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Abstract
Introduction: Viral warts are a group of dermatological diseases caused by human papillomavirus (HPV). Several studies have demonstrated an association between HPV infections and oxidative stress. Thiols are important components of cellular redox homeostasis as antioxidant molecules in the organism. Aim: This study aimed to investigate the role of oxidative stress in patients with HPV infection by analyzing native thiol/disulfide homeostasis. Material and Methods: Forty-two patients with HPV infection and 40 healthy subjects were analyzed for the levels of native thiols, total thiols, and disulfide. Disulfide/native thiol, disulfide/total thiol, and native thiol/total thiol ratios were also calculated. Results: Disulfide and total thiol levels were higher in the patients compared to the healthy controls. The disulfide/native thiol ratio was also higher in the patient group. Native and total thiol levels decreased with the increasing duration of the disease. Conclusion: The native thiol/disulfide homeostasis was shifted toward disulfide in the patients' group, indicating the existence of oxidative stress in HPV infection.
Keywords: Human papillomavirus infection, oxidative stress, thiol/disulfide homeostasis, thiols, viral warts
Introduction 
Viral warts are a group of dermatological diseases caused by human papillomavirus (HPV) that affect the skin and mucous membranes. HPV infections have many clinical forms, mainly including verruca vulgaris, verruca plantaris, verruca planus, verruca anogenitalis, and epidermodysplasia verruciformis.[1] HPV is a double-stranded DNA virus that has more than 100 subtypes. Skin lesions are usually caused by HPV-1, HPV-2, HPV-4, and HPV-10, whereas HPV-6, HPV-11, HPV-16, and HPV-18 are detected in anogenital lesions. The HPV-16 and HPV-18 subtypes are referred to as high-risk HPV because of their association with intraepithelial neoplasia.[2] HPV lesions usually regress spontaneously within a few years due to innate immunity. However, they can sometimes become chronic and even progress to cancer rarely.
Like other pathogens, HPV also triggers an immune response, leading to inflammation and oxidative stress in the infected organism.[3] Recent studies have demonstrated the association between HPV infections and oxidative stress.[4],[5] Under oxidative stress, the endogenous antioxidant system minimizes the deleterious effects by counteracting the free radicals. It has been suggested that the balance between oxidants and antioxidants controls the spontaneous regression or chronification of HPV infection, as well as cancer development.[6]
In recent years, studies have shown the association between inflammation, oxidative stress, and thiol metabolism.[7],[8],[9] Thiols are a class of organic compounds containing a sulfhydryl group (–SH) on their active region. They play a key role in the eradication of the reactive oxygen species (ROS) via enzymatic and non-enzymatic pathways.[10],[11] Under oxidative stress, the sulfhydryl groups of thiols are oxidized and form disulfide bonds. Thus, they protect the tissue from oxidative damage by acting as ROS scavengers. This reaction is reversible and disulfide bonds are reduced back. In this way, the dynamic thiol/disulfide balance that is critical for the cellular redox homeostasis is maintained. Dynamic thiol/disulfide homeostasis also has crucial roles in apoptosis, detoxification, stabilization of protein chemical structures, and cellular signaling.[12]
An increase in the oxidant levels and a decrease in the thiol levels have been demonstrated in some viral and bacterial diseases.[13],[14],[15] However, the serum thiol levels in HPV infections have not been investigated yet.
The aim of this study was to investigate the dynamic thiol/disulfide balance in patients with HPV infection and evaluate the association between HPV and oxidative stress in terms of this balance.
Materials and Methods This study is a single-center, prospective, cross-sectional study. Forty-two patients over the age of 18 who applied to our dermatology outpatient clinic and were diagnosed with multiple verrue and 40 healthy controls were included in the study. The control group was selected from people who applied for cosmetic purposes. Patients' age, gender, verrue type and localization, presence of systemic disease (diabetes, hypertension, liver and kidney disease, immune deficiency, cancer, etc.), and family history of verrue were questioned and recorded. Those who had systemic disease, who took medication in the last 3 months, and those who used cigarettes and alcohol were not included in the study. The presence of systemic disease was determined according to the history and recent tests (whole blood, biochemistry). Patients were randomly selected from the outpatient clinic. The work was completed in 6 months. The permission was obtained from the local ethics committee before the study. It was carried out in accordance with good clinical practices and the Declaration of Helsinki. Consent was obtained from the patient and control groups.
Blood samples were taken from all subjects into EDTA tubes following 8–10 h of fasting. The samples were immediately centrifuged at 1500 rpm for 10 min and stored at − 80°C. The serum native thiol and total thiol levels were analyzed in all samples simultaneously using the fully automated spectrophotometric method as described by Erel and Neşelioğlu.[7] The difference between total thiol and native thiol levels was divided by two for calculating dynamic disulfide levels. Disulfide/native thiol, disulfide/total thiol, and native thiol/total thiol ratios were also calculated. The correlations of thiol and disulfide levels with clinical parameters were analyzed.
Statistical analysis
Statistical analysis of the study was performed using SPSS for Windows 20 (IBM SPSS Inc., Chicago, IL) program.
A statistical comparison between the groups was conducted using the t test, Pearson's correlation analysis, and Chi-square test. The Chi-square test was performed for qualitative variables. The relationship between the parameters was assessed using Pearson's and Spearman's correlations and analysis. P < 0.05 were considered statistically significant. Categorical variables were expressed as numbers and percentages.
