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

In inflammatory states, nitric oxide (. NO) may be synthesized from pr ecursor 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 iro n-containing enzymes or initiation of DNA single-strand breaks. In con trast to molecular targets of . NO, considerably less is known regardi ng mechanisms by which cells become resistant to . NO. Metallothionein (MT), the major protein thiol induced in cells exposed to cytokines a nd 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-nitro soacetylpenicillamine (SNAP), and to . NO released from cells (NM 3T3- DFG-iNOS) after infection with a retroviral vector expressing human iN OS gene. There was a 4-fold increase in MT in cells transfected with t he 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) ex posure 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 paramagne tic 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/T M cells, These data are consistent with a role for cytoplasmic MT in i nteracting with . NO and reducing . NO-induced cyto- and nuclear toxic ity.