RESUMEN
Nitrogen mustard (NM) is a known surrogate of sulfur mustard, a chemical-warfare agent that causes a wide range of ocular symptoms, from a permanent reduction in visual acuity to blindness upon exposure. Although it has been proposed that the two blistering agents have a similar mechanism of toxicity, the mode of NM-induced cell death in ocular tissue has not been fully explored. Therefore, we hypothesized that direct ocular exposure to NM in mice leads to retinal tissue injury through chronic activation of the unfolded protein response (UPR) PERK arm in corneal cells and VEGF secretion, eventually causing cell death. We topically applied NM directly to mice to analyze ocular and retinal tissues at 2 weeks postexposure. A dramatic decline in retinal function, measured by scotopic and photopic electroretinogram responses, was detected in the mice. This decline was associated with enhanced TUNEL staining in both corneal and retinal tissues. In addition, exposure of corneal cells to NM revealed 228 differentially and exclusively expressed proteins primarily associated with the UPR, ferroptosis, and necroptosis. Moreover, these cells exhibited activation of the UPR PERK arm and an increase in VEGF secretion. Enhancement of VEGF staining was later observed in the corneas of the exposed mice. Therefore, our data indicated that the mechanism of NM-induced ocular toxicity should be carefully examined and that future research should identify a signaling molecule transmitted via a prodeath pathway from the cornea to the retina. SIGNIFICANCE STATEMENT: This study demonstrated that NM topical exposure in mice results in dramatic decline in retinal function associated with enhanced TUNEL staining in both corneal and retinal tissues. We also found that the NM treatment of corneal cells resulted in 228 differentially and exclusively expressed proteins primarily associated with ferroptosis. Moreover, these cells manifest the UPR PERK activation and an increase in VEGF secretion. The latter was also found in the corneas of the cexposed mice.
Asunto(s)
Sustancias para la Guerra Química , Gas Mostaza , Animales , Ratones , Mecloretamina/toxicidad , Mecloretamina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Neuropatía Óptica Tóxica , Córnea , Sustancias para la Guerra Química/toxicidad , Gas Mostaza/toxicidad , Gas Mostaza/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
The chemical warfare agent sulfur mustard and its structural analog nitrogen mustard (NM) cause severe vesicating skin injuries. The pathologic mechanisms for the skin injury following mustard exposure are poorly understood; therefore, no effective countermeasure is available. Previous reports demonstrated the protective activity of carvedilol, a US Food and Drug Administration (FDA)-approved ß-blocker, against UV radiation-induced skin damage. Thus, the current study evaluated the effects of carvedilol on NM-induced skin injuries in vitro and in vivo. In the murine epidermal cell line JB6 Cl 41-5a, ß-blockers with different receptor subtype selectivity were examined. Carvedilol and both of its enantiomers, R- and S-carvedilol, were the only tested ligands statistically reducing NM-induced cytotoxicity. Carvedilol also reduced NM-induced apoptosis and p53 expression. In SKH-1 mice, NM increased epidermal thickness, damaged skin architecture, and induced nuclear factor κB (NF-κB)-related proinflammatory genes as assessed by RT2 Profiler PCR (polymerase chain reaction) Arrays. To model chemical warfare scenario, 30 minutes after exposure to NM, 10 µM carvedilol was applied topically. Twenty-four hours after NM exposure, carvedilol attenuated NM-induced epidermal thickening, Ki-67 expression, a marker of cellular proliferation, and multiple proinflammatory genes. Supporting the in vitro data, the non-ß-blocking R-enantiomer of carvedilol had similar effects as racemic carvedilol, and there was no difference between carvedilol and R-carvedilol in the PCR array data, suggesting that the skin protective effects are independent of the ß-adrenergic receptors. These data suggest that the ß-blocker carvedilol and its enantiomers can be repurposed as countermeasures against mustard-induced skin injuries. SIGNIFICANCE STATEMENT: The chemical warfare agent sulfur mustard and its structural analog nitrogen mustard cause severe vesicating skin injuries for which no effective countermeasure is available. This study evaluated the effects of US Food and Drug Administration (FDA)-approved ß-blocker carvedilol on nitrogen mustard-induced skin injuries to repurpose this cardiovascular drug as a medical countermeasure.
