Tobacco Smoke Exposure Biomarkers, 1st Edition

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Additional Contributions From

Xiaotao Zhang
Jianjun Xia
Wei Xiong
Huan Chen
Xiaojing Zhang
Yongfeng Tian
Chunlei Ren
Yaning Fu
 Tobacco
Smoke Exposure
Biomarkers

Qingyuan Hu • Hongwei Hou

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Contents
Preface.......................................................................................................................xi

Chapter 1 Smoking and Health..............................................................................1

1.1 Global Tobacco Epidemic...........................................................1
1.2 Smoke-Related Diseases............................................................2
1.3 Smoke Constituents of Concern................................................. 3
1.3.1 TSNAs...........................................................................6
1.3.2 B[a]P..............................................................................6
1.3.3 Volatile Organic Compounds........................................6
1.3.4 Aromatic Amines..........................................................7
1.3.5 Catechol and Hydroquinone..........................................8
1.3.6 Hydrogen Cyanide, Carbon Monoxide,
and Nitrogen Oxides...................................................... 8
1.3.7 Inorganic Compounds................................................... 8
1.4 Biomarkers.................................................................................9
1.4.1 Classification of Biomarkers....................................... 10
1.4.1.1 Biomarkers of Exposure.............................. 10
1.4.1.2 Biomarkers of Potential Harm..................... 10
1.4.1.3 Biomarkers of Susceptibility....................... 10
1.5 Biomarkers of Exposure to Environmental
Tobacco Smoke........................................................................ 11
1.6 Biomarkers Application in Tobacco Regulation and
Health Policy............................................................................ 11
References........................................................................................... 12

Chapter 2 Biomonitoring and Biomarkers........................................................... 17

2.1 Introduction.............................................................................. 17
2.2 Preparation of Samples of Biological Materials....................... 18
2.2.1 Sampling of Biological Material................................. 19
2.2.2 Sample Storage............................................................20
2.2.3 Techniques of Sample Preparation prior
to Analysis................................................................ 20
2.2.3.1 Liquid–Liquid Extraction............................20
2.2.3.2 Solid-Phase Extraction................................ 21
2.2.3.3 Solid-Phase Microextraction....................... 21
2.2.3.4 Supercritical Fluid Extraction..................... 21
2.3 Methodology of Determination of Biomarkers........................ 21
2.3.1 Colorimetric Method................................................... 22
2.3.2 Test Strip Assay........................................................... 22
2.3.3 Immunoassay............................................................... 22

v
vi Contents

2.3.4 Gas Chromatography/Gas Chromatography–

Mass Spectrometry...................................................... 23
2.3.5 Liquid Chromatography/Liquid
Chromatography–Mass Spectrometry........................ 23
2.4 Biomarkers in Tissues and Body Fluids...................................24
2.4.1 Urine............................................................................24
2.4.2 Blood...........................................................................25
2.4.3 Saliva........................................................................... 26
2.4.4 Tissues.........................................................................26
2.5 Conclusions............................................................................... 26
References........................................................................................... 27

Chapter 3 Biomarkers of Pyridine Alkaloids: Nicotine and Cotinine................. 33

3.1 Introduction.............................................................................. 33
3.2 Toxicological Evaluation.......................................................... 33
3.3 Metabolic Pathway of Nicotine in the Body............................34
3.4 Samples Used........................................................................... 36
3.4.1 Urine............................................................................ 36
3.4.2 Blood........................................................................... 36
3.4.3 Hair and Nails............................................................. 37
3.5 Analytical Technology............................................................. 37
3.5.1 GC-MSn....................................................................... 37
3.5.2 LC–MSn....................................................................... 38
3.5.3 Other Analytical Technologies.................................... 38
3.6 Metabolites of Nicotine............................................................ 39
3.6.1 Cotinine....................................................................... 39
3.6.2 3-Hydroxycotinine....................................................... 39
3.6.3 Nicotine Glucuronide..................................................40
3.6.4 Cotinine Glucuronide..................................................40
3.6.5 3-Hydroxycotinine Glucuronide.................................. 41
3.6.6 Nornicotine.................................................................. 41
3.6.7 Norcotinine.................................................................. 41
3.6.8 Nicotine-N-Oxide........................................................ 41
3.6.9 Cotinine-N-Oxide........................................................ 42
3.7 Application of Nicotine Biomarkers on Biomonitor................ 42
3.7.1 Cotinine....................................................................... 42
3.7.2 3-Hydroxycotinine....................................................... 43
3.7.3 Nicotine Glucuronide.................................................. 43
References...........................................................................................44

