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Tobacco-specific nitrosamines in smokeless tobacco products marketed in India
Irina Stepanov
1
, Stephen S. Hecht
1
*, Sreevidya Ramakrishnan
2
and Prakash C. Gupta
3
1
The Cancer Center, University of Minnesota, Minneapolis, MN, USA
2
Tata Memorial Centre, Mumbai, India
3
Tata Institute of Fundamental Research, Mumbai, India
Smokeless tobacco products are a known cause of oral cancer in
India. Carcinogenic tobacco-specific nitrosamines in these prod-
ucts are believed to be at least partially responsible for cancer
induction, but there have been no recent analyses of their
amounts. We quantified levels of 4 tobacco-specific nitrosamines,
N0-nitrosonornicotine (NNN), N0-nitrosoanatabine (NAT), N0-
nitrosoanabasine (NAB) and 4-(methylnitrosamino)-1-(3-pyridyl)-
1-butanone (NNK), in 32 products marketed currently in India.
Levels of nitrate, nitrite and nicotine were also determined. The
highest levels of tobacco-specific nitrosamines were found in cer-
tain brands of khaini, zarda and other smokeless tobacco prod-
ucts. Concentrations of NNN and NNK in these products ranged
from 1.74–76.9 and 0.08–28.4 lg/g, respectively. Levels of tobacco-
specific nitrosamines in gutka were generally somewhat lower
than in these products, but still considerably higher than nitros-
amine levels in food. Tobacco-specific nitrosamines were rarely
detected in supari, which does not contain tobacco, or in tooth
powders. The results of our study demonstrate that exposure to
substantial amounts of carcinogenic tobacco-specific nitrosamines
through use of smokeless tobacco products remains a major prob-
lem in India.
'2005 Wiley-Liss, Inc.
Key words: tobacco-specific nitrosamines; Indian tobacco; oral
cancer; gutka; khaini; zarda; mishri
Oral cancer is one of the most common cancers in India with
rates among the highest in the world.
1
In many regions of India,
oral cancer incidence rates exceed 6 per 100,000 males and in
some parts they are as high as 10.8 per 100,000.
1
Smokeless
tobacco products (products in which there is no combustion or
pyrolysis at the time of use) account for over one-third of all
tobacco consumed in India. There are approximately 100 million
users of smokeless tobacco products in India and Pakistan. Tradi-
tional forms of smokeless tobacco include betel quid containing
tobacco, tobacco with lime and tobacco tooth powder but there are
also new products with increasing popularity.
2
Chewing of betel
quid containing tobacco is a well-established cause of oral cancer
in India.
2–4
Oral leukoplakia and submucous fibrosis, likely pre-
cursor lesions to oral cancer, are also strongly linked to smokeless
tobacco use. In India and other parts of southern Asia, smokeless
tobacco use is a major public health problem.
Tobacco-specific nitrosamines are the most prevalent strong
carcinogens in smokeless tobacco products and are widely
believed to play a significant role as causes of oral cancer in peo-
ple who use these products.
5–11
These carcinogens are formed
from tobacco alkaloids during the curing and processing of
tobacco. Vast amounts of data convincingly demonstrate their
presence in various forms of smokeless tobacco, but products
available in India have been examined in only scattered studies
and there have been no reports since 1989.
6,12–17
In view of the
variety of new smokeless tobacco products now available in India
and the widespread use of these products, it is important to obtain
current data on levels of tobacco-specific nitrosamines. Such data
are critical in approaches to the control and regulation of smoke-
less tobacco products in India, and ultimately to prevention of
oral cancer. Therefore, we analyzed a variety of products for N0-
nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-
1-butanone (NNK), N0-nitrosoanabasine (NAB) and N0-nitrosoana-
tabine (NAT).
