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ORIGINAL ARTICLE
Year : 2022  |  Volume : 35  |  Issue : 2  |  Page : 445-450

Evaluation of glutathione peroxidase enzyme in patients with melasma: a clinical and immunohistochemical study


1 Department of Dermatology, Andrology and STDs, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission26-Sep-2021
Date of Decision22-Nov-2021
Date of Acceptance23-Nov-2021
Date of Web Publication27-Jul-2022

Correspondence Address:
Wesam S. A. El Ashkar
Berkit Elsabaa, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_183_21

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  Abstract 


Objective
To evaluate the role of glutathione peroxidase (GPX1) in melasma.
Background
Melasma is a common acquired disorder of hyperpigmentation. GPX1 is an intracellular enzymatic antioxidant, and its levels are used as a major parameter representing oxidative damage that occurs in melasma. However, the clear association between GPX1 expression and melasma remains unknown.
Patients and methods
This prospective case–control study was conducted on 20 cases having melasma and 20 normal age-matched and sex-matched healthy volunteers. All sections were immunohistochemically stained for GPX1 antibody.
Results
There was significant difference between melasma cases and normal skin as regards GPX1 epidermal distribution (P = 0.001) and intensity (P = 0.005). Also there was a statistically significant association between epidermal intensity and distribution of GPX1 and the severity of melasma assessed by the modified Melasma Area and Severity Index (MASI) score. Moreover, there was statistically significant difference between epidermal intensity and distribution of GPX1 and the degree of dermal inflammation. Also, there was a tendency of melasma cases receiving hormonal therapy to show positive dermal expression of GPX1 than cases not receiving therapy (P = 0.07).
Conclusion
GPX1 was expressed diffusely in the epidermis of all control cases (100%), eight of them were of mild intensity and 12 were of moderate intensity while expressed in most of melasma cases in a focal form with mild intensity expression.

Keywords: glutathione peroxidase, immunohistochemistry, melasma, oxidative stress


How to cite this article:
Khaled HN, El Ashkar WS, Abdou AG. Evaluation of glutathione peroxidase enzyme in patients with melasma: a clinical and immunohistochemical study. Menoufia Med J 2022;35:445-50

How to cite this URL:
Khaled HN, El Ashkar WS, Abdou AG. Evaluation of glutathione peroxidase enzyme in patients with melasma: a clinical and immunohistochemical study. Menoufia Med J [serial online] 2022 [cited 2024 Mar 28];35:445-50. Available from: http://www.mmj.eg.net/text.asp?2022/35/2/445/352140




  Introduction Top


Melasma is a common acquired disorder of hyperpigmentation[1], which is characterized by symmetric, brownish-gray macules and patches on the face and sometimes the neck, chest, and the forearm[2]. Melasma can be further characterized by the depth of involvement, and divided into four categories: epidermal, dermal, mixed, and indeterminate[3].

There is currently no definite etiology except for numerous elements including bright radiation, hormonal modifications inside the estrogen or progesterone pathways, hereditary inclination, and additionally aggravation that have all been involved[4]. Also, melasma has a vascular segment as certain investigations have discovered melasma-influenced skin to have expanded vascularity[5].

The common outcome from these diverse triggers is an increased synthesis of melanosomes in melanocytes and an increased transfer of melanosomes to keratinocytes[3].

Free radical injury has been proposed as a potential pathogenic incentive in the causation of melasma. Glutathione peroxidase (GPX1) is an intracellular enzymatic antioxidant, and its levels are used as a major parameter representing oxidative damage. GPX1 is the key enzyme for the neutralization of hydrogen peroxide responsible for the clearance of free oxygen radicals. It converts hydrogen peroxide to oxygen and water, thus attenuating the harmful effects of hydrogen peroxide[6].

The aim of this work was to evaluate the role of GPX1 in melasma.


  Patients and methods Top


The study had been conducted on 40 individuals divided into two groups consisting of 20 patients presented with melasma and 20 normal age-matched and sex-matched healthy volunteers. Patients had been selected from the Dermatology Outpatient Clinic, Faculty of Medicine, Menoufia University Hospital between December 2019 and March 2020. Control group had been selected from the Plastic Surgery Department, Faculty of Medicine, Menoufia University.

