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1.
Toxicol Appl Pharmacol ; 455: 116258, 2022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36174671

RESUMO

A characteristic of cytochrome P450 (CYP) enzymes is their ability to generate H2O2, either directly or indirectly via superoxide anion, a reaction referred to as "NADPH oxidase" activity. H2O2 production by CYPs can lead to the accumulation of cytotoxic reactive oxygen species which can compromise cellular functioning and contribute to tissue injury. Herein we determined if form selective CYP inhibitors could distinguish between the activities of the monooxygenase and NADPH oxidase activities of rat recombinant CYP1A2, CYP2E1, CYP3A1 and CYP3A2 and CYP1A1/2-enriched ß-naphthoflavone-induced rat liver microsomes, CYP2E1-enriched isoniazide-induced rat liver microsomes and CYP3A subfamily-enriched dexamethasone-induced rat liver microsomes. In the presence of 7,8-benzoflavone (2.0 µM) for CYP1A2 and 4-methylpyrazole (32 µM) or DMSO (16 mM) for CYP2E1, monooxygenase activity was blocked without affecting NADPH oxidase activity for both the recombinant enzymes and microsomal preparations. Ketoconazole (1.0 µM), a form selective inhibitor for CYP3A subfamily enzymes, completely inhibited monooxygenase activity of rat recombinant CYP3A1/3A2 and CYP3A subfamily in rat liver microsomes; it also partially inhibited NADPH oxidase activity. 7,8-benzoflavone is a type I ligand, which competes with substrate binding, while 4-methylpyrazole and DMSO are type II heme binding ligands. Interactions of heme with these type II ligands was not sufficient to interfere with oxygen activation, which is required for NADPH oxidase activity. Ketoconazole, a type II ligand known to bind multiple sites on CYP3A subfamily enzymes in close proximity to heme, also interfered, at least in part, with oxygen activation. These data indicate that form specific inhibitors can be used to distinguish between monooxygenase reactions and H2O2 generating NADPH oxidase of CYP1A2 and CYP2E1. Mechanisms by which ketoconazole inhibits CYP3A NADPH oxidase remain to be determined.


Assuntos
Citocromo P-450 CYP1A2 , Inibidores das Enzimas do Citocromo P-450 , Ratos , Animais , Inibidores das Enzimas do Citocromo P-450/farmacologia , Inibidores das Enzimas do Citocromo P-450/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Peróxido de Hidrogênio/metabolismo , NADP/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP3A/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Cetoconazol/farmacologia , Superóxidos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , beta-Naftoflavona/farmacologia , Fomepizol , Ligantes , Dimetil Sulfóxido , Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/metabolismo , Heme/metabolismo , Dexametasona/farmacologia , Oxigênio/metabolismo
2.
Chem Res Toxicol ; 35(4): 636-650, 2022 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-35312310

RESUMO

Cytotoxic blistering agents such as sulfur mustard and nitrogen mustard (HN2) were synthesized for chemical warfare. Toxicity is due to reactive chloroethyl side chains that modify and damage cellular macromolecules including DNA and proteins. In response to DNA damage, cells initiate a DNA damage response directed at the recruitment and activation of repair-related proteins. A central mediator of the DNA damage response is p53, a protein that plays a critical role in regulating DNA repair. We found that HN2 causes cytosolic and nuclear accumulation of p53 in HaCaT keratinocytes; HN2 also induced post-translational modifications on p53 including S15 phosphorylation and K382 acetylation, which enhance p53 stability, promote DNA repair, and mediate cellular metabolic responses to stress. HN2 also cross-linked p53, forming dimers and high-molecular-weight protein complexes in the cells. Cross-linked multimers were also modified by K48-linked ubiquitination indicating that they are targets for proteasome degradation. HN2-induced modifications transiently suppressed the transcriptional activity of p53. Using recombinant human p53, HN2 alkylation was found to be concentration- and redox status-dependent. Dithiothreitol-reduced protein was more efficiently cross-linked indicating that p53 cysteine residues play a key role in protein modification. LC-MS/MS analysis revealed that HN2 directly alkylated p53 at C124, C135, C141, C176, C182, C275, C277, H115, H178, K132, and K139, forming both monoadducts and cross-links. The formation of intermolecular complexes was a consequence of HN2 cross-linked cysteine residues between two molecules of p53. Together, these data demonstrate that p53 is a molecular target for mustard vesicants. Modification of p53 likely mediates cellular responses to HN2 including DNA repair and cell survival contributing to vesicant-induced cytotoxicity.


