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 Table of Contents  
ORIGINAL ARTICLE
Year : 2013  |  Volume : 8  |  Issue : 2  |  Page : 74-78

Effect of smoking on skin elastic fibers: a morphometric and immunohistochemical analysis


1 Department of Dermatology and Venereology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Dermatology and Venereology, National Research Centre, Cairo, Egypt
3 Department of Pathology, National Research Centre, Cairo, Egypt

Date of Submission02-Aug-2013
Date of Acceptance15-Sep-2013
Date of Web Publication27-Dec-2013

Correspondence Address:
Abdel Razik H Farrag
Department of Pathology, National Research Centre, Dokki, Zip Code: 12622; Cairo
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1687-4293.123790

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  Abstract 

Background/aim
Tobacco smoking is one of the major health problems in the world. This study was conducted to investigate the morphological changes of the dermal elastic tissue of sun-protected skin induced by smoking.
Participants and methods
Punch biopsies were obtained from the nonexposed skin of 15 smokers and 15 nonsmokers to evaluate the dermal elastic fibers using morphometric analysis and immunohistochemical techniques.
Results
Elastic fibers of the reticular dermis were more numerous, thickened and fragmented in smokers than in nonsmokers. Correlations between the cumulative tobacco dose and the morphology of the elastic fibers were highly significant. Immunohistochemical analysis for α-1-antitrypsin showed negative cytoplasmic staining for all samples of both smokers and nonsmokers.
Conclusion
This study indicates that smoking is one of the risk factors that lead to an increase in elastic fibers of the reticular dermis. This increase is due to elastic tissue degradation rather than by new synthesis, as in solar elastosis. Furthermore, smoking has multiplicative effects on facial ageing and could share, at least in part, a pathophysiological pathway.

Keywords: elastic fibers, immunohistochemistry, morphometry, skin, smoking


How to cite this article:
El-Kahky HM, Zuel-Fakkar NM, Ali DG, Mahdy AS, Farrag AR, Helmy NA. Effect of smoking on skin elastic fibers: a morphometric and immunohistochemical analysis . J Arab Soc Med Res 2013;8:74-8

How to cite this URL:
El-Kahky HM, Zuel-Fakkar NM, Ali DG, Mahdy AS, Farrag AR, Helmy NA. Effect of smoking on skin elastic fibers: a morphometric and immunohistochemical analysis . J Arab Soc Med Res [serial online] 2013 [cited 2020 Apr 2];8:74-8. Available from: http://www.new.asmr.eg.net/text.asp?2013/8/2/74/123790


  Introduction Top


Tobacco smoking is one of the major health problems in the world and is causally related to many chronic and malignant diseases [1] . Human skin is exposed to smoke directly through its irritant components on the epidermis and indirectly on the dermis through the bloodstream. It is not surprising therefore that smoking has many effects on the skin and is associated with significant morbidity of this organ [2] .

In the past three decades, several studies have shown that smoking is an independent risk factor for the development of premature facial wrinkling and skin aging [3],[4] . To establish the pathophysiological mechanisms underlying this premature wrinkling and on the basis of the finding that elastin is the major target in smoke-induced emphysema [5],[6] , several reports have used morphometry to analyze the elastic fibers of the skin in smokers and nonsmokers [7],[8] .

The results showed an increase in the number, the thickness, and the total area occupied by dermal elastic fibers in smokers compared with nonsmokers. Another study, however, shows contradictory findings that question the effects of smoking on elastic fibers of the skin [9] .

There is a hypothesis that elastin could also be the main target in smoke-induced wrinkles, as in emphysema. Lung function impairment with an obstructive pattern is associated with morphologic abnormalities in the reticular dermis appearing in the histological section as an increase in the percentage of the field filled by elastic fibers. This relationship depends on cumulative smoking and suggests a common effect of smoking on the elastic fibers in both the lung and the skin [10] .

This study was conducted to investigate the morphological changes of the dermal elastic tissue of sun-protected skin induced by smoking, and the possible mechanisms responsible for them.


  Participants and methods Top


This study included 15 cigarette smokers, selected from the Outpatient Clinic of Dermatology, Ain Shams University, during the period of February 2009 to August 2009. The study also included 15 nonsmokers as the control group. Only men were included in the study to avoid sex-related confounding factors. Patients in the age group of 30-50 years were included in our study. Patients were categorized as nonsmokers and current smokers who smoke more than 10 cigarettes per day for more than 10 years. The lifetime cumulative tobacco dose was expressed as pack-years (the average number of packs smoked per day multiplied by years smoked).

