RESUMEN
Following intramuscular injections of 0.1 mL, 3 mg kg-1 BW-1(1/10 LD50) T-2 toxin (T-2), the tissue concentration of T-2 in shrimp was quantitatively detected using LC-MS/MS. The biological half-time (t1/2) of T-2 in blood was 40.47 ± 0.24 min. The highest number of intramuscular T-2 shrimp could tolerate when given at blood t1/2 intervals was 4. The shrimps which were injected 5 T-2 died. The T-2 toxin highest accumulation was 0.471 ± 0.012 ng g-1 BW-1. The effect of toxic shrimp muscle subjected to different processing conditions (high pressure, trifluoroacetic acid, acid and alkali digestions, artificial digestive juice [to simulate exposure to gastric and intestinal juices]) on mouse macrophage cells (RAW267.4) were evaluated by the MTT assay. The inhibition ratio of 2% muscle extract on RAW267.4 was 85.70 ± 2.63%. The immunocytotoxicity of muscle extracts to RAW264.7 was highest in muscle extracts subjected to physical and chemical digestion (high pressure > NaOH > trifluoroacetic acid > 0.02 M HCl > 0.2 M HCl > controls), and also artificial digestion (artificial intestinal juice > artificial gastric juice > N type intestinal juice > N type gastric liquid > controls). Results showed that high-pressure and artificial intestinal juice were most effective in the release of modified T-2 to free T-2 thus enhancing toxicity. These results can be interpreted as measurement of T-2 in food being of little value because of enhanced toxicity of T-2-contaminated food as they pass through the gastrointestinal tract.
Asunto(s)
Macrófagos/efectos de los fármacos , Músculos/metabolismo , Penaeidae/metabolismo , Intoxicación por Mariscos , Mariscos/efectos adversos , Toxina T-2/toxicidad , Extractos de Tejidos/toxicidad , Animales , Supervivencia Celular/efectos de los fármacos , Digestión , Relación Dosis-Respuesta a Droga , Jugo Gástrico/metabolismo , Semivida , Inyecciones Intramusculares , Secreciones Intestinales/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Células RAW 264.7 , Medición de Riesgo , Toxina T-2/administración & dosificación , Toxina T-2/farmacocinética , Distribución TisularRESUMEN
Cadmium (Cd) has toxic and suppressive effects on the immune system, but the underlying mechanisms remain poorly understood. Here, we show that autophagy plays a critical role in regulation of Cd-induced immunosuppression in RAW264.7 cells. Cd decreased cell viability in a dose-dependent manner; cleaved caspase-8, caspase-3, and poly (ADP-ribose) polymerase (PARP)-1; increased DNA laddering; induced CCAAT-enhancer-binding protein homologous protein (CHOP); and reduced tumor necrosis factor (TNF)-α expression; indicating that caspase-dependent and endoplasmic reticulum (ER)-mediated apoptosis are involved in Cd-induced immunotoxicity. Furthermore, Cd induced autophagy, as demonstrated by microtubule-associated protein 1 light chain 3B (LC3B) plasmid DNA transfection and its conversion from LC3-I to the LC3-II form by autophagy inhibitors, via AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling. Pharmacological and genetic inhibition of autophagy suppressed Cd-induced apoptosis, as evidenced by inhibition of caspase-8, caspase-3, and PARP-1 cleavage, indicating that autophagy promotes apoptosis. The pan-caspase inhibitor zVAD inhibited Cd-induced apoptosis, but increased autophagy and decreased cell viability, indicating that autophagy can compensate for reduced apoptotic cell death. Calpain inhibitors blocked Cd-induced apoptosis and autophagy, indicating that calpain plays a critical role in Cd cytotoxicity. Treatment with Ca2+ chelators completely recovered Cd-induced cell viability and inhibited Cd-induced apoptosis and autophagy. Treatment with N-acetyl-l-cysteine (NAC) suppressed Cd-induced antioxidant enzyme levels, apoptosis, and autophagy. Collectively, Cd-induced oxidative stress triggers ER stress, leading to Ca2+-dependent calpain activation and subsequent activation of autophagy and apoptosis, resulting in immune suppression.