Waterpipe tobacco smoke toxicant
exposure and effects
Alan Shihadeh
American University of Beirut
1st International Conference on WTS
October 20-23, 2013
Abu Dhabi
Waterpipe toxicants & health effects
Molecule  Particle  Cell  Animal  Human individual  Population health
Does the smoke contain
toxicants?
• particle size
• chemistry
• biological activity
Does waterpipe smoking emit
toxicants?
sidestream
(SS)
SS +
EMS
inhaled
exhaled
Does the user absorb
toxicants?
biomarkers in: blood, breath,
urine
absorbed
What happens to the user?
• acute physiological effects
• BP, HR, inflammatory responses,
lung function…
http://firefighterparamedicstories.blogspot.com/2011_10
_01_archive.html
exhaled
mainstream
(EMS)
What happened to the smoker?
• long term mortality & morbidity
Narghile waterpipe
coal
head
mouthpiece
tobacco
hose
body
bowl
water
Photo credit: internet, source unknown
Narghile waterpipe
Differences with cigarette
tobacco burn not self-sustaining 
charcoal needed
coal
head
hose
Tobacco temperature < 500 C (Cig < 900 C)
“molasses” flavorings
mouthpiece
tobacco
body
order of magnitude higher flow rates/puff
volumes
long flow path with bubbler/humidifier
(produces cool “smooth” smoke)
bowl
water
Tobacco smoke constituents
N2, O2, CO2
Gas phase
Smoke
aerosol
CO
~ 90% wt
(invisible)
water vapor
Particle
phase
condensed water
10-1000 nm
(visible)
C2-C6 hydrocarbons,
aldehydes, nitriles …
nicotine
“tar”
4000+ compounds
PAH, nitrosamines,
metals,… contains
most important
carcinogens
Illustration from: N. deNevers Pollution Control
The “Hoffmann List“ of probable causative agents
in cigarette smoke-related disorders
disorder
contributing agents
possible enhancing
agents
tobacco dependence
major: nicotine
acetaldehyde
minor: secondary Nicotiana alkaloids,
flavor components
cardiovascular disease
major: carbon monoxide, nitrogen
oxides, hydrogen cyanide, tar
nicotine, alkylating
species
minor: cadmium, zinc
chronic obstructive
pulmonary disease
hydrogen cyanide, volatile aldehydes,
nitrogen oxides, carbon monoxide, tar
lung and larynx cancer
major: PAH, NNK
minor: 210polonium, formaldehyde,
acetaldehyde, butadiene, metals
(Cr, Cd, Ni)
oral cavity cancer
major: NNN, NNK
catechol, tumor promoters
acetaldehydes, diet,
alkylating species
herpes simplex, irritation
minor: PAH
esophageal cancer
NNN
ethanol, diet
pancreas cancer
NNK, NNAL
diet
Hoffmann et al, 2001; Hecht 1997
The “Hoffmann List“ of probable causative agents
in cigarette smoke-related disorders
disorder
contributing agents
possible enhancing
agents
tobacco dependence
major: nicotine
acetaldehyde
minor: secondary Nicotiana alkaloids,
flavor components
cardiovascular disease
major: carbon monoxide, nitrogen
oxides, hydrogen cyanide, tar
nicotine, alkylating
species
minor: cadmium, zinc
chronic obstructive
pulmonary disease
hydrogen cyanide, volatile aldehydes,
nitrogen oxides, carbon monoxide, tar
lung and larynx cancer
major: PAH, NNK
minor: 210polonium, formaldehyde,
acetaldehyde, butadiene, metals
(Cr, Cd, Ni)
oral cavity cancer
major: NNN, NNK
catechol, tumor promoters
acetaldehydes, diet,
alkylating species
herpes simplex, irritation
minor: PAH
esophageal cancer
NNN
ethanol, diet
pancreas cancer
NNK, NNAL
diet
*Hoffmann et al, 2001; Hecht 1997
Particle size & lung dosimetry
“Ultrafine
particles”
Hinds, Aerosol Technology (1999)
Human hair
Tobacco smoke particle
(100-200 nm)
Do waterpipes emit toxicants?
Do waterpipes emit toxicants?
What is in the
smoke?
