SMOKING AND SKIN
Comparison of the appearance, physical qualities, morphology,
collagen synthesis and extracellular matrix turnover of skin
in smokers and non-smokers
ANINA
RAITIO
Faculty of Medicine,
Department of Dermatology
and Venereology,
University of Oulu
OULU 2005
ANINA RAITIO
SMOKING AND SKIN
Comparison of the appearance, physical qualities,
morphology, collagen synthesis and extracellular matrix
turnover of skin in smokers and non-smokers
Academic Dissertation to be presented with the assent of
the Faculty of Medicine, University of Oulu, for public
discussion in the Auditorium 4 of Oulu University
Hospital, on August 19th, 2005, at 12 noon
O U L U N Y L I O P I S TO, O U L U 2 0 0 5
Copyright © 2005
University of Oulu, 2005
Supervised by
Professor Aarne Oikarinen
Reviewed by
Professor Ilkka Harvima
Professor Veli-Matti Kähäri
ISBN 951-42-7788-0 (nid.)
ISBN 951-42-7789-9 (PDF) http://herkules.oulu.fi/isbn9514277899/
ISSN 0355-3221
OULU UNIVERSITY PRESS
OULU 2005
http://herkules.oulu.fi/issn03553221/
Raitio, Anina, Smoking and skin. Comparison of the appearance, physical qualities,
morphology, collagen synthesis and extracellular matrix turnover of skin in smokers
and non-smokers
Faculty of Medicine, University of Oulu, P.O.Box 5000, FIN-90014 University of Oulu, Finland,
Department of Dermatology and Venereology, University of Oulu, P.O.Box 5000, FIN-90014
University of Oulu, Finland
2005
Oulu, Finland
Abstract
Numerous adverse effects and health problems are associated with smoking, but the mechanisms of
the adverse effects of smoking on skin are not well documented. The aim of the present study was to
elucidate the effects of smoking on the structure, metabolism and appearance of skin.
The study population consisted of 98 Finnish males, of whom 47 were current smokers and 51
non-smokers. The main parameters under evaluation were the appearance and physical qualities of
skin, including skin wrinkling, thickness and elasticity. Biochemical analyses were performed to
assess the rate of type I and III collagen biosynthesis as well as the degradation of the extracellular
matrix (ECM) of skin in terms of matrix metalloproteinase levels (MMPs). To compare the
morphology of skin between the groups, histological and immunohistological studies were
performed, including assessments of the proportional area and width of dermal elastic fibres.
The results revealed decreased synthesis of type I and III collagens in smokers as well as changes
in the regulatory mechanisms which control the turnover of these and other extracellular matrix
proteins. The level of matrix metalloproteinase -8 (collagenase-2), a protease degrading both type I
and type III collagen, in suction blister fluid was significantly higher in smokers, indicating enhanced
degradation of these collagens. In skin tissue samples, the levels of the active forms of MMP-8 and
MMP-9 were significantly lower in smokers compared to non-smokers. Serum levels of MMP-8 were
slightly but not significantly higher in smokers, whereas the levels of the matrix metalloproteinases
MMP-2 and MMP-9 (72-kDa and 92-kDa gelatinase, respectively) were significantly higher in
smokers compared to non-smokers. Salivary MMP-8 and MMP-9 were lower in smokers compared
to non-smokers, but only the latter showed a statistically significant difference. The levels of the
tissue inhibitor of matrix metalloproteinases (TIMP-1) were significantly lower in the suction blister
fluid of smokers compared to non-smokers. In general, there were no significant differences in skin
thickness and elasticity or regeneration of barrier function, nor in the amount or width of elastic fibres
between the groups. We did not observe significant differences in skin wrinkling between smokers
and non-smokers, but smokers looked older than their age compared to non-smokers.
It can be concluded that the rate of type I and III collagen synthesis in skin is decreased and the
regulation of ECM turnover is altered in smokers, which may lead to deterioration of the tensile
strength and resiliency of skin in the long term, even though no morphological changes were detected
in the present study.
Keywords: ageing, collagen, elastin, extracellular matrix, matrix metalloproteinases, skin,
smoking
Acknowledgements
This thesis project was carried out at the Department of Dermatology and Venereology,
University of Oulu, during the years 1998-2005. I wish to express my deepest gratitude to
my supervisor, Professor Aarne Oikarinen, M.D., Ph.D., Head of the Department of
Dermatology and Venereology, for having shared his expertise in the field of connective
tissue research as well as for his patient support and guidance throughout the study. I
would also like to thank Professor Jaakko Karvonen, M.D., Ph.D. for his positive attitude
towards this project.
I am grateful to my co-workers Professor Tuula Salo, D.D.S., Ph.D., from the
Department of Dentistry, Professor Juha Risteli, M.D., Ph.D., from the Department of
Clinical Chemistry, Professor Kirsi Vähäkangas, M.D., Ph.D., from the Department of
Pharmacology and Toxicology, and Docent Matti Kallioinen, M.D., Ph.D, from the
Department of Pathology, all of whom were willing to share their professional expertise
and their experience in scientific work and to help a young researcher with numerous
questions. I am indebted to Professor Timo Sorsa, D.D.S., Ph.D. for his collaboration and
valuable comments.
The reviewers of this work, Professor Ilkka Harvima, M.D., Ph.D. and Professor VeliMatti Kähäri, M.D., Ph.D. are thanked for the careful revision and valuable comments on
this work.
I am deeply grateful to all the volunteers who participated in this study. I am thankful
to co-workers Hans Tuomas and Nina Kokkonen from the Department of Dentistry and to
Professor Juha Röning, Dr. Tech., Jukka Kontinen M.Sc. and Mikko Rasi from the
Department of Electrical and Information Engineering as well as to Helmi Wirkkala,
Päivi Annala and Sirpa Kangas for their technical assistance. I also express my gratitude
to Risto Bloigu, M.Sc., for his crucial help in the field of statistics, Ms Sirkka-Liisa
Leinonen, Lic. Phil., for her prompt language revision and Ms Soili Manninen for her
secretarial assistance. I would like to thank Hannu Marjamaa and the staff of the
photography laboratory at Oulu University Hospital for their flexible collaboration
throughout the project. I wish to thank warmly Ms Seija Leskelä for her contribution in
creating graphics of good quality for the publications.
I wish to thank all my colleagues, especially Kirsi-Maria Haapasaari, Riitta Riekki,
and Katriina Lehtinen, for sharing the world of science and the life of a specialising
doctor in the recent years. I would also like to thank chief physician Timo Järvinen, M.D.,
Docent Kaisa Tasanen-Määttä, M.D., Ph.D. and Docent Arto Lahti, M.D., Ph.D. for
supporting my work in both clinical and scientific fields. I express my sincere gratitude to
all my friends, particularly Kati Martikainen, Rita Tarkka and Kaisu Järvinen for sharing
the happy and the sad turns of life with me, and for their interest in the development of
this thesis.
I would like to express my sincere thanks to my mother Maarit Raitio for her love,
patience and guidance in my lifetime. Without her gentle prodding this thesis would have
taken even longer to finish.
My deepest gratitude belongs to my dear husband Trent Fitzgibbon, who has brought
true love and joy into my life and shares my dreams for the future.
This work was financially supported by the University of Oulu, the Finnish Society of
Dermatology and the Finnish Medical Foundation, which are gratefully acknowledged.
Oulu, May 2005
Anina Raitio
Abbreviations
BCC
COPD
DU
ECM
kDA
MMP
MPa
mRNA
PINP
PIIINP
P53
RIA
SBF
SCC
SD
TGF-β
TEWL
TIMP
UV
Basal cell carcinoma
Chronic obstructive pulmonary disease
Densitometric unit
Extracellular matrix
kilo Dalton
Matrix metalloproteinase
Megapascal
messenger ribonucleic acid
Aminoterminal propeptide of type I procollagen
Aminoterminal propeptide of type III procollagen
53-kilodalton phosphoprotein
Radioimmunoassay
Suction blister fluid
Squamous cell carcinoma
Standard deviation
Transforming growth factor β
Transepidermal water loss
Tissue inhibitor of metalloproteinases
Ultraviolet
List of original publications
This thesis is based on the following publications, which are cited in the text by their
Roman numerals.
I
Raitio A, Kontinen J, Rasi M, Bloigu R, Röning J & Oikarinen A (2004) Comparison
of clinical analysis and computerized image analysis in the assessment of skin ageing
in smokers and non-smokers. Acta Derm Venereol 84: 422-427.
II
Knuutinen A, Kallioinen M, Vähäkangas K & Oikarinen A (2002) Smoking and skin:
a study of the physical qualities and histology of skin in smokers and non-smokers.
Acta Derm Venereol 82: 36-40.
III Knuutinen A, Kokkonen N, Risteli J, Vähäkangas K, Kallioinen M, Salo T, Sorsa T
& Oikarinen A (2002) Smoking affects collagen synthesis and extracellular matrix
turnover in human skin. Br J Dermatol 146: 588-594.
IV Raitio A, Tuomas H, Kokkonen N, Salo T, Sorsa T, Hanemaaijer R & Oikarinen A.
Levels of matrix metalloproteinase –2, -9 and -8 in the skin, serum and saliva of
smokers and non-smokers, submitted for publication
Anina Raitio née Knuutinen
Contents
Abstract
Acknowledgements
Abbreviations
List of original publications
Contents
1 Introduction................................................................................................................... 13
2 Review of the literature................................................................................................. 15
2.1 Tobacco .................................................................................................................15
2.1.1 Tobacco smoke ................................................................................................15
2.1.2 Nicotine and cotinine.......................................................................................16
2.2 Skin........................................................................................................................18
2.2.1 Collagen ..........................................................................................................19
2.2.2 Elastic fibres ....................................................................................................21
2.2.3 Matrix metalloproteinases in skin....................................................................22
2.2.4 Skin ageing ......................................................................................................24
2.3 Smoking and skin ..................................................................................................26
2.3.1 Smoking and skin diseases ..............................................................................26
2.3.2 Smoking and appearance .................................................................................28
2.3.3 Effect of smoking on skin connective tissue and wound healing ....................29
2.3.4 Oral manifestations of smoking.......................................................................30
2.4 Non-invasive methods in dermatological research -with special
reference to the methods used in this project.........................................................31
2.4.1 Evaluation of skin surface contour ..................................................................31
2.4.2 Skin thickness..................................................................................................31
2.4.3 Elasticity measurements ..................................................................................32
2.4.4 Suction blister method.....................................................................................33
2.4.5 Barrier function ...............................................................................................33
3 Aims of the study .......................................................................................................... 35
4 Material and methods.................................................................................................... 36
4.1 Subjects .................................................................................................................36
4.1.1 Smoking history ..............................................................................................36
4.1.2 Alcohol consumption.......................................................................................37
4.1.3 Sun exposure ...................................................................................................38
4.1.4 Medical history................................................................................................38
4.2 Methods.................................................................................................................40
4.2.1 Clinical status ..................................................................................................40
4.2.2 Clinical and computerized analyses of facial photographs (I).........................40
4.2.3 Skin thickness (II)............................................................................................41
4.2.4 Skin elasticity (II) ............................................................................................41
4.2.5 Suction blister technique (III, IV)....................................................................42
4.2.6 Transepidermal water loss ...............................................................................42
4.2.7 Immunohistochemistry (II,III).........................................................................42
4.2.8 Morphometric analyses of elastic fibres (II)....................................................43
4.2.9 Sampling for biochemical assays ....................................................................46
4.2.10 Biochemical methods (III, IV).......................................................................46
4.2.11 Statistical analysis..........................................................................................48
4.2.12 Ethical aspects ...............................................................................................49
5 Results........................................................................................................................... 50
5.1 Appearance and wrinkling (I)................................................................................50
5.2 Skin thickness and elasticity (II) ...........................................................................50
5.3 Biochemical analyses ............................................................................................51
5.3.1 Urinary nicotine metabolites ...........................................................................51
5.3.2 Procollagen propeptides in suction blister fluid and serum (III) .....................51
5.3.3 MMP-8 in suction blister fluid (III).................................................................53
5.3.4 TIMP-1 in suction blister fluid and serum (III) ...............................................54
5.3.5 Gelatinases and MMP-8 in serum (IV)............................................................54
5.3.6 Gelatinases and MMP-8 in skin tissue (IV).....................................................55
5.3.7 Salivary MMP-8 and MMP-9 (IV)..................................................................55
5.4 Skin histology and restoration of epidermal barrier function ................................56
5.4.1 Proportional area and width of elastic fibres (II).............................................56
5.4.2 Number of PINP-positive fibroblasts in papillary dermis (III)........................56
5.4.3 Epidermal thickness and number of Langerhans cells.....................................56
5.4.4 Number of vascular lumens in papillary dermis ..............................................56
5.4.5 Restoration of epidermal barrier function........................................................57
6 Discussion ..................................................................................................................... 58
6.1 Appearance and wrinkling (I)................................................................................58
6.2 Physical qualities of skin (II).................................................................................59
6.3 Collagen synthesis and ECM turnover (III, IV) ....................................................60
6.4 Histology ...............................................................................................................62
6.5 Study design and subjects......................................................................................63
6.6 Strengths and weaknesses of the study..................................................................64
7 Conclusions................................................................................................................... 67
References
1 Introduction
Despite the numerous known health hazards related to smoking, 47 % of men and 12 %
of women in the world smoke (WHO 1997), and tobacco is the leading cause of
preventable deaths in the United States (McGinnis & Foege 1993). A large Finnish
population study (Helakorpi et al. 1998) of the health behaviour of 5000 citizens aged 15
to 64 years showed that 30 % of males and 20 % of females in Finland were daily
smokers in 1998. The prevalence of smoking has decreased among males during the last
decades but increased among females during this time, especially in the younger age
cohorts (Helakorpi et al. 1998, Heloma et al. 2004). At the same time, the incidence of
lung cancer has been increasing among women, whereas the incidence of lung cancer
among males has been decreasing since the early 1970s (Heloma et al. 2004). Similar
trends have been observed in the United States, where women are beginning to smoke at
a younger age and tend to smoke more heavily than before (Bartecchi et al. 1994). In
Finland, 97 % of daily smokers reported smoking cigarettes, and only 3 % were daily
cigar smokers. 78 % of daily smokers expressed concern about the health consequences
of smoking, and 60% of men and 56% of women wished to quit. (Helakorpi et al. 1998)
Smoking is associated with numerous cancers, and chronic obstructive pulmonary
disease (COPD) is primarily a disease of smokers (Pride 1995, Hecht 1997, Hecht 1999).
Smoking increases the risk for cutaneous squamous cell carcinoma (De Hertog et al.
2001), and recurrences of SCC are more frequent in smokers and ex-smokers (Karagas et
al. 1992). Smoking is associated with skin diseases such as psoriasis, (Naldi et al. 1992,
Mills et al. 1992, Poikolainen et al. 1994) palmoplantar pustulosis (Akiyama et al. 1995,
Eriksson et al.1998) and infectious eczematous dermatitis (Karvonen et al. 1992).
Ultraviolet radiation is a well-known external factor causing skin damage, which has
been shown both clinically and at a histological level. Tobacco smoke is another external
factor that can potentially affect skin. Previous studies have shown increased wrinkling in
smokers compared to non-smokers, and the risk increases further when smoking is
combined with excessive sun exposure (Daniell 1971, Model 1985, Kadunce et al. 1991,
Ernster et al. 1995, Yin et al. 2001). Alterations in the elastic tissue of smokers have been
reported in both sun-protected skin (Francès et al. 1991) and sun-exposed skin (Boyd et
al. 1999), but the overall histology and metabolism of the skin of smokers compared to
non-smokers are not well documented.
14
The aim of the present study was to supply further knowledge of the basis of the
adverse effects of smoking on skin, such as premature wrinkling (Daniell 1971, Model
1985, Kadunce et al. 1991, Ernster et al. 1995,Yin et al. 2001), increased risk of
malignancies (Karagas et al. 1992, De Hertog et al. 2001), and aberrant wound healing
(Siana et al. 1989, Goldminz & Bennett 1991), which have all been previously reported
in smokers. In terms of the number of parameters evaluated, the present study is one of
the largest to date to compare the skin characteristics of smokers and non-smokers. Not
only were the appearance and qualities of skin surface investigated, but skin metabolism
at the biochemical level was also explored. The physical qualities of skin, such as
thickness and elasticity, were compared between smokers and non-smokers, as were skin
wrinkling and histology. At the biochemical level, the rates of ongoing collagen synthesis
and extracellular matrix (ECM) turnover of skin in vivo were compared between smokers
and non-smokers.
According to an American survey, nearly one fourth of smokers believe that most or
some smokers would quit if they knew that smoking increases facial ageing and
wrinkling (Demierre et al. 1999). The information of the adverse effects of smoking on
skin should be addressed especially at adolescents, since 90 % of smokers begin to smoke
during adolescence (Burns 1992, Skaar et al. 1997).