Patients reporting a history of connective tissue disease, treatment with D-penicillamine, colchicine, artificial ultraviolet radiation, and oral corticosteroids for more than months in their lifetime, BMI greater than 40 kgm 2 and pipe or cigar smokers were excluded from our study.

The study was approved by the ethical committee of the National Research Center and fulfilled all the ethical aspects required in human research. All participants gave an informed consent to participate in this study and the steps of the study were explained to all the participants.

Morphometric analysis

Skin biopsies were obtained from the upper inner arm of 15 cigarette smokers and 15 nonsmokers. These biopsies were fixed in 10% formalin and processed into paraffin sections of 3-μm thickness. They were stained with hematoxylin and eosin for the demonstration of the histological structure and orcein stain for the detection of elastic fibers.

Orcein-stained sections were examined using an image analysis system. This station included a Leica Q 500 IW (Leica DM LB, Cambridge, UK) photo microscope with position captors and a CCD video camera module N 50 (JVC TK-C 1380, Victor Company of Japan LT, Japan). Images were captured with a ×20 magnification objective, digitized after interactive light intensity equilibration, and analyzed as RGB 24-bit images. Five fields of papillary and reticular dermis were selected randomly from those that did not include large nonconnective tissue elements (i.e. hair follicles and sebaceous glands). From every section, an image of gray levels was obtained and converted into a binary image showing brown elastic fibers. This image was processed and the number of elastic fibers in every field and the area filled by them were determined, with the results expressed as elastic fibers mm 2 and the percentage of the field containing elastic fibers. Morphometric values were obtained in the five examined fields of the papillary and reticular dermis to determine the values for the elastic fiber content for each participant.

Immunohistochemistry for α-1-antitrypsin Ab-1

Immunohistochemical analysis was performed on routinely processed, formalin-fixed, paraffin-embedded tissues. Tissue sections were cut at 5 μm and mounted on poly-l-lysine-coated slides. After routine deparaffinization in xylene, the sections were hydrated through a series of graded alcohols, distilled water and PBS at pH 7.2-7.4. Antigen retrieval was performed using Tris-EDTA (pH 9) in a pressure cooker. The slides were incubated in a Dako aoutstainer S 3400, Dako North America Inc., Carpinteria, California, USA with monoclonal rabbit anti-human α-1-antitrypsin Ab-1 (RB-367-R7; NeoMarkers, Fremont, California, USA) (ready-to-use). The slides in the autostainer were removed and hematoxylin counterstaining was performed. Slides were dehydrated in ascending grades of alcohol and were cleared in xylene for three changes, and cover slips were placed. Histological assessment of the intensity of the staining was performed using a visual scale ranging from 0 to 2 (0, positive; 1, weakly positive; and 2, strongly positive).

Statistical analysis

All data were introduced in a database and analyzed using the SPSS statistical software package, version 11.5 (SPSS Inc., Chicago, Illinois, USA). Results for categorical variables were expressed as absolute and relative frequencies and results for continuous variables as mean and SD, or as the median and the interquartile range when the distribution was not normal.

Skin parameters in nonsmokers and smokers were compared (Student's t-test), and the correlation between skin morphology and the cumulative tobacco dosage was examined to determine as to which skin parameters were affected by the lifetime amount of tobacco consumed (Pearson's correlation coefficient). All statistical tests were two sided, and P value less than 0.05 was reported as statistically significant.


  Results Top


This study was carried out on 30 participants: 15 of them were active smokers and the remaining 15 patients were nonsmokers and taken as a control group. The age of all patients ranged from 30 to 50 years, with a mean ± SD of 37.8 ± 5.7 in smokers and 36.6 ± 6.6 in the control group. On comparing the smokers' group and the nonsmokers' group with regard to the age, there were no statistically significant differences (P < 0.05). The criterion of wrinkles in the face were detected in two of 15 smokers [Figure 1]. These criteria include prominent lines, wrinkles emanating from the corner of the eyes (crow's feet) extending on the cheeks, the cheeks appear sunken with prominence of the underlying bone, the skin has a dry and tough appearance and appear more atrophied than nonsmokers' skin [Figure 2]. The correlation between the morphological characteristics of dermal elastic fibers (including the number of fibers and the mean area filled by them) and age in the studied sample was not statistically significant (P < 0.05), confirming the homogeneity of the sample with respect to this parameter [Table 1].
Figure 1

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Figure 2

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Table 1: Correlation between the morphological characteristics of dermal elastic fibers with age