1) Find out how
people smoke
waterpipes
2) Program a
robot to smoke
the same way
3) Analyze the
smoke for
toxicants
Find out how people smoke
Shihadeh, Antonius, Azar, BRIMC,2005
Average puff
topography
• Volume
• Duration
• Frequency
• Number
Puff topography record
Field study of waterpipe users in a Beirut seaside café
Results
Field study of 52 café smokers in Beirut
mean age 21 years, 14 f/38 m
Puff parameter
Narghile
Cigarette
Shihadeh et al 2004
Djordjevic et al
2000
Number of puffs
171
12.1
Session smoking time, min
61
3.7
Puff volume, ml/puff
530
44.1
Puff duration, s/puff
2.6
1.5
Interpuff interval, s/cycle
17
18.5
90,600
523
Total volume, ml
A. Shihadeh et al, Biochemistry, Pharmacology, and Behavior, 79(1),75-82, 2004
“Beirut method” – a standard waterpipe smoking
machine protocol for generating WTS
puff regimen
• volume
• duration
• frequency
• number
Ma’ssel quantity
charcoal type and
application regimen
head preparation
the “Beirut method”
171 puffs, 530 ml, 2.6 s duration, 17 s IPI
10 g ma’assel tobacco mixture load
1 easy-light charcoal disk + 0.5 added at 105th puff
18-hole aluminum
perforation pattern
Program smoking robot & sample/analyze smoke
Shihadeh & Azar, JAM, 2006
Waterpipe smoke does contain toxicants (MS yields/unit)
T/N/CO
"Tar", mg
Nicotine, ug/10
Carbon monoxide, mg
Cigarette
PAHs
Benzo(a)pyrene, ng
Dibenz(a,h)anthracene, ng
Indeno(1,2,3-cd)pyrene, ng
Aldehydes
Formaldehyde, ug
Acetaldehyde, ug/10
Acrolein, ug
Heavy metals
TSNA
Waterpipe (AUB-BfR)
Arsenic, ng
Chromium, ng/10
Lead, ng/10
NAB (ng)
NNN (ng)
NNK (ng)
NAT (ng)
0
500
1000
Waterpipe smoke particles are small
Particle mass distribution
mass median dia mm
narghile
cigarette
0.61
0.33
0.74
0.21
1.02x1011
155
1.11x1011
123
Respirable mass
mg/puff
Nanoparticles
#/puff
count median dia, nm
Waterpipe smoke particles are biologically active
In vitro effects of WTS on
HEACs
• Oxidative stress
• Inflammation
• Cell cycle arrest
• Impaired vasodialation
• Impaired angiogenesis
 Plausible cellular mechanism for
vascular diseases
Rammah et al, 2013 Toxicology Letters Volume 219, Issue 2 2013 133 - 142
Impaired angiogenesis
Capillary tube formation of untreated and treated HAEC cells (mg/ml) WSC.
Rammah et al, 2013 Toxicology Letters Volume 219, Issue 2 2013 133 - 142
Waterpipe smoke particles are biologically active
Effects on lung epithelial (A549) and
endothelial cells and signaling mechanisms
 Plausible cellular mechanism for COPD
Hoechst staining of cells 72 hours post-treatment with
WSC. Pictures were taken using a 40x oil immersion lens.
Rammah et al, 2012 Toxicology Letters
Does the user inhale toxicants?
YES. Based on the Beirut Method:
• During a single WP use session the user inhales a large dose
of toxicants known to cause tobacco-related diseases
• WTS particle size distribution is similar to cigarette smoke
• WTS damages and interferes with repair mechanisms of lung
and vascular cells in culture
Q. Does the Beirut Method provide a
reasonable facsimile of real smoke?
Charcoal(!)
RESULTS
charcoal
electric
charcoal
More charcoal than ma’ssel is consumed
contribution
during a typical use
session 5.7
CO, mg
57.2
90%
PAHs, ng/mg TPM
Users continually0.219
“tune” it < 0.01
Benzo(a)pyrene
∑ 2- and 3-ring PAH
5.262
1.444
∑ 4- and 5-ring PAH
1.181
0.098-0.290a
charcoal
> 95%
73%
75-92%
electrical heater
temperature vs time
Monzer, B., Sepetdjian, E., Saliba, N. and Shihadeh, A. Charcoal combustion as a source of CO and carcinogenic PAH in
mainstream narghile waterpipe smok, Food and Chemical Toxicology, 2008
REALTIME sampling in the natural environment
Katurji et al, Inh Tox, 2010
How does Beirut Method smoke compare
to smoke made by people?
Nicotine
“Tar”
a) nicotine
b) “tar”
Beirut Method
Volume (l)
c) nicotine:tar
Nicotine:Tar
“tar” (mg)
Volume (l)
d) CO
CO
Volume (l)
Katurji et al, Inh Tox, 2010
Do waterpipe users absorb toxicants?
Do waterpipe users absorb toxicants?
Contains “tar”,
CO,
Nicotine,PAHs,
aldehydes….
What is in the
smoker?
