Prevalence of melanin pigmentation in a yemeni population and its relation to some risk factors

Objective: The present study aimed to explore the prevalence of melanin pigmentation in a sample of Yemeni population and its relation to some possible risk factors. Material and Methods: This crosssectional study was performed on 440 patients attending a private clinic. Printed questionnaires were introduced to the patients and a clinical examination was performed for each patient. The questionnaire included questions regarding demographic data, and questions regarding some common habits such as smoking, khat chewing and consuming of hot drinks. Melanin pigmentation was assessed regarding its presence, most affected areas and the numbers of affected quadrants. The data were managed and analyzed using SPSS software program. Descriptive statistics and inferential statistics were performed and the associations of melanin with risk factors were evaluated at P value < 0.05. Results: Four hundred and forty patients with mean age 29 ± 8.21 years were included in the study. Of them, 67.5% were fair-skinned, 26.8% were smokers, 48.2% were khat chewers and 33.6% were hot drinks consumers. The prevalence of melanin pigmentation was 62.7%, with class I represented 56.5% of cases. Males showed more prevalence (67.9%) of melanin pigmentation than females (57.7%) with no significant difference. Results also showed more prevalence of melanin pigmentation in patients > 25 years, and darkskinned patients. Regarding habits, smoking, khat chewing and hot drink consumption habits showed significant associations with melanin pigmentation. Whereas the association of khat chewing and hot drinks alone with melanin pigmentation showed no significant relationship with melanin pigmentation while, the merge effects of khat chewing and smoking habits together with melanin pigmentation showed RESUMO


O R I G I N A L A R T I C L E
Prevalence of melanin pigmentation in a yemeni population and its relation to some risk factors INTRODUCTION T he harmony of smile is determined not only by the shape, the position and the color of the teeth but also by the gingival tissue. Gingival health and appearance are essential components for an attractive smile, and removal of unsightly pigmented gingiva is the need for a pleasant and confident smile [1]. Normal physiologic color of gingiva is coral or salmon pink, with physiological variations that depend upon the degree of vascularization, epithelial thickness, the thickness of keratinized layer and the amount of melanin pigment [2]. Melanin, a brown pigment, is the most common natural pigment contributing to endogenous pigmentation of the gingiva. It is a non-hemoglobin-derived pigment formed by cells called melanocytes that are dendritic cells of neuroectodermal origin in the basal and spinous layers [1]. Oral pigmentation occurs in all races of man. There are no significant differences in oral pigmentation between males and females. The intensity and distribution of racial pigmentation of the oral mucosa is variable, not only between races but also between different individuals of the same race and within different areas of the same mouth [3,4]. The variation in color is due to amount, distribution and depth of melanin. Blue discoloration represents melanin deposits in the connective tissue whereas brown discoloration is associated with superficial deposition in the epithelium [5]. Attached gingiva is the most commonly effected intraoral site [6]. Gingival pigmentation often occurred in the labial area of anterior teeth. Excessive pigmentation in palatal mucosa as a result of tobacco smoke is a rare phenomenon, except in instances of reverse smoking.  Dummett, in 1945 [2] the degree of pigmentation is partially related to mechanical, chemical and physical stimulation. Melanin pigmentation in gingiva is correlated with active smoking: smokers displayed a greater propensity toward pigmentation than did nonsmokers. [3] Intake of antimalarial drugs including chloroquine and quinidine [7] is also associated with oral pigmentation. In darker skinned people, oral pigmentation increases but there is no difference in the number of melanocytes between fairskinned and dark-skinned individuals. The variation is related to differences in the activity of melanocytes [1]. Melanin pigmentation in human gingiva has been reported in several countries. To best of our knowledge, there is no published article about the prevalence of melanin pigmentation in Yemen. So, the present study was aimed to determine the prevalence of melanin (racial) pigmentation in a sample of Yemeni population and its relation with some suspected risk factors.

METHODOLOGY
A software program (G*Power v. 3.0.10, Los Angeles, CA, USA) was used to calculate the sample size. The power was set to be 90% using P value of 0.05. This revealed that 440 patients were needed for the study. Informed consents were obtained from all patients and printed questionnaire was introduced to each patient accompanied with clinical examination. The questionnaire included questions regarding demographic data (age and sex), questions regarding habits such as: smoking (prevalence and duration), khat chewing (prevalence, frequency, and duration), and consuming of hot drinks. Skin color was also evaluated through visual assessment as the visual assessment remains one of the "gold standard" methods of assessing skin color. [8]