Asunto(s)
Sustancias para la Guerra Química , Gas Mostaza , Animales , Ratones , Mecloretamina/toxicidad , Mecloretamina/metabolismo , Carvedilol/farmacología , Carvedilol/uso terapéutico , Carvedilol/metabolismo , Sustancias para la Guerra Química/toxicidad , Gas Mostaza/farmacología , Gas Mostaza/toxicidad , Piel , Antagonistas Adrenérgicos beta/farmacologíaRESUMEN
Bronchiolitis obliterans (BO) is a fibrotic lung disease characterized by progressive luminal narrowing and obliteration of the small airways. In the nontransplant population, inhalation exposure to certain chemicals is associated with BO; however, the mechanisms contributing to disease induction remain poorly understood. This study's objective was to use single-cell RNA sequencing for the identification of transcriptomic signatures common to primary human airway epithelial cells after chemical exposure to BO-associated chemicals-diacetyl or nitrogen mustard-to help explain BO induction. Primary airway epithelial cells were cultured at air-liquid interface and exposed to diacetyl, nitrogen mustard, or control vapors. Cultures were dissociated and sequenced for single-cell RNA. Differential gene expression and functional pathway analyses were compared across exposures. In total, 75,663 single cells were captured and sequenced from all exposure conditions. Unbiased clustering identified 11 discrete phenotypes, including 5 basal, 2 ciliated, and 2 secretory cell clusters. With chemical exposure, the proportion of cells assigned to keratin 5+ basal cells decreased, whereas the proportion of cells aligned to secretory cell clusters increased compared with control exposures. Functional pathway analysis identified interferon signaling and antigen processing/presentation as pathways commonly upregulated after diacetyl or nitrogen mustard exposure in a ciliated cell cluster. Conversely, the response of airway basal cells differed significantly with upregulation of the unfolded protein response in diacetyl-exposed basal cells, not seen in nitrogen mustard-exposed cultures. These new insights provide early identification of airway epithelial signatures common to BO-associated chemical exposures.NEW & NOTEWORTHY Bronchiolitis obliterans (BO) is a devastating fibrotic lung disease of the small airways, or bronchioles. This original manuscript uses single-cell RNA sequencing for identifying common signatures of chemically exposed airway epithelial cells in BO induction. Chemical exposure reduced the proportion of keratin 5+ basal cells while increasing the proportion of keratin 4+ suprabasal cells. Functional pathways contributory to these shifts differed significantly across exposures. These new results highlight similarities and differences in BO induction across exposures.
Asunto(s)
Bronquiolitis Obliterante , Diacetil , Humanos , Queratina-5/metabolismo , Diacetil/metabolismo , Mecloretamina/metabolismo , Mucosa Respiratoria/metabolismo , Bronquiolitis Obliterante/inducido químicamente , Bronquiolitis Obliterante/metabolismo , Células Epiteliales/metabolismoRESUMEN
The ocular surface comprises the wet mucosal epithelia of the cornea and conjunctiva, the associated glands, and the overlying tear film. Epitheliopathy is the common pathologic outcome when the ocular surface is subjected to oxidative stress. Whether different stresses act via the same or different mechanisms is not known. Dynasore and dyngo-4a, small molecules developed to inhibit the GTPase activity of classic dynamins DNM1, DNM2, and DNM3, but not mdivi-1, a specific inhibitor of DNM1L, protect corneal epithelial cells exposed to the oxidant tert-butyl hydroperoxide (tBHP). Here we report that, while dyngo-4a is the more potent inhibitor of endocytosis, dynasore is the better cytoprotectant. Dynasore also protects corneal epithelial cells against exposure to high salt in an in vitro model of dysfunctional tears in dry eye. We now validate this finding in vivo, demonstrating that dynasore protects against epitheliopathy in a mouse model of dry eye. Knockdown of classic dynamin DNM2 was also cytoprotective against tBHP exposure, suggesting that dynasore's effect is at least partially on target. Like tBHP and high salt, exposure of corneal epithelial cells to nitrogen mustard upregulated the unfolded protein response and inflammatory markers, but dynasore did not protect against nitrogen mustard exposure. In contrast, mdivi-1 was cytoprotective. Interestingly, mdivi-1 did not inhibit the nitrogen mustard-induced expression of inflammatory cytokines. We conclude that exposure to tBHP or nitrogen mustard, two different oxidative stress agents, cause corneal epitheliopathy via different pathologic pathways. SIGNIFICANCE STATEMENT: Results presented in this paper, for the first time, implicate the dynamin DNM2 in ocular surface epitheliopathy. The findings suggest that dynasore could serve as a new topical treatment for dry eye epitheliopathy and that mdivi-1 could serve as a medical countermeasure for epitheliopathy due to nitrogen mustard exposure, with potentially increased efficacy when combined with anti-inflammatory agents and/or UPR modulators.