Material and methods
Tobacco samples
Indian smokeless tobacco products were purchased from retail
stores in Gujarat, Karnataka, and Mumbai, India in October–
November 2003. The date and place of purchase and batch number
of each purchase was recorded. The 32 brands collected for analy-
sis represent products commonly used in India. Most of them (22
brands), such as zarda, gutka, khaini and mishri, are chewing
tobacco products that have become especially popular among
teenagers and young adults in many states of India. Other tobacco-
containing products were creamy snuff, a toothpaste, and moist
Swedish snuff that is being marketed in India under the brand
name Click. Three brands of tooth powder were of unknown
tobacco content, but suspected to contain tobacco on the basis of
previous analyses carried out in India. Five popular brands of
chewing mixtures that do not contain tobacco (supari) were also
included. University of Kentucky moist smokeless research
tobacco 1S3 was analyzed for comparison. For 24 hr before analy-
sis, the tobacco was conditioned in a chamber at a relative humid-
ity of 60%.
Apparatus
Tobacco-specific nitrosamines were analyzed by gas chroma-
tography with nitrosamine selective detection (GC-TEA) using a
model 5890 gas chromatograph (Hewlett Packard, Palo Alto, CA)
interfaced with a model 610 Thermal Energy Analyzer (Orion
Research, Beverly, MA). The GC was equipped with a DB-1301
capillary column (30 m 0.32 mm 0.25 mm) [6% (cyanopro-
pylphenyl)methylpolysiloxane; J&W Scientific, Folsom, CA] and
a2m0.53 mm deactivated fused silica precolumn. The flow
rate was 2.6 mL/min He; splitless injection port temperature was
2258C. The following oven temperature program was used: 808C
for 2 min, then 128C /min to 1508C, then 7 min at 1508C, then
128C /min to 2008C, then 10 min at 2008C.
GC-mass spectrometry (MS)-selected-ion monitoring analysis
for nicotine was carried out with a model 6890 GC equipped with
an autosampler and interfaced with a model 5973 mass-selective
detector (Agilent Technologies, Palo Alto, CA). The GC was
equipped with a DB-5MS fused silica capillary column (15 m
0.25 mm 0.25 mm). The splitless injection port temperature was
2508C; the oven temperature was 708C for 0.5 min, then increased
to 1808Cat108C/min, then held for 3 min, then 508C/min
Abbreviations: C5-NNK, 5-(methylnitrosamino)-1-(3-pyridyl)-1-penta-
none; GC-TEA, gas chromatography with nitrosamine selective detection;
5-MeNNN, 5-methyl-N0-nitrosonornicotine; MS, mass spectrometry; NAB,
N0-nitrosoanabasine; NAT, N0-nitrosoanatabine; NNK, 4-(methylnitrosa-
mino)-1-(3-pyridyl)-1-butanone; NNN, N0-nitrosonornicotine.
*Correspondence to: University of Minnesota Cancer Center, Mayo
Mail Code 806, 420 Delaware St. SE, Minneapolis, MN 55455, USA.
Fax:þ1-612-626-5135. E-mail: hecht002@umn.edu
Grant sponsor: U.S. National Cancer Institute; Grant number: CA-
81301; Grant sponsor: American Cancer Society; Grant number: RP-00-
138; Grant sponsor: Office of the World Health Organization Representa-
tive to India, New Delhi; Grant number: SE/02/232826.
Received 2 August 2004; Accepted after revision 12 November 2004
DOI 10.1002/ijc.20966
Published online 8 March 2005 in Wiley InterScience (www.interscience.
wiley.com).
Int. J. Cancer: 116, 16–19 (2005)
'2005 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
to 3008C, and returned to initial conditions. The flow rate was
1 mL/min He.
Nitrate and nitrite content were determined by ion chromatogra-
phy using a Dionex ICS-2000 Ion Chromatograph.
Reagents
Reference NNN, NNK, NAB, 5-methyl-N0-nitrosonornico-
tine (5-MeNNN), and 5-(methylnitrosamino)-1-(3-pyridyl)-1-pen-
tanone (C5-NNK) were synthesized as previously described.