A written consent form approved by the local ethics research committee in the Faculty of Medicine, Menoufia University was signed by every participant before the study initiation.

Two-millimeter punch biopsy had been taken under complete aseptic condition, preceded by an injection of 2% lignocaine local anesthesia from the involved skin of melasma patients and from matched site of control individuals.

Biopsies had been fixed in neutral formalin 10% and was submitted to routine tissue processing ending with paraffin-embedded blocks formation in the Pathology Department, Faculty of Medicine, Menoufia University.

Several paraffin sections, each 4 μm thick was cut from each block; one of them had been stained with hematoxylin and eosin for the evaluation of pathological changes, and other sections had been used for the immunostaining procedure for detection of GPX1 enzyme.

The technique utilized for immunostaining was a streptavidin-biotin-enhanced framework. The antibody used was rabbit polyclonal antibody, anti-GPX1, it was received as 80 μl concentrated and diluted to 1: 50 (Cat No.:abx034320) (Abbexa Ltd, Cambridge, UK).

Slides were subjected to deparaffinization and rehydration. Antigen recovery was performed by bubbling in citrate cushion saline (pH 6), trailed by cooling at room temperature. Endogenous peroxidase was blocked by incubation with H2O2, 3%. The primary antibody was incubated overnight at room temperature, and then the secondary antibody [ready-to-use, UltraVision Detection System anti-polyvalent HRP/DAB (Abbexa Ltd)] was applied with DAB as a chromogenic substrate and Mayer's hematoxylin as a counterstain.

Hepatocellular carcinoma was used as a positive control for GPX1 antibody. Substitution of the primary antibody in the staining method with a blocking buffer was incorporated as a negative control.

Statistical analysis

Data was analyzed using SPSS 23 IBM Corp., Armonk, NY and EpiCalc 2000 programs. Statistics was divided into two parts: (a) descriptive statistics: in the form of mean, SD, median, range, numbers, and percentages. (b) Analytical statistics: included χ2 test, Fisher's exact test, Student's t test, Mann–Whitney test (U), and Kruskal–Wallis test. P value less than 0.05 was considered statistically significant.


  Results Top


The age of patients ranged between 29 and 50 years, with a mean ± SD of 37.35 ± 5.96 years, while in the control group age ranged between 30 and 41 years with a mean ± SD of 35.6 ± 3.8 years.

The residence of melasma patients was divided into 15 patients living in rural areas representing 75% while five patients living in urban areas representing 25% of all cases, while residence of the control group was divided into 12 (60%) cases living in rural areas while eight (40%) cases were living in urban areas.

Regarding educational level, seven (35%) patients were highly educated while 13 (65%) patients were of low educational level, while in the control group eight (40%) cases were highly educated while 12 (60%) cases were of low educational level.

Regarding patient's occupation and site of work, 10 (50%) patients were more sun exposed while the rest of them were less sun exposed (50%), while in the control group 12 (60%) cases were more sun exposed while eight (40%) cases were less sun exposed.

The number of married cases was 19 (95%) patients, 12 of them were on hormonal therapy, and only one patient was not married, while in the control group 17 (85%) cases were married, eight of them were on hormonal therapy, and three (15%) were not married.

According to classification of their skin type, four (20%) patients were of skin type 3, 10 (50%) patients were of skin type 4, and six (30%) patients were of skin type 5, while in the control group nine (45%) cases were of skin type 4 while 11 (55%) were of skin type 5.

Regarding the modified MASI score, it ranged between 0.6 and 5.4 with a mean ± SD of 2.81 ± 1.53 and a median of 2.7.

Regarding the degree of dermal inflammation, most of the cases showed mild degree, 12 (60%) cases; moderate degree was seen in seven (35%) cases; and severe degree was seen in one (5%) case. Melanin in the epidermis was visible in basal layers in all cases (100%).