2 Review of the literature
2.1 Tobacco
2.1.1 Tobacco smoke
The tobacco plant belongs to the Solanaceae family. The species most commonly used in
the production of cigarettes, cigars, pipes and smokeless tobacco are Nicotiana tabacum
and Nicotiana rustica (Hoffman & Hoffman 1997). Tobacco smoke is an aerosol,
consisting of a gaseous phase and a particulate phase. The gaseous phase consists of
nitrogen, oxygen, carbon monoxide and numerous irritating agents (Burns 1992, Hecht
1999). Approximately 95 % of the weight of mainstream smoke comprises gaseous
compounds, and the remaining 5 % consists of particulates with over 3500 individual
components (Hecht 1999). The particulate matter of smoke that remains after the removal
of water and nicotine is called tar, and it contains numerous carcinogens, including
polynuclear aromatic hydrocarbons, N-nitrosamines and aromatic amines (Zevin et al.
1998). The portion of smoke that is inhaled is referred to as mainstream smoke, while the
portion emerging from the burning cone and the mouthpiece between inhalations is called
side-stream smoke. Even though the filter retains most of the particulate matter, both
gaseous compounds and particulates are inhaled by the smoker. (Pryor 1997) Cigarette
smoke is usually more acidic (pH 5.5) than smoke from cigars and pipes, which affects
both the absorption and the excretion of nicotine. The alkaline smoke of pipes and cigars
is absorbed more readily by the buccal mucosa, but at the level of the lower airways,
nicotine is absorbed rapidly regardless of the pH of smoke. (Benowitz 1988) As early as
the 1960s, it was known that tobacco contains both tumour-promoting and tumourinitiating agents, and the carcinogenicity of tobacco was reviewed by Wynder & Hoffman
(1968). More than fifty carcinogens have been identified in tobacco smoke. Though
polynuclear aromatic hydrocarbons and N-nitrosamines are considered the most
important carcinogens in tobacco smoke, several other constituents, such as polonium210, chromium, cadmium, lead, arsenic, nickel as well as aldehydes and aromatic amines,
16
are potential carcinogens (Table 1). (Hecht 1997, Hoffman & Hoffman 1997, Hecht
1999)
2.1.2 Nicotine and cotinine
Nicotine is a tertiary amine consisting of a pyridine ring and a pyrrolidine ring (Fig.1). It
is a weak base, which is able to cross cell membranes in its unionized form. Nicotine
binds to acetylcholine receptors in the autonomic ganglia, adrenal medulla,
neuromuscular junctions and brain. The stimulation of nicotinic receptors leads to a
release of catecholamines, dopamine, serotonin, vasopressin, growth hormone and
ACTH. (Benowitz 1988) The estimated nicotine yield per cigarette is 1.0-2.3 mg (Idle
1990). The blood nicotine concentration peaks approximately 10 minutes after the
smoking of a cigarette, after which it gradually declines, the average half-life being two
to three hours (range one to four hours) (Benowitz 1988).
Nicotine is highly addictive, and it is known that smokers can maintain a balanced
nicotine level in their circulation by adjusting the depth and frequency of puffs,
depending on the nicotine yield of the cigarette. Due to the fact that smokers smoke
several times a day, nicotine accumulates into the smoker`s body. (Benowitz 1988, Jacob
et al. 1999) Nicotine is mostly metabolized by the liver, partly via the cytochrome P450
pathway, and only 5 to 10 percent is excreted into urine as such (Vähäkangas & Pelkonen
1993, Benowitz 1996), (Fig. 1). The rate of renal elimination depends on pH and urine
flow in such a manner that acidification of urine increases excretion (Benowitz et al.
1983). Wald et al. (1984) reported urinary nicotine concentrations of 1393 ng/ml in
cigarette smokers, 1048 ng/ml in pipe smokers and less than 50 ng/ml in non-smokers.
The primary metabolites of nicotine are cotinine and nicotine-N-oxide (Fig.1)
(Benowitz 1988). Cotinine arises from enzymatic (P450) or auto-oxidation of nicotine,
and most of the further metabolites arise via cotinine (Gorrod 1993). An average of 70 to
80 percent of nicotine is estimated to be converted into cotinine, and most of cotinine is
further metabolized, leaving only a portion of 10 to 15 percent of cotinine to be excreted
in urine (Benowitz 1996). The estimated cotinine yield per cigarette is 9-57 μg, and the
estimated nornicotine yield 27-88 μg per cigarette. The average blood cotinine
concentration in smokers is approximately 300 ng/ml, but the range is wide, from 0 to
900 ng/ml. Cotinine is eliminated fairly slowly, the estimated half-life being
approximately 19 hours (Benowitz et al. 1983, Jacob et al.1999). Pipe smokers have been
found to have higher serum levels of cotinine (389 ng/ml) than cigarette smokers (306
ng/ml) or cigar smokers (121 ng/ml), but the urinary concentrations of nicotine and
cotinine seem to be fairly equal in cigarette smokers and pipe smokers (Wald et al. 1981,
Wald et al. 1984, Jacob et al. 1999). Previous cigarette smokers who have switched to
cigars or pipes have been shown to inhale the smoke, thus acquiring amounts of nicotine
and carboxyhemoglobin comparable to those found in cigarette smokers (Turner et al.
1977).
Cotinine levels can be measured from serum, saliva or urine, to evaluate the amount of
tobacco consumption. Salivary concentrations have been claimed to be unreliable, due to
the possibly increased concentration of cotinine in the salivary gland (Idle 1990).
17
Numerous confounding factors need to be taken into consideration when evaluating the
amount of tobacco consumption with laboratory methods based on assessment of the
amount of nicotine metabolites in body fluids. These possible confounding factors
include the dietary intake of nicotine (especially in vegetarians), the intersubject
variability in nicotine metabolism and cotinine metabolism and the rate of excretion of
the two substances (Idle 1990). However, environmental tobacco smoke is probably a
more significant source of nicotine in non-smokers (Benowitz 1996). Since self-reported
tobacco consumption tends to be underestimated, assessment of the concentrations of
cotinine and other nicotine metabolites in body fluids increases the reliability of the
studies comparing smokers, non-smokers and ex-smokers (Benowitz 1996). The time
needed for an average blood cotinine concentration of approximately 300 ng/ml in a
smoker to clear to the cut-off level suggested for non-smoking status (10 ng/ml) is
estimated to be usually 3.9 days, but it may be as long as a week in slow metabolizers,
and therefore at least one week`s abstinence from smoking is recommended before
assessment of smoking status by means of cotinine concentrations in smoking cessation
studies (Benowitz et al. 1983).
Fig. 1. Simplified representation of the main metabolic routes of nicotine. Modified from
Benowitz 1988.
18
Table 1. Important adverse effects of the main components of tobacco smoke. Modified
from Gupta et al. 1996 and Hoffman & Hoffman 1997.
Substance
Effects
Nicotine
Addiction, vasoconstriction, cardiovascular disease
Polycyclic aromatic hydrocarbons PAHs
Lung cancer,
Laryngeal cancer,
Cancer of the oral cavity
Tobacco-specific nitrosamines
Lung cancer,
Laryngeal cancer,
Cancer of the oral cavity,
Cancer of the pancreas,
Esophageal cancer
Tar
Cardiovascular disease,
Chronic obstructive pulmonary disease
Carbon monoxide and
Cardiovascular disease,
Nitrogen oxides
Chronic obstructive pulmonary disease
Aromatic amines
Cancer of urinary bladder
2.2 Skin
Human skin is composed of two layers, epidermis and dermis, each of which has its
specific functional importance (Fig. 2). Epidermis is a protective layer, which consists
mainly of keratinocytes and, to a lesser extent, melanocytes, Langerhans cells, Merkel
cells and unmyelinated axons. Dermis consists of eccrine and apocrine glands, hair
follicles, veins, nerves and a fine network of collagen fibres, elastic fibres, and other
components of the extracellular matrix (ECM). (Murphy 1997) ECM is mainly composed
of proteins and complex sugars, which form fibrillar networks and ground substance.
Collagen is an important structural component of skin connective tissue, giving tensile
strength to skin. Approximately 70 to 80 % of the dry weight of skin consists of collagen.
The most abundant collagen types in skin are the types I and III, the former of which
accounts for 80% of the total collagen content of skin and the latter for approximately 15
%. (Uitto et al. 1989) The other collagens found in dermis include type IV collagen,
which is abundant in the basement membrane, type V collagen, which is located
pericellularly, type VI collagen, which plays a role in matrix assembly and is present as
microfibrils between collagen fibers, and type VII collagen, which is a structural
component of anchoring fibrils. Elastin accounts for only about 1-2% of the dry weight of
skin. Elastic fibres provide skin with elasticity and resilience and are organized as a threedimensional net in dermis. Proteoglycans and glycosaminoglycans, such as hyaluronan,
are present in ECM in small quantities (0.1-0.3 % of the dry weight of skin) but are of
central importance in the maintenance of water balance in skin. (Oikarinen 1994,
Bernstein & Uitto 1996)
19
Epidermis
Dermis
Elastic fibres
Collagen fibres
Fig. 2. Skin biopsy sample stained with Verhoeff under 40 x magnification shows the collagen
fibres in grey and the elastic fibres in black colour.
2.2.1 Collagen
A collagen molecule consists of three α chains, which can be mutually similar or
dissimilar polypeptide chains. Type I collagen, for example, is composed of two identical
α1(I) chains, which are synthesized from the same gene, and an α2(I) chain, which is
synthesized from another gene, whereas type III collagen consists of three identical α1
(III) chains encoded by a single gene. (Prockop 1992) Each of these polypeptide chains
forms a leftward helix, and the three helical chains wrap around each other to form a
right-handed superhelix, which is stabilized in the extracellular space by cross-linking
between chains and molecules (Fig.3). The triple-helical conformation of the collagen
molecule requires the presence of glycine as every third amino acid in the polypeptide
chains, which results in a series of Gly-X-Y, where X and Y can be any amino acid except
glycine. The other amino acids essential for the triple-helical structure are proline and 4-
20
hydroxyproline. Proline is frequently found in the X position and 4-hydroxyproline in the
Y position of the amino acid sequence. Formation of 4-hydroxyproline and C-terminal
disulfide bonds is crucial for the formation of the triple helix. Lysine is an amino acid
also commonly found in the Y position, and it serves as a site for sugar attachment when
converted into hydroxylysine by a specific enzyme. (Prockop & Kivirikko 1984,
Burgeson & Morris 1987, Uitto et al. 1989)
Skin collagen synthesis takes place mainly in fibroblasts. The synthesis of collagen has
an intracellular and an extracellular phase, both of which involve post-translational
modifications crucial for the formation of stable triple-helical collagen molecules, with
appropriate cross-links (Fig. 3). Intracellular modifications include hydroxylation of
proline residues in the Y position into 4-hydroxyproline and of some proline residues in
the X position into 3-hydroxyproline as well as hydroxylation of lysine residues in the Y
position into hydroxylysine. (Prockop & Kivirikko 1984, Myllyharju & Kivirikko 2001)
The reactions are catalyzed by specific enzymes, prolyl-4-hydroxylase, prolyl-3hydroxylase and lysyl hydroxylase, respectively, in the presence of Fe2+, oxygen, 2oxoglutarate and ascorbate. Ascorbate is essential for the biosynthesis of collagen and
acts as a cofactor in the hydroxylation of proline and lysine. (Kivirikko & Myllylä 1982)
Glycosylation of hydroxylysine and asparagine residues also takes place intracellularly.
Both hydroxylation and glycosylation continue until triple-helical conformation of the
developing molecule is achieved.
Fig. 3. Biosynthesis of collagen. Modified from Oikarinen (1992).
The procollagen molecules synthesized intracellularly are excreted into the extracellular
space, where the large aminoterminal and carboxyterminal propeptides of the
procollagens are cleaved off en block by specific endoproteinases (Risteli et al. 1995).
21
Cleavage of the propeptides enables the initiation of fibril formation (Prockop & Fertala
1998). The molecular weights of the aminoterminal propeptides of type I and III
procollagens (PINP and PIIINP) are 35 000 and 42 000, respectively (Risteli et al. 1995,
Risteli et al. 1988). Since collagen is synthesized in a precursor form, and the cpropeptide and n-propeptide are cleaved off extracellularly, the amounts of procollagen
propeptides in serum and interstitial fluid reflect the rate of ongoing collagen synthesis
(Annala et al. 1993, Autio 1994, Oikarinen 1997). In adult human skin, the ratio of type I
to type III collagen is approximately 5-6:1 (Uitto et al. 1989), but there may be a
tendency towards an increased relative amount of type III collagen in the skin of the
elderly (Lovell et al. 1987).
Collagen metabolism is disturbed in numerous diseases, including diabetes (Seibold et
al. 1985), scleroderma (Rodnan et al.1979, Uitto et al. 1979), eosinophilic fasciitis
(Kähäri et al. 1990), osteogenesis imperfecta, Marfan syndrome and Ehlers-Danlos
syndrome, which has a wide variety of clinical manifestations, depending on the
underlying defect in collagen metabolism (Prockop et al. 1979, Prockop & Kivirikko
1984, Myllyharju & Kivirikko 2001). Several gene defects behind collagen-related
diseases have been elucidated (Prockop 1992), and cytokines, such as transforming
growth factor-β (TGF-β), are associated with increased synthesis of collagen and
formation of tissue fibrosis (Varga & Jimenez 1995).
2.2.2 Elastic fibres
Elastic fibres are crucial for the resilience and elasticity of skin, even though they make
up only 1-2 per cent of the dry weight of skin (Bernstein & Uitto 1996). Elastic fibres are
composed of elastin, which accounts for 90 % of the mature fibre, and of a microfibrillar
component, which consists of microfibrils, 10 to 12 nm in size, primarily located around
elastin but partly also interspersed within it. (Rosenbloom et al. 1993) Microfibrils
contain several glycoproteins, of which fibrillin has been studied in most detail
(Rosenbloom et al. 1993). Versican is a large glycoprotein, which occurs in both normal
and sun-damaged elastic fibres together with hyaluronic acid (Uitto & Bernstein 1998).
Elastic fibres are assembled in dermis as a three-dimensional net. Oxytalan fibres occur
perpendicular to epidermis and are connected to elaunin fibres, which run parallel to
epidermis (Lewis et al. 2004).
Elastin is a polypeptide approximately 70 kDa in size, which is encoded by a single
copy gene found in chromosome 7 (Christiano & Uitto 1994, Debelle & Tamburro 1999).
Elastin and microfibrillar proteins are synthesized primarily by fibroblasts (Sephel &
Davidson. 1986, Lewis et al. 2004), but recent knowledge indicates that keratinocytes
take part in the formation of microfibrils in papillary dermis, since they have been shown
to synthesize both fibrillin-1 and fibrillin-2 in vivo and in vitro (Haynes et al. 1997). The
elastin gene encodes tropoelastin, a precursor protein for elastin. Tropoelastin is
synthesized intracellularly and thereafter excreted into the extracellular space, where
cross-linking takes place. (Rosenbloom et al. 1993) A high degree of cross-linking is
characteristic of elastin, and the formation of desmosines is unique to it. A copperdependent enzyme, lysyl oxidase, is involved in the cross-linking of both collagen and
22
elastin. (Davidson 1987) In the cross-links of elastin, the lysine residues present as pairs
in polyalanine sequences in such a way that there are always two or three amino acids,
usually alanines, between two lysine residues, thus forming sequences of Lys-Ala-AlaLys, or Lys-Ala-Ala-Ala-Lys. These alanine-rich cross-linking domains have an α-helical
conformation. In addition to the cross-linking domains, elastin has hydrophobic domains
containing glycine, proline and valine residues. The mechanisms of elastic fibre assembly
are not well known, but microfibrils become visible first, after which elastin appears as
an amorphous material. The amorphous material coalesces and forms the core of the
fibre. Most microfibrils are transferred to the outer aspect of the fibre, where they remain
in mature tissue. (Rosenbloom et al. 1993, Christiano & Uitto 1994) Growth factors and
cytokines take part in the regulation of elastin gene expression and biosynthesis. Elastin
expression is upregulated in vitro by, for example, insulin-like growth factor I and
transforming growth factor β1 (Kähäri et al. 1992b, Debelle & Tamburro 1999). Other
cytokines, such as tumor necrosis factor α (TNF-α) and, to a lesser extent, interferon γ
(IFN γ) down-regulate the expression of elastin gene expression (Kähäri et al. 1992a).
Elastin is metabolized by proteolytic enzymes, such as serine-type elastases and matrix
metalloproteinases, of which stromelysin, macrophage metalloelastase (MMP-12),
matrilysin (MMP-7) and the gelatinases (MMP-2 and MMP-9) are the most active
towards elastic fibres (Lewis et al. 2004).
With increasing age, elastic fibres disintegrate especially in sun-exposed skin, in which
actinic damage is manifested as accumulated, thickened and tangled elastic fibres in light
microscopy (Braverman & Fonferko 1982, Bernstein et al. 1994). In photodamaged skin,
the microfibrils at the dermoepidermal junction and in the papillary dermis are reduced
and deformed (Watson et al. 1999). Abnormalities in elastic fibre morphology and
assembly are also seen in a number of congenital skin diseases, and specific gene defects
explaining genodermatosis have recently been found. Cutis laxa is a skin disease that
presents in mild cases as predominant wrinkling and in severe genetic cases as
widespread elastic fibre damage in skin and internal organs. Disturbed elastin crosslinking, due to defects in copper metabolism and/or function of lysyl oxidase, has been
suggested to cause X-linked cutis laxa (Debelle & Tamburro 1999), whereas defects in
the fibrillins 1 and 2 are found in Marfan syndrome and congenital contractural
arachnodactyly, respectively (Christiano & Uitto 1994).