Chapter 4 Biomarkers of Tobacco-Specific Nitrosamines................................... 51

4.1 Introduction.............................................................................. 51
4.1.1 Toxicological Evaluation............................................. 51
4.1.2 Metabolic Pathway in the Body.................................. 52
Contents vii

4.2 NNK......................................................................................... 53
4.2.1 NNAL.......................................................................... 53
4.2.2 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanol
Glucuronide (NNAL-Gluc)......................................... 55
4.3 N-Nitrosonornicotine................................................................ 56
4.4 TSNA Biomarkers in Others.................................................... 57
4.4.1 DNA Adducts Derived from TSNAs in Tissues......... 57
4.4.2 Protein Adducts and Hemoglobin Adducts
from TSNAs................................................................ 58
References........................................................................................... 59

Chapter 5 Biomarkers of Polycyclic Aromatic Hydrocarbons............................. 63

5.1 Introduction.............................................................................. 63
5.2 PAHs and PAH Exposure and Carcinogenesis......................... 63
5.3 Enzymes of Metabolic Activation and Detoxication
of PAHs.................................................................................64
5.3.1 Enzymes of Metabolic Activation............................... 65
5.3.2 Detoxication of PAH................................................... 67
5.3.2.1 UDP-Glucuronosyltransferases................... 68
5.3.2.2 Glutathione S-Transferases and
GSH Conjugates...........................................68
5.4 Biomonitoring PAHs and Their Metabolites in
Human Urine............................................................................ 69
5.4.1 B[a]P and 3-OH-B[a]P................................................. 69
5.4.2 PAH and 1-OH-Phenanthrene..................................... 70
5.5 PAH Biomarkers in Others....................................................... 71
5.5.1 Aromatic DNA Adducts.............................................. 73
5.5.2 Urinary Thioethers...................................................... 73
5.5.3 Urinary Mutagenicity.................................................. 73
5.5.4 Metabolites in Urine of the PAH Mixture.................. 73
5.5.5 PAH DNA Adducts in Human Tissues....................... 74
5.5.6 PAH Protein Adducts (Hemoglobin and Serum
Albumin Adducts)....................................................... 75
5.5.7 Biomonitoring of PAH in Hair.................................... 76
5.6 Discussion................................................................................. 77
5.7 Conclusion................................................................................ 78
References........................................................................................... 78

Chapter 6 Biomarkers of Volatile Organic Compounds...................................... 81

6.1 Introduction.............................................................................. 81
6.2 Toxicological Evaluation.......................................................... 81
6.3 Metabolic Pathway in the Body................................................ 81
6.3.1 Aldehydes.................................................................... 82
6.3.2 Formaldehyde.............................................................. 82
viii Contents

6.3.3 Acetaldehyde............................................................... 83
6.3.4 Acrolein.......................................................................84
6.3.4.1 3-HPMA...................................................... 85
6.3.5 1,3-Butadiene.............................................................. 87
6.3.5.1 Monohydroxybutenyl-Mercapturic
Acid, Dihydroxybutenyl-Mercapturic
Acid, and Trihydroxybutyl
Mercapturic Acid....................................... 87
6.3.5.2 1,3-Butadiene Adducts in Blood
and Tissue.................................................... 89
6.3.6 Benzene.......................................................................90
6.3.6.1 Benzene in Blood and Urine........................90
6.3.6.2 Urinary Metabolites..................................... 91
6.3.6.3 Benzene Adducts in Blood and Tissue........ 91
6.3.7 Acrylonitrile................................................................ 91
6.3.7.1 CEMA and HEMA......................................92
6.4 Discussion................................................................................. 95
6.5 Conclusion................................................................................ 96
References........................................................................................... 96

Chapter 7 Aromatic Amines.............................................................................. 105

7.1 Introduction............................................................................ 105
7.2 4-ABP..................................................................................... 105
7.2.1 Toxicological Evaluation........................................... 105
7.2.2 Metabolic Pathway in the Body................................ 106
7.2.3 Biomarker of 4-ABP................................................. 106
7.2.3.1 Urinary 4-ABP.......................................... 106
7.2.3.2 Hemoglobin Adducts................................. 107
7.2.3.3 DNA Adducts............................................. 108
7.3 2-NA....................................................................................... 108
7.3.1 Toxicological Evaluation........................................... 108
7.3.2 Metabolic Pathway in the Body................................ 109
7.3.3 Biomarkers of 2-NA.................................................. 109
7.4 Conclusion.............................................................................. 109
References......................................................................................... 110

