1. Effects of a Smokeless Tobacco,
Gutkha, on Neurotransmitter Levels
and Associated Parameters in the
Mouse Brain
Dana Lauterstein1, Carol Hoffman1, Muhammed Hossain3, Jason Richardson3, Francesca Gany2, Judith Zelikoff1
1
NYU School of Medicine, Tuxedo, NY; 2Memorial Sloan-Kettering Cancer Center, NY, NY; 3Rutgers University,
New Brunswick, NJ
2. Abstract
Many studies have been performed on the relationship between smoked tobacco and adverse effects on
health. However, fewer data are available on the toxicity of smokeless tobacco. Gutkha, a smokeless
tobacco (ST) product manufactured in India and readily available in the US (used extensively by South
Asian communities), is composed of powdered tobaccos, slake lime, and spices. To assess the effects of
Gutkha usage on the brain, adult male mice (B6C3F1) were exposed daily via the oral mucosa to water
(control), 50 μL of a 21 mg water-soluble Gutkha solution or of a 8 mg/kg nicotine solution for 1 or 7 days
and changes in brain levels of dopamine (DA), serotonin (5-HT), norepinephrine (NE) were assessed in the
striatum. Additionally, groups of mice were exposed to air (control), and mainstream cigarette smoke (CS)
via inhalation Monoamine oxidase B (MAOB) and tyrosine hydroxylase (TH) (enzymes important for DA
breakdown and synthesis, respectively) were assessed in the frontal cortex. Serum cotinine levels for all
the groups were analyzed upon sacrifice within 1 hr post-exposure; Gutkha-, nicotine and CS-exposed
mice had comparable cotinine levels ranging between 18-50 ng/mL, 20-60 ng/mL, and 11-32 ng/mL
respectively. HPLC studies measuring NT levels in the brain demonstrated that Gutkha-exposed mice had
a significant decrease in DA and 5-HT from the 1 day to 7 day exposure. The results here suggest that:
effects of Gutkha on certain brain parameters may be due to Gutkha-associated toxicants other than
nicotine; Gutkha may provide an additional biological stressor for the brain compared to nicotine alone;
and, Gutkha may be more addictive than nicotine alone or CS based on the rapid depletion rate of
catecholamines in the brain. Studies supported by funds from the Memorial Sloan Kettering Cancer Center
Pilot Projects and NYU NIEHS Center No. ES000260.
3. Introduction
Currently, smokeless tobacco (ST) products are being
advertised as a reduced harm strategy for cigarette
smokers. This perception, however, is misleading.
Although usage of ST in the U.S. is rapidly increasing,
particularly among adolescents, few studies have
examined health effects associated with its use.
More studies on these products are needed, particularly
in the area of their addictive nature.
Thus, the studies here examined the effects of an
ethnically-based ST product on brain pathways
important for addiction in a mouse model.
4. Gutkha
A form of smokeless tobacco that is consumed
(sucked or chewed) in large quantities by
adolescent and adult South Asians both in the U.S.
and abroad.
Made with dried tobacco, areca nut, catechu,
spices, and sweet or savory flavorings.
Contains numerous carcinogenic compounds
including tobacco-specific nitrosamines.
Each packet contains high levels of nicotine (~72
mg/4 g packet) which may make it highly addictive.
5. MATERIALS AND METHODS
Animals
7-8-wk-old B6C3F1 male mice (Jackson labs) were kept on a 12 hr light/dark cycle and housed 1 per cage for gutkha and nicotine
exposure; 4-5 animals were housed per cage for the cigarette smoke exposure. All animal procedures were conducted under an
animal protocol approved by the NYU IACUC.
Gutkha Exposure
Male mice were “painted” with a gutkha solution using a natural bristle paintbrush to coat the tongue and oral mucosa. The
gutkha solution was made by grinding the contents of 1-2 gutkha packets and dissolving the grounded product in water. The
resulting solution was filtered, frozen and lyophilized. Mice were exposed daily for a total of 1 or 7 days to 21 mg of the gutkha
lyophilate dissolved in a volume of 50 µl of water.
Nicotine Exposure
Male mice were “painted” with nicotine in the same manner as that used for Gutkha (i.e., using a natural bristle paintbrush to
coat the tongue and oral mucosa). The nicotine solution was prepared by dissolving commercially-available nicotine in water.
Mice were exposed daily to 8 mg/kg of nicotine in 50 µl of water. This concentration of nicotine yielded cotinine levels similar to
that produced by gutkha exposure.
Mainstream Cigarette Smoke Exposure
Adult male mice were exposed via whole body inhalation to mainstream cigarette smoke (MCS; Ng et al., 2006) generated from
3R4F reference cigarettes for 4 hours each day for a total of either 1 or 7 days.