Assuntos
Mecloretamina , Proteína Supressora de Tumor p53 , Cromatografia Líquida , Cisteína , Humanos , Queratinócitos , Mecloretamina/química , Espectrometria de Massas em Tandem
3.
Exp Mol Pathol ; 128: 104807, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35798063

RESUMO

Sulfur mustard (SM; bis(2-chloroethyl) sulfide) is a highly reactive bifunctional alkylating agent synthesized for chemical warfare. The eyes are particularly sensitive to SM where it causes irritation, pain, photophobia, and blepharitis, depending on the dose and duration of exposure. In these studies, we examined the effects of SM vapor on the corneas of New Zealand white male rabbits. Edema and hazing of the cornea, signs of acute injury, were observed within one day of exposure to SM, followed by neovascularization, a sign of chronic or late phase pathology, which persisted for at least 28 days. Significant epithelial-stromal separation ranging from ~8-17% of the epithelial surface was observed. In the stroma, there was a marked increase in CD45+ leukocytes and a decrease of keratocytes, along with areas of disorganization of collagen fibers. SM also disrupted the corneal basement membrane and altered the expression of perlecan, a heparan sulfate proteoglycan, and cellular fibronectin, an extracellular matrix glycoprotein. This was associated with an increase in basement membrane matrix metalloproteinases including ADAM17, which is important in remodeling of the basement membrane during wound healing. Tenascin-C, an extracellular matrix glycoprotein, was also upregulated in the stroma 14-28 d post SM, a finding consistent with its role in organizing structural components of the stroma necessary for corneal transparency. These data demonstrate that SM vapor causes persistent alterations in structural components of the cornea. Further characterization of SM-induced injury in rabbit cornea will be useful for the identification of targets for the development of ocular countermeasures.


Assuntos
Lesões da Córnea , Gás de Mostarda , Masculino , Coelhos , Animais , Gás de Mostarda/toxicidade , Proteoglicanas de Heparan Sulfato/metabolismo , Tenascina/metabolismo , Fibronectinas/metabolismo , Lesões da Córnea/induzido quimicamente , Lesões da Córnea/metabolismo , Membrana Basal/metabolismo , Membrana Basal/patologia , Matriz Extracelular/metabolismo , Alquilantes , Sulfetos/metabolismo , Colágeno/metabolismo
4.
Exp Mol Pathol ; 121: 104656, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34081961

RESUMO

Sulfur mustard (SM; bis (2-chloroethyl) sulfide) is a potent vesicant which causes irritation of the conjunctiva and damage to the cornea. In the present studies, we characterized the ocular effects of SM in New Zealand white rabbits. Within one day of exposure to SM, edema and hazing of the cornea were observed, followed by neovascularization which persisted for at least 28 days. This was associated with upper and lower eyelid edema and conjunctival inflammation. The conjunctiva is composed of a proliferating epithelium largely consisting of stratified columnar epithelial cells overlying a well-defined dermis. Superficial layers of the conjunctival epithelium were found to express keratin 1, a marker of differentiating squamous epithelium, while in cells overlying the basement membrane expressed keratin 17, a marker of stratified squamous epithelium. SM exposure upregulated keratin 17 expression. Mucin 5 ac producing goblet cells were interspersed within the conjunctiva. These cells generated both acidic and neutral mucins. Increased numbers of goblet cells producing neutral mucins were evident after SM exposure; upregulation of expression of membrane-associated mucin 1 and mucin 4 in the superficial layers of the conjunctival epithelium were also noted. These data demonstrate that ocular exposure of rabbits to SM causes significant damage not only to the cornea, but to the eyelid and conjunctiva, suggesting multiple targets within the eye that should be assessed when evaluating the efficacy of potential countermeasures.


Assuntos
Substâncias para a Guerra Química/toxicidade , Túnica Conjuntiva/patologia , Córnea/patologia , Epitélio/patologia , Células Caliciformes/patologia , Gás de Mostarda/toxicidade , Animais , Túnica Conjuntiva/efeitos dos fármacos , Túnica Conjuntiva/metabolismo , Córnea/efeitos dos fármacos , Córnea/metabolismo , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , Masculino , Mucina-1/metabolismo , Mucina-4/metabolismo , Coelhos
5.
Exp Mol Pathol ; 115: 104470, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32445752