Evaluation of melanin pigmentation
Melanin pigmentation was assessed in the oral cavity. Brownish or black pigmentation in gingiva was classified according to extent of pigmentation unit in the labial aspect of anterior teeth ( Figure 1). The clinical assessment of melanin pigmentation included its presence, most affected area and the numbers of affected quadrants. The method for the evaluation of the gingival pigmentation was followed according to modification of melanin index categories [3]. Upper right quadrant was coded as "1", upper left "2", lower left "3" and lower right "4" (Table I). Exclusion criteria included: a) Gingival pigmentation of hemoglobin, melanoid, melanoma and carotene [9], b) Gingival pigmentation adjacent to amalgam restoration, and c) Long-term usage of antimalarial drugs [7] and minocycline [10].
The data were managed and analyzed using SPSS software program for Windows. Descriptive statistics and inferential statistics were performed. Descriptive statistics were performed in terms of means, frequencies, and percentages for study variables. The association of melanin with risk factors were then evaluated. For comparison between both genders, age groups, khat chewing groups, smoking groups, and hot drinks consuming groups, non-parametric Mann-Whitney and Kruskal-Willis tests were used as appropriate. P value < 0.05 was set as significant for all tests. The Odds ratios were also calculated to find out the risk ratios for melanin pigmentation.

RESULTS
A total of 440 patients (225 "51.1%" males and 215 "48.9%" females) aged from 13 to 50 years with mean age 29 ± 8.21 years were included in the study. Of them, 297 (67.5%) were fair-skinned, 118 patients (26.8%) were smokers, 212 (48.2%) were khat chewers and 148 patients (33.6%) were hot drinks consumers. The prevalence of melanin pigmentation in the study population was found in 62.7% of the sample. Patients with class I pigmentation represented 56.5% and the four quadrants were affected in 40.6% of cases. More details are shown in Table II. Males showed more prevalence (67.9%) of melanin pigmentation than females (57.7%) with no significant difference (OR = 1.5; P = 0.313). Also no significant differences were found regarding type of melanin pigmentation and number of affected quadrants between males and females. Results also showed more prevalence of melanin pigmentation in patients older than 25 years compared to patients ≤ 25 years with significant difference (OR = 2.7; P = 0.018). However, class I pigmentation was more prevalent in patients ≤ 25 years. Dark-skinned patients were 3 times higher to had melanin pigmentation (79.7%) than fair-skinned patients (54.5%) with significant difference (OR = 3.3; P = 0.013). Class II was also associated with dark-skinned patients but with no significant difference (Table III).
Results showed that smoking habit had significant association with melanin Smokers also had more class II pigmentation while nonsmokers had more class I pigmentation with significant difference (P= 0.017). Smokers also had more affected quadrant than non-smokers but with no significant difference (P= 0.195). Duration of the smoking also had significant effect on oral melanin pigmentation. Smokers for more than 5 years had 98.6% prevalence of pigmentation with a significant difference than smokers less than 5 years. Also, longtime smokers had more prevalence of class II pigmentation (Table IV).
Khat chewing also showed significant association with melanin pigmentation, with more prevalence in khat chewers (OR = 4.1; P = 0.001). However, no significant difference in association with class I and II pigmentation or number of the affected quadrants (P > 0.05). Regarding frequency of khat chewing, there was no significant difference between daily or not-daily chewing, although the prevalence of melanin pigmentation was 2 times higher in khat chewers (OR = 2.0; P = 0.230). Duration of the khat chewing also had no significant effect on oral melanin pigmentation with more prevalence in long-time chewing (OR = 2.0; P = 0.405) ( Table V).
The effects of khat chewing alone with the exclusion of smoking and hot drink consumption showed that khat chewing only had no significant relationship with melanin pigmentation. Also, the effects of hot drinks consumption alone with the exclusion of smoking and khat chewing showed no significant relationship with melanin pigmentation although the hot drinks consumers had 1.8 times higher of melanin pigmentation. However, the effects of smoking and khat chewing with the exclusion of hot drink consumption showed significant relationship with melanin pigmentation (Table VI).
Results also showed that hot drinks consuming habit had significant association with melanin pigmentation (OR = 2.9; P = 0.022). However, hot drinks consumers didn't show significant difference regarding pigmentation types and number of affected quadrants (P > 0.05) (Table VII).