Chapter 8 Biomarkers of Catechol and Hydroquinone...................................... 113

8.1 Introduction............................................................................ 113
8.2 Toxicological Evaluation........................................................ 114
8.3 Biodegradation and Metabolism............................................ 115
8.4 Biomarkers of Hydroquinone and Catechol........................... 117
References......................................................................................... 117
Contents ix

Chapter 9 Biomarkers of Hydrogen Cyanide, Carbon Monoxide, and

Nitrogen Oxides................................................................................ 123
9.1 Introduction............................................................................ 123
9.2 Hydrogen Cyanide.................................................................. 123
9.2.1 Toxicological Evaluation........................................... 124
9.2.2 Metabolic Pathway in the Body................................ 124
9.2.3 Biomarkers................................................................ 125
9.3 Carbon Monoxide................................................................... 126
9.3.1 Toxicological Evaluation........................................... 126
9.3.2 Metabolic Pathway in the Body................................ 127
9.3.3 CO in Exhaled Air.................................................... 128
9.3.4 COHb in Blood.......................................................... 128
9.4 Nitrogen Oxides...................................................................... 129
9.4.1 Toxicological Evaluation........................................... 129
9.4.1.1 Nitric Oxide............................................... 129
9.4.1.2 Nitrogen Dioxide....................................... 130
9.4.2 Metabolic Pathway in the Body................................ 130
9.4.2.1 Nitric Oxide............................................... 130
9.4.2.2 Nitrogen Dioxide....................................... 131
9.4.3 Biomarkers................................................................ 131
9.4.3.1 8-Nitroguanine........................................... 131
9.4.3.2 Methemoglobin.......................................... 131
9.4.3.3 Urinary NO2− and NO3−............................. 131
9.4.3.4 Formation of N-Nitroso Compounds......... 132
9.5 Discussion............................................................................... 132
9.6 Conclusion.............................................................................. 133
References......................................................................................... 133

Chapter 10 Biomarkers of Heavy Metal Exposure.............................................. 137

10.1 Introduction............................................................................ 137
10.2 Metabolism Pathway.............................................................. 138
10.3 Cadmium................................................................................ 139
10.3.1 Occurrence, Exposure, and Dose.............................. 139
10.3.2 Physical Effects......................................................... 140
10.3.3 Cadmium in Oxidative Stress................................... 141
10.4 Arsenic................................................................................... 142
10.4.1 Occurrence, Exposure, and Dose.............................. 142
10.4.2 Physical Effects......................................................... 142
10.4.3 Metabolism Pathway................................................. 143
10.5 Mercury.................................................................................. 143
10.5.1 Occurrence, Exposure, and Dose.............................. 143
10.5.2 Physical Effects......................................................... 144
10.5.2.1 Inorganic Mercury..................................... 144
10.5.2.2 Organic Mercury....................................... 145
x Contents

10.6 Lead........................................................................................ 145

10.6.1 Occurrence, Exposure, and Dose.............................. 145
10.6.2 Physical Effects......................................................... 146
10.7 Discussion............................................................................... 147
10.8 Conclusion.............................................................................. 147
References......................................................................................... 148

Chapter 11 Application of Smoke Exposure Biomarkers in

Epidemiologic Research.................................................................... 151
11.1 Smoke Exposure Biomarker and Health Effects.................... 151
11.2 Application of Smoke Exposure Biomarkers in
Epidemiologic Research......................................................... 152
References......................................................................................... 153

Chapter 12 Suggestions for the Use of Specific Biomarkers of Exposure to

Cigarette Smoke for Regulation Purposes........................................ 155
12.1 Background............................................................................. 155
12.2 Measuring Exposure............................................................... 157
12.3 Measuring Biological Changes............................................... 158
12.4 Measuring Injury and Disease............................................... 159
12.5 Recommended Uses for Biomarkers of Exposure
and Effect............................................................................ 160
12.5.1 Improving the Accuracy of the Definition of
Current Tobacco Use Status...................................... 160
12.5.2 Evaluating the Intensity of Exposure to
Specific Constituents................................................. 161
12.5.3 Evaluating the Intensity of Exposure as a Proxy
for Total Tobacco Exposure...................................... 163
12.6 Measuring Reduced Injury or Harm...................................... 164
12.7 Summary of Biomarker Recommendations........................... 165
References......................................................................................... 166