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Síndromes de Ojo Seco , Hidrazonas , Mecloretamina , Naftoles , Quinazolinonas , Ratones , Animales , Mecloretamina/toxicidad , Mecloretamina/metabolismo , Síndromes de Ojo Seco/inducido químicamente , Síndromes de Ojo Seco/tratamiento farmacológico , Córnea , Lágrimas , DinaminasRESUMEN
Sulfur mustard (SM) and nitrogen mustard (NM) are vesicant agents that cause skin injury and blistering through complicated cellular events, involving DNA damage, free radical formation, and lipid peroxidation. The development of therapeutic approaches targeting the multi-cellular process of tissue injury repair can potentially provide effective countermeasures to combat vesicant-induced dermal lesions. MG53 is a vital component of cell membrane repair. Previous studies have demonstrated that topical application of recombinant human MG53 (rhMG53) protein has the potential to promote wound healing. In this study, we further investigate the role of MG53 in NM-induced skin injury. Compared with wild-type mice, mg53-/- mice are more susceptible to NM-induced dermal injuries, whereas mice with sustained elevation of MG53 in circulation are resistant to dermal exposure of NM. Exposure of keratinocytes and human follicle stem cells to NM causes elevation of oxidative stress and intracellular aggregation of MG53, thus compromising MG53's intrinsic cell membrane repair function. Topical rhMG53 application mitigates NM-induced dermal injury in mice. Histologic examination reveals the therapeutic benefits of rhMG53 are associated with the preservation of epidermal integrity and hair follicle structure in mice with dermal NM exposure. Overall, these findings identify MG53 as a potential therapeutic agent to mitigate vesicant-induced skin injuries.
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Irritantes , Mecloretamina , Ratones , Humanos , Animales , Mecloretamina/toxicidad , Mecloretamina/metabolismo , Irritantes/metabolismo , Queratinocitos/metabolismo , Cicatrización de Heridas/fisiología , Proteínas de la Membrana/metabolismoRESUMEN
Sulfur mustard gas (SM) is a vesicating and alkylating agent used as a chemical weapon in many mass-casualty incidents since World War I. Ocular injuries were reported in >90% of exposed victims. The mechanisms underlying SM-induced blindness remain elusive. This study tested the hypothesis that SM-induced corneal fibrosis occurs due to the generation of myofibroblasts from resident fibroblasts via the SMAD2/3 signaling pathway in rabbit eyes in vivo and primary human corneal fibroblasts (hCSFs) isolated from donor corneas in vitro. Fifty-four New Zealand White Rabbits were divided into three groups (Naïve, Vehicle, SM-Vapor treated). The SM-Vapor group was exposed to SM at 200 mg-min/m3 for 8 min at the MRI Global facility. Rabbit corneas were collected on day 3, day 7, and day 14 for immunohistochemistry, RNA, and protein lysates. SM caused a significant increase in SMAD2/3, pSMAD, and ÉSMA expression on day 3, day 7, and day 14 in rabbit corneas. For mechanistic studies, hCSFs were treated with nitrogen mustard (NM) or NM + SIS3 (SMAD3-specific inhibitor) and collected at 30 m, 8 h, 24 h, 48 h, and 72 h. NM significantly increased TGFß, pSMAD3, and SMAD2/3 levels. On the contrary, inhibition of SMAD2/3 signaling by SIS3 treatment significantly reduced SMAD2/3, pSMAD3, and ÉSMA expression in hCSFs. We conclude that SMAD2/3 signaling appears to play a vital role in myofibroblast formation in the cornea following mustard gas exposure.
Asunto(s)
Sustancias para la Guerra Química , Gas Mostaza , Humanos , Animales , Conejos , Gas Mostaza/toxicidad , Gas Mostaza/metabolismo , Miofibroblastos/metabolismo , Sustancias para la Guerra Química/toxicidad , Sustancias para la Guerra Química/metabolismo , Córnea/metabolismo , Mecloretamina/metabolismo , Mecloretamina/farmacología , Transducción de Señal , Proteína Smad2/metabolismoRESUMEN
A hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was developed for qualitative and quantitative analysis of ethanolamines (EAs), which are nitrogen mustard degradation products. With this method, the retention times of the highly hydrophilic EAs on the HILIC column were sufficient (retention times: methyl diethanolamine, 12.2 min; ethyl diethanolamine, 11.2 min; and triethanolamine, 9.5 min) and the EAs were analyzed more efficiently than with reported HILIC-MS/MS methods. The detection limits of methyl diethanolamine and ethyl diethanolamine in serum and urine using this approach were 15-20 ng/mL. The suitability of the method for real samples was evaluated via recovery tests involving urine and serum, and the method was validated. The MS/MS fragmentation of EAs was discussed based on density functional theory calculations.