DISCUSSION
The intensity and distribution of racial pigmentation of the oral mucosa is variable, not only between races but also between different individuals of the same race and within different areas of the same mouth [3,4]. Brownish or black pigmentation in human gingiva has been reported in several countries. To the best of our knowledge, no published studies discussing this topic in Yemen are available. In this study, the prevalence of melanin pigmentation in Yemeni population was 62.7%. Patients with class I represented 56.5% and class II represented 43.5%. These results contradict another study carried out in Sweden where melanin pigmentation was 9.9% and reached in smokers to 18.5%. We thought that this contradiction is axiomatical because Swedish is Scandinavian Nordic nations. Other studies showed that melanin pigmentation in the dark-skinned African population was 100% and in Asians was between 30% and 98%. [11] We suppose that the high percentage in Yemeni population might due to various habits practiced in the country which may be predisposing factors to melanin pigmentation. Regarding the number of the affected quadrants, results showed that the four quadrants were affected in 40.6% of cases. Furthermore, we tried to evaluate a possible relationship between any risk factor and most affected quadrant. Our results showed approximately equal distribution of melanin between all quadrants without any obvious relationship between risk factors and melanin distribution. To the best of our knowledge, no studies were found to evaluate these parameters. So that, comparison with the literature is limited. In earlier Swedish studies, smoker's melanosis was more frequent among women than men when smoking comparable amounts of cigarettes. This difference was credited to the female sex hormones, which are known to prime the melanocytes to melanin production. [12] In this study, males showed more prevalence (67.9%) of melanin pigmentation than females (57.7%) with no significant difference. This can be explained by the fact that men have more tendency to smoking and khat chewing than females in Yemeni society. It is worthy to mention that our results are consistent to with the study of Hedin CA and Axell T [13].
Epidemiological studies showed that the percentage of pigmented individuals is higher in adults than in juveniles. [14] It could be speculated upon the oral mucosa is exposed to more melanocyte stimulating factors such as benzopyrene and nicotine. [15] These results are consistent with the results of the present study in which people > 25 years old had more melanin pigmentation with more CL II pigmentation than patients ≤ 25 years old. This age is considered as the mean age of adulthood. The relationship of skin color with melanin pigmentation was also evaluated in this study where dark-skinned patients showed higher melanin pigmentation (79.7%) than fair-skinned patients (54.5%). Dark-skinned people had more CL II melanin pigmentation while fair-skinned people had more CL I pigmentation. These results are consistent with that of Dummett CO. [16] who showed that the distribution of oral pigmentation in black individuals was 60% in the gingiva.
Melanin pigmentation is probably genetically determined, but as suggested by Dummett in 1945 [2] the degree of pigmentation is partially related to mechanical, chemical and physical stimulation. The results of this study showed that the prevalence of melanin pigmentation was (55.2%) in nonsmokers and (80.5%) in smokers with more CL II pigmentation in smokers. These results are consistenet with Suraj Multani's study [17] in India, who reported melanin pigmentation in 73.4% non-smokers and 100% in smokers, and the study of Haresaku et al. who reported 87% of melanin pigmentation in smokers [18]. However, our results are not convergent with other studies where it was found that the prevalence of melanin pigmentation was 15% to 37% for non-smokers among Japanese workers [19]. On the other hand, prevalence was higher in Asian populations (34.6%) in comparison with Ashkenazi (0.8%) and Sephardic (11.1%) Jews. These differences may be due to ethnic differences and presence of different habits in each nation. In a Turkish study, the frequencies of melanin pigmentation were significantly higher in current smokers than in those without any smoking habits which is consistent with results of our study [20].
As noticed from our results, duration of the smoking also had significant association on oral melanin pigmentation. Smokers for more than 5 years had more prevalence of pigmentation with a significant difference than smokers less than 5 years. This results may be logical because of the length of smoking period the gingiva is exposed to more irritations. Regarding khat chewing habit, the percentage of khat chewers in Yemen population was 48.2% which in accordance to another study done in Kingdom of Saudi Arabia [21]. It was shown that melanin pigmentation was associated with 78.3% of khat chewers. This is also in agreement with other studies that attributed melanin discoloration to the chemical components of khat [22][23][24]. Duration of the khat chewing also had no obvious relationship with oral melanin pigmentation. Khat chewers for more or less than 5 years had approximate melanin pigmentation with no significant difference. Based on our knowledge, no documented studies in the literature discussing this topic.
We supposed that hot drinks may have thermal irritative effect to melanocytes resulting in increase of melanin pigmentation in the mouth. Our results showed that hot drinks consuming habit had an association with melanin pigmentation. In Yemeni community, tobacco smoking, khat chewing and hot drink intake are habitual to be consumed concomitantly. So, the presence of melanin pigmentation may be due the cumulative effects of these factors. Thus, we evaluated the effects of khat chewing and hot drinks alone in absence of other risk factors. The results showed that there were not relationships between khat chewing and hot drinks when consuming lonely with melanin pigmentation which emphasis our suggestion. Finally, because most Yemeni people accustomed to abuse khat and tobacco together, we evaluated the merged effect of both habits which showed strong relationship between smoking and khat chewing together with melanin pigmentation. So, it is recommended that furthers studies should be carried out to assess the lonely effects of khat chewing and hot drinks consumption with larger study samples with exclusion of other suspected risk factors.

CONCLUSION
Within the limitations of this study, it can be concluded that: