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1.
Chem Res Toxicol ; 30(1): 145-161, 2017 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-27802593

RESUMO

Acrolein is a highly toxic electrophile that participates in many diseases, yet efforts to delineate its precise mechanistic contributions to specific conditions are complicated by its wide distribution within human environments. This Perspective develops the proposal that due to its mixed status as environmental pollutant, metabolic byproduct, and endotoxicant which forms via ubiquitous pathophysiological processes, many diseases likely involve acrolein released from multiple sources. Although the category boundaries are indistinct, at least four identifiable exposure scenarios are identifiable. First, in some syndromes, such as those accompanying chronic or acute intoxication with smoke, whatever role acrolein plays in disease pathogenesis mainly traces to exogenous sources such as the combustion of tobacco or other organic matter. A second exposure category involves xenobiotics that undergo metabolism within the body to release acrolein. Still other health conditions, however, involve acrolein that forms via several endogenous pathways, some of which are activated upon intoxication with xenobiotics (i.e., Exposure Category 3), while still others accompany direct physical trauma to body tissues (Exposure Category 4). Further complicating efforts to clarify the role of endogenous acrolein in human disease is the likelihood that many such syndromes are complex phenomena that resemble "chemical mixture exposures" by involving multiple toxic substances simultaneously. This Perspective contends that while recent decades have witnessed much progress in describing the deleterious effects of acrolein at the cellular and molecular levels, more work is needed to define the contributions of different acrolein sources to "real-world" health conditions in human subjects.


Assuntos
Acroleína/toxicidade , Exposição Ambiental/efeitos adversos , Poluentes Ambientais/toxicidade , Acroleína/metabolismo , Animais , Doença/etiologia , Poluentes Ambientais/metabolismo , Humanos
2.
Chem Biol Interact ; 395: 111008, 2024 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-38636791

RESUMO

Oxidative protein damage involving carbonylation of respiratory tract proteins typically accompanies exposure to tobacco smoke. Such damage can arise via multiple mechanisms, including direct amino acid oxidation by reactive oxygen species or protein adduction by electrophilic aldehydes. This study investigated the relative importance of these pathways during exposure of a model protein to fresh cigarette emission extracts. Briefly, protein carbonyl adducts were estimated in bovine serum albumin following incubation in buffered solutions with whole cigarette emissions extracts prepared from either a single 1R6F research cigarette or a single "Heat-not-Burn" e-cigarette. Although both extracts caused concentration-dependent protein carbonylation, conventional cigarette extracts produced higher adduct yields than e-cigarette extracts. Superoxide radical generation by conventional and e-cigarette emissions was assessed by monitoring nitro blue tetrazolium reduction and was considerably lower in extracts made from "Heat-Not-Burn" e-cigarettes. The superoxide dismutase/catalase mimic EUK-134 strongly suppressed radical production by whole smoke extracts from conventional cigarettes, however, it did not diminish protein carbonyl adduction when incubating smoke extracts with the model protein. In contrast, edaravone, a neuroprotective drug with strong carbonyl-trapping properties, strongly suppressed protein damage without inhibiting superoxide formation. Although these findings require extension to appropriate cell-based and in vivo systems, they suggest reactive aldehydes in tobacco smoke make greater contributions to oxidative protein damage than smoke phase radicals.


Assuntos
Sistemas Eletrônicos de Liberação de Nicotina , Carbonilação Proteica , Soroalbumina Bovina , Fumaça , Superóxidos , Produtos do Tabaco , Superóxidos/metabolismo , Carbonilação Proteica/efeitos dos fármacos , Fumaça/efeitos adversos , Soroalbumina Bovina/química , Produtos do Tabaco/efeitos adversos , Bovinos , Animais , Nicotiana/química , Temperatura Alta
3.
Mol Pharmacol ; 82(5): 876-86, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22869587

