Tobacco Xenobiotics Release Nitric Oxide
© Lam et al; licensee BioMed Central Ltd. 2003
Published: 15 September 2003
Many xenobiotic compounds exert their actions through the release of free radicals and related oxidants [1, 2], bringing about unwanted biological effects . Indeed, oxidative events may play a significant role in tobacco toxicity from cigarette smoke. Here, we demonstrate the direct in vitro release of the free radical nitric oxide (•NO) from extracts and components of smokeless tobacco, including nicotine, nitrosonornicotine (NNN) and 4-(methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in phosphate buffered saline and human saliva using electron spin resonance and chemiluminescence detection. Our findings suggest that tobacco xenobiotics represent as yet unrecognized sources of •NO in the body.
Whether generated intracellularly, or exogenously delivered, the diatomic free radical nitric oxide (•NO) is rapidly disseminated throughout the body, affecting key biological processes. Supra-physiologic •NO concentrations favor the formation of a potent biological oxidant; peroxynitrite (ONOO-), the reaction product of •NO and the oxygen-centered free radical, superoxide, O2•- . Numerous cytotoxic lesions have been attributed to ONOO-, including lipid peroxidation, protein thiol oxidation, inhibition of Fe-S enzyme systems, and oxidative DNA lesions such as strand breaks and base modifications, to name some [4–6].
Of the over 30 carcinogens found in tobacco, the nitrosamine compounds, nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are thought to be the major contributors to the carcinogenic activity of nicotine and tobacco [7, 8]. NNN and NNK are formed during the curing, aging, and fermentation of tobacco, as well as during nicotine metabolism. Already, •NO generation has been demonstrated in cigarette smoke . The structural similarities between NNN and NNK, and other known therapeutic and experimental •NO-releasing compounds suggest that these nitrosamines may be novel • NO-releasing agents in tobacco [10, 11]. Indeed, NNK has been shown to generate DNA strand breaks, as well as induce the formation of DNA adducts, including methylated DNA [12, 13].
Here, we demonstrate, using both direct and indirect methods, the in vitro release of •NO from extracts and components of smokeless tobacco, including nicotine, and the nitrosamine metabolites of tobacco, nitrosonornicotine (NNN) and 4-(methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK).
Materials and methods
Tobacco xenobiotic preparations
Experiments were conducted in phosphate-buffered saline (PBS) at pH 7.4 or unstimulated human saliva obtained from healthy, non-users of tobacco, without clinical evidence of periodontal disease. We estimated the mass of a "pinch" of smokeless tobacco to be approximately 2.2 g, and suspended this (Copenhagen® brand, National Tobacco Co., Ltd., Pointe Claire, QB) in 4.4 mL of PBS or saliva. The amount of nicotine in this preparation has been determined previously to be 12 ± 0.7 mg per g tobacco . Therefore, 26.4 mg of nicotine (Sigma Chemical Co., St Louis, MO) was used for the assays. Ten mg of NNN and NNK (Midwest Research Institute, St. Louis, MO) was used for •NO determinations. Each of these prepared solutions was purged with argon gas, and incubated at 37°C for 20 min in an air-tight container before being assayed for •NO.
EPR spin trapping
Each xenobiotic preparation was incubated with a 10 mM solution of the iron (II)/N-methyl-D-glucamine dithiocarbamate, Fe2+(MGD)2, spin trap at 37°C for 20 min so that the final concentration of the spin trap was 1 mM . Each 500 μL solution was then quickly transferred to an argon-purged flat cell, and EPR spectra were collected with a Bruker (Billerica, MA, USA) X-band EMX spectrometer operating at 9.75 GHz, receiver gain of 2 × 104, modulation amplitude of 1 G, sweep time of 83 s, and a field center of 3418 G for •NO-Fe2+(MGD)2. Each spectrum represents the signal-averaged sum of 15 acquisitions.
Fifty μL of each xenobiotic solution was injected into a Sievers 280 Nitric Oxide Analyzer (Boulder, CO, USA) containing a reducing agent, KI, potassium iodide (5.9 mM) in glacial acetic acid . Standardization was accomplished by injecting various concentrations of a standard solution of NaNO2 into the same reducing environment. Samples were run in triplicate.
Results and Discussion
Chemiluminescent detection of •NO
Total •NO observed
in PBS (pH 7.4)
in human saliva
5 ± 1 μMa
Whole human saliva (WHS)
38 ± 17 μMa
Smokeless tobacco (ST)
1100 ± 50 μMa
2.53 ± 0.10 μmol/g ST
1380 ± 80 μMa
2.76 ± 0.16 μmol/g ST
< 0.02 μMb
150 ± 12 μMa
2.81 ± 0.23 nmol/mg nicotine
< 0.02 μMb
121 ± 6 μMa
.90 ± 0.30 nmol/mg NNN
4-(Methyl-N-nitrosamino)-1-(3- pyridyl)-1-butanone (NNK)
< 0.02 μMb
113 ± 5 μMa
5.45 ± 0.25 nmol/mg NNK
Although others have reported free radical, and in particular, O2•- production in cells exposed to smokeless tobacco and nicotine [17–19], none identified free radical release directly from smokeless tobacco xenobiotics. Tobacco xenobiotics represent as yet unrecognized sources of •NO in the body. Indeed tobacco-derived •NO may have widespread biological implications for tobacco users. Our results also lead us to speculate that •NO and nitrosative events may play a role in tobacco toxicity in the oral cavity and aerodigestive tract.
This work was supported by grants from the Canadian Institutes of Health Research and the Alberta Heritage Foundation for Medical Research to E.W.N.L., and the National Cancer Institute to G.R.B.
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