18–20
NAT was purchased from Toronto Research Chemicals Inc. (Tor-
onto, Ontario, Canada). [CD
3
]Nicotine was obtained from Sigma
Chemical Co. (St. Louis, MO).
Tobacco analyses
Tobacco-specific nitrosamines analyses was carried out by a
slight modification of a method described previously by Stepanov
et al.
20
Five-hundred milligram of humidity-conditioned tobacco
and 10 mL of citrate-phosphate buffer (pH ¼4.5) containing
ascorbic acid were added to a 30 mL Nalgene centrifuge tube
(Nalge Nunc International, Rochester, NY). Two-hundred nano-
grams of 5-MeNNN (internal standard for NNN, NAT, and NAB)
and C5-NNK (internal standard for NNK) were added. The sam-
ples were homogenized for 30 min with a Polytron tissue homoge-
nizer (Brinkmann Instruments, Westbury, NY) and sonicated for
1 hr. The buffer extracts were separated from the particles of
tobacco by high-speed centrifugation (15,000g, 10 min). The
extracts were filtered into 50 mL glass screw-top centrifuge tubes
(Kimble, Vineland, NJ), and the pH was adjusted to 7 by adding
100 mL of 10 N NaOH. Each sample was applied to a 20 mL
ChemElut cartridge (Varian, Harbor City, CA), eluted with 3
20 mL CH
2
Cl
2
, and the eluants were combined and concentrated
to dryness with a model SVT200H Speedvac concentrator
(Thermo Savant, Farmingdale, NY). Residues were dissolved in
0.5 mL of CH
2
Cl
2
and further purified by solid-phase extraction
using Sep-Pak Plus silica cartridges (Waters Corp., Milford, MA),
pre-equilibrated with CH
2
Cl
2
. The cartridges were washed with
5mLCH
2
Cl
2
/ethyl acetate: 50/50, and the tobacco-specific nitros-
amines were eluted with 10 mL of ethyl acetate. The ethyl acetate
eluants were concentrated to dryness (Speedvac). The dry residues
were transferred into GC-micro vials with 3 50 mL methanol,
dried, and re-dissolved in 100 mL of acetonitrile. Three microliters
of the prepared sample were injected into GC-TEA.
Nicotine analysis was carried out as described previously.
20
Fifty milligrams of humidity-conditioned tobacco and 20 mL of
methanol containing 50 mg of KOH were added to 30 mL Nalgene
centrifuge tubes. The samples were homogenized (Polytron) and
then sonicated for 3 hr. The methanol extracts were separated from
the tobacco by high-speed centrifugation. Methanol extracts
(200 mL) were transferred into a silanized 4 mL vial and 20 mLof
[CD
3
]nicotine internal standard was added. The samples were
transferred to GC-micro insert vials and analyzed by GC-MS-SIM.
For nitrate and nitrite analysis, 100 mg of humidity-conditioned
tobacco and 10 mL of reagent grade water (Milli-Q, Millipore
Corp.) were added to a 50 mL glass screw-top centrifuge tube
(Kimble) pre-washed with water. Two water negative controls and
three control solutions containing known concentrations of nitrate
and nitrite were included in the sample set. Tobacco was homo-
genized (Polytron), and the tubes were sonicated for 30 min. The
suspensions were centrifuged and the aqueous tobacco extract was
applied to a C-18 SPE cartridge (Waters Corp., Milford, MA) con-
ditioned with 2 mL of methanol. The first 5 mL of eluant was dis-
carded. The next 2 mL of eluant was collected in a prewashed
plastic tube and stored at 208C until analysis. The samples were
diluted 10-fold before analysis by ion chromatography. Conditions
were as follows: an AS14 anion exchange column and guard col-
umn were eluted with carbonate/bicarbonate using a 50 mL sample
loop and a flow rate of 1.0 mL/min. These analyses were carried
out at the University of Minnesota Geochemical Analysis Facility.
Statistical analysis
Pearson correlations were determined using Sigma Plot 2001, v.
7.101 (SPSS, Inc., Chicago, IL).
Results
A typical GC-TEA trace of tobacco-specific nitrosamines in
one of the smokeless tobacco products analyzed here is presented
in Figure 1.
Levels of tobacco-specific nitrosamines, nitrate, nitrite and nic-
otine in the products are summarized in Table I. Each value is the
mean of 2 analyses for tobacco-specific nitrosamines; the results
agreed on average within <10%. Recoveries of internal standards
averaged 42.8%. For nitrate and nitrite, each value represents the
mean of duplicate injections of the same sample, with the average
difference between the 2 values being <3%. For nicotine, a single
sample of each brand was prepared, and each value is the result of
a single injection.
On the basis of the tobacco-specific nitrosamine analyses, the
products can be divided into 3 groups. The first is products with
high levels (Raja and Hans Chhap khaini, Shimla zarda and Gai
Chhap tobacco). In these products, levels of NNN and NNK were
38.9 627.0 (SD) mg/g, range ¼19.2–76.9 mg/g and 8.99 6
13.0 mg/g, range ¼2.34–28.4 mg/g, respectively. The second
group comprises products with medium to low levels of tobacco-
specific nitrosamines. NNN and NNK in these products amounted
to 2.24 62.63 (SD) mg/g, range ¼0.09–8.36 mg/g (n¼20) and
0.71 60.86 mg/g, range 0.04-3.09 mg/g (n¼20), respectively.
In the third group (tooth powders and supari), tobacco-specific
nitrosamines were rarely detected.
FIGURE 1– Typical chromatogram obtained upon GC-TEA analysis
of tobacco-specific nitrosamines in a smokeless tobacco product
marketed in India.
17NITROSAMINES IN INDIAN TOBACCO
The highest levels of NNN and NNK, 76.9 mg/g and 28.4 mg/g,
respectively, were observed in Raja khaini. The second highest
NNN level, 39.4 mg/g, was observed in Hans Chhap khaini.
Among the products in which tobacco-specific nitrosamines were
commonly detected, the lowest levels were observed frequently in
different gutka brands.
Nitrite varied from non-detectable (<0.02 mg/g wet weight
tobacco) to 1,020 mg/g and 1,410 mg/g in Raja khaini and Hans
Chhap khaini, respectively. The average level of nitrate was
720 6870 (SD) mg/g, range ¼7.5–2950 mg/g (n¼32). Levels of
total tobacco-specific nitrosamines did not correlate with nicotine
or nitrate. A correlation was observed between total tobacco-spe-
cific nitrosamines and nitrite (r¼0.78, p<0.0001).
Discussion
We analyzed 32 Indian tobacco products, including smokeless
tobacco products, tobacco-free chewing products, creamy snuff,
tobacco toothpaste and tooth powder. These products were pur-
chased in 2003 in India and are used commonly in different parts
of the country.
Our study shows that the levels of tobacco-specific nitrosamines
in these products vary widely. Different brands of the same type
of product usually contain similar levels of tobacco-specific nitros-
amines, nitrate, nitrite and nicotine. This observation can be
explained by similarities in tobacco processing and is in agree-
ment with the general principle that yields of tobacco-specific
nitrosamines are influenced greatly by the processes involved in
the manufacturing of smokeless tobaccos.
