Tobacco-specific nitrosamines (TSNAs) are carcinogenic and are present in cured tobacco leaves. This study was designed to elucidate the mechanisms of TSNAs formation under warm temperature storage conditions. Results showed that nitrogen oxides (NOx) were produced from nitrate and nitrite in a short period of time under 45°C and then reacted with alkaloids to form TSNAs. Nitrite was more effective than nitrate in promoting TSNAs formation during 45°C storage which may be due to the fact that nitrite can produce a large amount of NOx in comparison with nitrate. Presence of activated carbon effectively inhibited the TSNAs formation because of the adsorption of NOx on the activated carbon. The results indicated that TSNAs are derived from a gas/solid phase nitrosation reaction between NOx and alkaloids. Nitrate and nitrite are major contributors to the formation of TSNAs during warm temperature storage of tobacco.
Tobacco-specific nitrosamines (TSNAs) are a group of important and toxic components of tobacco and tobacco smoke [
Proposed formation pathways of the major TSNAs found in cured tobacco leaves. Nitrosating agents can directly interact with nornicotine, anatabine, and anabasine to form NNN, NAT, and NAB, respectively. Nicotine is less susceptible to nitrosation; thus NNK is produced from pseudo-oxynicotine, an oxidized derivative of nicotine.
Many studies reported about the factors that influence TSNAs formation during air-curing [
The activated nitrosating agents participate in nitrosation of alkaloid to form TSNAs during different processes of air-cured tobacco production. Nitrate (
However, to our knowledge there are few reports available on the mechanisms of TSNAs formation during storage stage. TSNAs levels may increase several fold in comparison to the levels in freshly air-cured leaves [
Tobacco samples were grown in 2015 and cured locally. Leaves from the middle stalk positions were collected. Flue-cured tobacco (
The treatments included four levels of NaNO3 (10, 20, 30, and 40 mg/mL) and NaNO2 (5, 10, 15, and 20 mg/mL), which were from 10 mL solution each, equal to 73, 146, 219, and 292 mg of
A vacuum desiccator with a porcelain plate was used to form a closed system in which tobacco cuts and added nitrate and nitrite could be separated during storage. Five mL of each aqueous solution of NH4NO3, KNO3, NaNO3, and NaNO2 at 1 mol/L (equal to 310 mg of
Eight treatments in this experiment were divided to two groups: Group I: (1) tobacco; (2) tobacco and NaNO3; (3) tobacco and NaNO3 + 2.0 g activated carbon (AC); (4) tobacco and NaNO3 + 10.0 g AC and Group II: (1) tobacco; (2) tobacco and NaNO2; (3) tobacco and NaNO2 + 2.0 g AC; (4) tobacco and NaNO2 + 10.0 g AC. The amount of added NaNO3 or NaNO2 was 0.8 g and 0.4 g for treatments 2 to 4 (equal to 583 mg of
The AC used in the above experiments were taken out and then were placed in the new airtight desiccator, respectively. Two grams of AC was put into another container as control. All airtight desiccators were placed into a chamber at a temperature of 60°C and a relative humidity of 70% for 15 min and 90 min.
Twelve treatments in this experiment were divided into two groups: Group I: (1) tobacco (4°C); (2) tobacco (warm temperature control 45°C); (3) tobacco and NaNO3 separately; (4) tobacco and NaNO3 + 1.0 g AC separately; (5) tobacco and NaNO3 + 5.0 g AC separately; and (6) tobacco and NaNO3 + 10.0 g AC separately and Group II: (1) tobacco (4°C); (2) tobacco (warm temperature control 45°C); (3) tobacco and NaNO2 separately; (4) tobacco and NaNO2 + 1.0 g AC separately; (5) tobacco and NaNO2 + 5.0 g AC separately; and (6) tobacco and NaNO2 + 10.0 g AC separately. The amount of NaNO3 or NaNO2 added to each sample of treatments 3 to 6 was 0.3 g (equal to 219 mg of
In each experiment, tobacco samples were lyophilized, ground to powder, sieved through a 0.25 mm screen, and then measured for the content of NNN, NNK, NAT, and NAB. TSNAs contents were determined at the Beijing Cigarette Factory according to the method of SPE-LC-MS/MS [
NO3-N and NO2-N were quantified according to the method of Crutchfield and Grove [
The first step was diluting the standard gas with a dynamic gas calibrator (Model 146 i, Thermo Scientific, USA EPA) to give a concentration within the operational range of the instrument. The high purity nitric oxide (NO) and nitrogen dioxide (NO2) standards in N2 (component content: 69.8 ppm, gas sample number: L120911099, National Institute of Metrology/National Standard Material Research Center, Beijing, China) were configured into nitrogen oxides gas of a low concentration (1 ppm) by 146i calibrator. It was necessary to modify the original procedure by configuring the standard gas again if the levels of NOx exceeded the limit of detection. The NO and NOx in the air were defaulted to the zero point by the instrument.