Measure toxicant levels
in blood, breath, or
urine.
Studies: placebo control,
cigarette comparison,
observational.
CO and nicotine in blood
5
14
Carboxyhemoglobin (N=31)
Plasma nicotine (N=31)
12
4
10
8
ng/ml
Percent
3
2
6
4
1
2
0
0
0
5
15
30
Time relative to smoking onset
45
0
5
15
30
Time relative to smoking onset
Clinical setting, one 45 min WP session
Eissenberg & Shihadeh, American Journal of Preventive Medicine, 37, 518-523, 2009.
45
CO and nicotine in blood: compared to cigarettes
4
Cigarette
*
*
Waterpipe
3
Percent
14
Carboxyhemoglobin (N=31)
*
10
*
8
*
2
Plasma nicotine (N=31)
12
ng/ml
5
6
4
1
2
0
0
0
5
15
30
Time relative to smoking onset
45
0
5
15
30
Time relative to smoking onset
Eissenberg & Shihadeh, American Journal of Preventive Medicine, 37, 518-523, 2009.
45
CO and nicotine exposure over 24 hours
Plasma nicotine (n=13)
Exhaled breath CO (n=13)
• Hospital setting, cross-over design, N=13 dual users
• All day ad libitim cigarette smoking (11 cpd mean) versus 3 WTS use sessions
• Measurements on day 4 of 4-day protocol.
Jacob et al, 2013. Cancer Epidemiol Biomarkers Prev 2013;22:765-772.
Carcinogen exposure over 24 hours
Urinary NNAL (TSNA biomarker)
Urinary 1-HOP (PAH biomarker)
• Hospital setting, cross-over design, N=13 dual users
• All day ad libitim cigarette smoking (11 cpd mean) versus 3 WTS use sessions
• Measurements on day 4 of 4-day protocol.
Jacob et al, 2013. Cancer Epidemiol Biomarkers Prev 2013;22:765-772.
Carcinogens in populations of smokers
Urine samples collected from population in Aleppo.
Mean cigarette use 27 cigs per day, mean WTS 2 per day.
Total NNAL ng/g creatinine
*, **
100
80
60
*, **
40
20
0
CTRL (n=28)
Al Ali R, et al. 2013 Tob Control doi:10.1136/
WP (n=24)
CIG (n=23)
Do waterpipe users absorb toxicants?
YES!
• Clinical, hospital, natural environment findings consistent
with one another
• Generally consistent with what we know about toxicants
in smoke:
CO
PAH
TSNA
Nicotine
WP >> cig
WP > cig
WP < cig
WP ~ cig
What happens to the smoker?
Delivered nicotine is physiologically active
sympathetic activation
reduced complexity
Cobb et al., Inhalation Toxicology 2012
WP smoke suppresses “Urge to smoke” after 24
hour abstinence
Rastam et al., 2011
WP smoke compromises cardiac autonomic
regulation in human participants
(with or without nicotine!)
2
4
LF/HF
3
Tobacco
*
1
1
0.5
0
0
sympathetic activation
min
1515
min
*
*
1.5
2
end
end
Tobacco
Herbal
*
Herbal
baseline
baseline
SampEn
baseline
baseline
end
end
reduced complexity
Cobb et al., Inhalation Toxicology 2012
15 min
15 min
What happens to the smoker?
WP smoke inhalation induces inflammation and
oxidative stress in mice
Inflammatory markers
Oxidative stress
Khabour et al 2012 Inh Tox
WP smoke inhalation associated with:
• Genotoxicity (sister chromatid exchanges): WTS > CS > nonsmokers (Khabour et
al, 2011)
• Reduction in exercise capacity : VO2 1.86 vs. 1.7 l/min, pre- post (Hawari et al.,
2013)
• Reduction in lung function (Hakim et al., 2011; Mohammad et al., 2008; Koseoglu et
al., 2006; Kiter et al., 2000; Aydin et al. 2004; Al Mutairi et al., 2006, Al Fayez et al., 1988):
WTS compared to:
Parameter
a) non-smoker
FEV1
-4.04%
FVC
-1.38%
FEV1/FVC
-3.08%
b) cigarette smoker
ns
ns
ns
“WPS negatively affects lung function and may be as
harmful as cigarette smoking. WPS, therefore, is likely
to be a cause of COPD.” – Raad et al., 2011
What happened to the smoker?
What happened to the smoker?
Waked, Khayat, Salameh, 2012
What happened to the smoker?
(Jawad et al., 2013 based on Akl et al., 2010)
“A wide range of diseases have been associated with WTS, but research
in this area is relatively underdeveloped and a better evidence base is
needed.” - Akl et al., 2010
Does waterpipe smoking emit toxicants into the
environment?