RESUMO

Toxic carbonyls such as acrolein participate in many degenerative diseases. Although the nucleophilic vasodilatory drug hydralazine readily traps such species under "test-tube" conditions, whether these reactions adequately explain its efficacy in animal models of carbonyl-mediated disease is uncertain. We have previously shown that hydralazine attacks carbonyl-adducted proteins in an "adduct-trapping" reaction that appears to take precedence over direct "carbonyl-sequestering" reactions, but how this reaction conferred cytoprotection was unclear. This study explored the possibility that by increasing the bulkiness of acrolein-adducted proteins, adduct-trapping might alter the redistribution of chaperones to damaged cytoskeletal proteins that are known targets for acrolein. Using A549 lung adenocarcinoma cells, the levels of chaperones heat shock protein (Hsp) 40, Hsp70, Hsp90, and Hsp110 were measured in intermediate filament extracts prepared after a 3-h exposure to acrolein. Exposure to acrolein alone modestly increased the levels of all four chaperones. Coexposure to hydralazine (10-100 µM) strongly suppressed cell ATP loss while producing strong adduct-trapping in intermediate filaments. Most strikingly, hydralazine selectively boosted the levels of cytoskeletal-associated Hsp90, including a high-mass species that was sensitive to the Hsp90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin. Biochemical fractionation of acrolein- and hydralazine-treated cells revealed that hydralazine likely promoted Hsp90 migration from cytosol into other subcellular compartments. A role for Hsp90 mobilization in cytoprotection was confirmed by the finding that brief heat shock treatment suppressed acute acrolein toxicity in A549 cells. Taken together, these findings suggest that by increasing the steric bulk of carbonyl-adducted proteins, adduct-trapping drugs trigger the intracellular mobilization of the key molecular chaperone Hsp90.


Assuntos
Acroleína/toxicidade , Proteínas de Choque Térmico HSP90/metabolismo , Hidralazina/farmacologia , Estresse Oxidativo , Acroleína/metabolismo , Trifosfato de Adenosina/metabolismo , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citoproteção , Resposta ao Choque Térmico , Humanos , Filamentos Intermediários/efeitos dos fármacos , Filamentos Intermediários/metabolismo , Carbonilação Proteica/efeitos dos fármacos
4.
J Pharmacol Toxicol Methods ; 108: 106957, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33636341

RESUMO

INTRODUCTION: While cysteine thiol groups help to maintain the redox status of many proteins, they can be very susceptible to damaging oxidants. Despite broad interest in their antioxidant properties, whether tea polyphenols protect against protein thiol damage of this kind is unclear. This study sought to develop a simple immunoassay for use in screening tea extracts and other antioxidants for thioprotective efficacy at protein thiol groups. METHODS: Fresh aqueous extracts were prepared from commercially sourced green, white, black and red teas. Traut's reagent (2-iminothiolane) was used to prepare surface-thiolated bovine serum albumin for use as assay substrate in the protein oxidation assay. Oxidative damage was induced during a 15 min incubation with hydrogen peroxide (H2O2) in the presence of tea extracts and reference antioxidants. The substrate protein was then derivatised with dimedone before samples were loaded onto a nitrocellulose membrane housed within a Slot-Blot apparatus. After blocking nonspecific protein binding a commercially available antibody was used to detect dimedone-labelled groups. RESULTS: While the total phenol content of tea extracts typically correlated with their activity in lipid peroxidation and galvinoxyl radical-trapping assays, the former did not fully predict their abilities to suppress H2O2-induced cysteine oxidation, with black tea extracts displaying greater activity than the other teas and an apparent ability to reverse pre-existing cysteine oxidation. Among the model antioxidants tested, quercetin displayed a heightened ability to suppress cysteine oxidation. DISCUSSION: This slot-blot immunoassay is a convenient method that facilitates standardised comparisons between the thioprotective properties of structurally- and constitutively-diverse antioxidants.


Assuntos
Cisteína , Chá , Antioxidantes/farmacologia , Peróxido de Hidrogênio , Oxirredução , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio
5.
Cancer Res ; 67(10): 4751-8, 2007 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-17510403

RESUMO

Cutaneous squamous cell carcinomas (CSCC) are a common malignancy of keratinocytes that arise in sites of the skin exposed to excessive UV radiation. In the present study, we show that human SCC cell lines, preneoplastic solar keratoses (SK), and CSCC are associated with perturbations in glutathione peroxidase (GPX) activity and peroxide levels. Specifically, we found that two of three SKs and four of five CSCCs, in vivo, were associated with decreased GPX activity and all SKs and CSCCs were associated with an elevated peroxide burden. Given the association of decreased GPX activity with CSCC, we examined the basis for the GPX deficiency in the CSCCs. Our data indicated that GPX was inactivated by a post-translational mechanism and that GPX could be inactivated by increases in intracellular peroxide levels. We next tested whether the decreased peroxidase activity coupled with an elevated peroxidative burden might contribute to CSCC formation in vivo. This was tested in Gpx1(-/-) and Gpx2(-/-) mice exposed to solar-simulated UV radiation. These studies showed that Gpx2 deficiency predisposed mice to UV-induced CSCC formation. These results suggest that inactivation of GPX2 in human skin may be an early event in UV-induced SCC formation.