Cotinine Measurements
Serum cotinine levels in male mice were measured weekly using a commercially-available ELISA kit. Cotinine levels were
measured in the peripheral blood at the time of sacrifice which was 30 min or 1 hr after the final gutkha, nicotine or CS
exposure, and resulted in similar levels ranging between 18-50 ng/mL, 20-60 ng/mL, and 11-32 ng/mL, respectively.
HPLC
HPLC was used for neurochemical analysis of norepinephrine (NE), dopamine (DA), serotonin (5-HT), as well as DA metabolites
homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) on frozen half striatal samples recovered from each
treatment group of mice. Compounds were quantified by electrochemical detection using a glossy carbon working electrode (in
situ silver reference electrode [2.0 mm diameter]). To normalize the data, protein concentration for each sample was
determined using a commercially-available bicinchoninic acid (BCA) assay reagent kit.
Western Blots
Western blots, using commercially-available antibodies, were used to detect protein expression of monoamine oxidase B
(MAO B) and tyrosine hydroxylase (TH) in frontal cortex samples of each treatment group of mice.
Statistics
Biological parameters were analyzed by one-way analysis of variance (ANOVA) followed by post-hoc testing (when
appropriate). All statistical analyses were performed using Graph Pad software (San Diego, CA). Significance was accepted at p < 0.05.
6. Experimental Design
Gutkha, Nicotine, and Cigarette Smoke (CS) Exposure
Mice are
exposed to Acute Exposure Subacute Exposure
either
gutkha,
Striatum and Striatum and
nicotine or frontal cortex frontal cortex
CS tissues frozen in tissues frozen in
liquid nitrogen for liquid nitrogen for
HPLC and western Animals Sacrificed HPLC and western
Animals Sacrificed blot analysis after 7th day of blot analysis
after 1st exposure exposure
Blood taken for Blood taken for
measurement of measurement of
cotinine cotinine
Day 1- start of exposure Day 7
Acute exposure endpoint Subacute exposure endpoint
7.
8. Gutkha-exposed mice had the greatest increase in DA
levels after a 1-day exposure and have the most
dramatic decline from 1-7-day exposures.
9. Gutkha-exposed mice had the greatest increase in 5-HT
levels after a 1-day exposure and the most dramatic
decline from the 1 to 7-day exposures.
14. TH protein expression (by Western blot analyses) was
unaffected in any oral exposure group, but reduced
after 7 days exposure to CS.
15. MAOB protein expression (by Western blot analyses)
was reduced in mice exposed to gutkha at both time-
points.
16. DA metabolites (DOPAC and HVA) were
unchanged in any treatment group at either
time-point.
17. Other Pathways that Could Account for Decreased Dopamine
Levels in the Gutkha-Exposed Mice that Need to be Explored
18. Summary
A single exposure of adult male mice to gutkha increased DA and
5-HT levels compared to nicotine alone or CS.
DA and 5-HT levels in gutkha-exposed mice differed significantly
between days of exposure.
Neither gutkha nor nicotine exposure for 1 or 7 days altered NE
levels.
Monoamine oxidase B, an enzyme responsible for DA
metabolism, was reduced in mice exposed to gutkha, but not to
nicotine alone.
Tyrosine hydroxylase, an enzyme that catalyzes the conversion of
tyrosine to L-DOPA (a precursor to DA), was unaffected by
treatment with gutkha or nicotine alone for either 1 or 7 days.
DA metabolites, DOPAC and HVA, were unchanged following
exposure to either gutkha or nicotine alone.
19. Conclusions
The rapid drop in DA and 5-HT levels from 1 to 7-days observed in
gutkha-exposed mice could suggest that gutkha may be more
addictive than cigarettes or nicotine alone.
The increase in DA observed in mice exposed to gutkha for 1 day may
be (at least in part) due to a decrease in monoamine oxidase (MAO) B
activity.
The significant decrease in DA levels seen in gutkha-exposed mice
after 7 days of exposure are likely due to changes in pathways other
than those mediated by MAOB or TH.
NE levels suggest that the oral route of exposure used here may be
increasing animal stress and thus “masking” any effects of gutkha or
nicotine on this neurotransmitter.
Overall, smokeless tobacco products may prove to be more addictive
than cigarettes and pose an even greater threat for public heath and
thus should also be considered for increased regulation standards.
20. Future Studies
Further analysis of different dopaminergic pathways may
provide additional information concerning the underlying
mechanism(s) associated with the observed gutkha-induced
decrease.
Determine the individual gutkha constituents that may be
contributing to the observed effects on neurochemistry.
Evaluate the effects of long-term chronic exposure, a more
relevant exposure paradigm, to gutkha on additional
neuropharmacology parameters.
21. Acknowledgements
• Special thanks to Drs. Dan Willis, and Jason
Blum for technical assistance with the project.
• Studies supported by funds from Memorial
Sloan Kettering Cancer Center and NYU NIEHS
Center of Excellence Grant No. ES000260.