Does the user inhale
toxicants?
• particle size
• chemistry
• biological activity
Does waterpipe smoking emit
toxicants?
sidestream
(SS)
SS +
EMS
inhaled
exhaled
Does the user absorb
toxicants?
biomarkers in: blood, breath,
urine
absorbed
What happens to the user?
• acute physiological effects
• BP, HR, inflammatory responses,
lung function…
http://firefighterparamedicstories.blogspot.com/2011_10
_01_archive.html
exhaled
mainstream
(EMS)
What happened to the smoker?
• long term mortality & morbidity
Do waterpipe smoking emit toxicants?
• chamber based studies
• observational studies of cafés
Chamber study
Daher et. al, Atmospheric Environment, 44, 8-14, 2009.
Waterpipe emits more nanoparticles
Particle
concentration
(particles/cm3)
Daher et. al, Atmospheric Environment, 44, 8-14, 2009.
WP emits more of everything measured
mean±95% CI
Carbon monoxide, mg
waterpipe SS cigarette SS
N = 12
N=9
2269 ± 108
65.5 ± 5.5
PAH, ng
Total PAH
N = 11
1193 ± 226
Particle number emissions
ultrafine particles 5.6-99.5 nm, /1012
total particles 5.6-560 nm, /1012
count median diameter, nm
N=4
N=4
3.99 ± 0.60 0.639 ± 0.188
4.38 ± 0.66 1.68 ± 0.27
37.9 ± 4.1
130 ± 8
Volatile aldehydes, ug
Total aldehydes
N=3
305 ± 49
N=6
N=5
12000 ± 1610 2954 ± 416
Daher et. al, Atmospheric Environment, 44, 8-14, 2009.
What about per smoker-hour?
•
Waterpipe smokers release equivalent of 210 cigarette smokers per hour of smoking
Daher et. al, Atmospheric Environment, 44, 8-14, 2009.
Observational studies in WP cafés
Hammal et al., 2013
*
* 1-Hour PM2.5 exposure Guideline-Alberta
Observational studies in WP cafés
Zhang et al., 2013
Summary
WTS…
• Delivers a large dose of toxicants
• Elicits inflammatory responses and dysfunction in human cells
• Elicits immediate physiological changes in users, including
compromised lung function and autonomic function, subjective
effects
• Is addictive
• Is associated with elevated markers of genetic damage and
COPD
• Is associated with other long term health effects, but quality
of evidence is very weak.
• Emits large quantities of toxicants in second-hand smoke
Thanks for listening
RESULTS
CO, mg
PAHs, ng/mg TPM
Benzo(a)pyrene
∑ 2- and 3-ring PAH
∑ 4- and 5-ring PAH
base case
electrically heated
charcoal
contribution
57.2
5.7
90%
0.219
5.262
1.181
< 0.01
1.444
0.098-0.290a
> 95%
73%
75-92%
Monzer, B., Sepetdjian, E., Saliba, N. and Shihadeh, A. Charcoal combustion as a source of CO and carcinogenic PAH in
mainstream narghile waterpipe smok, Food and Chemical Toxicology, 2008
concentration, arbitrary units
base case
charcoal extract
R 2 = 0.94
charcoal grilling
(R2 = 0.96, Dyremark et al, 1995)
electrically heated, R 2 < 0.02
Relative PAH concentrations measured in the mainstream smoke of the base and
electrically heated conditions, and in extracts of the unburned charcoal. PAH
concentrations from smoke collected from a charcoal grill by Dyremark et al (1995) shown
for comparison. Correlation coefficients are shown relative to the base condition.
Unburned charcoal extract, charcoal grilling smoke, and base case narghile smoke exhibit
similar PAH patterns. Electrically heated condition produces a different PAH pattern.
Monzer, B., Sepetdjian, E., Saliba, N. and Shihadeh, A. Charcoal combustion as a source of CO and carcinogenic PAH in
mainstream narghile waterpipe smok, Food and Chemical Toxicology, 2008
Compromised cardiac autonomic regulation in human participants
Tobacco
Soex
Topography (N=32)
Total smoke volume (l)
Puffs drawn
Mean puff volume (ml)
Mean interpuff interval (s)
Mean puff duration (s)
31 (22)
95 (115)
420 (250)
45 (34)
2.3 (0.9)
57 (34)*
95 (58)
680 (330)*
35 (22)*
3.5 (2.0)*
Peak change from baseline plasma nicotine and CO
Nicotine (N=32)
CO% (N=29)
7.8 (8.0)
11.8 (7.7)
0.0 (0.0)*
30.9 (19.0)*
(N = 32)
HR (1/min)
LF (ms2)
Tobacco
Baseline
70.6
1400
15 min
72.2
1190
Soex
Baseline
72.8
1330
End
75.5*
2420*
End
71.5
3570*
15 min
68.1*
1610
HF(ms2)
1270
1230
1140
1410
1690
1400
LF/HF
SampEn
1.67
1.63
2.84*
1.48*
1.68
1.55
1.45
1.69
3.21*
1.52*
1.55
1.65
WP emits more of everything measured
mean±95% CI
Carbon monoxide, mg
waterpipe SS cigarette SS
N = 12
N=9
2269 ± 108
65.5 ± 5.5
PAH, ng
Total PAH
N = 11
1193 ± 226
Particle number emissions
ultrafine particles 5.6-99.5 nm, /1012
total particles 5.6-560 nm, /1012
count median diameter, nm
N=4
N=4
3.99 ± 0.60 0.639 ± 0.188
4.38 ± 0.66 1.68 ± 0.27
37.9 ± 4.1
130 ± 8
Volatile aldehydes, ug
Total aldehydes
N=3
305 ± 49
N=6
N=5
12000 ± 1610 2954 ± 416
Daher et. al, Atmospheric Environment, 44, 8-14, 2009.
Mass balance
For any
toxicant…
𝑡𝑠
𝑚𝑒 = 𝑉 𝐶 𝑡𝑠 − 𝐶 0 +
change of
suspended mass
within CV
𝐶 𝑡 𝑄𝑑𝑡 + 𝑚𝑑
0
advected
mass
wall losses
Sidestream emissions
mean±95% CI
Carbon monoxide, mg
PAH, ng
Total PAH
Particle number emissions
ultrafine particles 5.6-99.5 nm, /1012
total particles 5.6-560 nm, /1012
count median diameter, nm
Volatile aldehydes, ug
Total aldehydes
waterpipe SS
N = 12
2269 ± 108
cigarette SS
N=9
65.5 ± 5.5
N = 11
1193 ± 226
N=3
305 ± 49
N=4
N=4
3.99 ± 0.60
0.639 ± 0.188
4.38 ± 0.66
37.9 ± 4.1
1.68 ± 0.27
130 ± 8
N=6
12000 ± 1610
N=5
2954 ± 416
Daher et. al, Atmospheric Environment, 44, 8-14, 2009.
SS nanoparticle emissions: WP vs CIG
Particle
concentration
(particles/cm3)
Daher et. al, Atmospheric Environment, 44, 8-14, 2009.
Is robot-generated smoke
realistic?
Toxicants in robot-generated smoke track blood level
exposure in human participants
Blood CO (COHb, %)
16
Carbon
monoxide
12
8
P<0.01
R2>0.62
y = 0.018x + 1.470
R² = 0.622
4
0
0
200
400
600
800
Smoking machine CO (mg)
Shihadeh & Eissenberg, CEBP, 2011
Schubert et al., 2012
Environ Mol Mutagen. 2011 Apr;52(3):224-8. doi: 10.1002/em.20601. Epub 2010 Aug 25.
Assessment of genotoxicity of waterpipe and cigarette smoking in lymphocytes using the
sister-chromatid exchange assay: a comparative study.
Khabour OF, Alsatari ES, Azab M, Alzoubi KH, Sadiq MF.
Abstract
Tobacco smoking is a major world health problem. Recently, waterpipe smoking has become more popular in many countries.
Although the genotoxicity associated with cigarette smoking has been extensively investigated, studies evaluating such toxicity
in waterpipe users are still lacking. In this study, we examined the genotoxicity of waterpipe smoking in lymphocytes compared
with the genotoxicity of cigarette smoking. Genotoxicity was evaluated using the sister chromatid exchanges (SCEs) assay. Fifty
waterpipe smokers and 18 healthy nonsmokers participated in this study. Additionally, 18 heavy cigarette smokers (CS) were
recruited for comparison. The results show that waterpipe smoking and cigarette smoking significantly increase the frequencies
of SCEs (P < 0.01) compared with those of nonsmokers, indicating the genotoxic effect of tobacco smoking. In addition,
frequencies of SCEs were significantly higher among waterpipe smokers compared with CS (P < 0.01), indicating that waterpipe
smoking is more genotoxic than cigarette smoking. Moreover, the frequency of SCEs increased with the extent of waterpipe
use. In conclusion, waterpipe smoking is genotoxic to lymphocytes and the magnitude of its genotoxicity is higher than that
induced by regular cigarette smoking.