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Morphometric analysis

The elastic fibers of the reticular dermis were numerous, thickened and more fragmented in smokers than in nonsmokers [Figure 3], whereas there was no morphological difference in the papillary dermis between smokers and nonsmokers [Figure 4]. The total area of elastic fibers in smokers showed a significant increase as compared with nonsmokers (P < 0.05) [Table 2].
Figure 3

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Figure 4

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Table 2: Comparison between the total area of elastic fibers in smokers and nonsmokers


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Correlations between the cumulative tobacco dose and the morphology of the elastic fibers was highly significant (P < 0.01) with regard to the number and the mean of the area occupied by elastic fibers [Table 3] and [Figure 5] and [Figure 6].
Figure 5

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Figure 6

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Table 3: Correlations between the cumulative tobacco dose and the morphology of elastic fibers


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Immunohistochemical analysis for α-1-antitrypsin

All samples of both smokers and nonsmokers showed negative cytoplasmic staining for α-1-antitrypsin [Figure 7]a and b. There was no difference in the epidermal thickness between smokers and nonsmokers [Figure 8].
Figure 7

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Figure 8

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  Discussion Top


Our results support the concept that smoking increases the number, the thickness, and the total area occupied by elastic fibers in the reticular dermis of nonexposed skin, but not the papillary dermis in smokers compared with nonsmokers. This association is in agreement with earlier studies [7],[8],[11] , but not with the results reported by Knuutinen et al. [9] who showed contradictory findings.

The morphological changes of elastic fibers including the number and the mean area showed a significant correlation with the cumulative tobacco dose; these findings indicate that the morphological effects are dose related (P < 0.01; r = 0.733).

Similar findings are reported by Just et al. [11] who lend support to the fact that smoking is a risk factor for the development of facial wrinkling.

Smoking-induced changes in skin elastic fibers occur deeper in the dermis and are less intense than those associated with sun exposure, and must be considered as being related to cigarette smoke components that reach the reticular dermis through the bloodstream [12] . Hence, it is not surprising that smoking by itself is unable to change the macroscopic appearance of the sun-protected skin of smokers [13],[14] .

However, when the exposed skin was analyzed, a significant increase in the elastic tissue was found in both the papillary and the reticular dermis of smokers compared with nonsmokers [15] .

This explains, at least in part, the increase in facial wrinkling of smokers, which depends on the cumulative dose of tobacco consumed. This relationship is probably modulated by a genetic predisposition, because only one quarter of heavy smokers show abnormally severe wrinkling in their exposed skin [12],[16],[17] .

The increase in the number seems to be due to fiber fragmentation, as reported previously [8],[12] . The increase in the area occupied by elastic fibers in the reticular dermis could result from the synthesis of new normal or abnormal elastic tissue, from degradation of elastic fibers, from a decrease in dermal thickness or from external substance deposition.

This study showed that there is no difference in the epidermal or the dermal thickness between smokers and nonsmokers or external substance deposition in dermal elastic fibers of smokers, which is in agreement with other previous studies [8],[12],[18] .

Using immunohistochemistry, we found that all samples showed negative cytoplasmic staining for α-1-antitrypsin. Our result support the result found by Just et al. [10] who detected lack of immunostaining for plasma protease inhibitor reactivity in all the samples, which suggests the hypothesis that the increase in the area of elastic fibers in the reticular dermis in smokers is not due to newly synthesized elastic material, but due to their degradation, as in solar elastosis [19] .

The vascular structure of the skin may play a role in these elastotic changes. The cutaneous microvasculature is constricted by acute and long-term smoking. The decreased capillary and arteriolar blood flow may induce local ischemia and focal shunt of toxic substances to these tissues [20.

Smoker's elastosis may be partially due to biochemical effects on the elastic tissue. Previous studies have shown that cigarette smoke can increase the plasma neutrophils' elastase activity and the release of elastase from neutrophils [21] .

The presence of the criterion of wrinkles on the face in two of 15 smokers is an indicator that other factors contribute to its occurrence such as sun exposure and age of the participants. In addition, we cannot ignore the genetic factors.


