Controlled delivery of nitric oxide for cytotoxicity studies

Abstract

Endogenous synthesis of nitric oxide (NO) is essential for many physiological functions, including the immune defense. However, the sustained, high production of NO by immune cells (macrophages) that accompanies chronic inflammation may be cytotoxic, mutagenic and carcinogenic. To better understand the roles of NO and its various reactive derivatives (e.g., N203 and ONOO-) in cancer, it is important to know the NO concentration and the total NO dose that tissue cells are exposed to during inflammation. To obtain this quantitative information, methods are needed for exposing cells to physiological levels of NO and its derivatives over relatively long periods of time. This research has therefore focused on developing in vitro delivery systems that mimic the in vivo release of NO. To permit continuous NO exposures over lengthy periods, an apparatus was fabricated which utilizes gas-permeable polydimethylsiloxane tubing to supply both NO and 02 to a stirred, cylindrical vessel. Mass transfer in this system was characterized by measuring the delivery rates of NO or 02 alone, and of NO to air-saturated solutions. It was found that the total flux of nitrogen species into the liquid was 40-90% greater in the presence of 02, depending on the NO partial pressure in the gas. Also, the simultaneously measured mass transfer coefficients for NO and 02 differed greatly from the corresponding unreactive values. An analysis of the data using diffusion-reaction models showed that NO oxidation in the aqueous boundary layer contributed very little to the nitrogen flux increase or to variations in the mass transfer coefficients. However, the unusually strong dependence of the delivery rates on chemical reactions could be explained by postulating that partial oxidation of NO to NO2 occurred within the membrane, with a rate constant of ...

(cont.) Using the measured mass transfer coefficients, the aqueous NO concentrations could be accurately predicted for the case of simultaneous NO and 02 delivery. The NO delivery system was used first to study the kinetics of plasmid DNA base deamination under pathologically relevant conditions. Three nucleobase products, 2'- deoxyxanthosine (dX), 2'-deoxyinosine (dI), and 2'-deoxyuridine (dU), were formed from 2'- deoxyguanosine (dG), 2'-deoxyadenosine (dA), and 2'-deoxycytosine (dC), respectively, at constant rates under steady state concentrations of 1.2 tM NO and 190 !pM 02. Morpholine nitrosation rates were measured under similar exposure conditions. Using a kinetic model, along with the known morpholine deamination kinetics, the three rate constants for base deamination were found to be nearly identical ... The second application of the new NO delivery system was to study NO-mediated cyto- and genotoxicity in two human lymphoblastoid cell lines, TK6 (wild-type p53) and NH32 (p53-null but isogenic to TK6). The TK6 and NH32 cells were each exposed to several steady-state NO concentrations for varying lengths of time, so that the total dose (area under the concentration-time curve) covered a wide range. Endpoint assays, including lethality, apoptosis, mitochondrial damage, and mutation rate in the thymidine kinase (TK1) gene locus, were performed at different post-treatment times ...