Appendix A: G
 uideline Document for Ethics Review Concerning
Research Involving Intervention on Humans by the
European Commission.................................................................. 169
Appendix B: Declaration of Helsinki (V2008)................................................... 175
Appendix C: Protection of Animals Used for Scientific Purposes................... 181
Appendix D: G
 uidance for Industry Bioanalytical Method Validation by
the Food and Drug Administration.............................................. 247
Appendix E: Toxicity and IARC Classification of Targeted Constitutes........ 265
Preface
The causal relation between tobacco smoking and a variety of cancers is well docu-
mented and is attributable to the many carcinogens that smokers inhale. Federal
regulation of cigarettes and other tobacco products has been recommended as an
element of a broad-based approach to reduce the disease burden of tobacco use. One
component of federal regulation is likely to be the power to mandate limits of emis-
sions of tobacco smoke toxins. The Family Smoking Prevention and Tobacco Control
Act (FSPTCA) enables the Food and Drug Administration to establish tobacco prod-
uct standards. Similarly, Article 9 in the Framework Convention on Tobacco Control
(FCTC) describes the regulation of content and emissions of tobacco products.
Developing simple and robust analytical methods to measure toxicants in ciga-
rette smoke is of great importance for tobacco regulation. This has been an ongoing
requirement by the World Health Organization (WHO) under the FCTC. National
tobacco control organizations have been tasked to develop analytical methods to
support this initiative. Biomarkers allow for a quantitative linking of biological expo-
sure to tobacco or tobacco smoke to specific disease induction or progression prior to
the advent of the clinically apparent disease. It provides a strong foundation by which
to make scientific evaluations and regulatory decisions regarding tobacco products.
In this book, the toxicology, metabolic pathway, and biomarker of nicotine,
tobacco-specific nitrosamines, polycyclic aromatic hydrocarbons, volatile organic
compounds, aromatic amines, catechol and hydroquinone, hydrogen cyanide, carbon
monoxide and NOx, and heavy metals concerned by WHO are summarized, and their
application in exposure assessment and/or environmental epidemiology of cigarette
smoke exposure has been included. Ethics guidelines and guidelines for bioanalytical
method validation, toxicity, and classification of targeted constitutes are provided in
the appendices. The book will be very useful as a reference text for easy lookup of
information related to biomarkers of individual tobacco smoke components. It may
be useful for teaching graduate specialized courses and for professionals wishing to
transition their focus area to tobacco research.

Qingyuan Hu
Hongwei Hou
Zhengzhou, Henan, People’s Republic of China

xi
 1 Smoking and Health

1.1 GLOBAL TOBACCO EPIDEMIC

The World Health Organization (WHO) estimates that tobacco kills nearly 6 m ­ illion
people each year, and more than 5 million of those deaths are attributed to direct
tobacco use and more than 600,000 to indirect exposure through secondhand smoke.
This number could rise up to 8 million by 2030, according to the WHO. As of 2002,
approximately 5.5 trillion cigarettes are smoked annually around the world. The
global consumption of cigarettes in 2009, divided by the world’s population at that
time, results in an equivalent of 865 cigarettes smoked by each man, woman, and
child on the planet in 1 year (Wipfli, 2012).
Thousands of years ago, Native Americans rolled their own tobacco in corn or
palm leaves. The first factory-made cigarettes were produced 350 years later after
Europeans began colonizing the Americas. Factory-made cigarettes grew in pop-
ularity in the early twentieth century. Consumption doubled in the first decade,
between 1900 and 1910, from 50 to 100 billion cigarettes. By 1920, 300 billion
cigarettes were being consumed, and in 1940 consumption reached 1 trillion ciga-
rettes (Wipfli, 2012). The incidence of lung cancer also grew significantly in the
twentieth century, up until the early 1970s, particularly in the United States and in
the United Kingdom. The first paper on smoking and lung cancer was published by
Hoffmann in 1931 under the title “Cancer and smoking habits” (Hoffman, 1931).
In 1939, Mueller published Tabakmissbrauch und Lungencarcinom. Doll and Hill
and Wynder and Graham were among the first to investigate the association of lung
cancer with cigarette smoking after the Second World War. In 1950, Wynder and
Graham published a paper titled “Tobacco smoking as a possible etiologic factor in
bronchiogenic carcinoma,” which subsequently became a landmark in cancer epi-
demiology. Also in 1950, Doll and Hill published a preliminary report on smoking
and carcinoma of the lung in the United Kingdom. In 1954, they published a report
on their long-term cohort study of 40,000 British doctors (a cohort that could be fol-
lowed easily over time through the General Medical Council’s register), the results
of which led them to suggest that “tobacco has a significant adjuvant effect” (on
the steady increase in mortality they observed). In the United States, the Surgeon
General released his first report titled “Smoking and health” in 1964, in which he
concluded that smoking causes cancer (Surgeon General’s Advisory Committee on
Smoking and Health, 1964). In the United States, the consumption per capita of
factory-made cigarettes among the population at or above 18 years had more than
tripled from 1920 to 1950. Upon declining in 1954, following the publication of the
report by Doll and Hill, it rose again up to a record of 4345 cigarettes in 1963. From
1964 onward, it decreased gradually reaching 13% of the record incidence by 1981
(U.S. Department of Agriculture, 1981). This trend continued aided by aggressive
antitobacco campaigns, increased taxes on cigarettes, legislation banning smoking