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Técnicas de Química Analítica/métodos , Cromatografía Liquida , Espectrometría de Masas en Tándem , Etanolaminas/análisis , Interacciones Hidrofóbicas e Hidrofílicas , Límite de Detección , Mecloretamina/análisis , Mecloretamina/metabolismoRESUMEN
We report the quantitative analysis of 5-methylcytosine, a representative epigenetic modification in genomic DNA, with an enzyme-linked immunosorbent assay (ELISA). We synthesized a novel hetero-bifunctional linker molecule consisting of nitrogen mustard and biotin to capture DNA on the surface of biosensing devices. The molecule can successfully immobilize genomic DNA on a streptavidin coated 96-well microplate, which was then employed for immunochemical epigenetic assessment. We achieved the sensitive and quantitative detection of 5-mC in genomic DNA samples. The CpG methylation ratios obtained from our system for mouse brain and mouse small intestine genomes were 79% and 82%, respectively. These numbers are in good agreement with the previously reported methylation ratio of 75-85%, which was identified by whole genome bisulfite sequencing. Accordingly, the present technology using our novel bifunctional linker molecule provides a fast, easy, and inexpensive method for epigenetic assessment, without the need for any conventional bisulfite treatment, polymerase chain reaction (PCR), or sequencing.
Asunto(s)
5-Metilcitosina/análisis , Biotina/química , Ensayo de Inmunoadsorción Enzimática , Ácidos Nucleicos Inmovilizados/química , Mecloretamina/química , 5-Metilcitosina/inmunología , Animales , Biotina/metabolismo , Encéfalo/metabolismo , Metilación de ADN , Epigénesis Genética , Genoma , Ácidos Nucleicos Inmovilizados/metabolismo , Intestino Delgado/metabolismo , Mecloretamina/metabolismo , Ratones , Análisis de Secuencia de ADN , Estreptavidina/química , Estreptavidina/metabolismoRESUMEN
In this study, we propose a novel molecular platform-integrated fluorinated antitumor nitrogen mustards for 19 F-MRS assay of ß-galactosidase (ß-gal) activity. Following this idea, we have designed, synthesized, and characterized 2-fluoro-4-[bis(2'-chloroethyl)amino]phenyl ß-D-galactopyranoside 5, 2-fluoro-4-{bis[2'-O-(ß-D-galactopyranosyl)ethyl]amino}phenyl ß-D-galactopyranoside 8, 2-fluoro-4-{bis[[1â³-(ß-D-galactopyranosyl)-1â³, 2â³, 3â³-triazol-4â³-yl]methyl] amino}phenyl ß-D-galactopyranoside 14 and 2-fluoro-4-{bis[[1â³-(ß-D-glucopyranosyl)-1â³, 2â³, 3â³-triazol-4â³-yl]methyl]amino}phenyl ß-D-galactopyranoside 15 through glycosylation and click reaction strategies, and their structures were confirmed by NMR and HRMS or elemental analysis data. Among them, 2-fluoro-4-[bis(2'-chloroethyl)amino]phenyl ß-D-galacto-pyranoside 5 was found very sensitive to ß-gal (E801A) in PBS at 37°C with big ΔδF response. Here, we demonstrated the feasibility of this platform for assessing ß-gal activity in solution, and in vitro with lacZ-transfected human MCF7 breast and PC3 prostate tumor cells, by the characterization of ß-gal-responsive 19 F-chemical shift changes ΔδF and hydrolytic kinetics.