Assuntos
Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/etiologia , Glutationa Peroxidase/metabolismo , Neoplasias Induzidas por Radiação/enzimologia , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/etiologia , Ativação Enzimática , Glutationa Peroxidase/biossíntese , Glutationa Peroxidase/deficiência , Glutationa Peroxidase/genética , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , Queratinócitos/enzimologia , Queratinócitos/patologia , Queratinócitos/efeitos da radiação , Neoplasias Induzidas por Radiação/etiologia , Peróxidos/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Raios Ultravioleta
6.
Toxicol In Vitro ; 22(4): 844-53, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18282682

RESUMO

Acrolein is a toxic combustion product that elicits apoptotic and/or necrotic cell death depending on the conditions under which exposure occurs. As a strong electrophile, side-reactions with nucleophilic media constituents seem likely to accompany study of its toxicity in vitro, but these reactions are poorly characterized. We have thus examined the effect of media composition on the toxicity of acrolein in A549 cells. Cells were exposed to acrolein in either Dulbecco's buffered saline (DBS) or F12 supplemented with various concentrations of fetal bovine serum. Cell viability was assessed using the MTT assay, while heme oxygenase-1 (HO-1) and cytoplasmic cytochrome c were measured as respective markers of transcriptional response and apoptosis. Protein damage was evaluated using the protein carbonyl assay. Compared to F12 media (with or without serum), maximal cell death as evaluated using the MTT assay, as well as adduction of intracellular proteins, occurred when cells were exposed to acrolein in DBS. In contrast, cytochrome c release was maximal in cells exposed to acrolein in serum-containing F12, conditions which inhibited protein modification and overt cell death. These findings highlight the need for careful attention to experimental conditions when conducting in vitro toxicological studies of reactive substances.


Assuntos
Acroleína/toxicidade , Meios de Cultura/química , Citocromos c/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Alquilação/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Bovinos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Citoplasma/metabolismo , Heme Oxigenase-1/metabolismo , Humanos , Carbonilação Proteica/efeitos dos fármacos , Soro/metabolismo
7.
Biochem Pharmacol ; 154: 397-406, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29883705

RESUMO

During cellular metabolism, spontaneous oxidative damage to unsaturated lipids generates many electrophilic carbonyl compounds that readily attack cell macromolecules, forming adducts that are potential drivers of tissue dysfunction. Since such damage is heightened in many degenerative conditions, researchers have assessed the efficacy of nucleophilic carbonyl-trapping drugs in animal models of such disorders, anticipating that they will protect tissues by intercepting toxic lipid-derived electrophiles (LDEs) within cells. This Commentary explores recent animal evidence for carbonyl scavenger efficacy in two disparate yet significant conditions known to involve LDE production, namely spinal cord injury (SCI) and alcoholic liver disease (ALD). Primary emphasis is placed on studies that utilised hydralazine, a clinically-approved "broad-spectrum" scavenger known to trap multiple LDEs. In addition to reviewing recent studies of hydralazine efficacy in animal SCI and ALD models, the Commentary reviews new insights concerning novel lifespan- and healthspan-extending properties of hydralazine obtained during studies in model invertebrate organisms, since the mechanisms involved seem of likely benefit during the treatment of degenerative disease. Finally, noting that human translation of the histoprotective properties of hydralazine have been limited, the final section of the Commentary will address two obstacles that hamper clinical translation of LDE-trapping therapies while also suggesting potential strategies for overcoming these problems.


Assuntos
Elementos de Resposta Antioxidante/efeitos dos fármacos , Reposicionamento de Medicamentos/métodos , Hidralazina/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Doenças Neurodegenerativas/tratamento farmacológico , Pesquisa Translacional Biomédica/métodos , Animais , Elementos de Resposta Antioxidante/fisiologia , Reposicionamento de Medicamentos/tendências , Humanos , Hidralazina/uso terapêutico , Peroxidação de Lipídeos/fisiologia , Doenças Neurodegenerativas/metabolismo , Pesquisa Translacional Biomédica/tendências , Vasodilatadores/farmacologia , Vasodilatadores/uso terapêutico
8.
J Pharmacol Toxicol Methods ; 56(1): 18-22, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17395496