12,21–23
The highest
levels of tobacco-specific nitrosamines were observed in 2 different
brands of the same variety, khaini. Khaini is a mixture of tobacco,
lime and menthol or aromatic spices. The mode of tobacco proc-
essing that likely favors the reduction of nitrate to nitrite and nitro-
sating agents could be responsible for the high tobacco-specific
nitrosamine concentrations in these 2 brands. This seems reason-
able because the levels of nitrite in these 2 brands are the highest
TABLE I – TOBACCO-SPECIFIC NITROSAMINES, NITRATE, NITRITE, AND NICOTINE IN INDIAN SMOKELESS TOBACCO AND RELATED PRODUCTS
1
Product Tobacco-specific nitrosamines (mg/g)
2
NO
3
(mg/g)
3
NO
2
(mg/g)
3
Nicotine (mg/g)
4
NNN NAT NAB NNK
Khaini
Raja 76.9 13.8 8.83 28.4 705 1,020 21.3
Hans Chhap 39.4 4.83 3.78 2.34 1,090 1,410 19.6
Zarda
Goa 1000 8.36 1.98 0.48 3.09 966 2.20 14.6
Moolchand Super 6.47 0.64 0.46 1.64 1,320 ND 15.0
Sanket 999 7.77 1.51 0.36 1.99 1,910 2.08 65.0
Baba 120 4.81 1.40 0.19 1.07 1,700 1.63 44.2
Shimla 19.9 1.53 1.19 2.61 1,360 2.53 13.8
Other Tobacco
Hathi Chhap 2.75 1.53 0.23 0.85 2,760 1.97 39.5
Gai Chhap 19.2 11.9 1.57 2.61 2,950 8.40 47.8
Miraj 1.74 0.35 0.12 0.08 1,420 13.6 15.6
Mishri
Shahin 4.21 2.55 0.15 0.87 1,720 5.18 21.0
Gutka
Star 555 0.47 0.07 0.02 0.13 417 1.61 6.77
Manikchand 0.38 0.05 0.01 0.12 43.9 2.00 3.22
Zee 0.32 0.05 0.01 0.08 62.3 3.42 3.31
Tulsi Mix 0.69 0.07 0.02 0.31 184 2.58 5.67
Wiz 0.31 0.04 0.02 0.13 215 2.82 1.67
Kuber 0.32 0.03 0.01 0.13 47.3 4.50 1.23
Pan Parag 0.44 0.06 0.02 0.12 332 2.84 2.67
Zatpat 1.09 0.08 0.05 0.43 171 1.99 5.48
Vimal 0.09 0.01 ND 0.04 268 1.58 6.82
Josh 0.49 0.08 0.03 0.20 252 1.74 11.4
Supari
Goa ND ND ND ND 7.5 4.71 NA
Moolchand ND ND ND ND 8.5 2.48 NA
Rajanigandha ND ND ND ND 8.8 3.34 NA
Sanket ND ND ND ND 8.5 4.27 NA
Shimla ND ND ND ND 8.0 6.56 NA
Creamy snuff/ toothpaste
IPCO 3.32 0.53 0.11 1.31 580 ND 4.71
Dentobac 2.52 1.49 0.07 2.16 232 ND 7.71
Snuff
Click 0.56 0.38 0.02 0.24 2,260 ND 71.4
Tooth powder
Baidyanath 0.04 ND ND ND 48.6 ND 0.72
New Roshanjyot ND ND ND ND 11.6 1.25 0.25
Dabur 0.04 ND ND ND 27.6 ND 0.58
Reference snuff
Kentucky IS3 3.39 3.15 0.25 0.94 3.86 6.35 36.2
1
All data per gram wet weight. ND, not detected; detection limit 50 pmol/g tobacco; NA, not analyzed.–
2
Mean of duplicate analyses of prod-
uct from one package.–
3
Mean of duplicate injections of a single sample.–
4
Single determination.
18 STEPANOV ET AL.
in our study and, arguably, among the highest reported in smoke-
less tobacco products.
13
It should be mentioned that khaini is usu-
ally placed in the mouth and kept there. An extraordinarily high
amount of nitrite will then be released into saliva and swallowed.
As a result, additional amounts of N-nitroso compounds could be
formed endogenously.
