RESUMO
Sulfur mustard (SM) is a bifunctional alkylating agent. Its primary toxic consequence is severe skin damage with blisters, occurring after skin contact. These vesicant properties of SM have been linked to cell death of proliferating keratinocytes in the basal layer of the skin. Catalytic activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP-1) has been demonstrated to be a major event in response to high levels of DNA damage, and PARP-1 activation may be part of apoptotic signaling. In other contexts, overstimulation of PARP-1 triggers necrotic cell death because of rapid consumption of its substrate, beta-nicotinamide adenine dinucleotide (NAD+) and the consequent depletion of ATP. These findings prompted us to evaluate whether SM induces apoptosis in keratinocytes like HaCaT cells and to determine whether blocking of PARP enzyme activity with 3-aminobenzamide (3AB) can influence the mode of cell death. HaCaT cells were exposed to SM (10-1,000 microM; 30 min) and then cultivated in SM-free medium with or without 3AB for up to 48 h. This treatment resulted in a time and SM dose-dependent increase of apoptotic cell death characterized by PARP-1 cleavage and DNA fragmentation during the experimental period. After just 45 min of exposure to 1 mM SM, we observed a significant increase in PARP-1 activity in HaCaT cells. About 6 h after exposure, intracellular ATP levels were diminished by 22%, which seemed to be completely prevented by the addition of 3AB directly after exposure. However, 18 h later, this 3AB effect on the SM concentration-dependent loss of ATP was no longer detectable. Interestingly, the effect of SM on total cell viability was not changed by 3AB. However, the mode of cell death was influenced by 3AB exhibiting an increase of apoptotic cells and a concomitant decrease of necrotic HaCaT cells during the first 24 h after SM exposure. Our results indicate that SM concentrations of 1 mM or higher induce a prominent PARP activation leading to ATP depletion and necrosis. In contrast, lower concentrations of SM cause minor PARP activation and, especially, PARP-1 cleavage by caspase 3 without ATP depletion. Because ATP is required for apoptosis, we suggest that ATP acts as an early molecular switch from apoptotic to necrotic modes of SM-induced cell death, at least at high concentrations (> or =1 mM). Thus, the observed early proapoptotic effect of 3AB at lower SM concentrations may point to the influence of ATP-independent cell-death regulating mechanisms.
Assuntos
Carcinógenos/toxicidade , Morte Celular/efeitos dos fármacos , Substâncias para a Guerra Química , Inibidores Enzimáticos/farmacologia , Queratinócitos/efeitos dos fármacos , Gás de Mostarda/toxicidade , Inibidores de Poli(ADP-Ribose) Polimerases , Trifosfato de Adenosina/metabolismo , Antimetabólitos , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Bromodesoxiuridina , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Humanos , Cinética , Necrose , Dermatopatias/induzido quimicamente , Dermatopatias/patologiaRESUMO
The chemical warfare agent sulfur mustard (SM) is a strong alkylating agent that leads to erythema and ulceration of the human skin several hours after exposure. Although SM has been intensively investigated, the cellular mechanisms leading to cell damage remain unclear. Apoptosis, necrosis and direct cell damage are discussed. In this study we investigated apoptotic cell death in pulmonary A549 cells exposed to SM (30-1000 microM, 30 min). 24 h after SM exposure DNA breaks were stained with the TUNEL method. Additionally, A549 cells were lysed and cellular protein was transferred to SDS page and blotted. Whole PARP as well as PARP cleavage into the p89 fragment, an indicator of apoptosis, were detected by specific antibodies. SM concentration dependent increase in TUNEL positive cells and PARP cleavage showed that SM is an inducer of apoptosis. It has been previously suggested that AChE is activated during apoptotic processes and may be involved in apoptosis regulation. Therefore, we examined AChE activity in A549 cells upon induction of apoptosis by SM (100-500 microM). Increased AChE activity was found in SM treated A549 cell cultures examined as determined by the Ellman's assay and by western blot. AChE activity showed a strong correlation with TUNEL positive cells. However, the broad caspase inhibitor zVAD and the PARP-inhibitor 3-aminobenzamide had no protective effect on A459 cells measured with AChE activity and frequency of TUNEL positive cells. In summary, our studies demonstrate that AChE activity may be a potential marker of apoptosis in A549 cells after SM injury. To what extent AChE is involved in apoptosis regulation during SM poisoning has to be further investigated.