Results We evaluated 42 patients in this study. We diagnosed verruca vulgaris in 11 (26%), verruca plantaris in 15 (36%), verruca anogenitalis in 14 (34%), verruca planus in one (2%), and verruca filiformis in one (2%) of them. The mean duration of the disease was 19.6 ± 3.8 months. Demographic characteristics of the subjects are shown in [Table 1].
Table 1: Demographic characteristics of the HPV-infected patients and control groupThe thiol and disulfide levels and the calculated ratios are shown in [Table 2]. The mean disulfide and total thiol levels as well as disulfide/native thiol ratios were significantly higher in the patients as compared with the controls (P < 0.05 for all). There were no significant correlations between the age and the native thiol or total thiol levels in the control group (r: −0.28, P > 0.05 and r: −0.29, P > 0.05, respectively). A significant negative correlation was found between the age and the native or total thiol levels in the patients group (r: −0.47, P = 0.002 and r: −0.48, P = 0.001, respectively). This finding was concordant with the decrease in the native and total thiol levels by the duration of the disease (r: −0.40, P = 0.009 and r: −0.42, P = 0.005, respectively).
Table 2: Comparison of serum thiol and disulfide levels as well as ratios between the HPV infected patients and control group Discussion The HPV infection, like all the other viral infections, activates both humoral and adaptive immune responses, which trigger inflammation and oxidative stress in the organism.[2],[3],[16] During inflammation, leukocytes infiltrate the area of infection to eliminate it, accompanied by the release of a large number of chemokines.[2] Chronic inflammatory reaction increases the levels of reactive oxygen and nitrogen species (RONS), which oxidize proteins, lipids, and DNA.[16] In addition to these oxidant molecules, HPV early (E1–E7) and late (L1 and L2) proteins also play a role in the inflammatory process and oxidative stress induced by HPV. Oxidative stress is also required for viral genome encapsidation as well as for the elimination of viral infection.[6]
There must be a balance between oxidants and antioxidants, which is called cellular redox status. This homeostasis activates the antioxidant system when the cells are exposed to RONS and prevents oxidative damage to the cell. The RONS regulate cellular homeostasis and cell proliferation, differentiation, and signaling as secondary messengers in many signal transduction pathways.[16],[17] When the RONS levels are much higher than the enzymatic and nonenzymatic antioxidants, or if the antioxidant system is insufficient, oxidative stress status occurs. Prolonged oxidative stress leads to the oxidative damage of lipids, proteins, and nucleic acids, resulting in apoptosis, necrosis, and rarely, tumorigenesis.[16]
Enzymatic oxidants include superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT), while the most important non-enzymatic antioxidants are glutathione (GSH) and vitamin A, C, and E.[17] Previous studies have shown increased levels of CAT, SOD, glucose-6 phosphate dehydrogenase (G6PD), malondialdehyde (MDA), and paraoxonase-1 (PON1) in HPV patients with genital and non-genital warts that indicate oxidative stress.[4],[18],[19]
Thiols are physiological free-radical scavengers that eliminate RONS via enzymatic and non-enzymatic mechanisms. Plasma thiol pool consists of albumin, sulfur-containing amino acids such as cysteine (Cys) and homocysteine, GSH, and other sulfur-containing molecules.[10],[20] The GSH and Cys residues in the active regions of proteins such as thioredoxin, glutaredoxin, and peroxiredoxin detoxify RONS by oxidizing the thiol groups (RSH) into disulfide bonds (RS-SR).[11],[20]
Thiols are important molecules for viral infections in the organism. For instance, the fusion between the viral envelope and host cell is only possible in the presence of optimum thiol levels. Protein disulfide isomerase (PDI), which is an enzyme located on the surface of the cell, regulates the oxidized/reduced thiol (RS-SR/RSH) status of integral proteins. If this enzyme is blocked by specific antibodies, virus-host cell fusion can be prevented.[21] Similarly, an oxidative environment induces disulfide interactions between L1 proteins, resulting in HPV capsomere assembly.[22]
Cys residues are considerably reactive due to a nucleophilic characteristic of the thiolate group. Many HPV types contain high levels of Cys residues. Those in the E7 oncoprotein of HPV 11, HPV 16, and HPV 18 have been investigated experimentally. It is known that when Cys residues are exposed to ROS, they form disulfide bonds and regulate protein transport to the nucleus from the cytoplasm.[23]
In our study, serum thiol levels were measured and thiol/disulfide balance was evaluated in HPV patients. Disulfide and total thiol levels as well as disulfide/native thiol ratios were higher in the patient group.
Native thiol/disulfide homeostasis had been analyzed previously in various infectious diseases by using the same method.[13],[14],[15] In accordance with those studies, we found a shift in the native thiol/disulfide balance toward disulfides. These results indicate the presence of oxidative stress in HPV-infected patients. There was a negative correlation between the levels of native or total thiols and the duration of the disease. Whether lack of native thiol facilitates chronification of the disease or native thiol level decreases by consumption in chronic disease is unclear. Based on these results, local or systemic thiol-containing treatments can be considered for the management of HPV infections.
Limitations of the study
The number of lesions and thiol level could not be compared. HPV typing could not be performed. Thiol levels could not be measured in the tissue.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient (s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References 
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