2.2.3 Matrix metalloproteinases in skin
Three major families of proteases degrade components of the extracellular matrix. These
protease families are called serine, cysteine and metalloproteinases, and they are
important in tissue repair and inflammation as well as in tumour invasion and metastasis
(Mauch 1998). Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix
metalloproteinases (TIMPs) regulate the degradation of collagen, elastin and other
components of ECM. (Mauch 1998) Matrix metalloproteinases are zinc-dependent
neutral endopeptidases, which are divided into four main groups according to their
primary structure and substrate specificity: collagenases, gelatinases, stromelysins, and
membrane-type matrix metalloproteinases, as well as novel groups consisting of MMP-
23
19 and MMP-20, which do not fit into any of the above mentioned subgroups of MMPs
(Uitto et al. 1989, Kähäri & Saarialho-Kere 1999). The classification of MMPs is shown
in Table 2.
Knowledge of the function of MMPs is expanding rapidly, but the natural substrates of
various MMPs are still only partially known (Woessner 1998). The collagenases MMP-1,
MMP-8 and MMP-13 are the principal proteinases capable of initiating degradation of
the fibrillar collagens I, II, III and V, but the 72-kDa gelatinase (MMP-2) and MT-1 MMP
(MMP-14) are also able to cleave fibrillar collagen, whereas the 92-kDa gelatinase,
MMP-9, takes part in the final degradation of fibrillar collagens after their cleavage and
regulates re-epithelialization of skin (Aimes & Quigley 1995, Kähäri & Saarialho-Kere
1999, Mohan et al. 2002). MMP-1 degrades type III collagen at a faster rate than the
types I and II, whereas MMP-8 degrades type I collagen at a rate much faster than type
III (Jeffrey 1998, Nwomeh et al. 1999). Despite the name “neutrophil collagenase”,
MMP-8 has been shown also to be synthesized by other cells; at least chondrocytes,
endothelial cells and rheumatoid synovial fibroblasts are capable of synthesizing MMP-8
(Jeffrey 1998, Hanemaaijer et al. 1997). Elastin is a substrate for the 72-kDa and 92-kDa
gelatineses, MMP-3, MMP-7, MMP-10, and MMP-12. (Mauch 1998, Kähäri &
Saarialho-Kere 1999)
The wound healing process starts with the formation of a fibrin clot, followed by the
release of various growth factors from injured cells and ECM, inflammation, formation of
granulation tissue, epithelialization and finally matrix production and remodelling
(Mauch 1998. Ravanti & Kähäri 2000). Reepithelialization is initialized within hours
after tissue damage, and it is first manifested as proliferation of keratinocytes. Newly
formed epithelial cells migrate either on the basement membrane, if possible, or across a
transient matrix of fibrin and fibronectin while the basement membrane is under
construction. (Mauch 1998, Singer & Clark 1999) During remodelling, the temporary
ECM is degraded and replaced by collagen. MMP-1 and MMP-8 are particularly
important in the regulation of the wound healing process, but other MMPs, such as
MMP-2, MMP-9 and MMP-19, are also involved in wound repair (Oikarinen et al. 1993,
Pilcher at al. 1998, Mauch 1998, Nwomeh et al. 1999, Mohan et al. 2002, Hieta et al.
2003). MMP-1 is expressed by migrating basal keratinocytes in all types of cutaneous
wounds, and the completion of reepithelialization leads to decreased expression of MMP1 (Pilcher et al. 1998). Comparison of normally healing ulcers with nonhealing ulcers has
indicated that overexpression and activation of MMP-8 may be involved in the
pathogenesis of chronic ulcers (Nwomeh et al. 1999).
TIMPs are a family of proteins inhibiting MMPs. Up till now, four different TIMPs
have been identified. TIMPs regulate the activity of MMPs through non-covalent binding
to MMP molecules. TIMP-1 and TIMP-2 are capable of inhibiting almost all MMPs, but
TIMP-1 preferentially blocks the function of MMP1, whereas TIMP-2 is more important
in the inhibition of gelatinases A and B (Kähäri & Saarialho-Kere 1999). TIMP-1 and
TIMP-3 are found in the keratinocytes of normally healing wounds, but the expression of
both TIMP-1 mRNA and TIMP-3 mRNA has been reported to be totally absent in the
epithelial cells of chronic wounds, suggesting that an imbalance between MMPs and their
inhibitors leads to aberrant wound healing (Vaalamo et al. 1996, Saarialho-Kere 1998,
Vaalamo et al. 1999). The expression of MMPs in cells is also regulated by various
cytokines, such as interleukin -1 and tumour necrosis factor-α, and by growth factors,
24
which can induce rapid ne novo synthesis of MMPs (Mauch 1998). Cytokine actions are
transmitted through various signalling pathways, such as the ceramide pathway and the
mitogen-activated protein kinase pathways ERK ½, SAPK/JNK and p38 (Reunanen et al.
1998, Reunanen et al. 2002).
Table 2. Classification of human matrix metalloproteinases. Modified from Mauch 1998,
Kähäri & Saarialho-Kere 1999.
MMP subgroup
Enzyme
MMP
Substrate specificity for collagen and
elastin
Collagenases
Gelatinases
Fibroblast collagenase
MMP-1
Collagens I, II, III, II,VIII,X
Neutrophil collagenase
MMP-8
Collagens I, II, III
Collagenase-3
MMP-13
Collagens I, II, III, IV, IX, X, XI
Gelatinase A
MMP-2
Collagens I, IV, V, VII, X, XI, elastin
Gelatinase B
MMP-9
Collagens IV, V, XIV, elastin
Stromelysin-1
MMP-3
Collagens II, IV, IX, X
Stromelysin-2
MMP-10
Collagen IV
Stromelysin-3
MMP-11
Collagen IV
Metalloelastase
MMP-12
Collagen IV, elastin
Matrilysin
MMP-7
Collagen IV, elastin
Matrilysin-2
MMP-26
Membrane type
MT1-MMP
MMP-14
Collagens I, II, III
MMPs
MT2-MMP
MMP-15
Not known
MT3-MMP
MMP-16
Activates proMMP-2
MT4-MMP
MMP-17
Not known
Stromelysins
MT5-MMP
MT6-MMP
Novel MMPs
MMP-19, MMP-20, MMP-21,
Not known
MMP-23, MMP-27, MMP-28
2.2.4 Skin ageing
Skin ageing can be divided into intrinsic or chronological ageing and extrinsic ageing,
which is caused mainly by ultraviolet (UV) radiation. The ultraviolet radiation reaching
the earth’s surface consists of UVA (320-400 nm) and UVB (280-320 nm) radiation
(Mariéthoz et al. 1998). UVA penetrates deep into tissues and has direct effects on dermal
cells, including fibroblasts, whereas UVB has indirect effects on ECM turnover by
inducing the production of certain lymphokines and cytokines (Kligman 1989, Oikarinen
1997). Reactive oxygen species activated by UV radiation play an important role in UVinduced DNA damage, cellular senescence and ageing (Mariéthoz et al. 1998). Upon
ageing, the capacity to repair DNA decreases (Grossman & Leffell 1997). UVA radiation
has been shown to affect the function of transforming growth factor β (TGF-β) and to
downregulate TGF-β type II receptor, leading to reduced collagen and photoageing of
25
skin. (Yin, et al. 2003, Quan et al. 2004) UVB radiation induces the matrix
metalloproteinase mRNAs, proteins and activities in human skin within hours of UVB
exposure (Fisher et al. 1996).
Intrinsic or chronological ageing presents as fine, shallow lines and sagging of skin in
all body areas, as the elasticity of skin decreases, whereas extrinsic ageing or photoageing
is seen predominantly in the face and the dorsal sides of the hands. Both intrinsic and
extrinsic ageing occur on sun-exposed sites. Photoaged skin has coarse and fine wrinkles
and is rough with irregular pigmentation, teleangiectasiae, and various benign and
malignant tumors. (Kligman & Kligman 1986, Gilchrest 1989, Gilchrest & Yaar 1992,
Cook & Dzubow 1997) Skin wrinkling becomes evident gradually over age, especially in
the sun-exposed areas, such as the face.
Actual wrinkle formation is thought to be due to the combined effects of structural
changes in ageing skin, gravitational forces and the effects of facial muscle contractions,
which enable facial expressions (Lapière 1990). The authors of a study which failed to
differentiate wrinkles from the surrounding tissue by histological means suggested that
wrinkles are a result of long-term mechanical stress on skin without chemical or
architectural alterations (Kligman et al. 1985). Others (Contet-Audonneau et al.1999)
have found several distinct histological features within wrinkles, such as lack of the
otherwise typical age-related elastosis in the dermis underlying wrinkles as well as more
collagen atrophy under wrinkles than elsewhere, adding to wrinkle depth. Chondroitine
sulphates, which are essential for balanced skin hydration, were decreased in the papillary
dermis under wrinkles, as were the amounts of collagen types IV and VII and of oxytalan
fibres (Contet-Audonneau et al.1999).
Skin collagen synthesis declines upon ageing and due to such external factors as longterm sun exposure and medications, including D-penicillamine and topical corticosteroids
(Autio et al. 1994, Kang et al. 1997 Oikarinen et al. 1998, Oikarinen 1992, Haapasaari et
al. 1996, Haapasaari et al. 1997). Even short-term usage of topical corticosteroids
decreases collagen synthesis in skin (Oikarinen et al. 1998). This cannot be counteracted
with the use of topical tretinoin, and the recovery period needed for normalization of the
collagen synthesis rate is more than 2 weeks (Haapasaari et al. 1997, Haapasaari et al.
1996). Topical tretinoin has, however, proved effective in the treatment of photodamage
(Kligman & Kligman 1986, Griffiths et al. 1993).
Skin thickness remains quite constant between 10 to 70 years of age, after which a
marked decrease in skin thickness occurs (Escoffier et al. 1989, De Rigal et al. 1989).
Skin atrophy is common in the sun-protected skin of the elderly and in grossly sundamaged skin regions, whereas mildly or moderately photoaged skin is thickened.
(Kligman 1989, Takema et al. 1994). In ageing skin, collagen fibres become thicker and
less soluble (Fenske & Lober 1986). Precursors of both type I and III collagens also
decrease in photodamaged skin, and the degree of reduction in collagen production
correlates with the amount of photodamage (Talwar et al. 1995). UV radiation has been
shown to increase the expression of MMP-1, MMP-8 and MMP-9 in human skin (Fisher
et al. 1996, Fisher et al. 2001), and increased activity of gelatinases (MMP-2 and -9) has
been shown to be involved in the wrinkle formation of UVB-exposed mice skin (Inomata
et al. 2003). Topical tretinoin can inhibit UV-induced MMP activity, but does not
counteract the induction of TIMPs (1993, Fisher et al. 1997, Kang et al. 1997, Uitto
1997, Kang & Voorhees 1998). Recent evidence indicates that MMP-mediated collagen
26
damage in skin is at least partly responsible for the decreased collagen synthesis in
photoaged skin (Fligiel et al. 2003). Along with increasing age dermal elastic fibres
become thicker and fragmented and oxytalan fibres appear fragmented and shortened
(Gogly et al. 1997). Disintegration of elastic fibres is already seen in a minority of fibres
between the ages of 30 and 70 years, but the changes become more profound and
widespread after the age of 70 (Braverman & Fonferko 1982). As a result of the
decreased number of elastic fibres in aged skin, the elastic recovery of skin decreases in
the elderly (Bernstein & Uitto 1996). In photoaged skin, increased elastin mRNA levels
have been demonstrated, indicating transcriptional upregulation of the gene coding
elastin (Bernstein et al. 1994). Flattening of the dermo-epidermal junction is seen in both
sun-exposed and sun-protected skin of the elderly (Lavker 1979). In ageing skin,
epidermal thickness declines in sun-protected areas, whereas sun-exposed regions
develop an irregular epidermis with both thickened and atrophic regions (Marks &
Edwards 1992). A distinct feature of photoaged skin is a decrease in the ultrasound
echogenicity of the upper dermis (De Rigal et al. 1989, Gniadecka & Jemec 1998,
Pellacani & Seidenari 1999).
Several metabolic activities of skin and the skin immune system are also affected by
ageing. The number of Langerhans cells declines, especially in photoaged skin (Gilchrest
et al. 1983) but somewhat also in chronologically ageing skin, in which either the number
or the activity of Langerhans cells declines (Sunderkötter et al. 1997). The number and
activity of dermal fibroblasts decreases in chronologically ageing skin, whereas the
number of active fibroblasts increases in photoaged skin (Gilchrest et al. 1983, Gilchrest
& Yaar 1992). The rate of skin blood flow decreases with age, and the number and size of
capillaries in papillary dermis decline, especially in photodamaged skin (Marks &
Edwards 1992, Cook & Dzubow 1997). The number of eccrine glands in skin declines
and sebaceous glands become hypertrophic but less productive, adding to the dryness of
ageing skin (Fenske & Lober 1986).
2.3 Smoking and skin
2.3.1 Smoking and skin diseases
Smoking is a risk factor or a triggering agent in numerous skin diseases, including
psoriasis (Mills et al. 1992, Naldi et al. 1992, Poikolainen et al. 1994), infectious
eczematous dermatitis and vesicular palmar eczema (Karvonen et al. 1992, Edman 1988).
Men suffering from infectious eczematous dermatitis have been found to smoke more
than controls both before and during the disease (Karvonen et al. 1992). Heavy smoking
is also associated with the onset and exacerbation of pustulosis palmoplantaris, (Eriksson
et al. 1998) and a larger proportion of patients suffering from hidradenitis suppurativa are
smokers (89 percent) compared to patients with other dermatological diseases (46
percent) (Konig et al.1999). Smoking may also affect the efficacy of treatments on skin
diseases. A decreased response to bath-PUVA treatment of palmoplantar eczema has been
reported among patients who smoke (Douwes et al. 2000). According to an American
27
study (Mills et al. 1994), the prevalence of smoking among patients with atopic
dermatitis was similar to that seen in controls. The sample size of 127 individuals was,
however, fairly small for an epidemiological study. The results of a large population study
of 2776 current smokers, 1888 ex-smokers and 3680 non-smokers support the previous
data by demonstrating a lower prevalence of atopy and hay fever among smokers
(Wüthrich et al. 1996). However, children exposed to environmental tobacco smoke have
an increased risk for developing atopic eczema and sensitization against house dust mites,
especially if they also have a parental history of atopy in the family. (Kramer et al. 2004).
Tobacco has also proven to be a potential sensitizer. Patch test reactions have been
observed to cigarette components, such as tobacco, cigarette filter, cigarette ash, cigarette
paper, red match heads and phosphorous sesquisulfide, and there is a case report of
urticaria following exposure to nicotine in tobacco smoke. (Dawn et al. 1999, Lee et al.
1998)
Smoking affects the immune system in many ways. An increased risk of Crohn`s
disease and a protective effect against ulcerative colitis have been reported in smokers
(Cottone et al. 1994, Boyko et al. 1987, Motley et al. 1987), indicating a role of tobacco
in the onset and course of inflammatory diseases. It has been suggested that smoking
might have an anti-inflammatory effect on acne (Mills et al. 1993b), but a recent crosssectional study of 896 people in Germany showed a significantly higher prevalence of
acne in smokers (40%) compared to non-smokers (25%), with a significant dosedependent relationship between the prevalence of acne and cigarette consumption
(Schäfer et al. 2001). Weakened inflammatory responses of skin to external stimuli, such
as sodium lauryl sulphate and ultraviolet B irradiation, have been seen in smokers,
possibly due to a direct consequence of nicotine or some other component of cigarette
smoke (Mills et al. 1993a). Elevated serum IgE concentrations have been reported in
smokers (Wüthrich et al. 1996), but IgE levels may be biphasic in relation to the amount
of tobacco consumed, with elevated IgE levels in light smokers and low levels in heavy
smokers (Holt 1987). Exposure of mice to tobacco smoke condensate resulted in an
increased number and altered morphology and function of Langerhans cells in epidermis
(Zeid & Muller 1995). The activity of natural killer cells, which contribute to the
protection of the body from tumours and viral infections, is decreased in the peripheral
blood of heavy smokers and in patients with lung cancer, suggesting a possible link
between decreasing NK activity and the development of lung cancer (Phillips et al.
1985). Abstinence from smoking for a month has been shown to increase the activity of
natural killer cells (Meliska et al. 1995). Bronchoalveolar lavage of smokers shows a
dose-dependent increase in the concentrations of macrophages, neutrophils and
proinflammatory mediators, such as interleukin-1β (Kuschner et al. 1996).
Dietary antioxidants have been reported to exert a protective effect against
nonmelanocytic skin cancer (Sahl et al. 1995, Kune et al. 1992), but there is a
controversy about the effect of smoking. An important cancer-related gene is the p53 (53kd phosphoprotein) tumour suppressor gene, which is involved in the maintenance of the
stability of the genome by, for example, taking part in DNA repair and apoptosis
(Grossman & Leffell 1997, Hainaut & Vähäkangas 1997 Bennett et al. 1999). Mutations
in the p53 tumour suppressor gene are found in a large number of human cancers,
including lung cancer, and a high frequency of p53 mutations has been found in lung
cancer patients who smoke (Bennett et al. 1999) as well as in smokers with squamous
28
cell carcinomas of the head and neck (Brennan et al. 1995). Smoking has been shown to
be an independent risk factor for cutaneous squamous cell carcinoma after adjustment for
age, sex and sun exposure (De Hertog et al. 2001). The higher incidence of SCC in
smokers may be due to immunosuppression or direct carcinogenic effects of tobacco.