Asunto(s)
Pruebas de Enzimas/métodos , Mecloretamina/análogos & derivados , beta-Galactosidasa/metabolismo , Neoplasias de la Mama/enzimología , Línea Celular Tumoral , Femenino , Halogenación , Humanos , Masculino , Mecloretamina/síntesis química , Mecloretamina/metabolismo , Neoplasias de la Próstata/enzimología , beta-Galactosidasa/análisisRESUMEN
PURPOSE: The purpose of this study is to evaluate if the differential exchange rates with bulk water between amine and amide protons can be exploited using chemical exchange saturation transfer magnetic resonance (CEST-MR) to monitor the release of glutamate induced by carboxypeptidase G2 (CPG2), an enzyme utilized in cancer gene therapy. PROCEDURES: Z spectra of solutions of the CPG2 substrate, 3,5-difluorobenzoyl-L-glutamate (amide), and glutamate (amine) were acquired at 11.7 T, 37 °C, across different pH (5-8). The ability of CEST-MR to monitor CPG2-mediated release of glutamate was assessed in extracts of CPG2-expressing cancer cells and purified solution of CPG2. RESULTS: The addition of CPG2 to a solution containing 3,5-difluorobenzoyl-L-glutamate led to a marked and progressively increasing CEST effect (+3 ppm), concomitant with the time-dependent release of glutamate induced by CPG2. CONCLUSION: CEST-MR allows the detection of CPG2 activity in vitro and supports the translation of CEST-MRI to assess CPG2-based gene therapy in vivo.
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Espectroscopía de Resonancia Magnética/métodos , Profármacos/metabolismo , gamma-Glutamil Hidrolasa/metabolismo , Benzoatos/metabolismo , Línea Celular Tumoral , Humanos , Mecloretamina/química , Mecloretamina/metabolismo , Profármacos/química , Procesamiento de Señales Asistido por Computador , SolucionesRESUMEN
A potential anti-melanoma prodrug containing a phenolic activator, a hydrazine linker, and a nitrogen mustard effector - (N-{4-[bis-(2-chloroethyl)amino]benzoyl}-N'-(4-hydroxybenzyl)hydrazine) has been synthesized in seven steps. Spectrophotometric measurements of its oxidation by tyrosinase showed a rapid increase of absorbance at 337 nm. HPLC analysis demonstrated that two major products were formed. However, during the reaction one of the products was converted into the other. The stable product with a maximum of absorption at 337 nm was isolated and identified as 5,6-dihydroxy-1H-indazol-1-yl 4-[bis-(2-chloroethyl)amino]benzoate. It was formed by a cyclization of the enzymatically generated o-quinone. This reaction was unexpected, since the acylated hydrazine nitrogen atom should not be sufficiently nucleophilic to attack the o-quinone ring. This cyclization prevented the effector release from the enzyme-activated prodrug. As a result, the prodrug showed only limited specificity for B16-F10 murine melanoma cells compared to reference cell lines. When applied in solid tumors in mice it showed slightly higher activity than the parent mustard drug (4-[bis-(2-chloroethyl)amino]benzoic cid), but significantly lower activity than melphalan, a commercial mustard drug with a structure resembling tyrosine, occasionally used in the treatment of melanoma.
Asunto(s)
Hidrazinas/química , Hidrazinas/farmacología , Mecloretamina/química , Mecloretamina/farmacología , Melanoma Experimental/tratamiento farmacológico , Profármacos/química , Profármacos/farmacología , Animales , Línea Celular Tumoral , Ciclización , Hidrazinas/síntesis química , Hidrazinas/metabolismo , Mecloretamina/síntesis química , Mecloretamina/metabolismo , Ratones , Monofenol Monooxigenasa/metabolismo , Profármacos/síntesis química , Profármacos/metabolismoRESUMEN
Oxidative stress plays a key role in mechlorethamine (methylbis(2-chloroethyl)amine, HN2) toxicity. The thioredoxin system, consisting of thioredoxin reductase (TrxR), thioredoxin, and NADPH, is important in redox regulation and protection against oxidative stress. HN2 contains two electrophilic side chains that can react with nucleophilic sites in proteins, leading to changes in their structure and function. We report that HN2 inhibits the cytosolic (TrxR1) and mitochondrial (TrxR2) forms of TrxR in A549 lung epithelial cells. TrxR exists as homodimers under native conditions; monomers can be detected by denaturing and reducing SDS-PAGE followed by western blotting. HN2 treatment caused marked decreases in TrxR1 and TrxR2 monomers along with increases in dimers and oligomers under reducing conditions, indicating that HN2 cross-links TrxR. Cross-links were also observed in rat lung after HN2 treatment. Using purified TrxR1, NADPH reduced, but not oxidized, enzyme was inhibited and cross-linked by HN2. LC-MS/MS analysis of TrxR1 demonstrated that HN2 adducted cysteine- and selenocysteine-containing redox centers forming monoadducts, intramolecule and intermolecule cross-links, resulting in enzyme inhibition. HN2 cross-links two dimeric subunits through intermolecular binding to cysteine 59 in one subunit of the dimer and selenocysteine 498 in the other subunit, confirming the close proximity of the N- and C-terminal redox centers of adjacent subunits. Despite cross-linking and inhibition of TrxR activity by HN2, TrxR continued to mediate menadione redox cycling and generated reactive oxygen species. These data suggest that disruption of the thioredoxin system contributes to oxidative stress and tissue injury induced by HN2.
