl sulfide (CEES) or NM results in an inflammatory response including increased neutrophilic infiltration and MPO activity. To further define the role of neutrophil-derived MPO in NM-induced skin injury, here we used a genetic approach and examined the effect of NM exposure (12h and 24h) on previously established injury endpoints in C57BL/6J wild type (WT) and B6.129X1-MPOtm1Lus/J mice (MPO KO), homozygous null for MPO gene. NM exposure caused a significant increase in skin bi-fold thickness, epidermal thickness, microvesication, DNA damage and apoptosis in WT mice compared to MPO KO mice. MPO KO mice showed relatively insignificant effect. Similarly, NM induced increases in the expression of inflammatory and proteolytic mediators, including COX-2, iNOS and MMP-9 in WT mice, while having a significantly lower effect in MPO KO mice. Collectively, these results show that MPO, which generates microbicidal oxidants, plays an important role in NM-induced skin injuries. This suggests the development of mechanism-based treatments against NM- and SM-induced skin injuries that inhibit MPO activity and attenuate MPO-derived oxidants.
Assuntos
Apoptose/efeitos dos fármacos , Substâncias para a Guerra Química/toxicidade , Mecloretamina/toxicidade , Erros Inatos do Metabolismo/metabolismo , Pele/efeitos dos fármacos , Animais , Vesícula/induzido quimicamente , Vesícula/patologia , Ciclo-Oxigenase 2/metabolismo , Dano ao DNA/efeitos dos fármacos , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Mecloretamina/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/metabolismo , Peroxidase/genética , Pele/patologia , Fatores de TempoRESUMO
Sulfur mustard (SM) is a vesicant warfare agent which causes severe skin injuries. Currently, we lack effective antidotes against SM-induced skin injuries, in part due to lack of appropriate animal model(s) that can be used for efficacy studies in laboratory settings to identify effective therapies. Therefore, to develop a relevant mouse skin injury model, we examined the effects of nitrogen mustard (NM), a primary vesicant and a bifunctional alkylating agent that induces toxic effects comparable to SM. Specifically, we conducted histopathological and immunohistochemical evaluation of several applicable cutaneous pathological lesions following skin NM (3.2mg) exposure for 12-120h in SKH-1 and C57BL/6 mice. NM caused a significant increase in epidermal thickness, incidence of microvesication, cell proliferation, apoptotic cell death, inflammatory cells (neutrophils, macrophages and mast cells) and myleoperoxidase activity in the skin of both mouse strains. However, there was a more prominent NM-induced increase in epidermal thickness, and macrophages and mast cell infiltration, in SKH-1 mice relative to what was seen in C57BL/6 mice. NM also caused collagen degradation and edema at early time points (12-24h); however, at later time points (72 and 120h), dense collagen staining was observed, indicating either water loss or start of integument repair in both the mouse strains. This study provides quantitative measurement of NM-induced histopathological and immunohistochemical cutaneous lesions in both hairless and haired mouse strains that could serve as useful tools for screening and identification of effective therapies for treatment of skin injuries due to NM and SM.
Assuntos
Substâncias para a Guerra Química/toxicidade , Dermatite de Contato/patologia , Modelos Animais de Doenças , Mecloretamina/toxicidade , Pele/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Vesícula/induzido quimicamente , Vesícula/imunologia , Vesícula/metabolismo , Vesícula/patologia , Proliferação de Células/efeitos dos fármacos , Dermatite de Contato/enzimologia , Dermatite de Contato/etiologia , Dermatite de Contato/imunologia , Edema/induzido quimicamente , Edema/enzimologia , Edema/imunologia , Edema/patologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Pelados , Camundongos Endogâmicos C57BL , Peroxidase/metabolismo , Pele/enzimologia , Pele/imunologia , Pele/patologia , Dobras Cutâneas , Especificidade da EspécieRESUMO
Bifunctional alkylating agent sulfur mustard (SM) and its analog nitrogen mustard (NM) cause DNA damage leading to cell death, and potentially activating inflammation. Transcription factor p53 plays a critical role in DNA damage by regulating cell cycle progression and apoptosis. Earlier studies by our laboratory demonstrated phosphorylation of p53 at Ser15 and an increase in total p53 in epidermal cells both in vitro and in vivo following NM exposure. To elucidate the role of p53 in NM-induced skin toxicity, we employed SKH-1 hairless mice harboring wild type (WT) or heterozygous p53 (p53+/-). Exposure to NM (3.2mg) caused a more profound increase in epidermal thickness and apoptotic cell death in WT relative to p53+/- mice at 24h. However, by 72h after exposure, there was a comparable increase in NM-induced epidermal cell death in both WT and p53+/- mice. Myeloperoxidase activity data showed that neutrophil infiltration was strongly enhanced in NM-exposed WT mice at 24h persisting through 72h of exposure. Conversely, robust NM-induced neutrophil infiltration (comparable to WT mice) was seen only at 72h after exposure in p53+/- mice. Similarly, NM-exposure strongly induced macrophage and mast cell infiltration in WT, but not p53+/- mice. Together, these data indicate that early apoptosis and inflammation induced by NM in mouse skin are p53-dependent. Thus, targeting this pathway could be a novel strategy for developing countermeasures against vesicants-induced skin injury.