RESUMO

INTRODUCTION: The carbonyl content of cell proteins is a useful indicator of oxidative protein damage during drug- and chemically-induced toxicities. Since a range of lipophilic carbonylating agents is produced during the membrane peroxidation that accompanies chemically-induced oxidative stress, integral membrane proteins seem especially vulnerable to adduction by these species. To develop tools for assessing such damage, this work refined a popular spectrophotometric assay so that hydrophobic and hydrophilic proteins are separated prior to carbonyl analysis. METHODS: The low cloud point properties of Triton X114 were used to resolve mouse liver extracts into fractions comprising hydrophilic and hydrophobic proteins. Following phase separation, protein precipitation and removal of unwanted detergent was achieved via extraction with toluene containing 5% trichloroacetic acid. Carbonyl groups were derivatised using 2,4-dinitrophenylhydrazine and then quantified via spectrophotometry. RESULTS: Postmitochondrial fractions from mouse liver were incubated with azo initiators that generate peroxyl radicals in respective phases of biphasic systems, namely the water-soluble initiator 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) and the lipophilic initiator 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). Following resolution using Triton X114, the carbonyl content of hydrophilic proteins in controls was 1.5+/-0.1 nmol/mg protein, compared to 6.7+/-0.57 nmol/mg protein in the corresponding hydrophobic proteins (N=3, p<0.05). Exposure to AAPH for 2 h at 37 degrees C increased the carbonyl content of proteins in the hydrophilic and hydrophobic phases by 4.6- and 3.5-fold, respectively (p<0.05). AMVN produced a 3.3-fold increase in the carbonyl content of hydrophobic proteins (p<0.05) but did not alter that of aqueous phase proteins. DISCUSSION: This method allows facile resolution of proteins into hydrophobic and hydrophilic fractions prior to carbonyl determination. This modified assay method may facilitate studies of the role of oxidative protein damage in drug- and chemically-induced toxic syndromes.


Assuntos
Proteínas de Membrana/análise , Estresse Oxidativo , Carbonilação Proteica , Amidinas/química , Animais , Compostos Azo/química , Detergentes , Hidrazinas/química , Fígado/química , Masculino , Camundongos , Nitrilas/química , Octoxinol , Oxidantes/química , Oxirredução , Polietilenoglicóis , Espectrofotometria , Extratos de Tecidos/química
9.
ACS Med Chem Lett ; 8(7): 686-689, 2017 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-28740598

RESUMO

General levels of "pharmaceuticals literacy" are not high in contemporary societies. To address this educational need, in 2012 the University of Western Australia introduced an innovative multidisciplinary course for undergraduates within any degree program entitled PHAR1101: Drugs that Changed the World. Now ranking among the largest courses at the institution, PHAR1101 enrollments will likely approach 1000 students in 2017.

10.
Artigo em Inglês | MEDLINE | ID: mdl-27865847

RESUMO

INTRODUCTION: The aim of this study was to develop two dynamic ex vivo airway explant systems, a perfusion-superfusion system and a ventilation-superfusion system, for the study of toxic airborne substances, such as the prevalent smoke constituent acrolein. METHODS: Mouse isolated tracheal segments were perfused with physiological media or ventilated with humidified air at 37°C to mimic dynamic flow conditions, and superfused with media over the exterior surface. At selected time points, the histological and functional integrity of segments was evaluated. The perfusion-superfusion system was subsequently used to examine mucin secretory responses elicited by acrolein in airways in which mucous metaplasia had been induced with lipopolysaccharide (LPS; 1µgml-1) prior to 24h of media perfusion, followed by stimulation with acrolein or ATP for 15min. Epithelial mucin levels were determined by quantitative analysis of periodic acid-Schiff's reagent (PAS)-stained sections. RESULTS: Epithelial morphology was successfully preserved in the perfusion-superfusion and ventilation-superfusion systems for at least 24h and up to 18h, respectively. At these time points, the contractile and relaxation responses of perfused and ventilated tracheal segments to carbachol, the neuropeptide substance P, and the prostanoid PGE2 were also preserved. Using the perfusion-superfusion system, acute exposure to acrolein caused a dose-dependent reduction in the levels of PAS-positive mucin stores induced by LPS, consistent with mucin secretion. DISCUSSION: Both the perfusion-superfusion and ventilation-superfusion systems successfully preserved the viability of mouse isolated tracheal segments on a histological and functional level, and the perfusion-superfusion system was used to characterise the mucin secretory responses elicited by acrolein. Thus, this system may be a useful model through which to conduct further toxicological studies in mammalian airways.