(Smith & Fenske 1996) An Australian group investigated 88 patients with basal cell or
squamous cell carcinoma, but did not find a correlation between smoking or drinking and
these cancers (Kune et al. 1992). However, the number of cases was small and the control
population consisted of hospitalized veterans with a high prevalence of both smoking and
alcohol consumption, which may explain why this study failed to detect a causal
relationship between smoking and skin cancer. In a large study (n=1805) by an American
group (Karagas et al. 1992), the risk of subsequent basal and squamous cell skin
carcinoma was assessed within a 5-year follow-up period after the diagnosis of
nonmelanotic skin cancer. The rate of subsequent squamous cell skin cancer was found to
be higher among current and ex-smokers compared to never-smokers in a dose-depandent
manner, whereas no definite relationship between smoking and basal cell skin cancer was
found (Karagas et al. 1992). Smoking has not been shown to cause melanoma or to
increase the risk for it, but smokers are more likely to have an advanced stage of
melanoma than non-smokers (Van Durme et al. 2000, De Hertog et al. 2001).
2.3.2 Smoking and appearance
The prevalence of premature wrinkling has been found to be independently associated
with sun exposure and pack-years of smoking (Kadunce et al.1991, Ernster et al. 1995,
Chung et al. 2001, Yin et al. 2001, Koh et al. 2002). The first reports indicating increased
wrinkling in smokers appeared in the seventies and eighties (Daniell 1971, Model 1985).
In the more recent studies, possible confounding factors, such as age and sun exposure,
have been accounted for (Kadunce et al.1991, Ernster et al. 1995, Chung et al. 2001 Yin
et al. 2001, Koh et al. 2002). Kadunce et al. (1991) studied a series of 109 adult smokers
and 23 non-smokers, collecting data on the use of tobacco products and assessing the risk
of premature wrinkling. Heavy cigarette smokers were 4.7 times more likely to be
wrinkled than non-smokers, and for those with a history of abundant sun exposure, the
risk for excessive wrinkling was increased 3.1-fold (Kadunce et al. 1991). Another study
of facial wrinkling in 227 never-smokers, 456 former smokers and 228 current smokers
(Ernster et al. 1995) supported the finding of an increased risk of wrinkling in smokers.
After controlling for age, sun exposure and body mass index, the relative risk of moderate
or severe wrinkling for current smokers in comparison with never-smokers was 2.3
among men and 3.1 among women. The risk for wrinkling was also increased in women
who were former smokers. (Ernster et al. 1995) The possible association of the amount of
facial wrinkling in smokers with systemic side effects of smoking, such as stroke, has
also been evaluated. In a study of 40 smokers and 40 non-smokers, of whom half had
suffered a stroke, smokers were assigned significantly higher wrinkle scores than nonsmokers, but the degree of facial wrinkling did not correlate with the occurrence of
adverse cardiovascular events in either smokers or non-smokers (Aizen & Gilhar 2001).
29
In several studies, a significantly increased risk of wrinkling has been associated with
pack years of smoking (Kadunce et al. 1991, Yin et al. 2001, Koh et al.2002) Heavy
smokers with a high level of sun exposure have an even greater risk for acquiring
wrinkles, the relative risk being 11-12 times higher than that of non-smokers (Kadunce et
al. 1991, Yin et al. 2001). Contradictory results were published by O`Hare et al. (1999),
who had three dermatologists review photographs of 82 smokers and 118 non-smokers
and concluded that, despite the significant correlation between smoking and facial
wrinkling, the role of smoking as a cause of wrinkles is of minor importance. This study
was well arranged and controlled, and the authors criticized some earlier studies, e.g.
those performed by Daniell in 1971 and by Model in 1985, for their lack of blinding
techniques. A recent multicentre epidemiological study on 12,735 subjects also indicated
that smoking has only a minor effect on photoageing in women and no significant effect
in men (Malvy et al. 2000). It seems that, despite the strong body of evidence that
smoking increases the risk for premature wrinkling (Kadunce et al. 1991, Ernster et al.
1995, Chung et al. 2001, Yin et al. 2001, Koh et al. 2002), UV -radiation outweighs the
effects of smoking on skin ageing (O`Hare et al. 1999, Malvy et al. 2000).
An American survey of public awareness of the association between smoking and skin
ageing, based on telephone interviews (Demierre et al. 1999), indicated that neversmokers and former smokers were more likely to be aware of the effects of smoking on
physical appearance than current smokers. An interesting finding was that nearly one
fourth of smokers believed that most or some smokers would quit if they knew that
smoking increases facial ageing and wrinkling (Demierre et al. 1999). The authors
emphasized the health educational importance of these results and pointed out the unique
opportunity of dermatologists to take part in cancer prevention and smoking cessation.
(Demierre et al. 1999) Associations between smoking and premature grey hair and
baldness have also been reported, and it has been suggested that this information should
be added to the health education about the adverse effects of smoking (Mosley & Gibbs
1996). In mice, induction of alopecia and grey hair as well as massive apoptosis of hair
follicle cells at the edges of the alopecic patches were observed after 3 months of wholebody exposure to tobacco smoke (D´ Agostini et al. 2000).
2.3.3 Effect of smoking on skin connective tissue and wound healing
Abnormalities in the elastic fibres of heavy smokers were reported by a French group
(Francès et al. 1991), who found the elastic fibres in the skin of smokers to be more
numerous, wider and more fragmented than those in the skin of non-smokers. The
changes observed in elastic fibres resembled those seen in solar damage, which affects
the whole dermis, except that the papillary dermis in smokers remained unaffected.
According to the authors, the localization of elastic damage caused by smoking may be
related to the vascular distribution of the toxic substances of cigarette smoke. However,
the study sample was very small, with only 10 smokers and 10 non-smokers included,
which detracts from the reliability of the study. More recently, Boyd et al. (1999)
compared the skin of 17 smokers and 14 non-smokers and reported increased elastosis in
30
the sun-exposed skin of the forehead and cheeks in smokers compared to non-smokers
(Boyd et al. 1999).
The effect of tobacco smoke on the solubility and cross-linking of collagen has been
investigated previously. A dose-dependent decrease in collagen solubility as well as a
decrease in the lysine and hydroxylysine content, accompanied by a decrease in the
susceptibility of collagen to collagenase digestion, were observed (Rickert 1972, Rickert
& Forbes 1972). More recently, a Danish group (Jorgensen et al. 1998) reported
decreased collagen production in the skin of smokers. A subcutaneous
polytetrafluoroethylene wound healing model was used to assess the deposition of total
protein and mature collagen in subcutis. Smokers had a significantly smaller median
amount of hydroxyproline in their skin compared to non-smokers, and the deposition of
hydroxyproline correlated negatively with cigarette consumption (Jorgensen et al. 1998).
The volatile components of cigarette smoke extract have been shown to affect collagen
gel contraction in vitro, which could be a factor inhibiting wound repair in smokers
(Carnevali et al. 1998). Cell culture studies on human skin fibroblasts have shown
disturbances in ECM turnover after exposure to tobacco smoke extract, which induced
mRNA expression of the matrix metalloproteinase MMP-1 and MMP-3 and MMP-1
protein expression, but did not affect the expression of the tissue inhibitors of matrix
metalloproteinases TIMP-1 and TIMP-3. The production of type I and III collagens
decreased after exposure to tobacco smoke extract. (Yin et al. 2000) Imbalanced ECM
remodelling has also been reported in rat lungs after exposure to cigarette smoke.
Exposure to side-stream cigarette smoke resulted in elevated levels of MMP-1 mRNA
and TIMP-1 mRNA and declined levels of type I collagen mRNA and TIMP-2 mRNA in
the lung. (Morimoto et al. 1997). Recently, photohemolysis of human erythrocytes
exposed to tobacco smoke condensate and UV -radiation was reported, suggesting that
phototoxicity may be involved in the premature skin ageing in smokers (Placzek et.al.
2004).
In the skin of smokers, nicotine causes tissue hypoxia, and the observed decrease in
tissue oxygen tension in smokers may be present for a significant part of the day in heavy
smokers (Jensen et al. 1991). Due to the effects of tobacco on microcirculation and tissue
oxygenation, smoking affects wound healing. Adverse effects of smoking on the surgical
outcome of numerous procedures have been reported, including laparotomy, facelifts,
breast reconstructions and transfers of cutaneous flaps (Siana et al. 1989, Goldminz &
Bennett 1991, Chang et al. 1996).
2.3.4 Oral manifestations of smoking
Oral cancer, which is the sixth most common cancer in the world (Gupta et al.1996), is
strongly associated with all types of smoking, especially when linked with heavy alcohol
consumption. The oral manifestations seen mainly in smokers include leukoplakia,
leukokeratosis nicotina palati, or nicotine stomatitis, leukokeratosis nicotina glossi, or
smoker`s tongue, and acute necrotizing ulcerative gingivitis, which is a bacterial disease
(Smith & Fenske 1996).
31
Periodontitis is more common and more severe in smokers compared to non-smokers,
and smokers are less responsive to periodontal therapy (Johnson & Hill 2004). Higher
levels of salivary MMP-1 have been observed in patients with adult periodontitis
compared to healthy controls (Ingman et al. 1993), and other MMPs are also found in
saliva (MMP-2, MMP-9), in gingival crevicular fluid (MMP-8, MMP-13) and in gingival
tissue (MMP-8, MMP-13) in patients with various forms of periodontitis (Mäkelä et al.
1994, Ingman et al.1994 a & b, Kiili et al. 2002).
2.4 Non-invasive methods in dermatological research - with special
reference to the methods used in this project
2.4.1 Evaluation of skin surface contour
A magnifying lens is useful in the clinical assessment of skin contour, especially when
used together with immersion oil, which hinders the scattering of light and makes the
epidermis more translucent (Katz & Lindholm 1995). Various types of hand-held
dermatoscopes with adjustable magnifications have been developed for easy and quick
visualization of skin lesions and are widely used. They are especially suitable for the
assessment of lesions of vascular or malignant background or involving a variable texture
or colour pattern. (Westerhof 1995) Skin replication methods provide even more detailed
information of skin contour, including wrinkles or stratum corneum, when the skin
replica is studied under a light or scanning electron microscope (Forslind 1995) or with
profilometric techniques (Lévêque 1999, Marks 2000). Various scoring systems,
including Daniell`s wrinkle score, have been developed and used for the assessment of
facial wrinkling (Daniell 1971, Grove et al. 1989, Kadunce et al. 1991, Ernster et al.
1995, O`Hare et al. 1999).
2.4.2 Skin thickness
Skin thickness can be measured non-invasively by a one-dimensional A-mode, twodimensional B-mode, horizontal C-mode or three-dimensional ultrasound device. When
measuring skin thickness, ultrasound velocity of 1580 m/s and frequency of 20 MHz are
commonly used. (Serup et al.1995)
Dermascan A (Cortex technology, Hadsund, Denmark) is a 20 MHz one-dimensional
ultrasound device with a probe for transmitting and receiving ultrasound waves and a
built-in monitor (Fig. 4). The device enables visualization of a field 1.2 cm in size,
reaching a depth of 2cm. (Fornage et al. 1993) When ultrasound passes through tissue,
echoes responding to the structures passed are sent to the oscilloscope. The echoes
received by the probe produce electrical signals, which are displayed on the oscilloscope
as amplitudes and visualized as peaks. The velocity and reflection of ultrasound beams
are dependent on the medium through which the beams pass. Reflection of ultrasound
32
beams occurs when the acoustic impedance of the medium changes. High amplitudes on
the oscilloscope thus indicate marked differences in acoustic impedance in the different
parts of the examined tissue. In skin, for example, the difference in acoustic impedance
between dermis and subcutaneous fat results in a sharp demarcation of these structures by
ultrasound (Fornage et al. 1993). As ultrasound waves are reflected from skin, the
distance between two distinct peaks, that of the stratum corneum of epidermis and that of
the base of dermis, is reported as skin thickness in millimetres. Skin thickness can be
measured reliably when good contact between the probe and the skin surface is ensured
by immersing the probe in water and by keeping it perpendicular to the skin.
Environmental factors, such as changes in room temperature or humidity, do not affect
the reliability of ultrasound measurements, but biological factors, such as age, sex, body
region and weight, do influence ultrasonographic imaging. (Agner 1995, Serup et al.
1995)
2.4.3 Elasticity measurements
The static methods available for the measurement of skin elasticity include techniques
based on torsion, traction, suction, and uniaxial or biaxial extension (Marks & Edwards
1992). Torsional devices have been frequently used in the past, but have been replaced by
suction devices in the recent years. The basis of torsional methods was that skin was
rotated under a disc at a controlled torque, and the elongation of the skin surrounding the
disk was assessed (Agache 1995). Suction devices, such as Dermaflex® and Cutometer
SEM 474®, have been widely used. A vacuum of chosen intensity is applied to the skin
surface via a probe, and the mechanical response of skin to suction is evaluated by either
a strain versus time mode or a stress versus strain mode. The former presents the
deformation of skin as a function of time and the latter as a function of the vacuum
applied. (Barel et al. 1995, Barel et al. 1998)
Dermalab® is a suction device (Cortex technology, Hadsund, Denmark) consisting of a
main unit, a built-in printer and a probe (Fig. 4). The probe, which provides a vacuum
chamber with an aperture of 10 mm, is attached to the skin surface with adhesive tape
(Serup 2002). Elasticity is measured by applying suction to the skin surface and
calculating the differential force needed to elevate the skin surface 1.5 mm between two
infrared detection levels inside the probe chamber. The elasticity modulus, based on the
calculation of Young`s modulus, is reported as megapascals (MPas) and represents the
stiffness of skin (Serup 2002). The elasticity modulus originates from the first suction
cycle, and in the following cycles the elasticity modulus is expected to decrease, as skin
stiffness declines due to the effects of preliminary stretching (Serup 2002). Consecutive
measurements from a given spot require a period of at least half an hour between each
pair of measurements, in order to achieve total recovery of skin from the effects of
suction. (Dermalab Users Manual, Cortex Technology)
33
2.4.4 Suction blister method
The suction blister method was first described in 1964 by Kiistala and Mustakallio, and it
has since been used successfully for studying such phenomena as drug metabolism,
regeneration of barrier function and skin connective tissue metabolism (Kiistala &
Mustakallio 1964, Autio 1994, Haapasaari et al. 1997, Koivukangas & Oikarinen 1998).
Suction blisters are formed by applying negative pressure induced by a vacuum device to
separate epidermis from dermis. The vacuum device is attached to an adapter plate
(Ventipress, Lappeenranta, Finland), into which suction blisters develop gradually after
the negative pressure has been conducted to the skin (Fig. 4). The separation occurs
between the base of the basal cell layer and the basal lamina, and the latter forms the base
of the blister. (Kiistala 1968, Hunter et al. 1974) Originally, the vacuum pump Itka® was
used, but other vacuums can also be applied. The induction of blister formation is
promoted by the heat of an electric lamp placed approximately 15 cm above the skin area
where blisters are being made.
The suction blister fluid (SBF) acquired with this technique resembles interstitial fluid.
The protein level of SBF is approximately 30 % of that in serum (Oikarinen et al 1982).
The ratio of the protein concentrations found in SBF and serum depends on the molecular
weight of the proteins and of the law of diffusion. (Kiistala 1968, Herfst & van Rees
1978, Vermeer et al. 1979) Skin collagen synthesis can be quantified in vivo from SBF by
measuring the concentrations of procollagen propeptides in blister fluid. Less than 10 per
cent of PINP and PIIINP in SBF is derived from serum, whereas most of it is derived
from dermis, reflecting the ongoing collagen synthesis in skin (Oikarinen et al. 1992,
Autio & Oikarinen 1997). The epidermal roof of the blister has been used to study the
permeability of epidermis in vitro and to quantify epidermal bacteria (Kiistala 1968).
After removal of the blister roof, the base of the blister offers an opportunity to study the
regeneration of the epidermal barrier by comparing water evaporation from the base of
the blister at different time points during the healing process (Koivukangas & Oikarinen
1998). The suction blister method provides an almost painless technique to study skin
metabolism and healing. The healing process involves transient pigmentation of the
blister area. (Oikarinen et al. 1992, Annala et al. 1993, Autio & Oikarinen 1997)
2.4.5 Barrier function
Epidermal barrier function can be studied non-invasively by measuring transepidermal
water loss, which refers to the amount of water lost through skin without sweat gland
activity and reflects the integrity of skin (Marks 1998). TEWL measurements have been
used to assess barrier function in contact dermatitis and patch test reactions as well as to
evaluate the rate of reepithelization in wounds of partial thickness. Both intra- and
interindividual variations occur in TEWL values, which are affected by age and
anatomical site as well as by environmental factors, such as air convection, temperature
and humidity. (Pinnagoda & Tupker 1995)
TEWL can be measured with an Evaporimeter EP1 device (ServoMed, Stockholm,
Sweden), which is based on an open-chamber gradient estimation method. The device
34
consists of a main unit and two probes. Each probe is surrounded by a teflon capsule. At
each end of the capsule, there is a cylindrical, open measuring chamber 12 mm in
diameter with a pair of sensor units, which determine the relative humidity and
temperature at two levels above skin. The water vapor gradient between skin surface and
ambient air is displayed as g/m2 h. The sensor units are highly sensitive, and efforts
should be taken to minimize external confounding factors, e.g. draft or changes in room
temperature. (Blichmann & Serup 1987, Pinnagoda et al. 1990, Pinnagoda & Tupker
1995, Serup 1995) The study subjects are advised to rest for at least 15 minutes prior to
the measurements (Van Sam et al.1994). The recording of the TEWL value g/m2 h should
be done after approximately 30 seconds after the beginning of the measurement, when an
equilibrium in the rise of the TEWL value has been reached (Blichmann & Serup 1987).