After experiment, the airtight vacuum desiccators were taken out and then connected with a vacuum pump (DOA-P504-BN, GAST Manufacturing, A Unit of Idex Corporation, MI, USA) to extract the gas into a gas collecting bag. After 25 s, the gas bag was pulled out and connected with the NO-NO2-NO
Analysis of variance (ANOVA) and least significant difference (LSD) of TSNAs and NOx values were performed at the 0.05 level of significance. Data were statistically analyzed with SPSS 20.0. Figures were drawn with Origin 8.5. All treatments were randomly designed in triplicate.
The mean contents of nicotine, nornicotine, anabasine, and anatabine of tobacco used in this experiment were 19.8, 1.2, 0.3, and 1.5 mg/g, respectively, and the NO3-N and NO2-N content were correspondingly 119
Effects of added nitrate on TSNAs formation in flue-cured tobacco stored at 45°C for 15 d.
No significant change of NNN was observed in the tobacco samples when less than 0.2 g of NaNO3 (146 mg of
Table
Effects of added nitrite on TSNAs formation in flue-cured tobacco stored at 45°C for 15 d.
Storage conditions | Addition of |
NNN ( |
NAT ( |
NAB ( |
NNK ( |
Total TSNAs ( |
---|---|---|---|---|---|---|
Before storage | 0 |
|
|
|
|
|
45°C for 15 d | 0 |
|
|
|
|
|
33 | |
|
|
|
|
|
67 |
|
|
|
|
|
|
100 | |
|
|
|
|
|
133 | |
|
|
|
|
The higher TSNAs levels in burley tobacco are partly due to the relatively higher levels of TSNAs precursors, such as alkaloids and oxide of nitrogen, that are present in the leaf tissue [
TSNAs contents increased as the storage temperature increased [
After tobacco leaf treated with 1 mol/L NaNO2, total TSNAs content increased almost by 54 times compared with that in the control sample (Figure
Effects of indirect addition of three nitrate compounds and sodium nitrite on TSNAs formation in flue-cured tobacco during warm temperature storage.
Flue-cured tobacco leaves could generate trace concentrations of NOx under 45°C after 24 h treatment (Figures
Effect of indirect addition of nitrate and activated carbon (AC) on nitrogen oxides formation in a closed system with flue-cured tobacco.
Effect of indirect addition of nitrite and activated carbon (AC) on nitrogen oxides formation in a closed system with flue-cured tobacco.
It is interesting that when 2 g of AC was added to the system (the weight ratio of tobacco,
Table
The desorption rate of NOx at 60°C for different time.
Treatment | NO (ppm) | NOx (ppm) | |||
---|---|---|---|---|---|
15 min | 90 min | 15 min | 90 min | ||
AC (Control) | |
|
|
|
|
AC used in Figure |
Treatment 3 (2 g AC) | |
|
|
|
Treatment 4 (10 g AC) | |
|
|
|
|
AC used in Figure |
Treatment 3 (2 g AC) | |
|
|
|
Treatment 4 (10 g AC) | |
|
|
|
During flue-curing process, direct-fired systems allow combustion products, specifically NOx, to mix with the air and expose the green tobacco leaves to these gases [
Having a very porous structure and special surface properties, AC has been used to trap TSNAs in tobacco solution [
As shown in Figures
Effect of activated carbon (AC) on TSNAs formation in flue-cured tobacco in response to nitrate addition.
Effect of activated carbon (AC) on TSNAs formation in flue-cured tobacco in response to nitrite addition.
However, after adding 1 g of AC to the separating system of tobacco and
Decreasing NOx level by AC adsorption significantly reduced TSNAs formation of tobacco which indicated that the removal of NOx from storage environment could be an effective way to inhibit TSNAs formation in storing tobacco leaf. Therefore, controlling the storage environment and scavenging gaseous nitrosation agents would be crucial to reduce or inhibit TSNAs formation during leaf storage.
The results proved that TSNAs are derived from a gas/solid phase nitrosation reaction between NOx and alkaloids during storage. Nitrogen oxides produced from nitrate and nitrite are responsible for the formation of TSNAs during storage under warm temperature. Presence of activated carbon in the tobacco storage containers effectively inhibited the TSNAs formation due to the adsorption of NOx on the activated carbon.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
Jun Wang and Huijuan Yang contributed equally to this work and should be considered co-first authors.