RESUMO

Sulfur mustard (SM), a dermal vesicant that has been used in chemical warfare, causes inflammation, edema and epidermal erosions depending on the dose and time following exposure. Herein, a minipig model was used to characterize wound healing following dermal exposure to SM. Saturated SM vapor caps were placed on the dorsal flanks of 3-month-old male Gottingen minipigs for 30 min. After 48 h the control and SM wounded sites were debrided daily for 7 days with wet to wet saline gauze soaks. Animals were then euthanized, and full thickness skin biopsies prepared for histology and immunohistochemistry. Control skin contained a well differentiated epidermis with a prominent stratum corneum. A well-developed eschar covered the skin of SM treated animals, however, the epidermis beneath the eschar displayed significant wound healing with a hyperplastic epidermis. Stratum corneum shedding and a multilayered basal epithelium consisting of cuboidal and columnar cells were also evident in the neoepidermis. Nuclear expression of proliferating cell nuclear antigen (PCNA) was contiguous in cells along the basal epidermal layer of control and SM exposed skin; SM caused a significant increase in PCNA expression in basal and suprabasal cells. SM exposure was also associated with marked changes in expression of markers of wound healing including increases in keratin 10, keratin 17 and loricrin and decreases in E-cadherin. Trichrome staining of control skin showed a well-developed collagen network with no delineation between the papillary and reticular dermis. Conversely, a major delineation was observed in SM-exposed skin including a web-like papillary dermis composed of filamentous extracellular matrix, and compact collagen fibrils in the lower reticular dermis. Although the dermis below the wound site was disrupted, there was substantive epidermal regeneration following SM-induced injury. Further studies analyzing the wound healing process in minipig skin will be important to provide a model to evaluate potential vesicant countermeasures.


Assuntos
Gás de Mostarda/toxicidade , Pele/patologia , Cicatrização , Animais , Caderinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Epiderme/efeitos dos fármacos , Epiderme/patologia , Proteínas de Membrana/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Pele/efeitos dos fármacos , Suínos , Porco Miniatura , Cicatrização/efeitos dos fármacos
6.
Exp Mol Pathol ; 114: 104410, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32113906

RESUMO

Nitrogen mustard (NM) is a highly reactive bifunctional alkylating agent that induces inflammation, edema and blistering in skin. An important mechanism mediating the action of NM and related mustards is oxidative stress. In these studies a modified murine patch-test model was used to analyze DNA damage and the antioxidant/stress response following NM exposure in isolated epidermis. NM (20 µmol) was applied to glass microfiber filters affixed to a shaved dorsal region of skin of CD-1 mice. NM caused structural damage to the stratum corneum as reflected by increases in transepidermal water loss and skin hydration. This was coordinate with edema, mast cell degranulation and epidermal hyperplasia. Within 3 h of NM exposure, a 4-fold increase in phosphorylated histone H2AX, a marker of DNA double-stranded breaks, and a 25-fold increase in phosphorylated p53, a DNA damage marker, were observed in the epidermis. This was associated with a 40% increase in 8-oxo-2'-deoxyguanosine modified DNA in the epidermis and a 4-fold increase in 4-hydroxynonenal modified epidermal proteins. At 12 h post NM, there was a 3-75 fold increase in epidermal expression of antioxidant/stress proteins including heme oxygenase-1, thioredoxin reductase, superoxide dismutase, glutathione reductase, heat shock protein 27 and cyclooxygenase 2. These data indicate that NM induces early oxidative epidermal injury in mouse skin leading to an antioxidant/stress response. Agents that enhance this response may be useful in mitigating mustard-induced skin injury.


Assuntos
Antioxidantes/metabolismo , Epiderme/metabolismo , Mecloretamina/farmacologia , Estresse Fisiológico/genética , Alquilantes/farmacologia , Alquilantes/toxicidade , Animais , Apoptose/efeitos dos fármacos , Ciclo-Oxigenase 2/genética , Dano ao DNA/efeitos dos fármacos , Epiderme/efeitos dos fármacos , Glutationa Redutase/genética , Proteínas de Choque Térmico HSP27/genética , Heme Oxigenase-1/genética , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/patologia , Mecloretamina/toxicidade , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/metabolismo , Superóxido Dismutase/genética , Tiorredoxina Dissulfeto Redutase/genética
7.
Bioorg Chem ; 103: 104128, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32745761

RESUMO

A set of 4-(R2-imino)-3-mercapto-5-(R1)-4H-1,2,4-triazoles derivatives were synthesized, characterized and evaluated for their ability to inhibit nitric oxide (NO) production in PAM212 mouse keratinocytes, which led to the discovery and the subsequent evaluation of their growth inhibitory cytotoxic potency toward that same mouse cell line together with a number of human cells lines (PC3, HT-29 and HeLa). Some limited SAR could be established for both NO production inhibition potency and growth inhibition cytotoxicity. Noticeably, the compounds designed to be nitrofurantoin mimics were the most potent anti-neoplastic agents.