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Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/farmacología , Disulfuros/antagonistas & inhibidores , Células Epiteliales/efectos de los fármacos , Pulmón/citología , Pulmón/efectos de los fármacos , Mecloretamina/química , Mecloretamina/farmacología , Reductasa de Tiorredoxina-Disulfuro/antagonistas & inhibidores , Reductasa de Tiorredoxina-Disulfuro/química , Animales , Células Cultivadas , Reactivos de Enlaces Cruzados/metabolismo , Disulfuros/química , Disulfuros/metabolismo , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Humanos , Pulmón/enzimología , Pulmón/metabolismo , Masculino , Mecloretamina/metabolismo , Modelos Moleculares , Oxidación-Reducción/efectos de los fármacos , Ratas , Ratas Wistar , Reductasa de Tiorredoxina-Disulfuro/metabolismoRESUMEN
We investigated how asparagine mutagenesis of conserved aspartic acids in helix 2 (D2.50) and 3 (D3.32) of M1-M4 muscarinic receptors alters the irreversible binding of acetylcholine mustard and BR384 (4-[(2-bromoethyl)methyl-amino]-2-butynyl N-(3-chlorophenyl)carbamate), a nitrogen mustard derivative of McN-A-343 ([4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl] trimethylammonium chloride). The D2.50N mutation moderately increased the affinity of the aziridinium ions of acetylcholine mustard and BR384 for M2-M4 receptors and had little effect on the rate constant for receptor alkylation. The D3.32N mutation greatly reduced the rate constant for receptor alkylation by acetylcholine mustard but not by BR384, although the affinity of BR384 was reduced. The combination of both mutations (D2.50N/D3.32N) substantially reduced the rate constant for receptor alkylation by BR384 relative to that of wild type and mutant D2.50N and D3.32N receptors. The change in binding affinity caused by the mutations suggests that the D2.50N mutation alters the interaction of acetylcholine mustard with D3.32 of the M1 and M3 receptors but not that of the M4 receptor. BR384 exhibited the converse relationship. The simplest explanation is that acetylcholine mustard and BR384 alkylate at least two residues on M1-M4 receptors and that the D2.50N mutation alters the rate of alkylation of D3.32 relative to another residue, perhaps D2.50 itself.
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Acetilcolina/metabolismo , Asparagina/genética , Ácido Aspártico/genética , Mecloretamina/metabolismo , Receptores Muscarínicos/genética , Acetilcolina/análogos & derivados , Animales , Células CHO , Cricetinae , Cricetulus , Cobayas , Humanos , Cinética , Mutagénesis , N-Metilescopolamina/metabolismo , Receptores Muscarínicos/química , Receptores Muscarínicos/metabolismoRESUMEN
The major concern for anticancer chemotherapeutic agents is the host toxicity. The development of anticancer prodrugs targeting the unique biochemical alterations in cancer cells is an attractive approach to achieve therapeutic activity and selectivity. We designed and synthesized a new type of nitrogen mustard prodrug that can be activated by high level of reactive oxygen species (ROS) found in cancer cells to release the active chemotherapy agent. The activation mechanism was determined by NMR analysis. The activity and selectivity of these prodrugs toward ROS was determined by measuring DNA interstrand cross-links and/or DNA alkylations. These compounds showed 60-90% inhibition toward various cancer cells, while normal lymphocytes were not affected. To the best of our knowledge, this is the first example of H(2)O(2)-activated anticancer prodrugs.
