Objective: Many xenobiotic compounds exert their biological actions through the release of free radicals and related oxidants. The release of these species may bring about unwanted biological effects. Here, we demonstrate the in vitro release of the nitric oxide radical (·NO) from tobacco xenobiotics (TX) [smokeless tobacco, nicotine, and the nitrosamine metabolites, nitrosonornicotine (NNN) and 4-(methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)] using electron paramagnetic resonance (EPR) and the iron (II) N-methyl-D-glucamine dithiocarbamate, Fe2+(MGD)2 spin trap, and chemiluminescent detection. Methods: Experiments were conducted in phosphate-buffered saline (PBS) or fresh, unstimulated, whole human saliva (WHS) obtained from non-users of tobacco products without clinical evidence of periodontal disease. The EPR-silent spin trap strongly, and with a high degree of specificity, coordinates ·NO, forming a stable spin adduct, ·NO-Fe2+(MGD)2, which yields a characteristic three-line EPR spectrum. To confirm and further characterize the release of ·NO from TX, we used a chemiluminescent detection system. This protocol detects nitrite, the end-product of ·NO oxidation, which is then re-reduced to ·NO. Results: Our results demonstrate that smokeless tobacco, NNN and NNK spontaneously release ·NO in PBS, however this release is xenobiotic-dependent. In WHS, we observed markedly enhanced EPR signals from trapped ·NO. The strength of these EPR signals may reflect the contribution of ·NO generated from the reduction of salivary nitrite by cytochrome cd1 nitrite reductase found in some salivary bacteria. Using chemiluminescent detection, we confirmed nanomole to micromole release of ·NO per milligram weight TX, and show WHS to be an important mediator of this release. Conclusions: We suggest that TX represent as yet unrecognized sources of extracellular ·NO in the oral cavity, and speculate that oxidative events and ·NO may play a role in tobacco toxicity. This work was supported by grants from CIHR and AHFMR to EWNL, and NCI to GRB.
Division: AADR/CADR Annual Meeting
Meeting:2003 AADR/CADR Annual Meeting (San Antonio, Texas) Location: San Antonio, Texas
Year: 2003 Final Presentation ID:77 Abstract Category|Abstract Category(s):Pharmacology, Therapeutics, & Toxicology
Authors
Lam, E.w.n.
( University of Alberta, Edmonton, AB, Canada
)
Kelley, E.e.
( University of Iowa, Iowa City, IA, USA
)
Martin, S.m.
( University of Iowa, Iowa City, IA, USA
)
Buettner, G.r.
( University of Iowa, Iowa City, IA, USA
)