Safrole induced cytotoxicity, DNA damage, and apoptosis in macrophages via reactive oxygen species generation and Akt phosphorylation.

Safrole is a natural compound categorized as a group 2B carcinogen extracted from betel quid chewing, which is a common practice of psychoactive habits integrated into social and cultural ceremonies among serveral million people, especially in Southern or Southeastern Asia. Safrole is one of the major risk compunds for development of oral squamous cell carcinoma and hepatocellular carcinoma via DNA adduction. In innate immunity, macrophages are the predominant cells for non-specific first line defense against pathogens in oral tissue. Up to now, there is no evidence to implicate the potential toxicological effect of safrole on macrophages. In this study, we found safrole induced the generation of reactive oxygen species (ROS) and myeloperoxidase (MPO) in RAW264.7 macrophages in a concentration-dependent manner. Furthermore, cytotoxicity, DNA damage, and apoptosis were caused by safrole in a concentration-dependent manner. While the activation of antioxidative enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) was reduced, the phosphorylation of Akt was induced by safrole in a concentration-dependent manner. These results indicated that the induction of cytotoxicity, DNA damage, and apoptosis in macrophages by safrole was through generation of ROS and inhibition of antioxidative enzymes possibly via Akt phosphorylation.

Cadmium nitrate-induced neuronal apoptosis is protected by N-acetyl-l-cysteine via reducing reactive oxygen species generation and mitochondria dysfunction.

Cigarette smoking is a well-established risk factor for various diseases, such as cardiovascular diseases, neurodegeneration, and cancer. Cadmium nitrate (Cd(NO3)2) is one of the major products from the cigarette smoke. Up to now, no supporting evidence on Cd(NO3)2-induced apoptosis and its related working mechanism in neurons has been found. In present study, the mode of cell death, caspase activities, reactive oxygen species (ROS) generation, and mitochondrial dysfunction in N2a cells, which are neuron-like cells, were assessed by Annexin V-FITC and PI assays, caspase fluorometric assay, DCFH-DA fluorescence assay, and JC-1 fluorescence assay respectively. The results showed that not only Cd(NO3)2 induced apoptosis and necrosis but also the activities of caspase-3 and -9 expressed in a concentration-dependent manner. In addition, Cd(NO3)2 also induced both mitochondrial dysfunction and ROS generation in a concentration-dependent manner. All these indicated that in N2a cells parallel trends could be observed in apoptosis, caspase-3 and -9 activities, mitochondrial dysfunction, and ROS generation when induced by Cd(NO3)2. Furthermore, Cd(NO3)2-induced apoptosis, caspases activities, mitochondrial dysfunction, and ROS generation were reduced by N-acetyl-l-cysteine (NAC). These results indicated that Cd(NO3)2-induced neuronal apoptosis was reduced by NAC via intrinsic apoptotic caspase cascade activities and their up-stream factors, including mitochondrial dysfunction and ROS generation.