Assuntos
Alquilantes/toxicidade , Dermatite de Contato/metabolismo , Mecloretamina/toxicidade , Pele/efeitos dos fármacos , Proteína Supressora de Tumor p53/deficiência , Alelos , Animais , Apoptose/efeitos dos fármacos , Dermatite de Contato/etiologia , Dermatite de Contato/patologia , Camundongos , Camundongos Pelados , Peroxidase/metabolismo , Pele/metabolismo , Pele/patologia , Proteína Supressora de Tumor p53/genéticaRESUMO
A paucity of clinically applicable biomarkers to screen therapies in laboratory is a limitation in the development of countermeasures against cutaneous injuries by chemical weapon, sulfur mustard (SM), and its analog nitrogen mustard (NM). Consequently, we assessed NM-caused progression of clinical cutaneous lesions; notably, skin injury with NM is comparable to SM. Exposure of SKH-1 hairless and C57BL/6 (haired) mice to NM (3.2 mg) for 12-120 h caused clinical sequelae of toxicity, including microblister formation, edema, erythema, altered pigmentation, wounding, xerosis and scaly dry skin. These toxic effects of NM were similar in both mouse strains, except that wounding and altered pigmentation at 12-24 h and appearance of dry skin at 24 and 72 h post-NM exposure were more pronounced in C57BL/6 compared to SKH-1 mice. Conversely, edema, erythema and microblister formation were more prominent in SKH-1 than C57BL/6 mice at 24-72 h after NM exposure. In addition, 40-60% mortality was observed following 120 h of NM exposure in the both mouse strains. Overall, these toxic effects of NM are comparable to those reported in humans and other animal species with SM, and thus represent clinically-relevant cutaneous injury endpoints in screening and optimization of therapies for skin injuries by vesicating agents.
Assuntos
Biomarcadores/metabolismo , Irritantes/toxicidade , Mecloretamina/toxicidade , Dermatopatias/induzido quimicamente , Dermatopatias/patologia , Animais , Progressão da Doença , Edema/induzido quimicamente , Edema/patologia , Eritema/induzido quimicamente , Eritema/patologia , Camundongos , Camundongos Pelados , Camundongos Endogâmicos C57BL , Pele/efeitos dos fármacos , Pele/patologia , Dobras Cutâneas , Fatores de TempoRESUMO
Chemical warfare agent sulfur mustard (HD) inflicts delayed blistering and incapacitating skin injuries. To identify effective countermeasures against HD-induced skin injuries, efficacy studies were carried out employing HD analog 2-chloroethyl ethyl sulfide (CEES)-induced injury biomarkers in skin cells and SKH-1 hairless mouse skin. The data demonstrate strong therapeutic efficacy of silibinin, a natural flavanone, in attenuating CEES-induced skin injury and oxidative stress. In skin cells, silibinin (10 µM) treatment 30 min after 0.35/0.5 mM CEES exposure caused a significant (p<0.05) reversal in CEES-induced decrease in cell viability, apoptotic and necrotic cell death, DNA damage, and an increase in oxidative stress. Silibinin (1 mg) applied topically to mouse skin 30 min post-CEES exposure (2 mg), was effective in reversing CEES-induced increases in skin bi-fold (62%) and epidermal thickness (85%), apoptotic cell death (70%), myeloperoxidase activity (complete reversal), induction of iNOS, COX-2, and MMP-9 protein levels (>90%), and activation of transcription factors NF-κB and AP-1 (complete reversal). Similarly, silibinin treatment was also effective in attenuating CEES-induced oxidative stress measured by 4-hydroxynonenal and 5,5-dimethyl-2-(8-octanoic acid)-1-pyrolline N-oxide protein adduct formation, and 8-oxo-2-deoxyguanosine levels. Since our previous studies implicated oxidative stress, in part, in CEES-induced toxic responses, the reversal of CEES-induced oxidative stress and other toxic effects by silibinin in this study indicate its pleiotropic therapeutic efficacy. Together, these findings support further optimization of silibinin in HD skin toxicity model to develop a novel effective therapy for skin injuries by vesicants.