Assuntos
Apoptose/efeitos dos fármacos , Gás de Mostarda/farmacologia , Acetilcolinesterase/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Benzamidas/farmacologia , Caspase 3/metabolismo , Inibidores de Caspase , Extratos Celulares/análise , Linhagem Celular Tumoral , Substâncias para a Guerra Química/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Humanos , Hidrólise/efeitos dos fármacos , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
Lewisite is a highly toxic arsenic compound which can cause skin damage. In the present study effects of Lewisite on cell membrane integrity and energy metabolism as well as antidotal effects of DL-2,3-dimercaptopropanesulfonate (DMPS), and meso-2,3-dimercaptosuccinic acid (m-DMSA) were investigated in a keratinocyte derived cell line (SCL II) and primary human keratinocytes (HK). Cells were incubated in Lewisite (60 microM) containing medium for 5 min. During the following 6 h lactate dehydrogenase (LDH) activity in the supernatant, intracellular ATP content, tetrazolium reduction, glucose consumption and lactate formation were measured. Glucose consumption and lactate production were decreased in both cell lines after Lewisite exposure. In SCL II cells an increase of LDH activity in the supernatant, a decrease of ATP content, and an impaired ability to reduce tetrazolium was found 3 h after Lewisite exposure. In HK cultures tetrazolium reduction was significantly decreased already after 2 h, whereas LDH increase in the supernatant and ATP content decrease occurred only at 6 h after Lewisite exposure. When DMPS or m-DMSA was added directly after Lewisite exposure to SCL II cells, glucose consumption and lactate formation were restored and LDH leakage was prevented. SCL II cells might be more prone to membrane damage whereas in keratinocytes mitochondrial impairment seems to be the predominant effect of Lewisite.
Assuntos
Arsenicais/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Trifosfato de Adenosina/análise , Arsenicais/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Quelantes/farmacologia , Formazans/análise , Glucose/metabolismo , Humanos , Queratinócitos/metabolismo , L-Lactato Desidrogenase/análise , Ácido Láctico/análise , Succímero/farmacologia , Sais de Tetrazólio/análise , Fatores de Tempo , Células Tumorais Cultivadas , Unitiol/farmacologiaRESUMO
Oleanolic acid (OA) is a triterpenoid compound that has been shown to protect against some hepatotoxicants and is used in China to treat hepatitis. This study was conducted to examine the protective effects of OA against cadmium (Cd)-induced liver injury in mice and the mechanism of protection. OA (100 mg/kg x 3 days) pretreatment dramatically decreased Cd (3.7 mg/kg i.v.)-induced liver injury as indicated by decreased serum activities of alanine aminotransferase and sorbitol dehydrogenase, as well as by histopathological observation. To examine the mechanism of protection, the distribution of Cd to major organs and the hepatic subcellular distribution of Cd were determined 2 hr after 109Cd injection (3.5 mg/kg of Cd and 10 microCi/mg of Cd i.v.). OA did not reduce the amount of Cd in liver, but significantly altered the hepatic subcellular distribution of Cd, with more Cd in hepatic cytosol bound to metallothionein (MT), and with less Cd in other organelles and proteins. OA produced an approximately 30-fold increase in hepatic MT, but had no appreciable effects on MT levels of five other organs. Furthermore, OA increased both hepatic MT-I and MT-II levels, as determined by high-performance liquid chromatography/atomic absorption spectrophotometry. Northern blot analysis revealed that OA increases MT mRNA expression. In summary, OA pretreatment protects against Cd-induced hepatotoxicity by inducing MT. MT bound Cd in the cytosol, and thus decreased the amount of Cd in other critical organelles and proteins. OA is a hepatic MT inducer for both MT-I and MT-II isoforms, and this effect is due, at least in part, to an increased MT mRNA accumulation.
Assuntos
Cádmio/toxicidade , Hepatopatias/prevenção & controle , Metalotioneína/biossíntese , Ácido Oleanólico/uso terapêutico , Alanina Transaminase/sangue , Animais , Northern Blotting , Cádmio/farmacocinética , Doença Hepática Induzida por Substâncias e Drogas , Citosol/metabolismo , Relação Dose-Resposta a Droga , Isomerismo , L-Iditol 2-Desidrogenase/sangue , Fígado/anatomia & histologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Hepatopatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Tamanho do Órgão/efeitos dos fármacos , Sondas RNA , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismo , Distribuição TecidualRESUMO
Differences in the metabolite pattern were observed in previous experiments in guinea pig livers after a single injection or prolonged (5 days) treatment with AS2O3 (Reichl et al. 1988). To elucidate the underlying mechanism the effect of As2O3 on liver metabolism was therefore investigated. Male guinea pigs received either a single dose (s.d.) of As2O3 10 mg x kg-1 s.c. or repeated doses (r.d.) of 2.5 mg x kg-1b.i.d. on 5 consecutive days. One hour after the s.d. or 1 h and 16 h after the last injection in the r.d. groups the animals were sacrificed in anaesthesia. The livers were removed by freeze clamping for the determination of various metabolites. In the s.d. group a significant decrease in hydroxybutyrate, acetylCoA, adenosinemonophosphate and in the ratio of hydroxybutyrate/acetoacetate and an increase in pyruvate, citrate, malate, and adenosinetriphosphate were observed. A significant decrease in glycogen, pyruvate, alpha-ketoglutarate, acetylCoA, and acetoacetate and a significant increase in malate and in the ratios of lactate/pyruvate and hydroxybutyrate/acetoacetate were observed in the r.d.1-h group. In the r.d.16-h group a significant decrease in glycogen, pyruvate, lactate, and adenosinemonophosphate was found, but the values tended towards control values. The data are consistent with mechanisms of As2O3 toxicity in other species as PDH inhibition with consecutive citric acid cycle and gluconeogenesis inhibition and excessive carbohydrate depletion.