GPX1 was expressed diffusely in the epidermis of all control cases (100%), eight of them were of mild intensity, and 12 were of moderate intensity, while it was expressed in nine cases in the dermis of control [Figure 1].
Figure 1: Normal skin showing moderate cytoplasmic and diffuse epidermal GPX1 expression (immunohistochemical staining ×100). GPX1, glutathione peroxidase.

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GPX1 was expressed in the epidermis of 19 (95%) melasma cases out of 20 cases, which was focal in 16 (80%) cases and diffuse in three (15%) cases. The intensity of epidermal GPX1 expression was mild in 16 (80%) cases and moderate in three (15%) cases. Regarding GPX1 dermal expression, it was expressed in eight (40%) cases localized to dermal inflammatory cells [Figure 2].
Figure 2: Skin of melasma case showing mild focal cytoplasmic expression of GPX1 (immunohistochemical staining ×200). GPX1, glutathione peroxidase.

Click here to view


There was significant difference between melasma cases and normal skin as regards GPX1 epidermal distribution (P = 0.001) and intensity (P = 0.005), as diffuse expression and moderate intensity were in favor of the control group compared with melasma cases [Table 1].
Table 1: Comparison between melasma and control cases regarding glutathione peroxidase expression

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There was a statistically significant association between epidermal distribution of GPX1 and severity of melasma assessed by the modified MASI score, as a high mean and median score was associated with diffuse epidermal expression compared with cases showing focal expression (P = 0.02); moreover, mild and moderate inflammation were associated with focal GPX1 expression compared with cases showing severe inflammation which were associated with diffuse epidermal expression of GPX1 [Table 2].
Table 2: Relationship between epidermal distribution of glutathione peroxidase and clinicopathological data

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On the other hand, no significant association was found between GPX1 epidermal distribution and other parameters.

There was a statistically significant association between epidermal intensity of GPX1 and severity of melasma assessed by the modified MASI score, as high mean and median scores were associated with moderate epidermal intensity compared with cases showing mild intensity (P = 0.02); moreover, there was a statistically significant difference between epidermal intensity of GPX1 and the degree of dermal inflammation, as mild and moderate inflammation were associated with mild epidermal intensity of GPX1 compared with cases showing severe inflammation which were associated with moderate epidermal intensity of GPX1 (P = 0.035); however, no significant relations were detected regarding other parameters.

There was a tendency of melasma cases receiving hormonal therapy to show positive dermal expression of GPX1 than cases not receiving therapy (P = 0.07); however, no significant relationships were detected regarding other parameters.

There was no significant association between epidermal and dermal expression of GPX1 in melasma cases.


  Discussion Top


Melasma is a characteristic example of facial hyperpigmentation, occurring fundamentally on the brows, cheeks, and chin in a mask-like distribution. Women are the most affected sex especially darkly pigmented races. The pathogenesis of melasma is not completely seen, but ultraviolet light exposure, pregnancy, estrogen ingestion, and family history are well-recognized associations[7].

Antioxidants are often reducing agents that exist both intracellularly and extracellularly and have the ability to respond to free radicals and reactive species, limiting their actions and in this way delaying or preventing oxidative stress[8].

GPXs are individuals from a group of antioxidant enzymes that scavenge hydrogen peroxide in the presence of reduced glutathione (GSH). GPX1 is a selenium-dependent enzyme that contains a selenium atom incorporated within the selenocysteine residue[9].

All of these GPX enzymes catalyze the reduction of H2O2 or organic hydroperoxide (ROOH) to water (H2O) and alcohol (ROH), respectively, using reduced GSH or in some cases thioredoxin or glutaredoxin as the electron donor. At the point when GSH is the electron donor, it gives a pair of hydrogen ions and GSH is oxidized to glutathione disulfide (GSSG)[10].

In the current study, we aimed to investigate the role of GPX1 in the pathogenesis of melasma through their immunohistochemical localization in skin lesions of this disease and to correlate their expression with the clinical and the histopathological information.

In the present study, the age of the studied patients ranged between 29 and 50 years, with a mean ± SD of 37.35 ± 5.96 years. This was in agreement with Al-Hamdi et al.[7], where melasma cases were more prevalent in the third and fourth decades of life.

Siddique et al.[11] reported that the mean age of their studied patients was 32.43 ± 6.70 years which is nearly similar to ours.