Assuntos
Músculo Liso/fisiologia , Perfusão/métodos , Traqueia/fisiologia , Acroleína/farmacologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Perfusão/instrumentação , Traqueia/efeitos dos fármacos
11.
J Toxicol Environ Health A ; 68(9): 739-53, 2005 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-16020200

RESUMO

Cylindrospermopsin (CYN) is a cyanobacterial toxin found in drinking-water sources world wide. It was the likely cause of human poisonings in Australia and possibly Brazil. Although CYN itself is a potent protein synthesis inhibitor, its acute toxicity appears to be mediated by cytochrome p-450 (CYP450)-generated metabolites. CYN also induces genotoxic effects both in vitro and in vivo, and preliminary evidence suggests that tumors are generated by oral exposure to CYN. To understand the role of CYP450-activated CYN metabolites on in vitro genotoxicity, this study quantified the process in primary mouse hepatocytes using the COMET assay in both the presence and absence of CYP450 inhibitors known to block acute CYN cytotoxicity. CYN was cytotoxic at concentrations above 0.1 microM (EC50 = 0.5 microM) but produced significant increases in Comet tail length, area, and tail moment at 0.05 microM and above; hence genotoxicity is unlikely to be secondary to metabolic disruption due to toxicity. The CYP450 inhibitors omeprazole (100 microM) and SKF525A (50 microM) completely inhibited the genotoxicity induced by CYN. The toxin also inhibits production of glutathione (GSH), a finding confirmed in this study. This could potentiate cytotoxicity, and by implication genotoxicity, via reduced reactive oxygen species (ROS) quenching. The lipid peroxidation marker, malondialdehyde (MDA) was quantified in CYN-treated cells, and the effect of the reduced glutathione (GSSG) reductase (GSSG-rd.) inhibitor 1,3-bis(chloroethyl)-l-nitrosourea (BCNU) on both MDA production and lactate dehydrogenase (LDH) leakage was examined. MDA levels were not elevated by CYN treatment, and block of GSH regeneration by BCNU did not affect lipid peroxidation or cytotoxicity. It therefore seems likely that CYP450-derived metabolites are responsible for both the acute cytotoxicity and genotoxicity induced by CYN.


Assuntos
Sistema Enzimático do Citocromo P-450/fisiologia , Hepatócitos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Uracila/análogos & derivados , Uracila/toxicidade , Alcaloides , Animais , Toxinas Bacterianas , Ensaio Cometa , Toxinas de Cianobactérias , Inibidores das Enzimas do Citocromo P-450 , Hepatócitos/enzimologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Testes de Mutagenicidade , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo
12.
Biochem Pharmacol ; 93(4): 519-26, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25557294

RESUMO

The airway epithelium is an important source of relaxant mediators, and damage to the epithelium caused by respiratory tract viruses may contribute to airway hyperreactivity. The aim of this study was to determine whether influenza A-induced epithelial damage would modulate relaxation responses evoked by acrolein, a toxic and prevalent component of smoke. Male BALB/c mice were inoculated intranasally with influenza A/PR-8/34 (VIRUS-infected) or allantoic fluid (SHAM-infected). On day 4 post-inoculation, isometric tension recording studies were conducted on carbachol pre-contracted tracheal segments isolated from VIRUS and SHAM mice. Relaxant responses to acrolein (30 µM) were markedly smaller in VIRUS segments compared to SHAM segments (2 ± 1% relaxation vs. 28 ± 5%, n=14, p<0.01). Similarly, relaxation responses of VIRUS segments to the neuropeptide substance P (SP) were greatly attenuated (1 ± 1% vs. 47 ± 6% evoked by 1 nM SP, n=14, p<0.001). Consistent with epithelial damage, PGE2 release in response to both acrolein and SP were reduced in VIRUS segments (>35% reduction, n=6, p<0.01), as determined using ELISA. In contrast, exogenous PGE2 was 2.8-fold more potent in VIRUS relative to SHAM segments (-log EC50 7.82 ± 0.14 vs. 7.38 ± 0.05, n=7, p<0.01) whilst responses of VIRUS segments to the ß-adrenoceptor agonist isoprenaline were similar to SHAM segments. In conclusion, relaxation responses evoked by acrolein were profoundly diminished in tracheal segments isolated from influenza A-infected mice. The mechanism through which influenza A infection attenuates this response appears to involve reduced production of PGE2 in response to SP due to epithelial cell loss, and may provide insight into the airway hyperreactivity observed with influenza A infection.