1
2 Tobacco Smoke Exposure Biomarkers

in public areas, restriction to youth access, provisions for tobacco dependence

treatment, and prevention education (Bonnie, 2007). From 2000 to 2011, the sales
volume of all combustible tobacco decreased from 450.7 billion cigarette equiva-
lents to 326.6, a 27.5% decrease. The per capita sales of all combustible tobacco
products declined from 2148 to 1374, a 36.0% decrease. However, while sales of
cigarettes decreased 32.8% from 2000 to 2011, sales of cigars and loose tobacco
increased 123.1% over the same period. As a result, the percentage of total combus-
tible tobacco sales composed of cigars and loose tobacco increased from 3.4% in
2000 to 10.4% in 2011. The data suggest that certain smokers have switched from
cigarettes to other combustible tobacco products, most notably since a 2009 increase
in the federal tobacco excise tax that created tax disparities between product types
(MMWR, 2012). The United States have made historic progress in combating the
epidemic of tobacco-caused illness and death. Despite this progress, tobacco use
remains the leading cause of preventable death in many countries all over the world.
At present, it is estimated that tobacco is used by more than one-seventh of the
global population, including 1200 million smokers, and hundreds of millions of
smokeless tobacco (SLT) users (Khariwala et al., 2012).
Low- and middle-income economies, once mainly affected by basic food and
sanitation challenges and infectious diseases, have started to face a double burden
of disease and are increasingly suffering from noncommunicable diseases (NCDs),
such as chronic respiratory illnesses, cancer, diabetes, and cardiovascular disor-
ders. The health profile of low- and middle-income countries is therefore chang-
ing rapidly, precipitating increases in health-care costs and depriving families of
income. Tobacco is one of the main risk factors driving this change. It is the single
greatest preventable cause of NCDs, which are now the number one cause of pre-
mature death and disability worldwide, killing more people than HIV, tuberculosis,
and malaria combined.

1.2 SMOKE-RELATED DISEASES

A variety of cancers are attributable to tobacco use, including those of the lung,
head and neck, bladder, ureter, kidney and cervix, and myeloid leukemia. There are
69 identified carcinogens in tobacco smoke (Hoffmann and Hoffmann, 2001) and
no less than 16 in processed tobacco leaf. Among these, tobacco-specific nitrosa-
mines (TSNAs) (such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone [NNK]
and N′-nitrosonornicotine [NNN]), polycyclic aromatic hydrocarbons (PAHs)
(such as benzo[a]pyrene (B[a]P)), and aromatic amines (AAs) (such as 4-amino-
biphenyl) are among the most potent carcinogens (Hecht, 2003). Lung cancer and
head and neck cancer have similar genotoxic risk factors. While the vast majority
of lung cancers are associated with cigarette smoking alone, smoking in combina-
tion with heavy drinking is the main etiological agents of head and neck cancer
(Hunter et al., 2005).
The constituents of tobacco smoke most likely to promote carcinogenesis in dif-
ferent organs have been identified on the basis of the organ tropism observed in
occupationally induced cancers and of the sites of tumor formation observed in ani-
mal studies. Thus, Hoffmann and Hecht (1990) postulated PAHs, acetaldehyde, and
Smoking and Health 3