When the evaporimeter EP1 is used accordingly, good technical accuracy and
repeatability are achieved (Blichmann & Serup 1987). Recently, a closed unventilated
chamber technique has been developed for TEWL measurements, with good precision
and ability to avoid the artefacts caused by external air currents (Nuutinen et al. 2003).
Fig. 4. Devices used in this project. A) Dermascan-A ultrasound, B) Dermalab device for
measuring skin elasticity, C-D) suction blisters.
3 Aims of the study
The aim of the study was to find out more about the mechanisms of the adverse effects of
smoking on skin by comparing the appearance, physical qualities, histology and
metabolism of skin between smokers and non-smokers. Of special interest were the
comparison of the rates of collagen synthesis and the levels of proteases involved in the
degradation of ECM in smokers and non-smokers.
The parameters of central importance were:
1. Comparison of skin wrinkling by both clinical and computerized methods in smokers
and non-smokers.
2. Skin thickness and elasticity as well as the histology of elastic fibres in smokers and
non-smokers.
3. Synthesis rates of type I and III collagens in the skin and serum of smokers and nonsmokers.
4. Assessment of ECM turnover in terms of MMPs and TIMP-1 in the skin and other
tissue compartments of smokers and non-smokers.
4 Material and methods
4.1 Subjects
Volunteers for the study were recruited from among the staff of ENSO Fine Papers,
Kemira Chemicals and Oulu University Hospital. An advertisement of the study was
published in a local newspaper. The aim was to recruit 50 smokers and 50 non-smokers.
Only males were eligible. The exclusion criteria included diagnoses of diabetes,
rheumatoid arthritis, psoriasis and other diseases requiring long-term corticosteroid
treatment. The final study series consisted of 98 men compatible with these inclusion and
exclusion criteria. The mean age of the study subjects was 52 years (range 34-71, SD=
8.5). The mean age of the smokers was 50 years (SD=8.5) and that of the non-smokers 53
years (SD=8.3).
4.1.1 Smoking history
Non-smokers were defined as men who had never been habitual smokers. Some had tried
the taste of cigarettes in their teens or during military service, but had not smoked since.
All smokers were current smokers and had been smoking for at least 15 years. The mean
number of years smoked was 33 years (range 15-56, SD=8.4). Pack years of smoking
averaged 30 years. Pack-years of smoking were calculated using the following formula:
number of years smoked multiplied by the number of packs smoked per day. Most of the
smokers smoked cigarettes, three were pipe smokers and two smoked cigars. All current
pipe and cigar smokers were previous cigarette smokers. The average amount smoked per
day was 19 cigarettes (range 5-40, SD=6.6). Approximately 70 % of the smokers had
tried to quit smoking, and only one third reported never having tried to quit. Of those who
had attempted to quit, 17 % had had a total of six or more months of abstinence, 40 %
had managed less than six months without tobacco, and 13 % did not report the length of
abstinence. Passive exposure to tobacco smoke at home was reported by two percent of
the non-smokers and nine percent of the smokers. Passive exposure to tobacco smoke at
work was also reported by two percent of the non-smokers and nine percent of the
37
smokers. The smoking status of both the smokers and the non-smokers was confirmed by
assessing the urinary concentrations of cotinine and other nicotine metabolites and
controlling for the urinary creatinine concentration (Fig. 5).
Fig. 5. Correlation between the amount of smoking and urinary nicotine metabolites.
4.1.2 Alcohol consumption
The frequency of alcohol consumption did not differ significantly between the smokers
and non-smokers, but the smokers tended to drink more heavily when they used alcohol.
The difference in the amount of alcohol consumed per occasion was statistically
significant (p<0.001, Table 3). One alcoholic drink was defined as one bottle of beer, one
glass of wine or 4 cl of spirits.
38
Table 3. Self-reported average amount of alcohol consumed per occasion.
Average amount of alcohol consumed per occasion
Non-smokers
Smokers
(N=51)
(N=47)
None
12 %
4%
< 5 drinks
71 %
32 %
≥ 5 drinks
14 %
64 %
Question not answered
4%
* A drink was defined as one bottle of beer, one glass of wine, or 4 cl of spirits.
4.1.3 Sun exposure
A present outdoor occupation was reported by 7 (15%) smokers and 14 (28%) nonsmokers. In the past, 10 (21%) smokers and 5 (10%) non-smokers had been outdoor
workers Most of both smokers (30 persons, 64%) and non-smokers (32 persons, 63%)
were currently working indoors. There was no statistical difference in the types of
occupation reported by the two groups (p>0.1).
Frequent holidays in southern countries were reported by 12 (26%) smokers and 15
(29%) non-smokers. One smoker (2%) reported travelling to the south often, whereas 16
(34%) smokers and 24 (47 %) non-smokers reported travelling to south seldom, and 18
(38%) smokers and 12 (24%) non-smokers did not spend holidays in southern countries
at all. No statistically significant difference was found in sun exposure due to travelling
(p>0.1).
Sun exposure during the Finnish summer months was recorded as no exposure, 1-2
weeks, 3-4 weeks, 5-8 weeks or 9-12 weeks of exposure. The summer season was defined
as starting from the beginning of June and lasting until the end of August. The study
subjects were asked to estimate how much time they spend outdoors and exposed to
sunshine during that time. Two (4%) smokers and 8 (16%) non-smokers reported no sun
exposure during the summer, while 12 (26% ) smokers and 8 (16%) non-smokers
reported 1-2 weeks of sun exposure, 19 (40%) smokers and 24 (47 %) non-smokers
reported 3-4 weeks of sun-exposure, 7 persons in both groups (15 % of the smokers and
14 % of the non-smokers) reported 5-8 weeks of sun exposure, and 7 (15%) smokers and
4 (8%) non-smokers reported 9-12 weeks of sun exposure during the summer months.
The differences in the amount of sun exposure during the summer months were not
statistically significant (p>0.1).
4.1.4 Medical history
Skin disease of some kind was reported by 36 % of the smokers and 20 % of the nonsmokers, the difference being statistically non-significant (Table 4). The data presented
here are based on the information supplied by the patient questionnaires, on the patient
39
records of Oulu University hospital and on the clinical diagnoses available at the time of
the recruitment. The diversity of skin symptoms is demonstrated in Table 4. It must be
noted that some patients had had more than one skin problem, and the percentages of skin
diseases in smokers and non-smokers therefore cannot be obtained directly from the
table. Occasional usage of topical corticosteroids was reported by 30 % of the smokers
and 37 % of the non-smokers. A treatment protocol of 1 to 2 weeks up to twice a year was
reported by 9% of the smokers and 2 % of the non-smokers. Two smokers (4 %) used
topical corticosteroids more often. The differences between the groups were not
statistically significant. None of the subjects had been using topical corticosteroids at the
measurement or biopsy sites prior to the study.
Cardiovascular disease was present in 11 (23 %) smokers and 7 (14 %) non-smokers,
neurological disease in 2 smokers (convulsions due to alcohol, trigeminus neuralgy) and
3 non-smokers (Menier`s disease, cephalgia vascularis, epilepsy) and respiratory disease
in 2 smokers (tuberculosis in the past, chronic bronchitis) and 2 non-smokers (both were
cases of mild asthma with no current medication). One smoker had a history of
ventricular carcinoma, which had been operated. In addition, solitary cases of various
miscellaneous symptoms and diseases were reported, with minor relevance to the present
study.
Of the nevi and skin tumours, which were removed from the study subjects, all but one
were benign. One smoker had a facial basalioma.
Table 4. Anamnestic skin diseases in the study population.
Skin disease
Smokers
Non-smokers
Total (N=98)
N (%)
N (%)
N (%)
Constitutio atopica
4 (9%)
0
4 (4%)
Eczema nummulare / infectiosum
5 (11%)
2 (4%)
7 (7%)
Nonspecific eczema
4 (9%)
3 (6%)
7 (7%)
Neurodermatitis
1 (2%)
1 (2%)
2 (2%)
Pruritus
0
2 (4%)
2 (2%)
Mycosis
0
8 (16%)
8 (8%)
1 (2%)
0
1 (1%)
Acne
Alopecia areata
0
1 (2%)
1 (1%)
Lichen planus
1 (2%)
0
1 (1%)
Miscellaneous*
4 (9%)
6 (12%)
11 (10%)
* 3 condyloma accuminatum, 1 verruca vulgaris, 1 cold urticaria, 1 erysipelas, 1 herpes zoster, 1 pityriasis
versicolor, 1 skin infection, 1 scaling feet.
40
4.2 Methods
4.2.1 Clinical status
The appearance of skin in the face, trunk and extremities was checked, and the skin
phototype was assessed according to the system of skin phototype grading proposed by
Fitzpatrick (1988), where phototype I is characterized by white skin, which burns easily
and never tans, phototype II presents as white skin which burns easily and tans
minimally, phototype III as white skin which burns minimally and tans gradually,
phototype IV as light brown skin which burns minimally and tans well, phototype V as
brown skin which rarely burns but tans profusely and phototype VI as dark brown skin
which never burns but tans profusely. Most of the study subjects (91 % of smokers and
86 % of non-smokers) had skin phototype III, and only a minority had other skin types.
The main methods and the numbers of patients evaluated by these methods are shown in
Table 5.
4.2.2 Clinical and computerized analyses of facial photographs (I)
A panel consisting of 3 dermatologists, 3 interns specialising in dermatology and 2
medical students performed a standardized assessment of the facial wrinkling, smoking
status and age of 41 current smokers and 48 never-smokers. One to two panellists at a
time were shown the slides of the study subjects at a fixed distance of 2 meters. The
slides were photographs presenting a frontal view of the face and a close-up of the temple
area. The camera used was a Canon EOS 650 with a Canon EF Zoom Macro objective,
35-105 mm, 1:3.5-4.5. A macro setting of 105 mm and a 1:8 ratio to life size was used for
the frontal pictures. A Canon T 500 close-up lens in a 1:4 ratio to life size was used for
the close-up of the temple region. Two Pro 5 1200 Ws studio flashes were used with a
fixed angle of 40 degrees.
A frontal view was shown first, and the panellists were asked to estimate the age and
smoking status of the person in question. The panellists recorded skin wrinkling after
having seen both the frontal and the temporal views by using Daniell´s score, which was
explained to every panel member before starting the task (I, Fig. 1). Daniell´s score
grades wrinkling from 1 to 6 in the following manner:
− Grade 1 - Facial skin essentially unwrinkled. Two or three short (< 1.5 cm) shallow
lines may be present in the temple region.
− Grade 2 - Several, usually 2 to 6 significant wrinkles up to 3 cm long may be seen on
the temples.
− Grade 3 - Several prominent wrinkles 3 to 4 cm in length, on the temples together with
smaller wrinkles. Increased wrinkling on the forehead but not on the cheeks.
− Grade 4 - Wrinkles extend from the temples towards the forehead and the cheek,
usually 5 cm or more, or if exceptionally deep, 4 cm in length. Wrinkles extend over
the zygomatic ridge.
41
− Grade 5 - Wrinkles extend superiorly and inferiorly from the temples and are
prominent on the forehead and cheeks.
− Grade 6 - Essentially wrinkled. Profound wrinkling over most of the face.
Facial photographs of 26 smokers and 31 non-smokers were assessed by computerized
image analysis at the Department of Electrical and Information Engineering, University
of Oulu. The original digitized images were first preprocessed by cropping and colour
feature counting. The preprocessed images were then analyzed with self-developed
algorithms, which detect wrinkles from an image by using a line matching technique and
then count the percentage area of wrinkle involvement. The final wrinkle percentage of
each patient was calculated as an average of the two cropped images. The images were
cropped in such a way as to exclude confounding areas, such as the eyes and the
eyebrows. The need to maintain comparability between the results of different patients
was the reason to standardize the cropping of the images. The image in the RGB (Red,
Green, Blue) model is based on three independent primary colours, and the value of each
component strongly depends on the light intensity of the image. In the present study, the
rgb system (normalized colours: red, green, blue) was used, which minimizes errors
caused by irregular light intensity in the image. Normalized blue colour (b) was counted
from the original colour image with the formula b=B:(R+G+B), where R, G and B were
normalized to be in the range of 0-1.
The final wrinkle assessment is based on a line pattern matching technique, where the
cropped gray-scale image is scanned to detect local line patterns. When a local line
pattern is found, the algorithm makes a connected components analysis relative to its
neighbouring pixels and, depending on the previously given parameters, either approves
or rejects them as part of the wrinkle. The manually given parameters are threshold,
length and minimum size. Threshold is a gray-scale value that determines whether a pixel
is part of a wrinkle or normal skin. Length is the minimum length of a local line pattern.
Minimum size is the smallest number of pixels in an approved connected component.
After wrinkle assessment of the image, the software calculates the area of wrinkle
involvement as a percentage of total skin area.
4.2.3 Skin thickness (II)
Skin thickness was measured with a 20 MHz Dermascan A ultrasound device (Cortex
Technology, Hadsund, Denmark) from five different sites: upper inner arm, dorsal
forearm, cheek, temple and abdomen. Three measurements were made in each area, and
means were calculated.
4.2.4 Skin elasticity (II)
Elasticity was measured with a Dermalab device (Cortex Technology, Hadsund,
Denmark) from five different sites: upper inner arm, dorsal forearm, cheek, temple and
abdomen. The probe was placed perpendicular to the skin, in order to avoid the impact of
42
gravitation on the measurements. Three measurements were made in each skin region,
slightly changing the placement of the probe each time, and means of the three
measurements were calculated. Five suction cycles were used with all measurements to
control the reliability of the method. During the measurements, the study subjects were
immobile in a supine position.
4.2.5 Suction blister technique (III, IV)
Suction blisters were induced on the sun-protected skin of the upper inner arm with a
Cuub MS 20 device (Finnomedical, Helsinki, Finland) and adapter plates supplied by
Ventipress (Lappeenranta, Finland). The pressure was set at -60 KPa for approximately
40 minutes and at -40 KPa for approximately 20 minutes. An electric lamp was placed 15
cm above the adapter plate to help induce blister formation with the heat of the lamp.
Suction blister fluid was collected into an Eppendorf tube with a needle and a syringe and
the samples were stored at -20 ° C until analyzed.
4.2.6 Transepidermal water loss
EP1 (ServoMed, Stockholm, Sweden) was used to study epidermal barrier regeneration in
terms of transepidermal water loss. Measurements were made from the bases of the
suction blisters formed in the upper inner arm skin. After blister formation, tissue fluid
was collected from each blister with a needle and a syringe. The blister roofs were then
carefully removed with tweezers, to expose the bases of the blisters. The first
measurements were made on the day the suction blisters were induced and the second
measurements four days later. Three measurements were made from the base of each
blister, after which means were calculated. TEWL was recorded until an equilibrium in
the rise of the values was reached. TEWL was automatically displayed as g/m2 h. All
measurements were made at room temperature, with the air conditioning apertures closed
in the vicinity of the study subjects. Preceding the first measurements, the study subjects
had been resting for at least an hour. Preceding the second measurements the study
subjects rested for a while, to avoid false results caused by sweating. TEWL was also
measured from healthy skin in the vicinity of the suction blister area, to further control
the reliability of the results.
4.2.7 Immunohistochemistry (II,III)
Skin samples from the upper inner arms of 42 smokers and 39 non-smokers were
available for histological and immunohistological evaluations. Immunohistochemical
staining was performed at the Department of Pathology, University of Oulu. Visualization
of elastic fibres was performed by staining with Verhoeff, after which the width and
43
proportional area of elastic fibres were assessed with a digital image analyzer (see
paragraph 4.2.8.).
Skin biopsy specimens for the calculation of PINP-positive fibroblasts were stained
with an anti-PINP antibody (Melkko et al. 1996), which was supplied by Orion
Diagnostica (Oulunsalo, Finland). Fibroblasts showing cytoplasmic staining with the
PINP antibody were considered PINP-positive and were calculated from five consecutive
fields from papillary dermis using an Olympus BH 2 microscope and an objective with
40 x magnification.
Skin biopsy specimens for the calculation of vascular lumens were stained with an
endothelial antibody CD34 (Immunodiagnostic OY, Hämeenlinna, Finland), which
detects CD34 antigen in endothelial cells (Elenitsas et al. 1997). There was no difficulty
in identifying the vascular lumens, which were counted from five consecutive fields from
papillary dermis using an Olympus BH 2 microscope and an objective with 40 x
magnification. Follicular regions were avoided when counting both PINP-positive
fibroblasts and vascular lumens.