Assuntos
Acroleína/toxicidade , Vírus da Influenza A/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Infecções por Orthomyxoviridae , Traqueia/efeitos dos fármacos , Animais , Embrião de Galinha , Relação Dose-Resposta a Droga , Vírus da Influenza A/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Relaxamento Muscular/fisiologia , Músculo Liso/fisiologia , Músculo Liso/virologia , Técnicas de Cultura de Órgãos , Traqueia/fisiologia , Traqueia/virologia
13.
Toxicology ; 181-182: 229-36, 2002 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-12505316

RESUMO

Elevated levels of reactive alpha,beta-unsaturated aldehydes (e.g. malondialdehyde, 4-hydroxynonenal and acrolein) in the affected tissues of various degenerative conditions suggest these substances are active propagators of the disease process. One experimental approach to attenuating damage by these intermediates employs 'aldehyde-sequestering drugs' as sacrificial nucleophiles, thereby sparing cell macromolecules and perhaps slowing disease progression. Drugs with demonstrated trapping activity toward lipid-derived aldehydes include various amine compounds such as aminoguanidine, carnosine and pyridoxamine. We have focused on identifying scavengers of acrolein, perhaps the most toxic aldehyde formed during lipid peroxidation cascades. Various phthalazine compounds (hydralazine and dihydralazine) were found to trap acrolein readily, forming hydrazone derivatives in a rapid Schiff-type reaction. These compounds strongly protect against acrolein-mediated toxicity in isolated hepatocytes.


Assuntos
Aldeídos/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Proteínas/química , Acroleína/metabolismo , Animais , Carnosina/farmacologia , Guanidinas/farmacologia , Humanos , Hidralazina/farmacologia , Oxirredução , Piridoxamina/farmacologia
14.
Chem Biol Interact ; 145(2): 201-11, 2003 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-12686496

RESUMO

Glucuronidation of a number of carboxyl-containing drugs generates reactive acyl glucuronide metabolites. These electrophilic species alkylate cell proteins and may be implicated in the pathogenesis of a number of toxic syndromes seen in patients receiving the parent aglycones. Whether acyl glucuronides also attack nuclear DNA is unknown, although the acyl glucuronide formed from clofibric acid was recently found to decrease the transfection efficiency of phage DNA and generate strand breaks in plasmid DNA in vitro. To determine if such a DNA damage occurs within a cellular environment, the comet assay (i.e. single-cell gel electrophoresis) was used to detect DNA lesions in the nuclear genome of isolated mouse hepatocytes cultured with clofibric acid. Overnight exposure to 50 microM and higher concentrations of clofibric acid produced concentration-dependent increases in the comet areas of hepatocyte nuclei, with 1 mM clofibrate producing a 3.6-fold elevation over controls. These effects closely coincided with culture medium concentrations of the glucuronide metabolite formed from clofibric acid, 1-O-beta-clofibryl glucuronide. Consistent with a role for glucuronidation in the DNA damage observed, the glucuronidation inhibitor borneol diminished glucuronide formation from 100 microM clofibrate by 98% and returned comet areas to baseline levels. Collectively, these results suggest that the acyl glucuronide formed from clofibric acid is capable of migrating from its site of formation within the endoplasmic reticulum to generate strand nicks in nuclear DNA.


Assuntos
Ácido Clofíbrico/efeitos adversos , Ácido Clofíbrico/análise , Ácido Clofíbrico/metabolismo , Dano ao DNA , Glucuronídeos/análise , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Hepatócitos/metabolismo , Animais , Canfanos/farmacologia , Morte Celular , Ácido Clofíbrico/análogos & derivados , Ensaio Cometa , Relação Dose-Resposta a Droga , Hepatócitos/enzimologia , Masculino , Camundongos , Fatores de Tempo
15.
Toxicology ; 319: 44-52, 2014 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-24594012

RESUMO

The combustion product acrolein is a key mediator of pulmonary edema in victims of smoke inhalation injury. Since studying acrolein toxicity in conventional in vitro systems is complicated by reactivity with nucleophilic culture media constituents, we explored an exposure system which delivers airborne acrolein directly to lung cell monolayers at the air-liquid interface. Calu-3 lung adenocarcinoma cells were maintained on membrane inserts such that the basal surface was bathed in nucleophile-free media while the upper surface remained in contact with acrolein-containing air. Cells were exposed to airborne acrolein for 30 min before they were allowed to recover in fresh media, with cell sampling at defined time points to allow evaluation of toxicity and protein damage. After prior exposure to acrolein, cell ATP levels remained close to controls for 4h but decreased in an exposure-dependent manner by 24h. A loss of transepithelial electrical resistance and increased permeability to fluorescein isothiocyanate-labeled dextran preceded ATP loss. Use of antibody arrays to monitor protein expression in exposed monolayers identified strong upregulation of phospho-keratin-8 (Ser(73)) as an early consequence of acrolein exposure. These changes were accompanied by chemical damage to keratin-8 and other intermediate filament family members, while acrolein exposure also resulted in controlled ubiquitination of high mass proteins within the intermediate filament extracts. These findings confirm the usefulness of systems allowing delivery of airborne smoke constituents to lung cell monolayers during studies of the molecular basis for acute smoke intoxication injury.