Assuntos
Antineoplásicos/farmacologia , Inibidores do Crescimento/farmacologia , Iminas/farmacologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Triazóis/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores do Crescimento/síntese química , Inibidores do Crescimento/química , Iminas/síntese química , Iminas/química , Camundongos , Estrutura Molecular , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/química
8.
Chem Res Toxicol ; 32(6): 1123-1133, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-30964658

RESUMO

Nitrogen mustard, mechlorethamine (bis(2-chloroethyl)methylamine; HN2), and sulfur mustard are potent vesicants that modify and disrupt cellular macromolecules including DNA leading to cytotoxicity and tissue injury. In many cell types, HN2 upregulates DNA damage signaling pathways including ataxia telangiectasia mutated (ATM), ataxia telangiectasia mutated- and Rad3-related (ATR) as well as DNA-dependent protein kinase (DNA-PK). In the present studies, we investigated crosstalk between the HN2-induced DNA damage response and cell cycle progression using human A549 lung epithelial cells. HN2 (1-20 µM; 24 h) caused a concentration-dependent arrest of cells in the S and G2/M phases of the cell cycle. This was associated with inhibition of DNA synthesis, as measured by incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into S phase cells. Cell cycle arrest was correlated with activation of DNA damage and cell cycle checkpoint signaling. Thus, HN2 treatment resulted in time- and concentration-dependent increases in expression of phosphorylated ATM (Ser1981), Chk2 (Thr68), H2AX (Ser139), and p53 (Ser15). Activation of DNA damage signaling was most pronounced in S-phase cells followed by G2/M-phase cells. HN2-induced cell cycle arrest was suppressed by the ATM and DNA-PK inhibitors, KU55933 and NU7441, respectively, and to a lesser extent by VE821, an ATR inhibitor. This was correlated with abrogation of DNA damage checkpoints signaling. These data indicate that activation of ATM, ATR, and DNA-PK signaling pathways by HN2 are important in the mechanism of vesicant-induced cell cycle arrest and cytotoxicity. Drugs that inhibit activation of DNA damage signaling may be effective countermeasures for vesicant-induced tissue injury.


Assuntos
Antineoplásicos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Substâncias para a Guerra Química/farmacologia , Dano ao DNA , Mecloretamina/farmacologia , Células A549 , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Substâncias para a Guerra Química/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Mecloretamina/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de Tempo , Células Tumorais Cultivadas
9.
Bioorg Med Chem Lett ; 29(4): 619-622, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30638875

RESUMO

Linear furocoumarins, also known as psoralens, are clinically useful photo-activated pharmaceuticals employed to address hyperproliferative skin diseases. Seven diverse cytotoxic pharmacophores have been synthetically attached to 8-methoxypsoralen via a 5-amino functionality. The resulting unique set of compounds was evaluated for dark and light toxicity against PAM212 keratinocytes in culture.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Escuridão , Luz , Metoxaleno/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Células Cultivadas , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Metoxaleno/química , Fármacos Fotossensibilizantes/química , Dermatopatias/patologia
10.
Bioorg Chem ; 89: 103014, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31170642

RESUMO

Seventy-one 7-oxycoumarins, 66 synthesized and 5 commercially sourced, were tested for their ability to inhibit growth in murine PAM212 keratinocytes. Forty-nine compounds from the library demonstrated light-induced lethality. None was toxic in the absence of UVA light. Structure-activity correlations indicate that the ability of the compounds to inhibit cell growth was dependent not only on their physiochemical characteristics, but also on their ability to absorb UVA light. Relative lipophilicity was an important factor as was electron density in the pyrone ring. Coumarins with electron withdrawing moieties - cyano and fluoro at C3 - were considerably less active while those with bromines or iodine at that location displayed enhanced activity. Coumarins that were found to inhibit keratinocyte growth were also tested for photo-induced DNA plasmid nicking. A concentration-dependent alteration in migration on neutral gels caused by nicking was observed.


Assuntos
Cumarínicos/farmacologia , Queratinócitos/efeitos dos fármacos , Fármacos Fotossensibilizantes/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cumarínicos/síntese química , Cumarínicos/química , Relação Dose-Resposta a Droga , Camundongos , Estrutura Molecular , Processos Fotoquímicos , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/química , Relação Estrutura-Atividade
11.
Toxicol Appl Pharmacol ; 359: 102-107, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30222979

RESUMO

NADH cytochrome b5 reductase mediates electron transfer from NADH to cytochrome b5 utilizing flavin adenine dinucleotide as a redox cofactor. Reduced cytochrome b5 is an important cofactor in many metabolic reactions including cytochrome P450-mediated xenobiotic metabolism, steroid biosynthesis and fatty acid metabolism, hemoglobin reduction, and methionine and plasmalogen synthesis. Using recombinant human enzyme, we discovered that cytochrome b5 reductase mediates redox cycling of a variety of quinones generating superoxide anion, hydrogen peroxide, and, in the presence of transition metals, hydroxyl radicals. Redox cycling activity was oxygen-dependent and preferentially utilized NADH as a co-substrate; NADH was 5-10 times more active than NADPH in supporting redox cycling. Redox cycling activity was greatest for 9,10-phenanthrenequinone and 1,2-naphthoquinone, followed by 1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone (menadione), nitrofurantoin and 2-hydroxyestradiol. Using menadione as the substrate, quinone redox cycling was found to inhibit reduction of cytochrome b5 by cytochrome b5 reductase, as measured by heme spectral changes in cytochrome b5. Under anaerobic conditions where redox cycling is inhibited, menadione had no effect on the reduction of cytochrome b5. Chemical redox cycling by cytochrome b5 reductase may be important in generating cytotoxic reactive oxygen species in target tissues. This activity, together with the inhibition of cytochrome b5 reduction by redox-active chemicals and consequent deficiencies in available cellular cytochrome b5, are likely to contribute to tissue injury following exposure to quinones and related redox active chemicals.


Assuntos
Benzoquinonas/metabolismo , Citocromo-B(5) Redutase/metabolismo , Nitrofurantoína/metabolismo , Radicais Livres/metabolismo , Humanos , Cinética , Microssomos Hepáticos , NADP/metabolismo , Oxirredução , Consumo de Oxigênio , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo
12.
Chem Res Toxicol ; 30(7): 1406-1418, 2017 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-28595002

RESUMO

Reactive carbonyls such as diacetyl (2,3-butanedione) and 2,3-pentanedione in tobacco and many food and consumer products are known to cause severe respiratory diseases. Many of these chemicals are detoxified by carbonyl reductases in the lung, in particular, dicarbonyl/l-xylulose reductase (DCXR), a multifunctional enzyme important in glucose metabolism. DCXR is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. Using recombinant human enzyme, we discovered that DCXR mediates redox cycling of a variety of quinones generating superoxide anion, hydrogen peroxide, and, in the presence of transition metals, hydroxyl radicals. Redox cycling activity preferentially utilized NADH as a cosubstrate and was greatest for 9,10-phenanthrenequinone and 1,2-naphthoquinone, followed by 1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone (menadione). Using 9,10-phenanthrenequinone as the substrate, quinone redox cycling was found to inhibit DCXR reduction of l-xylulose and diacetyl. Competitive inhibition of enzyme activity by the quinone was observed with respect to diacetyl (Ki = 190 µM) and l-xylulose (Ki = 940 µM). Abundant DCXR activity was identified in A549 lung epithelial cells when diacetyl was used as a substrate. Quinones inhibited reduction of this dicarbonyl, causing an accumulation of diacetyl in the cells and culture medium and a decrease in acetoin, the reduced product of diacetyl. The identification of DCXR as an enzyme activity mediating chemical redox cycling suggests that it may be important in generating cytotoxic reactive oxygen species in the lung. These activities, together with the inhibition of dicarbonyl/l-xylulose metabolism by redox-active chemicals, as well as consequent deficiencies in pentose metabolism, are likely to contribute to lung injury following exposure to dicarbonyls and quinones.


Assuntos
Células Epiteliais/metabolismo , Pulmão/patologia , Desidrogenase do Álcool de Açúcar/metabolismo , Células A549 , Relação Dose-Resposta a Droga , Células Epiteliais/enzimologia , Humanos , Pulmão/enzimologia , Pulmão/metabolismo , Estrutura Molecular , Oxirredução , Quinonas/química , Quinonas/farmacologia , Relação Estrutura-Atividade , Desidrogenase do Álcool de Açúcar/antagonistas & inibidores , Desidrogenase do Álcool de Açúcar/genética
13.
Toxicol Appl Pharmacol ; 305: 1-11, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27212445

RESUMO

Most mortality and morbidity following exposure to vesicants such as sulfur mustard is due to pulmonary toxicity. Acute injury is characterized by epithelial detachment and necrosis in the pharynx, trachea and bronchioles, while long-term consequences include fibrosis and, in some instances, cancer. Current therapies to treat mustard poisoning are primarily palliative and do not target underlying pathophysiologic mechanisms. New knowledge about vesicant-induced pulmonary disease pathogenesis has led to the identification of potentially efficacious strategies to reduce injury by targeting inflammatory cells and mediators including reactive oxygen and nitrogen species, proteases and proinflammatory/cytotoxic cytokines. Therapeutics under investigation include corticosteroids, N-acetyl cysteine, which has both mucolytic and antioxidant properties, inducible nitric oxide synthase inhibitors, liposomes containing superoxide dismutase, catalase, and/or tocopherols, protease inhibitors, and cytokine antagonists such as anti-tumor necrosis factor (TNF)-α antibody and pentoxifylline. Antifibrotic and fibrinolytic treatments may also prove beneficial in ameliorating airway obstruction and lung remodeling. More speculative approaches include inhibitors of transient receptor potential channels, which regulate pulmonary epithelial cell membrane permeability, non-coding RNAs and mesenchymal stem cells. As mustards represent high priority chemical threat agents, identification of effective therapeutics for mitigating toxicity is highly significant.


Assuntos
Substâncias para a Guerra Química/toxicidade , Irritantes/toxicidade , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/terapia , Gás de Mostarda/toxicidade , Animais , Fibrina/metabolismo , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Lesão Pulmonar/metabolismo , Metaloproteinases da Matriz/metabolismo , Células-Tronco Mesenquimais , RNA não Traduzido , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
14.
Toxicol Appl Pharmacol ; 303: 30-44, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27125198

RESUMO

Vesicants including sulfur mustard (SM) and nitrogen mustard (NM) are bifunctional alkylating agents that cause skin inflammation, edema and blistering. This is associated with alterations in keratinocyte growth and differentiation. Endogenous cannabinoids, including N-arachidonoylethanolamine (anandamide, AEA) and 2-arachidonoyl glycerol (2-AG), are important in regulating inflammation, keratinocyte proliferation and wound healing. Their activity is mediated by binding to cannabinoid receptors 1 and 2 (CB1 and CB2), as well as peroxisome proliferator-activated receptor alpha (PPARα). Levels of endocannabinoids are regulated by fatty acid amide hydrolase (FAAH). We found that CB1, CB2, PPARα and FAAH were all constitutively expressed in mouse epidermis and dermal appendages. Topical administration of NM or SM, at concentrations that induce tissue injury, resulted in upregulation of FAAH, CB1, CB2 and PPARα, a response that persisted throughout the wound healing process. Inhibitors of FAAH including a novel class of vanillyl alcohol carbamates were found to be highly effective in suppressing vesicant-induced inflammation in mouse skin. Taken together, these data indicate that the endocannabinoid system is important in regulating skin homeostasis and that inhibitors of FAAH may be useful as medical countermeasures against vesicants.


Assuntos
Alquilantes/toxicidade , Substâncias para a Guerra Química/toxicidade , Irritantes/toxicidade , Mecloretamina/toxicidade , Gás de Mostarda/toxicidade , Pele/efeitos dos fármacos , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Pelados , PPAR alfa/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Pele/metabolismo
15.
Exp Mol Pathol ; 100(3): 522-31, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27189522

RESUMO

Nitrogen mustard (NM) is a bifunctional alkylating agent that is highly reactive in the skin causing extensive tissue damage and blistering. In the present studies, a modified cutaneous murine patch model was developed to characterize NM-induced injury and to evaluate the efficacy of an indomethacin pro-drug in mitigating toxicity. NM (20µmol) or vehicle control was applied onto 6mm glass microfiber filters affixed to the shaved dorsal skin of CD-1 mice for 6min. This resulted in absorption of approximately 4µmol of NM. NM caused localized skin damage within 1 d, progressing to an eschar within 2-3 d, followed by wound healing after 4-5 d. NM-induced injury was associated with increases in skin thickness, inflammatory cell infiltration, reduced numbers of sebocytes, basal keratinocyte double stranded DNA breaks, as measured by phospho-histone 2A.X expression, mast cell degranulation and increases in inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Wound healing was characterized by epidermal hyperplasia and marked increases in basal cells expressing proliferating cell nuclear antigen. A novel indomethacin-anticholinergic prodrug (4338) designed to target cyclooxygenases and acetylcholinesterase (AChE), was found to markedly suppress NM toxicity, decreasing wound thickness and eschar formation. The prodrug also inhibited mast cell degranulation, suppressed keratinocyte expression of iNOS and COX-2, as well as markers of epidermal proliferation. These findings indicate that a novel bifunctional pro-drug is effective in limiting NM mediated dermal injury. Moreover, our newly developed cutaneous patch model is a sensitive and reproducible method to assess the mechanism of action of countermeasures.


Assuntos
Anti-Inflamatórios/farmacologia , Indometacina/análogos & derivados , Mecloretamina/toxicidade , Pró-Fármacos/farmacologia , Pele/efeitos dos fármacos , Alquilantes/toxicidade , Animais , Anti-Inflamatórios/química , Antagonistas Colinérgicos/química , Antagonistas Colinérgicos/farmacologia , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/química , Inibidores de Ciclo-Oxigenase/farmacologia , Dano ao DNA , Feminino , Histonas/metabolismo , Imuno-Histoquímica , Indometacina/química , Indometacina/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo II/metabolismo , Pró-Fármacos/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Pele/lesões , Pele/patologia , Fatores de Tempo , Cicatrização/efeitos dos fármacos
16.
J Pharmacol Exp Ther ; 352(3): 529-40, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25550200

RESUMO

Sepiapterin reductase (SPR) catalyzes the reduction of sepiapterin to dihydrobiopterin (BH2), the precursor for tetrahydrobiopterin (BH4), a cofactor critical for nitric oxide biosynthesis and alkylglycerol and aromatic amino acid metabolism. SPR also mediates chemical redox cycling, catalyzing one-electron reduction of redox-active chemicals, including quinones and bipyridinium herbicides (e.g., menadione, 9,10-phenanthrenequinone, and diquat); rapid reaction of the reduced radicals with molecular oxygen generates reactive oxygen species (ROS). Using recombinant human SPR, sulfonamide- and sulfonylurea-based sulfa drugs were found to be potent noncompetitive inhibitors of both sepiapterin reduction and redox cycling. The most potent inhibitors of sepiapterin reduction (IC50s = 31-180 nM) were sulfasalazine, sulfathiazole, sulfapyridine, sulfamethoxazole, and chlorpropamide. Higher concentrations of the sulfa drugs (IC50s = 0.37-19.4 µM) were required to inhibit redox cycling, presumably because of distinct mechanisms of sepiapterin reduction and redox cycling. In PC12 cells, which generate catecholamine and monoamine neurotransmitters via BH4-dependent amino acid hydroxylases, sulfa drugs inhibited both BH2/BH4 biosynthesis and redox cycling mediated by SPR. Inhibition of BH2/BH4 resulted in decreased production of dopamine and dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, and 5-hydroxytryptamine. Sulfathiazole (200 µM) markedly suppressed neurotransmitter production, an effect reversed by BH4. These data suggest that SPR and BH4-dependent enzymes, are "off-targets" of sulfa drugs, which may underlie their untoward effects. The ability of the sulfa drugs to inhibit redox cycling may ameliorate ROS-mediated toxicity generated by redox active drugs and chemicals, contributing to their anti-inflammatory activity.


Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/metabolismo , Pterinas/antagonistas & inibidores , Pterinas/metabolismo , Sulfassalazina/farmacologia , Sulfatiazóis/farmacologia , Oxirredutases do Álcool/química , Animais , Humanos , Camundongos , Oxirredução/efeitos dos fármacos , Células PC12 , Estrutura Secundária de Proteína , Pterinas/química , Ratos , Sulfatiazol
17.
Toxicol Appl Pharmacol ; 288(1): 114-20, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26212258

RESUMO

Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation.


Assuntos
Inibidores da Colinesterase/toxicidade , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Inseticidas/toxicidade , Fígado/efeitos dos fármacos , Intoxicação por Organofosfatos/prevenção & controle , Paration/toxicidade , Vitamina K 3/farmacologia , Acetilcolinesterase/metabolismo , Ativação Metabólica , Animais , Inibidores da Colinesterase/metabolismo , Inibidores das Enzimas do Citocromo P-450/metabolismo , Citoproteção , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Proteínas Ligadas por GPI/metabolismo , Humanos , Inseticidas/metabolismo , Fígado/enzimologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , NADP/metabolismo , Intoxicação por Organofosfatos/enzimologia , Intoxicação por Organofosfatos/etiologia , Oxirredução , Paraoxon/metabolismo , Paraoxon/toxicidade , Paration/metabolismo , Ratos Long-Evans , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Vitamina K 3/metabolismo
18.
Chem Res Toxicol ; 28(11): 2091-103, 2015 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-26451472

RESUMO

The thioredoxin (Trx) system, which consists of Trx and thioredoxin reductase (TrxR), is a major cellular disulfide reduction system important in antioxidant defense. TrxR is a target of mechlorethamine (methylbis(2-chloroethyl)amine; HN2), a bifunctional alkylating agent that covalently binds to selenocysteine/cysteine residues in the redox centers of the enzyme, leading to inactivation and toxicity. Mammalian Trx contains two catalytic cysteines; herein, we determined if HN2 also targets Trx. HN2 caused a time- and concentration-dependent inhibition of purified Trx and Trx in A549 lung epithelial cells. Three Trx cross-linked protein complexes were identified in both cytosolic and nuclear fractions of HN2-treated cells. LC-MS/MS of these complexes identified both Trx and TrxR, indicating that HN2 cross-linked TrxR and Trx. This is supported by our findings of a significant decrease of Trx/TrxR complexes in cytosolic TrxR knockdown cells after HN2 treatment. Using purified recombinant enzymes, the formation of protein cross-links and enzyme inhibition were found to be redox status-dependent; reduced Trx was more sensitive to HN2 inactivation than the oxidized enzyme, and Trx/TrxR cross-links were only observed using reduced enzyme. These data suggest that HN2 directly targets catalytic cysteine residues in Trx resulting in enzyme inactivation and protein complex formation. LC-MS/MS confirmed that HN2 directly alkylated cysteine residues on Trx, including Cys32 and Cys35 in the redox center of the enzyme. Inhibition of the Trx system by HN2 can disrupt cellular thiol-disulfide balance, contributing to vesicant-induced lung toxicity.


Assuntos
Reagentes de Ligações Cruzadas/toxicidade , Mecloretamina/toxicidade , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo , Linhagem Celular Tumoral , Dissulfetos/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Insulina/metabolismo , Pulmão/citologia , Modelos Moleculares , Oxirredução , Tiorredoxinas/química , Tiorredoxinas/genética
19.
Bioorg Med Chem Lett ; 25(23): 5609-12, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26510670

RESUMO

Novel ethynylphenyl carbonates and carbamates containing carbon- and silicon-based choline mimics were synthesized from their respective phenol and aniline precursors and screened for anticholinesterase and anti-inflammatory activities. All molecules were micromolar inhibitors of acetylcholinesterase (AChE), with IC50s of 28-86 µM; the carbamates were two-fold more potent than the carbonates. Two of the most potent AChE inhibitors suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation by 40%. Furthermore, these molecules have physicochemical properties in the range of other CNS drugs. These molecules have the potential to treat inflammation; they could also dually target Alzheimer's disease through restoration of cholinergic balance and inflammation suppression.


Assuntos
Acetilcolinesterase , Anti-Inflamatórios/síntese química , Carbamatos/síntese química , Carbonatos/síntese química , Inibidores da Colinesterase/síntese química , Acetilcolinesterase/química , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Carbamatos/química , Carbamatos/farmacologia , Carbonatos/química , Carbonatos/farmacologia , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Humanos , Concentração Inibidora 50 , Estrutura Molecular
20.
J Biol Chem ; 288(26): 19221-37, 2013 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-23640889

RESUMO

In the lung, chemical redox cycling generates highly toxic reactive oxygen species that can cause alveolar inflammation and damage to the epithelium, as well as fibrosis. In this study, we identified a cytosolic NADPH-dependent redox cycling activity in mouse lung epithelial cells as sepiapterin reductase (SPR), an enzyme important for the biosynthesis of tetrahydrobiopterin. Human SPR was cloned and characterized. In addition to reducing sepiapterin, SPR mediated chemical redox cycling of bipyridinium herbicides and various quinones; this activity was greatest for 1,2-naphthoquinone followed by 9,10-phenanthrenequinone, 1,4-naphthoquinone, menadione, and 2,3-dimethyl-1,4-naphthoquinone. Whereas redox cycling chemicals inhibited sepiapterin reduction, sepiapterin had no effect on redox cycling. Additionally, inhibitors such as dicoumarol, N-acetylserotonin, and indomethacin blocked sepiapterin reduction, with no effect on redox cycling. Non-redox cycling quinones, including benzoquinone and phenylquinone, were competitive inhibitors of sepiapterin reduction but noncompetitive redox cycling inhibitors. Site-directed mutagenesis of the SPR C-terminal substrate-binding site (D257H) completely inhibited sepiapterin reduction but had minimal effects on redox cycling. These data indicate that SPR-mediated reduction of sepiapterin and redox cycling occur by distinct mechanisms. The identification of SPR as a key enzyme mediating chemical redox cycling suggests that it may be important in generating cytotoxic reactive oxygen species in the lung. This activity, together with inhibition of sepiapterin reduction by redox-active chemicals and consequent deficiencies in tetrahydrobiopterin, may contribute to tissue injury.


Assuntos
Oxirredutases do Álcool/química , Células Epiteliais/citologia , Pulmão/citologia , Animais , Biopterinas/análogos & derivados , Biopterinas/química , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Vetores Genéticos , Humanos , Camundongos , Modelos Químicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredução , Estresse Oxidativo , Quinonas/química , Espécies Reativas de Oxigênio , Proteínas Recombinantes/química
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