Assuntos
Antídotos/farmacologia , Substâncias para a Guerra Química/toxicidade , Inflamação/prevenção & controle , Gás de Mostarda/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Silimarina/farmacologia , Pele/efeitos dos fármacos , 8-Hidroxi-2'-Desoxiguanosina , Aldeídos/química , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dano ao DNA , Desoxiguanosina/análogos & derivados , Desoxiguanosina/antagonistas & inibidores , Desoxiguanosina/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Inflamação/patologia , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Pelados , Gás de Mostarda/toxicidade , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Silibina , Pele/citologia , Pele/metabolismoRESUMO
There are no effective and approved therapies against devastating ocular injuries caused by vesicating chemical agents sulfur mustard (SM) and nitrogen mustard (NM). Herein, studies were carried out in rabbit corneal cultures to establish relevant ocular injury biomarkers with NM for screening potential efficacious agents in laboratory settings. NM (100nmol) exposure of the corneas for 2h (cultured for 24h), showed increases in epithelial thickness, ulceration, apoptotic cell death, epithelial detachment microbullae formation, and the levels of VEGF, cyclooxygenase-2 (COX-2) and matrix metalloproteinase-9 (MMP-9). Employing these biomarkers, efficacy studies were performed with agent treatments 2h and every 4h thereafter, for 24h following NM exposure. Three agents were evaluated, including prescription drugs dexamethasone (0.1%; anti-inflammatory steroid) and doxycycline (100nmol; antibiotic and MMP inhibitor) that have been studied earlier for treating vesicant-induced eye injuries. We also examined silibinin (100µg), a non-toxic natural flavanone found to be effective in treating SM analog-induced skin injuries in our earlier studies. Treatments of doxycycline+dexamethasone, and silibinin were more effective than doxycycline or dexamethasone alone in reversing NM-induced epithelial thickening, microbullae formation, apoptotic cell death, and MMP-9 elevation. However, dexamethasone and silibinin alone were more effective in reversing NM-induced VEGF levels. Doxycycline, dexamethasone and silibinin were all effective in reversing NM-induced COX-2 levels. Apart from therapeutic efficacy of doxycycline and dexamethasone, these results show strong multifunctional efficacy of silibinin in reversing NM-induced ocular injuries, which could help develop effective and safe therapeutics against ocular injuries by vesicants.
Assuntos
Substâncias para a Guerra Química/toxicidade , Doenças da Córnea/tratamento farmacológico , Dexametasona/farmacologia , Doxiciclina/farmacologia , Silimarina/farmacologia , Animais , Antibacterianos/farmacologia , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Doenças da Córnea/induzido quimicamente , Doenças da Córnea/patologia , Ciclo-Oxigenase 2/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Quimioterapia Combinada , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/patologia , Técnicas In Vitro , Irritantes/toxicidade , Mecloretamina/toxicidade , Gás de Mostarda/toxicidade , Coelhos , Silibina , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Head and neck squamous cell carcinoma (HNSCC) accounts for 6% of all malignancies in USA and unfortunately the recurrence of secondary primary tumors and resistance against conventional treatments decrease the overall 5 year survival rate in HNSCC patients. Thus, additional approaches are needed to control HNSCC. Here, for the first time, employing human HNSCC Detroit 562 and FaDu cells as well as normal human epidermal keratinocytes, we investigate grape seed extract (GSE) efficacy and associated mechanism in both cell culture and nude mice xenografts. GSE selectively inhibited the growth and caused cell cycle arrest and apoptotic death in both Detroit 562 and FaDu cells by activating DNA damage checkpoint cascade, including ataxia telangiectasia mutated/ataxia telangiectasia-Rad3-related-checkpoint kinase 1/2-cell division cycle 25C as well as caspases 8, 9 and 3. Consistent with these results, GSE treatment resulted in a strong DNA damage and a decrease in the levels of DNA repair molecules breast cancer gene 1 and Rad51 and DNA repair foci. GSE-caused accumulation of intracellular reactive oxygen species was identified as a major mechanism of its effect for growth inhibition, DNA damage and apoptosis, which was remarkably reversed by antioxidant N-acetylcysteine. GSE feeding to nude mice decreased Detroit 562 and FaDu xenograft tumor growth by 67 and 65% (P < 0.001), respectively. In immunohistochemical analysis, xenografts from GSE-fed groups showed decreased proliferation but increased DNA damage and apoptosis. Together, these findings show that GSE targets both DNA damage and repair and provide mechanistic insights for its efficacy selectively against HNSCC both in cell culture and mouse xenograft, supporting its translational potential against HNSCC.