Assuntos
Acroleína/toxicidade , Poluentes Atmosféricos/toxicidade , Filamentos Intermediários/metabolismo , Queratina-8/metabolismo , Vimentina/metabolismo , Brônquios/citologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , Ubiquitinação
16.
Biochem Pharmacol ; 89(1): 148-56, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24561178

RESUMO

Airway sensory C-fibres express TRPA1 channels which have recently been identified as a key chemosensory receptor for acrolein, a toxic and highly prevalent component of smoke. TRPA1 likely plays an intermediary role in eliciting a range of effects induced by acrolein including cough and neurogenic inflammation. Currently, it is not known whether acrolein-induced activation of TRPA1 produces other airway effects including relaxation of mouse airway smooth muscle. The aims of this study were to examine the effects of acrolein on airway smooth muscle tone in mouse isolated trachea, and to characterise the cellular and molecular mechanisms underpinning the effects of acrolein. Isometric tension recording studies were conducted on mouse isolated tracheal segments to characterise acrolein-induced relaxation responses. Release of the relaxant PGE2 was measured by EIA to examine its role in the response. Use of selective antagonists/inhibitors permitted pharmacological characterisation of the molecular and cellular mechanisms underlying this relaxation response. Acrolein induced dose-dependent relaxation responses in mouse isolated tracheal segments. Importantly, these relaxation responses were significantly inhibited by the TRPA1 antagonists AP-18 and HC-030031, an NK1 receptor antagonist RP-67580, and the EP2 receptor antagonist PF-04418948, whilst completely abolished by the non-selective COX inhibitor indomethacin. Acrolein also caused rapid PGE2 release which was suppressed by HC-030031. In summary, acrolein induced a novel bronchodilator response in mouse airways. Pharmacologic studies indicate that acrolein-induced relaxation likely involves interplay between TRPA1-expressing airway sensory C-fibres, NK1 receptor-expressing epithelial cells, and EP2-receptor expressing airway smooth muscle cells.


Assuntos
Acroleína/farmacologia , Músculo Liso/efeitos dos fármacos , Traqueia/efeitos dos fármacos , Canais de Potencial de Receptor Transitório/fisiologia , Animais , Dinoprostona/farmacologia , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Músculo Liso/fisiologia , Substância P/farmacologia , Canal de Cátion TRPA1 , Traqueia/fisiologia
18.
Toxicol Lett ; 212(3): 241-51, 2012 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-22705057

RESUMO

The combustion product acrolein contributes to several smoke-related health disorders, but whether this immunomodulatory toxicant alters pulmonary susceptibility to viruses has received little attention. To study the effects of prior acrolein dosing on the severity of influenza A viral infection, male BALB/c mice received acrolein (1mg/kg) or saline (control) via oropharyngeal aspiration either 4- or 7-days prior to intranasal inoculation with either influenza A/PR/8/34 virus or vehicle. At 0, 2, 4 and 7 days post-inoculation, lung samples were assessed for histological changes while pulmonary inflammation was monitored by estimating immune cell numbers and cytokine levels in bronchoalveolar lavage fluid (BALF). After viral challenge, animals that were exposed to acrolein 4 days previously experienced greater weight loss and exhibited an accelerated inflammatory response at 2 days after viral inoculation. Thus compared to saline-pretreated, virus-challenged controls, BALF recovered from these mice contained higher numbers of macrophages and neutrophils in addition to increased levels of several inflammatory cytokines, including IL-1α, IL-1ß, IL-6, TNF, IFN-γ, KC, and MCP-1. The acrolein-induced increase in viral susceptibility was suppressed by the carbonyl scavenger bisulphite. These findings suggest acute acrolein intoxication "primes" the lung to mount an accelerated immune response to inhaled viruses.