formaldehyde (with polonium-210 as a possible minor factor) are the main carcino-
gens involved in the development of cancer of the lung and of the larynx. Bladder
cancer is mainly attributable to AAs. Cancers of the esophagus and pancreas are
attributable to TSNAs, and PAHs and nitrosamines are involved in the development
of cancer of the oral cavity (Phillips, 1996).
Extensive scientific research has been conducted for decades in the field
of tobacco carcinogenesis aimed at understanding the mechanisms by which
tobacco products induce carcinoma (Ding et al., 2008; LeMarchand et al., 2008).
Meanwhile, it has been demonstrated that tobacco carcinogens and their metabo-
lites can bind covalently to DNA, resulting in the formation of DNA adducts. DNA
adducts, if unrepaired, can cause miscoding and permanent mutations, which can
activate oncogenes such as K-ras or inactivate tumor suppressor genes such as p53
(Figure 1.1; Ding et al., 2008; Hecht, 1999, 2003). These advances paved the way
for the use of tobacco carcinogen and toxicant metabolites and DNA adducts as
biomarkers of exposure to, and metabolism of, tobacco smoke constituents and/or
tobacco leaf components (Hecht et al., 2010).

1.3 SMOKE CONSTITUENTS OF CONCERN

Tobacco smoke is a very complex mixture of chemicals. By 1959, some 400 chem-
icals were known to be present in tobacco smoke. Meanwhile, more than 6000
tobacco smoke constituents and more than 2000 tobacco leaf components have
been identified. Moreover, 69 smoke constituents have been classified as carcino-
gens by the International Agency for Research on Cancer (IARC) (IARC Working
Group on the Evaluation of Carcinogenic Risks to Humans, 2007). In addition,
tobacco smoke contains many free radicals—those in the gaseous phase being
short lived and those in the particulate phase relatively long lived. In general, envi-
ronmental tobacco smoke (ETS) contains higher levels of identified carcinogens
than mainstream smoke (MSS) (which is directly inhaled by smokers) (Narkowicz
et al., 2013).
In 2008, the working group tasked with the elaboration of the Guidelines for
Implementation of Articles 9 and 10 of the WHO’s Framework Convention on
Tobacco Control (FCTC) identified nine cigarette smoke constituents for which
methods for testing and measuring in MSS (analytical chemistry) should be vali-
dated as a priority (WHO Framework Convention on Tobacco Control, 2008). Those
nine chemicals are included in the initial list of priority toxicants recommended by
the WHO Study Group on Tobacco Product Regulation (TobReg) for reporting and
regulation, also in 2008 (Series, 2008; Table 1.1). In 2009, Xie et al. (2009) devel-
oped a novel hazard index of mainstream cigarette smoke (MSS) for risk evalu-
ation of cigarette products. The yields of 29 toxic chemicals (4 TSNAs, 3 PAHs,
8 carbonyls, 7 phenolic compounds, HCN, NO, NOx, NH3, CO, Nic, and tar) in
MSS were determined. The toxicity of cigarette smoke was measured employing a
bacterial mutagenicity assay (Ames Salmonella mutagenicity assay) with cigarette
smoke condensate (CSC), the MTT cytotoxicity assay with CSC, the micronucleus
assay in vivo with CSC, and a mouse inhalation study. The results indicate that
contributions of seven toxic chemicals (CO, HCN, NNK, NH3, B[a]P, phenol, and
 4

Akt, PKA Apoptosis ↓

Receptor activation and Angiogenesis ↑
binding other changes Transformation ↑

Cigarette Metabolic Loss of normal

Smoking initiation/ Carcinogens DNA Persistence Mutations in RAS,
smoking growth control Cancer
nicotine addiction activation adducts miscoding TP53, and other genes mechanisms
Metabolic Repair
detoxification
Excretion Normal DNA Apoptosis

Cocarcinogens Tumor suppressor

tumor promoters gene inactivation Enhanced
Gene promoter methylation and other changes carcinogenicity

FIGURE 1.1 The development of carcinoma after carcinogenic damage induced by tobacco consumption. (From Khariwala, S.S. et al., Head Neck,
34, 441, 2012.)
Tobacco Smoke Exposure Biomarkers