Langerhans cells were stained with a polyclonal S-100 antibody (Dako A/S, Glostrup,
Denmark), which binds to the S-100 protein present in Langerhans cells (Murphy 1997).
S-100 protein is an acidic protein, which is found in the cytoplasm and nucleus of a large
variety of cells. In epidermis it is found in melanocytes and in Langerhans cells. The S100 antibody has high sensitivity but low specificity. (Elenitsas et al. 1997) Langerhans
cells were counted from five consecutive epidermal fields using an Olympus BH 2
microscope and an objective with 40 x magnification. Cells with distinct nuclear staining
or distinct dendritic staining with star-like appearance but no visible nucleus were defined
as Langerhans cells and counted. Solitary non-specific dendritic structures were not
counted. Melanocytes were differentiated from Langerhans cells by their location and
shape.
4.2.8 Morphometric analyses of elastic fibres (II)
Computerized image analysis is useful when studying elastic fibres or other unevenly
distributed materials. The image analyzer enables detection of Verhoeff-stained dermal
elastic fibres (Uitto et al. 1983, Flotte et al. 1989). The slides are placed under a
microscope, and the image is transferred via a camera and a computer to the screen. The
image is first focused and digitized, after which the desired qualities, e.g. proportional
areas of the target of interest, can be counted. In the process of digital image analysis, the
information in an image can be emphasized by increasing contrast and extracting data
values such as density from the image. The process begins with digitization, i.e.
transformation of such image features as density and colour into discrete digital values.
The image is broken into small elements, pixels, which are stored within the processor.
Thus, each pixel represents a digitally coded image element of a certain location and
density or colour. Most image analyzers include a host computer and an imaging board.
MCID uses a complex imaging board, with a large capacity to store images
(Fundamentals of image analysis, Imaging research Inc.1997).
44
In this study, image analysis was used to compare the proportional area and the width
of elastic fibres in the skin of smokers and non-smokers. Punch biopsies were obtained
from the upper inner arm and stained with Verhoeff for visualization of elastic fibres. The
image analyzer used was a MCID/M4 3.0 Rev 1.1 (Imaging Research Inc.) with a Nikon
TMD Optiphot light microscope and a 40 x Plan Nikon objective. In the assessment of
the proportional area occupied by elastic fibres, four fields from papillary dermis and four
fields from reticular dermis were analyzed. In the assessment of the width of the elastic
fibres, three fields from reticular dermis were analyzed. All visible elastic fibres within
each field were analyzed.
TEWL by Evaporimeter EP1
Computerized analyses
Facial photographs
6 mm punch biopsies
Skin biopsies
Canon EOS 650
4 mm punch biopsies
Cuub MS 20 suction device
(ServoMed, Stockholm, Sweden)
Suction blisters
Barrier function
elevate the skin surface 1.5 mm between two infrared detection levels
self-developed algorithms.
The images were processed and the wrinkles were analyzed with
smokers and non-smokers.
Panellists assessed the age, smoking status and wrinkling of the
zymography of MMP-2 and MMP-9.
Biochemical assays, such as Western blotting of MMP-8 and
fibres, vascular lumens, fibroblasts and Langerhans cells.
Assessment of skin histology and immunohistochemistry of elastic
the dermo-epidermal junction.
Negative pressure conducted to the skin induces blister formation at
Rate of water evaporation through skin.
inside the probe chamber.
Elasticity is calculated on the basis of the differential force needed to
Dermalab (Cortex technology, Hadsund,
Denmark)
dermis-hypodermis boundary represents skin thickness in mm.
(Cortex technology, Hadsund, Denmark)
Skin elasticity
The distance between the ultrasound echoes from epidermis and the
Dermascan A
Skin thickness
Description of the method
Method / Device
Parameter
26
41
37
42
47
20
47
47
Smokers
31
48
31
39
51
20
51
51
Non-smokers
Table 5. Summary of the patients, the main methods used and the numbers of patients evaluated by these methods in this study.
45
46
4.2.9 Sampling for biochemical assays
The SBF samples were obtained from the upper inner arm and stored at -20° C until
analyzed.
Skin biopsy samples were obtained from the upper inner arm under local anesthesia,
frozen with liquid nitrogen and stored at –70 °C. Venous blood samples (2 x 10 ml) were
drawn from the antecubital vein and kept at -70° C until analyzed. Salivary samples were
obtained from 40 smokers and 43 non-smokers over a collection time of 3 minutes,
during which time the participants chewed a paraffine gum to induce saliva formation.
The mean amount of saliva collected was 8.3 ml (SD 4.1) in the smokers and 7.8 ml (SD
3.9) in the non-smokers (P=0.598). Urinary samples were collected from each participant
in order to evaluate their smoking status by performing urine assays of cotinine and other
nicotine metabolites.
4.2.10 Biochemical methods (III, IV)
The smoking status of both smokers and non-smokers was evaluated by assays of urinary
cotinine and other nicotine metabolites with a double antibody nicotine metabolite kit,
which is commercially available (Diagnostic Products Corporation, Los Angeles, CA,
USA). The method is a liquid-phase RIA, in which radioactively labelled cotinine
competes for antibody sites with cotinine and other nicotine metabolites for an incubation
time of 40 minutes at room temperature, after which the antibody-bound fraction is
separated, precipitated and counted. The assays were performed at the Department of
Pharmacology and Toxicology, University of Oulu.
Sequential radioimmunoassay (RIA) and competitive RIA were used to assess the
levels of PINP and PIIINP in SBF and serum, respectively (Oikarinen et al. 1992, Risteli
et al. 1988). The method is a rapid equilibrium assay based on the use of highly purified
human serum antigen and optimized reaction conditions (Risteli et al. 1988). The assay
requires a tracer and an antiserum, which are diluted with phosphate buffer. Kaolin bound
anti-rabbit-IgG is used as a second antibody. The method is commercially available and it
was supplied by Orion Diagnostica, Oulunsalo, Finland. The assays were made at the
Department of Clinical Chemistry, Oulu University Hospital, as described previously.
(Risteli et al. 1988) The reference range of PINP in the serum of adult males is 10-77
μg/l, and the reference range of serum PIIINP in adults is 1.7-4.2 μg/l (Melkko et al.
1996, Risteli et al. 1988). A solid-phase enzyme immunoassay for the assessment of
TIMP-1 levels in serum and SBF was supplied and performed by SBA Sciences, Oulu,
Finland.
47
4.2.10.1 Time-resolved immunofluorometric assay (IFMA) of MMP-8 in
SBF, serum and saliva
The levels of MMP-8 in SBF, serum and saliva were assessed by using a time-resolved
immunofluorometric assay (IFMA) based on MMP-8- specific tracer and catching
antibodies, as described by Hanemaaijer et al. (1997). The method is based on two
monoclonal MMP-8-specific antibodies, which are used as a catching antibody and a
labelled tracer antibody. The assay buffer contained 20 mM Tris-HCl, pH 7.5, 0.5 M
NaCl, 5 mM CaCl2, 50 µM ZnCl2, 0.5% bovine serum albumin, 0.05 % sodium azide and
20 mg/ 1 DPTA. At first, the samples were diluted in the assay buffer and then incubated
for one hour. Then, incubation was continued for one hour with the tracer antibody, after
which enhancement solution was added and fluorescence was measured after 5 minutes
by a 1234 Delfia Research Fluorometer (Wallac, Turku, Finland).
4.2.10.2 Western blot analysis of MMP-8 from skin samples
The presence of MMP-8 isoforms in skin tissue was assessed by Western blotting. The
frozen skin samples were weighed and suspended in 1 x sample buffer (1.25 M Tris, pH
6.8, 10 % SDS, 10 % glyserol, 37 μM bromophenol blue) containing 15 mg dry weight of
tissue in 50 μl buffer. The samples were incubated for one hour at room temperature and
kept at +4 °C until further diluted and incubated at +60 °C for 20 min, after which they
were run on Brilliant Blue –stained 12 % SDS-polyacrylamide gel for visualization of the
protein content. The samples were then transferred onto polyvinylidene fluoride (PVDF)
microporous membrane (Immobilon P Transfer membrane, Millipore), washed and
incubated with 1 x TBS (10 x TBS= 0.5 M Tris, 1.5 M NaCl, pH 7.6) supplemented with
5 % non-fat dry milk for 60 min. The filters were washed with TBS-Tween-20 and
incubated for one hour with the primary antibody (1 μg/ml mouse monoclonal antibody,
8073, 1:1000) against MMP-8, then for one hour with the anti-mouse IgG secondary
antibody (1:1000, DAKO, A/S, Glostrup, Denmark) and, finally, for 45 min with avidinperoxidase complex (DAKO, A/S, Glostrup, Denmark). The ECL Western Blotting
detection kit (Amersham Pharmacia Biotech, Buckinghamshire, UK) was used as
described in the product protocol. The chemiluminescence reaction produced by ECL
reagents was detected by autoradiography. The Western blotting immunoreactive bands
and total protein contents were quantified for each sample using software for image
processing and analysis (ScionImage PC, Scion Corporation, Frederick, MD, USA). The
molecular forms of the complex, PMN tot, PMN pro, PMN act, Non-PMN pro and NonPMN act forms of immunoreactive MMP-8 were quantified separately (Prikk et al. 2001,
Prikk et al. 2002).
4.2.10.3 Capture activity assay of salivary MMP-9
The levels of MMP-9 in saliva were assessed by capture captivity assay, enabling
detection of the total and endogenously active forms of MMP-9 by using a monoclonal
48
mouse antihuman MMP-9 antibody (Fuji Chemical Industries, Toyama, Japan) and
modified urokinase as a substrate. Diluted saliva samples (1:5 in PBS) were incubated at
4ºC for 16 hours. After the non-bound proteins had been washed off, the bound MMP-9
was incubated in 50 mM Tris-HCl, pH 7.6, containing 150 mM NaCl, 5 mM CaCl2, 1 µM
ZnCl2 and 0.01% (v/v) Brij-35 with or without 0.5 mM APMA for 2 hours at 37ºC. Then,
15 µl of modified urokinase was added (UKCOL, final concentration 5 µg/ml), and the
aminolytic activity of UKCOL was determined at 37ºC after the addition of 0.4 mM
chromogenic substrate S-2444 (Chromogenix, Mölndal, Sweden). Absorbance changes
were measured at 405 nm at different time intervals using a Titertek multiscan 8-channel
photometer (Konttinen et al. 1998, Vuotila et al. 2002).
4.2.10.4 Zymography of MMP-2 and MMP-9 in serum and skin tissue
Zymography was used to assess the presence of MMP-2 and MMP-9 in the serum
samples of 47 smokers and 50 non-smokers as well as the levels of MMP-2 and MMP-9
protein in the frozen skin samples of 18 smokers and 21 non-smokers. The protein
content of the serum samples was measured by the Bio-Rad (Bio-Rad, Hercules, CA,
USA) protein assay kit following the manufacturer’s instructions, and 30 µg of total
protein in sample buffer was analyzed by zymography. We used 10% SDS-PAGE
containing 1 mg/ml fluorescently labelled gelatin (2-methoxy-2,4-diphenyl3(2H)furanone as a label) (Fluka, Ronkonkoma, NY, USA) by the method of O’Grady et
al. (1984) to mark the degradation of gelatin. The standard lane was loaded with lowrange prestained SDS-PAGE standards (Bio-Rad, Hercules, CA, USA). A control sample
was used for each. The electrophoresis was run for 2 h and 30-45 min with 120V, after
which the gels were washed in 2.5% Triton X-100 for 2x15 min to remove SDS and
incubated at +37°C overnight in 50 mM Tris-HCl buffer (pH 7.8, 150 mM NaCl, 5 mM
CaCl2, 1 µM ZnCl2). The gels were photographed in ultraviolet light, and the photographs
were scanned and analyzed by Scion Image software (Scion Corporation, Frederick, MD,
USA).
4.2.11 Statistical analysis
The data were analyzed with the SPSS software with the help of a biostatistician, Medical
Informatics Group, University of Oulu. The statistical methods included histograms and
box plots to show the distributions of the parameters. Means and standard deviations
(SDs) were calculated with independent samples t-tests and one-way variance analyses
(one-way ANOVA). When dealing with a Gaussian distribution, the p-values were
obtained from independent samples t-tests. Parameters with skewed distributions were
assessed with non-parametric Mann-Whitney tests. In some cases with a skewed
distribution, logarithmic transformation was performed to achieve a more Gaussian
distribution, after which independent samples t-tests were performed. For the comparison
of TEWL values, the average differences between the measurements of the days 0 and 4
were statistically compared using the paired samples t-test. Socioeconomic parameters
49
were analysed with cross-tabulation. Correlations were estimated with Pearson`s
correlation coefficient in the case of Gaussian distributions, and with Spearman`s
correlation coefficient in the case of skewed distributions. Fisher`s exact t-test was
applied when comparing the panellists` estimation of the ages of the smokers and nonsmokers, and confidence intervals were observed when comparing the ability of the
panellists to identify smokers and non-smokers. Statistical analyses were performed with
the groups of smokers and non-smokers as such, and some analyses were done with the
groups subdivided into younger (<50 years) and older (> 50 years) age groups. Statistical
significance was set at p< 0.05 throughout the study.
4.2.12 Ethical aspects
All study subjects were volunteers who agreed to participate after having been informed
about the study protocol in person and having given written informed consent. The study
protocol was approved by the ethical committee of the Medical Faculty of the University
of Oulu. The measurements of skin thickness, elasticity and blood flow were all noninvasive. The suction blister method is minimally invasive and almost painless. During
blister formation itching or tickling of the blister area was frequently reported. The
healing process involves a period of skin pigmentation, which lasts for some months, but
the blister area heals gradually without scarring. The skin punch biopsies of 6 mm and 4
mm in size were obtained from the upper inner arm under local anesthesia. Some study
subjects preferred not to have biopsies taken, and no pressure was put on those who
refused or hesitated.
5 Results
5.1 Appearance and wrinkling (I)
The panellists identified 68 % of the smokers correctly as smokers, whereas 40 % of the
non-smokers were falsely estimated to be smokers, the difference in the percanteges
being statistically significant based on the 95% confidence interval (95%CI= 0.09-0.49).
The smokers were estimated to be an average of 2.1 years older than their age, whereas
the non-smokers were estimated to be an average of 0.7 years younger than their age
(p=0.005; 95% CI of the difference 0.88-4.69). Daniell´s scores of I to II were assigned to
49% of the smokers and 46% of the non-smokers, and scores above III were assigned to
51% of the smokers and 54% of the non-smokers. The mean percentage of wrinkles by
computerized image analysis was 2.6 % for the smokers and 2.3 % for the non-smokers
(p=0.35). On the whole, the clinical wrinkle scores correlated with the percentages of
wrinkles obtained by computerized image analyses (r = 0.63, p<0.001) (I, Fig. 3). There
was a tendency towards higher Daniell´s scores in the smokers with higher pack year
values (r=0.53, p<0.001), whereas the correlation between pack years and the percentages
of wrinkles obtained by image analysis was weaker (r=0.37, p=0.06). Neither the wrinkle
scores nor the wrinkle percentages by image analysis differed significantly between the
groups with high (at least 5 weeks) versus low (<5 weeks) levels of sun exposure
(p=0.113 and p=0.089, respectively).
5.2 Skin thickness and elasticity (II)
A statistically significant difference in skin thickness between the groups was found only
in the cheek. Skin thickness in the other body regions did not differ significantly between
the groups (Table 6). Skin elasticity did not show significant differences between the
groups. The elasticity modulus on the upper inner arm was slightly decreased in the
smokers (Table 6).
51
Table 6. Skin thickness (mm) and elasticity (MPa) values. Mean, (SD).
Measurement
Upper inner arm
Dorsal
Temple
Cheek
Abdomen
1.80 mm
forearm
Skin thickness
Smokers
Skin thickness
non-smokers
1.07 mm
1.42 mm
1.65 mm
1.86 mm*
(0.1)
(0.2)
(0.2)
(0.2)
(0.2)
1.11 mm
1.44 mm
1.60 mm
1.71 mm
1.84 mm
(0.1)
(0.2)
(0.2)
(0.2)
(0.2)
Elasticity
9.83 MPa**
18.90 MPa
4.55 MPa
7.67 MPa
5.68 Mpa
Smokers
(3.4)
(2.3)
(3.3)
(3.6)
(3.0)
Elasticity
11.23 Mpa
18.96 MPa
4.70 MPa
7.78 MPa
6.06 MPa
(4.0)
(1.9)
(3.3)
(3.1)
(3.3)
non-smokers
Standard deviations (SDs) are presented in brackets. * significant difference p< 0.001 ** p=0.07
5.3 Biochemical analyses
5.3.1 Urinary nicotine metabolites
The smoking status of both the smokers and non-smokers was confirmed based on the
urinary concentrations of cotinine and other nicotine metabolites and controlled for the
urinary creatinine concentration. The mean urinary concentration of nicotine metabolites
was 465.4 ng/nmol (range 48.8-1100.0, SD=252.5) in the smokers and 8.8 ng/nmol
(range 3.0-29.2, SD=5.1) in the non-smokers. The levels of nicotine metabolites in the
non-smokers were within the range suggested to be common for non-smokers with or
without exposure to environmental tobacco smoke (Benowitz 1996, Curvall &Vala 1993).