Assuntos
Carcinoma de Células Escamosas/patologia , Dano ao DNA , Neoplasias de Cabeça e Pescoço/patologia , Extratos Vegetais/química , Espécies Reativas de Oxigênio/metabolismo , Sementes/química , Vitis/embriologia , Animais , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/metabolismo , Divisão Celular/efeitos dos fármacos , Imunofluorescência , Fase G2/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Células Tumorais CultivadasRESUMO
Employing mouse skin epidermal JB6 cells and dermal fibroblasts, here we examined the mechanisms of DNA damage by 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of sulfur mustard (SM). CEES exposure caused H2A.X and p53 phosphorylation as well as p53 accumulation in both cell types, starting at 1h, that was sustained for 24h, indicating a DNA-damaging effect of CEES, which was also confirmed and quantified by alkaline comet assay. CEES exposure also induced oxidative stress and oxidative DNA damage in both cell types, measured by an increase in mitochondrial and cellular reactive oxygen species and 8-hydroxydeoxyguanosine levels, respectively. In the studies distinguishing between oxidative and direct DNA damage, 1h pretreatment with glutathione (GSH) or the antioxidant Trolox showed a decrease in CEES-induced oxidative stress and oxidative DNA damage. However, only GSH pretreatment decreased CEES-induced total DNA damage measured by comet assay, H2A.X and p53 phosphorylation, and total p53 levels. This was possibly due to the formation of GSH-CEES conjugates detected by LC-MS analysis. Together, our results show that CEES causes both direct and oxidative DNA damage, suggesting that to rescue SM-caused skin injuries, pleiotropic agents (or cocktails) are needed that could target multiple pathways of mustard skin toxicities.
Assuntos
Fibroblastos/efeitos dos fármacos , Gás de Mostarda/análogos & derivados , Pele/efeitos dos fármacos , Animais , Linhagem Celular , Cromanos/farmacologia , Dano ao DNA , Relação Dose-Resposta a Droga , Fibroblastos/metabolismo , Glutationa/farmacologia , Histonas/metabolismo , Camundongos , Camundongos Pelados , Gás de Mostarda/farmacologia , Oxirredução , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Pele/citologia , Pele/metabolismo , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/metabolismoRESUMO
Bifunctional alkyalating agent, sulfur mustard (SM)-induced cutaneous injury is characterized by inflammation and delayed blistering. Our recent studies demonstrated that 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of SM that can be used in laboratory settings, induces oxidative stress. This could be the major cause of the activation of Akt/MAP kinase and AP1/NF-κB pathways that are linked to the inflammation and microvesication, and histopathological alterations in SKH-1 hairless mouse skin. To further establish a link between CEES-induced DNA damage and signaling pathways and inflammatory responses, skin samples from mice exposed to 2 mg or 4 mg CEES for 9-48 h were subjected to molecular analysis. Our results show a strong CEES-induced phosphorylation of H2A.X and an increase in cyclooxygenase-2 (COX-2), inducible NOS (iNOS), and matrix metalloproteinase-9 (MMP-9) levels, indicating the involvement of DNA damage and inflammation in CEES-induced skin injury in male and female mice. Since, our recent studies showed reduction in CEES-induced inflammatory responses by glutathione (GSH), we further assessed the role of oxidative stress in CEES-related DNA damage and the induction of inflammatory molecules. Oral GSH (300 mg/kg) administration 1h before CEES exposure attenuated the increase in both CEES-induced H2A.X phosphorylation (59%) as well as expression of COX-2 (68%), iNOS (53%) and MMP-9 (54%). Collectively, our results indicate that CEES-induced skin injury involves DNA damage and an induction of inflammatory mediators, at least in part via oxidative stress. This study could help in identifying countermeasures that alone or in combination, can target the unveiled pathways for reducing skin injury in humans by SM.