In this work, all the studied melasma cases were females. Similarly, females were the dominant sex in other studies such as those of Rokhsar and Fitzpatrick[12], Dogra et al.[13], and Al-Hamdi et al.[7].

We found that the incriminated triggering factors were sunlight exposure (50%), pregnancy (95%), and oral contraceptive pills (29.4%). This was in agreement with the study carried out by Guinot et al.[14], where the main triggering factors were sunlight, pregnancy, and oral contraceptive pills in the same order.

Moreover, we found that most of the cases showed a mild degree of dermal inflammation (60%), while in 35% of cases a moderate degree of inflammation was seen and 5% of cases showed severe degree of inflammation. Melanin in the epidermis was visible in the basal layer in all cases (100%).

This goes with Pandya et al.[15] and Prignano et al.[16], who reported that melasma usually starts as epidermal inflammation and hyperpigmentation and after sometime, as a result of factors that damage the basement membrane, the melanin drops down to the dermis. Therefore, epidermal melasma will be transformed to dermal or combined type with persistent dermal melanophages Also Sarkar et al.[17] who studied melasma in men stated that chronic inflammatory infiltrate was seen in 30% of melasma cases.

In addition, there was a statistically significant relationship between epidermal intensity of GPX1 and the degree of dermal inflammation, as mild and moderate inflammation were associated with mild epidermal intensity of GPX1 compared with cases showing severe inflammation, which were associated with moderate epidermal intensity of GPX1 (P = 0.035). Also, there was a statistically significant relationship between epidermal GPX1 distribution and the degree of dermal inflammation (P = 0.035) as mild and moderate inflammation was associated with focal GPX1 epidermal expression compared with cases showing severe inflammation, which were associated with diffuse epidermal expression of GPX1.

The association between intense GPX1 expression and dermal inflammation, detected in the present work, can be explained by the association of GPX1 with inflammatory and immune responses that was implicated in the pathogenesis of a wide range of human skin diseases[18].

In addition, there was a significant correlation between epidermal distribution of GPX1 and severity of melasma assessed by the modified MASI score (P = 0.02), as a high mean and median score was associated with diffuse epidermal expression compared with cases showing focal expression. Also, there was a statistically significant association between epidermal intensity of GPX1 and severity of melasma assessed by the modified MASI score (P = 0.02).

In the current study, GPX1 was expressed diffusely in the epidermis of control cases more than of the dermis (P > 0.001). This finding was consistent with Shindo et al.[19] who measured enzymic and nonenzymic antioxidants in human epidermis and dermis. They found that among the enzymatic antioxidants, the activities of GPX were higher in the epidermis compared with the dermis by 61%.

In this way the antioxidant capacity of the human epidermis is far more noteworthy than that of dermis. As the epidermis makes the outermost 10% of the skin and acts as the initial barrier to oxidant assault, it is may be not unexpected that it has higher levels of antioxidants.

The present work demonstrated that GPX1 was focally expressed with mild intensity in the epidermis of 16 melasma cases representing 80% and diffusely expressed with moderate intensity in the epidermis of three cases representing 15%.

Limitation and recommendation

Our results should be viewed in the light of the limited number of patients in our study. However, further studies on larger scales are recommended to expand our findings, investigate the role of GPX1 in different pathological types (epidermal, dermal, and mixed) and different clinical patterns (malar, centrofacial, and mandibular) of melasma, and to clarify the association between GPX1 and dermal inflammation and study the expression of GPX1 in different age groups of melasma cases.


  Conclusion Top


GPX1 was expressed diffusely in the epidermis of all control cases; eight of them were of mild intensity and 12 were of moderate intensity while in most of melasma cases it is focally expressed with mild intensity. These results may suggest the protective effect of GPX1 against oxidative stress which occurs in melasma. Further studies are recommended to clarify the exact role of GPX1 in melasma.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Pandya A, Berneburg M, Ortonne JP, Picardo M. Guidelines for clinical trials in melasma. Pigmentation disorders academy. Br J Dermatol 2006; 156:21–28.  Back to cited text no. 15
    
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    Figures

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