5.3.2 Procollagen propeptides in suction blister fluid and serum (III)
The levels of the precursors for type I and type III collagens, PINP and PIIINP, in the
SBF of the smokers were lower by 18 % and 22 %, respectively compared with the nonsmokers, but the difference was statistically significant only for PIIINP (p< 0.05) (III,
Table 1). The levels of PIIINP were also significantly lower in the serum of the smokers
compared to the non-smokers (p<0.001). The levels of the procollagen propeptides PINP
and PIIINP in SBF but not those in serum correlated inversely with the amount of urinary
nicotine metabolites (r = -0.2, p<0.05 and r = -0.3, p<0.05 for PINP and PIIINP
respectively) (Fig. 6-7). PIIINP in SBF also showed an inverse correlation with the
number of cigarettes smoked per day (r = -0.3, p<0.05) and with the number of years
smoked (r = -0.2, p<0.05). There was no significant correlation between age and the
levels of procollagen propeptides in SBF or serum.
52
The total protein content in SBF was 24.0 μg/μl in the smokers and 23.6 μg/μl in the
non-smokers, which difference was not statistically significant (p=0.7), suggesting that,
despite the vasoconstrictive effects of nicotine, the diffusion of proteins from the vessels
into SBF is not affected in smokers.
Fig. 6. Correlation between PIIINP in SBF and urinary nicotine metabolites.
53
Fig. 7. Correlation between PINP in SBF and urinary nicotine metabolites.
5.3.3 MMP-8 in suction blister fluid (III)
The mean MMP-8 concentration in SBF was significantly higher (16 μg/l) in the smokers
compared to the non-smokers (8 μg/l), p<0.001 (Fig. 8). There was a significant
correlation between the amount of urinary nicotine metabolites and the levels of MMP-8
in SBF (r = 0.3, p<0.01). Mean MMP-8 was significantly higher in the young smokers
(21 μg/l) compared to the young non-smokers (7 μg/l) and in the older smokers (13 μg/l)
compared to the older non-smokers (9 μg/l) (III, Fig. 1C).
54
100
80
MMP8 in the SBF ug/l
60
40
20
0
-20
N=
49
47
non-s mokers
smokers
Smoking status:
Fig. 8. Distributions of MMP-8 levels in the SBF of smokers and non-smokers.
5.3.4 TIMP-1 in suction blister fluid and serum (III)
The mean TIMP-1 concentration was significantly lower in the SBF of the smokers
compared to the non-smokers (185 ng/ml and 215 ng/ml, respectively, p<0.01). TIMP-1
in SBF correlated weakly with age (r = 0.2, p<0.05) and showed an inverse correlation
with the amount of smoking (number of years smoked r = -0.2, p<0.05 and number of
cigarettes smoked per day r = -0.3, p<0.05) The mean serum concentrations of TIMP-1
were 318 ng/ml in the smokers and 312 ng/ml in the non-smokers, the difference being
not statistically significant (p=0.64).
5.3.5 Gelatinases and MMP-8 in serum (IV)
The mean level of MMP-8 in serum was slightly higher in the smokers than the nonsmokers, but the difference was not statistically significant (p>0.1; 95%CI=-7.1-2.0). The
serum levels of MMP-8 did not correlate with age, the amount of urinary nicotine
55
metabolites or the number of years smoked. The mean amount of MMP-9 in serum was
significantly higher in the smokers compared to the non-smokers (p<0.001; 95 % CI 0.24- -0.09). The mean amount of MMP-2 in serum was also higher in the smokers than
in the non-smokers (p= 0.001; 95 % CI -0.24- -0.06). Serum MMP-9 had a moderate
correlation (r=0.4, p<0.001) and serum MMP-2 had a weak correlation (r=0.3, p<0.05)
with the amount of urinary nicotine metabolites but neither of them correlated with age.
5.3.6 Gelatinases and MMP-8 in skin tissue (IV)
The mean levels of both the proactive form and the active form of MMP-9 were
significantly lower in the skin of the smokers compared to the non-smokers. Pro MMP-9
was found in the skin of 9 smokers and 8 non-smokers, with mean levels of 4139 DU and
8894 DU, respectively (p=0.002). Act MMP-9 was found in 9 smokers and non-smokers,
with mean levels of 5015 DU and 9175 DU, respectively (p=0.002). The mean levels of
both pro MMP-2 (8533 DU vs 9062 DU) and act MMP-2 (9409 DU vs 9862 DU) were
slightly lower in the skin of the smokers compared to the non-smokers, but the
differences were not statistically significant. The amounts of both PMN-type active
MMP-8 (PMN act) and Non-PMN-type active MMP-8 (Non-PMN act) were lower in the
skin of the smokers compared to the non-smokers (p=0.03 and p=0.05, respectively). The
expression levels of the other molecular forms of MMP-8 did not differ significantly
between the smokers and non-smokers.
5.3.7 Salivary MMP-8 and MMP-9 (IV)
The mean concentration of MMP-8 in salivary samples was 66 μg/l (SD 92.4) in the
smokers and 85 μg/l (SD 93.4) in the non-smokers, but the difference was not statistically
significant (p>0.1). The levels of salivary MMP-8 did not correlate with age, the amount
of urinary nicotine metabolites or the number of years smoked.
The mean salivary concentrations of total MMP-9 in the smokers and non-smokers
were 156.0 units/ml (SD 179.2) and 223.9 units/ml (SD 208.0), respectively, the
difference being statistically significant (p=0.032). The mean salivary concentrations of
active MMP-9 did not differ significantly between the smokers (68.3 units/ml; SD 64.7)
and non-smokers (66.1 units/ml; SD 68.3) (p=0.348). Total salivary MMP-9 had a weak
inverse correlation with the amount of urinary nicotine metabolites (r =- 0.2, p=0.09) and
with the number of years smoked (r = -0.2, p=0.09), whereas active MMP-9 did not
correlate with these parametres.
56
5.4 Skin histology and restoration of epidermal barrier function
5.4.1 Proportional area and width of elastic fibres (II)
Computerized image analysis was used to assess the proportional area of elastic fibres
from Verhoeff-stained skin biopsy specimens. Four consecutive fields from papillary
dermis and four fields from reticular dermis were analyzed, avoiding follicular and
damaged regions. The mean proportional area of elastic fibres in papillary dermis was
6.57 per cent (SD 2.0) in the smokers and 6.51 per cent (SD 1.8) in the non-smokers, the
difference being not statistically significant (p=0.9). The mean proportional area of elastic
fibres in reticular dermis was 8.67 per cent (SD 3.0) in the smokers and 9.08 per cent (SD
3.0) in the non-smokers, the difference being not statistically significant (p=0.5).The
width of elastic fibres was measured from three fields of reticular dermis. The average
width of elastic fibres was 1.8 μm in both the smokers and non-smokers.
5.4.2 Number of PINP-positive fibroblasts in papillary dermis (III)
The number of PINP-positive fibroblasts was counted from five consecutive fields of
papillary dermis. The mean number of PINP-positive fibroblasts was 7.9 per field (SD
6.5) in the smokers (N=39) and 9.2 per field (SD 5.8) in the non-smokers (N=39), the
difference being not statistically significant. The number of PINP-positive fibroblasts did
not correlate with age.
5.4.3 Epidermal thickness and number of Langerhans cells
Three measurements representing the average areas of epidermis were made on each skin
specimen, and the means were calculated and compared. The mean epidermal thickness
was 41.2 μm (SD 7.9) in the smokers and 40.4 μm (SD 6.4) in the non-smokers, the
difference being not statistically significant.
Five consecutive fields of epidermis were analyzed for the assessment of the number
of epidermal Langerhans cells. The mean number of Langerhans cells was 10 per field
(SD 2.9) in the smokers (N=42) and 10 per field (SD 3.2) in the non-smokers (N=39).
There was a statistically significant inverse correlation between the amount of epidermal
Langerhans cells and age (r = -0.3, p<0.01).
5.4.4 Number of vascular lumens in papillary dermis
The number of vascular lumens was calculated from five consecutive fields of papillary
dermis. The mean number of lumens was 11 per field (SD 2.3) in the smokers (N=42) and
57
11 per field (SD 2.2) in the non-smokers (N=38). There was a weak negative correlation
between age and the number of vascular lumens (r = -0.2, p= 0.06).
5.4.5 Restoration of epidermal barrier function
Transepidermal water loss (TEWL) was measured on the day suction blisters were
induced and four days later from the bases of the blisters. In the smokers, the mean
TEWL was101 g/m2h on day one and 29 g/m2h on day four. In the non-smokers, the
mean TEWL was 100 g/m2h on day zero and 29 g/m2h on day four. The average decline
in the transepidermal water loss was 72 g/m2h in the smokers and 68 g/m2h in the nonsmokers, the difference being not statistically significant.
6 Discussion
6.1 Appearance and wrinkling (I)
The panellists identified 68 % of the smokers correctly as being smokers, which is more
than suggested by Model (1985), who reported that 50 % of smokers could be identified
by their facial features. In the present study, 60 % of the non-smokers were correctly
estimated to be non-smokers, while 40 % were falsely estimated to be smokers.
Furthermore, the smokers were estimated to be older than their age, whereas the nonsmokers were estimated as younger than their age. Since neither the clinical nor the
computerized assessment of skin wrinkling revealed significant differences between the
groups, other facial features than wrinkles perhaps enable the correct recognition of a
smoker and explain the more aged appearance of smokers, as suggested by Model (1985),
who listed such features as skin colour, prominence of facial bones and sinking of cheeks
as being typical for a smoker´s face (Model 1985). In a more recent study (Kennedy et al.
2003), smoking was strongly associated with elastosis among both females and males and
with the amount of facial telangiectasia among male smokers.
There are convincing studies showing an increased risk of premature wrinkling in
smokers, with an additional impact when smoking is accompanied by excessive sun
exposure (Kadunce et al. 1991, Ernster et al. 1995, Chung et al. 2001). However, the
importance of smoking as a causative agent for wrinkling seems to be smaller than that of
UV radiation (O´Hare et al.1999, Malvy et al. 2000). The older studies on wrinkling in
smokers were not controlled for sun exposure (Daniell 1971, Model 1985), and the
results may therefore have been affected by the environment in which these studies were
performed. The studies that have most convincingly reported an increased risk of facial
wrinkling in smokers were also carried out in regions with a high level of sun exposure,
but the effect of sun exposure was controlled for (Kadunce et al. 1991, Ernster et al.
1995, Chung et al. 2001). Since almost two thirds of the smokers in this study were
correctly identified as being smokers by their appearance, it would be an interesting
prospect for the future to further examine the appearance of facial skin in terms of
complexion, facial contours, fine and deep wrinkles as well as dermal components, such
as the levels of glycoproteins and hyaluronic acid, in smokers and non-smokers.
59
6.2 Physical qualities of skin (II)
Skin thickness varied in different anatomical sites, as has been reported previously
(Fornage et al. 1993, Takema et al. 1994). In the present study the mean thickness of skin
was 1.78 mm on the cheek, 1.43 mm on the dorsal forearm and 1.09 mm on the upper
inner arm, which findings are in concordance with the earlier reports by Fornage et al.
(1993), who reported a forearm skin thickness of 1.4 mm ( +/- 0.3) in 10 healthy
volunteers whose mean age was 33 years, and by Takema et al. (1994), who reported an
average skin thickness of 1.72 mm (+/- 0.20) on the cheek and 0.95 mm (+/-0.11) on the
ventral forearm. In the present study, only the thickness of cheek skin differed
significantly between the smokers and the non-smokers, but the reason for the increased
thickness of cheek skin in smokers remains unclear. The levels of past recreational sun
exposure reported by the two groups did not differ significantly, but the amount of sun
exposure to facial skin specifically was not recorded, and it therefore remains a possible
source of error when evaluating previous sun exposure as a confounding effect, which
could affect the thickness, elasticity and wrinkling of facial skin. Since smoking and sun
exposure potentiate each other`s effects on skin ageing (Kadunce et al. 1991), the
observed difference in skin thickness in cheek skin between the groups might be due to
the combined effects of smoking and UV radiation.
Overall, skin thickness in various body regions did not differ significantly between the
groups, which is in line with previous data (Whitmore & Sago 2000). When comparisons
of skin thickness of the volar forearm were made in black and white women, and
smoking was taken into account as a possible confounding factor, the thickness of skin
was not found to be affected by smoking (Whitmore & Sago 2000). Correlation
coefficient analysis did not show significant correlations between age and skin thickness
in any of the studied regions in the present study, contrary to Pellacani & Seidenari
(1999), who observed thickening of facial skin along with age in all facial regions except
the infraorbital area. This is probably due to the age distribution of 34 to 71 years, since
skin thickness remains quite constant between 10 to 70 years, after which a marked
decrease in skin thickness takes place (Escoffier et al. 1989). Skin thickness declines over
age in sun-protected and severely photodamaged skin areas, but increases in the early
stages of actinic damage. (De Rigal et al. 1989, Takema et al. 1994, Oikarinen 1994)
The elasticity of facial, abdominal and forearm skin was fairly similar in smokers and
non-smokers. The elasticity modulus of upper inner arm was slightly but not significantly
smaller in smokers compared with non-smokers. The skin of the upper inner arm was of
special interest, since it is a sun-protected area. It was also an interesting site because skin
biopsies were obtained from the same area, and thus both non-invasive elasticity
measurements and the histological appearance and number of elastic fibres in the skin
samples could be evaluated. Studies with Twistometer® and Cutometer SEM 474® have
shown an age-associated decline in skin elasticity and an increase in extensibility
(Adhoute et al. 1992, Takema et al. 1994, Piérard et al. 1998). In the present study, a
linear correlation between age and elasticity modulus was only found in the upper inner
arm (r =0.2, p=0.05) and abdomen (r = 0.3, p<0.01), indicating increased skin stiffness in
these areas. Previously, an age-associated decrease in extensibility and an increase in
stiffness have been observed in the sun-protected skin of the ventral forearm (Agache et
60
al. 1980, Escoffier et al. 1989, Pedersen et al. 2003). There are also site-dependent
variations in skin elasticity. In the present study, skin was stiffer (elasticity modulus
higher) in the upper extremities than in the abdomen, as previously noted by Serup &
Northeved (1985). Due to variations in the methodology and the wide variety of devices
used for assessing skin elasticity, it is difficult to compare the results of different studies,
as also pointed by others (Escoffier et al. 1989, Marks & Edwards 1992).
6.3 Collagen synthesis and ECM turnover (III, IV)
This study demonstrated decreased skin collagen synthesis in smokers in terms of both
type I and type III collagens. The mean concentration of the precursor for type III
collagen, PIIINP, in SBF was 22 % lower in the smokers compared to the non-smokers,
the difference being statistically significant, and the mean PINP concentration in SBF
was 18 % lower in the smokers compared to the non-smokers, but the difference was not
statistically significant, possibly at least in part due to the considerable interindividual
variation in the propeptide concentrations, as shown by the high standard deviations in
both groups. However, the concentrations of the procollagen propeptides PINP and
PIIINP were systematically lower in the smokers even when these groups of smokers and
non-smokers were subdivided into younger and older age groups and then compared. The
total protein content in SBF did not differ significantly between the smokers and nonsmokers, which suggests that the diffusion of proteins from the vessels into SBF is not
affected by smoking.
The clinical relevance of the observed decrease in skin collagen synthesis in smokers
is unknown, but it can be assumed to potentially affect the mechanical strength and
wound healing of skin, even though the physical qualities of skin as well as the rate of reepithelialization remained unaffected in the present group of smokers. The results are in
concordance with an earlier study, in which wound healing and collagen production were
assessed in 19 smokers and 19 non-smokers, and decreased collagen synthesis was
observed in smokers in the form of lowered subcutaneous hydroxyproline concentrations
(Jorgensen et al. 1998) and with a cell culture study, which showed decreased production
of type I and III collagens in human skin fibroblasts after exposure to tobacco smoke
extract (Yin et al. 2000). The ratio of PINP/PIIINP in SBF was 3.5:1 in smokers and 3:1
in non-smokers, being slightly smaller than the estimated average for adults (Uitto et al.
1989). The ratio of PINP/PIIINP in serum was 13:1 in smokers and 11.5:1 in nonsmokers. The levels of procollagen propeptides in SBF and serum did not correlate with
age, but there was a significant negative correlation between the levels of both PINP and
PIIINP in SBF and the amount urinary nicotine metabolites.
MMP-8 levels were significantly higher in the SBF of smokers, indicating that not
only collagen synthesis is affected in smokers, but also collagen degradation, which leads
to decreased amounts of collagen in skin and could affect the tensile strength of skin in
smokers. In skin tissue samples, the mean levels of both the proactive form and the active
form of MMP-9 as well as of active MMP-8 (PMN and Non-PMN types) were
significantly lower in the smokers compared to non-smokers, whereas the levels of
MMP-2 did not differ significantly between the groups. The levels of both MMP-2 and -9
61
were significantly higher in the serum of smokers compared to non-smokers, whereas the
levels of MMP-8 were slightly but not significantly higher in the serum of smokers
compared with the reference group. It is possible that some of the MMP-8 in SBF could
originate from serum. MMPs -1, -2, -8 and -13 can cleave fibrillar collagens, whereas
MMP-9 cannot (Mohan et al. 2002). Chronic ulcers have been shown to express
significantly higher levels of MMP-1 and-8 compared to normally healing wounds, and
these collagenases were usually in the inactive form in normally healing wounds,
whereas the active forms were common in chronic ulcers (Nwomeh et al. 1999). Even
though some MMPs, including MMP-8 and -9, for example, are stored in cells within
tissues, the baseline expression of most MMPs in cells is low, but their expression
increases rapidly in response to various external stimuli, such as cytokines and growth
factors (Kähäri & Saarialho-Kere 1999).