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Antineoplásicos Alquilantes/farmacología , Diseño de Fármacos , Peróxido de Hidrógeno/metabolismo , Sustancias Intercalantes/farmacología , Mecloretamina/farmacología , Profármacos/farmacología , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/metabolismo , Línea Celular Tumoral , ADN/metabolismo , Humanos , Sustancias Intercalantes/química , Sustancias Intercalantes/metabolismo , Mecloretamina/química , Mecloretamina/metabolismo , Neoplasias/tratamiento farmacológico , Profármacos/química , Profármacos/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Nitrogen mustards (NMs) are known to have DNA alkylation and strong vesicant properties. Their availability to terrorist organizations makes them a potential choice for chemical attacks on civilian populations. After an exposure, it is difficult to measure NMs directly because of their rapid metabolism in the human body. Therefore to determine an individual's level of exposure to NMs, it is necessary to analyze for NM metabolites being excreted by the body. The metabolites of NMs are generated by a hydrolysis reaction, and are easily detectable by liquid chromatography tandem mass spectrometry (LC-MS/MS). This work is focused on the development of a high-throughput assay for the quantitation of N-ethyldiethanolamine (EDEA) and N-methyldiethanolamine (MDEA) metabolites of bis (2-chloroethyl) ethylethanamine (HN1) and bis (2-chloroethyl) methylethanamine (HN2), respectively. The method uses automated 96-well plate sample preparation of human urine samples and a 2-position 10-port switching valve to allow for simultaneous regeneration of the liquid chromatography (LC) columns. Using this method, over 18 h was saved through the reduction of sample preparation and analysis time when compared to a conventional method for 96 samples. The validated method provided excellent accuracy for both EDEA (100.9%) and MDEA (100.6%) with precision better than 5.27% for each analyte.
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Cromatografía Liquida/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Mecloretamina/metabolismo , Mecloretamina/orina , Espectrometría de Masas en Tándem/métodos , Cromatografía Liquida/instrumentación , HumanosRESUMEN
A gas chromatographic-mass spectrometric method was developed, validated and demonstrated by measuring the levels of nitrogen mustard hydrolysis products in the urine collected from dosed rats. The recovery values for trimethylsilyl derivatives of EDEA and MDEA are between 82-95% and 88-112%, respectively. In vivo studies performed by using three different doses (0.5 mg/kg, 1.0 mg/kg, and 2.0 mg/kg) of HN2 base of nitrogen mustard. MDEA concentrations were between 43.1-232.2 ng/mL. The limit of detection (S/N = 3) values are 2.5 ng/mL and 1.6 ng/mL for EDEA and MDEA, respectively, and the precision of the method in terms of RSD is between 5-8%.
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Etanolaminas/orina , Cromatografía de Gases y Espectrometría de Masas/métodos , Mecloretamina/orina , Animales , Estabilidad de Medicamentos , Masculino , Mecloretamina/metabolismo , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Investigating how a test drug alters the reaction of a site-directed electrophile with a receptor is a powerful method for determining whether the drug acts competitively or allosterically, provided that the binding site of the electrophile is known. In this study, therefore, we mutated nucleophilic residues near and within the orthosteric pockets of M(1) and M(2) muscarinic receptors to identify where acetylcholine mustard and 4-[(2-bromoethyl)methyl-amino]-2-butynyl-N-(3-chlorophenyl)carbamate (BR384) bind covalently. BR384 is the nitrogen mustard analog of [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride (McN-A-343). Mutation of the highly conserved aspartic acid in M(1) (Asp105) and M(2) (Asp103) receptors to asparagine largely prevented receptor alkylation by acetylcholine mustard, although modest alkylation still occurred at M(2) D103N at high concentrations of the mustard. Receptor alkylation by BR384 was also greatly inhibited in the M(1) D105N mutant, but some alkylation still occurred at high concentrations of the compound. In contrast, BR384 rapidly alkylated the M(2) D103N mutant. Its affinity was reduced to one tenth, however. The alkylation of M(2) D103N by BR384 was competitively inhibited by N-methylscopolamine and allosterically inhibited by gallamine. Mutation of a variety of other nucleophilic residues, some in combination with D103N, had little effect on M(2) receptor alkylation by BR384. Our results suggest that BR384 alkylates at least one residue other than the conserved aspartic acid at the ligand-binding site of M(1) and M(2) receptors. This additional residue seems to be located within or near the orthosteric-binding pocket and is not part of the allosteric site for gallamine.