Assuntos
Acroleína/toxicidade , Poluentes Atmosféricos/toxicidade , Pulmão/patologia , Infecções por Orthomyxoviridae/patologia , Pneumonia/patologia , Administração por Inalação , Animais , Líquido da Lavagem Broncoalveolar , Citocinas/sangue , Suscetibilidade a Doenças , Esquema de Medicação , Sequestradores de Radicais Livres/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/patogenicidade , Vírus da Influenza A/fisiologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/patologia , Infecções por Orthomyxoviridae/complicações , Infecções por Orthomyxoviridae/imunologia , Pneumonia/imunologia , Pneumonia/virologia , Sulfitos/farmacologia
19.
Chem Biol Interact ; 183(3): 416-24, 2010 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-20015449

RESUMO

The noxious 3-carbon electrophile acrolein forms on combustion of diverse organic matter including synthetic polymers such as polyethylene. While known to play a key role in smoke inhalation injury (SII), the molecular basis for the pulmonary toxicity of high dose acrolein-containing smoke is unclear. As a result, drug interventions in SII are poorly directed against pathogenetic smoke toxicants such as acrolein. The first aim of this study was to confirm a role for acrolein in the acute toxicity of smoke extracts towards A549 lung cells by monitoring adduction of known acrolein targets and the expression of acrolein-inducible genes. A second aim was to evaluate carbonyl scavengers for their abilities to protect cell targets and block smoke extract toxicity. Extracts were prepared by bubbling smoke released by smouldering polyethylene through a buffered saline-trap. Acrolein levels in the extracts were estimated via HPLC after derivatisation with 2,4-dinitrophenylhydrazine. Extracts were highly toxic towards A549 cells, eliciting greater ATP depletion than an equivalent concentration of acrolein alone. The toxicity was accompanied by pronounced carbonylation of several cytoskeletal targets, namely vimentin and keratins-7, -8 and -18. Western blotting revealed that polyethylene combustion products also upregulated several acrolein-responsive protein markers, including GADD45beta, NQO1, HMOX, Hsp70, Nur77 and Egr1. Several carbonyl scavengers (bisulfite, d-penicillamine, hydralazine and 1-hydrazinoisoquinoline) strongly attenuated smoke extract toxicity, with bisulfite suppressing both the adduction and cross-linking of intermediate filament targets. Bisulfite also suppressed the cytotoxicity of smoke extracts when detected using real-time monitoring of cellular impedance. These findings confirm a key role for acrolein in smoke cytotoxicity and suggest drugs that block acrolein toxicity deserve further investigation as possible interventions against SII.


Assuntos
Acroleína/toxicidade , Sequestradores de Radicais Livres/metabolismo , Fumaça , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Heme Oxigenase-1/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Queratina-18/metabolismo , Queratina-7/metabolismo , Queratina-8/metabolismo , Neoplasias Pulmonares , NAD(P)H Desidrogenase (Quinona)/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Polietileno/toxicidade , Carbonilação Proteica/efeitos dos fármacos , Vimentina/metabolismo , Proteínas GADD45
20.
Antioxid Redox Signal ; 12(3): 337-47, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19686041

RESUMO

Extensive protein carbonylation accompanies cellular exposure to acrolein, a ubiquitous smoke constituent implicated in life-threatening pulmonary edema in fire victims, a condition involving rapid erosion of the "watertight" properties of respiratory epithelium. Since the identities of lung epithelial proteins that sustain carbonylation by acrolein are unknown, we sought to identify significant targets in subcellular fractions from A549 cells after 30 min exposure to either subtoxic or acutely toxic acrolein concentrations (60 or 360 fmol acrolein/cell). The lower concentration mainly modified cytosolic proteins while the higher concentration also damaged nuclear, membrane, and cytoskeletal proteins. The multifunctional intermediate filament proteins vimentin, keratin-18, keratin-7 and keratin-8, were conspicuous targets. Consistent with their mechanical functions, a loss of cellular adhesive strength accompanied adduction of the two most abundant intermediate filaments in A549 cells, keratins-8 and -18. Acrolein also elicited redistribution of several chaperones (Hsp40, -70, -90, and -110) to intermediate filament fractions, suggesting chaperone-mediated autophagy contributes to the triage of acrolein-adducted proteins. The carbonyl scavenger bisulfite suppressed acrolein toxicity, intermediate filament adduction, vimentin cross-linking, Hsp90 redistribution, and loss of cellular adhesive strength, while also suppressing vimentin hyperphosphorylation. These novel observations identify intermediate filaments as key targets for the reactive smoke constituent acrolein.


Assuntos
Acroleína/toxicidade , Filamentos Intermediários/metabolismo , Carbonilação Proteica/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP110/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Queratina-18/metabolismo , Queratina-7/metabolismo , Queratina-8/metabolismo , Chaperonas Moleculares/metabolismo , Fosforilação/efeitos dos fármacos , Vimentina/metabolismo
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