METALLOTHIONEIN PROTECTS AGAINST THE CYTOTOXIC AND DNA-DAMAGING EFFECTS OF NITRIC-OXIDE

In inflammatory states, nitric oxide (. NO) may be synthesized from precursor L-arginine via inducible . NO synthase (iNOS) in large amounts for prolonged periods of time. When . NO acts as an effector molecule under these conditions, it may be toxic to cells by inhibition of iron-containing enzymes or initiation of DNA single-strand breaks. In contrast to molecular targets of . NO, considerably less is known regarding mechanisms by which cells become resistant to . NO. Metallothionein (MT), the major protein thiol induced in cells exposed to cytokines and bacterial products, is capable of forming iron-dinitrosyl thiolates in vitro. Therefore, we tested the hypothesis that overexpression of MT reduces the sensitivity of NIH 3T3 cells to the . NO donor, S-nitrosoacetylpenicillamine (SNAP), and to . NO released from cells (NM 3T3-DFG-iNOS) after infection with a retroviral vector expressing human iNOS gene. There was a 4-fold increase in MT in cells transfected with the mouse MT-I gene (NIH 3T3/MT) compared to cells transfected with the promoter-free inverted gene (NIH 3T3/TM). NIH 3T3/MT cells were more resistant than NIH 3T3/TM cells to the cytotoxic effects of SNAP (0.1-1.0 mM) or . NO released from NIH 3T3-DFG-iNOS cells. A brief (1 h) exposure to 10 mM SNAP caused DNA single-strand breaks that were 9-fold greater in NIH 3T3/TM compared to NIH 3T3/MT cells. Electron paramagnetic resonance spectroscopy of NIH 3T3 cells revealed a greater peak at g = 2.04 (e,g., iron-dinitrosyl complex) in NIH 3T3/MT than NIH 3T3/TM cells, These data are consistent with a role for cytoplasmic MT in interacting with . NO and reducing . NO-induced cyto- and nuclear toxicity.