Asunto(s)
Cloruro de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamonio/metabolismo , Acetilcolina/análogos & derivados , Mutagénesis Sitio-Dirigida , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M2/metabolismo , Cloruro de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamonio/análogos & derivados , Acetilcolina/química , Acetilcolina/metabolismo , Sitio Alostérico/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Células CHO , Cricetinae , Cricetulus , Humanos , Mecloretamina/química , Mecloretamina/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida/métodos , Unión Proteica/genética , Receptor Muscarínico M1/genética , Receptor Muscarínico M2/genéticaRESUMEN
A novel liquid chromatography-multiple reaction monitoring (LC-MRM) procedure has been developed for retrospective diagnosis of exposure to different forms of mustard agents. This concise method is able to validate prior exposure to nitrogen mustards (HN-1, HN-2, and HN-3) or sulfur mustard (HD) in a single run, which significantly reduces analysis time compared to separate runs to screen for different mustards' biomarkers based on tandem mass spectrometry. Belonging to one of the more toxic classes of chemical warfare agents, these potent vesicants bind covalently to the cysteine-34 residue of human serum albumin. This results in the formation of stable adducts whose identities were confirmed by a de novo sequencing bioinformatics software package. Our developed technique tracks these albumin-derived adduct biomarkers in blood samples which persist in vitro following exposure, enabling a detection limit of 200 nM of HN-1, 100 nM of HN-2, 200 nM of HN-3, or 50 nM of HD in human blood. The CWA-adducts formed in blood samples can be conveniently and sensitively analyzed by this MRM technique to allow rapid and reliable screening.
Asunto(s)
Cromatografía Liquida/métodos , Monitoreo del Ambiente/métodos , Compuestos de Mostaza/análisis , Espectrometría de Masas en Tándem/métodos , Alquilación , Biomarcadores/sangre , Cromatografía de Afinidad , Biología Computacional/métodos , Exposición a Riesgos Ambientales/análisis , Humanos , Mecloretamina/análisis , Mecloretamina/metabolismo , Compuestos de Mostaza/metabolismo , Gas Mostaza/análisis , Gas Mostaza/metabolismo , Compuestos de Mostaza Nitrogenada/análisis , Compuestos de Mostaza Nitrogenada/metabolismo , Reproducibilidad de los Resultados , Albúmina Sérica/química , Albúmina Sérica/aislamiento & purificación , Albúmina Sérica/metabolismo , Programas Informáticos , Extracción en Fase SólidaRESUMEN
Covalent binding of various clinically important nitrogen mustards to the cysteine-34 residue of human serum albumin, in vitro and in vivo, is demonstrated. A rapid method for detection of these adducts is presented, based on liquid chromatography-tandem mass spectrometry analysis of the adducted tripeptide Cys*-Pro-Phe after digestion of the protein with Pronase.
Asunto(s)
Cisteína/metabolismo , Monitoreo de Drogas/métodos , Compuestos de Mostaza Nitrogenada/metabolismo , Albúmina Sérica/metabolismo , Sitios de Unión , Clorambucilo/metabolismo , Ciclofosfamida/metabolismo , Humanos , Técnicas In Vitro , Mecloretamina/metabolismo , Melfalán/metabolismo , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Expansion of the triplet repeat DNA sequence d[CGG]n.d[CCG]n is a characteristic of Fragile X syndrome, a human neurodegenerative disease. Stable intrastrand conformations formed by both d[CGG]n and d[CCG]n, and involving G-G and C-C mismatch pairs, respectively, are believed to be of importance in the development of the disease. We have shown previously that C-C mismatch pairs can be crosslinked covalently by mechlorethamine, a nitrogen mustard alkylating agent, and hence this reaction may be of value as a probe for conformers of d[CCG]n. To characterize the mechlorethamine C-C crosslink reaction further, here we report the kinetics and sequence dependence of formation of the crosslink species, using a series of model duplexes. The rate of reaction depends on the base sequence proximal to the C-C mismatch pair. Hence, in 19mer duplexes containing a central d[M4M3M2M1Cn1n2n3n4].d[N4N3N2N1Cm1m2m3m4] sequence, where M-m and N-n are complementary base pairs, the amount of crosslink increased with increasing G-C content of the eight base pairs neighboring the C-C mismatch and with the proximity of the G-C pairs to the C-C mismatch. Molecular dynamics simulations of the solvated duplexes provided an explanation of these data. Hence, for a C-C pair flanked by G-C base pairs the mismatched cytosine bases remain stacked within the duplex, but for a C-C pair flanked by A-T base pairs, the simulations suggested local opening of the duplex around the C-C pair, making it a less effective target for mechlorethamine.