  Acknowledgements Top


Conflicts of interest

None

 
  References Top

1.Wolf R, Lo Schiavo A, Ruocco V. Smoking out the skin. J Appl Cosmetol 1995; 13:1-14.  Back to cited text no. 1
    
2.Kennedy C, Bastiaens MT, Bajdik CD, Willemze R, Westendorp RGJ, Bouwes Bavinck JN. Effect of smoking and sun on the aging skin. J Invest Dermatol 2003; 120:548-554.  Back to cited text no. 2
    
3.Daniell HW. Smoker's wrinkles. A study in the epidemiology of 'crow's feet'. Ann Intern Med 1971; 75:873-880.  Back to cited text no. 3
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4.Leung W-C, Harvey I. Is skin ageing in the elderly caused by sun exposure or smoking? Br J Dermatol 2002; 147:1187-1191.  Back to cited text no. 4
    
5.Wright JL, Churg A. Smoke-induced emphysema in guinea pigs is associated with morphometric evidence of collagen breakdown and repair. Am J Physiol 1995; 268(Pt 1):L17-L20.  Back to cited text no. 5
    
6.Vlahovic G, Russell ML, Mercer RR, Crapo JD. Cellular and connective tissue changes in alveolar septal walls in emphysema. Am J Respir Crit Care Med 1999; 160:2086-2092.  Back to cited text no. 6
[PUBMED]    
7.Frances C, Boisnic S, Hartmann DJ, Dautzenberg B, Branchet MC, Le Charpentier Y, Robert L. Changes in the elastic tissue of the non-sun-exposed skin of cigarette smokers. Br J Dermatol 1991; 125:43-47.  Back to cited text no. 7
    
8.Lister RK, Barnes, R, Khorshid M, Cerio, R. Structural changes in nonsun-exposed skin of smokers: histological assessment of dermal thickness and elastin. Br J Dermatol 1999; 141(Suppl 55):24.  Back to cited text no. 8
    
9.Knuutinen A, Kallioinen M, Vähäkangas K, Oikarinen A. Smoking and skin: a study of the physical qualities and histology of skin in smokers and non-smokers. Acta Derm Venereol 2002; 82:36-40.  Back to cited text no. 9
    
10.Just M, Monsó E, Ribera M, Lorenzo JC, Morera J, Ferrandiz C. Relationships between lung function, smoking and morphology of dermal elastic fibres. Exp Dermatol 2005; 14:744-751.  Back to cited text no. 10
    
11.Just M, Ribera M, Monsó E, Lorenzo JC, Ferrándiz C. Effect of smoking on skin elastic fibres: morphometric and immunohistochemical analysis. Br J Dermatol 2007; 156:85-91.  Back to cited text no. 11
    
12.Frances C. Smoking and the skin. Int J Dermatol 1992; 31:779-780.  Back to cited text no. 12
    
13.Allen HB, Johnson BL, Diamond SM. Smoker's wrinkles? J Am Med Assoc 1973; 225:1067-1069.  Back to cited text no. 13
    
14.O'Hare PM, Fleischer Jr AB, D'Agostino Jr RB, Feldman SR, Hinds MA, Rassette SA, et al. Tobacco smoking contributes little to facial wrinkling. J Eur Acad Dermatol Venereol 1999; 12:133-139.  Back to cited text no. 14
    
15.Boyd AS, Stasko T, King LE, Jr Cameron GS, Pearse AD, Gaskell SA. Cigarette smoking-associated elastotic changes in the skin. J Am Acad Dermatol 1999; 41:23-26.  Back to cited text no. 15
    
16.Kadunce DP, Burr R, Gress R, Kanner R, Lyon JL, Zone JJ. Cigarette smoking: risk factor for premature facial wrinkling. Ann Intern Med 1991; 114:840-844.  Back to cited text no. 16
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17.Chung JH, Lee SH, Youn CS, Park BJ, Kim KH, Park KC, et al. Cutaneous photodamage in Koreans: influence of sex, sun exposure, smoking, and skin color. Arch Dermatol 2001; 137:1043-1051.  Back to cited text no. 17
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18.Whitmore SE, Sago NJG. Caliper-measured skin thickness is similar in white and black women. J Am Acad Dermatol 2000; 42:76-79.  Back to cited text no. 18
    
19.Mera SL, Lovell CR, Russell Jones R, Davies JD. Elastic fibres in normal and sun-damaged skin: an immunohistochemical study. Br J Dermatol 1987; 117:21-27.  Back to cited text no. 19
    
20.Tur E, Yosipovitch G, Oren-Vulfs S. Chronic and acute effects of cigarette smoking on skin blood flow. Angiology 1992; 43:328-335.  Back to cited text no. 20
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21.Weitz JI, Crowley KA, Landman SL, Lipman BI, Yu J. Increased neutrophil elastase activity in cigarette smokers. Ann Intern Med 1987; 107:680-682.  Back to cited text no. 21
[PUBMED]    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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