Another tobacco product with relatively high tobacco-specific
nitrosamine levels is zarda, which is usually chewed or kept in the
mouth. To produce zarda, tobacco leaf is boiled in water with lime
and spices until evaporation. The residual particles are then dried
and colored with vegetable dyes. Four brands of this product (Goa
1000, Moolchand Super, Sanket 999, Baba 120) contain an aver-
age of 6.85 61.55 (SD) mg/g NNN. The fifth brand (Shimla) is
relatively high in NNN content (19.9 mg/g), even though the levels
of nitrite and nicotine are similar to the other zarda brands.
The ‘‘other tobacco products’’ (Table I) that are used for chew-
ing may be processed or unprocessed. It is interesting to note that
the brand Gai Chhap, which is made from unprocessed tobacco,
contains the highest tobacco-specific nitrosamine levels of this
group. Clearly, factors other than processing can influence nitros-
amine levels in these products.
Mishri is a powdered form of tobacco that is used primarily for
cleaning teeth. It is prepared by baking tobacco on a hot metal
plate until it becomes uniformly black. The brand Shahin mishri
was found to contain 4.21 mg/g NNN and 0.87 mg/g NNK. As with
some of the other products studied here, nitrosamine uptake from
mishri may increase when it is used habitually (i.e., being placed
and retained in the mouth several times a day).
Gutka usually contains powdered tobacco, betel nut, catechu,
lime and flavors. It has been commercialized since 1975, having
originally been available custom mixed from pan-vendors. The
use of these products is strongly associated with oral cancer.
2–4
The levels of tobacco-specific nitrosamines in gutka were lower
than those in many of the other products examined here, but were
still considerably higher than nitrosamine levels in food and other
common products, which are typically in the low ppb range.
11,24
Supari, which is similar to gutka but does not contain tobacco, did
not have detectable levels of tobacco-specific nitrosamines.
Tobacco is not mentioned as an ingredient of red tooth powders.
Small amounts of nicotine and trace amounts of tobacco-specific
nitrosamines were observed in these products, however, raising
concerns about their safety. Considerable levels of tobacco-
specific nitrosamines and nicotine were found in Dentobac, a
tobacco-containing toothpaste. It is remarkable that a product con-
taining relatively high levels of carcinogens and an addictive agent
is marketed for the purpose of dental hygiene.
The levels of tobacco-specific nitrosamines in 3 of the products
analyzed (Raja khaini, Hans Chhap khaini, Gai Chhap tobacco)
are considerably higher than those found in most smoke-
less tobacco products marketed in Europe and North America,
where the total amounts of these compounds are usually
<10 mg/g.
12,13,25
Levels of these carcinogens in Kentucky refer-
ence smokeless tobacco 1S3 are <8mg/g (Table I). Our results
serve to emphasize the potential hazards of these products
marketed in an area of high oral cancer incidence.
To our knowledge, there have been no published reports on
tobacco-specific nitrosamines in Indian smokeless tobacco prod-
ucts in the past 15 years. Brunnemann et al.
15
reported levels of
tobacco-specific nitrosamines in tobacco used in betel quid. The
amounts were similar to those reported here in gutka. Nair et al.
14
found high levels of tobacco-specific nitrosamines in Indian chew-
ing tobacco and creamy snuff. Tricker and Preussmann
16,17
reported levels of tobacco-specific nitrosamines in zarda tobacco
similar to those found here and also observed relatively high levels
in Kiwam tobacco. It seems that little has changed in the past
15 years with respect to levels of these carcinogens in tobacco
products marketed in India. High exposure to tobacco-specific
nitrosamines in smokeless tobacco products is likely a major fac-
tor in the continuing epidemic of oral cancer in India. Immediate
public health measures are urgently needed to decrease morbidity
and mortality associated with the use of these products.
Acknowledgements
We thank S.G. Carmella for his advice and J. Hodge for techni-
cal assistance.
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19NITROSAMINES IN INDIAN TOBACCO