The relevance of the variable expression of different molecular forms of gelatinases
and MMP-8 in the healthy skin tissue of smokers and non-smokers is unknown, but it
seems that smoking affects the metabolism of skin in many ways. The fact that the serum
levels of both 72-kDa and 92-kDa gelatinases were higher in the present smokers
compared to non-smokers indicates that the effects of smoking on ECM are not confined
to skin but are likely to affect several tissues in the body. Since osteoclasts express both
MMP-2 and MMP-9, it is plausible that gelatinases in serum are partially derived from
bone (Andersen et al. 2004). Cigarette smoke is distributed directly into the lung and,
through circulation, into numerous other organs as well. Recently, it was shown that
alveolar macrophages from patients with COPD released more MMP-9 with greater
enzymatic activity than those from healthy smokers or non-smokers, and cigarette smokeconditioned culture medium showed an increase in MMP-9 and TIMP-1 in all of the
groups (Russell et al. 2002). TIMP-1 in SBF was 14 % lower in smokers compared to
non-smokers. Since TIMP-1 is an important inhibitor of many MMPs (Kähäri &
Saarialho-Kere 1999), the result indicates changes in the regulation of ECM turnover due
to smoking, as previously reported based on in vitro findings (Yin et al. 2000).
The salivary concentrations of MMP-8 were lower in smokers than in non-smokers,
but despite the marked difference in the mean concentration of salivary MMP-8 between
the two groups, the difference was not statistically significant, probably due to the great
interindividual variations in MMP-8 levels in both groups. The levels of total but not
active MMP-9 were significantly lower in the smokers compared to the non-smokers.
Previously, MMP-8 and MMP-13 have been detected in the inflamed gingival tissue of
adult periodontitis patients (Ingman et al. 1994 a&b, Kiili et al. 2002), and salivary
MMP-1 was increased in patients with periodontitis (Ingman et al. 1993). In a recent
study, MMP-9 seemed more important in periodontal remodelling than MMPs -2, -8 or 13 (Apajalahti et al. 2003). In the past, MMP-8 has been regarded as a neutrophil
collagenase and thought to be synthesized by polymorphonuclear neutrophils only.
However, there is evidence that other cell lines, such as endothelial cells and rheumatoid
synovial fibroblasts, are capable of synthesizing MMP-8 in vivo (Hanemaaijer et al.
1997). In the present study, skin tissue samples from both smokers and non-smokers
expressed both PMN type MMP-8 (60-80 kDa) and non-PMN type MMP-8 (38-45 kDa),
which is suspected to originate from fibroblasts.
62
6.4 Histology
No significant differences were seen in the number or width of elastic fibres between the
smokers and non-smokers, contrary to the results published by Francès et al. (1991) and
Boyd et al. (1999), who found changes in both the number and the structure of elastic
fibres in smokers. The former group reported mean relative areas of 19 % and 9 % of
elastic fibres in the sun-protected skin of smokers and non-smokers, respectively
(p<0.01) and the latter group reported mean relative areas of 25 % and 20 % of elastic
fibres in the sun-exposed facial skin in smokers and non-smokers, respectively (p<0.05).
Both of the previous studies were, however, based on notably smaller study populations.
Francès et al. (1991) based their findings on morphometric assessments of 10 smokers
and 10 non-smokers aged 60, and Boyd et al. (1999) studied 17 smokers and 14 nonsmokers aged 59 to 65. The study subjects were somewhat older and had smoked more in
terms of pack-years than those in the present study, which may partially explain the
difference in the results. The study setting used by Boyd et al. (1999) was blinded,
whereas Francès et al. (1991) do not mention whether or not their assessments were
blinded. In the present study, the proportional area occupied by elastic fibres accounted
for approximately 6.5 percent of papillary dermis and 9 percent of reticular dermis, being
in concordance with the previous findings on sun-protected skin (Gogly et al. 1997).
There was an age-associated increase in the amount of elastic fibres in both papillary
dermis (r =0.2, p<0.05) and reticular dermis (r =0.3, p<0.05), as reported previously, but
the width of elastic fibres did not correlate with age, as opposed to the previous results by
Gogly et al. (1997).
The number of PINP-positive fibroblasts was counted, to further assess the rate of
collagen synthesis in skin and to find out whether the number of PINP-positive
fibroblasts is affected in a similar fashion as the levels of procollagen propeptides. In in
vitro studies, nicotine has been shown to have toxic effects on cardiac fibroblasts,
affecting the gene expression for type I collagen, collagenase activity and DNA synthesis
of cardiac cells (Tomek et al. 1994). The average number of dermal PINP-positive
fibroblasts was slightly but not statistically significantly smaller in smokers than in nonsmokers. The number of PINP-positive fibroblasts did not correlate with age or smoking
history.
The number of epidermal Langerhans cells was counted to search for mechanisms
behind the altered immunological function of skin in smokers (Mills et al. 1993a).
Normally, Langerhans cells represent approximately 4 % of epidermal cells, but their
number and antigen-presenting function are known to decrease after exposure to UV
radiation (Koulu & Jansén 1980, Räsänen et al. 1989). It was expected that long-term
exposure to a toxic substance like tobacco might have a similar effect as UV radiation
and decrease the number of epidermal Langerhans cells. However, no differences in the
number of Langerhans cells between the smokers and non-smokers emerged. The
observed decline of Langerhans cells with age is in concordance with the earlier reports
(Sunderkötter et al. 1997). Previously, morphologic and functional alterations in the
Langerhans cells of mice exposed to tobacco smoke condensate have been reported (Zeid
& Muller 1995). It would be interesting future research to evaluate the functional
capacity of Langerhans cells in smokers. There is also a large open field for further
immunological studies.
63
To assess the possible effects of smoking on the vascular functions in skin, all separate
vascular lumens of papillary dermis were counted within five separate fields, since it was
impossible to estimate from the non-three-dimensional microscopic image how each
blood vessel runs in dermis. Another possibility would have been to measure the
diameters of blood vessels, but the measurements would have suffered from the same
problem of dimensionally partial visibility. The numbers of vascular lumens did not differ
between the groups. There was a negative correlation between the number of vascular
lumens and age, which supports the previous findings (Marks & Edwards 1992).
Smoking history (number of years smoked and number of cigarettes smoked per day) did
not correlate with the number of papillary lumens, nor did the amount of urinary nicotine
metabolites.
The restoration of epidermal barrier function was studied by measuring transepidermal
water loss from the base of the suction blisters right after they were induced and four
days later. The smokers and non-smokers did not differ in respect of the normalization of
transepidermal water loss. The TEWL values on day 0 and on day 4 were similar to those
reported by Koivukangas & Oikarinen (1998), who used the suction blister model to
study the recovery of barrier function after UV- exposure. Earlier reports (Siana et al.
1989, Goldminz & Bennett 1991) have shown postoperative wound healing to be
compromised in smokers. However, surgical wounds are much deeper and wider than the
experimental wound in this study. The present results indicate that superficial skin
wounds heal equally well in smokers and non-smokers.
6.5 Study design and subjects
This study was a case-control study of Finnish male smokers and non-smokers. The study
population represented a fairly homogeneous group of men from the provinces of Oulu
and Lapland in Northern Finland. Most of the study subjects (65 %) participated in
response to an advertisement, which was published in the largest local newspaper of the
provinces of Oulu and Lapland. Approximately 12 % of the participants came from the
companies ENSO Fine Papers and Kemira Chemicals, 10 % from within Oulu University
Hospital and 12 % from elsewhere. Both smokers and non-smokers enrolled into the
study from the factories as well as from the hospital, which means that these sources are
not likely to have caused bias. Non-smokers were more keen to participate, possibly due
to having a predisposition towards healthier skin because of their presumably healthy
life-style.
Since sun exposure is an important confounding factor when assessing skin wrinkling
and histology, the fact that this study was carried out in Northern Finland, where sun
exposure during most of the year is low, minimised the confounding effect of sun
exposure on the skin of the study subjects and might also explain why the results are
different from the studies that have been made closer to the Equator (Daniell 1971). The
anamnestic questionnaire included questions about the frequency of holidays in more
southern countries and about the amount of sun exposure during the Finnish summer
months. The questionnaire did not include a direct question about the amount of
sunbathing and about the amount of sun exposure of facial skin. Therefore, it is
64
impossible to make definite conclusions about whether the groups might have differed in
their attitude towards sun exposure. Based on the available data, the amount of sun
exposure between the smokers and non-smokers did not, however, differ significantly.
There were slightly more patients with dermatological diseases in the group smokers, but
the difference was not statistically significant, and none of the participants used systemic
steroids, which are known to affect collagen synthesis (Oikarinen et al. 1992), whereas
localized skin diseases do not seem to alter the serum markers of collagen synthesis or
degradation (Autio et al. 1993). Two patients with a diagnosis of mild asthma were
accepted into the study because they did not use inhaled corticosteroids. No patients with
diabetes, COPD, rheumatoid arthritis or psoriasis were included in the study population,
to prevent the confounding effects of these diseases or their treatments on the results
concerning collagen synthesis and ECM metabolism (Seibold et al. 1985, Oikarinen et al.
1986).
All but five of the smokers smoked cigarettes. All of the current pipe and cigar
smokers were previous cigarette smokers and therefore likely to inhale pipe and cigar
smoke in amounts comparable to cigarette smoke (Turner et al. 1977). The smokers had
been smoking for an average of 33 years, and the average smoker smoked almost a pack
per day (range 5-40 cigarettes). Approximately 70 % of the smokers had tried to quit
smoking, which is a similar proportion to the percentages previously reported from a
Finnish population, in which 60% of male smokers and 56% of female smokers
expressed a wish to quit (Helakorpi et al. 1998), and from the United States, where
approximately 70 % of smokers were estimated to have a desire to quit (Skaar et al.
1997). Passive exposure to tobacco smoke at home or at work was reported by two
percent of the non-smokers and nine percent of the smokers.
6.6 Strengths and weaknesses of the study
Having a group of males under evaluation involves both benefits and disadvantages.
Males do not generally use hormonal treatments, and there are more male smokers than
female smokers. It can also be suspected that males are not equally concerned about
having skin biopsies taken as females, who might be more worried about scarring.
However, since smoking among young girls is increasing in Finland (Helakorpi et al.
1998), and women are more concerned about the effects of smoking on appearance than
men (Rundmo et al. 1997), it would have been beneficial to study the effects of smoking
in females, too, especially since premature wrinkling is thought to appear earlier in
females who smoke than in males (Ernster et al. 1995). Since power analyses were not
made prior to the recruitment of study subjects, the results showing no difference
between the smokers and non-smokers may be due to insufficient sample sizes, which
should be borne in mind when interpreting the results. The results concerning skin
wrinkling differ from previous, larger studies (Kadunce et al. 1991, Ernster et al. 1995),
which could be due to an insufficient sample size as well as to environmental factors.
Since this study was carried out in northern Finland, the confounding effects caused by
solar irradiation are minimised. The smoking status of the study subjects can be
considered reliable, since nicotine metabolites were assessed from the urinary samples of
65
all participants. The amount of alcohol consumed was not verified by laboratory methods,
since tobacco was the main substance of interest. If there is some bias in the reporting of
drinking habits, one would expect the bias to be similar in both groups. Ernster et al.
(1995) reported alcohol as not having a significant impact on the risk of moderate to
severe wrinkling.
Some of the methods used in this study assessed skin as a whole, such as the skin
thickness measurements and the evaluations of appearance and wrinkling of skin.
Epidermal functions were studied in terms of transepidermal water loss, which enables
assessment of the regeneration of epidermal barrier function. Non-functional
measurements of epidermis included comparison of epidermal thickness and the number
of Langerhans cells in the epidermis of smokers and non-smokers. No significant
differences were observed between the groups concerning either the morphology or the
functionality of epidermis. The morphology and function of the dermal layer was of
special interest, since dermal blood vessels enable tobacco smoke to reach dermis. To
evaluate the effects of smoking on dermis, the synthesis and degradation of collagen was
assessed by measuring the levels of the procollagen propeptides PINP and PIIINP and the
levels of MMPs in SBF and skin tissue. Also, the numbers of dermal PINP-positive
fibroblasts and skin elasticity were compared between the smokers and non-smokers.
Furthermore, we compared the levels of gelatinases and MMP-8 in the different tissue
compartments of the smokers and non-smokers, i.e. in SBF, serum, saliva and skin
samples. Morphological studies of dermis included comparisons of the quantity and
quality of elastic fibres and of the number of vascular lumens in smokers and nonsmokers.
Of the methods, the laboratory methods can be considered to be of high standard,
reliable and unbiased. Almost all measurements were performed by the author, but the
technical assistance of a trained nurse was used on some occasions. All measurements
were done in the same room either in the morning or early afternoon, with the ventilation
apertures in the vicinity of the patient closed. For the assessments of skin thickness,
elasticity and TEWL, the same devices and methodologies were used in all subjects. Ascan ultrasound has proved highly reproducible in the assessment of skin thickness and
has been widely used for years (Tan et al. 1982, Agner 1995, Serup et al. 1995). The
Dermalab device has proved useful compared to another suction cup method, Dermaflex,
although a higher coefficient of variation was found for the Dermalab device (Pedersen et
al. 2003). TEWL is also well documented in the assessment of the physical qualities of
skin (Blichmann & Serup 1987, Pinnagoda et al. 1990, Pinnagoda & Tupker 1995). The
suction blister method has been well described and widely used previously in assessments
of both ECM turnover and the healing process of superficial wounds (Kiistala
&Mustakallio 1964, Kiistala 1968, Autio & Oikarinen 1997, Koivukangas & Oikarinen
2003). The assessment of facial photographs is affected by the scattering of light despite
standardized photographic methods, which could affect the ability to detect fine wrinkles.
Daniell´s score as a scoring system is well defined and has been used by others in the
evaluation of smokers´wrinkles (Daniell 1971, Kadunce et al. 1991, Yin et al.2001, Koh
et al. 2002).
The histological evaluations were made in a blinded fashion, and the code was opened
after the data had been saved on the computer. There are, however, sources of error in the
assessment of histological samples. The quality of the original sample, the handling of the
sample while cutting it and the quality of staining in immunohistochemistry all affect the
66
assessment of skin samples. Furthermore, the microscopic image of skin is practically
two-dimensional, which affects the evaluation of skin morphology, especially of such
structures as elastic fibres, which form a three-dimensional fibrillar network in dermis,
and of dendritic cells, such as Langerhans cells. Despite the possible sources of error due
to partial visibility of these and other skin components, computerized image analyses
have proved useful in the assessment of elastic fibres (Uitto et al. 1983, Flotte et al.
1989). The validity and reliability of the computerized image analysis method has shown
good accuracy and reproducibility in the assessment of elastic tissue. When the reliability
of the method was tested in four different ways (same sections analyzed at different
times, adjacent sections analyzed at different times, adjacent sections with variable
staining lots and analyses of adjacent biopsy sites), all reliability tests showed good
consistency of the acquired results. (Flotte et al. 1989) However, accurate assessment of
the structure of elastic fibres is demanding and remains qualitative. Morphological
studies comparing dermal elastic fibres of smokers and non-smokers might benefit from a
combined study protocol of computerized image analysis and biochemical analyses of
desmosine or elastin or measurement of elastin mRNA. In this study, image analysis of
Verhoeff-stained skin biopsies was chosen to study the morphology of elastic fibres. The
other possibility would have been to use immunohistochemistry, but this alternative was
abandoned because the Verhoeff stain is more sensitive in analyses involving all elastic
fibres. Biochemical methods for the assessment of elastin or desmosine were not readily
available and were therefore not used.
7 Conclusions
The majority of smokers were identified as smokers by their appearance, and smokers
were estimated older than their age, even though they were not considered to be more
wrinkled than non-smokers by either clinical or computerized assessments. The physical
qualities of skin, such as skin thickness, elasticity and regeneration of epidermal barrier
function, did not generally differ between smokers and non-smokers, but differences at
the biochemical level were observed between these groups. Skin collagen synthesis,
measured in vivo by the levels of the procollagen propeptides PINP and PIIINP, was
decreased in smokers compared with non-smokers, and the MMP-8 levels in SBF were
elevated in smokers, indicating increased degradation of type I and III collagens in skin.
TIMP-1 levels were lower in the SBF of smokers than non-smokers, not compensating
for the increased collagen loss by MMP-8 induction. The serum levels of MMPs -2, -9
and -8 were also higher in smokers. The decreased synthesis rates of type I and III
collagens and the observed disturbances in the amounts of proteins regulating ECM
turnover provide evidence of altered structure and metabolism of skin due to smoking
and partly explain the pathogenesis of premature wrinkling and impaired wound healing
previously reported in smokers.
The adverse effects of smoking on the appearance and function of skin could be used
in health education, to discourage people from starting to smoke and to incourage current
smokers to quit.
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